Human Germline Gene Therapy: Scientific, Moral and Political Issues (Tissue Engineering Intelligence Unit)

MEDICAL I N T E L L I G E N C E U N I T

9

David B. Resnik • Holly B. Steinkraus Pamela J. Langer

Human Germline Gene Therapy: Scientific, Moral and Political Issues

R.G. LANDES C O M P A N Y

MEDICAL INTELLIGENCE UNIT 9

Human Germline Gene Therapy: Scientific, Moral and Political Issues David B. Resnik, Ph.D. East Carolina University

Holly B. Steinkraus, Ph.D. University of Wyoming

Pamela J. Langer, Ph.D. University of Wyoming

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

MEDICAL INTELLIGENCE UNIT 9 Human Germline Gene Therapy: Scientific, Moral and Political Issues R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1999 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-586-0

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Resnik, David. Human germ-line therapy : scientific, moral, and political issues David Resnik, Pamela J. Langer, Holly B Steinkraus. p. cm. -- (Medical intelligence unit) Includes bibliographical references and index. ISBN 1-57059-586-0 (alk. paper) 1. Gene therapy. 2. Gene therapy--Social aspects. 3. Human reproductive technology. 4. Human reproductive technology--Social aspects. I. Langer, Pamela J. II. Steinkraus, Holly B. III. Series. [DNLM: 1. Gene Therapy. 2. Germ-line Mutation. 3. Ethics, Medical. 4. Public Opinion. QH 442 R434h 1999] RB155.8.R48 1999 616'.042--dc21 DNLM/DLC 98-43505 for Library of Congress CIP

MEDICAL INTELLIGENCE UNIT 9 PUBLISHER’S NOTE

Human Germline Gene Therapy: Scientific, Moral and Political Issues

R.G. Landes Company produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental. The authors of our books are acknowledged leaders in their fields. Topics are unique; almost without exception, no similar books exist on these topics. Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience. Most of our books are published 90 to 120 days of receipt of East Carolinawithin University the manuscript. We would like to thank our readers for their continuing interest and welcome any comments or suggestions they may have for future books.

David B. Resnik, Ph.D.

Holly B. Steinkraus, Ph.D. University of Wyoming

Stephanie Stewart

Production Manager Pamela J. Langer, Ph.D. R.G. Landes Company University of Wyoming

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

CONTENTS 1. From Genes to Disease .............................................................................. 1 Scope of the Term Gene Therapy ........................................................... 1 Some Common Misconceptions ............................................................ 2 Gene Expression ...................................................................................... 4 Genes, Mutations and Inheritance ......................................................... 8 Summary and Conclusion .................................................................... 14 2. Alternatives to Human Germline Gene Therapy .................................. 17 Introduction........................................................................................... 17 Genetic Disease and Testing ................................................................. 18 When In Vitro Fertilization Is Not an Acceptable Option .................. 20 When In Vitro Fertilization and Embryo Selection Are Acceptable Options .................................................................... 24 Some Methods of Analysis in Single-cell Genetics .............................. 35 Prenatal Diagnosis ................................................................................. 39 When Germline Genetic Manipulation Is an Option ......................... 41 Summary and Conclusions ................................................................... 42 3. Gene Delivery Systems ............................................................................ 47 Introduction........................................................................................... 47 Targets of Gene Therapy ....................................................................... 47 Transgene Destination and Expression ................................................ 50 Consequences of Transgene Destination and Expression in SGT or HGLGT ............................................................................. 55 Methods of Gene Transfer .................................................................... 56 Summary and Conclusions ................................................................... 66 4. Challenges of Human Germline Gene Therapy .................................... 71 HGLGT Targets ..................................................................................... 71 Transgenic Animals ............................................................................... 72 Examples of Approaches in HGLGT .................................................... 73 Technical Obstacles in HGLGT ............................................................ 79 SGT Obstacles That Are Not HGLGT Obstacles ................................. 82 Summary and Conclusions ................................................................... 83 5. Therapy vs. Enhancement and Other Pertinent Distinctions .............. 85 Some Ethical Questions in Embryo Selection and HGLGT ................ 86 Gene Therapy vs. Genetic Enhancement ............................................. 86 Parental Choice vs. State Controls ........................................................ 89 Genetic Determinism ............................................................................ 89 Moral and Political Decision Making ................................................... 90 6. Potential Benefits and Harms of Human Germline Gene Therapy ..... 93 The Logic of Benefit/Harm Arguments ................................................ 93 Medical Benefits/Harms ........................................................................ 95 Evolutionary Benefits/Harms ............................................................. 101

Psychosocial Benefits/Harms .............................................................. 105 Economic Benefits/Costs .................................................................... 107 Conclusion: Optimism, Pessimism, or Prudence? ............................ 109 7. Human Germline Gene Therapy, Rights and Responsibilities .......... 113 What Are Rights and Responsibilities? ............................................... 113 Parental Rights ..................................................................................... 116 HGLGT and Harms to Unborn Children .......................................... 117 Is Nonexistence a Harm? ..................................................................... 121 Harms to Future Generations ............................................................. 122 Other Rights Considerations .............................................................. 124 Conclusion ........................................................................................... 126 8. Human Germline Gene Therapy and Justice ...................................... 129 What Is Justice? .................................................................................... 129 HGLGT and Human Equality ............................................................ 131 Equality of Opportunity ...................................................................... 133 Two Objections to Defining a Normal Range of Variation .............. 137 Genetic Discrimination ....................................................................... 140 Conclusion ........................................................................................... 142 9. Human Germline Gene Therapy and Our Humanness ...................... 145 Human Germline Gene Therapy and Our Humanness .................... 145 HGLGT’s Effects on Our Humanness ................................................ 148 Changing our Humanness .................................................................. 149 HGLGT and Natural Law .................................................................... 150 HGLGT and “Playing God” ................................................................ 152 The “Who Decides?” Question ........................................................... 154 HGLGT and Human Perfection ......................................................... 154 Conclusion ........................................................................................... 155 10. Public Policy Issues ............................................................................... 157 Science and Public Policy .................................................................... 157 Research Restrictions ........................................................................... 159 HGLGT Funding ................................................................................. 160 Privacy .................................................................................................. 162 Discrimination and Bias ...................................................................... 162 HGLGT Patents ................................................................................... 162 International Cooperation .................................................................. 164 Monitoring Research ........................................................................... 165 Welfare and Enhancement .................................................................. 165 Conclusion ........................................................................................... 166 Glossary ............................................................................................................ 169 Index ................................................................................................................ 183

PREFACE

R

elatively little controversy surrounds the issue of whether we should use ge– netic interventions to treat diseases in existing people. Controlled clinical studies involving genetic manipulation of human somatic cells (cells that are not destined to carry genetic information to future generations) have taken place since the early 1990s. Somatic gene therapy (SGT) techniques are rapidly being extended to treat a variety of diseases, ranging from cystic fibrosis to cancer and AIDS. SGT is regarded by many people as a new and promising approach to treating human diseases. The most serious question raised by SGT thus far is whether it is a medically safe and effective procedure for patients. These are important questions and are similar to concerns raised by non-genetic therapies or treatments. In contrast, human germline gene therapy (HGLGT), i.e., genetic manipulation of germ cells that are destined to pass on genetic information to future generations, raises profound moral, political, philosophical, and theological questions. At the moment, HGLGT is not an option for both technical and ethical reasons. Most scientists agree that we do not understand human genetics and genetic manipulation enough to even attempt HGLGT on human beings. However, the successful production of animals and plants with genetically altered germlines (so-called “transgenic organisms”) and the recent advances in animal cloning suggest that HGLGT may be a technical possibility in the not too distant future. While discussions of the genetic engineering of human beings are only now becoming more than pure science fiction, for a few hundred years writers have anticipated these developments and speculated about their possible consequences. In the 18th century, Johann Wolfgang von Goethe’s epic tale Faust depicted a man who sold his soul to the devil for scientific knowledge and technological power.1 The idea of a “Faustian” bargain still haunts all discussions of new scientific and technological developments, including human genetics. During the 19th century, Mary Wollstonecraft Shelley’s novel Frankenstein warned of the dangers of “playing God” by attempting to create life. Victor Frankenstein had hoped to fabricate the “perfect” man, but his experiment went awry and produced a being that he regarded as a hideous monster.2 In his book Walden, Henry David Thoreau expressed his disdain for the locomotive and its intrusion into nature and human life. He also warned that modern society and technology can turn men into machines.3 In the 20th century, Aldous Huxley’s novel Brave New World envisioned a society where the state controls human reproduction and manufactures people to perform various social roles. The state’s eugenics program clones human zygotes and uses “Boskanovsky’s Process” to produce five genetic castes: alphas, betas, gammas, deltas, and epsilons. People gestate in the artificial wombs of the state hatcheries, most adults are infertile—sex is for pleasure, not for procreation—and “pregnancy” is a dirty word. Since its publication in 1932, Huxley’s anti-utopia has served as a warning against the genetic engineering of

human beings and has shaped many of our social policies relating to human genetics.4 Human history also offers us a grave reminder of the dangers of genetic control: During Adolph Hitler’s rule, Nazi Germany launched its own eugenics program in an attempt to create and propagate a “master” race. Many Western nations, including the United States, have conducted involuntary sterilization programs in order to rid the population of people perceived to be mentally or physically defective. 5 During Huxley’s time and for many decades since then, virtually no one thought that we would actually be able to engineer human beings the way we design automobiles. But all of that has changed in the last decade, as human genetic engineering has moved out of the realm of science fiction into the purview of science fact. Given the astonishing advances in research on human and animal genetics, recombinant DNA technology, germline gene manipulation in plants and animals, animal cloning, in vitro fertilization in human beings, preimplantation genetic diagnosis and embryo selection, and SGT, many scientists, physicians, ethicists, and policy analysts are beginning to address the prospects of HGLGT and its moral, ethical, social, legal, and political ramifications. Does HGLGT represent a Faustian bargain? Is it an arrogant attempt to “play God?” Will we repeat the mistakes of the past by refusing to learn from Nazi Germany’s eugenics programs or will we use HGLGT as an important tool in the prevention of genetic diseases? This book will attempt to provide a framework for addressing these important questions. Given the West’s current emphasis on individual freedom and autonomy, our Brave New World may not arise from state intervention but from parental choices. Imagine a world where parents can purchase services to modify or enhance the genomes of their offspring. Parents who want to buy their children the best education, toys, health care, and clothing, may also want to buy their future children the “best” genes. Over time, the genetically rich could get genetically richer, and a genetic caste system could emerge. People with “inferior” genes may have fewer opportunities and rights, may occupy lower social positions, and may be treated as “inferior.” The intended genetic enhancements might lead to unintended behavioral changes that could have serious negative effects in society. This nightmare may arise not by the deliberate actions of the state, but from the aggregate actions of many individuals. In this genetic “tragedy of the commons”, the sum of many different individual actions might lead to results that no one anticipates or intends.6,7 Some optimists might suggest that parents will not succumb to the temptations of genetic enhancement since anyone in their right mind would find such interventions morally abhorrent. No one would want to break an implicit taboo against manipulating the human genome. No one would “play God” with human genes. But all it takes are a few parents who try to artificially produce children with “superior” genes to open the door to a market for genetic enhancement. Once the technology exists, some people will try to use it. History teaches us that the free market system often accelerates technological develop-

ment, and this lesson also applies to HGLGT. Of course, even if HGLGT is an illegal practice available only on a black market, legal restrictions usually do not prevent people from trying to obtain their desires, and many parents desire to have “perfect” children.8 Although eugenics policies and issues have been debated in the public sphere for many years, the prospect of HGLGT brings an added dimension and urgency to these and other controversies concerning human reproduction. While we still must overcome many scientific and technological obstacles before HGLGT becomes a reality, we need to discuss its implications while we still have the luxury of careful reflection. If we wait too long, the urgency of the moment will force us to make hasty, unsystematic, and unsound decisions. Consider, for example, the lessons we can learn from computers and information technology. History reminds us that very few people in the 1950s, including the so-called experts, thought that the computer would have a significant impact on daily life.9 Many people viewed computers as immense, expensive, dumb, calculating machines that would probably be used only to guide missiles, control satellites, or “crunch numbers” for scientists. Consequently, little discussion took place during the 1950s on the social impacts of advances in computing technology, and the result is that we are now trying to make our policies keep pace with this rapidly advancing technology. But policy lags behind technology in this case, and we now have a hodgepodge of hastily made rules and laws pertaining to computer and information technologies. We still need to do a great deal of thinking about important issues like privacy, security, censorship, honesty, intellectual property rights, and a whole host of issues surrounding the use (and abuse) of computers.10 Wouldn’t it have been much easier to manage these new technologies if we had at least laid more of a foundation for discussion and analysis? Clearly, developments in the sciences and technologies relating to human reproduction and genetics will have profound impacts on our society. We have already seen the impact of genetics on our overall understanding of human health and disease. We now understand the genetic basis of many diseases and this knowledge has played an essential role in diagnosis, prognosis, and treatment. By the beginning of the 21st century the Human Genome Project will have achieved its goal of sequencing the entire human genome.7 But the potential impacts of medical genetics go far beyond the scope of medicine and have already generated concerns about privacy, discrimination, bias, parental rights, gene patents, autonomy, justice, and the meaning of human life. HGLGT will generate similar social concerns and will raise some additional questions. One reason it is so difficult for us to understand or discuss the implications of HGLGT is that during our entire history we have made assumptions about human reproduction and genetics. These assumptions, sometimes called the “natural lottery” in social and political philosophy, generally hold that we use culture and technology to affect social changes but we do not (significantly) modify human biology.11 Conditions of our birth and our genetic endowments are chance events beyond our control. As a result of our common

genetic heritage, each of us is basically the same kind of being, MEDICAL INTELLIGENCE UNIT 9 even though we have different medical, social, economic, and psychological characteristics. But Human Germline Therapy: Scientific, anddemise Political these assumptions mayGene not hold in the future, Moral and the of Issues the natural lottery could have profound effects on the way we think of human society. For many years genetic engineering of human beings was somewhat of a taboo topic in biomedical ethics.12 For many people, the very idea of genetic engineering was so revolting that they would not take the time to consider its moral and political dimensions. In discussions of human genetics, phrases like “that would of course lead to a Brave New World,” or “that would be eugenics,” or “that would be playing God,” would serve as trump cards to defeat any argument in favor of some form of genetic engineering. In these preliminary debates, many organizations and prominent scholars condemned germline therapy unconditionally.13-17 As long as genetic engineering remained in the realm of science fiction, few people would bother to probe the conundrums that lurked behind this moral revulsion. But genetic engineering of human beings is quickly becoming a scientific fact and we can no longer shroud it in mystery and malevolence. Scientists have already succeeded in introducing genes into the germline of mice, and the step from an engineered mouse to an engineered human is not as great as one might think.18 In the last decade, scholars throughout the world have begun to break the conversational taboo surrounding genetic engineering and have discussed the moral, social, and political issues raised by this new kind of technology. ISBN: 1-57059-586-0 This book will attempt to contribute to this important discussion and try to lay a foundation for further debates and public policy decisions. We do not intend to pass judgment upon this new technology. We simply hope to provide a sober assessment of the possible benefits and risks implicated by genetic engineering. In order to carry out this plan, we begin with a description of the current state of medical genetics and technologies pertaining to HGLGT and we offer some speculation on future developments. Drs. Langer and Steinkraus drafted the first four chapters, which provide the scientific background for subsequent chapters which were drafted by Dr. Resnik. However, all authors had substantial involvement in preparing the final versions of all chapters Library of Congress Cataloging-in-Publication Data in the book. The first four chapters are written for readers with a basic background in the bioResnik, David. logical sciences and should serve as a review of basic concepts as well as an Human germ-line therapy : scientific, moral, and political issues introduction to new methods. For example, we are presuming that readers are David Resnik, Pamela J. Langer, Holly B Steinkraus. familiar with the terms chromosome, DNA (deoxyribonucleic acid), DNA sep. cm. -- (Medical intelligence unit) quence, base pair (two bases of DNA paired—A with T and G with C—in a Includes bibliographical references and index. double strand of DNA), gene, mutation, RNA (ribonucleic acid), protein, and ISBN 1-57059-586-0 (alk. paper) cell. The technical terms used in this book are defined in the glossary. For a 1. Gene therapy. 2. Gene therapy--Social aspects. 3. Human reproductive general review of molecular and cellular mechanisms, several technology. 4. Human reproductive technology--Social aspects. excellent texts have been assembled. 19-21 I. Langer, Pamela J. II. Steinkraus, Holly B. III. Series. In chapter 1 we introduce many of the relevant concepts in molecular [DNLM: 1. Gene Therapy. 2. Germ-line Mutation. 3. Ethics, Medical. genetics which are essential to an in-depth understanding of HGLGT. In chap4. Public Opinion. QH 442 R434h 1999] ter 2 we review current and future options for couples making reproductive RB155.8.R48 1999 choices. We discuss embryo selection, a technique which is used to choose an 616'.042--dc21 DNLM/DLC for Library of Congress

98-43505 CIP

embryo without a genetic defect in the absence of any genetic engineering. In chapter 3 we discuss current methods for gene delivery which will form the basis for research on and development of HGLGT techniques. We also stress the many potentially negative consequences if a gene is allowed to go into a chromosome at random. In chapter 4 we outline potential approaches for carrying out HGLGT and some technical obstacles impeding HGLGT development. In chapters 5 through 9 we address moral, social and political issues in HGLGT. In the last chapter, we address some public policy recommendations based on the analysis in chapters 1 through 9. We would like to thank our families for their supportive help during the completion of this project. We would also like to thank all those who shared their ideas and opinions with us throughout the development of this book and those who reviewed various portions of the book, including experts in the fields of molecular biology, molecular genetics, gene therapy, developmental biology, plant biology, virology, philosophy, nursing, patent law and gynecology, as well as medical students, undergraduates and friends. In particular we would like to thank Drs. Mary Forrester, Minx Fuller, Theo Hanekamp, Don Jarvis, Flora Katz, Kay Kohler, Glenn McGee, Cynthia Peterson, Don Roth, Mary Thorsness, Peter Thorsness and Hana VanCampen as well as Jill Adamski, John Flanagan, Scott O’Brien, Summer Abdel-Megeed and David Stephenson, all of whom read a section of the book, but none of whom read the entire book. We apologize to the many authors whose ideas about gene therapy and contributions to the field were not included directly and to those who were not cited in the text because of the need to reduce our originally very extensive reference lists. We also apologize to those readers with a more advanced understanding of these concepts for any oversimplification or generalizations that we felt were necessary to bring this information to a wider readership. We have tried to be as accurate and up to date as possible in every aspect of this book, on both the scientific and the philosophical side. However, we realize that it is impossible to include all recent advances in a field as quickly moving as gene therapy. We do not expect that all readers will agree with our conclusions and opinions, but we do hope that readers will join us in a rational and open discussion of these momentous questions. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Goethe J. Faust. Translated by P. Wayne. Baltimore: Penguin, 1959. Shelley M. Frankenstein. New York: Oxford University Press, 1969. Thoreau D. Walden and Civil Disobedience. Paul S, ed. Boston: Houghton Mifflin, 1957. Huxley A. Brave New World. New York: Harper and Rowe, 1932. Paul D. Controlling Human Heredity. Atlantic Highlands, NJ: Humanities Press International, 1994. Gardener W. Can genetic enhancement be prohibited? J Med Phil 1995; 20: 65–75. Kitcher P. The Lives to Come. New York: Simon and Schuster, 1997. Chadwick R. The perfect baby: An introduction. In: Chadwick R, ed. Ethics, Reproduction, and Genetic Control. London: Routledge, 1992. Volti R. Society and Technological Change. 3rd ed. New York: St. Martin’s, 1995. Johnson D (ed.). Computer Ethics, 2nd ed. Englewood Cliffs, NJ: Prentice Hall, 1994.

11. Buchanan A. Equal opportunity and genetic intervention. Soc Phil Pol 1995; 12: 105-35. 12. Fletcher J, Anderson W. Germline therapy: A new stage of debate. Law Med Hea Car 1992; 20: 26-39. 13. Council for Responsible Genetics. Position paper on germ line manipulation. Hum Gen Ther 1993; 4: 35-37. 14. Congregation for the Doctrine of Faith. Instruction on respect for human life in its origin and on the question of procreation: Replies to certain questions of the day. Rome: The Roman Catholic Church, 1987. 15. Suzuki D, Knutdson P. Genethics. Cambridge, MA: Harvard University Press, 1989. 16. Kass L. Toward and More Natural Science: Biology and Human Affairs. New York: The Free Press, 1985. 17. Rifkin J. Resolution. Washington: Foundation on Economic Trends, 1983. 18. Anderson W. Human gene therapy: Why draw a line? J Med Phil 1989; 14: 81-93. 19. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. Molecular Biology of the Cell. 3rd ed. Robertson M, ed. New York: Garland Publishing,1994. 20. Lewin, B. Genes VI. Oxford: Oxford University Press, 1997. 21. Lodish H, Baltimore D, Berk A, Zipursky SL, Matsudaira P, Darnell J. Molecular Cell Biology. 3rd ed. New York: WH Freeman and Co., Scientific American Books, 1995.

ABBREVIATIONS AAV ADA APC ARS BAC CEA CF CFTR CVS DNA ES FDA FISH GIFT HAC HGLGT HIV HR IVF kb LCR LDL mRNA neo NHR NIH ORDA PAC PCR PGD PKU RAC RNA rRNA SGT snRNA tk tRNA YAC ZIFT

adeno-associated virus adenosine deaminase adematous polyposis coli autonomous replicating sequence bacterial artificial chromosome carcinoembryonic antigen cystic fibrosis cystic fibrosis transmembrane regulator chorionic villus sampling deoxyribonucleic acid embryonic stem Food and Drug Administration fluorescence in situ hybridization gamete intrafallopian transfer human artificial chromosome human germline gene therapy human immunodeficiency virus homologous recombination in vitro fertilization kilobase locus control region low density lipoprotein messenger RNA neomycin phosphotransferase nonhomologous gene replacement National Institutes of Health Office of Recombinant DNA Activities phage P1 artificial chromosome polymerase chain reaction preimplantation genetic diagnosis phenylketonuria Recombinant DNA Advisory Committee ribonucleic acid ribosomal RNA somatic gene therapy small nuclear RNA thymidine kinase transfer RNA yeast artificial chromosome zygote intrafallopian transfer

CHAPTER 1

From Genes to Disease I

n the past few years, “gene” has become a household word. We know that we inherit genes from our parents, that genes are made up of DNA (deoxyribonucleic acid), and that changes in DNA can cause disease. The popular media has gone a long way in educating the public about the connection between abnormal genes and disease. Unfortunately, for many genetic diseases, current medical treatments are inadequate. The use of genes in the treatment of disease provides a whole new type of medical therapeutics. Gene therapy could alleviate or even cure a currently untreatable disease. Alternatively, it could provide a more effective treatment where some therapeutic options already exist. Gene therapy is unique in that it has the potential for removing the cause of the disease rather than treating only the symptoms. With the promise of gene therapy on the horizon, the seriously ill have renewed hope that they might live to profit from the new technology. For a patient, it may not be necessary to understand the details of gene therapy as long as health care professionals can provide reliable estimates of the success or failure of a procedure. However, as a voting citizen or one involved with formulating policy, an appreciation for the limitations and consequences of gene therapy is essential for decision making. In this chapter, we introduce the scientific concepts that will allow a greater understanding of the technical barriers and potential advantages or harms in gene therapy. This chapter is not meant to be a comprehensive review of molecular genetics, but is limited to the information required for a basic understanding of the processes of gene transfer and gene expression. Much of our information on gene transfer techniques has come from extensive research in model organisms, where the creation of genetically altered animals is a well studied process. Consequently, analyses of some of the predicted effects of gene transfer into humans are predominantly based on data from human cells and mice. Many principles introduced here are essential for understanding problems in two basic types of human gene therapy: 1. Human germline gene therapy (HGLGT), where transferred genes will eventually reside in most cells of the body, including the germ cells (eggs and sperm); or 2. Somatic gene therapy (SGT), where genes are introduced into somatic (not germline) cells and are not destined to pass on genetic information to future human generations. In this chapter we predominantly treat both types of gene therapy as a unified topic and distinguish between SGT and HGLGT in chapters 3 and 4.

Scope of the Term Gene Therapy The term “human gene therapy” describes medical procedures that use DNA in the therapeutic treatment of human disease. The DNA that is introduced into a cell may be a Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus and Pamela J. Langer. ©1999 R.G. Landes Company.

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Human Germline Therapy: Scientific, Moral and Political Issues

gene, a gene plus additional sequences required for its proper function, or other DNA. The use of DNA for altering traits unrelated to disease is distinct from gene therapy and is designated genetic enhancement. For example, the use of genetic manipulation to affect the height of a future child would be termed genetic enhancement, unless it is used to prevent dwarfism. The distinction between gene therapy and genetic enhancement is not always easy to make. We discuss this issue in chapter 5. The term “gene therapy” was originally used to describe procedures in which a normal gene was introduced into a cell in an attempt to restore a cell function that was lacking because of a defective gene. With recent technological advances, gene therapy now refers to an array of potential therapeutic procedures. First, gene therapy can be used to introduce a foreign gene that is not part of the human genome. For example, genes which are called “suicide genes” can be introduced into cancer cells. When the suicide gene is activated, it causes death of the cancer cells harboring the suicide gene. Second, gene therapy can be used to introduce genes into unaffected, healthy cells as a means to produce a desired product. The development of DNA vaccines relies on this principle, where a foreign protein expressed in a cell may be able to vaccinate a person against a pathogen. 1,2 Third, a gene therapy procedure may cause a change in the expression of specific genes. For example in anti-sense therapy, a piece of introduced DNA is designed to bind to specific RNA sequences, thereby inhibiting production of a particular gene product.3 Fourth, a gene therapy treatment may activate dormant genes to replace the function of a defective gene. For instance, the muscle protein utrophin may be able to replace the function of the defective dystrophin protein in patients with a muscle disorder called Duchenne type muscular dystrophy. Also, induction of the expression of fetal hemoglobin in an adult can partially compensate for the absence of adult hemoglobin in a blood disorder called thalassemia.4 The long term clinical usefulness of each of these new approaches has yet to be evaluated.

Some Common Misconceptions A commonly held synopsis of gene therapy is: “You take the bad gene out and put a good gene in.” A statement such as this exposes several important misunderstandings about gene therapy. In order to address some of these misconceptions, we first review some principles that are essential to the understanding of gene therapy: 1. Genes are not necessarily “good” or “bad;” 2. More than one gene can be involved in a process or manifestation of a disease; 3. A new gene can be added to a cell (temporarily or stably) without replacing the original defective copy of a gene; 4. SGT and HGLGT are fundamentally different with respect to long term consequences.

Misuse of the Terms “Good” and “Bad” Sorting genes into two piles of good and bad is simplistic. What may appear to be a “bad” disease-causing gene may have unknown value. In a population, a gene occurs in many different forms called the alleles of a gene. A person carries two alleles of most genes, one on each member of a chromosome pair. The fact that several defective or “bad” alleles are present in the population at high frequency suggests that they may have an evolutionary advantage, in former times or at present, when present in combination with a “good” allele (in a heterozygous state). For example, hemoglobin is a blood protein which carries oxygen around the body. Hemoglobin is made up of α and β subunits, and globin genes encode these protein subunits. A person who has two “bad” β-S alleles encoding the β-S globin subunit is homozygous for the defect and produces a defective hemoglobin called

From Genes to Disease

3

HbS, instead of the normal HbA hemoglobin. The person with HbS develops sickle cell anemia. However, a heterozygous individual who has one defective β-S allele and one normal β-A allele is protected from malaria.5 Other examples of such a heterozygote advantage are less well characterized. In general, we avoid the use of the terms “good” and “bad” since they are often value judgments based on incomplete knowledge. Instead, we refer to normal alleles, which are found at a high frequency in the population and do not appear to cause disease, and defective alleles, which are responsible for causing disease. An abnormal allele appears at a low frequency in the population but would not necessarily lead to disease.

Involvement of One or More Genes Genetic defects can be congenital (present at birth) or can be acquired during a person’s lifetime. Most of the characterized genetic disorders originate from defects in a single gene and are called monogenic or single-gene disorders. With some monogenic disorders, defects in one of several different genes can give rise to the disease. For example, xeroderma pigmentosum, a condition which causes extreme sensitivity to ultraviolet radiation from the sun, may be caused by mutations in any one of seven different genetic regions (genetic loci).6 Gene therapy can potentially be used to introduce a single gene to correct a monogenic defect. In contrast to monogenic disorders, polygenic or multigenic disorders are ones which are affected by multiple genes. Since more than one gene, as well as the environment, contribute to the manifestation of polygenic diseases, these disorders are also called multifactorial disorders. For example, the development of certain types of colon cancer is affected by multiple genetic defects.7 Many of the polygenic disorders will be more difficult to treat using gene therapy, partially because it is more difficult to trace the contributing genetic factors. Once “disease-contributing” alleles are identified, one or more alleles may have to be replaced or added in order to lower an individual’s chance of developing the polygenic disorder. If alteration of one gene is sufficient to significantly affect the polygenic disorder, then the gene transfer goal would be similar to that for monogenic disorders. It is also important to identify “disease-contributing” environmental parameters. In most monogenic and polygenic disorders, the environment can be an important factor in the manifestation of disease (see the section below on Genes, Mutations and Inheritance and chapter 5).

Gene Replacement vs. Gene Addition To aid our subsequent discussion, we introduce the terms “resident gene” and “resident chromosome” to identify a gene or chromosome that exists in a cell before any genetic manipulation is attempted. Another name for a resident gene would be an “endogenous” gene, while an introduced gene would be an “exogenous” gene. In current gene therapy protocols, a defective resident gene is not replaced with a normal one. Rather, the procedure involves adding a normal gene to cells that persist in harboring the defective allele(s). For example, the introduced gene may be located on a plasmid, a circular piece of DNA that replicates independently outside of the chromosomes. It may also be carried by an episome, a plasmid that can be inserted randomly into a chromosome. Furthermore, other gene delivery systems cause random insertion of the gene into chromosomal DNA via a mechanism of nonhomologous recombination (NHR). These gene addition methods are sometimes called genetic augmentation,8 because the genetic content of a cell is expanded by the addition of a normal copy of the gene. Since the term genetic augmentation is ambiguous in some ethical contexts, we refer to the process of adding genes to a cell as gene addition.

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Human Germline Therapy: Scientific, Moral and Political Issues

In contrast, when referring to a gene that exactly replaces a resident allele via a mechanism of homologous recombination (HR), we use the terms homologous gene replacement or simply allele replacement. A gene transfer protocol involving allele replacement would be preferable to one employing gene addition; however SGT protocols to date have used only gene addition methods. Since HR has recently been achieved in human somatic cells, the design of some of the SGT protocols may change in the near future.9 Some researchers reserve the term “transgene” for a gene added to a cell via NHR. That is, a gene introduced via allele replacement would not be considered a transgene. However, for the purposes of this book, we have chosen to use the term “transgene” as an umbrella term to refer to all genes introduced into a cell by a gene delivery system, whether or not they cause allele replacement. We believe that this broader definition of transgene will facilitate a better general public discussion of HGLGT. Understanding the difference between allele replacement and gene addition is central to comprehending the biological consequences of gene therapy. If a defective gene is replaced exactly with a transgene (by allele replacement), the transgene will most likely be stably integrated into the host genome, function normally, and not alter the function of other genes in the genome. In contrast, if introduction of the transgene results in gene addition, the transgene may have only a temporary effect on cell function, especially if it resides extrachromosomally on a plasmid. Such a transient existence of a transgene in a cell may be insufficient for correcting the genetic defect. Furthermore, if gene addition results from insertion of the transgene by NHR, it could interfere with the function of other genes in the genome (i.e., affect nonallelic gene expression). Depending on its point of insertion in the genome, the transgene may cause deleterious effects on cell function (see chapter 3). Many of the potentially undesirable consequences of gene therapy protocols are associated with transgene addition.

Somatic vs. Germline Gene Therapy Many people are aware of SGT, where a transgene is transferred to nonreproductive cells (such as those of the blood or airway epithelium). In SGT, the genetic intervention affects only a subset of cells in the patient and does not directly affect any descendents. However, in HGLGT, the transgene would be introduced into cells which have the potential for being the progenitors of future generations. We consider SGT and HGLGT as separate methods, but it is theoretically possible that a transgene destined for a somatic cell would accidentally be delivered to a germline cell during an in vivo delivery of a transgene (see chapter 3). Thus, in extremely rare instances SGT could unintentionally become a case of HGLGT.10

Gene Expression The presence of a gene in a cell does not necessarily mean that it is expressed. Many genes are intermittently turned on or off during development or in cells in the mature organism. Although muscle cells and nerve cells have the same set of genes, they are different because each cell type expresses different subsets of genes. Several events are orchestrated in the cellular decision of whether or not to express a gene. The timing and selectivity of gene expression controls embryonic development, cell differentiation, cell communication, or the response of cells to a changing physiological environment (e.g., hormonal responses, immune responses, etc.). We first discuss the process of normal gene expression and then describe some aspects of these mechanisms to consider when introducing transgenes into a cell.

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Normal Gene Expression The end product of gene expression can be a protein or a functional RNA. In order to describe a few basic steps in gene expression, we use the example of a gene encoding a protein. In gene expression, the information contained in the gene is decoded in the processes of transcription and translation (Figure 1.1): 1. Transcription: The information in the gene is used to make a ribonucleic acid (RNA) copy of the DNA. This RNA is called the “primary RNA transcript.” Many primary transcripts are further modified. 2. RNA processing: Depending on the organism and the gene in question, a primary RNA transcript may be a precursor RNA (pre-RNA) which is further processed to a mature, functional form. These mature forms include messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or other small RNA molecules. One example of a processing event is the removal of introns from pre-mRNA. Introns are noncoding RNA sequences (between the coding regions or exons of a gene) that are spliced out in order to form a continuous coding sequence in the mature mRNA. Another processing event is the addition of a stretch of A (adenine) nucleotides at the end of most mature mRNAs. Some RNAs do not encode proteins but are used directly in a cellular function. For example, rRNA is a component of ribosomes. 3. Translation: The mature mRNA sequence is decoded by tRNAs, rRNA, ribosomal proteins and other factors to produce proteins in a process called “translation.” One mRNA molecule can be translated multiple times into a polypeptide chain (a string of amino acids), which is destined to become a mature protein. 4. Protein processing: In order for a protein to function normally, it must be properly modified, folded into the correct conformation, and sent to the correct place in the cell. Gene expression denotes more than gene transcription. A gene is “expressed” when it generates a functional protein. Many layers of regulation surround each step of gene expression, only some of which are characterized. The ultimate health of a cell depends not only on the processes of transcription and translation, but also on the interaction of proteins with other proteins and with other molecules in the cell.

Differential Gene Expression Although most of the cells in the body have the same DNA content, each cell type expresses its own subset of genes in order to carry out its specific function. No matter what cell type it is, a minority of the total number of genes is expressed at any one time. Liver cells synthesize one subset of proteins necessary for their function, while cells in the pancreas synthesize a different subset of proteins required for their function. Moreover, within one organ there are different cell types, each specialized for a particular function, and each expressing a different subset of genes. In an organ like the pancreas, some cells are involved in secreting digestive enzymes that end up in the gut, while other cells participate in glucose regulation and secrete precursors for the hormones insulin or glucagon. In general, genes which are needed for basic cell functions, so-called housekeeping genes, are expressed in most cells, while genes encoding proteins involved in more specialized functions are expressed in only a limited number of cells. If factors such as specific amount, time, or place of gene expression are critical to the proper function of a gene, the gene is said to be tightly regulated. Genes or transgenes that are expressed in the wrong tissues at the wrong time or wrong developmental stage may have severe negative effects on the proper function of the cell, tissue or organ.

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Fig. 1.1. The flow of genetic information. This diagram illustrates a typical eukaryotic gene encoding a protein. The gene consists of exons containing coding sequence (slashed boxes), introns containing intervening sequence (open boxes), a poly(A) addition site (gray diamond), and other sequences upstream and downstream of the coding sequence but still within the transcribed region. In this diagram, promoter and enhancer sequence elements are located upstream of the gene (enhancer elements may be found upstream, downstream or within the coding region of a gene). Transcription begins at the transcription start site and results in a primary transcript (pre-mRNA) that is frequently processed to mature RNA by adding a cap structure (filled semi-circle) to one end, a poly(A) sequence to the other end, and by removing intron sequences. The mature mRNA transcript is translated into protein that may be further processed before performing its cellular function.

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Fig. 1.2. Representation of a mitotic chromosome. In this diagram, the DNA double helix has already been replicated in preparation for cell division (mitosis). The mottled appearance of the chromosome arms emanates from the DNA-protein complexes wrapping into coil upon coil. The resulting supercoiled structure is highly condensed. The central constricted region of a chromosome, the centromere, is indicated. (In reality the chromosome arms may be juxtaposed more closely.)

Chromosomal Effects on Gene Expression A chromosome is often visualized as a linear piece of DNA containing genes and other DNA sequences in between the genes. However, a chromosome has many microenvironments that affect the expression of a gene or transgene. An inappropriate level of transgene expression may have severe consequences to the cell and may even cause cell death (see chapter 3). DNA exists predominantly as two antiparallel strands wrapped around one another in the famous double helix structure. Double-stranded DNA, in association with proteins, is wrapped into coils and then into coiled coils. The result is a supercoiled structure that contains a highly organized, highly packed form of DNA that can fit into a cell. During the period immediately before the chromosomes divide in mitosis, they become condensed to an even greater degree (Figure 1.2). At other times, only parts of chromosomes contain very highly condensed DNA regions, called heterochromatin. Heterochromatic regions, like those at the constricted region of a chromosome called the centromere, do not contain DNA which is transcribed, but rather harbor DNA sequences which might function in chromosome structure.11 In contrast, an entire chromosome may become heterochromatic during the phenomenon of X-inactivation, where one of the two X chromosomes in a female is inactivated. Euchromatin, i.e., the non-heterochromatic regions of a chromosome, contains the expressed genes as well as other DNA. Although the heterochromatin/euchromatin classification may sound like an off/on switch, only a small proportion of genes located in the euchromatic regions are expressed at any given time.11 Different regions of a chromosome display different properties; however in most cases we do not know the molecular basis for the variation.11 For example, after staining condensed chromosomes with the chemical dye Giemsa, one can observe a series of bands. The basis for differential chromosome staining is not known. It is thought to derive from different modifications or structural differences of various parts of a chromosome. The understanding of the molecular features dictating the microenvironments of a chromosome remains one of the frontiers in biological discovery.

The Boundaries of a Gene For discussions of gene transfer, it is useful to understand what genetic information is included in a gene. Originally, a gene was defined as a functional unit of heredity. People envisioned genes as blocks that passed from one generation to the next. Genes were thought to be made up of the coding DNA that specifies or encodes a single protein. Now we know that in some genes, the coding regions or exons are interspersed with a significant amount of non-coding DNA or introns, which are located in between the exons of a single gene (see Fig. 1.1). The definition of a gene that we use is the DNA region which is used as a template in the synthesis of a primary RNA transcript. By this definition, a gene would include the

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region from the DNA encoding the transcription start site to the end of the unprocessed precursor RNA (see Fig. 1.1). Sequences that are involved in regulating the transcription of a gene are therefore not part of the gene per se. Sequence elements such as promoters (regions required for the initiation of transcription), enhancers (regions that affect the level of transcription) or silencers (“negative enhancers” that inhibit transcription) are generally located outside of a gene, i.e., they are “extragenic.” (Enhancers may also be located within a gene).12,13 In order for a gene or transgene to function properly, it must be linked to regulatory sequences. When a transgene and the associated DNA (a “transgene construct”) is being designed, regulation of the transgene is a primary consideration. A transgene construct is often part of a recombinant vector such as a virus, which is designed to deliver the transgene to a designated target cell in vitro or in vivo. If a transgene participates in allele replacement, the transgene might be regulated by sequences in the resident chromosome or in the transgene construct, depending on what is included in the transgene construct. In contrast, with gene addition, when a transgene inserts randomly into the genome or is located in an episome, it is essential to include regulatory sequences along with the transgene. Regulation of transgene expression and the impact of transgene insertion on the function of other genes is discussed in chapter 3.

Genes, Mutations and Inheritance In the previous sections, we discussed the process of normal gene expression and the importance of regulating this mechanism correctly. We limited our discussion to molecular considerations in the absence of environmental influences. However, the environment in which we live and the changing physiology of a person certainly affect gene expression. Furthermore, genes operate in a genetic environment. The products of different alleles of one gene can interact in a variety of ways, and the activities of other, nonallelic genes can modify the expression of a gene. Issues of genetic interactions are important considerations in gene therapy. In this section we briefly review some basic genetic concepts, including genotype and phenotype, genetic mutations, recessive and dominant genes, autosomal and sex-linked genes, and the effect of the environment and other genes on the function of a gene.

Genotype and Phenotype The nuclear genetic material in a normal human cell consists of 46 chromosomes, including 22 pairs of autosomes and two sex chromosomes (two X chromosomes for a female or an X and Y chromosome for a male.) The DNA in this set of chromosomes, along with mitochondrial DNA, constitutes the genome of an individual. Thus, the genotype of an organism refers to the genetic constitution of an organism. The phenotype is the expression or manifestation of the genotype, resulting in observable traits at a cellular or organismal level. Humans are diploid because they have two copies of each chromosome (as opposed to haploid, where an organism has only one copy of each chromosome). The DNA content of a diploid human cell is approximately six billion nucleotide pairs.14

Genetic Mutations Although elaborate DNA repair mechanisms exist in cells to ensure the maintenance of our genetic information, some errors escape the surveillance mechanisms, giving rise to mutations. A genetic mutation is a change in the DNA sequence of an organism. Genetic mutations can have a positive, neutral or negative effect on the phenotype. A positive mutation may persist through the process of evolution. A neutral mutation may be silent, i.e., one that causes no change or only an innocuous change in the phenotype. Deleterious

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mutations can cause disease or may be lethal, resulting in termination of the organism before or after birth. Many different things can cause a person to have a mutation. Mutations can be inherited, can arise spontaneously, or can be induced by environmental agents, including ionizing radiation, chemicals or infectious agents. A genetic disorder can result from a mutation as small as a change in a single nucleotide or from major alterations to the genome. Surprisingly, the severity of a disease does not correlate with the size of a mutation. Disorders resulting from single nucleotide mutations can be far more devastating than having an extra copy of chromosome 21, leading to Downs’ syndrome, characterized by various degrees of mental retardation. Single nucleotide changes in a hexosaminidase gene can cause the infantile form of Tay-Sachs disease, where the patient usually does not live past two to four years of age. If an abnormality is present at birth, it is called a “congenital abnormality”, whether or not it has a genetic cause. A congenital disorder could have many different origins. It could arise from mutant genes present in the parental genomes or it may be acquired by parental germ cells during their development. Alternatively, a defect could be acquired during fetal development from environmental agents (e.g., drugs, alcohol) or through spontaneous genetic mutation. Thus, not all congenital abnormalities are inherited from the parents’ DNA.

Recessive, Dominant and Codominant Genes Genes encode gene products that are involved in cell function. Different allelic forms of a gene encode variants of the gene product. Since humans are diploid (two copies of each chromosome), they normally have two copies of each autosomal gene (two alleles). In some cases, there is more than one copy of a gene on a chromosome, but we are not considering these cases for this discussion. If two alleles are identical, the gene products will be the same. However, if two alleles are different and are expressed simultaneously, the cell will contain a mixed pool of gene products. Alternatively, if one allele produces a functional product and the other allele is not even transcribed, then only one type of gene product will appear in the cell. Having different types of gene products in a cell raises the issue of which product has a dominant function with respect to the other. The dominant allele of a gene gives rise to a gene product that determines the phenotype when a second, recessive allele is present. When two recessive alleles are present, the recessive phenotype is observed. The terms dominant and recessive are also used in reference to genetic mutations. In most cases, a recessive mutation is one which prevents the gene product from being made or from functioning properly. A functional allele would be dominant with respect to a mutated (recessive) allele that is not expressed into a gene product. At a physiological level, the terms dominant or recessive refer to characteristics of the phenotype associated with a particular allele. However, the dominant/recessive demarcation is not always so clear. First, an allele may exhibit both recessive and dominant phenotypes. For example, heterozygous carriers of the sickle cell allele do not have sickle cell anemia. Therefore, the sickle cell allele is recessive with respect to the manifestation of the disease. However, these heterozygous carriers are more resistant to malaria infections. Thus, the sickle cell allele is dominant with respect to conferring partial protection against infection with malarial parasites.15 Second, alleles may be codominant if both gene products effect a cellular phenotype independently (such as A and B blood group proteins). Third, alleles may be partially dominant (semidominant) if the affects of two alleles are synergistic. An example of the latter is observed in familial hypercholesterolemia, a disorder caused by mutations in low density lipoprotein (LDL) receptors. Defective LDL receptors promote the development of vascular disease eventually leading to heart attacks. A person

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with two partially dominant mutant LDL receptor alleles develops symptoms much earlier than a person with only one mutant allele. Although we will continue using the terms dominant and recessive, these states can be viewed as relative and not absolute, representing a relative point on a continuum of effects. In terms of gene therapy (SGT or HGLGT), it is important to consider whether a defective gene is dominant or recessive. For example, if a disorder occurs with homozygous recessive mutations, introduction of a normal transgene would be expected to restore the normal or so-called “wild type” phenotype. On the other hand, if the disease is caused by a dominant mutation, a transgene would restore the normal phenotype only if the transgene replaces the dominant mutant allele. If the transgene is added to the cell by a means other than allele replacement, the normal transgene product would not overshadow the effect of the dominant mutant gene product (i.e., a dominant mutant gene product is “dominant” with respect to a normal or wild type gene product.) When considering HGLGT alteration of an early embryonic genome which is homozygous for a defect, there is a significant difference between homozygous recessive and homozygous dominant situations. With a homozygous recessive defect, allele replacement of one recessive allele should be sufficient to avoid the disease. However, with a homozygous dominant defect, replacement of one mutant allele would not be adequate since the effect of the remaining dominant allele would overshadow the effect of the normal transgene. Two-allele replacement would be required to completely remove the dominant defective genes. However, since inherited dominant mutations are much rarer than recessive ones, a hypothetical mating of two individuals, both of whom are homozygous for the same dominant defect and had lived to reproductive age, would be extremely rare. Thus the important situation to consider is the one where two people who are both homozygous for a recessive defect (e.g., cystic fibrosis patients) would want to use HGLGT to avoid transmitting the defective alleles to prospective children. At the moment, this also seems like an uncommon scenario; however, with improvements in the therapeutic management of cystic fibrosis, the situation may be less rare. It is important to keep in mind that even if a single-allele replacement prevents a disease in the original HGLGT-altered individual (the proband), this normal allele may not be the one that is transmitted to the next generation. That is, there is only a 50% chance that a child of the proband (the “F1”) would receive the normal allele and a 50% chance that the F1 would receive the defective allele. Thus, the defect may have been corrected in the proband, but it would not remove the defective allele from the gene pool of the descendents if the introduced transgene does not pass to the next generation. However, if HGLGT is designed to replace two defective alleles, a successful procedure would prevent the defect from passing on to the next generation. Various scenarios associated with potential medical benefits and harms of HGLGT to the proband and F1 are presented in chapter 6.

Autosomal or Sex-linked Genes Genes are classified as either autosomal or sex-linked, depending on whether they are located on an autosome or on one of the sex chromosomes, respectively. Although a sexlinked gene is found on a sex chromosome, it does not imply that this gene plays a role in sex determination. For example, the gene encoding hypoxanthine phosphoribosyltransferase (HPRTase) is a gene found on the X chromosome but has no effect on sex determination. HPRTase is involved in recycling building blocks of DNA and RNA. On the other hand, some genes involved in sex determination are found on sex chromosomes. For example, when the Sry gene, located on the mouse Y chromosome, was expressed in genetically female (XX) mouse embryos, they developed into phenotypically male mice.16

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Males have only one X chromosome and will therefore have only one allele for HPRTase (they are hemizygous for the gene). The phenotype of a male will be directed by whatever HPRTase allele is present. If a mutant HPRTase allele is present on a male’s X chromosome, he will develop Lesch-Nyhan syndrome, a recessive X-linked recessive disorder, characterized by mental retardation and a tendency toward self-mutilation. (The HPRTase gene is a candidate for use in gene therapy.) Since females have two X chromosomes, they will have two HPRTase alleles. A female carrying one normal and one mutant HPRTase allele does not exhibit Lesch-Nyhan syndrome. A female can carry an X-linked recessive trait without phenotypic effect. The location of a gene on an X chromosome subjects it to the phenomenon of X-inactivation. When there are two X chromosomes in a cell, one of the X chromosomes is inactivated. Once X-inactivation is established in a cell, the same X chromosome is inactivated in daughter cells. Thus, if a female is a heterozygous carrier of a defective HPRTase allele, approximately half of her cells will be producing a functional HPRTase. Fortunately, this level of expression of HPRTase is sufficient to avoid the clinical symptoms associated with Lesch-Nyhan syndrome. There are significant differences in the patterns of inheritance and expression for autosomal or sex-linked genes (Figs. 1.3 and 1.4.) An autosomal dominant allele will determine the phenotype. Autosomal recessive alleles determine the phenotype when the person carries two recessive alleles (e.g., the person is homozygous for the recessive gene). With sex-linked genes, phenotypic determination is more complicated. Lesch-Nyhan is considered an X-linked recessive disorder because heterozygous female carriers do not exhibit a disease state. Moreover, X-linked dominant disorders also exist but are relatively rare. One example of an X-linked dominant disorder is Vitamin D-resistant rickets (X-linked hypophosphatemia), which causes bone malformation. This disorder is considered to be X-linked dominant because in females the dominant allele is responsible for the phenotype. At a cellular level, the dominant allele is responsible for the cellular phenotype only when present on an active X chromosome (as opposed to an inactivated X chromosome). Although both sexes show disease symptoms, the disease usually has a more severe presentation in males, possibly due to the fact that approximately half of the female cells will not be expressing the trait because of X-inactivation.17

Penetrance and Expressivity Some genes consistently exhibit a particular phenotype while others exhibit a great deal of variation in their phenotypic expression. In a population there are two parameters which can vary: 1. The proportion of individuals who display any phenotypic features arising from the presence of a given gene (called the penetrance of a gene); and 2. The degree to which the phenotype is expressed in individuals carrying a given gene (called the expressivity of a gene).15 Penetrance can be incomplete and expressivity can be variable. A gene shows incomplete penetrance when a phenotypic manifestation does not appear in all individuals who have the gene.18 Variable expressivity occurs when a range of different phenotypes result from having a particular gene. The extreme of variable expressivity is when the gene is inpenetrant. Some diseases exhibit both variable expressivity and incomplete penetrance. For example, Huntington’s disease, a degenerative brain disorder caused by autosomal dominant mutations in the huntingtin gene, has a variable onset from childhood to over age 70.19 This is a case of variable expressivity of a gene. Some people who have a mutant Huntingtin protein never show symptoms of the disease but die of old age or other causes. Thus, the huntingtin gene also shows incomplete penetrance.

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Fig. 1.3. Inheritance of an autosomal recessive gene. In this diagram, both parents are heterozygous carriers for an autosomal recessive defect. Each child has a 25% chance of being a non-carrier, a 50% chance of being an unaffected heterozygous carrier, and a 25% chance of being affected by the disorder. Shading indicates the presence of the defective allele in the person. Letters under the figures indicate the genotype. A, normal allele of gene A; a, defective allele of gene A.

Environmental factors can affect gene penetrance and expressivity. For example, acute intermittent porphyria is an autosomal dominant disorder characterized by attacks of abdominal pain, limb cramps, muscle weakness and psychiatric disturbances. The clinical expression of this type of porphyria can be precipitated by drugs, diet or steroid hormones.20 Likewise, α1-antitrypsin deficiency is an autosomal recessive disorder affecting lung and liver function. The development of emphysema in this disorder is hastened by smoking.15

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Fig. 1.4. Inheritance of an X-linked recessive gene. In this diagram, the mother is a heterozygous carrier for an X-linked recessive defect. A female child has equal probabilities of being either a non-carrier or unaffected carrier. Since a male child receives only a single X chromosome, he has equal probabilities of being a non-carrier or affected with the trait. Shading indicates the presence of the defective allele in the person. Letters under the figures indicate the chromosomes carried by the individuals. X, X chromosome carrying a normal allele of gene X; X', X chromosome carrying a defective allele of gene X; Y, Y chromosome.

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Genetic factors also affect the penetrance and expressivity of a gene. First, allelic differences can cause variation in penetrance and expressivity. Some of this genetic variation may cause subtle differences in expression, possibly significant only on an evolutionary scale. Other allelic differences lead to more overt variations in expression. For example, in the case of the huntingtin gene discussed above, different mutant alleles have different lengths of additional DNA sequence in a certain region of the gene. A longer stretch of additional DNA in a mutant allele may be correlated with an earlier age of onset of Huntington’s disease.21 In cystic fibrosis, alleles carrying different mutations in the cystic fibrosis transmembrane regulator (CFTR) protein are linked to different clinical symptoms and different molecular events.22,23

Monogenic and Polygenic Traits in Disease It is relatively simple to consider single genes encoding single proteins with single functions. However, many phenotypic manifestations of the genome are more complicated than that. For example, a single gene product (such as a hormone) can affect many different cell functions. In this case, the gene has a pleiotropic effect. Alternatively, the interactions of multiple genes or gene products can contribute to the development of a polygenic trait such as height or a polygenic disorder such as diabetes mellitus, essential hypertension, gout, coronary heart disease or cancer. The more genes that contribute to a polygenic trait, the more variation is observed. The polygenic trait may be further modified by environmental influences (consider the effect of diet on the development of heart disease). We are just beginning to understand how gene products interact and are still very far from defining all the genes that contribute to a polygenic trait such as intelligence. Even if we could list all the genes that were involved in producing “intelligence” of a particular kind, it would be difficult to predict how alteration of any of those contributing genes would affect intelligence. In addition, with such a genetic alteration, the effects on other parts of a person’s personality would be incalculable. When a person’s unpredictable life experience is then thrown into the equation, the number of possible outcomes is infinite. Although it is conceivable that a trait such as intelligence could selectively be altered, it is impossible to predict how such an alteration would truly affect a person’s life, positively or negatively.

Summary and Conclusion Gene therapy refers to the transfer of DNA in the therapeutic or preventative treatment of medical conditions. The use of gene transfer techniques for genetically altering traits which are not associated with recognized disease is a separate category of genetic manipulation; however the distinctions between the two are not clearly defined by society. Either could be considered eugenics, defined as the use of genetics to promote the occurrence of desirable traits in a population. There are several basic features of the process and limitations of gene therapy which are not generally understood. For example, current SGT methods involve the addition of a gene to a cell rather than the replacement of a defective gene with a normal gene. Gene addition is an acceptable design for SGT, where a potentially harmful effect on one of millions of cells is usually inconsequential. However with HGLGT, allele replacement would be preferred in order to minimize (or eliminate) the potential for any deleterious effect on the expression of nonallelic genes and to maximize the probability of appropriate transgene expression. HGLGT has not been attempted for technical and ethical reasons. However, SGT protocols are being used in increasing numbers in clinical trials. Although none of the SGT protocols are a proven cure for a genetic disease, the results from clinical trials are very promising and many new SGT technologies are currently being developed (see chapter 3).

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Basic genetic and molecular biological concepts must be understood for an in-depth appreciation of the advantages, disadvantages, methodology and technical obstacles in the field of gene therapy. In this chapter we discussed basic issues such as the factors affecting the expression of a transgene, the dominant or recessive nature of a mutation, and the complex nature of genetic and environmental factors which ultimately contribute to the phenotype of an individual. Although we view SGT as an attainable goal and an incredible breakthrough in medical science, the technology required to carry out HGLGT safely has not been developed. The genetic, ethical, financial, social and political costs involved in HGLGT raise serious questions. Furthermore, the usefulness of HGLGT in preventing disease would be limited even if it were deemed ethically acceptable. Most genetic diseases known today are caused by single-gene defects. As we discuss in the following chapter, the technique of genetic embryo selection would provide a much safer and reliable alternative to HGLGT in eliminating a single disease-causing gene. However difficult it might appear from our point in time, HGLGT is a theoretical possibility. Some societies (or individuals) might consider using it for reasons other than the elimination of disease-causing genes, such as for adding “desirable” traits to a future human being. Some scientists may consider using a process of random insertion of transgenes into a genome in HGLGT. As discussed in chapter 3, random transgene insertion in HGLGT would be highly risky because of the many potential negative consequences that could occur. If genetic alteration affected a characteristic such as a personality trait, the permutations of genetic and environmental interactions would be complicated and unpredictable. For example, if genetic manipulations were designed to alter basic intelligence, how would the genetic alteration affect the person’s motivation, compassion, stamina and all the other things that are involved in achieving a goal or being successful according to societal standards? Because HGLGT is a real technical possibility, we believe it is necessary to grapple with the issue of HGLGT now so that we as a world community can make informed decisions and regulations before HGLGT is attempted. In the following chapters, we review many of the methodologies that need to be perfected before HGLGT is considered from a technical standpoint. The ethical, moral and political issues surrounding HGLGT are discussed in the later chapters of this book.

References 1. Chattergoon M, Boyer J, Weiner DB. Genetic immunization: A new era in vaccines and immune therapeutics. FASEB 1997; 11:753-763. 2. Donnelly JJ, Ulmer JB, Shiver JW, et al. DNA vaccines. Annu Rev Immunol 1997; 15:617-648. 3. Hess P. Gene therapy; A brief review. Clin Lab Sci 1996; 16:197-211. 4. Roush W. Backup gene may help muscles help themselves. Science 1997; 276:35. 5. White NJ, Breman JG. Malaria and other diseases caused by red blood cell parasites. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine. 14th ed. New York: McGraw-Hill, 1998:1182. 6. Cleaver JE, Kraemer KH. Xeroderma pigmentosum and Cockayne syndrome. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine. 14th ed. New York: McGraw-Hill, 1998:4403. 7. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. Cancer. In: Robertson M, ed. Molecular Biology of the Cell. 3rd ed. New York: Garland Publishing, Inc., 1994:12891290. 8. Verma IM. Gene therapy. Sci Amer 1990; 263:68-72.

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9. Russell DW, Hirata RK. Human gene targeting by viral vectors. Nat Genet 1998; 18:325-330. 10. Rehmann-Sutter C, Muller H. Ethik und Gentherapie; zum praktischen diskurs um die molekulare medizin. Tubingen: Attempto Verlag, 1995: 11. Lewin B. Chromosomes. In: Genes VI. Oxford: Oxford University Press, 1997:743-767. 12. Lewin B. Isolating the gene. In: Genes VI. Oxford: Oxford University Press, 1997:139. 13. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. Control of gene expression. In: Robertson M, ed. Molecular Biology of the Cell. 3rd ed. New York: Garland Publishing, 1994:456-457. 14. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. The cell nucleus. In: Robertson M, ed. Molecular Biology of the Cell. 3rd ed. New York: Garland Publishing, 1994:335-399. 15. Beaudet AL. Genetics and disease. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine. 14th ed. New York: McGraw-Hill, 1998:365-395. 16. Koopman P. The molecular biology of SRY and its role in sex determination in mammals. Reprod Fertil Dev 1995; 7:713-722. 17. Rasmussen H, Tenenhouse HS. Mendelian hypophosphatemias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 7th ed. New York: McGraw-Hill, 1995:3727-3729. 18. Lerner IM. Heredity, Evolution and Society. San Francisco, CA: WH Freeman and Co, 1968: 19. Bird TD. Alzheimer’s disease and other primary dementias. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine. 14th ed. New York: McGraw-Hill, 1998:2354. 20. Desnick RJ. The porphyrias. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine. 14th ed. New York: McGraw-Hill, 1998:2154. 21. Brinkman RR, Mezei MM, Theilmann J, et al. The likelihood of being affected with Huntington disease by a particular age, for a specific CAG size. Am J Hum Genet 1997; 60:1202-1210. 22. Boucher RC. Cystic fibrosis. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine. 14th ed. New York: McGraw-Hill, 1998:1448-1449. 23. Shalon LB, Adelson JW. Cystic fibrosis: Gastrointestinal complications and gene therapy. Pediatr Clin N Am 1996; 43:157-195.

CHAPTER 2

Alternatives to Human Germline Gene Therapy Introduction

T

here are many reasons why individuals seek medical assistance with reproduction. Motivating circumstances may exist when two prospective parents have a fertility problem, if they already have a child affected with a disease, if they are known carriers of a genetic defect, or if they are planning to conceive when there is advanced maternal age. With the recent advances in reproductive technology and diagnosis, these couples now have many choices to help minimize the chance that a future child will have a serious genetic disease. Human germline gene therapy (HGLGT) is only one (theoretical) approach which could be used. There is a significant difference between HGLGT and other technologies aimed at preventing the birth of a child with a genetic disease. With HGLGT, the DNA in cells would be manipulated to correct a genetic defect. Other technologies rely on the principles of selection and termination rather than genetic manipulation. That is, information from genetic diagnosis of immature eggs, embryos or fetuses is used in the decision of whether or not to implant an embryo or to allow a pregnancy to continue. Although HGLGT employs a different approach to avoiding genetic disease, many of the techniques described here as alternatives to HGLGT would also be part of the HGLGT procedure. For example, if an embryo were modified by HGLGT, the genetic changes would be checked by analyzing the genome of the resulting embryo before implantation. This method of genetic diagnosis on embryos, described in this chapter, is also used when selecting embryos for implantation in the absence of HGLGT. Since HGLGT would be a complicated procedure involving many techniques, a complete analysis of HGLGT involves consideration of safety and ethical issues surrounding all the methods associated with the HGLGT procedure. In writing this chapter we had two main goals: First, we wished to provide background information needed to fully appreciate what might be involved in an HGLGT protocol; and, second, we hoped to make the point that HGLGT would not be the recommended method for preventing the transmission of single-gene defects from parent to offspring in most cases or for avoiding certain chromosomal abnormalities in children. In presenting various reproductive choices, we have grouped procedures according to their common ethical considerations. For example, artificial insemination with sperm does not involve fertilization outside the body and does not involve intentional termination of embryos. In contrast, genetic diagnosis of an embryo before implantation involves selection of some embryos, termination of others, and may also involve prenatal testing of a fetus to confirm or reject the earlier diagnosis. As exploration of the ethical issues surrounding these specific procedures is beyond the scope of this book, we refer interested readers to other resources.1,2 However, we present some scientific aspects of the issues so that readers can use this information in an overall evaluation of methods used in these various procedures. Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus, Pamela J. Langer. ©1999 R.G. Landes Company.

18

Human Germline Gene Therapy: Scientific, Moral and Political Issues

To tie parts of our discussion to a real life scenario, we have chosen to envision two prospective parents who are known carriers of cystic fibrosis, an autosomal recessive disorder. Single-gene defects, the most common class of genetic disorder, are precisely the type of genetic defects which could be prevented without using HGLGT if at least one of the parents is a heterozygous carrier of the defective gene. In order to choose among the various reproductive options available, the couple would need to evaluate the risks and ethical considerations involved in each step of a procedure and be willing to accept the consequences. Although HGLGT might be one future option for correcting a genetic defect, the alternative reproductive choices may provide safer, more economical, and more reliable ways to achieve the couple’s goal of having a child without cystic fibrosis. All of the reproductive technologies described in this chapter have been performed and many are now widely available. In contrast, for many technical and ethical reasons discussed in later chapters, HGLGT has not yet been done.

Genetic Disease and Testing Categories of Genetic Disease Genetic disorders can be grouped into three main categories: 1. Chromosomal abnormalities; 2. Polygenic diseases; and 3. Monogenic diseases. Chromosomal Abnormalities Chromosomal abnormalities occur when a large amount of chromosomal DNA is either absent, in excess, rearranged or moved to a different chromosome. A chromosomal abnormality usually affects the function of many genes. Types of chromosomal abnormalities include: aneuploidy, when there is either one additional or one missing chromosome; polyploidy, when there are one or more extra sets of all the chromosomes (e.g., triploidy would mean there are three complete sets of chromosomes instead of two); translocations, when one part of a chromosome is moved to another part of the same chromosome or different chromosome; inversions, when a region of DNA is turned around or “inverted;” or duplications, where a region of DNA is duplicated. The incidence of aneuploidy is higher in pregnancies when there is advanced maternal age. Thus, aneuploidies such as trisomy 21 (three copies of chromosome number 21 resulting in Down’s syndrome) are more common in pregnancies involving women over 40. Polygenic Diseases Polygenic diseases are also called “multigenic diseases,” “complex disease traits” or “multifactorial disorders.” Although each of the names have slightly different connotations, the basic idea is that the development of such a disease involves multiple genes and is often greatly influenced by the environment. Although disorders such as some forms of diabetes mellitus, gout, heart disease or cancer can already be assigned to this class, we do not yet understand all of the contributing factors. Information from the Human Genome Project combined with population-based and familial studies should greatly enhance our understanding of polygenic diseases in the future. Monogenic Disorders Monogenic disorders, also called “single-gene” or “Mendelian disorders,” are determined primarily by a single gene, but can also be influenced by the environment. They are caused by autosomal recessive, autosomal dominant, or X-linked genes (see also chapter 1). It is

Alternatives to Human Germline Gene Therapy

19

estimated that about 1% of live-born children have a disorder attributable to a monogenic defect.3,4 Some of the more commonly known (but not necessarily the most frequently occurring) monogenic disorders include: 1. Autosomal recessive disorders such as cystic fibrosis, sickle cell anemia, β-thalassemia, homocystinuria, phenylketonuria, Lesch-Nyhan syndrome, and α1-antitrypsin deficiency; 2. Autosomal dominant disorders such as Huntington’s disease, familial hypercholesterolemia, familial polyposis, and breast cancer associated with the BRCA1 or BRCA2 genes; and 3. X-linked disorders such as Duchenne muscular dystrophy, fragile-X syndrome, and color blindness. Although there are approximately 4500 known human diseases that are caused by a monogenic defect,4 detection is limited to the most common genetic diseases for which diagnostic tests have been developed. Because of the rapid pace of cloning human disease genes, many more genetic diagnostic tests could become available in the near future, as long as there is sufficient demand to make their development economically feasible.

Genetic Testing and Risk Assessment When considering whether or not to conceive a child, some prospective parents choose to undergo genetic testing. Frequently their interest in genetic testing is prompted by the fact that one of the parents, a previous child or other close blood relative is afflicted with a specific genetic disorder. The parents we use as an example in this chapter are known carriers of an autosomal recessive defect in a gene called the cystic fibrosis transmembrane regulator (CFTR) gene. For simplicity, we refer to this CFTR gene as a “CF gene” since it is responsible for causing cystic fibrosis when a child receives two defective alleles. Cystic fibrosis is a disease which can lead to dysfunction of the lungs, pancreas, intestine, urogenital system and sweat glands. Cystic fibrosis patients frequently secrete a thick dehydrated mucus in the lungs. This mucus is difficult to clear and it traps microorganisms and promotes infections. Certain bacteria colonizing the lung may further exacerbate the condition by stimulating mucus formation by lung epithelial cells.5 The incidence of cystic fibrosis varies widely among different ethnic groups.6 An average estimate of the incidence of cystic fibrosis in the United States is approximately 1/3600. The frequency of presence of a defective CF allele (i.e., the allelic frequency ) is 1/30. To date, there are over 400 different CF gene mutations known.6,7 However, some CF mutations have a higher incidence than others. The most common defect, present on 66% of chromosomes carrying a CF defect, is known as ∆-F508.6 This particular mutation is a 3 base pair deletion in the CF gene which results in a CF protein lacking an amino acid called phenylalanine at position 508 in the protein. Whenever a specific genetic defect is very common, that is, when it has a high allelic frequency, that defect is the primary suspect in genetic testing. However, in the case of cystic fibrosis it is clear that many defective alleles would be missed if genetic testing identified only the ∆-F508 mutation. When a more complete test for CF gene defect carriers is desired, tests for the next most common group of mutations are used, and so on. Of course, with the increase in number of genetic tests performed comes a large increase in the financial cost to the patient. If a close family member already has cystic fibrosis, an alternative to testing for specific mutant alleles is to test for the presence of other DNA regions that are next to the mutant allele. This process is referred to as genetic linkage analysis. Genetic linkage analysis is based on the fact that adjacent DNA regions have a good chance of traveling together from parent to child. So, in the case of an unknown CF gene mutation, if one can follow the inheritance

20

Human Germline Gene Therapy: Scientific, Moral and Political Issues

of DNA associated or “linked” with the unknown mutation, one can predict, with a certain degree of expected error, whether an individual carries the unknown mutation. Linkage analysis does not work in all genetic situations and is not very useful unless there is already an affected family member (the proband). However, linkage analysis can provide information, with a certain probability of being accurate, at a lower cost than testing for specific mutant alleles. As more mutations are characterized and genetic testing methods become more rapid and accurate, the usefulness of linkage analysis will probably decline.

Genetic Counseling Suppose that our prospective parents carrying cystic fibrosis defects request genetic counseling in hopes of learning about their various options. Since they are each a heterozygous carrier for a CF defect, they have a 25% chance of having a child afflicted with cystic fibrosis, a 50% chance of having a heterozygous carrier, and a 25% chance of having a child born free of a defective CF allele (Fig. 1.3). They are advised that they have several prenatal and postnatal options. Since the couple wishes to evaluate various procedures based on risk estimates and on ethical considerations, they would like to know which methods are used in each of the various procedures. The type of classification shown in Table 2.1 may help clarify the ethical decisions involved when evaluating various procedures.

When In Vitro Fertilization Is Not an Acceptable Option If for ethical or religious reasons, our cystic fibrosis carrier couple does not want fertilization to occur in vitro, they have several options to consider. They could eliminate their chances of having a child with cystic fibrosis by choosing not to conceive. However, any method of birth control other than abstinence or hysterectomy has a certain chance of being ineffective. They could choose to conceive naturally and plan to treat a cystic fibrosis child with conventional treatments or somatic gene therapy (SGT). Alternatively, they could adopt, use donated sperm or eggs, or employ prenatal genetic testing with the option of pregnancy termination. Since options involving germline genetic manipulation or potential interruption of pregnancy involve another set of ethical issues, we will discuss these options in later sections of this chapter.

Postnatal Treatments Despite the fact that the pathology in some genetic disorders can be managed quite well with medication, for others a satisfactory treatment plan has not been devised. The most common treatment for cystic fibrosis is conventional medical therapy, including antibiotics to manage bacterial colonization in the lungs, daily physical therapy to help break up mucus in the lungs, and dietary supplements to make up for deficient secretion of pancreatic digestive enzymes.8 If conventional therapy is followed, the average life expectancy for a cystic fibrosis patient is on the order of 30-40 years.8,9 However, the burden of the intensive therapy could be lessened for the patient and caregivers with a more effective treatment for cystic fibrosis, which could vastly improve their quality of life. Patients will greatly benefit from the development of alternative therapies such as SGT, when the treatment protocols are more effective. The treatment of cystic fibrosis is one of the prime targets for SGT. However, as in many other genetic diseases, in cystic fibrosis the genetic defect affects the physiology of several cell types, including epithelial cells in the lung, pancreatic duct, sweat duct and liver biliary duct. To address the complete clinical picture with SGT, many cells at several different locations in the body would eventually have to be altered genetically.

Alternatives to Human Germline Gene Therapy

21

Adoption Another possibility for our couple is adoption. In this case the couple will (usually) not be genetically related to the child, they would not experience the pregnancy and birth, and they may have to wait an extended period of time due to the high demand for children to adopt. Furthermore, adoption of an infant does not guarantee that the child will be free of a genetic disorder. Adoption only eliminates the parents' role in passing on defective alleles to a child.

Gamete Donation The donation of gametes (sperm or immature eggs called oocytes) to our cystic fibrosis carrier couple reduces the possibility of having a child afflicted with cystic fibrosis but it does not eliminate it. The chance of having a cystic fibrosis child using gametes donated by a person in a particular ethnic group can be estimated. Statistical Risks in Gamete Donation For the following calculations we assume that gamete donors and recipients are within a population where the allelic frequency of a defective CF allele is 1/30. (The reported incidence of cystic fibrosis in the Caucasian population in the United States would suggest an allelic frequency in this range.)10 In the case of donation to our heterozygous female carrier, the chance of having a child with cystic fibrosis is determined by multiplying the following probabilities: The chance that the female recipient has a cystic fibrosis allele (= 1); the chance that the female will pass on a defective CF allele (= 1/2); the chance that the male donor has a CF allele (= 1/30); the chance that the male donor will pass on a defective CF allele if he has one (= 1/2). Thus, there is a 1/120 (1 x 1/2 x 1/30 x 1/2) chance that the child will have cystic fibrosis. This 1/120 chance of having cystic fibrosis is much less than the 1/4 (1 x 1/2 x 1 x 1/2) chance if the heterozygous parents choose to conceive without using donor gametes. If the child is born without cystic fibrosis after single gamete donation, the child still has a high chance of being a carrier for a defective CF allele, since the child has a 50% chance of receiving it from the heterozygous mother. If the couple accepts donations of both sperm and oocytes, the chance of having a cystic fibrosis child is now (1/30 x 1/2 x 1/30 x 1/2 =) 1/3600. If genetic testing of a gamete donor demonstrated the absence of a defective CF allele, the risk of having a child with cystic fibrosis would be significantly less. Still, it would not be a zero chance, since not all defective CF alleles are detected in the standard genetic tests. Furthermore, with gamete donation, the couple must still understand that although they are reducing their risk of having a child afflicted with cystic fibrosis, the risk of having a child with other genetic disease still remains. In Vivo Conception with Donated Gametes Although in vitro fertilization (IVF) can be used with gamete donation, it is also possible to allow conception to take place inside the body. Artificial insemination with donor is a technically simple procedure and poses little risk to donor or recipient. The donated are administered to the female recipient at the proper time of her menstrual cycle, thereby optimizing conditions for successful fertilization. Depending on her medical history with respect to fertility, the physician may advise a drug regime which will stimulate ovulation. This is called controlled ovarian hyperstimulation or superovulation, since multiple oocytes may be ovulated during the process. Various drugs, including clomiphene citrate, gonadotropin releasing hormone or human menopausal gonadotropin, in combination with human chorionic gonadotropin, may be used to promote the development of the oocytes.11 In most cases superovulation is achieved with

Adoption or conventional medical treatments Artificial insemination Gamete intrafallopian transfer (GIFT)3 Preconception genetic diagnosis Preimplantation genetic diagnosis (PGD) Prenatal diagnosis: (CVS or amniocentesis) Somatic gene therapy (SGT) Human germline gene therapy (HGLGT)

PROCEDURES

N

N

N Y/N5 Y

N

N Y

Y4

Y

Y

N

N

Y Y

N

N

Y

N

N

Y/N

N

Embryo biopsy

N

In Vitro fertilization

N

Superovulation

Y

N

N

Y

Y/N5

N

N

N

Embryo selection1

Y

N

N

Y

Y/N5

N

N

N

Embryo transfer and storage

C

N

Y

Y

N

N

C6

C

N

N

N

N

Genetic manipulation of somatic cells

C6

C

C

N

Potential interruption of pregnancy2

METHODS

Table 2.1. Procedures involved in various reproductive or medical choices

Y

N

N

N

N

N

N

N

Cellular expansion of early embryonic cells

Y

N7

N

N

N

N

N

N

Genetic manipulation of germline cells

22 Human Germline Gene Therapy: Scientific, Moral and Political Issues

Alternatives to Human Germline Gene Therapy

23

Table 2.1. Table legend. The likelihood that a particular method will be employed in various procedures is indicated: (N), method is usually not involved; (Y), method is usually involved; (C), patient may choose the use of the method, depending on the physician’s recommendation and other circumstances. 1 Embryos are clinically selected in two ways: (i) on the basis of morphological characteristics, and (ii) on the basis of genetic testing. Embryo selection consequently results in rejection of some embryos so that genetic abnormalities, spontaneous abortions and intentional interruptions of pregnancy can be minimized. 2 If multiple embryos develop, multifetal pregnancy reduction may be recommended by the physician in order to increase the chances of one or more of the fetuses being born healthy. This category also includes selective termination after genetic testing in a multifetal pregnancy or complete abortion of a pregnancy. 3 In gamete intra-fallopian transfer (GIFT), usually a mixture of sperm and oocytes are introduced into the end of the fallopian tube. The oocytes may be from the female partner or from a donor female. A modification of this procedure involves separation of oocytes and sperm until inside the female recipient. 4 Either an oocyte donor or the female patient may undergo superovulation to stimulate the release of multiple oocytes to be used in assisted reproductive techniques. 5 If the selected oocytes are transferred to the female before conception (e.g., with a modification of the GIFT procedure), IVF and embryo selection would not be involved. If the oocytes are fertilized in vitro while the genetic tests are being performed on the first polar bodies, embryo selection, transfer and storage may be involved. Alternatively, the zygote(s) may be transferred. 6 Since errors can occur in pre-conception or pre-implantation genetic diagnosis (see text), the clinician may recommend that prenatal testing be used to confirm the fetal genotype. If a couple does not want to increase the risk of spontaneous abortion with amniocentesis or CVS, or has decided that they are not willing to consider interrupting the pregnancy under any circumstances, they may refuse this further testing. 7 There is a theoretical chance that germline cells could unintentionally be genetically altered during somatic gene therapy procedures.

one of these protocols, and it is followed by intrauterine insemination. Superovulation can be monitored with ultrasound to assess the number of oocytes developing,12 and insemination is avoided in a cycle when a large number of oocytes are detected. However, even if relatively few oocytes are ovulated, there is still a chance of multiple fetuses developing, should fertilization occur. Reducing the number of fetuses using the technique of multifetal pregnancy reduction may be recommended by the physician in order to increase the chance of giving birth to at least one healthy infant.13 In contrast to the relatively easy procedure of artificial insemination, oocyte donation is a much more invasive procedure for both the donor and the recipient. The donor must be superovulated according to a protocol designed to stimulate the development of many oocytes.14 The oocytes are collected by transvaginal aspiration with ultrasound guidance.15 If five or more oocytes can be retrieved after superovulation, the ovulation and collection aspects of the procedure are considered successful.16 Excess oocytes may be frozen for future use. If our cystic fibrosis carrier couple desires an in vivo conception with donated oocytes, the next step is a variation of a procedure called gamete intrafallopian transfer or GIFT. In a typical GIFT procedure, sperm and oocytes are combined and introduced into the end of the fallopian tube within 30 minutes of oocyte recovery.17 However, in order to accommodate a couple’s wish for an in vivo conception, individual fluids containing sperm or oocytes can be separated by an air bubble, thus delaying any potential union between sperm and oocyte until inside the woman’s body. Alternatively, donated oocytes could be transferred

24

Human Germline Gene Therapy: Scientific, Moral and Political Issues

to the fallopian tubes first, followed by a later intrauterine insemination with sperm. The in vivo conceptions using GIFT are approved by the Catholic Church as acceptable means of assisted reproduction.18 Some authorities suggest that there might be a greater pregnancy rate with GIFT as compared with IVF. It is estimated that 20-30% of IVF patients and 30-40% of GIFT patients will become pregnant when two or three embryos are transferred.19 In contrast, others suggest that studies comparing GIFT, IVF or superovulation with intrauterine insemination are still inadequate for drawing conclusions about the relative efficacy of these procedures.20

When In Vitro Fertilization and Embryo Selection Are Acceptable Options In this section we examine options for our cystic fibrosis carrier couple when they are willing to consider procedures involving IVF and embryo selection. In most cases fertilized oocytes (zygotes) are cultured in vitro until the embryos reach a stage where their suitability for transfer can be assessed. As with in vivo conceptions, many abnormalities in fertilization and embryonic maturation can occur in vitro .19 It is almost inevitable that some zygotes or embryos will die or be judged morphologically abnormal. Also, if embryos are analyzed for genetic abnormalities, they could be damaged in the process or could be rejected if they are diagnosed with a serious genetic defect. Selected embryos are transferred to the female or frozen for potential future use. Frozen embryos are also at risk for termination because of potential loss of viability during freezing and thawing or lack of use if the parents so choose.21 Thus, the following procedures can be considered only if the couple can accept the process of intentional or unintentional pre-pregnancy embryo termination.

Fertilization and Embryo Development To facilitate the subsequent discussions, we first review the normal developmental scheme for gametes and early embryos and the processes of IVF and embryo transfer. Gamete Development Even before a female is born, her immature eggs are well on their way in their development. Immature eggs develop through a series of stages, starting with primordial germ cells which migrate into the developing gonads, where they form oogonia. Oogonia undergo normal cell division in a process called mitosis (Fig. 2.1). At some point between 3 and 8 months of gestation,22 oogonia duplicate their chromosomes for the last time in fetal development. Now called primary oocytes, the cells enter into the first part of a unique type of cell division called meiosis (meiotic division I). Although the two chromosome homologs have been duplicated, the cell does not divide. Instead, the maternal and paternal chromosome homologs line up next to one another and begin the process of exchanging similar regions of their chromosomes. This exchange of DNA, which occurs because one region of DNA “crosses-over” to the other chromosome homolog, is responsible for making new chromosomes that differ from those inherited from the parents. These new chromosomes are the ones which are eventually passed on to the future children of the developing fetus. The chromosomes we inherit from our parents normally stay intact inside our somatic cells. It is predominantly in our germ cells that crossing-over takes place between the chromosomes we inherited from our parents. Shortly after birth, the process of crossing-over in females is usually complete. Still without dividing, the primary oocytes enter a resting phase within the diplotene stage of meiotic division I until they undergo oocyte maturation after the female reaches sexual

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maturation.23 Under proper hormonal stimulation during a menstrual cycle, a primary oocyte completes meiotic division I by dividing asymmetrically into a small, first polar body and a larger cell called the secondary oocyte. The result is a reductive division where each member of a chromosome pair is distributed to one of the two cells. The final division of the oocyte, called meiotic division II, does not occur until after fertilization. Meiotic division II is also an asymmetric division resulting in a small second polar body and a larger mature egg, each of which now has only one copy of each chromosome homolog. Genetic exchange by crossing-over occurs at different times in the female and male. In the female, chromosomes in the primary oocyte are involved in the process of crossing-over in fetal life. In the male, genetic exchange during meiotic division I occurs in developing sperm which are continuously being generated throughout adult life.24 Fertilization As the primary oocyte matures, it develops a protective coat called the zona pellucida (Fig. 2.2). To achieve fertilization, the sperm must bind to a component of the zona pellucida on the secondary oocyte, penetrate the zona pellucida, fuse with the oocyte surface membrane, and release the sperm nucleus into the oocyte cytoplasm. The fusion of the sperm membrane with the oocyte plasma membrane initiates an oocyte response designed to block the entry of additional sperm. If more than one sperm does fuse with the oocyte membrane, a condition called polyspermy will result. With polyspermy there can be abnormal segregation of chromosomes, which will usually cause the embryo to die. The arrival of a sperm also stimulates the oocyte to complete meiotic division II, after which the second polar body and the mature egg are formed. The sperm and egg nuclei, called pronuclei, duplicate their chromosomes. The two pronuclei approach each other and the nuclear membranes interdigitate and break down. The pronuclear membranes do not fuse directly in mammals as they do in many other species.22 The fertilized egg (zygote) proceeds through a mitotic division, resulting in two cells called “blastomeres.” The polar bodies may persist for the first few cell divisions of the embryo, but they soon degenerate. In vitro fertilization of oocytes with sperm has long been used as a means of assisted reproduction for infertile couples. Although there are many variations in procedure, the basic steps are to superovulate the female, collect immature oocytes using transvaginal aspiration guided by ultrasound, incubate the oocytes to allow further development (0 to 8h), and then combine oocytes and sperm in vitro. The probability that conception will occur in one cycle of IVF is 20-30%.25 Variations of this scheme have been developed to circumvent some of the problems with fertilization and implantation. For example, to increase the likelihood of sperm penetration, sperm may be injected beneath the zona pellucida. Alternatively, sperm may be injected directly into the oocyte cytoplasm, in which case the procedure is called “intracytoplasmic sperm injection.”26 For any method of IVF, one of the most critical factors, in addition to the viability and activity of the sperm, is the developmental status of the oocyte to be fertilized.27 As a consequence of superovulation, oocytes in various stages of development are collected. It is of utmost importance to be able to classify the degree of oocyte maturity before continuing with IVF. If an immature oocyte is fertilized prematurely, abnormalities in fertilization or embryonic development may ensue.19 For example, if dispermy occurs (fertilization with two sperm) and the abnormality is not detected, the triploid embryo will usually not survive to the fetal stage. A minimum criterion for an oocyte to be used for IVF is that it is a secondary oocyte, as evidenced by the appearance of the first polar body.19 If the oocytes are not sufficiently mature, they may be incubated for a longer time in culture before combining them with sperm.

26

Human Germline Gene Therapy: Scientific, Moral and Political Issues

Fig. 2.1. Human egg development. Nuclei of developing eggs, from the oogonium stage to the mature egg, are shown. Only one pair of chromosomes is drawn. Two lines of descent are shown to illustrate the distribution of “A” or “a” alleles, with or without crossing-over. Oogonia divide mitotically in the developing gonad until between 3 and 8 months of gestation.22 After the last DNA replication in fetal development, an oogonium becomes a primary oocyte which will enter into the process of meiosis. In prophase of meiotic division I, crossing-over between chromosome homologs takes place. The primary oocytes then “rest” in the diplotene stage of meiotic prophase I until the female reaches sexual maturity, when they undergo oocyte maturation. The first meiotic division is a reductive division resulting in a secondary oocyte and a first polar body, each of which receives only one chromosome of each chromosome pair. After fertilization, the second meiotic division takes place, resulting in a mature egg and a second polar body, each of which has a haploid chromosomal DNA content.

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Early Embryonic Development At the start of embryogenesis, the blastomeres rely on reserves of materials synthesized by the egg. The embryonic genome becomes activated between the 4 and 8 cell stage.28 Depending on the animal species studied, the first few cell divisions result in blastomeres which are totipotent, meaning that each cell has the full potential for developing into an animal. For example, single blastomeres from an 8 cell stage pig,29 rabbit30 or mouse embryo31 have each developed into normal animals. No one has ever reported experimentally splitting a human embryo into blastomeres and allowing each to develop into a child (this would be considered “cloning.”) However, about one third of monozygotic (identical) twins originate from an embryo split before the development of the trophoblast layer of the blastocyst (see Fig. 2.3). This fact is highly suggestive that these early cells are totipotent.32 Furthermore, since we know that the 8 cell human embryo can lose some of its blastomeres and still develop normally (see below), even 8 cell stage blastomeres are not yet irreversibly committed to a developmental fate. Blastomere loss could occur naturally or as a result of in vitro manipulations. When human embryos are frozen, some of the blastomeres may degrade. Although many embryos that lose blastomeres are not viable, embryos with some blastomere loss can implant and result in a live birth.33 Furthermore, one or two of the blastomeres may be intentionally removed at the 8 cell stage for diagnostic purposes (blastomere biopsy). Following removal of a blastomere, an 8 cell embryo is still capable of growing into a normal fetus and child. However, blastomere removal before the 8 cell stage can result in reduced embryo viability due to the sensitivity of the human embryo to micromanipulation at this time in development.34 Between the 8 and 16 cell stage, the blastomeres go through a process called compaction, in which the cells become more tightly associated. The cells sort into an external cell region surrounding a few internal cells in the 16 cell morula stage (Fig. 2.3). By about the 64 cell stage, the embryo develops into a blastocyst, which consists of an inner cell mass destined to become the fetus, an inner fluid-filled cavity called the blastocoel, and the outer trophoectodermal layer containing trophoblasts (Fig. 2.3).19 Data from experiments with mice suggest that only 3 or 4 cells from the 32 to 64 cell stage are destined to become the cells in the inner cell mass.35 The majority of early embryonic cells become part of extraembryonic tissue, starting with the trophoectoderm. This later develops into the chorion, which is part of the placental tissue. Once the blastocyst sheds the zona pellucida in a hatching process, implantation may occur, usually between 5 and 8 days after fertilization.36 One technique in assisted reproduction is directed toward increasing the chance that an embryo will hatch out of a thickened zona pellucida. A hole drilled chemically in the zona pellucida will assist hatching and increase the probability of implantation.19 Embryo Selection The term “embryo selection” is usually used to refer to the process of choosing certain embryos for uterine transfer after genetic diagnosis. However, there are actually three types of embryo selection: natural selection of embryos after in vivo or in vitro conceptions, embryo selection based on morphological criteria and embryo selection based on genetic criteria. For simplicity, we will refer to these as “natural embryo selection,” “morphological embryo selection,” and “genetic embryo selection,” respectively. After in vivo or in vitro conceptions, natural embryo selection in the uterus occurs because only some embryos develop normally, implant and develop into fetuses. Problems with fertilization, such as polyspermy, or genetic abnormalities could arrest development of the embryo. It is estimated that for in vivo conceptions, 20-25% of embryos

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Human Germline Gene Therapy: Scientific, Moral and Political Issues

Fig. 2.2. Formation of a human zygote. Immature eggs (called oocytes) develop through a series of stages into mature eggs. The fate of one pair of homologous chromosomes in the primary oocyte is indicated. As the primary oocyte matures, it synthesizes a protective egg coat called the zona pellucida. Upon proper hormonal stimulation, the primary oocyte divides asymmetrically, giving rise to a small first polar body and a larger secondary oocyte. During fertilization, the sperm binds to the zona pellucida and the sperm membrane fuses with the cytoplasmic membrane of the secondary oocyte. After sperm entry, the sperm head decondenses78 and the secondary oocyte completes its maturation by dividing asymmetrically to form a second polar body and the mature egg. The sperm and egg pronuclei contained within the egg cytoplasm approach each other, the nuclear membranes disintegrate and a mitotic spindle is formed in the zygote. In contrast with Figure 2.1, in which only the nuclei are shown, in this figure both nuclei (shaded area) and cytoplasm (clear area) are illustrated.

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have chromosomal abnormalities that either prevent development to the implantation stage or cause spontaneous abortions during the first or second trimester.37 This number varies with maternal age since in vitro (and in vivo) fertilized embryos from women over 40 years old exhibit a significantly higher number of chromosomal abnormalities.38 The most common type of genetic abnormality is an aneuploidy where there is one extra or one missing chromosome. Some types of aneuploidy can result in viable fetuses. For example, one extra copy of chromosome number 21 (trisomy 21) causes Down’s syndrome. Some other autosomal trisomies are also viable. Additional X or Y sex chromosomes produce viable offspring, such as in Kleinfelter’s syndrome, where the male has an XXY genotype and is usually infertile. Aneuploidy in which there is one missing chromosome (monosomy) does not produce a viable fetus, except in the case where there is only one X chromosome and no Y chromosome. Although the XO genotype is found in 1-2% of all pregnancies, less than 1% of these produce a live-born female (a sterile Turner’s syndrome female).39 Morphological embryo selection routinely occurs during an IVF and embryo transfer procedure. Embryos are rated in order to determine which embryos are candidates for embryo transfer or storage. Clinicians assess the rate of embryo growth, the regularity and symmetry of the blastomeres, the proportion of fragmented cells, and the clarity of blastomere cytoplasm.19 Morphologically abnormal embryos are discarded, since most would never be able to develop into a normal fetus. Genetic embryo selection uses information from preimplantation genetic diagnosis (PGD), as discussed below. Genetic embryo selection must be coupled with morphological embryo selection to maximize the chance that the selected embryo will implant and develop normally. When morphological embryo selection alone is used, such as in conventional IVF, many of the normal-appearing embryos may actually harbor major chromosomal abnormalities. Since some chromosomal abnormalities can impede or terminate embryonic development, morphologically normal embryos do not necessarily have equal chances of survival. Thus, although it may appear that genetic embryo selection is used to select certain embryos from a group of morphologically normal embryos with equal chances of survival, this is not the case, for the reason stated above. The distinction between embryo selection based on morphology or genetics is important, since genetic selection of embryos raises ethical concerns, such as eugenics, which will be discussed later. Consequently, the applications of embryo selection must be closely monitored by regulatory agencies. In the United Kingdom, the Human Fertilization and Embryology Authority oversees the application of embryo selection in order to avoid the use of this technology for selecting desirable traits in an attempt to carry out a genetic enhancement.37 Embryo Transfer Usually after one or two cleavage divisions, a chosen number of the healthiest embryos are transferred to the uterus to allow implantation and initiation of pregnancy. In the absence of a need for genetic diagnosis, embryos are normally transferred from culture to the uterus 24-48h after fertilization (before the 8 cell stage) (Fig. 2.3).19 If the embryos are grown to the 8 cell stage for genetic diagnosis, the selected embryos will be transferred to the uterus, preferably the same day. Although up to three embryos may be transferred,19 many physicians choose not to transfer more than two in order to minimize complications in pregnancy.37 Approximately 20% of all embryos transferred, considering multiple IVF procedures, will result in the birth of a child.40 If multiple embryos survive, in order to avoid obstetric complications the physician may advise a multifetal pregnancy reduction to increase the chances that the remaining fetuses will survive to term and be born healthy.13

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Human Germline Gene Therapy: Scientific, Moral and Political Issues

As an alternative to embryo transfer, a single celled zygote can be transferred to the fallopian tube within 24 hours of conception.11 This procedure is called zygote intrafallopian transfer or ZIFT. In terms of the pregnancy success rate, ZIFT may have some advantages as compared with transferring a more developed embryo.19 While ZIFT allows for selection of a normally fertilized egg, the zygote does not remain in culture as long as a more developed embryo. On the other hand, allowing a few cell divisions to take place provides an opportunity to select embryos which are developing normally and allows for genetic screens to be performed. Cryopreservation Embryos can be frozen at various developmental stages ranging from the pronuclear one cell stage to the blastocyst stage.41-43 The methods of freezing and thawing of embryos, embryo culture systems, and embryo transfer techniques have advanced rapidly in the past 10 years. In some centers, the implantation rate of cryopreserved embryos approaches that of fresh embryos.41 Collected unfertilized oocytes can also be cryopreserved with an increasing degree of success; however, there are currently many more technical difficulties with freeze-thawing oocytes than with embryos.41,44,45 Approximately 60% of cryopreserved oocytes are viable upon thawing.46 Over the years, there have been many discussions concerning the rights and ownership of frozen embryos. Since this topic is beyond the scope of this book, we refer readers to other sources.1,47,48

Preimplantation Genetic Diagnosis The overall goal of preimplantation genetic diagnosis (PGD) is to provide a new diagnostic option for detecting genetic defects before pregnancy and to promote the health of future human beings in a fundamental way by eliminating a particular disease rather than treating it postnatally. In the case where a couple plans to act on the results of genetic testing in order to avoid giving birth to a child with a serious genetic defect, PGD would offer several advantages over other forms of prenatal testing. The major advantage of PGD vs. prenatal testing is that embryos which do not have the disease-causing genes can be identified before pregnancy is initiated, i.e., before implantation. Although the process of genetic embryo selection is controversial, it is generally accepted from a medical standpoint that embryo selection before implantation is more desirable than termination of an established pregnancy.1,37 Since large population-based surveys have not yet been conducted on the relative desirability of embryo termination vs. fetal abortion as a means for avoiding the birth of a child with a serious genetic disorder, we cannot yet assess general public opinion. The results from one small survey of carriers of recessive disorders suggests that both PGD and elective abortion would be considered valuable reproductive options.49 There are other advantages of PGD. First, if PGD is highly accurate, it could reduce the chance of unintentional fetal loss associated with prenatal diagnostic procedures such as chorionic villus sampling or amniocentesis (see chapter 2). Second, if a clinician is able to select for embryos without serious chromosomal imbalances, there may be a greater chance of implantation, a lower risk of miscarriage, and thus a greater chance of a successful pregnancy. Third, a greater chance of a successful pregnancy would also be beneficial to the prospective mother. It would reduce the number of times she would undergo the physical and emotional distress of the first few months of pregnancy and loss of the fetus. It could also be argued that PGD promotes life, since people who elect to use PGD could actually end up with a greater number of children than they would otherwise because they would be able to procreate with only a minimal fear of passing on a genetic defect to

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APPROXIMATE NUMBER OF DAYS AFTER FERTILIZATION

Zygote

0

mitotic spindle zona pellucida

2-cell stage

1 - 1.5

4-cell stage

2

8-cell stage

2.5 - 3

Morula

3-4

compaction

inner cell mass blastocoel

Blastocyst

4-5

trophoblast

Fig. 2.3. Early human embryonic development. The early stages in embryogenesis are illustrated, starting with the 1 cell zygote and ending with the blastocyst. Whole (shaded) cells are shown for the 1 , 2 , 4 and 8 cell cleavage stage embryos and crosssections are shown for the morula and blastocyst stages. The zona pellucida (light gray) surrounds the embryo until it hatches. See text for more detail. their future children. (This argument assumes that financial cost is not a factor for the couple.) However, if one round of PGD could result in selection of an embryo with a genotype which gives it a greater chance to be born, the couple may save the considerable cost of multiple rounds of IVF attempts. One of the prerequisites for PGD is to be able to analyze the genetic material in a single cell (single-cell genetics). Techniques involving amplification of DNA with a polymerase chain reaction (PCR) or direct visualization of chromosomes with fluorescence in

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Human Germline Gene Therapy: Scientific, Moral and Political Issues

situ hybridization (FISH) have allowed the analysis of DNA from a single cell. Although it is beyond the scope of our discussion to review all of the techniques used in PGD, the PCR and FISH methods and potential sources of diagnostic error are described below. All of the PGD techniques except for one are carried out after IVF. The exception is a newly-developed approach called “preconception genetic diagnosis,” which analyzes the DNA in the first polar body. Post-fertilization techniques include two-step oocyte genetic analysis, where the first and second polar bodies are sequentially analyzed, or embryo biopsy, where one or more cells are removed from the embryo for analysis. If a pregnancy is achieved after PGD, it is generally recommended that the patient undergo later fetal prenatal diagnosis to confirm the PGD results;50 however, this is the patient’s choice. Preconception Genetic Diagnosis Preconception genetic diagnosis is used to determine whether a secondary oocyte is a carrier for a particular genetic disorder. Preconception genetic diagnosis involves the analysis of the first polar body produced after the first meiotic division (Fig. 2.1). Since the first meiotic division sorts one chromosome of each pair into either the first polar body or the secondary oocyte, the genotype of the secondary oocyte may be inferred from the genotype of the first polar body. Selection of unaffected secondary oocytes is then followed by either IVF and embryo transfer or the GIFT procedure, where sperm and selected oocytes are combined and transferred to the fallopian tube (see above). In order to describe the advantages and limitations of preconception genetic diagnosis, we will use the following example: Consider a female carrier of an autosomal recessive disease (like cystic fibrosis) who has one defective allele which we will call “a” and one normal “A” allele. Before the first meiotic division, the replicated chromosomes in the primary oocyte will have two copies of the “A” allele on one chromosome and two copies of the “a” allele on the homologous chromosome (see Fig. 2.1). In the absence of crossing-over, the two “a” alleles will travel together and the two “A” alleles will travel together at meiotic division I. If the first polar body receives the two “A” alleles, the secondary oocyte will receive the two defective “a” alleles (left side of Fig. 2.1). Conversely, if the first polar body receives the two defective “a” alleles, the secondary oocyte will receive the two normal “A” alleles (not shown in Fig. 2.1). Another distribution of alleles must be considered since crossing-over may take place 50% of the time between the chromosome homologs before completion of meiotic I division. (Frequency of the crossing-over event depends on the location of the gene on the chromosome; however, for our purposes, we will consider the crossing-over frequency to be 50%.) With crossing-over, the first polar body and the secondary oocyte will each contain one defective “a” allele and one normal “A” allele (right side of Fig. 2.1). With preconception genetic diagnosis, only a subset of the secondary oocytes can be used for fertilization,51 namely the 25% that have the “AA” genotype (Table 2.2). The “aa” secondary oocyte is homozygous defective and the “Aa” secondary oocyte has equal chances of yielding a mature egg with the defective “a” allele or the normal “A” allele. If only a few oocytes are collected (five or less, for example), candidate secondary oocytes may be few or may not even be available from one superovulation of the female. Preconception genetic diagnosis has the unique advantage that oocytes can be screened prior to conception. The manipulation of the unfertilized oocyte is minimal and does not appear to have any deleterious effects on the developing embryos.51 If the selected oocytes are subsequently used in an IVF procedure, there will still be a morphological embryo selection. However, preconception genetic diagnosis avoids genetic selection of embryos and does not involve cell removal from early embryos for testing. The only cell that is analyzed is

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the first polar body which is naturally destined to die. Thus, where there are individual or societal restrictions on genetic embryo selection, preconception genetic diagnosis could still be a viable option. Preconception genetic diagnosis can also be used to detect chromosome number abnormalities in the secondary oocyte. For example, analysis of the first polar body would provide an indication of whether an oocyte would be destined to give rise to a trisomic fetus after fertilization. That is, if the first polar body lacked a chromosome 21, the oocyte would have two copies of chromosome 21. After the second meiotic division, the contribution of two copies of chromosome 21 from the egg and one from the sperm would yield a trisomy 21 embryo. Selection of oocytes with a normal chromosomal content may be used to reduce the number of children born with aneuploidies and the number of spontaneous or intentional fetal abortions resulting from chromosome abnormalities. (See below for a method for detecting chromosomal abnormalities in single cells.) This procedure may be particularly useful for couples when there is advanced maternal age, a situation where there is a higher incidence of chromosomes not sorting properly into cells. Although an overall estimate of the incidence of embryo aneuploidy is 20-25%,52 the incidence of aneuploid embryos is age related, increasing from less than 10% in women under 40 to approximately 34% in women over 40.38,40,52 There are also serious disadvantages with preconception genetic diagnosis. Only 25% of the original oocytes can be used for fertilization and it is possible that none of the collected oocytes will qualify for use. As a diagnostic technique it is limited, in that only maternal defects are detected.34 Furthermore, it is not highly reliable since only a single cell is available for genetic analysis. Testing two cells dramatically increases the statistical chances that a diagnosis will be accurate.53 Consequently, a new technique for analyzing polar bodies, called two-step oocyte genetic analysis, is being developed to address some of the disadvantages of preconception genetic diagnosis. Two-step Oocyte Genetic Analysis This PGD procedure is called a two-step analysis because genetic material of the first and second polar bodies is analyzed. Since the second polar body is not extruded from the oocyte until after fertilization, this is not a preconception diagnostic procedure. With first polar body analysis, the genotype of only 50% of the mature eggs, on average, can be predicted (see Table 2.2). With analysis of only the second polar body, none of the genotypes of the mature eggs can be determined. That is, if the second polar body carries the “a” allele, there is only a 50% chance that the mature eggs will have the “A” allele (Table 2.2). However, if both the first and second polar bodies are analyzed, the genotype of all of the mature eggs can theoretically be predicted. This additional genetic information increases the number of usable oocytes from 25%, with analysis of only the first polar body, to 50%, with analysis of the first and second polar bodies. The real number of selected oocytes is necessarily less than these theoretical percentages because of ambiguous experimental results. To increase the accuracy of two-step oocyte genetic analysis, new methods are being developed which include analysis of the gene of interest as well as additional DNA regions.53 Cleavage Stage Embryo Biopsy Approximately two and a half days following IVF, human embryos can be biopsied. This is accomplished by making a hole in the zona pellucida surrounding the embryo and removing one or two blastomeres from the 8 cell cleavage stage embryo. Blastomere removal does not appear to hinder the development of the embryo, and there are no reports of serious abnormalities in children which were born after cleavage stage embryo biopsy.37,52 However, there is a risk of damaging the embryo or losing the blastomere during removal of

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Table 2.2. Pre-implantation genetic diagnosis using polar bodies Primary oocyte genotype

First polar body

Secondary oocyte

Second polar body

Mature egg

Status of mature egg

egg carries defect egg does not carry defect

(25%) AA

+

aa

(100%) a

+

a

(25%) aa

+

AA

(100%) A

+

A

(50%) A

+

a

egg carries defect

(50%) a

+

A

egg does not carry defect

AA aa

(50%) Aa

+

Aa

Genetic diagnosis of polar bodies can be used to infer the genotype of the developing oocyte (see text). For the example illustrated in this table, a heterozygous primary oocyte has two normal “A” alleles on one (duplicated) chromosome and two defective “a” alleles on the homologous chromosome. When the primary oocyte divides, the distribution of these alleles will be affected by whether or not crossing-over has taken place between the chromosome homologs. (See Fig. 2.1 for a diagrammatic representation of the distribution of these alleles.) Assuming that crossing-over occurs approximately 50% of the time, 50% of the secondary oocytes will have an “Aa” chromosome, 25% will have an “AA” chromosome and 25% will have an “aa” chromosome. For each possible genotype of the first polar body/secondary oocyte pair, the possibilities for the second polar body/mature egg genotypes are indicated. With analysis of only the first polar body, the genotype of 50% of the eggs can be predicted (namely, the products of the “AA” secondary oocytes or products of the “aa” secondary oocytes) and only 25% of the secondary oocytes can be used. In contrast, with analysis of the first and second polar bodies using two-step oocyte genetic analysis, theoretically all of the mature egg genotypes can be predicted. In this case, 50% of the originally collected oocytes can be diagnosed as having the normal “A” allele.

the cells. Embryos that do not carry a particular genetic defect (or that are heterozygous carrier embryos if no unaffected embryos are available) are transplanted within 8-12h following embryo biopsy.52 Embryos that are affected or otherwise abnormal are terminated, and excess unaffected or carrier embryos may be frozen. The first account of embryo biopsy and selection being used to produce a child free of a single gene defect was from a small study in England. A healthy non-carrier girl was born to one of three sets of parents undergoing embryo biopsy to screen for cystic fibrosis.50 Although PGD has proven to be successful in producing children without a particular genetic defect, diagnostic problems are still a serious concern. For example, sometimes the blastomeres in an embryo have different genotypes because of abnormalities in chromosome sorting (the embryos are mosaic). Some studies suggest that there is a fairly high incidence of embryonic mosaicism with respect to chromosome number.52,54,55 Since the fetus is derived from only a subset of the blastomeres at the 8 cell stage, the fetal genotype can differ from the biopsied blastomere genotype.52 It has also been suggested that a cell with an abnormal chromosome number may migrate to the trophoectoderm as opposed to becoming part of the inner cell mass which is destined to become the fetus.37 Therefore,

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even if an embryo biopsy indicates an abnormal genotype, the fetus could still have a normal chromosomal content. A second concern in embryo biopsy is that technical errors can lead to erroneous interpretations (see below). Given the ambiguities which may arise in the genetic diagnosis of a single blastomere, the availability of two blastomeres greatly increases the level of accuracy and reliability which may be achieved in the diagnosis. These types of problems are a central focus of current research to improve PGD.37 Because of the potential for diagnostic inaccuracies, it is usually recommended that the patient confirm the fetal genotype with a prenatal diagnostic procedure (see below). Using a variety of methods to detect genetic mutations, PGD has now been used to screen human embryos for autosomal recessive, autosomal dominant, X-linked recessive, and X-linked dominant disorders as well as abnormalities in chromosomal number.37 Despite some of the reported errors and misdiagnoses,52 PGD has been used successfully in the detection of several monogenic disorders such as cystic fibrosis,50 Duchenne muscular dystrophy,56 and Tay-Sachs disease.57 Although PGD is not widely available in the United States, there are several centers worldwide which offer this service. A recent summary of PGD and pregnancy data from 14 centers indicated that out of 171 embryos transferred, 50 pregnancies resulted, ending up with 34 babies born in 28 deliveries.58 Although removal of a blastomere usually does not harm the early development of the embryo, there is relatively little data analyzing development in the first trimester of pregnancy.59 Although randomized studies with large sample sizes have not yet been done, preliminary estimates of PGD accuracy and the rate of success in producing a healthy child look promising. The success rates at each stage of the process will undoubtedly increase as technology improves and as more centers are involved in PGD. It is likely that embryo biopsy would be used in HGLGT. If the genome of a zygote were genetically manipulated, it would be highly desirable to check the developing embryo to verify the presence of the genetic change before implantation. Blastocyst Biopsy Biopsy of a blastocyst approximately 5 days after IVF involves the removal of trophoblasts from the outer trophoectodermal layer, leaving the inner cell mass of the embryo intact (Fig. 2.3).60,61 This diagnostic technique theoretically could provide some advantages over other PGD techniques in that more cells could be removed, thereby increasing the chance of an accurate diagnosis. Furthermore, additional biochemical tests could be used, since embryonic gene expression would be more advanced at this stage. 34 For example, mRNA from the trophoblasts could be used as a substrate for analysis, as has been done in PGD with earlier embryonic cells.62 The ethical considerations for blastocyst biopsy would be somewhat different than with 8 cell embryo biopsy, since the cells analyzed would be those destined to become part of the placenta and not the fetus. On the other hand, later stage preimplantation embryos would be terminated if the genetic tests so indicated. As yet, this diagnostic technique is not being used clinically, since very few embryos reach the blastocyst stage in culture and the rate of implantation of transferred blastocysts is low.52 Consequently, the 8 cell embryo is the stage used clinically for genetic diagnosis of preimplantation embryos. With advances in early embryo techniques, blastocyst biopsy may become more feasible in the future.

Some Methods of Analysis in Single-cell Genetics The need to analyze a sample as small as the DNA from a single cell has prompted the development of extremely sensitive techniques. Although PGD has employed a number of molecular techniques,37 the two techniques which have had the most impact on PGD are

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Human Germline Gene Therapy: Scientific, Moral and Political Issues

the polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). Starting with a small quantity of DNA such as would be found in a single cell, PCR is used to make copies of DNA regions to generate a sufficient amount for analysis (Figure 2.4). For example, the DNA from particular genes can be “amplified” with PCR and then further analyzed with a variety of techniques. FISH utilizes fluorescent probes to identify the presence of a particular chromosome in a single cell without having to propagate the cells in culture. Each of these methods will be described briefly below. Polymerase Chain Reaction The discovery of a way to amplify a specific DNA region using PCR has fundamentally advanced many areas of molecular biology, including molecular medicine and diagnostics. Many of the single-cell PGD analyses are completely dependent on a successful PCR step. Although PCR has provided a very powerful means of analyzing minute quantities of DNA, there can be errors in the technique and consequently in the diagnosis. When PGD involves a PCR step, whether in the genetic selection of embryos or in the testing of geneticallyengineered embryos in HGLGT, there are potential sources of error. First we will describe the basic PCR reaction and then review some sources of error and misdiagnosis in techniques where PCR is employed. With PCR, a specific region of DNA can be amplified as long as some sequence of the adjacent or flanking DNA is known. For genes which have been analyzed in a number of patients, sufficient sequence data is available so that small synthetic pieces of DNA can be made that have a high probability of matching the DNA regions immediately outside of the region to be amplified. These small pieces of DNA are called “primers” and are used in the PCR reaction. PCR consists of three main steps which are repeated over and over again in an automated process (Figure 2.4A). In the first step, double stranded DNA is heated so that the two strands come apart. In step two, the separated DNA strands are allowed to cool in the presence of the primers which will then stick or hybridize to the single DNA strands. In the last step, a protein called DNA polymerase attaches to the primers and synthesizes a second strand of DNA using the original strand as a guide. These three steps when taken together are referred to as a cycle. After each PCR cycle, the amount of DNA present will be double that which was present at the end of the previous cycle. In this first round of PCR, 20-30 cycles are typically performed to amplify the DNA. Occasionally the DNA region of interest fails to amplify in the PCR reaction. The range of PCR failure rates in PGD is reported to be from 2-21% in the sex determination of embryos (used in the analysis of embryos potentially carrying a sex-linked disorder) and from 7-36% in the detection of single gene defects causing cystic fibrosis or sickle cell anemia.63 When using PCR in PGD, there are multiple sources of potential error.40 For example, a blastomere may be retrieved which is missing a nucleus (and therefore would not have any chromosomal DNA), the sample may be handled improperly, the wrong piece of chromosomal DNA may be amplified, or a sample could become contaminated with DNA from other samples in the laboratory. Since PCR is such a sensitive process, even a tiny amount of contaminating DNA in the reaction can completely alter the results. Although extreme precautions are taken to minimize the chance of sample contamination, additional methods have been developed to combat problems with contamination and sensitivity of the technique. Frequently, a second round of PCR is performed after the first round described above. These two rounds taken together are referred to as nested PCR because the second round of PCR amplifies a region nested within the first amplified DNA (Figure 2.4B). This second round of PCR increases the sensitivity and specificity of the reaction. In other words, the

Alternatives to Human Germline Gene Therapy

Fig. 2.4. The polymerase chain reaction. Basic steps in the polymerase chain reaction (PCR) are shown. Each rectangle represents a single strand of DNA. (A) First round of PCR. A single cycle of the PCR reaction is illustrated. The original DNA strands (solid shading) contain the “region of interest” to be amplified. Double-stranded DNA is denatured by heating, and short primers are allowed to stick or “anneal” to the single strands. Using the primers as a starting point, DNA polymerase synthesizes new DNA. The entire process is repeated 20 to 30 times, in order to obtain an adequate amount of amplified DNA in the final product for performing analyses or for continuing with a second round of PCR (in panel B). (B) Second round of PCR. After the first round of PCR (panel A), a second round of PCR, called “nested PCR,” is performed using the amplified DNA from the first round as a starting point. Primers for the second round nested PCR step anneal to a DNA sequence within the originally-amplified DNA region.

37

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Human Germline Gene Therapy: Scientific, Moral and Political Issues

sequential use of two sets of primers greatly increases the probability that the correct piece of DNA from the sample will be amplified. The use of nested PCR is now standard in PGD; however, newer techniques may eliminate the need for it (discussed below).63 Unfortunately, nested PCR does not eliminate all problems with single-cell genetic diagnosis. Whenever the PCR reaction is supposed to amplify two alleles of a gene, as it would be in the diagnosis of any autosomal gene defect, it is possible that the PCR reaction will amplify only one of the two alleles. Some of the reasons that this could occur are that the DNA from one of the chromosomes might be degraded or the DNA strands may not separate during the PCR reaction, thereby preventing the primers from sticking to singlestranded DNA. This phenomenon of amplifying only one of the two alleles is called “selective allelic amplification” or “allele drop-out” and may confuse the results. For example, consider the situation where an embryo is heterozygous for a genetic defect. If only the normal allele is amplified, the embryo will appear to be homozygous for the normal allele. If only the defective allele amplifies, the embryo will appear to be homozygous for the defective allele. Depending on the type of genetic defect, a misdiagnosis may or may not have a serious consequence. With a recessive condition, a heterozygous embryo misdiagnosed as being homozygous for the normal allele would develop into a carrier child. This type of misdiagnosis would not have severe consequences, since the child would not have the disease. However, with a disorder caused by a dominant allele, if the dominant allele was the one that “dropped out” of the analysis and was not amplified, the embryo would appear to be homozygous for the normal allele even though it was carrying the dominant defective allele. Clearly, the latter misdiagnosis would have a more deleterious consequence. Fortunately, methods are being developed to address the problem of allele drop-out. For example, if DNA from other regions of the genome are also PCR amplified, the clinician can distinguish between an amplification failure of a specific region of DNA and total failure of the PCR reaction (because of lack of DNA, etc.). To ensure that enough DNA is available for these multiple tests on the DNA from a single cell, one approach is to amplify many regions of the genome first and then use this amplified DNA as a substrate for amplifying several specific regions.64 Alternatively, the sensitivity of detection of an amplified DNA product may be increased by using a fluorescent PCR method.63 With an increase in sensitivity, PCR products that have undergone little amplification can still be detected as opposed to appearing as an allelic drop-out. The development of approaches such as these will allow the testing of several genetic markers in one cell. Advances in detection methods should substantially increase the reliability and accuracy of diagnosis in both polar body and blastomere biopsy, especially when more than one cell is analyzed.53 Once the DNA regions of interest are amplified with PCR, there are a number of ways to analyze the DNA to determine whether it came from a normal or mutant allele. Since a full description of these methods is beyond the scope of our discussion, we refer interested readers to other reviews of these techniques.37,40,52 Fluorescence In Situ Hybridization Fluorescence in situ hybridization (FISH) is a method used to detect the presence of certain chromosomes or genes in a single cell. The basic procedure involves attaching a single cell, such as a blastomere or polar body, to a microscope slide (the cell dies in the process). A DNA probe, attached to a color-producing chemical, will stick (hybridize) specifically to a certain DNA region on a chromosome. Using multiple probes in multi-color FISH, the presence of several different DNA regions can be ascertained in the same sample. For example, it is possible to determine the presence or absence of several different chromosomes in a single cell sample. Using X- and

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Y-chromosome probes in FISH, the sex of embryos can be determined.65 (Embryo sexing can also be done with a modification of PCR.)63 The sex of an embryo is important information when there is a medical reason motivating a couple’s desire to have a child of a particular sex. For example, when the embryo is at risk for an X-linked recessive disorder which would affect only male children, the couple may want to have only female embryos implanted. Another application is in the identification of aneuploid embryos. Some reports point to the age-related increase in aneuploid embryos as one of the reasons for decreased fertility with advancing maternal age.66 FISH can be used to screen embryos for trisomies.67,68 Therefore, FISH analysis followed by embryo selection may significantly increase the pregnancy rate and reduce miscarriages in older females who are planning to use IVF in their attempts to become pregnant.37,40,52 The FISH technique has many advantages as compared with some other methods of PGD; however, the information that can be obtained is limited. One important advantage of FISH is that it is not subject to the problems of contamination associated with PCR analysis. With FISH, errors in interpretation could arise from chromosome loss during sample processing or if one chromosome were lying on top of another in a way that would prevent or obscure the binding of the DNA probe. However, these types of technical difficulties do not appear to be major reported problems.40 Since FISH can be used to detect the presence of a chromosome whether or not it is in a dividing cell, the technique presents several advantages over traditional karyotype analysis, which is not practical for use in PGD.52 In traditional karyotype analysis, it is necessary to have a population of dividing cells which can be arrested in the stage where chromosomes can be stained and observed in their condensed form (in metaphase). With FISH, cells do not have to be propagated, a feature which also avoids the potential problem of individual cells having a different genotype than the biopsied cell. Furthermore, with FISH, results can be obtained in two hours, allowing transfer of an embryo the same day as the analysis.69 Traditional karyotype analysis could take several weeks. While FISH is not yet being used to identify single gene defects in PGD, it is likely that this will be used in the future, since FISH is used to identify specific gene sequences in basic research.70

Prenatal Diagnosis Prenatal diagnosis has been used for over 30 years for gathering genetic information about the fetus before birth. The information is used to aid management of pregnancy and delivery, to enable parents to prepare for the care of a special-needs child after birth, or to provide data which will contribute to a parental decision to interrupt a pregnancy if the data indicates that the child will be born with a serious abnormality. In view of our previous discussion, prenatal diagnosis would also be recommended after preconception genetic diagnosis or PGD, since the accuracy of PGD is not 100%. Given that errors could also occur if HGLGT were ever attempted, both PGD and prenatal diagnosis would be advised. However, the range of theoretical errors that could occur in HGLGT is beyond that which could be detected with any of the preimplantation or prenatal diagnostic techniques (see chapter 4). Current methods of prenatal diagnosis include chorionic villus sampling in the first trimester of pregnancy and amniocentesis in the first or second trimester. Each of the procedures is associated with a certain degree of inaccuracy and fetal loss. As mentioned earlier, preimplantation analysis and selection of embryos without major chromosomal abnormalities could minimize fetal loss associated with prenatal diagnostic procedures and provide a genetic screening option for those couples for whom fetal abortion is not an option.

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Chorionic Villus Sampling After PGD, the next window for prenatal testing is in the first trimester at around 9 weeks gestation. One of the methods used for early prenatal diagnosis is chorionic villus sampling (CVS). In early fetal development, the fetus is suspended in the amniotic cavity, which is surrounded by an outer chorionic membrane. The external surface of the chorion has threadlike projections called villi. By about 9-12 weeks gestation, the chorionic villi have degenerated over most of the surface of the chorion. However, one area of the chorion, called the chorion frondosum, still contains villi with dividing cells. Between 10 and 13 weeks gestation, a sample of the chorionic villi is removed, the actively dividing cells are used directly for karyotype analysis (the “direct method”), and other cells may be cultured for later analysis (the “culture method”).35 It is recommended that both the direct and culture methods be used, since different cell types will be analyzed by each method and abnormalities in either can provide valuable information.35 If the first attempt to retrieve chorionic villi fails, a second attempt is usually made. However, more than two attempts can result in a pregnancy loss at the rate of up to 10%.35 Depending on patient or physician preference, or on certain clinical circumstances, CVS is performed using an ultrasound-guided transabdominal or transcervical route.35 Some studies comparing these two methods indicate a somewhat higher risk of fetal loss with transcervical as opposed to transabdominal CVS.71,72 It has been suggested that the experience of the operators performing the procedure is also an important factor, since fetal loss rate can be 2-3 times higher when operators have performed fewer than 100 cases as compared with those who have performed more than 1000 procedures.35 Other data, from prospective randomized trials comparing transabdominal and transcervical CVS, suggest that there is no difference in post-procedure pregnancy loss between the two approaches (2.3 vs. 2.5%, respectively).35 It is possible that when the experience of centers is equivalent, the risk of pregnancy loss may also be equivalent when comparing the two CVS approaches and second-trimester amniocentesis, described below.35 One of the serious concerns with all types of prenatal diagnosis is whether the genotype of an analyzed cell is identical to the fetal genotype. With CVS, the true karyotype of the fetus could differ from that of the biopsied sample for two different reasons. First, maternal cells could have contaminated the sample during retrieval. Second, the genotype of a chorionic villus cell could be different from the fetal genotype. This discrepancy in genotypes among fetal and chorionic villi tissue could have arisen from abnormal events in the distribution of chromosomes in early development. For example, the fetus could have a normal genotype while the chorionic villus cell could have a trisomy.73 When the preplacental chorionic villi cells harbor a chromosomal abnormality (such as aneuploidy) that is not present in the fetus, it is called “confined placental mosaicism.”73 Although confined placental mosaicism is observed in about 1% of all CVS cases, the mosaicism is confirmed in the fetus in only 10-40% of these cases. Thus, the majority of CVS diagnoses of mosaicism would be classified as a false positive, since the chromosomal abnormality is confined to the placenta and is not a reflection of the true fetal genotype.

Amniocentesis Amniocentesis involves the surgical removal of amniotic fluid and cells from the amniotic cavity during the first or second trimester. The amniotic fluid contains cells (amniocytes) which have sloughed off from the respiratory and gastrointestinal tract and skin of the fetus. Both cells and fluid are used for analysis in amniocentesis, whereas in CVS only cells are available for analysis. First trimester or early amniocentesis is done at approximately 10-14 weeks gestation while second or midtrimester amniocentesis is performed between 15-17 weeks gestation.74 The amount of amniotic fluid removed depends on the age of the

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fetus, with a lesser amount being removed in early amniocentesis. Although the success rate in removing a sample and obtaining a diagnosis is high for either type of amniocentesis (98-100%), there are still problems associated with the procedures.74 As with CVS, genotypic mosaicism can occur with amniocytes. Mosaicism is observed in approximately 0.1-0.3% of amniotic samples and is confirmed in the fetus in 70% of the cases. Therefore, compared with CVS, amniocentesis is a more reliable method for detecting true fetal mosaicism (70% confirmation of amniocentesis diagnosis vs. 40% confirmation of CVS diagnosis).35 As with CVS, the safety of amniocentesis has increased over time with improvements in procedure and is also affected by operator experience. A current analysis of procedurerelated fetal loss rate indicates that the loss rate is significantly higher after early amniocentesis (2-3%) than after midtrimester amniocentesis (0.2-0.5%).74,75 Minor differences in loss rates between midtrimester amniocentesis and CVS are not statistically significant.35 Prenatal diagnosis has been performed successfully in twin and triplet pregnancies, where a dye is injected during the procedure to identify which fetus is being tested.76 If only one of the multiple fetuses is diagnosed with a genetic disorder, then the couple is faced with the decision of whether or not to proceed with a selective termination of one fetus, with the accompanying risk of losing a normal fetus.13 With any of the prenatal diagnostic procedures, their usefulness is limited by the tests available. That is, methods exist for detecting only certain substances and only certain genes and mutations. As our information about genes and their mutations increases, there will be a concomitant increase in the availability of probes and analytical methods. However, no matter how many detection methods are available in the future, there will be some genetic events that will go undetected.

When Germline Genetic Manipulation Is an Option The manipulation of germline DNA in the prevention of a medical disorder presents another option for our cystic fibrosis carrier couple. HGLGT would most likely not be considered in the case of heterozygous parents carrying defective cystic fibrosis genes, since embryo selection would provide a simpler means of preventing a monogenic defect. However, if both parents had cystic fibrosis, that is, if they were both homozygous recessive for CF defective alleles, HGLGT would be their only option if they both wanted to be genetically related to their future child. This scenario is very unlikely at present, since patients with cystic fibrosis are often infertile. However, if management of cystic fibrosis increases their reproductive health, a couple in which both partners have cystic fibrosis might seek a way to have an unaffected child.77 If the genetic disease in question were a polygenic disease, embryo selection may or may not be an option, depending on the particulars of the disease. If more than one gene would need to be altered to avert the disease, then embryo selection would probably not be an option, since there would be such a low frequency of embryos having the correct combination of genes. In this case, HGLGT might be the only way to eliminate multiple defective genes from the embryo. On the other hand, if selection of a single allele would be effective in decreasing the chance that the polygenic disease would develop, then embyro selection would be useful in the following cases: 1. If the “beneficial” allele is dominant and is present in at least one of the parents; or 2. If the “beneficial” allele is recessive and is present in two of the parents. For a couple to elect HGLGT as the procedure for preventing a disease with a genetic basis, they would have to understand and evaluate their participation in many procedures and be willing to accept the potential consequences of altering the genome of the future child. The HGLGT protocol is likely to utilize many of the procedures discussed in this

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chapter. For example, oocytes would be collected after superovulation and frozen or used directly. Fertilization would occur in vitro. In some cases, PGD might be used to select oocytes or embryos for use in the HGLGT protocol. In the absence of a 100% success rate for achieving the desired genetic changes in embryos, HGLGT-engineered embryos would be analyzed using PGD and selected using genetic criteria. Morphological criteria would be used before selecting embryos for uterine transfer or cryopreservation. Inaccuracies in PGD would dictate that prenatal testing by CVS or amniocentesis should also be done. If many transferred embryos develop, the physician may advise a multifetal pregnancy reduction to improve the chances of giving birth to at least one healthy child. Even SGT might be used if something goes wrong with HGLGT genetic manipulation, resulting in a secondary genetic disease that could be treated with SGT. In the case of an HGLGT-engineered fetus, the general health and development of the HGLGT fetus could be assessed using prenatal diagnostic procedures. However, there are many unpredictable genetic events that could theoretically occur and not be detectable. Genetic analysis could readily determine whether a transgene had inserted in the genome by allele replacement. However, if the transgene inserts randomly in the fetal genome, it would be difficult or impossible to predict the ultimate effect of this insertion on the phenotype of the fetus. In the next chapter we discuss methods being developed for transferring genes in SGT and the obstacles involved. These somatic gene transfer techniques are providing the experimental background which would be used in development of an HGLGT protocol, two examples of which are discussed in chapter 4.

Summary and Conclusions If a couple wishes to minimize the chance that their offspring will have a specific genetic abnormality, there are many real and theoretical reproductive options. Manipulation of the germline genes is only one theoretical way to eliminate defective genes. HGLGT would not be the procedure of choice for avoiding the transmission of chromosomal abnormalities or monogenic defects to progeny, in most cases. Various techniques involving genetic diagnosis and selection are available, some on a very limited basis. To reduce the chance of aneuploidy, especially in pregnancies when there is advanced maternal age, biopsy of one or two polar bodies could be used. However, polar body biopsy is limited to detection of defects in the maternal genetic contribution. To reduce the chance of a fetus having a specific monogenic defect contributed by either parent, embryo biopsy and embryo selection could be used. To assess the health and genetic status of a fetus, CVS or amniocentesis could be used. If a couple chooses not to have PGD or prenatal diagnosis, then adoption, gamete donation, conventional treatments, or newer therapies such as SGT are their options. It is safe to assume that HGLGT will be a complicated protocol and will involve many of the procedures and techniques discussed in this chapter. HGLGT, if it were ever deemed a safe and reliable procedure sanctioned by society, may be considered when it is the only way to prevent a genetic disorder in a future child. (Use of HGLGT for genetic enhancement is discussed in other chapters.) Also, HGLGT would only be used when the couple does not want to consider adoption, gamete donation or treatment at the somatic level. Cases where HGLGT would be the only way to engineer a genetic change at the germline level are summarized in chapter 4. Since HGLGT represents a major step beyond any form of genetic manipulation or prenatal diagnosis which has been used to date, the primary concern in evaluating HGLGT is the issue of altering genes in a future human being. The consequences to the individual, the individual’s descendents, the gene pool, and society must all be analyzed. However, a complete evaluation of HGLGT includes an assessment of all the proce-

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dures involved, each with their own set of advantages, inaccuracies, risks and ethical considerations. Technological advances in many of the procedures described in this chapter would be critical to the development of HGLGT.

References 1. Robertson J. Children of choice: Freedom and the new reproductive technologies. Princeton: Princeton University Press, 1994. 2. Robertson JA. Ethical and legal issues in preimplantation genetic screening. Fertil Steril 1992; 57:1-11. 3. Beaudet AL. Genetics and disease. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine. 14th ed. New York: McGraw-Hill, 1998: 365-395. 4. Beaudet AL, Scriver CR, Sly WS, Valle D. Genetics, biochemistry, and molecular basis of variant human phenotypes. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 7th ed. New York: McGraw-Hill, 1995: 53-228. 5. Li JD, Dohrman AF, Gallup M, et al. Transcriptional activation of mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease. PNAS 1997; 94:967-972. 6. Walsh MJ, Tsui L-C, Boat TF, Beaudet AL. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 7th ed. New York: McGraw-Hill, 1995:3799-3878. 7. Boucher RC. Cystic fibrosis. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine. 14th ed. New York: McGraw-Hill, 1998:1448-1449. 8. Alton EWFW, Geddes DM. Gene therapy for cystic fibrosis: A clinical perspective. Gene Ther 1995; 2:88-95. 9. Shalon LB, Adelson JW. Cystic fibrosis: Gastrointestinal complications and gene therapy. Pediatr Clin N Am 1996; 43:157-195. 10. Walsh MJ, Tsui L-C, Boat TF, Beaudet AL. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 7th ed. New York: McGraw-Hill, 1995:3848. 11. Kaplan LA, Pesce AJ. Clinical chemistry: Theory, analysis, and correlation. 3rd ed. St. Louis: Mosby-Year Book, Inc., 1996:908. 12. Coetsier T, Dhont M. Complete and partial luteinized unruptured follicle syndrome after ovarian stimulation with clomiphene citrate/human menopausal gonadotrophin/human chorionic gonadotrophin. Hum Reprod 1996; 11:583-587. 13. Berkowitz RL, Lynch L, Stone J, et al. The current status of multifetal pregnancy reduction. Am J Obstet Gynecol 1996; 174:1265-1272. 14. Filicori M, Cognigni GE, Arnone R, et al. Role of different GnRH agonist regimens in pituitary suppression and the outcome of controlled ovarian hyperstimulation. Hum Reprod 1996; 11:123-132. 15. Carr BR, Bradshaw KD. Disorders of the ovary and female reproductive tract. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Nedicine. 14th ed. New York: McGraw-Hill, 1998:2109. 16. Calderon I, Healy D. Endocrinology of IVF. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1993:7. 17. Osborn J. Oocyte retrieval and maturation. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1993:28. 18. Dawson K. Ethical aspects of IVF and human embryo research. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1993:290. 19. Trounson A, Osborn J. In vitro fertilization and embryo development. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1993:57-84. 20. Dawood MY. In vitro fertilization, gamete intrafallopian transfer, and superovulation with intrauterine insemination: Efficacy and potential health hazards on babies delivered. Am J Obstet Gynecol 1996; 174:1208-1217.

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21. Shaw JM, Oranratnachai A, Trounson A. Cryopreservation of oocytes and embryos. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1993:214. 22. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. Germ cells and fertilization. In: Robertson M, ed. Molecular Biology of the Cell. 3rd ed. New York: Garland Publishing, 1994:1011-1035. 23. Baker TG. Oogenesis and ovulation. In: Austin CR, Short RV, eds. Reproduction in Mammals. Book 1, Germ Cells and Fertilization. 2nd ed. Cambridge: Cambridge University Press, 1982:24. 24. Byskov AG. Primordial germ cells and regulation of meiosis. In: Austin CR, Short RV, eds. Reproduction in Mammals. Book 1, Germ Cells and Fertilization. 2nd ed. Cambridge: Cambridge University Press, 1982:3. 25. Feichtinger W. Results and complications of IVF therapy. Curr Opin Obstet Gynecol 1994; 6:190-197. 26. Trounson A, Sathananthan AH. Fertilization using micromanipulation techniques. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1993:132-147. 27. Osborn J. Oocyte retrieval and maturation. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1993:18-32. 28. Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 1988; 332:459-461. 29. Niemann H, Reichelt B. Manipulating early pig embryos. J Reprod Fertil Suppl 1993; 48: 75-94. 30. McLaren A. The embryo. In: Austin CR, Short RV, eds. Reproduction in Mammals. Book 2, Embryonic and Fetal Development. 2nd ed. Cambridge: Cambridge University Press, 1982:9. 31. Kelly SJ. Studies of the developmental potential of 4- and 8-cell stage mouse blastomeres. J Exp Zool 1977; 200:365-376. 32. Gilbert S. Developmental Biology. 4th ed. Sunderland, MA: Sinauer Associates, Inc., 1994:184. 33. Van den Abbeel E, Camus M, Van Waesberghe L, et al. Viability of partially damaged human embryos after cryopreservation. Hum Reprod 1997; 12:2006-2010. 34. Tarin JJ, Trounson A. Embryo biopsy for preimplantation diagnosis. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1993: 117-119. 35. Wapner RJ. Chorionic villus sampling. Fetal diagnosis and therapy 1997; 24:83-110. 36. Kaplan LA, Pesce AJ. Clinical chemistry: Theory, analysis, and correlation. 3rd ed. St. Louis: Mosby-Year Book, Inc., 1996:794. 37. Handyside AH, Delhanty JDA. Preimplantation genetic diagnosis: Strategies and surprises. Trends in Genetics 1997; 13:270-275. 38. Munne S, Sultan KM, Weier HU, et al. Assessment of numeric abnormalities of X, Y, 18, and 16 chromosomes in preimplantation human embryos before transfer. Am J Obstet Gynecol 1995; 172:1191-1199. 39. Willard HF. The sex chromosomes and X chromosome inactivation. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 7th ed. New York: McGraw-Hill, 1995:731. 40. Grifo JA, Tang YX, Munne S, et al. Update in preimplantation genetic diagnosis: Successes, advances, and problems. Curr Opin Obstet Gynecol 1996; 8:135-138. 41. Tucker MJ, Morton PC, Sweitzer CL, et al. Cryopreservation of human embryos and oocytes. Curr Opin Obstet Gynecol 1995; 7:188-192. 42. Garrisi GJ, Navot D. Cryopreservation of semen, oocytes, and embryos. Curr Opin Obstet Gynecol 1992; 4:726-731. 43. Freitas S, Le Gal F, Dzik A, et al. Value of cryopreservation of human embryos during the blastocyst stage. Contracept Fertil Sex 1994; 22:396-401.

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44. Gelety T, Surrey E. Cryopreservation of embryos and oocytes: An update. Curr Opin Obstet Gynecol 1993; 5:606-614. 45. Trounson AO. Cryopreservation. Br Med Bull 1990; 46:695-708. 46. Toth TL, Baka SG, Veeck LL, et al. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril 1994; 61:891-894. 47. Dawson KJ. The storage of human embryos. Hum Reprod 1997; 12:6. 48. Englert Y, Revelard P. Isn’t it ‘who decides’ rather than ‘what to do’ with spare embryos? Hum Reprod 1997; 12:8-9. 49. Snowdon C, Green JM. Preimplantation diagnosis and other reproductive options: Attitudes of male and female carriers of recessive disorders. Hum Reprod 1997; 12:341-350. 50. Handyside AH, Lesko JG, Tarin JJ, et al. Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. N Engl J Med 1992; 327: 905-909. 51. Verlinsky Y, Ginsberg N, Lifchez A, et al. Analysis of the first polar body: Preconception genetic diagnosis. Hum Reprod 1990; 5:826-829. 52. Handyside AH. Preimplantation genetic diagnosis today. Hum Reprod 1996; 11:139-151. 53. Verlinsky Y, Rechitsky S, Cieslak J, et al. Preimplantation diagnosis of single gene disorders by two-step oocyte genetic analysis using first and second polar body. Biochem Mol Med 1997; 62:182-187. 54. Munne S, Lee A, Rosenwaks Z, et al. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod 1993; 8:2185-2191. 55. Delhanty JD, Harper JC, Ao A, et al. Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum Genet 1997; 99:755-760. 56. Liu J, Lissens W, Van Broeckhoven C, et al. Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion. Prenat Diagn 1995; 15:351-358. 57. Gibbons WE, Gitlin SA, Lanzendorf SE, et al. Preimplantation genetic diagnosis of Tay Sachs disease; successful pregnancy after pre-embryo biopsy and gene amplification by polymerase chain reaction. Fertil Steril 1995; 63:723-728. 58. Harper JC. Preimplantation diagnosis of inherited disease by embryo biopsy: An update of the world figures. J Assist Reprod Genet 1996; 13:90-95. 59. Soussis I, Harper JC, Kontogianni E, et al. Pregnancies resulting from embryos biopsied for preimplantation diagnosis of genetic disease: Biochemical and ultrasonic studies in the first trimester of pregnancy. J Assist Reprod Genet 1996; 13:254-258. 60. Dokras A, Sargent IL, Ross C, et al. Trophectoderm biopsy in human blastocysts. Hum Reprod 1990; 5:821-825. 61. Muggleton-Harris AL, Glazer AM, Pickering SJ. Biopsy of the human blastocyst and polymerase chain reaction (PCR) amplification of the beta-globin gene and a dinucleotide repeat motif from 2-6 trophectoderm cells. Hum Reprod 1993; 8:2197-2205. 62. Eldadah ZA, Grifo JA, Dietz HC. Marfan syndrome as a paradigm for transcript-targeted preimplantation diagnosis of heterozygous mutations. Nat Med 1995; 8:798-803. 63. Findlay I, Quirke P, Hall J, et al. Fluorescent PCR: A new technique for PGD of sex and single-gene defects. J Assist Reprod Genet 1996; 13:96-103. 64. Snabes MC, Chong SS, Subramanian SB, et al. Preimplantation single cell analysis of multiple genetic loci by whole genome amplification. PNAS 1994; 91:6181-6185. 65. Griffin DK, Handyside AH, Penketh RJ, et al. Fluorescent in-situ hybridization to interphase nuclei of human preimplantation embryos with X and Y chromosome specific probes. Hum Reprod 1991; 6:101-105. 66. Munne S, Alikani M, Cohen J, et al. Implantation failure of morphologically normal human embryos is due largely to aneuploidy. Fertil Steril 1995; 64:382-391. 67. Verlinsky Y, Cieslak J, Freidine M, et al. Pregnancies following preconception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Hum Reprod 1995; 10:19231927. 68. Verlinsky Y, Cieslak J, Freidine M, et al. Polar body diagnosis of common aneuploidies by FISH. J Assist Repro Genet 1996; 13:157-162.

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69. Harper JC, Coonen E, Ramaekers FC, et al. Identification of the sex of human preimplantation embryos in 2 hours using an improved spreading method and fluorescent in-situ hybridization (FISH). Hum Reprod 1994; 9:721-724. 70. Milot E, Strouboulis J, Trimborn T, et al. Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription. Cell 1996; 87:105-114. 71. Silver RK, MacGregor SN, Muhlbach LH, et al. A comparison of pregnancy loss between transcervical and transabdominal chorionic villus sampling. Obstet Gynecol 1994; 83: 657-660. 72. Fortuny A, Borrell A, Soler A, et al. Chorionic villus sampling by biopsy forceps. Results of 1580 procedures from a single centre. Prenat Diagn 1995; 15:541-550. 73. Kalousek DK, Vekemans M. Confined placental mosaicism. J Med Genet 1996; 33: 529-533. 74. Reece EA. Early and midtrimester genetic amniocenteses. Fetal Diagnosis and Therapy 1997; 24:71-80. 75. Valle D. Treatment and prevention of genetic disease. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s principles of internal medicine. 14th ed. New York: McGraw-Hill, 1998:408-409. 76. Pergament E, Schulman JD, Copeland K, et al. The risk and efficacy of chorionic villus sampling in multiple gestations. Prenat Diagn 1992; 12:377-384. 77. Sawyer SM. Reproductive health in young people with cystic fibrosis. Curr Opin Ped 1995; 7:376-380. 78. Payne D, Flaherty SP, Barry MF, et al. Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum Reprod 1997; 12:532-541.

CHAPTER 3

Gene Delivery Systems Introduction

M

ajor challenges in the development of gene therapy lie in designing appropriate systems for delivery and expression of a transgene. Some of the technical obstacles hindering development of gene therapy are unique to somatic gene therapy (SGT) or human germline gene therapy (HGLGT). However, much of the technology used in SGT will be applicable to HGLGT, if its development is deemed worthwhile. Since it is not possible to estimate the pace of discovery and technological advance, it is not possible to predict which procedure will be technically easier to develop, SGT or HGLGT. Currently, the development of HGLGT is not being promoted because of difficult ethical and political issues. However, given the extensive research effort in the field of SGT, the technology transfer to HGLGT in the future may have fewer obstacles than one might imagine. In this chapter, we discuss several parameters that are considered in the development of gene therapy protocols. To provide the framework for evaluating the advantages and disadvantages of various types of gene delivery systems, we provide an overview of several current gene transfer systems, including viral, non-viral, chemical and physical delivery systems. This review is not comprehensive and does not include several novel systems such as the use of a baculovirus (an insect virus) or Shigella (an intracellular bacterium) as gene delivery vehicles.1,2 In SGT, gene transfer systems are chosen and modified to meet the particular needs of the therapeutic target. However, at this point in time, it is not possible to predict which gene delivery systems will be the first to be used for HGLGT. One possibility is that a totally new system will be developed. Given the current work in SGT, it is also likely that an HGLGT vector would be a modified form of one of the systems described in this chapter. Whatever systems are chosen for potential HGLGT approaches, several features of the system will undoubtedly be developed first in SGT. Unfortunately, it is not possible to “perfect” an HGLGT system in SGT. Even with a seemingly flawless system, there will be hidden risks if the technology is transferred to HGLGT. With HGLGT, genes would be introduced into a different human with a different genome each time. This is a far different scenario than using experimental animals with relatively well-characterized genetic backgrounds.

Targets of Gene Therapy Although gene therapy is predicted to have many benefits over conventional therapies, it cannot cure all genetic disorders. Major chromosome imbalances cannot be corrected by gene therapy, since entire chromosomes cannot be added or deleted from cells. For example, it is not possible to delete a copy of chromosome 21 from cells in an attempt to correct trisomy 21 (Down’s syndrome). Likewise, gene therapy cannot add an entire X chromosome to a person with Turner’s syndrome who is monosomic for the X chromosome. As described in chapter 2, it is possible to reduce the chance of having certain Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus, Pamela J. Langer. ©1999 R.G. Landes Company.

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major chromosomal abnormalities by using preimplantation genetic diagnosis (PGD) and embryo selection. However, once a person is born with a chromosome imbalance, there are no foreseeable ways to treat the chromosome disorder with SGT. The most straightforward use of is in the correction of single-gene disorders. While it might be possible to treat a polygenic disorder by introducing a single transgene, most of the early targets for are monogenic disorders.

Criteria for Selecting Targets for SGT Factors which enter into the choice of SGT targets include the severity and frequency of the disease, lack of alternative treatments, nature of the genetic defect, presence of an appropriate animal model for preclinical research and presence of an appropriate gene transfer system for the cell type affected. The most promising initial candidates for SGT are recessive, monogenic disorders.3 The gene causing the disorder must first be identified and isolated using molecular techniques so that this so-called “cloned gene” may be given to the patient as a transgene. Moreover, the gene’s intracellular function must be well understood. Currently, SGT research is limited to diseases which are lethal or which cause a dramatic loss in the quality of the patient’s life.4 These diseases typically have rather ineffective or costly traditional treatments or lack any form of conventional treatment. For example, adenosine deaminase (ADA) deficiency, a rare disorder of the immune system, is caused by a defective ADA gene. The preferred treatment for this immunodeficiency disorder is a bone marrow transplant that cures 70-90% of treated ADA-deficient children. However, it is estimated that only 15-25% of ADA-deficient patients have a sibling or other donor with appropriately matched bone marrow.5 The remaining 75-85% of ADA deficient patients must rely on a transplant with partially matched bone marrow, which results in a substantially lower success rate (as low as 40%).5 If the bone marrow transplant fails, the only widely available treatment is the administration of bovine ADA, which may cost as much as $20,000 per month.6 Given the low success rates and high costs of managing ADA deficiency alone, it is clear that SGT would be a preferable form of treatment. Once a disorder is determined to be a suitable candidate for SGT, the chance of successful gene therapy is also influenced by various additional criteria. Not surprisingly, SGT tends to be more successful if the target tissue is readily accessible.4 It is also advantageous if expression of the transgene does not have to be tightly regulated.4,7 For example, ADA expression may vary between 10% of normal to 50 times the normal amount of ADA in the body without adversely affecting patients.5 Furthermore, gene therapy is also more successful if the genetically altered cells possess a selective advantage over non-altered cells. A selective advantage of cells containing the transgene should increase the chance that they, rather than the unaltered cells, will survive in the body.4,7,8

Current Targets of Gene Therapy The first gene therapy trial in humans took place in 1990 when Drs. Blaese, Culver, and Anderson treated two young girls suffering from ADA deficiency.9 The ADA enzyme is normally produced continually in a cell, and the expression of the ADA enzyme is not tightly regulated. The wide range of acceptable expression of the ADA transgene suggested that ADA deficiency would be a good target for SGT. White blood cells called T lymphocytes were removed from the patients, grown in the laboratory and used as recipients of a normal ADA transgene. The genetically altered T lymphocytes were then reintroduced into the patients. The entire procedure was repeated on each patient every 1-2 months. After two years, 50% of the lymphocytes in one of the patients expressed normal ADA.6 This was described as a “normal immune state”.10 The other patient had only 0.1-1% ADA-positive lymphocytes.11 Since discontinuation of standard treatment would have been unacceptable, given

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Table 3.1. Current targets of somatic gene therapy Monogenic defects

Cancer

• • • • • • • •

• • • • • • •

Alpha-1-antitrypsin deficiency Canavan disease Chronic granulomatous disease Cystic fibrosis Familial hypercholesterolemia Fanconi anemia Leukocyte adherence deficiency Lysosomal storage diseases • Gaucher disease • Hunter syndrome • Partial ornithine transcarbamylase deficiency • Severe combined immunodeficiency • Adenosine deaminase deficiency • Purine nucleoside phosphorylase deficiency • X-linked severe combined immunodeficiency Infectious diseases • Human immunodeficiency virus (HIV) Other diseases/disorders • Arterial restenosis • Coronary artery disease • Cubital tunnel syndrome • Peripheral artery disease • Rheumatoid arthritis

• • • • • • • • • • • • • • • •

Adenocarcinoma Astrocytoma Bladder cancer Brain cancer Breast cancer CEA-expressing malignancies Central Nervous System malignancies Chronic myelogenous leukemia Colon cancer Colorectal cancer Gastrointestinal tract cancer Glioma Head and neck squamous cell cancer Leptomeningeal carcinomatosis Lung cancer (small cell and nonsmall cell) Lymphoma Melanoma Mesothelioma Neurobastoma Non-Hodgkin’s B-cell lymphoma Ovarian cancer Prostate cancer Renal cell cancer

The information presented in this table was compiled from the List of Human Gene Therapy Protocols, Office of Recombinant DNA Activities, National Institutes of Health, in May 1998,12 and other sources.3,8,55–57

the possibility of failure of the SGT protocol, the standard supplementation with ADA protein was continued throughout the trial.11 Numerous other recessive monogenic disorders are currently being considered as candidates for SGT. Currently, over 200 SGT protocols have been approved (by the Recombinant DNA Advisory Committee (RAC), Office of Recombinant DNA Activities (ORDA)/ National Institutes of Health (NIH) or Food and Drug Administration (FDA)). Most commonly, protocols utilize SGT for various forms of cancer (approximately 70%), HIV (12%) and cystic fibrosis (8%).12 Some of the current SGT protocols are listed in Table 3.1. This list does not yet include projected SGT target diseases such as hemoglobinopathies (structural hemoglobin variants, e.g., sickle cell anemia), Lesch-Nyhan syndrome (HPRTase deficiency), phenylketonuria (PKU); glycogen storage disease Ia (von Gierke disease); hemophilia A or B; Duchenne muscular dystrophy and Huntington’s disease.13 Some of the work on these SGT targets is currently in preclinical studies.

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Human Germline Gene Therapy: Scientific, Moral and Political Issues

Transgene Destination and Expression The goal of a gene transfer system is to have the transgene functioning appropriately in the correct cells in the body without causing any harm to the person. If a transgene replaces a defective gene via a homologous recombination (HR) mechanism, there is a good chance that the transgene will be expressed correctly. If the defect is successfully corrected at the beginning of development with HGLGT, that would theoretically be the end of the treatment (as long as secondary effects of the genetic manipulation do not cause medical problems). In contrast, SGT protocols are directed at many different tissue targets, depending on the particular genetic disease being treated. Thus in SGT, gene delivery systems must be designed to fit each situation. Factors involved in the choice of an SGT gene delivery system include: 1. The physiological accessibility of the target cells, affecting the choice of ex vivo or in vivo therapy; 2. The mitotic status of the target cells (dividing or nondividing cells); 3. The desired location of the transgene within the cell (integrated into the genome or existing extrachromosomally); and 4. The specific requirements for transgene regulation. Issues involved in transgene delivery and expression are discussed below. Specific examples of gene delivery systems are presented in the section on Methods of Gene Transfer in this chapter.

Ex Vivo or In Vivo Methods In SGT, transgenes can be delivered to the patient by three different means. The most common method, called the “ex vivo” approach, is used when the target cells are physiologically accessible. Cells are removed from the patient, and the vector containing the transgene construct (termed a recombinant vector) is introduced into the cells in the laboratory. These genetically modified cells are then reintroduced into the patient.14,15 In contrast, with the “in vivo” approach, the recombinant vector is injected directly into the patient.15 The vector then travels through the body until it “finds” the tissue to which it is directed. When the recombinant vector is injected directly into the specific tissue that is being targeted, the approach is called “in situ.” Ex vivo methods have been used much more extensively than either in vivo or in situ methods for several reasons. First, when a recombinant vector is injected into the body or a specific tissue, the vector:target cell ratio is low because of the large number of cells that may take up the vector. In ex vivo approaches, the vector:target cell ratio is higher, since there are only a limited number of cells in the laboratory dish which are exposed to the transgene. Consequently, ex vivo therapy tends to be a more efficient procedure than in vivo therapy. Second, ex vivo approaches allow for the selection of only those cells that have taken up the transgene.8 After incubating cells and recombinant vector together in the laboratory, the researcher may add a drug that will allow growth of only those cells that have taken up the transgene. Any non-modified cells in the culture would die. Thus, only cells that have been genetically modified by the introduction of the transgene would be reintroduced into the patient. Third, the ex vivo approach eliminates the need for incorporating features in the vector that target it specifically to a certain tissue in the body. Fourth, the ex vivo approach eliminates the chance of accidental gene transfer to the germline. Since in vivo techniques permit movement of recombinant vector throughout the body, there is always a chance that the vector will infect a germline cell instead of the cell that is being targeted.8,16 In fact, a somatic gene transfer experiment in unborn lambs apparently produced a lamb whose offspring contained the transgene, suggesting that the transgene was also inserted into the lamb’s germline cells.17

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In vivo approaches also have advantages. This methodology is less invasive to the patient, as compared with ex vivo techniques that require cell removal and reintroduction. Additionally, not all cells in the body are amenable to existence and growth outside of the body for the period of time necessary for transgene insertion.18 One of the main hurdles to in vivo gene transfer is the challenge of correctly targeting the gene delivery system to the desired tissue. Several vector targeting approaches are currently being developed. In situ approaches bypass some of the problems with vector targeting, since the transgene vector is introduced directly to the target tissue. For example, a vector carrying a normal CFTR gene is administered to the airways of cystic fibrosis patients via nebulization.19 In an attempt to make in vivo approaches more feasible, an effort is being made to engineer recombinant vectors so that they are more tissue specific.20 For example, viruses complexed with specific molecules increase the chance that they will attach to specific cell surface receptor proteins (see below).

Replication Status of Target Cells Many of the retroviral vectors (see below) require dividing cells for the delivery of a transgene. Since many of the cells in the body are not actively dividing at any given time, in vivo application of retroviral vectors is usually not practical. Therefore, these vectors can only be used if the target cells are capable of surviving and dividing outside of the body, thereby making them amenable to ex vivo therapy. If, however, the target cells are not easily removed from the body and cultured in the laboratory (such as is the case with brain cells), then gene delivery vehicles which do not require dividing cells should be used.

Transgene Location in a Cell The means by which a transgene is introduced into a cell will affect its ultimate destination and whether it is integrated as a single or multiple copies. In turn, its location in the cell will affect its stability and function. Listed below are various ways a transgene can be maintained in a cell: 1. The transgene could be located outside of a resident chromosome as part of a plasmid that has a temporary existence or as part of an artificial chromosome designed to have a stable existence in a cell; 2. The transgene could be integrated into a resident chromosome at a nonallelic site (via a nonhomologous recombination (NHR) mechanism), at a designated site (which would be a well characterized, nonallelic “transgene acceptor site”) or at the exact location of the original defective gene, resulting in allele replacement. Extrachromosomal Maintenance of a Transgene A transgene can be maintained outside the resident chromosomes on a recombinant plasmid vector that exists independently in the cell. In bacteria, plasmid stability is sometimes regulated by a set of genes.21 In a lower eukaryotic parasite, a recombinant plasmid vector can be retained throughout many cell divisions, even including cell divisions within an animal.22,23 In other systems, the plasmid, and therefore transgene, existence is only transient in the cell. After one or more cell divisions, the transgene-carrying plasmid may be completely eliminated from the cell. Although temporary expression of a transgene is not a desirable feature of a gene delivery system, one of the advantages of a non-integration vector is that random integration into the genome is avoided, along with the potentially negative effects discussed below. Another possible location for a transgene is on a human artificial chromosome. This system may provide a way to stably maintain a transgene without integration into the resident genome.

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Human Germline Gene Therapy: Scientific, Moral and Political Issues

Chromosomal Integration of a Transgene Transgenes can be integrated as a single gene or as multiple, tandemly-linked arrays. Certain viral vectors promote integration of a single copy of a transgene via NHR. However, if DNA is microinjected into a cell, transgenes often integrate as tandem arrays ranging from one to several hundred copies. In some cases it has been observed that the copy number of a transgene does not significantly influence the level of gene expression. Rather, the site of transgene integration dictates transgene expression.24 Depending on the gene delivery system used, the transgene could enter chromosomal DNA at a random site via an NHR mechanism or a specific site via an HR mechanism. With NHR, the expression of the transgene and possibly neighboring genes in the recipient chromosome can be affected in unpredictable ways. In order to achieve a site-specific integration of a transgene, an HR mechanism would be required. Since HR occurs in only a small percentage of random transgene integration events (1:25 to 1:5,000,000 integration events),25 specific methodologies must be used to intentionally direct HR (described in chapter 4). Two types of site-specific transgene integration can be envisioned: 1. Site-specific integration resulting in allele replacement; and 2. Site-specific chromosome integration at a (hypothetical) “transgene acceptor site.” Allele replacement would result in removal of a resident allele. Site-specific chromosome integration at a transgene acceptor site would require that such an acceptor site had been identified and characterized. The target acceptor site would have to be chosen such that it would stably retain the transgene, allow appropriate transgene expression and allow transgene function without causing any negative effects on expression of other resident genes. However, site-specific insertion of a transgene in such an acceptor site would not eliminate the original defective gene from the genome. Potential Effects of Transgene Insertion via Nonhomologous Recombination on Nonallelic Gene Expression The consequences of transgene insertion via NHR can range from benign to lethal. In a best case scenario, the insertion of a transgene via NHR would result in an appropriate level of transgene expression and no deleterious effect on the expression of any other genes in the genome (nonallelic genes). Some potential consequences of random transgene insertion on nonallelic gene function are listed below: 1. The transgene may have no effect on other, nonallelic genes or on chromosome structure/function (which could affect gene expression). 2. The transgene may have a minor effect on the expression of a nonallelic gene, and the cell might have a slightly altered function. 3. The transgene insertion could disrupt a gene, thereby causing an insertional mutation. In this case, the disrupted gene is nonessential for cell survival. 4. The transgene may cause an insertional mutation in a nonessential gene; however, the cell may exhibit altered function under certain circumstances (e.g., when its physiological environment changes). 5. The transgene may cause an insertional mutation in an essential gene or regulatory sequence and the cell would die. 6. The transgene insertion could result in the activation of a cancer-causing gene (oncogene) or inactivate a gene protecting the cell from becoming cancerous (tumor-suppressor gene).6,26,27

Transgene Expression For a transgene to be effective, it must be expressed in the right place in the body, at the right time, and in the right amount. The understanding of intricate gene regulatory mecha-

Gene Delivery Systems

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nisms is critical to the eventual success of gene therapy. Inappropriate transgene expression could fail to reverse a disease state or could disrupt the delicate interplay of macromolecules in a cell. For example, an overexpressed transgene product could be toxic to a cell. Expression of a transgene can be long term (stable) or short term (transient). The stability of transgene expression usually goes hand in hand with transgene integration: If a transgene is integrated into chromosomal DNA, it tends to be stably expressed; if a transgene is maintained extrachromosomally as an episome, it tends to be transiently expressed. In most cases, one would prefer to have stable expression of a transgene. Stable expression eliminates the need for repeated administration of the transgene (which if done in vivo, may induce an immunologic response against the transgene vector) and it minimizes the surgical procedures a patient must endure. On the other hand, since there is always a chance that a transgene could be randomly integrated into an inappropriate genomic site (e.g., a site that induces tumor formation), transient expression may be desirable as a “safer” alternative. If transgene integration occurs via allele replacement, the transgene is likely to be expressed appropriately. Although HR would be the ideal mechanism for introducing transgenes, current gene delivery systems in humans are limited to extrachromosomal maintenance or insertion of transgenes via NHR. In both cases, the regulation of transgene expression is a serious consideration, since the transgene will not be in its natural location in the genome. Position Effects The location of a transgene on a chromosome can affect its level of expression (a position effect). Instead of being expressed at a normal level, the transgene might be overexpressed, underexpressed or not expressed at all. If a transgene inserts into or near one of the heterochromatic regions of a chromosome such as the centromere, it is likely that it will not be expressed. If the transgene locates in an euchromatic region, many factors could affect its expression, such as adjacent regulatory elements, modification mechanisms for that portion of the chromosome, or other unknown influences of the microenvironment of the chromosome. A transgene construct, including the transgene and regulatory sequences, might be designed to circumvent the effect of potentially disruptive regulatory signals in the recipient DNA. For example, the transgene construct might include its own regulatory sequences as well as some insulation from the silencing mechanisms of nearby heterochromatin.28 DNA Modification A common modification of DNA is methylation, i.e., adding methyl (CH3) groups to nucleotides. The methylation of DNA is known to play a role in gene expression.29 If the transgene is methylated differently than a resident gene, the transgene may not function properly. Furthermore, insertion of an inappropriately methylated transgene could disturb the expression of neighboring genes and even alter chromosome structure.30 Therefore, even if a transgene is located in its natural site in the genome, it could malfunction if it contains an improper modification. For this reason, it can not be assumed that exact replacement of a defective gene with a normal transgene will cause no molecular problems. For argument’s sake, in later chapters (e.g., chapter 6) we refer to allele replacement as our example of the most problem-free method for HGLGT; however, we do not mean to imply that we think this approach is without potential problems.

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Human Germline Gene Therapy: Scientific, Moral and Political Issues

Additional Regulatory Elements The difficulty in regulating transgenes integrated via NHR may be overcome if entire functional chromosomal domains are stably transferred along with the transgene.28 Functional chromosomal domains may consist of regulatory switches (such as promoters, enhancers and silencers) and additional regulatory elements (such as “insulators” and “locus control regions”).28,29 The concept of including a chromosomal functional domain in a transgene construct has led to the development of vectors which could carry such a large amount of DNA, namely artificial chromosome vectors. Insulators function in two ways: 1. When an insulator element is located between an enhancer and a promoter for a specific gene, the insulator element “insulates” that promoter from the effects of its enhancer.29 Consequently, the enhancer is not able to stimulate the promoter. 2. Insulators located between a gene and heterochromatic region of a chromosome insulate the gene from the inactivating effects of heterochromatin.29 In this way, insulators shield genes from the effects of the surrounding genetic environment. By positioning an insulator upstream of the gene of interest in a transgene construct, it should be possible to reduce the position effect sometimes observed when genes are inserted via an NHR mechanism. However, the addition of such an insulator could also affect the expression of adjacent resident genes, which may not be a desirable consequence. Locus control regions (LCRs) function as super-enhancers28 as well as insulators.29 They are known to regulate the expression of certain clusters of differentially expressed genes28 by allowing the chromosomal domain to “open up” so that the DNA is available for transcription.29,31 The first LCR to be characterized was isolated from a human β-globin gene locus.32 The β-globin LCR, located far upstream of the β-globin gene locus,33 is essential for the transcription and replication of the human β-globin domain.33 Only a relatively small number of gene clusters controlled by an LCR have been characterized so far (e.g., genes for α- and β-globin). Targeting and Timing of Transgene Expression If a transgene should be expressed only in a certain tissue, it is essential to deliver the transgene to the correct cell type. An additional technique for increasing the chance that a transgene will be expressed in the correct place in the body is to add a promoter designed to operate only in the target tissue. Such a tissue-specific promoter would mediate transcription of the transgene only in the appropriate cell type.20 The most straightforward type of gene to be used in gene therapy is one whose expression is not tightly regulated, such as a gene that is expressed continually. If continual expression of a transgene is either ineffective or detrimental to a patient, tighter regulation of transgene expression is essential. If the timing of gene expression is critical, such as with a gene that is turned on in response to a stimulus or with a developmentally regulated gene, the goal is to identify all the relevant regulatory signals before designing the transgene construct. These regulatory elements may be located in a large region of DNA. Therefore, in some cases distant regulatory elements may not have been identified; this could lead to problems with transgene expression. One point worth mentioning here is that every human genome is different and thus will provide a different genetic background for every SGT or HGLGT procedure. Even if a transgene replaces a defective allele by HR, the combination of the particular introduced allele and resident regulatory elements may result in inappropriate transgene expression. Under somatic conditions, the effect of a distant regulatory sequence may be slight. However, during prenatal development, the timing and level of expression of a particular gene may be much more critical to the proper development of the fetus.

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Consequences of Transgene Destination and Expression in SGT or HGLGT In the previous section we discussed three major considerations when introducing transgenes: 1. The destination of the transgene; 2. The potential effects of transgene introduction on the function of nonallelic genes; and 3. The regulation of transgene expression. Here we evaluate these parameters in terms of their consequences in SGT and HGLGT. In SGT, many cells are the recipients of a transgene. In HGLGT, at the very beginning of development one original cell receives the transgene that is destined to be present in all nucleated cells. If a transgene inserts via HR in either SGT or HGLGT, it would have the best chance of being expressed appropriately and causing minimal disturbance to the expression of other genes. One concern with a protocol designed to direct HR is that the transgene may erroneously insert via an NHR mechanism, as frequently occurs in nonhuman experimental systems. Many of the potential problems of SGT or hypothetical HGLGT protocols are associated with transgenes inserted via NHR in the genome. While many transgene locations may be acceptable for SGT, most are inappropriate for HGLGT. For example, if the transgene is located in an episome, there is the potential for transgene loss. The genetic “fix” is only temporary, a situation which is not appropriate when the goal is to alter the genetic material in all cells of an organism. Transgene insertion via NHR is an unacceptable method for HGLGT because of the unpredictability of genetic consequences. Random transgene insertion in HGLGT theoretically could result in early death of the embryo or fetus, physical deformities, the creation of other genetic disease(s) or cancer. With SGT, since millions of cells receive the transgene, the death of a few target cells is inconsequential to the human recipient. However, if transgene insertion causes a cell to become cancerous, this would clearly be an undesirable consequence in SGT or HGLGT. So, even for SGT, a transgene insertion method employing HR is preferable, although immensely more technically difficult to engineer with current technology. Methodologies are being developed in hopes of bypassing some of the current problems with NHR in SGT. For example, if a large piece of DNA is used to carry the transgene, the transgene will be well separated from neighboring genes and is less likely to affect the expression of neighboring genes. However, a large transgene construct could still cause an insertional mutation. One way to avoid gene disruption would be to have a human artificial chromosome carry the transgene construct. However, the consequences of adding a human artificial chromosome to a germ cell in HGLGT would be unpredictable and hazardous. In SGT, the inappropriate expression of a transgene is less critical than in HGLGT. The death of a few cells in SGT is trivial in comparison with death of an entire embryo in HGLGT if there is a toxic effect of transgene expression. As discussed previously, in SGT the most effective transgenes are likely to be those encoding proteins that can be present in vastly different amounts in a cell, such as with ADA. In contrast, if gene expression has to be tightly regulated, the challenge to produce a properly functioning transgene with SGT is much greater. That is, if the gene is supposed to be expressed only in certain tissues (in a tissue-specific manner) or only at certain times, the regulation will be more complicated. If a gene delivery system other than one directing HR were ever considered for HGLGT, the transgene construct would have to include the DNA sequence involved in correctly regulating the gene in every cell and throughout the life of the person. Thus, regulatory instructions in an HGLGT transgene construct may need to cover a greater variety of circumstances as compared with SGT transgene constructs. There are many unknowns with respect

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Human Germline Gene Therapy: Scientific, Moral and Political Issues

to minor and temporary expressions of genes and the interaction of gene products in development. Since it would be an almost impossible task to characterize all the effects of a gene in early development, introduction of a transgene regulated by non-native regulatory elements would present an unacceptable risk in human development. Homologous gene replacement would clearly be the best choice for directing the appropriate expression of a transgene in HGLGT.

Methods of Gene Transfer A number of different gene delivery systems have been developed. When a method is first tested as a means for gene transfer, it must pass many rigorous tests before being approved for SGT trials. Protocols are tested first for their efficiency of gene delivery in mammalian cells grown in the laboratory. These early tests typically involve the use of a reporter gene which encodes a protein that is easily detected. For example, a reporter construct may encode a fluorescent protein such as luciferase (found in fireflies), while another may encode an enzyme (β-galactosidase) whose expression can cause a cell to turn blue if given a certain chemical. Following testing in laboratory-grown cells, a gene transfer system is tested for safety in animals (typically mice) prior to human testing. Only after the gene delivery system passes these rigorous tests is it submitted to the RAC for approval for use in gene therapy trials. The methods currently available for gene transfer may be classified in four major categories: 1. Viral vectors (both RNA and DNA viruses); 2. Non-viral vectors (including naked DNA, artificial chromosomes and liposomes); 3. Chemical methods; and 4. Physical methods. To date, there is no ideal method that is able to deliver transgenes to all desired tissues with 100% efficiency. Each vector has its advantages and limitations that will be briefly discussed below (summarized in Table 3.2). So far, only some of these gene delivery systems have been used in human gene therapy trials.

Viral Vectors Viruses are particles that consist of a nucleic acid genome contained within an outer coat of proteins. Many viruses have been modified for various scientific uses such as vaccines. For gene transfer, researchers place transgene constructs inside these viral vectors and allow them to infect (or transduce) the desired cells. Viral vectors have been used to introduce transgenes into cells more commonly than non-viral vector systems.8 If a virus cannot replicate, it is called a “replication incompetent virus.” Two types of viruses have been used as gene transfer vehicles: RNA viruses, containing an RNA genome; and DNA viruses, containing a DNA genome. A type of RNA virus called a “retrovirus” contains RNA that is copied (reverse transcribed) into DNA, which is then incorporated into the host cell genome. Types of DNA viruses that are used in gene transfer protocols include adenoviruses, adeno-associated viruses (AAV), and herpes simplex viruses. All of the viral vectors used in gene transfer protocols have been disabled to minimize replication of the virus in the target cell and minimize development of virally-caused disease, both features of which would be highly undesirable. Retroviruses Of all the viral vectors used in gene transfer experiments, replication incompetent retroviruses have been the most extensively studied and utilized. Retroviruses are highly efficient in their gene transfer capabilities.6,8,14 Up to 100% of the target cells can

Herpes virus

no no

transient

transient/ stable12

no/yes11

no/yes, random10

Adenoviralretroviral chimeras

stable9

no8

yes, random

transient

no

no3

fairly stable

fairly stable

Stable or transient expression?

no

yes

Cell division necessary?

yes, random

yes, random

Integration into the genome?

Adeno-associated virus (AAV)

Adenovirus

HIV-based vectors

VIRAL-BASED DELIVERY Retrovirus

VECTOR FEATURES

Table 3.2. Features of selected gene delivery systems

yes/no13

no

no14

no

yes

—

unlikely

yes6

no

no2 yes5

yes

Risk of vector reproduction?

no2

Is vector immunogenic?

no

no

no4

no

no1

Specific cell target?

yes

—

no

yes7

no

no

Cell toxicity?

Gene Delivery Systems 57

Microinjection

yes, random18

yes, random17

no

DNA-coated pellets

Synthetic DNAligand complexes (molecular conjugates)16

no

no

Integration into the genome?

Naked plasmid DNA

NON-VIRAL BASED DELIVERY Liposomes

VECTOR FEATURES

—

no

no

no

no

Cell division necessary?

stable

transient

transient

transient

transient

Stable or transient expression?

Table 3.2. Features of selected gene delivery systems (cont.)

n/a

yes

no

no

no

Specific cell target?

no

no15

no

no

no

no

no15

no15

no

Risk of vector reproduction?

no

Is vector immunogenic?

no

no

no

no

no

Cell toxicity?

58 Human Germline Gene Therapy: Scientific, Moral and Political Issues

Information concerning numerous transgene vectors is indicated. Some of the listed vectors are currently being used in gene therapy protocols, whereas others have yet to be used in clinical trials. 1 Attempts are being made to make tissue–specific retroviral vectors. 2 Vectors would be non–immunogenic if used to deliver genes in an ex vivo approach, and viral proteins were not expressed by the transduced cells in vivo. 3 Viral DNA exists as nuclear episomes. 4 Adenovirus has a wide cellular host range. Epithelial cells from the eye, and respiratory, gastrointestinal and urinary tract cells are most permissive. 5 The effects of adenovirus therapy persist an average of 6 weeks and repeated dosing is not possible due to an anti–adenovirus immune response. 6 Adenoviral vectors are not totally replication defective. 7 At least one viral structural protein expressed late in the replication cycle is toxic to cells. 8 AAV can enter nondividing cells. It is not known whether they can integrate into the genome of nondividing cells or if they integrate only upon cell replication. 9 Expression is stable since the immune system does not eliminate AAV–infected cells. 10 DNA integration does not occur in the transient retroviral producer cell; however, random DNA integration does occur in the retrovirally infected neighboring cells. 11 Cell division is not necessary in the transient producer cell, but it is necessary in the retrovirally infected neighboring cells for DNA integration. 12 The producer cells are transient, the retrovirally infected neighboring cells are stably transduced. 13 The retroviral producer cells are most likely immunogenic; however, the retrovirally–infected neighboring cells should not be immunogenic. 14 Herpes viruses exhibit a propensity (tropism) for infecting neural cells (the virus is neurotropic). 15 Naked DNA is relatively non–immunogenic, but it can stimulate the immune system to react to substances (i.e., act as an adjuvant).58 16 Components include a ligand for a specific cell receptor and a DNA binding moiety (a polycation such as polylysine) that links the DNA encoding the gene of interest to the ligand. The polylysine helps to target the DNA to the nucleus because the nuclear–targeting signals are often lysine–rich. 17 DNA taken up by cells exists mainly as nuclear extrachromosomal material, which can be incorporated into the genome in some rapidly–dividing cells.20 18 The DNA usually inserts into the genome by nonhomologous recombination (NHR) as a variable–length tandem gene array.

Table 3.2. Features of selected gene delivery systems. (cont.)

Gene Delivery Systems 59

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be transduced by retroviruses in a gene transfer experiment.7,14 Retroviruses have the distinct advantage that the transferred DNA is stably integrated into host chromosomal DNA.8,15 Furthermore, the transgene is typically integrated in the genome as a single copy, in contrast to the multiple head-to-tail arranged copies that are found after chemical and physical gene transfer techniques.7 A disadvantage of retroviral vectors is that transgenes are integrated via NHR, potentially leading to negative effects on expression of the transgene or nonallelic genes, as discussed previously.6-8 Second, a great limitation of some retroviral vectors is that they require a dividing target cell.6,8 The majority of the currently available retroviruses can only integrate into host chromosomal DNA when the nuclear membrane dissolves during cell division.15 The requirement for host cell division greatly limits the application of this vector. For this reason, the majority of gene transfer procedures that use retroviruses utilize ex vivo approaches, since it is possible to induce some nondividing cell types to divide in the laboratory. Generally speaking, retroviruses are not well suited for in vivo gene transfer techniques for the following reasons: 1. Most of the cells in the body are not actively dividing at a high enough rate for retroviral integration of the transgene; 2. Retroviruses are relatively unstable in blood, as compared with other viral vectors,14 since they are rapidly inactivated by human complement (specific protein components in the blood);34 3. The ability of a retrovirus to bind to and enter a target cell is dependent on the presence of an appropriate viral receptor on the surface of the target cell.14 Generally, if a target cell does not possess the appropriate receptor, viral vectors cannot deliver the transgene. On the other hand, if multiple cells in addition to the desired target cell possess the appropriate receptor, delivery of the transgene would be nonspecific. The most common retrovirus vectors currently used in gene transfer approaches are replication incompetent vectors based on Moloney murine leukemia virus. However, retroviral vectors based on human immunodeficiency virus (HIV) are also being developed.35 HIV and other lentiviruses have an advantage over Moloney murine leukemia virus in that they do not require cell division to integrate into host genome. 36 Adenoviruses Adenoviruses are DNA viruses that infect humans, usually causing minimal clinical symptoms.37 Occasionally they cause more serious illnesses such as acute respiratory infection or conjunctivitis. It has been estimated that 75% of young people have been infected with an adenovirus.6 Although adenoviruses have a wide range of cells which they can infect, they show a preference for epithelial cells of the eye, and for cells of the respiratory, gastrointestinal, and urinary tracts.8,37 Retroviruses and adenoviruses can accommodate inserts in the 7-8 kb range.8,37 Compared with retroviruses, adenoviruses have several advantages. Adenoviruses are more stable than retroviruses and they can deliver a transgene to nondividing cells. Thus, adenoviruses can be used in ex vivo and in vivo approaches.6,8,14 Once the transgene is delivered to a target cell, it is maintained as an episome in the nucleus rather than being integrated into a resident chromosome.8,14,38 Nonintegration of the transgene may be viewed as an advantage in that it minimizes the risk of interfering with resident gene function.37 Adenoviruses also have their limitations. The episomal existence of the transgene allows only transient expression of the desired protein. As a result, additional treatment of the patient with adenoviral preparations is typically required.8,15,38 Since adenoviral proteins

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are immunogenic in humans, an immune response could result in inflammation and killing of cells that express adenoviral genes.6,15,37 Due to the induced immune response, repeated dosing with the recombinant adenoviral vector is a serious problem. Another concern is that replication deficient adenoviruses used in gene transfer procedures may actually replicate.6,14,38 Proteins essential for replication that are missing from an adenoviral vector could be provided by a wild type adenovirus or another virus in the patient.37 Lastly, at least one adenovirus structural protein has been demonstrated to be toxic to cells.39 Adenoviral-retroviral Chimeras Recently, a chimeric vector has been developed that combines the most favorable attributes of adenoviruses and retroviruses.40 These new adenoviral vectors induce target cells to become transient retroviral-producing cells in vivo. The retroviruses produced by these adenoviral-infected target cells are then able to infect locally dividing cells without having to traverse the bloodstream, which is laden with complement components known to inactivate retroviruses. The new adenoviral/retroviral vectors have many advantages. They are delivered to target cells with the same high level of efficiency that is observed with adenoviruses and they allow the in vivo administration of retroviruses. Furthermore, the transgene carried by this chimera is stably integrated into the chromosomal DNA of the cells infected by the resultant retroviruses. Adeno-associated Viruses Adeno-associated viruses (AAV) are defective DNA viruses that normally replicate in conjunction with a helper virus.8,41,42 In the absence of a helper virus, the wild type AAV integrates into a specific location on human chromosome 19.14 A deletion mutant of AAV, used in gene transfer procedures, has been found to integrate at random into chromosomal DNA by NHR.38,42 However, an AAV system was recently shown to direct HR at a high rate relative to other viral or nonviral systems (up to 0.7% of cells exhibited an HR event, depending on the cell type).43 If modifications can be made so that AAV can mediate HR at an even higher rate, this would have profound implications on the field of gene therapy, for both SGT and HGLGT applications. In addition to their potential for mediating HR events, AAV vectors possess numerous other advantages. Similar to HIV-based vectors, AAV vectors are capable of infecting nondividing cells.8,42 AAV vectors are very stable14 and nonimmunogenic,42 thus providing a significant advantage over adenoviral vectors. Since no human diseases have been linked to AAV infection, this vector also poses a lower risk to patients than some of the other viral vectors.15,41,42 AAV systems possess relatively few disadvantages. The greatest limitation is that they are difficult to grow in the laboratory41 and they have a relatively small cloning capacity compared to other viral vectors.15,41 Current AAV vectors can hold only 4.5 kb of DNA, a size which would be too small to carry many of the human genes plus regulatory regions.42 Herpes Viruses Herpes viruses preferentially infect neural cells (they are neurotropic) and thus may be a good vector choice for gene targeting procedures involving the central nervous system.8,14 Like adenoviruses and AAV, herpes viruses do not require target cell division for transgene expression.8 Since they do not integrate the transgene into the chromosomal DNA, their expression is transient. They are also non-immunogenic since they exist in neural tissue, where molecules are less likely to stimulate the immune system.18 Unfortunately, laboratory

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preparation of replication incompetent herpes vectors is difficult,14 and a viral protein that is produced in both wild type and replication incompetent herpes viruses is toxic to cells.14 Adenoviral Dodecahedrons An adenoviral dodecahedron consists only of specific adenoviral proteins; it does not contain the viral genome.44 Since the vector is not large enough physically to contain DNA, the plasmid containing the transgene is attached to the outside of the adenoviral dodecahedron.44 As compared with adenoviral vectors, the adenoviral dodecahedrons are less immunogenic, safer (since they cannot replicate), and apparently can deliver a transgene with an efficiency equal to that of the adenovirus vectors.44

Non-viral Vectors The second most popular means by which transgenes are transferred in gene therapy trials involve the use of non-viral vectors. This class of vectors is very broad, ranging from uncomplexed, naked DNA to DNA that is enclosed in complex lipid structures. Non-viral vectors are generally considered to be safer than viral vectors, since there is no risk of viral replication. Unfortunately, non-viral vectors are not as efficient in their gene transfer abilities when compared with their viral counterparts. Liposomes Liposomes are small hollow spheres comprised of a lipid (fatty) membrane, the inner portion of which is filled with an aqueous solution. Liposomes, which can form spontaneously when specific lipids are added to an aqueous solution, may be filled with plasmid DNA or a drug to be delivered to a cell.45 These “loaded” liposome vectors are capable of fusing with cellular membranes, a process that facilitates delivery of the liposome contents into the interior of the cell. In the first generation of liposome vectors (called simple liposomes), the interior diameter was quite small, resulting in a very low efficiency of encapsulation of plasmid during liposome preparation.45 In an attempt to improve the efficiency of liposome loading with DNA, some of the standard lipids were replaced with lipids possessing a positive charge on the portion of the lipid facing the interior of the sphere.45 In theory, the positively charged end of the lipids would be able to better associate with the negatively charged DNA backbone. These positively charged liposomes, or “cationic liposomes,” package plasmids more efficiently and accommodate much greater amounts of DNA.45 Due to the variable structures formed by cationic liposomes and their packaged plasmids, these structures have been given the name “lipoplexes”45 (see Figure 3.1). Simple liposomes and lipoplexes have many advantages in common with other nonviral gene delivery vehicles. Since they do not contain any viral genes, they do not cause disease and do not replicate.15 In addition, they are not immunogenic and have very low levels of cell toxicity.6 Lastly, simple liposomes and lipoplexes do not require target cell replication and can deliver genes to nondividing cells. A disadvantage of simple liposomes and lipoplexes is that they possess a rather low delivery efficiency as compared with viral vectors.8,15 Furthermore, transgene expression tends to be transient, thereby requiring repetitive dosing. Non-mammalian Artificial Chromosomes For many applications of gene therapy, a large amount of DNA will be needed in the transgene construct. Delivery of a very large gene plus regulatory regions would require a vector with a high capacity for carrying foreign DNA. Vectors that possess the largest cloning capacities are artificial chromosome vectors. Several different types of non-mammalian

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Fig. 3.1. An example of DNA–lipid interactions. A double–stranded DNA helix is positioned between two lipid bilayers. The portion of the lipid molecules depicted by a sphere is hydrophilic (water–loving) while the wavy tails are hydrophobic. The hydrophobic tails of the lipid molecules associate with each other, thereby spontaneously forming a lipid bilayer when present in a water–based solution. DNA molecules interact with the hydrophilic “heads” of the lipid molecules, thereby becoming “sandwiched” in between two lipid bilayers. Simple liposomes consist of a lipid bilayer sphere that may be filled with DNA molecules. The more complex lipoplexes may be structured as tubes filled with DNA, a section of which is depicted in this figure. The DNA–filled tubes apparently can wrap up to form a larger particle with a lipid wall.45

artificial chromosomes have been developed, including yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and bacteriophage P1-derived artificial chromosomes (PACs). These artificial chromosomes exist as “chromosomes” in yeast (YACs) or bacteria (BACs and PACs). However, once they are transferred to mammalian cells, they can insert into the mammalian genome via NHR, potentially resulting in deleterious consequences to the recipient cell. Two main advantages of these artificial chromosome vectors are their DNA carrying capacity and the fact that the large DNA flanking the transgene may eliminate the positional effects frequently encountered with transgene insertion via NHR. Of these artificial chromosomes, YACs have the largest cloning capacity. A YAC contains two chromosome “arms” with telomeres (the ends of chromosomes), a centromere (the center of a chromosome that is important in the segregation of a chromosome during cell division) and an autonomous replicating sequence (ARS) which allows the YAC to replicate. The maximum cloning capacity for YACs is approximately 1 million bases (1 megabase),46 although their inserts are usually in the length range of 500,000 to 600,000 bases (500-600 kilobases).47 Unfortunately, YACs tend to be rather unstable48 and may undergo sequence rearrangements.49 YACs can be transferred to mammalian cells by three different means:48

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1. Yeast-embryonic cell fusion, where YAC-containing yeast cells are fused directly with mouse embryonic cells; 2. “Lipofection” of embryonic cells, where YACs are incorporated into a liposome which is used to introduce the DNA into embryonic cells; and 3. Microinjection of YACs into mouse zygote pronuclei. Since the isolation of intact YAC DNA has proven to be very difficult because of their large size,47 one would think that the yeast-embryonic cell fusion method should be preferred, since purification of YAC DNA is not necessary with this technique. Unfortunately, when the yeast cell is fused to the embryonic cell, yeast genomic DNA is also introduced. The effects of introducing this large amount of yeast DNA into the embryonic cells are unknown.48 Currently, YACs are not being used in SGT; however, they have been used to generate transgenic mice since 1993.48 Upon examining transgenic mice, it was determined that the introduced YAC DNA is integrated into the mouse genome at a single site and is present in single or multiple copies.48 Occasionally, a YAC copy is fragmented at the ends; however, the internal portions of the YACs are usually intact.48 BACs are based on the bacterial fertility plasmid (the F-factor)46 and have cloning capacities up to 300-350 kb.47,48 To transfer a BAC to a mouse zygote, purified BAC DNA is injected into the pronucleus where it will integrate by NHR. BACs have several advantages as compared with YACs. BACs are more stable than YACs. In some cases, even after 100 generations DNA rearrangements have not been observed.47 Intact BAC DNA is also much easier to isolate than YAC DNA.47 PACs were designed by combining features of the bacteriophage P1 system and BACs.46 Like BACs, PACs have a maximal cloning capacity of 300 to 350 kb, although the normal insert size ranges from 100-300 kb.46,48 PACs are introduced into bacterial cells by electroporation (exposing the bacteria to an electric field) and they are easier to isolate in an intact form than YAC DNA.46 Furthermore, DNA inserts in PACs are as stable as those cloned into BACs.46 The largest drawback to PACs is that a system to modify their inserts has not yet been developed. Human Artificial Chromosomes Recently, researchers have succeeded in generating the first completely synthetic, selfreplicating human chromosome.50 The artificial microchromosome, only one fifth to one tenth the size of a normal human chromosome, was generated by allowing three different types of DNA to self-assemble: telomeric DNA, alpha-satellite DNA (a portion of a centromere), and a human gene. The DNA pieces interacted with one another and generated functional human artificial chromosomes (HACs) ranging in size from 6 million to 10 million bases. The HACs were capable of replication and segregation during cell division and persisted for as long as six months intracellularly. HACs have several key advantages over other currently available gene delivery vehicles. Since they do not integrate into the native chromosomes, the chance of insertional gene inactivation is eliminated. Also, one would expect the transgene to be as stable as the presence of the HAC. Expression of a transgene should be more predictable, since it would be independent of endogenous regulatory sequences in the resident chromosomes. Lastly, HACs would allow for the delivery of larger genes as well as more regulatory elements since they have a large cloning capacity. One predicted obstacle in the use of HACs is that it may be difficult to deliver such a large amount of DNA to a target cell.

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Receptor-mediated Gene Transfer Receptor-mediated gene transfer is a very appealing technique for SGT, since this methodology permits the delivery of a transgene to a cell that possesses specific surface receptors.20 This technique takes advantage of a normal cell process that involves a molecule, generically called a “ligand,” which specifically binds to a cell surface receptor. A transgene may be chemically linked to a ligand via a positively charged molecule such as polylysine (a string made up of several lysines, an amino acid) to form a transgene-ligand complex or “molecular conjugate.” The positive charges interact well with the negatively charged DNA, thereby causing compaction and precipitation of the DNA, which promotes internalization by the target cell.10 Once the transgene is internalized, it is temporarily stored in a sac-like structure called an endosome. The DNA encoding the transgene can subsequently escape from the endosome and move to the nucleus, where it will be transcribed. In the nucleus, the transgene typically remains extrachromosomal and is therefore transiently expressed.20 In rapidly dividing cells, however, the transgene may be integrated into the chromosomal DNA.20 A complication of this gene delivery system is that the endosomes may fuse with lysosomes (sac-like organelles that contain degradative processes), resulting in the degradation of the endosomal contents. Therefore, the level of expression of a transgene depends on its ability to escape the endosome and locate in the nucleus. If a replication-deficient adenovirus is introduced with the molecular conjugate, the virus can cause disruption of the endosome and improve the gene delivery efficiency.51 Molecular conjugates have the distinct advantage that they allow one to customize the gene delivery system to allow introduction of a transgene to a select cell population.20 As a result, this gene delivery system is highly applicable to in vivo gene transfer procedures. Molecular conjugates are not infectious, not very immunogenic (as are adenoviruses for example) and do not require a dividing target cell population.20 Aside from the problem that the transgene may be degraded in the lysosome,8 other disadvantages of molecular conjugates include the variable and transient expression of the transgene.20

Chemical Means Transgene introduction into cells can also be mediated chemically using a method involving calcium phosphate. An aggregate or “precipitate” of DNA and calcium phosphate adheres to the cell surface and the cell takes up the DNA. Once inside the cell, the transgene can be expressed in a stable manner.8,14 Due to the nature of the procedure, this gene delivery technique is only applicable to ex vivo gene transfer approaches. The efficiency of DNA uptake has been found to depend on many factors, including the type of cells being transduced .7 Several modifications have improved the original procedure so that, depending on the cell type, up to 10% of the cells will take up the DNA.52

Physical Means As suggested by their categorical name, these gene delivery systems rely on physical techniques to introduce a transgene to a cell. Tissue Injection One surprisingly successful gene transfer technique involves injecting naked DNA directly into a target tissue such as muscle. The injection process utilizes a needle or jet injectors to force small volumes of a DNA solution into animal tissues.53 The exact mechanism by which the injected DNA is taken up by cells is unknown. However, it has been postulated that the process may be aided by the small amount of tissue damage or slight increase in pressure that occurs as a consequence of the injection process.45 Delivering a transgene construct in the form of naked DNA is advantageous in that it is devoid of a biological

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vector and nonimmunogenic.6 One drawback is that the delivery efficiency is lower than with viral vectors.6 It has been reported that administration of a transgene via tissue injection yields transgene expression levels that were similar to or greater than the level of expression when liposomes were the gene delivery vehicle.45 Particle Bombardment An unusual gene delivery system termed “particle bombardment” involves the use of a “gene gun”. This procedure gained notoriety when it was used to transfect plants where their rigid cell wall precluded other gene transfer protocols. In the gene gun method, the targeted tissue is bombarded with gold particles that have been coated with the transgene construct.10 This technique, which may be utilized for in vivo and ex vivo approaches, has been used to a limited extent in SGT approaches. Microinjection This technique involves the injection of a transgene construct directly into a single cell using a very thin glass pipette or needle to pierce the cell and nuclear membranes. Transgene delivery to a cell by microinjection is efficient, as compared with other nonviral techniques, in that up to 20% of the cells injected can be transfected.3,7 However, this technique tends to be very labor intensive, since only one cell at a time can be injected.3,7,8 Consequently, microinjection is not used as a means to introduce a transgene in SGT, since it would be nearly impossible to inject sufficient numbers of cells in order to see a clinical effect in the patient. Microinjection is frequently used in the creation of transgenic animals such as mice. A plasmid transgene construct is injected into one of the two pronuclei of a recently fertilized egg.54 The microinjected egg is then transferred to a pseudopregnant female for gestation.54 In a fraction of the injected fertilized eggs, the transgene will integrate randomly into the chromosomal DNA in multiple (one to several hundred) head to tail tandem repeats. The tandem array of transgenes is relatively stable; however, rearrangements and deletions have been documented.24 It might be expected that the level of transgene expression would be influenced by the number of transgenes in the tandem array (e.g., the greater the gene number, the more protein that would be expressed). However, it appears that the genomic site of the transgene array integration is more critical than gene number in determining the level of gene expression.24 Electroporation Electroporation is the process by which DNA is introduced into a cell with the aid of an electrical current. The administration of an electric shock renders a cell temporarily permeable to DNA contained in the medium surrounding the cells.3 This gene transfer technique is slightly more efficient than calcium phosphate precipitation; however, the overall gene delivery efficiency is still low (approximately 1% of electroporated cells are transfected). For obvious reasons, this method is only applicable to ex vivo approaches and has the disadvantage that the electric shock could damage the cells being transfected.3,8

Summary and Conclusions Many gene delivery systems are being developed, each of which must be adapted to the particular goal of the gene therapy procedure. All of the techniques have advantages and disadvantages. For example, stable expression of a transgene is better than transient expression, but stable maintenance of a transgene often involves random integration into the genome via NHR mechanisms. Transgene integration by NHR has many potential problems, including inappropriate expression of the transgene or deleterious effects on the expression of other genes in the genome. The problems of stable transgene expression and insertion via

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NHR may be solved by using human artificial chromosome vectors (HACs). Recombinant HAC vectors are large enough to carry a transgene plus extensive regulatory regions and they do not insert into resident chromosomes. However, it may be difficult to introduce such a large piece of DNA into a cell without fragmenting the DNA. Microinjection of DNA is a very direct method for introducing DNA into a cell, but it is an impractical method when the target is more than a few cells. Liposomes are not immunogenic like some of the viral vectors, but liposome delivery of a transgene may not be as efficient as some of the viral vectors. Ex vivo introduction of a transgene is a more efficient method than in vivo, but ex vivo therapy may be a more invasive procedure for the patient and can only be used if the target cells can be cultured outside the body. In vivo therapy may be used when it is not practical to use an ex vivo approach, but the transfer efficiency can be very low, and there is the theoretical chance that the transgene will be incorporated into the genome of germline cells. If HGLGT is to be used to prevent a genetic disease, a gene delivery system which directs homologous gene replacement via HR is likely to be the most effective method and may be the only acceptable one from the standpoint of genetic risk to the future person. With SGT protocols, a method employing HR would be desirable, but insertion of a transgene via NHR or extrachromosomal maintenance of a transgene are currently acceptable goals, although these methods are not without risk. Even if a gene delivery system is developed that can successfully direct allele replacement, a process of selecting the correctly modified cells may still be required. However, it is quite possible that some clever modification of almost any of the systems described here will tweak the cell machinery in such a way that transgene insertion via HR will no longer be a rare event.

References 1. Boyce FM, Bucher NLR. Baculovirus-mediated gene transfer into mammalian cells. PNAS 1996; 93:2348-2352. 2. Sizemore DR, Branstrom AA, Sadoff JC. Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization. Science 1995; 270:299-302. 3. Verma IM. Gene therapy. Sci Amer 1990; 263:68-72. 4. Valerio D. Retrovirus vectors for gene therapy procedures. In: Grosveld F, Kollias G, eds. Transgenic Animals. San Diego: Academic Press, Inc., 1992:225-227. 5. Culver KW. The first human gene therapy experiment. In: Sharrer GT, ed. Gene therapy: A Primer for Physicians. 2nd ed. Larchmont: Mary Ann Liebert, Inc., 1996:47-55. 6. Marshall E. Gene therapy’s growing pains. Science 1995; 269:1050-1055. 7. Anderson WF. Prospects for human gene therapy. Science 1984; 226:401-409. 8. Lee J-H, Klein HG. Cellular gene therapy. Hema/Oncol Clin N Amer 1995; 9:91-113. 9. Blaese RM. Development of gene therapy for immunodeficiency: Adenosine deaminase deficiency. Ped Res 1993; 33 (Suppl):S49-S53. 10. Yaron Y, Kramer RL, Johnson MP, et al. Gene therapy: Is the future here yet? Ob Gyn Clin N Amer 1997; 24:179-199. 11. Blaese RM, Culver KW, Miller AD, et al. T lymphocyte-directed gene therapy for ADASCID: Initial trial results after 4 years. Science 1995; 270:475-480. 12. List of Human Gene Therapy Protocols, Office of Recombinant DNA Activities, National Institutes of Health. (http://www.nih.gov/od/orda/protocol htm) 1998. 13. Beaudet AL, Scriver CR, Sly WS, Valle D. Genetics, biochemistry, and molecular basis of variant human phenotypes. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 7th ed. New York: McGraw-Hill, 1995:53-228. 14. Mulligan RC. The basic science of gene therapy. Science 1993; 260:926-932. 15. Friedmann T. Overcoming the obstacles to gene therapy. Sci Amer 1997; June:96-101.

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16. Rehmann-Sutter C, Muller H. Ethik und Gentherapie; zum praktischen diskurs um die molekulare medizin. Tubingen: Attempto Verlag, 1995: 17. Gavaghan H. Fetal gene therapy under the microscope. Nature 1994; 372:490. 18. Culver KW. Methods for gene transfer and repair. In: Sharrer GT, ed. Gene Therapy: A Primer for Physicians. 2nd ed. Larchmont: Mary Ann Liebert, Inc., 1996:19-45. 19. Caplen NJ, Alton EW, Middleton PG, et al. Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nat Med 1995; 1:39-46. 20. Perales JC, Ferkol T, Molas M, et al. An evaluation of receptor-mediated gene transfer using synthetic DNA-ligand complexes. Eur J Biochem 1994; 226:255-266. 21. Middaugh CR. Analysis of plasmid DNA from a pharmaceutical perspective. J Pharm Sci 1998; 87:130-146. 22. Tobin JF, Reiner SL, Hatam F, et al. Transfected Leishmania expressing biologically active IFN-gamma. J Immunol 1993; 150:5059-5069. 23. Kaye PM, Coburn CM, McCrossan M, et al. Antigens targeted to the Leishmania phagolysosome are processed for CD4+ T cell recognition. Eur J Immunol 1993; 23:23112319. 24. Camper SA, Saunders TL, Kendall SK, et al. Implementing transgenic and embryonic stem cell technology to study gene expression, cell-cell interactions and gene function. Biol Reprod 1995; 52:246-257. 25. Doi S, Campbell C, Kucherlapati R. Directed modification of genes by homologous recombination in mammalian cells. In: Grosveld F, Kollias G, eds. Transgenic Animals. San Diego: Academic Press, Inc., 1992:40-41. 26. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. The molecular genetics of cancer. In: Robertson M, ed. Molecular Biology of the Cell. 3rd ed. New York: Garland Publishing, 1994:1277. 27. Doerfler W. The insertion of foreign DNA into mammalian genomes and its consequences: A concept in oncogenesis. Advances in Cancer Research 1995; 66:313-345. 28. Sippel AE, Saueressig H, Winter D, et al. The regulatory domain organization of eukaryotic genomes: Implications for stable gene transfer. In: Grosveld F, Kollias G, eds. Transgenic Animals. San Diego: Academic Press, Inc., 1992:1-26. 29. Lewin B. Regulation of transcription. In: Genes VI. Oxford: Oxford University Press, 1997:847-883. 30. Doerfler W, Schubbert R, Heller H, et al. Integration of foreign DNA and its consequeces in mammalian systems. TIBtech 1997; 15:297-301. 31. Milot E, Strouboulis J, Trimborn T, et al. Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription. Cell 1996; 87:105-114. 32. Grosveld F, Dillon N, Higgs D. The regulation of human globin gene expression. Baillieres Clin Haematol 1993; 6:31-66. 33. Grosveld F, Blom van Assendelft G, Greaves DR, et al. Position-independent, high-level expression of the human β-globin gene in transgenic mice. Cell 1987; 51:975-985. 34. Welsh RM J., Cooper NR, Jensen FC, et al. Human serum lyses RNA tumour viruses. Nature 1975; 257:612-614. 35. Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 272:263-267. 36. Lewis P, Hensel M, Emerman M. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J 1992; 11:3053-3058. 37. Randrianarison-Jewtoukoff V, Perricaudet M. Recombinant adenoviruses as vaccines. Biologicals 1995; 23:145-157. 38. Verma IM. Gene therapy: Hopes, hypes, and hurdles. Interdisciplinary Science Reviews 1996; 21:96-98. 39. Valentine RC, Pereira HG. Antigens and structure of the adenovirus. J Mol Biol 1965; 13:13-20. 40. Feng M, Jackson WH Jr, Goldman CK, et al. Stable in vivo gene transduction via a novel adenoviral/retroviral chimeric vector. Nat Biotechnol 1997; 15:866-870. 41. Smith AE. Viral vectors in gene therapy. Ann Rev Microbiol 1995; 49:807-838.

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42. Berns KI, Linden RM. The cryptic life style of adeno-associated virus. BioEssays 1995; 17:237-245. 43. Russell DW, Hirata RK. Human gene targeting by viral vectors. Nat Genet 1998; 18:325330. 44. Fender P, Ruigrok RWH, Gout E, et al. Adenovirus dodecahedron, a new vector for human gene transfer. Nat Biotechnol 1997; 15:52-56. 45. Felgner PL. Nonviral strategies for gene therapy. Sci Amer 1997; June:102-110. 46. Ioannou PA, Amemiya CT, Garnes J, et al. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nature Genetics 1994; 6:84-89. 47. Yang XW, Model P, Heintz N. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol 1997; 15:859-865. 48. Peterson KR, Clegg CH, Li Q, et al. Production of transgenic mice with yeast artificial chromosomes. Trends in Genetics 1997; 13:61-66. 49. Simon MI. Dysfunctional genomics: BACs to the rescue. Nat Biotechnol 1997; 15:839. 50. Harrington JJ, Van Bokkelen G, Mays RW, et al. Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nat Genet 1997; 15:345-355. 51. Wagner E, Zatloukal K, Cotten M, et al. Coupling of adenovirus to transferrin-polylysine/ DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. PNAS 1992; 89:6099-6103. 52. Kingston RE, Chen CA, Okayama H, Rose JK. Introduction of DNA into mammalian cells. In: Ausubel FM, Brent R, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current Protocols in Molecular Biology. 1998th ed. John Wiley & Sons, Inc., 1998:9.1.1-9.1.11. 53. Furth PA, Shamay A, Hennighausen L. Gene transfer into mammalian cells by jet injection. Hybridoma 1995; 14:149-152. 54. Gordon JW, Scangos GA, Plotkin DJ, et al. Genetic transformation of mouse embryos by microinjection of purified DNA. PNAS 1980; 77:7380-7384. 55. Hess P. Gene therapy; a brief review. Clin Lab Sci 1996; 16:197-211. 56. Culver KW. Gene therapy: A primer for physicians. 2nd ed. Larchmont: Mary Ann Liebert, Inc., 1996:145-159. 57. Miller DA. Human gene therapy comes of age. Nature 1992; 357:455-460. 58. Tighe H, Corr M, Roman M, et al. Gene vaccination: Plasmid DNA is more than just a blueprint. Immunol Today 1998; 19:89-97.

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Challenges of Human Germline Gene Therapy

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CHAPTER 4

Challenges of Human Germline Gene Therapy A

lthough the stage has been set with advances in molecular biology, embryo manipulation techniques, animal cloning and somatic gene therapy (SGT), human germline gene therapy (HGLGT) is not yet a technical reality. Drawing on the material presented in the last three chapters, we now discuss possible uses of HGLGT, foreseeable methodologies for achieving HGLGT and obstacles impeding the development of HGLGT. Although experience with SGT will provide valuable information and lay the foundation for HGLGT research, many of the technical difficulties with SGT are not factors which need to be considered in HGLGT development. Conversely, parameters which are critical to the success of HGLGT may not be so important for achieving effective SGT. Given the unpredictable pace of scientific advances, it is not possible to estimate a time frame in which SGT will be perfected or in which HGLGT will be possible. Alteration of many somatic cells would be required for SGT, at one or more sites in the body, in order to reverse the effects of a genetic defect. Repeated SGT procedures may also be necessary to sustain the therapeutic benefit. HGLGT, on the other hand, would be the more efficient means of genetic correction, since the correction is intended to be a one-time procedure. With HGLGT the correction will be propagated in every cell and the transgene may be passed on to the next generation. However, if something goes wrong with the HGLGT procedure, a negative effect of the genetic change will also be widespread and not localized to a subpopulation of cells. SGT might even be required to reverse an HGLGT error at a somatic level. Furthermore, a negative consequence of an HGLGT procedure could be perpetuated in future generations. The potential effects of an HGLGT procedure are incalculable and dramatically more significant than with SGT. For HGLGT, the ideal would be to develop a procedure which is 100% efficient and accurate before using it the first time in a human. This requirement may be justifiable, but would certainly leave little room for clinical trials. With any new therapy there is an uncertainty as to the outcome, which is the reason for having clinical trials. One of the outstanding questions to be answered by society is whether HGLGT “experiments” are substantively different from other types of human experiments carried out in clinical trials.

HGLGT Targets The choice of gene targets for HGLGT will be influenced by different considerations than those used in selecting SGT targets. For example, since tissue targeting is not an issue in HGLGT development, HGLGT targets are not limited to defective genes affecting organs that are easily accessible to a transgene vector. Furthermore, genetic defects that can be avoided by using preimplantation genetic diagnosis (PGD) and embryo selection would not be appropriate targets for HGLGT. Thus, widespread application of HGLGT would probably not be used to correct single gene defects (passed from heterozygous parents) or Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus, Pamela J. Langer. ©1999 R.G. Landes Company.

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chromosomal disorders such as aneuploidies (addition or deletion of an entire chromosome). For the purposes of discussion, situations where society might consider using HGLGT for genetic change are divided into four categories listed below: 1. If both parents are homozygous for a monogenic defect, embryo selection cannot be used, since neither parent has a normal allele. HGLGT would be needed to add a normal transgene to the genome (by allele replacement). 2. If a couple wishes to alter multiple unlinked single-gene traits, it would be difficult or impossible to select for an embryo with the desired combination of genes because of the large number of embryos which would have to be screened. The use of HGLGT might be considered in some cases. 3. If an attempt were being made to alter several genes contributing to a polygenic trait, embryo selection could not be used, and HGLGT would be required to introduce several transgenes with the desired composition. (Note that alteration of only one gene contributing to a polygenic trait may also have a pronounced effect on the phenotype.) 4. If the goal were to add a new gene to the germline, HGLGT would be the only way to do this. If parents wish to replace a potential child’s gene with an allele which neither of them carried, HGLGT would be required. For example, they might desire introduction of an allele engineered to reduce the chance of developing cancer. This specially-designed allele might be considered a “superallele” because of its phenotypic value. A second example would be if parents wished to introduce a foreign gene from another organism (we refer to this as a “xenogene”) into their future child’s genome. Perhaps the xenogene would immunize the child against a virulent pathogen. In this latter case, since there would not be a homologous resident gene to replace, it would not be possible to introduce the transgene by allele replacement. Here, the best option would be to design the transgene construct to include sequences which are homologous with DNA in a designated site that we term a “transgene acceptor site.” So far the concept of a transgene acceptor site is still theoretical.

Transgenic Animals The technology developed to create genetically altered animals and plants will serve as the foundation from which transgenic human technology will grow, if the development of HGLGT is sanctioned by society. Most of the early research directed toward finding ways to promote gene insertion by HR has been done in mice for the purpose of determining the function of a gene. In these cases, the gene is inactivated by one of two mechanisms: 1. Deletion of the gene sequence (called a gene deletion); or 2. Insertion of a mutant, disrupted gene into the middle of the gene of interest, thereby inactivating the gene of interest (this process is called a “gene knockout”). In this section we describe a method for construction of transgenic mice which employs HR.

Drug Selection of Homologous Recombination Events Achieving a high frequency of HR during an experiment is not as critical if the system is designed to select cells that have undergone HR. One experimental design for HR includes the use of two foreign genes: the neo gene and the tk gene. The neo gene is a bacterial drug resistance gene encoding neomycin phosphotransferase, an enzyme which confers resistance to the neomycin analog called “G418.” Since mammalian cells do not have a neo gene, they will normally die if exposed to G418. Thus, cells which have received and are

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expressing a neo gene can be selected by growing them in media containing G418. The neo gene is called a “positive selectable marker” since cells with the neo gene will survive in media containing the G418 drug. The thymidine kinase or “tk gene” is a herpes simplex viral gene. The viral tk gene is called a negative selectable marker since cells harboring the viral tk gene will die if grown in the presence of the drug gancyclovir. The viral thymidine kinase enzyme will convert gancyclovir to a lethal nucleotide analog which will kill the cells when it is incorporated into their DNA. In engineering the targeting construct the neo gene is inserted into the middle of a gene of interest, resulting in a gene disruption (see Figure 4.1). The tk gene flanks the coding region of the disrupted gene. Once the targeting construct is introduced into the cell, only the cells that have the neo gene but do not have the viral tk gene will grow in the media containing G418 and gancyclovir. In order to have the neo gene but lack the viral tk gene, the targeting construct must have been inserted by HR. This results in inactivation of the resident gene. This type of insertion would be called a knockout because a resident gene is “knocked out” when replaced by the disrupted gene.1

Generation of Homozygous Altered Mice The experimental scheme described in the last section is directed toward replacing one resident gene with a disrupted gene. However, the goal of these types of experiments is to analyze the function of a gene by studying the phenotype when gene function is lost. In order to completely destroy gene activity, both alleles must be nonfunctional. The following procedure is used to generate mice that are homozygous for the gene knockout and therefore have both alleles of the gene inactivated. Three concepts are important to the understanding of how the homozygous transgenic mice are generated, as described in detail in the legend to Figure 4.2. First, the procedure takes advantage of the fact that the presence of a single “agouti” gene will cause mouse hair to be brownish in color. Thus, if a mouse has one agouti gene and one black gene, brown coloration is dominant over black and the mouse coat color will be brownish. Second, cells called embryonic stem cells or ES cells can be removed from the inner cell mass of a blastocyst, grown in vitro, and transfected with the targeting construct, as described above. Third, the genetically manipulated ES cells from the agouti mouse can be introduced into a blastocyst with two black alleles. If some of the cells of the resultant mouse originate from the host blastocyst cells and some originate from the ES cells, the mouse will be a chimera of black with brown stripes. The hope is that the chimeric mouse will also have germ cells that originated from the ES cells. However, the proportion of germ cells derived from the ES cells would be variable among experiments. To generate a mouse which is homozygous for the gene disruption, a series of matings must take place, as described in Figure 4.2. Once homozygous mutant mice are identified, they are examined for any physical or behavioral differences, as compared with normal mice. Any differences that are observed will give clues as to the function of the gene which was knocked out. One disadvantage of this procedure is that the neo gene used for positive selection of ES cells remains in the genome of modified mice. Methods to eliminate this neo gene from the genome do exist; however, using the classical method described here, it is not possible to select for HR without leaving some foreign DNA in the genome of the mouse.

Examples of Approaches in HGLGT Although a method designed to introduce DNA via HR might be similar in mice and humans, there are several significant differences. First, in humans, one would not be using knockout approaches since the goal is not to destroy the function of a gene, but rather to replace the defective allele with a normal one. Second, the entire genetic change in humans

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Fig. 4.1. An experimental design for homologous recombination. Cells which have undergone HR during transgene insertion are selected based on their survival in specialized media containing drugs (see text for explanation). In the experimental design, a targeting vector is constructed: a neo gene is inserted into the unaltered gene A and a tk gene is inserted outside of gene A, thereby generating a targeting vector with gene A disrupted. Upstream of the transgene and between the transgene and the tk gene are DNA sequences that are homologous to the identical region in the genome. Once the targeting construct is introduced into the cells, three different scenarios are possible: (1) Insertion of the disrupted gene A by HR, resulting in replacement of the resident gene with the disrupted gene; (2) Insertion of the targeting construct by NHR, resulting in retention of the neo and tk genes in the genome; and (3) No insertion of the transgene but possible maintenance of the transgene construct extrachromosomally. In the presence of the drugs G418 and gancyclovir, only the cells which have inserted the transgene by HR survive, while the other two categories of cells will die. Slashed bars, resident gene A or unaltered gene A; solid bars, neo gene; wavy lines, tk gene; diamond pattern, gene at a nonallelic genomic site.

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isolate blastocysts

agouti mouse

remove ES cells

blastocyst

cell growth

add targeting construct

drug selection

cell transfer

implantation

surrogate mother

chimeric blastocyst

chimera

coat color: gene A:

BI+/BI+ +

A /AI

+

black mouse

Ag+/BI+ +

A /A

+

Ag+/BI+ +

Ag+/BI+

-

A+/A-

A /A

25% A+/A+ 50% A+/A25% A-/A-

Fig. 4.2. Generation of homozygous transgenic mice. See text for background information. Embryonic stem (ES) cells are retrieved from the inner cell mass of a blastocyst which is homozygous for the agouti gene (one agouti allele will cause the mouse to be brownish in color). The ES cells are grown in the laboratory and the targeting construct described in Figure 4.1 is introduced into the cells. (The targeting construct carries a disrupted gene A, designated “A–”.) ES cells which incorporate the A– gene by HR are selected using the drugs G418 and gancyclovir, as described in Figure 4.1 and the text. The altered ES cells are injected into a black mouse blastocyst, which is subsequently transplanted into a surrogate mother. The offspring are examined to identify black and agouti chimeras which have originated from a mixture of altered ES cells and host blastocyst cells. Mature chimeric mice are mated with normal black mice. From this mating, agouti mice (in the F1 generation) will have received genetic material from ES–derived germ cells. Of these agouti F1 mice, half will also carry the (A–) gene. Those F1 sibling mice which harbor the (A–) gene are then mated to each other. Approximately 1/4 of the resultant F2 mice will be homozygous for the (A–) gene.

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would obviously have to take place in the original embryo, unlike in mice where they are bred to homozygosity. Thus, methodology would be designed to replace either one or two human alleles, as appropriate and as technically achievable. Third, it would be undesirable to introduce extra nonhomologous DNA such as a drug resistance gene into a human. If an HGLGT method is employed which does not involve drug selection of the HR events, efficient screening approaches using PCR technology would have to be designed to identify the correctly altered embryos before implantation.2 The goal in HGLGT would be to correct a genetic defect without disturbing the expression of any other genes. The most straightforward way to envision accomplishing this goal is to introduce DNA via an HR mechanism. The fact that HR can occur in human mitotic (somatic) cells is centrally important to the goals of allele replacement technology. However, there are other possible approaches that might be considered in HGLGT. First, a molecular mechanism which could generate the same result as HR is referred to as gene correction or gene conversion. In gene conversion, an introduced gene can be used by the cell as a guide for changing the resident gene sequence. As long as the introduced gene sequence was used to correct the defective gene sequence and not vice versa, the result of the gene conversion process would be tantamount to homologous gene replacement. With this caveat in mind, we will continue to refer to allele replacement caused by HR as a first choice method for HGLGT. Second, a vector could be designed which would introduce a transgene into an hypothetical transgene acceptor site in the genome (also by HR). This method may be a more technically efficient way to design HGLGT approaches as compared with constructing a gene delivery vehicle for each different transgene. One obvious disadvantage of this latter approach is that the defective gene(s) would not be eliminated from the genome during the procedure. Third, it would also be possible, and technically easier, to design a protocol that allowed random transgene insertion using an NHR mechanism. Because of the numerous potentially negative consequences arising from the random insertion of a transgene, a protocol intentionally introducing a transgene by NHR should probably not be attempted in HGLGT for any foreseeable reason short of preservation of the species. In order to provide some examples of how HGLGT might be done, we describe two theoretical approaches. Our intention here is not to outguess technological developments, but solely to provide two somewhat feasible approaches. In the first method, a transgene would be introduced directly into the pronucleus of a zygote. (For simplicity, we do not elaborate an HGLGT protocol where the transgene is introduced into the nucleus of an oocyte.) In the second method, genetically engineered cells would be fused with an oocyte from which the original nucleus was removed (an enucleated oocyte). We stress once more that the complete technology to proceed with either of these hypothetical approaches does not exist; however, many of the steps in the procedures have been developed. Furthermore, these are certainly not the only approaches which could be attempted. They merely provide contrasting examples to illustrate some of the obstacles in HGLGT. No matter what approach is used, it would be essential to use genetic diagnosis to monitor any changes in the preimplantation embryo or fetus (see below for a discussion of diagnostic inaccuracies).

Alteration of a Zygote One approach for carrying out HGLGT would involve introduction of the transgene directly into a pronucleus of a zygote, similar to what is done in the creation of transgenic mice. Allele replacement in a zygote would be feasible only if a transgene could be introduced by HR at a very high rate, without concomitant NHR. Various schemes have been developed for increasing the chance of an HR event. For example, it was determined in the

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early days of HR experiments that an increase in length of a region of homology caused an increase in HR events (within the size range of 2-15 kb).3 If it turns out that the relationship between the length of the homologous region and the HR rate holds for larger pieces of DNA, a very long transgene construct might have a high chance of being introduced by HR. It is also worth noting that a small amount of heterology, i.e., differences between the resident gene and transgene, may help rather than hinder the process.2 Currently, several groups are attempting to develop new targeting vectors that would encourage HR without the introduction of drug resistance genes.4,5 These new vectors, based on artificial chromosomes from either yeast (YACs) or bacteria (BACs), can accommodate large inserts up to 1 Mb and 300-350 kb, respectively. Design of the YAC or BAC vectors is based on the premise that including long regions of homology will increase the tendency of the transgene to recombine by HR. One difficulty with these new systems appears to be the preparation of such large inserts. A second consideration touches on the arguments over how much change in the human genome is acceptable using HGLGT. For example, in constructing a YAC transgene construct, the DNA flanking the transgene would presumably be derived from a living or deceased individual. Thus, during the HR event, much of the DNA flanking the transgene would also be transferred to the altered cell. This may cause replacement of several normal genes flanking the defective gene. One way to potentially avoid such a large genetic exchange would be to construct the replacement vector using the flanking DNA from another embryo which was the product of the parental germ cells. Although the flanking DNA in the construct would then be from a “sibling” embryo, there would be a relatively high chance that this flanking DNA would be the same as that in the embryo to be altered (but not a 100% chance, given meiotic crossing-over events; see chapter 2). Such an individualized tailoring of a transgene construct would be difficult, time consuming, costly and impractical at best. A serious obstacle in the alteration of an oocyte or zygote with a large transgene construct is that the frequency of successful HR would have to be high enough so that it could be used when only a limited number of oocytes are available. To gain an appreciation of how many oocytes one might need to collect for such an HGLGT procedure, we offer an example employing hypothetical success rates at each step of the procedure in Figure 4.3 (see chapter 2 for additional information on these procedures). From the calculation presented in Figure 4.3, it can be seen that in order to end up with at least three HGLGTmodified embryos which can be transferred to the uterus, many oocytes must be collected from the female, who must undergo many superovulation procedures over an extended period of time. Although we have based most of these estimates of success on current data, the one very optimistic estimate is the future achievable rate of HR. If this estimate is wildly overestimated, then many more than 126 oocytes would need to be collected. After transgene introduction into mammalian cells (by various methods), estimates of the ratio of HR to NHR events range from 1 in 15 to 1 in 5 million,2 as compared with the 1 in 10 chance used in this example. However, since the stable transfection efficiency is approximately 10-20% for microinjected DNA (this includes HR and NHR events),2 it seems reasonable to expect that a similar success rate can be achieved with a mechanism which employs HR exclusively. In a recent study using adeno-associated virus (AAV) to direct targeted gene replacement, researchers achieved an HR rate as high as 1% in the somatic cells used. This new approach may accelerate the work directed toward using HR in gene therapy.6 However, without a scheme to select cells in which HR has occurred, the approach outlined here remains relatively impractical until additional technology emerges. Given the risk of error in the various steps of HGLGT, it would be impossible to carry out HGLGT without using genetic embryo selection and embryo termination. It is conceivable that an HGLGT procedure could involve a gamete intrafallopian transfer

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Fig. 4.3. Oocyte requirement in hypothetical HGLGT procedure. In this example, oocytes are collected from the prospective mother in a series of superovulation procedures over an extended period of time, frozen, thawed and used for IVF. The transgene construct is introduced into one of each zygote’s pronuclei, and the resulting embryos are selected based on morphological criteria. After embryo biopsy, PGD is used to assess the ploidy of the embryos (euploid, aneuploid or polyploid) and whether or not the transgene was inserted via HR. The euploidy rate for total fertilized embryos is estimated at 75% for this example, but this number is age–dependent (see chapter 2). Since many of the embryos with chromosome number imbalances will be among the morphologically abnormal embryos, we arbitrarily chose a euploidy rate of 90% among the morphologically normal embryos. Three embryos, in which the transgene replaced a resident gene by HR, were used for uterine transfer. The goal of the procedure is to produce one healthy child. The required number of starting oocytes depends on the success rates for the various procedures. Estimated success rates are drawn from various reports15-18 (with one exception) and are indicated to the right of each arrow. The rate of HR is set at 10% for this example. We assume that a 10% HR success rate is a realistic future expectation for this procedure; however, this has not yet been achieved. IVF, in vitro fertilization; PGD, preimplantation genetic diagnosis.

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(GIFT) procedure instead of in vitro fertilization (IVF) (see chapter 2) or that transgene vectors would be targeted to germ cells in vivo, but these options are still in the realm of science fiction. Furthermore, it could also be argued that it would be completely irresponsible to conduct HGLGT without checks on the continued presence of the transgene by using genetic embryo selection and CVS or amniocentesis, resulting in termination of the embryo or pregnancy if the results indicated an error in transgene location in the genome.

Nuclear Transfer from Altered Embryonic Stem Cells A second hypothetical approach for HGLGT relies on the ability to transfer a human cell nucleus to an egg without a nucleus and succeed in generating an embryo with the potential for developing into a healthy human. This procedure has features in common with the recent generation of the cloned sheep “Dolly”7 and would be considered human cloning (see Figure 4.4 for description). In this highly controversial procedure, ES cells removed from an embryo would be grown in vitro. Those cells which had incorporated the transgene by HR would be selected. The nucleus from a correctly altered ES would be used to create a diploid egg which has the potential to develop into a human without the genetic disease. The human developing from this diploid egg would have essentially the same genotype as the original embryo, with the exception that a defective allele was replaced with a normal transgene. (This would be true when only the defective gene was replaced and not a large region of flanking sequence.) The unique genome of the original embryo would be retained with the exception of one small region of DNA. The human resulting from this HGLGT procedure would be a clone of the original embryo, with a single gene difference. From our previous discussions of embryo transfer, readers may quickly deduce that it would be medically prudent to use more than one embryo for uterine transfer. Therefore, if more than one ES cell nucleus were used and more than one embryo transferred, the resulting humans would be clones of one another.8 For the parents this would be roughly equivalent to having identical twins, if two children are born. There is a way around having children who are identical twins, however. If more than one original embryo is used as sources for individual ES cell cultures, and the procedures are done in parallel, the two to three embryos used for uterine transfer could be fraternal siblings. Although this may be desirable for parents who wish not to have identical siblings, some parents may not choose this approach because of the added expense and difficulty in timing genetic corrections on multiple ES cell lines.

Technical Obstacles in HGLGT In the last section we provided two potential methods for HGLGT, assuming that technical obstacles had been overcome. In this section we highlight some of the major technical hurdles which must be passed before HGLGT could be achieved.

High Efficiency Transgene Introduction by Homologous Recombination A goal for HGLGT is to replace a defective allele with a normal transgene by HR without any auxiliary molecular events that could cause additional insertions by NHR. We have considered two scenarios for transgene insertion: In the first, the transgene construct is introduced directly to the oocyte or zygote. The vector or methodology for achieving HR by this route has not been developed. Note that this approach does not take advantage of any selection or enrichment for HR events but relies on identification of HR events by screening. The HR event would have to occur at a sufficiently high frequency so that the number of required starting oocytes is in a realistic range (see Figure 4.3). In the second scenario, drug selection is used to eliminate all cells which have not incorporated the transgene by

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Both parents are affected Targeting vector

In vitro fertilization

Grow in cell culture Remove cells from the inner cell mass

original embryo (blastocyst stage)

Altered and unaltered cells

Egg with nucleus removed

Altered cell

Select cells which have undergone homologous recombination

Fuse cells or transfer nucleus Altered diploid egg

Altered clone of original embryo

Carrier, unaffected child

Fig. 4.4. Generation of an unaffected child from two homozygous defective parents. In this example, two homozygous recessive parents with cystic fibrosis wish to have an unaffected child. Oocytes are collected from the superovulated female and fertilized in vitro. ES cells are removed from the inner cell mass of a blastocyst and maintained in vitro. After introduction of the targeting construct, the ES cells are grown in specialized media to select for those cells which have integrated the transgene by allele replacement, similar to the rationale described in Figures 4.1 and 4.2 for the mouse experiments. A successfully altered ES cell, harboring one copy of the normal transgene, is used as a nuclear donor for an enucleated oocyte from the female. The technology might involve physical transfer of an ES cell nucleus to the enucleated oocyte, or the ES cell may be fused with the oocyte. The resulting cell is a diploid egg which carries the nucleus from the successfully altered ES cells. Two or three diploid eggs from this procedure may be implanted. If the pregnancy is successful, an unaffected child who is heterozygous for the CF allele will be born. Although not illustrated in this figure, if two defective CF alleles are replaced, the child would be homozygous normal and would not be a potential carrier of that same CF gene defect to the next generation.

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HR. Still, to avoid growing the ES cells for many generations to yield high numbers of starting cells (see next section), the HR event would also have to occur at a high frequency.

Cell Manipulations A number of cell techniques would have to be established before HGLGT could proceed. First, the culturing of human ES cells would have to be routine and designed to minimize promotion of genetic changes resulting in chromosome number imbalances or loss of pluripotency or totipotency of the ES cells. Second, techniques for enucleating human oocytes would have to be perfected. Third, in order to achieve nuclear transplantation of an altered ES cell nucleus, a method would have to be developed for fusing ES cells with the enucleated oocyte or for physically transferring an ES cell nucleus to the enucleated oocyte. In the creation of several cloned sheep, embryo-derived cells, fetal cells and adult udder cells were used as nuclear donors. The famous sheep Dolly was derived from the fusion of an adult udder cell with an enucleated, unfertilized oocyte, and the fusion was mediated by electrical pulses.7

Resetting the Genetic Clock The technology developed for sheep cloning might suggest that we are well on the way to being able to use ES cell nuclei to direct oocyte development. However, it may not be a simple case of technology transfer. In humans, the embryo proceeds to the 4 to 8 cell stage without activating the embryonic genome.9 These early blastomeres survive on materials supplied by the oocyte. If a more mature nucleus, such as one from an ES cell, is transferred to an enucleated oocyte, the genome of the transplanted nucleus must be reset from the configuration found in the more developed cells to that which is appropriate for a zygote. This process of changing DNA modifications (such as methylation) and possibly altering chromosome structure takes time. If the transplanted ES cell nucleus can be reprogrammed before the normal time of activation of the embryonic genome, there is a good chance that an ES cell nucleus can direct the development of a human oocyte. The success in sheep cloning is partially attributed to the fact that the sheep embryonic genome is not activated until the 8 to 16 cell stage10 and that the researchers found a way to aid the reprogramming of the transplanted nuclear machinery (i.e., by starving the cells prior to nuclear transfer).7,11 Since the mouse cell genome is activated in the late 2 cell stage, even earlier than the human embryonic genome, the mouse system may not provide an adequate experimental model for testing approaches in the development of HGLGT.12

Accuracy in Genetic Testing For evaluating the success of an individual HGLGT procedure, one option is to apply the criterion that occurrence of any gene insertion event other than allele replacement is unacceptable. Early preimplantation diagnosis of the error would be preferable; however, later prenatal genetic diagnosis using CVS or amniocentesis might also be recommended to monitor any genetic changes. As discussed in chapter 2, the CVS and amniocentesis procedures also carry with them a certain risk of spontaneous fetal loss. A major problem with genetic diagnosis at any stage is that all diagnostic procedures are subject to a certain degree of error. The errors derive from two major sources: 1. Only a subset of cells are biopsied and their genotype may not be representative of the majority of cells in the embryo or fetus (see chapter 2); 2. Technical errors can cause misdiagnosis or a lack of diagnosis. What chance of diagnostic error is acceptable when monitoring the development of an HGLGT-engineered embryo? One might argue that the diagnostic procedures should be

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100% accurate and reliable before even considering an HGLGT procedure because of the risks of diagnostic errors. As more information about the genome becomes available from the Human Genome Project and subsequent studies of gene function, the criteria for embryo or fetal termination in an HGLGT procedure may be more lenient. For example, if accidental NHR occurred in an HGLGT procedure, the decision of whether or not the pregnancy should continue may depend on where the transgene was secondarily inserted.

SGT Obstacles That Are Not HGLGT Obstacles The current difficulties with many aspects of potential HGLGT procedures may provide reassurance that we are not scientifically close to being able to conduct an HGLGT experiment, legally or illegally. One might think that we will go on improving SGT and perfect this procedure and then sometime in the very distant future we will be able to start thinking about whether or not we should condone and develop HGLGT procedures. But viewed from another perspective, there are so many technical difficulties impeding highly successful SGT therapy that one might conjecture that safe and successful HGLGT would be easier to achieve. That is, many of the SGT obstacles (listed below) do not have to be overcome before successful HGLGT could be achieved.

Tissue Targeting with In Vivo SGT Since gene transfer in HGLGT would most likely be done by an ex vivo approach, the development of elaborate tissue targeting vectors is not an HGLGT issue. Also, with SGT there remains the challenge to achieve a high local concentration of targeting vector so that an adequate number of cells can be altered. Moreover, the SGT risk that a vector will inappropriately insert the transgene into germline cells is certainly not an issue with HGLGT since germline intervention would be intential.

Cell Manipulation in Ex Vivo SGT One limitation with SGT is that ex vivo approaches can only be done with cells that are easily removed from the body, can be cultured in the laboratory and can be reintroduced efficiently into the patient. Furthermore, certain SGT protocols require dividing cell populations, since some transgene vectors can infect only dividing cells and a proliferating population of altered cells is desirable for establishing the physical manifestation of the genetic alteration. With HGLGT, early embryonic cells are easily obtained, are ideal dividing cell populations, and embryo transfer back to the female is already a well established procedure.

Immunological Response to Transgene Products If the targeted cells in SGT are not stem cells, then it will be necessary to administer the vector regularly if using in vivo approaches. However, repeated exposure of the immune system to the vector can lead to an immunological memory response and thus result in rapid removal of the vector before it can deliver the DNA to a target cell. Delivery of the gene is not the only immunological obstacle facing SGT. Once the new gene is established and expressed, the immune system might recognize the gene product as foreign and destroy the cell expressing the gene. SGT is analogous to DNA vaccination because in both scenarios DNA from outside sources is expressed in a cell that has never expressed the gene product before. The goal of DNA vaccination is to generate an immune response against the foreign gene product, for example an influenza (flu virus) surface protein. The immune response that is stimulated by DNA vaccination can destroy the cells expressing the foreign gene product.13 If an individual is treated by SGT, the cells expressing

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the new gene product may be susceptible to the same type of immunological destruction found in DNA vaccination. HGLGT provides a way to get around the immune system. First of all, no in vivo vectors would be required, so the gene products can be safely delivered to the target cell. Second, because the new gene product is present during the development of the immune system the gene product is recognized as normal and subsequently ignored by the immune system.

Problems Associated with NHR in SGT Assuming that an HGLGT procedure would not even be considered unless the transgene were inserted by HR, the concerns over consequences of NHR such as insertional mutagenesis or inappropriate transgene or nonallelic gene expression would not be an issue. However, if the possibility exists that at some low frequency an NHR event is associated with transgene insertion by HR, then NHR remains a serious concern for HGLGT as well as SGT. Furthermore, if the transgene has different modifications (e.g., too much or too little methylation) as compared with a normal resident gene, nonallelic gene expression could be disturbed even when the transgene is inserted via HR. However, one possibility is that changes in methylation are more of a problem when introducing a transgene into a somatic cell than into a zygote or early embryonic cell since in early embryogenesis there may be a better chance to correctly reset the patterns of modification.14

Summary and Conclusions In this chapter we have reviewed some of the research and experimental ideas which are laying the foundation for HGLGT. We have repeatedly pointed out the dangers which could stem from random transgene insertion by NHR and emphasize the many advantages of transgene insertion by HR mechanisms. At this point we cannot foresee which type of transgene vector or gene transfer methodology would be used to accomplish the goal of allele replacement. However, no matter which avenue is pursued in the development of HGLGT technology, there are still many scientific barriers impeding progress. Although the development of SGT is paving some of the way for HGLGT, many of the challenges of HGLGT are different, from a scientific and ethical perspective. In the rest of the book, we weave the scientific considerations presented in the first four chapters together with moral and political issues in HGLGT.

References 1. Capecchi MR. Targeted gene replacement. Sci Amer 1994; March:52-59. 2. Doi S, Campbell C, Kucherlapati R. Directed modification of genes by homologous recombination in mammalian cells. In: Grosveld F, Kollias G, eds. Transgenic Animals. San Diego: Academic Press, Inc., 1992:40-41. 3. Capecchi MR. Altering the genome by homologous recombination. Science 1989; 244:12881292. 4. Yang XW, Model P, Heintz N. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol 1997; 15:859-865. 5. Peterson KR, Clegg CH, Li Q, et al. Production of transgenic mice with yeast artificial chromosomes. Trends in Genetics 1997; 13:61-66. 6. Russell DW, Hirata RK. Human gene targeting by viral vectors. Nat Genet 1998; 18:325-330. 7. Wilmut I, Schnieke AE, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385:810-813. 8. Mirsky S, Rennie J. What cloning means for gene therapy. Sci Amer 1997; June:122-123.

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9. Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 1988; 332:459-461. 10. Crosby IM, Gandolfi F, Moor RM. Control of protein synthesis during early cleavage of sheep embryos. J Reprod Fertil 1988; 82:769-775. 11. Campbell KH, McWhir J, Ritchie WA, et al. Sheep cloned by nuclear transfer from a cultured cell line. Nature 1996; 380:64-66. 12. Bolton VN, Oades PJ, Johnson MH. The relationship between cleavage, DNA replication, and gene expression in the mouse 2-cell embryo. J Embryol Exp Morphol 1984; 79: 139-163. 13. Tighe H, Corr M, Roman M, et al. Gene vaccination: Plasmid DNA is more than just a blueprint. Immunol Today 1998; 19:89-97. 14. Doerfler W. The insertion of foreign DNA into mammalian genomes and its consequences: A concept in oncogenesis. Advances in Cancer Research 1995; 66:313-345. 15. Calderon I, Healy D. Endocrinology of IVF. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1993:7. 16. Toth TL, Baka SG, Veeck LL, et al. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril 1994; 61:891-894. 17. Feichtinger W. Results and complications of IVF therapy. Curr Opin Obstet Gynecol 1994; 6:190-197. 18. Handyside AH, Delhanty JDA. Preimplantation genetic diagnosis: Strategies and surprises. Trends in Genetics 1997; 13:270-275.

CHAPTER 5

Therapy vs. Enhancement and Other Pertinent Distinctions I

n the last four chapters, we provided a scientific and technical background for understanding the obstacles in developing human germline gene therapy (HGLGT) and the potential molecular and phenotypic consequences of HGLGT. We also discussed other options for couples making reproductive decisions, such as embryo selection. In the second half of this book we employ empirical facts (and speculations) in our discussion of the ethics of HGLGT. The arguments we examine in the remainder of this book make use of empirical assumptions as well as moral, political, or religious concepts and principles. Before explicating and evaluating these arguments, we discuss some preliminary questions that will set the stage for our subsequent discussion. We also describe our overall approach to resolving ethical dilemmas and issues in biomedicine. In chapter 2 we argued that embryo selection would be the most safe and effective way of preventing genetic diseases that result from single genetic defects or certain chromosomal abnormalities. Although the majority of genetic diseases result from a monogenic recessive defect, some diseases that have a genetic basis, such as various forms of cancer, diabetes and heart disease, are caused by interactions among many different gene products as well as strong environmental influences. Some polygenic diseases would be difficult to treat with somatic gene therapy (SGT), would not be preventable with embryo selection, and would require HGLGT if the goal were to correct the genetic defects before birth. However, it might take decades before our science and technology have advanced to the point where we could understand and manipulate polygenic traits. Currently, SGT methods do not involve replacement of defective human genes but only the addition of normal transgenes. The best chance for appropriate transgene expression without disrupting expression of any other genes would be when the transgene is introduced into the genome by homologous recombination (HR), i.e., where the transgene exactly replaces a defective allele (allele replacement). However, other scenarios for HGLGT are possible, such as allowing a transgene to insert randomly into the genome via nonhomologous recombination (NHR), a method which would have a high risk of having seriously negative consequences, as discussed in chapter 3. We would hope that HGLGT will not be considered, from a scientific/medical standpoint, until a method employing allele replacement is an established technique. Unfortunately we have to consider both ends of the spectrum: those situations where HGLGT may be deemed “safe” and those where it would be a high risk procedure. Given the variety of values in the world community and the unpredictable nature of future biological events which could warrant a high risk procedure, both ends of the HGLGT safety spectrum are real possibilities. If we limit our thinking to situations where HGLGT would be deemed a “safe” procedure, HGLGT might be used to prevent monogenic disorders in very rare cases or to replace a single disease-contributing gene (potential uses of HGLGT are discussed in chapter 4.) Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus, Pamela J. Langer. ©1999 R.G. Landes Company.

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Alternatively, HGLGT could be used to replace a normal allele with a more desirable allele in an attempt to create an individual with an enhanced genome. The latter situation would be called “genetic enhancement”; sometimes this is referred to as “positive eugenics.” As we discuss in later sections, the distinction between “positive” and “negative” eugenics is not always clear.

Some Ethical Questions in Embryo Selection and HGLGT The embryo selection techniques we discussed are far from uncontroversial, since they involve in vitro fertilization and the selective termination of embryos. We do not discuss the ethics of in vitro fertilization or selective termination of embryos here, but refer the reader to useful analyses of these issues.1-4 We will assume, for the sake of this book, that anyone who engages in preimplantation genetic diagnosis (PGD) authorizes in vitro fertilization and selective embryo termination based on morphological or genetic criteria (morphological embryo selection or genetic embryo selection; see chapter 2). It almost goes without saying that someone who is morally opposed to a reproductive technology such as in vitro fertilization, which necessarily involves morphological embryo selection, would also be opposed to genetic embryo selection. If any of these procedures are illegal, then other complications arise that we will not address here, such as the morality of lawbreaking. HGLGT technologies will necessarily employ in vitro fertilization and selective termination of embryos or fetuses, in addition to cloning of early embryonic stem cells if certain HGLGT methods are used (see chapter 4). The cloning of human embryos (or adults) is one of the most controversial bioethics issues of our times, and many scholars, politicians, religious leaders, and interest groups oppose the cloning of human beings for any reason.5,6 Again, we will not review the arguments for or against the cloning of human beings, but we will refer the reader to some illuminating discussions of the issue.7-9 Selective abortion would be used in HGLGT in order to terminate fetuses for which the gene transfer procedure had failed or for fetuses with severe genetic defects created by the genetic interventions, if these genetic defects were detected (see chapter 4). Selective abortion is another controversial issue that we will not address here, although we will assume that anyone who is willing to conduct HGLGT would also authorize selective abortion in order to avoid harmful genetic consequences of the procedure. One could conduct HGLGT without practicing selective abortion, but this policy would be reckless, since it could allow severely defective children to be born. Presumably any person seeking HGLGT would want to avoid that outcome. We refer the reader to some useful discussions of selective abortion as well.1-4 It is worth noting that there are morally significant differences between genetic embryo selection and HGLGT. Although embryo selection involves termination of embryos, it does not involve genetic manipulation of genome. HGLGT would involve manipulation of the genome in a way that avoids the “natural gene lottery” of mating. Although both processes interfere with natural, human reproduction, one might regard embryo selection as “less unnatural” than HGLGT, since embryo selection does not involve the manipulation of the human genome. We will explore questions of the unnaturalness of HGLGT in more detail later on in the book.

Gene Therapy vs. Genetic Enhancement Many writers who discuss the moral and political issues in human genetics distinguish between germline therapy and germline enhancement.10,11 While both processes involve manipulations of the human germline, the goal of germline therapy is to cure or eliminate a genetic disease and the goal of germline enhancement is to improve or enhance human offspring. Genetic alteration of one individual does technically alter the gene pool; however, whether this is a significant effect is another matter which we discuss in chapter 6. Thus, the

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intended alteration of an individual may have an unintended effect on the gene pool if the transgene is passed on to offspring for several generations. This is different from the type of genome intervention where the focus is not on the individual but on a deliberate program to change the gene pool by selective breeding, selective abortion or genetic manipulation. All germline interventions can be regarded as a type of eugenics, i.e., the attempt to control the genetic composition of an individual or the human population. For our purposes we will distinguish between medical and nonmedical eugenics. “Medical eugenics” occurs when we manipulate a human genome or the human gene pool in order to prevent diseases in human populations; nonmedical eugenics occurs when we manipulate a human genome or the gene pool in order to achieve other goals, such as increased height or longevity. Using these definitions, introduction of a gene which would significantly lower the chance of developing cancer would fall under the classification of medical eugenics (or gene therapy) while manipulating the germline in order to produce a child with blonde hair would be nonmedical eugenics (or genetic enhancement). (Other writers use the phrases “negative eugenics” and “positive eugenics” to distinguish between two different forms of eugenics, but we will not use this terminology, since in some contexts these terms are vague and value laden.) Is the distinction between therapy and enhancement valid and clear?12,13 On the surface, this seems to be a worthwhile distinction: There are qualitative differences between manipulating the germline in order to prevent a disease and manipulating the germline in order to produce children with blue eyes or blonde hair. However, since we are analyzing this distinction in terms of medical vs. nonmedical interventions, one might challenge the soundness of the distinction by raising questions about the concept of disease. Although the concept seems clear and objective, one might argue that the concept is neither clear nor objective. Many writers argue that diseases have important psychosocial and cultural dimensions.14,15 This point seems to apply most clearly to psychosocial diseases, such as schizophrenia. In our modern era, people who hear voices or see visions are regarded as mentally ill, but they might have been treated as prophets in medieval times or in some contemporary, non-Western cultures. Since it appears that there may be a genetic basis for schizophrenia,16 it is not at all clear that an attempt to avoid this disease via genetic engineering should be viewed as gene therapy (medical eugenics). If one considers deafness to define membership in a culture or community, then even congenital deafness might not be considered a genetic disease by members of that culture or community. While the non-deaf may regard deafness as a disease, many deaf people view it as a basis for belonging to a language community.17 Other conditions with a possible genetic basis also have strong psychosocial and cultural components, such as alcoholism, depression, homosexuality, and male pattern baldness. The traditional reply to this social critique of the concept of disease is to seek an objective foundation for human health in biology. One popular biological notion of disease employs the idea of biological norms, which are understood in a statistical sense: Normal phenotypes are defined as having properties that fall within a statistically normal range of variation, e.g., within two standard deviations of the mean for a property.18 Thus, if the normal blood cholesterol level for a human being ranges between 120 and 280 mg/dl, then a person who has a blood cholesterol level below 120 or above 280 mg/dl can be viewed as having a disease. There is an evolutionary explanation for the statistical norms we observe in phenotypes: Phenotypes occur in populations because they have played a key role in reproduction and survival. Traits that fall within these statistical norms are functioning properly, and deviations from these norms are dysfunctions.19 An organism is functioning properly when its traits are performing (or capable of performing) the functions that they were designed

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by natural selection to perform. Natural selection “designs” traits through the differential survival and reproduction of organisms that possess those traits. Traits that evolve by the process of natural selection are therefore designed to perform functions that contribute to the fitness of organisms that possess them. For example, the function of the heart is to pump blood, because blood pumping contributed to the reproduction and survival of organisms with hearts. A heart that does not pump blood well is not functioning properly and is therefore a diseased heart. Genetic intervention that attempts to prevent heart disease could be viewed as gene therapy, not genetic enhancement, on this understanding of disease.10 We think that the biological concept of disease can provide an objective basis for many genetically-based maladies, such as juvenile development of heart disease (with hypercholesterolemia), Tay-Sachs disease, cystic fibrosis, adenosine deaminase (ADA) deficiency, hemophilia, and other dysfunctional traits. These and many other diseases are clearly maladaptive in the evolutionary sense of the term. Humans with diseases like these are very likely to fare poorly in terms of reproduction and survival if the disease is not treated medically. However, this approach does not work for all genetic conditions. First, the adaptiveness of a trait always depends on environmental factors, including the individual’s biological and social environment. For example, the presence of sickle cell anemia may be adaptive in one environment but maladaptive in a different environment. Sickle cell anemia patients have a high mortality rate because of the blood disorder, but they are resistant to malaria. Since malaria causes a significant amount of death in Africa, sickle cell anemia could therefore be viewed as adaptive in climates where malaria is common. Certainly the presence of a single sickle cell allele is adaptive, since people carrying the defect in a heterozygous condition are usually clinically silent and also have a higher than normal resistance to malarial infection. Second, many genetic conditions do not manifest their symptoms until a subject is past reproductive age (see chapter 1). A late-onset Huntington’s disease may have no “bad” evolutionary consequences, yet we would still consider it a disease. Third, many conditions, such as color blindness or albinism, may have only limited effects on reproduction and survival, even though they are deviations from a population’s statistical norms. Fourth, some conditions, such as certain mental illnesses, have a genetic basis but also have a strong psychosocial component. Even though these conditions have a genetic basis, social factors play a key role in determining whether we decide to call them diseases and how we treat them. Fifth, some traits, such as height, vary considerably across populations. A child born in the United States who grows to be four feet tall would be considered a “dwarf;” the same child born in a pigmy tribe would be considered normal. The upshot of this discussion, we believe, is that there is a theoretical basis for a biologically grounded distinction between germline therapy and germline enhancement, and that this distinction is based on the concept of disease. However, since the distinction between health and disease is not absolute, the distinction between gene therapy and enhancement will often be difficult to apply, since conditions present in the social or biological environment will determine the boundaries between health and disease. Thus, it may be difficult to decide (in practice) whether a particular form of genetic manipulation should be treated as germline therapy or germline enhancement. The therapy/enhancement distinction in genetics leaves room for many borderline cases. We have stressed the distinction between gene therapy and enhancement because we believe that it is essential to understand this issue before addressing arguments for or against HGLGT, since most of these discussions assume that we can make this distinction. Indeed, many writers argue that therapy is morally and politically acceptable although enhancement is not.20,21 This position loses its cogency once we see that the distinction between therapy and enhancement is often not clear cut. The choices we face are much more complex than “therapy = good, enhancement = bad.” First, as we shall see in the next chapter,

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not all genetic therapies are “good.” Some therapies may pose unacceptable risks for patients. Second, there may be some types of enhancement that could have a sound ethical basis, if they are proven to be medically safe and effective. It may be possible to manipulate the germline to make people resistant to viruses such as HIV,22 Ebola, or even more virulent pathogens to be encountered in the future. It may also be possible to manipulate the germline to allow people to have better defenses against cancer,23 a goal which could be considered either therapy or enhancement. Genetic enhancement could be an immunization technology for the 21st century.12,13 Already scientists are designing DNA vaccines at the somatic (not germline) level. Here, DNA encoding a protein is introduced into a cell in order to provide resistance against infection with a pathogen.24 None of these reflections imply that the therapy vs. enhancement distinction has no place in the HGLGT debate. But, in order to understand the morality of HGLGT, we need to go beyond this distinction and reflect on deeper moral and political concerns, such as harms vs. benefits, justice, human rights, and human nature. Our central question will not be, “Is a particular form of human genetic engineering therapy or enhancement?” but “Is a certain form of human genetic engineering morally or politically acceptable?”

Parental Choice vs. State Controls It is worth noting that there are morally and politically significant differences between parental decisions involving HGLGT and state-mandated decisions.25 State-mandated HGLGT decisions occur when a government encourages or requires couples to use HGLGT to produce children. For instance, a government might require a couple to conceive a child via HGLGT in order to participate in an experiment or to prevent the couple from producing a child with a genetic disease that cannot be prevented by other means. Very often when people envision nightmare eugenics scenarios, such as Aldous Huxley’s Brave New World, they are most concerned about state-mandated reproductive choices. One might argue that state-mandated choices are more problematic from a moral and political point of view than individual choices, since governmental control of HGLGT implies coercion and the state’s intrusion into private affairs. We will return to these questions later on in the book. For now, we note the importance of the distinction between parental and state decisions, and unless we state otherwise, whenever we are discussing HGLGT decisions, we will be discussing decisions made by prospective parents, not decisions made by governments.

Genetic Determinism Many writers have criticized genetic interventions, including genetic testing and gene therapy, on the grounds that they assume that genes causally determine human phenotypes.26 Although we will discuss genetic interventions in this book, we do not assume the truth of genetic determinism. We recognize that the vast majority of traits are caused by a complex interplay between genes and the environment and that very few phenotypes are determined entirely by single genes.27 Genetic interventions can still be useful and significant in their effect on polygenic traits, since genes are statistically relevant to phenotypic outcomes of polygenic traits. Genes can predispose people to express or acquire certain traits even if genes do not causally determine those traits. For example, a gene might increase a person’s chances of developing colon cancer even if the cancer will not develop unless the person is exposed to certain environmental carcinogens. One might acknowledge the points about the contribution of genes to polygenic traits but argue that, since the environment plays a much larger role in some human diseases and traits, focusing on genetic causes while ignoring environmental factors constitutes a particular form of bias or prejudice.26 This type of prejudice, which we will label “genocentrism,” would wrongly emphasize genetic explanations and causes over environmental ones. Our

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culture has become infatuated with human genetics. Where we used to look for environmental causes we now look for genetic ones. We ignore mountains (the environment) in order to focus on molehills (genes). One could argue that our time, effort, and energy might be better spent by addressing environmental factors, not genetic ones. We believe the best approach to human diseases and traits is to address both genetic and environmental causes. An effective anticancer program would address exposure to carcinogens as well as the genetic basis of cancer. Genetic interventions can and should be combined with policies and procedures that address environmental factors.25 Thus, none of our comments in this book should be construed as a commitment to genetic determinism or genocentrism.

Moral and Political Decision Making Since this book will discuss the moral and political arguments for and against HGLGT in order to provide a framework for decisions and policies, a few words about how we approach moral and political decisions are in order. Moral or political decisions involve choices about the kinds of lives we should live, the kinds of people we should strive to be, the kinds of goals to which we should aspire, the kind of society that we should build. These decisions pertain to our conduct, our ideals, and our values. Moral decisions tend to focus on the conduct of individuals, while political decisions tend to focus on the conduct of groups. However, there is no sharp distinction between morality and politics, since collective actions result from individual choices and individual choices can and should be influenced by political considerations.28 Many social issues, such as abortion, can be evaluated from both a moral and political perspective and have significance for both the conduct of individuals and groups. Since it would be virtually impossible to write a book on the morality of HGLGT without addressing political questions, our book will address questions that span across morality and politics. Many writers distinguish between ethics and morality: Morals are society’s general standards of conduct and ethics are special standards (or codes) that govern conduct in specific professions, disciplines, or institutions. Ethics and morality are similar in that they both prescribe standards of conduct, even though ethics can be viewed as “morality in context” or “applied morality.” Thus, we can speak of “medical ethics,” “business ethics,” “sports ethics,” and so on. While we recognize that many people distinguish between “morality” and “ethics,” we will not place a great deal of weight on this distinction, since many people use the words “ethical” and “moral” more or less interchangeably and because “ethics” and “morality” both address standards of conduct. Although we believe that there are some moral standards that apply to most situations, we also acknowledge that factors inherent in different situations play a key role in determining how we should interpret and apply these principles. We agree with the slogan “ethics are situational,” while acknowledging that this does not mean “anything goes” in moral decision making. The slogan should be understood as implying that information about a particular situation as well as our emotional responses, can and should make a difference in how we respond to that situation. Even if ethics are situational (in some sense), we believe that there are some general principles that can and should guide moral and political decisions. These principles are rules, values, or standards that reflect our common sense intuitions and can be justified from a wide variety of theoretical viewpoints.28 Thus, many different people (and many different moral theories) can agree on common principles like “don’t murder, steal, lie, or cheat,” “help others,” “respect human rights,” “be fair,” etc. These general principles can guide our conduct by prescribing particular courses of action in specific situations. They apply to many different cases and have validity that transcends any particular situation or context.

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Morality and politics are situational in the sense that one needs to understand the unique features of a situation or case in order to apply moral and political principles.28 Conflicts among different principles can produce moral and political dilemmas and questions. For example, a physician might face a conflict between benefiting a patient and respecting her right to autonomous decision making. When the patient asks the physician about some alternative, unproven breast cancer treatments, the physician knows about these treatments but may wonder whether he should tell his patient about them. The patient has an excellent chance of recovering from cancer through conventional means, but the patient is still interested in alternative treatments. Should the physician tell her about these alternative treatments? Should he give the patient the plain facts about the alternative therapies without providing his judgment or opinion? In order to answer these questions, the physician needs to examine the relevant moral (or ethical) principles in light of the unique features of this case. The solution he reaches should be based on a critical, reflective, “all things considered,” examination of the relevant facts and values. In making his decision, the physician may choose to give more weight to a specific moral or political principle even though he might give less weight to this principle in a different case. The physician’s decision will be in some sense “situational,” since particular features of the situation should influence the choice he makes. We believe that moral and political questions about HGLGT are like the physician’s dilemma discussed above and that they should be solved in the same fashion. In order to justify particular HGLGT decisions or social policies, one needs to understand the relevant facts and moral and political principles. Answers to issues and questions pertaining to HGLGT should result from critical, reflective, “all things considered” assessments. In this book we therefore attempt to provide readers with a balanced discussion of the questions and issues so they can reach their own decisions. But our book is not completely neutral, since we do make recommendations and take stands on controversial issues. The views we defend are based on our own critical, reflective, assessment of the facts and values. Those who disagree with us should at least be able to understand our reasoning process and some of our assumptions.

References 1. McGee G. The Perfect Baby. Lanham, MD: Rowman and Littlefield, 1997. 2. Alpern K, ed. The Ethics of Reproductive Technology. New York: Oxford University Press, 1992. 3. Rothman B. The Tentative Pregnancy. New York: Viking Penguin, 1986. 4. Robertson J. Children of Choice. Princeton, NJ: Princeton University Press, 1994. 5. Kolata G. With cloning of a sheep, the ethical ground shifts. New York Times, 24 February 1997: A1, B8. 6. Elmer-Dewitt P. Cloning: Where do we draw the line? Time Magazine, 8 November 1993: 64-70. 7. Macklin R. Embryo splitting on the slippery slope: Ethics and public policy. Ken In Eth J 1994; 4: 209-225. 8. Pence G. Who’s Afraid of Human Cloning? Lanham, MD: Rowman and Littlefield, 1998. 9. National Bioethics Advisory Commission. Cloning Human Beings: Report and Recommendations. Rockville, MD: National Bioethics Advisory Commission, 1997. 10. Berger E and Gert B. Genetic disorders and the ethical status of germ-line gene therapy. J Med Phil 1991; 16: 667-83. 11. Zimmerman B. Human germ-line therapy: The case for its development and use. J Med Phil 1991; 16: 593-612. 12. Juengst E. Can enhancement be distinguished from prevention in genetic medicine? J Med Phil 1997; 22: 125-142.

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13. Resnik D. The moral significance of the therapy/enhancement distinction in human genetics. Cam Quar Hcr Eth (in press). 14. King L. What is disease? Phil Sci 1954; 21: 193-200. 15. Pellegrino E, Thomasma D. For the Patient’s Good. New York: Oxford University Press, 1988. 16. Weinberger D. The biological basis of schizophrenia: New directions. J Clin Psych 1997; 58 (suppl 10):22-27. 17. Davis D. Genetic dilemmas and the child’s right to an open future. Has Cen Rep 1997; 27:7-15. 18. Daniels N. Just Health Care. Cambridge: Cambridge University Press, 1985. 19. Boorse C. Health as a theoretical concept. Phil Sci 1977; 44: 542-573. 20. Anderson W. Human gene therapy: Why draw a line? J Med Phil 1989; 14: 81-93. 21. Suzuki D and Knudtson P. Genethics. Cambridge, MA: Harvard University Press, 1989. 22. O’Brien S and Dean M. In search of AIDS-resistance genes. Sci Am 1997; 277: 44-53. 23. Weinberg R. How cancer arises. Sci Am 1996; 275: 62-71. 24. Chattergoon M, Boyer J and Weiner DB. Genetic immunization: A new era in vaccines and immune therapeutics. FASEB J 1997; 11:753-763. 25. Kitcher P. The Lives to Come. New York: Simon and Schuster, 1997. 26. Nelkin D and Lindee S. The DNA Mystique. New York: WH Freeman, 1995. 27. Beaudet AL. Genetics and disease. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine, 14th ed. New York: McGraw Hill, 1998, p. 377. 28. Beauchamp T and Childress J. Principles of Biomedical Ethics, 2nd ed. New York: Oxford University Press, 1994.

CHAPTER 6

Potential Benefits and Harms of Human Germline Gene Therapy The Logic of Benefit/Harm Arguments

H

aving addressed some important preliminary questions, we turn to a closer examination of benefit/harm reasoning about human germline gene therapy (HGLGT). The basic structure of a benefit/harm argument is consequentialist (or forward looking). The moral or political features of an action (or decision, technology or social policy) depend on its short and long term consequences (good vs. bad) for individuals and for society. In moral theory, the most popular form of this approach goes by the name of “utilitarianism.” In economic theory we have “cost/benefit analysis;” in the technological arena the analog is “technology assessment;” and in the health and environmental realm we have “risk assessment.” In any case, the basic structures of benefit/harm arguments are the same: The arguments attempt to determine whether the benefits of something outweigh its harms (or costs). There are three basic moral maxims related to benefit/harm reasoning in bioethics:1 1. Nonmalificence: We should avoid harming individuals. 2. Beneficence: We should benefit individuals. 3. Utility: We should maximize benefits/harms for all people in society. Most people would agree that these are sound moral (or ethical) principles, but disputes arise when we apply them to practical problems, such as HGLGT. For instance, arguments in favor of HGLGT focus on its potential benefits for individuals and society, while arguments against HGLGT dwell on its potential harms. Readers should take heed that benefit/harm arguments are fraught with many methodological and philosophical difficulties. We shall mention only two key ones here. First, it is often very difficult to reliably predict the effects of any given policy, technology, social practice, decision or action. Short term effects are sometimes predictable, but long term effects often puzzle even the most bold prognosticators: Who could have known in 1950 (or in 1970 for that matter!) that the computer would lead to a complete revolution in the way we live and work? We shall call this first problem the “predictability problem.” The second problem is that there may be some fundamental disagreements about how we should balance good or beneficial consequences against bad or harmful ones. Is economic prosperity worth the price of pollution? Are the medical benefits of a new drug worth the side effects? We find these kinds of questions discussed over and over again in matters of public policy, and there are no definite answers in sight. We shall call this second problem the “comparability problem,” since it has to do with how different individuals in society compare (or should compare) the moral/political/social worth of different outcomes. The comparability problem also introduces uncertainty into decision making, although the uncertainty is moral/ political in nature rather than scientific/empirical. Despite the difficulties inherent in benefit/harm arguments, many writers maintain that we still must carry out some sort Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus, Pamela J. Langer. ©1999 R.G. Landes Company.

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of prudential calculus in thinking about moral and political questions, since no social or political policy can afford to ignore risks, benefits, and consequences. In assessing the benefits and harms of HGLGT, we need to distinguish between actual and potential benefits and actual and potential harms. An actual benefit is something that promotes an individual’s (or a group’s) interest(s); an actual harm is something that adversely affects an individual’s (or a group’s) interest(s). Potential benefits (or opportunities) are things that might result in benefits to an individual or a group; potential harms (or risks) are things that might cause harm to an individual or a group. The modality “might” here is often assigned a probability based on evidence. Thus, it is commonplace to speak of cigarette smoking as increasing one’s risk (or probability) of developing lung cancer, or to speak of education as increasing one’s opportunity (or probability) for obtaining employment. Although there are some disputes about how to rank benefits—“Is health more important than economic prosperity?”—most people agree about the kinds of things that should be regarded as benefits. A benefit can be thought of as something that most people would have an interest in having, and harm can be thought of as the denial of something that most people have an interest in having.2 Pain is a harm in that most people have an interest in not feeling pain; the same point applies to injury, dysfunction, loss of privacy, and damage to or loss of property. The concepts of benefit and harm need not be limited to individuals, since social groups, institutions, and societies can have interests too. For example, a church benefits from donations and is harmed by fraud. In order to avoid confusions in our discussion of benefits and harms, we need to distinguish between the nouns benefit and harm and the verbs to benefit and to harm. To benefit a person is to be causally responsible for the person’s having a benefit; to harm a person is to destroy or threaten benefits to that person or to act negligently toward that person. A person acts neglectfully when they fail to fulfill a duty to benefit or prevent harm. For example, someone who refuses to donate bone marrow to a cancer patient does not harm that patient, since that person did not cause the cancer and his refusal does not constitute negligence. By refusing to donate bone marrow, the person acts selfishly, perhaps, but he does not act neglectfully, since he does not have a duty to donate bone marrow. However, a father who let his one year old daughter drown in a bathtub while he watched television would have harmed that child through his negligence, since the father has duties to prevent harm to the child. Thus, although the nouns benefit and harm can be treated as logical opposites, the verbs to benefit and to harm are not logically opposed in this way, since there are shades of gray between benefiting and harming people, such as negligence, selfishness, and callousness. Although failure to benefit a person may sometimes be just as morally unconscionable as actually harming them, sometimes the choice to refuse to benefit a person is on sound moral ground. We need to pay attention to these shades of gray in order to avoid the spurious form of argument that claims that failing to benefit a person is the same thing as harming them. Following distinctions made in the law, we should note that there is a difference between harm, hurt and offense.2 Hurt and offense both depend on the subjective, psychological states of the person who is hurt or offended: Both of these concepts are related to personal feelings or judgments of being hurt or offended in some way. The concept of harm is not subjective in this way, however, since a person can be harmed even if they do not feel harmed, hurt or offended. Since hurt and offense are highly subjective concepts, we shall omit them from our discussion of HGLGT and stick to the more objective concepts, harm and benefit. However, we do not mean to imply that psychological harms are not real, since many are, but a person can be hurt or offended without being psychologically harmed.

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In the following sections we will address four different types of possible benefits and harms that may result from HGLGT: medical benefits/harms, evolutionary benefits/harms, psychosocial benefits/harms, and economic benefits/harms. This list is not exhaustive, but we believe that it is fairly comprehensive. This list is also not unique to HGLGT, because many of the reproductive technologies that we have discussed in this book, such as embryo selection and selective abortion, can also result in similar benefits and harms. However, HGLGT does carry its own special risks and possibilities, and we will focus on these wherever appropriate.

Medical Benefits/Harms One of the main arguments for HGLGT is that it can provide us with great medical benefits by helping us to eliminate certain genetic diseases.3-5 Scientists have found a strong genetic basis for over 6000 diseases and it is likely that we will find even more genetic diseases as we learn more about human genetics.6 HGLGT or embryo selection can be viewed as the ultimate form of preventive medicine in that we can potentially prevent certain types of diseases even before a person is born. From the point of view of medicine, prevention of a disease is usually preferable to treatment after the onset of disease, since prevention usually results in no disease symptoms, including pain and suffering. By developing and using HGLGT or embryo selection (whichever method is most applicable), we could prevent Huntington’s disease, cystic fibrosis, hemophilia, diabetes, and perhaps even many forms of cancer, according to this argument. The advent of genetic medicine could represent one of the most important strides in the treatment and prevention of diseases. The medical benefit argument for HGLGT depends on the assumption that two of medicine’s primary goals are the promotion of human health and the mitigation of suffering. These goals generate a prima facie duty to prevent and treat diseases, since diseases adversely affect health and cause suffering.3 If we assume that it is rational to pursue the most effective means to one’s goals, and if HGLGT is the most effective way of eliminating or ameliorating certain medical conditions, then there is a strong case for developing and applying HGLGT. This is the basic structure of the medical benefit argument. We agree with the logic of this argument: If HGLGT is the most effective way of preventing certain genetic diseases, then we have a sound medical justification for developing and using HGLGT. But this is a big “if.” According to our earlier analysis, HGLGT is likely to be the most effective way of avoiding genetic-based disease in only a limited number of cases. Many genetic diseases can be treated by conventional means, may be treatable with somatic gene therapy (SGT) in the future, or may be avoided by employing embryo selection techniques before implantation of the embryo. There are, however, some cases where HGLGT might be the best form of disease prevention. These include cases where the prospective parents are both homozygous for a genetic defect or when the goal is to use genetic intervention to reduce the risk of developing a polygenic disease (see chapter 4 for list of potential categories of HGLGT use). Since the technology for introducing single genes by homologous recombination (HR) is just being developed now, the engineering of multiple genes is well beyond the scope of current technologies. Another futuristic use of HGLGT might be as a way of immunizing people against certain diseases, if we can isolate genes that confer immunity to pathogens such as the virus that causes AIDS (HIV virus).7 The logic that supports conventional immunizations would also seem to justify genetic ones: If our aim is disease prevention, then we should develop and use effective means to this goal. If HGLGT immunizations prove to be an effective way of preventing disease, then we have medical justification for developing this kind of therapy. By preventing diseases, HGLGT could also prevent the suffering they create. Genetic (and non-genetic) diseases cause many different types of suffering, ranging from chronic

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pain and dysfunction to paralysis and premature death. Although we recognize that the concept of suffering, like the concept of quality of life, has psychological and social components, many types of suffering have a strong biomedical basis because they result from biological dysfunction.8 For example, the suffering of a person with cystic fibrosis results from a dysfunction of the lungs and pancreas.9 The suffering of a person with male-pattern baldness is socially constructed because it is not caused by a dysfunction. A person with malepattern baldness may feel tremendous anguish and misery over being bald, but their anguish and misery results from their failure to meet social expectations and values. If people valued baldness and bald people were viewed as beautiful, then people with male-pattern baldness would not be as likely to suffer. Their suffering is socially constructed in that it rests on social values and preferences. However, people born with cystic fibrosis will suffer regardless of the social circumstances; they would still have to experience all the symptoms of this disease even if people idolized cystic fibrosis. We do not claim that a life marred by suffering is not worth living, since each person must determine whether his (or her) life is worth living. We only claim that the elimination of some kinds of suffering is a possible benefit of HGLGT. Our decisions about the usefulness of HGLGT as a form of medical treatment should be guided by a standard method of decision making in medicine, which holds that we should consider several different factors in assessing treatment options, including medical efficacy, the benefit/harm ratio, cost, and pain.10 In making these comparisons we will need to ask the following questions once the technology for HGLGT exists: How effective is HGLGT? Is there evidence that it works? What are the potential risks for different HGLGT methods? What is the ratio of expected benefits to risks of HGLGT for the patient? How expensive is HGLGT? How much pain or suffering is HGLGT likely to incur? At this point we stare the potential harms of HGLGT squarely in the face. In thinking about medical risks of HGLGT, it will be useful to think of HGLGT as like any other medical procedure, except that in this case the patients at risk are unborn children and the medical treatment involves manipulation of their genome. Since the methodology of HGLGT has not been developed, we can only predict what the various classes of harms would be to the individual who is the initial recipient of the transgene (the “proband,” “index case,” or “patient”) and to his or her first generation descendants (the “F1”). The use of the terms “proband” and “F1” are not meant to depersonalize the individuals involved; the terms are simply used for clarity in discussion. We use the term F1 to refer to a single individual in the example below. We will not consider second generation descendents (F2) since the same reasoning would apply. (See below for a discussion of potential effects of HGLGT on the human gene pool in general.) Note also that an introduced transgene only has a 50% probability of being transmitted to an F1 child if it is located on an a chromosome(assuming only one transgene is inserted in the genome). Below we outline some potential classes of genetic and medical harms of HGLGT. This is not a complete list of all possible genetic outcomes of HGLGT; rather, it is a list of only some of the scenarios related to one specific example. Our example case involves prospective parents who are homozygous for an autosomal recessive disorder, such as cystic fibrosis, and wish to have a child without the disease. As medical treatments for certain disorders improve, there will be a greater chance of a scenario such as this occurring.9 At this point we are not commenting on whether or not HGLGT should be developed to address such a rare situation; we are only outlining potential harms from a genetic and medical standpoint. In our example, the goal of the procedure is to replace one defective cystic fibrosis (CF) allele with one normal CF transgene using HGLGT. If the transgene is inserted via HR, resulting in an allele replacement, we will assume that the transgene is expressed appropriately and that its insertion has no effect on expression of nonallelic genes (we emphasize that this is

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definitely an assumption which cannot be taken for granted). Since a transgene could be introduced via nonhomologous recombination (NHR) unintentionally, we must formally include this possibility. (Since we consider the intentional use of NHR for transgene insertion to be a haphazard technique for HGLGT with an unacceptable level of risk, we will not consider these cases). For the cases below, we describe a genetic situation which would fall into each category.

Procedure and Assumptions HGLGT is used in an attempt to replace one defective CF allele so that the future child of cystic fibrosis parents (who are both homozygous for a CF defect) will not have the disease but will still be a carrier of a single defective CF allele. Being free of cystic fibrosis is a benefit. Failure to receive a defective CF allele is also considered a benefit to the F1 since it may affect her reproductive choices. Furthermore, a person could benefit from a procedure but also incur a secondary harm. Here we do not consider “nonexistence of the F1” as a harm. Case 1: Failure to benefit proband; no harm to proband; no F1 • The normal CF transgene is introduced into the proband genome, but the transgene is subsequently lost from the genome during development. • Transgene insertion or loss did not affect or change the state of the genome in this case. • Prenatal genetic tests indicated the loss of the CF transgene from the potential proband, and the parents elected not to terminate the pregnancy. • This would be a failure to benefit the proband rather than a harm. The proband develops cystic fibrosis and does not have children. Case 2: Benefit to proband; no harm to proband; failure to benefit F1; no harm to F1 • A CF transgene replaces a defective CF allele in the proband genome without any adverse effects. • The proband is a carrier for one defective CF allele and one CF transgene. • Proband does not have cystic fibrosis. • The F1 does not receive the transgene but receives the defective CF allele from the proband instead. Case 3: Benefit to proband; no harm to proband; benefit to F1; no harm to F1 • The CF transgene is introduced into the proband genome by allele replacement as in Case 2, but the transgene is transmitted to the F1. • Proband does not have cystic fibrosis • The F1 is not a carrier for the proband’s defective CF allele. • There are no adverse effects on nonallelic gene expression in the F1. Case 4: Benefit to proband; no harm to proband; failure to benefit F1; harm to F1 • The CF transgene is introduced into the proband genome by unintentional NHR. This would result in gene addition rather than allele replacement. The proband embryo is not terminated by choice or because of diagnostic error. • The two defective CF alleles remain in the proband genome, resulting in a total of three copies of the gene (two defective CF alleles, one normal CF transgene). • The functional CF transgene provides relief from cystic fibrosis in the proband. • There are no adverse effects on gene expression at nonallelic sites in the proband genome.

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• There is harm to the F1 since his particular genetic background or physical environment is such that the abnormal location of the CF transgene causes inappropriate transgene expression, and there is an adverse effect on nonallelic gene expression in the F1. • In summary, there is failure to benefit the F1 and harm to the F1, since the F1 inherits a defective CF allele as well as the CF transgene which is not functioning properly and causing secondary harm to nonallelic gene expression in the F1. Case 5: Benefit to proband; harm to proband; failure to benefit F1; no harm to F1 • The CF transgene is introduced by accidental NHR, resulting in random transgene insertion in the proband genome. • The CF transgene functions appropriately and prevents development of cystic fibrosis in the proband (benefit to proband.) • In this case, the CF transgene causes a adverse affect on nonallelic gene expression in the proband (harm to proband). • There is no harm to the F1 since the F1 does not receive the chromosome containing the randomly inserted CF transgene. • Since the F1 receives a defective CF allele but not the mislocated CF transgene, there is a failure to benefit the F1, with respect to inheritance of the CF transgene. However, the F1 also escapes potential harm from the random transgene insertion. Case 6: Benefit to proband; harm to proband; no harm to F1 Proband does not have cystic fibrosis Transgene passed to F1 Benefit to F1; no harm to F1 • The CF transgene introduced by NHR prevents development of cystic fibrosis in the proband but causes secondary harm to the proband, as in Case 5. • The F1 receives the CF transgene, but the mislocated transgene does not cause harm in the F1 because of differences in his genetic and physical environment. Case 7: Benefit to proband; harm to proband; harm to F1 • The CF transgene introduced by NHR prevents development of cystic fibrosis in the proband but causes secondary harm to the proband, as in Cases 5 and 6. • Here there is also harm to the F1, resulting from an adverse effect of the CF transgene on nonallelic gene expression in the F1. Case 8: Benefit to proband; harm to proband; no harm to F1 • The CF transgene introduced by NHR prevents development of cystic fibrosis in the proband but causes secondary harm to the proband, as in Cases 5, 6 and 7. • A harm from HGLGT is reversed in the F1. Using preimplantation genetic diagnosis and genetic embryo selection, those embryos which are not carrying the chromosome with the random transgene insertion are chosen for implantation. • This would be a reversal of the original genetic manipulation and parallels Case 5, where there is no harm to the F1 who does not receive the chromosome containing the mislocated CF transgene.

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Case 9: Benefit to proband; no harm to proband; no harm to F1 • The CF transgene is introduced into the proband by allele replacement and it is passed on to future generations without adverse effect. This would ostensibly be a successful case of HGLGT. If this same normal CF transgene construct were used every time a defective allele for this gene was replaced with HGLGT, one might question if there would be an overrepresentation of this normal CF allele in the population (as compared with the allelic frequency of this allele in the absence of genetic manipulation). In the extreme, one might be concerned about the phenomenon of genetic bottlenecking, where genetic variability at a locus is reduced when one allele of a gene is highly overrepresented in the population. However, as we argue later, the chances of a transgene copy used in HGLGT becoming the predominant one in the world is insignificant as long as there is not widespread use of HGLGT.

Risk Assessment Tied to each of the representative cases above are the potential risks, each dependent on the particulars of the HGLGT method used. We can safely state that medical risks in HGLGT would range from neutral to unacceptable. The degree of risk depends on the type of genetic manipulation undertaken, the extent of manipulation (for example if multiple genes would be altered), the timing of transgene expression, the degree to which the transgene product will interact with other cellular molecules, and many other factors discussed in previous chapters. Genetically engineered children could have severe physical deformities or diseases, or they could benefit from the procedure and suffer few side effects. HGLGT could prevent some genetic diseases, promote human health, and minimize human suffering. But it could create new genetic diseases and adversely impact human health and the quality of life of a patient or future generations. If HGLGT is likely to have these adverse impacts, then it may be wise to avoid this type of medicine, even if it has well documented medical benefits to the original recipient of a transgene. In medicine, sometimes the cure is worse than the disease, and in genetic medicine, the cure may be much worse than the disease. It is of course very difficult to estimate, at this point, the probabilities of various harmful outcomes, but we should note at this point two unique features of HGLGT that affect the potential severity of its harms and make it different from ordinary, non-genetic, medical interventions:11 1. Effect on future generations: HGLGT can potentially affect future generations beyond those who are the initial products of HGLGT. Most ordinary medical interventions affect the patient but not her children or her children’s children, and so on. 2. Systematicity: Since the genome influences development, growth, and cell regulation in individuals, HGLGT can have wide ranging, systematic, and (virtually) permanent effects, in contrast with most ordinary medical therapies. These two features make germline therapy completely unlike other types of medical therapies previously employed, and they also confound our ability to estimate potential harms. Data from animal studies, computer models, embryo manipulation, and SGT will yield information with significant import for evaluating various germline therapies. However, these data sources, if used in prospective analysis of HGLGT, are still inadequate if current standards for evaluating medical procedures are applied to the assessment of HGLGT.11 In order to proceed with widespread application of a medical procedure, we

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need to have data from exhaustive, controlled, clinical trials. This type of data on HGLGT may not be available for decades or centuries. We may find ourselves in a situation where we have the ability to perform germline manipulations but we have no reliable data for predicting the results of these manipulations. Even our best and safest germline manipulations might still be “shots in the dark.” One more possible medical harm worth mentioning addresses the accidental creation of new forms of life, such as viruses or other pathogens, that could wreak havoc on the human race or destroy other species or ecosystems. For example, new pathogens could be accidentally created as a result of altering viruses or bacteria for the purpose of delivering transgenes to human genomes. Or, latent viruses in the human genome could be activated. The new pathogens could infect the patients as well as other people in society and future generations. Many people worried about genetic accidents when recombinant DNA technology was first introduced in the early 1970s. At that time, molecular biologists and science policy analysts engaged in thoughtful, reflective discussions about the biohazards of genetic engineering.12 The prevailing consensus that emerged from these discussions was to go ahead with genetic engineering but to monitor and control research in order to manage these risks. So far, no genetic accidents have happened that could threaten the human race or other species, and all the evidence indicates that there is very little chance that any of these accidents will wander out of the laboratory to destroy human beings or other forms of life. Although we should not ignore the biohazards of genetic engineering, the fact that they might occur should not stop us from conducting responsible research and development. Given our current state of ignorance about the effects of HGLGT, one might argue that it would be unwise and immoral to subject future generations to these kinds of medical interventions for the foreseeable future. However, since our goal in this book is to attempt to anticipate possible future developments for the purposes of discussing moral and political issues and formulating policy, we need to consider the possibility that we will, at some point in time, acquire enough data about HGLGT to make clinical applications of this technology both reasonable and practical. If this is the case, it seems reasonable to conclude that HGLGT will be the best form of therapy for some diseases but not for others. HGLGT would seem to be medically recommended only when: 1. There are no other treatments available; 2. It has been proven effective or at least more effective than other therapies; 3. The potential benefits of HGLGT (for the patient) outweigh the risks; 4. HGLGT inflicts less pain or suffering than other forms of treatment. However, medical recommendations are often not the sole determining factor in establishing a treatment plan for a patient or society. This will be the case as long as we recognize that there are some things that are at least as valuable as physical health, such as human rights, justice, economic well-being or other nonmedical, social goods. For example, suppose that the most effective treatment for fetal alcohol syndrome is to mandate that all women avoid alcohol during pregnancy and to incarcerate women who violate this law. One might regard this type of treatment as unethical in that it violates women’s rights to autonomy, even though it might be medically justified.13 No physician should treat an elderly man for lung cancer if that man does not want treatment, for example, since this treatment would violate the man’s autonomy and waste money, even if the treatment might be medically indicated. The main point here is that medical considerations constitute powerful arguments in favor of treatment and prevention, but even medical judgments must be balanced against other moral and political principles and values.1

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Evolutionary Benefits/Harms In the previous section we considered the medical effects of HGLGT on individual patients and future generations. In this section we move beyond individuals directly affected by HGLGT and consider this technology’s effects on the human gene pool. Since the use of HGLGT to avoid genetic disease in offspring of two homozygous recessive parents would be extremely rare, we do not consider these cases to have any significant affect on the gene pool. What we address in this section is the use of HGLGT to reduce the risk of polygenic diseases, such as heart disease or cancer, or to enhance the genome with genes that are not a normal part of the human gene repertoire. We predict that reduction of single gene defects from the gene pool will occur mainly with genetic embryo selection and not HGLGT.

The Eugenics Argument One popular argument for various forms of eugenics, including HGLGT, is that they can be used to make the human population genetically stronger: By eliminating maladaptive, weak, and undesirable genes from the human gene pool, and by promoting adaptive, strong, and desirable ones, we can improve the genetic constitution of our species. This kind of argument—one that appeals to the genetic benefits of HGLGT—is not too far removed from arguments made by Nazis and the Social Darwinists. The Nazis believed that we should eliminate various populations in order to make a stronger, more intelligent, superior human race.14 The Social Darwinists argued that the entire human species will benefit if we follow “survival of the fittest” in social policy and refuse to help the “less fit” members of the population, i.e., the poor, the sick, the hungry, and the mentally disabled.15,16 Those who do not accept these arguments may still worry that our species will go into a genetic decline if the educationally or economically well off people in the world continue to reproduce at a lower rate than those people who are educationally or economically worse off. The argument assumes, of course, that those who are well off come from genetically “superior” stock. Arguments that attempt to show how various eugenics policies can benefit the human race can be rhetorically effective and emotionally appealing, but they are not based on fact. There is no solid evidence that we can promote the genetic well-being of our species through eugenics policies. In fact, one might argue that any attempt to control the genetic composition of our species could result in genetic/evolutionary harms, rather than genetic/evolutionary benefits.16 Furthermore, there are no data to suggest that we are in danger of genetically declining as a species.16,17 It is unlikely that there has been any significant change in the gene pool of our species over the last few hundred years, i.e., within the time period when increased food production and health care has allowed the economically worse off members of our species to reproduce and survive in greater numbers.16,17 To frame the questions on the effects of eugenics, we should ask the following questions: How important is genetic diversity to the success of human evolution? What factors could affect the gene pool? What factors will cause a significant effect on the gene pool? Although we will not directly answer these questions, we will present some views on these questions and discuss some of the factors which could affect genetic diversity. Although we will maintain our focus on HGLGT, it should be noted that many types of “interference” with reproduction could have an influence on the transmission of genes to future generations. For example, genetic counseling, availability of adoption, assisted reproductive technologies, HGLGT, preconception genetic diagnosis and oocyte selection, genetic embryo selection, prenatal diagnosis and abortion all have an effect on the transmission of normal and defective genes. Whether any of these interventions have a significant effect on the gene pool is a separate issue. Many of the arguments addressing the effect

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of HGLGT on genetic diversity can also be applied to these other technologies. The key to the effect of all of these technologies is the extent of their use in society. In the following sections, we review some of the arguments which attempt to evaluate the importance or irrelevance of genetic diversity to human evolutionary success and survival.

Importance of Genetic Diversity According to several writers, genetic diversity is considered to be critical to human evolutionary success and survival.17-19 The argument is developed as follows: If we eliminate genetic diseases or enhance various human traits, it is possible that we will decrease the genetic variability of the human population. This could happen because the human population might converge on various genetic norms relating to health and disease. If we reduce the genetic variability of the human population, then the entire human species could be doomed, since it will be more difficult for our species to adapt to changing environmental conditions with a more genetically homogeneous population. The genes we eliminate today, even those that cause diseases, could be useful tomorrow.20,21 For example, the gene that causes sickle cell anemia also protects people against malaria. In support of the argument for retaining genetic diversity, some writers employ the “lessons” from reductions in agricultural diversity through human manipulation.17 For example, through cultivation and controlled breeding, humans reduced the variability of the gene pool in corn (Zea mays). Human beings have produced different varieties of Zea mays over time by selecting different characteristics, such as taste and productivity. Although maize grows at a fast rate and produces a great deal of food for human consumption, the lack of genetic diversity in the maize gene pool has had some undesirable consequences: Maize is susceptible to many different diseases. Corn blights can spread at a rate of eighty kilometers a day and devastate harvests. For all of its productivity, the common corn plant’s Achilles’ heel could be seen as its genetic homogeneity.17 Some writers argue that these concerns about genetic diversity demonstrate that we should never tamper with the human gene pool, no matter what the possible benefits may be; the risks are simply too great.18 However, even when evaluating the effect of genetic manipulation on maize, it is important to balance the benefits (yield, quality of product) with the potential harms (reduction of genetic diversity, increased susceptibility to disease). Maize production has been significantly increased in spite of its increase in genetic vulnerability. Human ingenuity has in some senses overridden the negative effects of genetic vulnerability since additional human interventions, such as planting different genotypes of maize in adjacent fields, have diminished the spread of infectious disease in maize production.

Irrelevance of Genetic Diversity The evolutionary success of a population (or species) depends on its genetic diversity when the population must rely on its genetic diversity in order to adapt to a changing environment. For most biological populations this assumption is correct. But humans have largely freed themselves from environmental demands and pressures during the last few thousand years. Competition, predation, climate, resources, and even disease do not currently pose a significant threat to the survival of Homo sapiens. One could argue that human survival depends far more on human ingenuity and cultural adaptation than on genetic diversity.22 Even if the diversity of our gene pool decreases as a result of genetic interventions, it is not at all likely that this change will lead to the extinction of our species. Indeed, the perennial threat of nuclear war indicates that we pose the greatest threat to our own survival. Although the Cold War has ended and the nuclear powers are coexisting peacefully, as long as we have nuclear weapons and nuclear technology, the path to nuclear war will remain open. No amount of genetic diversity could save us from extinction if a global, nuclear war erupts.

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Although the human race is not likely to be eradicated by predators, pathogens such as parasites, viruses and bacteria still pose a serious threat. The nightmare scenario worth considering might be the emergence of some new pathogens, e.g. virus strains, that threaten to destroy the human race.23,24 The viruses could infect various parts of the human body, including the germline. We might also suppose that they could be transmitted by casual contact and would result in a pandemic. A blight or plague could sweep through the human population the way that blights sweep through corn fields. Given this horrible scenario, would genetic variability help our race survive? Perhaps. Some people might be naturally resistant to these hypothetical, devastating diseases, and the survival of our species might depend on their genes. For example, it is now known that there are allelic differences in the human population that determine resistance to viral infections such as HIV. However, it is also possible that our survival would depend on our ability to respond to pandemics with new medicine and medical technologies. Therefore, if we would use science and technology to ward off these science fiction diseases, it would not be wise to ban HGLGT research, developments, or applications, since we might need to take advantage of HGLGT science and technology. Faced with the destruction of the human race, we might have no alternative but to explore all possible ways of saving our species, including somatic and germline gene therapy. We might use HGLGT to enhance the human immune system to create a new generation of people who can resist deadly diseases, for instance. We admit that this scenario seems like science fiction but it does have some basis in science fact. New pathogens have emerged throughout the history of our species and we often are not prepared for them when they do. Consider, for example, the emergence of the HIV virus during the 1980s and the recent outbreaks of the Ebola virus. Indeed, evolutionary biologists have begun to understand how the immune system and various pathogens have evolved in response to each other over millions of years and how these two sides are engaged in a perennial arms race.25 There is also mounting evidence that new pathogens have caused the extinction of species that failed to evolve in response to their threat. On the other hand, research also indicates that diseases tend to become more benign through evolution, since the survival of a disease tends to depend on the survival of its hosts. However, there are factors that can break the link between disease and host survival, such as diseases that occupy different species and biological warfare.

Effect of HGLGT on Genetic Diversity The question of the importance of genetic diversity to our survival as a species is fraught with speculation and uncertainty. However, if we assume that genetic diversity is an important factor, even for survival of the human species, we now address these questions: How will the manipulation of genes with HGLGT affect human genetic diversity? Will HGLGT decrease diversity, increase diversity or have no effect on the human gene pool? Will the use of HGLGT adversely affect the gene pool by significantly decreasing the representation of an allele? First, as stated earlier, we do not predict that HGLGT will be the primary way that the frequency of an allele will be reduced in a population. Since other interventions in reproduction are likely to be more safe and effective than HGLGT, they are likely to play a larger role in preventing inheritance of disease-causing genes. However, HGLGT may be used: 1. To replace several genes with transgenes (multigene replacement); 2. To replace a gene with an allele discovered to or engineered to confer specific advantages on an individual (such as decreased chance of developing cancer; we refer to this as a “superallele”); or

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3. To add a foreign gene, e.g. originating from another organism, to a human genome (we designate this foreign gene a “xenogene”). An example of this use might be in the introduction of a DNA vaccine type gene. For these cases it is valid to consider the potential effect of such actions on the gene pool. Let us contrive an example where a superallele is designed to lower the risk of developing cancer if the superallele replaces a “disease-contributing allele.” Will the replacement of the disease-contributing allele with the superallele significantly change the frequency of the disease-contributing allele in the gene pool? Note that “an effect” is substantially different from “a significant effect.” Given the limited extent with which such an HGLGT procedure would be used, the deletion of a few disease-contributing genes from a relatively small number of people will not significantly affect the total number of these genes in the population. The number of heterozygous carriers of these disease-contributing genes will far exceed the number of superalleles introduced into the gene pool. Furthermore, the chance that the superallele would be maintained in the gene pool over several generations is small (see next section). Therefore, our view is that the primary effects of HGLGT will be on the individual who is the product of the genetic manipulation and possibly a few descendants. On the evolutionary scale that we are dealing with, the repertoire of genes in the human gene pool will not be significantly affected.26 Genetic intervention would reduce diversity only if intervention is widespread or global and if a society imposes highly restrictive, genetic norms on the gene pool. On the other hand, one might argue that the introduction of new genes into the human population could increase human genetic diversity. If an artificially engineered superallele or even a xenogene is introduced into a human genome, then human genetic diversity has technically increased. However, this may be a temporary state, since the person may not transmit that transgene to children either because the gene was not passed on to a child or the person did not have children. Based on Mendelian patterns of inheritance, the chance of a single autosomal transgene surviving four generations, if there is one offspring in each generation, is 1/16. Of course if there are more offspring, this will affect the probability that the transgene will be retained in the gene pool. Even if the engineered superallele or xenogene is retained in the human gene pool for several generations, it is unlikely to significantly affect the composition of the gene pool.26 However, under some special circumstances, the consequence of introducing a new transgene to the human gene pool could have significant and profound effects. Five scenarios worth considering are: 1. Controlled interbreeding. People with the new transgene interbreed by personal choice or state mandate in order to increase the number of people with that gene. 2. Cloning of people with the new transgene. Embryos or adults with new transgene could be cloned for eugenic or other purposes. 3. Accidental interbreeding. People with the new transgene interbreed due to historical, economic, or political circumstances. For example, people with the new gene might all live in the same geographic region and only breed with people within that region. 4. Controlled, genetic reengineering. Successive generations could take steps to intentionally reengineer the gene in each generation. 5. Accidental genetic reengineering. Genetic engineering accidents or biological weapons could reengineer the gene in each generation if, for example, a new gene could be carried by a genetically engineered virus that becomes widespread in the human population. If the virus delivers the gene to members of the population, the gene could eventually become widespread.

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We will not comment on the likelihood of these different scenarios, since it is not possible to reliably predict their occurrence. However, we will note that all of the scenarios, with the exception of scenario #5, presuppose the widespread use of HGLGT. If HGLGT is not widely used, its effects on the human gene pool are likely to be insignificant. Therefore, it is unlikely that HGLGT or other forms of genetic intervention would significantly affect the gene pool, if used on a small scale. For the foreseeable future HGLGT will be too unsafe, ineffective, and expensive to use on a large scale. However, it is worth considering science fiction worlds where much is known about polygenic traits and when it would be possible to alter not one, but many, polygenic traits simultaneously. Under these futuristic circumstances, HGLGT might be used for nonmedical eugenics. By picking designer genes, people might build a portfolio of hair color, eye color, skin color, body size and shape, talents and abilities in the same way that they currently design and select different kinds of clothing, automobiles, or music. Some writers propose that if we allow people to express their values and tastes in reproductive choices, then HGLGT could increase genetic variability.21 The hypothesis is that the gene pool could become so diversified that genetically distinct castes, races, or subspecies will emerge from the species Homo sapiens.27 Before this could happen, HGLGT use would have to be routine and/or governmentally dictated, a scenario that we deeply hope would never occur. The possibility of social or governmental intervention on a large scale raises the question of who would decide the design of the “perfect” human form.28-30 On the other hand, one might argue that the widespread use of HGLGT would lead to a decrease in genetic diversity. This argument would be developed as follows: A decrease in genetic diversity could occur in two different ways. If we consider parental control of HGLGT, one might argue that there will be many traits that all people will find desirable or useful, especially if other members of the population have these traits. Most people will avoid genetic diseases and many people will want to enhance some of the same physical and psychological traits. Even if no government or other authority attempts to impose genetic standards, the proposal is that a standardized human form will emerge as a result of the cumulative choices of many people over time. Social pressures and expectations would compel people to make their offspring conform to social standards. Though individuals may resist this social inertia for a time, most people will eventually conform. If we consider state control of HGLGT, then governments could impose genetic norms on society in order to promote health, safety, or other goals. Governments could regulate the production of people in the same way that they regulate the production of automobiles or houses. In sum, we believe that HGLGT will not significantly affect the human gene pool if it is used on a small scale. We will return to issues involving the more significant impacts of widespread use of HGLGT in chapters 8 and 9.

Psychosocial Benefits/Harms New reproductive technologies, including HGLGT, could lead to a variety of psychosocial benefits or harms. As in our previous discussions, we will attempt to focus on the unique problems raised by HGLGT while we recognize that other technologies, such as embryo selection, raise similar issues and concerns.

Benefits/Harms to Children There are a variety of ways that HGLGT could confer psychosocial benefits on children who are created using this technology (the probands). If HGLGT is used for medical purposes, then children could benefit from having a life that is not marred by disease and suffering. If health is a key component of human happiness, then HGLGT could promote happiness in the proband by promoting health. If HGLGT is used for nonmedical purposes,

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then it could confer a variety of benefits on the proband. Although we can only speculate about these possible benefits, they might include enhanced physical strength, vigor, height, intelligence, or musical ability. One might argue that people who enjoy these benefits from the nonmedical uses of HGLGT would also have enhanced “happiness” or self-esteem.31 (All of these nonmedical benefits depend on our ability to successfully manipulate polygenic traits, a possibility that is still in the realm of science fiction.) On the other hand, HGLGT, whether used for therapy or enhancement, could cause a variety of psychosocial harms in the proband as well. Some of these include: 1. HGLGT mistakes that adversely affect the emotional stability/balance, cognitive function, intelligence, or other psychological characteristics of the proband. The possibility of making a Frankenstein monster looms large.32 2. Feelings of inferiority resulting from social stigmas against genetically engineered children; 3. Difficulty in social integration as a result of being “different” in some way; 4. Overly demanding parental (or social) expectations to have a special talent or ability, to pursue a specific career, to be healthy, to be “perfect;”33 5. Having a future that has been “closed” as a result of parental decision, e.g. having a genetic “destiny” to be a Mozart or a great athlete.33,34 It should be mentioned that most of these psychosocial harms can occur with or without the development and use of HGLGT. For example, children that are produced via in vitro fertilization also face possible feelings of inferiority and possible difficulties in social integration.35 Virtually all children face the possible burden of meeting parental or social expectations or having a future that is “closed” as a result of parental decisions.33 Children born by normal means also face the possibility of suffering from birth defects that harm cognitive or emotive functions. Although none of these concerns are unique to HGLGT, HGLGT could produce more dramatic and devastating psychosocial harms than existing reproductive technologies. For example, although human beings have struggled with imposing too many expectations on children for years, we have not been able to “design” children in the same way we can design cars. HGLGT would amplify and exacerbate parental and social expectations. While a small percentage of children are born with mental or emotional disabilities, HGLGT could increase the odds of giving birth to children with cognitive or emotive dysfunction. This could occur from an HGLGT mistake or an intended genetic change. For example, the alteration of a hypothetical “intelligence-contributing gene” is likely to cause different effects in individuals with different genetic backgrounds, environmental exposure and life experiences. Before closing this discussion, we will also note that HGLGT could harm children that are not the products of HGLGT. People who do not benefit from medical or nonmedical uses of HGLGT might be viewed as “less than perfect” or as “damaged goods.” Thus, HGLGT could create new kinds of discrimination and bias.36 We recognize the importance of this problem, but we also note that it is not unique to HGLGT; embryo selection and selective abortion raise similar concerns. In an era when many genetic diseases will be preventable, children that are born with genetic diseases may be viewed as “mistakes” or “damaged goods.” But is this a good argument for not developing a new technology? When the polio vaccine was first developed, polio victims were viewed as “tragedies” or “mistakes.” It would be absurd to argue that we should not have developed this vaccine out of concern for those who have not been or would not be vaccinated, since the vaccine conferred significant benefits on its recipients. One might take the same attitude toward the development of reproductive technologies that can prevent diseases.33 But what about the nonmedical uses of HGLGT? Wouldn’t this latter argument provide us with good reasons not to use HGLGT for enhancement purposes? Once again, let us

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consider an example. Education can be a form of non-genetic enhancement: Parents who can afford private schools, piano lessons, tutors, computers, laboratory equipment, and exotic vacations can produce children with abnormally high intelligence or other abilities. Should we not allow rich parents to benefit their children because we are afraid that these benefits will incur psychosocial harms on poor children? Should we not allow rich children to have these benefits? One might argue that if we allow parents to purchase non-genetic enhancements for their children, we should also permit genetic enhancements, assuming that these enhancements are safe and effective.33 (However, the rich vs. poor problem also raises concerns about justice that we will consider in chapter 8).

Benefits/Harms to Society One might argue that HGLGT can benefit society in many ways. If the use of HGLGT can promote human health and “happiness”, then society benefits from a healthier, “happier” population. If we can successfully use HGLGT for nonmedical purposes, such as enhancement, then society may benefit from having people who have enhanced physical abilities or special talents.31 Don’t we all benefit from having more Einsteins, Beethovens, Mother Teresas, and Michael Jordans? We grant that HGLGT may one day be useful as a form of genetic medicine and that it may benefit society by promoting human health. However, we remain skeptical of the social benefits of HGLGT’s nonmedical uses. Although society might benefit from genetic enhancements, these possible benefits will remain in the realm of science fiction for years to come, since they depend on our ability to manipulate traits that have many different genetic and environmental causes, such as intelligence or athletic ability. One might argue that HGLGT could harm society in many different ways. Some possible social harms include:36-41 1. The exacerbation of existing racial and ethnic prejudices and biases; 2. Increased genetic discrimination; 3. Loss of genetic privacy; 4. A widening rich/poor gap, medical/genetic injustice; 5. A loss of phenotypic diversity resulting from a loss of genetic diversity; 6. An increase in human perfection as an ideal; 7. Genetic warfare Once again, we stress that most of these consequences could occur with or without the development and use of HGLGT. For example, writers have raised similar concerns about the Human Genome Project, genetic testing, embryo selection and recombinant DNA technology.17,20,21,32,38-40 Most of these disturbing social consequences will occur as a result of current and anticipated developments in reproductive and genetic technologies. Although HGLGT could exacerbate many of these possible problems, it is very difficult to estimate HGLGT’s possible contribution to these undesirable outcomes. Instead of exploring these possible consequences here, we will discuss some of them in more detail in chapters 8 and 9.

Economic Benefits/Costs Some writers argue that HGLGT will provide society with important economic benefits,3,5 while others have argued that HGLGT could be a very costly and economically inefficient medical procedure.41 To understand these economic questions, it will be useful to provide an overview of these costs and benefits. Possible economic benefits of HGLGT include: EB 1. Reduced costs of genetic and other diseases; EB 2. Increased human productivity and economic output. Concerning EB 1, as we mentioned earlier, HGLGT could be the ultimate form of preventive medicine for many diseases, and from an economic viewpoint, an ounce of preven-

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tion is worth a pound a cure. Thus HGLGT could help save a great deal of money that we currently spend on treating diseases with a genetic component, possibly even cancer. It might also help us save money if it plays a role in immunizing people against specific pathogens. Concerning EB 2, this benefit could occur if HGLGT is used for enhancement purposes: If we can design people who are faster, stronger, smarter, and more durable, then they should also be economically efficient workers. These possible economic benefits need to be balanced against the possible costs of HGLGT, which may include: EC 1. Costs of research and development; EC 2. Costs of clinical applications; EC 3. Costs of mistakes and other unintended costs. Concerning EC 1, it is likely to take many years and billions of dollars to develop HGLGT to the point where it would be safe and effective enough for clinical applications. For comparison, it usually takes a decade of research and several hundred million dollars to bring a new drug from the laboratory to the clinic.42 Since HGLGT is perhaps the most complex medical technology ever conceived, its costs would far exceed the costs of developing a new drug by an order of magnitude or more. Turning to EC 2, HGLGT is likely to be very expensive when (or if) it becomes available. For the foreseeable future, only the very rich would be able to afford HGLGT. But costs could go down as the technology develops. Computers can serve as a good example here. In the 1950s, the idea of an affordable home computer was unthinkable. As this technology has advanced, the costs of computers have gone down tremendously and they continue to fall, while the benefits have skyrocketed with no end in sight. Scientific and technological advances, industrial developments and applications, and the free-market economy have made computers cost effective in only a few decades.43 A similar causal network could reduce the costs of HGLGT. It is not unrealistic to think that HGLGT could eventually be no more expensive than in vitro fertilization or other reproductive technologies. However, HGLGT may not follow the same path as computing technology and we acknowledge that it is very difficult to make plausible predictions about technological developments and economic changes. Many goods and services do not become more economically efficient over time.43 It could be the case that HGLGT becomes cost effective, but then again, it might not become cost effective. Or it could be the case that relatively simple, lower risk genetic interventions, e.g. homologous gene replacement, become cost effective, while other, more complex and risky interventions, e.g. the introduction of a superallele, do not become cost effective. Finally, the costs associated with EC 3 must also be considered. For example, suppose clinical applications of HGLGT result in genetic and developmental mistakes that create people with mental, physical, or emotional dysfunction. The economic costs of taking care of people who are born with these “man-made” genetic diseases could outweigh any economic benefits we obtain from preventing genetic diseases. Like other new technologies, HGLGT might have some other unintended costs that are difficult to anticipate at this time. For example, the respirator is an important medical technology; it saves many lives each year. However, it also has generated some unintended costs: We now spend millions of dollars a year keeping people alive who would have died by natural causes before the invention of the respirator. Although it is difficult to anticipate some of the unintended costs of HGLGT, there are two added costs worth considering. First, HGLGT could be used to increase the human lifespan. The average life span might one day be over a hundred years as a result of tinkering with genes relating to aging and cell repair. What would be the economic consequences of a longer lifespan? The answer to this question depends, in part, on the quality of that longer lifespan—will people with extended lifespans have extra years of life at a high or low functional level? Obviously, these questions

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cannot be answered here, but we can at least speculate that a longer lifespan would probably result in a larger, more elderly population. In the last two decades, the United States has observed some of the economic consequences of its own aging population, which include increased health care and social security costs.44 The use of genetic engineering to extend the human lifespan could mean that we would spend even more money to care for what could become a very old population. Given the potentially dramatic effects of extending the human lifespan, even the most ardent defender of HGLGT should endorse prudence and caution when it comes to creating genetic Methuselahs. Second, in order to promote social justice, we may need to make HGLGT available to all people, not to just those who people can afford it.45 Many other medical technologies have created these additional costs. For example, in order to promote justice, we have programs such as Medicaid and Medicare that make hearing aids available to people who otherwise might not be able to afford them. The costs of “genetic welfare” might skyrocket as we use technology to battle standard diseases or enhance humans. These questions about justice take us beyond the scope of this chapter, but we will return to them chapter 8. In concluding this section, we recognize that HGLGT will generate a variety of economic benefits and costs if it is developed and used. However, at this point in time we can do little more than speculate about HGLGT’s economic impact, since we still do not know how this technology might be developed or applied.

Conclusion: Optimism, Pessimism, or Prudence? The possible benefits and harms discussed in this chapter range from the mundane to the bizarre; their scope and profundity are limited only by our imagination. At the outset of our discussion we drew attention to two problems with benefit/harm arguments, the predictability problem and the comparability problem. Our discussion in this chapter indicates that any assessment of the benefits/harms of HGLGT suffers from both of these difficulties. We cannot predict the short term or long term consequences of tampering with the human germline with any confidence or reliability at this point in time. However, it is worth noting that some benefit/harm assessments would seem to be more tractable than others. It may be possible to develop a better understanding of medical benefits and harms over time as we learn more from germline experiments in animals, in vitro fertilization in humans, somatic gene therapy experiments in humans, human genetics, molecular biology, and even germline gene therapy trials in humans. Since it is possible to increase our understanding of the medical benefits and harms of other, non-genetic therapies through controlled experiments and scientific procedures, it should also be possible to increase our knowledge of the medical benefits and harms of HGLGT. The only epistemological difference between HGLGT and other medical procedures is that it is more complex and difficult to understand. But there are no insurmountable barriers to improving our predictions about HGLGT’s medical effects. Thus, some parts of the predictability problem could be solved as time goes on. On the other hand, we do not have the same hope for reduction of uncertainty when it comes to HGLGT’s biological, psychosocial, or economic benefits and harms. These questions involve so many different, interacting causal factors, and so many different values and assumptions, that carefully controlled studies are out of the question. We might be able to build a database of relevant information, but we will still face many uncertainties. But this should come as no surprise to those familiar with the history of science and technology, since we are rarely able to reliably predict the psychosocial, economic, or ecological effects of even the simplest technologies or scientific discoveries.43 Even if we make some headway in solving the predictability problem, we will still need to come to terms with the comparability problem, since HGLGT creates possible conflicts

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between different social, political, and moral values. We have already seen how we need to compare different possible benefits and harms in assessing HGLGT. In the next three chapters, we will see how HGLGT forces us to compare other competing values, such as social utility, parental rights, the rights of unborn children, justice, and respect for the natural order. Given all the scientific, technical, social, moral, economic, and political uncertainties surrounding HGLGT, at this point in time we can do little more than speculate, discuss scenarios, or offer caveats. One might conclude that it is pointless to try to assess HGLGT’s possible harms and benefits. It is better to wait and say nothing at this point in time than to be proven wrong in a few years. The problem with this suggestion is that we cannot afford to wait. In the next few years society will face a number of different public policy decisions relating to HGLGT. Some of these include: 1. HGLGT research: Will we allow people to conduct research on HGLGT? Who will conduct the research? Will research be restricted or banned, widely distributed or censored or classified? Will it be publicly or privately funded? 2. HGLGT applications/development: Will we allow people to perform HGLGT? Who will be the test subjects? How will clinical trials be approved or regulated? What kinds of HGLGT applications will be approved, if any? Who will pay for HGLGT? Who will monitor HGLGT? Will HGLGT technologies be patentable? 3. HGLGT consequences/liability: Who will be liable if there is an HGLGT mistake? For what period of time will the person, group of people, government, or company be liable? Until birth? Until puberty? Until the HGLGT-engineered person dies? Until the transgene is no longer transmitted to descendants? These are decisions that we cannot avoid. We can no longer pretend that human genetic engineering is imaginative science fiction, since it is fast becoming science fact. We cannot refuse to make a decision or formulate a policy, since such deliberate inaction would be irresponsible and would probably be worse than plodding ahead in the face of uncertainty. In thinking about some of these policy decisions, there are three different attitudes one may take toward HGLGT in light of its possible benefits and harms. They are:

Pessimism HGLGT creates new moral, political, technological, and scientific possibilities that will lead us down a “slippery slope” toward our worst science fiction nightmares.18,46,47 We should place a moratorium on HGLGT research and development in order to avoid doom and despair.

Optimism HGLGT will bring many important benefits and will not create the nightmares envisioned by pessimists. We should continue to develop HGLGT and use it when it becomes safe and effective.5,31

Prudence HGLGT will create benefits and harms, dreams and nightmares. As HGLGT develops, we should take a cautious, moderate path guided by common sense and a healthy respect for the power and danger of science and technology.48-50 We accept the prudent view, since the optimistic view underestimates the important harms that can be created by HGLGT, and the pessimistic view underestimates the important benefits. The pessimist fears scientific and technical progress and the optimist wel-

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comes it. The prudent person, however, recognizes that progress has both good and bad effects as well as advantages and disadvantages. Prudence instructs us to proceed with optimism tempered by caution and circumspection. In our last chapter we will return to these themes when we consider specific policy issues in more detail.

References 1. Beauchamp T and Childress J. Principles of Biomedical Ethics, 2nd ed. New York: Oxford University Press, 1994. 2. Feinberg J. Social Philosophy. Englewood Cliffs, NJ: Prentice-Hall, 1973. 3. Munson R and Davis L. Germline gene therapy and the medical imperative. Ken Inst Eth J 1992; 2: 137-158. 4. Berger E and Gert B. Genetic disorders and the ethical status of germline therapy. J Med Phil 1991; 16: 667-683. 5. Zimmerman B. Human germline therapy: The case for its development and use. J Med Phil 1991; 16: 59-612. 6. McKusick V, Francomano C, Antonarakis S, Pearson P. Mendelian Inheritance in Man: A Catalog of Human Genes and Genetics Disorders,11th ed. Baltimore, MD: Johns Hopkins University Press, 1994. 7. Yang A, Bai X, Huang X, et al. Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection. Proc Natl Acad Sci 1997; 94:11567-11572. 8. Cassell E. Recognizing suffering. Has Cen Rep 1991; 21(3): 24-31. 9. Sawyer, S. Reproductive health in young people with cystic fibrosis. Curr Opin Ped 1995; 7, 376-380. 10. Albert D, Munson R, Resnik M. Reasoning in Medicine. Baltimore: Johns Hopkins University Press, 1988. 11. Greenwell P. Germline gene therapy — changing future generations. Int J Biosci Law 1997; 1: 217-226. 12. Jackson D and Stich S, eds. The Recombinant DNA Debate. Englewood Cliffs, NJ: Prentice Hall, 1979. 13. Annas G. Pregnant women as fetal containers. Has Cen Rep 1986; 16(6): 16-17. 14. Aly G, Chroust P, Pross C. Cleansing the Fatherland. Cooper B, trans. Baltimore, MD: Johns Hopkins University Press, 1994. 15. Paul D. Controlling Human Heredity. Atlantic Highlands, NJ: Humanities Press International, 1995. 16. Kevles D. In the Name of Eugenics. New York: Alfred A. Knopf, 1985. 17. Suzuki D and Knutdson P. Genethics. Cambridge, MA: Harvard University Press, 1989. 18. Rifkin J. Algeny. New York: Viking Press, 1983. 19. Hirschhorn K. Practical and ethical problems in human genetics. Bir De 1972; 3: 22-28. 20. Kitcher P. The Lives to Come. New York: Simon and Schuster, 1997. 21. Holtug N. Altering humans—the case for and against human gene therapy. Cam Quart Hcr Eth 1997; 6: 157-174. 22. Leslie J. The End of the World. London: Routledge, 1996. 23. Preston R. The Hot Zone. New York: Doubleday, 1996. 24. Garrett L. The Coming Plague. New York: Penguin, 1995. 25. DuPasquier L. The evolution of the immune system. In: Paul W, ed. Fundamental Immunology, 3rd ed. New York: Raven Press, 1993. 26. Davis B. Germline gene therapy: Evolutionary and moral considerations. Hum Gene Ther 1992; 3:361-363. 27. Resnik D. Debunking the slippery slope argument against human germline gene therapy. J Med Phil 1994; 19: 23-40. 28. Murphy T and Lappe M, eds. Justice and the Human Genome Project. Berkeley, CA: University of California, 1994. 29. Boone C. Bad axioms in genetic engineering. Has Cen Rep 1988; 18(4): 9-13.

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30. Robertson J. The question of human cloning. Has Cen Rep 1994; 24(2): 6-14. 31. Glover J. What Sort of People Should There Be? New York: Penguin Books, 1984. 32. Gaylin W. The Frankenstein factor. New Eng J Med 1977; 297(12):665-666. 33. McGee G. The Perfect Baby. Lanham, MD: Rowman and Littlefield, 1997. 34. Davis D. Genetic dilemmas and the child’s right to an open future. Has Cen Rep 1997; 27(2): 7-15. 35. Morin N et al. Congenital malformations and psychosocial development in children conceived by in vitro fertilization. J Ped 1989; 115: 222-227. 36. Kass L. Toward a More Natural Science. New York: The Free Press, 1985. 37. Kevles D. Social and ethical issues in the Human Genome Project. Nat For 1993; 77 (2): 18-21. 38. Wilkie T. Perilous Knowledge. London: Faber and Faber, 1993. 39. Kellert S. The Value of Life. Washington, DC: Island Press, 1996. 40. Santos M. Genetic and Man’s Future. Springfield, IL: Charles Thomas, 1981. 41. Juengst E. Germline gene therapy: back to basics. J Med Phil 1991; 16: 587-582. 42. Bowie N. University-Business Partnerships. Lanham, MD: Rowman and Littlefield. 43. Volti R. Society and Technological Change, 3rd ed. New York: St. Martin’s, 1995. 44. Posner R. Aging and Old Age. Chicago: University of Chicago Press, 1995. 45. Miringoff M. The Social Costs of Genetic Welfare. New Brunswick, NJ: Rutgers University Press, 1995. 46. Science for the People. Biological, social, and political issues in genetic engineering. In: Jackson D and Stich S, eds. The Recombinant DNA debate. Englewood Cliffs, New Jersey: Prentice Hall, 1979. 47. Council for Responsible Genetics. Position paper on germ line manipulation. Hum Gen Ther 1993; 4(35-37). 48. Ledeberg J. Gene splicing: Will fear rob us of its benefits? In: Jackson D and Stich, S, eds. The Recombinant DNA debate. Englewood Cliffs, New Jersey: Prentice Hall, 1979. 49. Cohen C. When may research be stopped? In: Jackson D and Stich S, eds. The Recombinant DNA debate. Englewood Cliffs, New Jersey: Prentice Hall, 1979. 50. Macklin R. Moral issues in human genetics: Counseling or control? Dialogue 1977; 16(3): 386-96.

CHAPTER 7

Human Germline Gene Therapy, Rights and Responsibilities I

n the last chapter we discussed the potential benefits and harms of human germline gene therapy (HGLGT) to individuals and society. In this chapter we will examine the issue from the perspective of rights and responsibilities. The rights and responsibilities we will consider here include the rights and responsibilities of parents, potential children and future generations.

What Are Rights and Responsibilities? Responsibilities Before exploring the different rights and responsibilities pertaining to HGLGT, it will be useful to briefly review the concepts of moral or political rights and responsibilities so that we may better understand how to think about these issues. We shall discuss the concept of responsibilities first, since this concept requires less exposition. People often use the term “moral responsibility” as a synonym for “moral duty” or “moral obligation.” Thus, the sentence: “Joe has a responsibility to be a good father,” is roughly equivalent to: “Joe has a duty to be a good father,” or “Joe has an obligation to be a good father.”1 A responsible person, in this sense, is someone who generally fulfills his or her obligations or responsibilities. A list of moral responsibilities would thus resemble a list of moral obligations or moral duties; therefore we use these terms interchangeably. In the previous chapter we discussed three principles relating to benefits and harms, i.e., beneficence, non-maleficence, and utility. Three other moral principles worth mentioning at this point include:1 1. Autonomy: We should allow rational (or competent) individuals to make their own decisions and act on them. 2. Justice: We should be fair and just in our actions and policies. 3. Privacy: We should protect personal privacy and confidentiality. In this chapter we will focus on the principle of autonomy insofar as it provides a basis for individual rights. We will discuss the confidentiality of genetic information as well, and we will see how other principles may be invoked to restrict autonomy. In the next chapter we will address the principle of justice in more detail. We can use the concept of responsibility discussed here to make sense of the widely accepted claim that people should be responsible for the consequences of their actions. What this claim amounts to is simply the following: 1. Our actions and policies have consequences that we can often anticipate; and 2. We have moral duties relating to these consequences, i.e., we should try to maximize good outcomes and minimize bad ones so long as we do not violate other accepted moral principles. Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus, Pamela J. Langer. ©1999 R.G. Landes Company.

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A person who fails to take responsibility, in this sense, is someone who fails to understand the moral consequences of her actions or fails to take appropriate steps in light of those consequences. We should also distinguish, at this point, between two types of responsibilities: basic responsibilities and acquired responsibilities. Our basic moral and political responsibilities are those duties or obligations shared by all rational agents. These include the obligations covered by the principles of beneficence, nonmalificence, utility, autonomy, and justice. However, we can also acquire additional responsibilities as a result of marriage, reproduction, employment, social roles, and personal relationships.1 For instance, a father’s obligation to care for his son is an acquired obligation; he would not have this obligation if he had not become a father to the boy.

Rights The concept of a moral right is used (and overused) in many different contexts and to many different ends. For the purposes of this book, we can think of a right as a claim that a person can make against other people or social institutions.2,3 Rights generate various obligations among people and social institutions. For instance, if I have a right to life, then other people have a duty not to kill me. We can distinguish between different kinds of rights or claims, depending on the obligations and values in question: A legal right is a legal claim that generates legal obligations; a moral right is a moral claim that generates moral obligations; and a political right is a political claim that generates political obligations. For the purposes of this book we will discuss only moral or political rights, since we are concerned with moral and political issues in HGLGT, not with legal ones. However, we should note that arguments for legal rights are usually based on moral or political considerations, and moral and political rights often have analogs in the legal realm.2 The main function of rights is to protect individuals or promote their welfare.2,3 Some writers assert that rights can be extended beyond individuals to encompass species, corporations, and ecosystems, but for the purposes of this book we will not explore these controversial views, but address only the rights of individuals. Individuals may have rights even if they do not have moral responsibilities; for instance, one might assert that an infant has a right not to be killed even though the infant has no duties or responsibilities. Individuals that have both rights and responsibilities are sometimes called moral agents; individuals that have rights but no moral responsibilities are sometimes called moral patients.4,5 It is generally assumed that in order to be a moral agent, an individual must be capable of: 1. Following moral rules; 2. Understanding and responding to moral arguments; and 3. Making competent decisions.1 Although most writers agree that infants and children should have the status of moral patients, there is considerable disagreement about extending moral patienthood (and moral rights) to other types of individuals, such as animals, human fetuses, and unborn generations.5 Questions about the rights of future generations and fetuses loom large in moral controversies over HGLGT. Although some philosophers argue that human rights are natural and conceptually basic,3 most philosophers argue that rights can be derived from other moral concepts and principles.2 For the purposes of this work, we will assume that moral and political rights can be founded on the concept of individual worth (or value). The idea here is quite simple: Individuals have an inherent moral worth that should not be sacrificed or traded for some greater good.2 Since individuals have inherent moral worth, we have obligations to respect individuals, and these obligations give rise to moral and political rights.2 Moreover, all individuals are equally worthy, in some moral sense, even if they differ in their talents, wealth,

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physical attributes, and so on. Since individuals have equal moral worth, the rights possessed by individuals are held equally.2 A president’s right to life has no more (or less) moral weight than a pauper’s right to life. The idea of dignity or moral worth also provides a foundation for the principle of autonomy we mentioned earlier. If we take an individualistic perspective on rights, we can think of rights as moral or political trump cards.2,3 If an individual has a right to do or have something, then we need a special justification in order to prevent that individual from doing or having the thing in question. For example, if we believe in a right to free speech, then we should not restrict this right without some morally or politically sound justification.2 This gives a key insight into the logic of rights-based arguments: When we accept a rights claim as valid, then we should not allow this right to be restricted or violated without good reasons.2,3 Thus, in order to evaluate debates about moral rights, we will need to determine the nature of the rights in question and consider reasons for violating or restricting those rights.

Restrictions on Rights What counts as a sound justification for violating or restricting individual rights is a matter of heated dispute, but the harm principle is one of the commonly accepted justifications for restricting individual rights. According to this principle, individual rights many be restricted in order to prevent demonstrable harms or risks to other individuals.2 For example, the right to free speech does not allow people to yell “Fire!” in a crowded movie theater. However, it may not always be easy to apply the harm principle to rights disputes, since controversies may arise over the assessment of risk, which is a function of the probability and severity of harms. The debate over smoking in public illustrates this point. Those who want to restrict the rights of smokers to smoke in public have gained moral and legal victories as they have produced evidence to document the risks of passive smoke, but those who defend smokers rights continue to dispute these claims.6 Once we move beyond the harm principle, we find that there are very few areas of agreement about restrictions on individual rights. We often place restrictions on an individual’s rights when their rights conflict with someone else’s rights. But it is often very difficult to settle conflicting rights claims. For instance, in the battle over pornography on the internet, we have a conflict between the rights of those who want to disseminate or read pornography and the rights of those who want to use the internet but also protect their children from indecent or offensive material.7 Pornography on the internet renews the perennial debate over justifiable limits on the right to free expression. Because it is often difficult to settle conflicting rights claims, many ethicists now argue that we should de-emphasize “rights talk” and focus instead on “duties and responsibilities.” Rights talk, according to many writers, is inherently divisive because it pits individual interests against each other at the expense of a common consensus or meaningful debate.8 We recognize some of the problems inherent in rights-based arguments, but we shall include these arguments in our discussion of the morality of HGLGT, since some of the controversial questions addressed in this book are best viewed as limitations on individual rights or conflicts of rights. A final distinction worth mentioning at this point is the distinction between negative and positive rights: Negative rights are rights to noninterference; positive rights entail something much more than noninterference. For example, if we interpret the right to life in a purely negative sense, it is simply a right not to be killed and it implies that people have obligations not to kill individuals who have this right. But if we interpret it in a more positive sense, the right to life could be taken to imply that other people have duties to protect and promote the lives of people who have a right to life. Questions about positive vs. negative rights, like questions about limitations on rights and conflicts of rights, are also controversial. Some ethicists reject the distinction between positive and negative rights altogether.9

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However, we will make use of this distinction because many of the important rights issues with regard to HGLGT can be understood as questions about positive vs. negative rights.

Parental Rights Having made these general remarks about rights and responsibilities, we can consider how rights claims might apply to HGLGT. We shall first consider the issue from the perspective of parents and their rights to reproduction and child rearing (parental rights).10-12 According to a standard approach to parental rights, parents have two kinds of basic rights: rights to reproduction and rights to child rearing. Although reproductive and child rearing rights are conceptually distinct, we consider these rights as linked together here because it would be almost useless to have rights to reproduction without rights to child rearing (or vice versa): A person who can have a child but who has no rights when it comes to raising it is as oppressed as a person who can raise a child but has no rights to create or choose a child. Furthermore, many of our child rearing practices now begin in utero or before; maternal diets and genetic counseling can have as much (or more) of an effect on a child’s welfare as education. In our culture and in many other societies we find a very strong commitment to parental rights, and we usually do not interfere with reproduction and child rearing. The United States’ legal system, for example, recognizes both a right to procreative liberty and a right to privacy that extends to child rearing.12 Parental rights can be based on a broader notion of a right to self-determination or autonomy. The right to self-determination gives competent (or rational) individuals the right to make decisions and act on them.13,14 Some of the most important decisions people make in life involve reproductive and child rearing choices, e.g., whether to have children, who to have children with, how many children to have, whether to seek assistance in reproduction, how to educate children, and so on. Reproductive choices are important because they can affect an individual’s life plans or her health and well-being. Having and raising children are two of the most significant and meaningful activities in human existence. Given the importance of reproductive and child rearing choices, there is a strong justification for parental rights. Parental rights may not be absolute, but it takes very compelling reasons to violate or restrict these rights. Indeed, the complete denial of parental rights by the state was one of the horrors of Huxley’s Brave New World: Sex was for pleasure, not for reproduction. Nearly all ethical theorists and most laypeople agree that parental rights deserve to be recognized, even if they disagree on their priority and centrality in morality and politics. Parental rights could have both positive and negative connotations. Negative rights would include freedom from interference in reproductive or child rearing choices. For instance, freedom from interference would support arguments for free choice in birth control, abortion, education, and health care. Positive rights would include the promotion of government assistance in reproduction or child rearing. A person who asserts that the government should fund reproductive services as part of Medicaid might base these claims on positive, parental rights. It almost goes without saying that parents also have moral duties to their children. At a bare minimum, parents have duties to promote and protect the interests of their children.13 A child’s interests include food, clothing, shelter, health, well-being, education, opportunities, love and a stable home environment. Parents have not only a right but a duty to cultivate the best interests of their children. Parents that fail to promote and protect the best interests of their children may be judged as irresponsible, neglectful or abusive. The next step in this rights-based argument is to assert that parents have a right to HGLGT.15 These rights are based on reproductive and child rearing rights: HGLGT could be viewed as a matter of reproductive choice on a par with in vitro fertilization, artificial insemination, and other forms of technologically aided reproduction. The argument

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further states that these rights should include rights to use HGLGT for therapy or enhancement purposes. According to Burke Zimmerman, parents have a right to embryo screening and selection as well as HGLGT, in order promote the genetic health of their offspring.15 One might even extend this argument a bit further and argue that parents might one day have a duty to use HGLGT in reproduction if this procedure can be shown to be in the best interests of their children. If HGLGT ever becomes safe and effective, it might be similar to other kinds of medical procedures. Parents who produce children with preventable genetic diseases could even be regarded as irresponsible or neglectful if there are some ways of preventing these diseases, such as embryo selection, selective abortion, or HGLGT. Given the importance of parental rights and responsibilities and HGLGT’s natural alignment with those rights and responsibilities, proponents of this new technology would appear to have a strong argument against state restrictions on its development. According to the parental rights argument we present here, restrictions on HGLGT (research or applications) would be analogous to restrictions on the development of new methods of birth control or other new reproductive services.

HGLGT and Harms to Unborn Children One might argue that there are some sound arguments for restricting parental rights and responsibilities in the case of HGLGT. For the purposes of this chapter, we will focus only on decisions concerning the clinical applications of HGLGT, since decisions relating to HGLGT research involve not only the rights of parents but also the rights and freedoms of researchers. (We will discuss research policies in chapter 10.) Earlier in this chapter we mentioned two rationales for restricting or violating individual rights, i.e., in order to prevent harm to others or in order to respect the rights of others. In the case of parental rights to use HGLGT, the relevant “others” are the children who would be the products of HGLGT and future generations. (HGLGT might harm women who carry genetically engineered children, of course, but this is a slightly different consideration, since our rights to harm ourselves are much stronger than our rights to harm other people. Case in point: cigarette smoking.) Thus, one might argue that parental rights to HGLGT can be restricted in order to prevent harm to unborn children or to protect their rights. (See the discussion of medical risks of HGLGT in chapter 6.)

Three Types of Unborn Children In order to understand these concerns, it is important to distinguish between three kinds of unborn children: UB 1. A fetus, embryo or zygote developing in the uterus or fallopian tube (for example, in a pregnancy without reproductive assistance or after an HGLGT proband has been transferred to the uterus); UB 2. An embryo or zygote that has not yet been transferred to the uterus or fallopian tube (for example, a frozen embryo or an unimplanted HGLGT proband embryo developing outside the body (in tissue culture); UB 3. A potential child that has not yet been conceived (future generations, F1, F2, etc.). These are important distinctions to make, we believe, since there are some reasons for thinking that our obligations to these different types of unborn children are not identical. One might argue that we have stronger obligations to an embryo that has implanted and is developing normally than to a frozen embryo, since the former has a good chance of developing normally and becoming an adult, while the latter will not implant or develop unless we take steps to bring about its implantation and development. If we simply do not interfere with a UB 1 unborn child, there is a high probability that this child will be born. As we shall

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see in a subsequent section, our obligations to future generations may be even weaker than our obligations to embryos or zygotes. The questions before us are as follows: Do any of these types of unborn children have rights?16 Can they be harmed by our actions? Do we have a duty not to harm them? One might argue that we cannot answer these questions without entering the abortion debate, since the two extreme sides of this issue offer some clear and simple answers to these questions. If we follow the conservative view and assume that conception marks the beginning of a human person, then children who are UB 1 or UB 2 have rights—they are moral patients. If we follow the most liberal view and assume that personhood does not begin until after birth, then children who are UB 1 or UB 2 have no rights. We do not intend to take a stance on this divisive issue or add more fuel to this volatile debate.17 However, we believe that we do not need to say whether zygotes, embryos and fetuses have rights in order to tackle the important questions raised by HGLGT. For the purposes of our discussion, we shall set aside the question “Do unborn children have rights?,” and ask a slightly different one, “Do we have a moral obligation not to harm unborn children?” Although this sounds like only a subtle twist of wording, there is an important difference between these two questions: The first one addresses the rights of a zygote, embryo or fetus who will be the proband or direct result of HGLGT engineering; the second one addresses our moral obligations to promote the welfare of children who may be born in the future.

Obligations to Zygotes, Embryos, and Fetuses To help make sense of the questions before us, consider a hypothetical case of a child who is born with mental retardation as a result of his mother’s decision to have natural childbirth. During a difficult and long labor, the woman’s physicians recommend that she take medicine to assist labor, and if that does not work, that she have a Cesarean birth in order to insure that her child is born healthy. The mother refuses both of these interventions, the child suffers from oxygen deprivation, and is born mentally retarded. Did the child’s mother have a moral obligation to do her best to insure that her child would be born in good health? We think that most people would answer “yes” to this question. We can put the argument this way: Every child has an interest in being born healthy, and actions that adversely affect the health of children, whether in utero or earlier, can harm children. In this case before us, the child has an interest in being born healthy, and the woman subverted this interest by refusing to comply with the physicians’ recommendations.18,19 This child would still have these interests even if it were an early stage embryo, a zygote, or a frozen embryo. We can use this scenario to address harms to other unborn children as well: We need not claim that zygotes or fetuses have rights in order to assert that potential children can be harmed by events or decisions occurring in utero or earlier. Recalling the link between harms and interests discussed in the last chapter, we can put it this way: We can harm potential children by taking actions that would adversely affect their interests, if they are born. If we assume that all children have interests in health and well-being, then reproductive choices and decisions can cause harm to potential children by adversely affecting their health and well-being.18,19 Thus, we can appeal to the harm principle to limit parental rights to HGLGT. Parents should not be allowed to use HGLGT if it imposes an unjustifiable risk on the health and well-being of their children. Indeed, one could carry this argument further and claim that parents have a moral responsibility not to give birth to children who could have significantly diminished life prospects, due to genetic or other abnormalities.19 One might argue that it is wrong to bring a child to term if that child is likely to have a short life marked by

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severe pain and/or dysfunction. For example, one might argue that it would be wrong to give birth to a child if you know that the child has Tay-Sachs. The same point would hold whether the abnormality is the result of HGLGT mistakes or occurs naturally.

Restricting Parental Rights If we understand the argument this way, then we can view the harm principle as an application of the principle of nonmalificence that was mentioned in the previous chapter. In this case, the principle of nonmalificence imposes obligations on parents that restrict their rights and generates parental duties and responsibilities. Before proceeding further with this line of reasoning, we need to distinguish between two different requirements imposed by this principle. First, the principle requires that we do not intentionally harm others; second, the principle requires that we do not impose unjustifiable risks on others.1 An unjustifiable risk would be one where the goals (or good outcomes sought) do not merit the risks taken (or possible bad outcomes). Examples of intentional harm would include theft, murder or physical assault; examples of unjustified risk impositions might include driving too fast, leaving children alone at home, or testing an unknown food additive on an infant.1 In some cases the line between intentional harm and unjustifiable risk is murky: If the risk is known to be sufficiently great, an action that produces harm would seem to be intentional. For example, if I blindfold myself and drive down a busy street and hit a pedestrian, wouldn’t we consider this harm intentional and not merely unjustified risk imposition? In any case, the main import of the harm principle for parents is that they have a duty not to impose unjustifiable risks on children or potential children. To strengthen this argument for the restriction of parental rights to HGLGT, we can appeal to debates about reproductive technologies that are already in use, such as in vitro fertilization, since HGLGT exposes many of the same, key issues that existing technologies already raise.11,20,21 Ever since the first “test tube baby” was conceived and born, physicians, ethicists and policy makers have been concerned about potential psychosocial harms to children conceived in vitro. These issues of harm concerning test tube babies continue to weigh heavily on policy decisions relating to clinical applications of in vitro fertilization.21 At present, we have some but not enough data on the psychosocial and medical consequences of in vitro techniques. If we appeal to the harm principle (as applied to potential children) as a valid reason for restricting parental rights to HGLGT, then we return to some of the questions discussed in the last chapter: Will HGLGT harm children? What is the probability that it will cause harm? How significant will the harms be? What are the possible benefits of HGLGT? How important (or significant) are those benefits? Do the benefits justify risk imposition? At present, the genetic manipulation required for HGLGT would be a very risky procedure, although it may become safer in the future. Since HGLGT would be such a risky procedure, one might argue that parents do not presently have the right to use this technology for therapeutic or non-therapeutic purposes. The argument against the use of HGLGT can be taken a step further: The use of HGLGT at this point in time would be morally irresponsible and ought to be illegal. Clearly, the use of HGLGT (or any other risky medical procedure for that matter) would not be in the best interests of the children who would be the products of genetic engineering, according to this argument. One does not promote a child’s (or a potential child’s) interests by gambling with his or her health.

Justifiable Risks? On the other hand, one might argue that in some cases the benefits of HGLGT are worth the risks, and that gambling would therefore sometimes be in a child’s best interests.

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The use of HGLGT for therapeutic purposes might be justifiable risk imposition in some cases because the goal—a healthy child—might be worth the risk.15,22 If HGLGT presents the only hope for having a healthy child, the decision to take a “gamble” might make sense, since the child and parents have a great deal to gain and not much to lose. Even if HGLGT fails to stop the disease or has some adverse side effects, this outcome might not be much worse than the disease itself. Though we agree with the logic of this argument for the therapeutic use of germline manipulation, we must emphasize that the above argument would be most pertinent to cases where the genetic disease is life-threatening, severely disabling or debilitating in early childhood. Genetic alterations which would statistically modify the chance of getting cancer at age 80 would not fall into this category. Many of the serious early childhood diseases, like Tay-Sachs disease, Lesch-Nyhan syndrome or cystic fibrosis, are monogenic disorders. Here we return to our earlier point that HGLGT will be useful for avoidance of monogenic diseases only in some rare (homozygous) cases where genetic embryo selection can not be used to prevent the genetic disease. Since genetic embryo selection is likely to be a safer, more effective and less complicated procedure than HGLGT, parents who seek to prevent their children from being born with monogenic diseases should use this reproductive technology, wherever possible. If we think of this problem in terms of rights and responsibilities, then parents may sometimes have a right and a responsibility to gamble with a potential child’s health (i.e., in the rare cases where both parents are homozygous for a genetic defect causing a life-threatening, severely disabling or debilitating disease). If parents have a right and a responsibility to promote the best interests of their potential children, then HGLGT is justified if it is the only viable option that could produce an outcome that is in a potential child’s best interests. The use of HGLGT in these rare cases would be like trying an experimental drug on a child with an incurable illness. Even if the experimental drug causes harm, the parents who seek this experimental treatment do not violate the harm principle since they would be attempting to save their child from other harms, including death. Thus, the use of HGLGT to prevent devastating genetic diseases might be viewed as justifiable risk imposition in some rare cases. However, the problem changes considerably when we consider using HGLGT to enhance a child that we have every reason to believe would be healthy, or when we consider using HGLGT to prevent tolerable genetic diseases, such as color blindness. In these types of cases, the wisest choice would seem to be not to use HGLGT. Only the most optimistic, risktaking parents would take a shot at producing a child with enhanced height. Once again, a great deal depends on how we think about the outcomes in these cases, but one might argue that genetic intervention would be unwise (and perhaps irresponsible) in such situations. Potential children have a great deal to loose and not much to gain from the decision to pursue genetic enhancement or therapy for tolerable diseases. Parents who seek to use HGLGT on children who are likely to be healthy (or who will have only minimal health damage) would be violating the harm principle by taking actions that count as unjustified risk imposition. The goals are simply not worth the risks.

Therapy, Enhancement, and Justifiable Risks To summarize, the decision to use HGLGT to prevent devastating genetic diseases may be justified in some rare cases where it is the only option, but HGLGT is not justified to enhance children or eliminate tolerable genetic diseases, given our level of technology for the foreseeable future. There may be some very good reasons for restricting parental rights with respect to genetic enhancement or gene therapy for tolerable genetic diseases, since parents seeking to use HGLGT for these purposes would be gambling with the health and

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well-being of their potential children. But there may be no good reasons for restricting parental rights with respect to genetic therapy in rare cases where it is needed to prevent devastating genetic diseases, since parents would be risking very little in order to gain a great deal. Like the parents who consent to experimental treatments for children with incurable diseases, parents who use HGLGT in some cases would be choosing the only path that offers hope and a chance at good health. In addition to ethical considerations, there are also scientific reasons why the first HGLGT trials, if any, would be directed toward replacement of single defective genes causing devastating disease: 1. These are the genes that researchers are already studying because of the genetic disease; and 2. Protection from a disease using an HGLGT replacement of a single defective gene would be much less complex than replacing one or more genes that affect a polygenic disease. In chapter 5 we tried to debunk the therapy/enhancement distinction, but the above conclusions suggest that therapies are often on sounder moral ground than enhancements. But, the reason why this is so relates to morality of risk imposition; it has nothing to do with the “sacredness” of the germline, or the immorality of genetic enhancement. If genetic enhancement becomes a “good gamble” for parents and their offspring, then this risk imposition argument for prohibiting enhancements would weaken. If HGLGT becomes safe and effective, then parents might be justified in using the technology in order to benefit their children. The key factors in this kind of reasoning are the risk to the child and the potential benefits. For the foreseeable future, HGLGT poses enormous risks on children, so it would be justified only in rare cases. In the distant future, HGLGT might be justified in more types of cases as it becomes more safe and effective. If we learn more about the effects of HGLGT and we are able to develop safe and effective interventions, then HGLGT may be justified in other types of cases, including for enhancement purposes. At this point in time, however, the harm principle provides a strong rationale for restricting parental rights to HGLGT, even for therapeutic purposes.

Is Nonexistence a Harm? Before continuing further with our discussion, we need to address an objection to the view we have just defended. According to some writers, nonexistence is itself a kind of harm that is always (or almost always) worse than existence. In its most extreme version, this view holds that life is always a blessing, no matter how much suffering that life may involve. No child that is lucky enough to be born can be harmed by the circumstances of his or her birth. In its less extreme version, this view holds that a child is only harmed by the circumstances of his birth if the child’s life is so miserable that he would be better off having never been born.23 Both versions of this view pose serious problems for restricting parental rights based on harms to potential children, since these harms will almost never be worse than not being born.12 Even if we admit that parents can take actions to harm their potential children, this harm is usually less than the harm of not being born, so parents usually do not violate the harm principle when making reproductive choices. This line of reasoning has become known as the Interest in Existing argument.11 According to this argument, only if the outcomes of HGLGT might be a fate worse than nonexistence would parents be violating the harm principle or the nonmalificence principle. It makes little difference whether parents use HGLGT for therapy or enhancement: In either case the chance at existence would be worth the risks, from the point of view of the unborn child. (This argument assumes that the parents will not have the child unless they can use reproductive technologies, such as HGLGT.) If we accept the

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idea that nonexistence is a very undesirable possible outcome for a child, then the argument has the implication that parents can virtually do no wrong when it comes to reproducing, since very few lives will be so bad that they are not worth living.21 The Interest in Existing argument even has the abhorrent implication that it would be morally acceptable for parents to use HGLGT for the purpose of creating genetic “freaks,” so long as their malformed offspring do not prefer death to life. There are, we believe, at least two serious problems with the Interest in Existing argument. The first problem is that it claims that we can make comparative judgments about existence and nonexistence. We think that it is not possible to know whether existence is better than nonexistence, since no one knows what nonexistence is like. We can make value judgments about different kinds of lives, but we cannot compare life and nonlife. Indeed, one might argue that it is impossible to ever know what nonexistence is like, since knowledge must be based on experiences and no one experiences nonexistence. A second problem with the Interest in Existing argument is that it presupposes an unusual metaphysics in which children can exist in state of limbo, waiting to be born. These children might exist as “souls” waiting to join a body, for example. Although some religions accept this sort of view, since this book aims to develop a public policy framework, we will not make any tendentious religious assumptions. (We will discuss the body/soul connection in chapter 9, however.) Thus, risk imposition arguments should be based on comparing different possible lives of the children who might exist; they should not be based on comparing the states of existence and nonexistence for potential children.21 Hence, the Interest in Existing argument does not pose a serious objection to our reasoning about limiting parental rights.

Harms to Future Generations Thus far we have considered the rights of those people who will be most directly affected by HGLGT and we have argued that parents have a duty not to use HGLGT when it imposes unjustified risks on their potential children. But what about the generations of children born after the first genetically engineered progeny? Should parents avoid harming these future generations as well? What about a genetic defect caused by HGLGT that is not expressed for several generations or that gets worse with each generation? Should parents try to avoid these kinds of harms? These questions raise issues about the rights of future generations and our duties toward them, concerns that also play a key role in current debates about population control, global warming, and natural resource use. In a sense, a zygote, embryo, or fetus can be a member of a future generation, but the future generations we have in mind are the generations much further removed from the parents, such as grandchildren, great grandchildren, and so on. We have set aside the question of whether unborn children have rights, but we claimed that it makes sense to assert that people have obligations to unborn children. We will use this same strategy in our discussion of future generations: Our main question will be: “Do we have obligations to future generations?” We believe that we have obligations to future generations, although our obligations to them are not as strong as our obligations to zygotes, embryos, fetuses, or members of currently living generations. Three considerations support this view. First, future generations have not even been conceived. Although parents may sometimes have the intention to conceive or give birth to a child in the future, the unconceived child’s existence is highly precarious and contingent.24 Two people may say that they want children or grandchildren, but many different factors and conditions must come together before these unconceived children are born. Second, since future generations are so much

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further away from us in time, our actions do not have as much of a direct effect on them as they do on embryos, fetuses or currently living people. It is also much more difficult to know how our actions will affect future generations than it is to know how our actions will affect these earlier generations.24 Third, although we can be fairly certain about some of the interests of future generations, such as interests in health or happiness, we are less certain about their other interests, because we do not know what the world will be like when they exist, how human societies and technologies will have changed, what resources will be available, and so on.24,25 This third point implies that it will be difficult to know how best to promote or protect the interests of future generations, although we can be fairly certain that future generations will still have an interest in having the basic necessities of life.24 From these considerations some people draw the conclusion that we have no obligations to future generations. But we believe that this conclusion is short sighted and goes against most people’s moral sensibilities. So long as we accept the idea that people should be responsible for the consequences of their actions, and we realize that these consequences can affect future generations, then we should admit that people have responsibilities (obligations) toward future generations.24 These obligations are not as demanding or stringent as our duties to existing people or potential children, but we at least have obligations to avoid causing harm to future generations and to promote their welfare.24,25 Given this perspective on our obligations to future generations, we think that the principle of nonmalificence applies to our conduct toward future generations. We should avoid imposing unnecessary, known risks on future generations, such as contaminating drinking water with carcinogens or storing nuclear waste in unsafe containers. On the other hand, we would make little scientific and technological progress and take very few risks if we constantly worried about how our actions will affect future generations. If we had given heavy moral and political weight to our duties to avoid harming future generations, then we might not have developed automobiles, airplanes, and electric power on the grounds that these inventions could impose severe risks on future generations. Our main responsibility is to concern ourselves with what lies close at hand rather than to worry about what lies dimly in the distance. How do these points apply to HGLGT decisions? Some would argue that future generations need to be considered in such decisions and that society needs to take steps to prevent obvious, unjustified risks to future generations. For example, it would be wrong to introduce a gene into the human population that scientists have good reasons to believe could lead to a new, devastating genetic disease. However, one might argue that there is little point in allowing our concerns about harms to future generations hinder the development of potentially beneficial genetic and reproductive technologies. People need to take some risks and have faith that future generations will have the ability to compensate for many of the problems created by scientific and technological advances. Human ingenuity can provide solutions to many of the problems created by new technologies, including HGLGT. For comparison, consider automobiles. During this century, automobiles have created many problems, such as pollution and congested roads, but humans have developed new technologies and modes of living in response to these problems.26 HGLGT is also likely to create many problems, but it is also possible that human beings will be able to respond to them in the same way that they have responded to other technologies. Thus, the long record of human ingenuity and adaptability gives us reasons not to worry too much about harms to future generations, even though one must recognize that any responsible policy needs to avoid imposing obvious risks and harms on generations that follow.

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Other Rights Considerations Informed Consent Before concluding this chapter, we would like to consider three other human rights issues raised by HGLGT. First, one might argue that HGLGT would violate medicine’s principle of informed consent, since the subjects of HGLGT experiments, potential children and future generations, could not, in principle, give consent.27 Informed consent is a legal and ethical doctrine developed during the last fifty years in response to the needs for standards of human experimentation.1 According to the Nuremburg Code, a widely recognized code of research ethics, research subjects must give their voluntary and informed consent before participating in an experiment; subjects must also understand the information that they receive.28 Clearly, potential children and future generations do not satisfy any of these three requirements. However, when subjects cannot give informed consent, proxy consent is sometimes morally acceptable.1,13 In the case of HGLGT, parents could give proxy consent for their potential children. In giving proxy consent, parents should attempt to promote the best interests of the human subjects.13 If it would be in the subject’s best interests to participate in an experiment, then the parents can give proxy consent for the experiment; if it would not be in the best interests of the subject, parents should not give consent to the experiment. As we argued earlier, the question of whether HGLGT is in the best interests of research subjects depends on its benefits and risks. Currently, HGLGT would be in the best interests of a research subject only in some very rare cases; in the future HGLGT might be in the best interests of subjects in many cases. To give proxy consent, parents must satisfy the requirements mentioned above. Even if parents can give voluntary consent, it is not likely that this consent will be fully informed, since we are not likely to know all the possible medical and psychosocial consequences of HGLGT. In addition, even where we have adequate information about HGLGT, consenting parents may not understand genetic engineering’s highly complex procedures and processes. However, the mere fact that parental consent will probably be far less than perfect in many cases should not pose a moral barrier to parental consent to HGLGT, since we already condone other kinds of experiments where consent is not accompanied by complete understanding or full information. For instance, we allow parents to consent to experimental treatments for children with cancer and we allow AIDS patients to consent to highly experimental drugs. The important consideration for consent is that whoever gives consent has enough information and understanding to make a competent decision.13 We can (and often do) make competent decisions despite lacking a great deal of understanding and information. When we buy a car or house, or choose a career or a mate, we often lack a great deal of information and understanding, but we can still make competent decisions. Although the notion of proxy consent can be applied to HGLGT experimentation on potential children, it is difficult to apply to HGLGT experimentation on future generations, i.e., grandchildren, great grandchildren, etc. As far as these subjects are concerned, no one can even be considered their legal or moral proxy, since these unborn children do not yet have any parents, guardians, or other proxies. Does HGLGT (or any kind of experimentation) on future generations violate their rights to informed consent? If we set aside the question of whether future generations have rights and focus on our duties toward them, as we suggested earlier, our main concern should be to understand whether we have any duties to provide some kind of proxy consent for future generations. We think that current generations have at least a minimal moral responsibility to provide some sort of proxy consent for future generations. A proxy need only be an institution, a group of people, or a person who serves as an advocate for future generations. The voice of future generations needs to

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be heard in our public policy debates even if it is not as loud as the voice of current generations. At the very least, we need to provide a forum for those who attempt to foresee how the consequences of actions and policies are likely to affect the welfare of future generations. For example, environmentalists provide a voice for future generations when they raise questions about global warming, pollution, overpopulation, and other environmental problems related to human activities.

The Right to Natural Birth The next human rights consideration sometimes discussed is the claim that children have a so-called “right to be born by natural means.” One might argue that all people have a right to be born by natural means, wherever possible, and that HGLGT therefore violates this right.29 (Many other reproductive technologies, such as in vitro fertilization, also violate this right.) Arguments like this one often appeal to the moral or religious sanctity of human nature, human existence, and human biology.20 We will address concerns about the unnaturalness of HGLGT in chapter 9. For the purposes of this chapter, we can rephrase this question as: “Do we have a moral obligation to produce children by natural means?” since we are not assuming that potential children or future generations have rights. Even if we do not accept the idea that future generations have rights, we still have obligations to unborn children and future generations, and we need to consider how unnatural reproduction may affect their welfare. Can “unnatural birth” itself be a harm? Even if a child is born in good health, could the circumstances of his birth cause the child some harm? While we do not view unnaturalness itself as harm, we recognize that unnatural births can have harmful psychosocial effects. Children can be harmed by the unnaturalness of their procreation in various ways. A child born unnaturally might regard herself as a mere artifact of genetic technology, as different from other human beings, as a mere instrument for fulfilling her parent’s wishes, and so on. People who discover the conditions of the child’s birth might discriminate against the child, treat the child disrespectfully, mock the child, etc. Moreover, a disturbing feature of our cultural myths about genetic technology may contribute to the psychological damage that children created by HGLGT may suffer: Our society now tends to view genetics as providing an explanation for virtually any human trait or behavior, from alcoholism to intelligence, from crime to musical ability, even if the suspected genetic basis is completely unsubstantiated by scientific data (see also the discussion on genocentrism in chapter 5). The gene has taken on a meaning and significance in our culture way out of proportion to its actual causal role in human development and behavior.30 Although genetics provides us with a powerful explanatory framework, even the most enthusiastic genetic determinists admit that most human traits result from three main causes: the genome, the environment, and genome-environment interactions. In light of the gene’s role as a cultural icon, society should make special efforts to prevent psychological harm to children who are the products of HGLGT, such as education and counseling. Although children created via unnatural means may suffer a great deal of psychosocial harm, we should also keep in mind that many of these types of harms may occur with or without HGLGT. Even children born by natural means often suffer from psychosocial harms due to the circumstances of their birth. Many of the harms we have discussed also occur when children are adopted, born out of wedlock, have parents of two different races, immigrate to a new country, and so on. None of our comments should be interpreted as an underestimation of the harms caused by unnatural births, but they point to the need to study these potential harms in more depth.21 (On the other hand, HGLGT people could be revered.)

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Genetic Confidentiality The final issue we would like to discuss in this section concerns the confidentiality of genetic information relating to HGLGT. Who should be allowed to have access to this information? Do individuals have a right to genetic confidentiality? Do individuals have a right not to know information about their genetic conditions? These questions surfaced decades ago with the advent of the first genetic tests. Since then, businesses, insurance companies, government agencies, and many other organizations have sought to obtain genetic information about individuals, and questions about the confidentiality of genetic information have remained at the forefront of our discussions of legal and ethical issues in human genetics.30-35 We do not intend to settle questions about confidentiality in this book, but we would like to discuss confidentiality issues raised by genetic engineering of human beings. As in our previous discussions, we will not assume that unborn children have a right to confidentiality. However, we can still have duties to protect the confidentiality of genetic information about unborn children. For example, if a scientist obtains genetic information about a zygote in order to perform HGLGT, she has a duty to keep that information in confidence to protect the potential child’s welfare. One important concern about HGLGT is that children born via this process, unlike currently living adults, have no choice about submitting to genetic tests. The very act of creating these people will require us to obtain a great deal of genetic information about them. Since so much information will be obtained and presumably kept, the duty to protect privacy and confidentiality takes on added importance. Thus, people who have access to this information have a duty to protect its confidentiality. Children who are the products of HGLGT and their parents should have access to this information, but in the absence of a very strong justification, it should not be shared with employers, insurance companies, government agencies, or other interested third parties. The burden of proof for such a justification falls on those who would seek to obtain genetic information, not on those who seek to protect its privacy.31,33 If children produced by HGLGT have a right to obtain genetic information about themselves, do they also have a right not to obtain or know this information? This is a tricky question. One might argue that the right to autonomy gives people a right not to know certain things about themselves. Some people might not want to know whether they carry an allele which predicts a high chance of developing Huntington’s disease or Alzheimer’s disease. People have a right to remain ignorant about certain things so that they may live a life that they find meaningful and enjoyable. On the other hand, a person may make a poor decision due to their ignorance: A person who carries a genetic disease and does not know this fact may pass on that disease to a child. Earlier we mentioned that the harm principle can serve as a valid reason for restricting autonomy. If we apply this point to the “right not to know” genetic information, it would follow that people have a right to remain ignorant unless their ignorance is likely to cause harm to other people or impose unjustifiable risks on others. Thus, the “right not to know” is not absolute, and genetic information may be revealed to people in order to prevent them from harming others through reproductive choices. However, when individuals are given genetic information about themselves, those who disclose this information should make sure that the information is both useful and reliable, since it is irresponsible to burden a person with a belief that turns out to be untrue, inaccurate, or misleading.32,34 More harm than good may be done by telling a person that they carry an allele that is linked to breast cancer if it turns out that this allele is not a very good predictor for susceptibility to cancer.

Conclusion In conclusion, we have discussed the moral and political rights and responsibilities of parents, potential children, and future generations in the context of HGLGT. Our main

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point is that parents have a right to use HGLGT only in some rare cases, because parental rights are limited by the duty to avoid imposing unjustifiable risks on potential children. Parents have a right to HGLGT only when potential children have a great deal to gain and not much to loose from genetic engineering. For the foreseeable future, these types of cases will be very rare, since embryo selection will be more safe and effective than HGLGT in preventing devastating monogenic diseases. Furthermore, HGLGT technology will not be safe or effective enough to justify its use for other purposes, such as enhancement or the elimination of tolerable diseases. Parents would seem to have a right to use HGLGT only when they cannot use other, less risky technologies to prevent their children from being born with devastating genetic diseases. Of course, the situation could change if HGLGT advances to the point where it offers great benefits with minimal risks. It is possible that parents will one day have a right to use HGLGT for enhancement purposes. In this chapter we have also argued that the users of HGLGT technology have obligations to future generations. HGLGT users should try to anticipate the consequences of their actions and policies and avoid causing preventable harms, such as the introduction of manmade genetic diseases into the human population. We shall return to some of the issues mentioned here later on the book. In the next chapter, we evaluate HGLGT from the point of view of justice.

References 1. Beauchamp T and Childress J. Principles of Biomedical Ethics, 2nd ed. New York: Oxford University Press, 1994. 2. Feinberg J. Social Philosophy. Englewood Cliffs, NJ: Prentice-Hall, 1973. 3. Thomson J. The Realm of Rights. Cambridge, MA: Harvard University Press, 1990. 4. Regan T. The Case for Animal Rights. Berkeley, CA: University of California Press, 1983. 5. Forrester M. Persons, Animals, and Fetuses. Dordrecht: Kluwer, 1996. 6. Goodin R. The ethics of smoking. In: Arthur J, ed. Morality and Moral Controversies, 4th ed. Upper Saddle River, NJ: Prentice-Hall, 1996. 7. Kapor M. Civil liberties in cyberspace. In: Johnson D and Nissenbaum, eds. Computers, Ethics, and Social Values. Upper Saddle River, NJ: Prentice-Hall, 1995. 8. Glendon M. Rights Talk. New York: Free Press, 1991. 9. Shue H. Basic Rights. Princeton, NJ: Princeton University Press, 1980. 10. Page E. Parental rights. J App Phil 1984; 1: 187-203. 11. Robertson J. Children of Choice. Princeton, NJ: Princeton University Press, 1994. 12. Robertson J. Noncoital reproduction and procreative liberty. In: Alpern K, ed. The Ethics of Reproductive Technology. New York: Oxford University Press, 1992, 249-258. 13. Buchanan A and Brock D. Deciding for Others. Cambridge: Cambridge University Press, 1989. 14. Dworkin G. The Theory and Practice of Autonomy. Cambridge: Cambridge University Press, 1988. 15. Zimmerman B. Human germ-line therapy: The case for its development and use. J Med Phil 1991; 16: 593-612. 16. Thomson J. A defense of abortion. Philosophy and Public Affairs 1971; 1(1): 47-66. 17. Callahan D. The abortion debate: Can this chronic public illness be cured? Clin Obst Gyn 1992; 35: 783-791. 18. Purdy L. Genetics and reproductive risk: Can having children be immoral? In: Munson R, ed. Intervention and Reflection, 5th ed. Belmont, CA: Wadsworth, 1995. 19. Steinbock B and McClamrock R. When is birth unfair to the child? Has Cen Rep 1994; 24: 16-22. 20. Kass L. Toward a More Natural Science. New York: Free Press, 1985. 21. Cohen C. “Give me my children or I shall die!” New reproductive technologies and harm to children. Has Cen Rep 1996; 26: 19-27.

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22. Munson R and Davis L. Germline therapy and the medical imperative. Ken Inst Eth J 1992: 2: 137-58. 23. Feinberg J. Wrongful life and counterfactual elements in harming. Soc Phil Pol 1988; 4: 145-78. 24. Baier A. For the sake of future generations. In: Regan T, ed. Earthbound: New Introductory Essays in Environmental Ethics. New York: Random House, 1984. 25. Golding M. Obligations to future generations. The Monist 1972; 56: 85-99. 26. Volti R. Society and Technological Change, 3rd ed. New York: St. Martin’s Press, 1995. 27. Lappe M. Ethical issues in manipulating the human germ line. J Med Phil 1991; 16: 621-39. 28. The Nuremburg Code. In: Beauchamp T and Childress J. Principles of Biomedical Ethics. New York: Oxford University Press, 1979. 29. Commission of the European Community. Adopting a Specific Research and Technological Development Programme in the Field of Health. Brussels: Commission of the European Community, 1989. 30. Nelkin D and Lindee S. The DNA Mystique. New York: W.H. Freeman, 1995. 31. Resnik D. Genetic privacy in employment. Pub Aff Quar 1993; 7: 47-56. 32. Kitcher P. The Lives To Come. New York: Simon and Schuster, 1997. 33. Draper E. Risky Business. Cambridge: Cambridge University Press, 1991. 34. Murray T. Ethical issues in human genome research. FASEB J 1991; 5: 55-60. 35. Elias S and Annas G. Somatic and germline therapy. In: Elias S and Annas G, eds. Gene Mapping: Using Law and Ethics as Guides. New York: Oxford University Press, 1992.

CHAPTER 8

Human Germline Gene Therapy and Justice I

n the last chapter we examined the rights and responsibilities of parents, possible children, and future generations with regard to the issue of human germline gene therapy (HGLGT). While the last chapter took more of a micro perspective by addressing individual rights and obligations, this chapter will take more of a macro perspective by exploring questions relating to justice and fairness. Once again, we include an introduction to some of the basic concepts and principles that we will employ in this chapter.

What Is Justice? Of all the moral and political ideas we have discussed so far, the concept of justice is by far the most multifaceted, context-sensitive, and intricate.1 We appeal to justice in debates about punishment, jurisprudence, affirmative action, taxation, social welfare, health care rationing, environmental regulation, and many other social issues. Two common threads run through these different debates. The first thread is that all of these issues involve questions about giving people their deserts. Justice, at its barest minimum, involves giving people what they are due, and it is intimately connected to a slightly different idea, the idea of fairness. Thus, treating people justly, giving them what they are due, is roughly equivalent to treating them fairly. Although some writers distinguish between “justice” and “fairness,” we will treat these concepts as more or less interchangeable for the purposes of this book. The second thread among these different topics is that they all address macro-level questions about the arrangement of social institutions and human societies.1,2 As we noted earlier, we do not maintain that there are sharp distinctions between moral and political philosophy, since many controversial topics, including HGLGT, have both moral (or ethical) and political dimensions.1,2 Even though the distinction between macro-level questions relating to justice and fairness and micro-level questions about rights and duties is not absolute, it can still be useful in helping us organize our thinking about morality and politics. Having muddied the waters a bit, we will distinguish between several different kinds of justice and explicate several different principles of justice. The most basic division in types of justice is between procedural and distributive justice.3,4 Procedural justice has to do with the fairness of a procedure, decision, practice, or process. Distributive justice has to do with the fairness of outcomes under conditions of scarcity. Since outcomes may be benefits such as jobs, money and health care, or burdens such as unemployment, punishment, impoverishment and disease, distributive justice addresses the distribution of benefits and burdens in society.1 A procedure may be just or fair even though its outcomes are not, and vice versa. For instance, consider five people who play poker one evening. As long as the players follow the rules—they do not cheat—then their games are fair even if one of the players ends up with Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus, Pamela J. Langer. ©1999 R.G. Landes Company.

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most of the money. We might say that the player with most of the money acquired his money “fair and square” so long as he played according to the rules. Although the games result in a very unequal distribution of scarce resources, this does not affect the fairness of the game itself. On the other hand, consider four friends dividing a pizza. A fair division of the pizza might be to divide the pizza into eight equal slices and give each friend two slices (one fourth of the pizza). In seeking fairness in dividing up the pizza, we want the outcome to produce a fair or just distribution, given that there is a scarce resource, one pizza. Even if the process of slicing the pizza was, in some sense, unfair or unjust, e.g., one person sliced the pizza while the others watched, the outcome was fair and just. (Notice that there is not a distribution problem when the friends can have as much pizza as they want, although there still will be procedural questions.) It is important that we understand the distinction between these two types of justice, since our questions about justice will often reflect these different concerns. For instance, in discussing a criminal trial we may ask whether “justice was served.” In examining the justice or fairness of the trial, we might focus on two different questions: 1. Was there due process in this case? 2. Did the defendant get what he deserved? The first question focuses on the procedural justice; the second, distributive justice. Readers will notice that these same kinds of questions can also be raised about HGLGT: 1. What makes the process of implementing HGLGT fair/just? 2. What makes the outcome of this process (the distribution of social/genetic resources) fair/just? We will discuss these questions later on in this chapter. According to many writers, principles of justice consist of a formal principle and several material principles. The Formal Principle of Justice, which most philosophers attribute to Aristotle, can be stated as follows:1,3,4 The Formal Principle of Justice: Treat equals equally; treat unequals unequally. This principle is a purely formal schema that provides a conceptual framework for other principles but lacks substantive content. The mere fact that the principle lacks substantive content should not diminish its importance, however, since it insures that other principles of justice meet standards of integrity, impartiality, and consistency. Material principles of justice actualize the Formal Principle by elaborating on what is meant by “equal,” “unequal,” “equal treatment,” and so on.3,4 For example, consider justice in college admissions. The Formal Principle of Justice would require that in college admissions we should treat equals equally; unequals unequally. It is important for the college to meet this condition in order to avoid making capricious, biased, or arbitrary admissions decisions. But what makes two applicants equal or unequal? Computerized test scores? Grades? Extracurricular activities? Race, ethnicity or gender? And what counts as equal treatment? Are we treating applicants unequally if we admit one applicant but not another? In order to answer questions like these, we need to employ some substantive principles of justice. Thus, we need to follow both formal and material principles of justice in order to make just decisions. For the purposes of our discussion, we will introduce five material principles of justice:3,4 1. Equality: Benefits and burdens should be distributed equally. 2. Need: Benefits should be distributed according to need. 3. Merit: Benefits and burdens should be distributed according to merit and achievement. 4. Utility: Benefits and burdens should be distributed according to a policy that produces the greatest balance of benefits over burdens for all members of society.

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5. Free Choice: Benefits and burdens should be distributed according to the choices people make when rights are not violated.5 Although these principles are stated as principles of distributive justice, they have procedural analogs. For instance, a principle of procedural equality might require that people have equal rights and equality of opportunity, even though it would not require that these conditions produce equality of results.1 A principle of procedural need might require that needs be addressed as they arise, although it might allow that some needs are not met. A principle of procedural free choice might specify rules for acquiring and transferring property, yet not require that property have any distribution pattern.5 A principle of utility might support a triage policy in medicine. Different theories of justice develop one or more of these principles in more detail by placing them in a larger moral/political/economic perspective. For instance, egalitarian theories elaborate on the principle of equality; Marxist theories explicate the principle of need; libertarian theories emphasize merit, achievement, and free choice; and utilitarian theories endorse a variety of principles in order to maximize social utility.3 Although different theories of justice disagree on the relative importance of these different principles, each of these principles would appear to offer just or fair guidance under certain conditions. For example, a principle of equality should govern the allocation of voting rights: One person, one vote. A principle of need would seem to provide the most just allocation of unemployment benefits; people who need these benefits—the unemployed—should get them. When grading exams, it makes sense to follow a principle of merit: The best exams should get the best grades. In playing a game of poker, we should employ a principle of free choice: Winnings should be distributed based on the choices made by players in the game, so long as players do not violate each other’s rights by cheating. Since different principles may fit different situations, many writers argue that justice requires that we employ a combination of different principles in our decision making.3,4 In order to interpret and apply any of these principles, one needs to have a fuller understanding of their key terms and concepts, such as “equality,” “need,” “merit,” and so on. Although this task falls beyond the scope of our book, we will provide an expanded discussion of the concept of “equality” as it pertains to the genetic engineering of human beings. Thus, we will attempt to develop an egalitarian approach to HGLGT, although we leave the defense of egalitarianism to other writers.1,2 We focus our discussion on equality because we believe that some of the most profound fairness issues raised by HGLGT relate to questions about human equality.6-9

HGLGT and Human Equality To focus our discussion, we shall develop two different scenarios describing the possible future evolution of our species. (We mentioned both of these scenarios in our preface.) The first scenario, which we shall call the “Fascist Scenario,” resembles Huxley’s Brave New World. Suppose that one or two renegade nations develop and control various reproductive technologies, including HGLGT, to promote their national interests. Nations that find this practice morally repugnant eventually succumb to eugenic temptations in order maintain genetic, strategic, and economic parity with the renegade nations, and a genetic arms race ensues. Eventually, all industrialized nations employ reproductive technologies, including HGLGT, to design and manufacture different types of human beings for various purposes, such as warfare, management, research, athletics, manual labor, and so on. These humans reproduce with other humans who belong to the same social class, and genetically enhanced specialization continues. As a result, different genetic castes emerge in these societies. These castes differ genetically, politically, economically, intellectually, and socially. The nations that do not have access to HGLGT, or those that choose to refrain from using it,

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eventually disperse or dissolve in the turbulent genetic seas. As this process continues, genetic castes propagate throughout the world, Homo sapiens splits into different subspecies, and new species emerge. The second scenario, which we shall call the “Capitalist Scenario,” unfolds in a slightly different way. In this case we imagine that renegade parents who can afford HGLGT use this technology to provide their children with genetic advantages. Even if many countries initially ban HGLGT, this technology might still be available on the black market (or in more permissive countries) for parents who wish to use it. Parents who already are willing to spend money on other kinds of advantages for their children, such as training for taking tests, a new computer, or an Ivy League education, will be equally motivated to spend money on HGLGT. These parents could attempt to buy therapies to enhance any trait that may have an enhanceable, genetic component, such as height, intelligence, eye color, skin color, musculature, musical ability, and so on.7,8 At first only the very rich will be able to afford HGLGT, but the cost of this technology will start to decline as an HGLGT industry develops in order to meet parental demands for HGLGT. This industry fuels HGLGT development by investing money in research and applications; more parents demand new HGLGT goods and services; HGLGT becomes more affordable, and so on. Of course, many parents will have some moral reservations about using HGLGT, but others will succumb to the temptations of HGLGT in order to “keep up with the Joneses.” If we suppose that genetic enhancement occurs over many generations and that the richest parents can afford the best enhancements, then the rich will get richer, and the genetically rich will get genetically richer, and so on. People who do not have access to HGLGT or do not wish to use it for moral reasons will soon find that they and their progeny are genetically, economically, and socially worse off than those who have access to HGLGT. Eventually, children that do not benefit from genetic enhancement will be at a distinct disadvantage. Once again, something like a caste system emerges from this process, carried out over time, and Homo sapiens branches into subspecies and new species. Although these scenarios envision two different mechanisms for bringing about dramatic changes to the human population, i.e., state control vs. free market control, they both predict a similar outcome: Homo sapiens, like many species of ants, will display caste polymorphism, and new species of the genus Homo will emerge.10 Given that organizing production and labor in human societies appears to be the triumph of the capitalist system, we speculate that the Capitalist Scenario is more likely to occur than the Fascist Scenario, unless we see a resurgence of strong, centralized, nationalistic governments. Most writers who condemn eugenics and HGLGT express concerns about state control over the gene pool and point to the Nazis as the embodiment of this kind of evil. However, an unregulated capitalist system can produce the same results. It should also be noted that some combination of capitalistic and governmental mechanisms could also bring about these shocking social consequences.11 Of course, both of these scenarios are highly speculative and one might challenge their cogency. First, as we noted in chapter 6, HGLGT is not likely to have a significant impact on the gene pool unless it is widely used. For the purposes of these scenarios, however, we are considering a world where HGLGT has become safe and effective enough to be widely used. Second, one might argue that the scenarios are not likely to occur because we have had the ability to control human reproduction for hundreds of years and we have not significantly changed the genetic composition of the human population. Government-backed eugenics programs have not succeeded at achieving their objectives and private citizens have not managed to have a significant impact on the genetic composition of our species through their reproductive choices. HGLGT increases our ability to control human reproduction, but its impact will probably be marginal.

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We do not find this objection convincing because it fails to appreciate the historical and technological uniqueness of HGLGT. HGLGT is not like other forms of genetic control in many respects: It is more powerful, more dangerous, and more profound than any previous reproductive technology. Other technologies and social policies have attempted to control the union of human gametes or interfere with human development through selective abortion or infanticide. No other reproductive technologies or social policies have aimed at altering the human genome itself. Thus, we believe that HGLGT’s impact can be more than marginal and that it could be one of the most significant technologies ever developed. Other readers may object that our scenarios will not occur in the space of a few thousand years (or less). Evolution is a slow process that takes millions of years, not thousands or hundreds. The problem with this objection is that it fails to recognize the evolutionary uniqueness of HGLGT. Ordinary Darwinian evolution takes a long time, but HGLGT allows human beings to circumvent natural selection. HGLGT undercuts two assumptions made by the theory of natural selection, i.e., that variation of traits is random with respect to their adaptive value and that genetic changes and adaptations are gradual. When we bring genetic “self-engineering” into the evolutionary process, we raise the possibility that organisms can change their own genotypes and phenotypes in order to meet environmental demands (the environment includes both the physical and the social environment). Evolution is no longer truly Darwinian at all. Human evolution, under this model, bears a closer resemblance to technological development than to biological evolution. New technologies respond to the needs and demands of users and designers and change as rapidly as time, energy, and human ingenuity permit. Less than a decade after human beings invented recombinant DNA technology, they used it to design and manufacture genetically altered bacteria, the first man-made organisms. Can man-made people be far behind? The last objection to our scenarios is that they simply will not occur at all. People will refrain from performing HGLGT for moral, religious, medical, technological, political, legal, and economic reasons. HGLGT will always be a big gamble that will offer little in the way of payoffs. Even if HGLGT offers reliable and significant results, which is highly questionable at this point in time, it will most certainly be banned throughout the world. It will always be an impractical, unrealistic, taboo technology. This last objection is little more than wishful thinking. In many ways the world might be a better place without HGLGT, and one might hope that people never use or develop this powerful technology. The world might also be a better place without nuclear weapons. But there are also profound benefits from such technology and wishful thinking cannot make new technologies go away. Prudence, realism, and moral responsibility require that we anticipate the possible consequences of genetic research and technology and that we do not deceive ourselves into believing that these disturbing scenarios could never happen. Even the strongest opponents of germline manipulations must come to terms with mankind’s desire for power, choice, and opportunity. Humans find it very difficult to resist temptations, and HGLGT is one of the most profound temptations to ever emerge. It is not our aim in this book to serve as prophets. We introduce these science fiction nightmares in order to develop some policies designed to prevent them from occurring. At this stage of HGLGT’s development, we believe that the future is open and that the human race could take radically different paths, depending on the choices we make during the next century. In order to understand these choices, we discuss HGLGT’s possible impacts on human equality and distributive justice.

Equality of Opportunity Both the Fascist and Capitalist Scenarios described above portray societies with very unequal distributions of benefits and burdens. Those with “superior” genes have more

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benefits such as wealth, health, opportunities, privileges, and social status. Those who have “inferior” genes have fewer benefits and more burdens such as poverty and poor health. The rich get richer and the genetically rich get genetically richer. Moreover, those members of society born in the lower genetic classes have virtually no chance of obtaining the social, economic, and political status of those born in the upper classes. What is wrong with this picture? In a word, inequality. When a caste system exists (or a functional equivalent) people are not created equal. Ever since the American and French revolutions, Western nations have stressed the importance of human equality. However, it is important to distinguish between different kinds of equality, since it may be morally acceptable for human beings to be equal in some respects though unequal in others. For the purposes of this discussion, we shall distinguish between several different types of equality: 1. Moral Equality (ME), i.e., equal dignity, equal moral rights; 2. Political Equality (PE), i.e., equality under the law, equality in voting; 3. Social Equality (SE), i.e., equality in social standing, power; 4. Economic Equality (EE), i.e., equality in wealth 5. Biomedical Equality (BE), i.e., equality in natural abilities and health. Although we distinguish among these different types of equality, it is worth noting that there are important causal connections between these different equalities. For example, political inequalities can cause social and economic inequalities; biomedical inequalities can lead to social and economic inequalities, and so on. Despite these causal connections, we can treat these types of equalities as conceptually distinct. It is important to understand how these equalities can be conceptually distinct, since one might support or value one type of equality but not another type. For example, Western, liberal democracies attempt to uphold moral and political equality, but they allow for great differences in income, wealth, power, social standing, and health. Socialistic nations, on the other hand, strive for economic, political, and moral equality. We will assume that most readers of this book will agree that human beings should be morally and politically equal. So, a caste system is unjust in that it violates standards of moral and political equality. This much seems to be fairly uncontroversial. However, one might argue that even a caste system that maintains moral and political equality would still be unjust because it would not uphold a kind of social equality, equality of opportunity.1,2 Equality of opportunity requires that a person’s life prospects should depend on factors that are within their own control, such as motivation, character, skill and merit. In the United States, a large part of the American dream is the belief that any person in society can succeed at what they choose to do, if they have enough ability and determination. In order to promote equality of opportunity, liberal democracies have various policies to compensate people for social, economic, and natural inequalities such as public education, scholarships, public health clinics, progressive taxes, and antidiscrimination laws. Rules aimed at promoting equality of opportunity help to insure that a person’s life prospects do not depend on factors that are arbitrary from a moral point of view, such as natural endowments (the “natural lottery”) or parentage.1 So what does all of this have to do with HGLGT? If HGLGT creates a caste system or its functional equivalent, then HGLGT will have unjust social consequences. HGLGT could have this dire result by exacerbating genetic inequalities.6,7,9 When people are born with radically different genetic endowments, they do not have the same life prospects. A person born with Down’s syndrome, for example, does not have the same life prospects as a person born with the normal number of human chromosomes. Likewise, a person born with “superalleles” for enhanced intelligence would not have the same life prospects as someone born with normal genes.11

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One might object at this point that people are already born genetically unequal and that this does not undermine equality of opportunity. We use social redress policies to compensate people for the misfortunes of the natural lottery and to promote equality of opportunity. So HGLGT raises no significant or novel concerns, since we can continue to enact social policies to compensate people for genetic differences. This objection is simply another version of the marginal impact argument discussed earlier. People have been born genetically unequal for thousands of years, but these inequalities have, for the most part, fallen within a normal range for our species. We have been, for the most part, monomorphic, not polymorphic. We have not been a race of giants and dwarves, geniuses and morons, gods and monsters; we have been a race of human beings. Genetically speaking, members of our species are more like each other than they are like members of any other species. But HGLGT could change this entire picture of the human race and expand the range of genetic and phenotypic variation. Thus, we should be concerned about HGLGT’s potential impacts on equality of opportunity. If one accepts the idea that we should be concerned about HGLGT’s potential impacts on equality of opportunity, the next question to ask is how we should go about safeguarding equality of opportunity. To address this issue, let us return to the therapy vs. enhancement distinction discussed in chapter 6. One might argue that therapies can promote equality of opportunity by preventing genetic diseases. All diseases, including genetic maladies, undermine equality of opportunity by limiting our life prospects. Our life prospects depend on a normal opportunity range of human health, and sick people have a reduced range.2 Thus, HGLGT, viewed as a form of health care, can promote equality of opportunity through the prevention of genetic diseases.6 Moreover, if HGLGT is used for therapeutic purposes it would also seem to promote the principle of need discussed earlier if we make this technology available to those who need it. If we view health as a basic, human need, then health care should be distributed to people who have health needs that must be met. Just as a sick person has health care needs, unborn children may have health care needs. We meet the needs of an auto accident victim and an unborn child in different ways, but the concern for need is the same.12 We agree with the logic of these arguments, but once again question HGLGT’s usefulness in preventing diseases. HGLGT will be useful only in preventing diseases that cannot be prevented by other methods that are more safe and effective, such as genetic embryo selection or conventional medicine. If these cases turn out to be very rare, given our level of technological development, then we will not have a very strong justification for developing HGLGT to address medical needs or promote biomedical equality. It is far more likely that people would use HGLGT to enhance human traits. At the moment it is difficult to imagine such a deliberate and predictable alteration of polygenic traits such as height. However, if we assume a day when monogenic and polygenic traits alike can be altered to achieve a phenotypic result that can be predicted with high accuracy, we can ask the following question: Would the use of HGLGT to enhance human traits promote or undermine equality of opportunity? Genetic enhancement could threaten equality of opportunity by generating social, economic, and biomedical inequalities. Is it possible that these inequalities could become so extreme that a de facto caste system emerges? In order to promote equality of opportunity, we need to take some steps to insure that HGLGT does not have these dire social consequences. If we decide to follow this course of action, there seem to be three basic options: 1. Forbid genetic enhancement; 2. Allow genetic enhancement within a genetically “normal” range; 3. Allow genetic enhancement with no restrictions.

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Option 1 would appear to be both unrealistic and wasteful. It is unrealistic in that a complete ban on genetic enhancement is unenforceable.8 In the future, governments and private citizens are likely to have very strong interests in genetic enhancement, and we cannot expect a ban on this use of genetic technology to be very effective for an indefinite period of time. Option 1 is also wasteful, because genetic enhancements could yield social benefits.11,13 Enhanced individuals could benefit their fellow citizens through their talents and abilities. It makes little sense, from a utilitarian point of view, to ban a use of a new technology that could contribute to the social good. Option 3 should be rejected because it could undermine equality of opportunity by generating extreme inequalities. Option 2 would appear to be preferable to options 1 and 3, but it has its own drawbacks: How do you define a “normal” range of genetic variation? How do you enforce limits on genetic variation?12,13 We will consider three different principles which might be used to formulate a definition of a “normal” range of human variation: 1. Species Normality: All people, including genetically-engineered offspring, should belong to the species Homo sapiens. Genetic variation should not permit the development of new species of “super” humans or “sub” humans. 2. Genetic Equality: All people should be essentially genetically equal; there should be no significant increase in the range in human genetic variation. 3. Genetic Minimum/Maximums: Human genetic variation should fall within a circumscribed range of variation; humans should not fall below or rise above these lower and upper limits. First, defining a “normal” range of variation using the “Species Normality” criteria would still allow for tremendous differences among human beings and would not even rule out a caste system. In biology, a species is defined as a population of interbreeding organisms, and so long as all people could interbreed, they would still fall within this normal range. But organisms can manage to interbreed despite great phenotypic differences. Poodles, dalmatians, terriers, and German shepherds can all interbreed, after all. Allowing the amount of variation in humans that we find in domesticated dogs would undermine equality of opportunity.13 Second, the use of the “Genetic Equality” principle errs in the direction of too little variation and too much homogeneity. Cloning humans on a massive scale would move the population toward genetic equality; however, the loss of variation in the human gene pool would produce a dangerous level of homogeneity in an evolutionary sense. The human population would be subject to the phenomenon of genetic bottlenecking (see chapter 6). Socialistic approaches to justice, which emphasize economic, social, and political equality above all other values, would also imply a commitment to genetic equality. During this century some states have sought to promote equality by banning private property, controlling artistic expression, determining career choices, standardizing clothing, and so on. Only technological limitations would seem to prevent states from taking the step from uniform dress styles to uniform genotypes, since complete equality at all costs provides the rationale for both policies. If we pursued these socialistic eugenics policies, there could indeed be drastic evolutionary/genetic/medical consequences for the human race, because we need a certain amount of genetic diversity in the human population in order to respond to environmental changes and demands. In chapter 6, we argued that the Zea mays story does not apply to our species because individual, genetic choices would result in a tolerable amount of genetic diversity and because, optimistically, our species will hopefully have enough scientific and technical knowledge to respond to most environmental changes and demands. But our argument assumed that people and/or governments would not pursue genetic homogeneity as a mat-

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ter of social policy and that individuals would be able to freely choose their offspring’s genotypes. If we drop these assumptions, then the Zea mays story could foretell the fate of the human race. A second reason why we should not seek genetic homogeneity as a matter of policy is that human phenotypic diversity has moral value.14 One reason why we need at least some phenotypic diversity is that diversity helps develop our sense of uniqueness, which plays an important role in our conception of self-worth. Imagine how difficult it would be to develop a sense of uniqueness and self-worth in a world where physical and psychological traits are nearly uniform. Few people would want to live in a world where everyone has the same eye color, skin color, hair color, height, and so on. Since phenotypic diversity depends, in part, on genetic diversity, we should avoid genetic homogeneity if we value phenotypic diversity. We should not threaten human dignity in order to pursue equality or other moral/ political values. Since too much genetic diversity undermines equality of opportunity and too little diversity threatens human uniqueness and the survival of our species, perhaps a third alternative that pursues a path between these two extremes would be wiser. This option, governed by the “Genetic Minimum/Maximum” principle, would specify a genetically normal range of variation for a variety of genotypes in the human population. Genetic enhancement would be controlled so that parents would not be allowed to produce children that are predicted to exceed limits of height, strength, longevity, and other traits with a genetic basis. Genetic counseling, embryo selection, HGLGT, and other technologies would be available to parents who want to insure that their children do not fall below certain standards for health, intelligence, and so on. This socially acceptable range of variation might be analogous to specifications for the design of new automobiles sold to the public: We find a great deal of variety in new cars, but they all must meet minimum standards of safety and fuel efficiency, and they are not allowed to exceed some standards for speed and size. The idea of specifying a normal range of human variation promotes equality of opportunity without placing too many restrictions on genetic diversity. In theory, a normal range of variation sounds like a plausible idea. However, some difficult questions emerge when it comes to putting this theory into practice. Which genes or genotypes would be regulated? Can phenotypic outcomes be accurately predicted? What limits on variation would be set? Is ten feet too tall for a human being? Is two feet tall too short? Should people be allowed to live two hundred years? Would the government fund reproductive services for the poor to insure that their children will not fall below the genetic minimum mark? Would genetic limits be “set in stone” or would it be possible for the normal range of genetic variation to change as mankind evolves? We will not attempt to answer these difficult questions in this chapter, but we will assume that they can be answered (in principle). Appropriate public policies aimed at controlling genetic variation will depend on our abilities to manipulate the human genome. Our main goal in this chapter is to argue for the theoretical plausibility of controlling human genetic diversity, and our main goal in this book is to provide a foundation for policy decisions. Practical solutions will emerge through technological changes and further policy debates.

Two Objections to Defining a Normal Range of Variation We realize that some people may disagree with our view on theoretical, not just practical grounds, since the very idea of government control of human genetics brings to mind Nazi eugenics programs, Huxley’s Brave New World, and other nightmares. To address these concerns, we will discuss two objections to our view.

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The Slippery Slope The first objection to the idea of setting a “normal” range of human genetic variation is that this policy sets in place a rationale for tightening this range and for attempting to create “perfect” human beings. We might start out using a range of genetic variation to promote equality of opportunity, human health, and other legitimate moral concerns, but by pursuing these policies we would soon establish eugenic controls and regulations that stamp out genetic diversity and “abnormality” and seek to mass produce “perfect” humans. Given the nefarious history of eugenics movements in Germany and the United States and the lessons from socialistic governments, we should be wary of policies that seek to control the genetic constitution of the human species.15 Those who do not understand the history of eugenics movements are doomed to repeat it. The legacy of Nazism, Social Darwinism, and other eugenics movements will haunt our future attempts to control human genetics, and these ghosts will serve as a constant reminder of the errors of the past. However, as we stressed earlier, the failure to regulate human genetics could also have some drastic social costs that cannot be ignored. Human history also includes a legacy of other types of social injustice and inequality that rivals the evils of Nazism and Social Darwinism. Throughout history we have seen and continue to see great disparities in wealth, health care, social status, legal rights, and political power among human beings. Perhaps the choice is between the lesser of two evils or between two different readings of human history. We believe that it is possible to avoid sliding down the slope toward too much genetic control if we adopt policies that allow for a great deal of genetic diversity and remind ourselves of the legacies of Nazism and Social Darwinism. For example, the state may regulate the use of HGLGT, as it may regulate any new technology, but it may not engage in coercive eugenics policies, such as forced sterilization or abortion. The state may fund reproductive services, but it should not dictate private, reproductive choices.

Freedom and Fairness Throughout the thread of our argument runs a key assumption associated with the American philosopher John Rawls, i.e., social institutions should be designed to protect and promote equality of opportunity.1 Rawls’ theory offers an egalitarian approach to justice in that it stresses the importance of equality of moral and political rights and opportunities. Egalitarians believe that the government should protect moral and political rights and that it may also promote equality of opportunity through regulating businesses, providing social services, levying progressive taxes, and other policies. At the beginning of this chapter we mentioned that there are other approaches to social justice and that these approaches emphasize different principles of justice and moral and political values. We will now entertain an objection to socially imposed limits on genetic variation based on an alternative approach to justice known as libertarianism. Libertarians place human rights and liberties above all other moral and political values. They equate “justice” with “fair process” instead of “fair results,” and they hold that the sole function of government is to protect rights and liberties. The government is not justified in taxing rich citizens in order to redistribute wealth to poor ones and it is not justified in enforcing a right to equality of opportunity or happiness.5 So long as a rich person acquires his wealth through a fair process—he does not acquire wealth by violating the rights of other people—then he may amass as much wealth as he can. He can also transfer that wealth to other people, and the government should not meddle in these transfers, as long as they do not involve theft, fraud, manipulation, or other violations of human rights. For libertarians, autonomy and freedom are paramount values that can only be limited by the

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harm principle: Our basic rights and liberties can be limited only by the injunction not to violate the rights and liberties of others. Given this thumbnail (and some might argue oversimplified) sketch of libertarianism, how might this view approach questions about genetic control? A pure libertarian, one who is true to her philosophy, would hold that the government should not interfere in parental, reproductive choices, including the decision to use HGLGT, so long as those choices do not violate human rights. A libertarian might allow some government regulation of HGLGT in order to prevent unjustifiable harms or risks to potential children, but libertarians would not impose any upper limits on genetic diversity. So long as people acquire their genetic resources through a fair process, there should be no limits on genetic wealth. If this process, carried out over time, results in people who are vastly stronger, taller, healthier, and smarter than the genetically less fortunate people, then this result is just and fair, so long as it does not violate anyone’s rights. Moreover, the government is not justified in taking away wealth from people in order to prevent other people from being born with genetic disadvantages or in order to help other people at the bottom of the social order. If libertarianism permits the rich to get richer, then it should also allow the genetically rich to get genetically richer. The view would even seem to imply that governments are not justified in taking steps to prevent the emergence of new species or subspecies, so long as individuals are born into these populations through a fair process. An adequate response to this objection to establishing a normal range of genetic variation requires a deeper exploration of libertarian and egalitarian theories of justice. We will not undertake that task here, although we would like to point out that we find the libertarian approach to HGLGT to be morally and politically unacceptable because it would completely undermine equality of opportunity. We realize that this claim sounds dogmatic and unreflective, since we do not offer a fuller defense of equality of opportunity in this book. We defer the defense of this principle to other writers.1 But we do stand by this commitment and we think it helps explain our common moral revulsion to a genetic caste system. Moreover, we wonder whether a process that could permit the emergence of such vastly different kinds of human organisms, should even be considered a “fair process.” Consider the “fairness” of a road race. A race between two healthy humans could be considered “fair” even if one of the humans happened to have social advantages that allowed him to receive better training. But what about a race between a hare and a tortoise or between a human and a gazelle? If HGLGT creates radically different kinds of humans, we wonder whether social interactions that involve competition for jobs, resources, power, and opportunities would be fair. To return to the concerns we expressed earlier in this chapter, we believe that justice and fairness require us to prevent the emergence of a caste system. If libertarian genetics policies would permit the emergence of social castes or new human species or subspecies, so much the worse for libertarianism. Thus, perhaps even libertarians might need to impose some control on HGLGT in order to insure the fairness of processes of wealth accumulation and transfer, competition for jobs, and so on. Theories of justice that emphasize merit and liberty need to have at least some semblance of a “level playing field;” otherwise, merits will not be deserved and liberties will not be equal. One final aspect of the libertarian view that should be addressed concerns its emphasis on individual rights. As we noted earlier, libertarians accept the harm principle as a legitimate restriction on rights. The concerns about the emergence of a caste system or new human species imply that restrictions on human rights can and should go beyond the harm principle. In the “tragedy of the commons” the sum of many individual actions leads to undesirable results that no one anticipates or intends. Although no single person makes a choice that harms another person, the aggregate choices of different people produce a great deal of harm. For instance, when people choose to drive their cars to work instead of biking

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or using mass transit, their collective actions can produce smog, congested freeways, and other problems of urban living. In order to promote harmonious, balanced, and clean urban environments, the government may constrain individual liberties by imposing pollution controls on automobiles, enacting zoning restrictions and so on.16 We believe that the same kind of policy applies to the freedom to employ HGLGT: In order to promote social justice and prevent harm, the government may constrain reproductive choices. Philosophical theories that demand an unwavering commitment to individual rights and liberties must yield to the moral and political dilemmas created by the dynamics of science, technology, and society.16 We agree with libertarians that individual rights need to be respected. Parents should have a right to make reproductive choices and they should not be forced to receive genetic counseling or testing, but these rights are not absolute or unconditional.

Genetic Discrimination Some writers have attacked HGLGT and other genetic technologies on the grounds that they imply genetic discrimination. To get a better understanding of this issue, we will distinguish between two senses of “discrimination,” a neutral sense and pejorative sense.17 In the neutral sense of this word, “to discriminate” is to make a choice or select something. We discriminate when we buy apples, choose picnic sites, switch television stations, and so on. But this is not the kind of discrimination people have in mind when they speak of discrimination in a public policy debate. The pejorative sense of “discrimination” refers to a kind of choice that we would call unjust (or unfair). Discrimination is unjust when it violates the Formal Principle of Justice and one or more of the material principles. The Formal Principle of Justice implies that individuals who are the same in all relevant respects should receive equal treatment, and we might violate this principle if we make decisions based on irrelevant characteristics. We need to appeal to material principles of justice, however, in order to determine which characteristics are relevant to the decisions we make. For example, refusing to hire somebody to teach philosophy because they have blue eyes is unjust according the principle of merit because eye color has nothing to do with one’s ability to teach. On the other hand, refusing to hire somebody because they do not have a Ph.D. in philosophy would not violate the principle of merit because having a Ph.D. in philosophy is (somewhat) related to one’s ability to teach the subject. Discrimination can also violate equality of opportunity by preventing individuals from obtaining offices, positions, careers and opportunities that would normally be open to them. If one holds that individuals have a right to equal treatment or a right to equality of opportunity, then discrimination (in the pejorative sense) also violates the rights of individuals.1,17 Many forms of discrimination focus on phenotypic characteristics, not genetic ones. For the purposes of this book we define genetic discrimination as discrimination based on actual or presumed genetic differences as opposed to discrimination based on phenotypes.18 To this point, discussions of genetic discrimination have focused on two main areas of concern, employment and insurance. Employers have used genetic tests to determine whether prospective employees are susceptible to occupational hazards and have based employment decisions on the results of those tests.19 Insurance companies have used genetic tests to determine whether people have pre-existing genetic conditions, and they have used the results of these tests to refuse to pay for insurance claims or to charge customers higher insurance rates.18 However, there is evidence that blood banks, the armed services, educational institutions, and medical professionals have also practiced genetic discrimination.18 Is genetic discrimination unjust? It certainly could be in many circumstances. If a person is denied employment based on genetic conditions that are not relevant to her employment qualifications, then genetic discrimination would be unjust in this case. For example, suppose an employer decides that it does not want to hire people who carry a particular

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huntingtin gene allele which will predispose a person for developing Huntington’s disease, and it requires job applicants to undergo genetic tests. If the company fails to hire a woman who tests positive for a particular allele, one might argue that this would be an unjust employment decision, because having a genetic allele predisposing someone for developing Huntington’s disease should not disqualify a person from an employment opportunity. Some kinds of genetic discrimination may be justified, provided that the genotypes in question are relevant to the decisions being made. For example, one might argue that a woman who carries an allele associated with a high risk of developing breast cancer should not be an egg donor on the grounds that parents who are seeking this kind of assistance in reproduction would not want to pass this gene on to their children. The key problem in genetic discrimination is proving that information gained by genetic tests is indeed relevant to the decision being made, since a great deal of genetic information may not be relevant. For example, a genetic test that predicts that a person has a 50-99% chance of developing Alzheimer’s disease in old age would not be relevant in college admissions, since the person may get the disease after admission to college, if at all. Our popular culture currently has such a poor understanding of human genetics that the chances of abusing or misinterpreting genetic information are very great. Since we do not intend to write a treatise on genetic discrimination in general, we will refer the reader to some other sources for further discussion about these basic issues.18-21 Could HGLGT be another form of genetic discrimination? Consider the following possible use of HGLGT: A couple decides they want to have a child that is seven feet tall, so they insert one or more superalleles designed to promote height into their embryos. Would this choice be a form of discrimination against short people? Should their actions be considered unjust? We should note that these same kinds of discrimination issues already arise when people decide to abort fetuses on the basis of sex or other characteristics detectable in utero, such as Down’s syndrome. Embryo selection would also raise discrimination issues if we use the techniques to select against specific genotypes.22 But there is an important difference between embryo selection, selective abortion, and the creation of a zygote. The first two choices raise questions about how to treat an embryo or fetus; the third kind of choice raises questions about making an individual. Couples who select various genotypes for their zygotes do discriminate in the non-pejorative sense—they make preconception choices-but they do not discriminate against individuals, since these individuals do not yet exist in even a potential sense of existence. These kinds of choices may be viewed as discrimination against genotypes, but they do not constitute (pejorative) discrimination against individuals based on their genotypes. However, one might object to this line of reasoning on the grounds that HGLGT is still a form of genetic discrimination against individuals, because individuals are composed of genotypes and to discriminate against a genotype is equivalent to discriminating against individuals with that genotype. Prospective parents who attempt to create a tall child commit a kind of genetic discrimination: by deciding to have a tall child, they discriminate against children who would lack the “tall” superallele. This argument makes sense, as far as it goes, but what is the status of the children who would lack this superallele? These children are not actual or even potential children but only possible ones. If one holds that we can discriminate against possible people, then many absurd consequences follow. Any time we make choices that affect reproductive outcomes, we would be discriminating against some possible children who do not get to exist as a result of our choices. It would even follow that the choice of a mate involves genetic discrimination: A man who is homozygous for blue eyes who mates with a homozygous, blue-eyed woman would be discriminating against children with brown eyes since his choice prevents possible brown-eyed children from being born. But this is absurd. People should be allowed to choose their mates without being

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accused of practicing a kind of unjust discrimination.5 If mate choice is a form of discrimination, then virtually all of our actions are discriminatory, since nearly all of our actions have differential effects on possible or actual individuals. We take this implication to be a reductio ad absurdum of the claim that HGLGT is a form of discrimination. Thus, we stand by our original assertion that HGLGT is not a form of genetic discrimination. Although it involves the selection of genotypes, it does not imply unjust discrimination against existing individuals. However, as we noted in the last chapter, HGLGT could encourage genetic discrimination, bias, prejudice, and hatred. First, HGLGT choices may reveal something about the people who would use this technology to create children: They like some traits and do not like others. HGLGT, like other reproductive technologies, could exacerbate this bias and prejudice.23-25 Second, as we noted in chapter 6, many traits could be viewed as “inferior” as we develop the ability to choose traits through HGLGT, embryo selection, and other reproductive technologies. Children born with genetic diseases or even those who do not benefit from genetic enhancements could be viewed as unfortunate mistakes who do not pass genetic muster.23-25 Third, parents who fail to produce children that live up to various genetic standards might feel guilt, remorse, and shame, and they might be the victims of discrimination and bias. Finally, HGLGT (and other technologies) could exacerbate existing racial and ethnic discrimination if it turns out that specific “undesirable” genes are associated with particular racial or ethnic groups. For example, the gene that causes Tay-Sachs is more common among Jews of European origin and the gene that causes sickle cell anemia is more common among people of African origin.21,25,26 On the other hand, while HGLGT could have many harmful, psychosocial consequences, including discrimination and bias, we should not let these concerns weigh too heavily in our assessments of this technology. Suppose someone had mounted the same kind of argument against the polio vaccine: We should not develop this vaccine because those children who do not benefit from the vaccine will suffer a great deal of psychological harm, will be regarded as “freaks,” and so on. Parents who do not provide the vaccine for their children will feel guilt and remorse and the children will suffer from discrimination and bias. Ethnic groups that have a higher rate of polio from lack of treatment will be discriminated against, and so on. One might make a similar argument against developing personal computers and other technologies that can aid education. But now we can understand these arguments for what they really are: They are arguments that appeal to envy and resentment. These arguments would not allow one person to have “good fortune” so long as other people may not benefit from this “good fortune.” If this kind of reasoning has little credence when it comes to developing a polio vaccine or personal computers, then it should also carry little weight in our thinking about HGLGT and related reproductive technologies. Given the increasing use of genetic tests, the future of genetic research, and our culture’s infatuation with human genetics, it is likely that genetic discrimination and bias will occur regardless of whether we genetically engineer human beings.26,27 However, since HGLGT can contribute to genetic discrimination and bias, we may need to enact some regulations and laws to counteract or prevent genetic discrimination. We may also need to take extra steps to educate people about human genetics and reproductive technologies so that they understand the significance of genetic differences and appreciate our common humanity.7,21

Conclusion In this chapter we have argued that justice requires some governmental control of HGLGT. Completely unregulated human genetic engineering would lead to the antiutopias envisioned by Huxley and other writers. In order to promote egalitarian ideals, the government is justified in placing limits on the use of HGLGT for human enhancement, in

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subsidizing genetic counseling, embryo selection and other technologies used to prevent people from being born with genetic disadvantages, and in taking steps to prevent or counteract genetic discrimination and bias.13 More coercive government controls, such as forced sterilization or abortion, are not justified. In chapter 10 we discuss some of these policy implications in more depth.

References 1. Rawls J. A Theory of Justice. Cambridge, MA: Harvard University Press, 1971. 2. Daniels N. Just Health Care. Cambridge: Cambridge University Press, 1984. 3. Beauchamp T and Childress J. Principles of Biomedical Ethics, 2nd ed. Oxford: Oxford University Press, 1994. 4. Feinberg J. Social Philosophy. Englewood Cliffs, NJ: Prentice-Hall, 1973. 5. Nozick R. Anarchy, State and Utopia. New York: Basic Books, 1974. 6. Buchanan A. Equal opportunity and genetic intervention. Soc Phil Pol 1995; 12: 105-35. 7. Resnik D. Debunking the argument against human germline gene therapy. J Med Phil 1994; 19: 23-40. 8. Gardner W. Can genetic enhancement be prohibited? Med Phil 1995; 20: 65-75. 9. Wilkie T. Perilous Knowledge. London: Faber and Faber, 1993. 10. Holldobler B and Wilson E. The Ants. Cambridge, MA: Harvard University Press,1990. 11. Glover J. What Sort of People Should There Be? New York: Penguin Books, 1984. 12. Holtug N. Altering humans—the case for and against human gene therapy. Cam Quart Healthcare Eth 1997; 6: 157-174. 13. Resnik D. Human genetic engineering and social justice: A Rawlsian approach. Soc Theory Prac 1997 23(3): 427-448. 14. Kellert S. Value of Life: Biological Diversity and Human Society. Washington, DC: Island Press, 1996. 15. Paul D. Controlling Human Heredity: 1865 to the Present. Atlantic Highlands, NJ: Humanities Press International, 1995. 16. McGinn R. Technology, demography, and the anachronism of traditional rights. J App Phil 1994; 11: 57-70. 17. De George R. Business Ethics, 4th ed. New York: MacMillan, 1995. 18. Geller L. et al. Individual, family, a societal dimensions of genetic discrimination: A case study analysis. Sci Eng Ethics 1996; 2: 71-78. 19. Draper E. Risky Business. Cambridge: Cambridge University Press, 1991. 20. Gostin L. Genetic discrimination: the use of genetically based diagnostic and prognostic tests by employers and insurance. Am J L Med 1991; 17:109-144. 21. Kitcher P. The Lives To Come. New York: Simon and Schuster, 1997. 22. Rothman B. The Tentative Pregnancy. New York: Viking Penguin, 1986. 23. Kass L. Toward a More Natural Science. New York: The Free Press, 1985. 24. Heyd D. Genethics. Berkeley: University of California Press, 1992. 25. McGee G. The Perfect Baby. Lanham, MD: Rowman and Littlefield, 1997. 26. Nelkin D and Lindee S. The DNA Mystique. New York: W.H. Freeman, 1995. 27. Marteau T and Richards M, eds. The Troubled Helix. Cambridge: Cambridge University Press, 1996.

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CHAPTER 9

Human Germline Gene Therapy and Our Humanness O

ne of the lessons that people usually derive from Mary Shelley’s classic horror tale, Frankenstein, is that man should not “play God” by trying to create life. Only God can create life, and people who challenge this prerogative will bring disaster upon themselves and mankind. Humans may have the technical skills and scientific knowledge to construct or reconstruct life, but they lack the wisdom to understand or control their creative acts. A second lesson that one might draw from this story is that people should not try to alter the human form because is it morally or theologically sacred. Scientists who tamper with the design of the human body may create unnatural, inhuman monsters, and terrible consequences or God’s wrath will follow such transgressions. Frankenstein is the paradigmatic case of scientific hubris in the face of natural wonder, and many works of science fiction have repeated this theme.1 This horror story has obvious implications for human germline engineering; we need only change the names and places to update the tale for the 21st century.1,2 In the modern version of this parable, Dr. Frankenstein is a geneticist who uses human germline engineering to create inhuman monsters. The rest of the tale follows according to a familiar script. In this chapter we will explore some of the moral issues raised by the attempt to interfere with the natural, human form.

Human Germline Gene Therapy and Our Humanness In order to make sense of the issues discussed in this chapter, it will be useful to explore the question: “What is a human being?” since we can use answers to this query as a basis for understanding the terms “humanness,” “humanity,” and “human nature.” The question, “What is a human being?” has been asked by philosophers, theologians, and scientists for thousands of years and we are still looking for answers. The Greek philosopher Aristotle gave an answer that many people still accept. According to Aristotle, all living things have a soul, but man’s function is rational activity of the soul.3 For Aristotle, reason was not only man’s function but also man’s essence; man is the rational animal.3 In Aristotle’s view, all physical objects have essential and accidental properties. An object’s essential properties are those properties that make it the kind of thing that it is, and we refer to these properties in defining objects of its type. That is, essential properties provide us with necessary and sufficient conditions for classifying things. Accidental properties are those properties possessed by objects that do not make them the kind of things that they are. For example, a human male might have brown hair, straight teeth, and brown eyes; but these properties would only be his accidental features. He would still be a human being even if his hair turned gray, his teeth fell out, and he went blind. So long as he remains a rational animal, he is human in the Aristotelian view of humanity. 2500 years of criticism have unearthed many difficulties with Aristotle’s position. First, the division of traits into essential and accidental properties is not supported by our current Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus, Pamela J. Langer. ©1999 R.G. Landes Company.

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knowledge of genetics. Both essential and accidental properties have a genetic basis, however many accidental properties are influenced by “extragenetic” factors such as our chemical and physical environment and life experience. Second, many philosophers have argued that things do not have essences, and in particular, man does not have an essence.4 Most philosophers now accept the idea that we can use lists of relevant criteria, rather than necessary and sufficient conditions, to classify objects. Objects that fall within a given classification will typically have most, but not necessarily all, of the properties described by the criteria on the list. Thus, there is no human essence, even though there might be a collection of properties that we agree are possessed by nearly all creatures that we would call human.4 Third, philosophers and scientists have also challenged the idea that “man is the rational animal” and have offered other definitions of “human being.” Even if we reject Aristotelian essentialism, most of us will agree that our hair color has very little to do with our status as human beings and that certain traits, such as cognition and emotion, play a vital role in making us who we are. Instead of attempting to define the concept of a “human being,” we will discuss some of the different characteristics that contribute to our humanness. These include biological, psychological, and social characteristics.4 Biological characteristics could include the various anatomical traits that we find in human populations, such as having two arms, two legs, a four-chambered heart, an opposable thumb, and bipedal locomotion; developmental traits such as a nine month gestation period, live births and puberty between ten and fourteen years of age; and phylogenetic traits such as having a 23 pairs of chromosomes, a human genome and belonging to the species we call Homo sapiens. Psychological characteristics might include cognitive traits such as consciousness, intelligence, rationality and learning; emotive traits such as anger, fear, sympathy and sexual desire; and behavioral traits such as voluntary movement, aggressive displays, parental care for offspring, and so on. Social characteristics might include living in societies, cooperation, competition, language use, social rules and institutions, family or tribal structures, a division of labor, etc. Biological characteristics provide the most basic contribution to our humanness. According to a popular definition, an animal species is defined as a distinct, interbreeding population.5 (This definition applies to most, but not all, species.) According to this account, so long as two animals can breed and produce fertile offspring, they belong to the same species. Thus, all domestic dogs belong to the same species, Canis domesticus, despite significant physiological and behavioral differences. While horses and donkeys can breed, they do not belong to the same species because their offspring are not fertile. Using the biological species concept to define humanness, our humanness would be based on the genetic, behavioral, developmental, and physiological mechanisms that prevent gene flow between the human population and other animal populations.5 However, humanness consists in more than the ability to breed successfully in the human population. For example, suppose a population of people is exposed to some radiation that creates an unusual mutation in their offspring: The offspring can breed with each other but they cannot breed with people outside of this population. These offspring are a distinct, interbreeding population. Are they no longer human even if they look like human beings, talk like human beings, think like human beings, and walk like human beings? We submit that they would they be considered a new emerging human species of the genus Homo. Or suppose a female is born without a brain. If this being were kept alive, then she could be a member of our breeding population if we were to harvest her eggs, fertilize them in vitro, and implant them in a surrogate mother. She would be a human according to the definition of Homo sapiens, but she would lack many of the qualities we associate with humanness.

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Humanness also consists in more than having a functioning human body. Consider a healthy male who is in a car accident, incurs significant brain damage, and lapses into an irreversible coma or persistent vegetative state. He would still be a human being in that he is a member of the species Homo sapiens and has a functioning human body, but as a result of his dysfunctional brain he would lack many of the psychological and social characteristics we associate with humanness, such as rationality, voluntary movement, and language abilities. According to many philosophers and theologians, our humanness consists in characteristics commonly associated with the word “soul.”6 By this account, humanness amounts to a variety of psychological and social characteristics: The soul guides our behavior, shapes our emotional responses, and provides us with awareness of our place in the world. The idea of a “soul” is not the same thing as a “mind” although the two ideas are related. We tend to associate the mind with cognitive activities such as thinking, perceiving, judging and knowing, but the soul is much broader than the mind; the soul also encompasses emotive functions such as feeling and wanting, as well as behavioral ones such as acting, willing, judging, deciding, and personality traits such as extroversion, introversion, self-confidence, humor, and so on. For our purposes, we can think of “soul” as referring to many of the human features that we tend to view as non-corporeal, including cognitive, emotive, and behavioral traits. Although many people may not feel comfortable using a word like “soul” in our modern age, the word provides us with a useful way of organizing and discussing those aspects of our humanness that are psychosocial instead of biological in nature. Characteristics that we associate with the “soul” are key contributions to humanness rather than properties that define humanness. The distinction between body and soul has a long tradition in philosophical and theological discussions of our humanity.6 We find this separation of the body and soul in the writings of Aristotle, since rationality is a property of the soul, not a property of the body. Aristotle’s teacher, Plato, also defended a metaphysical dualism of body and soul and valued the soul over the body. The father of modern philosophy, Rene Descartes, identified the human self (or soul) with thinking, wanting, willing, judging, perceiving, and believing, and he argued that his mind/soul could exist apart from his body. Perhaps the most influential moral philosopher, Immanuel Kant, argued that human beings have moral dignity and worth because the human mind can recognize moral laws, and the human will can be motivated to follow moral imperatives. Neither the mind nor the will should be understood in a purely corporeal sense, according to Kant. Christian theologians, such as St. Augustine, St. Thomas Aquinas, and Karl Barth, have defended the place of body/soul dualism in Christian doctrine. We also find the body/soul dualism in other religious traditions as well, including Islam, Hinduism, Buddhism, and Native American and Bantu religions. This non-corporeal perspective on our humanness has some important implications for human germline gene therapy (HGLGT) if it were to be used to assess whether or not HGLGT would affect our humanness. The argument is developed as follows:6,7 1. Our humanness consists in those various traits that we equate with the human soul. 2. The human body and soul represent distinct human features of human beings. 3. HGLGT affects only the human body. 4. Thus, HGLGT does not affect the human soul. 5. Thus, HGLGT does not affect our humanness; it can have an effect only on incidental, “non-essential” human features. This argument makes several mistakes that need to be addressed in order to understand the relation between HGLGT and our humanness. For the moment, let us accept something like the first premise, at least for the sake of argument. Whatever our humanness is, it cannot be defined only in terms of the human

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body. If we have learned anything from the history of racism, we have learned why people should not be dehumanized on the basis of physical characteristics such as skin color, hair color, height, and so on. The characteristics that we associate with the human soul are what make us human beings. However, even if we recognize this point, we should acknowledge that there is a relationship between the human body and the human soul. Modern science has taught us that many of our psychological and social traits have a biological basis. Psychology has provided us with some insight into how the brain’s structural organization and chemistry play an important role in determining cognitive functions such as perception, memory and judgment, as well as emotive ones such as aggression, fear and sympathy. Sociobiology has allowed us to begin to understand the biological basis for social characteristics such as cooperation and language. In short, the human body and the human soul are intimately connected, and changes in the human body therefore affect the human soul.6 However, we do not have to assume that biology completely determines behavior in order to reach this conclusion. “Free” human choices as well as cultural practices and traditions can have an important impact on our behavior, even if biology provides some direction and guidance. Nature and nurture both play a role in shaping the human soul.6 So long as we reject naïve, Cartesian dualism, we can recognize that there are intimate connections between the human body and the human soul. Body and soul are conceptually distinct even though they are conjoined in concreto.6

HGLGT’s Effects on Our Humanness If nature and nurture play a role in shaping the human soul, and our humanness includes characteristics that we associate with the human soul, then HGLGT (which affects nature) can affect the human soul and it can affect our humanness. If genetic manipulations can have a significant influence on cognitive or emotive functions of the human brain, then HGLGT could most definitely affect the range of characteristics found in the human population. That is, the “composition” of the human population could theoretically change if new phenotypes or genotypes are introduced via HGLGT. It could allow societies to create beings, such as a race of docile “slaves,” that lack some of the traits that we associate with humanness, or to create beings that have been altered or “enhanced” in various ways, such as a race of “supermen.” HGLGT could allow parents to create children who have enhanced physical abilities or it could be used to introduce special disease resistance genes into the human population. In theory, HGLGT could even be used to extend the average human lifespan to over a hundred years. It might even be possible to use HGLGT to create people who would be considered a distinct, biological species. One can see that questions like these go on indefinitely when we think about HGLGT’s possible effects on our humanness. On the other hand, one might object that this whole query overestimates the influence of genetics and genetic engineering on the human body and soul, as well as our ability to understand and control the human genome. The view assumes naïve genetic determinism, genetic hegemony and scientific and technological omnipotence.8,9 One might argue that many traits are influenced by such a wide variety of genetic, developmental, and environmental factors and that genetic changes will often have little effect. Human intelligence, for example, involves so many genetic, environmental, and developmental factors that we may never be able to genetically engineer superintelligent beings in a predictable way. Moreover, even if a human trait does have a genetic basis, our understanding of human genetics will not currently allow us to implement these genetic manipulations and it is unlikely that we will ever have this God-like power over the composition of the human race. Human genetics is so complex and befuddling that we will never be able to design human beings the way we design cars. Hence, we do not really need to be concerned that HGLGT will affect our humanness or allow us to create nonhuman beings.10

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We recognize that one should not overestimate the importance of genetics or our scientific/technological capabilities. Most genetic manipulations of human beings still belong in the realm of science fiction, not science fact. We must await the results of further developments in science and technology to know how much of an influence HGLGT could have on human characteristics. However, in order to understand the moral and political issues that besiege HGLGT, we must be willing to address science fiction scenarios, since these situations can shape our judgments of right and wrong and they may one day actually occur. It is always important to remind ourselves of our current limitations, but responsible science policy requires us to address the possible shape of things to come. We can only speculate about these effects at this point in time, but if we have learned anything from the history of science and technology, we have learned that those who say, “This can’t be done,” or “This will never happen,” often end up looking like fools.11

Changing our Humanness HGLGT, if developed and applied, will have some affect on the range of genotypes and phenotypes that we find in the human population; it will change those characteristics that we associate with humanness. The changes brought about by HGLGT may range from trivial to profound. These reflections bring us to a central question of this chapter: Is altering our humanness inherently wrong? Is there something immoral in the very act of tampering with the human genome?

The Utilitarian or Consequentialist Perspective The answers to the questions about the morality of HGLGT depend, in large part, on our moral principles, theories and assumptions. If we approach these questions from a utilitarian or consequentialist perspective, then the morality of tampering with the human genome boils down to how we assess the possible benefits and harms. Using a utilitarian basis for decisions, it would be morally permissible to alter our humanness as long as the benefits outweigh the harms for all persons in society. Indeed, utilitarianism might even require us to redesign human beings and reengineer our race in order to promote the most good for the most people. The act of changing or manipulating the human genome would not be itself immoral. For utilitarians, the social ends justify genetic means. However, utilitarianism does not imply that “anything goes” when it comes to creating people or reengineering the human race, since some actions could have very bad consequences. Utilitarians would not reengineer the human form if it turns out that altering the human form would produce more bad consequences than good ones. Utilitarians would not endorse the use of HGLGT when it is neither safe nor effective, nor would they endorse the creation of people whose lives would be marked by hardship and suffering. Though these actions and policies might confer substantial benefits on society, they would also produce some very bad social consequences that would outweigh the good results. Utilitarians also would not favor the creation of people who would pose a grave risk to society, such as people who are highly aggressive, people who lack any compassion, or people who are so intellectually or physically talented that they believe they are “supermen” who are beyond the common morality. Utilitarians might not endorse using HGLGT to extend the human lifespan, since longer lives might also involve more suffering. If those who live longer are not healthy or happy, then increased human longevity can have adverse social and economic consequences. Our perspective on the utilitarian approach to these questions is very conjectural and provisional, of course, since possible utilitarian solutions depend on our knowledge of the consequences of various actions and policies involving the use of HGLGT. Until we know

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more about our abilities to alter the human genome and the consequences of such genetic manipulations, we can only speculate about how one might maximize benefits and harms.

The Rights-based Perspective If we approach questions about altering the human form from a rights-based perspective, we obtain results that are similar to the results derived from the utilitarian perspective. That is, under a rights-based perspective, the use of HGLGT does not, by its very nature, violate individual rights. Since unborn people and future generations will be the most adversely affected by HGLGT, we need to ask whether HGLGT would violate the rights of unborn people or future generations. In chapter 7 we resisted assigning rights to potential people or future generations and we will continue to avoid this philosophical quagmire. However, we can still address the interests of unborn children and future generations by asking whether we have a moral duty to prevent harm to those beings. Although some writers maintain that any genetically engineered person would be harmed or violated by the mere fact of being genetically engineered,12 another view is that one must examine the type of change in characteristics in order to judge the nature of the harm. If a person is born with physical deformities, psychological disabilities, and other maladies, then HGLGT could harm the person by causing them pain or by denying them dignity or self-respect. A person might also suffer if they have enhanced traits that make them so different from the rest of the human race that they cannot fit into human society, they feel lonely, and so on. Superior physical or intellectual abilities could be a burden, not a blessing. One might also argue that we can harm individuals by curtailing their opportunities: The person who is engineered to be a slave or super-soldier has limited opportunities and a closed future.13 In any case, the main concern should be whether the use of HGLGT would violate the engineered person’s moral worth, life opportunities, and self-respect. Thus, we have a moral obligation not to use HGLGT to create people who would be adversely harmed or violated by their genetically engineered traits. The morality of our actions depends, in large part, on how we use HGLGT to create people. Some uses are immoral because they harm, demean, or violate individuals; other uses may not have these results. Although this conclusion sounds like the utilitarian position discussed above, it is not, because utilitarians are willing to harm some individuals for the good of many individuals. In our view, we do not need to ascribe rights to unborn people or future generations in order to recognize that we have moral obligations to promote the welfare and dignity of every individual. To summarize our arguments to this point, two of the most influential moral theories, the utilitarian and rights-based approaches to morality, hold that tampering with the human genome is not inherently immoral. Both of these views would allow us to change the composition of the human race under certain conditions. Neither of these well known moral traditions imply that the human form—our humanness—is morally or theologically sacred.

HGLGT and Natural Law In order to address the moral concerns raised by genetic tinkering with the human form more fully, we need to introduce a different moral tradition into the analysis. This moral tradition, known as “natural law,” has its origins in Aristotelian thought and has been incorporated into various religious doctrines. According to the natural law approach to morality, moral principles and standards have a basis in nature.14 The moral laws that govern human beings are based on humanity’s inherent nature, our human nature. Actions that are “natural” are right; actions that are “unnatural” are wrong. For example, many natural law theorists maintain that homosexuality and sodomy are wrong because these actions are

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unnatural.15 The view also maintains that some things are naturally good or have natural, moral worth. An action that destroys something having natural worth is wrong. For example, many natural law theorists maintain that euthanasia and abortion are morally wrong because human life has inherent, moral worth.16 What is the ultimate basis for these natural, moral laws? There are two different answers to this question, a secular answer and a religious one. According to the secular answer, which was defended by Aristotle, moral laws are based on mankind’s natural functions and purposes, and these natural functions and purposes are based on human biology. According to the religious answer, which was defended by St. Thomas Aquinas, the natural law is based on God’s will or God’s commands. The religious version of the natural law view is based on the divine command theory in ethics.14 We mention this theory now because it would appear to be a moral tradition that would hold that HGLGT is inherently immoral if it amounts to an interference with the natural, human form (or humanness). Two other influential theories, as we have seen, do not view HGLGT as inherently immoral but hold that its morality depends on its relation to other moral considerations such as rights, benefits and harms, and justice. Our humanness, according to the natural law view, is naturally good and it is against nature and morality to change it. It is unnatural for people to be ten feet tall, to live to be 200 years old, to run as fast as a horse, and so on. It makes little difference to the natural law theorists whether HGLGT could have some good consequences for individuals or society or even whether prospective parents have a right to HGLGT. HGLGT is wrong because it is contrary to nature. The very idea of genetic meddling with the human form is morally repugnant, in this view.16 Indeed, natural law theorists oppose most forms of reproductive technology, including in vitro fertilization and surrogate pregnancy, on the grounds that these procedures are unnatural. There is no difference, in the natural law view, between Dr. Frankenstein and future genetic engineers. In both cases, the moral transgression occurs when science oversteps its bounds and interferes with the natural order of things.12,17 Despite the visceral appeal of the natural law position, it has some serious drawbacks that undermine its stance on genetic engineering. Many medical procedures are unnatural and interfere with the human form. It is unnatural to remove an appendix, to administer chemotherapy for cancer, or to delivery a baby through Cesarean section. But these medical procedures benefit patients, despite their unnaturalness. Some medical procedures that benefit patients also change or alter their human characteristics, such as amputation, sterilization, surgical removal of the larynx or breasts, and sex change surgery. But these procedures can benefit patients and so are regarded as morally justifiable. So why shouldn’t we treat genetics and reproduction like we treat oncology, plastic surgery and trauma care? If HGLGT can benefit patients without producing unacceptable harms—and this is of course a big “if ” at this point in time—then it should not matter whether it is unnatural. If people want to deviate from the natural, human form, and these deviations provide them with important benefits, then why should we stand in their way? Although we believe that the natural law position does not provide us with good reasons for refraining from genetic engineering—one cannot equate “immoral” with “unnatural”—we do recognize that some changes could be so unnatural that they should be forbidden. A person could be born so radically different from the rest of the population that they feel lonely, lack self-respect, and so on. A person created for the sole purpose of serving the state as a specially designed soldier might lack dignity, opportunities and self-respect. However, these extremely unnatural interventions would be immoral not because they are unnatural, but because they harm, violate or demean people. On the other hand, one might argue that some medical interventions harm, violate or demean people because they are unnatural. Leon Kass argues that reproductive technolo-

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gies dehumanize and depersonalize human reproduction by making reproduction into a manufacturing process where we attempt to perfect human beings.17 Human reproduction occurs naturally within a framework of love, spiritual connection, sex, marriage, and family. This framework constitutes part of our humanness. Reproductive technologies, including HGLGT, violate nature and our humanness and violate our human dignity. Kass articulates two reasons why reproductive technologies, including HGLGT, are immoral: 1. They violate the natural process of male/female procreation; 2. They make human life artificial.17 We believe that neither of these reasons show that reproductive technologies are dehumanizing or immoral. Without a doubt, reproductive technologies, including HGLGT, interfere with the natural process of reproduction and involve the fabrication of human life. But interfering with natural reproduction need not destroy the most important moral value shared by a man and woman in marriage, love. Couples who rely on in vitro technologies can certainly still love one another and achieve the “mysterious union” of marriage. They can still produce their own genetic progeny and cooperate together to raise their children.18 It is also true that human life takes on an artificial dimension when we modify, shape, and engineer human beings. But this element of artificiality need not destroy human dignity, self-respect, or self-worth. In a sense, many medical interventions introduce an element of artificiality into the human form. Today, human beings have artificial limbs and organs, metal plates in their heads, steel pins in their tibias, eyeglasses, hearing aids, pacemakers and so on. We have already crossed the boundary between man-made and naturemade; genetic engineering just takes us a step further. If patients with artificial hearts can still have self-respect and moral worth, then why can’t genetically engineered children still have self-respect and moral worth? Once again, we recognize that some interventions might be so unnatural that they would violate human dignity, but the “artificiality” of human life does not violate human dignity, per se.18

HGLGT and “Playing God” Although our main intent in this book is to address moral and political issues in HGLGT, in order to come to a fuller understanding of the natural law position we need to address some of the theological and religious questions raised by genetic engineering. Since the natural law approach looms as the strongest critic of any attempt to tamper with the human form, this theory deserves a full and fair hearing. In order to do this, we need to discuss the theological basis of natural law. As we noted earlier, many natural law theorists hold that moral principles are God’s commands and that some things in nature have moral value because God made them. This position places human beings in a subservient place in the natural order: Only God has the authority to make the natural laws and natural goods, and the main purpose of human existence is to obey these laws and promote these goods. Human beings who challenge natural laws or try to change natural goods commit the transgression known as “hubris:” Like Prometheus stealing fire, genetic engineers overstep their place in the world order and attempt to make decisions that properly belong to a divine being. Genetic tinkering is immoral and sinful because it is “playing God.” It is based on human arrogance, pride and selfishness.12,17,19 We examined a very different version of the “playing God” argument in chapter 6, a version that focuses on ignorance, not hubris. According to this other version of the argument, our lack of knowledge and wisdom requires us to refrain from trying to manipulate the human germline.2,12 Genetic engineering is immoral because only someone with God’s knowledge and wisdom could avoid its pitfalls and follies. According to the theological version of this argument, genetic engineering would still be immoral even if we had a complete

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understanding of human genetics and a powerful and infallible genetic technology, because we would be usurping God’s dominion and authority. Since arguments like these are based on a premise that cannot be rationally proven, one might argue that it is easy to dismiss them. The existence of God is a belief based on faith, not reason. Thus, arguments that assume the existence of God have no place in rational debates about morality, politics, law, and social policy. Since religion is based on faith or personal convictions, it has no place in public debates, one might argue. (We will not defend these views on the nature of religious belief here, but mention them as a reason why some people believe that religion should be excluded from public debates.) However, one does not need to exclude religion from the debate about HGLGT in order to address some of the concerns raised by the theological version of the “playing God” argument. One could accept the existence of God and challenge the natural law theorist’s claim that God would not want human beings to change the human form. One might argue that human beings can use science and technology to realize God’s will. Physicians who use medical technology to save or prolong lives are often accused of “playing God,” since only God has the authority to decide when we die. One way to respond to this charge is to argue that God would want us to use medical technology to save or prolong lives in many cases. Human hands and tools could implement God’s commands and decisions. Indeed, many religions defend the idea that we may use technology to improve on nature.20 But how do we know that God would want us to use technology to prolong lives? How do we know how we should improve on nature? Many physicians answer questions like these by appealing to commonly accepted moral principles, such as beneficence, nonmalificence, and autonomy. If a patient makes a rational decision to forego life-prolonging treatments or therapies, then physicians should respect that choice. God would not want us to use technology to prolong a person’s life if that person has decided to let nature take its course, one may argue. If a patient is brain dead and cannot benefit at all from the continuation of life-prolonging technologies, then physicians should withdraw those technologies. God would not want us to keep a brain dead body on a respirator, one might argue. The main lesson to draw from this discussion of “playing God” in medicine is that one may use moral and political concepts and principles to at least speculate about God’s will. We can attempt to “speak for God” even if we have no direct knowledge of His will.21 Indeed, this method of “speaking for God” would seem to be the only reasonable way of bringing religion into public debates. Although religious texts can offer moral insight and guidance, most are fraught with inconsistencies and must be interpreted in order to give practical solutions to today’s moral problems. But in interpreting religious texts, people invariably appeal to moral and political concepts and principles. If one wants to know whether The Bible permits euthanasia, it is not enough to simply cite biblical passages; one must also make sense of those passages, and “making sense” of scripture involves interpretation in light of reasoning about moral and political standards. Of course, some people claim to have a direct knowledge of God’s will through their personal communications with the Almighty. But how do we know that these people who claim to have a special revelation are not deluded, crazy or simply lying? What do we do when different people claim to receive conflicting messages from God? For these and other reasons, appeals to a special revelation have no place in public debates. If one extends this line of reasoning to HGLGT, then it follows that questions about “playing God” in the realm of human genetics should be answered by reasoning about problems and dilemmas in light of various moral and political principles and concepts. One might find that God’s will could be brought about through genetic technologies. If it is possible to follow God’s will in medical interventions, such as decisions concerning the

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prolongation of life by artificial means, then it should also be possible to follow God’s will in genetic interventions, such as decisions concerning the use of reproductive technologies to prevent diseases. In order to make decisions like these, one must go beyond dogmatic assertions like “all genetic engineering is playing God” and engage in serious moral and political inquiry and debate. This is the kind of dialogue that we have tried to encourage in this book.

The “Who Decides?” Question Having addressed the natural law tradition’s perspective on HGLGT, we will examine some other arguments against HGLGT that question the morality of changing the human form. Concerns about changing the human form often include a series of rhetorical questions asking, “Who decides?”2 Who decides what traits should be eliminated or promoted? Who decides what will count as a normal or healthy human? One way of construing the “Who decides?” argument is that it asks whether the government or some other powerful social institution should be allowed to genetically engineer a population. Thus, the argument may be suggesting that genetic engineering could lead to a Brave New World scenario where a totalitarian government shapes the human population. Or, perhaps the argument expresses the worry that the human population’s genetic composition will be chosen by politically or economically dominant groups in society. In either case, the argument would be wondering what could happen to individual rights and liberties if genetic engineering becomes a reality. We have addressed these points in earlier chapters. If these rhetorical questions constitute an argument for allowing individuals to make their own reproductive decisions and for avoiding socially imposed, genetic norms, then we concur.13 People should be allowed to make their own reproductive choices as long as these decisions do not harm children, they do not undermine equality of opportunity, and they do not threaten human genetic and phenotypic diversity. The “Who decides?” argument could also be construed as an attack on the distinction between medical and nonmedical eugenics or between therapy and enhancement. The argument could be suggesting that once we begin deciding which kind of people are “fit” or “unfit,” then people who lack genetic enhancements or who are less than perfect will come to be viewed as “diseased” or “unfit.” Although we agree that there is not a sharp distinction between therapy and enhancement, we believe that this distinction has a basis in human biology (see chapter 5).

HGLGT and Human Perfection One final point we would like to discuss in this chapter on our humanness is the idea that genetic engineering involves an attempt to perfect the human race or produce “perfect” people (we mentioned this concern briefly in chapter 6). The topic of human perfection reminds us of a tale by Nathaniel Hawthorne, “Dr. Heidegger’s Experiment.” In this story, Dr. Heidegger falls in love with a woman who is “perfect” in every way except for a heartshaped beauty mark. He succeeds in removing the beauty mark and in “perfecting” her beauty, but the experiment kills her.22 One might argue that people who seek to engineer human beings are like Dr. Heidegger and that such attempts will only lead to disaster and folly. There are two different versions of the perfection argument. According to the theological version, we should accept the human form as it is without attempting to improve it or make it better because it has been designed by God, and only God has the wisdom and authority to make human beings.19 According to the secular version, the human form is the result of millions of years of evolution—it has been “designed” by natural selection—and we lack the wisdom to tamper with its structure or organization.12,23 Both of these arguments against attempts to “improve” or change the human form suffer from some difficulties. The theological argument overlooks the fact that people are

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born with genetic diseases. Even if we accept the idea that we do not have the wisdom or authority to alter the “normal” human form, we should be allowed to prevent or treat diseases, unless one takes the extreme view that genetic diseases are God’s “punishment.” The secular argument assumes a Panglossian view of evolution, i.e., natural selection produces organisms that are perfectly adapted to their environments. But this idea is little more than a neo-Darwinian myth, since we know that many organisms are born with maladaptive traits, many traits result from genetic drift, and other traits are adaptively neutral. Natural selection “tinkers” with existing organisms but it does not “design” perfect organisms.24 Thus, there would seem to be some room for improving the human form or for at least eliminating nature’s various errors and mistakes, so long as one realizes that evolution is not perfect. However, we do agree that there are some serious problems with any attempt to “perfect” the human race. In chapter 6 we discussed some of the serious evolutionary and psychosocial harms that genetic manipulations could cause. A serious attempt to “perfect” the human gene pool could have a very negative effect on the genetic variability of our species,25 and viewing genetically “imperfect” people as “damaged goods” contributes to discrimination, prejudice and bias. 26 Thus, the notion that humans could be “perfected” in any sense of this word is a fallacy, and this idea needs to be dispelled. We agree with the moral of Hawthorne’s tale: People cannot be “perfected” any more than anything in this world can be “perfected.” Just as there is no perfect car, there is not, nor will there ever be, a perfect human being. Indeed, one might argue that the notion that we are “imperfect” is part of what makes us human. By recognizing that humans are imperfect, we are able to develop moral virtues such as forgiveness, tolerance, patience, cooperation, sympathy, courage and humility. By struggling with our own imperfections and the imperfections we find in others, we develop many of the psychological and social traits that contribute to our humanness. A genetically engineered, “perfect” being who did not understand what it means to be an imperfect human would therefore lack some important human qualities and might not able to participate in our moral and social practices. (We note that the question of cloning human beings raises similar questions about perfecting humans, but we will not explore this topic here.27) However, even though we should not regard humans as perfectible artifacts, we can recognize that imperfect beings can and should strive toward certain goals or ideals, even if many of these goals will always be beyond reach. Accepting our imperfections does not mean an end to the quest for excellence. Some things can be improved in certain respects, and so can human beings.28,29 Just as we can eliminate safety problems from automobile designs, we can also eliminate genetic diseases. We could, in principle, enhance human beings in various ways. However, all attempts to change humanity should be buffered by humility and realism: We can strive for progress, not perfection. We must accept our limitations and not succumb to Faustian delusions of grandeur.

Conclusion In this chapter we have examined several different arguments relating to HGLGT’s affects on our humanness. We have argued that HGLGT can change our humanness but that there is nothing inherently immoral in changing the human form. Whether it is immoral or unjust to reengineer the human population depends on how these changes affect the structure of society and the health and well-being of our future progeny. Without a doubt, we can make changes in our humanity that would cause devastating harms to individuals, society and future generations, and these kinds of changes should not be allowed. Since even “minor” genetic manipulations can have significant effects, it is important to

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carefully scrutinize any attempt to alter a human genome. Any tinkering with the human form must be made with a keen eye to benefits, harms, utility, human rights and justice.

References 1. Gaylin W. The Frankenstein factor. New Eng J Med 1977; 297 (12): 665-666. 2. Boone C. Bad axioms in genetic engineering. Has Cen Rep 1988; 18 (4): 9-13. 3. Aristotle. Nichomachean Ethics. Irwin T, tran. Indianapolis: Hackett Publishing CO, 1985, 1098a20. 4. English J. Abortion and the concept of a person. Can J Phil 1975; 5(2):233-243. 5. Mayr E. Animal Species and Evolution. Cambridge, MA: Harvard University Press, 1963. 6. Parens E. Taking behavioral genetics seriously. Has Cen Rep 1996; 26(4): 13-18. 7. Anderson W. Genetic engineering and our humanness. Hum Gen Ther 1994; 5: 755-760. 8. Hubbard R and Wald E. Exploding the Gene Myth. Boston: Beacon Press, 1993. 9. Nelkin D and Lindee S. The DNA Mystique. New York: WH Freeman, 1995. 10. Davis B. Evolution, epidemiology, and recombinant DNA. In: Jackson D and Stich S, eds. The Recombinant DNA Debate. Englewood Cliffs, New Jersey: Prentice-Hall, 1979; 25-35. 11. Stich S. The recombinant DNA debate: Some philosophical considerations. In Jackson, D and Stich S, eds. The Recombinant DNA Debate. Englewood Cliffs, New Jersey: PrenticeHall, 1979; 78-90. 12. Rifkin J. Algeny. New York: Viking Press, 1983. 13. Kitcher P. The Lives to Come. New York: Simon and Schuster, 1997. 14. Barcalow E. Moral Philosophy. Belmont, CA: Wadsworth, 1994. 15. Vatican declaration on some questions of sexual ethics. In: Mappes T and Zembaty J, eds. Social Ethics, 3rd ed. New York: McGraw-Hill, 1984. 16. Sullivan J. The immorality of euthanasia. In: Kohl M, ed. Beneficent Euthanasia. Buffalo, NY: Prometheus Books, 1975. 17. Kass L. Toward a More Natural Science. New York: The Free Press, 1985. 18. Robertson J. Children of Choice. Princeton: Princeton University Press, 1994. 19. Ramsey P. Fabricated Man. New Haven: Yale University Press, 1970. 20. Cole-Turner R. Genes, religion and society: The developing views of the churches. Sci Eng Eth 1997; 3: 273-288. 21. Peters T. Playing God. New York: Routledge, 1997. 22. Hawthorne N. Complete Short Stories. Garden City, NJ: Hanover House, 1959. 23. Chargaff E. On the dangers of genetic meddling. Science 1976; 192: 904-938. 24. Gould S. and Lewontin R. The spandrels of San Marco and the Panglossian paradigm: A critique of the adaptationist programme. Proc Roy Soc Lon 1978; 205: 581-98. 25. Suzuki D and Knudtson P. Genethics. Cambridge, MA: Harvard University Press, 1989. 26. Council for Responsible Genetics. Position paper and germ line manipulation. Hum Gen Ther 1993; 4: 35-37. 27. Robertson J. The question of human cloning. Has Cen Rep 1994; 24 (2): 6-14. 28. Glover J. What Sort of People Should There Be? New York: Penguin Books, 1984. 29. McGee G. The Perfect Baby. Lanham, MD: Rowman and Littlefield, 1997.

CHAPTER 10

Public Policy Issues S

o far we have provided a scientific and technical background to human germline gene therapy (HGLGT) in chapters 1 through 4 ,and have examined moral and political issues relevant to HGLGT in chapters 5 through 9. In this final chapter, we examine social policy questions.

Science and Public Policy Before tackling some of these policy issues, we need to discuss the relation between science and public policy. Policy debates are concerned with formulating rules, regulations, and laws that govern the conduct of individuals and institutions. These debates make use of facts as well as values. The “facts” in policy debates often include relevant information from science, medicine, engineering, and technology; “values” often include moral, political, religious, philosophical, social, and legal norms and standards.1 We have included discussions of both facts and values in this book. Even though most people accept a distinction between “facts” and “values,” this distinction is not as absolute or clear cut as one might expect, since moral, political, social and religious norms can influence scientific and technological decisions.2 Even though we recognize that science is sometimes not as objective (or value-free) as one might expect, this does not mean that we should not distinguish between science and values, or that scientists should not strive to be objective. Indeed, social policy formation would grind to a halt if we did not assume that there are facts that can be employed in public debates.3 If we did not make this assumption, policy debates would be nothing more than moral, political, religious and philosophical quagmires. Facts play a key role in policy formation by serving as islands of agreement in the seas of controversy. To give an example of how this combination of facts and values interacts in policy formation, consider regulations on smoking in public places.4 The facts would include information about the effects of secondhand smoke on human health, as well as information about ways of disposing of smoke, and information about the economic impact of smoking on restaurants, bars, sports arenas, airlines, and so on. The values would include laws pertaining to individual liberties, as well as moral norms such as the principle of autonomy, the harm principle, and the principle of utility. One can see that facts and values are essential to an effective and rational solution to this problem, since someone who insisted that there should be no restrictions on smoking in public places could be accused of either ignoring the facts (smoking imposes risks on others) or denying important values (the harm principle). In resolving an issue like smoking in public, policy makers need to balance competing rights, values, and interests (smokers, nonsmokers, businesses) in light of scientific, medical, technological, and economic facts. The rules and regulations that emerge from this policy formation process aim to enact workable compromises rather than partisan solutions. In this book, we have discussed scientific, moral, and political issues relevant to HGLGT policy formation, but we have not discussed legal issues. However, we need to discuss the relation between morality/politics and the law in order to give some indication of how policy Human Germline Gene Therapy: Scientific, Moral and Political Issues, by David B. Resnik, Holly B. Steinkraus, Pamela J. Langer. ©1999 R.G. Landes Company.

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debates relate to laws. Policy debates often lead to legal actions such as the enactment of statutes, institutional guidelines, civil codes, or the formation of judicial decisions. For example, many cities have passed ordinances banning smoking in various kinds of public places as a result of public policy debates like the one discussed above.4 Obviously, HGLGT policy questions are far more complex than questions about smoking in public, but policy formation should follow the same kind of procedure that we can use in regulating smoking in public. Now, one might object to the suggestion that social policies can or should lead to the formation of laws on the grounds that “You can’t legislate morality.” Many people would argue that moral, religious, and other values should be a matter of free choice. Values are private; laws are public. The state should not use its coercive power to intrude into private, personal matters. This objection has some merit, but it misunderstands the relation between morality/politics and the law. Many of our laws are based on strongly held moral and political convictions. For instance, laws against murder are based on the respect for human life; laws against stealing are based on the respect for property; laws permitting free speech are based on an understanding of the value of freedom; and so on. A nation’s laws should reflect the moral and political judgments of its citizens. Where a society has solid agreement on moral and political values, these values can and often should become laws. Indeed, one might argue that the sole function of the legal system is to protect social order and to promote social values.5 One can extend this line of argument to international laws: These laws should also reflect a moral and political consensus reached by different nations. Policy debates can provide some guidance for the law by allowing people to express their opinions on issues that have moral and political dimensions. Sometimes those debates result in a convergence of views, and sometimes they do not. If a nation enacts a law, even though its people do not agree on the moral or political basis of the law, then many people will disobey the law and it will be ineffective. People need a moral or political rationale for obeying the law.5 For example, one might argue that the US government should not make abortion illegal on the grounds that there is not sufficient public consensus on this issue. If abortion were made illegal, then it is likely that many women and physicians would disobey this law, “back alley” abortions would proliferate, etc.6 Therefore, according to this argument, abortion in the US should be a private decision. This does not mean that abortion should never be illegal, of course, since US citizens may reach sufficient agreement on the question of banning abortions. Many of the points we have made in the last few paragraphs also apply to HGLGT policy debates. The relevant facts should include information from the biomedical and social sciences as well as information about genetic and reproductive technologies. Does society currently have all the “facts” it may require? Of course not. But ignorance should not prevent us from formulating some preliminary policies that would be revised in light of new information. We would never be able to formulate any effective policies if we refused to have public debates until “all the facts are in.” Sometimes we can afford to wait for additional information, but we usually do not have this luxury. In the case of HGLGT, rapid advances in science, medicine, and genetic and reproductive technologies require us to make some policy decisions before we have a complete understanding of all the relevant facts. The values pertaining to HGLGT policies include the various moral and political principles and concepts that we have discussed in this book, such as the principles of beneficence, nonmalificence, autonomy, utility, justice, and the concepts of harm, benefit, equality of opportunity, individual rights, and so on. Since this book is not intended to discuss legal issues per se, we will not discuss the laws that have some bearing on HGLGT, although we will make some recommendations for people who debate, discuss and make laws. We will

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now discuss some specific policy questions in light of our assessment of the facts and values pertaining to HGLGT.

Research Restrictions When research is controversial or dangerous, societies often take steps to restrict it. Some options for restricting research are as follows: 1. Impose a ban or moratorium on research, e.g., Italy and other European countries banned Copernican astronomy for several centuries; 2. Allow research to take place under highly controlled or regulated conditions, such as research on fissionable materials or narcotics; 3. Allow research to take place with moderate controls, such as research on human subjects; 4. Allow research to take place with minimal controls, such as research on computer software. Questions about restricting research relating to HGLGT are by no means remote, since research has, in many senses, already begun. Here it will be useful to distinguish between direct and indirect research: direct research would be an actual clinical trial involving human subjects (parents and probands); indirect research would include research that is likely to have important impacts on our understanding of HGLGT, such as genetic engineering in animals, somatic gene therapy (SGT) in humans, research on cloning mammals, preimplantation genetic diagnosis and embryo selection, the Human Genome Project, and so on. Although we are not aware of any direct HGLGT research, a great degree of indirect research goes on every day. In thinking about whether HGLGT research should be restricted, it is important to note that restrictions on research can threaten scientific progress. First, restrictions can clamp down progress on specific research questions or problems (such as HGLGT). From the 1930s to the 1960s, the Soviet Union banned research on Mendelian genetics because one of the key assumptions of Mendelian genetics—the idea that many traits have genetic causes— threatened that government’s Marxist ideology. As a result of this ban, Soviet biology lagged behind Western biology for many years.7,8 Second, broad restrictions can have unintended, negative affects on progress. For example, many scientists worry that a ban on human cloning would also prevent useful and uncontroversial research, such as research on cloning cells for use in human tissue regeneration.9 Third, restrictions can have a “chilling” effect on the research environment. Science generally thrives in nations that value freedom of inquiry; it languishes in nations that maintain tight control over ideas and communication.7,10 Finally, we should also mention that restrictions on research can threaten moral and political values, such as individual autonomy and liberty of thought and expression.7 Since research restrictions can threaten scientific progress and moral and political values, any proposed restriction must be backed by solid moral or political arguments. Are there some good reasons for banning HGLGT research at present? We believe that there are. First, HGLGT currently poses unjustifiable risks to unborn children; it is neither safe nor effective. Society should refrain from performing this research until it is less risky. Second, people have not had enough time to discuss the complex social issues surrounding HGLGT, such as genetic enhancement and altering the human form. It could be useful to have a “cooling off ” period while people think about the ethical, political and social aspects of this research. Thus, there is a strong justification for a moratorium on all direct HGLGT research. A moratorium should exist until HGLGT becomes more safe and effective and society has resolved some of the moral and political questions surrounding HGLGT.

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Although human germline gene therapy is more complex than animal germline gene transfer, progress on genetic engineering in animals gives us some reasons to expect that requests for HGLGT clinical trials may not be as far away as one might think. If these clinical trials take place, researchers should follow the legal and ethical regulations that apply to other kinds of research on human subjects, including informed consent, preservation of confidentiality, preliminary animal studies, sound research design, data monitoring, and so on.11,12 The first HGLGT clinical trials should be conducted for medical reasons on subjects who have very much to gain and not much to lose, such as the use of homologous gene replacement to prevent a future child of two parents with cystic fibrosis from being born with cystic fibrosis. Only after scientists have had success with the procedures that are less risky and controversial should we even consider clinical trials for more risky or controversial procedures, such as HGLGT for polygenic diseases or HGLGT for nonmedical reasons. Although we recommend a moratorium on direct HGLGT research, we do not recommend a moratorium on indirect HGLGT research, since this ban would place unjustifiable restrictions on research and hamper important and useful developments in molecular biology, genetics, medicine, and biotechnology. Although indirect HGLGT research may provide the technical and scientific background for conducting direct HGLGT research, this is the price we must pay for obtaining the benefits of the genetic revolution. In addressing our fears about the genetic engineering of human beings, we should not kill the goose that lays the golden egg. In order to make sure that a ban on direct research does not adversely impact useful indirect research, any restrictions must be worded carefully, since laws and regulations that are too broad might prevent scientists from conducting useful research. For example, researchers are developing a procedure that uses embryonic germ cell precursors to repair dysfunctional tissues.13 The germ cell precursors may have the potential for developing into many kinds of tissues. Furthermore, to avoid tissue rejection, it might be possible to genetically engineer the cells so that they would match the recipient’s immunologic characteristics.13 Research like this could provide valuable new approaches in treating Parkinson’s disease, Huntington’s disease, diabetes, and a variety of illnesses where dysfunctional tissues need to be replaced or regenerated.13 If this research were conducted on humans, it might be classified as a type of HGLGT since it would involve transferring modified germline cells to another human, even though the germ cells would not be destined to produce a new individual. Thus, even a ban on HGLGT must be worded in such a way that it does not stop potentially useful, safe, effective and uncontroversial research.

HGLGT Funding A less extreme, though often effective way of controlling research is to deny government funding. The denial of funding can be an effective deterrent to research, since scientific research usually requires a great deal of money, and governments fund the majority of basic research conducted in Western nations. In recent years, the US government used this ploy to deter research on fetal tissue transplants and the cloning of human embryos.7,14 We recommend that governments do not provide funding for direct HGLGT research because this research is still very risky and people need more time to think about the social issues it raises. On the other hand, we do not recommend that governments deny funding to indirect HGLGT research, since it is important to fund research in genetics, molecular biology, medicine and biotechnology, even if this research may have some implications for HGLGT. For example, germline research in animals has some important implications for HGLGT, but we should continue funding research involving the construction of transgenic animals in order to reap the benefits of genetic engineering, such as the production of human hor-

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mones and enzymes in animal “bioreactors”. One might propose funding restrictions on only certain types of genetic research with clear and obvious implications for the genetic engineering of human beings. However, this kind of policy would probably have to extend to research on SGT in humans and germline gene transfer experiments in animals. Any funding restrictions on indirect research would require that scientists curtail or stop ongoing research programs that benefit people and contribute to the advancement of science. Like it or not, scientists around the world are now conducting research with applications for the genetic engineering of human beings, and it is too late to close Pandora’s box. It is possible, of course, that direct HGLGT research might one day merit government funding. However, decisions to fund medical research should reflect society’s medical needs, goals, and priorities. If scientists and the public decide that some other areas of research take precedence over HGLGT, then HGLGT should have a lower ranking in the research budgets of those branches of government that would fund this work, such as the National Institutes of Health. If direct HGLGT research becomes safe, effective, and economically sound, one might still claim that this research should not be funded by the government, on the grounds that it is controversial. One might argue that government funding of research should reflect the “will” of the people, and if a majority (or a highly vocal minority) of a nation’s citizens object to a particular area of research, then it should not be funded. This argument has some merit, we believe. Certainly public funds should not be spent on projects that lack public support. But how do we know when research lacks public support? If interest groups or concerned citizens object to the research, does this mean that the research should be not be funded? If we refused to fund research that is opposed by various interest groups, then we would stop funding all research that uses animal subjects, since animal rights activists oppose this kind of testing on moral grounds. We would also refuse to fund research in evolutionary biology, since many religious groups object to the theory of evolution. Although there are sometimes sound moral and political arguments for refusing to fund certain areas of research, we need to be careful not to play political games with our science policy. Science has always met with moral and political opposition, and this will continue to be the case for the foreseeable future. A government that funded only those areas of research that have a great deal of public support would end up funding very little scientific research. Since government funding of scientific research benefits science and society, and since science does not flourish in an environment where it is constantly subject to intense moral and political scrutiny, most research should not have to pass moral and political litmus tests. Political and moral opposition to a study can provide a compelling reason for denying funding to the study, but funding should be denied on this basis only when opposition is strong and united. Of course, scientists should not have a “blank check” to pursue any kind of research that they find interesting, since all research still must go through several levels of peer and institutional review before it is funded. However, there are appropriate and inappropriate ways to bring moral and political considerations to bear on funding decisions. Even if governments do not fund direct HGLGT research, it is conceivable that business and industry, e.g., biotechnology companies, might fund this kind of research, since direct HGLGT research could result in products, services, and technologies that satisfy consumer demands at some point in time.15 Even if some countries outlaw direct HGLGT research, private corporations could still conduct this research in countries that have not banned this research. Thus, any effective ban on direct HGLGT research would require the voluntary cooperation of business and industry. Private corporations might agree to refrain from conducting direct HGLGT research on a temporary basis, but this policy could change if HGLGT becomes a profitable venture. Even if private corporations fund direct HGLGT

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research, a ban on the use of government funds for this type of research could have some symbolic and political importance. Our approach to restrictions on research reflects a policy of prudence rather than optimism or pessimism, which we discussed in chapter 6. The extreme pessimist would try to stop all research that could lead to HGLGT; the optimist would promote all genetic research with little thought to its consequences. We believe that at the present time some types of research should be banned, some types of research should be denied funding, some types of research should be permitted, and some types of research should be permitted and funded. We also believe that the scientific community and society at large should continually reexamine funding priorities and research policies in light of new developments in science and technology.

Privacy In chapter 7 we argued for protecting the confidentiality of genetic information for people who are the products of HGLGT, and we reiterate this point here. Genetically engineered children will be especially vulnerable to invasions of privacy because the very act of creating these children will require scientists to obtain genetic information about them. This genetic information should belong to parents and their children and should not be made available to insurance companies, employers, and other interested parties without a compelling rationale. We can translate this moral stance into a public policy by demanding legal protections for confidential, genetic information. Since we already have laws protecting the confidentiality of medical records, it would make sense to extend these laws to HGLGT records. What counts as a “legitimate use” of genetic information is not an easy question to answer, of course, and we will not attempt to resolve this issue here. Obviously, confidentiality should not be invaded or broken for trivial reasons, and one would need a very important, overriding reason to gain access to genetic information.16,17

Discrimination and Bias In chapters 8 and 9 we argued that we need to take various steps to protect people from discrimination, prejudice and bias, and we reemphasize this point here. Society should protect people who are the products of HGLGT as well as people who are not “lucky” enough to benefit from this technology. Currently existing discrimination laws can and should play an important role in protecting people against genetic discrimination.18 These laws need to be updated and reinterpreted in order to provide people with sufficient protection against genetic discrimination. But discrimination is more than just a legal problem; it also has cultural and psychological components. In order to address these social dimensions of discrimination and bias, society also needs to educate people about human genetics, genetic differences, and genetic similarities.17 Although education is not a magic cure for social problems, it can help people overcome their biases and prejudices and understand their similarities, differences, opportunities and shortcomings.

HGLGT Patents Ever since the US Patent and Trademark Office (PTO) awarded a patent for a form of gene therapy technology several years ago, a dispute has raged over the legality and morality of patenting human genes.19 Defenders of human gene patents argue that patenting encourages commercial investment in genetic research, which will lead to medical, scientific and technological advances. These advances, in turn, will have beneficial social consequences, such as improved health and well-being, economic growth, and so on. Although we do not believe that any patents relating directly to HGLGT are currently pending, many patents

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relating to indirect research have been awarded. This raises the issue: If HGLGT technologies are ever developed, should they be patentable? To answer this question, we will give a brief summary of US patent laws (many countries have similar laws). According to US laws, a patent is granted by the PTO and gives the patent holder exclusive rights to make, use or sell an invention for a 20 year period. To obtain a patent, one must submit an application to the PTO. The application must contain enough information for someone who is skilled in the relevant technical field to make and use the invention. According to US laws, a patent must demonstrate that the invention has novelty, utility and nonobviousness.20 Scientific discoveries (e.g., data, concepts or principles) or naturally occurring phenomena are not patentable.19 According to US patent laws, there would be a legal basis for owning the following HGLGT technologies: 1. Genetic technologies, i.e., processes, methods, or procedures for analyzing, cloning, sequencing, mapping, transferring or manipulating genes 2. Artificial genes, i.e., new genes or combinations of genes, such as superalleles or xenogenes (see chapter 6), or new genomes. There would not be a legal basis for owning naturally occurring human or animal genes, according to US patent laws, unless such naturally occurring genes are altered or otherwise made to exist in a form not found in nature.19 Even if there is a legal basis for patenting HGLGT technologies, one might still object to the patenting of these inventions on moral grounds. We shall consider two main arguments against patenting related to HGLGT: 1. No one should be able to patent human genes because treating genes as intellectual property objectifies and demeans human life; 2. If genes can be viewed as property, they should be treated as common property, belonging to no particular person or corporation.19 Although we have not discussed the morality of human gene patents to this point in the book, we will comment on these issues since they have important implications for HGLGT policy. To understand the first objection, we need to introduce the Kantian tradition in ethics, which holds that individual human beings have inherent (or intrinsic) moral worth and should not be treated as having only extrinsic worth. People should not be bought, sold, objectified, manipulated, or demeaned. On the surface, it seems like human gene patenting could objectify and demean human life by treating human beings as commodities. We agree with the Kantian view that people should not be treated as having only extrinsic worth and we would object to any form of ownership that objectifies or demeans human life. However, most forms of gene patenting would not treat people as having only extrinsic value. Gene patents only allow individuals or corporations to own genetic technologies or artificial genes. Ownership of a genetic technology, such as a process for transferring a gene, is analogous to ownership of any other medical technology. Unless one holds that patents on stethoscopes, HIV tests, CAT scanners, and flu vaccines objectify or demean people, then one should not claim that patents on genetic technologies objectify or demean people. Ownership of an artificial gene would be analogous to ownership of an artificial body part, such as an artificial heart or hip. Thus, ownership of a single artificial gene or even a gene sequence does not constitute ownership of a human being any more than ownership of a bolt or wheel constitutes ownership of a car. If we think of genes as like other body parts, then ownership of artificial human genes should be no more objectionable than ownership of blood, blood products, or artificial body parts.19 However, there are some forms of ownership that would objectify or demean people. For example, biotechnology companies now own patents on genetically engineered mice.19

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These mice are regarded as inventions because they have artificial genotypes and phenotypes, such as genes that predispose them to specific forms of cancer. This kind of patenting would objectify and demean people and should not be permitted. Thus, HGLGT probands should not be patentable. Ownership of an entire human genome by one company or person would be morally objectionable. Suppose that a company, an individual, or conglomeration of companies invents a human genome and markets it to prospective parents. By owning an entire genome, the company would have ownership over the production of a human being. Although the human body and person are not identical, they are so intimately connected that ownership of a human genome is tantamount to ownership of a human being.19 According to the second objection, human genes should be treated as common property, not as personal property. Human genes belong to all of humanity, one might argue, because we share so many genes and this sharing of genes prevents ownership. We can no more claim personal ownership of genes than we can claim personal ownership of the air. The problem with this argument against gene patenting is that it is based on a misunderstanding of human gene patents. Those who attempt to patent genes do not attempt to own original, human genes; they attempt to own genetic technologies or artificial genes. These inventions are not shared among all people of the world, even if many of the original genes are shared. As an analogy, no one claims to own carbon, which is common property, although companies can own processes for making, analyzing, or purifying carbon. Companies may also own artificial compounds made out of carbon, such as plastic. The same point applies to human genes. So, patents on inventions related to human genes can be viewed as personal rather than common property. Thus, patent laws should permit the patenting of genetic technologies or artificial genes, but they should not permit the ownership of an entire human genome or an HGLGT proband by one individual, company, or conglomeration of companies. If private corporations or individuals develop inventions related to HGLGT, then they should be able to patent those inventions, provided that these inventions are on sound moral and legal ground. Of course, one might object that patent laws would be difficult to enforce, since they would require a great deal of international cooperation. This concern brings us to the next topic of this chapter.

International Cooperation In the not too distant future, the knowledge gained from the Human Genome Project and other scientific programs relating to human genetics will be available all over the world. Genetic technologies and biotechnological industries will soon spread from Western to nonWestern nations and span the globe. Thus, a great deal of international cooperation will be required to enforce any policies relating to human genetics. For example, it would take substantial international cooperation to enforce a moratorium on direct HGLGT research, since HGLGT researchers could migrate to those countries that do not ban HGLGT research. Many countries have already initiated cooperative, bioethics efforts through international conferences, societies, meetings, committees, policy boards, and so on.21,22 These efforts should not only continue, but they should be expanded in response to the ongoing advances in human genetics. All of the policy issues we have discussed would require some kind of international cooperation. Unilateral actions and policies are not likely to be very effective in a world where information, markets, corporations, technologies and social institutions cross international boundaries. However, even if people from different nations come together to discuss policies and laws relating to human genetic engineering, it would be naïve to expect that a global consensus would emerge. Just as we have seen international controversies over the

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environment, human rights, economic development, trade, intellectual property and arms control, we should also expect to see disagreements over human genetic engineering, if HGLGT is developed and proves to be useful. Some nations may oppose all forms of HGLGT, while others may not; some nations may allow companies to develop human genes or genetic technologies for use in genetic engineering while others may not. The possibility of fundamental disagreements over HGLGT policies does not mean, of course, that we should not try to promote international cooperation on issues in human genetics. However, we should have realistic expectations about the outcomes of these efforts. It may not be possible to ratify or enforce international agreements on human genetic engineering even if a majority of the world’s nations accept similar policies or have similar laws. A significant minority of the world’s countries may not accept the views of the majority, and some countries may want to assert their national sovereignty and independence. We need look no further than to disputes about nuclear weapons proliferation, global warming, population control or free trade to understand the difficulties of obtaining international cooperation.

Monitoring Research Since human genetic engineering raises so many important moral, political, and social issues, the scientific community should carefully monitor and study new developments in HGLGT research and applications. The world needs to know whether any scientists are conducting germline therapy on human beings, whether any nations allow or even fund HGLGT clinical trials, and whether any companies are sponsoring HGLGT research. In order to monitor new developments in HGLGT research and applications, it would be wise to establish a genetic engineering “watchdog” group similar to the Worldwatch Institute, which provides an annual summary of population growth, resource depletion, topsoil erosion, pollution, and other factors relating to the “physical health” of our planet.23 A watchdog group could provide people with the information they need in order to formulate sound social policies pertaining to HGLGT. Since HGLGT raises issues that concern all people in the world, this watchdog group should have an international composition and constituency. It should share its information with concerned citizens, the media, government officials, policy analysts, scientists, clergy, corporations, and other people who have a stake in HGLGT. Even those people who are strongly opposed to HGLGT would agree that the world needs to form a watchdog group, since we cannot regulate or prevent what we do not understand. The analogy with nuclear weapons fits here: People on all sides of the nuclear weapons issue can agree on the need to gather information about weapons proliferation, nuclear arsenals, nuclear tests, research and development programs, and so on.

Welfare and Enhancement The last policy issue we will discuss relates to questions about justice that we discussed in chapter 8. In that chapter we argued that governments should take some steps to promote equality of opportunity. First, governments may need to subsidize genetic counseling, embryo selection and other technologies used to prevent people from being born with genetic disadvantages. Genetic counseling and other reproductive services should be optional; governments should not use coercive means to promote equality of opportunity. A “genetic welfare” program would help to insure that the genetic revolution does not exclude the poorer members of society. People should not be born with genetic diseases because their parents could not afford genetic counseling. If wealthy people are allowed to prevent genetic diseases, this privilege should be extended to the poor. Second, if HGLGT is developed and widely used, governments may need to place restrictions on genetic enhancements in order to prevent parents from producing genetic “supermen.” It may be necessary to prevent people from exceeding some maximum limits

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in order to insure the survival of an egalitarian society. Although genetic enhancement remains in the realm of science fiction, society may need to impose some restrictions on the use of HGLGT for enhancement purposes at some point. Of course, any attempt by the government to control the gene pool is a form of governmental eugenics, and we recognize the problems and dangers of this type of policy. But totally unregulated genetic engineering could undermine our notions of justice, fairness, and human equality; it could lead to the end of human society as we know it and the emergence of a Brave New World.

Conclusion HGLGT raises more policy issues than the ones we discuss here, of course, and there are many additional issues that will not emerge until the manipulation of human genes has advanced well beyond its current state. We have tried to address some of the important policy concerns in this book, but we cannot hope to cover every problem or question relating to HGLGT. As we remarked in the beginning of this book, we also do not expect that all readers will agree with our conclusions and opinions. We have tried to present a fair and balanced discussion of the scientific, moral and political issues in HGLGT, not the last word on this complex subject. Hopefully, those who have read this book now have a deeper, clearer, and more comprehensive understanding of these issues, which will enable them to engage in rational debates and discussions about this fascinating and powerful technology.

References 1. Buchanan A and Brock D. Deciding for Others. Cambridge: Cambridge University Press, 1989. 2. Longino H. Science as Social Knowledge. Princeton, NJ: Princeton University Press, 1990. 3. Bimber B. The Politics of Expertise in Congress. Albany, NY: State University of New York Press, 1996. 4. Goodin R. The ethics of smoking. In: Arthur J, ed. Morality and Moral Controversies, 4th ed. Upper Saddle River, NJ: Prentice-Hall, 1996. 5. Hart H. The Concept of Law. Oxford: Clarendon Press, 1961. 6. Callahan, D. The abortion debate: Can this chronic public illness be cured? Clin Obst Gyn 1992; 35(4): 783-91. 7. Resnik D. The Ethics of Science. London: Routledge, 1998. 8. Dickson D. The New Politics of Science. Chicago: University of Chicago Press, 1984. 9. Marshall E. Biomedical groups derail fast-track anticloning bill. Science 1998 279: 1123-1124. 10. Merton R. The Sociology of Science. Chicago: University of Chicago Press, 1973. 11. Anderson W. Human gene therapy: Scientific and ethical considerations. J Med Phil 1985; 10: 275-91. 12. Zimmerman B. Human germ-line therapy: The case for its development and use. J Med Phil 1991; 16: 593-612. 13. Beardsley T. Culturing new life. Sci Am 1998; 278 (6): 11-12. 14. Kolata G. With cloning of a sheep, the ethical ground shifts. New York Times, 24 February 1997: A1, B8. 15. Gardner W. Can genetic enhancement be prohibited? J Med Phil 1995; 20: 65-75. 16. Resnik D. Genetic privacy in employment. Pub Aff Quar 1993; 7(1): 47-56. 17. Kitcher P. The Lives to Come. New York: Simon and Schuster, 1997. 18. Natowicz M, Alper JK, Alper JS. Genetic discrimination and the law. Am Jour Hum Gen 1992; 50: 465-75. 19. Resnik D. The morality of human gene patents. Ken Inst Eth Jour 1997; 7(1): 31-49. 20. Flanagan JK. Gene therapy and patents. J Patent Trade Off Soc 1998; 80(10):739-751.

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21. Inaugural Conference-International Association of Bioethics. Bioethics News 1992; 12(1): 11. 22. Deadlock broken as first international bioethics treaty is signed. Lancet 1996; 347(9016): 1686. 23. State of the World. Washington, DC: Worldwatch Institute, 1996.

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GLOSSARY Important note: The definitions supplied in this glossary are closely linked to the usage in the text and are not necessarily meant to be a comprehensive definition of the term. For terms which we are introducing in this book or for ones which are not in common usage, the definition contains the phrase “we define...” A brief definition is sometimes followed by a phrase illustrating a specific use of a word. Simplified terms rather than technical terms are used in the definitions wherever possible. Acquired trait: a trait that appears in the phenotype during the postnatal life of the person; an acquired trait may or may not have a genetic origin. Acute intermittent porphyria: an autosomal dominant disorder resulting from a mutation in the porphobilinogen deaminase gene, encoding an enzyme required in the synthesis of the heme part of hemoglobin. The disorder is characterized by attacks of abdominal pain, limb cramps, muscle weakness, and psychiatric disturbances. Adeno-associated virus (AAV): a DNA virus that normally requires a helper virus in order to replicate. Adenosine deaminase (ADA) deficiency: a severe combined immunodeficiency disorder caused by an autosomal recessive mutation in the adenosine deaminase gene. Adenoviral dodecahedron: a vector consisting of a subset of adenoviral proteins. A transgene construct can be attached to the outside of this vector. Adenovirus: a DNA virus that infects humans, usually causing minimal symptoms but occasionally causing respiratory and other diseases. Agouti gene: a gene whose product allows mice to be brownish in color when present in either a homozygous or heterozygous state. Allele: one form of a gene found at a particular genetic locus; one gene may have many different allelic forms. Allele drop-out: a consequence of a failed amplification of one of the parental alleles during a polymerase chain reaction. Allele replacement: a process in which a resident gene is replaced by another allele during a genetic manipulation procedure. An allele replacement requires a homologous recombination mechanism. Allelic frequency: the frequency at which a particular allele is present in a specific population. Alpha1-antitrypsin deficiency: an autosomal recessive disorder resulting from a defect in the gene encoding alpha1-antitrypsin, a proteinase inhibitor. The disorder primarily affects lung and liver function. Smoking exacerbates the disorder and accelerates development of emphysema. Amino acid: the basic unit of a protein or polypeptide chain; a gene sequence encodes information directing synthesis of a polypeptide chain. Amniocentesis: a method of prenatal testing where fetal cells and amniotic fluid are collected and used in laboratory analysis; second trimester amniocentesis is performed at approximately 15-18 weeks gestational age; early amniocentesis is done at approximately 12-14 weeks gestational age. Amniocytes: cells present in amniotic fluid. Aneuploidy: genetic condition resulting from a chromosome number imbalance caused by either the loss or addition of a chromosome(s), relative to the normal diploid state.

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Anti-sense therapy: introduction of a piece of DNA that is designed to bind to and therefore inactivate a specific RNA sequence. This process is aimed at shutting off the expression of a target gene. Artificial insemination: the process by which collected sperm are introduced into the vagina or cervix during an assisted reproduction procedure. Autonomous replicating sequence (ARS): a DNA sequence containing a yeast origin of replication; an ARS enables a YAC vector to replicate as an independent chromosome. Autosomal gene: a gene located on any chromosome that is not a sex chromosome. Autosome: any chromosome that is not a sex chromosome; humans normally have 22 autosomal pairs of chromosomes (44 total) plus 2 sex chromosomes for a total of 46 chromosomes in the diploid genome. Blastocoel: the fluid-filled cavity of a blastocyst. Blastocyst: an early mammalian embryo consisting of an outer layer of trophoblast cells, an inner cell mass and a fluid-filled cavity called the blastocoel. Blastomere: cell in an early embryo; the 2-cell stage following cleavage of a zygote consists of two blastomeres. Carrier: an individual carrying a specific allele. Cationic liposome: see “lipoplex.” Centromere: the constricted central region of a chromosome, heterochromatic in nature, where the two sister strands (chromatids) are held together. CF gene: abbreviated terminology for the CFTR gene that is defective in cystic fibrosis patients. CFTR: cystic fibrosis transmembrane regulator; a defective CFTR protein, encoded by a mutant CFTR gene, is found in cystic fibrosis patients. Chimera: an organism containing cells of different genotypes; for example, the coat color of a chimeric mouse may be brown and black if the mouse originates from a mixture of cells in the early embryonic blastocyst stage, some of which are from a black mouse and others of which are from injected embryonic stem cells derived from an agouti (brownish-colored) mouse (see chapter 4 for further explanation). Chimeric vector: a vector generated by combining parts of more than one vector; e.g. adenoviral-retroviral chimeric vectors contain components of adenoviral and retroviral vectors. Chorion: the outermost extra-embryonic membrane. Chorionic villus sampling (CVS): a method of prenatal testing using fetal cells obtained via sampling of the chorionic villi; performed at approximately 10-12 weeks gestational age. Chromosomal abnormality: a genetic situation in which the cell or organism does not have the normal chromosomal DNA content. Chromosomal abnormalities include aneuploidy, polyploidy, translocations, inversions and duplications. Chromosome: a supercoiled strand of DNA containing genes complexed with proteins. Cleavage stage biopsy: see “embryo biopsy.” Cloned gene: a gene joined to vector DNA to form a recombinant vector; the gene can be obtained in large amounts by growing the recombinant vector DNA in appropriate host cells. Coding DNA: the region of a gene that encodes a gene product. Codominant allele: an allele encoding a product that contributes to the phenotype along with another product of a codominant allele. Complement: a group of eleven proteins, found in normal blood, which are involved in various biological activities including removal of pathogenic organisms.

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Complex disease traits: disease traits influenced by more than one gene; also called “polygenic trait” or “multifactorial trait.” Confined placental mosaicism: a condition characterized by a difference in genotypes between some preplacental chorionic villi cells and the fetus. Congenital trait: a trait present at birth; a congenital abnormality may or may not result from an inherited genetic defect. Crossing over: reciprocal exchange of material between homologous chromosomes; occurs during meiosis in developing gametes and rarely in mitotic cells. Cryopreservation: the process by which embryos or germ cells are preserved by freezing. Denatured DNA: DNA in a single-stranded rather than in the normal double-stranded configuration; denaturation is frequently accomplished by heating is an essential step in the polymerase chain reaction (PCR). Deoxyribonucleic acid (DNA): the primary carrier of genetic information (except for RNA viruses); composed of sequences of four deoxyribonucleotides containing adenine (A), thymine (T), guanine (G) and cytosine (C). Diploid: a genome comprised of two copies of each chromosome. Disease-contributing allele: we define a disease-contributing allele to be an allele which contributes to the development of a polygenic disease. DNA amplification: the process by which the amount of a DNA molecule can be increased; this process can occur in a cell or in the laboratory; laboratory amplification of DNA is accomplished by using the polymerase chain reaction (PCR). DNA repair: the repair of damage or mistakes in DNA. Dominant allele: gene that gives rise to a product that determines the phenotype displayed by the person when the dominant allele is present in a heterozygous state with a recessive allele. That is, a dominant allele is “dominant” with respect to a normal allele or a recessive defect. Down’s syndrome (trisomy 21): chromosomal abnormality characterized by the presence of three copies of chromosome 21, resulting from three individual copies of chromosome 21 or from two individual copies of chromosome 21 plus a translocation of chromosome 21 onto chromosome 14; Downs’ syndrome is characterized by mental deficiency and cardiac abnormalities. Electroporation: the process in which DNA is introduced into a cell with the aid of an electrical current that renders the cell temporarily permeable. Embryo biopsy: the process in which one or two cells (blastomeres) are removed from a 6to 8-cell stage embryo (in humans); the blastomeres are analyzed for the presence of a genetic disorder. Embryonic stem cells (ES cells): cells of the inner cell mass of a blastocyst. Enhancer: regulatory DNA sequence that can increase the utilization of some gene promoters in the presence of appropriate proteins; enhancers are located on the same doublestranded DNA molecule as the gene but may be upstream, downstream, in either orientation in the DNA, and at a varying distance from the promoter. Enucleated oocyte: an oocyte that has had its nucleus removed. Enzyme: a protein that is capable of catalyzing a chemical reaction. Episome: a circular piece of DNA (plasmid) that can replicate independently and which sometimes integrates into the resident chromosome. Epistasis: the phenomenon whereby expression of a gene at one genetic locus obscures the phenotypic expression of a nonallelic gene. Frequently epistatic genes encode proteins in the same biochemical pathway. Euchromatin: regions of chromosomes that are less condensed than heterochromatin; euchromatic regions contain transcriptionally active genes as well as other DNA, how-

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ever only a subset of genes residing in euchromatin are transcriptionally active at any given time. Eugenics: the control of reproduction to improve hereditary characteristics; “negative eugenics” is concerned with preventing genetic transmission of undesirable traits; “positive eugenics” is concerned with promoting genetic transmission of desirable traits. Eukaryote: a single-celled or multicellular organism whose cells contain a distinct nucleus. Euploid: a genetic condition where the chromosomal content of a cell or organism is comprised of the correct number of chromosomes for that organism. Ex vivo: “outside of the body”; ex vivo somatic gene therapy approaches involve removing the cells to be altered from the patient, introducing a transgene into the cells in the laboratory, and reintroducing the altered cells into the patient. Exon: the region of a gene that contains coding DNA. Expressivity of a gene: the degree to which the phenotype is displayed. Extragenic: DNA region outside of a given gene. Familial hypercholesterolemia: disorder caused by mutations in the low density lipoprotein (LDL) receptor; the disorder is characterized by elevated levels of serum cholesterol, secondary to increased LDL, and early death from a heart attack. Familial adematous polyposis coli: an autosomal dominant disorder resulting from mutations in the adematous polyposis coli or APC gene. Over 90% of APC mutations result in truncation of the APC protein. This disorder is characterized by the development of multiple polyps throughout the large bowel portion of the intestine. If not surgically removed, the patient usually develops colorectal cancer. Fanconi anemia: an autosomal recessive disorder that leads to bone marrow failure. Fluorescence in situ hybridization (FISH): a procedure that utilizes fluorescent probes to identify particular chromosomal regions. Fragile-X syndrome: a genetic disorder resulting from the presence of numerous trinucleotide repeats ((CGG)n) upstream of an X-chromosome gene called FMR1, whose function is currently being investigated. The pattern of inheritance is complicated and does not follow the rules of Mendelian inheritance. This syndrome is characterized by mental retardation. G418: an aminoglycoside drug used in the selection for cells harboring a neomycin phosphotransferase (neo) gene. Gamete: a specialized reproductive cell that is haploid; oocyte and sperm cells are gametes. Gamete intra-fallopian transfer (GIFT): an assisted reproduction technique in which sperm and oocytes are transferred to the fallopian tubes. Gene: the basic unit of heredity; it includes the DNA region that is transcribed into RNA but not the regulatory sequences located outside of this transcribed region. Gene addition: a process in which a gene is added to a genome, in contrast with a process where an allele is replaced. Gene cloning: the process of isolating a gene and incorporating it into a vector in order to manipulate the gene and to produce identical copies of a gene in a laboratory. Gene construct: a DNA molecule that contains a gene of interest plus associated sequences which may be required for regulation of the gene or for maintenance of the gene in a foreign cell. Gene conversion: the process by which an introduced transgene is used by the cell as a guide to change the resident gene sequence; the process can also work in reverse, resulting in an altered transgene instead of an altered resident gene. Gene deletion: elimination of part or all of a gene from the genome. Gene expression: the process of using genetic information to make a functional gene product. Transcription is only one step in gene expression.

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Gene knock-out: the process in which a resident gene is inactivated by the insertion of another disrupted, mutant allele; the disrupted gene is typically constructed by insertion of a drug resistance gene into the middle of the gene of interest. Gene pool: the complete set of genetic information in a population; the gene pool includes all alleles in a population. Gene product: the product encoded by a gene; the final product of gene expression may be either protein or RNA, such as rRNA, tRNA, etc. Gene therapy: the manipulation of genetic material in order to treat human disease; the procedure changes either the genetic makeup of a cell or the expression of specific genes contained within the cell. Genetic augmentation: see “gene addition.” Genetic bottlenecking: a phenomenon in population genetics which occurs when one allele is highly overrepresented in a population, causing reduced genetic variability at a given genetic locus. Genetic embryo selection: we define genetic embryo selection as the selection of embryos based on certain genetic criteria. Genetic enhancement: the use of gene transfer for non-disease-related purposes. See text for discussion of difficulties in categorizing a procedure as gene therapy or genetic enhancement (see chapter 5). Genetic locus: the position of a gene on a chromosome; different alleles of a gene can be found at the same genetic locus. Genetic screening: a process in which a population of individuals is subjected to genetic testing in order to analyze the frequencies of certain alleles of a gene; in genetic screening of embryos, several embryos are biopsied in order to reveal genetic characteristics. Genetic testing: an individual’s DNA is analyzed for the presence of certain alleles of a gene. Genocentrism: a particular form of bias or prejudice which focuses on genetic explanations and causes while ignoring environmental factors contributing to a phenotype (see chapter 5). Genome: the nucleic acid sequences comprising all the genetic information of a cell or organism; the genome includes nuclear and mitochondrial DNA in eukaryotes. Genotype: the genetic make-up of a cell or organism. Germ cell: reproductive cells (oocytes and sperm in humans) that are destined to carry genetic information to descendents; germ cells have a haploid DNA content in humans. Germline: genetic material passed from parent to offspring via the germ cells. Glycogen storage disease type Ia: also known as von Gierke disease; the disorder results from a mutation in the gene encoding glucose 6-phosphatase. The disorder is characterized by abnormal metabolism of glycogen (the storage form of glucose), resulting in problems associated with the liver and other organs. Haploid genome: the DNA content in a cell when it has one representative of each autosome plus one sex chromosome (in humans); a haploid genome is found in a human sperm cell. Hemizygous: a state in which only one copy of a particular gene exists in a cell; males are hemizygous for genes contained on their single X-chromosome since a second copy of those genes is not present on the Y-chromosome. Hemoglobin: a protein, found in blood, which transports oxygen. It is composed of a heme moiety and alpha- and beta-globin polypeptides.

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Hemoglobinopathies: inherited disorders that result in structurally abnormal hemoglobin; the clinical manifestations are determined by which protein subunit (which globin subunit) is affected. Sickle cell anemia is an example of a hemoglobinopathy. Hemophilia A or B: disorders characterized by deficiency of blood clotting factors VIII or IX, respectively. Herpes virus: a DNA virus; herpes viruses possess a natural tropism for neural cells. Heterochromatin: tightly folded regions of chromosomes that remain transcriptionally inactive while in this highly condensed state; regions of chromosomes may be temporarily heterochromatic or whole chromosomes can be stably heterochromatic, as in X-inactivation. Heterozygous: a diploid genome of a cell or individual that contains two different alleles of a particular gene. Heterozygote advantage: a situation where carrying a defective allele in a heterozygous state with a normal allele confers an advantage to the organism in a given environment. Homocystinuria: a family of disorders characterized by increased levels of the amino acid homocystine in the urine, most commonly caused by a mutation in the cystathionine beta-synthase gene. Homologous chromosome: a chromosome carrying the same (or most of the same) genetic loci as another chromosome. Homologous gene replacement: a process in which an original gene is replaced by another form of the gene; also called “allele replacement.” Homologous recombination (HR): a process in which two DNA molecules with similar sequences are aligned, cut, and rejoined such that the sequences of the two DNA molecules are exchanged but not altered in the process. Homozygous: a diploid genome of a cell or individual that contains two identical alleles of a particular gene; the term homozygous is also used to refer to a situation where a cell or individual has two normal alleles or two defective alleles of a gene, whether or not the normal alleles or two defective alleles are identical. For example, a person with cystic fibrosis who has two different defective alleles would also be classified as being homozygous for the CF defect. Housekeeping gene: a gene that encodes a protein that is expressed in all cell types in an organism and is essential for the basic function of the cell. Human artificial chromosome (HAC): an artificially constructed chromosome that replicates in human cells. Human germline gene therapy (HGLGT): the manipulation of genes destined to be part of germ cells in order to prevent the occurrence of a disease with a genetic contribution; problems with making the distinction between human germline gene therapy and human germline genetic enhancement is discussed in chapter 5. Huntington’s disease: a degenerative brain disorder caused by an autosomal dominant mutation in the huntingtin gene; the disorder shows both variable expressivity as well as incomplete penetrance (see chapter 1). Hybridize: complementary pairing of two DNA strands or an RNA and a DNA strand; the strands are held together by hydrogen bonds as well as other forces. Hydrophilic: “water-loving” chemical or portion of a molecule that is more likely to associate with water than lipid. Hydrophobic: “water-hating” chemical or portion of a molecule that is more likely to associate with lipid than water. Immunogenic: a characteristic of a substance which provokes a response by the immune system.

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In situ: “in a tissue”; in situ somatic gene therapy approaches involve injecting a recombinant vector containing a transgene directly into the specific tissue that is being targeted. In vitro: literally “within a glass”; term used to refer to a process that occurs in an artificial environment such as a laboratory, as opposed to within an organism. In vitro fertilization (IVF): the process in which oocytes collected from a female are fertilized in the laboratory by sperm; resultant embryos are transferred to the uterus to initiate pregnancy. In vivo: “inside the body”; in vivo somatic gene therapy approaches involve injecting a recombinant vector containing a transgene into the patient; sometimes features are included in the vector which target it to specific cell types. Inner cell mass: the cluster of cells found at one end of the blastocyst from which the embryo proper is derived. Insertional mutation: a mutation caused by the insertion of foreign DNA; a large DNA insertion usually causes gene disruption and inactivation of a gene. Insulator: a DNA sequence element that protects or insulates a DNA region from the regulatory effects of outlying DNA regions. Intracytoplasmic sperm injection: sperm are injected directly into the oocyte cytoplasm in an in vitro fertilization procedure. Intron: also called intervening sequence; the region of a gene that is located between coding DNA regions (the exons); transcribed intron sequence is removed from pre-RNA during RNA processing. Karyotype: the chromosomal make-up of an individual. Kilobase (kb): a polynucleotide containing 1000 base pairs or 1000 nucleotides. Kleinfelter’s syndrome: a chromosomal abnormality in which an XXY genotype results in a male who is normally infertile. Lesch-Nyhan syndrome: an X-linked recessive disorder caused by a mutation in the hypoxanthine phosphoribosyltransferase (HPRTase) gene; this disorder is characterized by mental retardation and a propensity for self-mutilation. Ligand: a molecule that binds to a specific region on another molecule, for example on a cell surface receptor. Lipoplex: a liposome which is composed of lipids that possess a positive charge on the portion of the lipid facing the interior of the sphere; these liposomes form many variable structures and are therefore termed lipoplexes; also called cationic liposomes (see chapter 3) Liposome: a small hollow sphere that is comprised of a lipid (fatty) membrane and an inner portion that is filled with an aqueous solution. Locus control region: DNA region involved in the regulation of the transcriptional activity of a genetic locus. Lysosome: sack-like organelle, found in eukaryotic cells, that contains degradative enzymes. Medical eugenics: we define medical eugenics as the manipulation of a human genome or the human gene pool in order to prevent diseases in an individual human or in human populations. Meiosis: a special type of eukaryotic cell division involving one replication of chromosomes and two successive cell and nuclear divisions producing four genetically non-equivalent haploid cells (gametes). Methylation (of DNA): the addition of a methyl (-CH3) group to DNA; implicated in regulation of gene expression. Microinjection: the process in which a transgene or other substance is injected directly into a single cell.

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Mitosis: a type of eukaryotic cell division whereby a cell and nucleus divide to produce two genetically equivalent descendant cells. Molecular conjugate: a complex used to target a transgene to a particular cell; comprised of a transgene, a ligand for the particular cell receptor that is being targeted, and a DNA binding moiety that links the transgene to the ligand; see chapter 3. Monogenic disorder: a disorder originating from a single gene or alleles of a single gene. Monogenic trait: a phenotypic trait resulting from a single gene (or the combined effects of alleles of a single gene). Monosomy: the state of having one copy of a chromosome instead of the normal two in a diploid organism. Morphological embryo selection: we define morphological embryo selection as embryo selection based on morphological criteria such as the rate of embryo growth, regularity and symmetry of the blastomeres, etc.; embryos are subjected to morphological embryo selection during in vitro fertilization procedures. Morula: the mulberry-shaped cluster of cells in an 8- to 16-cell embryo. Mosaicism: the condition when an individual is derived from two or more cell lines that have different genotypes. Multifactorial disorder: another name for a polygenic or multigenic disorder; the name reflects the fact that multiple genes and the environment may contribute to the development of the genetic disorder. Multifetal pregnancy reduction: the process by which the number of fetuses in a multifetal pregnancy is reduced in an attempt to minimize maternal and fetal health problems often associated with multifetal pregnancies, and to maximize the chance that at least one child will be born healthy. Mutation: a change in a DNA sequence; mutations may have neutral, deleterious or beneficial effects. Naked DNA: pure DNA that is not complexed with any proteins, ligands, viral particles, etc. Natural embryo selection: we define natural embryo selection as embryo selection that occurs naturally in the uterus because of chromosomal abnormalities or other genetic cause terminating the development of abnormal embryos. Nebulization: delivery of a liquid in the form of a fine spray; e.g. asthma patients use nebulizers to inhale medication. Negative selectable marker: a gene whose product, when expressed in a cell, will kill that cell when grown under selective conditions such as in the presence of a specific drug; the herpes virus tk gene is often used as a negative selectable marker when cells are grown in the drug gancyclovir. neo gene: a bacterial drug resistance gene that encodes neomycin phosphotransferase; the neo gene product confers resistance to the drug G418 and is commonly used as a positive selectable marker. Nested PCR: the process in which DNA is amplified in two separate rounds of PCR amplification; the second round of amplification utilizes primers which bind to the previously amplified DNA. Nonallelic gene: a gene that is not an allelic form of the gene of interest. Noncoding DNA: any DNA sequence that is not part of the coding DNA; noncoding DNA may be part of a gene (e.g. the intron) or may be located outside of a gene. Nonhomologous recombination (NHR): a process in which a gene is inserted into a genome by a mechanism other than homologous recombination; random transgene insertion occurs by nonhomologous recombination.

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Non-medical eugenics: we define nonmedical eugenics as the manipulation of a human genome or the human gene pool in order to achieve goals other than disease prevention in an individual human or in human populations. Normal allele: an allele that is found at a high frequency in the population and that does not appear to cause disease when functioning properly. Normal transgene: a transgene that encodes a gene product that falls within a range defined as normal; see “normal allele.” Nuclear transplantation: the process of transferring a nucleus from one cell to another; accomplished either by fusing a nucleated cell with an enucleated cell or by removing a nucleus from one cell and inserting it into a second enucleated cell. Nucleated cell: a cell containing a nucleus; a red blood cell is a cell that does not have a nucleus although the precursors in its developmental pathway do have a nucleus. Nucleotide: the building blocks of DNA and RNA; comprised of a purine or pyrimidine base, a ribose or deoxyribose sugar, and a phosphate group. Nucleus: eukaryotic organelle containing chromosomes. Oncogene: a gene that contributes to the process of turning a normal cell into a cancerous one; an oncogene is typically a mutant form of a proto-oncogene. Oocyte: a developing egg; a primary oocyte divides to give rise to a first polar body and a secondary oocyte, which upon fertilization divides to give rise to a second polar body and a mature egg. Ovarian hyperstimulation (controlled): see “superovulation.” Partial dominance: also called semi-dominance or incomplete dominance. Partial dominance describes a situation when one allele is not completely dominant with respect to the other allele in a diploid cell or organism; the manifestation of the heterozygous state differs from the homozygous dominant phenotype; partial dominance may also describe a condition where the effects of two dominant alleles are synergistic (e.g. LDL receptor alleles); see chapter 1. Particle bombardment: the process in which a transgene is delivered to a cell with the use of a “gene gun” which “shoots” transgene-coated gold particle “bullets.” Penetrance of a gene: the proportion of individuals displaying the expected phenotype. Phenotype: the physical manifestation of the information encoded in the genome of a cell or organism. Phenylketonuria (PKU): an autosomal recessive disorder usually caused by a mutation in the gene encoding phenylalanine hydroxylase. The disorder is characterized by an increase in phenylalanine and its derivatives in the blood and urine and severe mental retardation if not treated in infancy; treatment involves instituting a diet low in phenylalanine. Plasmid: an extrachromosomal circular DNA molecule that is capable of replicating; plasmids which can be integrated into a chromosome are classified as “episomes.” Pleiotropic effect: a single gene that affects many different traits. Pluripotent: cells that are capable of developing into several different types of cells. Polar body (first and second): small cells resulting from primary and secondary oocyte division, respectively; they are contained within the zona pellucida and eventually degenerate. Polygenic disorder: a disorder originating from two or more different genes; also called multigenic or multifactorial disorder. Polygenic trait: a phenotypic trait originating from two or more different genes. Polymerase chain reaction (PCR): a method for amplifying DNA; see chapter 2 for details. Polynucleotide: a chain of nucleotides; DNA and RNA are polynucleotides. Polypeptide: a chain of amino acids; a protein is a polypeptide.

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Polyploidy: the presence of two or more extra sets of the haploid number of chromosomes of a diploid organism; e.g. in humans, triploidy is a type of polyploidy where there are 3 complete sets of chromosomes. Polyspermy: fertilization of a secondary oocyte by more than one sperm. Positive selectable marker: a gene whose product, when expressed in a cell, allows the cell to grow in the presence of a specific drug (e.g. the neo gene is a positive selectable marker). Preconception genetic diagnosis: the process of determining if an unfertilized secondary oocyte contains a mutant allele; see chapter 2. Preimplantation genetic diagnosis (PGD): the diagnosis of a genetic disorder before embryo transfer and implantation in the uterus; see chapter 2. Precursor RNA (pre-RNA): the RNA product of transcription before it is completely processed into mature RNA; also called heterogeneous nuclear RNA; pre-RNA includes RNA in all intermediate stages of processing, including the initial RNA product (the primary transcript). Prenatal testing: the process in which a fetus in an established pregnancy is tested for the presence of a genetic disorder; this testing involves either chorionic villus sampling or amniocentesis. Primer (oligonucleotide primer): a short DNA or RNA sequence that “primes” the synthesis of a DNA strand. Proband: a person upon whom a hereditary or genetic study is based; in terms of human germline gene therapy, the proband would be the product of the initial genetic engineering procedure. Progeny: the descendents of a cell or organism. Prokaryote: a living organism (e.g. bacteria) that lacks a distinct nucleus. Promoter: nucleotide sequence to which an RNA polymerase binds at the start of synthesis of a strand of RNA (an RNA polymerase synthesizes RNA). Pronucleus: the nucleus of a sperm or egg cell; pronuclei (pl.). Protein: a molecule comprised of amino acids linked to one another in a specific sequence. Proteinase: an enzyme that degrades proteins. Proto-oncogene: gene that may cause cancer when mutated or overexpressed in cells. Receptor: a protein that binds to a specific ligand; receptors may be on the cell surface or inside the cell. Recessive allele: a gene whose phenotypic effect is obscured when the recessive allele is present in a heterozygous state with a dominant allele; a mutant allele which is not expressed at all would also be considered “recessive.” Recombinant vector: a vector containing a foreign gene. Replication incompetent virus: a virus that has been altered so that it is no longer capable of replicating. Resident chromosome: we define a resident chromosome to be a chromosome that normally resides in the cell as opposed to an artificial chromosome that is added to a cell; a resident chromosome is an endogenous chromosome. Resident gene: we define a resident gene to be a gene normally residing in a cell as opposed to a transgene added to the cell; a resident gene is an endogenous gene. Retrovirus: an RNA virus that converts its RNA genome into double-stranded DNA during its reproductive cycle; the double-stranded DNA version of the retroviral genome has the capability of integrating into the DNA genome of the host cell it has infected. Reverse transcription: the process of synthesizing DNA using an RNA template, catalyzed by the enzyme reverse transcriptase.

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179

Ribonucleic acid (RNA): nucleotide chain composed of sequences of four ribonucleotides containing adenine (A), uracil (U), guanine (G) and cytosine (C). Ribosomal RNA (rRNA): RNA that is part of the structure of a ribosome. Ribosome: particle composed of rRNA and protein that is involved in the synthesis of polypeptide chains. RNA processing: the conversion of a precursor RNA molecule into a mature, functional form. Selective allelic amplification: see “allele drop-out.” Selective termination: the process by which an embryo or fetus is selectively terminated following genetic analysis. Sequence element: region of DNA that has a functional role such as a regulatory role in gene expression. Sex chromosome: the X or Y chromosome. Sex-linked gene: a gene that is located on one of the sex chromosomes (X or Y). Sickle cell anemia: a recessive disorder caused by a mutation in the beta-globin subunit of hemoglobin; the mutated allele is called the beta-S allele; the disorder is characterized by sickle-shaped red blood cells and anemia. Silencer: DNA sequences that inhibit transcription; also called negative enhancers. Simple liposome: see “liposome.” Single-cell genetic diagnosis: the analysis of DNA originating from a single cell; this DNA is used to analyze the genetic make-up of the cell. snRNA: small nuclear RNA; a component of the RNA splicing machinery. Somatic cell: all cells that are not destined to become eggs or sperm; somatic cells are normally diploid in humans. Somatic gene therapy (SGT): the use of transgene introduction into somatic (non-reproductive) cells in the therapeutic treatment of a medical condition. Suicide gene: a gene that mediates cell death when present in a cell; a suicide gene may encode an enzyme which converts an inactive drug to a form that is toxic to a cell; a commonly used suicide gene is the gene encoding thymidine kinase from herpes simplex virus type 1; see chapter 4. Superallele: we define a superallele to be an allele that is engineered to enhance certain phenotypic characteristics. Supercoiled DNA: DNA where the double helix is twisted upon itself (see Figure 1.2). Superovulation: the process in which a female is stimulated hormonally to ovulate multiple oocytes that may be collected for in vitro fertilization attempts. Tandem gene array: a cluster of multiple copies of a gene arranged in series. Tay-Sachs disease: a lysosomal storage disease caused by a defective hexosaminidase gene; causes severe mental retardation. Telomere: the DNA region at the end of a chromosome. Thalassemia: a family of blood disorders that result from decreased synthesis of one or more hemoglobin polypeptides. Tissue-specific mechanism: a mechanism that is operative in a specific tissue as opposed to one found in all tissues. Tissue-specific promoter: a promoter sequence that regulates gene expression so that the gene is expressed only in certain tissues. tk gene: herpes simplex virus thymidine kinase gene. The tk gene product is capable of converting the prodrug gancyclovir into a nucleotide analog which, when incorporated into a cell’s DNA, causes termination of DNA synthesis and thus kills the cell; the viral tk gene is commonly used as a negative selectable marker.

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Totipotent cells: cells that have the ability to develop into any cell type of the adult organism. Transcription: the synthesis of an RNA molecule from a DNA template. Transduction: the introduction of genetic material into a target cell by a virus-mediated method. Transfection: the introduction of genetic material into a target cell by non-viral means. Transfer RNA (tRNA): RNA molecule that is involved in the process of using the information in mRNA to direct synthesis of polypeptide chains (process is called translation). Transgene: we use the term transgene to refer to any cloned gene that is introduced to a cell; a transgene may or may not be a gene from the same species. See chapter 1 for explanation of an alternate usage of the word transgene which we have not used in this text. Transgene acceptor site: we define a transgene acceptor site to be an hypothetical location in the genome that would be a designated site into which transgene constructs could be inserted via homologous recombination. No such site has been identified since most methods directing transgene introduction into human cells have not employed homologous recombination mechanisms. Transgene expression: the expression of a transgene into a functional gene product. Transgenic human: a person who carries a transgene in all the nucleated cells in their body, including germline cells. Transgenic organism: an organism that carries a transgene in all the nucleated cells in their body, including germline cells; the transgene may reside transiently or stably in the cells. Translation: the process in which information contained in mRNA is used in the synthesis of a polypeptide. Translocation: the movement of a segment of DNA from one site in the genome to another. Trisomy: the state of having three copies of a chromosome instead of the normal two in humans. Trisomy 21: the presence of three copies of chromosome number 21, results in Down’s syndrome; see also Down’s syndrome. Trophectoderm: the outer cell layer of the blastocyst. Trophoblast: an individual cell of the trophectoderm. Tumor suppressor gene: a gene encoding a protein which, when inactivated, leads to the formation of tumors. Turner’s syndrome: a chromosomal abnormality resulting in a female who possesses only one X chromosome (an XO genotype) and is sterile. Two-step oocyte genetic analysis: a process in which the genetic make-up of the first and second polar bodies is examined sequentially. Uterine transfer: transfer of an embryo to the uterus. Vector: plasmid or viral DNA into which a gene may be inserted; the resultant “recombinant vector” may be used for transfection (with a plasmid) or transduction (with a virus) of target cells. Virus: a particle consisting of a nucleic acid genome (either DNA or RNA) within an outer protein coat. Vitamin D-resistant rickets (X-linked hypophosphatemia): an X-linked dominant disorder resulting in bone malformation. Wildtype gene or phenotype: a normal gene or phenotype X-inactivation: the random inactivation and heterochromatization of one of the two X chromosomes in a mammalian female; X-inactivation occurs early in the develop-

Glossary

181

ment of an embryo; all descendent cells maintain the same inactive X chromosome as the parental cells. Xenogene: we define xenogene as a foreign gene originating from a different species; a subclass of transgenes. Xeroderma pigmentosum: a rare autosomal recessive condition that causes extreme sensitivity to ultraviolet radiation from the sun; several different genetic defects can cause the disorder. Zona pellucida: a glycoprotein (protein linked to sugars) coat that protects an oocyte from damage and is a partial barrier against sperm of other species. Zygote: a diploid cell resulting from fusion of a male and female germ cell (gamete); a singlecelled embryo. Zygote intra-fallopian transfer (ZIFT): the assisted reproduction procedure in which a zygote is transferred to the fallopian tube within 24 hours of in vitro fertilization.

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Glossary

183

INDEX A α1-antitrypsin deficiency 19 Abortion 23, 29, 30, 33, 39, 90, 101, 116, 118, 138, 143, 151, 158 selective abortion 86, 87, 95, 106, 117, 133, 141 selective termination 23, 41, 86 Adeno-associated virus (AAV) See Virus 56, 57, 59, 61, 77 Adenosine deaminase (ADA) deficiency 48, 49, 55, 88 Adenovirus See Virus 56, 59-62, 65, 77 Adoption 12, 21, 42, 101 agouti 73, 75 AIDS See HIV 95, 124 Allele codominant 9 disease–contributing 85, 104 dominant 9-11, 38 drop–out 38 partially dominant 9, 10 semidominant 9 recessive 9-11 replacement 4, 8, 10, 42, 51-53, 67, 72, 76, 81, 83, 85, 96, 97, 99 selective allelic amplification 38 superallele 72, 103, 104, 108, 134, 141, 163 Allelic frequency 19, 21, 99 Amniocentesis 30, 39-42, 79, 81 Anti-sense therapy 2 Aquinas, T 147, 151 Aristotle 130, 145, 147, 151 Artificial insemination 17, 21, 23, 116 Autonomy 100, 113-116, 126, 138, 153, 157-159

B Beneficence 93, 113, 114, 153, 158 Bias 89, 106, 107, 130, 142, 143, 155, 162

Biohazard 100 Blastocoel 27 Blastocyst biopsy 35 Blastomere 25, 27, 29, 33-36, 38, 81 Brave New World 89, 116, 131, 137, 154, 166 BRCA 19

C Cancer 2, 3, 14, 18, 19, 49, 52, 55, 72, 85, 87, 89-91, 94, 95, 100, 101, 103, 104, 108, 120, 124, 126, 141, 151, 164 Capitalist Scenario 132, 133 Carrier 9, 11, 17-21, 23, 24, 30, 32, 34, 38, 41, 97, 104 Caste system 132, 134-136, 139 Chimera 61, 73 Chorionic villus sampling (CVS) 30, 39-42, 79, 81 Chromosome artificial chromosomes bacterial artificial chromosome (BAC) 62-64, 77 human artificial chromosome (HAC) 51, 55, 64, 66 phage P1 artificial chromosome (PAC) 62-64 yeast artificial chromosome (YAC) 62-64, 77 autosomal 8-12, 18, 19, 29, 32, 35, 38, 96, 104 chromosomal abnormalities 17, 18, 27, 29, 33, 39, 42, 47, 85 crossing–over 24, 32, 77 euchromatin 7 heterochromatin 7, 53, 54 homologous 32, 50-52, 56, 67, 72, 76, 77, 79, 85, 95 karyotype 39, 40 resident 3

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Chromosome (cont.) sex 8, 10, 29 structure of centromere 7, 53, 63, 64 telomere 63 telomeric 64 translocation 18 Cloning human cloning 79, 104, 159 Closed future 150 Compaction 27, 65 Comparability problem 93, 109, 110 Confidentiality See Privacy 113, 126, 160, 162 Confined placental mosaicism 40 Conflicts 91, 110, 115 of rights 115 Corn See Maize, Zea mays 102, 103 Cystic fibrosis 10, 14, 18-21, 23, 24, 32, 34-36, 41, 49, 51, 88, 95-98, 120, 160

D Darwinian evolution 133 Darwinism 138 Deoxyribonucleic acid (DNA) 1 amplification 31, 38 calcium phosphate transfection of 65, 66 coding DNA 7 DNA polymerase 36 DNA repair 8 DNA vaccine 2, 89, 104 flanking DNA 36, 77 naked DNA 56, 62, 65 non-coding DNA 7 supercoiled 7 Descartes 147 Diabetes mellitus 14, 18 Discrimination 106, 107, 134, 140-143, 155, 162 Dolly 79, 81 Down’s syndrome (trisomy 21) 18, 29, 47, 134, 141

Duchenne type muscular dystrophy 2, 19, 35, 49 Dystrophin 2

E Ebola 89, 103 Economic benefits 95, 107-109 Economic costs 108 Education 94, 101, 107, 116, 125, 132, 134, 140, 142, 162 Egalitarianism 131, 138, 139, 142, 166 Electroporation 64, 66 Embryo biopsy 32-35, 42, 78 selection genetic embryo selection 15, 27, 29, 30, 33, 77, 79, 86, 98, 101, 120, 135 morphological embryo selection 27, 29, 32, 86 natural embryo selection 27 sexing 39 transfer 24, 29, 30, 32, 79, 82 Embryonic stem cells (ES cells) 73, 75, 79-81, 86 Enhancer 6, 8, 54 Episome 3, 8, 53, 55, 59, 60 Equality of opportunity 131, 133-140, 154, 158, 165 Essentialism 146 Ethics 85, 86, 90, 93, 124, 151, 163, 164 Eugenics medical eugenics 87, 105, 154 nonmedical eugenics 87, 105, 154 Evolution 2, 8, 14, 87, 88, 93, 95, 101-104, 131, 133, 134, 136, 155, 160, 161, 165 Expressivity 11, 12, 14 Extinction 102, 103

F F1 10, 75, 96-99, 117 F2 75, 96, 117 Faith 123, 153 Familial polyposis coli 19

Glossary Index

Fanconi anemia 49 Fascist Scenario 131, 132 Faust 155 Fluorescence in situ hybridization (FISH) 31, 32, 36, 38, 39 Food and Drug Administration (FDA) 49 Formal Principle of Justice 130, 140 Fragile–X syndrome 19 Frankenstein 106, 145, 151 Free choice 116, 131, 158 Freedom 116, 117, 138, 140, 158, 159 Funding 160-162 Funding priorities 162 Future generations 4, 71, 99-101, 113, 114, 117, 118, 122-127, 129, 150, 156

G Gamete donation 21, 42 Gamete intrafallopian transfer (GIFT) 22-24, 32, 77 Gametes See Oocyte, Sperm 21, 24, 133 Gene artificial gene 163, 164 conversion 76 correction 76 deletion 19, 61, 66, 72, 104 delivery 3, 4, 47, 50-53, 55-59, 62, 64-67, 69, 76 dormant 2 gene construct 8, 50, 53-56, 62, 65, 66, 72, 74, 77-79, 99 gene knockout 72, 73 gun 66 lottery 86 oncogene 52 patents 162-164 penetrance 11, 12, 14 pool 10, 42, 86, 87, 96, 101-105, 132, 136, 155, 166 reporter gene 56 suicide gene 2 therapy 1-4, 8, 10, 11, 14, 15, 17, 20, 47, 48, 53, 54, 56, 61, 62, 66, 71, 77,

185

85-89, 93, 95, 103, 109, 120, 129, 145, 147, 159, 160, 162 tumor–suppressor gene 52 xenogene 72, 104, 163 Genetic accidents 100 augmentation 3 bottlenecking 99, 136 determinism 89, 90, 148 diversity 101-105, 107, 136-139 engineering 87, 89, 100, 104, 109, 110, 119, 124, 126, 127, 131, 142, 148, 151-154, 159-161, 164-166 enhancement 2, 29, 42, 86-89, 107, 120, 121, 132, 135-137, 142, 154, 159, 165, 166 equality 136 linkage analysis 19 reengineer 104, 149, 155 screening 39 systematicity 99 testing 19-21, 23, 30, 35, 81, 89, 97, 107, 126, 140-142 therapy 121 variability 99, 102, 103, 105, 155 warfare 107, 109, 165 Genocentrism 89, 90, 125 Glycogen storage disease 49 Government funding 160, 161

H Happiness 105-107, 123, 138 Harm principle 115, 118-121, 126, 139, 157 Hawthorne, Nathaniel 154, 155 Hemoglobin 2, 3, 49 Hemophilia 49, 88, 95 Herpes virus 57, 59, 61 Heterozygote advantage 3 HGLGT (Human germ-line gene therapy) applications 61, 110 consequence 110 development 71, 132

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HGLGT (cont.) procedure 17, 54, 71, 77-79, 81-83, 104 method 76, 86, 96, 99 protocol 17, 41, 42, 55, 76 research 71, 103, 110, 117, 159-161, 164, 165 target 71 HIV (human immunodeficiency virus) See AIDS 49, 57, 60, 61, 89, 95, 103, 163 Homo sapiens 102, 105, 132, 136, 146, 147 Homocystinuria 19 Homologous gene replacement 4, 56, 67, 76, 108, 160 Homologous recombination (HR) 4, 50-55, 59, 61, 67, 72-74, 76, 77, 79, 81, 83, 85, 95-97 Housekeeping gene 5 Human being 15, 30, 42, 86, 87, 100, 102, 106, 123, 125, 126, 131, 133-138, 142, 145-149, 151-155, 160, 161, 163-165 Human experimentation 124 Human Genome Project 18, 82, 107, 159, 164 Human nature 89, 125 Humanness 145-152, 154, 155 huntingtin 11, 14, 141 Huntington’s disease 11, 14, 19, 49, 88, 95, 126, 141, 160 Huxley, Aldous 89, 116, 131, 137, 142 Hypercholesterolemia 9, 19, 49, 88

I Immune response 4, 59-61, 82 Immunization 89, 95 In vitro fertilization (IVF) 20, 21, 23-25, 29, 31-33, 35, 39, 78, 79, 86, 106, 108, 109, 116, 119, 125, 151 Indirect research 159-161, 163 Informed consent 124, 160 Inner cell mass 27, 34, 35, 73, 75, 80, 81 Insertional mutation 52, 55

Insulator 54 Intellectual property 163, 165 Interbreeding population 146 Interest in Existing argument 121, 122 International cooperation 164, 165 Intracytoplasmic sperm injection 25 Intrauterine insemination 23, 24

J Justice distributive 129-131, 133 procedural 129, 130 Justifiable risks 118-120, 126, 127, 159

K Kantian 147, 163 Karyotype analysis 39, 40 Kleinfelter’s syndrome 29

L Law 47, 86, 94, 100, 134, 142, 147, 150-154, 157, 158, 160-165 Legal 82, 86 114-116, 119, 124, 126, 133, 138, 157, 158, 160, 162-164 Lesch-Nyhan syndrome 11 Liability 110 Libertarian 131, 138-140 Liposome 56, 58, 62, 63, 65, 67 Locus control region 54

M Maize See Zea mays, Corn 102 Malaria 3, 9, 88, 102 Male–pattern baldness 96 Marxist 131, 159 Material principles of justice 130, 140 Medical benefits 93, 95, 99, 106, 109 eugenics 87, 105, 154 harms 96, 100 Meiosis 24, 26 Methylation 53, 81, 83 Microinjection 58, 64, 66

Glossary Index

Mitosis 7, 24 Monogenic 3, 14, 18, 19, 35, 48, 49, 85, 120, 127, 135 Monogenic defect 3, 19, 41, 42, 49, 72 Monosomy 29 Moral agent 114 patient 114, 118 principles 113, 149, 150, 152, 153 responsibility 113, 118, 124, 133 values 110, 137, 152 worth 114, 115, 150-152, 163 Mosaicism 34, 40, 41 Multifactorial disorder See Polygenic disease 3, 18 Multifetal pregnancy reduction 23, 29, 42

N National Institutes of Health (NIH) 49, 161 Natural law 150-154 Natural lottery 134, 135 Natural selection 27, 88, 133, 155 Nazi Germany 101, 132, 137, 138 Negative rights 115, 116 Negligence 94 neo 72-74 New species 132, 136, 139 Nonexistence 97, 121, 122 Nonhomologous recombination (HR) 3, 4, 51-55, 59-61, 63, 64, 66, 67, 74, 76, 77, 79, 82, 83, 85, 97, 98 Nonmalificence 93, 114, 119, 121, 123, 153, 158 Normal range of variation 87, 137 Nuclear transplantation 81 Nuremburg Code 124

O Obligations 113-115, 117-119, 122, 123, 125, 127, 129, 150 Office of Recombinant DNA Activities (ORDA) 49 Oocyte

187

enucleated oocyte 76, 80, 81 mature egg 25, 26, 28, 32-34 oogonium 26 primary oocyte 24-26, 28, 32, 34 secondary oocyte 25, 26, 28, 32, 33 Oogonia 24, 26 Optimism 109-111, 162 Ovarian hyperstimulation 21

P Pandemic 103 Panglossian 155 Parental choices 89 rights 110, 116-122, 127 Patents 110, 162-164 Penetrance 11, 12, 14 Perfectionism 107, 154, 155 Pessimism 109, 110, 162 Phenotype 8-11, 15, 42, 72, 73, 87, 89, 133, 140, 148, 149, 164 Phenylketonuria (PKU) 19, 49 Plasmid 3, 4, 51, 58, 62, 64, 66 Playing God 152-154 Ploidy aneuploid 18, 29, 33, 39, 40, 42, 72, 78 diploid 8, 9, 79-81 euploid 78 polyploid 18, 78 triploid 18, 25 Pluripotency 81 Polar body 25, 26, 28, 32-34, 38, 42 Political values 137, 138, 158, 159 Politics 90, 91, 116, 129, 153, 158 Polygenic disease 3, 18, 41, 85, 95, 101, 121, 160 polygenic disorder 3, 14, 48 Polymerase chain reaction (PCR) 31, 32, 36-39, 76 allele drop–out 38 fluorescent PCR 38 nested PCR 36-38 selective allelic amplification 38

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Porphyria 12 Position effects 53, 54, 63 Preconception genetic diagnosis 32, 33, 39, 101 Predictability problem 93, 109, 110 Preimplantation genetic diagnosis (PGD) 22, 29-36, 38-40, 42, 48, 71, 78, 86, 98, 159 Prejudice 89, 107, 142, 155, 162 Prenatal diagnosis 22, 32, 39-42, 101 Privacy See Confidentiality 94, 107, 113, 116, 126, 162 Proband 10, 20, 96-99, 105, 106, 117, 118, 159, 164 Promoter 6, 8, 54 Proxy consent 124 Prudence 109-111, 129, 133, 162 Psychological characteristics 106, 146 Psychosocial benefits 95, 105 harms 106, 107, 119, 125, 155 Public policy 93, 110, 122, 125, 140, 157, 158, 162

R Race 3, 47, 48, 64, 100, 101, 103, 105, 125, 130, 131, 133, 135-137, 139, 148-150, 154, 155 Racism 148 Random transgene insertion 15, 52, 55, 76, 83, 98 Receptor 9, 10, 51, 59, 60, 64 Recombinant DNA Advisory Committee (RAC) 49, 56 Religion 122, 147, 153 Replication incompetent virus 56 Responsibility 113, 114, 120, 123 Ribonucleic acid (RNA) 2, 5-8, 10, 35, 56 messenger RNA (mRNA) 5, 6, 35 ribosomal RNA (rRNA) 5 RNA processing 5 transfer RNA (tRNA) 5 Rights conflicts of 115

negative 115, 116 positive 115, 116 rights of unborn children 110

S Selective advantage 48 Self–determination 116 Shigella 47 Sickle cell anemia 3, 9, 19, 36, 49, 88, 102, 142 Silencer 8, 54 Slippery slope 110, 138 Social characteristics 146-148 Darwinism 138 integration 106 policies 91, 93, 101, 133, 135, 137, 153, 157, 158, 165 stigma 106 Sociobiology 148 Somatic gene therapy (SGT) 1, 2, 4, 10, 14, 15, 20, 22, 23, 42, 47-50, 54-56, 61, 64, 66, 67, 71, 82, 83, 85, 95, 99, 159, 161 Soul 122, 145, 147, 148 Sperm 1, 17, 20, 21, 23-25, 28, 32, 33 Sry 10 State controls 89, 105, 132 Statistical norms 87, 88 Subspecies 105, 132, 139 Suffering 48, 95, 96, 99, 100, 105, 106, 121, 149 Suicide gene 2 Superallele 72, 103, 104, 108, 134, 141, 163 Supermen 148, 149, 165 Superovulation 21, 23-25, 32, 41, 77, 78, 80 Surrogate pregnancy 151

T Tay-Sachs disease 9, 88, 119, 120, 142 Thalassemia 2, 19 The "who decides?" question 154

Glossary Index

Tissue injection 65 Tissue targeting 71, 82 tk 72-74 Totipotent 27 Tragedy of the commons 139 Trait complex disease traits 18 Transduce 56, 59, 65 Transfect 66, 73, 77 Transgene acceptor site 51, 52, 72, 76 chromosomal integration of 52 construct 8, 50, 53-56, 62, 65, 66, 72, 74, 77-79, 99 expression 7, 8, 14, 52-55, 61, 62, 65, 66, 85, 98, 99 position effects 53, 54, 63 Transgenic human 72 Trisomy 21 (Down's syndrome) 18, 29, 33, 47 Trophoblast 27, 31, 35 Trophoectoderm 27, 34, 35 Turner’s syndrome 29, 47 Two-allele replacement 10

U Unborn children 96, 110, 117, 118, 122, 124-126, 135, 150, 159 possible people 141 potential child 72, 113, 117-127, 139, 141 Unintended cost 108 Utilitarianism 93, 149 Utility 93, 110, 113, 114, 130, 131, 156-158, 163 Utrophin 2

V Values 85, 90, 91, 96, 100, 105, 109, 110, 114, 136-138 Vector recombinant vector 8, 50, 51

189

Virus adeno-associated virus (AAV) 56, 57, 59, 61, 77 adenovirus 56, 59-62, 65, 77 baculovirus 47 herpes virus 57, 59, 61 HIV (human immunodeficiency virus) 49, 57, 60, 61, 89, 95, 103, 163 Moloney murine leukemia virus 60 replication incompetent virus 56 retrovirus 56, 60, 61 Vitamin D-resistant rickets (X-linked hypophosphatemia) 11

W Watchdog group 165 Welfare 109, 114, 116, 118, 123, 125, 126, 129, 150, 165 Wild type 10, 61

X X–linked disorder 11, 19, 35, 39 X-inactivation 7, 11 Xenogene 72, 104, 163 Xeroderma pigmentosum 3

Y Yeast autonomous replicating sequence (ARS) 63

Z Zea mays See Corn, Maize 102, 136, 137 Zimmerman, Burke 117 Zona pellucida 25, 27, 28, 31, 33 Zygote intrafallopian transfer (ZIFT) 30