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4 Human Gene Therapy IN THE EARLY 1970s an American researcher named Stanfield Rogers infected three German girls who lacked the enzyme arginase with Shope papilloma virus, hoping that the virus would transfer to the girls the gene for the missing enzyme. In 1980 Martin Cline of the University of California at Los Angeles exposed the bone marrow of two patients from Italy and Israel who suffered from beta-thalassemia (a blood disorder resembling sickle cell anemia) to recombinant DNA coding for the blood protein hemoglobin, hoping that the bone marrow would incorporate the new genes and alleviate the patients' disease. Neither of these first two attempts at human gene therapy had an effect on the patients involved. But they dramatically affected the biomedical community and, especially in Cline's case, the public. They demonstrated that attempts to alter the genetic constitution of human beings were not a distant prospect, sufficiently far off to leave years for exploring their scientific and ethical implications, but a present real- ity. In the past few years the advent of successful human gene therapy has come even closer. Since 1980 the replacement of defective genes has been accomplished in fruit flies and mice. At least six major research centers in the United States are working to develop the This chapter includes material from the presentations by W. French Anderson, Leroy B. Walters, Jr., and Albert Gore, Jr., at the symposium. 43
44 BIOTECHNOLOGY techniques that will permit gene therapy in humans. The first clinical trials of these techniques in human subjects are expected to begin in 1986. There are many different types of human gene therapy, each with its own set of scientific and ethical questions. The kind of gene therapy now being pursued by researchers is far removed from the kinds of gene therapy that many people, for a variety of reasons, have come to fear. The first sanctioned attempts at gene therapy will involve the insertion of a single gene into a limited subset of a patient's cells to palliate a severe genetic disorder. The gene will not be able to spread beyond those cells, and it will not be passed on to the patient's offspring. Gene therapy of this type closely resembles other types of medical proce- dures, such as transplants or drug treatment, and a consensus has gradually emerged that it presents no new ethical problems. Other kinds of human gene therapy can be envisioned, but formi- dable technical difficulties make it hard to imagine when, if ever, they may become feasible. For instance, researchers have succeeded in changing the genetic constitution of mice so that new genes are passed down from generation to generation. But it is not now possible to do this with humans, and technical and ethical problems inherent in such work are likely to keep it from being attempted for years. Any endeavor to genetically engineer human beings to enhance certain characteristics (versus repairing an inborn genetic defect) would of course raise difficult questions of ethics and safety. For instance, it is impossible to predict how the introduction of one or a few "enhancing" genes into the body's cells would affect the health of either an individual cell or an entire person. Such complex human attributes as intelligence, character, and physical appearance are undoubtedly controlled by many genes inter- acting among themselves and with innumerable environmental influ- ences. It is difficult to conceive of how genetic engineering could ever be used to affect these complex human traits. But that does not mean that the broad moral and social implications of human gene therapy should not be the subjects of continuing reflection. As the experiments of Rogers and Cline demonstrate, events in biomedical research have often leapt ahead of their ethical and philosophical underpinnings. Somatic Cell Gene Therapy The kind of gene therapy now being studied by researchers involves the insertion of one or a handful of genes into somatic cells in the body. Somatic cells include all the body's cells except for sperm cells, egg
HUMAN GENE THERAPY 45 cells, and the cells that give rise to them, which are collectively known as germline cells. Because somatic cell gene therapy does not affect the germ line, the genes conveyed through the procedure will not appear in the recipients' offspring. The first diseases selected for treatment with somatic cell gene therapy will share several characteristics, according to W. French Anderson of NIH's National Heart, Lung, and Blood Institute. First, they will arise from a defect in a single gene causing the loss of an enzyme with potentially lethal consequences. "Those genetic disorders that are serious but not lethal are unlikely to be the first candidates," says Anderson. Defects in single genes cause more than 200 known human disorders, including muscular dystrophy, sickle cell anemia, cystic fibrosis, and hemophilia, and there are more than 2,000 known genetic diseases. But only a few of the genes responsible for single-gene disorders have so far been isolated and reproduced through genetic engineering so that copies of them can be inserted into cells. Second, the diseases will be treatable through the genetic manipu- lation of bone marrow cells, because techniques have been developed to remove these cells from the body, transform them with recombinant DNA, and reintroduce them into the body. Perhaps in the future it will be possible to genetically manipulate skin cells and even tissues and whole organs, but for now bone marrow cells are the only cells conducive to this kind of treatment. Finally, the genes responsible for the diseases will have a fairly simple kind of regulation. It was originally thought that the various diseases caused by defects of hemoglobin, such as sickle cell anemia and beta-thalassemia, would be the first disorders to be treated with gene therapy. However, the regulation of hemoglobin production has turned out to be unusually complicated, involving several different genes on different chromosomes. Thus, the first genes to be inserted into human cells will be those with a simple "always-on" type of regulation. Given these constraints, the initial candidates for human gene therapy are the genes coding for the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT), the absence of which results in Lesch-Nyhan disease, a lethal neurological disorder that can lead to uncontrollable self-mutilation; adenosine deaminase (ADA), the ab- sence of which causes a severe combined immunodeficiency disease so that victims have to live in totally sterile environments; and purine nucleoside phosphorylase (PNP), the absence of which leads to another form of severe immunodeficiency disease. Approximately 200 new cases of Lesch-Nyhan disease are reported in the United States each
46 BIOTECHNOLOGY Individuals lacking the enzyme adenosine deaminase (ADA) have such severely impaired immune systems that they must live in totally sterile environments to survive. For instance, David, the famous "Bubble Boy" (shown here at age 5), lived 12 years in isolated living quarters. By infecting the defective bone marrow cells of such patients with genetically engineered viruses containing the gene that codes for ADA, researchers hope to cure the disease. year, making this the most common of the initial candidates for gene therapy. However, the neurological component of Lesch-Nyhan disease is caused by a lack of HPRT in the brain, and it is not known if supplying the enzyme from the bone marrow will overcome this deficit. The other two diseases are much rarer: only 40 to 50 cases of ADA deficiency and 9 cases of PNP deficiency are known worldwide. The key step in the treatment of these diseases will be the insertion of genetically engineered copies of the respective genes into bone marrow cells removed from the patient's body. Researchers have been investigating several ways of introducing DNA into animal cells, including microinjection, chemically or electrically induced uptake, or fusion of the cells with vesicles containing the new DNA. But the most
HUMAN GENE THERAPY 47 promising technique, and the one now being developed for human gene therapy, is the infection of the cells with genetically engineered retroviruses. As described in Chapter 3, retroviruses can insert a single copy of a DNA strand into the cells they infect. By attaching the appropriate regulatory signals to the inserted DNA, the gene can also be made to function within its new host. However, the position of insertion of the foreign gene into the host's DNA is random. After the bone marrow cells have been transformed, they will be reimplanted into the patient. A limited number of studies have THE GENETIC ENGINEERING OF HUMAN CELLS Retrovirus Human Cell with Functional Gene Â» DNA Equivalent of Retroviral Â» RNA with Major Genes Deleted DNA -Functional Gene Recombinant Retrovirus Bone Marrow Cell with Defective Gene Bone Marrow Cell Containing Functional Gene Upcoming attempts at human gene therapy will use retroviruses, infectious agents that have the ability to insert a single strand of DNA into the DNA of cells they infect. The genetic material of a retrovirus consists of RNA, which is enzymatically copied into its close chemical cousin DNA when the virus invades the cell. The major genes in a DNA copy of the retroviral RNA can be deleted and replaced with the desired gene from a human cell, along with the appropriate regulatory signals to ensure the expression of the gene. The bioengineered retroviruses can then be used to infect bone marrow cells withdrawn from a patient with a defective gene. The retroviruses insert the functional gene into a random location in the cells' DNA, and the transformed cells are reimplanted into the patient.
48 BIOTECHNOLOGY suggested that bone marrow cells that can produce HPRT and ADA have a growth advantage over bone marrow cells that cannot. If so, they will eventually come to predominate over a patient's defective bone marrow cells. If not, the defective bone marrow may have to be weakened or destroyed, through irradiation or other means, so that the transformed bone marrow cells can proliferate. According to Anderson, several conditions must be met before such a procedure will be ethically permissible in human beings. The new genes must enter the proper cells and remain stable in those cells long enough to have the desired effect. They must also express their products at a level that will ameliorate the disease. Researchers are subjecting both of these conditions to rigorous study in tissue culture and laboratory animals to demonstrate their feasibility. A final, more demanding condition is that the procedure not harm the cells to which it is applied or, by implication, the person receiving those cells. For example, a major concern is that the viral DNA used to transform bone marrow cells might naturally recombine with other pieces of DNA in the cell to form new infectious viruses, which could then spread to other cells. Researchers are looking for such recombi- nant viruses in tissue culture and laboratory animals to determine if this is possible. Other researchers are working to genetically engineer safeguards into the genetic material of retroviruses so that such recombinations cannot occur. These conditions, which essentially amount to demands of delivery, expression, and safety, are no more than would be required of any new drug treatment or surgical procedure, and for a good reason. Somatic cell gene therapy differs little in its practical application from these more traditional treatments. "Somatic cell gene therapy is not funda- mentally different from other kinds of medical care," says Leroy B. Walters, Jr., of Georgetown University's Center for Bioethics. "In particular, it is very similar to transplantation techniques, and espe- cially to bone marrow transplantation techniques." Consequently, a consensus has been growing among those who have studied human gene therapy that it would be unethical to deny this treatment to desperately ill patients once the basic conditions of delivery, expres- sion, and safety have been satisfied. Nevertheless, a thorough review process has been set up to monitor the initial attempts at human gene therapy. After review by local Institutional Review Boards and Institutional Biosafety Committees, the research protocols for human gene therapy will have to be approved by a working group of NIH's Recombinant DNA Advisory Committee (RAC), by the committee itself, and by the director of NIH. (See
HUMAN GENE THERAPY 49 REGULATORY APPROVAL STEPS FOR HUMAN GENE THERAPY Institutional review board and institutional biosafety committee of researcher's home institution Institutional review board and institutional biosafety committee of the institution where research will be conducted Investigational new drug notice, filed with Food and Drug Administration Working Group on Human Gene Therapy of the Recombinant DNA Advisory Committee, National Institutes of Health Entire Recombinant DNA Advisory Committee, National Institutes of Health Director, National Institutes of Health Proposed interagency coordinating committee on biotechnology Proposed national commission on bioethics Approval Possibly Required Experiments involving human gene therapy will require approval at several different levels before they can proceed. The review boards and biosafety commit- tees of the researcher's home institution and the institution where the work is to be conducted must first approve the research protocol. The researcher must also file an investigational new drug notice (IND) with the Food and Drug Administration, although the agency does not have to approve the IND before the experiment can begin. The protocol then has to be approved at three separate levels within the National Institutes of Health. Finally, one or more other groups or individuals may have to approve the protocol, including the secretary of Health and Human Services, the commissioner of the Food and Drug Administration, the interagency coordinating committee proposed by the Cabinet Council Working Group on Biotechnology (discussed in Chapter 6), and the national commission on bioethics proposed by Senator Albert Gore, Jr.
50 BIOTECHNOLOGY Chapter 6 for a detailed discussion of the history and function of the RAC.) The Food and Drug Administration will regulate human gene therapy under the same regulations that it applies to clinical trials of any new drug or biologic (also discussed in Chapter 6). As somatic cell gene therapy evolves and new procedures are developed, other concerns will come to the fore. For instance, it may someday be possible to conduct gene therapy on cells within the body as well as on cells withdrawn from the body. If genetically engineered retroviruses could be designed that home in on certain types of somatic cells when injected into the body, DNA could be delivered to specific body tissues. It appears, however, that a cell must be dividing for retroviral DNA to be incorporated into its chromosome, which would preclude the use of this technique for mature brain or nerve cells. Also, the introduction of viruses into the body would undoubtedly raise a host of additional questions about safety. Germline Gene Therapy Much more controversial than the replacement of a defective gene in somatic cells is the replacement of a defective gene in germline cellsâ the cells that contribute to the genetic heritage of offspring. In this case, gene therapy has the potential to affect not only the individual undergoing the treatment but his or her progeny as well. Germline gene therapy would change the genetic pool of the entire human species, and future generations would have to live with that change, for better or worse. Germline gene therapy has been accomplished in laboratory ani- mals. The mice described in Chapter 3 that were transformed by an inserted growth gene passed the gene on to their offspring, demonstrat- ing that the gene had been inherited as a stable genetic trait. But a number of technical difficulties make it extremely unlikely that germline gene therapy will be attempted in humans in the near future, if ever. First, the procedure has a very high failure rate. Most fertilized mouse eggs are so damaged by the microinjection and transfer that they never develop into live animals. Furthermore, a good laboratory can get the foreign gene into the germ line of only about 10 to 30 percent of the mice that are born. This degree of success occurs in mice that have been carefully inbred to give good results in this procedure. It would probably be even lower in genetically heteroge- neous human cells. A second major barrier is that none of the methods of inserting foreign DNA into cells offers any control over where the DNA will
HUMAN GENE THERAPY 51 integrate into the chromosome. A foreign gene may be inserted into the middle of a critical gene in the cell, blocking that gene's function. The insertion of a foreign gene can also turn other genes in the cell on or off, causing metabolic imbalances that harm the cell. If this happens in a few of the bone marrow cells treated by somatic cell gene therapy, the consequences might go unnoticed. If it happens in a germline cell, the consequences are more likely to be severe. The inability to control the chromosomal location of insertion points toward a much more fundamental problem that affects both somatic cell and germline gene therapy. The control of a gene in a cell depends on a number of regulatory factors, including position, and many of these regulatory influences are at this point not understood. It is impossible to insert the new gene into the exact position of the defective gene, because the defective gene is already there, and there are no techniques available for deleting or repairing a defective gene. Foreign genes have been genetically engineered to carry their own regulatory signals so that they are expressed in the cell. But these regulatory systems are crude compared with the precise regulatory systems of the cell. Some of the mice treated with growth hormone genes suffered from gigantism, so that parts of their bodies grew disproportionately large, and the metabolic imbalances caused by the inappropriate expression of growth hormone left nearly all the genet- ically engineered female mice sterile. Because of these and other technical difficulties, there are no plans now being made to attempt germline gene therapy in human beings, and the prospects for any such attempts in at least the near future look dim. Genetic Engineering to Enhance Human Traits All the procedures discussed in the preceding sections are designed to insert into cells a normal gene corresponding to a defective gene. However, another kind of gene therapyâwhich Anderson feels is more properly termed genetic engineeringâcan also be imagined. This is genetic manipulation that seeks to insert genes into either somatic or germline cells in a way that alters or improves normal human attributes. An example of such genetic engineering is the introduction into human cells of a gene that would produce elevated levels of growth hormone. This is not now possible with humans, but it is being attempted with livestock animals to increase their production of meat or milk. Given the present level of understanding, the physiological
52 BIOTECHNOLOGY consequences of such genetic engineering cannot be predicted. A person who produces excess growth hormone would probably be taller, but the debilitating symptoms and disfiguration of people who naturally pro- duce too much growth hormone, including a susceptibility to diabetes and heart disease and an appearance characteristic of gigantism, would probably also be present. There are other examples of enhancement genetic engineering in which the issues are less clear-cut. Anderson points out the case of a gene that would produce more receptors for low-density lipoproteins on the surfaces of cells, reducing the level of cholesterol in the blood. Such a gene could bring the cholesterol level of people at the greatest risk of atherosclerosis down to its lower ranges. "This is an issue that doesn't need to be discussed in the immediate future," says Anderson. "But it is something that might very well come up in later years." This ability to change specific physiological indexes should be sharply distinguished from the type of genetic engineering that has generated the most concern among the public, according to Anderson. This latter type of genetic engineering involves changing complex human traitsâlike intelligence, character, and physical appearanceâ that are shaped by a subtle interplay of many interacting genes and environmental influences. Such "eugenic" genetic engineering "really is a fantasy at the present time," says Anderson. "Any of these characteristics involves hundreds or thousands of genes interacting in completely unknown ways. How to be able to go in and insert one gene or two genes and in any way predictably change these enormously complex polygenic characteristics is totally unknown." It is difficult to assess in scientific terms the likelihood of eugenic genetic engineering, because, as Anderson puts it, there simply isn't any science to discuss. But the ethical issues surrounding germline gene therapy or more straightforward forms of enhancement genetic engineering should not be slighted simply because these capabilities are not yet in hand. Technical advances are occurring at an increasing rate in molecular biology, and it is almost impossible to predict what eventually will or will not be doable. Walters feels that the premature attempts at human gene therapy have given rise to "a very meaningful process of public reflection and discussion." In 1982 the President's Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Re- search released a report that clarified many of the basic issues surrounding the different types of human gene therapy, drawing on the expertise of not only scientists and physicians but philosophers, soci- ologists, and theologians as well. Since then several other hearings,
HUMAN GENE THERAPY 53 symposia, and reports have elaborated on these issues, and they have received considerable attention from the more general media. To continue this ongoing discussion, Senator Albert Gore, Jr., has proposed the creation of a national commission to monitor develop- ments in biotechnology that affect human genetic engineering. The commission would be interdisciplinary and nonregulatory in nature, with the main goal of rendering advice and recommendations about the ethical implications of new capabilities. "We are at the present time woefully unprepared to grapple with the serious ethical choices with which the new technology will confront us," says Gore. "The very power to bring about so much good will also open the door to serious potential problems. If we are not careful, we may well cross the line separating the two. Knowing where that line exists is the challenge that we face." Additional Readings W. French Anderson. 1984. "Prospects for Human Gene Therapy." Science 226 (October 26): 401-409. Yvonne Baskin. 1984. The Gene Doctors. New York: William Morrow. Office of Technology Assessment. 1984. Human Gene TherapyâBackground Paper. Washington, D.C.: U.S. Government Printing Office. President's Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research. 1982. Splicing Life: A Report on the Social and Ethical Issues of Genetic Engineering with Human Beings. Washington, D.C.: U.S. Government Printing Office.