Journal of Assisted Reproduction and Genetics, VoL 14. No. 6, 1997 NEWS AND VIEWS REPRODUCTIVE HEALTH CARE POLICIES AROUND THE WORLD Genetic Engineering" Moral Aspects and Control of Practice INTRODUCTION outline the concept of gene therapy with regard to the ethical aspects involved. Since the time of Hippocrates, physicians have sought to understand the'mechanisms of disease development for the purposes of developing more HISTORY effective therapy. The application of molecular biol- ogy to human diseases has produced significant The following is a concise review of the history discoveries about some of these disease states. of gene therapy. Extensive reviews can be found Gene transfer involves the delivery to target cells elsewhere (1). The human species has for many of an expression cassette made up of one or more generations practiced genetic manipulation on genes and the sequence controlling their expres- other species. Examples can be seen in the breed- sion. The process is usually aided by a vector that ing of animals and plants for specific intent, such helps deliver the cassette to the intracellular site as, corn, roses, dogs, and horses. The term "genet- where it can function appropriately. Human gene ics" was first used in 1906, and the term "genetic therapy has moved from feasibility and safety stud-. engineering" originated in 1932. Genetic engi- ies to clinical application more rapidly than was neering was then defined as the application of expected. The development of recombinant DNA genetic principles to animal and plant breeding. technology brought science to the brink of curing The term "gene therapy" was adopted to distin- previously untreatable diseases in a relatively short guish itself from the ominous, germ-line perception period of time. The ability to characterize gene associated with the term "human genetic engi- defects at the DNA level, and the availability of neering" (1). vectors for the insertion of genetic information into "Gene therapy can be defined as the application normal and abnormal tissues of the human body, of genetic principles to the treatment of human has made possible the initiation of genetic therapy disease" (1). The 1944 discovery by Avery and col- of human disease. The Human Genome Project is leagues that a gene can be transferred within expected to promote additional scientific ad- nucleic acids is an important landmark (2). The vances. This raises the fear of much to rapid devel- capacity of viruses to transmit genes was first dem- opment leading to uncontrollable gene transfers onstrated in Salmonella species (3). Viral genomes being performed. It is the purpose of this article to were discovered to be able to integrate into cell genomes (4) and, later, were found to be responsi- ble for cell transformation (5,6). The elucidation of the structure of DNA in 1953 and the subsequent The opinions presented in this column are those of its authors and do not necessarily reflect those of the journal and its editors, discoveries of mRNA increased progress in the field publisher, and advertisers. of genetic research (7). In the interval preceding 297 I058-0468/9710700-0297512.50/0© 1997 Plenum Publishing Corporation 298 EISENBERG AND SCHENKER the recombinant DNA era, key aspects of gene lings with arginase deficiency were injected with therapy were elaborated. In 1966, Tatum predicted the Shope virus, without any effect on their arginine that viruses could be used to transduce genes (8). levels (16). In 1980 Martin Cline attempted gene Another important milestone is the successful repli- transfer of the p-globin gene into human bone mar- cation of DNA in a test tube by Kornberg (9). By row cells and their transplantation into patients with the late 1960s and early 1970s, gene therapy thalassemia. The study, which was not approved became the subject of many articles and meetings. by the Institutional Review Board, was severely criti- In 1970 Davis discussed human genetic engi- cized for scientific, ethical and procedural reasons neering and explored the feasibility and ethics of (17). The indirect result of this was the NIH decision somatic and germ cell alterations, cloning of that all future gene therapy must be approved by humans, genetic modification of behavior, sex pre- the NIH Recombinant DNA Advisory Committee selection, and selective reproduction. He stated (RAC) (1). Unsound practices in these early studies then that "control of polygenic behavioural traits is in cell culture, animals, and humans made both the much less likely than cure of monogenic dis- experimental results and the entire approach of eases" (10). gene therapy seem suspect, even though some of Studies in the late 1950s and early 1960s the basic concepts and approaches upon which revealed that cultured cells could take up radioac- the studies were based were eventually proven cor- tive DNA and that the DNA could enter into the rect (1). nucleus of the cells. Naked viral DNA or RNA was Major progress was made when early transfec- demonstrated to be ineffective when applied to tion techniques and selection systems for cultured cells. Uptake of cellular or viral polynucleotides cells were combined with recombinant DNA tech- could be improved by complexing with various pro- nology. The isolation of a single gene enabled both teins, such as protamine (11). In the 1960s several greater efficiency and better documentation of its studies asserted changes in cellular phenotype by transfer. Studies demonstrated that any gene can the transfer of nonviral genes. Later, cell lines con- be transferred into mammalian cells along with a taining defined enzymatic defects and setectable selectable marker (18). The early 1980s brought systems were established, and thus, started the forth the development of retroviral vectors (19). In era of gene transfer (1). Later developments spe- 1983 The Banbury Gene Therapy meeting set the cialized a method of calcium phosphate-mediated course to further research into safe gene delivery transfection which became widely used (12). applications (20). Another important development The earliest predecessor of a direct in vivo was that of the HPRT gene transfer by Szybalski approach to gene transfer was the use of vaccines (21). After several disease-related genes were with attenuated viruses, which permanently modify transferred into various cells in culture, the possibil- the body's response to infection and may persist ity of efficient gene transfer into mammalian cells for a long term (1). The ease of administratior~ for the purpose of gene therapy became widely relative cheapness, and long-lasting effect of vac- accepted (1). In 1989 the first approved human cines are ideal qualities to which proponents of gene therapy trial was initiated (22). direct gene therapy aspire. An early attempt at treat- The idea of gene therapy developed shortly after ing bacterial infections by the injection of bacterio- the discovery of molecular genetics, however, phages (13) was later dropped with the ascent of advancement was for a long period of time hin- antibiotics. Other studies explored the ability of dered by poorly designed studies. Progress has DNA to transfer the neoplastic state. The phenotype accelerated in recent years, as the field has gained for neoplastic transformation was reliably trans- greater credibility. The Human Genome Project is ferred from mammalian DNA into cells in culture expected to advance the field further. (14). The maturation of plasmid expression vectors, reporter genes, and better in situ detection systems prompted further attempts-at direct in vivo gene transfer. POSSIBLE APPLICATIONS OF GENE In the late 1960s, Rogers injected the shope papi- THERAPY AND CURRENT TRIALS Ioma virus into patients with arginase deficiency, based upon studies indicating that the virus con- There are four potential levels for the application tained an arginase gene (15). However, three sib- of gene transfer techniques. Journal of Assisted Reproduction and Genetics, Vol. 14, No. 6, 1997 GENETIC ENGINEERING 299 1. Somatic cell gene therapy involves the correc- fication. However, these vectors must inte- tion of a genetic defect in somatic cells. This grate into the dividing cell in order to expand, type of gene therapy is beneficial primarily so that if a mutation occurs there will be per- for the replacement of a defective or missing manent damage. The early cells that have enzyme or protein or a deficient circulating the greatest potential to sustain the long term protein. changes are often quiescent and nondividing. 2. Germline alteration requires the insertion of a The genes, once inserted, do not always gene into the reproductive tissue of the remain transcriptionally active. In vivo selec- patient, resulting in the correction of the disor- tion of cells with functionally active retroviral der in the offspring as well. genes may assist in solving these problems. 3. Enhancement genetic engineering involves The risks are the potential for toxicity associ- the insertion of a gene with the intent to ated with chronic overexpression or inser- enhance a certain characteristic, such as tional mutagenesis. For example, if the height. proviral DNA randomly disrupts a tumor sup- 4. Eugenic genetic engineering involves an pressor gene or activates an oncogene. attempt to alter or improve complex human 2. Adenoviruses infect epithelial cells at a high traits, which are usually polygenic, such as frequency but they do not require a cell for personality or character. proliferation. Adenoviruses do not infect the Since presently only somatic cell gene therapy bone marrow, and they are immunogenic. Fur- is feasible, the following discussion centers around thermore, they exist only temporarily in the it. The upcoming ethical discussion includes all the cytoplasm of the host cell. Thus, adenoviruses above aspects and their ir:nplications. are more suitable for populations of quiescent It is presently possible to insert a single gene, cells, especially in cases when a transient thus, recessive disorders are amenable to treat- expression of the transgene is sufficient to ment.