Gene Therapy and the Germ Line
Authors: Trisha Gura
Journal: Molecular Therapy (2002)

Like a specter from the swamps, the issue of accidental germline transmission has resurfaced. Beginning last August, pediatrician Mark Kay, at Stanford University in California, and hematologist Katherine High, at Children's Hospital of Philadelphia in Pennsylvania, watched with horror as signature sequences from their gene delivery vector (adeno-associated virus or AAV) showed up in the semen of two hemophilia patients undergoing gene transfer. Although Kay and High never detected the vector in the patients' sperm cells, and semen samples cleared of virus within 12 weeks after AAV injection, the incident has reignited a decades-old discussion. Given that researchers are designing more aggressive vectors that can reach higher concentrations for longer periods of time, what exactly are the risks of getting a renegade vector into an egg or sperm? Kay and High's experience has brought this concern one biological compartment closer to reality. "How prepared are we to accept the possibility that we may alter the germ line?" asks ethicist Mark Frankel, director of the program on scientific freedom, responsibility, and law at the American Association for the Advancement of Science in Washington, D.C. "Until very recently, nobody was giving much thought to the matter." As early as 1972, gene therapy pioneers such as Theodore Friedmann at the University of California at San Diego and ethicist Leroy Walters at Georgetown University in Washington, D.C., took up the issue: Could gene therapy vehicles—and the loads they carried—land in the genome controls of egg and sperm cells? [1Walters L. The ethics of gene therapy.Nature. 1986; 320: 225-227Crossref PubMed Scopus (64) Google Scholar, 2Friedmann T. Roblin R. Gene therapy for human genetic disease?.Science. 1972; 175: 949-955Crossref PubMed Scopus (518) Google Scholar]. Then, and now, no one had developed a means to control exactly where the vectors will hunker down—perhaps into the middle of a master switch that controls embryonic development. Such genetic consequences would permanently nest in the biological tree of life, investigators cautioned. But over time, researchers seemed to put the issue to bed with tables of probabilities, animal data, and results of early clinical trials which pointed to a very low—some say ridiculously low—risk of actually contaminating the human germ line. Then came a promising hemophilia trial. Katherine High and her colleagues wanted to inject an AAV vector carrying factor IX into the leg muscles of patients. Mark Kay wanted to do something similar, except with a direct liver route of administration. The two joined forces and started with a muscle-delivery protocol. In preclinical studies conducted throughout the late 1990s, High's group began injecting the AAV-factor IX construct into the leg muscles of mice and, later, rabbits. The vector ended up in the animals' gonads, rising in concentration in parallel with increasing doses. Vector DNA never showed up in semen, however. "Initially, you have vector in the testes," High explains, "then the vector washes out." When the investigators moved on to muscle injection in dogs, the results came up negative in both semen (measured between 7 and 90 days postinjection) and testes (observed at 5 months after injection) [3Arruda V.R. Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.Mol. Ther. 2001; 4: 586-592Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. Those data paved the way for the first clinical trial by muscle delivery, begun in June 1999. None of the eight patients given the AAV construct at two dose levels tested showed any vector sequences in their semen. But High's colleagues could not get vector concentrations high enough to demonstrate a clear effect on patients' factor IX blood levels. So the gene therapists temporarily halted the muscle trial and set their sights on the liver-delivery protocol developed by Kay, which uses an AAV-factor IX vector manufactured by Avigen in Alameda, California. By August 13, the Food and Drug Administration (FDA) gave Kay and his colleagues the go-ahead to inject their first patient, a 60-year-old hemophiliac. That event brought a startling revelation. "To our surprise, the patient's semen tested positive," High says. That raised everyone's apprehension. Researchers began digging back into old data to get a better sense of what the risks actually are. According to Frankel, much of that was sadly lacking. Immediately Kay, High, and researchers at Avigen notified the FDA and postponed injection into the second patient. Upon further testing of the first, the investigators found that DNA persisted in the 63-year-old man's semen for up to 10 weeks. Sperm cells, on the other hand, always came up negative. At the FDA's request, High set up rabbit experiments testing infusion of AAV vector directly into the animals' liver. These animals showed a dose-dependent increase of vector in the basement membrane of the testes and in the semen. Animals' sperm cells, as in patients', tested negative after amplification of DNA by polymerase chain reaction. In fact, Kay and High's group isolated the sperm of mice in culture and pipetted in high titers of AAV-factor IX (transduced at a multiplicity of infection of 5000). The sperm cells still did not take up the vector DNA. Based on these data, on January 29, the FDA gave Kay's team permission to inject the second patient. But again, the germline issue reared its ugly head: The vector showed up immediately in the patient's semen and waned again within 10 weeks of injection. The FDA, concerned, officially put the entire trial on hold. Meanwhile, researchers at Chiron were having their own problems with a clinical gene transfer trial in hemophilia patients with a retroviral vector carrying factor VIII sequences. There, the vector initially appeared in the first patient's semen but did not show up in a second test, according to John Gallagher, director of media relations for Chiron. The company later halted the trial as it refocused its priorities, Gallagher says. Next, the Biological Response Modifiers Advisory Committee (BRMAC), set up by the FDA, stepped in. Headed by Daniel Salomon at Scripps Research Institute in La Jolla, the BRMAC arranged for a meeting on May 9–10 in Gaithersburg, MD, to discuss problems with the liver-delivery hemophilia trial as well as another germ cell modification procedure taking place in in vitro fertilization clinics. The committee dealt with two main issues, says Salomon: How likely is this particular vector to target a stem cell in the ovary or testes and, given that risk, what kind of tests are required to monitor patients undergoing gene transfer? "Essentially," Salomon says, "we were asking what is the risk-versus-benefits." Kay provided some numbers from animal studies. The concentrations of vector in semen after liver administration were "minuscule," he says. The vector simply was not detectable in the sperm, with a sensitivity of one copy of vector per 30,000 sperm. In addition, the direct incubation of sperm and vector previously showed that the sperm themselves have some sort of natural barrier against vector attack. There is also the very nature of AAV. In its wild-type state, AAV will find a specific site on chromosome 19 and hop in. But the modified vector manufactured by Avigen integrates very inefficiently into chromosome 19, or anywhere in the genome, for that matter. Unlike retroviruses and lentiviruses which do take up residence directly in the genome, a positive result for AAV in the sperm, if it did happen, would not necessarily indicate that viral sequences were on their way into an offspring. AAV isn't very promiscuous either. Phillipe Moullier's group at INSERM in Nantes Cedex France reported in Molecular Therapy that they had injected the muscles of eight macaques with a recombinant AAV vector carrying factor IX, modeling the hemophilia study. The French group found vector in various biological fluids for up to 6 days after infection. But those particles lost their infectious character by 72 hours postinjection. [5Favre D. Immediate and long-term safety of recombinant adeno-associated virus injection into the nonhuman primate muscle.Mol. Ther. 2001; 4: 559-566Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar] But there's another hook: A gene transfer patient could potentially send infectious particles into the eggs of his sexual partner. This so-called "horizontal transmission" was brought up at the BRMAC meeting and, without clear answers, the committee commissioned researchers to conduct further tests. Toward that end, transgenic mouse pioneer Jon Gordon, at the Mt. Sinai School of Medicine in New York, has set his focus on trying to answer some risk questions or perhaps, more practically, to set up tests of the "improbable but not impossible." For example, his team has both injected infectious vector particles into the ovaries of mice and soaked naked oocytes in vector particles an hour before in vitro fertilization. Using adenovirus constructs, Gordon, so far, has found that the vector cannot make it into the eggs [6Gordon J.W. Direct exposure of mouse ovaries and oocytes to high doses of an adenovirus gene therapy vector fails to lead to germ cell transduction.Mol. Ther. 2001; 3: 557-564Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Similarly, when Gordon injects high titers of adenovirus into the testes and epididymides of mice or incubates sperm cells with the vector before in vitro fertilization, the vector does not enter mature spermatocytes [7Hall S.J. Bar-Chama N. Ta S. Gordon J.W. Direct exposure of mouse spermatogenic cells to high doses of adenovirus gene therapy vector does not result in germ cell transduction.Hum. Gene Ther. 2000; 10: 1705-1712Crossref Scopus (44) Google Scholar]. It's a start. But there are less obvious possibilities to consider. Take, for example, the case of immature sperm and egg precursors within infected testes or ovaries. Can those take up vector? Gordon is now testing this scenario by injecting large amounts of vector into testicular ducts and tubules. And there are techniques in assisted reproduction in which the outer covering of the egg is stripped off to help sperm get in, or in which sperm and DNA are mixed together and used to fertilize eggs. Those techniques could potentially increase the likelihood of infecting germ cells with vector if laboratories are not kept scrupulously clean or if technicians, who are working with eggs and sperm, have undergone gene therapy and somehow contaminate the eggs with vector. Many of these scenarios, however contrived, have not even been thought about—let alone tested. Nor have the increased risks of many new modified vectors that are emerging. Why not? "Because the gene therapy community does not know much about gametogenesis and early embryo development," says Gordon. "And the in vitro fertilization community does not know much about gene therapy." So as the science solidifies, the BRMAC concluded that during the course of High and Kay's hemophilia trial the vector probably did not get into the patient's sperm cells and recommended to the FDA that the trials could go on. But the gene therapists had to alter their consent form to notify patients of the possibilities for inadvertent germline transmission and then counsel patients about sperm banking and barrier contraception use until semen clears. For up to a year after injection, patients' semen has to be monitored for vector contamination. A positive result, persisting for a year, would result in a "working presumption" of permanent germline modification. Then, the trial would be halted while studies to prove gene transfer were undertaken, for example testicular biopsy or direct sperm DNA sequencing. There is a bigger problem for testing females, however. So far, physicians have no noninvasive means to test for oocyte transduction. Rather than prohibiting women from undergoing gene therapy, the BRMAC instead charged researchers with developing animal models to better assess the risks to oocytes. Although the committee agreed that gene transfer cannot be limited to males, gender equity issues were beyond the scope of the meeting. "We realize that there is quite a bit of science left to be done," Salomon says. Of course, "the real crunch will come when and if somebody finds evidence of vector in the sperm or ova itself," says Friedmann, who is chairman of the National Institutes of Health's Recombinant DNA Advisory Committee. Gordon agrees. But until then, "I think the overall message is don't panic and don't put a halt to gene therapy over what we regard as improbable event," he says. "But don't put your head in the sand either."