- Email: [email protected]
Gene Therapy and Biosecurity
commentary
© The American Society of Gene Therapy
Gene Therapy and Biosecurity Michael J Imperiale1 doi:10.1038/sj.mt.6300136
T
he gene therapy field holds tremendous promise of curing or preventing a myriad of diseases. Gene therapy is by nature translational, and investigators in the field are committed to the betterment of human, animal, and plant health. Indeed, most researchers in the biological sciences would like to see their research results put to beneficent use. Unfortunately, not all humans are similarly driven; there are nefarious individuals who would rather do harm than good. Some of these individuals will use a gun, or develop an improvised explosive device, or spread computer viruses over the Internet. There are also those who would use biological agents to inflict harm on others. For example, the Rajneeshee cult tried to fix the results of an election in Oregon in 1984 by contaminating salad bars with Salmonella enterica. Perhaps most chilling, given its timing, was the distribution in 2001 of Bacillus anthracis spores through the US Postal Service on the heels of the September 11 attacks. Since the anthrax mailings, the US government has become acutely sensitized to the potential misuse of the reagents, technologies, and information that are being derived using biotechnology. The issue was brought to the forefront by a report published in 2004 by the National Research Council, “Biotechnology Research in an Age of Terrorism.” This document (also called the Fink Report, because the committee that produced it was chaired by Gerald Fink at the Massachusetts Institute of Technology) outlined the potential threats posed by possible misuse of biological research results and made a series of recommendations for minimizing the threat. The com1
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA Correspondence: Michael J Imperiale, Department of Microbiology and Immunology, University of Michigan, 1500 E Medical Center Drive, Ann Arbor, Michigan 48109, USA. E-mail: [email protected]
648
mittee recognized the so-called dual-use nature of biological research—namely, that the same information that would be used by most individuals for the good of mankind might be misused by someone wishing to inflict harm—and devised a series of recommendations on how to deal with it. One of the recommendations that has been adopted by the federal government is the creation of the National Science Advisory Board for Biosecurity (NSABB). The board was initially charged with five tasks: (1) define dual-use research of concern, (2) devise a code of conduct for the life sciences, (3) develop guidelines for publication of results that might be of concern, (4) develop recommendations for involving the international community in preventing misuse, and (5) examine whether the emerging fields of synthetic genomics and synthetic biology require special consideration. The NSABB has made significant progress, including presenting formalized versions of the first three proposals to the public at its July 2006 meeting and the first recommendations about synthetic genomes in November 2006. At the July meeting, a sixth task was defined: developing guidelines for the oversight of dual-use research of concern, which has been defined by the board as “research that, based on current understanding, can be reasonably anticipated to provide knowledge, products, or technologies that could be directly misapplied by others to pose a threat to public health, agriculture, plants, animals, the environment, or materiel.” Feedback from the scientific community was sought throughout the deliberation process of NSABB and will continue to be as its discussions continue. The areas of bioterrorism and gene therapy have an important interface, with many investigators interested in developing vaccines and therapeutics to fight pathogens that have high degrees of morbidity and mortality. Scientists who work with dangerous pathogens are well aware of the risks these organisms pose, and use appropriate
containment facilities to protect laboratory workers, the public, and the environment from exposure. Indeed, even investigators who use viruses simply as vectors understand the need for preventing unwanted exposure to those viruses. But there are ways in which gene therapy research presents potential dual-use concerns, and those who work with viral vectors ought to be alert to this potential. The issue at hand is that we are manipulating viruses, many of which are serious human or animal pathogens. Probably the area of greatest potential concern is attempts to alter the tropism of viruses, either by changing the cell-type specificity or by designing viruses that will replicate only in certain cell types by transcriptional targeting. Although these modifications are based on sound theoretical bases (e.g., the receptor for ligand A on the surface of an engineered virus is found only on cell type B, or promoter X is only active in cell type Y), until one actually builds the construct and produces infectious virus, there is no way of knowing whether it will behave as predicted. If such a virus is able to infect new cell types and somehow becomes replication competent, perhaps by recombination with a wild-type virus in the environment, or is in and of itself a conditionally replicating virus, it has the potential to cause disease in persons or animals exposed to the virus. Similarly, certain transgenes might affect the behavior of the virus, as was evidenced by the experiment in which investigators tried to enhance a contraceptive vaccine by inserting the gene that encodes interleukin-4 into a mousepox virus but ended up producing a highly virulent strain of virus that defeated even pre-existing immunity. It doesn’t take very much imagination to come up with more possible scenarios. Although in most cases the risk of creating something dangerous is very low, it is certainly not zero. Does this mean that we should not build these viruses and try to advance the field of gene therapy so as to realize its full potential? Definitely not, because the potential benefits are enormous. What it does mean is that we must be aware of these potential risks and ensure that if a virulent agent arises during the course of an experiment the possible ramifications are carefully considered. Therefore, as we design, carry out, www.moleculartherapy.org vol. 15 no. 4 april 2007
© The American Society of Gene Therapy
and analyze the results of our experiments, we should keep these concerns in mind. It would be preferable to demonstrate to the public that we are being responsible than to have Congress impose restrictions on what we do should something bad happen. As NSABB continues its work, it will be developing educational materials to inform the
commentary
life sciences research community on how to think about these issues as well as guidelines for reducing the potential risks. In the meantime, your institutional biosafety committee or biosafety officer can probably provide sound advice. For more information about the work of the NSABB, please visit http://www.biosecurityboard.gov.
See page 792
Immune Responses to AAV Capsid: Are Mice Not Humans After All? Roland W Herzog1 doi:10.1038/sj.mt.6300123
A
deno-associated viruses (AAVs) have been favored for in vivo gene transfer of therapeutic genes because of their low immunogenicity, strong safety record, and high efficiency of transduction of a number of cell types in animal models.1 However, in a recent gene therapy trial involving delivery of human coagulation factor IX (F.IX) for the treatment of hemophilia, two patients developed a Tcell response to AAV-2 capsid, which was not predicted from preclinical studies in animals. Importantly, transgene expression in the two patients declined to pretreatment levels, suggesting that a cytotoxic Tlymphocyte (CTL) response to the capsid may have been responsible for a loss of transgene-expressing cells. In this issue, Li et al. now provide a detailed analysis of the effect of CTL responses to AAV capsid in mice.2 The results highlight a problem facing the field: the lack of an appropriate and predictive animal model for immune-mediated loss of hepatocytes after transduction with AAV. Enthusiasm for the AAV vector system grew tremendously approximately a decade 1
Division of Molecular and Cellular Therapy, University of Florida, Cancer and Genetics Research Complex, Gainesville, Florida, USA Correspondence: Roland W Herzog, Division of Molecular and Cellular Therapy, University of Florida, Cancer and Genetics Research Complex, 1376 Mowry Road, Room 203, Gainesville, Florida 32610, USA. E-mail: [email protected]
Molecular Therapy vol. 15 no. 4 april 2007
ago, when several laboratories demonstrated sustained expression of a bacterial LacZ gene without activation of CTLs after intramuscular injection of recombinant AAV (serotype 2).3–5 Similarly, T-cell responses and innate responses to AAV capsid were consistently low in animal studies. In other experiments, sustained expression of a F.IX gene over several years in animal models of the X-linked bleeding disorder, hemophilia B, has been documented for muscle- and liver-directed gene transfer.6,7 These observations led to two phase I/II clinical trials. In the muscle-directed approach, gene transfer was long lasting and safe but subtherapeutic.8 A major concern in gene therapy for hemophilia is the risk of antibody formation to the coagulation factor transgene product. Therefore, subjects enrolled in the muscle trial were limited to those with F.IX missense mutations. Interestingly, liver-directed gene transfer showed higher efficacy than intramuscular injection, and the hepatic route was found to induce immune tolerance to F.IX and other transgene products.9 Consequently, hopes were high for the liver trial, and the only major immunological concern was that preexisting immunity in the form of neutralizing antibodies might prevent gene transfer, because most humans are seropositive for AAV-2. As predicted from studies in large animals, the first human subject with low preexisting neutralizing antibodies in the highest vector dose cohort exhibited therapeutic
F.IX expression (>10%) after infusion of the AAV-2 vector into the hepatic artery.10 However, F.IX activity declined to pretreatment levels within 2 months, a result quite different from the years of expression observed in animals. A transient elevation of liver transaminase enzymes (transaminitis) was found to coincide with the loss of transgene expression.10 No antibody to F.IX was found in the affected patient, and causes for the transaminitis not related to the gene transfer were ruled out. In a subject treated subsequently with a slightly lower vector dose, transaminitis was again documented, and a detailed immunological analysis revealed a T-cell response to AAV-2 capsid. Computer software predicted the dominant epitope (initially identified by enzymelinked immunosorbent spot) to represent a major histocompatibility complex (MHC) class I–restricted CD8+ T-cell epitope.10 Ironically, a CTL response apparently had limited expression from a vector praised earlier for lack of CTL induction. Although there is no direct demonstration of a CTL response to AAV capsid proteins leading to human hepatocyte death, the data supporting this interpretation are compelling. The temporal pattern of the T-cell response can be superimposed onto the transaminitis, and both subjects experienced similar time courses for the transient increase of liver enzyme concentrations.10 An AAV capsid-specific CD8+ T-cell clone was expanded from one of the subjects and showed high cytolytic activity against cells that displayed capsid antigen.11 At the same time, AAV capsid T-cell epitopes were mapped in mice with the aim of developing an animal model to study these CTL responses.12,13 CD8+ T-cell epitopes in humans and mice are highly conserved among many AAV serotypes.10,13 The main question at this point is obviously why the duration of transgene expression was not limited by CTL responses to AAV capsid in any of the animal models. One potential explanation is natural infection in humans with AAV, which is not the case for murine and canine models (but does, however, occur in nonhuman primates). Upon natural infection in the presence of a helper virus, AAV capsid-specific memory T cells may be generated14. The kinetics of the transaminitis-CTL response in the documented human cases (4–6 weeks after gene transfer) may then be explained 649
© The American Society of Gene Therapy
Gene Therapy and Biosecurity Michael J Imperiale1 doi:10.1038/sj.mt.6300136
T
he gene therapy field holds tremendous promise of curing or preventing a myriad of diseases. Gene therapy is by nature translational, and investigators in the field are committed to the betterment of human, animal, and plant health. Indeed, most researchers in the biological sciences would like to see their research results put to beneficent use. Unfortunately, not all humans are similarly driven; there are nefarious individuals who would rather do harm than good. Some of these individuals will use a gun, or develop an improvised explosive device, or spread computer viruses over the Internet. There are also those who would use biological agents to inflict harm on others. For example, the Rajneeshee cult tried to fix the results of an election in Oregon in 1984 by contaminating salad bars with Salmonella enterica. Perhaps most chilling, given its timing, was the distribution in 2001 of Bacillus anthracis spores through the US Postal Service on the heels of the September 11 attacks. Since the anthrax mailings, the US government has become acutely sensitized to the potential misuse of the reagents, technologies, and information that are being derived using biotechnology. The issue was brought to the forefront by a report published in 2004 by the National Research Council, “Biotechnology Research in an Age of Terrorism.” This document (also called the Fink Report, because the committee that produced it was chaired by Gerald Fink at the Massachusetts Institute of Technology) outlined the potential threats posed by possible misuse of biological research results and made a series of recommendations for minimizing the threat. The com1
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA Correspondence: Michael J Imperiale, Department of Microbiology and Immunology, University of Michigan, 1500 E Medical Center Drive, Ann Arbor, Michigan 48109, USA. E-mail: [email protected]
648
mittee recognized the so-called dual-use nature of biological research—namely, that the same information that would be used by most individuals for the good of mankind might be misused by someone wishing to inflict harm—and devised a series of recommendations on how to deal with it. One of the recommendations that has been adopted by the federal government is the creation of the National Science Advisory Board for Biosecurity (NSABB). The board was initially charged with five tasks: (1) define dual-use research of concern, (2) devise a code of conduct for the life sciences, (3) develop guidelines for publication of results that might be of concern, (4) develop recommendations for involving the international community in preventing misuse, and (5) examine whether the emerging fields of synthetic genomics and synthetic biology require special consideration. The NSABB has made significant progress, including presenting formalized versions of the first three proposals to the public at its July 2006 meeting and the first recommendations about synthetic genomes in November 2006. At the July meeting, a sixth task was defined: developing guidelines for the oversight of dual-use research of concern, which has been defined by the board as “research that, based on current understanding, can be reasonably anticipated to provide knowledge, products, or technologies that could be directly misapplied by others to pose a threat to public health, agriculture, plants, animals, the environment, or materiel.” Feedback from the scientific community was sought throughout the deliberation process of NSABB and will continue to be as its discussions continue. The areas of bioterrorism and gene therapy have an important interface, with many investigators interested in developing vaccines and therapeutics to fight pathogens that have high degrees of morbidity and mortality. Scientists who work with dangerous pathogens are well aware of the risks these organisms pose, and use appropriate
containment facilities to protect laboratory workers, the public, and the environment from exposure. Indeed, even investigators who use viruses simply as vectors understand the need for preventing unwanted exposure to those viruses. But there are ways in which gene therapy research presents potential dual-use concerns, and those who work with viral vectors ought to be alert to this potential. The issue at hand is that we are manipulating viruses, many of which are serious human or animal pathogens. Probably the area of greatest potential concern is attempts to alter the tropism of viruses, either by changing the cell-type specificity or by designing viruses that will replicate only in certain cell types by transcriptional targeting. Although these modifications are based on sound theoretical bases (e.g., the receptor for ligand A on the surface of an engineered virus is found only on cell type B, or promoter X is only active in cell type Y), until one actually builds the construct and produces infectious virus, there is no way of knowing whether it will behave as predicted. If such a virus is able to infect new cell types and somehow becomes replication competent, perhaps by recombination with a wild-type virus in the environment, or is in and of itself a conditionally replicating virus, it has the potential to cause disease in persons or animals exposed to the virus. Similarly, certain transgenes might affect the behavior of the virus, as was evidenced by the experiment in which investigators tried to enhance a contraceptive vaccine by inserting the gene that encodes interleukin-4 into a mousepox virus but ended up producing a highly virulent strain of virus that defeated even pre-existing immunity. It doesn’t take very much imagination to come up with more possible scenarios. Although in most cases the risk of creating something dangerous is very low, it is certainly not zero. Does this mean that we should not build these viruses and try to advance the field of gene therapy so as to realize its full potential? Definitely not, because the potential benefits are enormous. What it does mean is that we must be aware of these potential risks and ensure that if a virulent agent arises during the course of an experiment the possible ramifications are carefully considered. Therefore, as we design, carry out, www.moleculartherapy.org vol. 15 no. 4 april 2007
© The American Society of Gene Therapy
and analyze the results of our experiments, we should keep these concerns in mind. It would be preferable to demonstrate to the public that we are being responsible than to have Congress impose restrictions on what we do should something bad happen. As NSABB continues its work, it will be developing educational materials to inform the
commentary
life sciences research community on how to think about these issues as well as guidelines for reducing the potential risks. In the meantime, your institutional biosafety committee or biosafety officer can probably provide sound advice. For more information about the work of the NSABB, please visit http://www.biosecurityboard.gov.
See page 792
Immune Responses to AAV Capsid: Are Mice Not Humans After All? Roland W Herzog1 doi:10.1038/sj.mt.6300123
A
deno-associated viruses (AAVs) have been favored for in vivo gene transfer of therapeutic genes because of their low immunogenicity, strong safety record, and high efficiency of transduction of a number of cell types in animal models.1 However, in a recent gene therapy trial involving delivery of human coagulation factor IX (F.IX) for the treatment of hemophilia, two patients developed a Tcell response to AAV-2 capsid, which was not predicted from preclinical studies in animals. Importantly, transgene expression in the two patients declined to pretreatment levels, suggesting that a cytotoxic Tlymphocyte (CTL) response to the capsid may have been responsible for a loss of transgene-expressing cells. In this issue, Li et al. now provide a detailed analysis of the effect of CTL responses to AAV capsid in mice.2 The results highlight a problem facing the field: the lack of an appropriate and predictive animal model for immune-mediated loss of hepatocytes after transduction with AAV. Enthusiasm for the AAV vector system grew tremendously approximately a decade 1
Division of Molecular and Cellular Therapy, University of Florida, Cancer and Genetics Research Complex, Gainesville, Florida, USA Correspondence: Roland W Herzog, Division of Molecular and Cellular Therapy, University of Florida, Cancer and Genetics Research Complex, 1376 Mowry Road, Room 203, Gainesville, Florida 32610, USA. E-mail: [email protected]
Molecular Therapy vol. 15 no. 4 april 2007
ago, when several laboratories demonstrated sustained expression of a bacterial LacZ gene without activation of CTLs after intramuscular injection of recombinant AAV (serotype 2).3–5 Similarly, T-cell responses and innate responses to AAV capsid were consistently low in animal studies. In other experiments, sustained expression of a F.IX gene over several years in animal models of the X-linked bleeding disorder, hemophilia B, has been documented for muscle- and liver-directed gene transfer.6,7 These observations led to two phase I/II clinical trials. In the muscle-directed approach, gene transfer was long lasting and safe but subtherapeutic.8 A major concern in gene therapy for hemophilia is the risk of antibody formation to the coagulation factor transgene product. Therefore, subjects enrolled in the muscle trial were limited to those with F.IX missense mutations. Interestingly, liver-directed gene transfer showed higher efficacy than intramuscular injection, and the hepatic route was found to induce immune tolerance to F.IX and other transgene products.9 Consequently, hopes were high for the liver trial, and the only major immunological concern was that preexisting immunity in the form of neutralizing antibodies might prevent gene transfer, because most humans are seropositive for AAV-2. As predicted from studies in large animals, the first human subject with low preexisting neutralizing antibodies in the highest vector dose cohort exhibited therapeutic
F.IX expression (>10%) after infusion of the AAV-2 vector into the hepatic artery.10 However, F.IX activity declined to pretreatment levels within 2 months, a result quite different from the years of expression observed in animals. A transient elevation of liver transaminase enzymes (transaminitis) was found to coincide with the loss of transgene expression.10 No antibody to F.IX was found in the affected patient, and causes for the transaminitis not related to the gene transfer were ruled out. In a subject treated subsequently with a slightly lower vector dose, transaminitis was again documented, and a detailed immunological analysis revealed a T-cell response to AAV-2 capsid. Computer software predicted the dominant epitope (initially identified by enzymelinked immunosorbent spot) to represent a major histocompatibility complex (MHC) class I–restricted CD8+ T-cell epitope.10 Ironically, a CTL response apparently had limited expression from a vector praised earlier for lack of CTL induction. Although there is no direct demonstration of a CTL response to AAV capsid proteins leading to human hepatocyte death, the data supporting this interpretation are compelling. The temporal pattern of the T-cell response can be superimposed onto the transaminitis, and both subjects experienced similar time courses for the transient increase of liver enzyme concentrations.10 An AAV capsid-specific CD8+ T-cell clone was expanded from one of the subjects and showed high cytolytic activity against cells that displayed capsid antigen.11 At the same time, AAV capsid T-cell epitopes were mapped in mice with the aim of developing an animal model to study these CTL responses.12,13 CD8+ T-cell epitopes in humans and mice are highly conserved among many AAV serotypes.10,13 The main question at this point is obviously why the duration of transgene expression was not limited by CTL responses to AAV capsid in any of the animal models. One potential explanation is natural infection in humans with AAV, which is not the case for murine and canine models (but does, however, occur in nonhuman primates). Upon natural infection in the presence of a helper virus, AAV capsid-specific memory T cells may be generated14. The kinetics of the transaminitis-CTL response in the documented human cases (4–6 weeks after gene transfer) may then be explained 649