cal trial phenomenaâ was the advice provided by. Jonathan ... two recent AAV gene therapy trials. One of .... research referred to as âbench to bedside and back.
editorial
© The American Society of Gene Therapy
doi:10.1038/sj.mt.6300279
Humility and Clinical Trials
“
One must be humble when investigating clinical trial phenomena” was the advice provided by Jonathan Yewdell at a symposium on immune responses to adeno-associated virus (AAV) vectors conducted by the National Institutes of Health Recombinant DNA Advisory Committee (RAC) on 19 June 2007. The symposium was organized in response to reports of apparent immune responses in two recent AAV gene therapy trials. One of these trials used an AAV serotype 2 (AAV2) vector expressing factor IX delivered to the livers of subjects with hemophilia B. One subject who received the highest dose of vector developed a transient elevation in serum transaminases concurrent with a loss of factor IX transgene expression. A second subject at that dose showed low but transient factor IX in blood without elevated transaminases. An additional subject, enrolled at a lower dose of vector, demonstrated transient transaminase elevation but no factor IX in the blood. In a splendid example of translational research, the investigators set out to determine why these unexpected events occurred. Possible explanations, including development of cellular or humoral immune responses to factor IX or an exacerbation of pre-existing hepatitis C virus infection, were ruled out. A key finding was the appearance of CD8+ T cells directed against the AAV capsid in the third subject. This led to the proposal that input vector capsids primed a capsid-specific T-cell response, such that transduced hepatocytes were targeted by cytotoxic T cells (called the capsid T-cell hypothesis). For this to occur, the exogenous capsid proteins would have to be shuttled into the major histocompatibility complex class I pathway via cross-presentation in both antigen-presenting cells for T-cell priming and hepatocytes for T-cell targeting. The second trial involved intramuscular administration of an AAV1 vector expressing lipoprotein lipase (LPL) in subjects with inherited hypertriglyceridemia. Key findings were a drop in serum triglycerides in all subjects, the appearance of capsid T cells in half of the subjects, and a transient lowlevel elevation of creatine phosphokinase (CPK) in the blood of one of the subjects who developed capsid T cells. Evidence of transgene expression could only be surmised by the drop in triglycerides, because Molecular Therapy vol. 15 no. 9 september 2007
gene transfer was not sufficiently robust to yield detectable LPL protein in blood. Extrapolating from the hemophilia trial, the investigators invoked the capsid T-cell hypothesis to explain findings in at least one of their subjects in which there was a concurrence of T cells to AAV1 capsid, elevation of CPK, and a decrease in transgene expression as measured by a return of triglycerides to baseline. Although these data must be carefully considered, they fall far short of an endorsement of the capsid T-cell hypothesis. For example, three of eight subjects had capsid T cells but no CPK elevations. In addition, correlations with transgene expression based on triglyceride levels were difficult because of substantial intrasubject variations. Validation of the capsid T-cell hypothesis and consideration of alternative mechanisms has been complicated by the fact that the clinical phenomena have not been fully reproduced in animals. Although several groups have shown activation of T cells in animals following AAV2 gene transfer, no one has provided convincing evidence that T cells directed against capsid target vectortransduced hepatocytes or muscle fibers in an antigen-specific manner. Hildegund Ertl described preliminary studies in mice implicating memory T cells directed against capsid in facilitating effector function, a finding that clearly deserves more study. Several participants pointed out that the onset of the presumed effector T-cell sequelae observed in the hemophilia trial (i.e., elevated liver function tests and loss of transgene expression) occurs later than would be expected from target-cell presentation of input capsid and that the amount of capsid presented in this way may not be enough to engage an effector response. Several alternative hypotheses explain capsidmediated activation of T cells and targeting of transduced cells. Nick Muzyczka pointed out that vector preparations are probably contaminated with cap sequences from the packaging constructs. In fact, vector-derived cap DNA has been detected in reasonably high amounts in monkey spleen and liver following systemic administration. However, cap transcripts have not been detected in mice after in vivo gene transfer. Vector-derived cap sequences could clearly play a role in T-cell 1571
© The American Society of Gene Therapy
editorial
activation, although their role in target-cell killing is less clear. One would need to propose a bystander effect to explain a total loss of transgene expression, because the numbers of captransduced cells are lower than those of transgene-transduced cells. Humans and monkeys harbor relatively large amounts of latent AAV genomes with low but detectable transcriptional activity. This establishes immunologic pre-exposure/memory that could confound host responses to vector that, if reactivated, could provide a source of antigen for T-cell activation or target-cell killing. Jude Samulski provided an intriguing hypothesis involving the potential expression of an open reading frame (ORF) from an internal ATG within the factor IX complementary DNA that is out of frame with factor IX. He argued that a T-cell response to a transgene-expressed antigen is more consistent with the time course of transaminitis and extinction of transgene expression observed in the hemophilia trial. It has been suggested that defective forms of newly synthesized proteins for which this ectopic ORF may qualify are the actual source of endogenous major histocompatibility complex class I ligands. In fact, Samulski has demonstrated T-cell activation in mice to an epitope inserted within the ectopic ORF of factor IX. If this hypothesis is to be relevant to gene therapy, one needs to envision the selective translation of this ORF from the transgene-derived messenger RNA, because it should be present in the endogenous messenger RNA, which if expressed would render the subject tolerant. I believe the community did heed Yewdell’s plea to proceed with humility during the deliberations. Phase I clinical studies
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are not designed necessarily to address mechanisms, and information is often anecdotal but nonetheless of potential importance. The RAC did a masterful job in capturing the spirit of open and objective scientific discourse regarding a topic of substantial importance to the field. One reason for this success was that the ad hoc participants were asked to discuss science rather than establish policy. It did not escape us, however, that our colleagues in the federal government responsible for policy and regulation of gene therapy were in attendance. In assessing the state of in vivo gene therapy with AAV vectors we have entered into the phase of translational research referred to as “bench to bedside and back.” The clinical phenomena described above have stimulated novel hypotheses and additional basic research. However, substantial gaps remain in our understanding of vector biology and host–vector interactions. The symposium enabled us to refine the questions that should help focus additional preclinical studies and develop better animal models. Until we know more about these basic issues, we should take seriously all the hypotheses in considering subsequent clinical trials. Potential considerations include increasing vector potency, decreasing immunogenicity of the capsid and vector preparations, reducing levels of cap sequences in the final products, eliminating potential cryptic ORFs, and avoiding modifications to the transgene cassette that may activate latent cryptic ORFs.
James Wilson Associate Editor
www.moleculartherapy.org vol. 15 no. 9 september 2007
