Autoimmunity, recessive diseases, and gene replacement therapy.

The next chapter in the complicated story of host immune responses to in vivo gene therapy has been written in the form of a report on adeno-associated vector (AAV)-mediated gene transfer to muscle of subjects with Duchenne's muscular dystrophy (DMD).1 In this phase I study, six boys with DMD received an intramuscular injection of an AAV vector encoding a truncated form of the dystrophin gene. An important conclusion of the study is that the AAV vector induced cytotoxic T lymphocytes (CTLs) against transduced cells, leading to loss of transgene expression. In two subjects, the studies also revealed the unexpected presence of dystrophin-specific T cells in peripheral blood mononuclear cells (PBMCs) harvested before gene transfer. Based on these findings, the authors speculate that the chronic inflammation observed in DMD may be due to T-cell targeting of revertant fibers. They also suggest that some subjects may have an enhanced CTL response to vector-transduced fibers due to the presence of dystrophin-specific T cells that have been activated by revertant fibers. In this Commentary, I evaluate the data on which these provocative conclusions are based. Many DMD patients have partial internal deletions in dystrophin that render downstream sequences out of frame, leading to premature termination of translation. All DMD patients should have circulating T cells specific for epitopes downstream of the premature stop codon because this portion of the dystrophin protein is not expressed and therefore would not allow for deletion of these T cells in the thymus. In the absence of peripheral stimulation with antigen, these T cells should be naive and found at low levels in the blood. Indeed, all subjects in the trial had partial deletions that rendered the dystrophin gene out of frame. Immunohistochemical analysis of muscle biopsies obtained after gene transfer detected transgene expression in only a limited number of muscle fibers of two of six subjects. This “failure to establish long-term transgene expression,” as the investigators described it, led them to evaluate PBMCs for evidence of T cells against the transgene product. Three subjects did in fact develop T cells specific to dystrophin that appeared subsequent to gene transfer. The authors claim that the well-described process of “reversion” of dystrophin expression that occurs in some DMD patients probably led to activation of the preexisting naive T cells. One proposed mechanism of reversion is exon skipping around the deletion in a way that reconstitutes the downstream open reading frame, thereby exposing the patient to epitopes recognized by naive T cells not deleted by the thymus (i.e., unless reversion occurs in epithelial cells responsible for presenting antigen in the thymus and negatively selecting the T cells). In assessing the validity of the conclusion that expression of the transgene was transient as a result of targeting of transduced muscle fibers by dystrophin-specific CTLs, it is critical to show that expression is indeed transient and that T cells with appropriate effector functions are activated. One of the biggest challenges in evaluating the efficiency of transduction and stability of transgene expression in DMD is lack of noninvasive measures or surrogates of transgene expression. Expression of mini-dystrophin was evaluated via histochemical analysis of a single muscle biopsy obtained 1.5 or 3 months after gene transfer; as noted above, these studies were essentially negative. The absence of detectable expression at a single time point subsequent to gene transfer may be consistent with CTL-mediated clearance; however, there are a number of more likely explanations, such as the fact that there was little transduction to begin with. Clinical models of gene therapy that provide more quantitative, specific, and noninvasive measures of transgene expression, such as hemophilia and α-1-antitrypsin deficiency, are more useful for answering this question.2,3 The primary assay for detecting T cells specific to dystrophin was enzyme-linked immunosorbent spot (ELISPOT) analysis of PBMCs, which measured the number of cells that secreted interferon-γ (IFN-γ) in response to dystrophin peptides. One of the six subjects (i.e., subject 2) clearly demonstrated a T-cell response to dystrophin in terms of its magnitude and kinetics. Two others showed delayed, sporadic, and low-level ELISPOT results that were less convincing. The demonstration of T-cell responses against a non-self-transgene product following AAV gene transfer to muscle in humans is not unexpected given findings with large-animal models such as the canine model of DMD.4,5 What is not clear from the DMD clinical trial is the function of the ELISPOT-positive T cells and their clinical consequences. The key question is whether the T cells exhibited effector functions such as cytolytic activity and were trafficked into the muscle so as to target the transduced fibers. The investigators did successfully isolate clones of dystrophin-specific T cells from PBMCs through a process of repeated cycles of in vitro stimulation, which provided an opportunity to more extensively characterize their properties. These studies helped map epitopes and major histocompatibility complex class I restriction, but they did not shed light on effector activity because only one CD8 T-cell clone was identified (in subject 4), and its effector functions were not characterized. In conducting future clinical gene transfer studies with AAV, it will be important to evaluate subjects for T-cell responses to both the transgene product and the capsid. The most commonly used assay is the ELISPOT because of its simplicity and sensitivity. However, one should not equate a positive ELISPOT signal with clinically meaningful CTL activity. In fact, it is being increasingly reported that AAV vectors can induce “dysfunctional” T cells that are capable of producing IFN-γ in response to antigen although they fail to proliferate or express genes necessary to traffic and target vector-transduced cells.6,7 More powerful techniques to characterize T cells, e.g., intracellular cytokine staining using multicolor flow cytometry, are being developed with sensitivities equal to that of ELISPOT assays, and these should be considered when analyzing gene therapy recipients. A more controversial aspect of the study by Mendell et al.1 is the hypothesis that revertant muscle fibers prime naive, dystrophin-specific T cells. The data to support this hypothesis are derived from ELISPOT analyses before and after gene transfer. The investigators were able to detect dystrophin-specific T cells at baseline in two subjects through direct analysis of PBMCs at a frequency higher than what would be expected if the cells were naive. In one subject, the signal was slightly above background. The other showed a more convincing ELISPOT result when a mapped peptide was used to stimulate the cells, although the signal was negative when pooled peptides were used. The second argument to support the existence of activated dystrophin-specific T cells was the rapid kinetics of the response observed in subject 2, which was claimed to be more consistent with a memory response. However, this conclusion is not supported by experience in animals that demonstrate similar kinetics from primary responses to AAV-encoded antigens.4,7 The notion that self-reactive T cells play a role in DMD pathogenesis is not new,8 but Mendell et al. suggest a potential mechanism by which this might occur. As is often the case in human pilot studies, the data are circumstantial and anectodal but nonetheless important in providing context for more extensive investigations. The study by Mendell et al. illustrates several principles of vector biology that are potentially relevant to recessive diseases other than DMD. Patients with two null alleles (i.e., do not express any protein) should be considered at risk of inducing T cells against the transgene product and, if possible, should be excluded from initial clinical trials of gene replacement therapies—diseases in which the target organ of gene transfer exhibits disease-related inflammation, such as skeletal muscle in DMD, are especially vulnerable to T-cell responses. Recipients should be carefully monitored for activation of T cells against the transgene product and vector capsid, although their presence in PBMCs does not necessarily mean that they are of clinical relevance. Finally, one should be circumspect about the existence of preexisting, primed T cells specific to the transgene product and their role in modulating vector–host responses until the hypothesis has been more extensively evaluated and corroborated. As pointed out by Mendell and colleagues, these issues are not unique to gene replacement and should be considered in any therapy that leads to the production of a functional version of the defective protein, including nonsense suppression with aminoglycosides and exon skipping with oligonucleotides.1