One method for gene therapy that targets problem genes in muscles did not require immunotherapy treatment. Removing the need for immunotherapy while still having effective gene therapy greatly reduces the cost and side effects of gene therapy.
Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1–AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.
Although AAT augmentation therapy can achieve effective serum levels of AAT, this therapy is not ideal due to the need for weekly intravenous infusions, high annual costs, and insufficient availability of product to treat all persons currently diagnosed with severe AAT deficiency, resulting in low physician motivation to accurately diagnose the vast majority of patients who remain unrecognized. If treatment with a rAAV vector expressing AAT can achieve similar serum AAT levels, it would provide a more readily available and convenient, potentially 1-time, treatment option.
In the 12-month follow-up period of this phase II gene transfer study for AAT deficiency, we observed persistent gene expression in the absence of immune suppression. The earlier time points within this study seemed to mimic those seen in other trials of both hepatic delivery and muscle delivery of rAAV vectors, with an anti-capsid effector T cell response within the first 30 days after administration, resulting in cellular infiltration at the injection sites, a transient rise in creatine kinase, and a partial decline in transgene expression. Remarkably, despite a persistence of peripheral anti-capsid T cells and local cellular infiltrates, transgene expression persisted for more than 1 year after vector administration, with no sign of diminution of expression levels. To the contrary, an upward trend was seen in each of the 3 patients in the cohort receiving the highest dose (those receiving doses of 6.0 × 1012 vg/kg). These findings call into question whether the cellular infiltrates present within the muscle were actually functionally cytotoxic in nature. A possible finding supporting this is the evidence of an expansion of Tregs found in situ. These results generally raise expectations about the long-term utility of muscle-directed rAAV gene therapy. The sustained levels seen here (17.7 ± 3.6 μg/ml) were relatively high in absolute terms for protein expression but were lower than the very high target level needed for correction of AAT deficiency, at 3% of the target of 572 μg/ml.
In comparing this study to other recent studies of rAAV-based gene therapy, it is important to point out that while there are important commonalities in the findings, none of the studies were designed precisely in the same manner as this one. All studies of liver and muscle delivery of rAAV have shown some evidence of anti-capsid T cell responses, often with some indication of cellular toxicity (elevation of transaminases or CK), and most showed at least some diminution of gene expression. In early studies of liver-directed therapy, patients were followed without immune suppression, but more recent studies have included some element of immune suppression, with initiation of such therapy either at the time of vector administration or in response to evidence of potential cytotoxicity manifested by elevation of serum enzymes. Thus, our study provides the unusual perspective of following the course after i.m. delivery without any additional intervention.
Clearly, the current study does not address the question of whether transgene expression might have been significantly higher if immune suppressive or antiinflammatory drugs had been used. In the recent report by Nathwani et al. (6), in a liver-directed hemophilia B trial, a modest immune modulation (60 mg prednisolone daily with tapering and discontinuation over 4 to 7 weeks) was sufficient to enhance gene expression and decrease transaminase elevation. This is very promising, since it could readily be incorporated into an AAT gene augmentation trial without a large increase in the risk to the study volunteers.
The current study’s results also do not predict whether or not further increases in the dose would continue to produce similar responses at proportionally higher levels. It was very encouraging that there was a linear dose-response relationship within the dose range used in this trial. However, since the current trial involved 100 individual i.m. injections of 1.35 ml each, further increases in the dose will likely require some form of regional vascular delivery (22, 23). This could potentially alter both the nature of distribution of the vector among the myofibers and the relative exposure of lymphoid tissues to the vector material. How this would affect the results is difficult to predict, although some preclinical data suggest that the amount of transgene expression observed for any given dose of vector could be higher with a regional vascular delivery method.
It has been generally accepted that, after cellular uptake, unprocessed rAAV vector capsid would be mostly degraded by the proteasomal machinery; however, in this study, we show the first published evidence of the persistence of intact capsid up to a year after administration in humans. This finding is consistent with an earlier study that documented the detection of rAAV particles up to 6 years after administration in the retina of dogs and nonhuman primates and may explain why Tregs would continue to reside within the injected muscle months after administration. This has important consequences for the design of future clinical trials, since the presence of viral antigens may not be as transient as once believed, thus influencing the timing and length of immunosuppressive strategies. Even more importantly, the persistence of capsid may mimic a chronic viral infection, which ultimately may limit excessive inflammation and allow persistence of transduced cells, as natural Tregs develop to protect against overexuberant immune responses and bystander killing of untransduced cells. It is known that natural Tregs (CD4+CD25+FOXP3+) arise in the thymus during development and are thought to possess T cell receptors specific for self antigens. However, it has also been well documented that these cells also suppress immune response to infectious agents. The mechanism by which this happens is still unclear, and it is largely unknown whether these natural Tregs require priming to recognize a viral or foreign antigen. Possible mechanisms that have been suggested include the nonspecific activation of Tregs through Toll-like receptor signaling or Treg stimulation by cross-reactive epitopes. The data presented here suggest that the Treg response is at least in part responsive to AAV capsid, as was seen by the capsid-induced activation. Taking into consideration that the Tregs detected in muscle had a demethylated TSDR and the capsid activation was observed among Tregs that were Helios+, it is likely that these are natural Tregs. Thus, the capsid-specific activation is not necessary a de novo Treg response but could also be explained by an expansion of a preexisting repertoire. While the mechanism remains elusive, it is clear that virus-specific natural Tregs allowing viral persistence have been observed in humans with chronic hepatitis C virus. Additionally, the expansion of Tregs in general has been also associated with the chronicity of HIV and papillomavirus infections. Thus, in this regard, the persistence of rAAV capsid and its epitopes could be mirroring these chronic viral infections. Interestingly, expression of PD-1 and PD-L1, which is associated with chronic viral infection and exhausted T cell responses, was also observed in many of the inflammatory foci of the rAAV-injected muscles. Consistent with our findings, not only does the PD-1/PD-L1 pathway limit T cell stimulation, but it also promotes the differentiation and maintenance of FOXP3+ Tregs. Finally, it should also be pointed out that while our data show an expansion of Tregs in the muscle, this is not necessarily the site in which the response arose. The levels of vector genomes in the blood after administration suggest that a substantial amount of vector may have transduced the liver or spleen, and it is possible that one of these sites could be playing a role in the induction of the Treg response.
Taken together, the results presented here are highly encouraging for the future of muscle-directed rAAV gene therapy. Persistent anti-capsid T cell responses were both well tolerated, in terms of clinical safety parameters, and did not inhibit persistent transgene expression at 1 year, and the potential mechanism for this was identified, namely, the presence of a T regulatory response. Thus, the anti-capsid responses might be considered to be a relatively manageable aspect of this therapy, at least within the dosage and time ranges tested. Indeed, some targets for serum replacement of other proteins might well have been reached with the methods used in the current study. AAT deficiency remains a very high target for full replacement, and many questions remain, as higher doses and immune suppression are contemplated for future trials. However, given the scalability of rAAV vector production (34), the availability of clinically tolerable limb infusion methods, and the relatively modest levels of immune suppression that may have a salient effect, it would seem that trials designed to achieve therapeutic levels of serum AAT should be feasible.