Self-Complementary AAV Vectors Delivered Intramuscularly Do Not Break Tolerance In a Partial Knockout Model Of Hemophilia B

Self-complementary adeno-associated viral (scAAV) vectors have become a popular tool for AAV gene therapies due to their increased transgene expression relative to single-stranded (ss)AAV vectors, at least for models in which the transgene can fit within these restrictive constructs. The most recent clinical trial for hemophilia B used a scAAV8 vector. However, recent reports by our lab and others have suggested that these vectors are also more immunogenic than their single-stranded brethren. We previously showed that, during hepatic gene transfer, the innate immune response is greater with a scAAV vector. Moreover, Wu et al. demonstrated an enhanced T and B cell response to a secreted form of HIV’s gag protein when it was delivered via scAAV. While this was beneficial for their vaccine model, it could potentially be problematic for therapeutic gene transfer applications. To investigate whether this phenomenon could deleteriously impact gene therapy for hemophilia B, we used a partial knockout model of hemophilia B. This mouse model has the murine factor IX gene knocked out, and then a truncated form of human factor IX (hF.IX) is inserted. Specifically, the mice we used have a late stop codon (LS) at amino acid 338; while hF.IX can be detected in the liver, this mutated protein is not released into circulation (crm^-). During muscle gene transfer with AAV2, LS mice have an interesting response: while they form antibodies to hF.IX, there is no CD8⁺ T cell infiltrate detected in the transduced tissue. We then set out to determine whether this partial tolerance could be disrupted using a scAAV vector. Using AAV1, which is a more relevant serotype for muscle gene therapy, we injected single-stranded and self-complementary vectors (10¹¹ vg/mouse) and measured the resulting immune responses. Surprisingly, unlike with AAV2, the LS mice completely tolerated AAV1-mediated intramuscular delivery of hF.IX. They did not make detectable anti-hF.IX IgG1, and their plasma had no Bethesda activity through 4 weeks post-injection. In contrast, hF.IX null mice (HBKO) made 10708±3869 ng anti-hF.IX IgG1 and developed inhibitory activity of 22.5±26 Bethesda units 4 weeks after delivery of a scAAV vector. Consequently, LS mice had circulating hF.IX levels of 148.9±18.64 (ssAAV1) and 60.12±8.71 ng/mL (scAAV1), while hF.IX was undetectable in the plasma of HBKO mice at this time point. The CD8 response was similarly lacking in LS mice, with splenic responses to hF.IX detected by ELISPOT reduced by ∼2-10 fold relative to HBKO mice. Infiltrating CD8 cells were detected in the muscle of HBKO but not LS mice. In conclusion, our data suggest that the underlying mutation within the patient is a more important risk factor for immune responses to the transgene than the molecular form of the AAV genome.