Background: Self-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for\r\ntherapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV\r\n(ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this\r\nstudy, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein,\r\nhuman factor IX (hF.IX).\r\nMethods: Hemophilia B mice were injected intramuscularly with ss or scAAV1 vectors expressing hF.IX. The\r\noutcome of gene transfer was assessed, including transgene expression as well as antibody and CD8+ T cell\r\nresponses to hF.IX.\r\nResults: Self-complementary AAV1 vectors induced similar antibody responses (which eliminated systemic hF.IX\r\nexpression) but stronger CD8+ T cell responses to hF.IX relative to ssAAV1 in mice with F9 gene deletion. As a\r\nresult, hF.IX-expressing muscle fibers were effectively eliminated in scAAV-treated mice. In contrast, mice with F9\r\nnonsense mutation (late stop codon) lacked antibody or T cell responses, thus showing long-term expression\r\nregardless of the vector genome.\r\nConclusions: The nature of the AAV genome can impact the CD8+ T cell response to the therapeutic transgene\r\nproduct. In mice with endogenous hF.IX expression, however, this enhanced immunogenicity did not break\r\ntolerance to hF.IX, suggesting that the underlying mutation is a more important risk factor for transgene-specific\r\nimmunity than the molecular form of the AAV genome.
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