615. A Self-Limiting but Prolonged Inflammatory Response to AAV-Factor IX Gene Transfer to Skeletal Muscle
Authors: L. Wang, R.W. Herzog
Journal: Molecular Therapy (2005)

Immune responses to the transgene product are a major concern in gene therapy for genetic disease. Previously, we have shown that AAV-mediated gene transfer to skeletal muscle may lead to antibody formation to coagulation factor IX (F.IX) in animals not tolerant to the transgene product. A T helper cell dependent adaptive immune response initiated locally in the transduced muscle resulted in CD4+ T cell and B cell activation in the draining lymph nodes. Both Th1 and Th2 subsets were involved in this process. However, activation of CD8+ cells appeared to be inefficient. Consequently, local transgene expression in the muscle persisted because of a lack of cytotoxic T lymphocyte (CTL) responses and lack of inflammation, even when systemic F.IX expression was neutralized by antibodies. For most of these experiments, we deliberately expressed a human F.IX (hF.IX) transgene in C57BL/6 mice as a model to study characteristics of an immune response to F.IX in the context of AAV gene transfer. When a more effective AAV serotype 1 vector was injected into skeletal muscle, anti-hF.IX formation was transient, and systemic expression emerged around 2 months after gene transfer. In a new set of experiments in outbred CD-1 mice, we injected AAV-2 vector at 3 different doses (2|[times]|1010 vg, 1|[times]|1011 vg, and 1|[times]|1012 vg per animal, 4 sites of injection) into skeletal muscle. Systemic hF.IX expression was transient, again limited by a neutralizing antibody response. Inhibitory anti-hF.IX IgG was measured at all 3 vector doses. Antibody titers persisted for >4 months, and no hF.IX was detected in circulation beyond day 14 at low and mid doses. However, hF.IX antigen re-emerged in plasma 1.5 months after high dose vector administration in 5 of 7 mice. Moreover, inhibitors to hF.IX became undetectable by 5 months at this dose. Coagulation assays on serial dilutions of plasma showed an increase in clotting activity in these animals at 5 months. This illustrates that persistent expression of F.IX from AAV-transduced skeletal muscle can down-regulate immune-mediated neutralization of the transgene product over time. Surprisingly, when we analyzed muscle tissues, marked inflammation was evident at mid and high doses starting at >2 month after gene transfer. This inflammatory response included a CD8+ cellular infiltrate and was still present at 4 months. The inflamed tissue included muscle fibers with central nuclei indicating destruction and regeneration of fibers. Interestingly, the number of hF.IX expressing fibers declined only marginally over time as determined by immunohistochemistry and image analysis. At late time points (3-4 months), a proportion of the transgene expressing fibers had central nuclei, raising the possibility that myofiber precursors had been transduced. These data show that depending on the genetic background (here CD-1 vs. C57BL/6 mice) and vector dose, CD8 T cell activation and inflammatory responses may occur in the context of AAV-F.IX gene transfer to skeletal muscle. However, these responses were delayed and ultimately failed to eliminate transgene expression. The inflammatory response was prolonged and self-limiting, perhaps because sustained transgene expression caused activation of regulatory T cells in addition to effector T cells.