Mechanism of immune response to muscle-directed AAV gene transfer

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Muscle-directed gene transfer is integral to the treatment of severe muscle degenerative disorders such as Duchenne muscular dystrophy (DMD). Adeno-associated viral (AAV) vectors represent the most advanced platform for in vivo gene delivery through intramuscular (IM) injection or systemic delivery to various types of muscles. A major hurdle for this approach is the potential for immune responses, which may limit the efficacy and duration of therapy and can also be a source of serious immunotoxicities. Cytotoxic T cell responses against viral capsid and transgene products and complement activation have been observed in patients. The latter is likely caused by antibody-capsid complexes that form within days after high-dose systemic delivery. Neutralizing antibodies (NAb) that form after vector administration persist long-term, tend to be cross-reactive with various serotypes, and preclude re-administration of the vector. Our most recent collaborative studies in the canine DMD models illustrate the potential for CD8+ T cell responses against Cas9 nuclease employed in gene editing to correct muscular dystrophy. It is therefore imperative that we better understand the immune response mechanisms in AAV muscle gene transfer. To take on this task, we formed a collaborative team that combines the expertise in basic immune mechanisms of AAV muscle gene transfer with expertise in translational research in animal models of DMD. Our preliminary studies directly support the hypothesis that innate immune sensing drives adaptive immunity against the transgene product upon muscle-directed AAV gene transfer, and in particular CD8+ T cell responses. Depending on vector dose, multiple innate signaling pathways have either critical or redundant roles. We further hypothesize that vector engineering combined with specific interventions minimizes deleterious immune responses, thereby preserving therapy. We specifically propose to i) define the mechanisms that link innate immune sensing to adaptive immune responses in AAV muscle gene transfer; ii) prevent deleterious immune responses against transduced/gene-edited muscle following systemic AAV vector delivery, and iii) develop a novel protocol for re-administration of systemic AAV delivery. We will continue to use a model antigen (ovalbumin) to dissect the response mechanisms in skeletal muscle upon genetic or pharmacological disruption of these pathways; combine engineering of the vector genome with targeted interventions; and define the impact of dystrophic muscle on immune responses. We will use our ovalbumin platform to determine if the mechanisms identified for intramuscular injection also apply to systemic delivery of liver-detargeted AAV vectors to skeletal muscle. Further, we will evaluate the B and T cell responses against therapeutic transgene products (micro-dystrophin and Cas9). Finally, we have developed a novel protocol based on transient antibody-mediated depletion of B cells and the B-cell growth factor BAFF.