Mechanism of immune response to muscle-directed AAV gene transfer

肌肉定向基因转移是治疗严重肌肉退行性疾病的组成部分， 杜氏肌营养不良症（DMD）。腺相关病毒（AAV）载体代表了最先进的 用于通过肌内（IM）注射或全身递送到各种类型的 肌肉.这种方法的一个主要障碍是潜在的免疫反应，这可能会限制疗效 和治疗持续时间，并且也可能是严重免疫毒性的来源。细胞毒性T细胞应答 已经在患者中观察到病毒衣壳和转基因产物以及补体激活。后者是 可能是由高剂量全身给药后数天内形成的抗体-衣壳复合物引起的。中和 在载体施用后形成的抗体（NAb）长期持续存在，倾向于与各种抗体交叉反应， 血清型，并排除载体的再施用。我们最近在犬DMD方面的合作研究 模型说明了针对基因编辑中使用的Cas9核酸酶的CD 8 + T细胞应答的潜力， 纠正肌肉萎缩症。因此，我们必须更好地了解免疫反应， AAV肌肉基因转移的机制。为了完成这项任务，我们组建了一个合作团队， 在AAV肌肉基因转移的基本免疫机制方面的专业知识，以及转化研究方面的专业知识 在DMD的动物模型中。我们的初步研究直接支持了先天免疫感应的假设， 在肌肉定向的AAV基因转移后驱动针对转基因产物的适应性免疫， 特别是CD 8 + T细胞反应。取决于载体剂量，多种先天信号传导途径 关键或冗余的角色。我们进一步假设，载体工程结合特定的干预措施， 最大限度地减少有害的免疫反应，从而保留治疗。我们具体建议：i）定义 在AAV肌肉基因转移中将先天免疫感应与适应性免疫应答联系起来的机制; ii） 预防针对转导/基因编辑的肌肉的有害免疫应答 iii）开发用于再施用全身性AAV递送的新方案。我们将继续使用 一种模型抗原（卵清蛋白），用于解剖骨骼肌中遗传或 这些途径的药理学破坏;将载体基因组的联合收割机工程化与靶向 干预措施;并确定营养不良的肌肉对免疫反应的影响。我们会用卵清蛋白 该平台用于确定肌内注射的机制是否也适用于全身递送 肝脏去靶向AAV载体至骨骼肌。此外，我们将评估B和T细胞对 治疗性转基因产物（微肌营养不良蛋白和Cas9）。最后，我们开发了一个新的协议， 对B细胞和B细胞生长因子BAFF的瞬时抗体介导的耗竭的影响。

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.