Development of optimized AAVrh74 vectors for gene therapy of muscular dystrophies

项目总结/摘要： 重组腺相关病毒（AAV）载体目前正在三个独立的临床试验中使用 用于Duchenne肌营养不良症（DMD）的基因治疗，由Solid Biosciences，Pfizer，Inc.，和 分别是萨雷普塔治疗学。第一代AAV 9载体在Solid Biosciences和Biochemistry中的使用 辉瑞的试验导致了一些严重的不良事件，包括辉瑞的一名患者死亡。 审判尽管在萨雷普塔试验中第一代AAVrh 74载体显示出良好的耐受性，但在第一代AAVrh 74载体中， 最近在DMD患者中进行的II期试验未能达到其主要功能终点。我们认为，基于 事实上，AAV是作为病毒进化的，而不是作为用于递送治疗基因的载体， 因此，第一代重组AAV载体的全部潜力不太可能实现。通过 通过修饰AAV衣壳蛋白，我们开发了下一代（“NextGen”）AAV载体， 在减少剂量的情况下有效性提高30倍。这些载体的免疫原性也较低。我们还 修饰AAV载体基因组以开发第X代（“GenX”）AAV载体。这些载体介导 到8倍增强的转基因表达。我们将这两种策略结合起来， （“Opt”）AAV血清型载体。这些载体在进一步降低的剂量下更有效20-30倍。我们有 还证明了在AAV反向末端重复序列中包含特异性调节元件可以 显著增加转基因表达水平。在本申请中，我们建议开发Opt AAVrh 74 （i）含有新的肌肉-骨骼肌融合蛋白的肝脏去靶向Opt AAVrh 74载体， 特异性增强子元件将导致转基因表达水平的进一步增加， （ii）Opt AAVrh 74载体可以以显著降低的剂量递送至靶肌肉细胞 在DMD的小鼠模型中体内全身给药后。以下三个具体目标将是 追求： 具体目标1：开发衣壳+基因组修饰的、肝脏去靶向Opt AAVrh 74载体用于 体外和小鼠模型中原代人骨骼肌细胞的高效转导 vivo. 具体目的2：开发具有肌肉特异性增强子元件的新型Opt AAVrh 74载体，以 在小鼠体内模型中以进一步降低的剂量增加转基因表达。 具体目标3：开发用于全身性免疫的新型Opt AAVrh 74-微肌营养不良蛋白载体（Opt 74-µDys） 在小鼠体内模型中的DMD的基因治疗。 开发在较低剂量下有效的优化的AAVrh 74载体可能会减少AAVrh 74的表达。 诱导宿主免疫应答的概率、载体生产成本以及经济成本 用于人类肌营养不良症的潜在基因治疗。

PROJECT SUMMARY/ABSTRACT: Recombinant adeno-associated virus (AAV) vectors are currently being used in three separate clinical trials for gene therapy of Duchenne muscular dystrophy (DMD), sponsored by Solid Biosciences, Pfizer, Inc., and Sarepta Therapeutics, respectively. The use of the first generation of AAV9 vectors in both Solid Biosciences and Pfizer trials have led to a number of serious adverse events, including the death of a patient in the Pfizer trial. Although the first generation of AAVrh74 vectors in the Sarepta trial were shown to be tolerated well, a recent Phase II trial in patients with DMD failed to meet its primary functional endpoint. We have argued, based on the fact that AAV evolved as a virus, and not as a vector for the purposes of delivery of therapeutic genes, and thus, the full potential of the first generation of recombinant AAV vectors is unlikely to be realized. By modifying the AAV capsid protein, we have developed the next generation (‘NextGen’) of AAV vectors, which are up to 30-fold more efficacious at reduced doses. These vectors are also less immunogenic. We have also modified the AAV vector genome to develop the generation X (‘GenX’) AAV vectors. These vectors mediate up to 8-fold enhanced transgene expression. We have combined both these strategies to generate the optimized (‘Opt’) AAV serotype vectors. These vectors are 20-30-fold more efficient at further reduced doses. We have also documented that inclusion of specific regulatory elements within the AAV inverted terminal repeats can significantly increase transgene expression levels. In this application, we propose to develop the Opt AAVrh74 vectors, and test the following hypotheses: (i) Liver de-targeted Opt AAVrh74 vectors containing novel muscle- specific enhancer elements will lead to further increased levels of transgene expression at significantly lower doses; and (ii) Opt AAVrh74 vectors can be delivered to target muscle cells at significantly reduced doses following systemic administration in a mouse model of DMD in vivo. The following three specific aims will be pursued: Specific Aim 1: Development of capsid+genome-modified, liver de-targeted Opt AAVrh74 vectors for high-efficiency transduction of primary human skeletal muscle cells in vitro, and in a mouse model in vivo. Specific Aim 2: Development of novel Opt AAVrh74 vectors with muscle-specific enhancer elements to augment transgene expression at further reduced doses in a mouse model in vivo. Specific Aim 3: Development of a novel Opt AAVrh74-micro-dystrophin vector (Opt74-µDys) for systemic gene therapy of DMD in a mouse model in vivo. The development of optimized AAVrh74 vectors that are effective at lower doses, are likely to reduce the probability of inducing the host immune responses, the vector production costs, as well as the economic cost per patient, for the potential gene therapy of muscular dystrophies in humans.