Muscle-specific AAV Vectors for DMD Gene Therapy

描述(申请人提供)：杜氏肌营养不良症(DMD)，每3500名男性中就有一名患者，由于肌肉组织中缺乏肌营养不良蛋白的表达，导致骨骼肌和心肌的进行性肌病和过早死亡。用一种功能性的、微型化的dystrophin基因替换有缺陷的蛋白质是治疗DMD的一种有前途的方法。目前正在使用AAV载体作为肌肉内递送剂进行人体临床试验，评估上述Minidystrophin转基因。目前的探索性/发展性研究方案集中在利用MDX小鼠模型设计和开发具有增强营养不良骨骼肌基因递送效率的新型AAV载体。翻译研究的探索性阶段的理论基础在于营养不良肌肉中整合素A7？1的过度表达，以及我们的开创性发现，AAV2衣壳上可能的整合素结合结构域的调节影响了它们转导正常骨骼肌的能力。我们根据上述发现制定了具体的设计策略，包括：(A)对整合素结合域进行合理的工程，以开发高效转导营养不良肌肉的整合素A7？1靶向AAV载体；(B)高效转导mdx小鼠营养不良肌肉的AAV载体的组合工程。在发育阶段，通过这一过程选择的新型AAV载体将被用于在MDX小鼠模型中传递用于治疗应用的增强型MIDDYROPHIN转基因。研究设计包括合理插入多肽配体以产生整合素A7？1靶向AAV载体，通过不同的给药途径将从AAV突变文库获得的营养不良肌肉特异性载体定向进化到MDX小鼠，以及增强型微型营养不良基因转基因构建物在MDX小鼠模型中的治疗应用。在这项建议中开发和优化的新型试剂将在更大的肌肉营养不良动物模型中进行进一步的治疗试验，并最终在不久的将来用于人类肌肉营养不良的临床试验。杜氏肌营养不良症(Duchenne Muscle Dystrophy，DMD)，每3500名男性中就有一人患病，由于肌肉组织中缺乏肌营养不良蛋白的表达，会导致骨骼肌和心肌的进行性肌病和过早死亡。目前的探索性/发展性研究方案集中在利用MDX小鼠模型设计和开发具有增强营养不良骨骼肌基因递送效率的新型AAV载体。在这项建议中开发和优化的新型试剂将在更大的肌肉营养不良动物模型中进行进一步的治疗试验，并最终在不久的将来用于人类临床试验。

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD), which affects one in 3500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death due to a lack of expression of the protein dystrophin in muscle tissues. Genetic replacement of defective protein with a functional, miniaturized form of dystrophin represents a promising approach for the treatment of DMD. The aforementioned minidystrophin transgene is currently being evaluated in human clinical trials using AAV vectors as agents for intramuscular delivery. The current exploratory/developmental research proposal is focused on the design and development of novel AAV vectors with enhanced gene delivery efficiency in dystrophic skeletal muscle using the mdx mouse model. Rationale for the exploratory phase of the translational research hinges on the overexpression of integrin a7¿1 in dystrophic muscle and our seminal finding that modulation of a putative integrin-binding domain on AAV2 capsids affects their ability to transduce normal skeletal muscle. We have formulated specific design strategies based on the aforementioned finding that include: (a) rational engineering of the integrin-binding domain to develop integrin a7¿1-targeted AAV vectors that transduce dystrophic muscle with high efficiency and (b) combinatorial engineering of AAV vectors that efficiently transduce dystrophic muscle in mdx mice. During the developmental phase, novel AAV vectors selected through this process will be utilized to deliver an enhanced minidystrophin transgene for therapeutic application in the mdx mouse model. Research design involves rational insertion peptide ligands to generate integrin a7¿1-targeted AAV vectors, directed evolution of dystrophic muscle-specific vectors obtained from an AAV mutant library administered into mdx mice through different routes of administration, and therapeutic application of an enhanced mini-dystrophin transgene construct in the mdx mouse model. The novel reagents developed and optimized in this proposal will be advanced for further therapeutic testing in larger animal models of muscular dystrophy and eventually in human muscular dystrophy clinical trials in the near future. Duchenne Muscular Dystrophy (DMD), which affects one in 3500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death due to a lack of expression of the protein dystrophin in muscle tissues. The current exploratory/developmental research proposal is focused on the design and development of novel AAV vectors with enhanced gene delivery efficiency in dystrophic skeletal muscle using the mdx mouse model. The novel reagents developed and optimized in this proposal will be advanced for further therapeutic testing in larger animal models of muscular dystrophy and eventually in human clinical trials in the near future.