Muscle-specific AAV Vectors for DMD Gene Therapy

描述（由申请人提供）：Duchenne Muscular Dystrophy (DMD)，影响3500名男性中的一人，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。用功能性、小型化形式的肌营养不良蛋白基因替代缺陷蛋白是治疗DMD的一种很有前途的方法。上述迷你肌萎缩蛋白转基因目前正在人类临床试验中进行评估，使用AAV载体作为肌肉内递送剂。目前的探索性/发展性研究计划主要集中在设计和开发新的AAV载体，在mdx小鼠模型中提高营养不良骨骼肌基因传递效率。翻译研究探索阶段的基本原理取决于营养不良肌肉中整合素7¿1的过度表达，以及我们的重大发现，即AAV2衣壳上一个假定的整合素结合域的调节会影响它们转导正常骨骼肌的能力。基于上述发现，我们制定了具体的设计策略，包括：(a)合理工程整合素结合域，以开发整合素7¿1靶向的AAV载体，高效转导营养不良肌肉；(b)组合工程AAV载体，高效转导mdx小鼠的营养不良肌肉。在发育阶段，通过该过程选择的新型AAV载体将用于在mdx小鼠模型中提供增强型迷你肌萎缩蛋白转基因。研究设计包括合理插入肽配体以生成整合素a7¿1靶向的AAV载体，通过不同的给药途径将从AAV突变文库中获得的营养不良肌肉特异性载体定向进化，以及在mdx小鼠模型中应用增强型微型肌营养不良蛋白转基因构建物进行治疗。在本提案中开发和优化的新试剂将在不久的将来在更大的肌肉萎缩症动物模型中进行进一步的治疗测试，并最终在人类肌肉萎缩症临床试验中进行。每3500名男性中就有一人患有杜氏肌营养不良症（DMD），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/发展性研究计划主要集中在设计和开发新的AAV载体，在mdx小鼠模型中提高营养不良骨骼肌基因传递效率。在本提案中开发和优化的新试剂将在不久的将来在更大的肌肉萎缩症动物模型中进行进一步的治疗测试，并最终进行人体临床试验。

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.