Engineering AAV for Enhanced Retrograde Transport

描述（由申请人提供）：基因疗法对神经退行性疾病的治疗具有重要的前景。然而，目前的基因传递技术必须经过改造，以提高传递效率，针对受影响的细胞类型，以及转导大且通常手术无法进入的组织区域的能力。最近的研究发现，AAV载体沿着轴突投射逆行运输，也提出了有效靶向和递送治疗有效载荷到脆弱且难以手术进入的细胞（如脊髓中的运动神经元）的有趣可能性。然而，我们发现AAV逆行运输的效率在小鼠模型中是有限的。因此，我们建议采用定向分子进化方法来开发具有增强体内逆行转运的新型AAV衣壳变体。我们预计这项工作可能会显著提高逆行递送生物制剂的治疗效果和成本效益，以靶向难以进入的神经系统区域。因此，AAV逆行转运能力的改善将使包括肌萎缩性侧索硬化症（ALS）和脊髓损伤在内的许多疾病更有效地向脊髓运动神经元输送。我们的具体目标是：1；确定定向进化是否能产生具有增强体内逆行运输特性的AAV变体。2. 表征增强逆行转运变异的转导效率，分析报告基因在体内基因传递的序列-功能关系。3. 目的：确定含有神经保护基因IGF-1的逆行转运变异体是否能增强ALS小鼠模型的治疗效果。我们相信我们的AAV突变体库的发展将为研究界提供新的试剂。

DESCRIPTION (provided by applicant): Gene therapy has significant promise for the treatment of neurodegeneration. However, current gene delivery technology must be engineered for improved delivery efficiency, targeting to affected cell types, and the ability to transduce large and often surgically inaccessible regions of tissue. The recent findings that AAV vectors undergo retrograde transport along axonal projections also raises the intriguing possibility for efficient targeting and delivery of therapeutic payloads to cells that are delicate and otherwise difficult to surgically access such as motor neurons residing in the spinal cord. However, we have found that the efficiency of AAV retrograde transport can be limiting in a mouse model. We therefore propose to employ a directed molecular evolution approach to develop novel AAV capsid variants with enhanced retrograde transport in vivo. We anticipate this work may significantly improve the therapeutic efficacy and cost effectiveness of retrogradely delivered biologics to target difficult to access areas of the nervous system. Improvements in the retrograde transport ability of AAV would therefore allow more efficient delivery to spinal cord motor neurons for numerous diseases including Amyotrophic Lateral Sclerosis (ALS) and spinal cord injury. Our Specific Aims are: 1. To determine whether directed evolution can yield AAV variants with enhanced retrograde transport properties in vivo. 2. To characterize the enhanced retrograde transport variants for transduction efficiency and analyze sequence-function relationships for in vivo gene delivery of reporter genes. 3. To determine whether retrograde transport variants harboring the neuroprotective gene IGF-1 can enhance therapeutic efficacy in a mouse model of ALS. We believe the development of our library of AAV mutants will provide for novel reagents to be provided to the research community.