Engineering AAV for Enhanced Retrograde Transport

描述(由申请人提供)：基因疗法在治疗神经退行性变方面有很大的前景。然而，目前的基因传递技术必须进行工程设计，以提高传递效率，针对受影响的细胞类型，以及转导组织的大片区域的能力，这些区域通常是手术无法到达的。最近的研究发现，AAV载体沿着轴突投影进行逆行运输，这也增加了有趣的可能性，即有效地靶向并将治疗有效载荷输送到脆弱的、以其他方式难以通过手术获得的细胞，如位于脊髓中的运动神经元。然而，我们发现，在小鼠模型中，AAV逆行运输的效率可能是有限的。因此，我们建议使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衣壳变异体。我们预计，这项工作可能会显着提高逆行输送的生物制品的治疗效果和成本效益，以瞄准神经系统难以到达的区域。因此，AAV逆行转运能力的提高将使许多疾病(包括肌萎缩侧索硬化症和脊髓损伤)更有效地输送到脊髓运动神经元。我们的具体目标是：1.确定定向进化是否能够在体内产生具有增强的逆行运输特性的AAV变体。2.鉴定增强的逆行转运变异体的转导效率，分析报告基因体内传递的序列-功能关系。3.确定携带神经保护基因IGF-1的逆行转运变异体能否提高ALS小鼠模型的治疗效果。我们相信，我们AAV突变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.