Whole-animal, high-resolution imaging of AAV tropism for the nervous system

项目摘要/摘要 腺相关病毒(AAV)是最常用的基因治疗载体，AAV技术是 准备为一系列以前无法治愈的疾病提供新的治疗方法。AAVs是非致病性的 并感染未分裂的细胞，使它们成为靶向调控基因表达的理想载体 人类中枢和外周神经系统(CNS/PNS)。这些极具吸引力的因素广泛地 对治疗性AAV的兴趣增加；然而，现有的AAV载体需要非常高的剂量才能表达 CNS/PNS中的转基因靶向于减轻病理变化的水平。由此产生的剂量依赖性毒性在其他 器官导致临床失败和不可预测的非目标递送，这是现代 基因疗法。迫切需要对病毒嗜性和病毒嗜性进行高通量、全面的剖析 可以加速安全有效的AAVs发展的转导效率。 AAV嗜性通常使用免疫组织化学技术来确定，这是诊断AAV的黄金标准 转基因表达的检测。然而，这些方法是劳动密集型的，并且只允许测试 少量预先确定的组织。高效和全面的AAV趋向性分析技术将 通过启用快速迭代测试和直接测试，帮助发现具有更大CNS/PNS取向的AAV变体 AAV突变载体的比较。我的长期目标是开发新的外周血细胞基因疗法 神经损伤。这项建议的目的是测试使用定制高光谱全身的可行性 冷冻巨噬细胞(BioSlice)成像，用于快速、自动评估神经系统的AAV趋向性。 我们提出的实验结合了两项强大的技术-转基因荧光报告鼠和 全身高分辨率高光谱广域成像-检测AAV转导并提供新的 AAV趋向性剖面图的探讨在目标1中，我们将使用BioSlice在Ai14小鼠中寻找非靶标递送 注射了三种已知的中枢神经系统/三叉神经节趋向性的AAV变体。在目标2中，我们将测试 BioSlice是否可以在同一动物中使用不同的荧光报告程序检测多个AAVs，以及 检查注射部位对AAV趋向性的影响。 完成这些目标将提供先进成像技术的创新应用，以解决 AAV发展中的一个根本瓶颈。如果成功，我们的工作将提供强有力的概念证明 BioSlice可用于严格评估用于治疗的临床前AAVs。这是跨学科的 该方法有可能通过对小鼠的所有组织进行无偏见的研究来发现趋向性， 从而准确地模拟跨AAV血清型的潜在的非靶标递送和负面副作用， 表达盒和注射部位。

PROJECT SUMMARY/ABSTRACT Adeno-Associated Viruses (AAVs) are the most commonly used gene therapy vector and AAV technology is posed to deliver new treatments for a wide range of previously incurable conditions. AAVs are non-pathogenic to humans and infect non-dividing cells, making them ideal vectors for targeted regulation of gene expression in the human central and peripheral nervous system (CNS/PNS). These highly attractive factors have broadly increased interest in therapeutic AAVs; however, existing AAV vectors require very high doses to express transgenes in CNS/PNS targets at levels that mitigate pathology. The resulting dose-dependent toxicities in other organs have led to clinical failures and unpredictable off-target delivery, which is a major limitation of modern gene therapy. There is a critical need for high-throughput, comprehensive profiling of viral tropism and transduction efficiency that could speed development of safe and effective AAVs. AAV tropism is typically determined using immunohistochemical techniques, which are the gold standard for detection of transgene expression. However, these methods are labor-intensive and permit testing of only a small number of pre-determined tissues. Efficient and comprehensive AAV tropism profiling technologies would facilitate discovery of AAV variants with greater CNS/PNS tropism by enabling fast iterative testing and direct comparison of AAV mutant vectors. My long-term goal is to develop novel gene therapy treatments for peripheral nerve injuries. The objective of this proposal is to test feasibility of using a custom hyperspectral whole-body imaging cryomacrotome (BioSlice) for rapid, automated evaluation of AAV tropism for the nervous system. Our proposed experiments combine two powerful technologies—transgenic fluorescent reporter mice and whole-body, high-resolution hyperspectral wide-field imaging—to detect AAV transduction and provide a new approach to AAV tropism profiling. In Aim 1, we will use the BioSlice to search for off-target delivery in Ai14 mice that have been injected with three AAV variants that have known tropism for the CNS/PNS. In Aim 2, we will test whether the BioSlice can detect multiplexed AAVs using distinct fluorescent reporters in the same animal, and examine effects on AAV tropism related to injection site. Completion of these aims will deliver an innovative application of an advanced imaging technology to address a fundamental bottleneck in AAV development. If successful, our work will provide robust proof-of-concept that the BioSlice can be used to rigorously evaluate pre-clinical AAVs for therapeutic use. This transdisciplinary approach has the potential to enable tropism discovery through unbiased investigation of all tissues in the mouse, thereby accurately modeling potential off-target delivery and negative side-effects across AAV serotypes, expression cassettes and injection sites.