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

项目总结/摘要 腺相关病毒（AAV）是最常用的基因治疗载体，并且AAV技术是最常用的基因治疗载体。 提出为广泛的以前无法治愈的疾病提供新的治疗方法。AAV是非致病性的 并感染非分裂细胞，使其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， 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来表达 CNS/PNS中的转基因靶向减轻病理的水平。在其他研究中产生的剂量依赖性毒性 但是，器官移植已经导致临床失败和不可预测的脱靶递送，这是现代医学的主要限制。 基因治疗迫切需要高通量、全面的病毒嗜性分析， 转导效率，可以加速安全和有效的AAV的开发。 AAV嗜性通常使用免疫组织化学技术来确定，其是确定AAV嗜性的金标准。 转基因表达的检测。然而，这些方法是劳动密集型的，并且仅允许测试 少量预定组织。有效和全面的AAV向性分析技术将 通过实现快速迭代测试和直接测序，促进具有更大CNS/PNS向性的AAV变体的发现。 AAV突变体载体的比较。我的长期目标是开发新的外周血基因治疗方法， 神经损伤本提案的目的是测试使用定制高光谱全身成像系统的可行性。 用于快速、自动化评价AAV对神经系统的向性的成像冷冻切片机（BioSlice）。 我们提出的实验结合了联合收割机两种强大的技术-转基因荧光报告小鼠和 全身，高分辨率高光谱宽场成像-检测AAV转导，并提供新的 AAV向性分析的方法。在目标1中，我们将使用BioSlice在Ai 14小鼠中搜索脱靶递送 已经注射了已知具有CNS/PNS向性的三种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基因检测多重AAV，以及 检查对与注射部位相关的AAV向性的影响。 这些目标的完成将提供先进成像技术的创新应用，以解决 这是AAV开发中的一个根本瓶颈。如果成功，我们的工作将提供强大的概念验证， BioSlice可用于严格评估临床前AAV的治疗用途。这种跨学科 该方法具有通过对小鼠所有组织的无偏研究来发现向性的潜力， 从而精确地模拟AAV血清型间潜在的脱靶递送和负副作用， 表达盒和注射位点。

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.