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

AAV 神经系统趋向性的全动物高分辨率成像

基本信息

批准号：
10573694
负责人：
Jennifer Hong
金额：
$ 44.39万
依托单位：
DARTMOUTH-HITCHCOCK CLINIC
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-26 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10573694
关键词：
3-Dimensional Address Anatomy Animals Automation Bar Codes Biodistribution Blindness Brain Capsid Cells Clinical Collaborations Consumption Custom DNA cassette Data Dependovirus Detection Development Dose Enterobacteria phage P1 Cre recombinase Evaluation Evolution FDA approved Failure Foundations Fright Future Gene Delivery Gene Expression Regulation Gene Transduction Agent Goals Gold Histology Hour Human Image Imaging technology Immunohistochemistry In Situ Hybridization Injections Injury Interphase Cell Investigation Isotopes Mediating Methods Modeling Modernization Mus Neoplasm Metastasis Nervous system structure Neurologic Neurosciences Research Nude Mice Oligonucleotides Organ Pathology Peripheral Nervous System Peripheral nerve injury Positron-Emission Tomography Preclinical Testing Rare Diseases Reporter Research Personnel Resolution Route Safety Serotyping Site Specimen Speed Spinal Muscular Atrophy System Tail Techniques Technology Testing Therapeutic Therapeutic Index Therapeutic Uses Time TimeLine Tissues Toxic effect Transcript Transgenes Transgenic Mice Transgenic Organisms Tropism Variant Veins Viral Visualization Work adeno-associated viral vector base body system clinical translation delivery vehicle experimental study feasibility testing gene therapy high resolution imaging improved innovation instrument interest mutant nervous system disorder next generation nonhuman primate novel novel strategies pre-clinical promoter protein expression reconstruction side effect single-cell RNA sequencing targeted delivery technology validation transduction efficiency transgene expression vector virus development virus tropism whole body imaging

项目摘要

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.

项目概要/摘要腺相关病毒（AAV）是最常用的基因治疗载体，AAV 技术是旨在为多种以前无法治愈的疾病提供新疗法的 AAV 是非致病性的。人类并感染非分裂细胞，使其成为靶向调控基因表达的理想载体这些极具吸引力的因素对人类中枢和周围神经系统（CNS/PNS）具有广泛的吸引力。人们对治疗性 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 中的转基因目标水平可减轻其他疾病中产生的剂量依赖性毒性。器官导致临床失败和不可预测的脱靶输送，这是现代技术的一个主要限制迫切需要对病毒趋向性进行高通量、全面的分析。转导效率可以加速安全有效的 AAV 的开发。 AAV 向性通常使用免疫组织化学技术来确定，这是 AAV 的金标准然而，这些方法是劳动密集型的并且只能检测转基因表达。少量的预先确定的组织，有效且全面的 AAV 趋向分析技术将通过实现快速迭代测试和直接检测，促进具有更强 CNS/PNS 向性的 AAV 变体的发现我的长期目标是开发新的外周基因治疗方法。该提案的目的是测试使用定制高光谱全身的可行性。成像冷冻切片机 (BioSlice) 用于快速、自动评估 AAV 对神经系统的趋向性。我们提出的实验结合了两种强大的技术——转基因荧光报告小鼠和全身高分辨率高光谱广域成像——检测 AAV 转导并提供新的 AAV 趋向性分析方法在目标 1 中，我们将使用 BioSlice 搜索 Ai14 小鼠中的脱靶递送。已注射了三种 AAV 变体，这些变体具有 CNS/PNS 的趋向性。在目标 2 中，我们将进行测试。 BioSlice 是否可以在同一动物中使用不同的荧光产生器检测多重 AAV，以及检查与注射部位相关的 AAV 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，以解决如果成功，我们的工作将提供强有力的概念验证： BioSlice 可用于严格评估临床前 AAV 的治疗用途。该方法有可能通过对小鼠所有组织的公正研究来发现趋向性，准确地模拟 AAV 血清型中潜在的脱靶递送和负面副作用，表达盒和注射位点。