Exploitation of the natural biodiversity of AAV to identify “super transducers"

利用 AAV 的自然生物多样性来识别“超级传感器”

• 批准号: 10674952
• 负责人: Dan Wang
• 金额: $ 54.44万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-09 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10674952
• 关键词: Address; Amaze; Amino Acids; Bar Codes; Biodiversity; Capsid; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Complex; Cryoelectron Microscopy; Dose; Ferrets; Gene Delivery; Gene Expression; Gene Transfer; Genes; Hepatocyte; Human; Hybrids; Immune; Immune response; Immunity; Immunologics; Length; Libraries; Liver; Mediating; Modeling; Mus; Outcome; Plasma; Population; Primates; Property; Recombinant adeno-associated virus (rAAV); Regulatory T-Lymphocyte; Serotyping; Tissues; Transducers; Tropism; Variant; Wild Type Mouse; adeno-associated viral vector; alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency; base editing; case-based; chimeric antigen receptor; clinical translation; clinically relevant; design; gene replacement; gene therapy; gene therapy clinical study; human tissue; humanized mouse; in vivo; meter; mouse model; neutralizing antibody; nonhuman primate; novel; permissiveness; pre-clinical; prime editing; promoter; reconstitution; single molecule real time sequencing; structural biology; structural determinants; tissue tropism; transgene expression; vector; vector induced

Project 3 - Project Summary: Liver is arguably the most permissive tissue for recombinant adeno-associated virus (rAAV)-mediated in vivo gene delivery, and liver transduction can be achieved by a simple systemic rAAV administration. However, liver-directed rAAV gene therapy for alpha-1-antitrypsin (AAT) deficiency (AATD) has met multiple challenges, demanding more efficient gene delivery and expression especially in the presence of pre-existing anti-AAV immunity. To address these challenges, one potential solution is to develop new AAV capsids that allow for more efficient gene delivery and/or transgene expression in the liver. We have recently employed single- molecule, real-time (SMRT) sequencing to discover novel full-length cap sequences from natural AAV proviral libraries from the human population to identify novel AAV variants with desirable tissue tropisms and vector properties. In this project, we will employ a high-throughput Illumina barcode sequencing approach to analyze a large collection of new AAV capsids identified from NHP and human tissues. The new capsid variants identified from this project collectively represent a valuable toolbox for liver-directed rAAV gene delivery to multiple species with favorable immunological profiles, and therefore are expected to have broad utilization in gene therapy applications. This project contains four specific aims designed to develop liver- tropic AAV capsids from our primate-derived library to support preclinical AATD gene therapy studies and clinical translation: Aim 1, To screen for mouse liver-targeting AAV capsid variants in wild-type (WT) mice; Aim 2, To screen for ferret liver-targeting AAV capsid variants in WT ferrets; Aim 3, To screen for human liver-targeting, NAB-escaping AAV capsid variants in humanized mice; and Aim 4, Structural-functional studies of new capsid variants. There is considerable interactivity between this project and the other three projects.

项目3--项目摘要： 肝脏可以说是重组腺相关病毒最容易感染的组织 (RAAV)介导的体内基因传递和肝转导可以通过简单的 全身性rAAV管理。然而，肝脏导向的rAAV基因治疗α-1抗胰蛋白酶 (AAT)缺乏症(AATD)已经遇到了多重挑战，需要更有效的基因传递 和表达，特别是在存在预先存在的抗AAV免疫的情况下。要解决这些问题 挑战，一个潜在的解决方案是开发新的AAV外壳，使其能够更有效地 肝脏中的基因传递和/或转基因表达。我们最近雇佣了单身人士- 分子实时(SMRT)测序以发现新的全长帽子序列 人类自然AAV前病毒文库用于识别新的AAV变异体 理想的组织取向和媒介特性。在这个项目中，我们将采用高吞吐量 用Illumina条形码测序方法分析大量新的AAV衣壳 从NHP和人体组织中鉴定。从这个项目中发现的新的衣壳变种 共同代表了肝脏导向的rAAV基因向多个物种传递的有价值的工具箱 具有良好的免疫学特性，因此有望在 基因治疗的应用。该项目包含四个具体目标，旨在开发肝脏- 我们灵长类动物来源的热带AAV衣壳蛋白支持临床前AATD基因治疗 研究和临床翻译：目的1，筛选小鼠肝脏靶向AAV衣壳变异体 野生型(WT)小鼠；目标2，筛选WT中雪貂肝靶向AAV衣壳变异体 雪貂；目标3，筛选人类肝脏靶向、NAB逃逸的AAV衣壳变异体 人源化小鼠；以及目标4，新衣壳变种的结构-功能研究。的确有 该项目与其他三个项目之间有相当大的互动性。