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

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 （r腺相关病毒）介导的体内基因递送和肝脏转导可以通过简单的方法实现 全身rAAV施用。然而，针对α-1-抗胰蛋白酶的肝脏定向rAAV基因疗法 (AAT)缺乏症（AATD）已经遇到了多重挑战，需要更有效的基因递送 和表达，特别是在存在预先存在的抗AAV免疫的情况下。解决这些 挑战，一个潜在的解决方案是开发新的AAV衣壳，允许更有效地 在肝脏中的基因递送和/或转基因表达。我们最近雇佣了一个- 分子，实时（SMRT）测序，以发现来自 来自人类群体的天然AAV前病毒文库，以鉴定新的AAV变体， 所需的组织向性和载体性质。在这个项目中，我们将采用高通量 Illumina条形码测序方法用于分析大量新的AAV衣壳 从NHP和人体组织中鉴定。从该项目中鉴定的新衣壳变体 共同代表了肝脏定向rAAV基因递送到多个物种的宝贵工具箱 具有良好的免疫学特性，因此预期在 基因治疗的应用该项目包含四个旨在开发肝脏的具体目标- 来自我们的灵长类衍生文库的嗜性AAV衣壳用于支持临床前AATD基因治疗 目的1、筛选小鼠肝靶向性AAV衣壳蛋白变异体 目的2，在野生型小鼠中筛选靶向雪貂肝的AAV衣壳变体 目的3，筛选人肝靶向的、NAB逃逸的AAV衣壳变体， 人源化小鼠;和目标4，新衣壳变体的结构-功能研究。有 这个项目和其他三个项目之间有很大的互动性。

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.