Identification of the AAVR-independent AAV entry pathway

项目概要 重组腺相关病毒 (rAAV) 载体已成为首选的基因递送剂之一 用于临床基因治疗。迄今为止，两种基于 rAAV 的药物 Luxturna 和 Zolgensma 已获得 FDA 批准 美国FDA和数百项使用rAAV载体进行人类基因治疗的临床试验已经进行或正在进行 正在进行中，有些已取得积极成果。但仍存在诸多障碍有待解决 特别是 AAV 载体的组织向性由初级附着聚糖受体和 蛋白质进入受体。 I 型跨膜蛋白 KIAA0319L，以下称为 AAV 受体 (AAVR)，已被鉴定为参与 rAAV1、2、3、5、6、8 转导的多血清型 AAV 受体。 和 9（进化枝 A-F 和 H AAV）。这些 AAV 衣壳在病毒叠加测定中直接与 AAVR 相互作用。 然而，Clade G AAV（AAV4 和 AAVrh32.33）不使用 AAVR 进行载体结合和转导， 而是通过所谓的 AAVR 独立条目。我们已经鉴定出两种 AAV4 结合蛋白 (AAV4-BP) 使用 rAAV4 或 rAAVrh32.33 载体进行病毒重叠测定，分别为 ~80-kDa 和 ~35-kDa。 重要的是，进化枝 A-F 和 G AAV，包括 AAV4 和 AAVrh32.33，但不包括进化枝 H AAV5，利用 跨高尔基体网络 (TGN) 定位的 GPR108（G 蛋白偶联受体样蛋白 108）用于 TGN 血管 在细胞内运输过程中逃逸。因此，我们假设 Clade G AAV 结合并使用 AAV4- BP 作为载体细胞进入和细胞内向 TGN 逆行运输的蛋白质受体 载体逃逸并进入细胞质进入核的转运途径。本期的主题 该提案旨在通过使用蛋白质组学方法来识别 AAV4-BP，并阐明与 AAVR 无关的 AAV 进入和 GPR108 依赖的细胞内运输途径，用于 Clade G AAV 的生产性转导。 我们的长期目标是确定限制 rAAV 转导并且可以改变的步骤，因此可以 在人类基因治疗应用中，可用于增强 rAAV 在各种细胞和组织中的转导。

PROJECT SUMMARY Recombinant adeno-associated virus (rAAV) vectors have emerged as one of the preferred gene delivery agents for clinical gene therapy. To date, two rAAV-based drugs, Luxturna and Zolgensma, have been approved by the US FDA, and hundreds of clinical trials of human gene therapy using rAAV vectors have been carried out or are ongoing, and some have yielded positive outcomes. However, various barriers still remain to be resolved, in particular the tissue tropism of AAV vectors controlled by the primary attachment glycan receptor and the proteinaceous entry receptor. A type I transmembrane protein, KIAA0319L, denoted hereafter as AAV receptor (AAVR), has been identified as a multiple serotype AAV receptor involved in transduction of rAAV1, 2, 3, 5, 6, 8, and 9 (Clades A-F and H AAVs). These AAV capsids directly interact with AAVR on virus overlay assays. However, Clade G AAVs (AAV4 and AAVrh32.33) do not use the AAVR for vector binding and transduction, rather through a so called AAVR-independent entry. We have identified two AAV4-binding proteins (AAV4-BPs) at ~80-kDa and ~35-kDa, respectively, on the virus overlay assays using the rAAV4 or rAAVrh32.33 vectors. Importantly, Clade A-F and G AAVs, including AAV4 and AAVrh32.33, but not the Clade H AAV5, utilize the trans-Golgi network (TGN)-localized GPR108 (G protein-coupled receptor-like protein 108) for TGN vascular escaping during intracellular trafficking. Therefore, we hypothesize that Clade G AAVs bind to and use the AAV4- BP(s) as a proteinaceous receptor for vector cell entry and intracellular trafficking towards the TGN retrograde transport pathway where the vector escapes and enters into the cytoplasm for nuclear entry. The subject of this proposal aims to identify the AAV4-BPs by using a proteomics approach and to elucidate the AAVR-independent AAV entry and GPR108-depedent intracellular trafficking pathway for productive transduction of Clade G AAVs. Our long-term goal is to identify steps that limit rAAV transduction and that can be altered, and, therefore, can be used to enhance rAAV transduction in various cells and tissue in human gene therapy applications.