Identification of the AAVR-independent AAV entry pathway

项目总结 重组腺相关病毒(Raav)载体已成为首选的基因载体之一。 用于临床基因治疗。到目前为止，两种基于rAAV的药物Luxturna和Zolgensma已获得美国食品和药物管理局的批准 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，有些已经产生了积极的结果。然而，仍有各种障碍有待解决。 尤其是由初级附着葡聚糖受体控制的AAV载体的组织趋向性 蛋白质类进入受体。一种I型跨膜蛋白KIAA0319L，以下称为AAV受体 (AAVR)是一种多血清型AAV受体，参与rAAV1、2、3、5、6、8、 和9个(A-F支和H型AAVs)。这些AAV衣壳在病毒覆盖试验中直接与AAVR相互作用。 然而，分支G AAVs(AAV4和AAVRh32.33)不使用AAVR进行载体结合和转导， 而是通过所谓的独立于AAVR的条目。我们鉴定了两个AAV4结合蛋白(AAV4-Bps) 用rAAV4或rAAVrh32.33载体进行病毒重叠检测，分别在~80 kDa和~35 kDa。 重要的是，分支A-F和G AAVs，包括AAV4和AAVrh32.33，而不是分支H AAV5，利用 跨高尔基网络(TGN)定位的GPR108(G蛋白偶联受体样蛋白108)对TGN血管的作用 在细胞内交易过程中逃脱。因此，我们假设G支路AAVs绑定并使用AAV4- BP(S)作为载体细胞进入和细胞内逆行转运TGN的蛋白受体 载体逃逸并进入细胞质以进入细胞核的运输途径。这个的主题是 该提案旨在通过蛋白质组学方法鉴定AAV4-BPS，并阐明AAVR非依赖性 AAV进入和GPR108抑制的细胞内转运途径对支链G AAVs的有效转导。 我们的长期目标是确定限制rAAV转导的步骤，这些步骤可以改变，因此可以 在人类基因治疗中用于增强rAAV在各种细胞和组织中的转导作用。

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.