Identification of the AAVR-independent AAV entry pathway

鉴定不依赖于 AAVR 的 AAV 进入途径

• 批准号: 10348981
• 负责人: Jianming Qiu
• 金额: $ 23.13万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10348981
• 关键词: Antibodies; Basic Science; Binding; Binding Proteins; Biochemistry; Biological Assay; Biology; Blood Vessels; CRISPR screen; Capsid; Cell Nucleus; Cell surface; Cells; Cellular Membrane; Clathrin; Clinical; Clinical Trials; Cytoplasm; Development; Directed Molecular Evolution; Electrophoresis; Endocytosis; FDA approved; G-Protein-Coupled Receptors; Gene Delivery; Gene Expression; Genetic Transcription; Genome; Goals; Human; Incubated; Integral Membrane Protein; Intervention; Knock-out; Link; Mediating; Medicine; Membrane; Membrane Proteins; Molecular Medicine; Mutagenesis; Nuclear; Organ; Outcome; Pathway interactions; Patients; Pharmaceutical Preparations; Polycystic Kidney Diseases; Polysaccharides; Prevalence; Primates; Proteins; Proteomics; Recombinant adeno-associated virus (rAAV); Role; Serotyping; Sialic Acids; Single-Stranded DNA; Testing; Tissues; Transportation; Tropism; Variant; Vesicle; Viral; Virus; adeno-associated viral vector; base; clinical practice; gene therapy; genome-wide; novel; polyacrylamide; polyvinylidene fluoride; receptor; retrograde transport; tissue tropism; trafficking; trans-Golgi Network; transduction efficiency; uptake; vaccine delivery; vector

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.

项目摘要 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。到目前为止，两种基于rAAV的药物Luxturna和Zolgensma已获得批准， 美国FDA，并且已经进行或正在进行数百项使用rAAV载体的人类基因治疗的临床试验。 有些正在进行，有些已取得积极成果。然而，各种障碍仍有待解决， 特别是由初级附着聚糖受体控制的AAV载体的组织嗜性，以及 蛋白质进入受体I型跨膜蛋白KIAA 0319 L，下文称为AAV受体 （AAVR），已被鉴定为参与rAAV 1，2，3，5，6，8， 和9（进化枝A-F和HAAV）。这些AAV衣壳在病毒覆盖测定中直接与AAVR相互作用。 然而，分化体G AAV（AAV 4和AAVrh32.33）不使用AAVR进行载体结合和转导， 而不是通过所谓的AAVR独立进入。我们已经鉴定了两种AAV 4结合蛋白（AAV 4-BPs）， 在使用rAAV 4或rAAVrh32.33载体的病毒覆盖测定中，分别在约80-kDa和约35-kDa处表达。 重要的是，分化体A-F和G AAV，包括AAV 4和AAVrh32.33，但不包括分化体H AAV 5，利用AAVrh32.33。 跨高尔基体网络（TGN）定位的GPR 108（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，我们假设分化体G AAV结合并使用AAV 4- BP（s）作为载体细胞进入和细胞内转运至TGN逆行的蛋白质受体 载体逃逸并进入细胞质以进入核的转运途径。主题的本 该提案旨在通过使用蛋白质组学方法鉴定AAV 4-BP，并阐明AAVR独立性。 用于分化体G AAV的生产性转导的AAV进入和GPR 108依赖性细胞内运输途径。 我们的长期目标是确定限制rAAV转导的步骤，并且可以改变，因此，可以 在人基因治疗应用中，可用于增强rAAV在各种细胞和组织中的转导。