Vector and Host Cellular Biology in AAV Vector Transduction in Vivo

描述（由申请人提供）：腺相关病毒（AAV）是一种非致病性的复制缺陷病毒。重组AAV （rAAV）显示出作为基因治疗病毒载体的潜力。rAAV结构简单，由3个衣壳蛋白和一个单链DNA基因组组成。它不表达病毒编码的蛋白质，并且缺乏野生型AAV的整合机制，但偶然地将其遗传物质通过未定义的机制插入宿主染色体DNA。尽管具有非致病性和复制缺陷的性质，但存在由病毒基因组DNA（即带发夹的单链DNA）介导的细胞内病毒-宿主相互作用。尽管在应用该载体系统治疗各种疾病方面已经取得了实质性进展，但细胞内病毒-宿主相互作用在rAAV载体生物学和体内（动物和人类）宿主细胞生物学中的作用仍然很大程度上未知。我们的最终目标是在实验动物的各种rAAV转导组织中，实质性地了解rAAV载体基因组与宿主细胞之间的生物学相互作用。这对于选择合适的靶组织、了解rAAV给药的潜在风险以及设计和开发更有效、更安全的基因传递系统用于临床应用至关重要。基于我们和其他人的观察，我们假设rAAV基因组通过病毒-宿主细胞相互作用激活多种DNA修复途径，并触发rAAV-rAAV和/或rAAV-宿主基因组重组。我们最近证明了一种特定的DNA修复内切酶打开rAAV载体基因组反向末端重复序列（ITR）发夹结构并触发rAAV基因组重组。在该项目中，我们将首先确定参与rAAV基因组加工的细胞因子并研究其作用过程（目标1）。其次，我们将研究rAAV基因组和细胞因子如何在网络中相互作用，以及rAAV载体如何激活该网络（目标2）。最后，我们将表征和量化rAAV在小鼠各种组织中的整合，并将识别参与rAAV整合的细胞因子，以了解rAAV在体内整合的机制（目标3）。拟议的项目应该对建立成功的人类基因治疗的智力基础作出实质性贡献。重组腺相关病毒（rAAV）是一种很有前途的人类基因治疗基因传递载体系统。本项目旨在阐明rAAV载体基因组、宿主细胞基因组和载体感染细胞中细胞蛋白之间的复杂相互作用，以更好地了解该载体系统。研究结果将有助于设计和开发更高效、更安全的rAAV载体，并对其安全性进行评估。

DESCRIPTION (provided by applicant): The adeno-associated virus (AAV) is a non-pathogenic, replication defective virus. Recombinant AAV (rAAV) shows promise as a viral vector for gene therapy. rAAV has a simple structure consisting of 3 capsid proteins and a single-stranded DNA genome. It does not express virally encoded proteins, and is devoid of the integration machinery of wild-type AAV but fortuitously inserts its genetic material into host chromosomal DNA by undefined mechanisms. Despite its non-pathogenic and replication defective nature, there are intracellular virus-host interactions mediated by viral genome DNA (i.e., single-stranded DNA with hairpins). Although there has been substantial progress in the application of this vector system to treat various diseases, the role of intracellular virus-host interactions in rAAV vector biology and host cellular biology in vivo (in animals and humans) remains largely unknown. Our ultimate goal in this proposal is to substantially understand the biological interactions between rAAV vector genomes and host cells in various rAAV-transduced tissues in experimental animals. This is key to selecting appropriate target tissues and understanding the potential risks of rAAV administration as well as designing and developing more efficient and safer gene delivery systems for clinical applications. Based on the observations by us and others, we hypothesize that rAAV genomes activate various DNA repair pathways via virus-host cell interactions, and trigger rAAV-rAAV and/or rAAV-host genome recombinations. We have recently demonstrated that a specific DNA repair endonuclease opens the rAAV vector genome inverted terminal repeat (ITR) hairpin structures and trigger rAAV genome recombinations. In the project, we will first identify cellular factors involved in rAAV genome processing and investigate their course of action (aim 1). Second, we will investigate how rAAV genomes and the cellular factors interact in a network and how rAAV vectors activate this network (aim 2). Finally, we will characterize and quantify rAAV integration in various tissues in mice and will identify cellular factors involved in rAAV integration to understand the mechanisms for rAAV integration in vivo (aim 3). The proposed project should substantially contribute to building an intellectual foundation for successful human gene therapy. Public Relevance Statement: Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector system for human gene therapy. This proposed project aims at elucidating complex interactions between rAAV vector genome, the host cellular genome and cellular proteins in vector-infected cells for a better understanding of this vector system. Results of the study will contribute to designing and developing more efficient and safer rAAV vectors and assessing the safety of the vector.