Vector and Host Cellular Biology in AAV Vector Transduction in Vivo

描述(申请人提供)：腺相关病毒(AAV)是一种非致病性、复制缺陷的病毒。重组AAV(RAAV)有望成为基因治疗的病毒载体。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，也缺乏野生型AAV的整合机制，但它的遗传物质以未知的机制插入到宿主染色体DNA中。尽管病毒具有非致病性和复制缺陷的性质，但存在由病毒基因组DNA(即带有发夹的单链DNA)介导的细胞内病毒与宿主的相互作用。虽然这种载体系统在治疗各种疾病方面已经取得了实质性的进展，但病毒与宿主之间的相互作用在体内(动物和人类)的rAAV载体生物学和宿主细胞生物学中的作用仍然很大程度上是未知的。我们在这项建议中的最终目标是实质上了解rAAV载体基因组与实验动物各种rAAV转导组织中宿主细胞之间的生物相互作用。这对于选择合适的靶组织和了解rAAV给药的潜在风险以及设计和开发更有效和更安全的临床应用基因递送系统是关键。根据我们和其他人的观察，我们假设rAAV基因组通过病毒与宿主细胞的相互作用激活了各种DNA修复途径，并触发了rAAV-rAAV和/或rAAV-宿主基因组重组。我们最近证明了特定的DNA修复内切酶打开rAAV载体基因组反向末端重复(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基因组处理的细胞因素，并研究它们的作用过程(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载体如何激活这个网络(目标2)。最后，我们将表征和量化小鼠不同组织中rAAV的整合，并将确定参与rAAV整合的细胞因素，以了解rAAV在体内整合的机制(目标3)。拟议的项目应该大大有助于为成功的人类基因治疗建立一个智力基础。公开相关声明：重组腺相关病毒(RAAV)是一种很有前途的人类基因治疗的基因递送载体系统。该项目旨在阐明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，以便更好地了解这一载体系统。研究结果将有助于设计和开发更高效、更安全的rAAV载体，并评估该载体的安全性。

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.