Mapping Adeno-associated Virus Capsid Structural and Dynamic Transitions

描述（由申请人提供）：腺相关病毒（rAAV）载体可以介导安全的基因转移，用于动物模型中遗传疾病的长期校正，并有效递送校正基因用于治疗人类疾病。它们是安全的，并且在递送至肺、窦、骨骼肌、脑和肝组织后在人体内持续存在。有效地扩增不同的细胞/组织群体以用于校正基因递送的能力已经引起了人们对理解其基础生物学的极大兴趣。这包括它们的衣壳结构、细胞向性和用于进入、运输、脱壳、复制、DNA包装、衣壳组装和抗体中和的相互作用。该项目的长期目标是获得关于高效细胞进入和细胞内运输到细胞核进行复制所需的AAV衣壳转换动力学的信息。将使用物理、生物化学和遗传方法来鉴定衣壳上参与细胞进入和随后通过内吞途径转运至细胞核期间发生的结构变化的位点。四种选择的AAV血清型（AAV 1、AAV 2、AAV 5和AAV 8）代表了迄今为止在灵长类AAV中观察到的序列和衣壳结构多样性的谱，将作为我们提出的研究的模型。具体目标1将利用溶液研究，采用有限的蛋白水解耦合质谱法来识别和测量动态蛋白质区域。具体目标2将专注于衣壳转换的晶体学可视化，提供一个3D平台，可以在其上注释目标1产生的数据。具体目标3将使用生物化学和遗传学方法验证具体目标1和2的观察结果。预期对AAV的更好的物理理解（这是该项目的主要目标）可以产生新一代校正病毒基因递送载体，其在组织向性和转导效率方面具有协同改进。公共卫生相关性几种腺相关病毒（rAAV）载体可以介导安全的基因转移，用于纠正遗传性疾病，并正在进行临床试验。然而，很少有信息是关于允许细胞感染和贩运到细胞核复制所需的蛋白质衣壳的物理转变。这些信息的可用性对于开发具有改进功效的下一代重组载体将是有价值的。这个项目的目的是填补我们的基础AAV生物学知识库的缺乏。

DESCRIPTION (provided by applicant): Adeno-associated viral (rAAV) vectors can mediate safe gene transfer for the long-term correction of genetic diseases in animal models and efficiently deliver corrective genes for the treatment of human diseases. They are safe and persist in humans following delivery to lung, sinus, skeletal muscle, brain, and liver tissue. The ability to efficiently transduce different cell/tissue populations for corrective gene delivery has generated significant interest in understanding their basic biology. This includes their capsid structure, cellular tropism and interactions for entry, trafficking, uncoating, replication, DNA packaging, capsid assembly, and antibody neutralization. The long- range goal of this project is to obtain information on the AAV capsid transition dynamics required for efficient cell entry and intracellular trafficking to the nucleus for replication. Physical, biochemical, and genetic approaches will be used to identify sites on the capsid that are involved in structural changes that occur during cell entry and subsequent transport to the nucleus via the endocytic pathway. Four selected AAV serotypes (AAV1, AAV2, AAV5, and AAV8) which represent the spectrum of sequence and capsid structural diversity so far observed for the primate AAVs, will serve as our models for the proposed studies. Specific aim 1 will utilize solution studies, employing limited proteolysis to coupled mass spectrometry to identify and measure dynamic protein regions. Specific aim 2 will focus on crystallographic visualization of capsid transitions, providing a 3D platform onto which the data resulting from aim 1 can be annotated. Specific aim 3 will validate the observations from specific aims 1 and 2 using biochemical and genetic approaches. It is anticipated that a better physical understanding of the AAVs, as is the main goal of this project, could give rise to a new generation of corrective viral gene delivery vectors with synergistic improvements in tissue tropism and transduction efficiencies. PUBLIC HEALTH RELEVANCE Several Adeno-associated viral (rAAV) vectors can mediate safe gene transfer for the correction of genetic diseases and are in clinical trials. However, very little information is available on the physical transitions of the protein capsid necessary for permissive cellular infection and trafficking to the nucleus for replication. The availability of this information will be valuable for the development of next generation recombinant vectors with improved efficacy. This project aims to fill this dearth in our knowledge base of basic AAV biology.