Determinants of AAV Lung Tropism

描述（由申请人提供）：呼吸道病原体，如人副流感病毒和SARS冠状病毒，通过利用气道细胞类型中遇到的多种细胞表面聚糖和糖蛋白受体，有效地感染人类气道。在腺相关病毒（AAV）的情况下，血清型1、5、6和最近，AAV9已被证明在体外和体内有效地转导气道，尽管具有显著的物种特异性和血清型特异性差异。本建议的目的是阐明AAV血清型气道趋向性的分子和细胞决定因素。为了实现这一目标，我们设计了一种综合的方法，该方法依赖于AAV衣壳的分子操纵，通过诱变，生化试剂鉴定介导AAV气道细胞进入的聚糖和共受体，以及人类呼吸道的相关体外模型。本文描述的策略将(a)在氨基酸水平上定义决定气道趋向性的AAV衣壳结构元件，以及(b)能够识别包括聚糖和整合素亚基在内的细胞表面成分，这些成分决定了不同气道细胞类型的AAV趋向性。拟议的研究将有助于全面了解AAV气道细胞进入的机制，并为AAV气道趋向性的物种特异性差异提供见解。如果成功，这一知识可能有助于改进AAV载体，以及设计专注于基因治疗气道疾病（如囊性纤维化）的临床前/临床研究。

DESCRIPTION (provided by applicant): Respiratory tract pathogens such as the human parainfluenza virus and SARS coronavirus effectively infect human airways by exploiting a diverse set of cell surface glycans and glycoprotein receptors encountered in airway cell types. In case of adeno-associated viruses (AAV), serotypes 1, 5, 6 and more recently, AAV9 have been shown to efficiently transduce airways in vitro and in vivo, albeit with striking species-specific and serotype-specific differences. The goal of this proposal is to elucidate molecular and cellular determinants of airway tropism in AAV serotypes. To achieve such, we have devised a comprehensive approach that hinges on molecular manipulation of AAV capsids through mutagenesis, biochemical reagents for identification of glycans and co-receptors that mediate AAV airway cell entry and relevant in vitro models of the human respiratory tract. The strategies described herein will (a) define AAV capsid structural elements at the amino acid level that determine airway tropism, and (b) enable identification of cell surface components including glycans and integrin subunits that dictate AAV tropism for different airway cell types. The proposed studies will help provide a comprehensive picture of the mechanisms underlying AAV airway cell entry as well as provide insight into species-specific differences in AAV airway tropism. If successful, this knowledge may facilitate improvements in AAV vectors as well as in the design of preclinical/clinical studies focused on gene therapy of airway diseases such as cystic fibrosis. Public Health Relevance: The goal of this proposal is to understand the molecular mechanisms exploited by AAV in infecting human airways. To achieve such, we will use a comprehensive approach including molecular virology techniques, novel biochemical reagents and a panel of different human airway cell types. If successful, this knowledge may facilitate improvements in AAV vectors as well as in the design of preclinical/clinical studies focused on gene therapy of airway diseases such as cystic fibrosis.