Determinants of AAV Lung Tropism

描述（由申请人提供）：呼吸道病原体，例如人类副流感病毒和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，已经显示AAV 9在体外和体内有效地阻断气道，尽管具有显著的物种特异性和亚型特异性差异。该建议的目的是阐明AAV血清型中气道嗜性的分子和细胞决定因素。为了实现这一点，我们已经设计了一种综合方法，该方法取决于通过诱变对AAV衣壳的分子操作、用于鉴定介导AAV气道细胞进入的聚糖和共受体的生化试剂以及人呼吸道的相关体外模型。本文所述的策略将（a）在决定气道向性的氨基酸水平上定义AAV衣壳结构元件，和（B）使得能够鉴定细胞表面组分，包括决定AAV对不同气道细胞类型的向性的聚糖和整联蛋白亚基。拟议的研究将有助于提供AAV气道细胞进入的机制的全面图片，以及提供深入了解AAV气道向性的种属特异性差异。如果成功的话，这些知识可能有助于AAV载体的改进以及专注于气道疾病（如囊性纤维化）基因治疗的临床前/临床研究的设计。 公共卫生相关性：该提案的目标是了解AAV感染人类气道的分子机制。为了实现这一目标，我们将使用一种综合的方法，包括分子病毒学技术，新型生化试剂和一组不同的人类气道细胞类型。如果成功的话，这些知识可能有助于AAV载体的改进以及专注于气道疾病（如囊性纤维化）基因治疗的临床前/临床研究的设计。

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.