Determinants of AAV Lung Tropism

描述(申请人提供)：呼吸道病原体，如人类副流感病毒和SARS冠状病毒，通过利用呼吸道细胞类型中遇到的一组不同的细胞表面多糖和糖蛋白受体，有效地感染人类呼吸道。对腺相关病毒(AAV)，血清型1、5、6和最近的研究表明，AAV9在体外和体内都能有效地转导呼吸道，尽管存在显著的物种特异性和血清型特异性差异。这项建议的目的是阐明AAV血清型中呼吸道趋向性的分子和细胞决定因素。为了实现这一目标，我们设计了一种全面的方法，该方法依赖于通过突变对AAV衣壳进行分子操作，用于鉴定多糖和介导AAV呼吸道细胞进入的辅助受体的生化试剂，以及相关的人类呼吸道体外模型。这里描述的策略将(A)在氨基酸水平上定义决定呼吸道趋向性的AAV衣壳结构元件，以及(B)能够鉴定细胞表面成分，包括决定不同类型呼吸道细胞的AAV趋向性的多糖和整合素亚基。拟议的研究将有助于全面了解AAV呼吸道细胞进入的机制，并提供对AAV气道趋向性的物种特异性差异的洞察。如果成功，这一知识可能会促进AAV载体的改进，以及专注于囊性纤维化等呼吸道疾病基因治疗的临床前/临床研究的设计。 公共卫生相关性：这项提案的目标是了解AAV感染人类呼吸道的分子机制。为了实现这一目标，我们将使用一种全面的方法，包括分子病毒学技术、新型生化试剂和一组不同类型的人类呼吸道细胞。如果成功，这一知识可能会促进AAV载体的改进，以及专注于囊性纤维化等呼吸道疾病基因治疗的临床前/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.