Virus-cell interactions necessary for adeno-associated virus vector-mediated gene delivery to the conducting airway epithelium.

摘要

Vectors based on adeno-associated virus (AAV) are promising candidates for gene therapy of a variety of genetic diseases due to their ability to provide safe and efficient delivery of therapeutic genes to multiple tissues. The airway epithelium is an important target for gene therapy of diseases affecting the lung, such as cystic fibrosis. In this dissertation, we sought to examine AAV vector-mediated transduction of the airway epithelium to identify important factors necessary for virus-cell interactions. Previously, it was observed that AAV serotype 6 (AAV6) demonstrates efficient transduction of both human airway epithelial cell cultures and the conducting airway epithelium of the mouse lung. Our first goal was to identify the domain of the AAV6 capsid that is responsible for conferring this favorable transduction phenotype. To address this, we constructed capsid chimeras between AAV6 and a serotype that does not target cells of the conducting airway, AAV9. By swapping specific capsid domains between these two serotypes, we discovered that incorporating variable regions IV-V of the AAV6 capsid into the homologous region of the AAV9 capsid conferred efficient conducting airway transduction onto AAV9. In addition to studying the capsid interactions necessary for AAV6 transduction, we also sought to understand the biology of AAV9 by identifying the cellular receptor for this serotype. Although AAV9 does not transduce conducting airway, it demonstrates efficient transduction of the alveolar epithelium of the lung, as well as many other tissues. Through a series of in vitro cell binding and transduction assays, we determined that AAV9 uses galactose as a receptor. This discovery led to a novel pharmacologic approach for enhancing AAV9 lung transduction. Pre-treating mice with an intranasal instillation of neuraminidase, to cleave sialic acid residues from the cell surface and expose the underlying galactose saccharides, led to robust conducting airway transduction by AAV9. Further, we mapped the galactose binding domain of AAV9 to five specific amino acids that form a pocket on the surface of the capsid. Overall, we discovered many important characteristics governing AAV capsid interactions with cells of the conducting airway, allowing for the potential engineering of improved lung gene therapeutics.