STRATEGIES TO IMPROVE AAV CFTR PERSISTENCE & EXPRESSION

描述（直接取自应用程序）

DESCRIPTION (Taken directly from the application) Adeno-associated virus (AAV) is a non-pathogenic human parvovirus which has been developed as a gene transfer vector for cystic fibrosis. The AAV life cycle includes a latent phase in which vector DNA is stably integrated into the host cell genome, usually within a specific region of chromosome 19 (the AAVSI site). This process appears to involve the AAV Rep proteins, Rep 68 and 78, which are capable of binding both the inverted terminal repeats (ITRs) of AAV and the chromosomal Sl sequence. Furthermore, AAV-CFTR vectors which lack the rep gene sequence have been found to have a decreased frequency and site-specificity of integration. Surprisingly, AAV-CFTR sequences have been found to persist and express for approximately 3 months in vitro, and for at least 6 months after in vivo delivery to the bronchial epithelium of rabbits and rhesus monkeys. In both settings, double-stranded episomal vector DNA sequences have been found. This suggests that if the latent phase integration process is aborted by the absence of Rep protein, that double-stranded intermediates may persist in cells for prolonged periods of time. This property is presumably conferred by the ITRs, since these are the only AAV sequences present in current AAV-CFTR vectors. This project seeks to further define the roles of AAV-ITRs and Rep proteins in the establishment of long-term AAV vector persistence. The ultimate goal of this project will be to develop newer AAV-based gene transfer technologies which will recapture the most unique and beneficial feature of wild-type AAV, its ability to persist in differentiated human cells without causing disease. We plan to make use of a novel non-viral delivery system, gelatin bead complexes, which are capable of incorporating both AAV-vector DNA sequences and Rep protein molecules in a form which is readily taken up by human bronchial epithelial cells. The goals of the project will be accomplished in four specific aims: (1) To characterize persistence of first generation AAV vector DNA sequences in a number of models, including the airway epithelium of rhesus monkeys and cystic fibrosis patients; (2) To determine the minimal cis-acting ITR sequence required for persistence and/or replication of vector sequences in cells after gel-bead or liposome-mediated transfection; (3) To determine the effects of AAV-Rep protein on the frequency and site-specificity of AAV integration and on episomal AAV vector replication and persistence; and (4) To test the ability of optimized integrating and episomal vector constructs to persist after in vivo administration to the primate airway epithelium.