STRATEGIES TO IMPROVE AAV CFTR PERSISTENCE & EXPRESSION

描述（直接取自应用程序） 腺相关病毒（AAV）是一种非致病性人细小病毒， 被开发为囊性纤维化的基因转移载体。 aav生命 周期包括潜伏期，其中载体DNA稳定地整合到 宿主细胞基因组，通常在染色体19的特定区域内（ AAVSI网站）。 该过程似乎涉及AAV Rep蛋白，Rep 68 和78，它们能够结合两个反向末端重复序列 AAV的ITR和染色体S1序列。 AAV-CFTR 已经发现缺乏REP基因序列的载体具有降低的 整合的频率和位点特异性。 令人惊讶的是，AAV-CFTR 已发现序列持续存在并表达约3个月 在体外，以及在体内递送至支气管后至少6个月 家兔和恒河猴的上皮。 在两种情况下，双链 已经发现了附加型载体DNA序列。这表明，如果 由于Rep蛋白的缺失，潜在的阶段整合过程中止， 双链中间体可以在细胞中持续存在， 一段时间。 这一属性大概是由ITR赋予的，因为 这些是目前AAV-CFTR载体中仅有的AAV序列。 这 该项目旨在进一步确定AAV-ITR和Rep蛋白在 AAV载体持久性的建立。 的最终目标 该项目将开发更新的基于AAV的基因转移技术 它将重新获得野生型的最独特和有益的特征， AAV，其在分化的人类细胞中持续存在而不引起 疾病 我们计划使用一种新型的非病毒传递系统，明胶 珠复合物，其能够掺入AAV-载体DNA 序列和Rep蛋白分子，其形式容易被 人支气管上皮细胞 该项目的目标将是 实现了四个具体目标：（1）描述持久性 第一代AAV载体DNA序列在许多模型中，包括 恒河猴和囊性纤维化患者的气道上皮;（2） 确定持久性所需的最小顺式作用ITR序列 和/或在凝胶珠粒化后在细胞中复制载体序列，或 脂质体介导的转染;（3）检测AAV-Rep 蛋白质对AAV整合的频率和位点特异性的影响，以及对 附加型AAV载体复制和持久性;以及（4）测试附加型AAV载体复制和持久性的能力。 优化的整合和附加型载体构建体的持续存在后， 对灵长类动物气道上皮的体内给药。

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.