Adeno-Associated Virus-5 for Gene Transfer to CF Airway

将基因转移到囊性纤维化(CF)患者的呼吸道可以代表这种致命疾病的主要治疗进展。重组腺相关病毒(AAV)载体为实现这一目标提供了几个优点，最近发现AAV5血清型靶向分化的呼吸道上皮细胞的顶面，使其成为一个特别有吸引力的载体。因此，该项目的总体目标是验证AAV5载体可以将CFTRcDNA转移到呼吸道上皮细胞并补充CF缺陷的假设。目的1.在AAV5载体中放置一个短的表达盒是否会驱动分化的呼吸道上皮细胞的表达？为CF开发AAV载体的主要挑战 基因转移是其有限的包装能力，这对表达盒的大小施加了严格的限制。我们将构建AAV5载体的调控序列，目标是获得一种短的启动子，在呼吸道上皮细胞中驱动转基因表达，产生持续表达，并抵抗炎症介质的衰减。我们将在AAV5和分化的人类呼吸道上皮细胞中测试载体。目的2.表达cftr的AAV5载体能弥补cf缺陷吗？使用另一种方法来满足包装大小限制，我们开发了一种包含R结构域部分缺失的CFTR(cftr-deltar)。我们的初步工作表明，含有短启动子和CFTR-deltar转基因的AAV5部分纠正了CF呼吸道上皮细胞的CFCl-转运缺陷，这让我们感到兴奋。这证明了我们的总体战略是正确的。然而，我们目前的AAV5载体有 限制，许多问题仍然存在。因此，我们将验证这样一种假设，即AAV5中的CFTR-deltar表达盒在体外可以纠正分化的CF气道上皮细胞的CFCl-转运缺陷。我们还将测试CFTR-deltar可以纠正CF小鼠临床肠道疾病的假设。目的3.AAV5载体能否在体内和体外对呼吸道上皮细胞产生持久的纠正作用？开发AAV5载体的一个重要原因是有可能产生长时间的转基因表达。我们将使用记者在分化的呼吸道上皮细胞和小鼠身上测试持久性，并测试AAV5载体将在体内长期补充CF小鼠鼻黏膜上皮中CFCl-转运缺陷的假设。这些研究将使我们更接近开发新的治疗方法的长期目标 患有慢性阻塞性肺疾病的人。

Gene transfer to the airways of patients with cystic fibrosis (CF) could represent a major therapeutic advance for this lethal disease. Recombinant adeno-associated viruses (AAV) vectors offer several advantages for achieving this goal, and the recent discovery that the AAV5 serotype targets the apical surface of differentiated airway epithelia makes this a particularly attractive vector. Thus, the overall goal of this project is to test the hypothesis that an AAV5 vector can transfer the CFTR cDNA to airway epithelia and complement the CF defect. Aim 1. Will a short expression cassette placed in an AAV5 vector drive expression in differentiated airway epithelia? A major challenge in developing an AAV vector for CF gene transfer is its limited packaging capacity, which places severe constraints on the size of the expression cassette. We will construct regulatory sequences for AAV5 vectors, with a goal of obtaining a promoter that is short, that drives transgene expression in airway epithelia, that generates persistent expression, and that resists attenuation by inflammatory mediators. We will test vectors in the context of AAV5 and in differentiated human airway epithelia. Aim 2. Will an AAV5 vector expressing CFTR complement the CF defect? Using an additional approach to meet packaging size constraints, we developed a CFTR containing a partial deletion of the R domain (CFTR-deltaR). We are excited by our preliminary work showing that an AAV5 containing a short promoter and a CFTR-deltaR transgene partially corrected the CF Cl- transport defect in CF airway epithelia. This validates our general strategy. However, our current AAV5 vectors have limitations, and many questions remain. Therefore, we will test the hypothesis that a CFTR-deltaR expression cassette in AAV5 will correct the CF Cl- transport defect in vitro in differentiated CF airway epithelia. We will also test the hypothesis that CFTR-deltaR can correct clinical intestinal disease in CF mice. Aim 3. Will an AAV5 vector generate long-lasting correction in airway epithelia in vivo and in vitro? An important reason to develop an AAV5 vector is the possibility of generating prolonged transgene expression. We will test persistence in differentiated airway epithelia and in mice using reporters, and we will test the hypothesis that an AAV5 vector will yield long-lasting complementation of the CF Cl- transport defect in vivo in nasal epithelia of CF mice. These studies will take us closer to our long-term goal of developing new therapies for people who suffer from CF.