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

基因转移到囊性纤维化（CF）患者的气道可能代表了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，并且最近发现AAV 5血清型靶向分化的气道上皮细胞的顶端表面使其成为特别有吸引力的载体。因此，该项目的总体目标是测试AAV 5载体可以将CFTR cDNA转移到气道上皮并补充CF缺陷的假设。目标1.将短表达盒置于AAV 5载体中会驱动分化的气道上皮细胞中的表达吗？开发用于CF的AAV载体的主要挑战 基因转移是其有限的包装能力，这对表达盒的大小有严格的限制。我们将构建AAV 5载体的调控序列，目标是获得短启动子，其驱动气道上皮中的转基因表达，产生持续表达，并抵抗炎症介质的衰减。我们将在AAV 5的背景下和在分化的人气道上皮中测试载体。目标2.表达CFTR的AAV 5载体会补充CF缺陷吗？使用另外的方法来满足包装尺寸限制，我们开发了包含R结构域的部分缺失的CFTR（CFTR-deltaR）。我们的初步工作显示，含有短启动子和CFTR-deltaR转基因的AAV 5部分纠正了CF气道上皮中的CF Cl-转运缺陷，这令我们感到兴奋。这证实了我们的总体战略。然而，我们目前的AAV 5载体具有 局限性，还有很多问题。因此，我们将检验以下假设：AAV 5中的CFTR-deltaR表达盒将在分化的CF气道上皮细胞中体外校正CF Cl-转运缺陷。我们还将测试CFTR-deltaR可以纠正CF小鼠中的临床肠道疾病的假设。目标3. AAV 5载体是否会在体内和体外气道上皮中产生持久的校正？开发AAV 5载体的一个重要原因是产生延长的转基因表达的可能性。我们将使用报告基因测试在分化的气道上皮和小鼠中的持久性，并且我们将测试以下假设：AAV 5载体将在CF小鼠的鼻上皮中产生CF Cl-转运缺陷的体内持久互补。这些研究将使我们更接近开发新疗法的长期目标。 患有CF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.