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

用于将基因转移至 CF 气道的腺相关病毒 5

• 批准号: 6853142
• 负责人: MICHAEL J. WELSH
• 金额: $ 20.15万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6853142
• 关键词: Adenoviridae; biotechnology; chloride channels; chloride ion; clinical research; cystic fibrosis; gene complementation; gene delivery system; gene expression; gene therapy; genetic promoter element; genetic transduction; genetically modified animals; human tissue; intestine disorder; intestines; ion transport; laboratory mouse; lung disorder; nonhuman therapy evaluation; recombinant virus; respiratory epithelium; transfection /expression vector

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.

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疗法。重组腺相关病毒（AAV）向量为实现这一目标提供了几个优点，并且最近发现AAV5血清型针对区分气道上皮的顶端表面，使其成为特别有吸引力的矢量。因此，该项目的总体目标是检验AAV5载体可以将CFTR cDNA转移到气道上皮并补充CF缺陷的假设。 AIM 1。在区分气道上皮中的AAV5矢量驱动器表达中放置短表达盒吗？为CF开发AAV矢量的主要挑战 基因转移是其有限的包装能力，它在表达盒的大小上施加了严重的限制。我们将构建AAV5载体的调节序列，其目标是获得短启动子，该启动子在气道上皮中驱动转基因表达，产生持续的表达，并抵制炎症介体的衰减。我们将在AAV5和分化的人类气道上皮中测试向量。 AIM 2。表达CFTR的AAV5向量会补充CF缺陷吗？使用其他方法来满足包装尺寸约束，我们开发了一个CFTR，该CFTR包含R域（CFTR-DELTAR）的部分删除。我们对我们的初步工作感到兴奋，表明包含短启动子和CFTR-DETTAR转基因的AAV5部分纠正了CF气道上皮中的CF CL传输缺陷。这证实了我们的一般策略。但是，我们目前的AAV5矢量有 局限性和许多问题仍然存在。因此，我们将测试以下假设：AAV5中的CFTR-DETTAR表达盒将在分化的CF气道上皮中纠正体外CF CL传输缺陷。我们还将检验以下假设：CFTR-DETTAR可以纠正CF小鼠的临床肠道疾病。 AIM 3。AAV5矢量会在体内和体外在气道上皮产生持久的校正吗？开发AAV5载体的一个重要原因是产生延长的转基因表达的可能性。我们将使用记者在分化气道上皮和小鼠中测试持久性，我们将测试以下假设：AAV5载体将在CF小鼠的鼻表上皮中产生CF Cl-Cl-转运缺陷的长期互补。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