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.

将基因转移到囊性纤维化（CF）患者气道可能代表这种致命疾病的重大治疗进展。重组腺相关病毒 (AAV) 载体为实现这一目标提供了多种优势，最近发现 AAV5 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检验 AAV5 载体可以将 CFTR cDNA 转移到气道上皮细胞并补充 CF 缺陷的假设。目标 1. 置于 AAV5 载体中的短表达盒是否会驱动分化的气道上皮细胞中的表达？开发 CF 的 AAV 载体的主要挑战 基因转移的最大缺点是其包装能力有限，这对表达盒的大小造成了严格的限制。我们将为 AAV5 载体构建调控序列，目标是获得一个短的启动子，该启动子可驱动气道上皮细胞中的转基因表达，产生持久的表达，并抵抗炎症介质的减弱。我们将在 AAV5 和分化的人类气道上皮细胞中测试载体。目标 2. 表达 CFTR 的 AAV5 载体能否弥补 CF 缺陷？使用另一种方法来满足包装尺寸限制，我们开发了包含部分 R 结构域删除的 CFTR（CFTR-deltaR）。我们对我们的初步工作感到兴奋，该工作表明含有短启动子的 AAV5 和 CFTR-deltaR 转基因部分纠正了 CF 气道上皮细胞中的 CF Cl-转运缺陷。这验证了我们的总体策略。然而，我们目前的 AAV5 载体有 局限性，并且仍然存在许多问题。因此，我们将测试以下假设：AAV5 中的 CFTR-deltaR 表达盒将在体外纠正分化的 CF 气道上皮细胞中的 CF Cl-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。目标 3. AAV5 载体是否会在体内和体外对气道上皮细胞产生持久的校正？开发 AAV5 载体的一个重要原因是能够产生延长的转基因表达。我们将使用报告基因测试分化的气道上皮和小鼠中的持久性，并且我们将测试 AAV5 载体将在 CF 小鼠鼻上皮体内产生持久的 CF Cl-转运缺陷补充的假设。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。