GENE TRANSFER TO HUMAN AIRWAY EPITHELIA IN VIVO

体内基因转移至人类气道上皮细胞

• 批准号: 6325956
• 负责人: PAUL B MCCRAY
• 金额: $ 27.25万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325956
• 关键词: Adenoviridae; athymic mouse; biotechnology; cell cycle; cell differentiation; chloride channels; cystic fibrosis; growth factor; human tissue; laboratory mouse; laboratory rat; respiratory epithelium; transfection /expression vector

Studies have shown that transfer of the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into cystic fibrosis (CF) epithelial corrects the defective cAMP-mediated chloride (CI-) transport that characterizes CF. However, no long term approach to somatic cell gene transfer has been developed. Gene transfer with integrating vectors such as retrovirus offers the exciting potential to provide long term correction. However, studies to date suggest that gene transfer with Moloney murine leukemia virus (MMLV) based vectors is inefficient in differentiated airway epithelia, in part because of the low rates of proliferation. Our recent demonstrate that we can overcome the limitation of low rates of cell division by stimulating cells with growth factors that cause differentiated epithelia to divide. Another limitation is the apparent lack of accessible receptors on the apical surface. We found that cells stimulated to divide with growth factors can be readily infected by applying the vector to the basolateral surface or by apply vector to the apical surface when tight junctions are transiently opened by Va/2+ chelation. An exciting recent development is the hybrid lentivirus-based vectors that can infect non- dividing cells. Our preliminary studies show these vectors share the same problem as MMLV with access to receptors from the apical surface. It is not yet clear if lentiviral vectors will be useful for gene transfer to airway epithelia. Now with this preliminary data and these insights into airway epithelial cell biology, we have the tools and reagents to address several important questions. In specific aims we will answer 3 questions: 1) Does infection of dividing cells with integrating vectors produce persistent expression and correction of the CF defect?, 2) Can an integrating vector target non-dividing cells and produce persistent expression and correction of the CF defect? 3) Can integrating vectors correct the CF defect in differentiated epithelia in vivo? The results from these studies are relevant to future work with any integrating vector.

研究表明，人囊性纤维化的转移 跨膜电导调节剂（CFTR）cDNA进入囊性纤维化 （cf）上皮纠正有缺陷的cAMP介导的氯化物（CI-） 特征CF的运输。但是，没有长期的方法 已经开发了体细胞基因转移。基因转移 集成载体（例如逆转录病毒）为 提供长期校正。但是，迄今为止的研究表明基因 基于Moloney鼠白血病病毒（MMLV）向量转移 差异化的气道上皮效率低下，部分原因是 较低的增殖率。我们最近的证明我们可以克服 通过用 导致上皮分裂的生长因子。其他 限制显然缺乏顶端的受体 表面。我们发现细胞被刺激与生长因子分裂 可以通过将矢量应用于基底外侧而容易感染 紧密连接时表面或通过将矢量施加到顶部表面 通过VA/2+螯合瞬时打开。令人兴奋的最近 开发是基于混合慢病毒的载体，可以感染非 - 分裂细胞。我们的初步研究表明，这些向量共享 与MMLV相同的问题，从顶部表面访问受体。 尚不清楚慢病毒载体是否对基因有用 转移到气道上皮。现在有了这些初步数据，这些 对气道上皮细胞生物学的见解，我们有工具和工具 解决几个重要问题的试剂。在特定目标中我们 将回答3个问题：1）是否会感染分裂的细胞 整合向量会产生持续的表达和校正 CF缺陷？，2）集成向量的靶标非分散细胞和可以 产生CF缺陷的持续表达和校正？ 3）可以 集成向量纠正分化性上皮中的CF缺陷 体内？这些研究的结果与未来的工作有关 任何集成向量。