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: 6581185
• 负责人: PAUL B MCCRAY
• 金额: $ 29万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6581185
• 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，人类囊性纤维化的转移 跨膜电导调节因子 (CFTR) cDNA 进入囊性纤维化 (CF) 上皮细胞纠正有缺陷的 cAMP 介导的氯化物 (CI-) CF 的传输特性。然而，没有长期的方法 体细胞基因转移已被开发出来。基因转移 整合逆转录病毒等载体提供了令人兴奋的潜力 提供长期修正。然而，迄今为止的研究表明，基因 使用基于莫洛尼鼠白血病病毒 (MMLV) 的载体进行转移是 分化的气道上皮细胞效率低下，部分原因是 增殖率低。我们最近证明我们可以克服 通过刺激细胞来限制细胞分裂率低 导致分化上皮细胞分裂的生长因子。其他 限制是顶端明显缺乏可接近的受体 表面。我们发现生长因子刺激细胞分裂 通过将载体应用到基底外侧可以很容易地被感染 表面或在紧密连接时将矢量应用于顶面 被 Va/2+ 螯合暂时打开。最近令人兴奋的 开发是基于混合慢病毒的载体，可以感染非 正在分裂的细胞。我们的初步研究表明这些载体共享 与 MMLV 存在同样的问题，即从顶端表面接触受体。 目前尚不清楚慢病毒载体是否对基因有用 转移至气道上皮。现在有了这些初步数据和这些 深入了解气道上皮细胞生物学，我们拥有工具和 试剂来解决几个重要问题。在具体目标中我们 将回答 3 个问题： 1) 分裂细胞是否感染 整合载体产生持久表达和校正 CF 缺陷？, 2) 整合载体能否靶向非分裂细胞和 产生持久的表达并纠正 CF 缺陷？ 3）可以 整合向量纠正分化上皮细胞中的 CF 缺陷 体内？这些研究的结果与未来的工作相关 任何积分向量。