Zabner J, Zeiher B G, Friedman E, Welsh M J
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242, USA.
J Virol. 1996 Oct;70(10):6994-7003. doi: 10.1128/JVI.70.10.6994-7003.1996.
The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia.
腺病毒介导的基因转移至气道上皮细胞的效率，将是决定重组腺病毒能否开发为载体，用于将囊性纤维化跨膜传导调节因子（CFTR）cDNA转移至囊性纤维化患者的一个重要因素。目前对囊性纤维化肺部疾病生物学的认识表明，载体应在成熟的、有纤毛的气道上皮细胞中表达转基因。我们评估了腺病毒介导的基因转移至正常和囊性纤维化患者人气道上皮细胞原代培养物的效率。我们的研究表明，气道细胞从接种后3天具有基底细胞特征性标志物且表面覆盖短微绒毛的未分化上皮，发展为10至14天后顶端表面覆盖纤毛的成熟上皮。腺病毒载体表达报告基因以及纠正囊性纤维化上皮细胞中缺陷性环磷酸腺苷刺激的氯离子转运的能力，与分化状态呈负相关。然而，当载体与上皮细胞的接触时间延长时，腺病毒介导的基因转移效率可得到部分纠正。延长接触后，我们观察到培养的分化型囊性纤维化气道上皮细胞中囊性纤维化氯离子转运缺陷以及纯合δF508突变小鼠鼻上皮细胞中氯离子转运缺陷得到完全纠正。基因转移至气道上皮细胞需要与载体长时间孵育这一事实，与在常用以研究腺病毒感染的细胞模型（如293和HeLa细胞）中观察到的快速感染形成对比。长时间孵育后观察到的基因转移可能源于与介导293细胞感染的机制不同的机制。这些观察结果表明，可能需要采取增加接触时间或改变上皮细胞或载体的干预措施，以促进基因转移至有纤毛的呼吸道上皮细胞。
