Harris C E, Agarwal S, Hu P, Wagner E, Curiel D T
Department of Pediatrics, University of North Carolina at Chapel Hill.
Am J Respir Cell Mol Biol. 1993 Oct;9(4):441-7. doi: 10.1165/ajrcmb/9.4.441.
A variety of methods have been utilized for gene transfer to the cells of the airway epithelium. These have included DNA-mediated mechanisms of gene transfer as well as recombinant viral vectors. Despite the availability of these methods, limitations in their utility warrant the development of alternate systems. As an alternative, receptor-mediated endocytosis using transferrin-polylysine conjugates has been shown to transduce immortalized airway epithelial cells efficiently via a physiologic pathway. When transferrin-polylysine conjugates were used to transduce airway epithelial cells grown in primary culture, however, gene transfer occurred inefficiently. Investigation into this relative inefficiency centered on endosomal entrapment of the conjugate-DNA complex. Pretreatment of the cells with chloroquine, which causes vacuolization and disruption of the endosome, or co-delivery of adenoviral particles, which serves to lyse the endosomal membrane, were both associated with greatly improved gene transfer efficiency. These studies established that the relative refractory state of the airway epithelial cells in primary culture was secondary to the retention of the internalized material within the endosome. We thus explored the efficiency of conjugates that possessed a mechanism to escape this endosomal entrapment; adenovirus-polylysine conjugates and transferrin-polylysine/adenovirus-polylysine conjugates were thus employed. Gene transfer efficiency improved significantly with the adenovirus-containing conjugates. These data support the concept that conjugates can be synthesized that mediate highly efficient gene transfer to airway epithelial cells in primary culture via the receptor-mediated endocytosis pathway.
人们已经采用了多种方法将基因导入气道上皮细胞。这些方法包括DNA介导的基因转移机制以及重组病毒载体。尽管有这些方法，但它们在实用性方面的局限性促使人们开发替代系统。作为一种替代方法，使用转铁蛋白-聚赖氨酸偶联物的受体介导的内吞作用已被证明可通过生理途径有效地转导永生化气道上皮细胞。然而，当使用转铁蛋白-聚赖氨酸偶联物转导原代培养的气道上皮细胞时，基因转移效率低下。对这种相对低效性的研究集中在偶联物-DNA复合物的内体截留上。用氯喹预处理细胞（氯喹会导致内体空泡化和破坏）或共同递送腺病毒颗粒（其作用是裂解内体膜），均与基因转移效率的大幅提高相关。这些研究表明，原代培养的气道上皮细胞的相对难治状态是由于内化物质保留在内体中所致。因此，我们探索了具有逃避这种内体截留机制的偶联物的效率；因此使用了腺病毒-聚赖氨酸偶联物和转铁蛋白-聚赖氨酸/腺病毒-聚赖氨酸偶联物。含腺病毒的偶联物使基因转移效率显著提高。这些数据支持这样一种概念，即可以合成通过受体介导的内吞作用途径介导高效基因转移至原代培养的气道上皮细胞的偶联物。
