Edwards R J, Carpenter D S, Minchin R F
Department of Pharmacology, University of Western Australia, Nedlands, Australia.
Gene Ther. 1996 Oct;3(10):937-40.
Receptor-mediated gene delivery has been reported for a number of different receptor systems although the intracellular fate of such systems has not been systematically investigated. In this study, we have determined the fate of a commonly used asialoglycoprotein (ASGP)-dependent DNA delivery system in isolated rat hepatocytes. ASPG-polylysine (PLL296) was ionically complexed with pSV-CAT DNA at a molar ratio of 10:1. The resulting complex inhibited 125I-ASGP binding to rat hepatocytes but ASGP only partially inhibited the binding of complex. The ASGP-independent binding was due to the interaction of the PLL component of the complex with plasma membranes and could be minimised by replacing PLL296 with PLL19. Following internalisation, ASGP was cleaved from the complex and translocated to the lysosomes where it was degraded. The DNA, however, remained in an intracellular compartment that cosedimented with plasma membranes in Percoll density gradients. This study shows first that hepatocytes do not process DNA internalised as ASGP complexes in a manner similar to ASGP itself, and second that the differential sorting of the two cleaved molecules leads to a rapid intracellular compartmentalisation of the DNA. Controlled release from this compartment may be a means for prolonged gene expression in gene therapy protocols.
尽管尚未对多种不同受体系统的细胞内命运进行系统研究，但已有关于受体介导的基因传递的报道。在本研究中，我们确定了一种常用的去唾液酸糖蛋白（ASGP）依赖性DNA传递系统在分离的大鼠肝细胞中的命运。ASPG-聚赖氨酸（PLL296）与pSV-CAT DNA以10:1的摩尔比进行离子络合。所得复合物抑制了125I-ASGP与大鼠肝细胞的结合，但ASGP仅部分抑制了复合物的结合。ASGP非依赖性结合是由于复合物的PLL成分与质膜的相互作用，通过用PLL19替代PLL296可将其最小化。内化后，ASGP从复合物中裂解并转运至溶酶体，在那里被降解。然而，DNA保留在一个细胞内区室中，该区室在Percoll密度梯度中与质膜共沉降。这项研究首先表明，肝细胞处理作为ASGP复合物内化的DNA的方式与ASGP本身不同，其次表明两种裂解分子的差异分选导致DNA在细胞内快速区室化。从该区室的控释可能是基因治疗方案中延长基因表达的一种手段。
