UPTAKE AND INTRACELLULAR TRAFFICKING OF ASIALOGLYCOPROTEIN-POLYLYSINE-DNA COMPLEXES IN ISOLATED RAT HEPATOCYTES

Receptor-mediated gene delivery has been reported for a number of diff erent receptor systems although the intracellular fate of such systems has not been systematically investigated in this study, we have deter mined the fate of a commonly used asialoglycoprotein (ASGP)-dependent DNA delivery system in isolated rat hepatocytes. ASPG-polylysine (PLL( 296)) was ionically complexed with pSV-CAT DNA at a molar ratio of 10: 1. The resulting complex inhibited I-125-ASGP binding to rat hepatocyt es 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 PLL(296) with PLL(19). Following internalisation, ASGP was cleaved fr om the complex and translocated to the lysosomes where it was degraded , The DNA, however, remained in an intracellular compartment that cose dimented with plasma membranes in Percoll density gradients. This stud y shows first that hepatocytes do not process DNA internalised as ASGP complexes in a manner similar to ASGP itself and second that the diff erential sorting of the two cleaved molecules leads to a rapid intrace llular compartmentalisation of the DNA. Controlled release from this c ompartment may be a means for prolonged gene expression in gene therap y protocols.