Improvement of receptor-mediated gene delivery to HepG2 cells using an amphiphilic gelling agent

Gene transfer was performed using asialo-orosomucoid-mucoid-polylysine (ASO R-PL) conjugates to allow targeted expression of the gene in cells of hepat ic origin, In a gel-electrophoretic analysis, the ASOR-PL conjugate produce d a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR-PL conjugate/pCMV beta-gal plasmid). The gene-transfer efficiency of the ASOR- PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein r eceptor and NIH 3T3 cells that do not. The expression was assayed by 5-brom o-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chloro phenol Red beta-D-galactopyranoside, When an expression vector for the tumo ur-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transf ected into HepG2 cells, the exogenously provided p53 gene was detected in t he HepG2 cells by PCR, To improve the efficiency of DNA delivery and expres sion of the therapeutic proteins poloxamer 407, a fusogenic peptide, influe nza-virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of beta-galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, wherea s the fusogenic peptide HA2 and chloroquine had no effects, When HepG2 cell s were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR, The curr ent findings open the possibility that a receptor-mediated gene-delivery sy stem for hepatic gene therapy using ASOR-PL conjugate in combination with p oloxamer 407 may be developed in the future.