Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro.
We have developed a system for targeting foreign DNA to hepatocytes in vitro using a soluble DNA carrier that takes advantage of receptor-mediated endocytosis to achieve internalization. The idea is based on the fact that hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo)glycoproteins. To create a targetable carrier system that could bind DNA in a nondeforming manner, we used poly(L-lysine) to bind DNA in a strong but noncovalent interaction. An asialoglycoprotein, asialoorosomucoid (AsOR), was chemically coupled to poly(L-lysine) to form an asialoorosomucoid-poly(L-lysine) conjugate. Various proportions of conjugate to DNA were tested to determine conditions that maximized DNA content in a soluble complex and that limited solubility of complexes. To test the targetable gene delivery system, AsOR-poly(L-lysine) conjugate was complexed to the plasmid pSV2 CAT containing the gene for chloramphenicol acetyltransferase (CAT) driven by an SV-40 promoter. We tested this complex using a model system consisting of human hepatoma cell line Hep G2 [asialoglycoprotein receptor (+)], hepatoma SK-Hep 1, IMR-90 fibroblasts, and uterine smooth muscle [receptor (-)] cells. Each cell line was incubated with 0.2 micron filtered AsOR-poly(L-lysine)-DNA complex or controls consisting of DNA plus AsOR, DNA plus poly(L-lysine), or DNA alone. Cells were assayed for the presence of CAT activity as a measure of gene transformation. SK-Hep 1, IMR-90, and smooth muscle [receptor (-)] cells produced no detectable acetylated chloramphenicol derivatives under any of these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Medicine, University of Connecticut School of Medicine, Farmington 06032.
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D000123:Acetyltransferases / Q000235:genetics*; D001212:Asialoglycoproteins; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase; D005260:Female; D005796:Genes; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D009130:Muscle, Smooth / Q000378:metabolism; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D010957:Plasmids; D011107:Polylysine / Q000378:metabolism; D014162:Transfection
D001212:Asialoglycoproteins; D009961:Orosomucoid; C017030:asialoorosomucoid; D011107:Polylysine; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase