Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes.
We have combined a receptor-mediated DNA delivery system with the endosomal lysis ability of adenovirus and shown that DNA can be delivered into primary hepatocytes, resulting in a high level of gene expression. When asialoorosomucoid conjugated with poly(L-lysine) was used to deliver the Escherichia coli beta-galactosidase gene into primary hepatocytes through binding with the hepatic asialoglycoprotein receptor, only a low level of beta-galactosidase was detectable, with less than 0.1% of the hepatocytes being transfected. This level of activity can be greatly enhanced by the cointernalization of the DNA.protein complex with a replication-defective adenovirus, resulting in 100% of the hepatocytes staining blue with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. Quantitative analysis of beta-galactosidase expression also showed a 1000-fold enhancement of activity. To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity. This method shows great promise for efficient gene delivery to the liver for correction of hepatic disorders.
Cristiano
R J
RJ
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Smith
L C
LC
Woo
S L
SL
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D001692:Biological Transport; D006528:Carcinoma, Hepatocellular; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage / Q000235:genetics*; D004337:Drug Carriers; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism / Q000502:physiology*; D008113:Liver Neoplasms; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D010661:Phenylketonurias / Q000235:genetics / Q000628:therapy*; D011971:Receptors, Immunologic / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D004337:Drug Carriers; D009961:Orosomucoid; D011971:Receptors, Immunologic; C017030:asialoorosomucoid; D004247:DNA; D010651:Phenylalanine Hydroxylase; D001616:beta-Galactosidase