Improvement of receptor-mediated gene delivery to HepG2 cells using an amphiphilic gelling agent.
Gene transfer was performed using asialo-oroso-mucoid-polylysine (ASOR-PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel-electrophoretic analysis, the ASOR-PL conjugate produced 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 receptor and NIH 3T3 cells that do not. The expression was assayed by 5-bromo-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chlorophenol Red beta-D-galactopyranoside. When an expression vector for the tumour-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza-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, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor-mediated gene-delivery system for hepatic gene therapy using ASOR-PL conjugate in combination with poloxamer 407 may be developed in the future.
Cho
C W
CW
Cellular Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Taejon 305-600, South Korea.
Cho
Y S
YS
Lee
H K
HK
Yeom
Y I
YI
Park
S N
SN
Yoon
D Y
DY
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D005697:Galactosides / Q000737:chemistry; D018014:Gene Transfer Techniques; D006801:Humans; D007211:Indoles / Q000737:chemistry; D008113:Liver Neoplasms / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010637:Phenolsulfonphthalein / Q000031:analogs & derivatives / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D020442:Poloxamer / Q000737:chemistry / Q000494:pharmacology; D011107:Polylysine / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D005697:Galactosides; D007211:Indoles; D009961:Orosomucoid; D011956:Receptors, Cell Surface; D016159:Tumor Suppressor Protein p53; C017030:asialoorosomucoid; D020442:Poloxamer; D011107:Polylysine; C007016:chlorophenol red; D010637:Phenolsulfonphthalein; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside