Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor.
The serpin enzyme complex receptor (SECR) expressed on hepatocytes binds to a conserved sequence in alpha 1-antitrypain (alpha 1-AT) and other serpins. A molecular conjugate consisting of a synthetic peptide (C1315) based on the SECR binding motif of human alpha 1-AT covalently coupled to poly-L-lysine was used to introduce reporter genes into hepatoma cell lines in culture. This conjugate condensed DNA into spheroidal particles 18-25 nm in diameter. When transfected with the SECR-directed complex containing pGL3, Hep G2 cells that express the receptor, but not Hep G2 cells that do not, expressed a peak luciferase activity of 538,731 +/- 144,346 integrated light units/mg protein 4 days after transfection. Free peptide inhibited uptake and expression in a dose-dependent manner. Complexes of DNA condensed with polylysine or LC-sulfo-N-succinimidyl-3-(2-pyridyldithio)propionate-substituted polylysine were ineffective. Transfection with a plasmid encoding human factor IX produced expression in Hep G2 (high) and HuH7 cells that express SECR but not Hep G2 (low) cells that lack the receptor. Fluorescein-labeled C1315 peptide labeled 9-31% of Hep G2 (high), 10-14% of HuH7, and 0.6-3.4% of Hep G2 (low) cells, and when the lac Z gene was transfected, only these cells expressed beta-galactosidase. SECR-mediated gene transfer gives efficient, specific uptake and high-level expression of three reporter genes, and the system merits further study for gene therapy.
Ziady
A G
AG
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Perales
J C
JC
Ferkol
T
T
Gerken
T
T
Beegen
H
H
Perlmutter
D H
DH
Davis
P B
PB
D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D003587:Cytomegalovirus / Q000235:genetics; D005164:Factor IX / Q000235:genetics; D018014:Gene Transfer Techniques; D006801:Humans; D007763:Lac Operon; D008024:Ligands; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics; D010957:Plasmids / Q000648:ultrastructure; D011107:Polylysine / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C064184:serpin-enzyme complex receptor; D011107:Polylysine; D005164:Factor IX; D008156:Luciferases; D001616:beta-Galactosidase