Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor

Abstract

The serpin enzyme complex receptor (SECR) expressed on hepatocytes binds to a conserved sequence in α 1 -antitrypsin (α 1 -AT) and other serpins. A molecular conjugate consisting of a synthetic peptide (C1315) based on the SECR binding motif of human α 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 Rep 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 dosedependent manner. Complexes of DNA condensed with polyly-sine or LC-sulfo-Nsuccinimidyl-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% ofHep G2 (low) cells, and when the lac Z gene was transfected, only these cells expressed β-galactosidase. SECRmediated gene transfer gives efficient, specific uptake and high-level expression of three reporter genes, and the system merits further study for gene therapy.

Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells

“…9 Complexes that contained conjugates with 7.8% of the lysines substituted with SPDP and approximately 40 C1315 ligands per DNA plasmid molecule, gave peak luciferase expression about 30-50% of peak luciferase activity obtained using lipofectin. Modulation of the protein portion of the complex while keeping the DNA portion constant produced complexes which gave peak luciferase expression eight-to 20-fold higher than expression from lipofectin-complexed plasmids.…”

“…[1][2][3][4][5][6][7][8][9] A carrier, usually composed of a polycationic protein chemically conjugated to a receptor-specific ligand, is used to condense DNA and present it to the target cell. Careful manipulation of the concentration of NaCl can produce complexes, containing a single plasmid DNA molecule, ranging in size from 10-25 nm in diameter.…”

“…Careful manipulation of the concentration of NaCl can produce complexes, containing a single plasmid DNA molecule, ranging in size from 10-25 nm in diameter. 2,3,5,9 Previously, we demonstrated that such DNA complexes could be targeted to hepatoma cells bearing the serpin enzyme complex receptor (SEC-R), using a synthetic peptide ligand (peptide C1315) based in sequence on amino acids 346-374 of human ␣ 1 -antitrypsin. 9 Gene transfer via this system resulted in expression of reporter genes for 6 to 8 days.…”

“…2,3,5,9 Previously, we demonstrated that such DNA complexes could be targeted to hepatoma cells bearing the serpin enzyme complex receptor (SEC-R), using a synthetic peptide ligand (peptide C1315) based in sequence on amino acids 346-374 of human ␣ 1 -antitrypsin. 9 Gene transfer via this system resulted in expression of reporter genes for 6 to 8 days. 9 The SEC-R is expressed on such cell types as mononuclear phagocytes, neutrophils, hepatocytes, the myeloid cell lines U937 and HL60, human intestinal epithelial cell line Caco2, mouse fibroblast L cells, rat neu-ronal cell line PC12 and human glial cell line U373MG.…”

“…9 Gene transfer via this system resulted in expression of reporter genes for 6 to 8 days. 9 The SEC-R is expressed on such cell types as mononuclear phagocytes, neutrophils, hepatocytes, the myeloid cell lines U937 and HL60, human intestinal epithelial cell line Caco2, mouse fibroblast L cells, rat neu-ronal cell line PC12 and human glial cell line U373MG. [10][11][12][13][14] Some of these cell types are good targets for therapeutic gene transfer.…”