Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro

Abstract

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)

Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes

“…Localized hepatic gene expression has also been achieved using direct hepatic DNA injection. 16 The feasibility of targeted gene delivery to hepatocytes through the ASGPr has been amply demonstrated by the work of Wu et al [17][18][19][20][21][22] In this system, DNA molecules are complexed with a stretch of polylysine (PLL), which is coupled to the asialoglycoprotein moiety. PLL residues are intended to interact with DNA and the asialoglycoprotein behaves as a homing device to target hepatocytes.…”

“…The resulting level is relatively low and restricted to the injection site, and sustained expression often implies previous partial hepatectomy. 19,21,32,33 Protamine sulfate, as a component of non-viral vectors, may promote the delivery of DNA from the cytoplasm into the nucleus. It has been proposed that this peptide increases lipid-mediated gene transfection by condensing DNA into a compact structure, which promotes cellular entry and stability of DNA.…”

Vector Gauge Boson Dark Matter for the SU(N) Gauge Group Model

“…The goal of the present paper is to investigate the possibility that be a stable neutral vector Cboson of mass 900 GeV (as predicted by the Wu mechanisms for mass generation of gauge fields: [46], [47], [48], [49], [50], [51]), proposed recently by the author [52], explains the existence of dark matter. According to the SM W, Z-bosons normally acquire their masses through their coupling with the SM Higgs boson, mass 125 GeV [53], [54], [55], [56], [57].…”

Inhibition of hepatitis B virus replication by targeted pretreatment of complexed antisense DNAin vitro

“…coli and isolated as described previously. 5 Preparation of Targetable Oligo DNA Complexes. A DNA Nonspecific, inhibitory effects of complexed antisense were examined using Huh7, 1.0 1 10 5 cells/dish incubated for 24 carrier targetable to hepatocytes was prepared by coupling asialoorosomucoid (AsOR) to poly L-lysine (molecular weight, hours with 25 mmol/L complexed antisense DNA or controls as described above, but in medium without fetal calf serum.…”

“…5 The titration point (representing the minimum proportion of conjugate required to gland) as described by the manufacturer. Using this protocol, contamination by fetal calf serum from the medium was decompletely retard the antisense DNA) occurred at a conjugate to antisense DNA molar ratio of approximately 1.5:1 (in termined to be negligible.…”