In vivo targeted gene transfer into liver cells mediated by a novel galactosyl-D-lysine/D-serine copolymer

Abstract

A novel synthetic polypeptide designed as a DNA bindingmice via the tail vein, high levels of luciferase and molecule for liver-specific, receptor-mediated, gene transenhanced green fluorescent protein, which were encoded fer was used to selectively introduce reporter genes into by the reporter genes, were observed in liver. In contrast, liver cells in the form of plasmid DNA-ligand complexes.luciferase activity in kidney, spleen, lung and heart was The polypeptide was a D-lysine/D-serine copolymer negligible. The high levels of gene expression obtained (Lys/Ser = 33/36 or 53/60) modified with a polyethylene with DNA/galactosyl-PLSP complexes were achieved withglycol 5000 at the carboxyl-terminus (PLSP). In addition, out partial hepatectomy or administration of lysosomothe lysine ⑀-amino groups were covalently bound to galactrophic agents. Thus, the synthetic Lys/Ser copolymer used tose (galactosyl-PLSP), a ligand for the asialoglycoprotein in the present study appears to be a promising new tool receptor on hepatocytes. Solutions of DNA-ligand complex which enhances the efficacy of receptor-mediated gene were prepared by adding solutions of DNA and galactosyltransfer into hepatocytes and which may provide another PLSP in a mixing ratio of DNA:galactosyl-PLSP = 1:3 step toward the clinical practice of organ-specific gene (w/w). Following injection of the DNA-ligand complex into therapy. Keywords: gene transfer; asialoglycoprotein receptor; liver; D-lysine/D-serine copolymerBecause its malfunction leads to critical metabolic damage affecting the entire body, the liver is a principle target organ for gene therapy. The efficacy of the therapy will depend in large part on the ability to target specifically liver cells for corrective gene transfer. Receptor-mediated gene transfer is one promising approach to the problem, provided the relevant receptors are selectively expressed by hepatocytes. Asialoglycoprotein receptors on the plasma membranes of hepatocytes have been a focus of research interest for receptor-mediated targeting since Wu and Wu first introduced DNA into hepatocarcinoma cells 1 and rat liver 2 in the form of a complex with asialoglycoprotein and polylysine. Receptor-mediated gene transfer does not pose the risk of adverse effects carried by some other methods (eg viral infection derived from viral vectors and cytotoxic effects from liposomes); thus, it would seem to be an attractive method for gene transfer in vivo. However, this approach has not yet become practical for gene therapy, because the methods developed so far do not effectively mediate DNA incorporation into target cells, and the expression of genes that are introduced is only transient. In addition, it is clear that preparation of DNA-ligand complexes remains a significant technical obstacle, and improved methods will likely be required To date, a variety of DNA-ligand complexes have been developed. [3][4][5][6][7][8][9] Polycations and proteins that carry numerous positive charges electrostatically bind polyanionic DNA to form condensed ...

Liver cell-targeted delivery of therapeutic molecules

“…Thus, several galactose-modified delivery systems show greater transfection efficiency in the liver than in other organs (e.g. lung, kidney, heart and spleen) (Hisayasu et al, 1999;Nishikawa et al, 2000). However, these results do not mean greater transfection efficiency for hepatocytes than other hepatic cells.…”

Targeting of Synthetic Gene Delivery Systems

“…For targeting of poly-L-lysine DNA-complexes to the liver, asialoorosomucoid [61, 62] and galactose [63, 64] have been used as ligands. While uptake in hepatocytes has been demonstrated for such systems, some of the liver targeting may be caused by the tendency of the liver, and specifically cells of the reticuloendothelial system (Kupfer cells), to “filter” out particulate drug carriers.…”

Inducing neutrophil recruitment in the liver of ICAM-1-deficient mice using polyethyleneimine grafted with Pluronic P123 as an organ-specific carrier for transgenic ICAM-1

“…For instance, folate receptor, integrins, growth factor receptors, and other cell surface molecules have been successfully used as targets for non-viral gene delivery. [32][33][34][35][36] However, the use of such targeting strategy could induce secondary effects related to biological activity of the targeting moiety, which may counterbalance the beneficial effect of gene therapy. Moreover, a considerable increase in cost is associated with manufacturing of such targeted carriers.…”