Biochemical and Functional Characterization of DNA Complexes Capable of Targeting Genes to Hepatocytes via the Asialoglycoprotein Receptor

Abstract

Functional genes can be introduced into mammalian cells in vitro by a variety of physical methods, including direct microinjection, electroporation, and co-precipitation with calcium phosphate. Most of these techniques, however, are impractical for delivering genes into tissues of intact animals. In contrast, receptor-mediated gene transfer has been shown to successfully introduce DNA into suitable recipient cells, both in vitro and in vivo (1-12). This procedure involves the formation of a complex between DNA and a polycation (such as poly-L-lysine), which bears a covalently linked ligand moiety specific for a given receptor on the surface of cells in the target tissue. The gene is internalized by the tissue, transported to the nucleus, and expressed in the cell for varying lengths of time (1,3,6,11). The overall level of expression of the transgene in the target tissue is dependent on several factors, such as the stability of the DNA/ligand⅐poly-L-lysine complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-DNA/ligand interaction, endocytosis of the DNA complex and the efficiency of gene transcription in the nucleus of the target cells.DNA in the nucleus of a higher eukaryote is intimately associated with basic nuclear proteins rich in lysine (i.e. histones) or arginine (i.e. protamines). The interaction of DNA with these basic proteins is responsible for the control of the condensation process that occurs upon chromosome formation during metaphase and is thought to play a role in the regulation of gene expression. DNA condensation, which occurs naturally in viruses, bacteria, and eukaryote nuclei, has been extremely difficult to reproduce in the laboratory (13,14). Due to the high negative charge of the DNA phosphate backbone, an increase in the degree of charge neutralization of the DNA theoretically results in extensive condensation and the separation of the DNA phase in the form of insoluble compact structures (15, 16). We have found, however, that the structure and stoichiometry of DNA⅐polycation complexes in solution can be manipulated by means of the process by which DNA⅐cationic polypeptide complexes are formed.Specific complexes of DNA (⌿-DNA) are formed with cationic homo-polypeptides (poly-L-lysine, poly-L-arginine, or poly-L-ornithine) after "annealing" both components in a step-down dialysis from NaCl concentrations of 3 to 0.010 M (11, 16). In contrast, direct addition of basic polypeptides to DNA at physiological salt concentrations results in reversible molecular aggregation and the formation of precipitates (7,17,18). Shapiro et al. (16) elucidated changes in DNA secondary structure in DNA⅐poly-L-lysine complexes prepared by directly mixing poly-L-lysine and DNA. The physical properties of the resulting soluble complexes were investigated by circular dichroism (CD) and optical rotatory dispersion. A change in the magnitude of the molar residue rotation was found, with a characteristic red shift and a strong negative rotatory transition center...

Stable polyplexes based on arginine-containing oligopeptides for in vivo gene delivery

“…Intriguingly, the size of peptide-derived polyplexes seems to be less sensitive toward the condensation conditions (eg DNA concentration, buffer, mixing protocol) than polymer-derived polyplexes. 11,34 At low charge ratios, polyplexes consisted of multiple small condensation nuclei on an uncondensed DNA strand, which transgressed into spherical particles at higher charge ratio, suggesting that unlike cationic polymers, 11,34 oligopeptides initiate DNA compaction at distinct condensation nuclei within the DNA strand.…”

Hepatocyte-targeted in vivo gene expression by intravenous injection of plasmid DNA complexed with synthetic multi-functional gene delivery system

“…The asialoglycoprotein receptor-mediated gene targeting hepatocytes using galactosylated polymers has been investigated as a promising approach to introduce genetic materials into hepatocytes in vivo. [26][27][28][29] To achieve hepatocyte-targeted delivery of DNA, however, not only the specific ligands (galactose) but also the overall physicochemical properties of DNA/carrier complexes should be optimized. In previous studies, 30,31 we have investigated the in vivo pharmacokinetics of DNA/galactosylated poly(L-lysine) (Gal-PLL) complexes and succeeded in delivering them to hepatocytes by controlling the physicochemical properties of the Gal-PLL and DNA/Gal-PLL complexes.…”

Supramolecular structure and nuclear targeting efficiency determine the enhancement of transfection by modified polylysines

“…8 Condensation of plasmid DNA by pLy appears to correlate with improved gene transfer efficiency. [9][10][11] Recently, we cross-linked peptides constituting the nuclear localization signal (NLS) of the simian virus SV40 large tumor antigen (T-ag) to pLy, in addition to internalisable ligands, and showed enhancement of gene transfer. 12 In this study, we examine the effect of complexation of DNA with pLy itself or with pLy to which NLS or mutant NLS peptides are cross-linked 12 at a range of different lysine/nucleotide (Ly/Nu) complexation ratios.…”