Formulation and in vivo analysis of a low-molecular-weight liver -targeted nonviral gene delivery system.

Abstract

Receptor mediated nonviral gene delivery systems incorporate ligands to target cell surface receptors. These ligands enhance gene delivery by binding to a specific receptor that internalizes the DNA carrier complex. In order to achieve specific targeting of DNA to liver hepatocytes the delivery system must condense the plasmid DNA into small particles, bind to the asialoglycoprotein receptor on hepatocytes, shield the positive charge of the DNA condensate, and protect the DNA from enzymatic destruction. A multi-component DNA delivery system was developed utilizing a combination of low molecular weight peptides optimized for use <italic>in vivo</italic>. Asialoglycoprotein receptor targeting was achieved by the conjugation of an N-linked triantennary oligosaccharide to a DNA condensing peptide with 18 lysine residues. The positive charge of the DNA condensates was shielded by the incorporation of polyethylene glycol (PEG) into the DNA condensate. DNA condensates were stabilized and protected from metabolism by glutaraldehyde cross-linking which reduces the dissociation of the peptide from the DNA. Plasmid DNA condensates were formed with different combinations of the triantennary glycopeptide (Tri-CWK₁₈) and the polyethylene glycol peptide (PEG-CWK₁₈) and stabilized by cross-linking with glutaraldehyde. <super> 125</super>I DNA condensates were used to determine the effect of formulation on the pharmacokinetics, metabolism, biodistribution, cell type targeting, and gene expression in mice. Optimal targeting to hepatocytes required the combined use of Tri-CWK₁₈ and PEG-CWK₁₈ to mediate specific recognition by the asialoglycoprotein receptor and reduce non-specific uptake by Kupffer cells. Glutaraldehyde cross-linking of DNA condensates reduced the liver elimination rate from a half-life of 0.8 hrs to 3.6 hrs. An optimized formulation targeted 60% of the dose to the liver with 80% of the liver targeted DNA localized to hepatocytes. This formulation also produced higher levels of human alpha1-antitrypsin in mouse serum over ten days compared to control formulations. The results demonstrate the application of formulation optimization to improve the targeting selectivity and gene expression of a peptide DNA delivery system.