Chronic viral hepatitis is a major clinical problem, with over half a billion persons infected worldwide. Current therapies, principally treatment with recombinant IFN-alpha protein, have limited benefit. Recent studies suggest that gene-based expression of IFN-alpha is a possible therapeutic alternative that may improve the effectiveness of treatment. Gene delivery to the liver and consequent IFN-alpha expression therein, has the potential to concentrate the protein at the target organ and provide more continuous exposure to the therapeutic agent. Other potential gene and nucleic acid therapeutics for viral hepatitis are also being investigated. Key to the deployment of these future therapies is a suitable method of gene delivery. Although recombinant viral vector systems, such as adenovirus, are currently the most effective means of gene delivery to the liver, their use presents many concerns. These include immune and inflammatory reactions to the viral vector and possible adverse interactions between the recombinant virus and the pre-existing viral infection. Non-viral gene delivery systems would be a preferred treatment modality. The efficiency of current non-viral systems is not adequate for systemically administered liver gene therapy. However, recent use of membrane permeabilisation techniques has shown that high efficiency non-viral gene transfer agents are possible. The future coupling of these improved delivery systems with gene- or nucleic acid-based therapeutics currently in development holds out great promise for new generations of antihepatitis therapies.

In vitro assays have demonstrated the capability of poly-L-lysine to protect plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysine-DNA polyplexes after intravenous injection into mice. Polyplexes consisted of 32P-radiolabeled plasmid DNA complexed with poly-L-lysine at specified charge ratios. Variations in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro studies, the systemically administered polyplexes exhibited marked DNA degradation in the vascular compartment within 5 min. Substitution of poly-L-lysine epsilon-amino sites with polyethylene glycol or hydrocarbon chains resulted in faster degradation even when complexed at higher charge (+/-) ratios. Use of excess cationic charge in the polyplexes (+/- 2.5) diminished degradation rates only slightly. An analysis was made of the strength of the poly-L-lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes with the strongest binding between conjugate and DNA in the competition assay were also the most stable in blood. However, tighter binding was not enough to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate with improved in vivo transfection efficiency.

Polycations have been used for gene delivery in vitro quite successfully, however, in vivo applications suffered from serum effects that lower the overall gene drug efficiency. PEG polymers have been used extensively to minimize serum effects and create "stealth liposomes", biocompatible materials, and proteins with extended circulation. Here, we report our efforts towards creating "stealth polyplexes". A comb-type polycation, poly-L-lysine-graft-PEG copolymers were successfully prepared by ring opening of PEG-epoxide with E-amino lysine groups of linear poly-L-lysine. The ratios of PEG-epoxide to poly-L-lysine, PEG-epoxide size (2K, 3K, and 5B), and poly-L-lysine size (10K, 26K, and 38K) were varied. Copolymers with as little as 2% grafted PEG chains sterically stabilized DNA/copolymer complexes (polyplexes) even at charge neutrality. These polyplexes, formed with copolymers with various size of PEG chains grafted on various lenghths of poly-L-lysine backbone, remained relatively small, approximately 100 nm in saline With higher degree of grafting, the binding was significantly diminished. In addition, the morphology of polyplexes changed from thoroidal to more elongated, worm-like forms. Some globular structures were detected in cases of a lower degree of grafting. Finally, DNA release form polyplexes when exposed to negatively charged macromolecules, poly-L-aspartic acid sodium salt, is very structure dependant. Enhanced levels of luciferase expression observed with PLL-PEC polyplexes versus either free DNA or PLL polyplexes are encouraging and warrant further optimization of the polymeric gene delivery system.