Given its central role in metabolism and in the secretion of circulating proteins, a large number of genetic or acquired disorders could be corrected by liver-directed gene therapy. However, a safe and effective delivery method for transferring nucleic acids molecules to hepatocytes remains to be perfected. From the Phase I studies, a synthetic peptide that enabled efficient hepatocyte targeted delivery in mice of a variety of proteins, and particles such as liposomes and most importantly, polycation-condensed DNA or siRNA particles was identified. Two properties are important for delivery of nucleic acids complexes: stabilization of nucleic acid particles using glutaraldehyde to cross-link polycations to themselves (termed caging) and PEGylation which shields surface charges. However, since particles are taken up by endocytosis, they must be released from endosome to gain entry to cytoplasm. To facilitate endosomal release, a new endosomolytic polycation developed by Mirus, termed pBAVE, was incorporated in particle formation. We were pleasantly surprised by the ability of these particles to enable functional delivery of siRNA (21-23 bp) and short hairpin RNA (shRNA) expression cassette (a 443 bp PCR construct). This breakthrough was accomplished by the combined effects of reducing the level of glutaraldehyde cross-linking and administering a separate pBAVE polymer injection for additional endosomal releasing power. However, attaining functional delivery with reduced cross-linking was at the expense of decreased transfection efficiency to hepatocytes due to a reduction in particle stability and delivery efficiency. From our preliminary studies, 40-50% gene knockdown (up to 60-70% in selected animals) can be achieved. Although highly significant, to realize therapeutic benefits, an optimized delivery method will be necessary to achieve 70-80% or higher of knockdown consistently in hepatocytes. The current project, taking advantage of our highly successful preliminary results in obtaining functional RNAi effects in vivo, will focus on optimizing delivery of siRNA and shRNA expression cassette to hepatocytes so that only a single injection is needed to achieve this goal. The first three Specific Aims will optimize complex formation by 1) using Mirus' proprietary acid labile technology to develop reversible cross-linkers that will enhance endosomal release, 2) develop reversible PEGylation molecules for surface charge shielding so that endosomolytic function of pBAVE in caged complex can be reactivated, and 3) incorporate these reagents to form hepatocyte targeted transfection competent complexes.
Specific Aim 4 will evaluate the ability of these complexes to enable therapeutic relevant levels of RNAi- mediated knockdown of target gene in normal and diseased mice. Given the immense commercial interest of delivering functional nucleic acids constructs, including RNAi molecules, to normal and diseased liver, successful development of these technologies may potentially revolutionize therapeutic strategies used to treat metabolic diseases, and other disorders specific to hepatocytes including cancer, hepatitis and cirrhosis. The goal of this study is to develop a safe and effective delivery system as a gene therapy tool to transfer nucleic acid molecules to hepatocytes in the liver. A large number of hereditary and acquired diseases can be treated by liver based gene therapy. Successful development of this technology will have great values both as a research tool and as a therapeutic delivery system. ? ? ? ? ?
