Site-specific correction of defective genes by homologous recombination has been achieved at only very low frequencies in gene therapy for inherited metabolic diseases. Recently, a synthetic RNA/DNA hybrid duplex oligonucleotide designed to align in perfect register with the homologous genomic sequence except for a single mismatch was shown to promote targeted single nucleotide (nt) conversion in genomic DNA inr at hepatocytes. The process exploits the cell's efficient endogenous DNA mismatch repair pathways, thereby making it a novel approach to gene therapy. The main objective of this research project is to evaluate the utility of these molecules in correcting in correcting single nt mutations in liver genomic project is to evaluate the utility of these molecules in correcting single nt mutations in liver genomic DNA responsible for metabolic diseases associated with mental retardation. This objective tests our hypothesis that gene correction in the liver will improve the phenotypic changes associated with the diseases. The first specific aim is to evaluate the capacity of these molecules to promote targeted single nt conversion to correct the ornithine transcarbamylase and phenylalanine hydroxylase mutations in hepatocytes isolated from their respective mouse models, sparse fur/ash and Pahenu2. In addition, we will target insertion of a single nt to correct the frameshift mutation in the beta- glucuronidase deficient will target insertion of a single nt to correct the frameshift mutation in the beta-glucuronidase deficient hepatocytes isolated from the gusmps murine model of Sly syndrome. Both non-viral liposomal and polycation delivery systems targeted to the unique hepatocyte asialoglycoprotein receptor will be utilized. The second specific aim is to then evaluate the correction of these three mutation in vivo in their respective mouse models. The relevant metabolic parameters will be monitored to quantitative the therapeutic effect of in situ genomic correction. Replicative stability of the corrected mutations will be evaluated using surgical resection of the liver to induce replication of the remaining hepatocytes. Optimization of the dosing regimen, as well as the delivery vehicle and route to administration will be established. The third specific aim is to evaluate the potential use of these molecules in promoting targeted single nucleotide alterations in genomic DNA of cultured skin fibroblasts and CD34+-enriched hematopoietic stem cells. Receptor-specific delivery systems will be developed with appropriate ligands to optimize transfection of the DNA/DNA oligonucleotides to these cells. The long term goal of this research proposal is to: (i) utilize hepatocyte specific delivery systems in vitro and in vivo, elucidate the optimal parameters for therapeutic correction of single nucleotide mutations using this technology in liver and (ii) to develop additional non-viral delivery systems that will promote the effectiveness of RNA/DNA oligonucleotides for correcting single nucleotide mutations in a variety of cells.
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