Site-specific correction of defective genes by homologous recombination has been achieved at only very low frequencies in the treatment of inherited metabolic diseases by gene therapy. Recently, a synthetic RNA/DNA hybrid duplex, oligonucleotide designed to align in perfect register with the homologous genomic sequence except for a single base mismatch was show to promote targeted single nucleotide (nt) conversion in genomic DNA in rat 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 t his research project is to evaluate the utility of these molecules in correcting the single nt mutations associated with hemophilia. This objective tests our hypothesis that gene correction in effected hepatocytes will improve the phenotype associated with the disease. The first specific aim is to optimize (1) in vitro our non-viral asialoglycoprotein receptor hepatocyte- specific delivery systems, and (ii) chimeric RNA/DNA oligo oligonucleotide design for maximal conversion of the G to A transition at nt 1477 in the hemophilia B factor IX gene expressed in the Chapel Hill strain of dogs. The second specific aim is to evaluate the capacity of these molecules/delivery systems to promote targeted single nt conversion to correct the G to A transition in the canine factor IX in vivo. The non-viral delivery systems and chimeric oligonucleotides identified in Specific Aim 1 will be utilized. The relevant metabolic parameters will be monitored to quantitate the therapeutic effect of in situ genomic correction. Optimization of the dosing regimen, as well as the delivery vehicle and route of administration will be established. The third specific aim is to evaluate the potential of this technology in altering the genomic factor VII gene to produce the optimized factor VIIa mutations devised in Project 2. The initial work will be performed in vitro using cultured hepatocytes to optimize the delivery and design of the chimeric oligonucleotides. The selected factor VIIa mutation will then be generated in vivo and evaluated in collected hemophilia A phenotype in a factor VIII deficient mouse model. The relevant metabolic parameters will be monitored to quantitate the therapeutic effect of in situ genomic correction. Optimization of the dosing regimen, as well as the delivery vehicle and route of administration will be established. The long term goal of this research proposal is to: (i) to optimize non-viral delivery systems and oligonucleotide design that will promote the utility of RNA/DNA oligonucleotides for correcting single nt mutations associated with hemophilia; (ii) elucidate the optimal parameters for in vivo therapeutic correction of single nt mutations using this technology in the Chapel Hill strain of hemophilia B dogs, and (iii) evaluate the use of this technology for creating factor VIIa variants in vivo and there therapeutic benefit. The comparison of the in vitro and in vivo correction results will establish the feasibility for in vivo gene therapy approach using this technology for treatment of hemophilia.
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