The process exploits the cell's efficient endogenous DNA replication and repair pathways to precisely correct the gene defect in situ. The main objective of this research project is to evaluate the utility of this non-viral gene therapy strategy in correcting inherited metabolic gene defects of liver ornithine transcarbamylase (OTC) both in utero and in the neonate. This objective tests our hypothesis that correction of the mutant gene in livers of affected sparse fur/ash (spf/ash) mice will improve the phenotype associated with the disease. The first specific aim is designed to evaluate SSO-mediated correction of the point mutation for OTC deficiency in adult and fetal hepatocytes isolated from the spfas"""""""" mouse, as well as Chinese hamster ovary (CHO) cells expressing a human R40H OTC mutant gene. A number of different SSO designs will be tested as well as non-viral polycation and nanocapsule delivery systems developed for use in vitro and in vivo. The second specific aim is designed to characterize the potential of SSOs for correcting the OTC deficiency of spf/ash mice by in utero and neonatal gene therapy. Genomic repair of the OTC point mutation in liver will be determined as well as the relevant metabolic parameters for OTC expression. The efficiency of in situ genomic correction will be correlated with changes in phenotype. Long-term studies will establish the stability of the corrected mutation in both quiescent and replicating hepatocytes after partial hepatectomy. In addition, we will optimize the dosing regimen, as well as the delivery vehicle, route and time of administration. Detailed short-and long-term toxicity/pathology profiles will be established to determine any potential adverse effects from either the delivery systems or oligonucleotides. These studies will provide novel and important new information on the potential problems and applications in using this non-viral strategy to genetically correct gene defects in utero. In addition, it will provide greater insight into the susceptibility of specific human mutations to site- specific gene repair. The long term goal of this research proposal is to develop, (i) optimal parameters for therapeutic correction of single base mutations in liver using non-viral strategies, and (ii) methods of neonatal and in utero gene correction for application to OTC and other inborn errors of metabolism in liver.
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