The long term goal of this proposal is to develop strategies for gene therapy of inborn errors in metabolism in the liver. Correction of a genetic defect in the liver using gene therapy requires (a) efficient transfer of the correct gene in the parenchymal liver cells, (b)long term gene expression at adequate levels in the correct liver zone, (c) regulation of gene expression and (d) safety of the methods used. Despite the progress in this field of research, a number of critical issues to increase not only gene transfer but also gene expression must be resolved. In this proposal we will focus on how replication incompetent retroviruses can be used as vehicles to introduce and express the human OTC gene into the liver of the spf-ash mice carrying a genetic defect for the urea cycle enzyme ornithine transcarbamylase. The ability of the developed strategy to treat a genetic defect will be determined in the deceased mice which have their livers reconstituted to express the healthy gene.
The specific aims are the following: (a) role of the age of the spf-ash mouse on the efficiency of gene transfer in the liver via retroviruses. The expression of genes for the viral receptors and the age (fetal, neonate and adult) of the mouse will be studied relative to the efficiency of gene transfer in the liver. (b) role of cis DNA sequences on the stable depression of healthy genes transferred into the diseased mouse liver. The expression of proviral genes in the liver of the spf-ash mice will be studied relative to the presence of liver specific promoter/enhancer sequences in the context of the provirus. (c) development of methods to increase the efficiency of gene transfer and expression in the diseased mouse liver. The ability of the liver to respond to toxic injury will be used as a mediator to increase gene transfer and expression of the OTC gene in the spf-ash mouse liver. More specifically, the following experimental plan will be followed: (i) constructing a replication incompetent retrovirus carrying both, the cDNA for human OTC and a gene conferring resistance to a hepatotoxic drug, G418, (ii) transferring via infection the two genes to a fraction of hepatocytes in live animals, (iii) administer G418 to destroy the hepatocytes that do not express the drug resistance gene and (iv) let the resistant hepatocytes repopulate the liver and express the OTC gene. The studies in this proposal will be a significant contribution for the widespread clinical application of gene therapy.
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