Ornithine transcarbamylase (OTC) is a mitochondrial enzyme of the urea cycle, encoded on the X chromosome, whose inherited deficiency leads to severe, often lethal, ammonia intoxication in affected humans. The proposed studies are directed 1) to understanding the system of mitochondrial compartmentation, whereby mitochondrial protein precursors, encoded in the nucleus and synthesized on cytoplasmic polyribosomes, are posttranslationally recognized by mitochondria, translocated across one or both membranes, and proteolytically processed to their active forms; and 2) to developing gene therapy for hepatic enzyme deficiencies. In both areas of investigation the experimental approaches taken will involve manipulation of the cloned biologically active cDNA sequence encoding human OTC. The studies of mitochondrial import are aimed at: definition of the sequences present in the cytoplasmically-synthesized precursor of OTC that are required for its import; study of putative cytoplasmic factors and outer membrane receptor molecules involved with recognition of the OTC precursor by mitochondria; and clarification of whether one or two NH2-terminal proteolytic processing steps are required to produce the mature OTC subunit. The coding sequence of the NH2-terminal leader portion, programmed for expression in eukaryotic cells, will be joined with that of a cytoplasmic enzyme to determine whether the leader alone is sufficient to direct mitochondrial import. The coding sequence of the entire OTC precursor, programmed for expression, will be subjected to deletion and point mutational alteration, and the effects upon import measured. The precursor coding sequence will also be programmed for high level expression in both eukaryotic and prokaryotic cells, enabling isolation of the precursor for use as a substrate in binding and proteolysis studies. Gene replacement experiments will aim to provide sufficient additional hepatic OTC activity, expressed from an introduced sequence, to permit correction of deficient nitrogen catabolism in OTC-deficient mice. The OTC cDNA sequence will be programmed for expression in a cloned retroviral genome that will be packaged into retroviral particles. The particles will be used to infect OTC-deficient mouse embryos and the liver of these animals analyzed for presence of the introduced sequence, and for enzymatically active human OTC.
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