Differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors

Mouse models of ornithine transcarbamylase (OTC) deficiency are being used to test the efficacy of viral vectors as possible vehicles for gene therapy . However, it has been demonstrated that virus containing the human OTC cDN A failed to express functional OTC enzyme in the recipient animals. Because functional OTC is assembled as a homotrimer in the mitochondria, there are at least two possible explanations for these results. Either endogenous mu tant protein coassembles with the human OTC and has a "dominant-negative ef fect," or the human version of the protein is not appropriately imported or processed in the mouse mitochondria. To test the importance of processing, which in rodents is thought to depend on the leader peptide, adenoviral ve ctors containing chimeric OTC cDNAs were prepared. These vectors were evalu ated in the OTC-deficient sparse fur mouse models. Although comparable leve ls of transgene expression were observed in all groups of mice, the only mi ce that had high levels of OTC activity and mitochondrial OTC immunoreactiv ity were those mice injected with the vectors containing the mouse leader p eptide (mouse OTC and a mouse-human chimera of OTC). To address possible do minant-negative effects, adenoviruses containing mutant human or mouse OTC cDNAs were prepared and evaluated in cell lines or normal C3H mice, respect ively. No inhibition of normal OTC activity was observed in either model sy stem. Together, these studies provide no evidence of a dominant-negative ef fect and suggest that the human and rodent enzymes responsible for transpor ting of OTC and possibly other mitochondrial proteins have different specif icity.