In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver.

Abstract

Mutant proteins have the potential to exert dominant-negative effects that might limit the therapeutic efficacy of their wild-type counterparts after gene transfer. For ornithine transcarbamylase (OTC) deficiency, in vitro studies have suggested the presence of dominant-negative effects, however, supporting in vivo studies have not been conducted. In this study, we exploited the capacity of recombinant adeno-associated virus (rAAV) 2/8 vectors to deliver transgenes to the mouse liver with high efficiency to determine whether expression of selected OTC mutant proteins exert inhibitory effects on endogenous wild-type OTC enzymatic activity. Using site-directed mutagenesis we constructed three OTC mutants with a theoretical or reported in vitro capacity to exert dominant-negative effects, and delivered these to the liver using rAAV2/8. Each mutation had been earlier identified in patients with OTC deficiency. Treated mice showed no increase in urinary orotic acid levels or reduction in OTC activity despite supra-physiological expression of the mutant proteins, consistent with an absence of dominant-negative effects. These data have important implications for the development of gene therapy strategies for OTC deficiency and validate a model system in which potential dominant-negative effects of specific mutations in prospective patients can be examined empirically before gene therapy.

• Authors: S L Ginn, S C Cunningham, M Zheng, A Spinoulas, K H Carpenter, I E Alexander
• Journal: Gene therapy (Gene Ther) Vol. 16 Issue 6 Pg. 820-3 (Jun 2009) ISSN: 1476-5462 [Electronic] England
• PMID: 19357713 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Orotic Acid
• Ornithine Carbamoyltransferase

Topics

• Adenoviridae
• Animals
• Blotting, Western
• Disease Models, Animal
• Enzyme Induction (genetics)
• Gene Expression (genetics)
• Gene Transfer Techniques
• Genetic Vectors
• Humans
• Liver (enzymology)
• Male
• Mice
• Mutagenesis, Site-Directed
• Mutation (genetics)
• Ornithine Carbamoyltransferase (biosynthesis, genetics)
• Ornithine Carbamoyltransferase Deficiency Disease (therapy)
• Orotic Acid (urine)