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- Title
In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver.
- Authors
Ginn, S. L.; Cunningham, S. C.; Zheng, M.; Spinoulas, A.; Carpenter, K. H.; Alexander, I. E.
- 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.Gene Therapy (2009) 16, 820–823; doi:10.1038/gt.2009.38; published online 9 April 2009
- Subjects
ORNITHINE carbamoyltransferase deficiency; TRANSGENES; GENETIC transformation; MUTAGENESIS; GENE therapy
- Publication
Gene Therapy, 2009, Vol 16, Issue 6, p820
- ISSN
0969-7128
- Publication type
Article
- DOI
10.1038/gt.2009.38