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.
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Acknowledgements
We thank Professor James M Wilson (Department of Pathology and Laboratory Medicine, University of Pennsylvania, USA) for kindly providing the AAV8 serotype plasmid p5E18-VD2/8, Professor R Jude Samulski (Gene Therapy Center, The University of North Carolina at Chapel Hill, USA) for providing the packaging plasmid pXX6 and Professor Matthew During (Department of Medical and Health Science, University of Auckland, New Zealand) for providing the pAM2 construct. We also thank Professor Nick Hoogenraad (Department of Biochemistry and Molecular Sciences, La Trobe University, Victoria, Australia) for providing the human OTC antibody, Allison Dane for help with injections and Grant Logan for critical reading of the manuscript. This work was supported by a project grant from the NHMRC (423400). Samantha L Ginn is the recipient of a fellowship honouring the memory of Noel Dowling.
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Ginn, S., Cunningham, S., Zheng, M. et al. In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver. Gene Ther 16, 820–823 (2009). https://doi.org/10.1038/gt.2009.38
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DOI: https://doi.org/10.1038/gt.2009.38