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

Gene Ther. 2009 Jun;16(6):820-3. doi: 10.1038/gt.2009.38. Epub 2009 Apr 9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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*
  • Ornithine Carbamoyltransferase / genetics*
  • Ornithine Carbamoyltransferase Deficiency Disease / therapy
  • Orotic Acid / urine

Substances

  • Orotic Acid
  • Ornithine Carbamoyltransferase