In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver.
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 Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
S L
SL
Ginn
S C
SC
Cunningham
M
M
Zheng
A
A
Spinoulas
K H
KH
Carpenter
I E
IE
Alexander
D000256:Adenoviridae; D000818:Animals; D015153:Blotting, Western; D004195:Disease Models, Animal; D004790:Enzyme Induction / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009954:Ornithine Carbamoyltransferase / Q000096:biosynthesis* / Q000235:genetics*; D020163:Ornithine Carbamoyltransferase Deficiency Disease / Q000628:therapy; D009963:Orotic Acid / Q000652:urine
D009963:Orotic Acid; D009954:Ornithine Carbamoyltransferase