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.
Ginn
S L
SL
Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
Cunningham
S C
SC
Zheng
M
M
Spinoulas
A
A
Carpenter
K H
KH
Alexander
I E
IE
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