Liver‐Directed Knockout and Transgene Delivery of Arginase‐1 in Mice

Arginase‐1 (Arg1) catalyzes the distal step in the liver‐based urea cycle that converts arginine to urea and ornithine. Genetic deficiency of this enzyme in humans, unlike proximal urea cycle disorders that lead to hyperammonemia and neonatal crisis, leads to neurological deficits (e.g. spastic diplegia) that manifest around 2–4 years of age and progress throughout childhood. We previously generated a tamoxifen‐inducible Arg1 deficient mouse model (Arg1‐Cre) that leads to a phenotype much more severe than in humans with lethality occurring ≈2 weeks after loss of enzyme function. The purpose of this study was to evaluate if liver‐selective Arg1 loss is sufficient to recapitulate the phenotype observed in global Arg1 knockout mice, as well as to gauge the effectiveness of a gene therapy protocol to rescue the phenotype. Liver‐selective Arg1 deletion was induced by using an adeno‐associated viral (AAV)‐thyroxine binding globulin (TBG) promoter‐Cre recombinase vector (1.5×10 11 genome copies (g.c.), intraperitoneally (i.p.) injected) administered to mice (n=6) with loxP sites flanking exons 7 and 8 of the Arg1 gene (Arg1 floxed mice; Arg1 fl/fl ). An AAV vector expressing an Arg1‐enhanced green fluorescent protein (Arg1‐eGFP) transgene from a strong promoter (hybrid cytomegalovirus enhancer/chicken β‐actin) was used in a gene therapy approach to “rescue” tamoxifen‐treated Arg1‐Cre mice. The results indicate that liver‐selective loss of Arg1 (>95 % deficient) leads to a phenotype resembling the whole body knockout of Arg1 with lethality around 2 weeks after Cre‐induced gene disruption in all mice. Delivery of Arg1‐eGFP AAV (n=10 male; 1.5×10 11 g.c. i.p. administered 2 weeks prior to tamoxifen treatment) rescues ≈ half of Arg1 global knockout mice (survival >4 months) but a significant proportion still succumb to the enzyme deficiency even though liver expression and enzyme activity of the fusion protein reach levels approaching that found in wild‐type animals. This raises a conundrum relating to liver‐specific expression of Arg1. On the one hand, loss of expression in this organ appears to be both necessary and sufficient to explain the lethal phenotype of the genetic disorder in mice. On the other hand, the gene therapy rescue studies suggest that loss of extra‐hepatic Arg1 expression factors into disease etiology. Further studies will be necessary to illuminate the detailed mechanisms for pathogenesis of Arg1‐deficiency. Support or Funding Information Canada Research Chairs Program