Minimal ureagenesis is necessary for survival in the murine model of hyperargininemia treated by AAV-based gene therapy

Abstract

Hyperammonemia is less severe in arginase 1 deficiency compared with other urea cycle defects. Affected patients manifest hyperargininemia and infrequent episodes of hyperammonemia. Patients typically suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, seizures, and intellectual disability; death is less common than with other urea cycle disorders. In a mouse model of arginase I deficiency, the onset of symptoms begins with weight loss and gait instability which progresses to development of tail tremor with seizure-like activity; death typically occurs at about two weeks of life. Adeno-associated viral vector gene replacement strategies result in long-term survival of mice with this disorder. With neonatal administration of vector, the viral copy number in the liver greatly declines with hepatocyte proliferation in the first 5 weeks of life. While the animals do survive, it is not known from a functional standpoint how well the urea cycle is functioning in the adult animals that receive adeno-associated virus. In these studies we administered [1-13C] acetate to both littermate controls and adeno-associated virus-treated arginase 1 knockout animals and examined flux through the urea cycle. Circulating ammonia levels were mildly elevated in treated animals. Arginine and glutamine also had perturbations. Assessment thirty minutes after acetate administration demonstrated that ureagenesis was present in the treated knockout liver at levels as low at 3.3% of control animals. These studies demonstrate that only minimal levels of hepatic arginase activity are necessary for survival and ureagenesis in arginase deficient mice and that this level of activity results in control of circulating ammonia. These results may have implications for potential therapy in humans with arginase deficiency.

Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells

“…Previously, we have shown in a murine model that with relatively low overall hepatic arginase activity establishing ureagenesis at 3.5–5% of normal led to long-term survival with controlled plasma arginine and ammonia; however these animals remained nitrogen vulnerable due to the low level of arginase expression. 7 LEAPR-corrected hepatocyte-like cells across all three lines demonstrated functionality of at least 40% compared with fetal liver; while the hEF1α promoter-based expression did decline with cellular differentiation, arginase expression in our studies remains well above the minimum threshold needed for survival and adequate nitrogen metabolism determined from the prior murine arginase studies. 7 While our present LEAPR construct contains a copy of the selection marker puromycin, an aminonucleoside antibiotic, before clinical applicability alteration of our donor construct to avoid integration of an antimicrobial resistance gene and its potential immunogenicity would be required.…”

“…7 LEAPR-corrected hepatocyte-like cells across all three lines demonstrated functionality of at least 40% compared with fetal liver; while the hEF1α promoter-based expression did decline with cellular differentiation, arginase expression in our studies remains well above the minimum threshold needed for survival and adequate nitrogen metabolism determined from the prior murine arginase studies. 7 While our present LEAPR construct contains a copy of the selection marker puromycin, an aminonucleoside antibiotic, before clinical applicability alteration of our donor construct to avoid integration of an antimicrobial resistance gene and its potential immunogenicity would be required. This would be accomplished by altering our donor construct by adding flanking lox P sites to the puromycin cDNA such that it could excised at the hiPSC stage prior to differentiation to the hepatocyte lineage.…”

“…36 In the former, the transient nature of expression led to loss of arginase function while in the later, long-term survival without neuropathology was achieved; however, with the loss of episomal adeno-associated virus genomes, the level of arginase expression was low and therefore, the animals remained nitrogen vulnerable. 7 However, these murine studies were able to determine that long-term survival 36 with normal behavior and learning 37 was possible and that only low levels of hepatic arginase activity, as low as 3.5–5%, were necessary to prevent brain injury and/or death from hyperammonemia or hyperargininemia. 7 Here, we developed a CRISPR/Cas9-based strategy to deliver a codon-optimized version of human arginase 1 into hiPSCs derived from patients with arginase deficiency.…”

“…7 However, these murine studies were able to determine that long-term survival 36 with normal behavior and learning 37 was possible and that only low levels of hepatic arginase activity, as low as 3.5–5%, were necessary to prevent brain injury and/or death from hyperammonemia or hyperargininemia. 7 Here, we developed a CRISPR/Cas9-based strategy to deliver a codon-optimized version of human arginase 1 into hiPSCs derived from patients with arginase deficiency. Arg1 was introduced into Exon 1 of the HPRT locus via a construct containing right and left homologous arms, the human elongation factor 1α promoter, a polyadenylation signal, and puromycin for selection (LEAPR), resulting in substantial arginase enzymatic activity in hiPSCs and in differentiated derivatives.…”

“…Patients who exhibit hyperargininemia typically present, after the neonatal period, with spasticity, seizures, spastic diplegia, and developmental regression, differing from the other UCDs. 5,6,7,8 Arginase 1 ( Arg1 ) is primarily located in the liver, hydrolyzing arginine to urea while regenerating ornithine to continue the cycle. 6,8 Loss of Arg1 activity results in an inability to remove nitrogen from arginine, but rarely causes symptoms of hyperammonemia.…”