Long-term Survival of the Juvenile Lethal Arginase-deficient Mouse With AAV Gene Therapy

Abstract

Arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia. Human patients suffer from neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation. In a murine model, onset of the phenotypic abnormality is heralded by weight loss beginning around day 15 with death occurring typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. The goal of this study was to address the development of a gene therapy approach for arginase deficiency beginning in the neonatal period. Lifespan extension, body weight, circulating amino acids and ammonia levels were examined as outcome parameters after gene therapy with an adeno-associated viral vector expressing arginase was administered to mice on the second day of life (DOL). One-hundred percent of untreated arginase-deficient mice died by DOL 24, whereas 89% of the adeno-associated virus (AAV)-treated arginase deficient mice have survived for >8 months. While animals at 8 months demonstrate elevated glutamine levels, ammonia is less than three times that of controls and arginine levels are normal. These studies are the first to demonstrate that AAV-based therapy for arginase deficiency is effective and supports the development of gene therapy for this and the other urea cycle disorders.

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

“…Previous gene therapy studies have demonstrated successful reversal of disease manifestations through either treatment of neonatal mice with a helper-dependent adenoviral vector 24 or with adeno-associated virus-expressing arginase. 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.…”

“…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.…”

“…Lysis buffer was prepared with 0.1% Triton X-100 and 1× HALT protease inhibitor cocktail (Thermo) and urea production was measured as previously performed. 36,54 Briefly, samples were centrifuged at 100 g for 5 minutes at 4°C and the supernatant was collected. An activated mixture, consisting of 12.5 µl of 50 mmol/l Tris-Cl (pH 7.5), 12.5 µl of 10 mmol/l MnCl, and 25 µl of the supernatant was incubated at 56°C for 10 minutes.…”

AAV-based gene therapy prevents neuropathology and results in normal cognitive development in the hyperargininemic mouse

“…7–8 The vector contains the ubiquitously-expressing chicken β-actin promoter/CMV enhancer; however it is unclear if functional protein can be produced in extrahepatic tissues. We have previously described using this vector for reporter gene studies 9 and long-term factor VIII expression using a similar neonatal approach in the murine model of hemophilia A 10 .…”

Development of operational immunologic tolerance with neonatal gene transfer in nonhuman primates: preliminary studies

“…Previous murine studies have demonstrated that neonatal IV administration of AAV results in high early expression in the liver and lower expression in myocytes 15 . With hepatic proliferation, substantial loss of expression occurs in the rapidly developing neonatal mouse liver 15, 18, 33 . Prior studies by Wilson et al with AAV8-mediated transduction in one-week-old rhesus monkeys were found to be safe and to produce high hepatic GFP transduction using the liver-specific promoter thyroxine binding globulin 35 .…”