Human Induced Pluripotent Stem Cell- and mRNA-based Gene Therapy Strategies for Treatment of Arginase Deficiency

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency is caused by biallelic mutations in ARG1, the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of circulating guanidino compounds while clinically is notable for developmental delays, spastic diplegia, psychomotor function loss, and uncommonly, death. There is currently no completely effective medical treatment available. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-Deficient Induced Pluripotent Stem CellsAdvances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.Lipid Nanoparticle Targeted mRNA Therapy as a Treatment for the Inherited Metabolic Liver Disorder Arginase DeficiencyWhile preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division and patient-specific genetically modified cell therapies suffer from lengthy preparation time where patients with urea cycle disorders (UCDs) are at continued risk for progressive intellectual decline. Recent advances in mRNA codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of rapidly applicable and safer, albeit temporary, treatments to restore liver metabolic function in patients with UCDs including arginase deficiency. In this study, we applied such technologies to successfully treat an arginase deficient murine model. Mice were administered LNP encapsulating human codon-optimized ARG1 mRNA every 3 days and demonstrated 100% survival with no evidence of hepatotoxicity. Plasma ammonia, arginine, and glutamine demonstrated good control and near-normal ureagenesis was measured. These results suggest that hARG1 mRNA delivery by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency