mRNA therapy restores ureagenesis and corrects glutathione metabolism in argininosuccinic aciduria

Argininosuccinate lyase (ASL) is a key enzyme integral to the hepatic urea cycle which is required for ammonia detoxification, and the citrulline-nitric oxide (NO) cycle for NO production. ASL deficient patients present with argininosuccinic aciduria (ASA), an inherited metabolic disease with hyperammonaemia and a chronic systemic phenotype with neurocognitive impairment and chronic liver disease. ASL deficiency as an inherited model of systemic NO deficiency, shows enhanced nitrosative and oxidative stress. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using ( S )-4-(3-18F-fluoropropyl)-L-glutamate ([18F]FSPG). Upregulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. hASL mRNA encapsulated in lipid nanoparticles corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis and glutathione metabolism and ameliorating chronic liver disease. We further present [18F]FSPG PET as a novel non-invasive diagnostic tool to assess liver disease and therapeutic efficacy in ASA. These findings support clinical translation of mRNA therapy for ASA. ### Competing Interest Statement The work was partly funded by Moderna Therapeutics. * ASA : Argininosuccinic Aciduria ASL : Argininosuccinate Lyase ASLD : Argininosuccinate Lyase Deficiency ASSD : Argininosuccinate Synthase Ddeficiency CyS : Cysteine CySS : Cystine GSH : Glutathione hASL : human Argininosuccinate Lyase HcyS : Homocysteine HcySS : Homocysteine HILIC : Hydrophilic Interaction Liquid Chromatography IP : Intraperitoneal IV : Intravenous IVIS : In vivo imaging system LNP : Lipid nanoparticle Luc : Luciferase MDA : Malondialdehyde NO : Nitric Oxide OTCD : Ornithine Transcarbamylase Deficiency PAS : Periodic Acid Schaff PET : Positron Emission Tomography ROI : Regions of Interest TBARS : Thiobarbituric Acid reactive substances assay WT : Wildtype xCT : Cystine/Glutamate antiporter [18F]FSPG : (4S)-4-(3-[18F]Fluoropropyl)-L-glutamate