1:Gene and Cell Therapy, Vall d’Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain; 2:Research Group on Neuromuscular and Mitochondrial Diseases, VHIR, UAB, and Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain; 3:Acuitas Therapeutics, Vancouver, BC, Canada; 4:Blood and Tissue Bank, Barcelona, Spain; 5:Transfusional Medicine, VHIR, UAB, Barcelona, Spain; 6:Biomedical Network Research Centre on Cardiovascular Diseases (CIBERCV); 7:Paediatric Neurology, Hospital Universitari Vall d'Hebron, VHIR, UAB, Barcelona, Spain

Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE) is a rare mitochondrial disease caused by mutations in TYMP, a nuclear gene encoding thymidine phosphorylase, which results in a systemic accumulation of nucleosides (thymidine and deoxyuridine) and neurologic and gastrointestinal manifestations. In this study, we achieved an efficient integration of a human TYMP transgene into introns of the Tymp and Alb loci of hepatocytes in a murine model of the disease through the coordinated action of CRISPR/Cas9 and a TYMP cDNA template. CRISPR/Cas9 was delivered either as mRNA using lipid nanoparticles (LNP) or in an AAV2/8 viral vector; the latter was also used to package the TYMP cDNA. Insertion of the templates downstream of the Tymp and Alb promoters resulted in efficient transgene expression. Mice treated with a single i.v. dose of LNP carrying the CRISPR/Cas9 mRNAs showed a permanent (lifetime) reduction in plasma nucleosides, which was associated with the presence of human TYMP mRNA and functional enzyme in the liver. In mice with the edited Alb locus, the transgene expressed a secreted hybrid Alb-hTP protein, that was functional, with supra-physiological levels of TP activity in plasma. Gene editing was also detected, to a lesser extent, in mice receiving only the AAV vectors containing DNA templates, in the absence of Cas9, although with no detectable impact on plasma nucleoside levels. These results demonstrate the feasibility of liver-directed genome editing in the metabolic correction of MNGIE. Molecular analysis of on-target gene editing in the liver cells of mice treated long-term is currently ongoing.