Off targets for Metabolic pathway reprogramming by CRISPR genomeEditing. OffTargetsMetPathway

Many metabolic liver disorders are refractory to conventional drug therapy and require orthotopic liver transplantation. Unfortunately, there are twice as many patients on the waiting list as there are available organs1. Moreover, transplantation carries the risk of life-long immunosuppression, with increased morbidity and mortality2. Here we propose an alternative therapeutic strategy called metabolic pathway reprogramming. The approach relies on the compensatory deletion of a critical metabolic gene in a disease-associated pathway: the resultant loss of function re-routes the metabolic pathway, leading to a benign phenotype. As a proof of principle, we tested this approach on hereditary tyrosinemia type I (fumarylacetoacetate deficiency, Fah-/-), which results in acute liver failure and premature death if untreated. Using CRISPR/Cas9 genome editing technology in vivo, we converted hepatocytes from tyrosinemia type I into the benign tyrosinemia type III (hydroxyphenylpyruvate dioxigenase deficiency, Hpd-/-). Edited, healthy hepatocytes (Fah-/-/Hpd-/-) displayed a growth advantage over non-edited, diseased hepatocytes (Fah-/-/Hpd+/+) and in some mice almost completely replaced them (>99%) within only 8 weeks. This metabolic pathway reprogramming from type I into type III tyrosinemia rescued the lethal phenotype of Fah-/- mice. Treated mice were healthy and asymptomatic for the duration of the experiment (4 months) without drugs or dietary restrictions. Genetic and metabolic analyses corroborated efficient and safe exon excision of the hydroxyphenylpyruvate dioxigenase and consecutive rerouting of the tyrosine catabolism. Metabolic pathway reprogramming is a novel, broadly applicable therapeutic strategy, which exploits the efficiency of the CRISPR/Cas9 system and reduces the risk associated with genome editing on critical disease-causing genes.