Preclinical Assessment of a Gene Editing Approach in a Mouse Model of MNGIE

Abstract

Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare and progressive disease caused by mutations in the TYMP gene, which encodes the enzyme thymidine phosphorylase (TP). This results in a systemic accumulation of nucleosides and mitochondrial toxicity. AAV-based liver-directed gene therapy has shown efficacy in preclinical studies, but it has limitations such as a decline of transgene expression long-term. We propose that gene editing can circumvent some of these problems. Methods: We performed a preclinical study in the murine model of MNGIE to assess that the coordinated delivery of CRISPR/Cas9 and a TYMP cDNA caused the efficient integration of a TYMP transgene into introns of the Tymp and Alb loci in hepatocytes. CRISPR/Cas9 was delivered either as mRNA using nanoparticles or in AAV2/8 viral vectors; the latter were also used to package the TYMP cDNA. We assessed the efficient integration of the templates into the liver cells, and monitored the nucleoside levels in plasma to detect the biochemical correction. Findings: The best results were obtained using lipid nanoparticles. The consistent and stable nucleoside reduction observed correlated with the presence of TYMP mRNA and functional enzyme in the liver cells. In mice with an edited Alb locus, the transgene produced a hybrid Alb-hTP protein that was secreted, with high levels of TP activity in plasma. Interpretation: Liver-directed gene editing is a feasible approach to achieve the long-term biochemical correction of the disease, with several advantages over previous methods. Funding Information: This project was made possible with financial support from the Instituto de Salud Carlos III/FEDER (grant PI15/00172) and from MINECO/FEDER (grant RTI2018-094734-B-C22). The support of the Agencia de Gestio d’Ajuts Universitaris i de Recerca (AGAUR) of the Generalitat de Catalunya, through SGR 2017 1559 grant, is also acknowledged. Declaration of Interests: S.H.Y.F. and Y.K.T. are employees of Acuitas Therapeutics, a company focused on the development of lipid nanoparticle delivery systems for nucleic-acid-based drugs. RM was the recipient of personal fees associated with advisory tasks and of financial support from Modis Therapeutics for research outside the submitted work. RM also holds a patent “Deoxynucleoside therapy for diseases caused by unbalanced nucleotide pools including mitochondrial DNA depletion syndromes” (PCT/US16/038110), with royalties paid to Modis Therapeutics. INCOMPLETE Ethics Approval Statement: All animal procedures were performed in accordance with European recommendations and were approved by our institutional ethics committee (Comite Etic d’Experimentacio Animal).