Hepatoencefalopatia deguda a mutacions en GFM1: obtenció d’un model de ratolí modificat genèticament i estudi preclínic de teràpia gènica amb vectors adenoassociats

Abstract

The hepatoencephalopathy due to mutations in GFM1, also known as combined oxidative phosphorylation deficiency type I (COXPD1) is a recessive mitochondrial disease caused by mutations in the nuclear gene encoding the mitochondrial translation elongation factor G1 (EFG1). Patients with COXPD1 usually develop a severe encephalopathy, sometimes combined with liver failure, with neonatal onset and rapid progression that normally leads to death during the first weeks or years of life. _x000D_ The study of COXPD1 has been so far performed mainly using in vitro models (cell lines and patient-derived biopsies). Although these experimental tools are useful, the lack of a suitable animal model has represented an important limitation to delve into the disease physiopathology and test potential therapeutic approaches. _x000D_ We have generated two mutant alleles for the Gfm1 murine gene: a knock-in allele (KI) harbouring the missense mutation p.R671C found in at least ten patients that survived more than one year, and a Gfm1 knock-out allele (KO). These alleles allowed us to obtain homozygous KI (Gfm1R671C/R671C) and compound heterozygous KI/KO (Gfm1R671C/-) mice._x000D_ The homozygous KO mutation was embryonically lethal, whereas Gfm1R671C/R671C mice were viable and showed normal growth. The p.R671C mutation entailed a drastic reduction of EFG1 mitochondrial protein levels in every analysed tissue, causing a decrease of the mitochondrial translation rate and the respiratory chain (RC) complex IV (CIV) enzyme activity in liver of 6-week-old Gfm1R671C/R671C mice. At 50 weeks of age, the mitochondrial translation was similarly reduced but CIV deficiency was no longer observed. _x000D_ The Gfm1R671C/- mouse model, which showed normal survival at least until 80 weeks of age, experienced a more pronounced decrease of EFG1 protein levels in liver and brain mitochondria, as compared with the Gfm1R671C/R671C mice. Consequently, 8 and 30-week-old Gfm1R671C/R671C animals developed a significant reduction of the mitochondrial translation rate in both tissues which, in turn, caused a clear OXPHOS system dysfunction: reduced levels of the assembled complexes I and IV, together with the consequent decrease of their enzymatic activities. Nevertheless, no neuromuscular or hepatic alterations were detected in association with the molecular COXPD phenotype in Gfm1R671C/- mice. Moreover, the increase of mitoriboproteins and ARN-mt levels in liver and brain from this mouse model could be part of molecular response mechanisms promoted to compensate the EFG1 depletion._x000D_ On the other hand, there is no currently available cure for COXPD1, and the nature of the disease (monogenic, autosomal and recessive) makes it a good candidate to be treated with gene therapy. In the second part of the thesis we initiated a preclinical gene therapy study using adeno-associated viral vectors (rAAV) to introduce the correct copy of the human GFM1 gene into Gfm1R671C/- mice. A single intravenous injection of an AAV9 carrying the GFM1 coding sequence under the control of the human alpha-1-antitripsin hepatic promoter (ssAAV9-ApoE-hAAT-GFM1) on 6-week-old Gfm1R671C/- mice (5E+12 vg/kg) partially recovered the mitochondrial EFG1 levels four weeks after treatment. Additionally, the treatment restored the levels of assembled complexes I and IV and their enzymatic activities in the target tissue, demonstrating the functionality of the EFG1 encoded by the therapeutic gene. _x000D_ Therefore, from the work performed in this thesis we obtained the Gfm1R671C/- mouse showing a clear dysfunctional molecular phenotype that makes it a suitable animal model for COXPD1. Moreover, the results of our gene therapy study show that using rAAV to provide the correct version of the GFM1 gene results in the restoration of the EFG1 function, pointing to gene therapy as a feasible and promising strategy for the treatment of COXPD1.

Date of Award	21 Jul 2023
Original language	Catalan
Supervisor	Ramon Antoni Martí Seves (Director) & Javier Torres Torronteras (Director)