Resumen de Avaluació preclínica d'un procediment d'edició gènica per mngie
MNGIE is a rare metabolic disease caused by recessive mutations in the TYMP gene, which encodes the enzyme TP. This causes a systemic accumulation of nucleosides which results in mitochondrial toxicity that is usually lethal during the first decades of life.
The goal of the present work was to achieve an efficient integration of the human TYMP cDNA on introns of the Tymp and Alb genes of murine hepatocytes by the coordinated action of two elements: CRISPR/Cas9 system and TYMP cDNA templates. These templates were designed to be integrated in a region downstream a genomic promoter, so that the TYMP cDNA expression will be under its control. In the case of the Alb locus the successful gene editing produced a hybrid Alb-hTP protein with a secretory ability. With this approach, hepatocytes would be permanently edited and the TYMP cDNA would be expressed long-term, thus overcoming the problem of loss of transgene expression often detected in conventional gene therapies. Two distinct strategies were tested. In the first one, CRISPR/Cas9 RNAs were delivered by nanoparticles (NPs). This allowed a high but transient expression. We tested NPs of two different sources: polymeric NPs (PNPs) of GEMAT-IQS, and lipid NPs (LNPs) of Acuitas Therapeutics®. In the second approach, CRISPR/Cas9 was delivered as DNA inside AAV vectors.
Both in vitro and in vivo results corroborated that the TYMP cDNA could be successfully inserted in both loci. We also assessed the presence of TYMP mRNA and functional TP protein in the edited cells. In vivo experimentation was performed with the mice model of MNGIE. The Thd and dUrd plasma levels of treated animals were monitored to assess the effectivity of the different genome editing approaches. The best results were obtained in animals treated with LNPs carrying the CRISPR/Cas9 RNAs and rAAV2/8 vectors carrying the DNA templates. These mice showed a consistent and stable biochemical correction that correlated with the presence of TYMP mRNA and functional TP enzyme in liver cells. Moreover, mice of the Alb locus gene editing group presented significant TP activity in plasma, which confirms that the hybrid enzyme can be successfully secreted and maintains its activity in plasma. However, the reduction of plasma nucleoside levels was not enough to reach the levels observed in WT mice, so there is room for optimization. Unexpectedly, in some cases genome edition was observed in the absence of CRISPR/Cas9, which suggests that the DNA templates alone could trigger the correct insertion through homologous recombination. All these results confirm that our genome editing approach is viable, although we need to increase the overall efficiency of the procedure to achieve a full biochemical correction in the murine model of MNGIE.
