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ESR 9 Project Novel insights into maintenance of mitochondrial genome WP4

Objectives: Mutations in POLG are associated with a spectrum of disease manifestations characterized by mtDNA instability. These include PEO, with multiple mtDNA deletions, transmitted as a dominant or recessive trait, recessive, juvenile-onset sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO), sometimes complicated by migraine and/or seizures with later development of myoclonus (spino-cerebellar ataxia and epilepsy, SCAE), infantile hepato-cerebral poliodystrophy with mtDNA depletion (Alpers-Huttenlocher syndrome). One of the most common mutations in POLG determines an A467T mutation, which is typically homozygous in SCAE or present in compound heterozygosity with a null allele in Alpers-Huttenlocher syndrome.

The reasons for such complexity, tissue specificity and peculiar genetic transmission are unclear.

We have recently developed a POLG knockin mouse by CRISPR/Cas9 technology expressing Polg1A449T variant, which is the murine equivalent to the human Polg1A467T. In addition, in collaboration with Partner 1 and 6 we will cross the Polg1A449T with the Polg1+/- mouse model to generate a double recombinant mutant reproducing the genotype often found in patients with Alper’s disease. Finally, a mouse model for a dominant mutation (for instance the Y955C mutation causing autosomal dominant PEO in patients) will be produced by CRISPR/Cas9 technology.

We will characterize the molecular and clinical phenotype of the double recombinant mice to understand the patho-molecular basis of the human disease. An array of in vivo (Digigait, rotarod, treadmill, etc.) and in vitro (assessment of mtDNA quantity and quality by real time PCR and/or Southern blot, analysis of the assembly and function of the respiratory chain, mtDNA synthesis rate, etc.) will be used.

Finally, therapeutically approaches will also be attempted. A first approach will be based on developing an adeno-associated viral vectors (AAV)-based gene therapy approach for the in vivo correction of point mutations in liver and other organs. This approach stems from the identification of Cas9 variants encoded by relatively small genes compatible with the limited cloning capacity of AAV vectors and from the development of new AAV serotypes targeting different organs. A second strategy will be based on the administration of deoxynucleotides, an approach that has been proved promising in other models of mtDNA instability, such as the thymidine phosphorilase knockout mouse and the thymidine kinase H126N knockin mouse.

Other strategies will eventually be considered during the progression of the project.

Expected Results: To clarify the still unexplained tissue specificity characterizing the clinical features of mitochondrial diseases associated with mitochondrial replication defects. To unravel the genetic, molecular and pathophysiological mechanisms for high variability of the phenotypes observed in POLG-related diseases.