FROM PATHOGENESIS TO TREATMENT OF MNGIE AND OTHER MITOCHONDRIAL DEPLETION SYNDROMES

L'Encefalomiopatia Neurogastrointestinale Mitocondriale (MNGIE) è una rara malattia dovuta a una mutazione del gene TYMP che determina perdita di funzione dell'enzima timidina fosforilasi (TP). Clinicamente è caratterizzata da dismotilità gastrointestinale, cachessia, ptosi e oftalmoparesi, neuropatia periferica e leucoencefalopatia. L'età media al decesso è 35 ani. Attualmente esistono 2 possibili cure: trapianto di cellule staminali ematopoietiche e terapia enzimatica sostitutiva con eritrociti incapsulati. Noi descriviamo in questa tesi le due metodiche terapeutiche e i risultati ottenuti nei nostri pazienti. Parallelamente abbiamo sviluppato un nuovo approccio terapeutico, basato sull'utilizzo di nucleosidi e inibitori farmacologici del loro catabolismo, utile sia per la MNGIE che per altre malattie mitocondriali caratterizzate da deplezione e delezioni multiple del DNA. Vengono descritti i risultati pre-clinici.

Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE) is a rare autosomal recessive disorder caused by mutations in the gene TYMP encoding thymidine phosphorylase (TP). Onset of symptoms occurs during the second or third decade of life and the clinical picture is characterized by external ophthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy and leukoencephalopathy. The natural course of the disease is progressive and devasting, and patients die at a mean age of 35 years; however, age at onset and clinical presentation may vary even among patients with identical TYMP mutations, and the rate of disease progression is not uniform. The pathogenesis of the disease is due to the nucleotide pool imbalance into the mitochondria, that determines development of point mutations, multiple deletions and depletion of mitochondrial DNA (mtDNA). Enzyme replacement therapy, based on carrier erythrocyte entrapped thymidine phosphorylase (EE-TP), and hematopoietic allogeneic stem cell transplantation (AHSCT) are currently available in clinical practice. MNGIE is a first of an expanding group of disorders characterized by depletion of mtDNA, in which common pathogenic mechanisms are the nucleotide pool imbalance and/or the defect in the intergenomic cross-talk (i.e. communication between nuclear and mitochondrial DNA into the cells). Deoxyribonucleoside triphosphates (dNTPs) are the building blocks of DNA, and a constant supply is essential for the synthesis and maintenance of both the nuclear and mitochondrial genomes. Inborn errors of nucleotide metabolism frequently cause dNTP pool imbalances, leading to depletion of mtDNA in non- replicating tissues. mtDNA depletion, in turn, causes failure of the mitochondrial respiratory chain, resulting in cellular energy depletion and cell death. We treated MNGIE patients with different approaches in order to assess the safety and efficacy of each treatment. We did an extensive clinical, biochemical and instrumental follow-up and we merge our results into a worldwide consortium that will establish benefits and risks of the different therapies. The mitochondrial salvage pathway enzymes are responsible for the intra-mitochondrial, stepwise phosphorylation of recycled deoxyribonucleosides to their respective dNTP’s. The first, rate limiting phosphorylation step is catalyzed by two constitutive mitochondrial deoxyribonucleoside kinases; thymidine kinase 2 (TK2) which catalyze the phosphorylation of deoxypyrimidines and deoxyguanosine kinase (dGK) which phosphorylates the deoxypurines. Mutations in both enzymes were identified in patients with MDS. A myopatic form of MDS was associated with altered TK2 and a hepatocerebral form, affecting both liver and brain was associated with defective dGK. We performed a series of experiments in order to verify if the nucleoside supplementation and/or the inhibition of nucleoside catabolism should restore, at least partially, the mtDNA level in some MDS. We tested different combinations of nucleosides and nucleotides monophosphates in vitro, and we treated a MNGIE mouse model with different pharmacologic compounds. AHSCT represents a promising effective treatment option in an otherwise unrelentingly progressive fatal disease, but the overall transplant related mortality still remains a major issue. Although EE-TP strongly reduced plasma nucleosides without eliminating them to undetectable levels, significant clinical improvements were noted in our treated patient, as observed in patients who underwent AHSCT, suggesting a greater muscle mitochondrial oxidative function. EE-TP should be considered as a rescue or maintenance therapy prior to the availability of a suitable AHSCT donor or as an alternative therapy for patients who have irreversible end-stage disease and are without an optimally matched donor. We showed that treatment with tetrahydrouridine (THU), a potent inhibitor of cytidine deaminase (CDA), increases deoxycytidine (dCtd) concentration and partially prevents the thymidine-induced mtDNA depletion in cultured quiescent fibroblasts (cellular model of MNGIE). When THU and dCtd are co-administered, a positive correlation between circulating dCtd and mitochondrial dCTP is evidenced in targeted tissues such as brain and liver, indicating that mitochondrial dCTP pool size can be manipulated in vivo. 
Additionally, we provide evidence that deoxyguanosine (dGuo), but not deoxyadenosine, supplementation is sufficient to completely prevent spontaneous mtDNA depletion in quiescent human dGK-deficient fibroblasts. Remarkably, we observe that the specific inhibition of purine nucleoside phosphorylase in fibroblasts by immucillin H prevents dGuo degradation and allows for mtDNA copy number stabilization at lower doses of the administered nucleoside. In light of the present results, we propose the use of deoxyribonucleosides and/or the specific inhibitors of their catabolism, as a potential pharmacological approach for treating MDDSs due to defects in dNTP homeostasis.