Terapia génica para el MNGIE: Estudio comparativo de diferentes vectores adeno-asociados en el modelo pre-clínico de la enfermedad

Abstract

MNGIE (Mitochondrial NeuroGastroIntestinal Encephalomyopathy) is a rare autosomal recessive disease characterized by muscular, neuronal and gastrointestinal symptoms. The average life-span of MNGIE patients is 37 years. The disease is caused by mutations in the nuclear gene TYMP, which encodes thymidine phosphorylase (TP). TP catalyzes the first step of thymidine (dThd) and deoxyuridine (dUrd) catabolism. In MNGIE patients, TP dysfunction results in systemic nucleoside accumulation and disturb mitochondrial deoxyribonucleoside triphosphate (dNTP) pools. Concretely, nucleoside overload leads to mitochondrial deoxythymidine triphosphate (dTTP) increase and deoxycytidine triphosphate (dCTP) decrease. This imbalance interferes with correct mitochondrial DNA replication thus producing depletion, multiple deletions and somatic point mutations and resulting in mitochondrial dysfunction. Despite the development of multiple therapeutic strategies based on the clearance of the toxic metabolites or in enzyme replacement with the aim of achieving systemic nucleoside clearance, only allogeneic hematopoietic stem cells transplantation and orthotopic liver transplantation have been effective in long-term biochemical correction. Nevertheless, both procedures are associated with non-negligible mortality rates as well as adverse complications that are compounded in MNGIE patients because of their poor medical condition at the time of treatment. Furthermore, compatible donors are required. In recent years, gene therapy has emerged as an alternative therapeutic approach for the treatment of MNGIE. Previous preclinical studies carried out in lymphoblastoid cell lines obtained from MNGIE patients and in the murine model of the disease, demonstrated that the use of lentiviral vectors targeted to hematopoietic stem cells is effective in the restoration of TP activity. However, due to the risk of oncogenic transactivation associated to integrative vectors, we later developed an alternative strategy based on adeno-associated (AAV) vectors transcriptionally targeted to the liver. In this work, we have studied the effect of some AAV2/8 vectors differing on the promoter (TBG, PGK, HLP and AAT) and the DNA configuration (single stranded, ssDNA, or self-complementary, scAAV) on the function of the TYMP transgene in the murine model of the disease. Specifically, we have treated double KO Tymp-/-Upp1-/- male mice with a single tail vein injection of the different vectors at different doses. Thirty-four weeks after treatment we have assessed TP activity, nucleoside concentration, TYMP copy number and mitochondrial dNTP content in different tissues. Despite the results show that all vectors provide TP activity to the liver, reduce systemic nucleoside overload and modulate mitochondrial dNTP concentration, the use of liver specific promoters (TBG, HLP and AAT) has been more effective than the use of the constitutive promoter PGK. The self-complementary configuration has not provided any significant improvements. We have not observed adverse side effects or signs of hepatocellular toxicity associated with the use of AAV vectors. The best results correspond to that obtained with the use of the TBG and AAT promoters (which are both ssDNA vectors). However, at the lowest tested dose (which is 5·1010 genome copies/kg), only the AAT vector has been effective in all mice. Based on this, our data suggest that the AAT vector should be chosen for the clinical use if AAV-based gene therapy is implemented for MNGIE.

Date of Award	24 Jul 2017
Original language	Spanish
Awarding Institution	Vall d'Hebron University Hospital (HUVH)
Supervisor	Miquel Vila Bover (Tutor)