Unbalanced deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphorylase-deficient mice

Abstract

Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP(-/-)UP(-/-)) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability.

Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE

“…Robust and stable TP expression in hematopoietic cells should clear the systemic accumulation of dThd and dUrd, as observed in MNGIE patients undergoing an allogeneic HSCT. 12 Here, we show that gene transfer using lentiviral vectors results in stable TP activity in cell lines from MNGIE patients and in a double knockout (Tymp À/À ; Upp1 À/À ) murine model of the disease, 10 reverting dThd and dUrd overload to normal levels. Our results suggest that gene therapy is a promising therapeutic approach for MNGIE.…”

“…Interestingly, wild-type (wt) mice had detectable but very low TP activity in the blood cell fraction (median: 0.04; range: undetectable to 0.10), in contrast to other tissues that show higher TP activities. 10 Circulating dThd and dUrd concentrations in untreated and sham-treated double KO mice were between two-and fivefold higher than those observed in the wt mice. After treatment with the p305-TP vector, double KO mice reduced their plasma dThd and dUrd concentrations to levels in the range of wt mice (Figures 7b and c).…”

“…p305-TP provided high levels of TP activity and reduced nucleoside overload in vivo To test the efficacy of our lentiviral vector in vivo, we used a double Tymp/Upp1 knockout murine model. 10 As the in vitro study had shown that EGFP was poorly expressed by the p305-sham vector ( Figure 1b and Table 1), we decided to use a modified version of the sham vector in the animal model (p-sham), identical to p305-sham but lacking the internal ribosomal entry site (IRES) (Figure 1a). Previous work in our laboratory showed that this vector resulted in higher EGFP expression levels than p305-sham.…”

“…[6][7][8] This is relevant from a pathogenic point of view as in vitro and in vivo studies have demonstrated that an excess of dThd induces an increase of thymidine triphosphate (dTTP), which interferes with the correct replication of mitochondrial DNA, causing mitochondrial dysfunction. 9,10 Therapeutic approaches have so far focused on reducing the systemic overload of these nucleosides. 7,[11][12][13][14] In recent years, several MNGIE patients have been treated with allogeneic hematopoietic stem cell transplantation (HSCT).…”

Mitochondrial ATP transporter Ant2 depletion impairs erythropoiesis and B lymphopoiesis

“…TP dysfunction results in a systemic increase of thymidine in plasma and selectively impairs mtDNA replication and repair. 35,36 Abnormalities of the digestive system are among the most common and severe features of MNGIE patients. The GI dysmotility, in which the muscles and nerves of the digestive system do not move food through the digestive tract efficiently, is a typical condition of the disease.…”