Age-related metabolic changes limit efficacy of deoxynucleoside-based therapy in thymidine kinase 2-deficient mice

Abstract

Background Thymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease. Methods We daily administered TK2 knockout mice ( Tk2 KO ) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism. Findings dThd+dCtd treatment extended average lifespan of Tk2 KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan. Interpretation dThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. Fund This study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness, the Spanish Instituto de Salud Carlos III, the Fundación Inocente, Inocente, AFM Téléthon and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Growth Differentiation Factor 15 is a potential biomarker of therapeutic response for TK2 deficient myopathy

“…In keeping with the extent of the clinical benefit, the rate of GDF-15 decrease was faster in children than in adults, in particular, in the most severe patients who are the ones that respond more markedly to the treatment in the form of extended survival, recovery from ventilatory support dependency, and regaining motor abilities. It has been recently reported that age-related metabolic changes in mice (namely increased deoxynucleoside catabolism and decreased anabolism with age) account, at least in part, for the limited efficacy of the treatment in this model 8 .…”

“…The 24 treated patients were grouped according to age at treatment initiation: group 1 (the paediatric group) started treatment before 16 years of age (P1 to P15), and group 2 (the adult group) started treatment after this age (P16 to P24). This grouping allowed us to compare GDF-15 and FGF-21 serum levels with aged-matched controls and takes into account the observation that age-related metabolic changes contribute to the efficacy of deoxynucleoside therapy 8 .…”

“…Although some patients started with taking oral deoxynucleotides (dTMP and dCMP), after the demonstration in vitro and in vivo 5,6 that deoxynucleosides are the active agents, the therapy was changed to deoxynucleosides (dThd and dCtd). The efficacy of oral deoxynucleoside treatment was demonstrated in pre-clinical studies 7,8 and in a group of 16 patients treated under a compassionate use program 9 . Without any major side effect, the therapy had striking effects on early-onset severe myopathy patients, such as improvement in muscle strength, reduction or discontinuation of mechanical ventilation and gastrostomy feeding, and regaining the ability to walk.…”

Strategies for fighting mitochondrial diseases

“…Supplementation with pyrimidine and purine nucleosides corrected ethidium bromide‐induced mtDNA depletion in human fibroblasts carrying mutations in RRM2B, the P53‐dependent subunit of riboside reductase, the enzyme converting ribonucleosides into deoxyribonucleosides. However, the corresponding monophosphate nucleotides did not correct mtDNA depletion in RRM2B deficient human myoblasts [91,92]. Finally, deoxycytidine or tetrahydrouridine corrected mtDNA depletion in a Tymp/Upp1 double knockout mouse model of MNGIE disease.…”

Synergistic effect of deoxynucleosides and AAV gene therapy for thymidine kinase 2 deficiency

“…TK2 deficiency manifests most frequently as a progressive and fatal mitochondrial myopathy in infants and children, although around 20% of patients develop the disease during adolescence or adulthood (Garone et al, 2018). Studies of Tk2 homozygous p.His126Arg knock-in (Tk2 -/-) mice indicate that onset and tissue-specificity of the disease are modulated by expression of thymidine kinase 1 (TK1), which catalyze the first step of the cytosolic thymidine salvage pathway (Akman et al, 2008, Blazquez-Bermejo et al, 2019, Dorado et al, 2011).…”