Bioavailability and cytosolic kinases modulate response to deoxynucleoside therapy in TK2 deficiency

Abstract

Background TK2 is a nuclear gene encoding the mitochondrial matrix protein thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial nucleotide salvage pathway. Deficiency of TK2 activity causes mitochondrial DNA (mtDNA) depletion, which in humans manifests predominantly as a mitochondrial myopathy with onset typically in infancy and childhood. We previously showed that oral treatment of the Tk2 H126N knock-in mouse model (Tk2 −/− ) with the TK2 substrates, deoxycytidine (dCtd) and thymidine (dThd), delayed disease onset and prolonged median survival by 3-fold. Nevertheless, dCtd + dThd treated Tk2 −/− mice showed mtDNA depletion in brain as early as postnatal day 13 and in virtually all other tissues at age 29 days. Methods To enhance mechanistic understanding and efficacy of dCtd + dThd therapy, we studied the bioavailability of dCtd and dThd in various tissues as well as levels of the cytosolic enzymes, TK1 and dCK that convert the deoxynucleosides into dCMP and dTMP. Findings Parenteral treatment relative to oral treatment produced higher levels of dCtd and dThd and improved mtDNA levels in liver and heart, but did not ameliorate molecular defects in brain or prolong survival. Down-regulation of TK1 correlated with temporal- and tissue-specificity of response to dCtd + dThd. Finally, we observed in human infant and adult muscle expression of TK1 and dCK, which account for the long-term efficacy to dCtd + dThd therapy in TK2 deficient patients. Interpretations These data indicate that the cytosolic pyrimidine salvage pathway enzymes TK1 and dCK are critical for therapeutic efficacy of deoxynucleoside therapy for Tk2 deficiency. Fund National Institutes of Health P01HD32062.

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

“…The activity of the required cytosolic deoxynucleoside kinases (TK1 and dCK) was also markedly altered with age, especially in brain and liver where both activities were significantly reduced, which could also contribute to the age-related loss of effect. Overall, our findings regarding these limiting factors are in agreement with the observations on a TK2 His126Asn knockin mice after IP and oral dThd+dCtd administration [16,17]. Finally, ontogenetic changes in other factors, such as dN transporters involved in intestinal absorption and cellular internalization, could further lessen the therapeutic potential of dNs in older mice.…”

“…Other routes of administration, such as intraperitoneal (IP) injection leads to higher bioavailability of dThd+dCtd. However, IP administration of dThd+dCtd to a TK2 His126Asn knockin mouse model yielded similar results on survival [17], so reduced efficacy of deoxynucleosides with aging is likely due to factors other than simply absorption at the level of small intestine. The activity of the required cytosolic deoxynucleoside kinases (TK1 and dCK) was also markedly altered with age, especially in brain and liver where both activities were significantly reduced, which could also contribute to the age-related loss of effect.…”

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

“…Nevertheless, response to dC+dT therapy in the Tk2 -/-mice is limited as it only delays onset and slow progression of the disease rather than halting or reversing the course. Several factors constrain the therapeutic response including: 1) rapid degradation of exogenously administered pyrimidine nucleosides by cytidine deaminase and thymidine phosphorylase (TP); 2) restricted delivery of dC and dT via nucleoside transporters into target tissues; and 3) cell-cycle and tissue-specific activities of deoxycytidine kinase (Dck) and thymidine kinase 1 (Tk1) (Blazquez-Bermejo et al, 2019, Lopez-Gomez et al, 2019. The nucleoside kinases are particularly critical in Tk2 -/-mice that develop early central nervous system manifestations, because expression of Tk1 in murine brain is low (Dorado et al, 2011).…”

“…Based upon these observations, a Phase 2 prospective, open-label clinical trialhas been initiated to further assess the safety and efficacy of deoxynucleoside treatment in TK2 deficient patients (NCT03845712). Nevertheless, results in Tk2 deficient mice showed limited efficacy, likely due to several factors tempering response to dC+dT(Blazquez-Bermejo et al, 2019, Lopez-Gomez et al, 2019. Potency of dC+dT therapy may be limited in encephalomyopathic forms of human TK2 deficiency, because low expression of TK1 in brain may contrain response of the encephalopathy to this therapy.…”

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

“…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.…”