Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency

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Encoded by the nuclear DNA gene, TK2, thymidine kinase 2 (TK2) is a mitochondrial matrix enzyme, which phosphorylates the nucleosides, deoxycytidine (dC) and deoxythymidine (dT), to generate deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP). These pyrimidine nucleoside monophosphates are subsequently converted to deoxynucleotide triphosphates (dNTPs) required for mitochondrial DNA (mtDNA) replication and maintenance. TK2 mutations were originally identified in 4 children with myopathic weakness and severe depletion of muscle mtDNA that began in the first 2 years of life and progressed rapidly to complete paralysis with respiratory insufficiency requiring mechanical ventilation or causing death by age 4 years.1 Subsequent clinical reports have expanded the clinical and molecular genetic spectrum of TK2 deficiency, leading to the recognition of three disease subtypes: (1) infantile‐onset myopathy with rapid progression to early death2; (2) childhood‐onset myopathy, which resembles spinal muscular atrophy type III, begins between ages 1 and 12 years with progression to loss of ambulation within few years3 (Garone et al, submitted); and (3) late‐onset myopathy starting at age 12 years or later with moderate‐to‐severe myopathy manifesting as either isolated chronic progressive external ophthalmoplegia (CPEO) or a generalized myopathy with CPEO plus facial and limb weakness, gradual progression, and, in some cases, respiratory failure and loss of ability to walk in adulthood.4 Molecular genetics defects in muscle include severe depletion of mtDNA in the infantile‐ and childhood‐onset forms as well as multiple deletions with or without depletion of mtDNA in late‐onset patients.
We previously reported that molecular bypass therapy using oral administration of the TK2 products, dCMP and dTMP, in our mouse model of TK2 deficiency (Tk2 H126N knock‐in [Tk2–/–] mice)6 delays onset of molecular and biochemical abnormalities, ameliorates the symptoms, and prolongs the life span of the animals by 2‐ to 3‐fold.7 We also observed that after administration, dCMP and dTMP were rapidly catabolized to dC and dT, suggesting that nucleosides, rather than nucleotides, are the major active therapeutic agents. Thus, we hypothesize that the increase of the nucleoside substrates enhances residual activity of TK1, TK2, or both, leading to correction of dNTP pool imbalances. Furthermore, dCMP+dTMP treatment increased levels of deoxyuridine in liver, indicating deamination of dC.
To further enhance this therapy, in this study, we have assessed two different approaches. First, because we have hypothesized that nucleosides are the active therapeutic compounds, we have tested the effects of oral dC+dT on the Tk2 H126N knock‐in mouse model. Second, we have assessed the effects of coadministration of tetrahydrouridine (THU), an inhibitor of the cytidine deaminase, with dCMP+dTMP to attempt to increase levels of dC. We have observed that oral dC+dT prolongs the life span of mutant animals by restoring mtDNA copy number and respiratory chain enzyme (RCE) activities and levels. In contrast, addition of THU unexpectedly reduces the life span of animals comparing to oral dCMP+dTMP therapy alone. These results reveal a novel nucleoside substrate enhancement therapy, which significantly ameliorates Tk2 deficiency in our mouse model.
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