Research Article
Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency
Caterina Garone1,2, Beatriz Garcia-Diaz1, Valentina Emmanuele1,3, Luis C Lopez4, Saba Tadesse1, Hasan O Akman1, Kurenai Tanji5, Catarina M Quinzii1 & Michio Hirano1,*
Abstract Autosomal recessive TK2 mutations cause severe mtDNA depletion and devastating neuromuscular diseases in infants and children, as Autosomal recessive mutations in the thymidine kinase 2 gene well as mtDNA multiple deletions and progressive external (TK2) cause mitochondrial DNA depletion, multiple deletions, or ophthalmoplegia in adults (Saada et al, 2001; Tyynismaa et al, both due to loss of TK2 enzyme activity and ensuing unbalanced 2012). deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 defi- To elucidate the molecular pathogenesis of TK2 deficiency, we ciency, we administered deoxycytidine and deoxythymidine mono- generated a homozygous Tk2 H126N knock-in mutant (Tk2 / ) / phosphates (dCMP+dTMP) to the Tk2 H126N (Tk2 ) knock-in mouse that manifests a phenotype strikingly similar to the human mouse model from postnatal day 4, when mutant mice are pheno- infantile encephalomyopathy (Akman et al, 2008). Between postna- typically normal, but biochemically affected. Assessment of tal day 10 and 13, Tk2 / mice rapidly develop fatal encephalomy- / 13-day-old Tk2 mice treated with dCMP+dTMP 200 mg/kg/day opathy beginning with decreased ambulation, unstable gait, coarse / each (Tk2 200dCMP/dTMP) demonstrated that in mutant animals, tremor, and growth retardation that rapidly progress to early death the compounds raise dTTP concentrations, increase levels of at age 14–16 days (Dorado et al, 2011). A similar phenotype was mtDNA, ameliorate defects of mitochondrial respiratory chain observed in the Tk2 knockout mouse (Zhou et al, 2008). enzymes, and significantly prolong their lifespan (34 days with In the Tk2 / mice, loss of Tk2 activity caused dNTP pool imbal- treatment versus 13 days untreated). A second trial of ances with low dTTP levels in brain and decreased dTTP and dCTP dCMP+dTMP each at 400 mg/kg/day showed even greater pheno- in liver, which, in turn, produce mtDNA depletion and defects of typic and biochemical improvements. In conclusion, dCMP/dTMP mitochondrial respiratory chain (RC) complexes I, III, IV, and V supplementation is the first effective pharmacologic treatment for containing mtDNA-encoded subunits, most prominently in the brain Tk2 deficiency. and spinal cord (Dorado et al, 2011). Based on the understanding of the pathogenesis of Tk2 defi- Keywords deoxycytidine monophosphate; deoxythymidine monophosphate; ciency, we have assessed a rationale therapeutic strategy to encephalomyopathy; therapy; thymidine kinase bypass the enzymatic defect with oral dCMP and dTMP supple- Subject Categories Genetics, Gene Therapy & Genetic Disease; Metabolism mentation. DOI 10.15252/emmm.201404092 | Received 20 March 2014 | Revised 22 May 2014 | Accepted 26 May 2014 | Published online 26 June 2014 EMBO Mol Med (2014) 6: 1016–1027 Results
dCMP/dTMP delays disease onset, prevents neuromuscular Introduction manifestations, and prolongs lifespan of Tk2-deficient mice
Encoded by the nuclear DNA gene TK2, thymidine kinase 2 (TK2) is Oral treatment with dCMP+dTMP 200 mg/kg/day each in milk a mitochondrial matrix protein that phosphorylates thymidine and (Tk2 / 200dCMP/dTMP) beginning at postnatal day 4 delayed disease deoxycytidine pyrimidine nucleosides to generate deoxythymidine onset to 20–25 days (Supplementary Video S1), when the mutant monophosphate (dTMP) and deoxycytidine monophosphate (dCMP), mice developed a mild tremor and stopped gaining weight. In the which, in turn, are converted to deoxynucleotide triphosphates fourth week, they manifested weakness and reduced movements. In (dNTPs) required for mitochondrial DNA (mtDNA) synthesis. contrast, Tk2 / mice treated from day 4 with dCMP+dTMP
1 Department of Neurology, Columbia University Medical Center, New York, NY, USA 2 Human Genetics Joint PhD Program, University of Bologna and Turin, Turin, Italy 3 Pediatric Clinic, University of Genoa, IRCCS G. Gaslini Institute, Genoa, Italy 4 Instituto de Biotecnología, Centro de Investigación Biomédica, Universidad de Granada, Parque Tecnológico de Ciencias de la Salud, Armilla, Spain 5 Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA *Corresponding author. Tel: +1 212 305 1048; Fax: +1 212 305 3986; E-mail: [email protected]
1016 EMBO Molecular Medicine Vol 6 |No8 | 2014 ª 2014 The Authors. Published under the terms of the CC BY 4.0 license Caterina Garone et al Bypass therapy for Tk2 deficiency EMBO Molecular Medicine