Thymidine kinase 2 (TK2), encoded by the nuclear gene, TK2, is a mitochondrial matrix protein that phosphorylates thymidine and deoxycytidine nucleosides to generate deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP), which in turn, are converted to deoxynucleotide triphosphates (dNTPs) required for mitochondrial DNA synthesis. Autosomal recessive TK2 mutations cause devastating neuromuscular weakness with severe depletion of mitochondrial DNA (mtDNA) in infants and children, as well as progressive external ophthalmoplegia with mtDNA multiple deletions in adults. The central nervous system is variably involved in these disorders. To determine the bases of disease onset and organ-specificity of TK2 deficiency, Michio Hirano, Drs. Orhan H Akman, and collaborators have generated a homozygous Tk2 H126N knock-in mutant (Tk2-/-) mouse that manifests a phenotype strikingly similar to the human infantile encephalomyopathy. Between postnatal day 10 and 13, Tk2-/- mice rapidly develop fatal encephalomyopathy characterized by decreased ambulation, unstable gait, coarse tremor, growth retardation, and rapid progression to early death at age 14 to 16 days. Molecular and biochemical analyses of the mouse model demonstrated that the pathogenesis of the disease is due to loss of enzyme activity and ensuing dNTP pool imbalances with decreased dTTP levels in brain and both dTTP and dCTP levels in liver, which, in turn, produces mtDNA depletion and defects of respiratory chain enzymes containing mtDNA- encoded subunits, most prominently in the brain and spinal cord. We have demonstrated that molecular bypass therapy with orally administered dCMP and dTMP ameliorates the abnormal phenotype and extends the lifespan of Tk2-/- mice by 2-3 fold, but does not cure the disease. Therefore, more effective therapies are needed to treat TK2 deficiency. Based on our unexpected observation that dTMP and dCMP supplementation increased blood and tissue levels of deoxythymidine (dT), we hypothesize that increasing dT and deoxycytidine (dC) substrates for TK2 will be therapeutic. An alternative treatment approach is gene therapy. Taking advantage of our Tk2 knockin mouse model, this proposal seeks to test both therapeutic approaches: 1) pharmacological treatment with deoxynucleosides and 2) gene therapy using adeno-associated virus (AAV) vector-mediated TK2 delivery.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Program Projects (P01)
Project #
Application #
Study Section
Developmental Biology Subcommittee (CHHD)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Columbia University (N.Y.)
New York
United States
Zip Code
Joseph, Leroy C; Barca, Emanuele; Subramanyam, Prakash et al. (2016) Inhibition of NAPDH Oxidase 2 (NOX2) Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes. PLoS One 11:e0145750
Barca, E; Musumeci, O; Montagnese, F et al. (2016) Cerebellar ataxia and severe muscle CoQ10 deficiency in a patient with a novel mutation in ADCK3. Clin Genet 90:156-60
Torres-Torronteras, Javier; Cabrera-Pérez, Raquel; Barba, Ignasi et al. (2016) Long-Term Restoration of Thymidine Phosphorylase Function and Nucleoside Homeostasis Using Hematopoietic Gene Therapy in a Murine Model of Mitochondrial Neurogastrointestinal Encephalomyopathy. Hum Gene Ther 27:656-67
Fryer, Robert H; Bain, Jennifer M; De Vivo, Darryl C (2016) Mitochondrial Encephalomyopathy Lactic Acidosis and Stroke-Like Episodes (MELAS): A Case Report and Critical Reappraisal of Treatment Options. Pediatr Neurol 56:59-61
Dalla Rosa, Ilaria; Cámara, Yolanda; Durigon, Romina et al. (2016) MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria. PLoS Genet 12:e1005779
Cloonan, Suzanne M; Glass, Kimberly; Laucho-Contreras, Maria E et al. (2016) Mitochondrial iron chelation ameliorates cigarette smoke-induced bronchitis and emphysema in mice. Nat Med 22:163-74
Engelstad, Kristin; Sklerov, Miriam; Kriger, Joshua et al. (2016) Attitudes toward prevention of mtDNA-related diseases through oocyte mitochondrial replacement therapy. Hum Reprod 31:1058-65
Barca, Emanuele; Tang, Maoxue; Kleiner, Giulio et al. (2016) CoQ10 Deficiency Is Not a Common Finding in GLUT1 Deficiency Syndrome. JIMD Rep 29:47-52
Sadat, Roa; Barca, Emanuele; Masand, Ruchi et al. (2016) Functional cellular analyses reveal energy metabolism defect and mitochondrial DNA depletion in a case of mitochondrial aconitase deficiency. Mol Genet Metab 118:28-34
Varma, Hemant; Faust, Phyllis L; Iglesias, Alejandro D et al. (2016) Whole exome sequencing identifies a homozygous POLG2 missense variant in an infant with fulminant hepatic failure and mitochondrial DNA depletion. Eur J Med Genet 59:540-5

Showing the most recent 10 out of 26 publications