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Nature Genetics Apr 2023Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed...
Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed genome-wide CRISPR-Cas9 functional telomere length screening and identified thymidine (dT) nucleotide metabolism as a limiting factor in human telomere maintenance. Targeted genetic disruption using CRISPR-Cas9 revealed multiple telomere length control points across the thymidine nucleotide metabolism pathway: decreasing dT nucleotide salvage via deletion of the gene encoding nuclear thymidine kinase (TK1) or de novo production by knockout of the thymidylate synthase gene (TYMS) decreased telomere length, whereas inactivation of the deoxynucleoside triphosphohydrolase-encoding gene SAMHD1 lengthened telomeres. Remarkably, supplementation with dT alone drove robust telomere elongation by telomerase in cells, and thymidine triphosphate stimulated telomerase activity in a substrate-independent manner in vitro. In induced pluripotent stem cells derived from patients with genetic telomere biology disorders, dT supplementation or inhibition of SAMHD1 promoted telomere restoration. Our results demonstrate a critical role of thymidine metabolism in controlling human telomerase and telomere length, which may be therapeutically actionable in patients with fatal degenerative diseases.
Topics: Humans; Telomerase; SAM Domain and HD Domain-Containing Protein 1; Nucleotides; Telomere Homeostasis; Thymidine; Telomere
PubMed: 36959362
DOI: 10.1038/s41588-023-01339-5 -
Nature Biotechnology Jul 2022Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors. Here, we describe a method to harness this abnormal splicing activity to drive...
Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors. Here, we describe a method to harness this abnormal splicing activity to drive splicing factor mutation-dependent gene expression to selectively eliminate tumor cells. We engineered synthetic introns that were efficiently spliced in cancer cells bearing SF3B1 mutations, but unspliced in otherwise isogenic wild-type cells, to yield mutation-dependent protein production. A massively parallel screen of 8,878 introns delineated ideal intronic size and mapped elements underlying mutation-dependent splicing. Synthetic introns enabled mutation-dependent expression of herpes simplex virus-thymidine kinase (HSV-TK) and subsequent ganciclovir (GCV)-mediated killing of SF3B1-mutant leukemia, breast cancer, uveal melanoma and pancreatic cancer cells in vitro, while leaving wild-type cells unaffected. Delivery of synthetic intron-containing HSV-TK constructs to leukemia, breast cancer and uveal melanoma cells and GCV treatment in vivo significantly suppressed the growth of these otherwise lethal xenografts and improved mouse host survival. Synthetic introns provide a means to exploit tumor-specific changes in RNA splicing for cancer gene therapy.
Topics: Animals; Antiviral Agents; Breast Neoplasms; Female; Ganciclovir; Genetic Therapy; Humans; Introns; Leukemia; Melanoma; Mice; Mutation; RNA Splicing Factors; Thymidine Kinase; Uveal Neoplasms
PubMed: 35241838
DOI: 10.1038/s41587-022-01224-2 -
Antiviral Chemistry & Chemotherapy 2023Brivudin, (()-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) can be considered the gold standard for the treatment of varicella-zoster virus (VZV) infections, such as herpes... (Review)
Review
Brivudin, (()-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) can be considered the gold standard for the treatment of varicella-zoster virus (VZV) infections, such as herpes zoster (shingles). It is available for clinical use in most European countries (except for the UK) and over the whole world (except for the US and Canada). Besides VZV its activity spectrum also includes various other herpesviruses, such as herpes simplex virus type 1 (HSV-1). Its activity against VZV and HSV-1 depends on phosphorylation by the virus-encoded thymidine kinase (TK). In its active form (BVDU TP or BVDU 5'-triphosphate), it can act as both substrate and inhibitor of the viral (i.e., HSV-1) DNA polymerase. It has proven to be effective against herpes zoster, including post-herpetic neuralgia (PHN). It is contra-indicated in patients concomitantly treated by 5-fluorouracil (FU), since its degradation product, ()-5-(2-bromovinyl)uracil, is inhibitory to the catabolism of FU, which may enhance the toxicity of the latter. A new compound, the bicyclic nucleoside analogue (BCNA) Cf-1743, has been described, which is a more potent inhibitor of VZV replication than BVDU and which does not interfere with the catabolism of FU. It is applicable orally, as its 5'-valine ester FV-100 (Fermavir), but has not (yet) been marketed for clinical use.
Topics: Humans; Antiviral Agents; Bromodeoxyuridine; Herpesvirus 3, Human; Herpes Zoster; Herpesvirus 1, Human; Fluorouracil; Thymidine Kinase
PubMed: 36710501
DOI: 10.1177/20402066231152971 -
Journal of Neuromuscular Diseases 2022Defects in the replication, maintenance, and repair of mitochondrial DNA (mtDNA) constitute a growing and genetically heterogeneous group of mitochondrial disorders.... (Review)
Review
Defects in the replication, maintenance, and repair of mitochondrial DNA (mtDNA) constitute a growing and genetically heterogeneous group of mitochondrial disorders. Multiple genes participate in these processes, including thymidine kinase 2 (TK2) encoding the mitochondrial matrix protein TK2, a critical component of the mitochondrial nucleotide salvage pathway. TK2 deficiency (TK2d) causes mtDNA depletion, multiple deletions, or both, which manifest predominantly as mitochondrial myopathy. A wide clinical spectrum phenotype includes a severe, rapidly progressive, early onset form (median survival: < 2 years); a less severe childhood-onset form; and a late-onset form with a variably slower rate of progression. Clinical presentation typically includes progressive weakness of limb, neck, facial, oropharyngeal, and respiratory muscle, whereas limb myopathy with ptosis, ophthalmoparesis, and respiratory involvement is more common in the late-onset form. Deoxynucleoside monophosphates and deoxynucleosides that can bypass the TK2 enzyme defect have been assessed in a mouse model, as well as under open-label compassionate use (expanded access) in TK2d patients, indicating clinical efficacy with a favorable side-effect profile. This treatment is currently undergoing testing in clinical trials intended to support approval in the US and European Union (EU). In the early expanded access program, growth differentiation factor 15 (GDF-15) appears to be a useful biomarker that correlates with therapeutic response. With the advent of a specific treatment and given the high morbidity and mortality associated with TK2d, clinicians need to know how to recognize and diagnose this disorder. Here, we summarize translational research about this rare condition emphasizing clinical aspects.
Topics: Animals; Child; DNA, Mitochondrial; Humans; Mice; Mitochondrial Myopathies; Muscular Diseases; Thymidine Kinase
PubMed: 35094997
DOI: 10.3233/JND-210786 -
Frontiers in Microbiology 2019Alpha-herpesvirus thymidine kinase (TK) genes are virulence-related genes and are nonessential for viral replication; they are often preferred target genes for the... (Review)
Review
Alpha-herpesvirus thymidine kinase (TK) genes are virulence-related genes and are nonessential for viral replication; they are often preferred target genes for the construction of gene-deleted attenuated vaccines and genetically engineered vectors for inserting and expressing foreign genes. The enzymes encoded by TK genes are key kinases in the nucleoside salvage pathway and have significant substrate diversity, especially the herpes simplex virus 1 (HSV-1) TK enzyme, which phosphorylates four nucleosides and various nucleoside analogues. Hence, the HSV-1 TK gene is exploited for the treatment of viral infections, as a suicide gene in antitumor therapy, and even for the regulation of stem cell transplantation and treatment of parasitic infection. This review introduces the effects of α-herpesvirus TK genes on viral virulence and infection in the host and classifies and summarizes the current main application domains and potential uses of these genes. In particular, mechanisms of action, clinical limitations, and antiviral and antitumor therapy development strategies are discussed.
PubMed: 31134006
DOI: 10.3389/fmicb.2019.00941 -
Annals of Neurology Oct 2021Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants...
OBJECTIVE
Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with pyrimidine deoxynucleosides deoxycytidine and thymidine ameliorates mitochondrial defects and extends the lifespan of Tk2 knock-in mouse (Tk2 ) and compassionate use deoxynucleoside therapy in TK2 deficient patients have shown promising indications of efficacy. To augment therapy for Tk2 deficiency, we assessed gene therapy alone and in combination with deoxynucleoside therapy in Tk2 mice.
METHODS
We generated pAAVsc CB6 PI vectors containing human TK2 cDNA (TK2). Adeno-associated virus (AAV)-TK2 was administered to Tk2 , which were serially assessed for weight, motor functions, and survival as well as biochemical functions in tissues. AAV-TK2 treated mice were further treated with deoxynucleosides.
RESULTS
AAV9 delivery of human TK2 cDNA to Tk2 mice efficiently rescued Tk2 activity in all the tissues tested except the kidneys, delayed disease onset, and increased lifespan. Sequential treatment of Tk2 mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2 mice dramatically improved mtDNA copy numbers in the liver and kidneys, animal growth, and lifespan.
INTERPRETATION
Our data indicate that AAV-TK2 gene therapy as well as combination deoxynucleoside and gene therapies is more effective in Tk2 mice than pharmacological alone. Thus, combination of gene therapy with substrate enhancement is a promising therapeutic approach for TK2 deficiency and potentially other metabolic disorders. ANN NEUROL 2021;90:640-652.
Topics: Animals; Compassionate Use Trials; DNA, Mitochondrial; Genetic Therapy; Humans; Mice; Mitochondria; Mitochondrial Myopathies; Mutation; Thymidine; Thymidine Kinase
PubMed: 34338329
DOI: 10.1002/ana.26185 -
International Journal of Molecular... Sep 2022Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and...
Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.
Topics: DNA, Mitochondrial; Electron Transport Complex IV; Humans; Mitochondrial Myopathies; Muscle, Skeletal; Myocardium; Succinate Dehydrogenase; Thymidine Kinase
PubMed: 36232299
DOI: 10.3390/ijms231911002 -
Thymidine Kinase 1 Drives Skin Cutaneous Melanoma Malignant Progression and Metabolic Reprogramming.Frontiers in Oncology 2022Thymidine kinase 1 (TK1) is a cell cycle-dependent kinase that catalyzes the addition of a gamma-phosphate group to thymidine. The protumorigenic role of TK1 has been...
BACKGROUND
Thymidine kinase 1 (TK1) is a cell cycle-dependent kinase that catalyzes the addition of a gamma-phosphate group to thymidine. The protumorigenic role of TK1 has been reported in various malignancies. However, the role of TK1 in skin cutaneous melanoma (SKCM) remains unclear. This study aimed to explore the molecular function of TK1 in SKCM progression.
METHODS
Bioinformatics data were acquired from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Subcutaneous xenografts were established to observe the effect of TK1 knockdown on the proliferation of SKCM cells . RNA sequencing (RNA-seq; deposited in Sequence Read Archive, SRX10950283-SRX10950285 for A375 control cells and SRX10950286-SRX10950288 for TK1-silenced A375 cells) and immunoprecipitation-mass spectrometry (IP-MS) were used to analyze TK1-related genes and pathways. Seahorse XF Cell Mito tests and glycolysis stress assays were conducted for metabolic testing.
RESULTS
TK1 was upregulated in malignant SKCM compared to that in normal tissues and cell lines. Elevated expression of TK1 was associated with poor prognosis. and assays demonstrated that TK1 promoted the proliferation and migration of SKCM cells. Moreover, TK1 was strongly associated with multiple intracellular metabolic pathways, facilitating cell mitochondrial respiration and glycolysis in SKCM malignant progression.
CONCLUSIONS
TK1 drives SKCM malignant progression and supports metabolic reprogramming, indicating that TK1 serves as a therapeutic target for SKCM.
PubMed: 35311151
DOI: 10.3389/fonc.2022.802807 -
Pharmaceutics Feb 2022Androgen deprivation therapy (ADT) is a systemic therapy for advanced prostate cancer (PCa). Although most patients initially respond to ADT, almost all cancers... (Review)
Review
Androgen deprivation therapy (ADT) is a systemic therapy for advanced prostate cancer (PCa). Although most patients initially respond to ADT, almost all cancers eventually develop castration resistance. Castration-resistant PCa (CRPC) is associated with a very poor prognosis, and the treatment of which is a serious clinical challenge. Accumulating evidence suggests that abnormal expression and activation of various kinases are associated with the emergence and maintenance of CRPC. Many efforts have been made to develop small molecule inhibitors to target the key kinases in CRPC. These inhibitors are designed to suppress the kinase activity or interrupt kinase-mediated signal pathways that are associated with PCa androgen-independent (AI) growth and CRPC development. In this review, we briefly summarize the roles of the kinases that are abnormally expressed and/or activated in CRPC and the recent advances in the development of small molecule inhibitors that target kinases for the treatment of CRPC.
PubMed: 35335873
DOI: 10.3390/pharmaceutics14030498 -
Journal of Inherited Metabolic Disease Mar 2021At present, there is just one approved therapy for patients with mitochondrial diseases in Europe, another in Japan, and none in the United States. These facts reveal an... (Review)
Review
At present, there is just one approved therapy for patients with mitochondrial diseases in Europe, another in Japan, and none in the United States. These facts reveal an important and significant unmet need for approved therapies for these debilitating and often fatal disorders. To fill this need, it is critical for clinicians and drug developers to work closely with regulatory agencies. In the United States, mitochondrial disease patients and clinicians, the United Mitochondrial Disease Foundation, and pharmaceutical industry members have engaged with the Food and Drug Administration to educate each other about these complex and heterogeneous diseases and about regulatory requirements to obtain approvals for novel therapies. Clinical development of therapies for rare diseases has been facilitated by the 1983 US Orphan Drug Act (ODA) and similar legislation in Japan and the European Union. Further legislation and regulatory guidance have expanded and refined regulatory flexibility. While regulatory and financial incentives of the ODA have augmented involvement of pharmaceutical companies, clinicians, with patient advocacy groups and industry, need to conduct natural history studies, develop clinical outcome measures, and identify potential supportive surrogate endpoints predictive of clinical benefit, which together are critical foundations for clinical trials. Thus, the regulatory environment for novel therapeutic development is conducive and offers flexibility for mitochondrial diseases. Nevertheless, flexibility does not mean lower standards, as well-controlled rigorous clinical trials of high quality are still required to establish the efficacy of potential therapies and to obtain regulatory agency approvals for their commercial use. This process is illustrated through the authors' ongoing efforts to develop therapy for thymidine kinase 2 deficiency.
Topics: Drug Approval; Humans; Mitochondrial Diseases; Orphan Drug Production; Rare Diseases; United States; United States Food and Drug Administration
PubMed: 33368420
DOI: 10.1002/jimd.12353