Deletion of the Viral Thymidine Kinase in a Meq-Deleted Recombinant Marek’s Disease Virus Reduces Lymphoid Atrophy but Is Less Protective

Marek’s disease (MD) is a ubiquitous disease of domesticated chickens and its etiologic agent is the Gallid alphaherpesvirus 2 (GaHV-2), also known as Marek’s disease virus (MDV). MD is currently controlled by vaccination using live attenuated strains of MDV (e.g., CVI988/Rispens), non-pathogenic serotypes of MDV (GaHV-3), or non-pathogenic strains of the related Melagrid alphaherpesvirus 1 (MeHV-1). One attractive strategy for the production of new vaccine strains is a recombinant MDV attenuated by the deletion of the major viral oncogene meq. However, meq-deleted variants of MDV cause atrophy of the bursa and thymus in maternal antibody-negative chickens, and the resulting immunosuppression makes them unsuitable. Herein we detail our attempt to mitigate the lymphoid atrophy caused by meq-deleted MDV by further attenuation of the virus through ablation of the viral thymidine kinase (tk) gene. We demonstrate that ablation of the viral tk from the meq-deleted virus rMd5B40/Δmeq resulted in a virus attenuated for replication in vitro and which spared chickens from atrophy of the lymphoid organs in vivo. When the rMd5B40/Δmeq/Δtk/GFP was used as a vaccine it was protective against challenge with the vv+MDV strain 686, but the protection was less than that provided by the CVI988/Rispens vaccine.

Thymidine Kinase 1: Making Its Mark In Immunotherapy

Cancer is one of the leading causes of death worldwide. In the U.S. alone, almost 2 million people will be diagnosed with cancer each year and just over a quarter of those diagnosed will pass away from the disease. Skin cancers are the most common forms of cancer. Early detection of cancer and cancer biomarkers enables clearer understanding of cancer progression in a patient and more effective treatments in response to the disease. Clinically relevant biomarkers are not only tools for early diagnosis of cancer but may also prove useful as immune targets for various immunotherapies, such as monoclonal antibody-based therapy and chimeric antigen receptor (CAR) T-cell therapy. This review provides a brief overview of the rescue pathway enzyme thymidine kinase 1 (TK1) and its history and biology, as well as discusses its role as a biomarker and potential immune target.

Neural Progenitor Cells Expressing Herpes Simplex Virus-Thymidine Kinase for Ablation Have Differential Chemosensitivity to Brivudine and Ganciclovir

Neural progenitor cell (NPC) transplants are a promising therapy for treating spinal cord injury (SCI), however, their long-term role after engraftment and the relative contribution to ongoing functional recovery remains a key knowledge gap. Selective human cell ablation techniques, currently being developed to improve the safety of progenitor cell transplant therapies in patients, may also be used as tools to probe the regenerative effects attributable to individual grafted cell populations. The Herpes Simplex Virus Thymidine Kinase (HSV-TK) and ganciclovir (GCV) system has been extensively studied in the context of SCI and broader CNS disease. However, the efficacy of brivudine (BVDU), another HSV-TK prodrug with potentially reduced bystander cytotoxic effects and in vivo toxicity, has yet to be investigated for NPC ablation. In this study, we demonstrate successful generation and in vitro ablation of HSV-TK-expressing human iPSC-derived NPCs with a >80% reduction in survival over controls. We validated an HSV-TK and GCV/BVDU synergistic system with iPSC-NPCs using an efficient gene-transfer method and in vivo ablation in a translationally relevant model of SCI. Our findings demonstrate enhanced ablation efficiency and reduced bystander effects when targeting all rapidly dividing cells with combinatorial GCV and BVDU treatment. However, for use in loss of function studies, BVDU alone is optimal due to reduced nonselective cell ablation.

Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma

Background Thymidine kinase 1 (TK1) plays a key role in the synthesis of deoxythymidine triphosphate (dTTP) and is thus important for DNA replication and cell proliferation. The expression of TK1 is highest during S-phase, and it is rapidly degraded after mitosis. In cancer cells, TK1 is upregulated, resulting in leakage of excess TK1 into the blood. Consequently, serum TK1 has been used as a diagnostic and prognostic cancer biomarker, mainly in human medicine. The aims of this work were to characterize equine TK1 and to evaluate its suitability as a serum biomarker for equine lymphoma. Results Equine TK1 was cloned, expressed in E. coli and affinity purified. The purified recombinant horse TK1 showed broad substrate specificity, phosphorylating pyrimidine deoxyribo- and ribonucleosides and, to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor. Serum TK1 activity was measured in samples collected from horses with confirmed or suspected lymphoma and control horses with and without concurrent diseases. Serum TK1 activity levels were significantly higher in horses with lymphoma (p < 0.0005) and suspected lymphoma (p < 0.02) and in tumour-free groups with diverse diseases (p < 0.03) than in controls without concurrent diseases. There was a significant difference between the lymphoma group and the tumour-free group with diverse diseases (p < 0.0006). Furthermore, receiver operating characteristic analysis revealed a sensitivity of 0.86, a specificity of 0.95 and an AUC (area under the curve) of 0.92 compared to the controls without concurrent diseases, with a sensitivity of 0.97, a specificity of 0.71 and an AUC of 0.88 when compared with the tumour-free group with diverse diseases. Conclusion Equine TK1 showed high specific activity and broader substrate specificity than human TK1. Anticancer and antiviral thymidine analogues were efficiently phosphorylated by horse TK1, suggesting that these analogues might be good candidates for chemotherapy in horses. Serum TK1 activity was significantly higher in horses with lymphoma than in controls. ROC analysis indicated that serum TK1 could serve as a promising cancer biomarker in horses.

Immunohistochemistry for Thymidine Kinase-1 (TK1): A Potential Tool for the Prognostic Stratification of Breast Cancer Patients

Breast cancer (BC) is the most frequent non-cutaneous malignancy in women. Histological grade, expression of estrogen and progesterone receptors (ER and PgR), overexpression/amplification of the human epidermal growth factor receptor 2 (HER2) oncogene, and proliferative activity measured with ki-67 provide important information on the biological features of BC and guide treatment choices. However, a biomarker that allows a more accurate prognostic stratification is still lacking. Thymidine kinase-1 (TK1), a ubiquitous enzyme involved in the pyrimidine nucleotide recovery pathway, is a cell-proliferation marker with potential prognostic and predictive impacts in BC. Eighty (80) cases of invasive BC with a long-term follow-up were retrospectively selected, and clinicopathological data were collected for each patient. TK1 tissue expression was evaluated immunohistochemically. Data suggested that TK1 expression levels are positively correlated with ER and PgR expression, and negatively correlated with HER2 status and the impact on patients' distant recurrence-free survival (DRFS): in detail, among patients undergoing adjuvant chemotherapy, lower TK1 levels are correlated with better DRFS. Therefore, these results contribute to furthering the knowledge of TK1, suggesting a possible and important role of this enzyme as a biomarker in the stratification of BC patients.

AVALIAÇÃO DA TERAPIA GÊNICA DO SUICÍDIO COM USO DE GENES SUICIDAS PARA O COMBATE AO CÂNCER: REVISÃO INTEGRATIVA

Introdução: Durante décadas os tratamentos quimioterápicos convencionais foram empregados no câncer devido à eficácia em matar as células tumorais. Diante disso, a terapia gênica com uso de genes suicidas surge como uma das abordagens mais inovadoras para o desenvolvimento de agentes antineoplásicos com maior seletividade tumoral. Assim, entre os genes suicidas disponíveis, a timidina quinase é investigada em vários modelos de tumor. Contudo, o uso dessa enzima apresenta limitações devido ao sistema de imunogenicidade. Recentemente, pesquisadores passaram a utilizar o sistema de gene suicida da Caspase-9 induzível, a qual apresentou resultados mais favoráveis se comparado à timidina quinase. Objetivo: Objetiva-se construir uma revisão integrativa da literatura sobre a terapia gênica do suicídio avaliando o uso de genes suicidas, sobretudo da timidina quinase e da caspase-9 induzível para o combate ao câncer. Material e Métodos: Foi realizada uma revisão integrativa de literatura por meio de uma pesquisa nos bancos de dados PubMed, Scopus e LILACS com os seguintes descritores “terapia gênica de genes suicidas”, “câncer”, “avaliação”, “timidina quinase” e “caspase-9 induzível” simultaneamente às correspondentes em inglês, em intervalo de 11 anos (2010-2021), com critérios de inclusão preestabelecidos. Para o cruzamento dos descritores, utilizou-se um protocolo com os seguintes booleanos: Gene Therapy of Suicide Gene AND Cancer AND Assessment AND Thymidine Kinase AND Inducible Caspase-9. Ao final, 4 artigos foram selecionados. Resultados: Foi selecionado 1 artigo (n = 25%) que abordava o tema da avaliação na terapia de genes suicidas. Em contrapartida, os critérios de exclusão foram estudos que não contemplavam o efeito avaliativo desses genes na terapia gênica, resumindo-se a 3 artigos selecionados (n = 75%). Com isso, observou-se que a avaliação do uso de genes suicidas ainda é uma abordagem recente, necessitando de mais estudos que contemplem essa temática. Conclusão: A avaliação da terapia gênica do suicídio pautada no uso de genes suicidas para o combate ao câncer é promissora, mesmo que recente e dos mínimos estudos publicados. Além disso, o sistema de entrega utilizando a caspase-9 induzível é mais viável do que o sistema de entrega da timidina quinase, já que esse sistema se limita à imunogenicidade do transgene viral.