Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Introduction of the Herpes simplex virus thymidine kinase (HSV-tk) gene into target cells renders them susceptible to killing by ganciclovir (GCV). We are studying the use of HSV-tk-transduced T lymphocytes in the context of hematopoietic stem cell transplantation. We have previously shown, in vitro and in vivo, the occurrence of transduced cells resistant to GCV due to a deletion within HSV-tk. This deletion, a consequence of the presence of cryptic splice donor and acceptor sites, originates in the retroviral producer cell. Here we adopt two different methods that introduce third-base degenerate changes at the cryptic splice sites and so prevent splicing. Consequently, the HSV-tk protein is unaltered and the sensitivity of the target cells to GCV is preserved. The use of this mutated HSV-tk should reduce the likelihood of the development of resistant genetically modified cells during clinical trials.
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PMID:Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene. 1148 86

Allogeneic donor T lymphocytes manipulated genetically to express the herpes simplex virus thymidine kinase (HSV-TK) gene have emerged as promising tools to alter the balance between graft versus host disease and graft versus leukemia after allogeneic stem cell transplantation, since they can be eliminated selectively in vivo with ganciclovir. Recently, it was reported that in SFCMM-3, an HSV-TK-encoding retroviral vector, two cryptic splice sites in the HSV-TK sequence led to the generation of an HSV-TK splice variant (deltaHSV-TK) that encodes a ganciclovir-resistant gene product. In order to quantify wtHSV-TK and deltaHSV-TK RNA levels we have developed two real time Taqman PCR assays. We demonstrate that the sensitivity of both PCR assays is 10(-4). It was found that the splice variant is generated in the packaging cell line and results in approximately 4.8+/-1.9% of virions that contain deltaHSV-TK RNA. After transduction of human T cells no significant increase in deltaHSV-TK RNA could be detected. Thus, at maximum 4.2+/-1.2% of T cells transduced with SFCMM-3 will be resistant to ganciclovir due to this mechanism only. Together, these assays provide a powerful method to monitor patients in future clinical trials.
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PMID:Development and application of quantitative real time PCR and RT-PCR assays that discriminate between the full-length and truncated herpes simplex virus thymidine kinase gene. 1271 Oct 61


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