The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors. The reaction kinetics of thymidine and thymidine analogues phosphorylation is complicated and only partly known. Nucleoside analogues are the major class of antiviral agents used for the management of Herpes Simplex Virus infections. Therefore, they are able to monophosphorylate certain nucleoside analogues whereas cellular nucleoside kinases cannot do so or only to a very limited extent. (adds one phosphate group) catalyzed by a virus-encoded enzyme thymidine kinase. Arvin A, Campadelli-Fiume G, Mocarski E, et al.
The HSV-TK enzyme has 1000 times more affinity for the substrate GCV than the host cell TK 11. HSV1-TK can also phosphorylate various nucleoside analogs of GCV such as acyclovir, ganciclovir, and penciclovir. HSV-1 encodes about 70 genes some are immediate early genes. The role of E-cadherin in suicide gene therapy is established in an in vivo model of pancreatic ductal adenocarcinoma. Following delivery of the gene encoding HSVTK to cancer cells, the prodrug is administered. Because HSVTK, but not the endogenous thymidine kinase (TK), is able to phosphorylate GCV, cytotoxicity is limited to the site of transfection. The thymidine kinase (TK) gene encoded by herpes simplex virus type 1 (HSV-1) is by far the most intensively studied suicide gene. In addition, HSV-1 TK is able to phosphorylate a wide range of pyrimidine and purine nucleoside analogs, which forms the basis for the selective therapy for herpesviral disease (De Clercq, 2000) and now, also, cancer gene therapy. It was demonstrated that the A167Y mutation resulted in the specific phosphorylation of purine nucleoside analogs but not pyrimidine nucleoside analogs, including the abundant natural substrate dThd.
The nucleoside analog ganciclovir (GCV) did not affect the growth of 143B EBV TK or 143B TK cells but effectively killed 143B HSV-1 TK cells. The EBV BamHI X fragment, containing the BXLFI ORF which encodes the EBV TK, was subcloned into pBluescript II SK (Stratagene, La Jolla, Calif. HSV TK is known to be a deoxypyrimidine kinase, able to phosphorylate both dC and thymidine. (1996) Analysis of phosphorylation pathways of antiherpesvirus nucleosides by varicella-zoster virus-specific enzymes. The BV enzyme phosphorylates some modified nucleosides and acyclonucleosides and l enantiomers of thymidine and related antiherpetic analogs. Interestingly, HSV TKs are not stereoselective, being able to phosphorylate both enantiomers of thymidine (TdR) and other TdR analogs. The HSV-1 TK phosphorylates ACV, PCV, and GCV to their monophosphate forms, which are subsequently converted to the diphosphorylated and triphosphorylated derivatives by cellular kinases. In contrast, BVDU and its closely related analogue BVaraU are phosphorylated to both their 5 monophosphate and 5 diphosphate metabolites by HSV-1 TK and its associated thymidilate kinase activity and are converted to the 5 triphosphate form by cellular kinases (25). The HSV TK gene coding region is 1,128 nucleotides (nt) in length and encodes a protein of 376 amino acids (aa) (Fig. 553), located downstream of the nucleoside-binding site of the enzyme.
Suicide Gene Therapy By Herpes Simplex Virus-1 Thymidine Kinase (HSV-tk)
(e) incubating said host cells harboring said vector at a temperature not permissive for growth of E. The method of any one of claims 1, 2, 9 or 17 wherein said host cells contain an additional gene encoding an enzyme able to phosphorylate nucleosides or nucleotides. Therefore, by rapid evolution of HIV, mutations in HIV RT arise frequently in infected individuals and render the virus resistant to nucleoside analogs and other antiviral therapies. Figure 5 illustrates induction of ddC and AZT sensitivity of E. coli by co-expression of HSV TK and HIV RT. In summary, we showed that the proteins were delivered in a functionally active form and that the sensitivity of the cell lines treated with the enzyme lipid complexes showed increased sensitivity to cytotoxic nucleoside analogues phosphorylated by the enzymes. The specific dThd kinase activity of the purified recombinant Dm-dNK was 64 nmol/ g/min. We were not able to induce complete killing of all of the cells in the cultures. The herpes simplex virus thymidine kinase gene type 1 (HSV-Tk) ganciclovir (GCV) system is a novel therapeutic strategy for the modulation of graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation (allo-SCT). The HSV-Tk gene renders the cells susceptible to killing by ganciclovir (GCV), a nucleoside analogue used in the treatment of herpesvirus infections.HSV-Tk phosphorylates GCV, converting it to a monophosphorylated form, which is phosphorylated further by cellular DNA kinases before it is incorporated into newly synthesized DNA. The HSV-Tk gene encodes an enzyme able to convert nontoxic prodrugs such as GCV and acyclovir to cytotoxic metabolites. 123-I-MIP-1095, a radiolabeled glutamate-urea-lysine analogue, selectively binds PSMA, which allows imaging of PSMA-expressing prostate cancer cells with gamma scintigraph. 131I-TM-601 binds to tumor cells of neuroectodermal origin and is internalized; administered once, it may be used as a radioimaging agent; repeated administration may result in a tumor-specific, cumulative radiocytotoxic dose of I 131. The dNKmu enzymes were expressed in the breast cancer cell lines MDA-MB-231 (ER-) and MCF7 (ER+). Our data suggest that the triple phosphorylated DFDC catalyzed by dNKmu inhibited the replication of adenovirus with a simultaneous positive therapeutic effect to cancer cells. Then we performed the tumor-bearing mice tests in vivo to observe the safety and sensitivity of SG500-dNKmu nucleotide analogs in animal experiments, and investigated the breast cancer cell killing effect of this treatment system to provide new ideas for breast cancer treatment. The viral E1B55-kDa gene was replaced by an expression cassette encoding dNKmu containing the sequence of CMV promoter + multiple clone site + SV40 poly A into plasmid SG500 to generate plasmid SG500-dNKmu (Fig.