Functional expression of thymidine kinase in human leukaemic and colorectal cells, delivered as EGFP fusion protein by herpesvirus saimiri-based vector.
Herpesvirus saimiri (HVS) has the capacity to incorporate large amounts of heterologous DNA and can infect many different human cell types. To develop its potential as a gene therapy vector, we cloned herpes simplex virus thymidine kinase (TK) gene into the HVS genome in the form of an enhanced green fluorescent protein (EGFP) fusion protein, using a cosmid-based approach. At multiplicity of infection = 100 over 90% of human leukemic K562 and Jurkat cells were transduced with HVS/EGFP-TK. Conditions of no selective pressure expression were maintained at > 92% per cell division. Expression of the EGFP-TK fusion protein rendered transfected leukaemic cells sensitive to cytotoxic treatment with the prodrugs ganciclovir (GCV) and (E)-5-(2-bromovinyl)-2'deoxyuridine (BVDU) at concentrations as low as 10 ng/ml. The viral vector was also screened against a panel of colorectal and pancreatic carcinoma cell lines. All cell lines were transduced but showed a range of sensitivity to infection. Three of the most easily transduced cell lines: Mia PaCa, HCT116 and SW948 transduced with HVS/EGFP-TK were effectively ablated by subsequent treatment with GCV or BVDU. Our results show that in its current form HVS/EGFP-TK could be utilized as an antitumour agent, or it could be developed further by inclusion of a therapeutic gene, with TK presence ensuring a mechanism of controlled removal of modified cells when no longer necessary. These results suggest that HVS/EGFP-TK has a great potential for a number of gene therapy applications.
Hoggarth
Jennifer H
JH
Molecular Medicine Unit, University of Leeds, St James' University Hospital, Leeds, UK. medjhh@leeds.ac.uk
Jones
Elena
E
Ensser
Armin
A
Meredith
David M
DM
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D016645:Aotidae; D001483:Base Sequence; D001973:Bromodeoxyuridine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002478:Cells, Cultured; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000628:therapy*; D015774:Ganciclovir / Q000494:pharmacology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D007668:Kidney / Q000378:metabolism; D007938:Leukemia / Q000378:metabolism / Q000628:therapy*; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000628:therapy*; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D000998:Antiviral Agents; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C020235:brivudine; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; D015774:Ganciclovir