Gene Therapy Strategies for the enhancement of Radiotherapy in Human Tumours (Dr.Gillian Ross)
2 year project with a grant of £79,652.00
1998 – 2000
This project termed “suicide gene therapy” comprises two steps. In the first step the brain tumour cells are injected with a gene that instructs the cell to make a foreign enzyme. The second step involves exposing the patient to an inactive anti-cancer drug, which when exposed to the enzyme, is transformed into a toxic drug that selectively kills only the cells of the brain tumour. The first stage of the project has already shown that the combination of the enzyme and anti-cancer drug kill the malignant cells. The second set of experiments focus on trying to assess how exact timing and exposure of the tumour to the inactive cancer drug must be to gain most effective tumour killing.
This project was voted one of the top three cancer research projects at the Royal Marsden for 1998, chosen out of 100 projects.
The genes for three “foreign” enzyme genes were transfected into the human glioma cell line U87. HSV-tk is widely utilized for GDEPT strategies, with the prodrug, ganciclovir. Two novel bacterial enzymes, carboxypeptidase G2 (CPG2) were developed that can activate nitrogen mustard prodrugs to alkylating agents and nitroreductase (NR) that can also activate mustard prodrugs.
Conclusion: The 2 years were spent optimizing the transfection of the 3 target enzyme genes, CPG2, NR and tk into glioma in vitro. This required molecular biological expertise. Following transient transfections, stable cell lines in U87 glioma cells expressing the 3 enzymes were selected. The enzymes were modified by mutation to be expressed either intracellularly or cell surface tethered or both. Biochemical analyses were performed on the stable cell lines to ensure that the enzyme activity was sufficiently high for subsequent prodrug activation. These analyses were repeated over time courses every month to ensure that there was no determioration in quality. Appropriate novel produgs were synthesized in house by chemists for activation by CPG2 and NR, since they are not available commercially.
Since tk has been used by other groups in the treatment of lioma clinically, it was interesting to note that 100% cell death could never be achieved when co-incubated with ganciclovir. By contrast, 100% cell death could always be achieved with CPG2 and NR and their relevant prodrugs. In accordance with findings on cytotoxicity, we found that we were unable to achieve 100% cell death in the bystander experiments with U87-tk cells in combination with ganciclovir.
Our conclusion from this are that localization therapy will be required to completely eradicate all cells for efficiency with the tk-ganciclovir GDEPT systems. By contrast, we would anticipate that U87-CPG2 or U87-NR could be used clinically without the need for additional irradiation treatment, thus simplifying these GDEPT systems.