Vector development for suicide gene therapy

Sammanfattning: Gene therapy is used to treat conditions that arise from errors in the genetic makeup of cells either congenital diseases resulting from a deletion or mutation in a gene or malignant diseases where genetic regulation mechanisms have been deranged. Suicide gene therapy is one of several gene therapeutic approaches to treat cancer. A suicide gene is a gene encoding a protein, frequently an enzyme, that in itself is non-toxic to the genetically modified cell. However, when the cell is exposed to a specific non-toxic prodrug, this is selectively converted by the gene product into toxic metabolites that kill the cell. The gene transfer technologies available today have limited effectiveness - only a fraction of target cells can be genetically modified. This can be compensated by several processes taking place after transgene expression and enzymatic conversion of the prodrug. In vivo, and also in cell culture, gap junction mediated intercellular communication (GJIC) enables the spread the converted metabolites. However, many tumours lacking connexin expression are not susceptible to this form of treatment. To bypass the requirement for gap junctions, one can link the HSV-tk gene to the gene of another herpes virus protein, VP22. The VP22 protein has been shown to pass freely between cells. We show the ability of VP22-GFP fusion protein to spread in cell culture and translocate into the nucleus of the recipient cells (Paper I), Also, VP22 fusion to TK (VP22-TK) is transferred to the surrounding cells in cell culture and in vivo in a mouse tumour model, in the absence of gap junction formation in a connexin negative tumour cell line and sensitises the tumours to GCV. However, relatively high proportion (50%) of VP22-TK expressing cells is necessary to exert a full effect (Paper II). Therefore, we have analysed the VP22 protein in detail. Mapping of functional domains revealed several independent functional domains and localisation signals in the VP22 polypeptide. These constructs can have a potential use in targeting defined functional peptides to specific subcellular locations (Paper III). Suicide gene technology can provide a safety switch for cytotoxic effector cells, to treat graft versus host disease (GvHD). In this application, it would be ideal to achieve 100% effectiveness of modification of the target cells. Different selection marker genes for selection have been utilised, and these fall into two categories: metabolic and physical selection markers. As there is requirement for a resistance marker better suited for selection of human cells, we have characterised the ouabain resistance gene (OuaR). OuaR belongs to the Na+ ,K+ -ATPase gene family - a housekeeping enzyme present in all mammalian cells. It maintains cellular homeostasis of Na+ and K+. Ouabain belongs to cardiac glucoside group of drugs. It inactivates the naturally occurring non-resistant Na+ ,K+ -ATPase which results in very rapid cell death. OuaR is a PCR-generated point mutant L799C that is completely resistant to inhibition by ouabain and can be used for background free selection in very short time - 12-36 hours, compared to 10- 15 days for classical antibiotic selection. We show the versatility of OuaR gene transfer by transient transfection and stable retrovirus-mediated integration in several cultured cell lines as well as primary human donor T-lymphocytes (Paper IV). We have prepared suicide gene therapy vectors containing HSV-tk and OuaR selection marker fusion gene in extensively optimised retroviral backbone SF91. The OuaR selection marker allows the transduced cells to be selected chemically in 36 hours, reducing the necessary time for in vitro culture to a week. The transduced T-cells display high sensitivity to gancyclovir. The rapid gene transfer and selection process prevents culture-related changes in T-cell function and helps to establish protocols to gain control over cytotoxic cells by suicide gene transfer (Paper V). In conclusion, we have characterised vectors for suicide gene therapy with the potential to achieve 100% efficiency of genetic modification of the target cells. VP22 protein vectors enable the spread of fusion protein to all surrounding cells after gene expression in the gene-modified cells. Viral or plasmid vectors containing the OuaR selection marker allow the gene- modified cells to be selected immediately after gene expression, reducing the time for selection and time-dependent in vitro cell culture-related sideeffects to minimum.