Optimization of suicide gene therapy in stem cell transplantation with special reference to the selection marker OuaSelect

Sammanfattning: For many patients suffering from leukemia and other hematological malignancies, allogeneic hematopoietic stem cell transplantation (HSCT) represents the only curative approach. However, an optimal HSCT procedure is yet to be developed. Transplant-related morbidity and mortality are severe complications arising from this treatment modality. Donor lymphocytes have been recognized to contribute to tumor cell eradication, a graft-versus-leukemia (GvL) effect. However, they can also cause graft-versus-host-disease (GvHD), a major contributor to transplant-related morbidity and mortality. A challenge of allogeneic HSCT is to prevent GvHD without loosing the GvL effect. Suicide gene modification of the donor lymphocytes has been proposed as a means of exploiting their beneficial effects, and allowing their safe elimination in the event of causing significant GvHD. In the ex vivo preparation of cells for suicide gene therapy, a selection step to purify genetically modified cells is necessary. The selection marker used in early gene therapy trials was the neomycin resistance gene (NeoR). Our first study aimed at marking the stem cell graft in autologous HSCT for multiple myeloma (paper I). The retroviral vector used encoded NeoR. The extension of ex vivo cell processing to encompass the seven days required for NeoR selection is not applicable for stem cell gene transfer protocols. Hence, selection was not performed. The results of this study showed that gene marking of stem cells was safe and marked cells were detected until end of study, 5 years after transplantation. In addition, the data suggested that it is the inability to achieve complete eradication of residual tumor cells after chemotherapy, rather than residual tumor cells contaminating the graft that constitute the source of malignant cells causing relapse after autologous HSCT. Selection of gene marked cells prior to transplantation could have strengthened the results, since it would have greatly increased the ratio of marked cells transplanted. In paper II the development of a new selection marker OuaSelect is described. OuaSelect is the human Na+, K+ ATPase alpha 1 subunit with two amino acid substitutions; Q118R and N129D. It confers two orders of magnitude increased resistance to the cardiac glycoside ouabain. In paper II and III we demonstrate that >98% pure populations of genetically modified cells can be obtained within only 48 hours of selection. OuaSelect uniquely combines affordable and highly efficient selection with high yield and short selection time. Moreover, the high similarity to the native human protein minimizes the risks for immunogenicity. Herpes simplex thymidine kinase (HSVtk)/ganciclovir (GCV) is the most frequently used suicide gene system. HSVtk expression of donor lymphocytes and GCV administration can abrogate GvHD in the setting of allogeneic HSCT. In paper III we show that protein fusion allows for highly correlated co-expression and function of OuaSelect and HSVtk in retroviral vector transduced lymphocytes. Limitations to the HSVtk/GCV system include toxicity at administered doses of GCV and a rather slow response rate to prodrug administration. In paper IV, we could demonstrate improvements of the HSVtk/GCV system with a codon optimized A168H HSVtk mutant (TK.007). We observed a higher rate of GCV induced cell death for TK.007 in hematopoietic cells. In addition, a significantly higher GCV sensitivity and bystander effect was observed in cancer cell lines, allowing equal efficiency at lower GCV doses. In summary, the TK.007 suicide gene potentially represents a valuable improvement to the clinical use of HSVtk.