Abstract
Donor T lymphocytes engineered to express a ‘suicide gene’ that facilitates negative selection following exposure to a non-toxic pro-drug provides an alternate strategy for the management of graft versus host disease occurring after allogeneic hematopoietic cell transplantation. The herpes simplex virus thymidine kinase (HSV-tk) gene remains the most widely studied suicide gene, but has limitations in this setting; these include restriction of ganciclovir (GCV) induced cytotoxicity to proliferating HSV tk+ cells, immunogenicity of HSV-tk, an inability to use acyclovir or GCV for viral therapy and acquired resistance to GCV. Cytosine deaminase (CD), an alternate suicide gene, converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). Sensitivity of cells to 5-FU can be further increased by exposure to a gene encoding uracil phosphoribosyl transferase (UPRT) which catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate (5-FUMP). Using a chimeric gene (NG/CD) expressing the truncated human nerve growth factor receptor for positive selection linked by a [Gly4Ser]2 linker to Sacchromyces CD gene, we examined strategies to achieve optimal CD and UPRT activity on a single retroviral vector. Human T-cells co-expressing NG/CD and UPRT gene achieved through sequential transduction with two independent vectors (NG/CD + U) were compared to 3 construct strategies using a single vector. The vector NG/CD-i-U utilizes an internal ribosomal entry site to express both genes from a single bicistronic message, and this was compared, to 2 viruses using direct fusions of the NG/CD and UPRT genes with or without a [Gly4Ser]2 linker termed NG/CD-L-U and NG/CD-U respectively. Parameters tested included expression based on flow cytometry and UPRT Western analysis, CD and UPRT activity by enzymatic determinations, cytotoxicity and the induction of apoptosis in proliferating and quiescent human T-cells. UPRT was found to enhance cytotoxicity to 5-FC in all constructs compared to the NG/CD alone (LD 90, 38.7 micrograms/ml). Cytoxicity was comparable in the NG/CD+U (double transductant) and NG/CD-i-U with a documented LD 90 of 0.13 micrograms/ml of 5-FC; in addition, the LD 90 in 5-FU was 0.03 and 0.01 micrograms/ml for the doubly transduced and NG/CD-i-U transduced cells respectively. In contrast, cytoxicity was decreased using the tri-chimeric contructs (LD 90, 6.4 micrograms/ml, and 10.9 micrograms/ml 5-FC respectively for NG/CD-L-U and NG/CD-U). Based on enzymatic testing, a decrease in both UPRT and CD is consistent with these differences. Cytotoxicity occurs through apoptosis, with increased annexin-V binding following exposure to 5-FC. Additionally, we demonstrated the ability to induce cytotoxicity and apoptosis in quiescent cell populations. In summary, we have demonstrated that in association with NG/CD, sufficient UPRT activity can be achieved using a single retroviral vector resulting in markedly enhanced cytotoxicity to 5-FC. The 5-FC concentration necessary to eliminate virtually 100% of transduced human T-cells can easily be achieved in human serum. This approach may have advantages over the HSV-tk system in eradicating T cells in a clinical setting.
Identification, purification, and characterization of truncated forms of the human nerve growth factor receptor