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Direct and Bystander Killing of Sarcomas by Novel Cytosine Deaminase Fusion Gene¹

Departments of Orthopedic Surgery [M. R., W. P., M. G., C. L., D. R. C.], Preventive Sciences [P. M.], and Pediatrics [P. O.], University of Minnesota, Minneapolis, Minnesota 55455, and Department of Pediatric Oncology, Alberta Children’s Hospital, Calgary, Alberta, T2T 5C7 Canada [V. L.]

Soft tissue and bone sarcomas of the extremities can be difficult to eradicate, and standard treatment may require limb amputation. New therapies to decrease tumor size could improve the effectiveness of treatment and decrease the frequency of limb amputation. Cytosine deaminase (CD)-based gene therapy has been shown to be effective in decreasing growth of solid tumors when animals with CD-expressing tumor cells are treated with 5 fluorocytosine (5FC), an inert prodrug that is converted to 5-fluorouracil (5FU) by CD. In this investigation, we used a novel CD-containing fusion gene to determine whether CD-based gene therapy affected soft tissue or bone sarcomas. The novel fusion gene (NGFR-CD) encodes for a protein with extracellular and transmembrane domains of human nerve growth factor receptor (NGFR) and cytoplasmic CD. Murine 2472 (2) sarcoma cells were transduced with fusion genes containing either the bacterial (NGFR-_bCD) or yeast (NGFR-_yCD) CD gene. 5FC treatment killed NGFR-_bCD- and NGFR-_yCD-transduced sarcoma cells in vitro through direct and bystander effects (P < 0.01). In contrast, 5FC treatment of mice with s.c. 2NGFR-_bCD or 2NGFR-_yCD tumors affected only 2NGFR-_yCD tumors. 5FC had no effect on growth of NGFR-_bCD tumors but caused significant decrease in the size of 2NGFR-_yCD tumors (51 ± 60 versus 938 ± 767 mm³, treated versus control, P < 0.01). Evaluation of bystander killing in vivo revealed significant tumor killing, with a 5-fold reduction in s.c. tumor volume evident in saline versus 5FC-treated mice when tumors were comprised of 90% 2472 cells and 10% 2NGFR-_yCD selected for fluorescence-activated cell sorting (P < 0.01). Bone sarcomas were eliminated in 9 of 10 5FC-treated mice, compared with 11.8 ± 6.0 mm² in saline-treated mice (P < 0.002). In addition, 5FC treatment of bone sarcomas caused a significant reduction in cancer-induced bone destruction (P < 0.002) and resulted in a reduction in the number of osteoclasts. Finally, 5FC treatment had no effect on animal weight or survival, whereas doses of 5FU providing equivalent tumor reduction as 5FC resulted in treatment-associated deaths and significant weight loss (P < 0.001).

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