462. Comparison of EMCV IRES and FMDV 2A Cleavage Factor for Expression of Canine MGMT and a Second Transgene Using HIV-1 Vectors

We previously described the cloning and expression of canine methylguanine-DNA methyl transferase (MGMT) using monocistronic HIV-1 and gamma-retroviral vectors (Zaboikin M et. al. (2004) Hum Gene Ther 15:383-392). In that study, we showed that the canine MGMT was as efficient as the human MGMT for providing resistance to nitrosourea drugs such as BCNU. Here, we describe the creation and testing of dual-gene expression HIV-1 vectors encoding canine MGMT and EGFP under control of the EF1α promoter. The two genes were expressed either using encephalomyocarditis virus internal ribosome entry site (EMCV IRES) or the foot and mouth disease virus (FMDV) 2A cleavage factor. Vector stocks were prepared by transient transfection of 293T cells and used for infection of canine thyroid-adenocarcinoma cells. The cells were then subject to selection with O 6 -benzylguanine (BG) and BCNU. Flow cytometry of cells prior to and following drug selection indicated that both configurations of vector were able to provide drug resistance and allowed enrichment of transduced cells to greater than 84%. The expression of EGFP in the IRES containing vector (pN-EF1α-cMGMT-IRES-EGFP) was reduced compared to vector in which both transgenes were expressed using the FMDV 2A cleavage factor (pN-EF1α-cMGMT-2A-EGFP). These studies pave the way for utilizing canine MGMT encoding vectors for genetic correction in the dog model.