Retrovector encoding a green fluorescent protein-herpes simplex virus thymidine kinase fusion protein serves as a versatile suicide/reporter for celland gene therapy applications

Expression vectors encoding herpes simplex virus thymidine kinase (HSVTK) h ave been extensively used in cell and gene therapy applications either as a nticancer "suicide" or as "self-destruct" transgenes in adoptive immunother apy applications. In both gene therapy applications, reliable detection of HSVTK transgene expression is required in genetically engineered cells, Dir ect fluorescent labeling of the HSVTK protein may be the remedy. We designe d a retrovector encoding a chimeric GFP-HSVTK fusion protein that can serve as a bifunctional suicide and reporter transgene. The fusion gene was inco rporated in a VSV G-pseudotyped retrovector (vGFPTKfus) and high-titer stab le retroviral producer was generated (similar to3 x 10(6) retroparticles/ml ). Tumor cell lines transduced at an MOI of 8 for 3 days led to >90% gene t ransfer efficiency. Southern blot analysis confirmed that unrearranged prov iral genomes integrated in chromosomal DNA. Protein extract immunoblot with HSVTK antisera revealed the presence of a 70-kDa protein consistent with t he predicted size of an HSVTK-GFP fusion protein. Fluorescence microscopy a nd FACS analysis revealed that GFPTKfus-mediated fluorescence was nuclear l ocalized and was 30-fold greater than that observed in a bicistronic HSVTK- GFP vector. Growth of cell lines expressing vGFPTKfus was significantly sup pressed in the presence of ganciclovir. The DA3 mouse mammary carcinoma cel l line was transduced with vGFPTKfus and implanted in syngeneic BALB/c mice . Preestablished tumors completely regressed in seven of nine mice treated with ganciclovir. Normal human peripheral blood T lymphocytes were transduced with vGFPTKfus and nucleus-restricted green fluorescence was observed. Sorting of green fl uorescent lymphocytes allowed for selection of engineered cells. In conclus ion, we demonstrate the utility of vGFPTKfus as a suicide/reporter transgen e in tumor cells in vitro and in vivo. Furthermore, its potential use as an analytical and therapeutic tool targeting human T lymphocytes is shown.