Tracking and quantitation of retroviral-mediated transfer using a completely humanized, red-shifted green fluorescent protein gene.

We have developed murine retroviral vectors (RVs) containing an optimized green fluorescent protein (GFP) gene to study retroviral gene transfer and expression in living cells. We used the codon "humanized", "red-shifted" GFP gene, hGFP-S65T, a gain of function variant of the wild-type GFP from the jellyfish Aequorea victoria. We cloned the hGFP-S65T gene into the RV plasmid pLNCX (pLNChG65T). A stable amphotropic RV-producer cell line (VPC), designated LNChG65T VPC, was generated that exhibited bright fluorescence in greater than 95% of the cells. Human A375 melanoma cells and IGROV ovarian carcinoma cells transduced from LNCh-G65T VPC demonstrated high levels of fluorescence. The expression of a single integrated hGFP-S65T gene in eukaryotic cells provides a powerful tool to study gene transfer, expression and functional studies in vitro and in vivo.

Retroviral transfer and expression of a humanized, red-shifted green fluorescent protein gene into human tumor cells.

Over two-thirds of the current gene therapy protocols use retroviral gene transfer systems. We have developed an efficient retroviral-based method that allows rapid identification of gene transfer in living mammalian cells. Cells were generated containing a gene for an improved (humanized, red-shifted) version of the Aequorea victoria green fluorescent protein (hRGFP) from a retroviral vector. The hRGFP gene was used to produce an amphotropic vector producer cell line that demonstrated vibrant green fluorescence after excitation with blue light. A375 melanoma cells transduced with the retroviral vector demonstrated stable green fluorescence. Both PA317 murine fibroblasts and A375 human cell lines containing the vector were easily detected by FACS analysis. These vectors represent a substantial improvement over currently available gene transfer marking systems. Bright, long-term expression of the hRGFP gene in living eukaryotic cells will advance the study of gene transfer, gene expression, and gene product function in vitro and in vivo particularly for human gene therapy applications.

Preliminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer.

A novel gene therapy strategy is the use of suicide genes that transfer a drug sensitivity to cancer cells. We present preliminary in vitro efficacy data and in vivo toxicity data using the herpes simplex thymidine kinase (HStk) gene for breast cancer. The long-term objective of this project is to develop novel approaches for the treatment of breast cancer using in vivo retroviral gene transfer. Intraductal breast cancer cell line HTB126 transduced by an HStk retroviral vector is efficiently inhibited in vitro after GCV exposure. Further analysis revealed a bystander effect through which nontransduced HTB126 cells were also inhibited by GCV when cocultured with HStk-transduced HTB126 cells. In cell mixture experiments if only 1% of the cells in culture contained the HStk gene, 83% of the culture could be destroyed. Next safety studies were performed. Vector producer cells (VPC) were implanted into the mammary fat pads of athymic nude mice. The mice were then treated with GCV and monitored for regression of the VPC. The injected VPC regressed rapidly in response to the GCV therapy and produced no evidence of local or systemic toxicity in the animals. These in vitro efficacy data and in vivo toxicity studies lend support to the further development of an in vivo therapy model to treat breast cancer.