Gene therapy shows promise for treating prostate cancer and has been successfully exploited in several clinical trials. A major hurdle is establishing a method of verifying transgene activity in situ. β-galactosidase (β-gal) has historically been the most popular reporter gene for molecular biology. We are designing non-invasive NMR approaches to reveal β-gal activity in vivo. 2-Fluoro-4-nitrophenol-β-D-galactopyranoside (OFPNPG) belongs to a novel class of NMR active molecules (fluorophenyl-β-D-galactopyranosides), which are highly responsive to the action of β-gal. OFPNPG has a single 19F peak at 55 ppm relative to aqueous sodium trifluoroacetate (NaTFA). Upon cleavage by β-gal, the pH sensitive aglycone OFPNP is observed at a chemical shift of 59-61 ppm. We now show the chemical shift response is sufficient to observe β-gal activity by NMR in PC3 human prostate tumor xenografts in mice. PC3/LacZ tumor cells were generated by recombinant plasmid phCMV/lacZ transfection and a high expressing clone selected. Cells were implanted in the flank of nude mice and allowed to grow to about 1cm3. When a solution of OFPNPG (4 mg in 50 μl aqueous DMSO) was injected intra-tumorally, 19F NMR signal was readily detected at 4.7 T. Over a period of 30min conversion of OFPNPG to product OFPNP was observed unequivocally demonstrating β-gal activity (see spectra). Tumor and tissues were also examined by Western blots and β-gal assay for activity. High β-gal activity was found in the tumor, with minimal activity in normal tissues. This approach directly reveals β-gal activity, which could be used in tandem with therapeutic genes to monitor therapy. As gene therapy becomes a reality, the ability to detect transgenic expression non-invasively will become increasingly important for treatment planning and optimization. The prototype gene reporter molecule (OFPNPG) releases a potentially toxic product (fluoronitrophenol) and we are also exploring whether this can serve as the basis for broad-spectrum chemotherapy. Meanwhile, we are also developing new generations of 19F NMR reporter designed to minimize toxicity. This research was supported by DOD PC031075 (LL), NCI pre-ICMIC CA86354 and P41-RR02584.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]