Choline kinase (Chk), an enzyme that converts choline to phosphocholine (PC), is overexpressed in several cancers, and provides a novel gene therapy target that can be imaged with magnetic resonance spectroscopy (MRS) . Here we report on the first study to silence Chk in a human breast cancer xenograft model using lentiviral vector. Gene delivery to cells and tumor xenografts was monitored by fluorescence microscopy imaging of the reporter gene, enhanced green fluorescent protein (EGFP), which is incorporated within the lentiviral construct. The effects of lentiviral-mediated Chk silencing were confirmed using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Chk downregulation in breast tumor xenografts following systemic injection of the lentivirus was also monitored using single-voxel 31P MRS in vivo to detect tumoral phosphomonoester (PME) and PC levels.

We initially cloned shRNA against choline kinase into the pRRL-U6-pGK-EGFP lentivirus vector. Pseudotyped lentiviral supernatant was tested for its efficacy to down regulate Chk in MDA-MB-231 cells. Following infection of MDA-MB-231 cells, cDNA was prepared and analyzed by qRT-PCR. Results obtained indicated an 80% reduction in the Chk transcript. Control shRNA against luciferase (luc) was used as the control for non-specific deregulation. Upon confirming the activity of the virus in tissue culture, we tested its activity in a human breast cancer xenograft model using MDA-MB-231 cells injected into the mammary fat pad of SCID mice. Two doses of 2.7x107 lentiviral particles in 200µl of PBS, at an interval of two days, were injected through the tail vein of mice bearing tumors with volumes of 300 mm3. To noninvasively monitor changes in tumoral PME and PC levels, in vivo single-voxel 31P MRS was performed pre- and up to 4 days post-viral injection using a 4.7T Bruker Biospec spectrometer. MR spectra were processed and analyzed with an in-house IDL program (Dr. D. C. Shungu), using gaussian multiplication and a combination of linear and nonlinear least-square fitting. In vivo 31P MRS detected a decrease in both PC and PME levels in tumors of animals treated with shRNA-Chk, compared to shRNA-luc treated control tumors, within 3 days of treatment. The decrease of PME and PC was consistent with regions of EGFP expression observed in fresh tissue tumor slices. At the end of the imaging study, the kidney, liver and tumor were excised from each animal and 1mm thick sections of the tissue samples were observed under a fluorescence microscope to detect EGFP expression.

.These data demonstrate that Chk silencing in tumors can be achieved in vivo through intravenous injection of lentiviral particles, which are capable of transducing cells to generate shRNA-Chk. Furthermore, the effects of Chk silencing can be noninvasively imaged with 31P MRS validating the functional effects of decreased choline kinase levels.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA