Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI

Abstract

In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year.

Figure 1

Synthetic scheme for 5-MDHT. The entire synthesis of 5-MDHT from thymidine as the starting material. Et₃N·3HF, triethylamine trihydrofluoride; Me, methyl.

Figure 2

CEST-MRI data acquisition and processing. (a) T₂-weighted image of a representative coronal slice of a mouse brain transplanted with 9L^wt and 9L^HSV1-tk cells, showing the ROI for CEST-MRI. (b) A series of CEST-weighted images acquired from +Δ to −Δfrequency offsets from the water resonance. (c) Voxel-by-voxel image analysis of the CEST data obtained at b. (d) CEST map (or MTR_asym map) obtained after processing the data from c overlaid on the anatomical image in a. Adapted with permission from ref. . 9L^wt, wild-type 9L rat glioma cells; 9L^HSV1-tk, HSV1-tk–expressing 9L cells.

Figure 3

Histological validation of HSV1-tk expression. Immunostaining of a perfused mouse brain coronal section. Staining for HSV1-tk (anti-V5 antibody in red) overlaid on DAPI staining (blue) at low (×4) and high (×40) magnifications. Adapted with permission from ref. .

Figure 4

In vivo detection of the imino proton in 9L^HSV1-tk. (a) ΔMTR_asym plots of 9L^wt (blue) and 9L^HSV1-tk (red) (mean ± s.d.; n = 8 mice). Arrow points to the local maximal Δ MTR_asym and represents the imino proton at 5 p.p.m. after accumulation of 2 in a 9L^HSV1-tk tumor. MTR_asym (MTR_asym = 100 × [S^−Δω − S^Δω]/S₀) was calculated from a complete CEST spectrum (from −6 p.p.m. to +6 p.p.m.; S₀ image without saturation). (b) P values for each Δω (Student’s t test, unpaired, two-tailed) comparing the ΔMTR_asym of 9L^wt or 9L^HSV1-tk tumors in the examined mouse brains. The lowest P value was obtained at 5 p.p.m. frequency offset from water (black arrow). Adapted with permission from ref. .