Cytoplasmically retargeted HSV1-tk/GFP reporter gene mutants for optimization of noninvasive molecular-genetic imaging

Abstract

To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a "cryptic" testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improved metabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [(131)I]FIAU and a gamma-camera.

Figure 1

Schematic structures of retroviral vectors for mammalian expression of the native and different HSV1-tk/GFP mutants: (A) native HSV1-tk/GFP; (B) mNLS-HSV1-tk/GFP with mutations in the N-terminal nuclear localization signal; (C) NES-HSV1-tk/GFP with the addition of the nuclear export signal from MAPKK; (D) NESmNLS-HSV1-tk/GFP with a combination of mutations in vectors (B) and (C); and (E) Δ45HSV1-tk/GFP with truncation of N-terminal sequence encoding the first 45 amino acids of the native protein.

Figure 2

Fluorescent photomicrographs of RG2 cells expressing the native and different HSV1-tk/GFP mutants: (A) native HSV1-tk/GFP; (B) mNLS-HSV1-tk/GFP with mutations in the N-terminal nuclear localization signal; (C) NES-HSV1-tk/GFP with the addition of the nuclear export signal from MAPKK; (D) NESmNLSHSV1-tk/GFP with a combination of mutations in vectors (B) and (C); and (E) Δ45HSV1-tk/GFP with truncation of N-terminal sequence encoding the first 45 amino acids of the native protein.

Figure 3

Detection and analysis of the native and different HSV1-tk/GFP mutants in transduced RG2 cells: (A) RT-PCR analysis; (B) Western blot using a monoclonal anti-HSV1-tk antibody; and (C) Western blot using a monoclonal anti-GFP antibody. Native—wt-HSV1-tk/GFP; with mutations in the N-terminal nuclear localization signal—mNLS-HSV1-tk/GFP; with the addition of the nuclear export signal from MAPKK—NES-HSV1-tk/GFP; with both the NLS mutations and the addition of NES from MAPKK—NESmNLSHSV1-tk/GFP; with truncation of N-terminal sequence encoding the first 45 amino acids of the native protein—Δ45HSV1-tk/GFP.

Figure 4

Relationship between two measurements of HSV1-tk/GFP expression—FACS measurement of GFP fluorescence (mean value) and ELISA assay of HSV1-tk/GFP protein levels. Strong linear relationship was observed in corresponding cell populations (paired t-test, P < .001).

Figure 5

A comparison of expression levels of the native (open circles) and different HSV1-tk/GFP mutants (closed squares) based on GFP domain fluorescence with their ability to accumulate FIAU in transduced RG2 cells. To achieve different levels of the native HSV1-tk/GFP expression levels, the transduced RG2 cells were mixed with nontransduced cells in percentages indicated on the plot above open circles. The data for HSV1-tk/GFP mutants were grouped together and reflect multiple populations of transduced RG2 cells with different levels of mutant HSV1-tk/GFP expression. The data sets for the native and different HSV1-tk/GFP mutants were fitted with a linear function to assess the relationship. The cell populations with similar levels of transgene expression (dotted rectangle) were later used to produce subcutaneous tumors for the in vivo imaging studies.

Figure 6

γ-Camera imaging of [¹³¹I]FIAU accumulation in rats bearing multiple subcutaneous tumors produced from RG2 cells expressing native HSV1-tk/GFP or different HSV1-tk/GFP mutants at similar levels based on GFP expression (see dotted cluster box in Figure 5) and from nontransduced RG2 cells (panel A). The levels of [¹³¹I]FIAU accumulation measured in tissue samples are expressed as percent injected dose per gram of tissue (panel B). Paired t-test of FIAU accumulation in tumors expressing different mutant HSV1-tk/GFP proteins versus tumors expressing native HSV1-tk/GFP protein (n = 6, *P < .05, **P < .01).