HSV1-tk is widely used in gene therapy studies as a suicide gene in combination with GCV and as a nuclear imaging reporter gene in combination with an appropriate reporter probe. Radiolabeled pyrimidine-based (124
F-FFEAU, and 18
) and acycloguanosine-based (18
F-FHBG, and 18
) nucleoside analogs have been used to image HSV1-tk and HSV1-sr39tk reporter gene expression with PET. HSV1-tk can also phosphorylate a variety of cytotoxic pyrimidine and acycloguanosine derivatives, including clinically used antiviral drugs such as GCV, ACV, and BVdU (25
). In a clinical setting in which a patient's condition requires the administration of antiviral drugs, cells expressing HSV1-tk as a reporter gene will be compromised by antiviral treatment. This will result in the negative selection of HSV1-tk–transduced cells and inaccurate imaging results with HSV1-tk–specific PET tracers. The objective of this work was to create a new acycloguanosine-specific mutant of the HSV1-tk reporter gene family that will allow for successful PET in patients undergoing therapy with pyrimidine-based nucleoside analogs. For example, noninvasive PET of acycloguanosine-specific HSV1-tk mutant expression with acycloguanosine-based radiotracers (e.g., 18
F-FHBG) will not be compromised by treatment with BVdU or other pyrimidine-based nucleoside analogs.
Recently, Degreve et al. showed that position 167 in the HSV1-tk protein is particularly favorable for discriminating between pyrimidine and acycloguanosine substrates (28
). In their studies, a mutant bearing the A167Y substitution in the nucleoside-binding region (amino acids 159–169) () showed a markedly decreased response to the antiviral pyrimidines (ARA-T and BVdU in particular), whereas the phosphorylation of GCV, ACV, and PCV was almost not affected by this mutation. These results were confirmed in our in vitro and in vivo studies (Figs. -). Moreover, we showed for the first time that cells transduced with the HSV1-A167Ytk mutant were not able to accumulate FEAU in vitro and in vivo, whereas the uptake of 3
H-GCV and 3
H-PCV in vitro and the uptake of 18
F-FHBG in vivo were found to be similar to those in cells transduced with wild-type HSV1-tk.
Several groups have performed genetic modifications of the wild-type HSV1-tk which resulted in mutants with enhanced phosphorylation kinetics for certain nucleoside derivatives. The widely used HSV1-sr39tk mutant, bearing 5 amino acid substitutions in the nucleoside-binding region of the enzyme () (36
), shows significantly improved activity with acycloguanosines, specifically GCV, ACV, and PCV. This mutant demonstrated a greater-than-100-fold reduction in the IC50
for GCV compared with wild-type HSV1-tk–expressing transfectants. When used in vivo for PET studies, HSV1-sr39tk–expressing tumors showed considerably increased uptake of the PCV analog 18
) compared with wild-type HSV1-tk–transduced xenografts. We confirmed these results in the present study. The IC50
for GCV with HSV1-sr39tk–transduced cells was 2 orders of magnitude lower than that with wild-type HSV1-tk–expressing transfectants (), and 18
F-FHBG accumulation was found to be ~6-fold higher in HSV1-sr39tk–expressing tumors than in wild-type HSV1-tk–expressing xenografts (Figs. and ). Also, in accordance with in vitro studies by Kang et al. (39
), we found that the level of 18
F-FEAU phosphorylation obtained in vivo with this mutant is slightly superior to that obtained with wild-type HSV1-tk (P
< 0.05). These results led us to conclude that the HSV1-sr39tk mutant exhibits a more favorable conformation of the nucleoside-binding pocket for both pyrimidine and acycloguanosine derivatives with a marginal increase in the rate of phosphorylation for the latter compared with the wild-type enzyme.
HSV1-sr39tk contains an alanine residue at position 167, similar to the wild-type protein (37
). On the basis of this observation, we hypothesized that the substitution of the alanine at position 167 with a pyrimidine-restrictive tyrosine within this mutant may lead to newly acquired specificity and high phosphorylation activity with acycloguanosine analogs without interference from pyrimidine-based derivatives. We were able to successfully develop and express in U87 cells a new supermutant of HSV1-tk, HSV1-A167Ysr39tk fused with GFP, at levels comparable to those of wild-type HSV1-tk and other mutants. In our experiments, FEAU accumulation in HSV1-A167Ysr39tk–transduced cells could not be detected in vitro () or in vivo (). Lack of pyrimidine phosphorylation by the HSV1-A167Ysr39tk supermutant was also confirmed with drug sensitivity assays, in which the IC50
s for ARA-T and BVdU were similar to those in nontransduced cells (). In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate GCV at a level similar to that of wild-type HSV1-tk (or HSV1-A167Ytk) () but showed enhanced activity with PCV in vitro (~10- to 15-fold) () and 18
F-FHBG in vivo (~2.5-fold) (Figs. and ). Interestingly, despite a substantial increase in 3
H-PCV and 18
F-FHBG uptake observed with HSV1-A167Ysr39tk relative to that observed with wild-type HSV1-tk or HSV1-A167Ytk, the IC50
s for GCV were comparable among wild-type HSV1-tk and both A167Y-containing mutants (). Thus, the A167Y substitution within the HSV1-sr39tk mutant led to a particular analog-specific enhancement of uptake within the acycloguanosine group.
The in vivo imaging results demonstrated the advantage of the new acycloguanosine-specific supermutant HSV1-A167Ysr39tk reporter gene over its predecessor, HSV1-A167Ytk. Both PET and direct measurement of radiotracer accumulation in tissue samples confirmed a much higher level of 18F-FHBG uptake in HSV1-A167Ysr39tk–expressing tumors than in HSV1-A167Ytk–expressing xenografts. On the basis of our in vitro and in vivo data, we are more than confident that our supermutant can be reliably imaged with PET and 18F-FHBG in patients treated with pyrimidine derivatives while providing an opportunity to eliminate transduced cells with GCV administration.
18F-FHBG is known to be cleared via the liver and kidneys, a property that makes it more challenging to elucidate HSV1-tk reporter gene expression in the lower gut. Therefore, the same issue of gastrointestinal clearance of 18F-FHBG remains to be addressed.
The alternative strategy of using a pyrimidine-specific mutant of the HSV1-tk reporter gene will be useful and will allow PET in patients undergoing therapy with acycloguanosine-based analogs. Studies on the development of such a mutant are under way.