A new acycloguanosine-specific supermutant of herpes simplex virus type 1 thymidine kinase suitable for PET imaging and suicide gene therapy for potential use in patients treated with pyrimidine-based cytotoxic drugs.
The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene is widely used as a suicide gene in combination with ganciclovir (GCV) and as a nuclear imaging reporter gene with an appropriate reporter probe. Wild-type HSV1-tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogs, including clinically used antiviral drugs. PET of HSV1-tk reporter gene expression will be compromised in patients receiving nucleoside-based antiviral treatment. With the use of an acycloguanosine-specific mutant of the enzyme, PET of HSV1-tk reporter gene expression can be successfully performed with acycloguanosine-based radiotracers without interference from pyrimidine-based antiviral drugs.


METHODS
The levels of expression of wild-type HSV1-tk and HSV1-A167Ytk, HSV1-sr39tk, and HSV1-A167Ysr39tk mutants fused with green fluorescent protein (GFP) and transduced into U87 cells were normalized to the mean fluorescence of GFP measured by fluorescence-activated cell sorting. The levels of enzymatic activities of wild-type HSV1-tk and its mutants were compared by 2-h in vitro radiotracer uptake assays with (3)H-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-ethyluracil ((3)H-FEAU), (3)H-pencyclovir ((3)H-PCV), and (3)H-GCV and by drug sensitivity assays. PET with (18)F-FEAU and (18)F-9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) was performed in mice with established subcutaneous tumors, expressing wild-type HSV1-tk and its mutants, followed by tissue sampling.


RESULTS
FEAU accumulation was not detected in HSV1-A167Ysr39tk-expressing cells and xenografts. Lack of conversion of pyrimidine derivatives by the HSV1-A167Ysr39tk supermutant was also confirmed by a drug sensitivity assay, in which the 50% inhibitory concentrations for thymine 1-beta-d-arabinofuranoside and bromovinyldeoxyuridine were found to be similar to those in nontransduced cells. In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate (3)H-GCV at levels similar to those of wild-type HSV1-tk and HSV1-A167Ytk but showed enhanced activity with (3)H-PCV in vitro and with (18)F-FHBG in vivo.


CONCLUSIONS
We developed a new reporter gene, HSV1-A167Ysr39tk, which exhibits specificity and high phosphorylation activity for acycloguanosine derivatives. The resulting supermutant can be used for PET with (18)F-FHBG and suicidal gene therapy protocols with GCV in patients treated with pyrimidine-based cytotoxic drugs.
Likar
Yury
Y
Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Dobrenkov
Konstantin
K
Olszewska
Malgorzata
M
Vider
Elena
E
Shenker
Larissa
L
Cai
Shangde
S
Pillarsetty
Nagavarakishore
N
Hricak
Hedvig
H
Ponomarev
Vladimir
V
D000212:Acyclovir / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D003603:Cytotoxins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015774:Ganciclovir / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D006147:Guanine / Q000031:analogs & derivatives; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D051379:Mice; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D049268:Positron-Emission Tomography / Q000379:methods*; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism*
C113308:9-(4-fluoro-3-hydroxymethylbutyl)guanine; D000998:Antiviral Agents; D003603:Cytotoxins; D011743:Pyrimidines; D006147:Guanine; D013937:Thymidine Kinase; C030986:pyrimidine; D015774:Ganciclovir; D000212:Acyclovir