PET imaging of HSV1-tk mutants with acquired specificity toward pyrimidine- and acycloguanosine-based radiotracers.
OBJECTIVE
The aim of this study was to create an alternative mutant of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene with reduced phosphorylation capacity for acycloguanosine derivatives, but not pyrimidine-based compounds that will allow for successful PET imaging.


METHODS
A new mutant of HSV1-tk reporter gene, suitable for PET imaging using pyrimidine-based radiotracers, was developed. The HSV1-tk mutant contains an arginine-to-glutamine substitution at position 176 (HSV1-R176Qtk) of the nucleoside binding region of the enzyme.


RESULTS
The mutant-gene product showed favorable enzymatic characteristics toward pyrimidine-based nucleosides, while exhibiting reduced activity with acycloguanosine derivatives. In order to enhance HSV1-R176Qtk reporter activity with pyrimidine-based radiotracers, we introduced the R176Q substitution into the more active HSV1-sr39tk mutant. U87 human glioma cells transduced with the HSV1-R176Qsr39tk double mutant reporter gene showed high (3)H-FEAU pyrimidine nucleoside and low (3)H-penciclovir acycloguanosine analog uptake in vitro. PET imaging also demonstrated high (18)F-FEAU and low (18)F-FHBG accumulation in HSV1-R176Qsr39tk+ xenografts. The feasibility of imaging two independent nucleoside-specific HSV1-tk mutants in the same animal with PET was demonstrated. Two opposite xenografts expressing the HSV1-R176Qsr39tk reporter gene and the previously described acycloguanosine-specific mutant of HSV1-tk, HSV1-A167Ysr39tk reporter gene, were imaged using a short-lived pyrimidine-based (18)F-FEAU and an acycloguanosine-based (18)F-FHBG radiotracer, respectively, administered on 2 consecutive days.


CONCLUSIONS
We conclude that in combination with acycloguanosine-specific HSV1-A167Ysr39tk reporter gene, a HSV1-tk mutant containing the R176Q substitution could be used for PET imaging of two different cell populations or concurrent molecular biological processes in the same living subject.
Likar
Yury
Y
Molecular Imaging Laboratory, Department of Radiology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, (Box 501) Z-2035, New York, NY, 10021, USA. likary@mskcc.org
Dobrenkov
Konstantin
K
Olszewska
Malgorzata
M
Shenker
Larissa
L
Cai
Shangde
S
Hricak
Hedvig
H
Ponomarev
Vladimir
V
D000212:Acyclovir / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D024882:Drug Resistance, Viral; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D051379:Mice; D009154:Mutation; D009369:Neoplasms / Q000000981:diagnostic imaging / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D049268:Positron-Emission Tomography; D011743:Pyrimidines / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism*; D011849:Radioactive Tracers; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism*
D011743:Pyrimidines; D011849:Radioactive Tracers; D013937:Thymidine Kinase; C030986:pyrimidine; D000212:Acyclovir