Application of a rapid, simple, and accurate adenovirus-based method to compare PET reporter gene/PET reporter probe systems

Abstract

Purpose: This study aims to use a simple, quantitative method to compare the HSV1sr39TK/(18) F-FHBG PET reporter gene/PET reporter probe (PRG/PRP) system with PRGs derived from human nucleoside kinases.

Procedures: The same adenovirus vector is used to express alternative PRGs. Equal numbers of vectors are injected intravenously into mice. After PRP imaging, quantitative hepatic PET signals are normalized for transduction by measuring hepatic viral genomes.

Results: The same adenovirus vector was used to express equivalent amounts of HSV1sr39TK, mutant human thymidine kinase 2 (TK2-DM), and mutant human deoxycytidine kinase (dCK-A100VTM) in mouse liver. HSV1sr39TK expression was measured with (18) F-FHBG, TK2-DM and dCK-A100VTM with (18) F-L-FMAU. TK2-DM/(18) F-L-FMAU and HSV1sr39TK/(18) F-FHBG had equivalent sensitivities; dCK-A100VTM/(18) F-L-FMAU was twice as sensitive as HSV1sr39TK/(18) F-FHBG.

Conclusions: The human PRG/PRP sensitivities are comparable and/or better than HSV1sr39TK/(18) F-FHBG. However, for clinical use, identification of the best PRP substrate for each enzyme, characterization of probe distribution, and consequences of overexpressing nucleoside kinases must be evaluated.

Fig. 1. Cloning and production of adenovirus vectors

Adenovirus vectors containing coding regions for TK2, TK2-DM, dCK-WT, dCK-DM, dCK-A100VTM, HSV1sr39TK (as a reference standard) and Luc2 as a negative control were constructed using Invitrogen’s Gateway^® Cloning System. The open reading frame of each reporter was inserted into an “entry” vector to create the pENTR-PRG plasmids, then transferred to the pAd/CMV/V5/DEST vector, using the LR recombination reaction, to create the seven pAd vectors. The adenoviral plasmids were linearized by PacI restriction enzyme digestion, purified, and transformed into HEK293A cells for vector rescue. After 100% cytopathic effect was observed, lysates were serially passaged on increasing numbers of HEK293A cells with each round of infection until sufficient vector was produced, following cesium chloride buoyant density ultracentrifugation and purification, for in vivo studies. Sites labeled attR1/attR2/attL1/attL2 are the initial recombination regions; attB1/attB2 are the recombination regions after the LR recombination reaction. Cm^R, the chloramphenicol resistance gene; Km^R, the kanamycin resistance gene; Ap^R, the ampicillin resistance gene; ccdB, the coding region for the cytotoxic protein CcdB, used as negative-selection marker in recombined clones; P_CMV, the CMV promoter; TKpA, the thymidine kinase polyadenylation signal; 5’ ITR, the viral 5’ inverted terminal repeats; wt Ad5 (DE3), Ad5 sequences that include a 3’ ITR and packaging signal.

Fig. 2. Viral titers, measured as Infectious Genome Units, for the Adenovirus vectors

IGU values for the seven adenovirus vector preparations were determined following HeLa cell transduction. Nuclei were harvested three hours after vector addition. Vector genomes were measured by quantitative PCR as described in Materials and Methods. Data are means ± S.E.M. of duplicate qPCR assays for duplicate transductions.

Fig. 3. Titration of Ad.HL.sr39TK for MicroPET/MicroCT quantification

(Panel a) Two mice per group were injected intravenously with 10¹¹, 3×10¹⁰, or 10¹⁰ IGU of Ad.HL.sr39TK or with 3×10¹⁰ IGU of Ad.HL.Luc2. After three days, mice were injected with ¹⁸F-FHBG (200 µCi) and subjected to microPET/microCT imaging. (Panel b) 2 mm ROIs were drawn within the liver of each mouse, and used to determine average %ID/g per liver. Data are means ± S.E.M.

Fig. 4. TK2 and dCK PRG efficacies, with L-FMAU as the PRP

(Panel a) Three mice for each adenovirus vector were injected with 5×10¹⁰ IGU of Ad.HL.TK2, Ad.HL.TK2-DM, Ad.HL.dCK-WT, Ad.HL.dCK-A100VTM, Ad.HL.sr39TK (as reference standard) or Ad.HL.Luc2 (as negative control). Four days later mice were injected with 200 µCi ¹⁸F-FHBG (Ad.HL.sr39TK) or 200 µCi ¹⁸F-L-FMAU (Ad.HL.TK2, Ad.HL.TK2-DM, Ad.HL.dCK-WT, Ad.HL.dCK-A100VTM, Ad.HL.Luc2) and subjected to microPET/microCT imaging. (Panel b) One day after imaging, mice were sacrificed. Viral and liver genomes were measured in liver extracts, to determine numbers of viral vectors in the livers. Each point is the mean of three liver samples per mouse. (Panel c) Four identical ROIs were drawn within the liver of each mouse and use to determine the %ID/g for liver. Values were then corrected by subtracting ROI values determined for the mice injected with Ad.HL.Luc2 virus and imaged with the appropriate PRP (negative control, bkg). Asterisk indicates statistically significant differences (p>0.05); individual p values follow: FHBG sr39TK vs. TK2-DM, p=0.23. FHBG sr39TK vs. dCK-A100VTM, p=0.01. TK2-DM vs. dCK-A100VTM, p=0.01. (Panel d). The [%IDg – bkg] in liver for each adenovirus vector (from panel c) was normalized for the number of viral genomes per liver (from panel b). To compare the efficacies of the experimental TK2, TK2-DM, dCK-WT and dCK-A100VTM/¹⁸F-L-FMAU PRG/PRP non-invasive imaging systems with the efficacy of the HSV1sr39TK/¹⁸F-FHBG PRG/PRP system, the values for the experimental systems using ¹⁸F-L-FMAU as the PRP are expressed as a percentage of the HSV1sr39TK/¹⁸F-FHBG PRG/PRP system. Asterisk indicates statistically significant differences (p>0.05); individual p values follow: FHBG sr39TK vs. TK2-DM, p=0.86. TK2-DM vs. dCK-A100VTM, p=0.08. FHBG sr39TK vs. dCK-A100VTM, p=0.03. Data for panels b, c and d are means ± S.E.M.