Comparative Analysis of Human Nucleoside Kinase-Based Reporter Systems for PET Imaging

PurposeRadionuclide-based reporter gene imaging has the sensitivity to monitor gene- and cell-based therapies in human subjects. Potential immunogenicity of current viral transgenes warrants development of human-based reporter systems. We compared human nucleoside kinase reporters to a panel of nucleoside analogs of FEAU, FMAU, and FIAU, including the first in vivo assessment of L-[18F]FEAU.ProceduresHuman isogenic U87 cell lines were transduced to express different human reporter genes including dCK-R104M/D133A (dCKDM), dCK-R104Q/D133N (dCKep16A), dCK-A100V/R104M/D133A (dCK3M), and TK2-N93D/L109F (TK2DM), and wild-type dCK (dCK) and herpes simplex virus type-1 (HSVTK) reporter gene as references. In vitro cell uptake assays were performed with [18F]FEAU, L-[18F]FEAU, [14C]FMAU, L-[18F]FMAU, and [124I]FIAU. Micro-positron emission tomography/X-ray computed tomography imaging of xenograft-bearing nu/nu mice was conducted with [18F]FEAU, L-[18F]FEAU, L-[18F]FMAU, and [124I]FIAU on consecutive days. A cell viability assay was also performed to assess sensitivities to gemcitabine and bromovinyldeoxyuridine (BVdU).ResultsIn vitro, dCKep16A and dCKDM with [18F]FEAU exhibited the highest sensitivity and selectivity of the human reporters, second only to HSVTK/[18F]FEAU. L-[18F]FEAU biodistribution in mice was on par with [18F]FEAU and L-[18F]FMAU. L-[18F]FMAU uptake in isogenic xenografts was highest for all human reporter genes. However, [18F]FEAU was the most selective of the short half-life reporter probes due to its minimal recognition by human dCK and relative sensitivity, whereas [124I]FIAU permitted imaging at a later time point, improving signal-to-background ratio. Of the human reporter genes, dCKep16A consistently outperformed the other tested reporters. Reporter genes of interest increased potency to the nucleoside analog prodrugs gemcitabine and BVdU.ConclusionsWe demonstrate that human nucleoside kinase reporter systems vary significantly in their sensitivity and selectivity for in vivo imaging. The sufficiently high signal-to-background ratios and enhanced suicide gene potential support clinical translation

Comparative Analysis of Human Nucleoside Kinase-Based Reporter Systems for PET Imaging

PURPOSE: Radionuclide-based reporter gene imaging has the sensitivity to monitor gene- and cell-based therapies in human subjects. Potential immunogenicity of current viral transgenes warrants development of human-based reporter systems. We compared human nucleoside kinase reporters to a panel of nucleoside analogs of FEAU, FMAU, and FIAU, including the first in vivo assessment of L-[(18)F]FEAU. PROCEDURES: Human isogenic U87 cell lines were transduced to express different human reporter genes including dCK-R104M/D133A (dCKDM), dCK-R104Q/D133N (dCKep16A), dCK-A100V/R104M/D133A (dCK3M), and TK2-N93D/L109F (TK2DM), and wild-type dCK (dCK) and herpes simplex virus type-1 (HSVTK) reporter gene as references. In vitro cell uptake assays were performed with [(18)F]FEAU, L-[(18)F]FEAU, [(14)C]FMAU, L-[(18)F]FMAU, and [(124)I]FIAU. Micro-positron emission tomography/X-ray computed tomography imaging of xenograft-bearing nu/nu mice was conducted with [(18)F]FEAU, L-[(18)F]FEAU, L-[(18)F]FMAU, and [(124)I]FIAU on consecutive days. A cell viability assay was also performed to assess sensitivities to gemcitabine and bromovinyldeoxyuridine (BVdU). RESULTS: In vitro, dCKep16A and dCKDM with [(18)F]FEAU exhibited the highest sensitivity and selectivity of the human reporters, second only to HSVTK/[(18)F]FEAU. L-[(18)F]FEAU biodistribution in mice was on par with [(18)F]FEAU and L-[(18)F]FMAU. L-[(18)F]FMAU uptake in isogenic xenografts was highest for all human reporter genes. However, [(18)F]FEAU was the most selective of the short half-life reporter probes due to its minimal recognition by human dCK and relative sensitivity, whereas [(124)I]FIAU permitted imaging at a later time point, improving signal-to-background ratio. Of the human reporter genes, dCKep16A consistently outperformed the other tested reporters. Reporter genes of interest increased potency to the nucleoside analog prodrugs gemcitabine and BVdU. CONCLUSIONS: We demonstrate that human nucleoside kinase reporter systems vary significantly in their sensitivity and selectivity for in vivo imaging. The sufficiently high signal-to-background ratios and enhanced suicide gene potential support clinical translation