Imaging reporter transgene expression in living subjects using positron emission tomography.

摘要

The goal of this investigation was to develop and validate approaches for non-invasive imaging of the biodistribution, magnitude, and time-variation of expression of therapeutic transgenes (TG) in living subjects. Positron emission tomography (PET) and PET reporter gene (PRG)/PET reporter probe (PRP) systems were used to develop indirect approaches for imaging TG expression, because these imaging systems have the potential for extension to clinical applications. At first, two PRG/PRP systems were used to validate the identical vector co-administration method. Two identical adenoviral vectors Bach carrying one of the PRGs, regulated by the constitutive Cytomegalovirus (CMV) promoter were co-administered in cell culture and in vivo and in both cases correlated expression of both PRGs was demonstrated. The same approach was then applied to link the expression of the p53 therapeutic transgene to the expression of a PRG in a prostate cancer cell model. Following observation of correlation between p53 and PRG expression in p53 null prostate cancer cells this approach was then validated in vivo by demonstrating correlated p53 protein levels and PRP accumulation in the livers of mice. In addition the linkage of p53 transgene expression with PRG expression was demonstrated in cell culture using a dual identical promoter bicistronic plasmid vector.; The PET reporter probe 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) that can detect the expression of the suicide/reporter transgene mutant herpes simplex virus 1 thymidine kinase (HSV1-sr39tk) was used to analyze the utility of PET in gene therapy. These studies demonstrated that [18F]FHBG can specifically detect cells susceptible to Ganciclovir (GCV) induced cell death, since HSV1-sr39TK enzyme converts GCV to a genotoxin. Therefore, [18F]FHBG can be used to determine when GCV treatment has effectively eradicated cancer cells expressing the HSV1-sr39tk gene. Finally, the biodistribution, pharmacokinetics and dosimetry of [18F]FHBG were analyzed in healthy human volunteers in order to examine how suitable [18F]FHBG will be for patient studies and develop a plan for monitoring gene therapy in patients using [ 18F]FEBG PET imaging. Our imaging approaches should be useful for improving gene therapy protocols and assessing selectivity and efficiency of targeting with various gene therapy vectors in pre-clinical animal models and patients.