306. Noninvasive Monitoring of Transgene Expression Using Surrogate Marker Peptides

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Our goal is to develop safe, reliable, convenient strategies for noninvasively monitoring transgene expression after in vivo gene delivery. This will enhance the information yield from preclinical gene therapy studies and will facilitate meaningful pharmacokinetic and pharmacodynamic studies in the course of phase I clinical testing of novel gene therapy agents. The underlying hypothesis for the current study is that the expression of a therapeutic transgene can be noninvasively monitored in vivo by measuring the concentration in body fluids of a soluble marker polypeptide which is nonimmunogenic to recipient animals, concordantly expressed from the same vector construct. To determine the optimal strategy for linking the expression of a transgene of interest to the expression of a soluble marker peptide, we studied the concordance of expression of two soluble marker genes (CEA and beta-hCG) that were linked through the encephalomyocarditis virus internal ribosome entry site (in lentiviral vectors) or driven individually from separate CMV promoters inserted at different sites in the vector genome (in adenoviral vectors). Concordance was tested by transducing a panel of cell lines representing different cell lineages or by direct intravenous vector administration to athymic rats, and comparing the concentrations of the two marker peptides in tissue culture supernatants or in rat body fluids. Concordance was poor when the transgenes were linked through the EMCV ires, with considerable variability in the relative rates of synthesis of the two peptides across different cell lineages, and as a function of the positioning of the two genes (before or after the ires). Furthermore, there was considerable random variation in the peptide ratios in supernatants of clonal populations of a single cell lineage with different proviral insertion sites. In contrast, a remarkably high level of concordance was observed when transgenes were expressed individually from CMV promoters at E1, E3 or E4 insertion sites in the adenovirus genome, regardless of cell lineage and transgene expression level. Moreover, in athymic rats, the concordance of expression from these adenoviral vectors was well maintained such that the expression of the CEA transgene was accurately reflected in the serum or urine measurements of beta-hCG (and vice versa). We conclude that it is feasible to utilize a surrogate marker peptide for accurate quantitative monitoring of the expression of a transgene of interest. However, the viability of the approach is dependent on the mechanism by which the expression of the two transgenes is linked with the use of separate transcription units with identical promoters being greatly superior to the use of internal ribosome entry sites.