Noninvasive quantitation of cytosine deaminase transgene expression in human tumor xenografts with in vivo magnetic resonance spectroscopy

Analysis of transgene expression in vivo currently requires destructive and invasive molecular assays of tissue specimens. Noninvasive methodology for assessing the location, magnitude, and duration of transgene expression in vivo will facilitate subject-by-subject correlation of therapeutic outcome s with transgene expression and will be useful in vector development. Cytos ine deaminase (CD) is a microbial gene undergoing clinical trials in gene-d irected enzyme prodrug gene therapy. We hypothesized that in vivo magnetic resonance spectroscopy could be used to measure CD transgene expression in genetically modified tumors by directly observing the CD-catalyzed conversi on of the 5-fluorocytosine (5-FC) prodrug to the chemotherapeutic agent 5-f luorouracil (5-FU). The feasibility of this approach is demonstrated in sub cutaneous human colorectal carcinoma xenografts in nude mice by using yeast CD (yCD). A three-compartment model was used to analyze the metabolic flux es of 5-FC and its metabolites. The rate constants for yCD-catalyzed prodru g conversion (k(1)(app)), 5-FU efflux from the observable tumor volume (k(2 )(app)), and formation of cytotoxic fluorinated nucleotides from 5-FU (k(3) (app)) were 0.49 +/- 0.27 min(-1), 0.766 +/- 0.006 min(-1), and 0.0023 +/- 0.0007 min(-1), respectively. The best fits of the 5-FU concentration data assumed first-order kinetics, suggesting that yCD was not saturated in vivo in the presence of measured intratumoral 5-FC concentrations well above th e in vitro K-m, These results demonstrate the feasibility of using magnetic resonance spectroscopy to noninvasively monitor therapeutic transgene expr ession in tumors. This capability provides an approach for measuring gene e xpression that will be useful in clinical gene therapy trials.