Imaging Transgene Activity In vivo

Abstract

The successful translation of gene therapy for clinical application will require the assessment of transgene activity as a measure of the biological function of a therapeutic transgene. Although current imaging permits the noninvasive detection of transgene expression, the critical need for quantitative imaging of the action of the expressed transgene has not been met. In vivo magnetic resonance spectroscopic imaging (MRSI) was applied to quantitatively delineate both the concentration and activity of a cytosine deaminase-uracil phosphoribosyltransferase (CD-UPRT) fusion enzyme expressed from a transgene. MRSI enabled the generation of anatomically accurate maps of the intratumoral heterogeneity in fusion enzyme activity. We observed an excellent association between the CD-UPRT concentration and activity and the percentage of CD-UPRT + cells. Moreover, the regional levels of UPRT activity, as measured by imaging, correlated well with the biological affect of the enzyme. This study presents a translational imaging paradigm for precise, in vivo measurements of transgene activity with potential applications in both preclinical and clinical settings. [Cancer Res 2008;68(8):2878-84]

Non-invasive molecular and functional imaging of cytosine deaminase and uracil phosphoribosyltransferase fused with red fluorescence protein

“…This finding is consistent with our results of the in vitro cytotoxicity assay shown in Figure 2, and also supports previous studies demonstrating the combination of CD and UPRT added an additional enzymatic activation which sensitized transduced cells to 5-FC [7,19]. In contrast to a relatively short tumor retention of 5-FU in Walker256 tumors [9], prolonged tumor retention of 5-FU and 5-FC were seen in the R3327 tumor models, suggestive of intratumoral trapping of 5-FU and 5-FC. Recently, using a CD/UPRT()/CD/UPRT( ) co-transplanted tumor xenograft, Gade et al showed that MRS imaging enabled the generation of anatomically accurate maps of the intratumoral heterogeneity in enzyme activity [9].…”

“…In contrast to a relatively short tumor retention of 5-FU in Walker256 tumors [9], prolonged tumor retention of 5-FU and 5-FC were seen in the R3327 tumor models, suggestive of intratumoral trapping of 5-FU and 5-FC. Recently, using a CD/UPRT()/CD/UPRT( ) co-transplanted tumor xenograft, Gade et al showed that MRS imaging enabled the generation of anatomically accurate maps of the intratumoral heterogeneity in enzyme activity [9].…”

“…The cDNA, encoding the CD gene (of Saccharomyces cervisiae) and CD/UPRT fusio gene (UPRT of Haemophilus influenzae), was obtained by PCR amplification of the SFG retroviral vector [9]. The PCR products were digested with ApaI and AgeI, separated in 1% agarose gel and purified with the Gel Extraction Kit (QIAGEN, Santa Clarita, CA).…”

“…Increased prodrug activation has also been monitored with 19 F MRS in tumors expressing CD/UPRT [19]. A recent report has demonstrated the feasibility of mapping the regions wherein CD/UPRT is expressed in a preclinical model [9].…”

Noninvasive detection of carboxypeptidase G2 activity in vivo

“…vivo, both preclinically and clinically, as there are no detectable endogenous fluorinated metabolites (7,8). 19 F MRS has been successfully exploited to monitor cytosine deaminase-mediated activation of 5-fluorocytosine into 5-fluorouracil in vivo, in both ADEPT and GDEPT strategies (9)(10)(11). A 19 F MRS reporter probe for b-galactosidase is currently under investigation to translate the well-established in vitro reporter gene LacZ in vivo, as well as to monitor b-galactosidase-based GDEPT.…”