645 Dual Therapeutic Reporter Genes Fusion for Enhanced Cancer Gene Therapy and Imaging Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S247[.]
CANCER-TARGETED GENE & CELL THERAPY II melanoma in an immune-competent mouse model Methods: p53responsive AdPG adenoviral vectors containing p19Arf or IFNβ were produced and expression verified by immunofluorescence and ELISA In vitro evaluation of the antiproliferative effect of single or co-transduction of B16F10 cells (B16, p53 wild-type) was done by FACS after annexin/PI staining C57/BL6 mice were injected subcutaneously with B16 cells and tumors were allowed to develop (∼0.5 cm2) At this point, three consecutive intra-tumor injections (3 day interval between injections) of individual or combined adenoviral vectors were performed Animals were then sacrificed and tumors collected and analyzed Results: We observed reliable expression of the transgenes in a p53-dependent manner Strikingly, the cotransduction with p19Arf and IFNβ vectors resulted in massive cell death consistent with apoptosis, whereas the transfer of individual genes was much less effective In situ gene therapy of subcutaneous tumors in an ectopic mouse model showed that p19Arf slowed, but did not prevent, tumor progression In contrast, IFNβ gene transfer, either alone or in combination with p19Arf, resulted in greatly reduced tumor volumes Moreover, the number of TUNEL-positive cells was greatly enhanced only when the combination of p19Arf and IFNβ was used as compared to either the controls or the single gene transfer conditions Conclusion: The combination of IFNβ and p19ARF proved to be more effective in inhibiting proliferation of B16 cells than either treatment alone, both in vitro and in vivo The use of p53responsive vectors for the restoration of p19ARF expression in the presence of IFNβ may represent a potential strategy for melanoma tumor suppression Work is underway to evaluate the potential of IFNβ to activate the immune system and prevent metastases in this treatment model This work was supported by FAPESP The conversion and entrapment is dynamically recorded using a time-lapsed microscopy Systemic and intratumoral delivery of CytoCy5S to NTR-expressing tumors in animals indicate steady and reproducible signals even 16 hours after-delivery 644 Imaging Nitroreductase (nfnB) Reporter Gene Expression in Living Animals Thillai V Sekar,1 Srabani Bhaumik,2 Jeannette Depuy,2 June Klimash,2 Ramasamy Paulmurugan.1 Dept.of Radiology, School of Medicine, Stanford University, Palo Alto, CA; 2GE Global Research, GE, Niskayuna, NY Gene directed enzyme prodrug therapy (GDEPT) is a promising suicide gene therapy approach to enhance selective cancer chemotherapy Nitroreductase (NTR)/CB1954 GDEPT system has been thoroughly investigated and is currently in phase I clinical trial However there is no non-invasive imaging method available to evaluate the therapeutic efficacy of this system in living animals Here, we developed a safe, sensitive and reproducible non-invasive imaging method to monitor NTR transgene expression that would allow quantitative assessment of both therapeutic efficacy and diagnostic outcome of NTR/CB1954 prodrug therapy The bacterial Nitroreductase gene (nfnB) cloned from E coli (K12) strain, in a eukaryotic expression vector under a constitutive CMV- promoter was used for the study NTR gene expression efficiency was evaluated in transient and stably transfected mammalian cell lines (LS174T, SKOV3, MCF7 and 293T) A novel fluorescent imaging dye CytoCy5S (a Cy5-labeled quenched substrate of NTR enzyme) was investigated on these cancer cell lines in vitro and in NTRtransfected tumor-bearing animals in vivo The substrate CytoCy5S produced Cy5-fluorescent signal only from cells transfected to express nitroreductase enzyme For this study we used different optical imaging systems both in vitro (Incell Analyzer 1000, Nikon Eclipse TE2000-U fluorescent microscope, Olympus 1X81 timelapsed microscope and FACS) and in vivo (IVIS and eXplore Optix) to evaluate NTR gene function Quantitative evaluation of fluorescent signal in stably transfected 293T cells after exposure to 100 ng/ml CytoCy5S for 60 showed a log-order elevation in fluorescent signal compared to corresponding control cells Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy The animals imaged at different time points following substrate injection via different routes showed a gradual increase in the signal level over time from NTR-bearing tumor site and not from NTR-negative control tumors Significant level of fluorescent signal is detected even after days post-implantation of NTR positive stable cells This is the first study to address visual monitoring and quantitative evaluation of NTR activity in small animals using CytoCy5S, and establishes the capability of NTR to function as an imageable reporter gene 645 Dual-Therapeutic Reporter Genes Fusion for Enhanced Cancer Gene Therapy and Imaging Ramasamy Paulmurugan,1 Thillai V Sekar,1 Srabani Bhaumik.2 Radiology, Stanford University, Palo Alto, CA; 2GE Global Research, Niskayuna, NY Gene directed enzyme prodrug therapy (GDEPT) is a promising suicide gene therapy approach to enhance selective cancer chemotherapy Herpes simplex virus thymidine kinase (TK) with ganciclovir (GCV), cytosine deaminase (CD) with 5-fluorocytidine (5-FC) and nitroreductase (NTR) with 5-(azaridin-1-yl)-2,4dinitrobenzamide (CB1954) are three most promising suicide gene/ prodrug combinations that are currently in different phases of clinical trials TK and CD sensitize prodrugs GCV and 5-FC respectively and block DNA synthesis, whereas the NTR activated prodrug CB1954 intercalate with DNA and develop cytotoxicity In this study we expressed TK and NTR in fusions to establish two different mechanisms of cell killing effect with their respective prodrugs in combinations to achieve efficient therapeutic value by lowering the toxicities associated with each prodrug The TK and NTR fusions in two different orientations (NTR-TK and TK-NTR) linked with a flexible 10aa linker (GGGGSGGGGS) were constructed and tested S247 NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY by the study The functional activities of TK and NTR were tested in HEK-293T cells stably expressing the fusion constructs of both orientations using 3H-PCV-uptake (TK) and, CytoCy5S (NTR) fluorescent substrate based cell sorter analysis and fluorescent microscopy Different dye based staining procedures (Calcein, PI and EtBr) were used for measuring the cell deaths and cell cycle arrests The results showed functional NTR only from cells expressing TKfused at the NH2-terminus of NTR gene TK was fully functional in TK-NTR fusion and ∼60% active in cells expressing NTR-TK fusion The HEK-293T cells stably expressing equal amount of functional NTR proteins from both NTR alone and TK-NTR-fusion constructs were used for further therapeutic evaluations using prodrugs GCV and CB1954 separately or in combinations By combining TK-NTR fusion we achieved significantly (p