1139 Antimetabolic Agents Improve Adenovirus Mediated Gene Expression by Increasing Viral mRNA Levels Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������®������������ �!����� ����[.]
GENE REGULATION: PROMOTER AND CONSTRUCT DESIGN doxorubicine (for both mutants) as selective agents ABCG expression was studied by flow cytometry using a monoclonal antibody recognizing an external domain (MAB4155, Chemicon) Different fluorescent dyes were assayed using efflux and retention experiments, both in the presence and in the absence of M fumitremorgin (FTC), a specific ABCG2 inhibitor Cells (105 cells/ ml) were loaded with different fluorescent probes for h at 37C Dye efflux was monitored for up to hours Results: R482 cells were able to efflux 800 nM mitoxantrone Mutant cells (both R482T and R482G) were able to efflux the following substrates: SYTO-13, Rhodamine 123, doxorubicine, daunorubicine, LDS-751, JC-1 and mitoxantrone at a concentration of 50 nM, 100 ng/ml, g/ml, 2.5 g/ml, 100 ng/ml, g/ml and 800 nM respectively The efflux of Hoechst 33342 was demonstrated using fluorescence microscopy and flow cytometry by means of competitive assays with Rho123 (or SYTO-13) Idarubicine, calceinAM, DRAQ5 and DiOC6 were not effluxed by mutants Dyeretention studies demonstrated the inhibitory effect of PSC833, FTC, cyclosporin A and verapamil into R482 cells, whereas verapamil did not have any effect in cells expressing mutant ABCG2 Conclusion: A variety of fluorescent dyes can be used to monitor the functional activity of the different ABCG2 forms Therefore, constructs carrying ABCG2 mutants may also help distinguishing the wild type ABCG2 activity in human hematopoietic cells or other targets for gene therapy Vectors carrying mutant versions of the ABCG2 gene can be used for in vivo or ex vivo selection and may enhance expression of therapeutic transgenes in human gene therapy protocols 1137 Gene Therapy ‘Switch’ Vectors Activated by Hypoxia and Ionizing Radiation Olga Greco,1 Brian Marples,1 Michael C Joiner,1 Simon D Scott.1 Radiation Oncology, Karmanos Cancer Institute/Wayne State University, Detroit, MI Intratumoral areas of low oxygen concentration are known to be refractive to radiotherapy treatment However, this physiological condition can be exploited for selective cancer gene therapy Consequently, we have developed a series of synthetic promoters selectively responsive to both hypoxia and ionizing radiation (IR) These promoters contain hypoxia regulatory elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase (PGK1) and vascular endothelial growth factor (VEGF) genes, and/or IRresponsive CArG elements from the Early Growth Response (Egr1) gene The HRE and CArG promoters were able to regulate expression of reporter and suicide genes in human tumor cells, following corresponding stimulation with hypoxia (0.1% O2) or Xirradiation (1 X 5Gy) [Greco et al, 2002, Gene Therapy 9:14031411] Furthermore, the chimeric HRE + CArG promoters could be activated by these stimuli independently, or significantly when given in combination, with the Epo HRE/CArG combination proving to be the most responsive and robust In order to amplify and maintain transgene expression, even following withdrawal of the triggering stimuli, we have now incorporated the HRE and CArG promoters into our previously described ‘molecular switch’ system [Scott et al, 2000, Gene Therapy 7:1121-1125] Importantly, the entire ‘switch’ has now been engineered as a single vector molecule The new series of HRE/ CArG switch vectors have been recently tested in an HSVtk/GCV suicide gene assay Results reveal that a) more tumor cell kill is achieved when the switch is employed compared with the HRE and CArG promoters driving HSVtk expression directly and b) the Epo HRE/CArG promoter appears to function as efficiently as the strong constitutive CMV IE promoter Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy 1138 AZT Enhances CMV-IE Promoter Driven Human Factor IX Gene Expression in HepG2 Cell Culture Fayaz R Khazi,1 Katherine A High.1 Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA Recombinant adeno-associated viral (rAAV) vectors have been used to treat hemophilia B by efficient transfer of Factor IX (hF.IX) into liver and muscle in animal models, and human trials are underway Since a substantial population of potential patients with severe hemophilia B is also infected with human immunodeficiency virus (HIV), it is important to study the effect of anti-retroviral drugs on the efficacy of rAAV-mediated hemophilia gene therapy 3´-Azido3´-deoxythymidine (AZT), used in the highly active antiretroviral therapy (HAART) against HIV, is a nucleoside analog that inhibits human immunodeficiency virus (HIV) reverse transcriptase activity AZT has also been implicated in inhibiting chromosomal DNA replication, altering mitochondrial structure and function, and contributing to accumulation of oxygen radicals in the host cells Previous studies conducted in our laboratory (Swanson et al, 1997) to study the effect of HAART drugs on transduction of rAAV vectors indicated enhanced transgene expression in the presence of AZT and 2´,3´-dideoxyinosine Here, we have used HepG2 and HEK 293 cell cultures to study the effect of AZT on rAAV-mediated transgene expression Cells pre-incubated for hour with conditioned medium containing 2, 25, 50, 100, 200 and 500 μM AZT, were transduced with 20x104 MOI rAAV vectors expressing hF.IX driven either by a CMV-IE enhancer/promoter or an EF1a promoter The cells were maintained in medium containing different levels of AZT for up to 72 hours Medium was collected from each treatment at different time points (3, 24, 36, 60 and 72h) and analyzed for secreted hF.IX expression using ELISA Our results indicate a 10-fold (5375ng/ml) increase in hF.IX expression in HepG2 cells over baseline following transduction with a CMV-IE driven hF.IX gene This increase in expression correlated with increasing AZT concentration However, HepG2 cells transduced with an EF1a driven gene maintained hF.IX levels similar to untreated controls (514ng/ml) Expression of hF.IX remained similar to untreated controls (429ng/ml) in HEK 293 cells transduced with a CMV-IE or an EF1a driven hF.IX gene The data suggest a role for AZT in inducing expression of proteins that specifically interact with the CMV promoter to enhance transgene expression Why this is observed in HepG2 cells but not in HEK 293 cells is not yet clear We are currently investigating a role for RelA/NF-kappaB in the induction of CMV-IE promoter 1139 Antimetabolic Agents Improve Adenovirus-Mediated Gene Expression by Increasing Viral mRNA Levels Xiaowu Huang,1 Theodore S Lawrence,1 Ming Zhang.1 Radiation Oncology, University of Michigan, Ann Arbor, MI, United States Efficiency and selectivity of transgene expression in tumors are major concerns in adenovirus gene therapy for cancer Our previous study demonstrated that adenovirus vector mediated yeast cytosine deaminase (yCD) gene expression selectively in human colon carcinoma cells by using an enhanced CEA promoter However, the overall yCD expression, thus, prodrug conversion (5FC to 5FU), was low in tumors In this study, we test whether the use of chemotherapeutic agents will enhance transgene expression in cells Adenovirus vector containing CMV-driven b-gal gene is used for infection in human colon carcinoma (LoVo), hepatocellular carcinoma (SMMC7721), and rat hepatocytes (WB) We found that b-gal activities are significantly enhanced in all cells treated by antimetabolic S439 GENE REGULATION: PROMOTER AND CONSTRUCT DESIGN agents (5.0-, 3.3- and 6.0-fold, respectively, at 50 mM 5FU and 6.6, 4.8-, and 8.0-fold, respectively, at mM hydroxyurea) but not by alkylating agents (100 mM carboplatinum and 100 mM cisplatinum) Viral uptakes in cells are determined by viral DNA contents using real-time PCR and appear to be upregulated by pre-treatment of cells with 5FU (2.0-, 1.5-, and 2.0-fold, respectively) and hydroxyurea (2.5-, 1.8-, and 2.1-fold, respectively) In addition, viral infections in cells prior to drug treatment result in greater induction for b-gal activities, suggesting that intracellular activation of viral gene is also involved By using real-time PCR, we found that viral DNA replicates in LoVo and SMMC7721 at 48 h after infection (5.3- and 5.7-fold, respectively) whereas no replication is detected in WB cells or drug-treated cells In contrast, mRNA levels for b-gal determined by real-time RT-PCR analysis are significantly elevated in LoVo, SMMC7721 and WB cells treated by 5FU (36.0-, 6.0-, and 5.0-fold, respectively) and hydroxyurea (4.0-, 11.0-, and 10-fold, respectively) as compared to non-treated control cells The level of mRNA for host gene a-actin remains unchanged, indicating these drugs may affect viral mRNA in dependent of cellular transcription Our findings provide scientific basis for the combination of enzyme/ prodrug adenovirus gene therapy and chemotherapy in improving selectivity and efficiency in cancer treatment 1140 Characterization of Sub-Nuclear and PostMitotic Plasmid Trafficking in Microinjected Cells Joshua Z Gasiorowski,1 David A Dean.1 Pulmonary Medicine, Northwestern University, Chicago, IL, United States One of the least understood aspects of non-viral gene therapy is the role of sub-nuclear plasmid localization Plasmids successfully delivered across the plasma membrane must still traffic through the cytoplasm and into the dense, highly organized nucleus before any transgene expression can occur Even if gene delivery is successful, episomal plasmids will cease to express if they fail to come into contact with the appropriate cellular transcription factors or are later excluded from the nucleus We used nuclear microinjection experiments in conjunction with several different fluorescent labeling techniques and in situ hybridizations to track sub-nuclear plasmid movement pEGFP-N1 injected directly into the nucleus moves into an organized speckle pattern over time After injection, most of these plasmid speckles co-localize with transcriptional machinery within 30-240 minutes whereas a plasmid containing no promoter or transgene (pBR322) shows little or no co-localization with the same factors The timeframe that transcriptional machinery colocalized with pEGFP-N1 also corresponded with the first visible detection of transgene Therefore, expression level may be linked to co-localization of the plasmid with a host of cellular proteins within discreet sub-nuclear regions It is currently unclear as to whether the plasmid actually moves into areas that are dense in transcriptional machinery or instead recruits the necessary cellular proteins to it We also tracked nuclear injected plasmids after a round of cell division using several different fluorescent labeling methods We show that the post-mitotic location of plasmids differed depending on the type of labeling method used Unlabeled plasmids detected by in situ hybridization or labeled with Cy3 protein nucleic acid (PNA) were distributed throughout the nuclei of divided cells while plasmids fluorescently labeled with commercial kits were excluded from the nucleus after cell division indicating that different fluorophores and labeling techniques may interfere with certain protein-DNA interactions that maintain the post-mitotic nuclear retention of episomal plasmids S440 1141 Insulin-Like Growth Factor-1 Enhances Plasmid Transgene Expression through a Phosphatidylinositol 3-Kinase-Dependent Pathway Edward Chaum,1,2,3 Xiuying Yang,1 Huaitao Yang.1 Ophthalmology; 2Pediatrics; 3Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, TN, United States Introduction The goal of our work is to modulate the phenotype of the human retinal pigment epithelium (RPE) using transgenic growth factors, to treat degenerative diseases of the retina We have previously shown that expression of an IGF-1 transgene enhances the proliferative potential of cloned RPE cells in vitro in a dosedependent manner Recent studies of these clones showed that IGF1-transduction also enhances transient transgene expression via a phosphatidylinositol 3-kinase (PI3-K)-dependent pathway Methods Naive human RPE cells and IGF-1-transduced RPE clones expressing increased levels of a biologically active IGF-1 transgene were transfected by lipofection with the pGeneGrip plasmid that encodes a CMV-promoted green fluorescent protein (GFP) The efficiency of plasmid-mediated transgene expression in the RPE was measured by quantifying GFP-fluorescence using flow cytometry 48 hours after transfection Transfection studies were performed in parallel under low-serum conditions, at confluence and in the presence of the PI3-K inhibitor, LY294002 Results IGF-1-treated RPE cells and IGF-1-transduced RPE clones c14, c62, and c49 demonstrated an enhanced and dosedependent increase in GFP transgene expression following transfection, compared to naive controls (treated, P < 0.001; c14, P < 0.003, c62, P < 0.001, c49, P < 0.001; paired samples, t-test) The enhanced efficiency of transgene expression was more pronounced in cycling subconfluent cells transfected under low-serum conditions, but was also seen in non-cycling, confluent RPE clones (c14, P < 0.038, c62, P < 0.003, c49, P < 0.007) Culturing the cells in the presence of the PI3-K inhibitor, LY294002 significantly inhibited the increased efficiency of transgene expression in naive cells and IGF-1-transduced RPE clones (naive, P < 0.022, c14, P < 0.006, c62, P < 0.008, c49, P < 0.006) Conclusions These studies show a direct and dose-dependent correlation between the level of IGF-1 expression and enhanced transgene expression following transient lipofection in RPE cells and IGF-1 transduced RPE clones Inhibition of the PI3-K pathway, a downstream arm of the IGF-1 signal transduction cascade, blocks this biological effect of IGF-1 gene expression Enhancement of transgene expression is a previously unrecognized biological effect of IGF-1 signal transduction Possible molecular mechanisms of action are discussed 1142 Regulation of Transgene Expression in Mouse Liver Mohammed Al-dosari,1 Guisheng Zhang,1 Joseph Knapp,1 Dexi Liu.1 Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, United States Understanding the mechanisms involved in regulating gene expression in a target specific manner is a critical part of developing practical applications in gene therapy Transgene expression was studied in the liver of mice transfected with various plasmid constructs by the hydrodynamics-based procedure Thirteen constructs were examined, including those containing viral promoters/ enhancer sequences (CMV, CMV/EBV, RSV), or those of liver specific regulatory sequences (human alpha 1-antitrypsin promoter, bovine serum albumin promoter, 5’-end flanking sequences of cytochrome P450 genes of 1A2, 2B10, 2C9, 2C18, 2D6, and 3A4) In addition, Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy ... IGF-1 transgene were transfected by lipofection with the pGeneGrip plasmid that encodes a CMV-promoted green fluorescent protein (GFP) The efficiency of plasmid -mediated transgene expression. .. by alkylating agents (100 mM carboplatinum and 100 mM cisplatinum) Viral uptakes in cells are determined by viral DNA contents using real-time PCR and appear to be upregulated by pre-treatment... mechanisms involved in regulating gene expression in a target specific manner is a critical part of developing practical applications in gene therapy Transgene expression was studied in the liver