774 A Functional Model System To Evaluate Spliceosome Mediated RNA Trans Spicing Using a LacZ Target Reporter Mouse Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© ���������[.]
CANCER TARGETED GENE THERAPY II sequence) and transcriptional silence in air across our cell line panel (up to fold reduction against various HRE constructs) TNFα has been placed under the transcriptional control of this construct and in vitro studies are ongoing In addition, we have cloned luciferase driven by our novel CA9-promoter in an adenoviral context and are currently evaluating whether the aerobic silencing apparent from in vitro studies translates to tumour specific expression when delivered systemically in nude mice Further refinement of this vector through the incorporation of SREs is anticipated to give robust radiation responsiveness resulting in the generation of a novel, tightly regulated, dual responsive vehicle for cancer gene therapy 772 Modulation of Transcriptional Activity of Egr-1 Promoter Mediated by GM-CSF in Human Gliomas Francisco Martinez-F,1,2 Erika Gomez,1 Joanne T Douglas,3 David T Curiel,3 Andres A Gutierrez.1 Cell Therapy Unit, Centro Nacional de Rehabilitacion, Mexico, DF, Mexico; 2Department of Pharmacology, School of Medicine UNAM, Mexico, DF, Mexico; 3Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, United States Introduction Suicide gene transfer mediated by adenoviral vectors is key factor for development of new treatments of gene therapy for human gliomas A crucial goal for this strategy is modulate the transcriptional process on the target cells for external factors such as cytokines, hormones or physical agents Based on this concept, we are testing several inductors to drive and modulate the transcriptional activity of the egr-1 promoter on the luciferase reporter gene Mueller et al., describe expression of Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) receptors in human glioma biopsies and autoregulated expression of receptors In other hand, Sakamoto et al describe egr1-promoter activity induced by GM-CSF in hematopoietic cells, fibroblasts and synovial cells Hereby, we test to GM-CSF and its value as inductor for gene therapy in different glioma cell lines Material and methods Adenoviral constructs have been generated based on the AdEasy system by homologous recombination in bacterial cells The –600 pb fragment of egr-1 Promoter was cloned upstream of the luciferase gene into the MCS of pShuttle plasmid Adenoviral plasmid generated was packaged into HEK293 cells, screened, purified for a large scale production and titer for further experiments Glioma Cell lines (CH235, D54 and U373) were cultured in standard conditions with D-MEM media containing 10% of FBS and 1% of Antibiotics 1x 105 cells were seeded and infected with 25 MOI for hours in low serum OPTIMEM media (Invitrogen) After that, cells were washed and maintained in D-MEM media containing % of FBS for 24 hrs before induction with nM and nM GM-CSF Stimulated cells with 20 % of FBS were used as positive control Proteins were obtained using Cell culture lysis (Promega Corp.) and stored at –20° C Luciferase Activity was quantified using Luciferase Assay System (Promega Corp.) in a Victor Wallac instrument according to manufacturer instructions All experiments were performed for triplicate and luciferase activity was corrected to protein amount Results and conclusions For CH235 cell line, maximum response was reached at six hours after induction with nm and nM of GM-CSF This response was similar or higher than positive control and compared to basal expression means 75% of luciferase activity In D54 cell line stimulated with nM of GM-CSF shows holds of luciferase activity compared to basal control at 12 hrs U373 Cell line shows 4.5 holds of luciferase activity at nM of GM-CSF and slightly upper than the positive control We are exploring the way to enhance this response and realizing comparative studies with other promoters and inductors S298 773 The TARP Promoter in Prostate Cancer Gene Therapy Wing-Shing Cheng,1 Valeria Giandomenico,1 Magnus Essand.1 Clinical Immunology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden A promoter, driving the transcription of a tissue specific gene can be used to restrict therapeutic gene expression in target cells from the same tissue Prostate cancer is a particularly appropriate target for such gene therapy approach since the prostate gland is nonessential for life and a number of prostate specific promoter and enhancer elements have been characterized TARP (T cell receptor γ chain alternate reading frame protein) is a protein that in males is uniquely expressed in epithelial cells within the acinar ducts of the prostate and in prostate cancer cells Here we describe the TARP promoter and its potential use in prostate cancer gene therapy We found that the proximal TARP promoter contains a functional androgen response element and that TARP expression is regulated by testosterone at the transcriptional level through androgen receptor activation Luciferase reporter gene assays revealed that the basal activity of the TARP promoter is higher than the activity of the PSA promoter Chimeric regulatory sequences containing the TARP promoter together with enhancers from prostate specific antigen (PSA) and/or prostate specific membrane antigen (PSMA) were constructed A regulatory sequence consisting of the TARP promoter and the PSA enhancer gives 20 times higher activity than a sequence consisting of the PSA promoter and PSA enhancer The TARP promoter/PSA enhancer regulatory sequence retains its prostate specificity, although its transcriptional activity is strictly controlled by testosterone A regulatory sequence consisting of the TARP promoter and the PSMA enhancer gives high prostate specific expression also under testosterone-depleted conditions Prostate cancer patients are often treated by androgen withdrawal In these cases it may be beneficial to the patient to have a gene therapy vehicle harboring high prostate specific gene expression that is not dependent on testosterone GENE REGULATION: TECHNOLOGY AND APPLICATIONS 774 A Functional Model System To Evaluate Spliceosome Mediated RNA Trans-Spicing Using a LacZ Target Reporter Mouse Xiaoming Liu,1 M Puttaraju,4 Gary S Mansfield,4 Meihui Luo,1 Ryan Driskell,1 Lloyd G Mitchell,4 John F Engelhardt.1,2,3 Department of Anatomy and Cell Biology, The University of Iowa, Iowa City, IA, United States; 2Internal Medicine, The University of Iowa, Iowa City, IA, United States; 3Center for Gene Therapy, The University of Iowa, Iowa City, IA, United States; Intronn Inc, Rockville, MD, United States The successful treatment of Cystic Fibrosis (CF) requires both functional correction and proper regulation of the Cystic Fibrosis Transmembrane Regulator (CFTR) protein In order to functionally test the in vivo potential of Spliceosome Mediated RNA Transsplicing (SMaRT) to repair the human CFTR gene, a transgenic animal model expressing the target region of the human gene in the context of a reporter was created Previous studies in human bronchial xenografts that model stem cell reconstitution of airway epithelia have demonstrated that expression of full length CFTR in dividing or differentiating cells causes a dramatic decrease in the clonal expansion of CFTR expressing cells This suggests that ectopic or overexpression of full length CFTR in human airway stem cells may confer a selective disadvantage in terms of proliferation and/or differentiation We have previously reported that SMaRT can correct the deltaF508 CFTR mutant protein in polarized airway models SMaRT reduced the level of CFTR-mediated developmental toxicity Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy GENE REGULATION: TECHNOLOGY AND APPLICATIONS leading to improved reconstitution of airway stem cells and partial restoration of CFTR-mediated Cl- permeability At present, the trans-splicing efficiency in vivo using SMaRT technology is not known To address transgene delivery and functional correction more closely, we developed a transgenic mouse that expresses a 3’ mutant LacZ mini-gene containing a mini-intron-9 of CFTR in the middle of the coding sequence, splitting the LacZ cDNA into two exons We also generated recombinant adenoviral and AAV vectors that encode pre-trans-splicing molecules (PTMs) designed to base-pair with human CFTR intron and trans-splice in a non-mutant 3’-LacZ exon PTMs delivered by both adenoviral and AAV vectors were capable of rescuing beta-galactosidase activity in cell lines harboring an integrated form of the LacZ mini-gene target Preliminary studies indicate that the functional correction of b-galactosidase can be quantified by histochemistry in the lung and skeletal muscle of transgenic mice after the administration of the PTM by adenoviral or AAV vector These results demonstrate that SMaRT technology can efficiently repair a mutant gene by trans-splicing into human CFTR intron 9, and that PTMs can be delivered in vivo by viral vectors to the appropriate tissues SMaRT offers the potential to treat a wide range of diseases caused by genes which are larger than the 10-fold in both of these stably transduced lines This level of repression was sufficient to generate a functional protein knock out, as demonstrated by the loss of the CHK2-dependent activation of p53 induced by DNAdamage The specificity of ZFP-TF driven repression was determined at the mRNA level using microarray technology We found that just a single gene, CHK2, was regulated by the ZFP-TF within the monitored genome (22,000 probes) Moreover, this result is independent of the cell type tested These data demonstrate the utility of ZFP-TFs for target validation and highlight the potential of these agents as therapeutic treatments in the clinic 777 A Novel Self-Inactivating Retroviral Vector Incorporating Retinoic Acid Responsive Elements Provides a Platform for Assessment of Transcriptional Responsiveness to Retinoids in Cancer Cell Lines Diana E Jaalouk,1 Laurence Lejeune,1 Milena Crosato,1 Sylvie Mader,2 Jacques Galipeau.1,3 Lady Davis Institute for Medical Research, McGill University, Montreal, QC, Canada; 2Department of Biochemistry, Universite de Montreal, Montreal, QC, Canada; 3Division of HematologyOncology, Jewish General Hospital, Montreal, QC, Canada All-trans retinoic acid (ATRA) has long been known as a transcriptional activator that inhibits cell proliferation and promotes differentiation of many cell types Recently, the drug’s efficacy in anticancer therapy has been broadened by reports suggesting a synergistic effect of ATRA to several cancer gene therapy approaches Our objective is to establish an efficient gene expression system that will provide a screening basis for retinoid responsiveness in cancer cell lines We hypothesize that cell lines transduced with retrovectors incorporating retinoic acid responsive elements (RAREs) will exhibit modulated transgene expression upon retinoid stimulation To test this hypothesis, we designed a self-inactivating retrovector by creating a 341 bp deletion in the U3 region of the Moloney Murine leukemia Virus 3’LTR We then incorporated into S299 ... Montreal, QC, Canada; 3Division of HematologyOncology, Jewish General Hospital, Montreal, QC, Canada All -trans retinoic acid (ATRA) has long been known as a transcriptional activator that inhibits... Liver Maider Zabala,1 Lin Wang,1 Cheng Qian,1 Jesus Prieto,1 M Gabriela Kramer.1 Internal Medicine, University of Navarra, Pamplona, Navarra, Spain Pharmacological control of gene expression can... designed to base-pair with human CFTR intron and trans- splice in a non-mutant 3’ -LacZ exon PTMs delivered by both adenoviral and AAV vectors were capable of rescuing beta-galactosidase activity