565 A Placebo Controlled Phase IIb Clinical Studyof TissueGene C (TG C) in Patients with Osteoarthritis Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & C[.]
DNA VECTOROLOGY & GENE TARGETING II provide a feasible strategy to design and make novel Ad vector for vaccine development, which will avoid spending too much time and cost to screen new Ad vectors based on the traditional way 563 Construction of a Novel Bicistronic Adenoviral Vector Feilong Jie,1 Shengyao Wang,2 Jie Luo,1 Na Zhang,3 Jingang Zhang,3 Hongwei Li.1 Southern Medical University, Guangzhou, Guangdong, China; 2Mulan Pharmaceuticals, Shenzhen, China; 3Institute of Transfusion Medicine, The Academy of Military Medical Sciences, Beijing, China Achieving consistent expression of two therapeutic proteins in the same cells is an important issue for gene therapy applications Currently, the most commonly employed strategies include the use of multiple promoters, or internal ribosome entry site (IRES) elements However, it has shown that the protein encoded downstream of the IRES in these vectors is not reliably expressed Meanwhile, the use of multiple promoters may be compromised by interference between promoters, promoter silencing, and vector rearrangements or deletions We have constructed a novel adenoviral vector, pShuttleCMV-GFP-2A-Cherry, with a shared CMV promoter and a 2A peptide encoding region from the foot-and-mouth disease virus (FMDV), which cleaves at its own C-terminus, thereby separating the “fusion protein” into two independent proteins Importantly, the restriction enzyme Apa I site at the 5’ end is designed to help replace Cherry with target proteins, resulting in the C site of cleaved fusion protein with an excess proline In our study, 293T cells were separately transfected by the calcium phosphate method with the plasmids pShuttle-CMV-GFP-2A-Cherry, pShuttle-CMV-GFP, or pShuttleCMV-Cherry while 293A cells were separately infected with Ad5CMV-GFP-2A-Cherry, Ad5-CMV-GFP or Ad5-CMV-Cherry We found that GFP or Cherry expression delivered by related plasmids or packaged viruses were almost at the same level no matter the cell type It indicates that the novel adenoviral vector can elicit bicistronic expression simultaneously and equivalently Moreover, the C end of cleaved fusion protein has only one excess amino acid of proline These results will greatly benefit the study on both structure and function of target protein and gene therapy 564 Ciprofloxacin Regulates Negatively Egr1 Transcriptional Activity in Human Primary Tenocytes Transduced with Ad-Egr-1/Luc Francisco Martinez-F,1,2 Araceli Barrera-Lopez,1 Hugo SandovalZamora,1 Karina Guzman-M,1 Alejandro Jimenez-Orozco,2 Christian Y Lomeli-R,1 David T Curiel,3 Rebecca E FrancoBourland.4 Molecular Biotherapeutic Program, Skin & Tissue Bank, National Institute of Rehabilitation Ministry of Health, Mexico City, DF, Mexico; 2Dapartment of Pharmacology, School of Medicine, National Univeristy of Mexico, Mexico City, DF, Mexico; 3Department of Radiation Oncology, School of Medicine, University of Washington., St Louis, MO; 4Department of Biochemistry, National Institute of Rehabilitation, Mexico Introduction: Tenocytes proliferation rate is an essential factor for remodeling and maintenance of tendon integrity Negative balance of the rhythm of cell proliferation is a key factor for inflammatory process and tendon rupture Helicases inhibitors such as fluoroquinolones have been reported as factor for tendon rupture Hereby, we analyze the effect of ciprofloxacin on rate of cell proliferation in human primary tenocytes and the effect on the transcriptional regulation of egr-1 promoter based on transduction of adenoviral vector Adegr1-Luc Materials and methods: Cells and adenoviral vectors: No replicative recombinant adenovirus (AdEgr1-Luc) were packaged at large scale Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy in HEK-293 cells and purified according to the current protocol based on cesium chloride gradient protocol for in vivo application Viral Stock was titled by plaque assay and OD Human primary tenocytes (HPT) were obtained based on collagenase digestion protocol and cultured in DMEM/F12 Media supplemented with 10% HI-FBS and antibiotics under standard culture conditions for week and stored for experimental procedure 5x104 Tenocytes were seeded/well After 12 hrs, cells were infected with AdEgr1-Luc at 50 MOI´s during two hrs in serum reduced media After infection, cells were keeped in 1% of FBS for 24 hours at environment standard conditions for culture and ciprofloxacin at a 10 μg/ml (15, 30, 60, 90 and 120 seg) Protein extraction was performed at 2, and 12 hrs based on Cell Glo Lysis Buffer (Promega corp.) and luciferase activity was quantified using a multidetector DTX-880 Results: Human tenocytes transduced with AdEgr1-Luc are positively responsive to (10%) of Egr-1 promoter (18,233 LC/s) Luminescent activity in presence of ciprofloxacin was observed at 2, and 12 hrs (1,490 LC/S; 1,704.66 LC/s and 1,851.33 LC/s, respectively) Conclussion Ciprofloxacin inhibits transcriptional activity of egr-1 promoter in human primary tenocytes However, this fact is actually studies to determinate the signal transduction of regulation in tenocytes and the effect on cell proliferation rate by Cell proliferation assays Acknowledgments: This research project is granted by the National Council of Science and Technology of México Grant FOSIS/ CONACYT-Salud-2011-1-161624 DNA Vectorology & Gene Targeting II 565 A Placebo Controlled Phase IIb Clinical Studyof TissueGene-C (TG-C) in Patients with Osteoarthritis Jung Jong Cho,1 Tae Won Kim,1 Yeo Myeong Park,1 Eu Gene Jeong,1 Moon Jong Noh,1 Kwan Hee Lee,1 Bum Sup Lee.1 Kolon Life Science, Inc., Gwacheon-Si, Gyeonggi-Do, Korea The objective of this study was to determine both safety and efficacy of TG-C in patients with knee osteoarthritis (OA) TG-C is a cell mediated gene therapy that contains non-transduced (hChonJ) and transduced (hChonJb#7) human allogeneic chondrocytes The hChonJb#7 cells were transduced with retrovirus encoding TGF-1 gene The study was a multicenter, randomized, single-blind, placebocontrolled phase IIb trial (NCT01671072) The OA patients (n = 54) were randomized into two groups; TG-C (n=27, 1.8x107cells, 3.5 ml/ knee) and placebo (n=27, normal saline, 3.5 ml/knee) TG-C or saline was injected directly to the knee joints and clinical evaluations were made at 12 and 24 weeks post treatment The primary evaluation endpoint was the changes of the International Knee Documentation Committee (IKDC) which measures pain, sports activities, and daily function Secondary evaluation endpoints were WesternOntario and MacMaster University (WOMAC) score, Knee Injury and Osteoarthritis Outcome Score (KOOS), and 100 mm Visual Analogue Scale (VAS) Blood samples were analyzed to detect the replication competent retrovirus (at 12 and 24 weeks post treatment), TGF-1 DNA and protein (starting from weeks up to months post treatment) TG-C treatment showed significant improvement in the primary and secondary clinical evaluations of IKDC (P