- Email: [email protected]
562. Development of a Novel Vaccine Vector Based on Synthesized Adenovirus Genome
ADENOVIRUS AND OTHER DNA VIRUS VECTORS II 561. Lister Strain Vaccinia Virus Demonstrates Favourable Anti-Tumor Efficacy and TumorSelectivity Compared to Western Reserve Strain
Jonathan Hughes,1 Pengju Wang,2 Ghassan Alusi,1 Vipul Bhakta,1 Yongchao Chu,2 Hailiang Shi,2 Iain McNeish,1 Andrea McCart,3 Istvan Fodor,4 Nicholas Lemoine,1 Yaohe Wang.1 1 Centre for Molecular Oncology, Barts Cancer Institute Queen Mary University of London, London, United Kingdom; 2SinoBritish Research Centre, Zhengzhou University, Zhengzhou, China; 3 Division of Experimental Therapeutics, Toronto-General Research Institute, Toronto, Canada; 4Center for Health Disparities & Molecular Medicine, Loma Linda University, Loma Linda.
560. Liver Retargeting of Adenovirus Using Robo4 Promoter and Hexon Substitution
Sergey A. Kaliberov,1 Lyudmila N. Kaliberova,1 Zhi Hong Lu,2 Meredith A. Preuss,3 Justin A. Barnes,4 Cecil R. Stockard,4 William E. Grizzle,4 Jeffrey M. Arbeit,2 David T. Curiel.1 1 Department of Radiation Oncology, School of Medicine, Washington University in St. Louis, St. Louis, MO; 2Department of Surgery, School of Medicine, Washington University in St. Louis, St. Louis, MO; 3Department of Medicine, University of Alabama at Birmingham, Birmingham, AL; 4Department of Pathology, University of Alabama at Birmingham, Birmingham, AL. Adenovirus serotype 5 (Ad5)-based vectors are well suited for gene therapy because they exhibit a favorable safety profile, have a high transgene capacity, and can be modified to infect target cells more readily. Despite these attributes, tissue-selective transduction by systemically administered Ad5-based vectors is currently confounded, in part, by sequestration of viral particles in the liver. Thus, a major goal in development of effective gene therapy vectors is to overcome the natural tropism of Ad5 for the liver and to optimize tissuespecific targeting. To address this issue, we have sought to retarget Ad using transductional and transcriptional methods. The purpose of this study was to demonstrate the utility of combined approaches for Ad5 targeting by using endothelial specific gene expression in combination with mitigation of hepatic sequestration of systematically administered Ad5 vectors. We have developed a panel of hexonmodified Ads including Ad5 vectors expressing a reporter transgene under control of the Roundabout 4 receptor (Robo4) promoter element for endothelial specific targeting. Hexon-modified Ad5 vectors expressing Luc gene under transcriptional control of the ubiquitous CMV or endothelial-specific Robo4 promoter were characterized by growth in cell culture, gene transfer in presence of human coagulation factor X, stability in vitro. Also, we have evaluated biodistribution of these Ad vectors in several animal models. Obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Also, substitution of the Ad5 hexon with the hypervariable region 7 from Ad3 hexon resulted in decreased liver tropism and improved gene expression in the pulmonary vasculature in comparison with Ad5. The results of these studies suggest that the combination of liver de-targeting using a genetic modification of the hexon protein with a transcriptional targeting approach using endothelium specific Robo4 promoter produces an additive effect in improving of Ad-mediated specific transgene expression to the lung endothelium.
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Vaccinia virus has many superior characteristics as a potential cancer therapeutic. There are several strains of vaccinia virus, some of which have established safety profiles following use as smallpox vaccines. The dominant strain in cancer research, Western Reserve, has been reported as the most potent oncolytic virus, but is a nonvaccine strain. Other strains, such as the European vaccine Lister strain, are largely untested. This study evaluated the anti-tumor potency and biodistribution of vaccinia virus strains using in vitro and in vivo models of cancer. Lister strain virus demonstrated superior anti-tumor potency and cancer-selective replication in vitro and in vivo, compared to Western Reserve strain, especially in human cancer cell lines and immunocompetent hosts. Studies examining the safety of the viruses following local and systemic delivery to immunocompetent mice bearing tumors revealed favourable viral biodistribution of the Lister strain, with higher viral titres in the tumors. Examination of serum from a mouse cancer model found lower levels of pro-inflammatory cytokines following Lister strain treatment, suggesting Lister strain may induce a diminished host inflammatory response. This study suggests that the Lister strain may be a more promising vaccinia virus strain than the Western Reserve strain for the development of cancer therapeutics.
562. Development of a Novel Vaccine Vector Based on Synthesized Adenovirus Genome
Yong Yang,1 Chao Zhang,1 Xiang Wang,1 Yudan Chi,1 Dongming Zhou.1 1 Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China. Adenoviruses (Ad) vectors have demonstrated great potential as vaccine-delivery vehicles by virtue of their ability to induce robust antigen-specific immune responses, which have been widely applied in various vaccine researches. However, clinical use of human adenoviral vectors for vaccine purposes is likely to be limited by preexisting immunity due to the previous human adenovirus infection. In order to circumvent this limitation, nonhuman adenovirus has been considered as an alternative choice, such as chimpanzee adenovirus. In our present study, we generated a novel strategy to create a totally artificial adenovirus that is similar to chimpanzee adenovirus (AdC). Based on the sequence alignment of non-human adenovirus genomes and the analysis of the organization and function of the AdC genomes, a novel artificial adenovirus genome sequence was designed, termed as AdS1. By dissection with several suitable unique restriction enzyme sites in the artificial genome sequence, AdS1 was divided into six fragments, namely EX, XB, BX, XS, SX, XH, which were synthesized by GeneScript Company. Then the six fragments were assembled into pNEB193 vector and the recombinant vector digested with Pac I was transfected into HEK 293 cells to rescue the recombinant adenovirus. To verify the efficiency and application of this novel Ad vector, several foreign genes of interest were inserted into the E1-deleted region of AdS1, respectively, and the recombinant viruses were rescued. Genetic stability of the vector and relative immune responses were tested to assess the potential of AdS1 as a vaccine vector. So we Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
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 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 1 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 1 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 5 a 10 μg/ml (15, 30, 60, 90 and 120 seg). Protein extraction was performed at 2, 6 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, 6 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 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 2 weeks up to 6 months post treatment). TG-C treatment showed significant improvement in the primary and secondary clinical evaluations of IKDC (P <0.05) and 100 mm VAS (P <0.05) compared to the placebo as shown in table 1. Serum TGF-1 protein levels were within normal ranges (65ng/ ml). RCR and TGF-1 DNA were not detected at the completion of the study.
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Jonathan Hughes,1 Pengju Wang,2 Ghassan Alusi,1 Vipul Bhakta,1 Yongchao Chu,2 Hailiang Shi,2 Iain McNeish,1 Andrea McCart,3 Istvan Fodor,4 Nicholas Lemoine,1 Yaohe Wang.1 1 Centre for Molecular Oncology, Barts Cancer Institute Queen Mary University of London, London, United Kingdom; 2SinoBritish Research Centre, Zhengzhou University, Zhengzhou, China; 3 Division of Experimental Therapeutics, Toronto-General Research Institute, Toronto, Canada; 4Center for Health Disparities & Molecular Medicine, Loma Linda University, Loma Linda.
560. Liver Retargeting of Adenovirus Using Robo4 Promoter and Hexon Substitution
Sergey A. Kaliberov,1 Lyudmila N. Kaliberova,1 Zhi Hong Lu,2 Meredith A. Preuss,3 Justin A. Barnes,4 Cecil R. Stockard,4 William E. Grizzle,4 Jeffrey M. Arbeit,2 David T. Curiel.1 1 Department of Radiation Oncology, School of Medicine, Washington University in St. Louis, St. Louis, MO; 2Department of Surgery, School of Medicine, Washington University in St. Louis, St. Louis, MO; 3Department of Medicine, University of Alabama at Birmingham, Birmingham, AL; 4Department of Pathology, University of Alabama at Birmingham, Birmingham, AL. Adenovirus serotype 5 (Ad5)-based vectors are well suited for gene therapy because they exhibit a favorable safety profile, have a high transgene capacity, and can be modified to infect target cells more readily. Despite these attributes, tissue-selective transduction by systemically administered Ad5-based vectors is currently confounded, in part, by sequestration of viral particles in the liver. Thus, a major goal in development of effective gene therapy vectors is to overcome the natural tropism of Ad5 for the liver and to optimize tissuespecific targeting. To address this issue, we have sought to retarget Ad using transductional and transcriptional methods. The purpose of this study was to demonstrate the utility of combined approaches for Ad5 targeting by using endothelial specific gene expression in combination with mitigation of hepatic sequestration of systematically administered Ad5 vectors. We have developed a panel of hexonmodified Ads including Ad5 vectors expressing a reporter transgene under control of the Roundabout 4 receptor (Robo4) promoter element for endothelial specific targeting. Hexon-modified Ad5 vectors expressing Luc gene under transcriptional control of the ubiquitous CMV or endothelial-specific Robo4 promoter were characterized by growth in cell culture, gene transfer in presence of human coagulation factor X, stability in vitro. Also, we have evaluated biodistribution of these Ad vectors in several animal models. Obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Also, substitution of the Ad5 hexon with the hypervariable region 7 from Ad3 hexon resulted in decreased liver tropism and improved gene expression in the pulmonary vasculature in comparison with Ad5. The results of these studies suggest that the combination of liver de-targeting using a genetic modification of the hexon protein with a transcriptional targeting approach using endothelium specific Robo4 promoter produces an additive effect in improving of Ad-mediated specific transgene expression to the lung endothelium.
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Vaccinia virus has many superior characteristics as a potential cancer therapeutic. There are several strains of vaccinia virus, some of which have established safety profiles following use as smallpox vaccines. The dominant strain in cancer research, Western Reserve, has been reported as the most potent oncolytic virus, but is a nonvaccine strain. Other strains, such as the European vaccine Lister strain, are largely untested. This study evaluated the anti-tumor potency and biodistribution of vaccinia virus strains using in vitro and in vivo models of cancer. Lister strain virus demonstrated superior anti-tumor potency and cancer-selective replication in vitro and in vivo, compared to Western Reserve strain, especially in human cancer cell lines and immunocompetent hosts. Studies examining the safety of the viruses following local and systemic delivery to immunocompetent mice bearing tumors revealed favourable viral biodistribution of the Lister strain, with higher viral titres in the tumors. Examination of serum from a mouse cancer model found lower levels of pro-inflammatory cytokines following Lister strain treatment, suggesting Lister strain may induce a diminished host inflammatory response. This study suggests that the Lister strain may be a more promising vaccinia virus strain than the Western Reserve strain for the development of cancer therapeutics.
562. Development of a Novel Vaccine Vector Based on Synthesized Adenovirus Genome
Yong Yang,1 Chao Zhang,1 Xiang Wang,1 Yudan Chi,1 Dongming Zhou.1 1 Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China. Adenoviruses (Ad) vectors have demonstrated great potential as vaccine-delivery vehicles by virtue of their ability to induce robust antigen-specific immune responses, which have been widely applied in various vaccine researches. However, clinical use of human adenoviral vectors for vaccine purposes is likely to be limited by preexisting immunity due to the previous human adenovirus infection. In order to circumvent this limitation, nonhuman adenovirus has been considered as an alternative choice, such as chimpanzee adenovirus. In our present study, we generated a novel strategy to create a totally artificial adenovirus that is similar to chimpanzee adenovirus (AdC). Based on the sequence alignment of non-human adenovirus genomes and the analysis of the organization and function of the AdC genomes, a novel artificial adenovirus genome sequence was designed, termed as AdS1. By dissection with several suitable unique restriction enzyme sites in the artificial genome sequence, AdS1 was divided into six fragments, namely EX, XB, BX, XS, SX, XH, which were synthesized by GeneScript Company. Then the six fragments were assembled into pNEB193 vector and the recombinant vector digested with Pac I was transfected into HEK 293 cells to rescue the recombinant adenovirus. To verify the efficiency and application of this novel Ad vector, several foreign genes of interest were inserted into the E1-deleted region of AdS1, respectively, and the recombinant viruses were rescued. Genetic stability of the vector and relative immune responses were tested to assess the potential of AdS1 as a vaccine vector. So we Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
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 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 1 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 1 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 5 a 10 μg/ml (15, 30, 60, 90 and 120 seg). Protein extraction was performed at 2, 6 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, 6 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 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 2 weeks up to 6 months post treatment). TG-C treatment showed significant improvement in the primary and secondary clinical evaluations of IKDC (P <0.05) and 100 mm VAS (P <0.05) compared to the placebo as shown in table 1. Serum TGF-1 protein levels were within normal ranges (65ng/ ml). RCR and TGF-1 DNA were not detected at the completion of the study.
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