- Email: [email protected]
489. Continuous Accumulation of Type II Collagen and Glycosaminoglycan without Scaffold Regenerates Cartilage
MESENCHYMAL STEM CELLS diagnostic) MSCs in inhibiting tumor growth was studied in nude mice. Xenograft tumors (n=10) were formed in the hind legs of nude mice followed by intratumoral injection of MSCs. Prodrug administration started 24 hours post MSC injection. MSCs without prodrug injection, prodrugs only and PBS were used as control groups. Tumor growths in treatment and control groups were monitored over a two months period. Results: The results demonstrated that the engineered stem cells are stably transfected and able to express luciferase and suicide genes. In the presence of prodrugs, among the suicide genes tested, the TK.007, SR39 and CD:UPRT genes showed maximum efficiency in killing stem cells. However, maximum bystander effect in killing SKOV-3 cells was observed with the prodrug (CB1954) corresponding to NTR and NTRO genes. The migration assay results illustrated that all engineered stem cells maintained their tumor tropic properties. While the in vivo studies are still in progress but preliminary results show that the engineered stem cells are able to significantly inhibit tumor growth in comparison to control groups. However, it is still too early to determine which suicide gene is more efficient than the others in killing SKOV-3 tumors. We expect to identify the most efficient suicide gene expressing MSCs as soon as the in vivo studies are finished. Acknowledgements: This work was made possible by the Rutgers University Funds for Research to A. Hatefi.
489. Continuous Accumulation of Type II Collagen and Glycosaminoglycan without Scaffold Regenerates Cartilage
Hyun Joo Yoon,1 Suk Bum Kim,2 Dhara Somaiya,1 Moon Jong Noh,1 Youngsuk Yi,1 Kwan Hee Lee.1 1 TissueGene Inc., Rockville, MD; 2Department of Orthopedice Surgery, Gil Medical Center, Gachon University, Incheon, Korea.
A localized non-surgical delivery of normal chondrocytes with irradiated genetically modified allogeneic human chondrocytes expressing transforming growth factor- β1 (TGF- β1) showed some efficacy in regenerating cartilage tissue. This successful regeneration of articular cartilage observed in our pre-clinical studies and human Phase I and II clinical trials led us to investigate molecular mechanisms of the cartilage repair. The amount of glycosaminoglycan (GAG) and type II collagen production were higher in chondrocytes when the cells were treated with TGF- β1. In addition, the TGFβ1 secreted to the culture supernatant of the genetically modified chondrocytes had shown the same effects in human chondrocytes. Attachment of chondrocytes to the cartilage defect as shown by the binding experiment, implies that the increased matrix performs the functions of a scaffold. In conclusion, irradiated genetically modified allogeneic human chondrocytes expressing TGF- β1, attached to the damaged cartilage area and produced TGF- β1, which activates the attached chondrocytes to produce type II collagen-GAG matrices, important components of hyaline cartilage, without the addition of scaffold.
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490. Baculovirus-Mediated Modification of Rodent and Human Adipose-Derived Stromal Cells for Cell Sheet Construction and VEGF165 Therapeutic Delivery
Pavel Makarevich,1,3 Evgeny Shevchenko,1 Anastasia Efimenko,3 Chia-Hsin Lu,2 Yu-Chen Hu,2 Yelena Parfyonova,1,3 Konstantin Dergilev.1 1 Laboratory of Angiogenesis, Russian Cardiology Research and Production Comples, Moscow, Russian Federation; 2Department of Chemical Engineering, National Tsing-Hua University, Hisnchu, Taiwan; 3Faculty of Medicine, Lomonosov Moscow State Univeristy, Moscow, Russian Federation. Modification of cells for therapeutic use has become a novel field in cell therapy and use of many DNA and RNA viral vectors has been tested to enhance cell regenerative potential. Baculovirus, which has been widely used for protein expression is a feasible tool for gene delivery characterised by non-pathogenecity and capability to infect a wide range of cell types. In this study we assessed application of baculovirus bi-component system for sustained over expression of human VEGF165 in primary adipose-derived stromal cells (ADSC) of rodent (mouse, rat) and human origin. In our experiments using GFP-reporter and sodium butyrate treatment to enhance transgene expression we have found, that efficacy of infection was up to 90% in mouse, 84% in rat and 76% in human ADSC. For therapeutic modification of ADSC we have used Bac-VEGFFRT-flanked and Bac-FLP recombinase co-infection to generate sustained VEGF-expressing ADSC. We found that VEGF expression was MOI-dependent and sodium butyrate treatment increased VEFGF concentration up to 1.2-1.4 fold in vitro. Concentration of VEGF was ranging from 5-12 ng/ml/10^5 cells after baculovirusmediated modification. We also didn’t find significant decrease of cell proliferation and viability 48 h post infection and sodium butyrate treatment. Next we seeded modified ADSC for cell sheet generation using from 5x10^5 to 1.5x10^6 cells. Cell sheets from mouse ADSC were transplanted subcutaneously into C57 mice with hindlimb ischemia for a short-term observation and comparison to simple injection of cell suspension. Using fluorescent-labeled ADSC we found transplanted cell sheets to be viable and retain subcutaneously for at least 96 h after transplantation. Serum concentration of human VEGF165 was below level of detection indicating minor to none system dissemination of protein. Moreover we have found explants of muscle injected with cell suspension or covered by modified cells sheets to produce VEGF165 into culture medium indicating cells were viable and capable to produce VEGF. As for limb perfusion measured by laser Doppler we haven’t found significant difference between cells suspension injection or cell sheet transplantation, possibly, due to short term of observation (5 days). Conclusion: Our preliminary data indicates that baculovirus is a powerful tool for modification of ADSC of both - rodent and human origin. Modified ADSC can be used for subcutaneous transplantation as cell sheets or intramuscular injection of cell suspensions providing VEGF165 delivery. Taking into account, that VEGF165 is a powerful growth factor, that can be used for treatment of ischemic disorders or neurodegenerative disease, developed system is an example of combined gene and cell therapy that has possible translational value in the future.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
489. Continuous Accumulation of Type II Collagen and Glycosaminoglycan without Scaffold Regenerates Cartilage
Hyun Joo Yoon,1 Suk Bum Kim,2 Dhara Somaiya,1 Moon Jong Noh,1 Youngsuk Yi,1 Kwan Hee Lee.1 1 TissueGene Inc., Rockville, MD; 2Department of Orthopedice Surgery, Gil Medical Center, Gachon University, Incheon, Korea.
A localized non-surgical delivery of normal chondrocytes with irradiated genetically modified allogeneic human chondrocytes expressing transforming growth factor- β1 (TGF- β1) showed some efficacy in regenerating cartilage tissue. This successful regeneration of articular cartilage observed in our pre-clinical studies and human Phase I and II clinical trials led us to investigate molecular mechanisms of the cartilage repair. The amount of glycosaminoglycan (GAG) and type II collagen production were higher in chondrocytes when the cells were treated with TGF- β1. In addition, the TGFβ1 secreted to the culture supernatant of the genetically modified chondrocytes had shown the same effects in human chondrocytes. Attachment of chondrocytes to the cartilage defect as shown by the binding experiment, implies that the increased matrix performs the functions of a scaffold. In conclusion, irradiated genetically modified allogeneic human chondrocytes expressing TGF- β1, attached to the damaged cartilage area and produced TGF- β1, which activates the attached chondrocytes to produce type II collagen-GAG matrices, important components of hyaline cartilage, without the addition of scaffold.
S188
490. Baculovirus-Mediated Modification of Rodent and Human Adipose-Derived Stromal Cells for Cell Sheet Construction and VEGF165 Therapeutic Delivery
Pavel Makarevich,1,3 Evgeny Shevchenko,1 Anastasia Efimenko,3 Chia-Hsin Lu,2 Yu-Chen Hu,2 Yelena Parfyonova,1,3 Konstantin Dergilev.1 1 Laboratory of Angiogenesis, Russian Cardiology Research and Production Comples, Moscow, Russian Federation; 2Department of Chemical Engineering, National Tsing-Hua University, Hisnchu, Taiwan; 3Faculty of Medicine, Lomonosov Moscow State Univeristy, Moscow, Russian Federation. Modification of cells for therapeutic use has become a novel field in cell therapy and use of many DNA and RNA viral vectors has been tested to enhance cell regenerative potential. Baculovirus, which has been widely used for protein expression is a feasible tool for gene delivery characterised by non-pathogenecity and capability to infect a wide range of cell types. In this study we assessed application of baculovirus bi-component system for sustained over expression of human VEGF165 in primary adipose-derived stromal cells (ADSC) of rodent (mouse, rat) and human origin. In our experiments using GFP-reporter and sodium butyrate treatment to enhance transgene expression we have found, that efficacy of infection was up to 90% in mouse, 84% in rat and 76% in human ADSC. For therapeutic modification of ADSC we have used Bac-VEGFFRT-flanked and Bac-FLP recombinase co-infection to generate sustained VEGF-expressing ADSC. We found that VEGF expression was MOI-dependent and sodium butyrate treatment increased VEFGF concentration up to 1.2-1.4 fold in vitro. Concentration of VEGF was ranging from 5-12 ng/ml/10^5 cells after baculovirusmediated modification. We also didn’t find significant decrease of cell proliferation and viability 48 h post infection and sodium butyrate treatment. Next we seeded modified ADSC for cell sheet generation using from 5x10^5 to 1.5x10^6 cells. Cell sheets from mouse ADSC were transplanted subcutaneously into C57 mice with hindlimb ischemia for a short-term observation and comparison to simple injection of cell suspension. Using fluorescent-labeled ADSC we found transplanted cell sheets to be viable and retain subcutaneously for at least 96 h after transplantation. Serum concentration of human VEGF165 was below level of detection indicating minor to none system dissemination of protein. Moreover we have found explants of muscle injected with cell suspension or covered by modified cells sheets to produce VEGF165 into culture medium indicating cells were viable and capable to produce VEGF. As for limb perfusion measured by laser Doppler we haven’t found significant difference between cells suspension injection or cell sheet transplantation, possibly, due to short term of observation (5 days). Conclusion: Our preliminary data indicates that baculovirus is a powerful tool for modification of ADSC of both - rodent and human origin. Modified ADSC can be used for subcutaneous transplantation as cell sheets or intramuscular injection of cell suspensions providing VEGF165 delivery. Taking into account, that VEGF165 is a powerful growth factor, that can be used for treatment of ischemic disorders or neurodegenerative disease, developed system is an example of combined gene and cell therapy that has possible translational value in the future.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy