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682. A Modular Vector System for Rreprogramming of Somatic Cells into Induced Pluripotent Stem (iPS) Cells
STEM CELL THERAPIES II ideally combine the catalytic activity of ribozymes with the stability of oligodeoxynucleotides, are easy to synthesize and less sensitive to chemical and enzymatic degradation than RNA-based reagents. Factors to influence the eventual therapeutic use of DNAzymes include its efficient cellular uptake, subcellular localization, and stability. A particularly important challenge to achieve the successful down-regulation of gene expression is to deliver DNAzymes efficiently to its intended site of action. Since various nanomaterials have unique, useful chemical, physical, and mechanical properties, they can be used for a wide variety of applications including nanobased biosensors, drug delivery devices, diagnostic tools, and means for tissue engineering and fundamental cell biology studies, etc. In this report, we describe synthesis and characterization of a multifunctional magnetic nanoparticle (MION) for both noninvasive in vivo imaging and delivery of DNAzyme to target organ for hepatitis C treatment. Hepatitis C is one of the infectious diseases in the liver caused by the hepatitis C virus (HCV), a small-sized, enveloped, positive sense single strand RNA virus. The multifunctional nanoparticles consist of magnetic nanoparticles labeled with near-infrared fluorescent dye and conjugated to a synthetic DNAzyme targeting a gene of interest. In addition, these nanoparticles are tailored with cellpenetrating peptides (CPPs) helping membrane translocation process. We first demonstrated the silencing effect of a gene of interest by Dz-loaded multifunctional nanoparticles in cultured human liver cells (Huh-7). For in vivo study in mice, we performed the alkaline phosphatase activity assay using sera to measure the efficiency of DNA transfection, and found that the hydrodynamic delivery of the reporter plasmid elicited and was maintained over-expression in mice for some periods. The delivery of the nanoparticles would be monitored in dual fashions in vitro and in vivo. We believe that our Dz-conjugated nanoparticles will be one of the widely applicable therapeutic options for the efficient HCV treatment in near future.
Stem Cell Therapies II 682. A Modular Vector System for Reprogramming of Somatic Cells into Induced Pluripotent Stem (iPS) Cells
Axel Schambach,1 Eva Warlich,1 Tobias Cantz,2,3 Martijn Brugman,1 Tobias Mätzig,1 Hans Schöler,3 Christopher Baum.1,4 1 Experimental Hematology, Hannover Medical School, Hannover, Germany; 2JRG Stem Cell Biology, Hannover Medical School, Hannover, Germany; 3Cell and Developmental Biology, MPI for Molecular Biomedicine, Münster, Germany; 4Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati. Recently, Yamanaka and colleagues showed that the transduction of somatic cells with 4 transcription factors (Oct3/4, Sox2, Klf4, c-Myc) resulted in reprogramming into induced pluripotent stem cells (iPS), which have morphological and functional properties of ES cells. This finding offers new perspectives for the generation of patient- or disease-specific iPS cells as well as new avenues for disease models, drug discovery and regenerative medicine. Here, we describe a new modular lentiviral vector system for these 4 reprogramming factors (either of human or murine origin). First, for constitutive expression we used the strong retroviral promoter SFFV, which mediates high transgene expression in a variety of cells and is frequently silenced in ES cells. Using this system, we induced endogenous Oct3/4expression with an efficiency of up to 14% with all 4 factors, or 7% with 3 factors (without c-myc), respectively. Second, we linked each transcription factor to a different fluorescent marker to be able to follow their gene marking and expression kinetics. This allowed us to monitor vector silencing in the process of reprogramming. Third, optionally we designed vectors under control of the tet system to mediate regulated expression of the reprogramming genes. Finally, S260
the same modular system can be used for transient, non-integrating delivery of the transcription factors using integrase-deficient retroand lentiviral vectors, potentially avoiding the risks of permanent transcription factor expression and insertional mutagenesis. In total, we derived >40 vectors encoding reprogramming factors, which are all validated for reprogramming efficiency using an Oct4-GFP reporter model and detailed ES cell marker analysis. We isolated >80 iPS clones, and performed further molecular and functional studies including vector copy number determination and teratoma formation with a subset of these. The described vector system may ease the generation of murine and human iPS-like cells and also contribute to the identification of the underlying mechanisms.
683. Gene-Enhanced Mesenchymal Stem Cells Improve Tendinitis Repair in a Large Animal Model
Alan J. Nixon,1 Lauren V. Schnabel,1 Maureen E. Lynch,2 Marjolein C. van der Meulen.2 1 Comparative Orthopaedics Laboratory, Cornell University, Ithaca, NY; 2Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY.
Introduction: Tendinitis remains a catastrophic injury amongst athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin-like growth factor-I (IGF-I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs as well as IGF-I gene-enhanced MSCs (AdIGF-MSCs) on tendon healing in vivo. Methods: Collagenase-induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Five days post collagenase injection (t=0), tendons were treated with 10 x 106 MSCs or 10 x 106 AdIGF-MSCs. Control limbs were injected with 1mL of PBS. Ultrasound examinations were performed at t=0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure, and healing lesions evaluated for biochemical composition and histologic characteristics. Quantitative RT-PCR for expression of collagen types I and III, IGF-I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), and aggrecanase-1 (ADAMTS-4) were performed as well as DNA, glycosaminoglycan, and total soluble collagen assays. Results: Both MSC and AdIGF-MSC intralesional injection resulted in significantly more normal tendon histological scores (Fig 1), and a trend toward improved biomechanical characteristics.
Levels of DNA, collagen, and tendon GAG content where similar bewteen MSC implanted and control tendons. There were Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Stem Cell Therapies II 682. A Modular Vector System for Reprogramming of Somatic Cells into Induced Pluripotent Stem (iPS) Cells
Axel Schambach,1 Eva Warlich,1 Tobias Cantz,2,3 Martijn Brugman,1 Tobias Mätzig,1 Hans Schöler,3 Christopher Baum.1,4 1 Experimental Hematology, Hannover Medical School, Hannover, Germany; 2JRG Stem Cell Biology, Hannover Medical School, Hannover, Germany; 3Cell and Developmental Biology, MPI for Molecular Biomedicine, Münster, Germany; 4Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati. Recently, Yamanaka and colleagues showed that the transduction of somatic cells with 4 transcription factors (Oct3/4, Sox2, Klf4, c-Myc) resulted in reprogramming into induced pluripotent stem cells (iPS), which have morphological and functional properties of ES cells. This finding offers new perspectives for the generation of patient- or disease-specific iPS cells as well as new avenues for disease models, drug discovery and regenerative medicine. Here, we describe a new modular lentiviral vector system for these 4 reprogramming factors (either of human or murine origin). First, for constitutive expression we used the strong retroviral promoter SFFV, which mediates high transgene expression in a variety of cells and is frequently silenced in ES cells. Using this system, we induced endogenous Oct3/4expression with an efficiency of up to 14% with all 4 factors, or 7% with 3 factors (without c-myc), respectively. Second, we linked each transcription factor to a different fluorescent marker to be able to follow their gene marking and expression kinetics. This allowed us to monitor vector silencing in the process of reprogramming. Third, optionally we designed vectors under control of the tet system to mediate regulated expression of the reprogramming genes. Finally, S260
the same modular system can be used for transient, non-integrating delivery of the transcription factors using integrase-deficient retroand lentiviral vectors, potentially avoiding the risks of permanent transcription factor expression and insertional mutagenesis. In total, we derived >40 vectors encoding reprogramming factors, which are all validated for reprogramming efficiency using an Oct4-GFP reporter model and detailed ES cell marker analysis. We isolated >80 iPS clones, and performed further molecular and functional studies including vector copy number determination and teratoma formation with a subset of these. The described vector system may ease the generation of murine and human iPS-like cells and also contribute to the identification of the underlying mechanisms.
683. Gene-Enhanced Mesenchymal Stem Cells Improve Tendinitis Repair in a Large Animal Model
Alan J. Nixon,1 Lauren V. Schnabel,1 Maureen E. Lynch,2 Marjolein C. van der Meulen.2 1 Comparative Orthopaedics Laboratory, Cornell University, Ithaca, NY; 2Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY.
Introduction: Tendinitis remains a catastrophic injury amongst athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin-like growth factor-I (IGF-I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs as well as IGF-I gene-enhanced MSCs (AdIGF-MSCs) on tendon healing in vivo. Methods: Collagenase-induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Five days post collagenase injection (t=0), tendons were treated with 10 x 106 MSCs or 10 x 106 AdIGF-MSCs. Control limbs were injected with 1mL of PBS. Ultrasound examinations were performed at t=0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure, and healing lesions evaluated for biochemical composition and histologic characteristics. Quantitative RT-PCR for expression of collagen types I and III, IGF-I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), and aggrecanase-1 (ADAMTS-4) were performed as well as DNA, glycosaminoglycan, and total soluble collagen assays. Results: Both MSC and AdIGF-MSC intralesional injection resulted in significantly more normal tendon histological scores (Fig 1), and a trend toward improved biomechanical characteristics.
Levels of DNA, collagen, and tendon GAG content where similar bewteen MSC implanted and control tendons. There were Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy