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707. Non-Viral Delivery of Basic Fibroblast Growth Factor Gene to Bone Marrow Stromal Cells
TISSUE ENGINEERING &OTHER CELL THERAPIES I AAV2-TGF-b1 may provide a novel strategy to enhance bone and cartilage formation of rAMSCs.
706. Design of 3D Culture Systems To Enhance In Vitro Gene Expression of Mesenchymal Stem Cells
Hossein Hosseinkhani,1 Mohsen Hosseinkhani.2 1 Center for Biomedical Engineering, Massachusetts Institute of Technology (MIT), Boston, MA; 2Center of Cancer Systems Biology, Tufts University School of Medicine, Boston, MA.
The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3-D) tissue engineered scaffolds and non-viral gene carrier. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran as a carrier of plasmid DNA encoded with BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a perfusion bioreactor. The level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2D (tissue culture plate) culture method.
707. Non-Viral Delivery of Basic Fibroblast Growth Factor Gene to Bone Marrow Stromal Cells
Basak Acan-Clements,1 Charlie Y. M. Hsu,2 Cezary Kucharski,3 Laura Rose,2 Xiaoyue Lin,3 Hasan Uludag.3 1 Pharmacy & Pharmaceutical Sciences, U. of Alberta, Edmonton, AB, Canada; 2Biomedical Engineering, U. of Alberta, Edmonton, AB, Canada; 3Chemical & Materials Engineering, U. of Alberta, Edmonton, AB, Canada.
Basic Fibroblast Growth Factor (bFGF) is capable of stimulating osteogenic differentiation of pre-osteoblast cells in vitro and can induce new bone tissue deposition in vivo as a result of multitude of actions on local cells. Delivering the bFGF gene, rather than the protein itself, can be more beneficial for bone repair since gene delivery obviates the need to produce the protein in pharmaceutical quantities. Bone marrow stromal cells (BMSC) will be the most likely targets for in vivo or ex vivo bFGF gene delivery and, accordingly, this study explored the feasibility of bFGF gene expression in BMSC. The studies to-date employed only viral vectors for BMSC modifications, and it was our intent to determine the efficiency of relatively safer non-viral methods for gene delivery to BMSC. Freshly isolated primary rat BMSC were transfected by using cationic polymers (25 kDa branched polyethylenimine and 22 kDa linear poly(L-lysine) substituted with palmitic acid) in vitro. After delivering a bFGFexpression plasmid (pFGF2-IRES-AcGFP) to BMSC, the presence of bFGF in culture supernatants was demonstrated by a commercial ELISA. As much as 0.3 ng bFGF/10(6) cells/day was obtained from the BMSC under optimal conditions. However, this secretion rate was approximately 150-fold lower than the secretion obtained from immortal, easy-to-transfect human 293T cells. The bFGF secretion rate obtained from the latter cells was similar to the secretion rates reported by others using viral vectors in transformed cells. Using the GFP reported gene, a significant difference in transgene expression was also obtained between the two cell types, suggesting that the differences between the two cell types were not specific to bFGF expression. Cationic polymers, therefore, appears to be sufficiently effective for bFGF gene delivery in transformed cells. Modification of BMSC with cationic polymers was also possible but significant improvements in transfection rate and transgene expression are needed for an effective bFGF therapy.
S270
708. Highly-Efficient Genome Editing in Human Stem Cells Using Engineered Zinc Finger Nucleases
Shuyuan Yao,1 Jianbin Wang,1 Gary Lee,1 Geoff Friedman,1 Nathaniel Wang,1 Kenneth Kim,1 James Li,1 Fyodor D. Urnov,1 Philip D. Gregory,1 Michael C. Holmes.1 1 R&D, Sangamo BioSciences, Inc., Richmond, CA.
Precise modification of human stem cells holds tremendous potential both in basic research and in the clinical application of stem cell therapies. For example, Mesenchymal Stem Cells (MSCs) can differentiate into a variety of cell types including fat, cartilage, bone, muscle, nerve and beta-pancreatic islets cells. MSCs can be isolated from different tissues and cultured for long periods in vitro without loss of differentiation potential, making them an ideal target for autologous cell/gene therapies or tissue engineering. Broad application of MSCs, both as potential therapeutic interventions and in basic research, is hampered by the lack of methods for efficient and specific engineering of the genome in living cells. Here we describe a general solution to this problem in human stem cells, namely, genome editing with engineered zinc finger nucleases (ZFNs). We show that ZFNs efficiently generate DSBs in vivo leading to a high frequency of target gene disruption (>10%), a process employing the cell’s own non-homologous end joining repair pathway. ZFN-modified MSCs stably maintained this high level of gene disruption when passaged for several weeks in culture, and importantly remained multipotent as demonstrated by their successful in vitro differentiation into osteocytes or adipocytes. Thus, ZFNs can be employed to knock out specific genes in MSCs. To extend these results beyond gene disruption, we next sought to achieve the targeted addition of gene-sized DNA sequences into a specific location in the human genome. To this end, for both ZFN target loci, we generated cognate homologous donor molecules encoding a GFP expression cassette flanked with target specific homology arms. MSCs transduced with the appropriate ZFN/homologous DNA donor vectors exhibited stable and uniform GFP expression, consistent with integration of the expression cassette into the target genomic location. High efficiency (>5%), targeted gene addition was confirmed by both PCR and Southern blot analysis. Stable GFP expression from the integrated reporter cassette was observed for several weeks in culture. Importantly, GFP+ve ZFN-modified cells also differentiated normally into both adipocytes and osteocytes, demonstrating that ZFN-modified cells remain multipotent.
709.
Meganucleases for Gene Therapy
Frédéric Pâques, Philippe Duchateau, Julianne Smith, Agnès Gouble, Christophe Perez, Jean-Pierre Cabaniols, Sylvestre Grizot, Roman Galetto, Fayza Daboussi, Fabien Delacôte, Frédéric Cedrone, Jean-Charles Epinat, Sylvain Arnould, Aymeric Duclert. Cellectis S.A., Romainville, Île de France, France. Most current gene therapy approaches for monogenic inherited diseases rely on gene transfer, by random integration into the genome of patient’s cells, of a functional copy of the mutated gene. However, a growing interest for targeted approaches, ranging from the targeted insertion of the functional gene into a chosen locus (“safe harbour” strategy) to the precise editing of the deleterious mutation (gene correction), is manifest. Such targeted approaches include the use of very specific endonucleases that can induce high frequencies of homologous gene targeting in the vicinity of their cleavage site. Natural meganucleases, also called Homing Endonucleases, are the most specific endonucleases in nature. Therefore, they represent ideal tools for targeted approaches. We have redesigned the DNAbinding interface of the I-CreI LAGLIDADG meganuclease, to create a series of endonucleases cleaving several human genes. Different engineered endonucleases allowed us to achieve percents of targeted recombination in human cells. Moreover, they displayed Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
706. Design of 3D Culture Systems To Enhance In Vitro Gene Expression of Mesenchymal Stem Cells
Hossein Hosseinkhani,1 Mohsen Hosseinkhani.2 1 Center for Biomedical Engineering, Massachusetts Institute of Technology (MIT), Boston, MA; 2Center of Cancer Systems Biology, Tufts University School of Medicine, Boston, MA.
The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3-D) tissue engineered scaffolds and non-viral gene carrier. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran as a carrier of plasmid DNA encoded with BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a perfusion bioreactor. The level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2D (tissue culture plate) culture method.
707. Non-Viral Delivery of Basic Fibroblast Growth Factor Gene to Bone Marrow Stromal Cells
Basak Acan-Clements,1 Charlie Y. M. Hsu,2 Cezary Kucharski,3 Laura Rose,2 Xiaoyue Lin,3 Hasan Uludag.3 1 Pharmacy & Pharmaceutical Sciences, U. of Alberta, Edmonton, AB, Canada; 2Biomedical Engineering, U. of Alberta, Edmonton, AB, Canada; 3Chemical & Materials Engineering, U. of Alberta, Edmonton, AB, Canada.
Basic Fibroblast Growth Factor (bFGF) is capable of stimulating osteogenic differentiation of pre-osteoblast cells in vitro and can induce new bone tissue deposition in vivo as a result of multitude of actions on local cells. Delivering the bFGF gene, rather than the protein itself, can be more beneficial for bone repair since gene delivery obviates the need to produce the protein in pharmaceutical quantities. Bone marrow stromal cells (BMSC) will be the most likely targets for in vivo or ex vivo bFGF gene delivery and, accordingly, this study explored the feasibility of bFGF gene expression in BMSC. The studies to-date employed only viral vectors for BMSC modifications, and it was our intent to determine the efficiency of relatively safer non-viral methods for gene delivery to BMSC. Freshly isolated primary rat BMSC were transfected by using cationic polymers (25 kDa branched polyethylenimine and 22 kDa linear poly(L-lysine) substituted with palmitic acid) in vitro. After delivering a bFGFexpression plasmid (pFGF2-IRES-AcGFP) to BMSC, the presence of bFGF in culture supernatants was demonstrated by a commercial ELISA. As much as 0.3 ng bFGF/10(6) cells/day was obtained from the BMSC under optimal conditions. However, this secretion rate was approximately 150-fold lower than the secretion obtained from immortal, easy-to-transfect human 293T cells. The bFGF secretion rate obtained from the latter cells was similar to the secretion rates reported by others using viral vectors in transformed cells. Using the GFP reported gene, a significant difference in transgene expression was also obtained between the two cell types, suggesting that the differences between the two cell types were not specific to bFGF expression. Cationic polymers, therefore, appears to be sufficiently effective for bFGF gene delivery in transformed cells. Modification of BMSC with cationic polymers was also possible but significant improvements in transfection rate and transgene expression are needed for an effective bFGF therapy.
S270
708. Highly-Efficient Genome Editing in Human Stem Cells Using Engineered Zinc Finger Nucleases
Shuyuan Yao,1 Jianbin Wang,1 Gary Lee,1 Geoff Friedman,1 Nathaniel Wang,1 Kenneth Kim,1 James Li,1 Fyodor D. Urnov,1 Philip D. Gregory,1 Michael C. Holmes.1 1 R&D, Sangamo BioSciences, Inc., Richmond, CA.
Precise modification of human stem cells holds tremendous potential both in basic research and in the clinical application of stem cell therapies. For example, Mesenchymal Stem Cells (MSCs) can differentiate into a variety of cell types including fat, cartilage, bone, muscle, nerve and beta-pancreatic islets cells. MSCs can be isolated from different tissues and cultured for long periods in vitro without loss of differentiation potential, making them an ideal target for autologous cell/gene therapies or tissue engineering. Broad application of MSCs, both as potential therapeutic interventions and in basic research, is hampered by the lack of methods for efficient and specific engineering of the genome in living cells. Here we describe a general solution to this problem in human stem cells, namely, genome editing with engineered zinc finger nucleases (ZFNs). We show that ZFNs efficiently generate DSBs in vivo leading to a high frequency of target gene disruption (>10%), a process employing the cell’s own non-homologous end joining repair pathway. ZFN-modified MSCs stably maintained this high level of gene disruption when passaged for several weeks in culture, and importantly remained multipotent as demonstrated by their successful in vitro differentiation into osteocytes or adipocytes. Thus, ZFNs can be employed to knock out specific genes in MSCs. To extend these results beyond gene disruption, we next sought to achieve the targeted addition of gene-sized DNA sequences into a specific location in the human genome. To this end, for both ZFN target loci, we generated cognate homologous donor molecules encoding a GFP expression cassette flanked with target specific homology arms. MSCs transduced with the appropriate ZFN/homologous DNA donor vectors exhibited stable and uniform GFP expression, consistent with integration of the expression cassette into the target genomic location. High efficiency (>5%), targeted gene addition was confirmed by both PCR and Southern blot analysis. Stable GFP expression from the integrated reporter cassette was observed for several weeks in culture. Importantly, GFP+ve ZFN-modified cells also differentiated normally into both adipocytes and osteocytes, demonstrating that ZFN-modified cells remain multipotent.
709.
Meganucleases for Gene Therapy
Frédéric Pâques, Philippe Duchateau, Julianne Smith, Agnès Gouble, Christophe Perez, Jean-Pierre Cabaniols, Sylvestre Grizot, Roman Galetto, Fayza Daboussi, Fabien Delacôte, Frédéric Cedrone, Jean-Charles Epinat, Sylvain Arnould, Aymeric Duclert. Cellectis S.A., Romainville, Île de France, France. Most current gene therapy approaches for monogenic inherited diseases rely on gene transfer, by random integration into the genome of patient’s cells, of a functional copy of the mutated gene. However, a growing interest for targeted approaches, ranging from the targeted insertion of the functional gene into a chosen locus (“safe harbour” strategy) to the precise editing of the deleterious mutation (gene correction), is manifest. Such targeted approaches include the use of very specific endonucleases that can induce high frequencies of homologous gene targeting in the vicinity of their cleavage site. Natural meganucleases, also called Homing Endonucleases, are the most specific endonucleases in nature. Therefore, they represent ideal tools for targeted approaches. We have redesigned the DNAbinding interface of the I-CreI LAGLIDADG meganuclease, to create a series of endonucleases cleaving several human genes. Different engineered endonucleases allowed us to achieve percents of targeted recombination in human cells. Moreover, they displayed Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy