705 AAV Ex Vivo Gene Therapy for Osteogenesis and Chondrogenesis Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S269 TISSUE ENGINEERING &OTHER CEL[.]
TISSUE ENGINEERING &OTHER CELL THERAPIES I Discussion: This study demonstrated that an ex vivo gene therapy using percutaneous injection of autologous DFb genetically engineered to secrete BMP2 were beneficial to accelerate and enhance bone healing An in vivo gene therapy using Ad-BMP2 also enhance the healing of medullary bone where many osteoprogenitor cells were readily available Both DFb-BMP2 and Ad-BMP2 induced a regional paracrine effect to stimulate adjacent bone activity DFb-mediated ex vivo BMP2 gene therapy may be considered as a potential treatment for osseous disorders including fractures 703 Fibronectin Films-Mediated SDF-1 Gene Release Recruits Stem Cells Weiwei Wang,1 Wenzhong Li,1 LeeLee Ong,1 Karola Lützow,2 Andreas Lendlein,2 Dario Furlani,1 Ralf Gaebel,1 Deling Kong,3 Jun Wang,4 Gustav Steinhoff,1 Nan Ma.1 Department of Cardiac Sugery, University of Rostock, Rostock, Germany; 2Institute of Polymer Research, GKSS Forschungszentrum, Teltow, Germany; 3Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Science, Nankai University, Nankai, China; 4Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, HeFei, China Gene activated matrix has wide potential utilization in tissue engineering It may genetically modify cells with plasmid DNA encoding therapeutic genes and allow sustained expression and release of the protein to surrounding tissues In this study, we assessed the feasibility of the local gene release from human fibronectin (HFN) substrate and the efficacy of local release of stromal cell-derived factor-1 (SDF-1) gene on c-kit+ cells homing Cationic polymer polyethyleneimine (25kDa PEI) was used as non-viral DNA vector Gene activated HFN (GAH) was prepared by mixing PEI/DNA complexes with HFN substrate The DNA retardation, complexes size and the DNA release speed from the GAH were studied The in-vitro transfection was optimized by luciferase expression and cell viability assay in COS7 cell line Localized gene expression in COS7 cells cultured on the GAH was assessed by LacZ and GFP-N3-SDF-1 marker genes c-kit+ cells homing was investigated in response to the local in-vitro SDF-1 expression from rat mesenchymal stem cells (RMSCs) cultured on GAH Results showed GAH allows long timesustained DNA release, localized gene delivery and high transfection efficiency Local SDF-1 expression with GAH is a promising method to induce targetable stem cells homing 704 Genetic Engineering of Non-Spontaneously Differentiating Pre-Osteoblasts Potentiates Mineralization by Sub-Optimal Doses of Recombinant Bone Morphogenetic Protein-2 Hui Liu,1 Manjula Viggeswarapu,1 Maggie Bargouti,1 Louisa F Titus,1,2 Scott D Boden.1,2 Orthopaedics, Emory University School of Medicine, Atlanta, GA; VA Medical Center, Decatur, GA MC3T3-E1 cells, like primary osteoblast cultures, require only serum and ascorbic acid (AA) to express a fully differentiated osteoblast phenotype Although originally isolated as a clonal cell line, MC3T3-E1 cells have become phenotypically heterogeneous as a result of prolonged passaging We took advantage of this heterogeneity and used clone # 24 which exhibits low mineralizing potential after growth in AA-containing medium for 10 days to investigate the role of recombinant human Bone Morphogenetic Protein-2 (rhBMP2) and LIM Mineralization Protein-1 (LMP1) on differentiation/mineralization BMPs stimulate osteoblast differentiation in a number of cell types including undifferentiated mesenchymal cells, bone marrow stromal cells, myoblast cell lines, Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy and pre-osteoblasts LMP-1 is an intracellular regulatory protein that can induce the expression of multiple BMPs and promote osteoblast differentiation in vitro and in vivo The purpose of this study was to determine if: 1) Treatment of clone # 24 cultures with rhBMP2 can induce osteoblast specific markers 2) Treatment of clone # 24 cultures with rhBMP2 can initiate differentiation/ mineralization in the presence of AA and beta glycerophosphate 3) Delivery of LMP-1 to clone #24 cultures can potentiate differentiation/mineralization by a sub-optimal dose of rhBMP2 MC3T3-E1 clone # 24 cells obtained from ATCC were seeded at 10,000/cm2 in a-MEM containing 10% non-heat-inactivated serum In order to establish a rhBMP2 dose response curve and determine the threshold dose, the cells were treated the next day with 1, 2.5, 5, 10, 20, 50 and 100ng/ml rhBMP2 The culture medium was supplemented with 50ug/ml ascorbic acid and 10mM b-glycerophosphate to support differentiation and the medium was changed every days for 17days Treatment of clone # 24 cells with rhBMP2 induced mineralization in a dose dependent manner BMP2 alone increased the expression of the bone specific gene, Runx2, Osteopontin and Bone Sialoprotein (BSP) in a dose dependent manner MC3T3-E1 clone #24 cells produce BMP2, which is essential for their differentiation Interestingly exogenous rhBMP2 treatment significantly stimulated endogenous BMP-2 gene expression in these cells Additional cultures were transduced with Ad5LMP-1 (10 pfu/cell) and grown in differentiation medium for 17days in the presence and absence of rhBMP-2 (0, 1, 5, 10ng/ ml) Treatment of cells with Ad5LMP-1 (10 pfu/cell) plus 10ng/ml rhBMP-2 dramatically induced mineralization observed by Alizarin red staining but neither rhBMP-2 (1,5,10ng/ml) nor LMP-1 alone induced mineralization in MC3T3-E1 cells clone # 24 on day 17 Thus, concurrent exposure of non spontaneously differentiating MC3T3-E1 clone # 24 cells to LMP-1 enabled an ineffective dose of BMP-2 to facilitate bone formation This key observation suggests that non spontaneously differentiating pre-osteoblasts can differentiate in response to rhBMP-2 and a non-osteoinductive dose of rhBMP2 can still have significant effects on pre-osteoblasts that have been genetically engineered to express LMP-1 705 AAV Ex Vivo Gene Therapy for Osteogenesis and Chondrogenesis Michael Y Mi,1,2 Ying Tang,1 Melessa N Salay,1 Guangheng Li,1 Johnny Huard,1 Freddie H Fu,1 Chritopher Niyibizi,3 Bing Wang.1,4 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 2Harvard College, Harvard University, Cambridge, MA; 3Orthopaedic and Rehabilitation College of Medicine, Pennsylvania State University College of Medicine, Hershey, PA; Neurology, University of Pittsburgh, Pittsburgh, PA To explore a new source of adult derived stem cells for musculoskeletal tissue repair and regeneration, we isolated rabbit adipose derived mesenchymal stem/progenitor cells (rAMSCs) from adult rabbit adipose tissue The isolated cells were shown to possess the potential to differentiate into cells of mesenchymal lineages that include osteoblasts and chondroblasts in vitro Then, rAMSCs were transduced by AAV2 vectors encoding human BMP-2 and human TGF-b1 genes, respectively, prior to implantation into hindlimb muscle of SCID mice (NOD.CB17 PRKDA SCID/J) for osteogenesis and chondrogenesis The results showed that AAV2-GFP could efficiently transduce rAMSCs compared with the other serotypes of AAV vector (AAV6) and that AAV2 transduced rAMSCs sustained to express BMP-2 and TGF-b1 proteins; furthermore, the AAV2 vector genetically modified rAMSCs differentiated towards osteoblasts and chondroblasts at the implanted sites 15 days after implantation The study demonstrates that rabbit adipose tissue contains cells with the potential to give rise to osteoblasts as well as chondroblasts, and that the AAV2 vector is suitable for genetic engineering of rAMSCs In addition, the data suggest that the combination of AAV2-BMP2 and S269 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 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 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 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, Ỵle 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 ... 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... 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... 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