575 iPS Cell Formation Invokes Shared Biological and Transcriptome Changes with Tumorigenesis Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therap[.]
STEM CELL THERAPIES II 573 Enhancement of Tumor-Killing Activity of TRAIL-Expressing MSCs by Using Trichostatin A Ryosuke Uchibori,1 Hiroyuki Mizuguchi,2 Tomonori Tsukahara,1 Masashi Urabe,1 Hiroaki Mizukami,1 Akihiro Kume,1 Keiya Ozawa.1 Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Shimotsuke-shi, Tochigi, Japan; Department of Biochemistry and Molecular Biology, Osaka University, Suita-shi, Osaka, Japan Background: Mesenchymal stem cells (MSCs) have a tendency to accumulate at the tumor sites and are particularly attractive for use in cell-based cancer gene therapies Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert cytotoxic activity selectively against many tumor cells but not normal cells Death receptor is a receptor for TRAIL Some studies have reported that histone deacetylase inhibitors (HDACis) such as trichostatin A (TSA), sodium butylate, and suberoylanilide hydroxamic acid (SAHA) sensitized various tumor cells to TRAIL through upregulation of DR5 expression In this study, we assessed therapeutic efficacy of TRAIL-expressing MSCs with the simultaneous administration of TSA Methods: A cDNA fragment encoding the extracellular domain (amino acid residues 114-281) of human TRAIL (TRAILex) was amplified by PCR To modify its structure to change soluble form and to enhance the tumor-killing activity, secretory signal sequence (N-terminal 28 amino acids of human fibrillin-1) fused with the isoleucine-zipper sequence was cloned into TRAILex The resulting plasmid was denoted pSCZT The SCZT-expressing adenovirus vector (AdK7) was generated by in vitro ligation method MSCs were transduced with AdK7-SCZT (MSC-SCZTs) The human colon adenocarcinoma SW480 cells were treated with TSA, and DR5 expression was measures by quantitative PCR MSC-SCZTs were cocultured with luciferase-expressing SW480 cells, and tumor killing activity of MSC-SCZTs was measured by luciferase assay To assess the accumulation of MSCs at the tumor sites, SW480 cells were subcutaneously injected into the nude mice, and luciferaseexpressing MSCs were injected into the left ventricular cavity of tumor-bearing mice Bioluminescence from MSCs was periodically measured by in vivo imaging system (IVIS) MSC-SCZTs were injected into the left ventricular cavity of tumor-bearing mice, and tumor volume was periodically measured Results: TSA significantly increased DR5 expression of SW480 sells TSA sensitized SW480 cells to SCZT and enhanced tumor-killing activity of MSC/SCZTs In vivo imaging revealed that administration of luciferase-expressing MSCs caused marked accumulation of the bioluminescence signal at the sites of tumors Moreover, the simultaneous administration of TSA significantly enhanced the tumor-suppressive activity of MSCSCZTs Conclusion: Our results suggest that MSCs have potential use as effective delivery vehicle for therapeutic genes in the treatment of tumors Furthermore, HDACis including TSA would work synergistically in enhancing TRAIL-induced apoptosis in tumors 574 Reprogramming of Adult Human Extrahepatic Biliary Cells into Functional Pancreatic Beta Cells Feorillo Galivo,1 Raymond Hickey,1 Kathryn Schubert,1 Rohan Manohar,2 Eric Lagasse,2 Markus Grompe.1 Oregon Stem Cell Center, Oregon Health & Science University, Portland, OR; 2McGowan Institute for Regenerative Medicine, Department of Pathology, University of Pittsburgh Medical School, Pittsburgh, PA Islet cell transplantation (ICT) is a promising alternative to insulin therapy for type I diabetes mellitus that can render a patient insulin-free and improve overall quality of life Although still largely experimental, ICT is limited by lack of donors, requirement S220 for or more pancreases and life-long immunosuppression Recent studies indicate that cells of non-pancreatic origin, such as liver and embryonic cells can be potential sources of renewable beta cells Given their interrelated ontogenies, we hypothesize that adult liver, gallbladder (GB), biliary ducts, and pancreas share molecular/ epigenetic signatures and their fundamental differences can be traced to differential expressions of multiple factors during development that determine their fates in an adult organism Studies showing successful transdifferentiation of liver, intrahepatic biliary cells, and exocrine pancreatic cells into functional insulin-producers suggest that an adult cell can be directly transformed into another cell type of similar developmental origin without reverting to a pluripotent state These direct cell conversions were accomplished by introducing factors relevant to the target cell’s ontogeny For the same reason, we focus on the GB and the cystic duct (CD) as potentially renewable and expandable sources of autologous pancreatic beta cells Normal tissue biopsies of GB and CD from human patients were enzymatically treated to obtain disaggregated cells for expansion and subsequent reprogramming in vitro To convert GB and CD cells into beta cells, these were transduced with replication-defective recombinant adenoviruses encoding for various transcription factors of significant roles in pancreatic beta cell differentiation and which were also shown to be operative in reprogramming of insulin-producing cells Our initial studies focused on the effects of Pdx1, NEUROG3, and MAFA on the gene transcription profiles of the reprogrammed cells in terms of pancreatic endocrine genes Adenoviral transduction had very low efficiency and was improved by pre-treating with DEAE-Dextran prior to incubation with human gallbladder and cystic duct cells Despite the low overall viral transduction, RNA transcripts of Pdx1, NEUROG3, and MAFA were significantly increased in transduced cultures More importantly, a significant induction of insulin expression was detected in transduced cells relative to mock and Ad-GFP controls Likewise, upregulation of glucagon, pancreatic polypeptide, and somatostatin genes was observed which can be enhanced by adding MAFB, NKX6.1, and PAX4-encoding adenoviruses These preliminary results indicate possible reprogramming of human gallbladder and cystic duct into pancreatic islet cells However, to fully assess the important determinants for full reprogramming, we are optimizing culture conditions, vector transduction, posttranscriptional and whole cell functionality assays, and testing combinations and stoichiometries of various regulators of pancreatic development 575 iPS Cell Formation Invokes Shared Biological and Transcriptome Changes with Tumorigenesis John Riggs,1 Paul S Knoepfler.1 Stem Cell Program, University of California Davis, Davis, CA There is growing concern about potential oncogenic events occurring during iPS cell formation We noted striking similarity between the methods employed for the production of induced pluripotent stem (iPS) cells and oncogenic foci (OF), a type of in vitro produced sarcoma cancer cell To test the hypothesis that iPS cells and OF are highly related cell types, we produced them in parallel from the same parental fibroblasts The end result is a system of iPS cells and cancer cells of identical genetic backgrounds We observed that strikingly similar transcriptional changes occur during iPS cell and OF production relative to the common starting fibroblasts The vast majority of genes repressed during iPS formation are also repressed in OF, including essentially identical cohorts of differentiation genes In addition iPS cells and OF share strong overlap in activated genes that form modules predominantly related to metabolism However, iPS and OF cells are distinct in specific ways iPS cells only form teratoma when injected into immunodeficient mice, while OF only form malignant sarcoma In addition, iPS cells activate a module of core pluripotency-related genes, while OF not In contrast, Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy STEM CELL THERAPIES II OF uniquely activate novel cellular damage and specific metabolic pathways Attempts to reprogram OF and other tumor cell lines into iPS cells were only partially successful We conclude that formation of OF and iPS cells are highly related, yet distinct processes Our findings support a model in which iPS cell formation and tumorigenesis are parallel processes that diverge in only very specific ways and may share a common transitional progenitor cell stage relative to parental fibroblasts These studies have critical implications for understanding stem cell biology, tumorigenesis including cancer stem cells, and regenerative medicine 576 Genetic Modifications at Safe Harbor Loci in Hematopoietic Stem Cells for Gene Therapy Laurent Poirot,2 Roman Galetto,2 Frédéric Lemaire,1 Frédéric Cédrone,1 Sylvain Arnoud,2 Agnès Gouble,2 Carole Desseaux,2 Julianne Smith,2 Frédéric Pâques.1 Cellectis, Romainville, France; 2Cellectis Therapeutics, Romainville, France Gene therapy for monogenic inherited diseases has generally relied on non-specific integration of genetic material into the genome of the target cells with potential risk of insertional mutagenesis In contrast, targeted approaches offer the possibility of precisely editing genomic sequences They therefore present less risk of adverse consequences but rely on the availability of accurate genomic tools Meganucleases are natural highly specific endonucleases capable of inducing high frequency of homologous recombination at their target site Redesigning their protein-DNA interface allowed us to engineer dozens of custom meganucleases with chosen specificity and ability to induce high levels of gene targeting We are developing a number of custom meganucleases targeting defined loci in the human genome where insertion of a gene expression cassette would have a better therapeutic benefit/risk ratio (called safe harbor loci) The definition of a potential safe harbor depends on the indication and on the cell type where the modification is made We characterize potential safe harbor meganucleases by measuring their ability to induce mutagenesis by non-homologous end joining or homologous recombination-mediated gene targeting in model cell lines In addition, we have set up initial assays to examine the safety and capacity for the locus to support transgene expression, essential characteristics for defining a safe harbor locus Because they can be manipulated ex vivo, cells of the blood lineage are an attractive target for gene therapy Among them, we focus our efforts on hematopoietic stem cells (HSCs) since they can differentiate into a variety of cell types and therefore could be of interest in a variety of indications such as immune deficiencies, hemophilia or thalassemia We will present our latest results on safe harbor meganucleases and on genetic modifications of HSCs 577 Efficient Generation of Vector/Transgene Free Human Induced Pluripotent Stem Cells from Intractable Disease Patients Using Non-Integrating Sendai Virus Vectors Noemi Fusaki,1,2 Hiroshi Ban,1 Toshiaki Tabata,1 Akihiro Iida,1 Mamoru Hasegawa,1 Hironobu Ihn,3 Takumi Era.4 DNAVEC Corporation, Tsukuba, Ibaraki, Japan; 2PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan; 3Department of Dermatology and Plastic Surgery, Kumamoto University, Kumamoto, Japan; 4Department of Cell Modulation, Institute of Molecular Embryology, and Genetics (IMEG), Kumamoto University, Kumamoto, Japan from numerous iPSC colonies Many methods have been developed to overcome the issue, however, they are suffering from low efficiency or need tedious repetitive transfection We have developed a novel and highly efficient method for generating human iPSCs by using Sendai virus (SeV) vector, an RNA replicon vector that carries no risk of integrating into host genome Here we applied this excellent method to generate patient-specific iPSCs Patient-specific iPSCs with intact genome would provide novel and considerable opportunities in the basic and pharmaceutical studies of the diseases, drug discovery and toxicology, and in regenerative medicine of coming age We have established 33 patient-derived iPSCs out of 48 intractable diseases and others are ongoing under the grant of Ministry of Health, Labour and Welfare, Japan [Methods and Results] Skin biopsy samples from 70 patients of 48 diseases were collected under the informed consent in the Hospital of Kumamoto University Examined intractable diseases included inherited (neural, muscular, metabolic and other types), skin, bone and connective tissue-derived diseases and disorder of chromosomes Fibroblasts from these patients were transduced with SeV vectors carrying the OCT3/4, SOX2, KLF4 and c-MYC gene and then cultured on MEF feeder cells We have successfully established iPSCs from all of the fibroblasts tested and they showed gene expression of human ES markers and viral/factor-free nature Clonal variation between the same patient-derived iPSCs was analyzed by the expression of specific genes and, very importantly, revealed that it was little in contrast to retrovially generated iPSCs We suggest that SeV vector is the most suitable tool for the study of disease specific iPSCs without any noise of integration 578 Towards Targeted Transgene Integration in Induced Pluripotent Stem Cells Ruan van Rensburg,1 Oleg Denisenko,2 Karol Bomsztyk,2 Ines Beyer,1 ZongYi Li,1 Andre Lieber.1 Division of Medical Genetics, University of Washington, Seattle, WA; 2UW Medicine Lake Union, University of Washington, Seattle, WA Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy A strategy to mediate site-specific integration involves site specific endonucleases As their disruption has no functional consequences, two genomic sites within the MBS85 and CCR5 genes (AAVS1 and CCR5 zinc finger nuclease site, respectively) have recently been suggested as potential target regions for integration Transgene integration can be affected by DNA accessibility of endonucleases and expression of the transgene is subjected to the epigenomic regulation of the region that contains the target site We therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent cell lines and pooled cord-blood derived CD34+ cells Matrix chromatin immunoprecipitation assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration and is transcriptionally inactive In contrast, the AAVS1 site was located within a transcriptionally active region and possessed an open chromatin configuration in both iPS cells and hematopoietic stem cells In addition, transductional studies comparing fiber-modified adenovirus vectors indicate that Ad5/35 effectively transduces iPS cells and that of the tested promoters, the ubiquitin gene promoter allows for efficient transgene expression in transduced cells Based on these results, we are currently developing a range of adenovirus vectors to facilitate targeted integration into the AAVS1 site on iPS cells [Introduction] Since the development of reprogramming somatic cells into induced pluripotent stem cells (iPSCs), a major limitation of current reprogramming strategies is the integration of vectors used to transduce the reprogramming factors As a result, it is necessary to select ‘elite’ iPSCs with silenced foreign genes of low copy number Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S221 ... successful We conclude that formation of OF and iPS cells are highly related, yet distinct processes Our findings support a model in which iPS cell formation and tumorigenesis are parallel processes...STEM CELL THERAPIES II OF uniquely activate novel cellular damage and specific metabolic pathways Attempts to reprogram OF and other tumor cell lines into iPS cells were only partially... patient-specific iPSCs Patient-specific iPSCs with intact genome would provide novel and considerable opportunities in the basic and pharmaceutical studies of the diseases, drug discovery and toxicology, and