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280 generation of integration free iPSCs from an x CGD patients blood cells as clinically relevant target for gene repair using designer ZFN or TALEN

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280 Generation of Integration Free iPSCs from an X CGD Patient''''s Blood Cells as Clinically Relevant Target for Gene Repair Using Designer ZFN or TALEN Molecular Therapy Volume 20, Supplement 1, May 20[.]

PLURIPOTENT STEM CELLS accumulation is responsible for cutaneous skin photosensitivity whose severity varies between diseases The only curative treatment for severe cases of EP is allogenic bone marrow transplantation (ABMT) that requires an HLA-matched donor Autologous hematopoietic stem cell (HSC) gene therapy represents an alternative to ABMT We have successfully used retroviral vectors for HSC gene therapy in congenital erythropoietic porphyria (CEP) and erythropoietic protoporphyria (EPP) mice However, due to multiple random integrations of integrative vectors, insertional mutagenesis represents a serious side effect that was observed in clinical trials Over the last few years, researchers were able to generate induced pluripotent stem cells (iPSCs) by reprogramming differentiated cells with retroviral expression of stem cell factors (Oct3/4, Sox2, Klf4 and c-Myc) These iPSCs cells shared many properties with embryonic stem cells (selfrenewal and totipotency) and give rise to many cell types, including HSC We have induced and characterised iPSCs lines from EP mice and evaluated their therapeutic potential after genetic correction and hematopoietic differentiation We have reprogrammed adult skin fibroblasts from EPP, CEP and wild type mice with a single integrative lentiviral vector expressing Oct3/4, Klf4 and Sox2 proteins (0.05% efficiency) We obtained iPSCs from the three mice models which presented ES-like morphology and expressed embryonic factors using RT-PCR and immunocytochemistry analysis LoxP sequences in the LTR of the provirus allowed us to remove the reprogramming vector by transient adenoviral expression of the recombinase CRE We showed that reprogramming vector-free iPSCs clones continue to express pluripotent markers and are able to form embryoid bodies in vitro and teratomas in immunodeficient mice For genetic correction of EP iPSCs cells, we used a lentiviral vector expressing either the ferrochelatase (FECH) or the uroporphyrinogen-III-synthase (UROS) cDNA from a chimeric erythroid-specific promoter We used the LAM-PCR to select for safe therapeutic proviral integration, far from known oncogenes The therapeutic efficiency was analysed after hematopoietic differentiation of corrected-iPSCs on OP9 cell stroma We obtained up to 48% CD41+ hematopoietic progenitors cells We are actually performing in vivo hematopoietic repopulation assays in EP mice to evaluate the therapeutical potential of our EPiPSCs-derived hematopoietic progenitor cells Transplanted EP mice will be monitored over time for metabolic and phenotypic correction These experiments represent important steps in the development of preclinical gene therapy protocols for erythropoietic porphyria 279 Derivation and Functional Analysis of Patient Specific Induced Pluripotent Stem Cells as an In Vitro Model of Chronic Granulomatous Disease Ulrich Siler,1 Yan Jiang,3 Sally A Cowley,2 Dario Melguzo,4 Katarzyna Tilgner,3 Cathy Browne,2 Angus deWilton,2 Stefan Pryzborski,5 Gabriele Saretzki,6 William S James,2 Reinhard A Seger,1 Janine Reichenbach,1 Majlinda Lako,1 Lyle Armstrong.1 Immunology, University Children’s Hospital Zürich, Switzerland; James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, United Kingdom; 3Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom; 4Centro de Investigacion Principe Felipe, Valencia, Spain; 5School of Biomedical Sciences, University of Durham, United Kingdom; 6Institute for Ageing and Health, Newcastle University, Newcastle, United Kingdom Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species In this study, we reprogrammed three normal unrelated patient’s fibroblasts (p47phox and gp91phox) to pluripotency by lentiviral transduction with defined pluripotency factors These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors S110 and posses high telomerase activity Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo They were isogenic with the original patient fibroblasts, exhibited normal karyotype and retained the gp47phox or gp91phox mutations found in the patient fibroblasts We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions Most importantly, CGD-patient specific iPSC derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modelling CGD disease phenotypes, screening candidate drugs and the development of gene therapy 280 Generation of Integration-Free iPSCs from an X-CGD Patient’s Blood Cells as Clinically Relevant Target for Gene-Repair Using Designer ZFN or TALEN Jizhong Zou,1 Colin L Sweeney,2 Harry L Malech,2 Linzhao Cheng.1 Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD; 2Laboratory of Host Defenses, NIAID/NIH, Bethesda, MD Human induced pluripotent stem cells (iPSCs) provided an unlimited cell resource for gene and cell therapy As an alternative approach to current viral vector-based, random integration-mediated gene transfer method, patient-derived iPSCs bearing defined diseasecausing mutations can be precisely corrected by homologous recombination mediated gene targeting, and then indefinitely expanded and differentiated into desired cell lineages for autologous transplantation Current genome engineering technologies offer two powerful DNA sequence-specific nucleases, zinc finger nuclease (ZFN) and transcriptional activator-like effector nuclease (TALEN) to create double-strand break and thus significantly stimulate the efficiency of homologous recombination For clinical applications of iPSCs it is advantageous to develop iPSC using reprogramming vectors and correction plasmids that either not integrate or that can be partially or completely excised following integration To develop clinically applicable iPSCs, we efficiently generated integrationfree patient-specific iPSCs from the CD34+ hematopoietic stem/ progenitor cells of an adult suffering X-chronic granulomatous disease (X-CGD), an inherited immunodeficiency disease caused by mutations in X-linked CYBB gene Using either a single polycistronic or a combination of three EBNA1/OriP-based episomal vectors, we delivered the reprogramming factors efficiently (>60%) to in vitro expanded CD34+ cells We obtained 3∼30 TRA-1-60+ colonies per million transfected CD34+ cells The derived integrationfree X-CGD iPSCs bearing a mutation in CYBB exon have normal ESC-like characteristics (such as AP+, SSEA-4+, OCT4+, NANOG+) and karyotype They form cystic embryoid body(EB) and teratoma containing three germ layer lineages during in vitro and in vivo differentiation, respectively Upon directed hematopoietic differentiation using a serum-free medium, 30∼50% CD34+CD45+ cells were detected 14 days after EB formation The episomal plasmids carrying reprogramming factors were undetectable after 10 passages following the iPSC derivation In order to enhance the gene targeting efficiency, both ZFNs and TALENs targeting the diseasecausing mutation were designed and synthesized Approximately 50% of context-dependent assembly (CoDA)-based designs generate active ZFNs that can boost gene targeting efficiency >100-fold TALEN offers more flexibility, therefore more candidates can be tested around Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy PLURIPOTENT STEM CELLS desired target sequence and offer more active DNA-cutting nucleases We will report our effort on the TALENs targeting CYBB mutation in X-CGD iPSCs and compare their efficacy with ZFNs 281 Rapid Generation of Induced Pluripotent Stem Cells (iPSCs) from the Urine of a Patient with Duchenne Muscular Dystrophy Xuan Guan,1 Yingai Shi,2 Chad D Markert,2 David L Mack,2 Tara N Jones,2 Emily C Moorefield,2 Yuanyuan Zhang,2 Martin K Childers.2 Department of Physiology and Pharmacology, School of Medicine, Wake Forest University, Winston-Salem, NC; 2Institute for Regenerative Medicine, Wake Forest University, WinstonSalem, NC Mature human somatic cells can be reprogrammed to a primitive stage, resembling human Embryonic Stem cells (hES) isolated from embryos These iPSCs acquire the same infinite self-renewal ability and pluripotent differentiation potential as ES cells Because of these extraordinary properties, iPSCs can provide virtually infinite numbers of multiple types of somatic cells Thus, iPSCs hold great potential for disease modeling, drug screening and regenerative medicine New emerging technologies, such as microRNA delivery, have vastly improved the efficiency of the reprogramming process However, iPSCs generation is still time-consuming (usually weeks) with relatively low efficiency Moreover, most iPSCs derived from skin fibroblasts or peripheral blood cells, require invasive collection procedures Here we show, for the first time, that iPSCs can be generated from the urine of a patient with Duchenne muscular dystrophy (DMD) Urine derived cells (UC) were expanded by culturing DMD patient urine sediment in tissue-culture treated plates Isolated UCs were fast-proliferating Moreover, they intrinsically expressed high levels of c-myc and klf4, two factors in the reprogramming cocktail For comparison, DMD patient UCs and normal human fibroblasts were seeded on Matrigel-coated plates and transduced with a polycistronic lentiviral vector expressing human oct4, sox2, klf4 and c-myc Virus-infected cells were maintained in original medium for days before switching to hES medium mTeSR ES-like colonies were observed around days from UCs Those colonies were alkaline phosphate positive, expressing the pluripotent surface markers ssea4 and Tra-1-81 Exogenous transgenes, as determined by red fluorescence reporter, started to silence around day and complete transgene silenced colonies were noted around day 10 to 14 Isolated large colonies could be manually picked and passaged by day 12 In contrast, fibroblast derived iPSC colonies generally require weeks RT-PCR array and immunostaining confirmed the expression of pluripotent markers in several UC iPSC lines An in vivo teratoma formation assay further confirmed the differentiation ability of iPSC to form three germ layers Together, these data demonstrate the feasibility of rapid iPSC generation from the urine of a DMD patient Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy S111 ... STEM CELLS desired target sequence and offer more active DNA-cutting nucleases We will report our effort on the TALENs targeting CYBB mutation in X- CGD iPSCs and compare their efficacy with ZFNs... Matrigel-coated plates and transduced with a polycistronic lentiviral vector expressing human oct4, sox2, klf4 and c-myc Virus-infected cells were maintained in original medium for days before switching... ability and pluripotent differentiation potential as ES cells Because of these extraordinary properties, iPSCs can provide virtually infinite numbers of multiple types of somatic cells Thus, iPSCs

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