- Email: [email protected]
46. Gene Therapy of Congenital Erythropoietic Porphyria Using Genetically Modified Induced Pluripotent Stem Cells
STEM CELL THERAPY included the ex vivo modification of autologous hematopoietic stem cells (HSC) with lentiviral vectors that express antigens involved in EAE/MS from a dendritic cell-specific promoter. After re-infusion, the modified HSC were to be progenitor to all cells of the immune system including antigen presenting dendritic cells. We hypothesized that the stable antigen presentation by these cells in thymus and periphery in a non-inflammatory condition would tolerize self-reactive T cells and, therefore, prevent/revert disease development. We demonstrated the effectiveness of this strategy for inducing myelin oligodendrocyte glycoprotein (MOG)-tolerance in an EAE model in mice. All mice which received HSC transduced with the MOG-expressing lentivirus vector were protected (clinical score 0) from EAE upon immunization, while 100% of mice that received HSC transduced with a control lentivirus vector developed EAE. No histological signs of EAE/MS (demyelination, axon damage and infiltration of macrophages and lymphocytes in brain, spinal cord and optical nerve) were observed in tolerized mice, whereas they fully developed in control mice. We also show that tolerance was concomitant with efficient deletion of MOG specific T cells and generation of Foxp3+ regulatory T cells in the tolerized mice. Tolerance induced in MOG-chimeras was antigen specific, as splenocytes from MOG-chimeras produced IFN-γ after in vitro re-stimulation with OVA peptide but not with MOG-peptide. Most importantly, tolerized mice did not develop any signs of autoimmune disease even when infused with pre-activated, MOG-specific effector T cells, which rapidly induced EAE in the control mice. The strategy presented here is particularly promising for clinical applications, since HSCs are modified for permanent and continuous output of genetically modified tolerogenic “steady-state” dendritic cells. Moreover, the ability to revert the pathogenic MOG T cells highlights the potential of this strategist in a therapeutic model.
absence of abnormality of iPSC clones. Gene correction of CEPderived iPSC was obtained by lentiviral transduction of a HAUPins containing UROS cDNA under the control of HS40-Ankyrin erythroid specific promoter and shield by two cHS4 insulators. After subcloning of iPSCs, the sequencing of proviral integration site (IS) by LAM-PCR allowed us to select two iPSC clones with single integration in non-oncogenic region. After hematopoietic differentiation, we obtained CD34+/CD45+ cells derived from all iPSC lines. In methyl cellulose assays, all type of CFCs was observed. In vitro erythroid differentiation was very efficient (more than 90% of CD71/glycophorin A positive cells). These erythroblasts contained principally HBF. Accumulation of porphyrins in erythroid cells derived from a non-corrected CEP-iPSC clone results in the presence of fluorocytes (47.3% ± 1.8). Metabolic correction was demonstrated by disappearance of fluorocytes in erythroid cells derived from two corrected CEP-iPSC clones (6.3% ± 3.5 and 4.3% ± 1.8, p<0.01 vs non-corrected CEP-iPSC). Conclusion: This study is the first report of gene therapy using iPSCs for porphyria. These data demonstrate that one proviral copy of HAUPins in each of the two CEP-iPSC clones allowed a metabolic correction of erythroid cells. These results are promising for IPSC-mediated gene therapy of CEP and more generally for genetic red blood cells disorders.
47. Robust Differentiation of Hematopoietic Progenitor Cells from Pigtail Macaque Induced Pluripotent Stem Cells toward Modeling Human Disease and Stem Cell Therapies In Vivo
46. Gene Therapy of Congenital Erythropoietic Porphyria Using Genetically Modified Induced Pluripotent Stem Cells
Jennifer L. Gori,1 Brian C. Beard,1,2 Sunita L. D’Souza,3 HansPeter Kiem.1,2 1 Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Medicine, University of Washington School of Medicine, Seattle, WA; 3Developmental and Regenerative Biology Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, NY.
Background: Congenital erythropoietic porphyria (CEP) is due to a deficiency in UROS enzymatic activity leading to porphyrin accumulation resulting in skin lesions and haemolytic anemia. CEP is candidate for gene therapy but recent reports of insertional leukemogenesis underscore the need for safer systems. The discovery of induced pluripotent stem cells (iPSCs) opens a new frontier in gene therapy. It could overcome the difficulty to obtain sufficient amount of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. The aim of this study was to derive human iPSC from CEP patients to propose a safe ex vivo gene correction. Results: Keratinocytes were isolated from normal donor and patient with CEP. iPSC generation was obtained by transduction with two SIN-lentivectors (OSK1 and Mshp53). OSK1 is an excisable single polycistronic vector co-expressing hOCT4, hSOX2 and hKLF4 cDNAs. Mshp53 express hc-Myc and a shRNA against TP53. iPSC colonies harvested were excised for exogene reprogramming factors by adenovirus-mediated CRE recombinase. Clones demonstrated characteristics of pluripotent stem cells: morphology, expression of pluripotent stem cell markers by immunocytochemistry, robust expression of endogenous pluripotency associated genes. iPSC injection into NSG mice resulted in the formation of teratomas with all 3 embryonic germ layers. Karyotypic analysis confirms the
Induced pluripotent stem cells (iPSCs) are currently considered for a number of therapeutic applications. To aid in the transition of iPSC technology into the clinic, we focused our attention on developing iPSCs from large animal models such as the pigtail macaque (Macaca nemestrina (Mn)). Nonhuman primate studies allow for the evaluation of iPSC-derived cell transplantation in an autologous setting and for the testing of clinical grade reagents prepared for use with human stem cells. We previously succeeded in deriving several MniPSC lines (Zhong, B. et al. Efficient generation of nonhuman primate induced pluripotent stem cells. Stem Cells Dev 20, 795-807 (2011)). To be able to safely control these lines in vivo, we also previously engineered MniPSC lines that express an inducible suicide gene (Zhong, B. et al. Safeguarding nonhuman primate iPS cells with suicide genes. Mol Ther 19, 1667-1675 (2011)). Next, we tested various hematopoietic differentiation protocols on these lines in vitro. We discovered that inclusion of prostaglandin E2 during the first week of differentiation supported a 3-fold increase in viable CD34+ cells, without loss of hematopoietic colony forming potential. Testing two MniPSC lines, the optimized protocol supported robust hematopoietic progenitor formation (30% CD34+CD45+). These progenitors also express CD133, CD43, CD31, ALDH and Flt3 and nonhuman primate hematopoietic-specific transcription factors (SCL/ Tal1, CDX1, CDX2, CDX4, GATA2, HOXA9). In addition, MniPSC CD34+ hematopoietic progenitors gave rise to both myeloid (CD33+, CD14+) and lymphoid (T lymphocyte, CD3+, CD4+, CD8+) progeny. These findings show that nonhuman primate iPSC are capable of giving rise to clinically relevant cell populations for transplantation. Finally, as we have previously shown with somatic CD34+ cells from pigtail macaques, using HIV-1 based lentivirus vectors we can also efficiently gene modify MniPSCs and MniPSC hematopoietic progeny with a variety of transgene cassettes, including the potent
Aurélie Bedel,1 Miguel Taillepierre,1 Pierre Dubus,3 Eric Lippert,1 Veronique Guyonnet-Duperat,2 Mautuis Thibault,1 Magalie Lalanne,1 Catherine Pain,1 Bruno Cardinaud,1 Cecile Ged,1 Benoit Rousseau,3 Yann Duchartre,1 Hubert de Verneuil,1 Emmanuel Richard,1 Francois Moreau-Gaudry.1,2 1 INSERM U 1035, University Bordeaux Segalen, Bordeaux, France, Metropolitan; 2Vectorology Plateform, University Bordeaux Segalen, Bordeaux, France, Metropolitan; 3University Bordeaux Segalen, Bordeaux, France, Metropolitan.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
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absence of abnormality of iPSC clones. Gene correction of CEPderived iPSC was obtained by lentiviral transduction of a HAUPins containing UROS cDNA under the control of HS40-Ankyrin erythroid specific promoter and shield by two cHS4 insulators. After subcloning of iPSCs, the sequencing of proviral integration site (IS) by LAM-PCR allowed us to select two iPSC clones with single integration in non-oncogenic region. After hematopoietic differentiation, we obtained CD34+/CD45+ cells derived from all iPSC lines. In methyl cellulose assays, all type of CFCs was observed. In vitro erythroid differentiation was very efficient (more than 90% of CD71/glycophorin A positive cells). These erythroblasts contained principally HBF. Accumulation of porphyrins in erythroid cells derived from a non-corrected CEP-iPSC clone results in the presence of fluorocytes (47.3% ± 1.8). Metabolic correction was demonstrated by disappearance of fluorocytes in erythroid cells derived from two corrected CEP-iPSC clones (6.3% ± 3.5 and 4.3% ± 1.8, p<0.01 vs non-corrected CEP-iPSC). Conclusion: This study is the first report of gene therapy using iPSCs for porphyria. These data demonstrate that one proviral copy of HAUPins in each of the two CEP-iPSC clones allowed a metabolic correction of erythroid cells. These results are promising for IPSC-mediated gene therapy of CEP and more generally for genetic red blood cells disorders.
47. Robust Differentiation of Hematopoietic Progenitor Cells from Pigtail Macaque Induced Pluripotent Stem Cells toward Modeling Human Disease and Stem Cell Therapies In Vivo
46. Gene Therapy of Congenital Erythropoietic Porphyria Using Genetically Modified Induced Pluripotent Stem Cells
Jennifer L. Gori,1 Brian C. Beard,1,2 Sunita L. D’Souza,3 HansPeter Kiem.1,2 1 Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Medicine, University of Washington School of Medicine, Seattle, WA; 3Developmental and Regenerative Biology Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, NY.
Background: Congenital erythropoietic porphyria (CEP) is due to a deficiency in UROS enzymatic activity leading to porphyrin accumulation resulting in skin lesions and haemolytic anemia. CEP is candidate for gene therapy but recent reports of insertional leukemogenesis underscore the need for safer systems. The discovery of induced pluripotent stem cells (iPSCs) opens a new frontier in gene therapy. It could overcome the difficulty to obtain sufficient amount of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. The aim of this study was to derive human iPSC from CEP patients to propose a safe ex vivo gene correction. Results: Keratinocytes were isolated from normal donor and patient with CEP. iPSC generation was obtained by transduction with two SIN-lentivectors (OSK1 and Mshp53). OSK1 is an excisable single polycistronic vector co-expressing hOCT4, hSOX2 and hKLF4 cDNAs. Mshp53 express hc-Myc and a shRNA against TP53. iPSC colonies harvested were excised for exogene reprogramming factors by adenovirus-mediated CRE recombinase. Clones demonstrated characteristics of pluripotent stem cells: morphology, expression of pluripotent stem cell markers by immunocytochemistry, robust expression of endogenous pluripotency associated genes. iPSC injection into NSG mice resulted in the formation of teratomas with all 3 embryonic germ layers. Karyotypic analysis confirms the
Induced pluripotent stem cells (iPSCs) are currently considered for a number of therapeutic applications. To aid in the transition of iPSC technology into the clinic, we focused our attention on developing iPSCs from large animal models such as the pigtail macaque (Macaca nemestrina (Mn)). Nonhuman primate studies allow for the evaluation of iPSC-derived cell transplantation in an autologous setting and for the testing of clinical grade reagents prepared for use with human stem cells. We previously succeeded in deriving several MniPSC lines (Zhong, B. et al. Efficient generation of nonhuman primate induced pluripotent stem cells. Stem Cells Dev 20, 795-807 (2011)). To be able to safely control these lines in vivo, we also previously engineered MniPSC lines that express an inducible suicide gene (Zhong, B. et al. Safeguarding nonhuman primate iPS cells with suicide genes. Mol Ther 19, 1667-1675 (2011)). Next, we tested various hematopoietic differentiation protocols on these lines in vitro. We discovered that inclusion of prostaglandin E2 during the first week of differentiation supported a 3-fold increase in viable CD34+ cells, without loss of hematopoietic colony forming potential. Testing two MniPSC lines, the optimized protocol supported robust hematopoietic progenitor formation (30% CD34+CD45+). These progenitors also express CD133, CD43, CD31, ALDH and Flt3 and nonhuman primate hematopoietic-specific transcription factors (SCL/ Tal1, CDX1, CDX2, CDX4, GATA2, HOXA9). In addition, MniPSC CD34+ hematopoietic progenitors gave rise to both myeloid (CD33+, CD14+) and lymphoid (T lymphocyte, CD3+, CD4+, CD8+) progeny. These findings show that nonhuman primate iPSC are capable of giving rise to clinically relevant cell populations for transplantation. Finally, as we have previously shown with somatic CD34+ cells from pigtail macaques, using HIV-1 based lentivirus vectors we can also efficiently gene modify MniPSCs and MniPSC hematopoietic progeny with a variety of transgene cassettes, including the potent
Aurélie Bedel,1 Miguel Taillepierre,1 Pierre Dubus,3 Eric Lippert,1 Veronique Guyonnet-Duperat,2 Mautuis Thibault,1 Magalie Lalanne,1 Catherine Pain,1 Bruno Cardinaud,1 Cecile Ged,1 Benoit Rousseau,3 Yann Duchartre,1 Hubert de Verneuil,1 Emmanuel Richard,1 Francois Moreau-Gaudry.1,2 1 INSERM U 1035, University Bordeaux Segalen, Bordeaux, France, Metropolitan; 2Vectorology Plateform, University Bordeaux Segalen, Bordeaux, France, Metropolitan; 3University Bordeaux Segalen, Bordeaux, France, Metropolitan.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
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