- Email: [email protected]
570. Efficient Generation of Induced Pluripotent Stem Cells by the Inhibition of PTEN Tumor Suppressor
STEM CELL BIOLOGY tested. The major conclusion of the study is that ZFN-mediated gene insertion is a very effective method to obtain high-level, quasi normal expression of globin therapeutic cassette.
569. Correction of Artemis Deficiency in Murine Hematopoietic Stem Cell by I-Sce1 Meganuclease and Artemis Recombination Matrix Mediated Homologous Recombination
Julie Riviere,1 Julia Hauer,2 Laurent Poirot,3 Julien Brochet,1 Agnès Gouble,3 Philippe Souque,4 Karine Courbeyrette,4 Pierre Charneau,4 Frédérique Pâques,3 Alain Fischer,1 Jean-Pierre de Villartay,1 Marina Cavazzana-Calvo.1 1 Hôpital Necker Enfants Malades, U768, Paris, France, Metropolitan; 2Dept of Pediatric Hematology and Oncology, Children’s University Hospital, Dusseldorf, Germany; 3Cellectis, Paris, France, Metropolitan; 4Hôpital Necker Enfants Malades, U768, Paris, France, Metropolitan.
Gene therapy allows correction of a number of monogenic diseases affecting the hematopoietic system. This approach can be conducted by two different strategies which are either semi-random integration of the therapeutical gene using self-inactivating (SIN) retroviral vectors or homologous recombination (HR). The feasibility and efficacy of the first approach has been demonstrated in two forms of severe combined immunodeficiency (SCID) but the occurrence of five severe adverse events leads to further consider the HR approach. HR is a highly specific DNA-repair mechanism occurring between two homologous DNA sequences. DNA double strand breaks (DSB) can enhance the frequency of HR. Meganucleases are endonucleases with long DNA-recognition sites (12-30bp). In view of that, engineering gene specific meganucleases represents a tempting novel approach for gene therapy and targeting. To further develop this approach, we chose the Artemis deficiency model. This variant of SCID combines the phenotype of radio-sensitivity and lymphocyte developmental arrest. In this model, successful gene targeting can thus be assessed by reversion of the pathological phenotype in a number of assays including T-cell development in vitro. The Artemis deficient mouse model was created by substitution of exon 12 of the murine Artemis gene by a restriction site for the I-Sce1. To deliver the I-Sce1 meganuclease and to provide the Artemis repair substrate in murine HSC we designed two SIN-integrase-defective lentiviral vectors (SIN-IDLV): IDLV-CMV-I-Sce1 and IDLV-Art. Reversion of the Artemis deficient phenotype indicating successful gene targeting of the Artemis exon 12 was observed in 2 out 10 experiments by means of appearance of CD4/CD8 double positive cells after co-culture on the OP9-DL1 system. Site specific insertion of Artemis exon 12 by HR was confirmed by nested-PCR and genomic DNA sequence analysis. These preliminary results demonstrate that meganucleases and repair matrixes can induce site specific DNA-repair by HR in HSC. Even though the efficiency of genomic reparation needs to be further improved and its potential genotoxicity remains to be evaluated, this new approach of gene therapies is very promising for the future.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
570. Efficient Generation of Induced Pluripotent Stem Cells by the Inhibition of PTEN Tumor Suppressor
Jiyuan Liao,1 Tomotoshi Marumoto,1,2 Saori Yamaguchi,1 Shinji Okano,3 Naoki Takeda,4 Chika Sakamoto,1 Hirotaka Kawano,1 Takenobu Nii,1 Yoko Nagai,1 Michiyo Okada,1 Yoshie Miura,1 Shinya Shimoda,1 Hiroyuki Inoue,1,2 Atsushi Takahashi,1 Masato Tanaka,5 Kaori Nagatoshi,5 Ken-ichi Yamamura,6 Kohichi Kawahara,7 Akira Suzuki,7 Kenzaburo Tani.1,2 1 Division of Molecular and Clinical Genetics, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Advanced Molecular and Cell Therapy, Kyushu University, Fukuoka, Japan; 3Division of Pathophysiological and Experimental Pathology, Kyushu University, Fukuoka, Japan; 4Resource Development and Analysis, Kumamoto University, Kumamoto, Japan; 5Laboratory of Embryonic and Genetic Engineering, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 6Developmental Genetics, Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan; 7Division of Cancer Genetics, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. The derivation of induced pluripotent stem cells (iPSCs) generated by the ectopic expression of defined transcription factors brings the regenerative medicine close to a real beneficial practice (Takahashi, and Yamanaka, Cell 126, 663-676, 2006). However the low efficiency (0.01∼0.1%) of iPSC generation from somatic cells is one of the major problems in this field. Phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal, proliferation and maintenance of embryonic stem cells (ESCs), but the contribution of this pathway or of its well-known negative regulator, Phosphatase and Tensin Homolog (Pten) in the process of reprogramming remain unclear. In this study we hypothesized that activation of PI3K pathway might facilitate the reprogramming process for iPSC generation. To test this we retrovirally transduced the expression of Oct3/4 (also called Pou5f1), Klf4, Sox2 and c-Myc (OKSM) in mouse embryonic fibroblasts (MEFs) lacking of Pten, and embryonic stem cells (ESCs) like round-shaped colonies were found 7 days after the transduction. The efficiency of iPSC generation was determined by alkaline phosphatase activity (AP). We found that significantly higher number of AP+ colonies was formed from Pten-/- MEFs compared to wild-type MEFs. We also transiently activated PI3K pathway by the use of Pten inhibitor, bpV(HOpic), during the generation of iPSCs (bpV-iPSCs). The efficiency of AP+ colony formation was approximately 9%, and this was twice higher than controls. These bpV-iPSCs expressed ES markers such as SSEA1and Nanog, and showed a normal karyotype. They had an ability of differentiation into three germ layers in immunodeficient mice. Moreover, bpV-iPSCs contributed to the germline-competent chimeras in mice. These results indicated that bpV-iPSCs had the pluripotency in vitro and in vivo. Notably we have also found that the use of bpV(HOpic) improved the efficiency of human iPSC generation. Overall activation of PI3K pathway by the use of Pten inhibitor, bpV(HOpic), improved the efficiency of iPSC generation. This simple method may offer a new approach to improve reprogramming process of iPSC generation in the future.
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569. Correction of Artemis Deficiency in Murine Hematopoietic Stem Cell by I-Sce1 Meganuclease and Artemis Recombination Matrix Mediated Homologous Recombination
Julie Riviere,1 Julia Hauer,2 Laurent Poirot,3 Julien Brochet,1 Agnès Gouble,3 Philippe Souque,4 Karine Courbeyrette,4 Pierre Charneau,4 Frédérique Pâques,3 Alain Fischer,1 Jean-Pierre de Villartay,1 Marina Cavazzana-Calvo.1 1 Hôpital Necker Enfants Malades, U768, Paris, France, Metropolitan; 2Dept of Pediatric Hematology and Oncology, Children’s University Hospital, Dusseldorf, Germany; 3Cellectis, Paris, France, Metropolitan; 4Hôpital Necker Enfants Malades, U768, Paris, France, Metropolitan.
Gene therapy allows correction of a number of monogenic diseases affecting the hematopoietic system. This approach can be conducted by two different strategies which are either semi-random integration of the therapeutical gene using self-inactivating (SIN) retroviral vectors or homologous recombination (HR). The feasibility and efficacy of the first approach has been demonstrated in two forms of severe combined immunodeficiency (SCID) but the occurrence of five severe adverse events leads to further consider the HR approach. HR is a highly specific DNA-repair mechanism occurring between two homologous DNA sequences. DNA double strand breaks (DSB) can enhance the frequency of HR. Meganucleases are endonucleases with long DNA-recognition sites (12-30bp). In view of that, engineering gene specific meganucleases represents a tempting novel approach for gene therapy and targeting. To further develop this approach, we chose the Artemis deficiency model. This variant of SCID combines the phenotype of radio-sensitivity and lymphocyte developmental arrest. In this model, successful gene targeting can thus be assessed by reversion of the pathological phenotype in a number of assays including T-cell development in vitro. The Artemis deficient mouse model was created by substitution of exon 12 of the murine Artemis gene by a restriction site for the I-Sce1. To deliver the I-Sce1 meganuclease and to provide the Artemis repair substrate in murine HSC we designed two SIN-integrase-defective lentiviral vectors (SIN-IDLV): IDLV-CMV-I-Sce1 and IDLV-Art. Reversion of the Artemis deficient phenotype indicating successful gene targeting of the Artemis exon 12 was observed in 2 out 10 experiments by means of appearance of CD4/CD8 double positive cells after co-culture on the OP9-DL1 system. Site specific insertion of Artemis exon 12 by HR was confirmed by nested-PCR and genomic DNA sequence analysis. These preliminary results demonstrate that meganucleases and repair matrixes can induce site specific DNA-repair by HR in HSC. Even though the efficiency of genomic reparation needs to be further improved and its potential genotoxicity remains to be evaluated, this new approach of gene therapies is very promising for the future.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
570. Efficient Generation of Induced Pluripotent Stem Cells by the Inhibition of PTEN Tumor Suppressor
Jiyuan Liao,1 Tomotoshi Marumoto,1,2 Saori Yamaguchi,1 Shinji Okano,3 Naoki Takeda,4 Chika Sakamoto,1 Hirotaka Kawano,1 Takenobu Nii,1 Yoko Nagai,1 Michiyo Okada,1 Yoshie Miura,1 Shinya Shimoda,1 Hiroyuki Inoue,1,2 Atsushi Takahashi,1 Masato Tanaka,5 Kaori Nagatoshi,5 Ken-ichi Yamamura,6 Kohichi Kawahara,7 Akira Suzuki,7 Kenzaburo Tani.1,2 1 Division of Molecular and Clinical Genetics, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Advanced Molecular and Cell Therapy, Kyushu University, Fukuoka, Japan; 3Division of Pathophysiological and Experimental Pathology, Kyushu University, Fukuoka, Japan; 4Resource Development and Analysis, Kumamoto University, Kumamoto, Japan; 5Laboratory of Embryonic and Genetic Engineering, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 6Developmental Genetics, Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan; 7Division of Cancer Genetics, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. The derivation of induced pluripotent stem cells (iPSCs) generated by the ectopic expression of defined transcription factors brings the regenerative medicine close to a real beneficial practice (Takahashi, and Yamanaka, Cell 126, 663-676, 2006). However the low efficiency (0.01∼0.1%) of iPSC generation from somatic cells is one of the major problems in this field. Phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal, proliferation and maintenance of embryonic stem cells (ESCs), but the contribution of this pathway or of its well-known negative regulator, Phosphatase and Tensin Homolog (Pten) in the process of reprogramming remain unclear. In this study we hypothesized that activation of PI3K pathway might facilitate the reprogramming process for iPSC generation. To test this we retrovirally transduced the expression of Oct3/4 (also called Pou5f1), Klf4, Sox2 and c-Myc (OKSM) in mouse embryonic fibroblasts (MEFs) lacking of Pten, and embryonic stem cells (ESCs) like round-shaped colonies were found 7 days after the transduction. The efficiency of iPSC generation was determined by alkaline phosphatase activity (AP). We found that significantly higher number of AP+ colonies was formed from Pten-/- MEFs compared to wild-type MEFs. We also transiently activated PI3K pathway by the use of Pten inhibitor, bpV(HOpic), during the generation of iPSCs (bpV-iPSCs). The efficiency of AP+ colony formation was approximately 9%, and this was twice higher than controls. These bpV-iPSCs expressed ES markers such as SSEA1and Nanog, and showed a normal karyotype. They had an ability of differentiation into three germ layers in immunodeficient mice. Moreover, bpV-iPSCs contributed to the germline-competent chimeras in mice. These results indicated that bpV-iPSCs had the pluripotency in vitro and in vivo. Notably we have also found that the use of bpV(HOpic) improved the efficiency of human iPSC generation. Overall activation of PI3K pathway by the use of Pten inhibitor, bpV(HOpic), improved the efficiency of iPSC generation. This simple method may offer a new approach to improve reprogramming process of iPSC generation in the future.
S221