- Email: [email protected]
211. Inducible Caspase 9-Safety Switch for Therapies Using Induced Pluripotent Stem Cells
PLURIPOTENT STEM CELL THERAPIES In summary, we generated murine Csf2rb-deficient iPSC lines, which upon hematopoietic differentiation recapitulated GM-CSF dependent functional defects characteristic of PAP. These cells – upon genetic correction - appear as a promising source to test future cell and gene therapy strategies.
211. Inducible Caspase 9-Safety Switch for Therapies Using Induced Pluripotent Stem Cells
Shigeki Yagyu,1 Valentina Hoyos,1 Francesca Del Bufalo,1 Maksim Mamonkin,1 Wade Tao,1 Carlos A. Ramos,1 Malcolm K. Brenner.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX. Human induced pluripotent stem cells (hiPSC) could be an unlimited source of material for autologous cell therapy and tissue engineering, since the cells can proliferate extensively in vitro and differentiate into multiple lineages. Unfortunately, undifferentiated hiPSC can cause teratomas after transplantation, so that complete removal of these tumorigenic cells is essential if hiPSC-based cell therapy is to be safe in clinical practice. To control tumorigenic hiPSC, we have stably transduced these cells with a lentiviral vector carrying an inducible caspase 9 (iC9) suicide gene (iC9-iPSC). The gene product can be dimerized by administration of a small molecule chemical inducer of dimerization (CID) that triggers apoptosis through the intrinsic (mitochondrial) pathway. The transfer and expression of iC9 to hiPSC did not induce significant apoptosis due to spontaneous dimerization and did not interfere with their selfrenewal and pluripotency. Following exposure to 10nM of CID, the iC9-iPSC rapidly apoptosed (residual live cells 24hrs after addition of CID = 0.75%±0.12% vs. 82.79%±0.41% in the absence of CID, p<0.01). The induction of iC9-iPSC cell death by CID exposure was completely inhibited by the pan-caspase inhibitor, qVD-Oph (% live cells; 93.62±0.97%), suggesting that this cytotoxic effect was caused by the enforced dimerization of caspase 9 by CID. We subsequently generated mesenchymal stromal cells from iC9-iPSC (iPSC-MSC), by culturing the iPSC with serum-free medium containing the TGFβ pathway inhibitor, SB-431542. Flow cytometric analysis showed these iPSC-MSCs expressed MSC phenotypic markers (positive for CD44, CD73, CD90, CD105 and negative for CD14, CD19, CD34, CD45, HLA-DR), and were also capable of adipocytic and osteoblastic differentiation in appropriate. Finally, iPS-MSCs carrying iC9 consistently and rapidly underwent robust apoptosis by the administration of CID (% of live cells; 3.37% in the presence of CID vs 61.03% in the absence of CID). In conclusion, we were able to genetically modify iPSC to express iC9 as a safety switch, without altering the self-renewal properties or the pluripotency of the cells. iC9-iPSC could be efficiently eliminated in the presence of CID either as primitive cells or following differentiation into MSC. Hence the iC9 suicide gene may allow the clinical benefits of hiPSC therapy while providing an additional safety mechanism to control adverse events.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
212. Disease Modeling of Late-Onset Pompe Disease-Specific Induced Pluripotent Stem Cells
Yohei Sato,1,2 Hiroshi Kobayashi,1,2 Takashi Higuchi,1 Hiroyuki Ida,1,2 Susumu Minamisawa,3 Yoshikatsu Eto,4 Takumi Era,5 Shigemi Kimura,6 Toya Ohashi.1,2 1 Gene Therapy, Jikei University School of Medicine, Tokyo, Japan; 2Pediatrics, Jikei University School of Medicine, Tokyo, Japan; 3Cell Physiology, Jikei University School of Medicine, Tokyo, Japan; 4Advanced Clinical Research Center, Institute of Neurological Disorders, Kanagawa, Japan; 5Department of Cell Modulation, Kumamoto University, Kumamoto, Japan; 6 Department of Child Development, Kumamoto University, Kumamoto, Japan.
Pompe disease is an inherited neuromuscular disorder caused by a genetic deficiency of acid-alpha-glucosidase (GAA). Based on the clinical phenotype, Pompe disease is classified as infantile or lateonset. Hypertrophic cardiomyopathy is usually high in infantile Pompe disease. In late-onset Pompe disease, cardiovascular complications such as cardiac hypertrophy and arrhythmia are clinically present; however, cardiac involvement is less frequent and milder than that in the infantile form. Patient/disease-specific induced pluripotent stem cells (iPSCs) have been used for disease modeling, drug screening, and cell therapy. Huang et al have reported that infantile Pompe disease iPSCs can be differentiated into cardiomyocyte-like cells (Hum Mol Genet. 2011) . We attempted to generate late-onset Pompe disease iPSCs, which could differentiate into cardiomyocytes, for disease modeling. We also attempted to generate gene-corrected Pompe disease iPSCs by using lentiviral gene transfer. GAA was cloned into cDNA-expressing third-generation lentiviral vector (CS2-EF1α-GAA). To assess the transfection efficacy, Venus, a YFP variant protein, was also cloned into the vector (CS2-EF1αVenus). Then, we transfected lentiviral vectors containing GAA and Venus into control iPSCs (TkDA3-4, healthy donor) at MOI 10, 50, and 100 to estimate the optimized titer for gene correction. Expressions of GAA and Venus in iPSCs were examined in a dosedependent manner. Pompe disease iPSCs (HPS0175, 0176, 0177) were generated from fibroblasts reprogrammed by the Sendai Virus acquired from lateonset Pompe disease patients. Control iPSCs (HPS0223) were also generated from healthy donors by using the Sendai Virus. Healthy control and Pompe disease iPSCs showed similar pluripotency characteristics, which were observed using immunohistochemistry and RT-PCR. Robust cardiomyocyte differentiation was induced according to the directed differentiation protocol using the GSK-3 and Wnt inhibitors. Beating cardiomyocytes were observed 10 days after the differentiation of healthy control (HPS0223) and Pompe disease iPSCs (HPS0175). Cardiomyocytes derived from Pompe disease iPSCs demonstrated disease-specific hallmarks such as high glycogen accumulation and lysosomal enlargement. In late-onset Pompe disease iPSCs, glycogen accumulation and lysosomal enlargement are sustained even after cardiomyocyte differentiation. Our results are compatible with those previously published, showing that cardiac hypertrophy sometimes accompanies late-onset Pompe disease. Pathological changes in differentiated cardiomyocytes might elucidate the cardiovascular complications of the late-onset Pompe disease. We would like to generate genecorrected iPSCs and differentiate into cardiomyocyte to evaluate the efficacy of gene transfer.
S81
211. Inducible Caspase 9-Safety Switch for Therapies Using Induced Pluripotent Stem Cells
Shigeki Yagyu,1 Valentina Hoyos,1 Francesca Del Bufalo,1 Maksim Mamonkin,1 Wade Tao,1 Carlos A. Ramos,1 Malcolm K. Brenner.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX. Human induced pluripotent stem cells (hiPSC) could be an unlimited source of material for autologous cell therapy and tissue engineering, since the cells can proliferate extensively in vitro and differentiate into multiple lineages. Unfortunately, undifferentiated hiPSC can cause teratomas after transplantation, so that complete removal of these tumorigenic cells is essential if hiPSC-based cell therapy is to be safe in clinical practice. To control tumorigenic hiPSC, we have stably transduced these cells with a lentiviral vector carrying an inducible caspase 9 (iC9) suicide gene (iC9-iPSC). The gene product can be dimerized by administration of a small molecule chemical inducer of dimerization (CID) that triggers apoptosis through the intrinsic (mitochondrial) pathway. The transfer and expression of iC9 to hiPSC did not induce significant apoptosis due to spontaneous dimerization and did not interfere with their selfrenewal and pluripotency. Following exposure to 10nM of CID, the iC9-iPSC rapidly apoptosed (residual live cells 24hrs after addition of CID = 0.75%±0.12% vs. 82.79%±0.41% in the absence of CID, p<0.01). The induction of iC9-iPSC cell death by CID exposure was completely inhibited by the pan-caspase inhibitor, qVD-Oph (% live cells; 93.62±0.97%), suggesting that this cytotoxic effect was caused by the enforced dimerization of caspase 9 by CID. We subsequently generated mesenchymal stromal cells from iC9-iPSC (iPSC-MSC), by culturing the iPSC with serum-free medium containing the TGFβ pathway inhibitor, SB-431542. Flow cytometric analysis showed these iPSC-MSCs expressed MSC phenotypic markers (positive for CD44, CD73, CD90, CD105 and negative for CD14, CD19, CD34, CD45, HLA-DR), and were also capable of adipocytic and osteoblastic differentiation in appropriate. Finally, iPS-MSCs carrying iC9 consistently and rapidly underwent robust apoptosis by the administration of CID (% of live cells; 3.37% in the presence of CID vs 61.03% in the absence of CID). In conclusion, we were able to genetically modify iPSC to express iC9 as a safety switch, without altering the self-renewal properties or the pluripotency of the cells. iC9-iPSC could be efficiently eliminated in the presence of CID either as primitive cells or following differentiation into MSC. Hence the iC9 suicide gene may allow the clinical benefits of hiPSC therapy while providing an additional safety mechanism to control adverse events.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
212. Disease Modeling of Late-Onset Pompe Disease-Specific Induced Pluripotent Stem Cells
Yohei Sato,1,2 Hiroshi Kobayashi,1,2 Takashi Higuchi,1 Hiroyuki Ida,1,2 Susumu Minamisawa,3 Yoshikatsu Eto,4 Takumi Era,5 Shigemi Kimura,6 Toya Ohashi.1,2 1 Gene Therapy, Jikei University School of Medicine, Tokyo, Japan; 2Pediatrics, Jikei University School of Medicine, Tokyo, Japan; 3Cell Physiology, Jikei University School of Medicine, Tokyo, Japan; 4Advanced Clinical Research Center, Institute of Neurological Disorders, Kanagawa, Japan; 5Department of Cell Modulation, Kumamoto University, Kumamoto, Japan; 6 Department of Child Development, Kumamoto University, Kumamoto, Japan.
Pompe disease is an inherited neuromuscular disorder caused by a genetic deficiency of acid-alpha-glucosidase (GAA). Based on the clinical phenotype, Pompe disease is classified as infantile or lateonset. Hypertrophic cardiomyopathy is usually high in infantile Pompe disease. In late-onset Pompe disease, cardiovascular complications such as cardiac hypertrophy and arrhythmia are clinically present; however, cardiac involvement is less frequent and milder than that in the infantile form. Patient/disease-specific induced pluripotent stem cells (iPSCs) have been used for disease modeling, drug screening, and cell therapy. Huang et al have reported that infantile Pompe disease iPSCs can be differentiated into cardiomyocyte-like cells (Hum Mol Genet. 2011) . We attempted to generate late-onset Pompe disease iPSCs, which could differentiate into cardiomyocytes, for disease modeling. We also attempted to generate gene-corrected Pompe disease iPSCs by using lentiviral gene transfer. GAA was cloned into cDNA-expressing third-generation lentiviral vector (CS2-EF1α-GAA). To assess the transfection efficacy, Venus, a YFP variant protein, was also cloned into the vector (CS2-EF1αVenus). Then, we transfected lentiviral vectors containing GAA and Venus into control iPSCs (TkDA3-4, healthy donor) at MOI 10, 50, and 100 to estimate the optimized titer for gene correction. Expressions of GAA and Venus in iPSCs were examined in a dosedependent manner. Pompe disease iPSCs (HPS0175, 0176, 0177) were generated from fibroblasts reprogrammed by the Sendai Virus acquired from lateonset Pompe disease patients. Control iPSCs (HPS0223) were also generated from healthy donors by using the Sendai Virus. Healthy control and Pompe disease iPSCs showed similar pluripotency characteristics, which were observed using immunohistochemistry and RT-PCR. Robust cardiomyocyte differentiation was induced according to the directed differentiation protocol using the GSK-3 and Wnt inhibitors. Beating cardiomyocytes were observed 10 days after the differentiation of healthy control (HPS0223) and Pompe disease iPSCs (HPS0175). Cardiomyocytes derived from Pompe disease iPSCs demonstrated disease-specific hallmarks such as high glycogen accumulation and lysosomal enlargement. In late-onset Pompe disease iPSCs, glycogen accumulation and lysosomal enlargement are sustained even after cardiomyocyte differentiation. Our results are compatible with those previously published, showing that cardiac hypertrophy sometimes accompanies late-onset Pompe disease. Pathological changes in differentiated cardiomyocytes might elucidate the cardiovascular complications of the late-onset Pompe disease. We would like to generate genecorrected iPSCs and differentiate into cardiomyocyte to evaluate the efficacy of gene transfer.
S81