The Experts below are selected from a list of 4929 Experts worldwide ranked by ideXlab platform
Sean Bong Lee - One of the best experts on this subject based on the ideXlab platform.
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ews wt1 oncoprotein activates neuronal Reprogramming Factor ascl1 and promotes neural differentiation
Cancer Research, 2014Co-Authors: Hong-jun Kang, Jun Hong Park, Weiping Chen, Soo Im Kang, Krzysztof Moroz, Marc Ladanyi, Sean Bong LeeAbstract:The oncogenic fusion gene EWS–WT1 is the defining chromosomal translocation in desmoplastic small round-cell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS–WT1 functions as an aberrant transcription Factor that drives tumorigenesis, but the mechanistic basis for its pathogenic activity is not well understood. To address this question, we created a transgenic mouse strain that permits physiologic expression of EWS–WT1 under the native murine Ews promoter. EWS–WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neural Reprogramming Factor ASCL1. Mechanistic analyses demonstrated that EWS–WT1 directly bound the proximal promoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS–WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal induction media increased neural gene expression and induced neurite-like projections, both of which were abrogated by silencing EWS–WT1. Taken together, our findings reveal that EWS–WT1 can activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT. Cancer Res; 74(16); 4526–35. ©2014 AACR.
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EWS–WT1 Oncoprotein Activates Neuronal Reprogramming Factor ASCL1 and Promotes Neural Differentiation
Cancer research, 2014Co-Authors: Hong-jun Kang, Jun Hong Park, Weiping Chen, Soo Im Kang, Krzysztof Moroz, Marc Ladanyi, Sean Bong LeeAbstract:The oncogenic fusion gene EWS–WT1 is the defining chromosomal translocation in desmoplastic small round-cell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS–WT1 functions as an aberrant transcription Factor that drives tumorigenesis, but the mechanistic basis for its pathogenic activity is not well understood. To address this question, we created a transgenic mouse strain that permits physiologic expression of EWS–WT1 under the native murine Ews promoter. EWS–WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neural Reprogramming Factor ASCL1. Mechanistic analyses demonstrated that EWS–WT1 directly bound the proximal promoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS–WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal induction media increased neural gene expression and induced neurite-like projections, both of which were abrogated by silencing EWS–WT1. Taken together, our findings reveal that EWS–WT1 can activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT. Cancer Res; 74(16); 4526–35. ©2014 AACR.
Luis F Z Batista - One of the best experts on this subject based on the ideXlab platform.
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in situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases
Stem Cells, 2011Co-Authors: Vittorio Sebastiano, John S. Hawkins, Morgan L Maeder, James Angstman, Bahareh Haddad, Cyd Khayter, Mathew J Goodwin, Cherie L Ramirez, Luis F Z Batista, Steven E ArtandiAbstract:The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of Reprogramming Factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications. STEM CELLS 2011;29:1717–1726
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in situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases
Stem Cells, 2011Co-Authors: Vittorio Sebastiano, John S. Hawkins, Morgan L Maeder, James Angstman, Bahareh Haddad, Cyd Khayter, Mathew J Goodwin, Cherie L Ramirez, Dana T Yeo, Luis F Z BatistaAbstract:The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of Reprogramming Factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.
John S. Hawkins - One of the best experts on this subject based on the ideXlab platform.
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Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell Reprogramming.
Stem cell research & therapy, 2017Co-Authors: Christopher E. Schmitt, Blanca M. Morales, Ellen M. H. Schmitz, John S. Hawkins, Carlos Lizama, Joan P. Zape, Edward C. Hsiao, Ann C. ZoveinAbstract:Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell Reprogramming. The episomal vectors carrying the “Yamanaka Reprogramming Factors” (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating Reprogramming of cells to a pluripotent state. However, the Reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for Reprogramming efficiency and for the ability to enrich for stoichiometric ratios of Factors. The reengineered vectors allow for cell sorting based on Reprogramming Factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the Reprogramming Factor dosage. Together, these modified vectors are a useful tool for understanding the Reprogramming process and improving induced pluripotent stem cell isolation efficiency.
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in situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases
Stem Cells, 2011Co-Authors: Vittorio Sebastiano, John S. Hawkins, Morgan L Maeder, James Angstman, Bahareh Haddad, Cyd Khayter, Mathew J Goodwin, Cherie L Ramirez, Luis F Z Batista, Steven E ArtandiAbstract:The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of Reprogramming Factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications. STEM CELLS 2011;29:1717–1726
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in situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases
Stem Cells, 2011Co-Authors: Vittorio Sebastiano, John S. Hawkins, Morgan L Maeder, James Angstman, Bahareh Haddad, Cyd Khayter, Mathew J Goodwin, Cherie L Ramirez, Dana T Yeo, Luis F Z BatistaAbstract:The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of Reprogramming Factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.
Vittorio Sebastiano - One of the best experts on this subject based on the ideXlab platform.
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in situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases
Stem Cells, 2011Co-Authors: Vittorio Sebastiano, John S. Hawkins, Morgan L Maeder, James Angstman, Bahareh Haddad, Cyd Khayter, Mathew J Goodwin, Cherie L Ramirez, Luis F Z Batista, Steven E ArtandiAbstract:The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of Reprogramming Factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications. STEM CELLS 2011;29:1717–1726
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in situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases
Stem Cells, 2011Co-Authors: Vittorio Sebastiano, John S. Hawkins, Morgan L Maeder, James Angstman, Bahareh Haddad, Cyd Khayter, Mathew J Goodwin, Cherie L Ramirez, Dana T Yeo, Luis F Z BatistaAbstract:The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of Reprogramming Factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.
Hong-jun Kang - One of the best experts on this subject based on the ideXlab platform.
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ews wt1 oncoprotein activates neuronal Reprogramming Factor ascl1 and promotes neural differentiation
Cancer Research, 2014Co-Authors: Hong-jun Kang, Jun Hong Park, Weiping Chen, Soo Im Kang, Krzysztof Moroz, Marc Ladanyi, Sean Bong LeeAbstract:The oncogenic fusion gene EWS–WT1 is the defining chromosomal translocation in desmoplastic small round-cell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS–WT1 functions as an aberrant transcription Factor that drives tumorigenesis, but the mechanistic basis for its pathogenic activity is not well understood. To address this question, we created a transgenic mouse strain that permits physiologic expression of EWS–WT1 under the native murine Ews promoter. EWS–WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neural Reprogramming Factor ASCL1. Mechanistic analyses demonstrated that EWS–WT1 directly bound the proximal promoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS–WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal induction media increased neural gene expression and induced neurite-like projections, both of which were abrogated by silencing EWS–WT1. Taken together, our findings reveal that EWS–WT1 can activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT. Cancer Res; 74(16); 4526–35. ©2014 AACR.
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EWS–WT1 Oncoprotein Activates Neuronal Reprogramming Factor ASCL1 and Promotes Neural Differentiation
Cancer research, 2014Co-Authors: Hong-jun Kang, Jun Hong Park, Weiping Chen, Soo Im Kang, Krzysztof Moroz, Marc Ladanyi, Sean Bong LeeAbstract:The oncogenic fusion gene EWS–WT1 is the defining chromosomal translocation in desmoplastic small round-cell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS–WT1 functions as an aberrant transcription Factor that drives tumorigenesis, but the mechanistic basis for its pathogenic activity is not well understood. To address this question, we created a transgenic mouse strain that permits physiologic expression of EWS–WT1 under the native murine Ews promoter. EWS–WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neural Reprogramming Factor ASCL1. Mechanistic analyses demonstrated that EWS–WT1 directly bound the proximal promoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS–WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal induction media increased neural gene expression and induced neurite-like projections, both of which were abrogated by silencing EWS–WT1. Taken together, our findings reveal that EWS–WT1 can activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT. Cancer Res; 74(16); 4526–35. ©2014 AACR.