Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model
Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler’s disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of co...
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| Format: | Article |
| Language: | English |
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Wiley
2019-01-01
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| Series: | Stem Cells International |
| Online Access: | http://dx.doi.org/10.1155/2019/6978303 |
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| author | Toshio Miki Ludivina Vazquez Lisa Yanuaria Omar Lopez Irving M. Garcia Kazuo Ohashi Natalie S. Rodriguez |
| author_facet | Toshio Miki Ludivina Vazquez Lisa Yanuaria Omar Lopez Irving M. Garcia Kazuo Ohashi Natalie S. Rodriguez |
| author_sort | Toshio Miki |
| collection | DOAJ |
| description | Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler’s disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from Idua knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neor) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neor sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neor removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients. |
| format | Article |
| id | doaj-art-64afa4737b334b2da07ac998db71302a |
| institution | Kabale University |
| issn | 1687-966X 1687-9678 |
| language | English |
| publishDate | 2019-01-01 |
| publisher | Wiley |
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| series | Stem Cells International |
| spelling | doaj-art-64afa4737b334b2da07ac998db71302a2025-02-03T06:08:03ZengWileyStem Cells International1687-966X1687-96782019-01-01201910.1155/2019/69783036978303Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse ModelToshio Miki0Ludivina Vazquez1Lisa Yanuaria2Omar Lopez3Irving M. Garcia4Kazuo Ohashi5Natalie S. Rodriguez6Department of Surgery, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 509A, Los Angeles, CA 90033-9141, USADepartment of Surgery, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 509A, Los Angeles, CA 90033-9141, USADepartment of Surgery, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 509A, Los Angeles, CA 90033-9141, USADepartment of Surgery, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 509A, Los Angeles, CA 90033-9141, USADepartment of Surgery, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 509A, Los Angeles, CA 90033-9141, USALaboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Surgery, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 509A, Los Angeles, CA 90033-9141, USAMucopolysaccharidosis type 1 (MPS-1), also known as Hurler’s disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from Idua knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neor) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neor sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neor removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients.http://dx.doi.org/10.1155/2019/6978303 |
| spellingShingle | Toshio Miki Ludivina Vazquez Lisa Yanuaria Omar Lopez Irving M. Garcia Kazuo Ohashi Natalie S. Rodriguez Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model Stem Cells International |
| title | Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model |
| title_full | Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model |
| title_fullStr | Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model |
| title_full_unstemmed | Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model |
| title_short | Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model |
| title_sort | induced pluripotent stem cell derivation and ex vivo gene correction using a mucopolysaccharidosis type 1 disease mouse model |
| url | http://dx.doi.org/10.1155/2019/6978303 |
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