title: Concurrent progress of reprogramming and gene correction to overcome therapeutic limitation of mutant ALK2-iPSC.
abstract: Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1, encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it, we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However, the mALK2 leads to inhibitory pluripotency maintenance, or impaired clonogenic potential after single-cell dissociation as an inevitable step, which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus, current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome, and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors, CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617G>A. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern, as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time, labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies, which is hampered by inhibitory pluripotency-maintenance requirements, or vulnerability of single-cell-dissociated iPSCs.
authors.affiliation: Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Yuseong-gu, Daejeon, South Korea.
authors.forename: Bu-Yeo
authors.initials: BY
authors.lastname: Kim
authors.affiliation: Medical Research Division, Korea Institute of Oriental Medicine, Yuseong-gu, Daejeon, South Korea.
authors.forename: SangKyun
authors.initials: S
authors.lastname: Jeong
authors.affiliation: Medical Research Division, Korea Institute of Oriental Medicine, Yuseong-gu, Daejeon, South Korea.
authors.forename: Seo-Young
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Medical Research Division, Korea Institute of Oriental Medicine, Yuseong-gu, Daejeon, South Korea.
authors.forename: So Min
authors.initials: SM
authors.lastname: Lee
authors.affiliation: Medical Research Division, Korea Institute of Oriental Medicine, Yuseong-gu, Daejeon, South Korea.
authors.forename: Eun Jeong
authors.initials: EJ
authors.lastname: Gweon
authors.affiliation: Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, South Korea.|Korea University of Science and Technology, Yuseong-gu, Daejeon, South Korea.
authors.forename: Hyunjun
authors.initials: H
authors.lastname: Ahn
authors.affiliation: Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, South Korea.|Korea University of Science and Technology, Yuseong-gu, Daejeon, South Korea.
authors.forename: Janghwan
authors.identifier: 0000-0003-3227-958X
authors.initials: J
authors.lastname: Kim
authors.affiliation: Medical Research Division, Korea Institute of Oriental Medicine, Yuseong-gu, Daejeon, South Korea.
authors.forename: Sun-Ku
authors.initials: SK
authors.lastname: Chung
mesh_terms: D030201:Activin Receptors, Type I / Q000235:genetics*; D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism; D000072669:Gene Editing; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D016513:Mice, SCID; D009154:Mutation; D009221:Myositis Ossificans / Q000235:genetics* / Q000628:therapy*; D020641:Polymorphism, Single Nucleotide; D059467:Transcriptome
chemical_list: C425423:ACVR1 protein, human; D030201:Activin Receptors, Type I