title: Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers.
abstract: Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model. A clinically relevant marker gene, human truncated CD19 (hΔCD19), or GFP was inserted into the AAVS1 site in RhiPSCs using the CRISPR/Cas9 system. Genetically modified RhiPSCs maintained normal karyotype and pluripotency, and these clones were able to further differentiate into all three germ layers in vitro and in vivo. In contrast to transgene delivery using randomly integrating viral vectors, AAVS1 targeting allowed stable transgene expression following differentiation. Off-target mutations were observed in some edited clones, highlighting the importance of careful characterization of these cells prior to downstream applications. Genetically marked RhiPSCs will be useful to further advance clinically relevant models for iPSC-based cell therapies.
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), NIH, Bethesda, MD 20892, USA. Electronic address: sogun.hong@nih.gov.
authors.forename: So Gun
authors.initials: SG
authors.lastname: Hong
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), NIH, Bethesda, MD 20892, USA.
authors.forename: Ravi Chandra
authors.initials: RC
authors.lastname: Yada
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), NIH, Bethesda, MD 20892, USA.
authors.forename: Kyujoo
authors.initials: K
authors.lastname: Choi
authors.affiliation: Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Carpentier
authors.affiliation: Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA.
authors.forename: T Jake
authors.initials: TJ
authors.lastname: Liang
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
authors.forename: Randall K
authors.initials: RK
authors.lastname: Merling
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
authors.forename: Colin L
authors.initials: CL
authors.lastname: Sweeney
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
authors.forename: Harry L
authors.initials: HL
authors.lastname: Malech
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), NIH, Bethesda, MD 20892, USA.
authors.forename: Moonjung
authors.initials: M
authors.lastname: Jung
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), NIH, Bethesda, MD 20892, USA; Multidisciplinar Center for Biological Research, University of Campinas, Campinas, SP 13083-877, Brazil.
authors.forename: Marcus A F
authors.initials: MAF
authors.lastname: Corat
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), NIH, Bethesda, MD 20892, USA; Department of Chemistry and Molecular & Cellular Biology, Georgetown University, Washington, D.C. 20057, USA.
authors.forename: Aisha A
authors.initials: AA
authors.lastname: AlJanahi
authors.affiliation: iPSC Core, Center for Molecular Medicine, NHLBI, NIH, Bethesda, MD 20892, USA.
authors.forename: Yongshun
authors.initials: Y
authors.lastname: Lin
authors.affiliation: iPSC Core, Center for Molecular Medicine, NHLBI, NIH, Bethesda, MD 20892, USA.
authors.forename: Huimin
authors.initials: H
authors.lastname: Liu
authors.affiliation: Systems Biology Core, Systems Biology Center, NHLBI, NIH, Bethesda, MD 20892, USA.
authors.forename: Ilker
authors.initials: I
authors.lastname: Tunc
authors.affiliation: Systems Biology Core, Systems Biology Center, NHLBI, NIH, Bethesda, MD 20892, USA.
authors.forename: Xujing
authors.initials: X
authors.lastname: Wang
authors.affiliation: Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Maryknoll
authors.initials: M
authors.lastname: Palisoc
authors.affiliation: Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Stefania
authors.initials: S
authors.lastname: Pittaluga
authors.affiliation: Laboratory of Cardiovascular Regenerative Medicine, NHLBI, NIH, Bethesda, MD 20892, USA.
authors.forename: Manfred
authors.initials: M
authors.lastname: Boehm
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), NIH, Bethesda, MD 20892, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Winkler
authors.affiliation: iPSC Core, Center for Molecular Medicine, NHLBI, NIH, Bethesda, MD 20892, USA.
authors.forename: Jizhong
authors.initials: J
authors.lastname: Zou
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), NIH, Bethesda, MD 20892, USA.
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
mesh_terms: D000818:Animals; D015415:Biomarkers; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D000072669:Gene Editing; D015870:Gene Expression; D018390:Gene Targeting; D056426:Genetic Loci; D005855:Germ Layers / Q000196:embryology / Q000378:metabolism*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D008253:Macaca mulatta; D009928:Organ Specificity / Q000235:genetics; D019076:Transgenes
chemical_list: D015415:Biomarkers
keywords: AAVS1; CRISPR/Cas9; gene editing; induced pluripotent stem cells; nonhuman primate; off-target mutations; rhesus macaque