title: Factor-induced Reprogramming and Zinc Finger Nuclease-aided Gene Targeting Cause Different Genome Instability in β-Thalassemia Induced Pluripotent Stem Cells (iPSCs).
abstract: The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in β-hemoglobin gene (HBB) that cause severe β-thalassemia (β-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in β-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected β-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting.
authors: From the Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors: Ning
authors: N
authors: Ma
authors: From the Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors: Yongli
authors: Y
authors: Shan
authors: From the Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors: Baojian
authors: B
authors: Liao
authors: Wuhan Frasergen Bioinformatics Company Limited, Wuhan 430075, China, and.
authors: Guanyi
authors: G
authors: Kong
authors: Wuhan Frasergen Bioinformatics Company Limited, Wuhan 430075, China, and.
authors: Cheng
authors: C
authors: Wang
authors: From the Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors: Ke
authors: K
authors: Huang
authors: From the Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors: Hui
authors: H
authors: Zhang
authors: From the Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors: Xiujuan
authors: X
authors: Cai
authors: From the Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors: Shubin
authors: S
authors: Chen
authors: From the Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors: Duanqing
authors: D
authors: Pei
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada chenn@sfu.ca.
authors: Nansheng
authors: N
authors: Chen
authors: From the Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, pan_guangjin@gibh.ac.cn.
authors: Guangjin
authors: G
authors: Pan
mesh_terms: D000483:Alleles; D000818:Animals; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D002875:Chromosomes / Q000648:ultrastructure; D055028:Comparative Genomic Hybridization; D056915:DNA Copy Number Variations; D004720:Endonucleases / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D004900:Erythroblasts / Q000166:cytology; D059472:Exome; D017353:Gene Deletion; D018390:Gene Targeting; D014644:Genetic Variation; D042822:Genomic Instability; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D051379:Mice; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D016335:Zinc Fingers / Q000235:genetics; D055544:beta-Globins / Q000235:genetics; D017086:beta-Thalassemia / Q000235:genetics
chemical_list: D055544:beta-Globins; D004720:Endonucleases; D004722:Endoribonucleases
keywords: Gene Therapy; Genomic Instability; Genomics; Induced Pluripotent Stem Cell (iPS Cell) (iPSC); Reprogramming