transcription activator-like effector nuclease (talen)-mediated
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Transcription Activator-like Effector Nuclease(TALEN)-mediated Gene Correction in Integration-free-Thalassemia Induced Pluripotent Stem Cells*SReceived for publication, June 24, 2013, and in revised form, October 21, 2013 Published, JBC Papers in Press, October 23, 2013, DOI 10.1074/jbc.M113.496174
Ning Ma1, Baojian Liao1, Hui Zhang, Linli Wang, Yongli Shan, Yanting Xue, Ke Huang, Shubin Chen,Xiaoxiao Zhou, Yang Chen, Duanqing Pei, and Guangjin Pan2
From the Key Laboratory of Regenerative Biology and the Guangdong Provincial Key Laboratory of Stem Cell and RegenerativeMedicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences, Guangzhou 510530, China and the School of Life Sciences, University of Science and Technology ofChina, Hefei, Anhui 230027, China
Background: Gene editing in human patient-specific iPSCs is critical for regenerative medicine.Results: Nonintegrating -thalassemia iPSCs corrected by TALENs display undetectable off targets and can be differentiatedinto erythroblasts expressing normal -globin.Conclusion: TALENs can be used for HBB correction efficiently in -thalassemia iPSCs with different types.Significance:Our study extends TALENs for gene correction in patient-specific iPSCs andmay have applications in cell therapy.
-Thalassemia (-Thal) is a group of life-threatening blooddisorders caused by either point mutations or deletions ofnucleotides in -globin gene (HBB). It is estimated that 4.5% ofthe population in the world carry -Thal mutants (1), posing apersistent threat to public health. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequentcorrection of the disease-causing mutations offer an ideal ther-apeutic solution to this problem. However, homologous recom-bination-based gene correction in human iPSCs remains largelyinefficient. Here, we describe a robust process combining effi-cient generation of integration-free -Thal iPSCs from the cellsof patients and transcription activator-like effector nuclease(TALEN)-based universal correction of HBB mutations in situ.We generated integration-free and gene-corrected iPSC linesfrom two patients carrying different types of homozygousmuta-tions and showed that these iPSCs are pluripotent and have nor-mal karyotype. We showed that the correction process did notgenerate TALEN-induced off targeting mutations by sequenc-ing. More importantly, the gene-corrected -Thal iPS cell linesfrom each patient can be induced to differentiate into hemato-poietic progenitor cells and then further to erythroblastsexpressing normal -globin. Our studies provide an efficientand universal strategy to correct different types of -globin
mutations in -Thal iPSCs for disease modeling andapplications.
-Thalassemia is a group of inherited genetic blood disor-ders caused by either point mutations or deletions of nucleo-tides in the -globin gene. These genetic defects result inreduced, abnormal, or no synthesis of -globin chains thatmake up hemoglobin. It is one of the most common geneticdiseases in the world, and patients with -Thal have severeanemia and a shortened life span (2). Hematopoietic stem celltransplantation is the only way to cure -thalassemia but ischallenged by the limited availability of human leukocyte anti-gen-matched healthy donors. Recently, the development ofgene therapy based on viral transduction of a normal HBB geneinto a patients own hematopoietic stem cells raised hopes forthose who do not have access to bone marrow transplantation(3, 4). However, it was still challenged by the concerns of longterm safety because of the viral integration. The somatic cells ofa patient can be reprogrammed back into pluripotent state asiPS3 cells that are capable of differentiation into any cells in thebody and thus could be potentially used for cell replacementtherapy (58). Generation of iPSCs from -Thal patients, cor-recting mutations, and subsequent differentiation into hema-topoietic stem cells raise great hopes for autologous transplan-tation to treat these inherited diseases (9). Moreover, iPS cellscan undergo indefinite self-renewal without losing the ability todifferentiate into all cell types and thus represent an ideal cellpopulation for in situ correction of the disease-causing muta-tions. However, gene targeting in human pluripotent stem cellsby standard homologous recombination is largely inefficient(10) and therefore hampers its extensive application in diseasemodels. Zinc finger nucleases (ZFNs) had been reported to sub-
* This work was supported by the National Basic Research Program of China,973 Program of China Grant 2012CB966503, Strategic Priority ResearchProgram of the Chinese Academy of Sciences Grant XDA01020202, 973Program of China Grant 2011CB965204, National S&T Major Special Projecton Major New Drug Innovation Grant 2011ZX09102-010, National NaturalScience Foundation of China Grants 31200970 and 91213304, Bureau ofScience and Technology of Guangzhou Municipality, China, Grant 2010U1-E00521 and funds from the Hundred Talents Program of the Chinese Acad-emy of Science (to G. P.).
S This article contains supplemental Table S1, Fig. S1, and supplementalinformation.
1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Guangzhou Institutes of
Biomedicine and Health, Chinese Academy of Sciences, Kaiyuan AvenueScience Park, Guangzhou 510530, China. Tel.: 86-020-32015213; E-mail:email@example.com.
3 The abbreviations used are: iPS, induced pluripotent stem; iPSC, iPS cell;-Thal, -Thalassemia; ZFN, zinc finger nucleases; HPC, hematopoietic pro-genitor cell; TALEN, transcription activator-like effector nuclease.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 48, pp. 3467134679, November 29, 2013 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
NOVEMBER 29, 2013 VOLUME 288 NUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 34671
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stantially enhance the homologous recombination efficiency byspecifically introducing a double-stranded DNA break at thetarget locus (1113). Gene targeting aided by ZFN in humaniPS or ES cells had been described to be highly efficient (1416).However, engineering ZFNs for a specific target is quiteinefficient and laborious, which largely hampers their wide-spread adoption. Recently, transcription activator-like effectornucleases (TALENs) had been described to recognize andcleave any given DNA sequences with high efficiency (1719).The DNA-binding domain of TALEN is unusual and containsmultiple units that arranged in tandem (TALE repeats). Eachindividual unit is composed of 34 amino acids with two highlyvariable amino acids to determine the unit to recognize oneDNA pair in the TALEN recognizing sequence (20). In theory,TALE repeat could be engineered and arranged to specificallyrecognize any given DNA sequence. TALEN-mediated genetargeting had been described in multiple species, includingzebrafish and human iPS, and ES cells (21, 22). Practically, com-pared with ZFN, TALEN is much more easy and convenientregarding the designing and constructing. Also, TALENsexhibited lower off target effects and reduced nuclease-associ-ated cytotoxicities compared with ZFNs (2325). In attempt toextend TALEN technology to gene correction for -Thal, wegenerated the-Thal iPS cells through a nonviral approach anddeveloped an efficient process to correct the mutations in-globin gene by designing and utilizing site-specific TALENs.
iPS GenerationThemethod of isolating amniotic fluid cellswas performed as previously described (26). For reprogram-ming, an oriP/EBNA1-based pCEP4 episomal vector contain-ingOCT4, SOX2, KLF4, and SV40LT genes (27) and miR-302367 (28) were co-transfected into amniotic fluid cells vianucleofection (Amaxa). The cells were then plated to Matri-gel-coated 6-well plates and cultured with reprogrammingmedium (mTeSR1). Themediumwas changed every 2 days andiPS-like colonies were picked onto newMatrigel plate for char-acterization. Cells of passages from 15 to 40 are used for thefollowing experiments.TALEN and Donor Vectors for Gene TargetingTALENs
were designed as described (17, 29). The full amino acidsequences of TALENs are given in the supplemental informa-tion. For donorDNA, left and right homology armswere ampli-fied from genomic DNA of healthy individual. A loxP-flankedPGK-puromycin cassette or loxP-flanked PGK-neomycin cas-settewere cloned between twohomology arms in the pMD-18Tvector. For targeting, 1 106 iPSCs were electroporated with 2g of donorDNA and 4.5g of each TALENplasmid. Then thecells were plated ontoMatrigel-coated 6-well plates in the pres-ence of Y-27632 (10 M; Sigma) for 1 day. Positive clones wereselected by puromycin (0.5 g/ml) or G418 (100 g/ml; Sigma)in mTeSR1. The selected colonies were verified by genomicPCR and Southern blot. All primers used are listed in supple-mental Table S1.GFP Reporter AssayGFP reporter activation was tested by
co-transfecting 293T cells with plasmids carrying TALENs andGFP reporters. 293T cells were seeded into 12-well plates theday before transfection. Approximately 24 h after initial seed-
ing, cells were transfected using calciumphosphate. For 12-wellplates, we used 1.5 g of each TALEN and 1 g of reporterplasmids/well. The cells were trypsinized from their culturingplates 48 h after transfection and resuspended in 800 l of PBSfor flow cytometry analysis. The flow cytometry data were ana-lyzed using C6 (BD Biosciences). At least 20,000 events wereanalyzed for each transfection sample.PCR Detection of Corrected ClonesPCR was performed
using High Fidelity Platinum Taq (Invitrogen)