Transcription Activator-like Effector Nuclease(TALEN)-mediated Gene Correction in Integration-free�-Thalassemia Induced Pluripotent Stem Cells*□S
Received 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 Ma‡§1, Baojian Liao‡§1, Hui Zhang‡§, Linli Wang‡§, Yongli Shan‡§, Yanting Xue‡§¶, Ke Huang‡§, Shubin Chen‡§,Xiaoxiao Zhou‡§, Yang Chen‡§, Duanqing Pei‡§, and Guangjin Pan‡§2
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 patient’s 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 (5–8). 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 protected].
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. 34671–34679, November 29, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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stantially enhance the homologous recombination efficiency byspecifically introducing a double-stranded DNA break at thetarget locus (11–13). Gene targeting aided by ZFN in humaniPS or ES cells had been described to be highly efficient (14–16).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 (17–19).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 (23–25). 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.
EXPERIMENTAL PROCEDURES
iPS Generation—Themethod 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-302–367 (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 Targeting—TALENs
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 2�g of donorDNA and 4.5�g 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 Assay—GFP 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 Clones—PCR was performed
using High Fidelity Platinum Taq (Invitrogen) according to themanufacturer’s instructions. 50–100 ng of genomic DNA tem-plates were used in all reactions. Primer set including P1 (onHBB locus, upstream of 5� homology arm) and P2 (in the drugresistance cassette) was used to amplify a 2.8-kb product of the5� junction of a targeted integration (illustrated in Fig. 2D). Aprimer set including P3 (on 5� homology arm) and P4 (3�homology arm) was used to amplify a 2-kb product or a 500-bpproduct to identify whether random integration occurred (seeFig. 2D). Primer IVS-654 and primer EXO-3-15 were used toamplify a 600-bp product including the mutant region of�Thal654_iPS, and the PCR products were sequenced to iden-tify the corrected clones. Primer AEXON-F and primerBEXON-Rwere used to amplify a 700-bp product including themutant region of �(�TCTT)_iPS, and the PCR products weresequenced to identify the corrected clones. All of the primersused are listed in supplemental Table S1.Southern Blot—To detect homologous recombination at
HBB locus, a 502-bpHBB specific probe in the 5� side of the lefthomology arm was synthesized by PCR amplification usingprimers 5�probe-F and 5�probe-R and a DIG-dUTP labeling kit(Roche Applied Science). Genomic DNAwas digested by BglII,and then standard Southern blotting was performed followingthe instruction manuals of DIG High Prime DNA labeling anddetection starter kit II (Roche Applied Science).Teratoma Formation and Analysis—Cells from a confluent
10-cm plate were harvested by 0.5 mM EDTA digestion, resus-pended inMatrigel, and injected subcutaneously into immuno-deficiency mice. Eight weeks after injection, teratomas weredissected, fixed in 4% paraformaldehyde, and processed forhematoxylin/eosin staining.ErythroblastDifferentiation ofHuman iPSCells—Human iPS
cells were harvested by treatment with 2 mg/ml dispase (Invit-rogen) and co-cultured with OP9 stromal cells at an approxi-mate density of 5 � 106/20 ml/10-cm dish in 20 ml of �-mini-mumEagle’smedium (Invitrogen) supplementedwith 10% FBS(HyClone, Logan, UT), 100 �M monothioglycerol (Sigma), and100�M vitamin C. The co-culture of OP9with pluripotent cellswere incubated for 8 days, with a half medium change on days 4and 6. Differentiated human iPSCs were harvested at day 8.CD34� cells were sorted out using a direct CD34 progenitorcell isolation kit (Milteneyi Biotech, Auburn, CA). Hematopoi-etic clonogenic assays were performed in 35-mm low adherentplastic dishes (Greiner Bio-one, Monroe, NC) using 2 ml/dishofMethoCultGF�H4435 semisolidmedium (StemCell Tech-nologies, Inc.) according to the manufacturer’s instructions.The number of sorted CD34� cells using magnetic activatedcell sorting for CFC assay was �5 � 105. Colonies were scoredafter 12–14 days of incubation. Colonies were picked individu-ally from methylcellulose cultures, washed in PBS contains 2%
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FBS, and spun onto slides with a cytospin apparatus (TXD-3).Cells were fixed and stained with Wright-Giemsa reagents(BASO Biotech, Auburn, CA).
RESULTS
Derivation and Characterization of iPSCs from Two Patientswith Different �-Thal Subtypes—To generate iPS cells, we iso-lated amniotic fluid cells from two fetuses with informed con-sents from their mothers. One was diagnosed with �-Thalmajor of IVS2–654, which carried two homozygous C 3 Tmutations at the second intron of HBB resulted in the forma-tion of an abnormally spliced mRNA and the deficiency of cor-rectly spliced�-globin transcript (30). The other was diagnosedwith �-Thal major of �41–42 (�TCTT), which carried two
homozygous TCTT deletions at the second exon caused aframeshift that generates a termination codon (TGA) in theposition of the new 59th codon (31). To obtain iPSCs that weremore close to clinical grade, we attempted to generate �-ThaliPSCs under nonviral, serum-free, and feeder-free conditions.In detail, we expanded the amniotic fluid cells and deliveredoriP/EBNA episomal vectors carrying a combination of repro-gramming factors including OCT4, SOX2, SV40LT, and KLF4(27) and miR-302–367 cluster (28) through electroporation.After �5 days growth in amniocyte medium, the cells weresubsequently plated on Matrigel-coated plate and cultured indefined mTeSR1medium (32) for further reprogramming (Fig.1A). The iPS-like colonies appeared at �25 days after electro-poration. For 1 � 106 starting amniotic fluid cells from these
FIGURE 1. Characterization of iPSCs from two �-thalassemia patients. A, top panel, schematic representation of the episomal-based iPSC generationprotocol. Bottom panels, bright field images of cultured human amniocytes and iPS cell colonies. Scale bars, 200 �m. B, flow cytometry expression analyses ofiPS cell-specific cell markers. Red, isotype control; blue, antigen staining for OCT4 or SSEA4. C, quantitative reverse transcription-PCR analysis of endogenousOCT4, SOX2, and NANOG expression in �Thal654_iPS cells and �(�TCTT)_iPS cells. The data are presented as means � S.D. from three assays. D, nonintegrationanalysis of episomal DNA in the iPS cells. Control, genome DNA extracted from amniocytes that were transfected with related episome vectors. E, normalkaryotypes of �Thal654_ iPS cells and �(�TCTT)_ iPS cells. F, hematoxylin/eosin staining of teratomas derived from indicated iPSCs. Scale bars, 200 �m. G, leftpanel, sequencing results of the C3 T mutation site in the second intron of HBB in �Thal654_iPS cells. Right panel, sequencing results of TCTT deletion in thesecond exon of HBB in �(�TCTT)_iPS cells. Orange boxes indicate exons of HBB.
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two patients for reprogramming, we usually obtained�50 alka-line phosphatase-positive iPSC-like colonies. We then pickedfour colonies that displayed typical iPSC morphology for fur-ther expanding and characterization. The clonally expanded�-Thal iPS cells exhibited typical human ES cell morphology(Fig. 1A) and expressed pluripotent markers such as OCT4 andSSEA4 homogenously (Fig. 1B). By quantitative RT-PCR, wedemonstrated that the endogenous pluripotent genes such asOCT4, SOX2, and NANOG were fully activated in �-Thal iPScells, as well as that in H1 ES cells (Fig. 1C). More importantly,by using transgene-specific primers to detect transgenes (27),we showed that both of these two �-Thal iPS cell lines harborneither the exogenous reprogramming factors such as OCT4,SOX2, and KLF4 nor the genes in episomal vector backbone(Fig. 1D). Moreover, these transgene-free �-Thal iPS cell linespossessed normal karyotype (Fig. 1E) and could form typicalteratomas containing three germ layers upon injection intoimmunodeficient mice, a well known assay for pluripotency(Fig. 1F). Furthermore, we confirmed that the two transgene-free �-Thal iPS cell lines carried the homozygous disease-causing mutations as diagnosed (Fig. 1G). Thus, based on theirdisease subtypes, we named them �Thal654_iPS and�(�TCTT)_iPS, respectively (Fig. 1G).Construction of Site-specific TALENs for �-Globin Cleavage—
We sought to correct these disease-causing mutations in�-Thal iPS cells through in situ gene targeting using TALENs(33). Based on previous studies (20), we designed a pair ofTALENs that could specifically recognize two adjacent 18-bpDNA sequences with a 17-bp spacer that was �600 bp down-stream of the last exon of HBB (Fig. 2A). To test the site speci-ficity and cleavage activity of the designed TALENs, we con-structed a TALEN-targeting plasmid that harbored a GFPreporter. Two 205-bp duplications of GFP coding sequencethat flank each side of TALEN-targeting site were introducedinto the middle of GFP coding region (illustrated in Fig. 2A).Once a break was introduced by TALENs cleavage, the dupli-cated homologous sequence will anneal together and recombi-nate into a full-lengthGFP and thus can be detected by FACS toevaluate the efficiency and specificity of newly designedTALENs (Fig. 2B). Thus, we transfected the TALEN-targetingGFP reporter together with the pair of TALENs (TALEN-L/R)into 293T cells and showed that the GFP signals were signifi-cantly increased compared with that GFP reporter transfectedalone orwith only oneTALEN, demonstrating that the cleavageactivity by the TALENs was high (Fig. 2B). To test their sitespecificity, we introduced different point mutations or dele-tions in TALEN-binding sequence. We showed that the GFPsignals were greatly reduced in reporters with mutated bindingsites compared with wild type control reporter (Fig. 2C). Thesedata demonstrated that the nuclease activity of the designedTALENs was highly specific to its targets and can be harnessedfor further genome editing.Efficient Correction of �-Globin Mutations Using TALEN-
mediated Gene Targeting—To correct the mutations in�Thal654_iPS and �(�TCTT)_iPS cells, we constructed adonor template centered on TALENs targeting site. The donortemplate contains a 2.4-kb 5� homology arm that harbors theentire wild type �-globin gene and a 1-kb 3� arm homology to
the downstream of �-globin gene (Fig. 2D). Upon cleavage byTALENs and subsequent homology recombination with thisdonor template, wild type �-globin gene in the donor willreplace the mutant one in �Thal654_iPS or �(�TCTT)_iPScells. Thus, we introduced the linearized donor plasmids andTALEN vectors into both �Thal654_iPS and �(�TCTT)_iPScells through electroporation. The positive colonies wereselected by puromycin (for �Thal654_iPS) or G418 (for�(�TCTT)_iPS). Drug-resistant colonies were manuallypicked and expanded for further characterization. To identifycorrectly targeting clones, we designed two pairs of PCR prim-ers as indicated in Fig. 2D and performed genomic PCR withthese primers (Fig. 2D). The correctly targeted clones would bepositive for both primers but with different sizes. By genomicDNA PCR, we found that the targeting efficiency was remark-able. In the case of �Thal654_iPS cells, of 37 picked clonescharacterized, 25 were double positive for both PCR primerpairs, with an efficiency of 68%. For �(�TCTT)_iPS cells, 4 of10 picked clones were identified as positive (supplemental Fig.S1).We then randomly expanded two positive clones fromeach�Thal iPS cell line to confirm the correction of mutant �-glo-bin. First, we confirmed that the correct gene targeting couldbe detected by genomic PCR with expected size using thedesigned two primer pairs (Fig. 2E). Second, we performedSouthern blot to further analyze the gene targeting.As shown inFig. 2F, both targeted cell lines showed the expected bands (Fig.2F). Lastly, through Sanger sequencing, we confirmed that both�Thal iPS cell lines were corrected at one allele of mutated�-globin gene (Fig. 2G), indicated as double peaks for�Thal654_corrected iPS and overlapping for �(�TCTT)_corrected iPS in Sanger sequencing map. Taken together, weshowed that TALEN-mediated gene targeting was highly efficientto correct different types of disease-causing mutations in HBBgene in �-Thal iPS cells.Characterization of Gene Corrected �-Thal iPSCs—To fur-
ther characterize the gene corrected �-Thal iPS cell lines, weshowed that typical pluripotent markers such as OCT4 andSSEA-4 were expressed homogenously in the two corrected�Thal654_iPS and �(�TCTT)_iPS cell lines, indicating thatthey remained pluripotent after correction (Fig. 3A). Further-more, both of them could differentiate into three germ layerlineages upon embryonic body formation (Fig. 3B). Also, thekaryotypes of the corrected iPS cells remain normal aftergenetic operation (Fig. 3C). Most importantly, upon injectioninto immunodeficient mice, the corrected �-Thal iPS cellscould form teratomas that contain all three germ layers (Fig.3D), demonstrating that the gene corrected �-Thal iPS cellskeep the pluripotency well upon gene targeting. One importantconcern in using nuclease to aid gene targeting is off targetcleavage because it might introduce extra mutations in thegenome. To address this question, we blasted TALEN-targetedsequences in the entire human genome and selected sixgenomic sites that were on the top in homology to the designedTALEN recognition sequence. We designed primers andamplified these regions from the genomic DNA extracted fromgene corrected �Thal654_iPS or �(�TCTT)_iPS cells and per-formed Sanger sequencing on those regions. We detected nomutations and deletions on these regions that are most poten-
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tial to be recognized and cut by TALENs (Fig. 3E). These dataindicated that off target cutting might not be introduced by thedesigned TALENs in gene targeting. To detect whether thegenetic modification process can introduce other mutationsaffecting the gene function, we amplified thewhole�-hemaglo-bin gene, and by sequencing we did not find any additionalmutations (supplemental information).In Situ Gene Correction Restores the Function of HBBGene in
�-Thal-iPSC-derived HPCs and Erythroblasts—To examinewhether the correction of disease-causing mutations in �-ThaliPSCs could restore normal expression of full-length �-globin,we attempted to differentiate these iPSCs into HPCs and then
erythroiblasts. OP9 mouse bone stromal cells had been shownto efficiently induce hematopoietic differentiation of humanpluripotent cells without additional cytokines (34); thus, weemployed the OP9 co-culture system to induce the hematopoi-etic differentiation of�-Thal iPSCs.UponOP9 co-culture, both�-Thal iPSCs cell lines, no matter whether corrected or uncor-rected, could differentiate rapidly and produce HPCs that weredetected as CD34�/CD31� (Fig. 4, A and B). These �-ThaliPSC-derived HPCs could further differentiate into variousblood lineages uponplating in semisolid culture to formvariousCFU-Cs, albeit with some efficiency variations between differ-ent cell lines and batches of experiments (Fig. 4C). We then
FIGURE 2. Site-specific gene correction of the �-thalassemia mutations using TALENs. A, top panel, the recognition sequence of TALENs 600 bp down-stream of the HBB gene. Middle panel, different mutation types in the recognition sequence of TALENs. Bottom panel, schematic of the GFP reporter assay. B,fluorescence images of 293T cells transfected with GFP reporter and TALENs. C, specificity of designed TALENs. 48 h after 293T cells transfected with GFPreporter containing different TALEN recognition sequence and TALENs, cells were harvested, and the GFP fluorescence was tested by flow cytometry. The pvalues were calculated by one-way analysis of variance. *** indicates �0.001. D, schematic overview of gene targeting strategy for the human HBB locus. Thedesired recombination event inserts a PGK promoter-puromycin resistance cassette or PGK promoter-neomycin resistance cassette flanked by loxP sites (blacktriangles) into the position 600 bp downstream of �-globin locus. The Southern blot probe is indicated by an arrow (5� probe), and PCR primers are indicatedby arrows (P1, P2, P3, and P4). E, representative PCR analysis of puromycin-resistant clone (�Thal654_corrected iPS) and neomycin-resistant clone(�(�TCTT)_corrected iPS). F, Southern blot of indicated iPSCs using the 5� probe. A HBB allele that has not undergone gene targeting gives a 5-kb band,whereas a targeted allele gives a 6.4-kb band. G, top panel, sequencing results of C3 T mutation site in the second intron of HBB in �Thal654_iPS cells and�Thal654_corrected iPS cells. Arrows indicate the location of the point mutation in the patient. Bottom panel, sequencing results of TCTT deletion site in thesecond exon of HBB in �(�TCTT)_iPS cells and �(�TCTT)_corrected iPS cells. A black box indicates the location of TCTT correction in the corrected line.
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manually picked the red blood lineage colonies (CFU-E) fromthe semisolid plate and examined the expression of globin geneHBB as well as the (fetal type) HBG gene as a control by quan-titative RT-PCR. We showed that the expression of HBB geneincreased �1000-fold more in CFU-E derived from both genecorrected �-Thal iPSCs than that from the uncorrected onesand that the levels were comparable with human ES cell (H1)-derived CFU-E (Fig. 4D). To the contrary, as the control, fetaltype globin gene, HBG expressed at similar levels in CFU-Esderived from either corrected or uncorrected �Thal iPSCs aswell as the human ES cells (Fig. 4D). Lastly, by using conven-tional RT-PCR and designing primers that could amply full-lengthHBB cDNA (the forward primerwas designed in the firstexon of HBB, and the reversed primer was designed in the thirdexon of HBB), we confirmed that the expressions of full-lengthHBB cDNA were successfully restored after gene correction
(Fig. 4E). Taken together, our data demonstrated that the in situcorrection of disease-causing mutations in �-Thal iPSCsrestored the function of HBB gene in the iPSC-derived HPCsand erythroblasts.
DISCUSSION
Here, we described an efficient nonintegrating process togenerate �-thalassemia iPS cells and subsequently correctedthe disease-causing mutations using TALEN-mediated genetargeting. For the purpose of future application, we excludedc-MYC as a reprogramming factor and avoided using serumand mouse feeder cells. The nonviral approach combined withthe defined condition employed in our system could be opti-mized further for generating clinical grade and safe iPSCs. Thetwo �-Thal iPS cell lines generated in this process remain nor-mal karyotype and stable in maintaining pluripotency. More
FIGURE 3. Characterization of gene-corrected iPSC clones. A, expression of human ES markers OCT4 and SSEA4 by flow cytometry. Purple, isotype control;green, antigen staining. B, quantitative reverse transcription-PCR analysis of pluripotency related genes including OCT4, SOX2, and NANOG expression anddifferentiation related genes including SOX17, PAX6, and MXS1 expression in embryonic bodies (EBs) from the two corrected induced pluripotent stem cells.The data are presented as means � S.D. from three assays. C, normal karyotypes. D, hematoxylin/eosin staining of teratomas derived from two corrected iPSCclones. Scale bars, 100 �m. E, no insertions and deletions (INDEL) were detected in the top six potential sites that might be recognized by TALENs in the genome.
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importantly, we did not detect any transgene integration inthese cell lines, which is essential for their further application.Gene targeting mediated by traditional homologous recom-
bination in human pluripotent stem cells is particularly difficultand inefficient. The recent developed TALEN technology pro-vides great help to solve this problem by enhancing the genetargeting efficiency. TALEN-mediated gene targeting has beenreported to be successful in multiple species (21, 22, 32, 35). Acritical point of TALEN application is its specificity to cleave agivenDNA sequence. By using a reporter assay, we showed thatthe cleavage activity of the designed TALENs is highly specific.Moreover, we failed to detect mutations caused by off targetcutting by TALENs in corrected �-Thal iPS cells. In sum,TALENs technology provides an efficient and cost-effective
way for disease-causing gene correction in human pluripotentstem cells, as we have demonstrated here. Further experimentsare needed to investigate genome widely whether TALEN-based gene targeting would cause genomic instability andwhether those potential genomic changes would be hazardousto further application.
�-Thal is the most common inherited genetic disease in theworld and contains over 200 different types of mutations in�-globin gene that could cause blood disorders. For practicalpurposes, it is essential to develop an efficient and universalprocess to correct most of the common type of mutations in�-globin gene. To achieve this goal, we selected 3� downstreamof �-globin as gene targeting site, because any little changeswithin the HBB gene region would affect its normal function
FIGURE 4. Erythroblasts differentiation of �-Thal iPSCs. A, top panel, schematic representation of in vitro hematopoietic differentiation. Bottom panel, brightfield images of �Thal654_iPS cells co-cultured with OP9 at days 0, 4, and 8 and Giemsa staining of differentiated erythroblasts. Scale bars, 200 �m. B, flowcytometric analysis of �Thal654_iPS, �Thal654_corrected iPS, �(�TCTT)_iPS, and �(�TCTT)_corrected iPS using surface markers CD34 and CD31. C, number ofdifferent types of colonies counted at day 21 after differentiation. E, erythroblasts; G, granulocyte; M, megakaryocyte; GM, granulocyte and megakaryocyte;EGMM, four types of colonies including erythrocyte, granulocyte, megakaryocyte, and macrophage. D, HBB and HBG gene expression (normalized to one copyof ACTIN) in erythroblasts derived from indicated iPSCs, H1(human embryonic stem cells), and core blood CD34� cells. The values are means � S.D. for triplicatesamples from a representative experiment. The p values were calculated by one-way analysis of variance. *** indicates �0.001. E, conventional RT-PCR thatamplifies HBB cDNA in erythroblasts derived from indicated cells. cDNA from undifferentiated H1 was used as a negative control.
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(14). Then, when we constructed the donor template forhomology recombination, we included the whole wild typeHBB gene in the 5� arm. Thus, for correction of mutations in�-Thal iPSCs, this template could be used to replace the endog-enous gene with mutations or deletions in any site within theHBB region. As shown here, the gene targeting in �-Thal iPSCswith either mutations or deletions was equally efficient, dem-onstrating that this approach is universal and could beemployed for other different types of �-Thal iPSCs. Indeed, thein situ correction of disease-causing mutations in two differenttypes of �-Thal iPSCs restored the function of theHBB gene intheir derived erythroblasts. Further studies are needed to eval-uate whether hematopoietic stem cells differentiated fromthese corrected �-Thal iPS cells are functional in vivo.
Acknowledgments—We thank the members of our lab for the kindhelp.
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Shubin Chen, Xiaoxiao Zhou, Yang Chen, Duanqing Pei and Guangjin PanNing Ma, Baojian Liao, Hui Zhang, Linli Wang, Yongli Shan, Yanting Xue, Ke Huang,
-Thalassemia Induced Pluripotent Stem CellsβCorrection in Integration-free Transcription Activator-like Effector Nuclease (TALEN)-mediated Gene
doi: 10.1074/jbc.M113.496174 originally published online October 23, 20132013, 288:34671-34679.J. Biol. Chem.
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