Gene Correction by TALEN

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TALEN-mediated gene correction in integration-free -thalassemia iPSCs *Ning Ma1,2, *Baojian Liao1,2, Hui Zhang1,2, Linli Wang1,2, Yongli Shan1,2, Yanting Xue1,2,3, Ke Huang1,2, Shubin Chen1,2, Xiaoxiao Zhou1,2, Yang Chen1,2, Duanqing Pei1,2 and Guangjin Pan1,2,#

1CAS Key Laboratory of Regenerative Biology. 2Guangdong Provincial Key Laboratory of Stem cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China. 3School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China.

*N.Ma and B.Liao contributed equally to this work.

#Corresponding Author: Guangjin Pan, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. Kaiyuan Avenue Science Park, Guangzhou, 510530. Phone: 86-020-32015213. E-mail: pan_guangjin@gibh.ac.cn

Running Title: Gene Correction by TALEN Key Words: TALEN, gene correction, -thalassemia, iPS, HBB

CAPSULE:

Background: Gene editing in human patient-specific iPSCs is critical for regenerative medicine. Results: Non-integrating iPSCs corrected by TALENs display undetectable off-targets and can be differentiated into 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 and may have applications in cell therapy.

ABSTRACT

-thalassemia (-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in -globin gene (HBB). It is estimated that 4.5% of the population in the

world carry -Thal mutants (1), posing a persistent threat to public health. The generation of patient specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However, homologous recombination-based gene correction in human iPS cells remains largely inefficient. Here, we describe a robust process combining efficient generation of integration-free -Thal iPSCs from patients cells and TALEN-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN induced off-targeting mutations by sequencing. More importantly, the gene-corrected -Thal iPS cell lines from each patient can be induced to

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.496174The latest version is at JBC Papers in Press. Published on October 23, 2013 as Manuscript M113.496174

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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Gene Correction by TALEN

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differentiate into hematopoietic progenitor cells (HPCs) and then further to erythroblasts expressing normal -globin. Our studies provide an efficient and universal strategy to correct different types of -globin mutations in -Thal iPSCs for disease modeling and applications.

-thalassemia is a group of inherited genetic blood disorders caused by either point mutations or deletions of nucleotides in -globin gene. These genetic defects result in reduced, abnormal or no synthesis of -globin chains that make up hemoglobin. It is one of the most common genetic diseases in the world and patients with -Thal have severe anemia and a shortened life span (2). Hematopoietic stem cell (HSC) transplantation is the only way to cure - thalassemia but is challenged by the limited availability of HLA-matched healthy donors. Recently, the development of gene therapy based on viral transduction of a normal HBB gene into patients own HSC raised hopes for those who do not have access to bone marrow transplantation (3,4). However, it was still challenged by the concerns of long-term safety because of the viral integration. Patients somatic cells can be reprogrammed back into pluripotent state as iPS cells that are capable of differentiation into any cells in the body, thus could be potentially used for cell replacement therapy (5-8). Generation iPSCs from -Thal patients, correcting mutations and subsequent differentiation into HSCs raise great hopes for autologous transplantation to treat these inherited diseases (9). Moreover, iPS cells can undergo indefinite self-renewal without losing the ability to differentiate into all cell types, thus represent an ideal cell population for in situ correction of the disease-causing mutations. However, gene targeting in human pluripotent stem cells by standard homologous recombination (HR) is largely inefficient (10) and therefore hampers its extensive application in disease models. Zinc

finger nucleases (ZFNs) had been reported to substantially enhance the homologous recombination efficiency by specifically introducing a double strand DNA break at the target locus (11-13). Gene targeting aided by ZFN in human iPS or ES cells had been described to be highly efficient (14-16). However, engineering ZFNs for a specific target is quite inefficient and laborious, which largely hampers their widespread adoption. Recently, transcription activator like effector nucleases (TALENs) had been described to recognize and cleave any given DNA sequences with high efficiency (17-19). The DNA binding domain of TALEN is unusual and contains multiple units that arranged in tandem (TALE repeats). Each individual unit is composed of 34 amino acids with two highly variable amino acids to determine the unit to recognize one DNA pair in the TALEN recognizing sequence (20). In theory, TALE repeat could be engineered and arranged to specifically recognize any given DNA sequence. TALEN mediated gene targeting had been described in multiple species, including zebrafish, human iPS and ES cells (21,22). Practically, compared with ZFN, TALEN is much more easy and convenient regarding the designing and constructing. Besides, TALENs exhibited lower off-target effects and reduced nuclease associated cytotoxicities compared with ZFNs (23-25). In attempt to extend TALEN technology to gene correction for -Thal, we generated the -Thal iPS cells through non-viral approach and developed an efficient process to correct the mutations in -globin gene by designing and utilizing site specific TALENs.

EXPERIMENTAL PROCEDURES

iPS generation The method of isolating amniotic fluid cells was

performed as previously described (26). For reprogramming, an oriP/EBNA1-based pCEP4

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episomal vector containing OCT4, SOX2, KLF4 and SV40LT genes (27) and miR-302-367 (28) were cotransfected into amniotic fluid cells via nucleofection (Amaxa). The cells were then plated to matrigel-coated 6 well plates and cultured with reprogramming medium (mTeSR1). The medium was changed every two days and iPS-like colonies were picked onto new matrigel plate for characterization. Cells of passages from 15 to 40 are used for the following experiments. TALEN and donor vectors for gene targeting

TALENs were designed as described (17,29). The full amino acid sequences of TALENs are given in supplementary information. For donor DNA, left and right homology arms were amplified from genomic DNA of healthy individual. A loxP-flanked PGK-puromycin cassette or loxP-flanked PGK-neomycin cassette were cloned between two homology arms in the pMD-18T vector. For targeting, 1x106 iPSCs were electroporated with 2 g of donor DNA and 4.5 g of each TALEN plasmid. Then the cells were plated onto matrigel-coated 6 well plates in the presence of Y-27632 (10 M, Sigma) for 1 day. Positive clones were selected by puromycin (0.5 g/ml) or G418 (100 g/ml, Sigma) in mTeSR1. The selected colonies were verified by genomic PCR and Southern blot. All primers used are listed in Supplementary Table1. GFP reporter assay

GFP reporter activation was tested by co-transfecting 293T cells with plasmids carrying TALENs and GFP reporters. 293T cells were seeded into 12 well plates the day before transfection. Approximately 24 hour after initial seeding, cells were transfected using Calcium phosphate. For 12-well plates we used 1.5 g of each TALEN and 1g of reporter plasmids per well. Cells were trypsinized from their culturing plates 48 h after transfection and resuspended in 800 l of PBS for flow cytometry analysis. The

flow cytometry data were analyzed using C6 (BD Biosciences). At least 20,000 events were analyzed for each transfection sample. PCR detection of corrected clones

PCR was performed using High Fidelity Platinum Taq (Invitrogen) according to the manufacturers instructions. 50-100 ng of genomic DNA templates were used in all reactions. Primer set including P1 (on HBB locus, upstream of 5 homology arm) and P2 (in the drug resistance cassette) was used to amplify a 2.8-kb product of the 5 junction of a targeted integration (illustrated in Figure 2D). Primer set including P3 (on 5 homology arm) and P4 (3 homology arm) was used to amplify a 2-kb product or a 500 bp product to identify whether random integration occurred (Figure 2D). Primer IVS-654 and primer EXO-3-15 were used to amplify a 600bp product including the mutant region of Thal654_iPS and the PCR products were sequenced to