Transcription Activator-like Effector Nuclease (TALEN)-mediated Gene Correction in Integration-free β-Thalassemia Induced Pluripotent Stem Cells

Ma, Ning; Liao, Baojian; Zhang, Hui; Wang, Linli; Shan, Yongli; Xue, Yanting; Huang, Ke; Chen, Shubin; Zhou, Xiaoxiao; Yang, Chen; Pei, Duanqing; Pan, Guoshun

doi:10.1074/jbc.m113.496174

Cited by 151 publications

(124 citation statements)

References 36 publications

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“…5E). Consistent with several reports (Chang et al 2006Zou et al 2011;Ma et al 2013), we also found that erythrocyte differentiation from iPSCs preferentially expressed fetal globin (hemoglobin gamma) more than 100 times greater than adult globin (Fig. 5E) even after gene correction, indicating the need for further research on improved culture conditions to foster erythrocyte development and adult globin-switching.…”

Section: Expression Of Hbb In Erythroblast Differentiation From Patiesupporting

confidence: 77%

“…In addition, their efficiency could be hampered by DNA methylation and histone acetylation in inactive chromatin (Miller et al 2007;Mussolino et al 2011;Sander et al 2011;Wood et al 2011;Sanjana et al 2012). In contrast, the Cas9 nuclease is not limited by these obstacles and is readily available, and the guide RNA containing 20 nt of homologous sequences is easy to synthesize onto the tracrRNA (Deveau et al 2010;Deltcheva et al 2011;Gasiunas et al 2012;Jinek et al 2012;Cong et al 2013;Hsu et al 2013;Ma et al 2013). The piggyBac construct with homologous genomic sequences on each side of the selection cassette allowed positive selection of homologous recombination events that corrected the two b-thalassemia mutations.…”

Section: Discussionmentioning

confidence: 99%

“…Site-specific DSBs can be achieved by introducing engineered nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), as well as the recently developed RNA-directed Cas9 nucleases (Miller et al 2007Cong et al 2013;Mali et al 2013). Corrections of the HBB mutation in b-thalassemia and sickle cell anemia using TALENs to introduce DSBs have been reported (Ma et al 2013;Sun and Zhao 2013).…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

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Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac

et al. 2014

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β-thalassemia, one of the most common genetic diseases worldwide, is caused by mutations in the human hemoglobin beta (HBB) gene. Creation of human induced pluripotent stem cells (iPSCs) from β-thalassemia patients could offer an approach to cure this disease. Correction of the disease-causing mutations in iPSCs could restore normal function and provide a rich source of cells for transplantation. In this study, we used the latest gene-editing tool, CRISPR/Cas9 technology, combined with the piggyBac transposon to efficiently correct the HBB mutations in patient-derived iPSCs without leaving any residual footprint. No off-target effects were detected in the corrected iPSCs, and the cells retain full pluripotency and exhibit normal karyotypes. When differentiated into erythroblasts using a monolayer culture, gene-corrected iPSCs restored expression of HBB compared to the parental iPSCs line. Our study provides an effective approach to correct HBB mutations without leaving any genetic footprint in patient-derived iPSCs, thereby demonstrating a critical step toward the future application of stem cell-based gene therapy to monogenic diseases.

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Section: Expression Of Hbb In Erythroblast Differentiation From Patiesupporting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

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Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac

et al. 2014

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“…Recent studies have shown that personalized iPSCs can be derived from b-thalassemia patient fibroblasts via the induction of transcription factors, and the mutations can be corrected using a transcription activator-like effector nuclease or clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 nuclease (CRISPR/Cas9) system [21][22][23]. However, these primed-state iPSCs derived from b-thalassemia patient fibroblasts have shown extremely low levels of single cell cloning efficiencies, thus impairing the subsequent targeting efficiencies.…”

Section: Introductionmentioning

confidence: 99%

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Yang

Zhang

et al. 2015

Stem Cells Translational Medicine

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Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self-renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with b-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross-species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene-correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:8-19 SIGNIFICANCEIn the present study, transgene-free naïve induced pluripotent stem cells (iPSCs) directly converted from the fibroblasts of a patient with b-thalassemia in a defined culture system were generated. These naïve iPSCs, which show ground-state pluripotency, exhibited significantly improved singlecell cloning ability, recovery capacity, and gene-targeting efficiency compared with conventional primed iPSCs. These results provide an improved strategy for personalized treatment of genetic diseases such as b-thalassemia.

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“…Induced pluripotent stem cells (iPSCs) derived from patient somatic cells, which can selfrenew indefinitely without losing the ability to differentiate into all cell types (5-9) and hold great promise for regenerative medicine, represent an ideal cell population for in situ correction of disease-causing mutations (10,11). Generation of ␤-Thal patient iPSCs, correction of the mutations housed in those iPSCs, and subsequent differentiation into HSCs offer an opportunity for autologous transplantation for disease treatment (12)(13)(14)(15)(16)(17)(18). Recently, the development of gene editing to correct the HBB mutation in ␤-Thal iPSCs followed by differentiation of the corrected iPSCs into HSCs offered a new therapeutic option for those who do not have a bone marrow match to that of potential donors (12,16,17).…”

mentioning

confidence: 99%

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells

Niu

Song

et al. 2016

Journal of Biological Chemistry

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␤-Thalassemia (␤-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific ␤-Thalinduced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin ␤ (HBB) gene CD41/ 42(؊CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in ␤-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in ␤-Thal iPSCs.

show abstract

Transcription Activator-like Effector Nuclease (TALEN)-mediated Gene Correction in Integration-free β-Thalassemia Induced Pluripotent Stem Cells

Cited by 151 publications

References 36 publications

Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac

Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells

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