One-Step Biallelic and Scarless Correction of a β-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection.
One-Step Biallelic and Scarless Correction of a β-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection.
复制标题
无需药物选择即可一步双等位基因和无疤痕校正患者特异性 iPSC 中的 β-地中海贫血突变。
DOI：
10.1016/j.omtn.2016.11.010
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发表时间：
2017-03-17
期刊：
影响因子：
--
通讯作者：
Fan Y
中科院分区：
文献类型：
--
作者：
Liu Y;Yang Y;Kang X;Lin B;Yu Q;Song B;Gao G;Chen Y;Sun X;Li X;Bu L;Fan Y
单基因突变引起的单基因疾病严重影响人类健康。其中，地中海贫血(β-地中海贫血，β-Thal)是由人类血红蛋白β(HbB)基因突变引起的最常见的遗传性血液病之一。诱导多能干细胞(IPSCs)和遗传矫正技术为包括β-Thal在内的MGDS的治疗提供了见解。然而，传统的纠正突变的方法效率低，并且会留下残留的足迹，这导致了临床应用中的一些安全问题。作为概念验证，我们利用单链寡核苷酸、高保真的CRISPR/Cas9核酸酶和小分子实现了对β地中海贫血患者特异性IPSCs中β-41/42(TCTT)缺失突变的无缝纠正，效率显著。此外，脱靶分析和全外显子组测序结果显示，校正后的细胞表现出最小的突变负荷，没有脱靶突变。当分化为造血祖细胞(HPC)，然后进一步分化为红细胞时，经过基因修正的细胞表达正常的β-珠蛋白转录本。我们的研究为IPSCs中β-41/42(TCTT)缺失的基因纠正提供了最有效和安全的方法，为进一步的β-Thal的潜在细胞治疗提供了一条潜在的治疗途径，为患者特定的IPSCMGD相关突变的基因纠正提供了一条潜在的治疗途径。
Monogenic disorders (MGDs), which are caused by single gene mutations, have a serious effect on human health. Among these, β-thalassemia (β-thal) represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin β (HBB) gene. The technologies of induced pluripotent stem cells (iPSCs) and genetic correction provide insights into the treatments for MGDs, including β-thal. However, traditional approaches for correcting mutations have a low efficiency and leave a residual footprint, which leads to some safety concerns in clinical applications. As a proof of concept, we utilized single-strand oligodeoxynucleotides (ssODNs), high-fidelity CRISPR/Cas9 nuclease, and small molecules to achieve a seamless correction of the β-41/42 (TCTT) deletion mutation in β thalassemia patient-specific iPSCs with remarkable efficiency. Additionally, off-target analysis and whole-exome sequencing results revealed that corrected cells exhibited a minimal mutational load and no off-target mutagenesis. When differentiated into hematopoietic progenitor cells (HPCs) and then further to erythroblasts, the genetically corrected cells expressed normal β-globin transcripts. Our studies provide the most efficient and safe approach for the genetic correction of the β-41/42 (TCTT) deletion in iPSCs for further potential cell therapy of β-thal, which represents a potential therapeutic avenue for the gene correction of MGD-associated mutants in patient-specific iPSCs.