Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation.
Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation.
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DOI：
10.1002/stem.1969
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发表时间：
2015-05
期刊：
影响因子：
--
通讯作者：
Cheng L
中科院分区：
文献类型：
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作者：
Huang X;Wang Y;Yan W;Smith C;Ye Z;Wang J;Gao Y;Mendelsohn L;Cheng L
人类诱导多能干细胞（iPSC）和基因组编辑提供了一种精确的方法来生成用于疾病建模和细胞治疗的基因校正细胞。从镰状细胞病（SCD）患者产生的人iPSC在编码成人β-珠蛋白的HBB基因中具有纯合错义点突变，并且用作模型系统以改进人基因治疗的策略。我们证明了CRISPR/Cas9系统设计者核酸酶在刺激人iPSC中SCD点突变附近的内源性HBB基因座的基因靶向方面比ZFN和TALEN有效得多。使用特异性引导RNA和Cas9，我们通过与含有野生型HBB DNA的供体DNA模板和随后被移除以避免HBB转录和翻译的可能干扰的选择盒的同源重组，容易地校正了人iPSC中SCD HBB基因的一个等位基因。我们选择具有一个校正的和一个破坏的SCD等位基因的靶向iPSC克隆用于红系分化测定，使用我们最近建立的改进的无异种和无饲养层的培养条件。来自校正的或其亲本（未校正的）iPSC系的红细胞以相似的效率产生。目前，约6%-10%的这些分化的红细胞确实缺乏细胞核，这是称为网织红细胞的进一步成熟的红细胞的特征。我们还检测了从基因组编辑的iPSC分化的红细胞中校正的HBB等位基因表达的16-kD β-珠蛋白蛋白。我们的研究结果代表了使用患者来源的iPSC进行基因组编辑的临床应用的重要一步，以产生用于细胞和基因治疗的无病细胞。
Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than ZFNs and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ~6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kD β-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step towards the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies.