Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells.
Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells.
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DOI：
10.1016/j.stemcr.2015.10.002
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发表时间：
2015-12-08
影响因子：
5.9
通讯作者：
Segovia JC
中科院分区：
文献类型：
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作者：
Garate Z;Quintana-Bustamante O;Crane AM;Olivier E;Poirot L;Galetto R;Kosinski P;Hill C;Kung C;Agirre X;Orman I;Cerrato L;Alberquilla O;Rodriguez-Fornes F;Fusaki N;Garcia-Sanchez F;Maia TM;Ribeiro ML;Sevilla J;Prosper F;Jin S;Mountford J;Guenechea G;Gouble A;Bueren JA;Davis BR;Segovia JC
丙酮酸激酶缺乏症（PKD）是一种罕见的红系代谢性疾病，由PKLR基因突变引起。来自PKD患者的红细胞显示能量不平衡，导致慢性非球形红细胞溶血性贫血，因为丙酮酸激酶缺陷损害红细胞中的ATP产生。我们通过非整合仙台病毒载体从PKD患者的外周血单个核细胞（PB-MNCs）产生PKD诱导的多能干细胞（PKDiPSC）。对PKDiPSC进行基因编辑，以通过TALEN介导的同源重组（HR）将部分密码子优化的R型丙酮酸激酶cDNA整合到PKLR基因的第二内含子中。值得注意的是，我们发现等位基因特异性的HR导致的单核苷酸多态性的存在。来源于基因编辑的PKDiPSC的大量红系细胞显示出能量失衡的校正，提供了一种校正代谢性红系疾病的方法，并证明了这种方法产生全面的生化和代谢性红系分析所需的大量细胞的实用性。通过非整合系统从PB-MNC产生患者特异性PKDiPSC对PKDiPSC进行基因编辑，以在TALEN介导的PKLR基因座中插入部分共RPK同源臂中的SNP导致等位基因特异性同源重组基因编辑的PKDiPSC产生大量代谢校正的红系细胞患者特异性诱导多能干细胞（iPSC）是研究红细胞代谢疾病和测试创新疗法的完美平台。Segovia，Quintana-Bustamante及其同事通过将iPSC和基因编辑技术相结合来纠正丙酮酸激酶缺乏症（PKD），并提供了一种方法来产生这种疾病及其治疗的全面生化和代谢分析所需的大量红系细胞。
Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses. Patient-specific PKDiPSCs are generated from PB-MNCs by a non-integrative system PKDiPSCs are gene edited to insert a partial co-RPK in the PKLR locus mediated by TALEN An SNP in the homology arm leads to allele-specific homologous recombination Gene-edited PKDiPSCs generate a high number of metabolically corrected erythroid cells Patient-specific induced pluripotent stem cells (iPSCs) are the perfect platform to study erythroid metabolic diseases and test innovative treatments. Segovia, Quintana-Bustamante, and colleagues showed the correction of pyruvate kinase deficiency (PKD) by combining iPSC and gene-editing technologies and provide an approach to generate the large number of erythroid cells required for comprehensive biochemical and metabolic analyses of this disease and its treatment.