Pyruvate Kinase Deficiency is an autosomal recessive disorder caused by mutations in the PKLR gene, causing chronic non-spherocytic hemolytic anemia. PKLR gene encodes for the erythroid pyruvate kinase protein (RPK) implicated in the last step of the anaerobic glycolysis in erythrocytes. The defective enzyme fails to maintain normal ATP levels, producing severe hemolytic anemia. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus. We combined CRISPR/Cas9 system and rAAVs to build an efficient and safe system to knock-in a therapeutic donor in the RPK isoform of human hematopoietic progenitors (HSPCs). Edited cells efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient recipients. Moreover, erythroid cells derived from edited PKD-HSPCs restored normal levels of ATP, concluding that our gene editing strategy may represent a lifelong therapy to restore RPK functionality in RBCs of patients and correct PKD.

中文翻译：

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造血干细胞治疗丙酮酸激酶缺乏症溶血性贫血的临床相关基因编辑

丙酮酸激酶缺乏症是一种由PKLR基因突变引起的常染色体隐性遗传疾病，可引起慢性非球囊性溶血性贫血。PKLR基因编码在红细胞中厌氧糖酵解的最后一步中涉及的类胡萝卜素丙酮酸激酶蛋白（RPK）。有缺陷的酶无法维持正常的ATP水平，从而导致严重的溶血性贫血。在这里，我们通过在PKLR内源基因座进行精确的基因编辑来解决PKD的校正问题。我们将CRISPR / Cas9系统和rAAV结合在一起，构建了一个有效且安全的系统，以敲除人类造血祖细胞（HSPC）RPK亚型中的治疗性供体。编辑的细胞在原发性和继发性免疫缺陷受体中有效地重建了人类造血功能。此外，源自编辑过的PKD-HSPC的类红细胞恢复了ATP的正常水平，