α-Thalassemia, a congenital hemoglobinopathy, is characterized by deficiency and/or reduced levels of α-globin chains in serious forms of α-thalassemia (HbH disease/Hb Bart’s). This research work deals with a Protein Replacement Therapy approach in order to manage α-thalassemia manifestations, caused by the excess of β-globin chain into HbH RBCs. The main goal was to produce the recombinant human α-globin chain in fusion with TAT, a Protein Transduction Domain, to ex vivo deliver it into HbH patients RBCs, to replace the endogenous missing α-globin chain. Cloning of the α-globin coding sequence, fused to the nucleotide sequence of TAT peptide was conducted and the human recombinant fusion proteins, 10xHis-XaSITE-α-globin-HA and 10xHis-XaSITE-TAT-α-globin-HA were produced. The ability of human recombinant 10xHis-XaSITE-α-globin-HA to interact in vitro with the previously produced 10xHis-XaSITE-TAT-β-globin-HA and form α-/β-globin heterodimers, was assessed and confirmed by size exclusion chromatography. The recombinant 10xHis-XaSITE-TAT-α-globin-HA was successfully delivered into human proerythroid K-562 cells, during the preliminary transduction evaluation experiments. Finally, the recombinant, TAT-fused α-globin was successfully transduced into RBCs, derived from HbH patients and reduced the formation of HbH-Inclusion Bodies, known to contain harmful β4-globin chain tetramers. Our data confirm the successful ex vivo transduction of recombinant α-globin chains in HbH RBCs to replace the missing a-globin chain and reduce the HbH-inclusion bodies, seen in α-thalassemias. These findings broaden the possibility of applying a Protein Replacement Therapy approach to module sever forms of α-thalassemia, using recombinant α-globin chains, through PTD technology.

中文翻译：

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在蛋白质替代疗法的框架内，PTD 介导的 α-珠蛋白链递送到 Κ-562 红白血病细胞和 α-地中海贫血 (HBH) 患者的红细胞中

α-地贫是一种先天性血红蛋白病，其特征是严重形式的α-地贫（HbH 病/Hb Bart's）中α-珠蛋白链缺乏和/或水平降低。这项研究工作涉及蛋白质替代治疗方法，以管理由 β-珠蛋白链过量进入 HbH RBC 引起的 α-地贫表现。主要目标是生产与 TAT（一种蛋白质转导结构域）融合的重组人 α-珠蛋白链，将其离体递送到 HbH 患者的红细胞中，以替代内源性缺失的 α-珠蛋白链。进行与 TAT 肽核苷酸序列融合的 α-珠蛋白编码序列的克隆，并产生人重组融合蛋白 10xHis-XaSITE-α-珠蛋白-HA 和 10xHis-XaSITE-TAT-α-珠蛋白-HA。人重组 10xHis-XaSITE-α-珠蛋白-HA 在体外与先前生产的 10xHis-XaSITE-TAT-β-珠蛋白-HA 相互作用并形成 α-/β-珠蛋白异二聚体的能力，通过尺寸排阻进行了评估和确认色谱法。在初步转导评估实验期间，重组 10xHis-XaSITE-TAT-α-珠蛋白-HA 成功递送到人类原红细胞 K-562 细胞中。最后，重组的、TAT 融合的 α-珠蛋白被成功转导到来自 HbH 患者的红细胞中，并减少了 HbH 包涵体的形成，已知包含有害的 β4-珠蛋白链四聚体。我们的数据证实了 HbH 红细胞中重组 α-珠蛋白链的成功离体转导，以取代缺失的 α-珠蛋白链并减少在 α-地中海贫血中看到的 HbH 包涵体。