Androulla N Miliotou, Dionysia Papagiannopoulou, Efthymia Vlachaki, Martina Samiotaki, Dimitra Laspa, Stamatia Theodoridou, Asterios S Tsiftsoglou, Lefkothea C Papadopoulou
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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.</p><p><strong>Results: </strong>Cloning of the α-globin coding sequence, fused to the nucleotide sequence of TAT peptide was conducted and the human recombinant fusion proteins, 10xHis-Xa<sub>SITE</sub>-α-globin-HA and 10xHis-Xa<sub>SITE</sub>-TAT-α-globin-HA were produced. The ability of human recombinant 10xHis-Xa<sub>SITE</sub>-α-globin-HA to interact in vitro with the previously produced 10xHis-Xa<sub>SITE</sub>-TAT-β-globin-HA and form α-/β-globin heterodimers, was assessed and confirmed by size exclusion chromatography. The recombinant 10xHis-Xa<sub>SITE</sub>-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 β<sub>4</sub>-globin chain tetramers.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":50251,"journal":{"name":"Journal of Biological Research-Thessaloniki","volume":null,"pages":null},"PeriodicalIF":1.9000,"publicationDate":"2021-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40709-021-00148-3","citationCount":"1","resultStr":"{\"title\":\"PTD-mediated delivery of α-globin chain into Κ-562 erythroleukemia cells and α-thalassemic (HBH) patients' RBCs ex vivo in the frame of Protein Replacement Therapy.\",\"authors\":\"Androulla N Miliotou, Dionysia Papagiannopoulou, Efthymia Vlachaki, Martina Samiotaki, Dimitra Laspa, Stamatia Theodoridou, Asterios S Tsiftsoglou, Lefkothea C Papadopoulou\",\"doi\":\"10.1186/s40709-021-00148-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>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). 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引用次数: 1
摘要
背景:α-地中海贫血是一种先天性血红蛋白病,在严重的α-地中海贫血(HbH病/Hb Bart's)中以α-珠蛋白链缺乏和/或水平降低为特征。这项研究工作涉及蛋白质替代疗法的方法,以管理α-地中海贫血的表现,过量的β-珠蛋白链进入HbH红细胞。主要目的是制备与TAT(一种蛋白质转导结构域)融合的重组人α-珠蛋白链,并将其体外输送到HbH患者的红细胞中,以取代内源性缺失的α-珠蛋白链。结果:克隆α-珠蛋白编码序列,与TAT肽核苷酸序列融合,获得重组融合蛋白10xHis-XaSITE-α-globin-HA和10xHis-XaSITE-TAT-α-globin-HA。人重组10xHis-XaSITE-α-球蛋白- ha与先前制备的10xHis-XaSITE- tat -β-球蛋白- ha在体外相互作用并形成α-/β-球蛋白异源二聚体的能力,通过尺寸排斥层析进行评估和确认。在初步转导评估实验中,重组蛋白10xHis-XaSITE-TAT-α-球蛋白- ha成功转染人原红细胞K-562细胞。最后,重组的、融合了tat的α-珠蛋白被成功地转导到来自HbH患者的红细胞中,并减少了已知含有有害的β4-珠蛋白链四聚体的HbH包涵体的形成。结论:我们的数据证实了重组α-珠蛋白链在HbH红细胞中的成功体外转导,以取代缺失的a-珠蛋白链并减少HbH包涵体,这在α-地中海贫血中可见。这些发现拓宽了通过PTD技术,利用重组α-珠蛋白链,将蛋白质替代疗法应用于不同形式α-地中海贫血的可能性。
PTD-mediated delivery of α-globin chain into Κ-562 erythroleukemia cells and α-thalassemic (HBH) patients' RBCs ex vivo in the frame of Protein Replacement Therapy.
Background: 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.
Results: 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.
Conclusions: 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.
期刊介绍:
Journal of Biological Research-Thessaloniki is a peer-reviewed, open access, international journal that publishes articles providing novel insights into the major fields of biology.
Topics covered in Journal of Biological Research-Thessaloniki include, but are not limited to: molecular biology, cytology, genetics, evolutionary biology, morphology, development and differentiation, taxonomy, bioinformatics, physiology, marine biology, behaviour, ecology and conservation.