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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 manifesta...

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Autores principales: Miliotou, Androulla N., Papagiannopoulou, Dionysia, Vlachaki, Efthymia, Samiotaki, Martina, Laspa, Dimitra, Theodoridou, Stamatia, Tsiftsoglou, Asterios S., Papadopoulou, Lefkothea C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8290593/
https://www.ncbi.nlm.nih.gov/pubmed/34284828
http://dx.doi.org/10.1186/s40709-021-00148-3
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author Miliotou, Androulla N.
Papagiannopoulou, Dionysia
Vlachaki, Efthymia
Samiotaki, Martina
Laspa, Dimitra
Theodoridou, Stamatia
Tsiftsoglou, Asterios S.
Papadopoulou, Lefkothea C.
author_facet Miliotou, Androulla N.
Papagiannopoulou, Dionysia
Vlachaki, Efthymia
Samiotaki, Martina
Laspa, Dimitra
Theodoridou, Stamatia
Tsiftsoglou, Asterios S.
Papadopoulou, Lefkothea C.
author_sort Miliotou, Androulla N.
collection PubMed
description 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-Xa(SITE)-α-globin-HA and 10xHis-Xa(SITE)-TAT-α-globin-HA were produced. The ability of human recombinant 10xHis-Xa(SITE)-α-globin-HA to interact in vitro with the previously produced 10xHis-Xa(SITE)-TAT-β-globin-HA and form α-/β-globin heterodimers, was assessed and confirmed by size exclusion chromatography. The recombinant 10xHis-Xa(SITE)-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(4)-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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40709-021-00148-3.
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spelling pubmed-82905932021-07-21 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 Miliotou, Androulla N. Papagiannopoulou, Dionysia Vlachaki, Efthymia Samiotaki, Martina Laspa, Dimitra Theodoridou, Stamatia Tsiftsoglou, Asterios S. Papadopoulou, Lefkothea C. J Biol Res (Thessalon) Research 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-Xa(SITE)-α-globin-HA and 10xHis-Xa(SITE)-TAT-α-globin-HA were produced. The ability of human recombinant 10xHis-Xa(SITE)-α-globin-HA to interact in vitro with the previously produced 10xHis-Xa(SITE)-TAT-β-globin-HA and form α-/β-globin heterodimers, was assessed and confirmed by size exclusion chromatography. The recombinant 10xHis-Xa(SITE)-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(4)-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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40709-021-00148-3. BioMed Central 2021-07-20 /pmc/articles/PMC8290593/ /pubmed/34284828 http://dx.doi.org/10.1186/s40709-021-00148-3 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Miliotou, Androulla N.
Papagiannopoulou, Dionysia
Vlachaki, Efthymia
Samiotaki, Martina
Laspa, Dimitra
Theodoridou, Stamatia
Tsiftsoglou, Asterios S.
Papadopoulou, Lefkothea C.
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
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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8290593/
https://www.ncbi.nlm.nih.gov/pubmed/34284828
http://dx.doi.org/10.1186/s40709-021-00148-3
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