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

Abstract

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 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.

Keywords: HbH thalassemic patients’ RBCs; Intracellular transduction via PTD; LC − MS/MS analysis; Size exclusion chromatography; TAT-α-globin.

Fig. 1

Schematic illustration of the cloning procedure, starting with RT-PCR from placenta tissue, derived from a healthy individual, followed by TA cloning of the a-globin CDS and then TA cloning of amplified TAT-α-globin-HA and α-globin-HA into pCRII-TOPO vector. Then, «sticky ends» cloning was conducted, with the restriction enzymes NdeI and XhoI, to generate the recombinant expression vectors pET-16b-TAT-α-globin-HA and pET-16b-α-globin-HA

Fig. 2

Western Blot of recombinant protein extracts, separated on 15% SDS-PAGE, transferred and immunoblotted with anti-His.IgG. A The bacterial-IBs’ protein extracts (in 1 M l-Arg, pH 8.0). Lane 1: protein molecular mass marker; Lane 2: 10xHis-Xa_SITE-TAT-α-globin-HA (~ 20.2 kDa); Lane 3: protein molecular mass marker; Lane 4: 10xHis-Xa_SITE-α-globin-HA (~ 18.7 kDa); B The soluble-purified by Ni²⁺-NTA chromatography-recombinant proteins, eluted with 10 mM imidazole. Lane 1: protein molecular mass marker; Lane 2: 10xHis-Xa_SITE-TAT-α-globin-HA (~ 20.2 kDa); Lane 3: 10xHis-Xa_SITE-α-globin-HA (~ 18.7 kDa). Higher bands at 29—41 kDa indicate the corresponding protein dimers, while lower bands may indicate proteolysis products

Fig. 3

Retrieval of recombinant α-globin fusion protein variants purified by affinity Ni²⁺-NTA chromatography under denaturing conditions and analyzed by 15% SDS − PAGE and Coomassie Blue staining: A SDS-PAGE analysis of the purification of 10xHis-Xa_SITE-α-globin-HA protein (~ 18.7 kDa). Lane 1: Crude extract of induced bacterial cells transformed by pET-16b-α globin-HA; Lane 2: Flow-through fraction of the Ni²⁺-NTA affinity column; Lane 3: Wash Buffer extract with 10 mM imidazole; Lane 4: Elution with 20 mM imidazole; Lane 5: Elution with 40 mM imidazole; Lane 6: protein molecular mass marker; Lane 7: Elution with 100 mM imidazole; Lane 8: Elution with 200 mM imidazole; Lane 9: Elution with 400 mM imidazole. B SDS-PAGE analysis of the purification of 10xHis-Xa_SITE-TAT-α-globin-HA protein (~ 20.2 kDa). Lane 1: Crude extract of induced bacterial cells transformed by pET-16b-TAT-α globin-HA; Lane 2: Flow-through fraction of the Ni²⁺-NTA affinity column; Lane 3: Wash Buffer extract with 10 mM imidazole; Lane 4: Elution with 20 mM imidazole; Lane 5: Elution with 40 mM imidazole; Lane 6: Elution with 100 mM imidazole; Lane 7: Elution with 200 mM imidazole; Lane 8: Elution with 400 mM imidazole; Lane 9: protein molecular mass marker

Fig. 4

Identification of recombinant 10xHis-Xa_SITE-TAT-α-globin-HA (TAT_HBA_HA) protein construct by LC − MS/MS. The highlighted peptides confidently identified cover 44.75% of the theoretical construct sequence

Fig. 5

Size exclusion HPLC chromatograms: A hemoglobin tetramer (64.5 kDa) (t_R = 21.1 min); B soluble (in PBS 1 ×) sample protein (∼36 kDa) (t_R = 23.628 min); C soluble monomer 10xHis-Xa_SITE-TAT-β-globin-HA (∼20.6 kDa), prior incubated with DTT for 48 h (t_R = 25.6 min); D soluble monomer 10xHis-Xa_SITE-α-globin-HA (∼18.7 kDa), prior incubated with DTT for 48 h (t_R = 25.6 min)

Fig. 6

Size exclusion HPLC chromatograms of reaction mixtures of α- and β-monomers at t = 0 h, t = 24 h and t = 48 h

Fig. 7

Western Blot of electrophoresed cell lysates derived from K-562 cells, transduced with 50 μg ml⁻¹ soluble 10xHis − Xa_SITE − TAT − α-globin − HA, immunoblotted using anti-HA.IgG. Lane 1: protein molecular mass marker; Lane 2: lysate from control (untreated) cells, 30 min; Lane 3: lysate from cells transduced with 10xHis − Xa_SITE − TAT − α-globin − HA, incubated for 30 min.; Lane 4: lysate from control (untreated) cells, 1 h; Lane 5: lysate from cells transduced with 10xHis − Xa_SITE − TAT − α-globin − HA, incubated for 1 h

Fig. 8

Western Blot of electrophoresed cell lysates derived from HbH patients’ peripheral RBCs (Patient 3), transduced with bacterial-IBs, enriched in 10xHis-Xa_SITE-TAT-α-globin-HA, immunoblotted using anti-His.IgG and anti-GAPDH.IgG. Patient 3: Lane 1: protein molecular mass marker; Lane 2: lysate from control (untreated) cells, 48 h; Lane 3: lysate from cells incubated with 1 M l-Arg, for 48 h; Lanes 4–6: lysate from cells transduced with bacterial-IBs, enriched in 10xHis-Xa_SITE-TAT-α-globin-HA, incubated for 48 h

Fig. 9

Methylene violet staining for the evaluation of HbH-IBs’ reduction, in HbH RBCs upon incubation with 10xHisXa_SITE-TAT-α-globin-HA. Patient 2: A Untreated HbH patient’s RBCs, and B HbH patient’s RBCs incubated with IBs, enriched in 10xHis-Xa_SITE-TAT-α-globin-HA, for 48 h. Patient 3: A Untreated HbH patient’s RBCs, B HbH patient’s RBCs incubated with 1 M l-Arg, for 48 h, C HbH patient’s RBCs incubated with IBs, enriched in 10xHis-Xa_SITE-TAT-α-globin-HA, for 48 h

Fig. 10

Diagrams for % positive for HbH-IBs in RBCs, derived from HbH patients (Patients 1–4), which were reduced after incubation with 10xHis-Xa_SITE-TAT-α-globin-HA, for 48 h