Production and Transduction of a Human Recombinant β-Globin Chain into Proerythroid K-562 Cells To Replace Missing Endogenous β-Globin

Abstract

Protein replacement therapy (PRT) has been applied to treat severe monogenetic/metabolic disorders characterized by a protein deficiency. In disorders where an intracellular protein is missing, PRT is not easily feasible due to the inability of proteins to cross the cell membrane. Instead, gene therapy has been applied, although still with limited success. β-Thalassemias are severe congenital hemoglobinopathies, characterized by deficiency or reduced production of the adult β-globin chain. The resulting imbalance of α-/β-globin chains of adult hemoglobin (α 2 β 2 ) leads to precipitation of unpaired α-globin chains and, eventually, to defective erythropoiesis. Since protein transduction domain (PTD) technology has emerged as a promising therapeutic approach, we produced a human recombinant β-globin chain in fusion with the TAT peptide and successfully transduced it into human proerythroid K-562 cells, deficient in mature β-globin chain. Notably, the produced human recombinant β-globin chain without the TAT peptide, used as internal negative control, failed to be transduced into K-562 cells under similar conditions. In silico studies complemented by SDS−PAGE, Western blotting, co-immunoprecipitation and LC−MS/MS analysis indicated that the transduced recombinant fusion TAT−β-globin protein interacts with the endogenous native α-like globins to form hemoglobin α 2 β 2 -like tetramers to a limited extent. Our findings provide evidence that recombinant TAT−β-globin is transmissible into proerythroid K-562 cells and can be potentially considered as an alternative protein therapeutic approach for β-thalassemias.

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

“…These include the successful delivery of human recombinant mitochondrial TAT-L-Sco2 fusion protein into the mitochondria of a cytochrome c oxidase (COX) deficient cell culture model bearing SCO2 mutations [ 1 , 16 , 17 ], the in vivo biodistribution of the radiolabeled recombinant TAT-L-Sco2 protein in mice [ 18 ] and the transduction of the human recombinant TAT-β‑globin chain into proerythroid K‑562 cells to replace the missing β‑globin as a therapeutic approach to β-thalassemia. The latter study confirmed the formation of hemoglobin α 2 β 2 -like tetramer, although to a limited extent [ 19 ]. The long-term goal of these studies has been to establish the PTD-mediated recombinant protein delivery as an alternative protein therapeutic approach for monogenetic disorders, including the β-thalassemias.…”

“…The cloning of the α-globin coding sequence (CDS) fused to the nucleotide sequence of the TAT peptide and the Hemagglutinin (HA) tag was carried out in both pCRII-TOPO vector (TOPO® TA Cloning® Kit with pCRII® TOPO®, Thermo Fisher Scientific, Massachusetts, United States) and the bacterial fusion expression vector pET-16b (Novagen, Darmstadt, Germany) [ 19 ].…”

“…Escherichia coli strain CD43 (DE3) was transformed by the pET-16b-recombinant prokaryotic plasmids to express 10xHis-Xa SITE -α-globin-HA and 10xHis-Xa SITE -TAT-α-globin-HA proteins, as previously described [ 17 – 19 ]. The fusion proteins were purified by affinity Ni 2+ -NTA column chromatography in order to be used in the Κ-562 transduction experiments, in the in vitro assembly of α- and β-monomers and in the size exclusion HPLC experiments.…”

In vivo biodistribution study of TAT-L-Sco2 fusion protein, developed as protein therapeutic for mitochondrial disorders attributed to SCO2 mutations

“…PTD technology permits intracellular delivery of therapeutic molecules, by facilitating penetration of almost all biological membranes, without interfering with the endogenous genetic information [ 2 , 20 ]. Our team has applied an in vitro PRT approach via the PTD technology, in the context of: a) β-thalassemia, with TAT-fused β-globin into K-562 cells to replace missing endogenous β-globin [ 13 ] and b) a mitochondrial disorder, due to SCO2 mutations [ 5 ].…”

“…Briefly, a selected colony of transformed by pET-16b-TAT-L-Sco2-HA bacterial E. coli BL21 (DE3) cells was cultured in LB (Luria-Bertani) medium, containing ampicillin (50 μg/mL). Induction was performed with 0.3 mM isopropyl-LD-thiogalactoside (IPTG) (PanReac, AppliChem), at 37 °C for 2 h, and bacterial pellet was harvested in order to collect inclusion bodies (IBs), which were further purified and dissolved either in l -Arginine ( l -Arg) [ 5 , 13 ] (Sigma-Aldrich) in 20 mM Tris–HCl, pH 7.5 [1–1.6 mg/mL protein] or in a salt buffer (300 mM NaCl, 50 mM NaH 2 PO 4 , pH 8.0) [0.3–1.2 mg/mL protein]. l -Arg solution has been found to facilitate folding of proteins [ 14 ].…”