Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in vivo

Abstract

High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic anemia. Thus, it is clear that therapy for chronic anemia would greatly benefit from an erythropoietin derivative with increased erythropoietic activity rather than the native endogenous hormone. In this report, the activity of a human Epo-Epo dimer protein, obtained by recombinant technology, is described and compared with its Epo monomer counterpart produced under identical conditions. Although monomer Epo and dimer Epo-Epo had similar pharmacokinetics in normal mice, the increase in hematocrit value was greater with the dimer than with the monomer. Moreover, in clonogenic assays using CD34 ؉ human hematopoietic cells, the human dimer induced a 3-to 4-fold-greater proliferation of erythroid cells than the monomer. Controlled secretion of dimeric erythropoietin was achieved in ␤-thalassemic mice by in vivo intramuscular electrotransfer of a mouse Epo-Epo plasmid containing the tetO element and of a plasmid encoding the tetracycline controlled transactivator tTA. Administration of tetracycline completely inhibited the expression of the mEpo dimer. On tetracycline withdrawal, expression of the Epo-Epo dimer resumed, thereby resulting in a large and sustained hematocrit increase in ␤-thalassemic mice. No immunologic response against the dimer was apparent in mice because the duration of the hematocrit increase was similar to that observed with the monomeric form of mouse erythropoietin. ( IntroductionErythropoietin (Epo) is a 34-kd glycoprotein produced mainly by kidney paratubular cells in response to reduced oxygen delivery. 1,2 Epo stimulates erythroid progenitor cell proliferation, differentiation, and maturation, and it inhibits cell apoptosis, which results in increased erythrocyte production. [3][4][5] The recombinant human erythropoietin (rhEpo) hormone is widely used to compensate for the reduced production of endogenous Epo in renal failure and to correct the associated anemia. Administration of rhEpo alleviates the necessity for blood transfusion and greatly improves the quality of life for patients. [6][7][8] Clinical studies have shown that rhEpo can also be effective in the treatment of other chronic anemias, 9 especially when endogenous Epo levels are inappropriately low. When injected into ␤-thalassemic mice, rhEpo has been shown to restore balanced globin chain synthesis and to improve the erythrocyte phenotype. 10 In patients with ␤-thalassemia, serum Epo levels are often lower than expected, 11 and injections of high doses of rhEpo have been shown to increase blood hemoglobin levels and occasionally to alleviate the need for transfusion. [12][13][14][15] Although high doses of Epo may be beneficial for patients with ␤-thalassemia, the cost of such a treatment prevents its use in long-term controlled therapeutic trials and in routine clinical practice. Consequently, various strategies have been pursued to increase the yield of Epo produced by gene transfer and to enhance its intrinsic activity. For i...

Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications

“…Such a construction was used to produce a dimeric Epo protein. In this study, the dimeric form has proven more active in vitro on erythroid cells and in vivo in electrotransferred mice than the monomeric form [72]. Even if kinetics of monomeric and dimeric proteins were the same with a rapid decrease, this improved effect of the dimer is due to a higher binding affinity because of the presence of two molecules of Epo.…”

“…A phenotypic correction of β-thalassemic mice following Epo-encoding plasmid electrotransfer has also been described [21]. In addition, a controlled expression of a dimeric form of Epo has been achieved by using an Epo-encoding plasmid containing the tetO element and a plasmid encoding the tetracyclinecontrolled transactivator tTA [72]. Hepatocyte growth factor (HGF) muscle secretion has recently shown cytoprotective activity in mice with acute liver injury [75].…”

Novel Erythropoiesis-Stimulating Agents

“…Recombinant EPO fusion proteins that contain additional peptides at the carboxy-terminus to increase in vivo survival have been expressed (25). Large EPO fusion proteins, of molecular weight 76 kD, have been designed from cDNA encoding two human EPO molecules linked by small flexible polypeptides (26,27). A single subcutaneous administration of this compound to mice increased red cell production within 7 d at a dosage at which epoetin was ineffective (26).…”

Death switch for gene therapy: application to erythropoietin transgene expression

“…Thus, millions of patients with chronic anemia caused by kidney diseases, cancer, www.bjournal.com.br Braz J Med Biol Res 43(7) 2010 AIDS, myelodysplasia, or even hemoglobinopathies (14)(15)(16)(17)(18) could benefit from the delivery of erythropoietin (Epo) by gene therapy, particularly in diseases where resistance to Epo is important and administration of large doses of this hormone is necessary in order to obtain a response. In view of its wide application, gene therapy using the Epo gene has been the aim of many in vitro and in vivo studies (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Sufficient gene expression for a long-term response has been easily obtained in most in vivo studies and the target of erythropoietin treatment is reached when the hormone is delivered to the plasma by intramuscular injection of the vector or implant of encapsulated transformed cells (19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”

“…In view of its wide application, gene therapy using the Epo gene has been the aim of many in vitro and in vivo studies (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Sufficient gene expression for a long-term response has been easily obtained in most in vivo studies and the target of erythropoietin treatment is reached when the hormone is delivered to the plasma by intramuscular injection of the vector or implant of encapsulated transformed cells (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). The success of this approach has been demonstrated directly by hematocrit increase as well as by measurement of Epo protein in cell culture or in the serum of tested animals, such as mice, monkeys, and cats.…”