erythropoietin   ( epo ) is a 34-kda glycoprotein hormone produced primarily by cells of the per tubular capillary endothelium of the kidney and regulates red blood cell production through stimulation of erythropoiesis [ 1 , 2 ] . epo synthesis in the kidney is increased following reduction in tissue oxygenation , it binds to specific receptors on red blood cell precursors in the bone marrow leading to proliferation , differentiation , and to an increase in haematocrit . biological responses associated with epo include activation of intracellular signaling molecules such as transcription factors like signal transducer and activator of transcription ( stat ) proteins leading to cellular growth and differentiation . epo receptor belongs to a family of homodimerization receptors where dimerization of the receptor is required to trigger the biological responses associated with epo [ 47 ] . anemia in patients with chronic kidney disease is due to a number of factors , the most common of which is abnormally low erythropoietin levels . anemia of epo deficiency is recognized in advanced renal failure but not in early renal disease . epo is heavily glycosylated with one o - linked and three n - linked oligosaccharide chains . it was found that o - linked oligosaccharide chain has no effect on secretion , receptor binding affinity , and in vitro or in vivo bioactivity . on the other hand , n - linked oligosaccharide- chains have no role in in vitro activity , but it is critical for in vivo bioactivity .       the gene encoding human erythropoietin was cloned in 1985 leading to the production of recombinant human epo ( rhuepo ) [ 9 , 10 ] . it has also been approved for the treatment of anemia associated with cancer , hiv infection , and in the surgical setting in order to reduce blood transfusions [ 1113 ] . one major issue regarding the clinical use of epo is its relatively short half - life in vivo due to its rapid clearance ( ~5 hours ) from the circulation when it is injected intravenously . thus , the clinical therapeutic protocols of available stimulating agents used in the treatment of patients require frequent injections of epo . the recommended therapy with rhuepo is 2 - 3 times per week by subcutaneous or intravenous injections . therefore , it can be anticipated that enhancing the in vivo half - life of epo would reduce the number of injections per week . previous studies indicated that there is a direct relationship between the sialic acid - containing carbohydrate content of the molecule and its serum half - life and in vivo bioactivity [ 1517 ] . it was shown that fusing the carboxyl - terminal peptide ( ctp ) of hcg subunit that associated with four sites of o - linked oligosaccharide chains to the c - terminal of fsh , tsh , gh , and epo cdna did not affect secretion , receptor binding affinity , and in vitro bioactivity . on the other hand , the addition of o - linked oligosaccharides to the backbone of the protein significantly increased the half - life and longevity in vivo [ 1822 ] . we hypothesis that the addition of 12 o - linked oligosaccharide chains to the backbone of epo will dramatically increase the longevity of epo . therefore , in the present study , three carboxyl - terminal peptides ( ctp ) of hcg subunit that each contains four o - linked oligosaccharide recognition sites was fused to n - terminal ( one ) and to the c - terminal ( two ) of human epo coding sequence , respectively . our results indicate that ligation of three ctps to the coding sequence of epo dramatically increased both in vivo potency and half - life in the circulation . enzymes used in the construction of dna vectors and constructs were purchased from new england biolabs ( beverly , mass , usa ) . cell culture media and reagents were obtained from biological industries ( beit haemek , israel ) . rabbit antisera against epo were purchased from fitzgerald ( concord , mass , usa ) . the eukaryotic expression vector ( pci - dhfr , dihydrofolate reductase ) into which the cdna encoding for the corresponding hepo variants were inserted was purchased from promega , ( san luis obispo , calif , usa ) . commercial human recombinant epo ( eprex ) was purchased from janssen - cilag ( north ryde , nsw , australia ) . the interaction between epo and its receptor was crystallized as described previously   by the department of structural biology , weizmann institute of science , rehovot , israel . a cassette gene containing the ctp of hcg was fused in tandem to the coding sequence of epo at the n - terminal ( one ctp ) and to the c - terminal end ( two ctps ) ( figure 1 ) . dna fragment containing sequences of hepo - cdna and coding sequence of ctp were synthesized by geneart ( regensburg , germany ) . the dna fragments contain the recognition sites of the restriction enzymes ; xba i ( in the n - terminal ) and not i ( in the c - terminal ) . fragment containing hepo and ctp sequences was completely sequenced to ensure that no errors were introduced during synthesis and ligated into the xbai  not i sites at the cloning site of the eukaryotic expression vector , pci - dhfr . similarly , cdna of human epo ( epo - wt ) was constructed into pci - dhfr vector . cells were cultured in mem- medium ( gibco brl , usa ) supplemented with penicillin ( 100  u / ml ) , streptomycin ( 100  mg / ml ) , l - glutamine ( 2  mm ) , and 10% heat - inactivated fetal bovine serum at 37c in humidified incubator containing 5% co2 . these cells were transfected with 2  g dna of plasmid by using fugene6 ( roche , mannheim , germany ) according to manufacturer protocol . cells were selected for insertion of the plasmid dna by growth in culture medium of cd dg44 without hypoxanthine and thymidine ( ht ) ( gibco brl , usa ) supplemented with 8 mm l - glutamine ( biological industries , beit haimic , israel ) and 18  ml / l of 10% pluronic f-68 solution ( gibco brl , usa ) . samples of condition medium which were collected from stable clones were electrophorised on denaturing 15% sds - polyacrylamide gels as described before . gels were allowed to equilibrate for 10  min in 25  mm tris and 192  mm glycine in 20% ( vol / vol ) methanol . proteins were transferred to a 0.2  m pore size nitrocellulose membrane ( sigma , saint louis , mo , usa ) at 250  ma for 3  h using a mini trans - blot electrophoresis cell ( biorad laboratories , richmond , ca ) according to the method described in the manual accompanying the unit . the nitrocellulose membrane was incubated in 5% nonfat dry milk for 2  h at room temperature . the membrane was incubated with epo antiserum ( 1  :  1000 titers ) for overnight at 4c followed by three consecutive washes in pbs containing 0.1% tween ( 10  min / wash ) . then , the membrane was incubated with secondary antibody conjugated to horse radish peroxidase ( hrp ) ( zymed , san francisco , ca ) for 2  h at room temperature followed by three washes . finally , the nitrocellulose paper was reacted with enhanced chemiluminescent substrate ( ecl ) ( pierce , rockford , ill , usa ) for 5  min , dried with whatman sheet and exposed to x - ray film . bioactivity of epo variants was assayed by testing the proliferation dependence of the human erythroleukemic cell line tf-1 ( kitamura ) ( dsmz ) in the presence of epo and epo variants . cultures were routinely grown at 37c , 5% co2 for 72  hrs in rpmi 1640 medium supplemented with 10% fetal bovine serum ( fbs),10  mm hepes , 1  mm sodium pyruvate , 2.5  g / l glucose , 2  mm glutamine , and 2  ng / ml rhgm - csf . before transferring the cells to 96-well plates , the tf-1 cells were washed three times with cold pbs and suspended in the assay medium ( 1640 medium supplemented with 10% fetal bovine serum ( fbs ) but without addition of rhgm - csf ) at a density of 200,000  cells / ml . the assay was performed in 96-well plates containing 50  l of cell suspension per well . 50  l of assay medium containing 3  iu of epo variants were then added to the wells of 96-well plates for 72  hrs . cell viability was measured using mtt reagent kit ( cell biolabs , san diego , ca ) according to manufacturer procedures . male icr mice were obtained from charles river laboratories , jerusalem , israel , and housed in air - conditioned quarters with a 12  h light / dark schedule . epo - wt , epo-(ctp)3 , or commercial rhuepo were injected to anesthetized animals as described in table 1 . the animals were weighed , and each received an identical amount ( 5 or 15  g / kg ) of epo variants by iv injections ( 0.2  ml / animal ) . the frequency of treatment was either thrice weekly ( days 1 , 3 , and 5 ) or once weekly . the level of haematocrit was determined three times a week and the experiment was stopped after three weeks . haematocrit was determined using blood samples obtained by filling two heparinized microhematocrit tubes from the inferior caval vein under anesthesia . in addition , reticulocyte counts were conducted since these cells are present in blood for 48 hours before developing into mature red blood cells . blood was obtained from each pup by cardiac puncture and placed in edta coated tubes . this was then mixed with brilliant cresyl blue and incubated for 20  min at 37c . the blood and stain were then smeared onto a glass slide and the number of reticulocytes assessed using a  100 oil objective lens . the metabolic clearance of epo - wt , aranesp , and epo-(ctp)3 was determined after iv injection of 20  iu / animal into male icr mice . at selected intervals after injection , statistical analysis of the data were performed using student 's t - test and 1-way multivariate analysis of variance ( anova1 ) to calculate p value . chrystallographic studies indicated that the n - terminal and c - terminal of epo are not involved in the binding of the hormone to the receptor ( figure 2 ) . therefore , we hypothesized that ligation of ctp to the n - terminal and to the c - terminal of epo will not affect receptor binding affinity and thus bioactivity . therefore , three ctps ( one in the n - terminal and two in the c - terminal ) were ligated to the coding sequence of epo . the cdna of human epo - wt and epo-(ctp)3   was   inserted into the eukaryotic expression vector , pci - dhfr , and transfected into cho cells . secretion of epo was assessed by western blot analysis under denaturing conditions using human epo - specific antiserum . epo-(ctp)3 exhibited high molecular weight ( 57  kda ) comparing to epo - wt ( 36  kda ) due to the addition of 84 amino acids and the o - linked oligosaccharides linked to ctp . these data may indicate that the o - linked glycosylation recognition site of the c - terminal region is preserved even though the sequence is fused to different proteins . levels of epo - wt and epo-(ctp)3 were quantities in condition medium by using a monoclonal antibody - based ria . the in vitro biological activity of epo analogs was demonstrated by measuring their ability to stimulate the proliferation of tf-1 cells as described under the activity of epo-(ctp)3 in tf-1 cell proliferation assay was similar to that of epo wild - type ( prepared by modigene tech ) and commercial rhuepo ( figure 4 ) . for further pharmacological evaluation of epo-(ctp)3 , comparative pharmacodynamic studies of epo-(ctp)3 and commercial rhuepo were performed in male icr mice ( n = 7/group ) using different frequencies and dose range as described in table 1 . the in vivo efficacy was obtained by measuring the mean values of haematocrit percentage in the blood . the results indicated that epo-(ctp)3 is significantly ( p < 0.001 ) more efficient than rhuepo when administered iv once a week with a dose of 15  g / kg ( figure 5 ) . epo-(ctp)3 can successfully increase the haematocrit when administered once a week with a dose of 15  g / kg ( figure 5 ) . once weekly dosing with the same concentration of commercial rhuepo or epo - wt was significantly ( p < 0.001 ) less efficient than once weekly dosing of epo-(ctp)3 . an interesting observation from the present study was the ability of a single injection once a week of epo - ctp ( 15  g / kg ) to increase dramatically ( 8 folds ) the levels of haematocrit . whereas administration of the same total dose of rhuepo administered three times a week as 5  g / kg per injection resulted in significantly ( p < 0.001 )   lower effect ( figure 5 ) . previously , we have shown that single injection once a week of epo - ctp , an epo that contains one ctp at the carboxyl - terminal end , ( 15  g / kg ) increased the level of haematocrit , whereas the same effect was achieved by administration of the same total dose of rhuepo administered three times a week as 5  g / kg per injection . these results indicated the importance of sustained blood levels , rather than total dose of epo . these findings are consistent with the hypothesis that the ability of a single injection of epo - ctp to increase haematocrit results from its increased stability in the circulation . effect of epo - wt , ( epo - ctp)3 , and aranesp in reticulocyte counts is shown in figure 6 . the results indicated that a single iv injection of 15  g / kg ( epo - ctp)3   dramatically increased reticulocyte number compared to rhuepo and to aranesp . the increased biopotency of the chimera may reflect a change in their in vivo longevity . epo - wt , aranesp , or epo-(ctp)3 was injected iv into immature male mice and ria monitored the plasma half - lives . the results indicated that epo-(ctp)3 has the highest half - life in circulation ( figure 7 ) . the estimated half - lives of epo - wt , aranesp , and epo-(ctp)3 are 4.4 , 10.8 , and 13.1 hours , respectively ( table 2 ) . these data suggest that the mechanism of epo clearance is affected by the presence of ctp . estimation of area under the curve ( auc ) and the maximal plasma concentration ( cmax ) of epo-(ctp)3 are higher than that of rhuepo and aranesp . however , the maximal concentration reached in plasma ( tmax ) is similar ( table 2 ) . previous studies indicated that the ctp sequence can be shuttled into different proteins and still be an acceptor for the o - linked oligosaccharides [ 1820 ] . it was postulated that the o - linked oligosaccharides add flexibility , hydrophilicity , and stability to the protein . this may explain the disinterference of ctp on the protein conformation and , thus , on receptor binding and bioactivity in vitro . on the other hand , it was suggested that the o - linked oligosaccharides play an important role in preventing plasma clearance and thus increasing the half - life of the protein in the circulation [ 18 , 21 , 22 ] . these roles have been postulated since the o - linked oligosaccharides are ended with sialic acid , which is negatively charged . it is known that negatively charged forms of the hormones are less cleared through the glomerular filtration . thus , addition of 12 o - linked oligosaccharide chains to the backbone of epo significantly decreased renal clearance ; the kidney is the main site of clearance for glycoprotein hormones and , thus , prolonged its half - life in the circulation . other studies described long acting hyperglycosylated epo analog that prepared by addition of n - linked oligosaccharides to the backbone of the protein . in order to add n - linked oligosaccharide chains , the dna sequence of the cloned human epo gene was modified by site - directed mutagenesis . this analog was 3-fold longer serum half - life and created in vivo potency comparing to human recombinant epo - wt . however , its relative affinity for the epo receptor was 4-fold lower than that of rhuepo . moreover , changing 5 amino acids in the backbone of the protein may increase the immunogenicity of the new derivative . addition of ctp to the coding sequence of hormones fsh , tsh , and gh , do not affect secretion , receptor binding affinity , or bioactivity in vitro [ 1822 ] . on the other hand , it was found that ligation of ctp to the coding sequence significantly increases the in vivo potency and half - life of the hormone . moreover , it was found that hormone bearing ctp is safe for use in human and not immunogenic [ 2931 ] . the present study describes a novel long - acting recombinant erythropoietin agonist designed by fusion of three ctp sequences to the coding sequence of epo . in contrast , addition of ctp sequences significantly increased the in vivo potency and half - life of epo . these data establish a rationale for using this chimera as a long - acting epo analog . human erythropoietin has a wide clinical use in the treatment of anemia associated with a renal failure , hiv , and chemotherapy [ 3236 ] . the therapeutic efficacy of this analog needs to be establishing in higher animals and in human clinical trials .