Modified human erythropoietin

Abstract

A modified human erythropoietin is provided that can maintain neuroprotective or neurogenic potential while suppressing erythropoietic activity. [Solution] The modified human erythropoietin of the present invention has an erythropoiesis-stimulating activity of less than 0.5% compared to human erythropoietin, and contains mutations in the binding site to homo- or heterodimeric receptors due to the addition of a consensus site for glycosylation, which extends the plasma half-life and can maintain neuroprotective or neurogenic ability while suppressing erythropoietic activity.

Description

[Technical field]

[0001] The present invention relates to modified human erythropoietin (hereinafter also referred to as "mhEPO"), which has neuroplastic and neuroprotective properties that are useful in the treatment of central nervous system diseases, such as cerebrovascular injury, neurotrauma, neuroinflammation, and neurodegeneration. [Background technology]

[0002] Erythropoietin (EPO) is a member of the type I cytokine superfamily, which is characterized by significant pleiotropic activity (Non-Patent Document 1). This cytokine is one of the major regulators of erythropoiesis, acting synergistically with other molecules to promote proliferation, differentiation, and survival of erythroid lineage cell progenitors and maintain the amount of circulating red blood cells (Non-Patent Document 2).

[0003] Human EPO (hEPO) is a highly glycosylated protein. Its molecular weight ranges from 30-39 kDa. It has three consensus N-glycosylation sites and one O-glycosylation site, which occur in all degrees of occupancy (Non-Patent Document 3). The sugar chain is composed of a variable sequence of monosaccharides and variable amounts of sialic acid (SA) (Non-Patent Documents 4, 5). The N-linked carbohydrate contains either two, three, or four branches, each of which terminates with a negatively charged SA molecule. As a part of it, the carbohydrate attached to the O-glycosylation site can contain up to two SA molecules (Non-Patent Document 6).

[0004] Glycosylation, especially the SA terminus of N-glycans, is fundamental for the in vivo biological activity of erythropoietin (EPO), but not for in vitro receptor binding (Non-Patent Documents 7, 8). This is why EPO molecules with low glycosidic content have high affinity for the EPO receptor (EPOR), but their in vivo biological activity disappears with increased plasma clearance (Non-Patent Document 8). The degree of glycosylation of these cytokines affects their production efficiency, affinity to the receptor, plasma half-life, secretion and protein stability. [Patent Document 1] US Patent Application Publication US2015119325 [Patent Document 2] US Patent Application Publication US2009170759 [Patent Document 3] US Patent Application Publication US2003130197 [Patent Document 4] US Patent Application Publication US2011008363 [Patent Document 5] US Patent Application Publication US2007027068 [Patent Document 5] US Patent Application Publication US2011003744 [Patent Document 6] PCT Publication WO2004 / 043382 [Patent Document 7] PCT Publication WO2005 / 025606(AI) [Patent Document 8] PCT Publication WO2006 / 127910 [Patent Document 9] PCT Publication WO2005 / 103076 [Patent Document 10] Mexican Patent Application Publication MXPA02011727 [Patent Document 11] Mexican Patent Application Publication MXPA05000063 [Patent Document 12] Spanish Patent Application Publication ES2457398(T3)

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[23] PJ Stohlawetz, L. Dzirlo, N. Hergovich, E. Lackner, C. Mensik, HG Eichler, E. Kabrna, K. Geissler, B. Jilma. Blood 95 (2000) 2983-2989.

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[25] Beguin Y., Haematologica (1999), 84:541-547.

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[26] M Leist, P Ghezzi, G Grasso, R Bianchi, P Villa, M Fratelli, C Savino, M Bianchi, J Nielsen, J Gerwien, P Kallunki, AK Larsen, L Helboe, S Christensen, LO Pedersen, M Nielsen, L Torup, T Sager, A Sfacteria, [ PubMed ] Erbayraktar S, Erbayraktar Z, Gokmen N, Yilmaz O, Cerami-Hand C, Xie QW, Coleman T, Cerami A, Brines M. Science 305 (2004) 239–242.

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[29] Erbayraktar, S. et al. Proc. Natl. Acad. Sci. (2003), USA 100: 6741–6746.

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[32] Das KP, Freudenrich TM, Mundy WR. Neurotoxicol Teratol (2004) 26:397-406

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[33] Rochefort NL, Konnerth A. EMBO Rep. (2012) Aug; 13 (8):699-708. DOI: 10.1038 / embor .2012.102.

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[35] Hutsler JJ1, Zhang H. Brain Res. (2010) Jan 14;1309:83-94. DOI: 10.1016 / j .brainres .2009.09.120.

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[36] Alfonso J., Fernandez ME, Cooper B., Flugge G. andFrasch AC Proc. Natl Acad. Sci. (2005) USA 102, 17196- 17201.

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[0037] Ippolito, DM, Eroglu, C., Journal of Visualized Experiments. (2010) Exp. 45:2270. DOI: 10.3791 / 2270.

[0005] Since the cloning of the hEPO gene in 1985, knowledge of cytokine biology has changed dramatically. One of the first advances was the discovery of new biological activities of the molecule described below, which expanded the capabilities of EPO to include several important physiological processes. Some of the most important are angiogenesis, arterial resistance regulation, and even more importantly, celluar protection (Non-Patent Documents 10, 11). Although the most important activity of EPO is hematopoiesis, the presence of EPO and its receptors in various tissues and non-EPO cells confirms the hypothesis that EPO has multiple functions. Among the most important are its cytoprotective function on the central nervous system, heart, kidney, gastrointestinal system, reproductive tract, and endothelium (Non-Patent Document 12), promotion of cell proliferation, angiogenesis, and prevention of cell apoptosis (Non-Patent Document 13). These discoveries have expanded the prospects for clinical use for the treatment of other pathologies. Other examples of pathologies include heart attacks, cerebrovascular accidents, and diseases related to neuroprotection (Non-Patent Document 14).

[0006] Neuroprotection can be defined as an approach to maintain and restore cellular interactions in the brain, resulting in maximum protection of neuronal function (Non-Patent Document 15). The goal of neuroprotection is to prevent pathological neuronal (hereinafter also referred to as "nerve") loss in central nervous system diseases. Examples of neuronal loss include cerebrovascular injury, neurotrauma, neuroinflammation, and neurodegeneration.

[0007] Neurodegenerative diseases are a group of disorders that affect the nervous system, causing cognitive and behavioral disorders and changes in the body's regulatory systems. They are characterized by chronic and acute progression. These diseases include Parkinson's disease, various types of dementia, Alzheimer's disease, multiple sclerosis, Huntington's disease, etc. These diseases create medical and health problems from a social / economic perspective that every country faces (Non-Patent Document 16). As the world's population ages, the impact of neurological disorders will increase in both developed and developing countries. The burden of neurological disorders will be significantly greater in countries where the proportion of people over 65 years of age is increasing. Looking only at Alzheimer's disease, statistics show that 44 million people worldwide suffer from dementia. This figure is predicted to reach 115 million in 2050. The reason is that the disease is considered a global epidemic (Non-Patent Documents 17-19). For this reason, many research groups around the world are engaged in research to provide treatments to control this type of neurodegenerative disease.

[0008] Collaborations of many research groups have provided new and promising background to the causes of these problems, bringing hope for early disease detection and finding specific ways to attack the disease. But even these efforts have failed to identify a biological therapy that can cure the disease. In this sense, the pharmaceutical market only offers drugs that supposedly slow the progression of these diseases once they are identified, but the benefits are not felt very much. This is because new treatments need to be developed. The success of addressing the health problems that neurodegenerative diseases present today depends, among other things, on treatments that have beneficial and long-lasting effects. Treatments should favorably affect the etiology or underlying disease, prevent or delay the onset of the disease, and safely, successfully, and effectively slow the deterioration of the disease. But now it is also necessary to come up with efficient medical technologies that are accessible to the masses. The main goal of the pharmaceutical industry these days is to find compounds that can attack common key points in the development of many diseases of the central nervous system. Examples of these diseases are apoptosis, oxidative stress, inflammation, metabolic dysfunction, and reduced neurogenesis.

[0009] In this research field, EPO plays a very important role, due to its ability to develop a wide range of cellular responses in the brain that are directly related to protection and repair of cellular damage (Non-Patent Document 20). EPO exerts neuroprotection, which is achieved through anti-inflammatory, antioxidant, anti-neurotoxic, angiogenic, neuroregenerative and anti-apoptotic mechanisms.

[0010] In this light, another very important aspect of EPO research is the identification of two different molecular sites related to erythropoietic and cytoprotective biological activities. These two activities are conferred by the binding of the cytokine to two different receptor systems: the homodimeric receptor (EPOR)2 involved in erythropoietic activity and the heterodimeric receptor (EPOR-pCR) involved in cytoactivities (Non-Patent Document 21). These discoveries are extremely important because they provide the information necessary to understand the pathways by which EPO develops its biological activity. This information, in turn, allows the selective modulation of its erythropoietic and cytoprotective biological responses, all while maintaining the objective of avoiding the side effects associated with the use of unselected drugs (EPO or EPO analogues).

[0011] In this sense, EPO is recognized as a safe and well-accepted drug in the treatment of anemia, but when EPO is used as a cytoprotectant in patients who have suffered a cerebrovascular or cardiac accident, i.e. when EPO is used as a neuroprotectant in non-anemic patients, its haematological effects should be considered as side effects, since EPO causes polycythemia, hypertension and prothrombotic phenomena (Non-Patent Documents 22-25). For this reason, the development of EPO analogs that selectively modulate its erythropoietic and cytoprotective roles is highly interrelated (Non-Patent Document 14). With this aim, various strategies have been undertaken, seeking to counteract the erythropoietic activity of the cytokine and maintain its neuroprotective capacity. This is done through the application of chemical modifications to the EPO molecule or through the manipulation of the glycoside content.

[0012] Regarding strategies to employ chemical modifications (hereinafter simply referred to as "modifications") to the EPO molecule, carbamylation of seven lysine residues to generate homocitrulline residues has been evaluated (Non-Patent Document 26). However, this method generates conformational changes in the protein that affect its function. Based on these results, various working groups have completely modified recombinant hEPO (hereinafter also referred to as "rhEPO") by carbamylation to obtain a new molecule called CEPO. This new molecule maintains cytoprotective activity but lacks erythropoietic activity (Non-Patent Documents 26-28). At the same time, high doses and multiple administrations are required to achieve the desired goals and maintain its therapeutic effect.

[0013] On the other hand, modifications have been made to rhEPO by manipulating its sialic acid (SA) content. In 2003, according to Non-Patent Document 29, the SA residues present at the termini of the EPO glycosidic chains were completely removed. Thus, the so-called asialoEPO was generated. This new EPO variant exhibits high in vitro activity. However, its in vivo neuroprotective activity was comparable to that obtained with rhEPO.

[0014] On the other hand, with the aim of extending the circulating half-life of erythropoiesis-stimulating cytokines, the erythropoiesis-stimulating protein NESP was developed according to Non-Patent Document 6. NESP is derived from hEPO and displays two additional N-glycosylation sites. This modification increased the plasma half-life and in vivo erythropoietic capacity three-fold.

[0015] By manipulating the glycoidic content, the inventors obtained a rhEPO variant with properties similar to cerebral EPO (rhNEPO). rhNEPO is a combination of less acidic isoforms of rhEPO. This rhEPO variant showed neuroprotective activity equivalent to rhEPO and showed less than 4% erythropoietic activity (Non-Patent Document 30). However, the rapid plasma clearance of rhNEPO is a disadvantage when proposing this molecule as a candidate for the treatment of chronic neurological diseases, which require plasma concentrations to be maintained over time and high enough to exert biological effects. Moreover, even if the glycoform combination maintains in vitro erythropoietic activity, high and frequent administration to achieve this goal carries the risk of producing hematological effects, which are considered as undesirable side effects.

[0016] The prior art describes many other efforts to obtain erythropoietin molecules or peptides derived therefrom, which show the presence of neuroprotective activity and the lack of erythropoietic activity. In this regard, US Pat. No. 5,399,436 describes asialoerythropoietin (asialo-rhEPO) produced in plants. US Pat. Nos. 5,499,435, 5,503,521 and 5,633,662 describe peptides that bind to the EPO receptor for the treatment of diseases related to the central nervous system.

[0017] Patent document 6 describes a variant of human erythropoietin (hEPO) polypeptide, which has an amino acid sequence with amino acid differences in multiple different EPO modification regions and has enhanced erythropoietic activity. The object of the present invention is to provide a human variant of erythropoietin polypeptide comprising an amino acid sequence with amino acid differences in multiple different EPO modification regions and having moderate erythropoietic activity associated with its cytoprotective function. to get It is.

[0018] Patent document 4 describes various mutants of EPO, in which 1-10 amino acids are deleted at the C-terminus of the protein. These are low molecular mass mutants that reduce erythropoietic activity and preserve neuroprotective action. US Patent No. 5,999,633 describes glycopegylated EPO peptides. One of the mutated EPO peptides has an amino acid sequence (SEQ ID NO: 73) and has at least one mutation selected from the following set: Arg <139> FromAla <139> , Arg <143> FromAla <143> , Lys <154> FromAla <154>

[0019] US Pat. No. 5,399,633 relates to polynucleotides encoding EPO variants. Patent document 7 describes modified EPO incorporating oligosaccharides, which increases erythropoietic activity and maintains cytoprotective activity.

[0020] Patent document 8 describes glycosylation that increases the production of red blood cells, which is said to be effective in treating neurodegenerative diseases because it performs a cytoprotective function.

[0021] US Patent No. 5,993,663 describes recombinant EPO (rEPO) for the treatment of neurodegenerative diseases. The document details modified amino acids to add glycosylation sites. Modifications of the following residues are highlighted: 87, 88, 90 / / 30, 32, 87, 88, 90 / / 24, 87, 88, 90 / / 38, 87, 88, 90 / / 83, 87, 88, 90.

[0022] The purpose is to incorporate glycans to increase erythropoietic activity and to increase the circulating half-life. A similar purpose exists for the modifications disclosed in US Pat. No. 5,313,666, which describes variants of EPO with additional glycosylation sites. Some of the sites described therein are 25, 30, 51, 57, 69, 88, 89, 136, and 138. In addition, the following substitutions are described: Asn <30> Thr <32> EPO; Asn <51> Thr <53> EPO; Asn <57> Thr <59> EPO; Asn <69> EPO; Asn <69> Thr <71> EPO; Ser <68> Asn <69> Thr <71> EPO; Val <87> Asn <88> Thr <90> EPO; Ser <87> Asn <88> Thr <90> EPO; Ser <87> Asn <88> Gly <89> Thr <90> EPO; Ser <87> Asn <88> Thr <90> Thr <92> EPO; Ser <87> Asn <88> Thr <90> Ala<l 62> EPO; Asn <69> Thr <72> Ser <87> Asn <88> Thr <90> EPO; Asn <30> Thr <32> Val <87> Asn <88> Thr <90> EPO; Asn <89> Ile <90> Thr <91> EPO; Ser <87> Asn <89> Ile <90> Thr <91> EPO; Asn <136> Thr <138> EPO; Asn <138> Thr <140> EPO; Thr <125> EPO; Pro <124> Thr <125> EpO .

[0023] US Patent No. 5,993,366 describes EPO variants having an even number of cysteine ​​residues. The even number of cysteine ​​residues is preferably 4 or less, more preferably 2 or less. The cysteine ​​residues are preferably at positions 7, 29, 33, 161, more preferably at positions 7 and 161. Additional variants provided by the present invention include any mutation by addition at any of the following positions: positions 6, 29, 33, 45, 47, 48, 49, 61, 64, 74, 88, 92, 107, 109, 133, 135, 154, 157, 158.

[0024] US Pat. No. 5,999,333 describes the formation of EPO, which is mediated by the enzyme in Through modified glycosidic residues, they contain polyethylene glycol-conjugated proteins or poly ethanolamine-conjugated proteins, which enhance their erythropoietic properties.

[0025] US Patent No. 5,399,663 describes a tissue-protective recombinant cytokine, which is devoid of erythropoietic activity at least on bone marrow. The tissue-protective recombinant cytokine is devoid of erythropoietic activity. Preferably, it is devoid of any erythropoietic activity on bone marrow. US Patent No. 5,399,663 describes modifications to amino acids, i.e. deletions or additions to EPO, occurring at one or more amino acids. In the examples, the tissue-protective recombinant cytokine is in the following regions: Either and has the modification. VLQRY (amino acids 11-15 of native hEPO: SEQ ID NO: 1); TKVNFYAW (amino acids 44-51 of native hEPO: SEQ ID NO:2); SGLRSLTTL (amino acids 100-108 of native hEPO: SEQ ID NO:3); SNFLRG (amino acids 146-151 of native hEPO: SEQ ID NO:4). Other mutations are given at amino acids 7, 20, 21, 29, 33, 38, 42, 59, 63, 67, 70, 83, 96, 126, 142, 143, 152, 153, 155, 156, and 161 of SEQ ID NO:10.

[0026] These other mutations are unique or in addition to the mutations in the regions described above. In one embodiment, the above amino acid changes in TKVNFYAW (amino acids 44-51 of native hEPO; SEQ ID NO: 2) result in a modified EPO with partial function (i.e., less erythropoietic activity than rhEPO). In another embodiment, the above amino acid changes in SGLRSLTTL (amino acids 100-108 of native hEPO; SEQ ID NO: 3) result in molecules lacking glycans or variants with carbohydrate modifications, such as sialic acid modifications, oxidation / reduction, and chemical modifications. Examples of chemical modifications are nitration, acylation, succinylation, biotinylation, iodination, and carbamylation. There is no mention of adding new glycans to the sites responsible for erythropoietic activity in order to reduce or prevent it. Summary of the Invention [Problem to be solved by the invention]

[0027] The efforts / achievements to date described in the prior art have not succeeded in obtaining an EPO exhibiting the presence of neuroprotective activity and the absence of erythropoietic activity. In contrast, the present invention presents novel hEPO muteins exhibiting neuroprotective and neurotropic effects. The hEPO muteins of the present invention are obtained by a unique method of modifying the hEPO molecule. The modification is achieved by creating new consensus sites for N-glycosylation in the hEPO molecular region. The hEPO molecular region is capable of expressing both homo- and hetero-glycosylation. of It is involved in binding to the dimeric receptor. However, the new hEPO muteins lack erythropoietic activity but have unchanged or even improved neuroprotective and neurogenic activities. As a result, they show improved pharmacokinetic properties. Modifications of the original EPO are minimal, thus preserving the structure and similarity of the natural protein. [Means for solving the problem]

[0028] The modified human erythropoietin (hEPO) described herein has absent or reduced erythropoietic activity and a long plasma half-life. Preferably, the reduction in erythropoietic activity is at most 0.5% compared to hEPO. The long plasma half-life allows for the maintenance of neuroprotective and neurogenic potential. The binding of the modified human erythropoietin to either homo- or heterodimeric receptors is partially or completely abolished or eliminated. This abolishment includes mutation of either the binding site to the homo- or heterodimeric receptor by incorporating a consensus site for N-glycosylation.

[0029] According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the mutations Tyr15Asn and Leul7Thr and comprises SEQ ID NO:2. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the mutations Lys45Asn and Asn47Thr and comprises SEQ ID NO:4. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the mutations Glu62Asn and Trp64Thr and comprises SEQ ID NO:8. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the mutations Gln65Asn and Leu67Thr and comprises SEQ ID NO:10. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the mutations Glu72Asn and Val74Thr and comprises SEQ ID NO:12. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the mutations Arg76Asn and Gln78Thr and comprises SEQ ID NO:14. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the Ala98Asn and Ser100Thr mutations and comprises SEQ ID NO:16. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has a Serl04Asn mutation and comprises SEQ ID NO:18. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the mutations Thrl06Asn and Leu108Thr and comprises SEQ ID NO:20. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the Leul49Thr mutation and comprises SEQ ID NO:22. According to one embodiment of the present invention, the modified human erythropoietin comprises: It has the mutations Glyl51Asn and Leul53Thr and comprises SEQ ID NO:24.

[0030] The modified human erythropoietin of the present invention has at most 1% of the erythropoietin activity compared to human erythropoietin. Preferably, it has at most 0.5% of the erythropoietin activity compared to human erythropoietin. More preferably, it has at most 0.5% of the erythropoietin activity compared to hEPO. and In comparison, it only has a maximum of 0.2%.

[0031] On the other hand, the present invention describes nucleic acids comprising the nucleotide sequence of the erythropoietin of the present invention. The DNA sequences encoding each of the described and generated muteins are outlined. These DNA sequences are any of the following: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23. According to another embodiment of the present invention, these nucleic acids are vectors for transforming cells, expression vectors or lentiviral vectors.

[0032] According to one embodiment of the invention, the nucleic acid sequence encoding the modified human erythropoietin is either: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23.

[0033] The present invention describes a genetically modified cell having any DNA sequence selected from the following set: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23. The genetically modified cell is capable of expressing a modified human erythropoietin of the present invention. According to one embodiment, the genetically modified cell is either a plant cell, an animal cell, or a cellular host capable of adding N-glycans. Preferably, the cell is derived from an animal cell line. Preferably, the cell line is selected from the set comprising: CHO K1, HEK293, NSO, BHK-21, HeLa.

[0034] On the other hand, the present invention describes a method for obtaining modified human erythropoietin (mhEPO). The method for producing mhEPO of the present invention comprises the following steps (A) to (H). (A) providing a nucleic acid sequence encoding the mhEPO; (B) constructing a packaging cell co-transfection vector; (C) co-transfecting the packaging cells; This produces lentiviral particles (hereinafter referred to as "lvp") containing a nucleic acid sequence encoding the mhEPO, (D) harvesting the lvp produced by the packaging cells of step (C); (E) transducing the mhEPO-expressing cells with the lvp from step (D); (F) selecting the cells of step (E) that contain a nucleic acid sequence encoding the mhEPO; (G) culturing the cells selected in step (F); As a result, they express the mhEPO, (H) isolating and purifying the mhEPO.

[0035] In said step (A), said nucleic acid sequence is selected from the following set: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23.

[0036] The vector in step (B) includes any one of the following vectors (1) to (4). (1) a vector that allows the lvp to enter a cell; (2) a vector encoding matrix protein, capsid, protease, reverse transcriptase and integrase; (3) a transfer vector comprising the mhEPO sequence; (4) A vector that induces nuclear export of the transfer vector. In the step (E), the cells are selected from the group consisting of the following (1) to (5). (1) CHO.K1, (2) HEK293, (3) NSO, (4) BHK-21, (5) HeLa.

[0037] The eluent is selected from the group comprising: glycine, acetic acid-NaCl, acetate, citric acid, phosphate, ethanol, isopropyl alcohol, dioxane, ethylene glycol, Tris-HCl, mixtures thereof. Further, the eluent is selected from the group comprising glycine, acetic acid-NaCl. The eluent is selected from the set comprising: 0.1 M glycine (pH=2), 0.15 M glycine (pH=2.5) and 0.2 M acetic acid, 0.15 M NaCl (pH=3). [Brief description of the drawings]

[0038] [Figure 1] FIG. 1 depicts a scheme of the sequential steps to obtain 12 hEPO muteins via site-directed mutagenesis. [Figure 2a] Diagram showing the analysis of the fragments obtained by PCR1 using an agarose gel. PCR1: introducing point mutations. [Figure 2b]Diagram showing the analysis of the fragment obtained by PCR2 using an agarose gel. PCR2: Synthesis of hEPO mutein sequence [Diagram 3] Diagram showing in silico analysis of potential antigenicity of hEPO muteins and unmodified molecules using the IEDB database. Potentially weak / low antigenicity, potentially strong / high antigenicity, antigenicity (in silico) [Figure 4] Western blot confirming the insertion of a novel N-glycosylation site. [Diagram 5] Diagram showing the elution capacity of rhEPO molecule and its muteins by ELISA sandwich technique. Vertical axis: protein binding, horizontal axis: elution solution ■: Protocol A (elution capacity after formation of Ag-Ac complex) ■: Protocol B (degree of preservation of binding capacity to the molecule of interest after treatment of mAb with various eluents) [Figure 6] Diagram showing purification of hEPO mutein Mut104 via immunoaffinity chromatography (IAC) using mAb2B2. A-Full chromatogram obtained during purification of hEPO mutein Mut104. B-Scale-up of chromatogram A eluent. [Figure 7] Figure 1. Evaluation of mutein Mut104 purification via immunoaffinity chromatography (IAC) where 0.15 M glycine (pH 2.5) was used as eluent. Samples: 1 - inoculum, 2 - flowthrough, 3 - 1st wash, 4 - 2nd wash, 5 - 3rd wash, 6 - mix of eluted fragments, 7 - rhEPO standard (Zellitek SA). [Figure 8] Purity analysis of eluents obtained after immunoaffinity chromatography (IAC) of hEPO muteins. Samples: 1 - molecular weight markers, 2 - rhEPO standard (Zellitek SA), 3 - eluate Mut45 47, 4 - eluate Mut104, 5 - eluate Mut151 153. [Figure 9] Diagram showing the determination of apparent molecular weight of hEPO muteins. MM: molecular weight marker [Figure 10] IEF of hEPO muteins purified by immunoaffinity chromatography (IAC). Samples: 1-Mut45,47, 2-Mut104, 3-rhEPO standard (Zellitek SA). [Figure 11] ECZ electropherograms obtained for rhEPO and its muteins. A: Mut45 47, B: -Mut104, C: rhEPO. The term "peak" corresponds to each peak assigned to the ECZ electropherogram. The term "area (%)" refers to the percentage of each isoform calculated by integrating the area under the curve of each peak. [Figure 12a] Comparison of specific erythropoietic biological activities evaluated in vitro of hEPO muteins. [Figure 12b] Evaluation of the in vivo erythropoietic biological activity of hEPO muteins. The in vivo activity of hEPO muteins was evaluated in normocytemic mice (n=4). The mice were treated with rhEPO mass or hEPOmut (apropriate) or with PBS (control). The percentage of reticulocytes after treatment was quantified for each treatment. ***p<0.001 and ns (not significant) represent the degree of statistical significance after ANOVA and post-ANOVA Tukey test (n=4). [Figure 13] Cyto / neuroprotective activity of rhEPO and Mut104, performed with cell cultures of neuronal origin SH-SY5Y. Vertical axis: cytotoxicity (% relative to CSTP). CSTP: control with added staurosporine STP, Ccel: cell control (no added STP or mutein). *p<0.05 and **p<0.01 represent the degree of statistical significance after ANOVA test and post-ANOVA Dunnett test (n=3). [Figure 14] Representative diagram of neuronal development in primary cultures of hippocampal neurons. [Figure 15a]Assessment of neurite outgrowth through murine N2a cells. Representative images of each of the analyzed groups. [Figure 15b] Evaluation of neurite formation through murine N2a cells. Graphical representation resulting from evaluation of neurite parameters (mean length, longest neurite extension, mean number of neurites per neuron). *p<0.05 and **p<0.01 represent the degree of statistical significance after ANOVA test and post-ANOVA Bonferroni test (n=3). [Figure 16] Assessment of filopodia density using primary cultures of hippocampal neurons from rat embryos. *p<0.05 and **p<0.01 represent the degree of statistical significance after ANOVA test and post-ANOVA Bonferroni test (n=3). [Figure 17] Evaluation of synapse formation using primary cultures of hippocampal neurons from rat embryos. *p<0.05 and ***p<0.001 represent the degree of statistical significance after ANOVA test and post-ANOVA Bonferroni test (n=3). [Figure 18] Evaluation of the erythropoietic biological activity of hEPO muteins on primary native cultures at 11 days in vitro (DIV). Vertical axis: apoptosis (%). The in vitro neuroprotective activity of hEPO muteins (Mut) was evaluated in primary cultures of hippocampal neurons from Sprague-Dawlet rats at 11 days (DIV). Cultures were treated with an equal amount of rhEPO or muteins as apropriates or with pBS as control. Apoptosis was then induced by incubation with STP. Immunofluorescene was performed with Hoescht and Phalloidin-FITC reagents and the percentage of apoptotic nuclei was analyzed. ***p<0.001 and **p<0.01 and ns (not significant) represent the degree of statistical significance after ANOVA test and post-ANOVA Bonferroni test (n=3). [Figure 19] Table I is shown. [Figure 20] Table II is shown. [Figure 21] Table III is shown. [Figure 22] Table IV is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] As explained in the prior art, the aim of the present invention is to develop various strategies to reduce the erythropoietic activity of a cytokine, but maintain its neuroprotective and neurogenic potential. This is based on the application of chemical modifications to the molecule or the manipulation of its glycoside content. However, the prior art documents do not describe the use of glycosylation (also called "glycosylation") as a mechanism for blocking the molecular moieties involved in the causative interactions of EPO.

[0040] Therefore, the present invention provides a new modified EPO that solves the problems of the prior art while simultaneously achieving the following: *HEPOT stops / inhibits erythropoiesis in patients who require neuroprotective therapy, where EPO has deleterious effects. *Extending the half-life of hEPO, which improves in vivo biological activity and reduces the frequency of dosing.

[0041] Thus, the present invention includes the use of hyperglycosylated muteins derived from hEPO for the preparation of pharmaceutical compositions for use in the prevention or treatment of diseases or for the prevention of genetic predisposition to such diseases, in which neurogenesis or neuroprotection have beneficial advantages. The agents of the present invention can be administered to patients with (1) neurodegenerative diseases, (2) motor neuron diseases, (3) disability disorders, (4) developmental disorders, and (5) neurological disorders. Examples of neurodegenerative diseases (1) include Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Examples of motor neuron diseases (2) include Huntington's disease, spinocerebellar atrophy, and Creutz-Jakob disease. Examples of disability disorders (3) include depression and schizophrenia. An example of developmental disorder (4) is Down's syndrome. An example of neurological disorders (5) is brain injury and craniocerebral trauma.

[0042] As used herein, "mutein" refers to a protein modified by a site-directed mutation that involves the substitution of one or two amino acid residues to introduce consensus and potential sites for N-glycosylation.

[0043] The creation or addition of a consensus site for N-glycosylation involves the mutation of an amino acid at the site or region of the molecule in question. For a cell to perform the addition of a glycan, it is necessary that an Asn-Xaa-Ser / Thr site exists, where Xaa is any amino acid except Pro. Thus, if an Asn residue exists at this site (the site where you want to add an N-glycosylation site), the amino acid at the +2 position relative to Asn is mutated to Thr. This is much more efficient compared to Ser. As a result, the Asn residue is occupied by the glycan. Alternatively, if the site to be modified has an amino acid at the +2 position, which is evidence of the presence of Ser or Thr, this amino acid is mutated to Asn. Thus, there is always a conservative change.

[0044] The present invention discloses hEPO having modified amino acid structures that can achieve the following (i)-(iii): (i) Conservation of amino acid residues that constitute sites essential for molecular conformation or for the development of neuroprotective / neurogenic activity. (ii) Amino acid modifications proposed to abolish or stop erythropoietic activity. (iii) Generation of consensus sites for N-glycosylation.

[0045] Twelve hEPO muteins have been constructed that incorporate single or double amino acid modifications to incorporate consensus sites for N-glycosylation, resulting in mutants with abrogated erythropoietic (hematopoietic) activity, preserved neuroprotective / neurogenic activity, and superior plasma half-life.

[0046] The assays carried out to obtain the molecules of the invention are described below and are illustrative of the invention.

[0047] 1. Neutralizes erythropoiesis-stimulating activity death, Design and obtainment of hEPO-derived muteins that preserve neuroprotective / neurogenic activity. Twenty-six oligonucleotides were designed, two of which would amplify the complete new EPO sequence, and the remaining 24 (forward and reverse oligonucleotides) would introduce 12 point mutations.

[0048] According to the present invention, a modification is understood as a substitution of an amino acid at a given position (hereinafter also referred to as "position") with another amino acid at the same position. Thus, taking the following mutation 1 as an example, a lysine residue at position 45 is understood as being changed to an asparagine residue at the same position, and an asparagine at position 47 is understood as being changed to a threonine at the same position. Mutation 1: (Lys45 -^Asn45) + (Asn47 -^Thr47) A schematic of the method for generating hEPO muteins by site-directed mutagenesis is shown in FIG.

[0049] Using hEPO DNA as a template, and oligonucleotides for each mutant, oligonucleotide pMATEPOF (GCCGTGAAGGCCACGTGTCTTGTCCA) and oligonucleotide PMATEPOR (AGGCCAGTCTTGTGCTCCAGGTACCG), which bind complementary to the ends of the hEPO sequence, a first round of PCR was carried out to introduce each of the above mutations, resulting in 24 fragments, corresponding to the 12 hEPO muteins. The nucleotide and amino acid sequences of the 12 synthesized hEPO muteins are shown in paragraph 0115.

[0050] These are shown in Figure 2a. Using these fragments as templates, a second PCR is performed. This second PCR uses only the oligonucleotides that bind to the terminals of hEPO to obtain the sequences of the 12 mutants (Figure 2b). After this PCR, the products corresponding to the 12 hEPO muteins were digested with the restriction enzymes Xbal / Sall (which flank the hEPO molecule) and cloned in the pLV-PLK vector, digested with the same restriction enzymes. The top 10 bacteria were transformed and selected with ampicillin. 3-4 colonies of each hEPO mutein were amplified in culture and mini-prepared with plasmid DNA. The presence of the insert was then confirmed by restriction enzyme digestion of a site present in the hEPO sequence but not in the vector sequence. Finally, the mini-prepared with plasmid DNA was sequenced to confirm the insertion of the mutations made on the hEPO molecule.

[0051] 2. ^2_ ^ ^i2ico_ ^ ^nal^sis__o^_^2EPO_^rvuteins_J3otential antigenicity Using the IEDB database (Immune Epitope Database and Analysis Resources), an in silico analysis was performed to compare the antigenic potential of the hEPO muteins with that of the unmodified molecule, and for all of them, prediction of T epitopes, recognized by the Class II Major Histocompatibility Complex (MHCII), was performed by analyzing the eight most represented alleles worldwide (shown below). (HUMAN, HLA-DR: DRB1*01.01, DRB1*03.01, DRB1*04.01, DRB1*07.01, DRB1*08.01, DRB1*11.01 , DRB1*13.01, DRB1*15.01). Antigenicity scores were obtained for each hEPO variant and compared with the score obtained for hEPO. Their degree of antigenic potential is shown in Figure 3. Two muteins with the same level as hEPO (Mut98 100 and Mut151 153) were observed. Two muteins with greater antigenic potential (Mut72 74 and Mut62 64) were observed. Finally, eight muteins with lesser antigenic potential (Mut45 47, Mut49, Mut15 17, Mut65 67, Mut49, Mut76 78, Mut104, and Mut106 108) were observed. These last two were less antigenic.

[0052] 3. Qbtaininc^_cel^_ ^ lines_ ^ tha^_joroduce_ ^ the_^rientioned muteins Lentiviral particles were assembled and then transduced into CHO.K1 cells. For this purpose, HEK293 T / 17 cells (packaging cells) were co-transfected with the four vectors to generate the corresponding lentiviral particles for each of the 12 muteins. These four vectors are: (1) pREV, (2) pVSVG, (3) pMDL, and (4) the transfer vector pLV-PLK-MutX. (1) pREV induces the nuclear export of the transfer vector and its packaging. (2) pVSVG encodes the VSV envelope G protein, which is required for transport of virus particles into cells. This G protein has a broad tropism. (3) pMDL encodes any of the following: matrix and capside proteins capable of packaging the expression vector, proteases, reverse transcriptases, and integrases (enzymes required for degradation of structural elements and integration into the cellular genome). (4) In the transfer vector pLV-PKL-MutX, all viral genes are deleted and replaced with the gene of interest corresponding to the hEPO mutein of the present invention. Two days after transfection of HEK293T / 17 cells, the supernatant of each cell containing mutX lentiviral particles was harvested. This was used to transduce CHO.K1 cells. Thus, 12 CHO.K1mutXhEPO lines were obtained. After 72 hours, the supernatant was harvested, stored, and later characterized. Similarly, to generate stable recombinant cell lines, they were compacted with increasing amounts of puromycin antibiotic to select cells that integrated the transgene. The compacted cell lines were expanded and cryopreserved in a cryogenic mixture (90% (v / v) FBS, 10% (v / v) DMSO) and stored in a liquid nitrogen tank.

[0053] Initially, concentrations were determined via a sandwich ELISA technique and antibodies specific for the hEPO molecule, using a culture of a cell line obtained by transfection. A monoclonal antibody was used as capture and a rabbit polyclonal antibody was used for detection. Both types of antibodies were developed in the applicant's laboratory. The concentrations obtained (Table I) were considered sufficient to continue the study.

[0054] Furthermore, culture supernatants were analyzed by Western Blot technique using polyclonal antibodies to detect all of the hEPO muteins, which could be visualized as exhibiting a larger molecular weight compared to unmodified hEPO, consistent with the insertion of a new N-glycosylation site into the molecular structure (Fig. 4).

[0055] Similarly, we performed an isoelectric focusing method followed by Western blot analysis to examine the number of glycoisoforms in each of the hEPO muteins expressed in CHO.K1 cell culture supernatants. The number of isoforms obtained for 11 of the 12 muteins examined was greater than that of unmodified hEPO, i.e., the number of isoforms ranged from 8 to 16, compared with 7 isoforms observed in the culture supernatant of unmodified hEPO.

[0056] The hEPO of the present invention has low or almost no erythropoietic activity, which can avoid cytokine-induced side effects when used as a potential candidate for neuroprotection / neurogenesis. For this reason, the in vitro activity was studied using TF-1 cells, whose proliferation or growth depends on the presence of hEPO. To measure the erythropoietic activity, the proliferation of these cells was evaluated after 96 hours of stimulation with hEPO standards of known biological activity. All of the developed hEPO muteins (except Mut49, which maintains its erythropoietic activity) clearly showed almost no or reduced erythropoietic activity when compared to the commercially available hEPO standard and the unmodified EPO molecule. The results are summarized in Table I. NDA: no detectable activity; rhEPO: recombinant human EPO; Mut: mutein; Table I: Characterization of the hEPO muteins, quantification of their concentrations in culture supernatants by sandwich ELISA, assessment of the number of isoforms measured by isoelectric focusing, and determination of their in vitro biological activity expressed as a percentage compared to the unmodified molecule.

[0057] The present invention provides hEPO muteins containing sites susceptible to N-glycosylation at predetermined locations that do not abolish the cytoprotective biological activity but abolish the hematopoietic activity. They were expressed in CHO.K1 cell culture supernatants and subsequently characterized. All of them exhibit a higher degree of glycosylation, achieving the goal of including additional sites of N-glycosylation that extend the plasma half-life. Similarly, modifications made to the molecule were confirmed to have an effect on the hematopoietic biological activity, either eliminating this effect in most of the muteins or dramatically reducing it in the rest.

[0058] 4. Purificatio ^_o^j2EPO^derive^j vuteins_Jo^^ r] rvunoaffinit^ chromatography The purification process of the muteins of the present invention is as follows. To facilitate understanding of this process, the following muteins (Mut45 47, Mut104, Mut151 153) are shown as examples. This process follows the scheme of immunoaffinity (IA) purification. First, the equivalent process performed with the IA matrix was simulated (Protocol A). The aim was to establish the most favorable conditions for eluting each of the hyperglycosylated EPO muteins captured with the anti-rhEPO monoclonal antibody developed in the Applicant's laboratory. Based on the above, a sandwich ELISA assay was performed to quantify the proportion of hEPO derivatives, which remain bound to mAb2B2 after exposing the antigen-antibody complex to each elution condition. Similarly, the impact / effect of the elution solution on the antibodies was evaluated (Protocol B), in view of the future reuse procedure of the IA matrix. This was done to determine whether the elution solution affects the binding capacity to the hEPO muteins. For this reason be. The elution doses of the following solutions 1-22 were evaluated. 1. Glycine 0.1M pH 2, 2. Glycine 0.15M pH 2.5, 3. Acetic acid 0.2M, NaCl 0.15M pH 2.5, 4. Glycine 0.15M pH 3, 5. Citric acid 0.1M pH 3, 6. Acetic acid 0.2M, NaCl 0.15M pH 3, 7. Glycine 0.15M pH 3.5, 8. Sodium acetate 0.1M pH 4, 9. Sodium acetate 0.1M / dioxane 10% (v / v) pH 4, 10. Sodium acetate 0.1M pH 5, 11. Sodium phosphate 0.1M pH 6, 12. Isopropyl alcohol 40% (v / v) in phosphate buffered saline (PBS) pH 7 13. PBS pH 7, 14. Ethanol 40% (v / v) in PBS pH 7; 15. Dioxane 10% (v / v) in PBS pH 7; 16. Ethylene glycol 40% (v / v) in PBS pH 7; 17. Tris / HCl 0.1M pH 8, 18. Glycine 0.1M pH 9, 19. Glycine 0.1M pH 10, 20. Glycine 0.1M pH 11, 21. Sodium phosphate 0.1M pH 11, 22. Sodium phosphate 0.1M pH 11.7

[0059] The absorbance values ​​obtained for the antigen-antibody complexes treated with PBS served as a control, which assumed 100% formation of the antigen-antibody complex, i.e. no absorption of rhEPO or its muteins. In protocol B, the absorbance values ​​obtained for the antibodies treated with PBS (i.e. before formation of the Ag-Ac complex) served as a control for the preservation of the binding capacity of the antibodies to the molecules under study. The results obtained for the rest of the solutions tested were then expressed relative to the control evaluated in PBS (Figure 5).

[0060] In all cases, the condition of glycine 0.1 M pH 2 (solution 1) was observed to have the highest desorption capacity for antigens compared to other solutions. However, the complex formation capacity was significantly reduced after the treatment with it. Therefore, the use of this eluent in immunoaffinity chromatography (IAC) is not feasible because it affects the reuse of the chromatographic matrix.

[0061] On the other hand, this assay allows the selection of two eluent solutions as candidates for use in IAC: glycine 0.15 M pH 2.5 (solution 2) and acetic acid 0.2 M, NaCl 0.15 M pH 3 (solution 6). Both solutions are able to dissociate the Ag-Ac complex and remove the protein of interest (Table II), and moreover, do not affect the ability of the antibody to bind to the antigen after previous treatment with them. Table II: Elution of hEPO muteins considering selected eluent solutions (1) Glycine 0.15M pH 2.5 (6) 0.2M acetic acid, 0.15M NaCl, pH 3

[0062] The 2B2 mAb used in the IA resin was previously purified by protein A affinity chromatography and dialyzed against carbonate solution. The resin was then coupled to CNBr-activated Sepharose 4B. The degree of coupling was calculated by measuring the concentration of immunoglobulin in the solution, both before and after the immobilization reaction. It resulted in 96%. The theoretical dose was thus 481 μg rhEPO per ml of gel.

[0063] Figure 6 shows an example of a chromatographic profile of the purification of mutein Mut104. This was performed using a glycine eluent solution of 0.15 M pH 2.5.

[0064] The presence of hEPO variants was assessed in different fractions (flow-through, wash, elution) of each chromatographic process using a sandwich ELISA technique, and parameters analysing the process performance were calculated.

[0065] The results obtained for the purification are shown in Table III. Under dynamic conditions, some differences were observed in the recovery efficiency of the target protein compared to the static conditions evaluated on the plate. Elution with pH 2.5 solution resulted in an increased recovery efficiency for the muteins Mut104 and Mut151 153 compared to pH 3 solution (54% vs. 19% and 55% vs. 21%, respectively). On the other hand, for the mutein Mut45 47 variant, a better recovery rate (49% vs. 34%) was obtained with pH 3 solution. SolI: acetic acid 0.2M, NaCl 0.15M pH 3: Solution 6 SolII: Glycine 0.15M pH 2.5: Solution 2 Table III: Purification parameters for hEPO muteins via IAC

[0066] When the various steps of the chromatographic process of the mutein Mut104 were analyzed by SDS-PAGE and subsequently stained with Coomassie Brilliant blue dye (Figure 7), it was observed that most of the contaminants present in the sample did not remain but were part of the flow-through and wash, thus resulting in a high purity product. This is observed in lane 6 of Figure 7.

[0067] Although IAC did not provide a high recovery rate for the target protein, it was optimal for the purification of hEPO muteins because it allowed 37-90-fold purification over the starting sample, resulting in high purity and a single chromatographic step.

[0068] The purity of the eluates corresponding to each of the hEPO muteins was assessed by band densitometry (FIG. 8).

[0069] Prior to the electrophoretic run, samples were concentrated 75-80 times using a diafiltration cartridge with a 10 kDa cut-off point. The purity obtained was greater than 89% for all muteins (Table IV), a characteristic value for the IA purification procedure. Table IV: Purity of eluents obtained from each IAC

[0070] The results of this IAC showed high purity of the hEPO mutein, which was deemed suitable for use in the following tests. Thus, this IAC was established as a suitable, simple and practical purification method for the hEPO mutein of the present invention. Ph^sicochemica^_characterizatioi^_o^j2EPO_jivuteins

[0071] 5.1 Determination of apparent molecular weights of various hEPO muteins Figure 9 shows a Western blot of the IAC-purified mutein, which was used to calculate the molecular weight of the mutein using known molecular weight markers. The apparent molecular weights of the hyperglycosylated muteins of hEPO were determined by interpolating the migration distances of the front and rear front of the bands corresponding to each variant on the variation curve of the migration distance of each marker, according to the logarithm of the molecular weight. The calculated apparent molecular weights for each variant are as follows: * Mut45-47 :34-66kDa *Mut104: 29-66kDa *Mut151-153: 35-45kDa * rhEPO: 31-43 kDa This determination confirms that additional N-glycosylation sites have been successfully incorporated into the hEPO molecule, since the muteins represent, on average, a fractional molecular weight greater than that of unmodified hEPO.

[0072] 5.2 Determination of isoform profiles by IEF To assess the heterogeneity of hEPO due to glycosylation, specifically the amount of sialic acid residues, samples were evaluated by IEF to determine isoforms with different PIs that produce hyperglycosylated variants of hEPO.

[0073] Figure 10 shows Mut45-47 Thirteen isoforms are shown for the mutants producing it, 14 for the Mut104 mutant and six for the rhEPO standard, a hormone produced as a biological therapeutic agent favoring erythropoiesis. It is enriched in the most acidic isoform of the hEPO molecule. This isoform was obtained after a purification process developed in four chromatographic steps. In addition, Mut45-47and Mut104 muteins show 5-7 more acidic isoforms than the rhEPO isoforms, representing a higher content of sialic acid in the molecule.

[0074] The data obtained with the determination of apparent molecular weight, together with the data from IEF, confirm that: The muteins produced have a higher level of glycosylation when compared to rhEPO.

[0075] 5.3 Analysis of isoform profiles of IAC-purified hEPO muteins by means of Capillary Zone Electrophoresis (CZE):

[0076] CZE was used to determine the isoforms of each hEPO variant considered as an example in this application. This process was performed on samples obtained from IAC purification of hEPO muteins. The samples were diafiltered with water and concentrated to approximately 1 mg / ml.

[0077] CZE provides quantitative data regarding the various isoforms observed. Thus, using the CZE data, isoforms were assigned to the peaks observed in each hEPO variant. Mut45-47 and Mut104 were assigned 1-11, and rhEPO was assigned 1-7. Isoform 1 was the isoform migrating along most of the capillary region (Figure 11). The percentage of each isoform was then calculated by integrating the area under each peak.

[0078] When comparing the electrophoretic mobility of each variant isoform with that of rhEPO, the latter isoforms showed Mut45-47 isoforms 1-7 of Mut104 and isoforms 3-9 of Mut104. Mut45-47has four more acidic isoforms compared to rhEPO, and Mut104 has two less acidic and two more acidic isoforms compared to rhEPO.

[0079] When the ratio of each isoform was evaluated, rhEPO had a high ratio of isoforms 3, 4, and 5. Mut45-47 Mut104 has a high proportion of isoforms 6, 7, 8, and 9, while Mut104 has a high proportion of isoforms 4, 5, 6, and 7. This reaffirms that the heterogeneity / heterogeneity of each hEPO variant with respect to the degree of glycosylation of rhEPO is due to the high content and proportion of more acidic isoforms.

[0080] When observing the electrophoresis of each hEPO variant, the presence of two additional peaks (two more isoforms) was visible in both cases, one in front of peak 1 and the other in front of peak 11. They were not precisely known, but this could be due to the small proportion of this isoform in the samples (which was below the detection limit of the system), which explains the difference between the number of isoforms detectable by IEF and CZE.

[0081] 5.4a Characterization of the biological activity of muteins in in vitro erythropoiesis Biological characterization of the purified and engineered hEPO muteins was performed. For this purpose, in vitro proliferation assays were performed using UT-7 cell line cultures to evaluate the erythropoietic biological activity of the muteins taken as examples to illustrate the present invention (AF), since the survival and proliferation of these cell lines is dependent on the presence of hEPO in the growth medium. Unlike the assay using the TF-1 cell line, the assay using the UT-7 cell line is characterized by a highly sensitive response and was therefore selected for this stage of the process.

[0082] Culture supernatants of all muteins expressed from each producer cell line and of the three IAC-purified muteins were analyzed as examples, and the proliferation developed with these molecules was compared to that with the rhEPO standard.

[0083] The hEPO muteins considered as examples (except Mut49) showed low or no ability to stimulate cell proliferation when tested at the same concentration as the rhEPO standard (Fig. 12a). To calculate the specific erythropoietic activity (SEA) evaluated in vitro, we worked with higher concentrations of the muteins (i.e., those present in the pure culture supernatant). Thus, the muteins Mut104 and Mut151-153 showed a complete loss of biological activity in erythropoiesis. Mut45-47 ,Mut62 64, Mut98-100 showed very little erythropoietic biological activity. Mut45 -47 Mut49, on the other hand, appears to maintain this activity (SEA=261Ul / μg). The SEA of Mut49 is increased, but this is due to the zone of the curve used to determine this parameter, provided that the slope of the linear response zone of the Mut49 curve is significantly different from that of the standard. Thus, the modifications made to obtain such muteins are not considered to be effective in blocking erythropoietic activity.

[0084] Furthermore, the biological activity of purified rhEPO muteins in in vitro erythropoiesis was analyzed as an example of the present invention. Evaluations were performed to study the hemo / cytoprotective activity and protective capacity of rhEPO and its muteins, that is, their ability to protect SH-SY5Y neuronal cells from the apoptotic / cytotoxic effects produced by staurosporine (STP). Thus, the cyto / neuroprotective assay consisted in protecting neuronal cells. This protection was achieved by adding rhEPO or its muteins 12 hours before inducing cell damage caused by STP. After the induction of cell damage, the viability of the cultures was evaluated by determining the metabolically active cells.

[0085] In this assay, both rhEPO and purified Mut104, an example of the present invention, were used at the same concentrations (FIG. 13).

[0086] The results obtained were statistically analyzed to compare each sample with CSTP using the ANOVA test followed by Dunnett's test. The percentage of cytotoxicity obtained for CSTP was considered as 100% cytotoxicity. Therefore, the cytotoxicity values ​​determined for each sample were determined relative to CSTP.

[0087] As shown in Figure 13, rhEPO reduced STP-induced cytotoxicity by 45% (p<0.05), whereas Mut104 significantly reduced STP-induced cytotoxicity by 65%, with a significance of 99% (p<0.01).

[0088] 5.4b Characterization of the biological activity of muteins on in vivo erythropoiesis

[0089] The muteins used as examples of the present invention have low or almost no in vivo biological activity. In contrast, extensive glycosylation confers the necessary pharmacokinetic properties for extended residence time in blood. These features allow the molecule to improve its low receptor interaction capacity and exhibit erythropoietic activity.

[0090] To assess such in vivo activity, normocytic mice were injected with rhEPO as standard, or with each of the three muteins used in the previous example, or with PBS as a negative control. In all cases corresponding to protein inoculation, a mass of protein equivalent to 80 IU of rhEPO was used. Blood was collected 96 hours after inoculation and the percentage of thiazole orange-labeled reticulocytes was determined by flow cytometry.

[0091] Figure 12b shows significantly different responses (p<0.001) of each mutein versus the negative control with respect to rhEPO. No significant differences were seen when comparing the reticulocyte responses generated by each mutein to the negative control. Both analyses confirmed the expected loss of erythropoietic activity of the muteins as a result of N-glyco-engineering by the hyperglycosylation approach.

[0092] 5.5 Effects of hEPO hyperglycosylated muteins on structural neurogenesis 5.5.1 Neurodevelopment (differentiation) and structural formation Structural neuroplasia includes processes that stimulate or promote neuronal development or differentiation. In this sense, chemical agents or compounds that promote the following functions are considered neurotorophic compounds: formation of neuronal processes or axonal growth, development of fibrous / dendritic spines, and increased synapse number. Primary cultures of neurons and neuronal cell lines are widely used for the in vitro study of such processes.

[0093] FIG. 14 shows the stages of neural development in primary cultures of hippocampal neurons. These stages are well characterized in the article by Dotti et al. (1998) (Non-Patent Document 31), which describes that after seeding, neurons develop lamellae. stomach Now the neurons are attached to the substrate (in vitro culture day 0 (DIV)). Then, during the first 1-2 days (1-2 DIV), immature neurites grow as extensions of the plasma membrane. During 2-3 days (2-3 DIV), one of these neurites extends beyond the remaining one and differentiates to form an axon. This axon has a triangular structure at its end, known as the axonal growth cone. The neurite then branches out to form a second neurite (3-4 DIV). Thin cytoplamic projections grow onto these neurites containing cross-linked actin filaments known as filopodia (4-5 DIV). The filopodia then form dendritic spines. These spines are the preferred sites for synaptic development (7-21 DIV). Part of this study evaluated the neurotropic activity of various hEPO hyperglycosylated muteins of the invention at various stages of neurodevelopment.

[0094] 5.5.2 Formation of neurites To determine whether the muteins of the present invention exert a neuritogenic effect (increasing the length and number of neurites per neuron), the N2a neuronal cell line (neuroblastoma mouse) was used. Cells (50,000 cells) were seeded onto glass culture plates (24 wells) and maintained in DMEM complete medium, supplemented with 20% fetal bovine serum (FBS) to block neuronal differentiation and gentamicin as an antibiotic. The culture medium was then replaced with medium without FBS and supplemented with different concentrations (50 and 300 ng / ml) of rhEPO or the muteins for 3 hours to induce neurite formation (Non-Patent Document 32). After this, cells were fixed with 4% paraformaldehyde (PFA) and 4% sucrose in phosphate-buffered saline (PBS) for 10 min at 4°C and permeabilized with 0.1% (v / v) Triton X-100 in PBS for 2 min. The fixed cells were then blocked with 3% (w / v) bovine serum albumin (BSA) in PBS for 1-2 h. This included mouse IgG in 1% (w / v) BSA. anti Neuronal processes were labeled with mouse anti-alpha-tubulin IgG monoclonal antibody (1:1000, Sigma) for 16 h at 4°C. The next day, after three washes with PBS, they were incubated with rhodamine conjugated phalloidin to label actin filaments (1:1000, Invitrogen) and goat anti-mouse antibody conjugated to Alex488 (1:1000, Invitrogen) for 1 h at room temperature. After three washes with cold PBS for 5 min, the glass was mounted in a Fluorsave (Calbiochem) instrument.

[0095] To quantify neurite outgrowth, images were taken with a Nikon TE2000 epifluorescence microscope. Quantification was performed by counting the number of neurites per image, their average length (neurite extension), and the length of the longest neurite (axonal growth) in at least 30 neurons per condition. This was performed using the NeurtoJ plug-in of ImageJ (NIH). Representative images were then processed in Adobe Photoshop and statistical analysis was performed with the software GraphPadPrism 5.

[0096] Figure 15a shows representative images of each group studied. The statistical analysis of each measurement is detailed on the right. The muteins of the invention have a significant, dose-dependent neurogenic effect, which is manifested by an increase in the number of neurites per neuron, an increase in the length of neurites, and axonal growth. These influences / effects are comparable, giving values ​​similar to those obtained for rhEPO. In other words, the muteins of the invention showed a neurotrophic effect similar to that observed with rhEPO in the N2a cell line.

[0097] 5.5.3 Filopoda density Filopodia and dendritic spines are membrane processes enriched with actin filaments that emerge from neuronal processes / dendrites and act as post-synaptic compartments. These post-synaptic compartments are abundant in excitatoril synapses of the central nervous system. The morphology of these spines is variable and classified according to their differential structure. Dendritic spines can change their shape / structure during neurodevelopment, which is favorable for neurogenesis (33). In this sense, certain neurodegenerative diseases are associated with changes in the shape and quantity (both increases and decreases) of dendritic spines. For example, it has been determined that in Down's syndrome, the amount of spines is significantly reduced (34). In contrast, a higher amount of spines has been found in autism spectrum disorders (35).

[0098] Therefore, the neuronal plasticity potential of the hyperglycosylated muteins of the present invention by promoting filopodia (fibroblast) formation was determined. Primary cultures of hippocampal neurons were exposed to various concentrations (50 and 300 ng / ml) of rhEPO and the muteins Mut104, Mut45-47, and Mur151-153 for 4 days (4DIV).

[0099] Neuronal cultures were prepared from rat embryos hippocampus (E19) as described in Non-Patent Document 36. Briefly, tissues were treated with trypsin-EDTA (0.25% (w / v)) at 37°C for 15 min. A solution of fully dispersed cells was prepared in Neurobasal Medium (NB, Invitron) supplemented with 2 mM glutamine, 100 units / ml penicillin (PEN), 100 μg / ml streptomycin (Strp), and 10% horse serum. 20,000–30,000 cells were seeded in 24-well culture plates that had been pretreated with 0.1 mg / ml poly-L-lysine hydrobromide (Sigma) and 20 mg / ml laminin (Invitrogen). After 2 h, the medium was replaced with the prescribed medium. The designated media were NB:N2 and B27 (serum-free supplements from Invitrogen) containing 1 g / l ovalbumin, and different concentrations (50 and 300 ng / ml) of rhEPO or muteins of the present invention were added and maintained for 4 DIV. Cells were fixed with 4% (w / v) PFA and 4% (w / v) sucrose in PBS for 10 min at 4°C. Afterwards, cells were permeabilized with 0.1% (v / v) Triton X-100 in PBS for 2 min. The fixed cells were then permeabilized with 3% (v / v) bovine serum albumin (BSA) in PBS. They were blocked for 1-2 h and then incubated with mouse anti-alpha-tubulin IgG monoclonal antibody in 1% (w / v) BSA (1:1000, Sigma) for 16 h at 4° C. The next day, after washing three times with PBS, they were incubated with rhodamine-conjugated phalloidin to label actin filaments (1:1000, Invitrogen) and a second goat anti-mouse antibody conjugated to Alex488 (1:1000, Invitrogen) for 1 h at room temperature.After washing three times for 5 min with cold PBS, the glass was mounted in a Fluorsave (Calbiochem) apparatus.

[0100] To quantify filopodia (fibrosprout) formation, images were taken with a Nikon E600 epifluorescence microscope. Quantification was performed by counting the number of fibrosprouts present within a 20 μm neurite, up to a distance of 50 μm from the neuronal soma. A filopodia (fibrosprout) is an actin-rich process extending from the membrane of a neurite. A neuronal soma is at least 3 neurites / neurons within 30 nerves. Representative images were then processed in Adobe Photoshop and statistical analysis was performed with the software GraphPadPrism 5.

[0101] The left panel of Figure 16 shows representative images of fibrosprout density, whereas the right panel details the statistical analysis of each group. The muteins of the invention significantly induce fibrosprout formation, depending on the dose, when compared to control neurons (PBS). Surprisingly, the muteins of the invention showed a neurotrophic effect on primary cultures of hippocampal neurons superior to that observed with rhEPO.

[0102] 5.5.4 Synapse A synapse is defined as a specialized contiguity relation (union) of membranes between two neurons. This union, known as the synaptic cleft, facilitates the conduction of electrical impulses and the passage of materials from one synapse (pre-synapse) to another (post-synapse). Various techniques have been developed to quantify the number of synapses. The currently accepted definition of a synapse is the co-occurrence of protein clusters exclusive from the pre-synaptic compartment and protein clusters exclusive from the postsynaptic compartment.

[0103] Following the study of the neurotropic effects of the muteins of the invention, the ability of the muteins of the invention to induce neuronal synapses was evaluated. For this purpose, immunodetection assays were performed in 15-day (15DIV) primary neuronal cultures, in which synaptogenesis was detected by overlaying pre- and postsynaptic markers.

[0104] Immediately, neurons (15,000 cells / well) were treated with different concentrations (50 and 300 ng / ml) of hEPO or rhEPO or PBA for 15 days (DIV). Then, neurons were cultured in a given solution. 10 min at 4°CThe specified solution was 90% (V / V) methanol and 10% (V / V) MES (100 mM MES pH 6.9, 1 mM MEGTA, and 1 mM MgCl2). Neurons were then washed three times for 5 min with filtered PBS containing Tween 20 (0.1% (v / v)). Blocking was performed with a solution of FBS containing Triton X-100 (10% (v / v) FBS and 0.1% (v / v) Triton X-100 in PBS) for 1 h at room temperature. Neurons were then incubated with a solution of 3% (w / v) BSA in PBS for 15 min at room temperature. Both blocking solutions were pre-centrifuged at maximum speed for 10 min. Neurons were then incubated with mouse anti-NMDA-RI antibody and rabbit anti-synaptophysin antibody for 12-16 h at 4°C. The latter is a post-synaptic marker available from the same company. Both antibodies were prepared in 1% (w / v) PBS-BSA solution and centrifuged at maximum speed for 10 min. After three washes with PBS, neurons were blocked again for 1 h at room temperature in 3% (w / v) BSA, 10% (w / v) FBS solution, Triton X-100 (0.1% (V / V) in PBS, centrifuged for 10 min. Then neurons were incubated with a second anti-mouse antibody conjugated to Alex647 and an anti-rabbit antibody conjugated to Alex568 prepared in 1% (w / v) BSA and centrifuged at maximum speed for 10 min. Neurons were then mounted with fluorescence preservation.

[0105] Pictures were taken using an Olympus Fv1000 confocal microscope associated with an Olympus 1x81 inverted microscope. The images were subsequently processed with the software Fluoview (version 3.3 Olympus 60X objective; AN 1.42: 0.066 μm / Pixel resolution), which meets the Nyquist criterion.

[0106] Synaptogenesis was measured as colocalization points within 25 μm of neuronal dendrites between pre- and post-synaptic markers in approximately 10-20 neurons per condition using three segments per neuron. Colocalization points were determined using the Puncta analyzer and Image J plugin (version 1.28U) (37).

[0107] Figure 17 shows representative images of each condition tested and the corresponding quantification. As with neurogenesis and fibroblast induction, it was observed that the muteins significantly induced the formation of new synapses in neuronal cultures, an effect similar to that observed with rhEPO.

[0108] Considering the above, there is strong experimental evidence that the novel hyperglycosylated hEPO muteins of the present invention have neurogenesis-facilitating effects at various stages of neural development / differentiation (from neurite outgrowth to synaptogenesis). These effects are comparable to those observed with EPO, and in some cases, these effects (fibrogenic effects) are even greater. These surprising, novel and innovative technical features of the present invention make the muteins of the present invention ideal for use in the treatment of diseases in which neurogenesis is reduced or in cases where there is a genetic predisposition to such diseases.

[0109] 5.6b Study of the neuroprotective biological activity of rhEPO in primary cultures of rat hippocampal neurons The evaluation of the anti-apoptotic effect of hEPO muteins on primary cultures of hippocampal neurons, to study the effects these compounds have in cells whose metabolism is not altered as in established cell lines, constitutes an interesting model because it more realistically resembles what occurs in vivo in the brain.

[0110] A measure of the neuroprotective activity of the hEPO and modified EPO of the present invention is the ability of such molecules to protect neuronal cells from apoptotic stimuli induced by treatment with staurosporine.

[0111] To assess such in vitro activity, primary cultures of hippocampal neurons from Sprague-Dawley rats were obtained. Eleven-day (11 DIV) cultures were pretreated for 24 hours with 400 ng / ml of the hEPO muteins of the present invention or with 400 ng / ml of rhEPO used in hematopoietic restoration therapy. Cells were then exposed to 30 nM STP in the presence of this molecule for 24 hours and finally fixed and stained with Hoechst fluorochrome and Phalloidin-FITC.

[0112] Figure 18 shows that treatment with various molecules significantly reduces apoptosis when compared to cell death control treated with STP only (**p<0.01, ***P<0.001), but reaches apoptosis values ​​similar to cell control without STP treatment. Moreover, most of the molecules of the present invention show an effect superior to that of rhEPO, in terms of protecting primary cultures of hippocampal neurons from the effects / impact of STP.

[0113] These results also correspond to those obtained in the study of the neuroprotective activity of the muteins on SH-SY5 neuronal cultures. Therefore, the addition of additional glycosylation sites to obtain the muteins of the invention makes it possible to block hematopoietic biological activities without modifying those related to neuroprotection.

[0114] The above description is for one embodiment of the present invention, and those skilled in the art may conceive of various modifications of the present invention, all of which are within the technical scope of the present invention. The numbers in parentheses following the components of the claims correspond to the part numbers in the drawings and are provided for easy understanding of the invention, and should not be used to interpret the invention in a restrictive manner. In addition, even if the numbers are the same, the names of parts in the specification and the claims are not necessarily the same. This is for the reasons described above. With regard to the term "or," for example, "A or B" includes the selection of "both A and B" rather than "only A" or "only B." Unless otherwise specified, the number of devices or means may be singular or plural.

[0115] MUTEIN 15-17(Tyr15->Asn15)+(Leu17->Thr17) The primers were: Mut15_17F : GTGCTGGAAAGAAACCTGACGGAAGCCAAA MUT15_17R : TTTGGCTTCCGTCAGGTTTCTTTCCAGCAC Nucleotide sequence of MUTEIN 15-17 (SEQ ID No. 1) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG AAACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGTGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 15-17 (SEQ ID No. 2) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERNLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR MUTEIN 45-47(Lys45->Thr45)+(Asn47->Thr47) The primers were: Mut45_47F : CCCGACACCAACGTGACCTTCTACGCC Mut45_47R: GGCGTGAAGGTCACGTTGGTGTCGGG Nucleotide sequence of MUTEIN 45-47 (SEQ ID No. 3) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAACGTGACCTTCTACGCCTGGAAGCGGATGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 45-47 (SEQ ID No. 4) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTNVTFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR MUTEIN 49(Tyr49->Thr49) The primers were: Mut49F: AAAGTGAACTTCACCGCCTGGAAGCGG Mut49R : CCGCTTCCAGGCGGTGAAGTTCACTTT Nucleotide sequence of MUTEIN 49 (SEQ ID No. 5) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCACCGCCTGGAAGCGGATGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 49 (SEQ ID No. 6) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFTAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR MUTEIN 62-64(Glu62->Asn62)+(Trp64->Thr64) The primers were: Mut62_64F : CAGGCTGTGAACGTGACGCAGGACTG MUT62_64R : CAGTCCCTGCGTCACGTTCACAGCCTG Nucleotide sequence of MUTEIN 62-64 (SEQ ID No. 7) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGTGG AAGTGGGCCAGCAGGCTGTGAACGTGACGCAGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 62-64 (SEQ ID No. 8) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNPYAWKRMEVGQQAVNVTQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLTTLRALGAQKEAISPPDAAASAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR MUTEIN 65-67(Gln65->Asn65)+(Leu67->Thr67) The primer is the following: Mut65_67F : GAAGTGTGGAACGGAACGCTCTGCTG MUT65_67R : CAGCAGAGCCGTTCCGTTCCACACTTC MUTEIN 65-67 の nucleotide seksei (SEQ ID No. 9) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGCTCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGATGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGAACGGAACGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 65-67 (SEQ ID No. 10) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWNGTALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR MUTEIN 72-74(Glu72->Asn72)+(Val74->Thr74) The primers were: Mut72_74F : CTGCTGAGCAACGCTACGCTGAGAGGA MUT72_74R : TCCTCTCAGCGTAGCGTTGCTCAGCAG Nucleotide sequence of MUTEIN 72-74 (SEQ ID No. 11) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGATGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCAACGCTACGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 72-74 (SEQ ID No. 12) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSNATLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR MUTEIN 76-78(Arg76->Asn76)+(Gln78->Thr78) The primers were: Mut76_78F : GCTGTGCTGAACGGAACGGCCCTGCTC MUT76 78R : GAGCAGGGCCGTTCCGTTCAGCACAGC Nucleotide sequence of MUTEIN 76-78 (SEQ ID No. 13) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGTGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAACGGAACGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 76-78 (SEQ ID No. 14) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLNGTALLVNSSQPWEPLQLHV DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR MUTEIN 98-100(Ala98->Asn98)+(Ser100->Thr100) The primers were: Mut98_100F: GTGGACAAGAATGTGACCGGCCTGAGATCC MUT98_100R: GGATCTCAGGCCGGTCACATTCTTGTCCAC Nucleotide sequence of MUTEIN 98-100 (SEQ ID No. 15) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGATGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGAATGTGACCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 98-100 (SEQ ID No. 16) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKNVTGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR Mutein 104 (Ser104 -> Asn104) The primers were as follows: Mut104F: TCCGGCCTGAGAAACCTGACCACCCTG MUT104R: CAGGGTGGTCAGGTTTCTCAGGCCGGA Nucleotide sequence of Mutein 104 (SEQ ID No. 17) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGATGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGAAACCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 104 (SEQ ID No. 18) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRNLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR MUTEIN 106-108 (Thr106 ->Asn106)+(Leu108->Thr108) The primers were: Mut106 108F : AGATCCCTGAACACCACGCTGAGAGCA MUT106 108R : TGCTCTCAGCGTGGTGTTCAGGGATCT Nucleotide sequence of MUTEIN 106-108 (SEQ ID No. 19) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGTGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGAACACCACGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 106-108 (SEQ ID No. 20) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLNTTLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR MUTEIN 149 (Leu149 -^Thr149) The primers were: Mut149F: TACTCCAACTTCACGCGGGGCAAGCTG MUT149R: CAGCTTGCCCCGCGTGAAGTTGGAGTA Nucleotide sequence of MUTEIN 149 (SEQ ID No. 21) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGTGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCACGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 149 (SEQ ID No. 22) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFTRGKLKLY TGEACRTGDR MUTEIN 151-153 (Gly151 - Asn151) + (Leu153 -^Thr153) The primers were: Mut151_153F : AACTTCCTGCGGAACAAGACGAAGCTGTAC MUT151_153R : GTACAGCTTCGTCTTGTTCCGCAGGAAGTT Nucleotide sequence of MUTEIN 151-153 (SEQ ID No. 23) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGATGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGAACAAGACGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of MUTEIN 151 - 153 (SEQ ID No. 24) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRNKTKLY TGEACRTGDR HUMAN EPO SEQUENCE Nucleotide sequence of human EPO (SEQ ID No. 25) ATGGGCGTGCACGAATGTCCTGCTTGGCTGTGGCTGCTGCTGTCCCTGCTGTCTCTGCCTC TGGGACTGCCTGTGCTGGGCGCTCCTCCTAGACTGATCTGCGACTCCCGGGTGCTGGAAAG ATACCTGCTGGAAGCCAAAGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCC CTGAACGAGAATATCACCGTGCCCGACACCAAAGTGAACTTCTACGCCTGGAAGCGGATGG AAGTGGGCCAGCAGGCTGTGGAAGTGTGGCAGGGACTGGCTCTGCTGAGCGAGGCTGTGCT GAGAGGACAGGCCCTGCTCGTGAACTCCTCCCAGCCTTGGGAACCCCTGCAGCTGCACGTG GACAAGGCTGTGTCCGGCCTGAGATCCCTGACCACCCTGCTGAGAGCACTGGGAGCCCAGA AAGAGGCCATCTCTCCACCTGACGCCGCCTCTGCTGCTCCTCTGAGAACCATCACCGCCGA CACCTTCAGAAAGCTGTTCCGGGTGTACTCCAACTTCCTGCGGGGCAAGCTGAAGCTGTAC ACCGGCGAGGCTTGCCGGACCGGCGACAGA Amino acid sequence of hEPO (SEQ ID NO:26) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCS LNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHV DKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLY TGEACRTGDR

Claims

1. In modified human erythropoietin (hereinafter referred to as "mhEPO"), Its erythropoiesis-stimulating activity is less than 0.5% compared to that of human erythropoietin (hereinafter referred to as "hEPO"), but it maintains its neuroprotective or neurogenic ability; Mutations in the binding sites for homodimeric or heterodimeric receptors by the addition of consensus sites for glycosylation, The binding site to the receptor is selected from a set of amino acids included in any of positions 45-47, 104, 151-153, and combinations thereof; The modified human erythropoietin comprises an amino acid sequence selected from the set comprising SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:

24. A modified human erythropoietin comprising:

2. In modified human erythropoietin (hereinafter referred to as "mhEPO"), Its erythropoiesis-stimulating activity is less than 0.5% compared to that of human erythropoietin (hereinafter referred to as "hEPO"), but it maintains its neuroprotective or neurogenic ability; Mutations in the binding sites for homodimeric or heterodimeric receptors by the addition of consensus sites for glycosylation, The binding site to the receptor is selected from a set of amino acids included in any of positions 45-47, 104, 151-153, and combinations thereof; The modified human erythropoietin is encoded by a nucleotide sequence selected from the set including SEQ ID NO:3, SEQ ID NO:17, and SEQ ID NO:

23. The modified human erythropoietin according to claim 1.

3. The nucleotide sequence selected from the set comprising the following SEQ ID NOs: SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:23 A nucleic acid encoding a modified human erythropoietin,

4. The nucleotide sequence selected from the set comprising the following SEQ ID NOs: SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:23 A transformation vector comprising a nucleic acid encoding a modified human erythropoietin,

5. The nucleotide sequence selected from the set comprising the following SEQ ID NOs: SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:23 1. An expression vector comprising a nucleic acid encoding a modified human erythropoietin.

6. The nucleotide sequence selected from the set comprising the following SEQ ID NOs: SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:23 A lentiviral vector comprising a nucleic acid encoding a modified human erythropoietin,

7. The nucleic acid molecule includes a nucleotide sequence selected from the set including the following SEQ ID NOs: SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:23 A genetically modified cell comprising a nucleic acid molecule encoding a modified human erythropoietin.

8. The genetically modified cells include any of the following cell hosts: animal cell lines, plant cell lines, and cell hosts that allow for the addition of N-glycans. The genetically modified cell of claim 7.

9. The genetically modified cells include animal cell lines. The genetically modified cell of claim 7.

10. The animal cell line is selected from the group consisting of (1) to (5) below: (1) CHO. K1, (2) HEK293, (3) NSO, (4) BHK-21, (5) HeLa The genetically modified cell of claim 9.

11. A method for producing the modified human erythropoietin (hereinafter referred to as "mhEPO") according to claim 1, comprising: (A) providing a nucleic acid sequence encoding the mhEPO; The nucleic acid sequence is selected from the set comprising SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:23; (B) constructing a packaging cell co-transfection vector; The co-transfection vector comprises: (B1) a vector that allows lentiviral particles to enter cells; (B2) vectors encoding matrix protein, capsid, protease, reverse transcriptase and integrase; (B3) a transfer vector comprising the mhEPO sequence; (B4) Any of the vectors that induce nuclear export of the transfer vector, (C) co-transfecting the packaging cells; This produces lentiviral particles containing a nucleic acid sequence encoding the mhEPO, (D) harvesting the lentiviral particles produced by the packaging cells of step (C); and (E) transducing the mhEPO-expressing cells with the lentiviral particles of step (D); (F) selecting the cells of step (E) that contain a nucleic acid sequence encoding the mhEPO; (G) culturing the cells selected in step (F); As a result, they express the mhEPO, (H) isolating and purifying the mhEPO. have A method for producing the modified human erythropoietin according to claim 1.

12. In the step (E), the mhEPO-expressing cells are selected from the following groups (1) to (5): (1) CHO. K1, (2) HEK293, (3) NSO, (4) BHK-21, (5) HeLa 12. The method of claim 11 .

13. Step (H) is an immunoaffinity purification involving an anti-rhEPO antibody and an eluent.

12. The method of claim 11 .

14. The eluent is selected from the group comprising: Glycine, Acetic acid-NaCl, Acetate, Citric acid, Phosphate, Ethanol, Isopropyl alcohol, Dioxane, Ethylene glycol, Tris-HCl, mixtures thereof, 14. The method of claim 13.

15. The eluent is selected from the group consisting of glycine, acetic acid-NaCl.

14. The method of claim 13.

16. The eluent is selected from the group consisting of: (1) 0.1 M glycine (pH = 2); (2) 0.15 M glycine (pH = 2.5); (3) 0.2 M acetic acid, 0.5 M NaCl (pH = 3) 14. The method of claim 13.

17. A method for treating a disease state selected from the following, comprising administering to a patient a therapeutically effective amount of the modified human erythropoietin according to claim 1 or 2: Disabling diseases including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, motor neuron disease, Huntington's disease, spinocerebellar atrophy, Creutzfeldt-Jakob disease, depression and schizophrenia, developmental disorders including Down's syndrome, neurological damage due to cerebrovascular disease, and craniocerebral trauma. A pharmaceutical composition comprising:

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