Erythropoietin variants

Abstract

The present invention relates to novel endogenous variants of erythropoietin (EPO) and their use for treatment or prevention of a condition associated with tissue damage due to cell death (apoptosis, necrosis) and inflammation, in particular for neuroprotection, e.g. treatment of acute (for example stroke) and chronic disease (for example ALS) of the nervous system.

What is claimed is: 
     
         1 . An erythropoietin (EPO) variant encoding polynucleotide or the complementary strand thereof, wherein the polynucleotide is selected from the group consisting of:
 (a) polynucleotides encoding at least the mature form of the polypeptides termed hs3, h1-4, h1-5, hs4, h1-1, h2-1, mS, mG3, mG5, m301, mK3, ha, hAma, hAmE, hA-10 and ha-sequence without leader having the deduced amino acid sequence as shown in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22, 50, 51, 52, 53 and 61 respectively;   (b) polynucleotides having the coding sequence, as shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 55, 56, 57, 58 and 60 encoding at least the mature form of the polypeptide;   (c) polynucleotide encoding a humanized version of the polypeptides mS, mG3, mG5, m301 and mK3 having the deduced amino acid sequence as shown in SEQ ID NOs 14, 16, 18, 20, and 22,   (d) polynucleotides encoding a polypeptide comprising an amino acid sequence selected from the group of amino acid sequences as shown in SEQ ID NO 24, 26, 28, and 30 fused to the N-terminus of an amino acid sequence selected from the group of amino acid sequences as shown in SEQ ID NO 32, 34, 36, and 38;   (e) polynucleotides comprising a polynucleotide sequence selected from the group of polynucleotide sequences as shown in SEQ ID NO 23, 25, 27, and 29 fused to 5′ of a polynucleotide sequence selected from the group of polynucleotide sequences as shown in SEQ ID NO 31, 33, 35, and 37;   (f) polynucleotides encoding a derivative of a polypeptide encoded by a polynucleotide of any one of (a) to (e), wherein in said derivative between 1 and 10 amino acid residues are conservatively substituted compared to said polypeptide, and said derivative comprises APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80);   (g) polynucleotides encoding a fragment of a polypeptide encoded by a polynucleotide of any one of (a) to (f), wherein in said fragment between 1 and 10 amino acid residues are N- and/or C-terminally deleted, and said fragment comprises APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80);   (h) polynucleotides which are at least 95 % identical to a polynucleotide as defined in any one of (a) to (e) and which code for polypeptide comprising APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80); and   (i) polynucleotides encoding an EPO variant polypeptide, which comprises the N-terminal part of full length EPO including helix A comprising APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80) and which lacks at least one of the following:
 (1) a fragment of at least 20 amino acids between helix C and D; or 
 (2) a fragment of at least 10 amino acids of helix D; 
   (j) polynucleotides encoding a derivative of a polypeptide encoded by a polynucleotide of any one of (i), wherein in said derivative between 1 and 10 amino acid residues are conservatively substituted compared to said polypeptide, and said derivative comprises APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80).   
     
     
         2 . The polynucleotide of  claim 1  which is DNA, genomic DNA or RNA. 
     
     
         3 . A vector containing the polynucleotide of  claim 1 . 
     
     
         4 . The vector of  claim 3  in which the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic and/or eukaryotic host cells. 
     
     
         5 . A method for the treatment or prevention of a condition associated with tissue damage due to cell death comprising using a polypeptide having the amino acid sequence encoded by a polynucleotide, wherein the polynucleotide is selected from the group consisting of:
 (a) polynucleotides encoding at least the mature form of the polypeptides termed hs3, h1-4, h1-5, hs4, h1-1, h2-1, mS, mG3, mG5, m301, mK3, ha, hAma, hAmE, hA-10 and ha-sequence without leader having the deduced amino acid sequence as shown in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22, 50, 51, 52, 53 and 61 respectively;   (b) polynucleotides having the coding sequence, as shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 55, 56, 57, 58 and 60 encoding at least the mature form of the polypeptide;   (c) polynucleotide encoding a humanized version of the polypeptides mS, mG3, mG5, m301 and mK3 having the deduced amino acid sequence as shown in SEQ ID NOs 14, 16, 18, 20, and 22,   (d) polynucleotides encoding a polypeptide comprising an amino acid sequence selected from the group of amino acid sequences as shown in SEQ ID NO 24, 26, 28, and 30 fused to the N-terminus of an amino acid sequence selected from the group of amino acid sequences as shown in SEQ ID NO 32, 34, 36, and 38;   (e) polynucleotides comprising a polynucleotide sequence selected from the group of polynucleotide sequences as shown in SEQ ID NO 23, 25, 27, and 29 fused to 5′ of a polynucleotide sequence selected from the group of polynucleotide sequences as shown in SEQ ID NO 31, 33, 35, and 37;   (f) polynucleotides encoding a derivative of a polypeptide encoded by a polynucleotide of any one of (a) to (e), wherein in said derivative between 1 and 10 amino acid residues are conservatively substituted compared to said polypeptide, and said derivative comprises APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80);   (g) polynucleotides encoding a fragment of a polypeptide encoded by a polynucleotide of any one of (a) to (f), wherein in said fragment between 1 and 10 amino acid residues are N- and/or C-terminally deleted, and said fragment comprises APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80);   (h) polynucleotides which are at least 95 % identical to a polynucleotide as defined in any one of (a) to (e) and which code for polypeptide comprising APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80); and   (i) polynucleotides encoding an EPO variant polypeptide, which comprises the N-terminal part of full length EPO including helix A comprising APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80) and which lacks at least one of the following:
 (1) a fragment of at least 20 amino acids between helix C and D; or 
 (2) a fragment of at least 10 amino acids of helix D; 
   (j) polynucleotides encoding a derivative of a polypeptide encoded by a polynucleotide of any one of (i), wherein in said derivative between 1 and 10 amino acid residues are conservatively substituted compared to said polypeptide, and said derivative comprises APPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80).   
     
     
         6 . A method according to  claim 5 , wherein said cell death is induced by ischemia, hypoxia, bacterial infection, viral infection, autoimmunologically, traumatically, chemically induced, or radiation induced. 
     
     
         7 . A method according to  claim 5 , wherein said condition is an acute or chronic neurodegenerative and/or neuroinflammatory disorder, is an acute or chronic disorder of the heart, lung, kidney, liver or pancreas or said condition is associated with an organ or cell transplantation. 
     
     
         8 . A method according to  claim 7 , wherein said acute neurodegenerative and/or neuroinflammatory disorder is selected from the group consisting of cerebral ischemia or infarction including embolic occlusion and thrombotic occlusion, reperfusion following acute ischemia, perinatal hypoxic-ischemic injury, cardiac arrest, intracranial hemorrhage, subarachnoidal hemorrhage and intracranial lesions, spinal cord lesions, intravertebral lesions, whiplash shaken infant syndrome, infectious encephalitis, meningitis, headache. 
     
     
         9 . Method according to  claim 7 , wherein said chronic neurodegenerative and/or neuroinflammatory disorder is selected from the group consisting of dementias, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multiple sclerosis, multiple system atrophy, chronic epileptic conditions associated with neurodegeneration, motor neuron diseases, degenerative ataxias, cortical basal degeneration, ALS-Parkinson's Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies, primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease/spinocerebellar ataxia type 3 and olivopontocerebellar degenerations, Gilles De La Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sach's disease, Sandhoff disease, familial spastic disease, spastic paraparesis, progressive multifocal leukoencephalopathy, familial dysautonomia (Riley-Day syndrome), polyneuropathies, prion diseases, addiction, affective disorders, schizophrenic disorders, chronic fatigue syndrome, chronic pain. 
     
     
         10 . A method according to  claim 5 , wherein said condition is aging. 
     
     
         11 . A method according to  claim 5 , wherein the medicament is administered prior to or after the onset of said condition.