Patent EA-003734-B1 (EA)
Title: Neurotrophic factors
Inventor: JOHANSEN TEIT E
IPC: C12Q1/6876
Filed: 1999-07-05
Abstract: 1. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: a) nucleotides 405 - 719 of SEQ ID NO. 1; b) a nucleotide sequence that hybridizes specifically to a complement sequence of nucleotides 405 - 719 of SEQ ID NO. 1 under high stringency conditions; wherein the nucleotide sequence codes for a neublastin polypeptide comprising the following: (i) seven conserved cysteine residues at positions 8, 35, 39, 72, 73, 101 and 103 when numbered in accordance with SEQ ID NO. 2; (ii) amino acid residues as follows: C at position 8, L at position 10, V at position 17, L at position 20, G at position 21, L at position 22, G at position 23, E at position 28, F at position 32, R at position 33, F at position 34, C at position 35, G at position 37, C at position 39, C at position 72, C at position 73, R at position 74, P at position 75, F at position 83, D at position 85, S at position 97, A at position 98, C at position 101 and C at position 103, each when numbered in accordance with SEQ ID NO. 2; (iii) an LGLG repeat, an FRFC motif, a QPCCRP motif, and a SATACGC motif; and (iv) an amino acid sequence having at least 90% sequence identity to AA1 - AA105 of SEQ ID NO. 2. 2. A nucleic acid comprising a nucleic acid sequence that is complementary to the nucleic acid of claim 1. 3. A vector comprising the nucleic acid of claim 1. 4. The vector of claim 3, wherein said vector is an expression vector. 5. A strain of cells transformed in vitro with the vector of claim 3 or 4. 6. The strain of claim 5, wherein said cells are selected from the group consisting of mammalian cells, fungal cells, yeast cells, insect cells and bacterial cells.] 7. The strain of claim 6, wherein said cells are Chinese hamster ovary cells. 8. The strain of claim 6, wherein said cells are cells derived from the mammalian central nervous system. 9. A nucleic acid molecule encoding a neublastin polypeptide, the nucleic acid molecule having the sequence set forth in SEQ ID NO. 29 or 30. 10. A vector comprising a nucleic acid of claim 9. 11. The vector of claim 10, wherein said vector is an expression vector. 12. A strain of cells transformed in vitro with the vector of claim 10 or 11. 13. The strain of claim 12, wherein said cells are bacterial cells. 14. An isolated neublastin polypeptide comprising the following: (a) seven conserved cysteine residues at positions 8, 35, 39, 72, 73, 101, and 103 when numbered in accordance with SEQ ID NO. 2; (b) amino acid residues as follows: C at position 8, L at position 10, V at position 17, L at position 20, G at position 21, L at position 22, G at position 23, E at position 28, F at position 32, R at position 33, F at position 34, C at position 35, G at position 37, C at position 39, C at position 72, C at position 73, R at position 74, P at position 75, F at position 83, D at position 85, S at position 97, A at position 98, C at position 101 and C at position 103, each when numbered in accordance with SEQ ID NO. 2; (c) an LGLG repeat, an FRFC motif, a QPCCRP motif, and a SATACGC motif; and (d) an amino acid sequence comprising at least 90% sequence identity to AA1 - AA105 of SEQ ID NO. 2. 15. The polypeptide of claim 14, wherein said polypeptide is glycosylated. 16. The polypeptide of claim 14, wherein said polypeptide is coded for by a nucleic acid of claim 1 or 2. 17. The polypeptide of any one of claims 14-16, wherein the polypeptide has a C-terminal amino acid sequence as set forth in AA72-AA105 of SEQ ID NO. 2. 18. The polypeptide of any one of claims 14-16, wherein the polypeptide has a C-terminal amino acid sequence as set forth in AA41-AA105 of SEQ ID NO. 2. 19. The polypeptide of any one of claims 14-16, wherein the conservative amino acid substitutions represent less than 10% of the total number of residues in the polypeptide. 20. The polypeptide of claim 19, wherein the conservative amino acid substitutions represent less than 2% of the polypeptide. 21. The polypeptide of claim 19 wherein the conservative amino acid substitutions represent a single amino acid substitution in the mature peptide sequence, wherein the both the substituted and replacement amino acid are non-cyclic. 22. A neublastin peptide having any of the following sequences: GPGSRARAAGARGC (AA 30-43 of SEQ ID NO. 9); LGHRSDELVRFRFC (AA 57-70 of SEQ ID NO. 9); CRRARSPHDLSL (AA 74-85 of SEQ ID NO. 9); LRPPPGSRPVSQPC (AA 94-107 of SEQ ID NO. 9); STWRTVDRLSATAC (AA 123-136 of SEQ ID NO. 9); CRLRSQLVPVRALGLGHRSDELVRFRFC (AA 43-70 of SEQ ID NO. 9); CRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC(AA 74-107 of SEQ ID NO. 9); CRPTRYEAVSFMDVNSTWRTVDRLSATAC (AA 108-136 of SEQ ID NO. 9); CRPTRYEAVSFMDVNST (AA 108-124 of SEQ ID NO. 9); and ALRPPPGSRPVSQPC (AA 93-107 of SEQ ID NO. 9). 23. A composition comprising the polypeptide of any one of claims 14-22 and a pharmaceutically acceptable carrier. 24. A method of administering the polypeptide of any one of claims 14-16, comprising the step of delivering said polypeptide to an in vitro cell culture or in vivo to a mammal. 25. The method of claim 24, wherein said administration comprises systemic administration. 26. The method of claim 24, wherein said mammal is afflicted with a condition selected from the group consisting of cerebral ischaemic neuronal damage, traumatic brain injury, peripheral neuropathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and memory impairment. 27. The method of claim 24, wherein said mammal is afflicted with a neuronal disorder of the peripheral nervous system, the medulla, or the spinal cord. 28. A use of the nucleic acid of claim 1 or 2 for the manufacture of a medicament for treatment of a neurodegenerative disease or disorder in an animal. 29. A use of the neublastin polypeptide of any one of claims 14-16 for the manufacture of a medicament for treatment of a neurodegenerative disease or disorder in an animal. 30. An antibody that specifically binds to any one of the polypeptides of any one of claims 14-16. 31. The antibody of claim 30, wherein said antibody is a monoclonal antibody.32. A method of determining whether a neurodegenerative disease or disorder in an animal is associated with an altered activity in a neublastin neurotrophic factor polypeptide, said method comprising the steps of: (a) contacting a biological sample from said animal with the antibody of claim 30 or 31, (b) determining whether an immune complex forms between said antibody and said protein, (c) comparing a level of said immune complex that forms in said sample with a level of said immune complex that forms in a corresponding biological sample from a patient lacking said neural condition, and (d) determining from said comparison whether said disease or disorder is associated with said altered level of activity in said neublastin neurotrophic factor polypeptide. 33. A strain of cells comprising an exogenous regulatory sequence cloned in operative association with an endogenous neublastin gene, said neublastin gene being capable of encoding the polypeptide of claim 14. 34. A method for producing a neublastin polypeptide of any one of claims 14-22, the method comprising growing cells of the strain of any one of claims 5-8, 12, 13 and 33 under conditions which allow expression of the polypeptide. 35. The method of claim 34, further comprising isolating the resulting polypeptide. 36. An isolated nucleic acid sequence comprising nucleotides 721-865 of SEQ ID NO. 1. 37. An antibody which specifically binds to any of the peptides of claim 22. 38. A kit comprising, in one or more containers, a substance selected from the group consisting of: (a) neublastin polypeptide according to any one of claims 14-22; (b) an antibody of any one of claims 30, 31 and 37; (c) one or more nucleic acid probes capable of hybridizing to neublastin RNA; or (d) at least one pair of nucleic acid primers capable of priming amplification of at least a portion of a neublastin gene. 39. A method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder characterized by an aberrant level of a neublastin polypeptide according to any one of claims 14-22, in a subject comprising measuring the level of said neublastin polypeptide, RNA encoding the neublastin polypeptide, or functional activity of the neublastin polypeptide in a sample derived from the subject in which an increase or decrease in the level of the neublastin polypeptide, neublastin RNA, or functional activity of neublastin polypeptide in the sample, relative to the level of the neublastin polypeptide, neublastin RNA or functional activity of neublastin found in an analogous sample not having the disease or disorder or a predisposition for developing the disease or disorder, indicates the presence of the disease or disorder or a predisposition for developing the disease or disorder. 40. A method for screening a purified neublastin polypeptide according to any one of claims 14-22, or derivative of fragment thereof, or a modulator of the activity of the foregoing, for activity in treating or preventing a disease, the method comprising: (a) measuring alterations in the phenotype, genotype, behavior, survival or proliferation of cells from a cell line or test animal, said cells or animal being derived from or display characteristics associated with the disease, and said cells or animals having been contacted with or administered with the neublastin polypeptide, derivative, fragment, or modulator, and (b) comparing said alterations with the phenotype, genotype, behavior, survival or proliferation in cells or animals not so contacted with or administered with the neublastin polypeptide, derivative, fragment, or modulator. 41. A method of treating peripheral neuropathies in a mammal, which method comprises administering a therapeutically effective amount of a neublastin polypeptide according to any one of claims 14-22, wherein said peripheral neuropathy is