Novel Neurotrophic Factor Protein and Uses Thereof

Abstract

The present invention discloses a novel neurotrophic factor protein, MANF2 and a genetic sequence encoding the same. The molecule will be useful in the development of a range of therapeutics and diagnostics useful in the treatment, prophylaxis and/or diagnosis of MANF2 dependent conditions. The molecule of the present invention is also a useful effector of primary and central neurons, especially dopaminergic neurons at the central nervous system and growth factor genes.

1 . An expression vector comprising a polynucleotide sequence comprising a promoter sequence capable of directing the expression of an operably linked polypeptide in a mammalian cell, said polypeptide comprising a MANF2 polypeptide or a functional fragment thereof. 
     
     
         2 . The vector according to  claim 1 , the vector being selected from the group consisting of HIV, SUV, FIV, EIAV, AAV, adenovirus, retrovirus, herpes virus. 
     
     
         3 . The vector according to  claim 2 , wherein the vector is a lentivirus vector. 
     
     
         4 . The vector according to  claim 1  for the treatment of treatment of chronic neurological disorders or a peripherial neuropathy including Alzheimer's disease, Parkinson's disease, epilepsy, drug addiction and ischemic brain injury. 
     
     
         5 . The vector according to  claim 1 , wherein said functional fragment is capable of preventing the 6-hydroxydopamine (6-OHDA)-induced degeneration of dopaminergic neurons. 
     
     
         6 . The vector according to  claim 5 , wherein said functional fragment is a MANF2 polypeptide without a signal peptide as shown in  FIG. 15  and has the amino acid sequence shown in SEQ ID NO:14. 
     
     
         7 . A pharmaceutical composition comprising the vector according to  claim 1  and one or more of pharmaceutically acceptable adjuvants, excipients, carriers and/or diluents. 
     
     
         8 . A method for treatment of chronic neurological disorders or a peripherial neuropathy including Alzheimer's disease, Parkinson's disease, epilepsy, drug addiction and ischemic brain injury, said method comprising administering to the central nervous system of an individual in need thereof a therapeutically effective amount of an expression vector, said vector comprising a promoter sequence capable of directing the expression of an operably linked polypeptide in a mammalian cell, said polypeptide comprising a MANF2 polypeptide or a functional fragment thereof. 
     
     
         9 . The vector according to  claim 8 , wherein said functional fragment is capable of preventing the 6-hydroxydopamine (6-OHDA)-induced degeneration of dopaminergic neurons. 
     
     
         10 . The vector according to  claim 8 , wherein said functional fragment is a MANF2 polypeptide without a signal peptide as shown in  FIG. 15  and has the amino acid sequence shown in SEQ ID NO:14 
     
     
         11 . A purified and isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2 of  FIG. 7  or a functional fragment thereof for use in gene therapy. 
     
     
         12 . The purified and isolated nucleic acid according to  claim 11 , wherein said gene therapy is for the treatment of a peripherial neuropathy including Alzheimer's disease, Parkinson's disease, epilepsy, drug addiction and ischemic brain injury. 
     
     
         13 . The purified and isolated nucleic acid according to  claim 11  comprising the nucleotide sequence shown in SEQ ID NO:1 of  FIG. 7 . 
     
     
         14 . The purified and isolated nucleic acid according to  claim 11 , wherein said functional fragment is a MANF2 polypeptide without a signal peptide as shown in  FIG. 15  and has the amino acid sequence shown in SEQ ID NO:14. 
     
     
         15 . An expression construct comprising the nucleic acid according to  claim 11  operatively linked to an expression control sequence, said expression construct capable of encoding a MANF2 polypeptide or functional fragments thereof for use in gene therapy of a peripherial neuropathy including Alzheimer's disease, Parkinson's disease, epilepsy, drug addiction and ischemic brain injury. 
     
     
         16 . An isolated and purified MANF2 polypeptide comprising the amino acid sequence of SEQ ID NO:2 of  FIG. 7  or a functional fragment thereof for the treatment of a peripherial neuropathy including Alzheimer's disease, Parkinson's disease, epilepsy, drug addiction and ischemic brain injury. 
     
     
         17 . The isolated and purified MANF2 polypeptide according to  claim 16 , wherein said functional fragment is a MANF2 polypeptide without a signal peptide as shown in FIG. 
     
     
         18 . Method of producing antibodies comprising:
 immunising a mammal with the isolated and purified MANF2 protein of  claim 16  or an antigenic fragment thereof.   
     
     
         19 . Use of the isolated and purified MANF2 protein of  claim 16  or an antigenic fragment thereof as an antigen. 
     
     
         20 . An antibody produced by the method of  claim 18 . 
     
     
         21 . The antibody of  claim 20  which is labeled with a detectable label. 
     
     
         22 . A kit of reagents for use in detecting the presence of MANF2 gene comprising a nucleotide sequence shown in SEQ ID NO:1 of  FIG. 7  or a functional fragment thereof in a biological sample, comprising
 a container; and in said container:   a compound, preferably labeled, capable of detecting said MANF2 gene; for the diagnosis of a peripherial neuropathy including Alzheimer's disease, Parkinson's disease, epilepsy, drug addiction and ischemic brain injury.   
     
     
         23 . The kit according to  claim 22 , wherein said compound is a primer or probe. 
     
     
         24 . The kit according to  claim 22 , wherein said compound is an antibody as defined in  claim 20 . 
     
     
         25 . The kit according to any one  claims 22 - 24  for assessing predisposition of an individual to a condition mediated by variation or dysfunction of MANF2. 
     
     
         26 . The kit according to  claim 22  further comprising instructions for using the kit. 
     
     
         27 . A transgenic non-human animal containing human or murine MANF2 gene as a transgene. 
     
     
         28 . A transgenic non-human animal containing a transgene or insertion disrupting expression of a MANF2 gene. 
     
     
         29 . A pharmaceutical compound comprising MANF2 nucleic acid molecule, MANF2 protein comprising the amino acid sequence of SEQ ID NO:2 of  FIG. 7  or a functional fragment thereof for the treatment of a peripherial neuropathy including Alzheimer's disease, Parkinson's disease, epilepsy, drug addiction and ischemic brain injury. 
     
     
         30 . The pharmaceutical compound according to  claim 29 , wherein said functional fragment is a MANF2 polypeptide without a signal peptide as shown in  FIG. 15 . 
     
     
         31 . Method for treatment of a condition dependent on MANF2 wherein a pharmaceutically effective amount of the compound of  claim 29  is administered to a patient in need of such treatment. 
     
     
         32 . The method according to  claim 31 , wherein said patient suffers or is at risk to suffer from a peripherial neuropathy. 
     
     
         33 . The method according to  claim 32 , wherein said peripherial neuropathy is associated with a systemic disease. 
     
     
         34 . The method according to  claim 31 , wherein said patient suffers or is at risk to suffer from Alzheimer's disease. 
     
     
         35 . The method according to  claim 31 , wherein said patient suffers or is at risk to suffer from Parkinson's disease. 
     
     
         36 . Method for affinity purification of receptor that binds to the MANF2 comprising the following steps: a) contacting a source of MANF2 receptor with an immobilized MANF2 under conditions whereby the MANF2 receptor to be purified is selectively adsorbed onto the immobilized MANF2; (b) washing the immobilized MANF2 and its support to remove non-adsorbed material; and (c) eluting the MANF2 receptor molecules from the immobilized MANF2 to which they are adsorbed with an elution buffer. 
     
     
         37 . Use of MANF2 nucleotide comprising the nucleotide sequence shown in SEQ ID NO:1 of  FIG. 7  or a functional fragment thereof for the manufacture of a gene therapy vector for the treatment of a peripherial neuropathy including Alzheimer's disease, Parkinson's disease, epilepsy, drug addiction and ischemic brain injury. 
     
     
         38 . Use of MANF2 polypeptide comprising the amino acid sequence of SEQ ID NO:2 of  FIG. 7  or a functional fragment thereof for the manufacture of a medicament for the treatment of a peripherial neuropathy including Alzheimer's disease, Parkinson's disease, epilepsy, drug addiction and ischemic brain injury.