Glial cell line-derived neurotrophic factor

Abstract

A novel neurotrophic factor referred to as glial derived neurotrophic factor (GDNF) has been identified and isolated from serum free growth conditioned medium of B49 glioblastoma cells. Rat and human genes encoding GDNF have been cloned and sequenced. A gene encoding GDNF has been subcloned into a vector, and the vector has been used to transform a host cell in order to produce biologically active GDNF in a recombinant DNA process.

1 . Substantially purified glial derived neurotrophic factor.  
     
     
         2 . The substantially purified glial derived neurotrophic factor of  claim 1  having a specific activity of at least about 12,000 TU/μg.  
     
     
         3 . The substantially purified glial derived neurotrophic factor of  claim 1  having a specific activity at least about 24,000 times greater than the specific activity of B49 conditioned medium.  
     
     
         4 . The substantially purified glial derived neurotrophic factor of  claim 1  having a molecular weight of about 31-42 kD on non-reducing SDS-PAGE.  
     
     
         5 . The substantially purified glial derived neurotrophic factor of  claim 4  comprising a dimeric polypeptide species having a specific activity of at least about 12,000 TU/μg.  
     
     
         6 . The substantially purified glial derived neurotrophic factor of  claim 1  having a molecular weight of about 20-23 kD on reducing SDS-PAGE.  
     
     
         7 . The substantially purified glial derived neurotrophic factor of  claim 6  comprising a monomeric polypeptide sequence.  
     
     
         8 . The substantially purified glial derived neurotrophic factor of  claim 1  comprising an amino acid sequence as follows: (Ser)-Pro-Asp-Lys-Gln-Ala-Ala-Ala-Leu-Pro-Arg-Arg-Glu-(Arg)-Asn-( )-Gln-Ala-Ala-Ala-Ala-(Ser)-Pro-(Asp)-(Asn)  
     
     
         9 . The substantially purified glial derived neurotrophic factor of  claim 1  comprising the amino acid sequence of mature rat glial derived neurotrophic factor as set forth in FIG. 14 (SEQ ID NO:4).  
     
     
         10 . The substantially purified glial derived neurotrophic factor of  claim 1  comprising the amino acid sequence of mature human glial derived neurotrophic factor as set forth in FIG. 19 (SEQ ID NO:5).  
     
     
         11 . A substantially purified glial derived neurotrophic factor characterized by the following: 
 (a) an apparent molecular weight of about 31-42 kD on non-reducing SDS-PAGE;    (b) an apparent molecular weight of about 20-23 kD on reducing SDS-PAGE; and    (c) a specific activity of at least about 12,000 TU/mg.    
     
     
         12 . The substantially purified protein glial derived neurotrophic factor of  claim 11  further characterized by comprising the amino acid sequence of mature rat glial derived neurotrophic factor as set forth in FIG. 14 (SEQ ID NO:4)  
     
     
         13 . The substantially purified protein glial derived neurotrophic factor of  claim 11  further characterized by comprising the amino acid sequence of mature human glial derived neurotrophic factor as set forth in FIG. 19 (SEQ ID NO:5).  
     
     
         14 . The substantially purified glial derived neurotrophic factor of  claim 11  produced by recombinant DNA technology.  
     
     
         15 . The substantially purified glial derived neurotrophic factor of  claim 1  produced by recombinant DNA technology.  
     
     
         16 . The substantially purified glial derived neurotrophic factor of  claim 15  comprising the amino acid sequence of mature rat glial derived neurotrophic factor as set forth in FIG. 14 (SEQ ID NO:4).  
     
     
         17 . The substantially purified glial derived neurotrophic factor of  claim 15  comprising the amino acid sequence of mature human glial derived neurotrophic factor as set forth in FIG. 19 (SEQ ID NO:5).  
     
     
         18 . A pharmaceutical composition comprising an effective amount of substantially purified glial derived neurotrophic factor in a pharmaceutically suitable carrier.  
     
     
         19 . The pharmaceutical composition of  claim 18  wherein said factor is comprised of the amino acid sequence of mature rat glial derived neurotrophic factor as set forth in FIG. 14 (SEQ ID NO:4).  
     
     
         20 . The pharmaceutical composition of  claim 18  wherein said factor is comprised of the amino acid sequence of mature human glial derived neurotrophic factor as set forth in FIG. 19 (SEQ ID NO:5).  
     
     
         21 . The pharmaceutical composition of  claim 18  wherein said factor is produced by recombinant DNA technology.  
     
     
         22 . The pharmaceutical composition of  claim 21  wherein said factor is comprised of the amino acid sequence of mature rat glial derived neurotrophic factor as set forth in FIG. 14 (SEQ ID NO:4).  
     
     
         23 . The pharmaceutical composition of  claim 21  wherein said factor is comprised of the amino acid sequence of mature human glial derived neurotrophic factor as set forth in FIG. 19 (SEQ ID NO:5).  
     
     
         24 . A method for obtaining substantially purified glial derived neurotrophic factor comprising: 
 preparing a serum-free growth-conditioned medium of B49 glioblastoma cells; concentrating the conditioned medium;    performing heparin sepharose chromatography on the concentrated conditioned medium;    performing fast protein liquid chromatography or fractions obtained from said heparin sepharose chromatography; and    performing reverse-phase high-performance liquid chromatography or fractions obtained from said fast protein liquid chromatography.    
     
     
         25 . The method of  claim 24  further comprising: 
 subjecting fractions obtained by reverse-phase high performance liquid chromatography to preparative SDS-PAGE; and  
 performing reverse-phase high-performance liquid chromatography on fractions obtained by preparative SDS-PAGE.  
 
     
     
         26 . A purified and isolated nucleic acid sequence encoding glial derived neurotrophic factor.  
     
     
         27 . The nucleic acid sequence of  claim 26  comprised of the rat nucleic acid sequence encoding mature rat glial derived neurotrophic factor as set forth in FIG. 13 (SEQ ID NO:3).  
     
     
         28 . The nucleic acid sequence of  claim 26  comprised of the human nucleic acid sequence encoding mature human glial derived neurotrophic factor as set forth in FIG. 19 (SEQ ID NO:5).  
     
     
         29 . A purified and isolated nucleic acid sequence encoding pre-pro glial derived neurotrophic factor.  
     
     
         30 . The nucleic acid sequence of  claim 28  comprised of the human nucleic acid sequence encoding pre-pro rat glial derived neurotrophic factor as set forth in FIG. 13 (SEQ ID NO:3).  
     
     
         31 . The nucleic acid sequence of  claim 28  comprised of the human nucleic acid sequence encoding pre-pro human glial derived neurotrophic factor as set forth in FIGS.  19  (SEQ ID NO:5) and  22  (SEQ ID NO:8).  
     
     
         32 . The nucleic acid sequence of  claim 26  encoding mature rat glial derived neurotrophic factor.  
     
     
         33 . The nucleic acid sequence of  claim 32  encoding the amino acid sequence of mature rat glial derived neurotrophic factor as set forth in FIG. 14 (SEQ ID NO:4).  
     
     
         34 . The nucleic acid sequence of  claim 26  encoding mature human glial derived neurotrophic factor.  
     
     
         35 . The nucleic acid sequence of  claim 34  encoding the amino acid sequence of mature human glial derived neurotrophic factor as set forth in FIG. 19 (SEQ ID NO:5).  
     
     
         36 . The nucleic acid sequence of  claim 26  encoding GDNF selected from the group consisting of: 
 (a) a nucleic acid sequence which encodes the amino acid sequence for pre-pro rat GDNF set forth in FIG. 13 (SEQ ID NO:3);  
 (b) a nucleic acid sequence which encodes the amino acid sequence for mature rat GDNF set forth in FIG. 13 (SEQ ID NO:3);  
 (c) a nucleic acid sequence which encodes the amino acid sequence for pre-pro human GDNF set forth in FIGS.  19  (SEQ ID NO:5) and 22 (SEQ ID NO:8);  
 (d) a nucleic acid sequence which encodes the amino acid sequence for mature human GDNF set forth in FIG. 19 (SEQ ID NO:5);  
 (e) a nucleic acid sequence which encodes an amino acid sequence with dopaminergic activity, and said amino acid sequence is recognized by an antibody which binds to a portion of GDNF; and  
 (f) a nucleic acid sequence which (1) hybridizes to the complementary sequence of (a), (b), (c) or (d) and (2) encodes an amino acid sequence with dopaminergic activity.  
 
     
     
         37 . A method for preventing or treating nerve damage which comprises administering to a patient in need thereof a therapeutically effective amount of glial derived neurotrophic factor.  
     
     
         38 . The method of  claim 37  wherein said nerve damage is Parkinson's disease.  
     
     
         39 . A pharmaceutical composition for preventing or treating Parkinson's disease comprising an effective amount of glial derived neurotrophic factor in a pharmaceutically suitable carrier.  
     
     
         40 . A pharmaceutical composition for preventing or treating damaged or improperly functioning dopaminergic nerve cells comprising an effective amount of glial derived neurotrophic factor in a pharmaceutically suitable carrier.  
     
     
         41 . A method for preventing damage to or treating damaged or improperly functioning dopaminergic nerve cells which comprises administering to a patient in need thereof a therapeutically effective amount of glial derived neurotrophic factor.  
     
     
         42 . A recombinant DNA molecule comprising expression regulatory elements operatively linked to a nucleic acid sequence encoding glial derived neurotrophic factor.  
     
     
         43 . A host cell transformed with the vector of  claim 42 .  
     
     
         44 . A recombinant DNA method for production of glial derived neurotrophic factor comprising: 
 (a) subcloning a DNA sequence encoding for glial derived neurotrophic factor into an expression vector which comprises the regulatory elements needed to express the DNA sequence;    (b) transforming a host cell with said expression vector;    (c) culturing the host cells under conditions for amplification of the vector and expression of glial derived neurotrophic factor; and    (d) harvesting the glial derived neurotrophic factor from the host cell culture.    
     
     
         45 . The recombinant DNA method of  claim 44  wherein said host cell is an animal cell.  
     
     
         46 . The recombinant DNA method of  claim 45  wherein said host cell is COS-7 cells.  
     
     
         47 . The recombinant DNA method of  claim 44  wherein said host cell is a bacterial cell.  
     
     
         48 . The recombinant DNA method of  claim 47  wherein said host cell is  E. coli.    
     
     
         49 . The recombinant DNA method of  claim 48  further comprising the step of refolding the harvested glial derived neurotrophic factor.  
     
     
         50 . A recombinant DNA method for production of glial derived neurotrophic factor comprising: 
 (a) culturing the host cell of  claim 43  under conditions for amplification of the vector and expression of glial derived neurotrophic factor; and    (b) harvesting the glial derived neurotrophic factor from the host cell culture.    
     
     
         51 . The recombinant DNA method of  claim 50  wherein said host cell is an animal cell.  
     
     
         52 . The recombinant DNA method of  claim 51  wherein said host cell is COS-7 cells.  
     
     
         53 . The recombinant DNA method of  claim 50  wherein said host cell is a bacterial cell.  
     
     
         54 . The recombinant DNA method of  claim 53  wherein said host cell is  E. coli.    
     
     
         55 . The recombinant DNA method of  claim 54  further comprising the step of refolding the harvested glial derived neurotrophic factor.  
     
     
         56 . Substantially purified glial derived neurotrophic factor prepared according to the method of  claim 24 .  
     
     
         57 . Substantially purified glial derived neurotrophic factor prepared according to the method of  claim 55 .  
     
     
         58 . Substantially purified glial derived neurotrophic factor prepared according to the method of  claim 44 .  
     
     
         59 . Substantially purified glial derived neurotrophic factor prepared according to the method of  claim 50 .  
     
     
         60 . Substantially purified antibodies that recognize glial derived neurotrophic factor.  
     
     
         61 . The antibodies of  claim 60  wherein said antibodies are monoclonal.  
     
     
         62 . The antibodies of  claim 60  wherein said antibodies are polyclonal.  
     
     
         63 . A method for preventing or treating nerve damage which comprises implanting cells that secrete glial derived neurotrophic factor into the body of patients in need thereof.  
     
     
         64 . The method of  claim 63  wherein said patient is suffering from Parkinson's disease.  
     
     
         65 . The method of  claim 63  wherein said cells are the cells of  claim 42 .  
     
     
         66 . The method of  claim 63  wherein said cells are naturally occurring cells that secrete glial derived neurotrophic factor.  
     
     
         67 . The method of  claim 63  wherein said cells are maintained within a biocompatible, semipermeable membrane.  
     
     
         68 . The method of  claim 63  wherein said glial derived neurotrophic factor is comprised of the amino acid sequence of mature human glial derived neurotrophic factor as set forth in FIG. 19 (SEQ ID NO:5).  
     
     
         69 . The method of  claim 63  wherein said glial derived neurotrophic factor is comprised of the amino acid sequence of mature rat glial derived neurotrophic factor as set forth in FIG. 14 (SEQ ID NO:4).  
     
     
         70 . A device for preventing or treating nerve damage by implantation in a patient comprising: 
 a semipermeable membrane; and    a cell that secretes glial derived neurotrophic factor encapsulated within said membrane,    said membrane being permeable to said glial derived neurotrophic factor, and impermeable to factors from said patents detrimental to said cells.    
     
     
         71 . The device of claim  70  wherein said cells are the cells of  claim 42 .  
     
     
         72 . The device of claim  70  wherein said cells are naturally occurring cells that secrete glial derived neurotrophic factor.  
     
     
         73 . The device of claim  70  wherein said glial derived neurotrophic factor is comprised of the amino acid sequence of mature human glial derived neurotrophic factor as set forth in FIG. 19 (SEQ ID NO:5).  
     
     
         74 . The device of claim  70  wherein said glial derived neurotrophic factor is comprised of the amino acid sequence of mature rat glial derived neurotrophic factor as set forth in FIG. 14 (SEQ ID NO:4).