Glial cell line-derived neurotrophic factor

• US 6,362,319 B1
• Filed: 05/26/1995
• Issued: 03/26/2002
• Est. Priority Date: 09/20/1991
• Status: Expired due to Term

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Lin, Leu-Fen H., Lile, Jack, Collins, Franklin D., Bektesh, Susan, Doherty, Daniel H.
• Primary Examiner(s): Kunz, Gary L.
• Assistant Examiner(s): HAYES, ROBERT CLINTON
• Application Number:
  US08/452,229
• Time in Patent Office: 2,496 Days
• Field of Search: 530/350, 530/402, 530/399, 514/2, 514/12, 514/8, 930/10
• US Class Current: 530/399
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 9/0085 Brain, e.g. brain implants; Spinal cord
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/26 Psychostimulants, e.g. nicotine, cocaine
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: Y10S 530/809 Fused cells, e.g. hybridoma
• CPC Class Codes: Y10S 530/839 Nerves; brain

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.

Claims

1. A purified and isolated glial cell line-derived neurotrophic factor (GDNF) polypeptide encoded by a polynucleotide, wherein said polynucleotide:

(a) comprises nucleotides 304 through 705 of SEQ ID NO;

3 or nucleotides 105 through 506 of SEQ ID NO;

5;

or

(b) encodes a polypeptide comprising an amino sequence set forth in SEQ ID NO;

4 or SEQ ID NO;

6;

or

(c) hybridizes to an oligonucleotide probe fully complementary to a polynucleotide encoding the amino acid sequence of SEQ ID NO;

4 under conditions comprising hybridizing in 6X SSPE, 0.1% SDS and 30% formamide at 42°

C., followed by washing in 2X SSPE and 0.1% SDS at room temperature and twice in 0.1 x SSPE, 0.1% SDS preheated to 50°

C. and wherein said polypeptide promotes dopamine uptake in dopaminergic neorons;

or

(d) hybridizes to an olignucleotide probe fully complementary to a polynucleotide encoding the amino acid sequence of SEQ. ID. NO;

6 under conditions comprising hybridizing in 6X SSPE and 0.1% SDS at 50°

C., followed by washing in 2X SSPE and 0.1% SDS at room temperature and twice in 0.1 x SSPE, 0.1% SDS preheated to 50°

C. and wherein said polypeptide promotes dopamine uptake in dopaminergic neurons.

2. A polypeptide according to claim 1 which is glycosylated.

3. A polypeptide according to claim 1 which is non-glycosylated.

4. A polypeptide according to claim 3, further comprising a methionine residue at the amino terminal when said polypeptide is expressed in a bacterial expression system.

5. A polypeptide according to claim 1 which is produced by recombinant technology or chemical synthesis.

6. A polypeptide according to claim 1 which is modified by attachment of one or more polymeric moieties.

7. A polypeptide according to claim 6, wherein the polymeric moiety is polyethylene glycol.

19. A composition comprising a glial cell line-derived neurotrophic factor polypeptide according to claim 1 or 8 and a pharmaceutically acceptable carrier or excipient.

20. The composition of claim 19 wherein said glial cell line-derived neurotrophic factor polypeptide is in the form of a dimer.

8. A purified and isolated glial cell line-derived neurotrophic factor polypeptide prepared according to the method comprising the steps of:

(a) culturing a host cell under conditions suitable for the expression of glial cell line-derived neurotrophic factor polypeptide by said host cell; and

(b) optionally, isolating said polypeptide;

wherein said host cell expresses a polynucleotide encoding said polypeptide, wherein said polynucleotide;

comprises nucleotides 304 through 705 of SEQ ID NO;

3 or nucleotides 105 through 506 of SEQ ID NO;

5;

or

(ii) encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO;

4 or SEQ ID NO;

6;

or

(iii) hybridizes to an oligonucleotide probe fully complementary to a polynucleotide encoding the amino acid sequence of SEQ ID NO;

4 under conditions comprising hybridizing in 6X SSPE, 0.1% SDS and 30% formamide at 42°

C., followed by washing in 2X SSPE and 0.1% SDS at room temperature and twice in 0.1 x SSPE, 0.1% SDS preheated to 50°

C. and wherein said polypeptide promotes dopamine uptake in dopaminergic neurons;

or

(iv) hybridizes to an oligonucleotide probe fully complementary to a polynucleotide encoding the amino acid sequence of SEQ. ID. NO;

6 under conditions comprising hybridizing in 6X SSPE and 0.1% SDS at 50°

C., followed by washing in 2X SSPE and 0.1% SDS at room temperature and twice in 0.1 x SSPE, 0.1% SDS preheated to 50°

C. and wherein said polypeptide promotes dopamine uptake in dopaminergic neurons.

9. A purified and isolated glial cell line-derived neurotrophic factor polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence comprising nucleotides 304 through 705 of SEQ ID NO:

3 or nucleotides 105 through 506 of SEQ ID NO;

5 wherein said polypeptide promotes dopaminc uptake in dopaminergic neurons.

10. A purified and isolated glial cell line-derived neurotrophic factor polypeptide comprising an amino acid sequence of SEQ ID NO:

4 or SEQ ID NO;

6 wherein said polypeptide promotes dopamine uptake in dopaminergic neurons.

14. A polypeptide according to claim 8, 9, 10, 12 or 13 further comprising a methionine residue at the amino terminal when said polypeptide is produced by means of a bacterial expression system.

15. A polypeptide according to claim 8, 9, 10, 12, or 13 which is glycosylated.

16. A polypeptide according to claim 8, 9, 10, 12 or 13 which is non-glycosylated.

17. A polypeptide according to claim 8, 9, 10, 12 or 13 which is modified by attachment of one or more polymeric moieties.

18. A purified and isolated glial cell line-derived neurotrophic factor polypeptide of claim 8, 9, 10, 12 or 13 wherein said polypeptide is in the form of a dimer.

21. A composition comprising a glial cell line-derived neurotrophic factor polypeptide according to claim 8, 9, 10, 12 or 13 and a pharmaceutically acceptable carrier or excipient.

22. The composition of claim 21 wherein said glial cell line-derived neurotrophic factor polypeptide is in the form of a dimer.

11. A purified and isolated glial cell line-derived neurotrophic factor polypeptide, comprising the amino acid sequence set forth in SEQ ID NO:

6 wherein said polypeptide promotes dopamine uptake in dopaminergic neurons.

12. A purified and isolated glial cell line-derived neurotrophic factor polypeptide comprising an amino acid sequence encoded by a polynucleotide which hybridizes to an oligonucleotide probe fully complementary to a polynucleotide encoding the amino acid sequence of SEQ ID NO:

4 under conditions comprising hybridizing in 6X SSPE, 0.1% SAS and 30% formamide at 42°

C., followed by washing in 2X SSPE and 0.1% SDS at room temperature and twice in 0.1 x SSPE, 0.1% SDS preheated to 50°

C. wherein said polypeptide promotes dopamine uptake in dopaminergic neurons.

13. A purified and isolated glial cell line-derived neurotrophic factor polypeptide comprising an amino acid sequence encoded by a polynucleotide which hybridizes to an oligonucleotide probe fully complementary to a polynucleotide encoding the amino acid sequence of SEQ. ID. NO:

6 under conditions comprising hybridizing in 6X SSPE and 0.1% SDS at 50°

C., followed by washing in 2X SSPE and 0.1% SDS at room temperature and twice in 0.1X SSPE, 0.1% SDS preheated to 50°

C. and wherein said polypeptide promotes dopamine uptake in dopaminergic neutrons.