United States Patent: 6,015,572
Inventors: Lin; Leu-Fen H. (Boulder, CO); Collins; Franklin D. (Agoura Hills, CA); Doherty; Daniel H. (Boulder, CO); Lile; Jack (Nederland, CO); Bektesh; Susan (Boulder, CO)Assignee: Amgen Inc. (Thousand Oaks, CA)
Appl. No.: 453176Filed: May 30, 1995
A novel neurotrophic factor referred to as glial cell line-derived neurotrophic factor (GDNF) has been identified and isolated from serum free growth 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. An implantable device containing recombinant GDNF secreting cells encapsulated in a semipermeable membrane may be used to treat nerve damage in patients suffering from disorders such as Parkinson's disease.
SUMMARY OF THE INVENTION
This invention relates to and claims substantially
purified glial cell lined derived neurotrophic factor (GDNF). In one
embodiment of this invention, substantially purified GDNF is obtained
having a specific activity at least about 24,000 times greater than the
specific activity of B49 conditioned medium. The substantially purified
GDNF has a specific activity of at least about 12,000 TU/.mu.g.
The substantially purified GDNF of the present invention has an apparent
molecular weight of about 31-42 kD on non-reducing SDS-PAGE, and about
20-23 kD on reducing SDS-PAGE. The substantially purified GDNF has an
amino terminal sequence comprised substantially of the amino acid sequence
(SEQ ID NO:1):
(Ser)-Pro-Asp-Lys-Gln-Ala-Ala-Ala-Leu-Pro-Arg-Arg-
Glu-(Arg)-Asn-( )-Gln-Ala-Ala-Ala-Ala-(Ser)-Pro-
(Asp)-(Asn).
The amino acid sequence of mature and "pre-pro" forms of rat GDNF is as set forth in FIGS. 13 and 14 (SEQ ID NO:3 and SEQ ID NO:4). The amino acid sequence of mature human GDNF is as set forth in the underlined portion of FIG. 19 (SEQ ID NO:6). The amino acid sequence of the pre-pro form of human GDNF is set forth in FIGS. 19 (SEQ ID NO:5) and 22 (SEQ ID NO:8).
One aspect of the invention is a method for obtaining purified GDNF comprising: 1) preparing a serum-free growth conditioned medium of B49 glioblastoma cells; 2) concentrating the conditioned medium; 3) performing heparin sepharose chromatography on the concentrated conditioned medium; 4) performing fast protein liquid chromatography on fractions obtained from said heparin sepharose chromatography; and 5) performing reverse-phase high-performance liquid chromatography on fractions obtained from said fast protein liquid chromatography. In-one embodiment, the method of obtaining purified GDNF is further comprised of the steps: 6) subjecting fractions obtained by reverse-phase high performance licuid chromatography to preparative SDS-PAGE; and 7) performing reverse-phase high-performance liquid chromatography on factions obtained by preparative SDS-PAGE.
Also described is the cloning of the rat GDNF gene from a cDNA library prepared from the B49 cell line. The nucleic acid sequence encoding mature and pre-pro rat GDNF is set forth in FIG. 13 (SEQ ID NO:3). The method for obtaining a human gene coding for GDNF is also disclosed. The nucleic acid sequence encoding mature human GDNF is as set forth in FIG. 19 (SEQ ID NO:5). The nucleic acid sequence encoding the first 50 amino acids of the pre-pro segment of human GDNF is as set forth in FIG. 22 (SEQ ID NO:8).
This invention also includes pharmaceutical compositions comprising an effective amount of purified GDNF in a pharmaceutically suitable carrier. Also described is a method for preventing or treating nerve damage which comprises administering to a patient in need thereof a therapeutically affective amount of GDNF. In preferred embodiments, the nerve damage is Parkinson's disease or damaged or improperly functioning dopaminergic nerve cells.
In the preferred embodiment of this invention, GDNF is produced by recombinant DNA methods, utilizing the genes coding for GDNF as described herein. The present invention includes a vector for use in producing biologically active GDNF comprised of expression regulatory elements operatively linked to a nucleic acid sequence coding for mature or pre-pro GDNF, and a host cell transformed by such a vector. Also included is a recombinant DNA method for the production of GDNF comprising: subcloning a DNA sequence coding for GDNF into an expression vector which comprises the regulatory elements needed to express the DNA sequence; transforming a host cell with said expression vector; culturing the host cells under conditions for amplification of the vector and expression of GDNF; and harvesting the GDNF.
A recombinant DNA method is described for the production of GDNF comprising: culturing the host cells of this invention under conditions for amplification of the vector and expression of GDNF; and harvesting the GDNF.
This invention includes substantially purified antibodies that recognize GDNF. Also included is a method for preventing or treating nerve damage which comprises implanting cells that secrete glial cell line derived neurotrophic factor into the body of patients in need thereof. Finally, the present invention includes a device for preventing or treating nerve damage by implantation into a patient comprising a semipermeable membrane, and a cell that secretes GDNF encapsulated within said membrane and said membrane being permeable to GDNF and impermeable to factors from the patient detrimental to the cells.
Claim 1 of 9 Claims
1. A device for treating nerve damage by implantation in a
patient comprising:
a membrane, and
one or more cells modified to express and secrete glial cell line-derived
neurotrophic factor encapsulated within said membrane, provided however,
said one or more cells are not B49 glioblastoma cells,
wherein said membrane is permeable to said glial cell line-derived
neurotrophic factor, and impermeable to immune system materials from said
patient which are detrimental to said cells, and
wherein said glial cell line-derived neurotrophic factor is a polypeptide
comprising the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:6
or a polypeptide which is at least 70% identical to an amino acid sequence
set forth in SEQ ID NO:4 or SEQ ID NO:6 when four gaps in a length of 100
amino acids may be introduced to assist in that alignment, and wherein
said glial cell line-derived neurotrophic factor has the ability to
promote the survival or function of nerve cells.
____________________________________________
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