U.S. patent application number 13/167419 was filed with the patent office on 2011-10-13 for methods of using polynucleotides encoding truncated glial cell line-derived neurotrophic factor.
This patent application is currently assigned to Amgen Inc.. Invention is credited to Shaw-Fen Sylvia Hu.
Application Number | 20110251267 13/167419 |
Document ID | / |
Family ID | 24135310 |
Filed Date | 2011-10-13 |
United States Patent
Application |
20110251267 |
Kind Code |
A1 |
Hu; Shaw-Fen Sylvia |
October 13, 2011 |
METHODS OF USING POLYNUCLEOTIDES ENCODING TRUNCATED GLIAL CELL
LINE-DERIVED NEUROTROPHIC FACTOR
Abstract
Disclosed are novel proteins, referred to as truncated glial
cell line-derived neurotrophic factor (truncated GDNF) proteins,
that promote dopamine uptake by dopaminergic cells and promote the
survival of nerve cells. Also disclosed are processes for obtaining
the truncated GDNF proteins by recombinant genetic engineering
techniques.
Inventors: |
Hu; Shaw-Fen Sylvia;
(Thousand Oaks, CA) |
Assignee: |
Amgen Inc.
Thousand Oaks
CA
|
Family ID: |
24135310 |
Appl. No.: |
13/167419 |
Filed: |
June 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10853930 |
May 25, 2004 |
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13167419 |
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09687993 |
Oct 13, 2000 |
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10853930 |
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08535681 |
Sep 28, 1995 |
6184200 |
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09687993 |
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Current U.S.
Class: |
514/44R ;
435/375 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/475 20130101; A61P 25/16 20180101; A61P 43/00 20180101;
A61P 25/00 20180101 |
Class at
Publication: |
514/44.R ;
435/375 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61P 25/16 20060101 A61P025/16; C12N 5/00 20060101
C12N005/00 |
Claims
1. A method for promoting the survival or function of dopaminergic
neurons comprising administering a polynucleotide encoding a
truncated glial cell line-derived neurotrophic factor (GDNF)
protein product comprising an amino acid sequence of Cys.sup.41
through Cys.sup.133 of SEQ ID NO:2.
2. A method according to claim 1 wherein said polynucleotide
encodes a truncated GDNF protein product of the formula:
X-[Cys41-Cys133]-Y wherein [Cys41-Cys133] is Cys41 through Cys133
of SEQ ID NO: 2, Y is absent or a carboxy-terminus amino acid
residue of Ile134, and X represents a methionylated or
nonmethionylated amine group of Cys41 or amino-terminus amino acid
residue(s) selected from the group: TABLE-US-00004 G RG NRG KNRG
(SEQ ID NO: 3) GKNRG (SEQ ID NO: 4) RGKNRG (SEQ ID NO: 5) QRGKNRG
(SEQ ID NO: 6) GQRGKNRG (SEQ ID NO: 7) RGQRGKNRG (SEQ ID NO: 8)
RRGQRGKNRG (SEQ ID NO: 9) G RRGQRGKNRG (SEQ ID NO: 10) KG
RRGQRGKNRG (SEQ ID NO: 11) GKG RRGQRGKNRG (SEQ ID NO: 12) RGKG
RRGQRGKNRG (SEQ ID NO: 13) SRGKG RRGQRGKNRG (SEQ ID NO: 14) NSRGKG
RRGQRGKNRG (SEQ ID NO: 15) ENSRGKG RRGQRGKNRG (SEQ ID NO: 16)
PENSRGKG RRGQRGKNRG (SEQ ID NO: 17) SPENSRGKG RRGQRGKNRG (SEQ ID
NO: 51) NPENSRGKG RRGQRGKNRG (SEQ ID NO: 18) ANPENSRGKG RRGQRGKNRG
(SEQ ID NO: 19) A ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 20) AA
ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 21) AAA ANPENSRGKG RRGQRGKNRG
(SEQ ID NO: 22) QAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 23) RQAAA
ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 24) NRQAAA ANPENSRGKG RRGQRGKNRG
(SEQ ID NO: 25) RNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 26)
ERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 27) RERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 28) RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID
NO: 29) P RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 30) LP
RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 31) VLP RRERNRQAAA
ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 32) AVLP RRERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 33) MAVLP RRERNRQAAA ANPENSRGKG RRGQRGKNRG
(SEQ ID NO: 34) QMAVLP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO:
35) KQMAVLP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 36)
DKQMAVLP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 37) and
PDKQMAVLP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 38)
3. A method according to claim 1 or 2 wherein X is selected from
the group consisting of SEQ ID NOS: 3, 7, 8, 14, 17, 18, and
24.
4. A method according to claim 1 or 2 wherein X is G, RG or
NRG.
5. A method according to claim 1 or 2 wherein said GDNF protein
product has the amino acid sequence of SEQ ID NO:42.
6. A method according to claim 1 or 2 wherein said GDNF protein
product has the amino acid sequence of SEQ ID NO:44.
7. A method according to claim 1 or 2, wherein said GDNF protein
product has the amino acid sequence of SEQ ID NO:46.
8. A method according to claim 1 or 2 wherein administering a
polynucleotide is ex vivo administering.
9. A method according to claim 4 wherein administering a
polynucleotide is ex vivo administering.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/853,930, filed May 25, 2004, now pending which is a
continuation of U.S. application Ser. No. 09/687,993, filed Oct.
13, 2000, now abandoned, which is a divisional of U.S. application
Ser. No. 08/535,681, now issued U.S. Pat. No. 6,184,200, which are
hereby incorporated by reference.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled A-357-US-CNT4_SeqList.txt, created Jun. 23, 2011,
which is 27,306 bytes in size. The information in the electronic
format of the Sequence Listing is incorporated herein by reference
in its entirety.
FIELD OF THE INVENTION
[0003] In general, the present invention relates to proteins,
referred to herein as glial cell line-derived neurotrophic factors
(also referred to as glial derived neurotrophic factor or GDNF),
that are characterized by the ability to promote dopamine uptake by
dopaminergic neurons and support the survival of the neurons that
die in Parkinson's Disease. The present invention more specifically
relates to a novel truncated GDNF proteins.
BACKGROUND OF THE INVENTION
[0004] Neurotrophic factors are proteins, found in the nervous
system or in non-nerve tissues innervated by the nervous system,
whose function is to promote the survival and maintain the
phenotypic differentiation of nerve and/or glial cells (Varon et
al., Ann. Rev. Neuroscience 1:327, 1979; Thoenen et al., Science
229:238, 1985). Because of this physiological role, neurotrophic
factors are useful in treating the degeneration of nerve cells and
the loss of differentiated function that occurs in a variety of
neurodegenerative diseases.
[0005] In order for a particular neurotrophic factor to be
potentially useful in treating nerve damage, the class or classes
of damaged nerve cells must be responsive to the factor. Different
neurotrophic factors typically affect distinctly different classes
of nerve cells. Therefore, it is advantageous to have on hand a
variety of different neurotrophic factors to treat each of the
classes of damaged neurons that may occur with different forms of
disease or injury.
[0006] Neurotrophic factors can protect responsive neurons against
a variety of unrelated insults. For example, nerve growth factor
(NGF) will rescue a significant portion of sensory neurons from
death caused by cutting their axonal processes (Rich et al., J.
Neurocytol 16:261, 1987; Otto et al., J. Neurosci. 83:156, 1987),
from ontogenetic death during embryonic development (Hamburger et
al., J. Neurosci. 4:767, 1984), and from damage caused by the
administration of taxol or cisplatin (Apfel et al., Ann. Neurol.
29: 87, 1991). This apparent generality of protection has led to
the concept that if a neurotrophic factor protects responsive
neurons against experimental damage, it may be useful in treating
diseases that involve damage to those neurons in patients, even
though the etiology may be unknown.
[0007] A given neurotrophic factor, in addition to having the
correct neuronal specificity, must be available in sufficient
quantity to be used as a pharmaceutical treatment. Since
neurotrophic factors are typically present in small amounts in
tissues (e.g., Hofer and Barde Nature 331:261, 1988; Lin et al.,
Science 246:1023, 1989), it would be inconvenient to prepare
pharmaceutical quantities of neurotrophic factors directly from
animal tissues. As an alternative, it is desirable to use a
recombinant expression system to produce the desired protein.
[0008] Lin et al. previously described a method for screening
biological samples for neurotrophic activity on the embryonic
precursors of the substantia nigra dopaminergic neurons (see U.S.
patent application Ser. No. 08/182,183 filed May 23, 1994, now U.S.
Pat. No. 7,226,758, issued Jun. 15, 2007, and its parent
applications; PCT/US92/07888 filed Sep. 17, 1992 (WO 93/06116); and
European Patent Application No. 92921022.7 (Publication No. EP 610
254); the disclosures of which are hereby incorporated by
reference). This bioassay is useful in identifying neurotrophic
factors which may be used in treating Parkinson's disease (Friedman
et al., Neuro. Sci. Lett. 79:65-72, 1987) as the disease is
characterized by the degeneration of dopaminergic neurons in the
midbrain that innervate the striatum.
[0009] Lin et al. further described the characterization of a new
neurotrophic factor that was purified from one such source, the
conditioned culture medium from a glioblastoma cell line, B49
(Schubert et al., Nature 249:224-27, 1974). The conditioned medium
from this cell line was previously reported to contain dopaminergic
neurotrophic activity (Bohn et al., Soc. Neurosci. Abs. 15:277,
1989). Prior to the disclosure of Lin et al., glial cell
line-derived neurotrophic factor (GDNF) had not been identified as
a discrete biologically active substance or isolated as a
substantially pure protein. In addition, Lin et al. described
processes for cloning human genes encoding GDNF, the nucleic acid
sequence of the human genes that encode GDNF and the amino acid
sequences of the GDNF protein. The GDNF gene was subcloned into an
expression vector, and the vector was used to express biologically
active GDNF. The GDNF protein is a homodimer composed of two 134
amino acid, 22 kDa, subunits joined by disulfide bond. The
description further included the use of GDNF for preventing and
treating nerve damage and nerve related diseases such as
Parkinson's disease.
[0010] GDNF therapy is helpful in the treatment of nerve damage
caused by conditions that compromise the survival and/or proper
function of one or more types of nerve cells. Such nerve damage may
occur from a wide variety of different causes. Nerve damage may
occur to one or more types of nerve cells by: (1) physical injury,
which causes the degeneration of the axonal processes and/or nerve
cell bodies near the site of injury; (2) temporary or permanent
cessation of blood flow to parts of the nervous system, as in
stroke; (3) intentional or accidental exposure to neurotoxins, such
as chemotherapeutic agents (e.g., cisplatinum) for the treatment of
cancer or dideoxycytidine (ddC) for the treatment of AIDS; (4)
chronic metabolic diseases, such as diabetes or renal dysfunction;
or (5) neurodegenerative diseases such as Parkinson's disease,
Alzheimer's disease, and Amyotrophic Lateral Sclerosis (ALS), which
result from the degeneration of specific neuronal populations.
[0011] GDNF therapy could be particularly helpful in the treatment
of neurodegenerative conditions involving the degeneration of the
dopaminergic neurons of the substantia nigra, such as Parkinson's
disease. The only current treatments for Parkinson's disease are
palliative, aiming at increasing dopamine levels in the striatum.
The expected impact of GDNF therapy is not simply to produce an
increase in the dopaminergic neurotransmission at the dopaminergic
nerve terminals in the striatum (which will result in a relief of
the symptoms), but also to slow down, or even stop, the progression
of the degenerative processes and to repair the damaged
nigrostriatal pathway and restore its function. GDNF may also be
used in treating other forms of damage to or improper function of
dopaminergic nerve cells in human patients. Such damage or
malfunction may occur in schizophrenia and other forms of
psychosis. The only current treatments for such conditions are
symptomatic and require drugs which act upon dopamine receptors or
dopamine uptake sites, consistent with the view that the improper
functioning of the dopaminergic neurons which innervate these
receptor-bearing neuronal populations may be involved in the
disease process.
SUMMARY OF THE INVENTION
[0012] In one aspect, the present invention provides novel
truncated glial cell line-derived neurotrophic factor (GDNF)
protein products. In one embodiment, truncated GDNF proteins are
produced by recombinant genetic engineering techniques. In an
alternative embodiment, the truncated GDNF proteins are synthesized
by chemical techniques, or a combination of the recombinant and
chemical techniques.
[0013] The truncated GDNF protein products of the present invention
include the proteins represented by the amino acid sequence
X-[Cys.sup.41-Cys.sup.133]-Y. The amino acid residue numbering
scheme of FIG. 1 (SEQ ID NO:2) is used to facilitate comparison to
the mature GDNF protein. [Cys.sup.41-Cys.sup.133] represents the
amino acid sequence of Cys.sup.41 through Cys.sup.133 as depicted
in FIG. 1 (SEQ ID NO:2). Y represents the carboxy terminal group of
Cys.sup.133 or a carboxy-terminus amino acid residue of
Ile.sup.134. X represents a methionylated or nonmethionylated amine
group of Cys.sup.41 or amino-terminus amino acid residue(s)
selected from the group:
TABLE-US-00001 G RG NRG KNRG (SEQ ID NO: 3) GKNRG (SEQ ID NO: 4)
RGKNRG (SEQ ID NO: 5) QRGKNRG (SEQ ID NO: 6) GQRGKNRG (SEQ ID NO:
7) RGQRGKNRG (SEQ ID NO: 8) RRGQRGKNRG (SEQ ID NO: 9) G RRGQRGKNRG
(SEQ ID NO: 10) KG RRGQRGKNRG (SEQ ID NO: 11) GKG RRGQRGKNRG (SEQ
ID NO: 12) RGKG RRGQRGKNRG (SEQ ID NO: 13) SRGKG RRGQRGKNRG (SEQ ID
NO: 14) NSRGKG RRGQRGKNRG (SEQ ID NO: 15) ENSRGKG RRGQRGKNRG (SEQ
ID NO: 16) PENSRGKG RRGQRGKNRG (SEQ ID NO: 17) NPENSRGKG RRGQRGKNRG
(SEQ ID NO: 18) ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 19) A ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 20) AA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 21)
AAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 22) QAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 23) RQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO:
24) NRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 25) RNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 26) ERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID
NO: 27) RERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 28) RRERNRQAAA
ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 29) P RRERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 30) LP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ
ID NO: 31) VLP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 32)
AVLP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 33) MAVLP
RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 34) QMAVLP RRERNRQAAA
ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 35) KQMAVLP RRERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 36) DKQMAVLP RRERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 37) PDKQMAVLP RRERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 38)
[0014] It is contemplated that such truncated GDNF protein products
would include truncated GDNF protein having the amino acid sequence
as represented by X-[Cys.sup.41-Cys.sup.133]-Y and variants and
derivatives thereof. Thus, the truncated GDNF protein products of
the present invention also include addition, substitution and
internal deletion variants and derivatives of the amino acid
sequences represented by X-[Cys.sup.41-Cys.sup.133]-Y. The
truncated GDNF protein products further include methionylated or
nonmethionylated forms as well as glycosylated or non-glycosylated
forms of truncated GDNF protein.
[0015] Exemplary truncated GDNF proteins of the present invention
include [Arg.sup.16-Ile.sup.134], [Asn.sup.22-Ile.sup.134],
[Pro.sup.23-Ile.sup.134], [Ser.sup.26-Ile.sup.134],
[Arg.sup.32-Ile.sup.134], [Gly.sup.33-Ile.sup.134],
[Lys.sup.37-Ile.sup.134] and [Asn.sup.38-Ile.sup.134] truncated
GDNF proteins, either methionylated or nonmethionylated, and
variants and derivatives thereof. Presently preferred truncated
GDNF proteins of the present invention include
[Lys.sup.37-Ile.sup.134] and [Asn.sup.38-Ile.sup.134] truncated
GDNF proteins, either methionylated or nonmethionylated, and
variants and derivatives thereof. Exemplary substitution variants
are the [Asn.sup.22.DELTA.Ser.sup.22-Ile.sup.134] and
[Pro.sup.23-Lys.sup.37.DELTA.Asn.sup.37-Ile.sup.134] truncated GDNF
proteins. An exemplary addition variant is the
Ser-[Pro.sup.23-Ile.sup.134] truncated GDNF protein.
[0016] In another aspect of the present invention, the truncated
GDNF proteins may be made in glycosylated or non-glycosylated
forms. Derivatives of truncated GDNF protein typically involve
attaching the truncated GDNF protein to a water soluble polymer.
For example, the truncated GDNF protein may be conjugated to one or
more polyethylene glycol molecules to decrease the precipitation of
the truncated GDNF protein product in an aqueous environment.
[0017] Yet another aspect of the present invention includes the
various polynucleotides encoding truncated GDNF proteins. These
nucleic acid sequences are generally used in the expression of
truncated GDNF in a eukaryotic or prokaryotic host cell, wherein
the expression product or a derivative thereof is characterized by
the ability to increase dopamine uptake by dopaminergic cells. The
polynucleotides may also be used in cell therapy or gene therapy
applications. Suitable nucleic acid sequences include those
specifically depicted in the Figures as well as additional
degenerate sequences and naturally occurring allelic
variations.
[0018] A further aspect of the present invention involves vectors
containing the polynucleotides encoding truncated GDNF proteins
operatively linked to amplification and/or expression control
sequences. Both prokaryotic and eukaryotic host cells may be stably
transformed or transfected with such vectors to express the
truncated glial derived neurotrophic factor. The present invention
further includes the recombinant production of a truncated GDNF
protein wherein such transformed or transfected host cells are
grown in a suitable nutrient medium, and the truncated GDNF
expressed by the cells is, optionally, isolated from the host cells
and/or the nutrient medium. The present invention further includes
the use of polynucleotides encoding truncated GDNF and vectors
containing such polynucleotides in gene therapy or cell
therapy.
[0019] In another aspect, the present invention involves a
recombinantly produced GDNF composition containing a mixture of a
mature GDNF protein and one or more truncated GDNF proteins derived
therefrom, wherein the mature GDNF protein has a molecular weight
of approximately 44 kDa, and wherein the truncated GDNF protein has
a molecular weight of approximately 36 to 40 kDa. The GDNF
composition may contain at least two truncated GDNF species wherein
a first species has a molecular weight of approximately 36 kDa and
a second species has a molecular weight of approximately 40 kDa.
The truncated GDNF species having a molecular weight of
approximately 40 kDa is a heterodimer of a GDNF monomer having a
molecular weight of approximately 22 kDa and a truncated GDNF
monomer having a molecular weight of approximately 18 kDa. It is
also contemplated that one or more of the truncated GDNF species
may be isolated from such a mixture for therapeutic use.
[0020] Another aspect of the present invention includes
pharmaceutical compositions containing truncated GDNF protein
product. Typically, the truncated GDNF protein product is
formulated in association with a pharmaceutically acceptable
vehicle. A variety of other formulation materials may be used to
facilitate manufacture, storage, handling, delivery and/or
efficacy. In another aspect of the present invention, truncated
GDNF protein products increase dopamine uptake and survival of
dopaminergic neurons. Thus, the truncated GDNF protein products are
particularly suitable for the treatment of damage to the nervous
system caused by injury or disease, such as Parkinson's
Disease.
[0021] Additional aspects and advantages of the invention will be
apparent to those skilled in the art upon consideration of the
following description, which details the practice of the present
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0022] Numerous features and advantages of the present invention
will become apparent upon review of the figures, wherein:
[0023] FIG. 1 depicts a nucleic acid sequence (SEQ ID NO:1)
encoding mature human glial cell line-derived neurotrophic factor
(hGDNF). Also depicted is the amino acid sequence (SEQ ID NO:2) of
the mature human GDNF protein.
[0024] FIG. 2 depicts a diagram of a plasmid construction made for
the expression of recombinant truncated GDNF proteins.
[0025] FIGS. 3A-B depict a restriction map of an alternative
nucleic acid sequence (SEQ ID NO:39) encoding GDNF and truncated
GDNF polynucleotides.
[0026] FIG. 4 depicts a restriction map of yet another nucleic acid
sequence (SEQ ID NO:40) encoding GDNF and truncated GDNF
polynucleotides.
[0027] FIG. 5 depicts a nucleic acid sequence (SEQ ID NO:41)
encoding [Pro.sup.23-Lys.sup.37.DELTA.Asn.sup.37-Ile.sup.134]
truncated GDNF protein substitution variant (SEQ ID NO:42). This
protein may also be described as a
Met-Ser-[Pro.sup.23-Lys.sup.37.DELTA.Asn.sup.37-Ile.sup.134]
truncated GDNF protein addition/substitution variant.
[0028] FIG. 6 depicts a nucleic acid sequence (SEQ ID NO:43)
encoding an [Arg.sup.32-Ile.sup.134] truncated GDNF protein (SEQ ID
NO:44).
[0029] FIG. 7 depicts a nucleic acid sequence (SEQ ID NO:45)
encoding a [Gly.sup.33-Ile.sup.134] truncated GDNF protein (SEQ ID
NO:46).
[0030] FIG. 8 depicts the amino acid sequence of mature hGDNF (SEQ
ID NO:47) in comparison to several exemplary truncated GDNF
proteins: Met-[Arg.sup.32-Ile.sup.134] (SEQ ID NO:48),
Met-[Gly.sup.33-Ile.sup.134] (SEQ ID NO:49) and
Met-Ser-[Pro.sup.23-Lys.sup.37.DELTA.Asn.sup.37-Ile.sup.134] (SEQ
ID NO:50).
DETAILED DESCRIPTION OF THE INVENTION
[0031] Human glial cell line-derived neurotrophic factor (hGDNF) is
synthesized as a precursor that is processed and secreted as a
mature protein of 134 amino acids. It was previously determined
that mature human GDNF has the amino acid sequence depicted in FIG.
1 (SEQ ID NO:2).
[0032] The present invention is based on the unexpected discovery
that the mature GDNF protein may be reduced in size (also referred
to herein as a "clipped" or "truncated" protein or truncated GDNF
protein) yet retain its biological activity. The clipped protein
was first discovered during the recombinant production of GDNF in
Chinese hamster ovary (CHO) cells. In brief, the recombinant human
GDNF (rhGDNF) was prepared as follows. A nucleic acid sequence
encoding the entire open reading frame of the mature human GDNF
protein was cloned into an expression plasmid. The nucleic acid
sequence was confirmed to be correct (by DNA sequencing as the
equivalent of the hGDNF sequence in GeneBank) and was translated to
an amino acid sequence identical to the published sequence for
mature human GDNF (Lin et al., Science 260, 1130-1132, 1993). The
plasmid DNA was linearized and transfected into dihydrofolate
reductase-deficient CHO cells (CHOd.sup.- cells) using the calcium
phosphate precipitation method. Transfected cells were cultured in
a selective medium, and those colonies that survived the selection
process were chosen for individual analysis of hGDNF
expression.
[0033] Serum-free conditioned media from the individual clones were
collected and subjected to Western blot analysis using antisera
specific for hGDNF. The antisera involved rabbit polyclonal
antibodies elicited from rabbits immunized with recombinant hGDNF
expressed in Escherichia coli. Under reducing conditions, the hGDNF
that was present in these samples was resolved into two major bands
having apparent molecular weights of approximately 22 kDa and 18
kDa. Each band consisted of a closely spaced doublet of
approximately 22+22.5 kDa and 18+17.5 kDa, respectively (for
simplicity, these doublets will be referred as the 22 kDa and 18
kDa bands or species).
[0034] GDNF had previously been reported to exist as a
disulfide-bonded homodimer composed of two identical subunits of
the mature GDNF protein having a molecular weight of approximately
20 to 22 kDa. When GDNF was analyzed under nonreducing conditions,
it was reported that a broad band of 32 to 42 kDa (Lin et al.,
Science 260, 1130-1132, 1993) or 33 to 45 kDa (Lin et al., J.
Neurochem. 63(2), 758-768, 1994) had been identified. The existence
of the range was interpreted as being due to the heterogeneity of
glycosylation on the mature monomers and was further substantiated
with de-glycosylation experiments.
[0035] While the present 22 kDa band corresponds to the mature GDNF
protein reported in the literature, the 18 kDa band has not
previously been reported. The relative amounts of the 22 kDa and
the 18 kDa protein varied in samples collected from individual
clones. In addition, it was found that multiple harvests from the
same clone showed a variable ratio of the two bands. Moreover, it
was found that storage of the CHO-expressed GDNF protein frequently
led to an increase in the presence of the 18 kDa band with a
concurrent decrease of the 22 kDa band.
[0036] When conditioned medium from the transformed CHOd.sup.-
cells was analyzed under nonreducing conditions by Western blots,
three well-resolved bands with apparent molecular weights of 36, 40
and 44 kDa were observed. This finding was also in contrast to
previous reports. The relative intensity of these bands was
variable, but they correlated well with the ratio of the 22 and 18
kDa monomer bands present in each of the samples. Upon further
analysis with monoclonal antisera, it was determined that the three
bands in the nonreducing gel corresponded to three possible dimers
composed of the two monomers. The largest 44 kDa protein is a dimer
of two 22 kDa mature GDNF proteins as previously reported. The
intermediate 40 kDa protein consists of a dimer in which one mature
protein has been reduced in molecular weight to an 18 kDa form. The
smallest 36 kDa dimer appears to contain two 18 kDa proteins, i.e.,
both 22 kDa forms have been reduced in molecular weight. This data
demonstrated for the first time not only the presence of a novel
form of GDNF monomer but also the presence of the clipped GDNF
protein in the dimeric configuration. It was also found that, when
stored, the monomer composition of the samples shifted towards that
of the clipped form and the corresponding dimer species, i.e., the
amount of the 36 kDa protein was seen to increase.
[0037] Studies were then performed to identify which part of the
protein was being eliminated or changed to cause of the reduction
in molecular weight in comparison to that of the previously
reported mature GDNF protein. It was first determined that the
reduction in molecular weight was not due to changes in
glycosylation.
[0038] GDNF contains two potential N-linked glycosylation sites and
has been reported to be glycosylated. The clipped protein, however,
is not simply the nonglycosylated or underglycosylated form of
mature GDNF. This was demonstrated in deglycosylation experiments
wherein samples were treated with N-glycanase, O-glycanase and
neuraminidase. On reducing gels, the 18 kDa protein was reduced to
a 13.5 kDa band by N-glycanase digestion indicating the presence of
an equivalency of 4.5 kDa of N-linked sugar. Treatment with
neuraminidase and O-glycanase caused the 18 kDa band to shift
slightly to 17 kDa. This indicated the presence of O-linked sugars
on the protein. The mature 22 kDa band has been reported to be
glycosylated and was also reduced to 18 kDa (i.e., also by 4.5 kDa)
by N-glycanase. This was further confirmed through the use of a
monoclonal antibody which is specific for the 22 kDa band on the
gel. The glycanase digestion pattern of the nonreduced dimer was
more complicated, but was interpretable and consistent with the
initial assignment of the three forms.
[0039] As a result, the 4.5 kDa reduction in molecular weight of
the protein was then viewed as resulting from the deletion of
approximately 30-35 amino acid residues rather than from changes in
glycosylation. The deletion was expected to most likely occur at
the amino-terminus of the mature GDNF protein for the following
reasons. Mature GDNF contains a total of seven cystines. If the
deletion were from the carboxyl terminus, 2 to 4 of the seven
cystines would be lost, and this would likely result in an inactive
protein. However, when a test sample consisting of predominantly
the clipped form was subjected to a bioassay to measure its
dopaminergic neuron neurotrophic activity, the sample demonstrated
comparable activity to a sample which contained proportionally more
of the mature form of GDNF.
[0040] The site of cleavage was then determined via amino acid
sequence analysis of the purified protein. Samples were sequenced,
according to manufacturer's instructions, using an Applied
Biosystems 494A protein sequencer for ten cycles. While amino acid
sequence analysis techniques and procedures are well known to those
skilled in the art, further descriptions of the sequencing of
proteins are provided in Fausset et al., Electrophoresis 12:22-27,
1991 and U.S. Pat. No. 576,316 filed Aug. 24, 1990 (abandoned)
(European Patent Application No. 90310899, Publication No. EP 423
980, filed Oct. 4, 1990, entitled "Stem Cell Factor") the
disclosures of which are hereby incorporated by reference. Upon
analysis, it was determined that the amino terminus of the clipped
protein was "RGQRGK" or Arg-Gly-Gln-Arg-Gly-Lys. SEQ ID NO: 52.
Therefore, the first 31 amino acids had been removed from the
mature protein in the conditioned media. The remaining amino acid
sequence of the clipped protein, beginning with amino acid
Arg.sup.32, was otherwise consistent with that of the mature GDNF
amino acid sequence depicted in FIG. 1 (SEQ ID NO:2).
[0041] The [Arg.sup.32-Ile.sup.134] truncated GDNF protein was
found to be active, on a qualitative basis, in a dopaminergic
neuron assay. The dopaminergic neurotrophic activity assay is used
to identify neurotrophic factors that may be beneficial in treating
Parkinson's disease. The assay is based on a previously described
assay (Friedman et al., Neuro. Sci. Lett. 79:65-72, 1987, the
disclosure of which is hereby incorporated by reference) and may
include modifications as described in Lin et al. (see U.S. patent
application Ser. No. 08/182,183 filed May 23, 1994, now U.S. Pat.
No. 7,226,758, issued Jun. 15, 2007, and its parent applications;
PCT/US92/07888 filed Sep. 17, 1992 (WO 93/06116); and European
Patent Application No. 92921022.7 (Publication No. EP 610 254)). A
detailed description of the assay is provided in Example 5,
below.
[0042] A subsequent purification procedure, followed by amino acid
sequencing, led to the discovery of another protein from which the
first 36 amino acid residues had been removed from the N-terminus
of mature GDNF: a [Lys.sup.37-Ile.sup.134] truncated GDNF protein,
with an N-terminal sequence of KNRG(C)VL--SEQ ID NO: 53. Again, the
remaining amino acid residues of the clipped protein were otherwise
consistent with those of the mature human GDNF amino acid sequence.
The [Lys.sup.37-Ile.sup.134] truncated GDNF protein was also
analyzed in the dopamine uptake bioassay. This truncated GDNF
protein was found to be active with an ED50 of about 50 pg/ml,
similar to that of purified recombinant E. coli-expressed, mature
GDNF.
[0043] It was further discovered that bacterially expressed mature
GDNF could be changed to a truncated form. Mature GDNF, expressed
in transformed E. coli (as described in Lin et al., U.S. patent
application Ser. No. 08/182,183, supra), was incubated with
CHO-derived conditioned media. Recombinant E. coli GDNF has an
apparent molecular weight of 17 kDa on reducing gel. When the
material was mixed with CHO cell conditioned media and incubated
for five days at 4.degree. C., the protein was clipped completely
to 12.5 kDa. This cleavage was less complete with one hour or 24
hours of incubation, suggesting a time-dependent process under such
conditions. It was also found that simply incubating recombinant E.
coli GDNF overnight with media containing 0.1% fetal bovine serum
did not generate the clipped form. Thus, the presence of live cells
in the culture seems to be necessary for the clipping process to
occur. It is possible, therefore, that the clipping event may also
occur in vivo within certain tissues.
[0044] In addition, it was found that derivatives of mature E.
coli-expressed hGDNF, such as pegylated GDNF (also described in Lin
et al., U.S. patent application Ser. No. 08/182,183, supra) may be
processed to a truncated form in the presence of CHO-derived
conditioned media. Mature GDNF may be pegylated at the amino
terminus in order to enhance its clearance time in circulation.
Pegylation increases the size of the protein, and the modified
mature GDNF migrates at about 45 kDa under reduced conditions. As
with the non-pegylated mature form, the incubation of pegylated E.
coli GDNF with CHO cell (untransfected) conditioned media generated
a 12.5 kDa band. In both cases, the 12.5 kDa species was present as
a disulfide-bonded dimer as shown on non-reducing gels. The
generation of this clipped form from the N-terminally pegylated
mature protein further demonstrated that the clipping event
occurred at the N-terminus of the protein since the pegylated
residue was lost during the clipping process.
[0045] Based on the these findings and because the clipping event
may also occur in vivo, a truncated form of the GDNF protein may be
the ultimate naturally processed form of hGDNF under physiological
conditions. Therefore, it was considered advantageous to produce a
truncated GDNF protein, or derivative thereof, for therapeutic use.
For example, a directly expressed or synthesized truncated GDNF
protein, such as the [Arg.sup.32-Ile.sup.134] truncated GDNF
protein, would be expected to be resistant to the above-described
proteolytic activity. Moreover, if it was desired to produce a
truncated GDNF derivative, such as a pegylated
[Arg.sup.32-Ile.sup.134] truncated GDNF protein, the resulting
derivative would be expected to have the advantage of not being
susceptible to the specific clipping which was observed with the
mature GDNF derivative.
[0046] Additional advantages can also be expected of truncated GDNF
protein products. First, the pI of a truncated protein, such as the
[Arg.sup.32-Ile.sup.134] truncated GDNF protein, will be reduced
from about 10 to about 8.0-8.5. This makes the protein
significantly less basic which could in turn provide beneficial
effects including better receptor binding and decreased
cytotoxicity at the site of administration, such as an intrathecal
injection site. Second, within the first 26 amino acids of the
mature GDNF amino acid sequence are two deamidation sites:
Arg-Asn-Arg (amino acids 14-16) and Glu-Asn-Ser (amino acids
24-26). The absence of one or both of these sites in a truncated
GDNF protein is expected to increase the stability of the
protein.
Truncated GDNF Protein Products
[0047] In a basic embodiment, the truncated GDNF proteins of the
present invention may be represented by the following amino acid
sequence wherein the amino acid residue numbering scheme of FIG. 1
is used to facilitate comparison to the mature GDNF protein:
X-[Cys.sup.41-Cys.sup.133]-Y
wherein
[0048] [Cys.sup.41-Cys.sup.133] represents the amino acid sequence
of Cys.sup.41 through Cys.sup.133 as depicted in FIG. 1 (SEQ ID
NO:2);
[0049] Y represents the carboxy terminal group of Cys.sup.133 or a
carboxy-terminus amino acid residue of Ile.sup.134; and
[0050] X represents a methionylated or nonmethionylated amine group
of Cys.sup.41 or amino-terminus amino acid residue(s) selected from
the group:
TABLE-US-00002 G RG NRG KNRG (SEQ ID NO: 3) GKNRG (SEQ ID NO: 4)
RGKNRG (SEQ ID NO: 5) QRGKNRG (SEQ ID NO: 6) GQRGKNRG (SEQ ID NO:
7) RGQRGKNRG (SEQ ID NO: 8) RRGQRGKNRG (SEQ ID NO: 9) G RRGQRGKNRG
(SEQ ID NO: 10) KG RRGQRGKNRG (SEQ ID NO: 11) GKG RRGQRGKNRG (SEQ
ID NO: 12) RGKG RRGQRGKNRG (SEQ ID NO: 13) SRGKG RRGQRGKNRG (SEQ ID
NO: 14) NSRGKG RRGQRGKNRG (SEQ ID NO: 15) ENSRGKG RRGQRGKNRG (SEQ
ID NO: 16) PENSRGKG RRGQRGKNRG (SEQ ID NO: 17) NPENSRGKG RRGQRGKNRG
(SEQ ID NO: 18) ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 19) A ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 20) AA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 21)
AAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 22) QAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 23) RQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO:
24) NRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 25) RNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 26) ERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID
NO: 27) RERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 28) RRERNRQAAA
ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 29) P RRERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 30) LP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ
ID NO: 31) VLP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 32)
AVLP RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 33) MAVLP
RRERNRQAAA ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 34) QMAVLP RRERNRQAAA
ANPENSRGKG RRGQRGKNRG (SEQ ID NO: 35) KQMAVLP RRERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 36) DKQMAVLP RRERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 37) PDKQMAVLP RRERNRQAAA ANPENSRGKG
RRGQRGKNRG (SEQ ID NO: 38)
[0051] As used herein, the term "truncated GDNF protein product"
includes biologically active synthetic or recombinant truncated
GDNF proteins, truncated GDNF proteins produced from mature GDNF,
biologically active truncated GDNF variants (including insertion,
substitution and deletion variants), and chemically modified
derivatives thereof. Also included are truncated GDNF proteins that
are substantially homologous to the human GDNF protein having the
amino acid sequence set forth in SEQ ID NO:2.
[0052] The term "biologically active" as used herein means that the
truncated GDNF protein demonstrates similar neurotrophic
properties, but not necessarily all of the same properties, and not
necessarily to the same degree, as the GDNF protein having the
amino acid sequence set forth in SEQ ID NO:2. The selection of the
particular neurotrophic properties of interest depends upon the use
for which the truncated GDNF protein product is being administered.
The truncated GDNF protein products are biologically active and
demonstrate dopaminergic neuron survival characteristics similar to
that demonstrated by mature GDNF protein using the evaluation of
dopamine uptake and tyrosine hydroxylase (TH) expression as an
exemplary bioassay, as discussed in the examples, below.
[0053] The term "substantially homologous", as used herein, means a
degree of homology to the human GDNF having the amino acid sequence
set forth in SEQ ID NO:2 that is preferably in excess of 70%, most
preferably in excess of 80%, and even more preferably in excess of
90% or even 95%. The percentage of homology as described herein is
calculated as the percentage of amino acid residues found in the
smaller of the two sequences which align with identical amino acid
residues in the sequence being compared when four gaps in a length
of 100 amino acids may be introduced to assist in that alignment as
set forth by Dayhoff, in Atlas of Protein Sequence and Structure
Vol. 5, p. 124 (1972), National Biochemical Research Foundation,
Washington, D.C., the disclosure of which is hereby incorporated by
reference. Also included as substantially homologous is any
truncated GDNF protein which may be isolated by virtue of
cross-reactivity with antibodies to the GDNF of SEQ ID NO:2 or
whose genes may be isolated through hybridization with the gene or
with segments of the gene encoding the GDNF of SEQ ID NO:1.
[0054] As will be apparent to those skilled in the art upon reading
the present description, substantially homologous proteins will
involve one or more deletions from, or additions or substitutions
to, the amino acid residues of the truncated GDNF protein
represented by X-[Cys.sup.41-Cys.sup.133]-Y. The production of such
variants is described in further detail below. It will be further
appreciated that because the present invention clearly addresses
"truncated" GDNF proteins, the amino-terminus addition variants are
contemplated as including the addition of a methionine residue, or
non-GDNF amino acid residue or sequence, but do not include the
addition of an amino acid residue(s) that would result in the
reconstruction of the mature GDNF protein. Truncated GDNF proteins
based upon naturally occurring allelic mutants or variants are also
within the scope of the present invention. The production of
variant truncated GDNF protein is described in further detail
below.
[0055] Lin et al. (U.S. patent application Ser. No. 08/182,183, now
U.S. Pat. No. 7,226,758, issued Jun. 15, 2007, supra) described the
truncation of mature GDNF at the carboxyl terminus by proteolytic
processing of the Lys-Arg residues which are the sixth and fifth
residues, respectively, from the carboxyl terminal end of mature
GDNF (i.e., Lys.sup.129-Arg.sup.130 according to the amino acid
residue numbering of FIG. 1 (also as in SEQ ID NO:1 or SEQ ID
NO:2). Such a truncation would eliminate two cysteine residues from
the mature GDNF protein. This would be likely to result in improper
folding of the protein, and therefore, would result in the
formation of an inactive protein. In contrast, the
X-[Cys.sup.41-Cys.sup.133]-Y truncated GDNF protein products of the
present invention retain the Cys.sup.131 and Cys.sup.133 residues
and are active proteins as determined by dopamine uptake assay.
[0056] In one embodiment of the present invention, preferred
truncated GDNF protein products lack one or more deamidation sites.
Such a lack of deamidation sites would result in the enhanced
biochemical stability of the purified protein and a decrease in
possible degradation products, thereby resulting in a more storage
stable protein. An exemplary truncated GDNF protein product is the
[Ser.sup.26-Ile.sup.134] truncated GDNF protein which lacks the
sites which otherwise may lead to deamidation of the mature
protein. Alternatively, the [Arg.sup.16-Ile.sup.134] truncated GDNF
protein would lack at least the first deamidation site otherwise
present in the mature protein.
[0057] A currently preferred truncated GDNF protein product is
[Arg.sup.32-Ile.sup.134] truncated GDNF protein. This truncated
GDNF protein lacks the site at or near which proteolytic clipping
of the mature protein occurs. Therefore, this truncated GDNF
protein is expected to be resistant to the processing event which
may also occur in vivo. Another currently preferred truncated GDNF
protein product is the [Lys.sup.37-Ile.sup.134] truncated GDNF
protein. This truncation would further reduce the pI of the
truncated protein, as would other truncations in which residues up
to and including Gly.sup.40 and Ile.sup.134 are removed from the N-
and C-terminals, respectively. The presently most preferred
truncated GDNF protein products retain all of the cysteine residues
found in mature GDNF protein, but lack any discernible sites for
rapid proteolytic processing of the truncated GDNF protein during
expression and manufacturing or following in vivo administration.
These preferred proteins include the [Arg.sup.32-Ile.sup.134],
[Gly.sup.33-Ile.sup.134], [Gln.sup.34-Ile.sup.134],
[Arg.sup.35-Ile.sup.134], [Gly.sup.36-Ile.sup.134],
[Lys.sup.37-Ile.sup.134], [Asn.sup.38-Ile.sup.134] and
[Arg.sup.39-Ile.sup.134] truncated GDNF protein products.
[0058] Similar to the results previously described for mature GDNF
by Lin et al. (U.S. patent application Ser. No. 07/855,413,
(abandoned), supra), the truncated GDNF proteins of the present
invention have demonstrated the ability to increase dopamine uptake
by the embryonic precursors of the substantia nigra dopaminergic
neurons. Bioassays of the truncated GDNF proteins are further
described in Example 4, below.
[0059] The novel truncated GDNF proteins are typically isolated and
purified to form truncated GDNF proteins which are substantially
free from the presence of other (non-GDNF) proteinaceous materials.
Preferably, the truncated GDNF protein products are about 80% free
of other proteins which may be present due to the production
technique used in the manufacture of the truncated GDNF protein
product. More preferably, the truncated GDNF protein products are
about 90% free of other proteins, particularly preferably, about
95% free of other proteins, and most preferably about >98% free
of other proteins. In addition, the present invention furnishes the
unique advantage of providing polynucleotide sequences for the
manufacture of homogeneous truncated GDNF proteins. For example,
the use of the polynucleotide sequence encoding the
[Arg.sup.32-Ile.sup.134] truncated GDNF protein allows the
recombinant production of the truncated GDNF protein in E. coli and
other appropriate expression systems. In other words, the novel
polynucleotides allow the production of truncated GDNF proteins
which are not susceptible to proteolytic processing, or which have
reduced susceptibility to such processing or other biochemical
processing effects as described above. Thus, the novel
polynucleotides make it easier to prepare and/or isolate single
species truncated GDNF proteins, and therefore, the truncated GDNF
proteins and/or products thereof do not contain or contain
decreased amounts of the above-described mixture of hetero- and
homodimers. It will be appreciated, however, that the final
truncated GDNF protein products may be combined with other factors,
chemical compositions and/or suitable pharmaceutical formulation
materials prior to administration, as described in further detail
below.
[0060] In one aspect of the present invention, the truncated GDNF
proteins are advantageously produced via recombinant techniques
because they are capable of achieving comparatively higher amounts
of protein at greater purity. Recombinant truncated GDNF protein
forms include glycosylated and non-glycosylated forms of the
protein, and protein expressed in bacterial, mammalian or insect
cell systems. Alternatively, the truncated GDNF proteins may be
chemically synthesized. Currently preferred production methods are
described in greater detail below.
Truncated GDNF Variants and Derivatives
A. Truncated GDNF Variants
[0061] Another aspect of the present invention includes variants of
truncated GDNF protein. The term "truncated GDNF protein products"
as used herein includes variant proteins in which amino acids have
been deleted from ("deletion variants"), inserted into ("addition
variants"), or substituted for ("substitution variants"), residues
within the amino acid sequence of naturally-occurring GDNF. Such
variants are prepared by introducing appropriate nucleotide changes
into the DNA encoding the protein or by in vitro chemical synthesis
of the desired protein. It will be appreciated by those skilled in
the art that many combinations of deletions, insertions, and
substitutions can be made provided that the final protein possesses
GDNF biological activity.
[0062] Mutagenesis techniques for the replacement, insertion or
deletion of one or more selected amino acid residues are well known
to one skilled in the art (e.g., U.S. Pat. No. 4,518,584, the
disclosure of which is hereby incorporated by reference.) There are
two principal variables in the construction of amino acid sequence
variants: the location of the mutation site and the nature of the
mutation. In designing truncated GDNF variants, the location of the
mutation site and the nature of the mutation will depend on the
biochemical characteristic(s) to be modified. The mutation sites
can be modified individually or in series, e.g., by (1)
substituting first with conservative amino acid choices and then
with more radical selections depending upon the results achieved,
(2) deleting the target amino acid residue, or (3) inserting amino
acid residues adjacent to the located site.
[0063] Amino acid sequence deletions generally range from about 1
to 30 amino acid residues, more usually from about 1 to 10
residues, and typically from about 1 to 5 residues. For example,
deletions in the "X" portion of the amino acid residues located
N-terminally to Cys.sup.41 may range from approximately 1 to 30
residues, while deletions between the cysteine residues of
[Cys.sup.41-Cys.sup.133] are typically from about 1 to 5 residues,
depending on the location, so as not to disrupt protein folding.
Deletions within the truncated GDNF proteins may be made in regions
of low homology with transforming growth factor-beta (TGF-.beta.)
family members. Deletions from truncated GDNF proteins in areas of
substantial homology with other TGF-.beta. family sequences will be
more likely to modify the biological activity more significantly.
The number of total deletions and/or consecutive deletions will be
selected so as to preserve the tertiary structure of truncated GDNF
protein in the affected domain, e.g., cysteine crosslinking.
[0064] Amino acid sequence additions may include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to one
hundred or more residues, as well as internal intrasequence
insertions of single or multiple amino acid residues. Internal
additions may range generally from about 1 to 10 amino acid
residues, more typically from about 1 to 5 amino acid residues, and
usually from about 1 to 3 amino acid residues. As described above,
the amino-terminus addition variants of the present invention are
contemplated as including the addition of a methionine (for
example, as an artifact of the direct expression of GDNF in
bacterial recombinant cell culture) or a non-GDNF amino acid
residue or sequence. Amino-terminus addition variants do not
involve the addition of an amino acid residue(s) that would result
in the reconstruction of the mature GDNF protein. A further example
of a terminal insertion includes the fusion of a heterologous
N-terminal signal sequence to the N-terminus to facilitate the
secretion of protein from recombinant host cells. Such signal
sequences generally will be obtained from, and thus be homologous
to, the intended host cell species. Insertions or additions may
also include amino acid sequences derived from the sequence of
other neurotrophic factors.
[0065] Another group of variants are amino acid substitution
variants. These variants have at least one amino acid residue in
the truncated GDNF protein removed and a different residue inserted
in its place. See, for example, FIG. 5 wherein naturally occurring
Asn.sup.22 was changed to Ser to facilitate further removal of the
Met residue. Using the X-[Cys.sup.41-Cys.sup.133]-Y amino acid
sequence representation and the present definition of truncated
GDNF protein products, such a truncated GDNF protein may be
referred to either as a substitution variant
Met-[Asn.sup.22.DELTA.Ser.sup.22-Ile.sup.134] truncated GDNF
protein or an addition variant Met-Ser-[Pro.sup.23-Ile.sup.134]
truncated GDNF protein. Substitution variants include allelic
variants, which are characterized by naturally-occurring nucleotide
sequence changes in the species population that may or may not
result in an amino acid change.
[0066] Specific mutations of the sequences of the truncated GDNF
proteins may involve modifications of a glycosylation site (e.g.,
serine, threonine, or asparagine). The absence of glycosylation or
only partial glycosylation may result from amino acid substitution
or deletion at any asparagine-linked glycosylation recognition site
or at any site of the protein that is modified by the addition of
an O-linked carbohydrate. An asparagine-linked glycosylation
recognition site comprises a tripeptide sequence which is
specifically recognized by appropriate cellular glycosylation
enzymes. These tripeptide sequences are either Asn-Xaa-Thr or
Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro. A
variety of amino acid substitutions or deletions at one or both of
the first or third amino acid positions of a glycosylation
recognition site (and/or amino acid deletion at the second
position) result in non-glycosylation at the modified tripeptide
sequence. Thus, the expression of appropriately altered nucleotide
sequences produces variants which are not glycosylated at that
site. Alternatively, the sequence may be modified to add
glycosylation sites to the truncated GDNF protein.
[0067] One method for identifying truncated GDNF amino acid
residues or regions for mutagenesis is called "alanine scanning
mutagenesis" as described by Cunningham and Wells (Science, 244:
1081-1085, 1989). In this method, an amino acid residue or group of
target residues are identified (e.g., charged residues such as Arg,
Asp, His, Lys, and Glu) and replaced by a neutral or negatively
charged amino acid (most preferably alanine or polyalanine) to
effect the interaction of the amino acids with the surrounding
aqueous environment in or outside the cell. Those domains
demonstrating functional sensitivity to the substitutions then are
refined by introducing additional or alternate residues at the
sites of substitution. Thus, the site for introducing an amino acid
sequence modification is predetermined, and to optimize the
performance of a mutation at a given site, alanine scanning or
random mutagenesis may be conducted and the variants are screened
for the optimal combination of desired activity and degree of
activity.
[0068] The sites of greatest interest for substitutional
mutagenesis include sites where the amino acids found in GDNF
proteins from various species are substantially different in terms
of side-chain bulk, charge, and/or hydrophobicity. Other sites of
interest include those in which particular residues of GDNF-like
proteins, obtained from various species, are identical. Such
positions are generally important for the biological activity of a
protein. Initially, these sites are modified by substitution in a
relatively conservative manner. Such conservative substitutions are
shown in Table 1 under the heading of preferred substitutions. If
such substitutions result in a change in biological activity, then
more substantial changes (exemplary substitutions) are introduced
and/or other additions/deletions may be made, and the resulting
products screened.
TABLE-US-00003 TABLE 1 Amino Acid Substitutions Original Residue
Preferred Substitutions Exemplary Substitutions Ala (A) Val Val;
Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Lys; Arg
Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly
(G) Pro Pro His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val;
Met; Ala; Phe; norleucine Leu (L) Ile norleucine; Ile; Val; Met;
Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe
(F) Leu Leu; Val; Ile; Ala Pro (P) Gly Gly Ser (S) Thr Thr Thr (T)
Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu
Ile; Leu; Met; Phe; Ala; norleucine
[0069] Conservative modifications to the amino acid sequence (and
the corresponding modifications to the encoding nucleic acid
sequences) are expected to produce truncated GDNF proteins having
functional and chemical characteristics similar to those of the
truncated GDNF proteins described in the Examples, below. In
contrast, substantial modifications in the functional and/or
chemical characteristics of truncated GDNF proteins may be
accomplished by selecting substitutions that differ significantly
in their effect on maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
protein at the target site, or (c) the bulk of the side chain.
Naturally occurring residues are divided into groups based on
common side chain properties:
[0070] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0071] 2) neutral hydrophilic: Cys, Ser, Thr;
[0072] 3) acidic: Asp, Glu;
[0073] 4) basic: Asn, Gln, H is, Lys, Arg;
[0074] 5) residues that influence chain orientation: Gly, Pro;
and
[0075] 6) aromatic: Trp, Tyr, Phe.
[0076] Non-conservative substitutions may involve the exchange of a
member of one of these classes for another. Such substituted
residues may be introduced into regions of the truncated GDNF
proteins that are homologous with other TGF-.beta. proteins, or
into the non-homologous regions of the protein.
B. Truncated GDNF Derivatives
[0077] Chemically modified derivatives of truncated GDNF or
truncated GDNF variants may be prepared by one skilled in the art
given the disclosures herein. The chemical moieties most suitable
for derivatization of truncated GDNF proteins include water soluble
polymers. A water soluble polymer is desirable because the protein
to which it is attached does not precipitate in an aqueous
environment, such as a physiological environment. Preferably, the
polymer will be pharmaceutically acceptable for the preparation of
a therapeutic product or composition. One skilled in the art will
be able to select the desired polymer based on such considerations
as whether the polymer/protein conjugate will be used
therapeutically, and if so, the desired dosage, circulation time,
resistance to proteolysis, and other considerations. The
effectiveness of the derivatization may be ascertained by
administering the derivative, in the desired form (i.e., by osmotic
pump, or, more preferably, by injection or infusion, or further
formulated for oral, pulmonary or other delivery routes), and
determining its effectiveness.
[0078] Suitable water soluble polymers include, but are not limited
to, polyethylene glycol (PEG), copolymers of ethylene
glycol/propylene glycol, monomethoxy-polyethylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyethylene glycol propionaldehyde, and
mixtures thereof. As used herein, polyethylene glycol is meant to
encompass any of the forms of PEG that have been used to derivatize
other proteins, such as mono-(C1-C10) alkoxy- or
aryloxy-polyethylene glycol. Polyethylene glycol propionaldehyde
may have advantages in manufacturing due to its stability in water.
The polymer may be of any molecular weight, and may be branched or
unbranched.
[0079] The present invention particularly relates to truncated GDNF
protein products involving truncated GDNF protein linked to at
least one PEG molecule. In another aspect, the present invention
relates to truncated GDNF protein attached to at least one PEG
molecule via an acyl or alkyl linkage.
[0080] Pegylation may be carried out by any of the pegylation
reactions known in the art. See, for example: Focus on Growth
Factors 3(2): 4-10 (1992); EP 0 154 316; EP 0 401 384; and Malik et
al., Exp. Hematol. 20: 1028-1035 (1992) (reporting pegylation of
GM-CSF using tresyl chloride). Preferably, the pegylation is
carried out via an acylation reaction or an alkylation reaction
with a reactive water soluble polymer. These preferred means for
derivatization are discussed in greater detail, below. For the
acylation reactions, the polymer(s) selected preferably have a
single reactive ester group. For the reductive alkylation
reactions, the polymer(s) selected preferably have a single
reactive aldehyde group. In addition, the selected polymer may be
modified to have a single reactive group, such as an active ester
for acylation or an aldehyde for alkylation, so that the degree of
polymerization may be controlled. Generally, the water soluble
polymer will not be selected from naturally-occurring glycosyl
residues since these are usually made more conveniently by
mammalian recombinant expression systems.
Acylation
[0081] In the present invention, pegylation by acylation generally
involves reacting an active ester derivative of polyethylene glycol
with a truncated GDNF protein. Any known or subsequently discovered
reactive PEG molecule may be used to carry out the pegylation
process. A preferred activated PEG ester is PEG esterified to
N-hydroxysuccinimide ("NHS"). As used herein, "acylation" is
contemplated to included without limitation the following types of
linkages between a truncated GDNF protein and a water soluble
polymer such as PEG: amide, carbamate, urethane, and the like. See
Bioconjugate Chem. 5: 133-140 (1994). Reaction conditions may be
selected from any of those known in the pegylation art or those
subsequently developed, but should avoid or limit exposure to
reaction conditions such as temperatures, solvents, and pH levels
that would inactivate the truncated GDNF protein to be
modified.
[0082] Pegylation by acylation will generally result in a
poly-pegylated truncated GDNF protein, wherein the lysine
.epsilon.-amino groups are pegylated via an acyl linking group.
Preferably, the connecting linkage will be an amide. Also
preferably, the resulting product ill be substantially only (e.g.,
.gtoreq.95%) mono-, di- or tri-pegylated. However, some conjugates
with higher degrees of pegylation may be formed in amounts
depending on the specific reaction conditions used. If desired,
more purified pegylated conjugates may be prepared from the mixture
by standard purification techniques, including, among others,
dialysis, salting-out, ultrafiltration, ion-exchange
chromatography, gel filtration chromatography and
electrophoresis.
Alkylation
[0083] In the present invention, pegylation by alkylation generally
involves reacting a terminal aldehyde derivative of PEG with a
truncated GDNF protein in the presence of a reducing agent.
Pegylation by alkylation can also result in a poly-pegylated
truncated GDNF protein. In addition, one can manipulate the
reaction conditions to favor pegylation substantially only at the
.alpha.-amino group of the N-terminus of the protein (i.e., a
mono-pegylated species). In either case of mono-pegylation or
polypegylation, the PEG groups are preferably attached to the
protein via a --CH.sub.2--NH-- group. With particular reference to
the --CH.sub.2-- group, this type of linkage is referred to herein
as an "alkyl" linkage.
[0084] Selective N-terminal chemical modification may be
accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, substantially
selective derivatization of the protein at the N-terminus with a
carbonyl group-containing polymer is achieved. For example, one may
selectively N-terminally pegylate the protein by performing the
reaction at a pH which allows one to take advantage of the pK.sub.a
differences between the .epsilon.-amino group of the lysine
residues and that of the .alpha.-amino group of the N-terminal
residue of the protein. By such selective derivatization,
attachment of a water soluble polymer to a protein is controlled:
the conjugation with the polymer takes place predominantly at the
N-terminus of the protein and no significant modification of other
reactive groups, such as the lysine side chain amino groups,
occurs. Using reductive alkylation, the water soluble polymer
preferably has a single reactive aldehyde for coupling to the
protein. Polyethylene glycol propionaldehyde, containing a single
reactive aldehyde, may be used.
[0085] The present invention includes pegylated truncated GDNF
proteins, wherein the PEG group(s) is (are) attached via acyl or
alkyl groups. As discussed above, such truncated GDNF protein
products may be mono-pegylated or poly-pegylated (e.g., containing
2-6, preferably 2-5, PEG groups). The PEG groups are generally
attached to the protein at the .alpha.- or .epsilon.-amino groups
of amino acids, but it is also contemplated that the PEG groups
could be attached to any reactive group of to the protein, which is
sufficiently reactive to become attached to a PEG group under
suitable reaction conditions. Thus, polyethylene glycol may be
covalently bound to a protein via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated PEG molecule may be bound. The amino acid residues having
a free amino group may include lysine residues and the N-terminal
amino acid residue. Those having a free carboxyl group may include
aspartic acid residues, glutamic acid residues, and the C-terminal
amino acid residue. Sulfhydryl groups may also be used as a
reactive group for attaching PEG molecule(s). For therapeutic
purposes, attachment at an amino group, such as attachment at the
N-terminus or lysine group is typically preferred. Attachment at
residues important for receptor binding should be avoided if
receptor binding is desired.
[0086] In one aspect, the present invention provides for a
substantially homogeneous preparation of mono-polymer/truncated
GDNF protein conjugate wherein a polymer molecule has been attached
substantially only (i.e., .gtoreq.95%) in a single location. More
specifically, if PEG is used, the present invention also provides
for pegylated a truncated GDNF protein lacking possibly antigenic
linking groups, and having the PEG molecule directly coupled to the
truncated GDNF protein.
[0087] In addition, derivatives may be prepared using glycosylated,
non-glycosylated or de-glycosylated truncated GDNF proteins.
Typically, non-glycosylated truncated GDNF proteins are used. For
example, the prokaryote-expressed [Arg.sup.32-Ile.sup.134]
truncated GDNF protein may be chemically derivatized to include
mono- or poly-, e.g., 2-4, PEG moieties, attached via an acyl or
alkyl group):
[0088] In general, chemical derivatization may be performed under
any suitable condition used to react a biologically active
substance with an activated polymer molecule. Methods for preparing
pegylated truncated GDNF proteins will generally comprise the steps
of (a) reacting a truncated GDNF protein with polyethylene glycol
(such as a reactive ester or aldehyde derivative of PEG) under
conditions whereby the truncated GDNF protein becomes attached to
one or more PEG groups, and (b) obtaining the reaction product(s).
In general, the optimal reaction conditions for the acylation
reactions will be determined case-by-case based on known parameters
and the desired result. For example, the larger the ratio of
PEG:protein, the greater the percentage of poly-pegylated product.
The optimum ratio (in terms of efficiency of reaction in that there
is no excess unreacted protein or polymer) may be determined by
factors such as the desired degree of derivatization (e.g., mono-,
di-, tri-, etc.), the molecular weight of the polymer selected,
whether the polymer is branched or unbranched, and the reaction
conditions used.
[0089] Reductive alkylation to produce a substantially homogeneous
population of mono-polymer/truncated GDNF protein conjugate will
generally comprise the steps of (a) reacting a truncated GDNF
protein with a reactive PEG molecule under reductive alkylation
conditions, at a pH suitable to permit selective modification of
the .alpha.-amino group at the amino terminus of the truncated GDNF
protein, and (b) obtaining the reaction product(s).
[0090] For a substantially homogeneous population of
mono-polymer/truncated GDNF protein conjugate, the reductive
alkylation reaction conditions are those which permit the selective
attachment of the water soluble polymer moiety to the N-terminus of
a truncated GDNF protein. Such reaction conditions generally
provide for pK.sub.a differences between the lysine amino groups
and the .alpha.-amino group at the N-terminus (the pK.sub.a being
the pH at which 50% of the amino groups are protonated and 50% are
not). The pH also affects the ratio of polymer to protein to be
used. In general, if the pH is lower, a larger excess of polymer to
protein will be desired (i.e., the less reactive the N-terminal
.alpha.-amino group, the more polymer needed to achieve optimal
conditions). If the pH is higher, the polymer:protein ratio need
not be as large (i.e., more reactive groups are available, so fewer
polymer molecules are needed). For purposes of the present
invention, the pH will generally fall within the range of 3-9,
preferably 3-6.
[0091] Another consideration is the molecular weight of the
polymer. In general, the higher the molecular weight of the
polymer, the fewer the number of polymer molecules which may be
attached to the protein. Similarly, branching of the polymer should
be taken into account when optimizing these parameters. Generally,
the higher the molecular weight (or the more branches) the higher
the polymer:protein ratio. In general, for the pegylation reactions
contemplated herein, the preferred average molecular weight is
about 2 kDa to about 100 kDa (the term "about" indicating that in
preparations of polyethylene glycol, some molecules will weigh
more, some less, than the stated molecular weight). The preferred
average molecular weight is about 5 kDa to about 50 kDa,
particularly preferably about 12 kDa to about 25 kDa. The ratio of
water-soluble polymer to truncated GDNF protein will generally
range from 1:1 to 100:1, preferably (for polypegylation) 1:1 to
20:1, and (for mono-pegylation) 1:1 to 5:1.
[0092] Using the conditions indicated above, reductive alkylation
will provide for selective attachment of the polymer to any
truncated GDNF protein having an .alpha.-amino group at the amino
terminus, and provide for a substantially homogenous preparation of
mono-polymer/truncated GDNF protein conjugate. The term
"mono-polymer/truncated GDNF protein conjugate" is used here to
mean a derivative containing a single polymer molecule attached to
a truncated GDNF protein. The mono-polymer/truncated GDNF protein
conjugate preferably will have a polymer molecule located at the
N-terminus, but not on lysine amino side groups. The preparation
will preferably be greater than 90% mono-polymer/truncated GDNF
protein conjugate, and more preferably greater than 95%
mono-polymer/truncated GDNF protein conjugate, with the remainder
of observable proteins being unreacted (i.e., protein lacking the
polymer moiety).
[0093] For reductive alkylation, the reducing agent should be
stable in aqueous solution and preferably be able to reduce only
the Schiff base formed in the initial process of reductive
alkylation. Exemplary reducing agents may be selected from the
group consisting of sodium borohydride, sodium cyanoborohydride,
dimethylamine borane, trimethylamine borane and pyridine borane. A
particularly preferred reducing agent is sodium cyanoborohydride.
Other reaction parameters, such as solvent, reaction times,
temperatures, etc., and means of purification of products, can be
determined case-by-case based on commonly available information
relating to derivatization of proteins with water soluble
polymers.
[0094] One may choose to prepare a mixture of polymer/protein
conjugates by acylation and/or alkylation methods, and the
advantage provided herein is that one may select the proportion of
mono-polymer/protein conjugate to include in the mixture. Thus, if
desired, one may prepare a mixture of protein having various
numbers of polymer molecules attached thereto (i.e., di-, tri-,
tetra-, etc.) and combine with the mono-polymer/protein conjugate
material prepared using the present methods, and have a mixture
with a predetermined proportion of mono-polymer/protein
conjugate.
Polynucleotides Encoding Truncated GDNF Proteins
[0095] The present invention further provides novel polynucleotides
which encode truncated GDNF proteins. When used as a hybridization
probe or amplification primer, the nucleic acid sequence will be
substantially free from all other nucleic acid sequences. For use
in recombinant protein expression, the nucleic acid sequence will
generally be substantially free from nucleic acid sequences
encoding other proteins, unless a fusion protein is intended. Based
upon the present description and using the universal codon table,
one of ordinary skill in the art can readily determine all of the
nucleic acid sequences which encode the amino acid sequences of
truncated GDNF proteins. Presently preferred nucleic acid sequences
include those polynucleotides encoding the
[Arg.sup.16-Ile.sup.134], [Ser.sup.26-Ile.sup.134],
[Arg.sup.32-Ile.sup.134], and [Lys.sup.37-Ile.sup.134] truncated
GDNF proteins. Examples of a variety of polynucleotides are
depicted in FIGS. 5, 6 and 7 as well as those portions of FIGS. 1,
3 and 4 which encode truncated GDNF proteins. It will also be
appreciated by those skilled in the art that the novel
polynucleotides which encode truncated GDNF proteins include those
nucleic acid sequences encoding variant truncated GDNF proteins,
whether man-made or naturally occurring.
[0096] Recombinant expression techniques, conducted in accordance
with the descriptions set forth below, may be followed to produce
these polynucleotides and express the various truncated GDNF
proteins. For example, by inserting a nucleic acid sequence which
encodes a truncated GDNF protein into an appropriate vector, one
skilled in the art can readily produce large quantities of the
desired nucleotide sequence. The sequences can then be used to
generate detection probes or amplification primers. Alternatively,
a polynucleotide encoding a truncated GDNF protein can be inserted
into an expression vector. By introducing the expression vector
into an appropriate host, the desired truncated GDNF protein may be
produced in large amounts.
[0097] As further described herein, there are numerous host/vector
systems available for the propagation of nucleic acid sequences
and/or the production of truncated GDNF proteins. These include,
but are not limited to, plasmid, viral and insertional vectors, and
prokaryotic and eukaryotic hosts. One skilled in the art can adapt
a host/vector system which is capable of propagating or expressing
heterologous DNA to produce or express the sequences of the present
invention.
[0098] By means of such recombinant techniques, the truncated GDNF
proteins of the present invention are readily produced in
commercial quantities. Furthermore, it will be appreciated by those
skilled in the art that, in view of the present disclosure, the
novel nucleic acid sequences include degenerate nucleic acid
sequences encoding the truncated GDNF proteins specifically set
forth in the Figures, variants of such truncated GDNF proteins, and
those nucleic acid sequences which hybridize, preferably under
stringent hybridization conditions, to complements of these nucleic
acid sequences (see, Maniatis et. al., Molecular Cloning (A
Laboratory Manual); Cold Spring Harbor Laboratory, pages 387 to
389, 1982.) Exemplary stringent hybridization conditions are
hybridization in 4.times.SSC at 62-67.degree. C., followed by
washing in 0.1.times.SSC at 62-67.degree. C. for approximately an
hour. Alternatively, exemplary stringent hybridization conditions
are hybridization in 45-55% formamide, 4.times.SSC at 40-45.degree.
C. DNA sequences which hybridize to the complementary sequences for
truncated GDNF protein under relaxed hybridization conditions and
which encode a truncated GDNF protein of the present invention are
also included herein. Examples of such relaxed stringency
hybridization conditions are 4.times.SSC at 45-55.degree. C. or
hybridization with 30-40% formamide at 40-45.degree. C.
[0099] Also provided by the present invention are recombinant DNA
constructs involving vector DNA together with the DNA sequence
encoding a truncated GDNF protein. In such DNA constructs, the
nucleic acid sequence encoding truncated GDNF protein (with or
without signal peptides) is in operative association with a
suitable expression control or regulatory sequence capable of
directing the replication and/or expression of the truncated GDNF
protein in a selected host.
Recombinant Expression of Truncated GDNF Protein
Preparation of Polynucleotides Encoding Truncated GDNF
[0100] A nucleic acid sequence encoding truncated GDNF, or a mature
GDNF starting material, can readily be obtained in a variety of
ways, including, without limitation, chemical synthesis, cDNA or
genomic library screening, expression library screening, and/or PCR
amplification of cDNA. These methods and others useful for
isolating such nucleic acid sequences are set forth, for example,
by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), by
Ausubel et al., eds (Current Protocols in Molecular Biology,
Current Protocols Press, 1994), and by Berger and Kimmel (Methods
in Enzymology: Guide to Molecular Cloning Techniques, vol. 152,
Academic Press, Inc., San Diego, Calif., 1987). Preferred nucleic
acid sequences encoding GDNF are mammalian sequences.
[0101] Chemical synthesis of a nucleic acid sequence which encodes
a truncated GDNF protein can also be accomplished using methods
well known in the art, such as those set forth by Engels et al.
(Angew. Chem. Intl. Ed., 28:716-734, 1989). These methods include,
inter alia, the phosphotriester, phosphoramidite and H-phosphonate
methods of nucleic acid sequence synthesis. The nucleic acid
sequence encoding the truncated GDNF protein will be several
hundred base pairs (bp) or nucleotides in length. Large nucleic
acid sequences, for example those larger than about 100 nucleotides
in length, can be synthesized as several fragments. The fragments
can then be ligated together to form a nucleic acid sequence
encoding truncated GDNF protein. A preferred method is
polymer-supported synthesis using standard phosphoramidite
chemistry.
[0102] Alternatively, a suitable nucleic acid sequence may be
obtained by screening an appropriate cDNA library (i.e., a library
prepared from one or more tissue source(s) believed to express the
protein) or a genomic library (a library prepared from total
genomic DNA). The source of the cDNA library is typically a tissue
from any species that is believed to express GDNF in reasonable
quantities. The source of the genomic library may be any tissue or
tissues from any mammalian or other species believed to harbor a
gene encoding GDNF or a GDNF homologue. The library can be screened
for the presence of the GDNF cDNA/gene using one or more nucleic
acid probes (oligonucleotides, cDNA or genomic DNA fragments that
possess an acceptable level of homology to the GDNF or GDNF
homologue cDNA or gene to be cloned) that will hybridize
selectively with GDNF or GDNF homologue cDNA(s) or gene(s) present
in the library. The probes typically used for such library
screening usually encode a small region of the GDNF DNA sequence
from the same or a similar species as the species from which the
library was prepared. Alternatively, the probes may be degenerate,
as discussed herein.
[0103] Library screening is typically accomplished by annealing the
oligonucleotide probe or cDNA to the clones in the library under
conditions of stringency that prevent non-specific binding but
permit binding of those clones that have a significant level of
homology with the probe or primer. Typical hybridization and
washing stringency conditions depend in part on the size (i.e.,
number of nucleotides in length) of the cDNA or oligonucleotide
probe, and whether the probe is degenerate. The probability of
obtaining a clone(s) is also considered in designing the
hybridization solution (i.e., whether a cDNA or genomic library is
being screened; if it is a cDNA library, the probability that the
cDNA of interest is present at a high level).
[0104] Where DNA fragments (such as cDNAs) are used as probes,
typical hybridization conditions include those as set forth in
Ausubel et al., eds., supra. After hybridization, the blot
containing the library is washed at a suitable stringency,
depending on several factors such as probe size, expected homology
of probe to clone, type of library being screened, number of clones
being screened, and the like. Examples of stringent washing
solutions (which are usually low in ionic strength and are used at
relatively high temperatures) are as follows. One such stringent
wash is 0.015 M NaCl, 0.005 M NaCitrate and 0.1% SDS at
55-65.degree. C. Another such stringent buffer is 1 mM
Na.sub.2EDTA, 40 mM NaHPO.sub.4, pH 7.2, and 1% SDS at about
40-50.degree. C. One other stringent wash is 0.2.times.SSC and 0.1%
SDS at about 50-65.degree. C.
[0105] There are also exemplary protocols for stringent washing
conditions where oligonucleotide probes are used to screen cDNA or
genomic libraries. For example, a first protocol uses 6.times.SSC
with 0.05 percent sodium pyrophosphate at a temperature of between
about 35 and 62.degree. C., depending on the length of the probe.
For example, 14 base probes are washed at 35-40.degree. C., 17 base
probes at 45-50.degree. C., 20 base probes at 52-57.degree. C., and
23 base probes at 57-63.degree. C. The temperature can be increased
2-3.degree. C. where the background non-specific binding appears
high. A second protocol uses tetramethylammonium chloride (TMAC)
for washing. One such stringent washing solution is 3 M TMAC, 50 mM
Tris-HCl, pH 8.0, and 0.2% SDS.
[0106] Another suitable method for obtaining a nucleic acid
sequence encoding a GDNF protein is the polymerase chain reaction
(PCR). In this method, poly(A)+RNA or total RNA is extracted from a
tissue that expresses GDNF. cDNA is then prepared from the RNA
using the enzyme reverse transcriptase. Two primers, typically
complementary to two separate regions of the GDNF cDNA
(oligonucleotides), are then added to the cDNA along with a
polymerase such as Taq polymerase, and the polymerase amplifies the
cDNA region between the two primers.
[0107] Where the method of choice for preparing the nucleic acid
sequence encoding the desired truncated GDNF protein requires the
use of oligonucleotide primers or probes (e.g., PCR, cDNA or
genomic library screening), the oligonucleotide sequences selected
as probes or primers should be of adequate length and sufficiently
unambiguous so as to minimize the amount of non-specific binding
that will occur during library screening or PCR amplification. The
actual sequence of the probes or primers is usually based on
conserved or highly homologous sequences or regions from the same
or a similar gene from another organism. Optionally, the probes or
primers can be fully or partially degenerate, i.e., contain a
mixture of probes/primers, all encoding the same amino acid
sequence, but using different codons to do so. An alternative to
preparing degenerate probes is to place an inosine in some or all
of those codon positions that vary by species. The oligonucleotide
probes or primers may be prepared by chemical synthesis methods for
DNA as described above.
[0108] Truncated GDNF proteins based on these nucleic acid
sequences encoding GDNF, as well as mutant or variant sequences
thereof, are also contemplated as within the scope of the present
invention. As described above, a mutant or variant sequence is a
sequence that contains one or more nucleotide substitutions,
deletions, and/or insertions as compared to the wild type sequence
and that results in the expression of amino acid sequence
variations as compared to the wild type amino acid sequence. In
some cases, naturally occurring GDNF amino acid mutants or variants
may exist, due to the existence of natural allelic variation.
Truncated GDNF proteins based on such naturally occurring mutants
or variants are also within the scope of the present invention.
Preparation of synthetic mutant sequences is also well known in the
art, and is described for example in Wells et al. (Gene, 34:315,
1985) and in Sambrook et al., supra.
Vectors
[0109] The cDNA or genomic DNA encoding a truncated GDNF protein is
inserted into a vector for further cloning (amplification of the
DNA) or for expression. Suitable vectors are commercially
available, or the vector may be specially constructed. The
selection or construction of the appropriate vector will depend on
1) whether it is to be used for DNA amplification or for DNA
expression, 2) the size of the DNA to be inserted into the vector,
and 3) the host cell (e.g., mammalian, insect, yeast, fungal, plant
or bacterial cells) to be transformed with the vector. Each vector
contains various components depending on its function
(amplification of DNA or expression of DNA) and its compatibility
with the intended host cell. The vector components generally
include, but are not limited to, one or more of the following: a
signal sequence, an origin of replication, one or more selection or
marker genes, enhancer elements, promoters, a transcription
termination sequence, and the like. These components may be
obtained from natural sources or synthesized by known procedures.
The vectors of the present invention involve a nucleic acid
sequence which encodes the truncated GDNF protein of interest
operatively linked to one or more of the following expression
control or regulatory sequences capable of directing, controlling
or otherwise effecting the expression of the truncated GDNF protein
by a selected host cell.
Signal Sequence
[0110] The signal sequence may be a component of the vector, or it
may be a part of GDNF DNA that is inserted into the vector. The
native GDNF DNA encodes a signal sequence at the amino terminus of
the protein that is cleaved during post-translational processing of
the protein to form the mature GDNF protein. Included within the
scope of this invention are truncated GDNF polynucleotides with the
native signal sequence and other pre-pro sequences as well as
truncated GDNF polynucleotides wherein the native signal sequence
is deleted and replaced with a heterologous signal sequence. The
heterologous signal sequence selected should be one that is
recognized and processed, i.e., cleaved by a signal peptidase, by
the host cell. For prokaryotic host cells that do not recognize and
process the native GDNF signal sequence, the signal sequence is
substituted by a prokaryotic signal sequence selected, for example,
from the group of the alkaline phosphatase, penicillinase, or
heat-stable enterotoxin II leaders. For yeast secretion, the native
GDNF signal sequence may be substituted by the yeast invertase,
alpha factor, or acid phosphatase leaders. In mammalian cell
expression the native signal sequence is satisfactory, although
other mammalian signal sequences may be suitable.
Origin of Replication
[0111] Expression and cloning vectors generally include a nucleic
acid sequence that enables the vector to replicate in one or more
selected host cells. In cloning vectors, this sequence is typically
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeasts, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria and various origins (e.g., SV40, polyoma,
adenovirus, VSV or BPV) are useful for cloning vectors in mammalian
cells. Generally, the origin of replication component is not needed
for mammalian expression vectors (for example, the SV40 origin is
often used only because it contains the early promoter).
Selection Gene
[0112] The expression and cloning vectors typically contain a
selection gene. This gene encodes a "marker" protein necessary for
the survival or growth of the transformed host cells when grown in
a selective culture medium. Host cells that were not transformed
with the vector will not contain the selection gene, and therefore,
they will not survive in the culture medium. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline; (b) complement auxotrophic deficiencies; or (c)
supply critical nutrients not available from the culture
medium.
[0113] Other selection genes may be used to amplify the gene which
will be expressed. Amplification is the process wherein genes which
are in greater demand for the production of a protein critical for
growth are reiterated in tandem within the chromosomes of
successive generations of recombinant cells. Examples of suitable
selectable markers for mammalian cells include dihydrofolate
reductase (DHFR) and thymidine kinase. The mammalian cell
transformants are placed under selection pressure which only the
transformants are uniquely adapted to survive by virtue of the
marker present in the vector. Selection pressure is imposed by
culturing the transformed cells under conditions in which the
concentration of selection agent in the medium is successively
changed, thereby leading to amplification of both the selection
gene and the DNA that encodes truncated GDNF. As a result,
increased quantities of truncated GDNF are synthesized from the
amplified DNA.
[0114] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate, a competitive antagonist
of DHFR. An appropriate host cell when wild-type DHFR is used is
the Chinese hamster ovary cell line deficient in DHFR activity
(see, for example, Urlaub and Chasin, Proc. Natl. Acad. Sci., USA
77(7): 4216-4220 (1980)). The transformed cells are then exposed to
increased levels of methotrexate. This leads to the synthesis of
multiple copies of the DHFR gene, and, concomitantly, multiple
copies of other DNA present in the expression vector, such as the
DNA encoding a truncated GDNF protein.
Promoter
[0115] The expression and cloning vectors of the present invention
will typically contain a promoter that is recognized by the host
organism and operably linked to the nucleic acid sequence encoding
the truncated GDNF protein. Promoters are untranslated sequences
located upstream (5') to the start codon of a structural gene
(generally within about 100 to 1000 bp) that control the
transcription and translation of a particular nucleic acid
sequence, such as that encoding truncated GDNF. Promoters are
conventionally grouped into one of two classes, inducible promoters
and constitutive promoters. Inducible promoters initiate increased
levels of transcription from DNA under their control in response to
some change in culture conditions, such as the presence or absence
of a nutrient or a change in temperature. A large number of
promoters, recognized by a variety of potential host cells, are
well known. These promoters are operably linked to the DNA encoding
truncated GDNF by removing the promoter from the source DNA by
restriction enzyme digestion and inserting the desired promoter
sequence into the vector. The native GDNF promoter sequence may be
used to direct amplification and/or expression of truncated GDNF
DNA. A heterologous promoter is preferred, however, if it permits
greater transcription and higher yields of the expressed protein as
compared to the native promoter, and if it is compatible with the
host cell system that has been selected for use.
[0116] Promoters suitable for use with prokaryotic hosts include
the beta-lactamase and lactose promoter systems; alkaline
phosphatase, a tryptophan (trp) promoter system; and hybrid
promoters such as the tac promoter. Other known bacterial promoters
are also suitable. Their nucleotide sequences have been published,
thereby enabling one skilled in the art to ligate them to the
desired DNA sequence(s), using linkers or adaptors as needed to
supply any required restriction sites.
[0117] Suitable promoting sequences for use with yeast hosts are
also well known in the art. Yeast enhancers are advantageously used
with yeast promoters. Suitable promoters for use with mammalian
host cells are well known and include those obtained from the
genomes of viruses such as polyoma virus, fowlpox virus, adenovirus
(such as Adenovirus 2), bovine papilloma virus, avian sarcoma
virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most
preferably Simian Virus 40 (SV40). Other suitable mammalian
promoters include heterologous mammalian promoters, e.g.,
heat-shock promoters and the actin promoter. A currently used
promoter in the production of GDNF proteins in CHO cells is
SR.alpha.. See Takebe et al., Mol. Cell. Biol. 8(1): 466-472
(1988). A suitable expression vector is pDSR.alpha.2, which is
further described below.
Enhancer Element
[0118] An enhancer sequence may be inserted into the vector to
increase the transcription of a DNA sequence encoding a truncated
GDNF protein of the present invention by higher eukaryotes.
Enhancers are cis-acting elements of DNA, usually about from 10-300
bp in length, that act on the promoter to increase its
transcription.
[0119] Enhancers are relatively orientation and position
independent. They have been found 5' and 3' to the transcription
unit. Several enhancer sequences available from mammalian genes are
known (e.g., globin, elastase, albumin, alpha-feto-protein and
insulin). Typically, however, an enhancer from a virus will be
used. The SV40 enhancer, the cytomegalovirus early promoter
enhancer, the polyoma enhancer, and adenovirus enhancers are
exemplary enhancing elements for the activation of eukaryotic
promoters. While an enhancer may be spliced into the vector at a
position 5' or 3' to truncated GDNF DNA, it is typically located at
a site 5' from the promoter.
Transcription Termination
[0120] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and occasionally 3'
untranslated regions of eukaryotic DNAs or cDNAs. These regions
contain nucleotide segments transcribed as polyadenylated fragments
in the untranslated portion of the mRNA encoding truncated
GDNF.
[0121] The construction of suitable vectors containing one or more
of the above-listed components together with the desired truncated
GDNF coding sequence is accomplished by standard ligation
techniques. Isolated plasmids or DNA fragments are cleaved,
tailored, and religated in the desired order to generate the
plasmids required. To confirm that the correct sequences have been
constructed, the ligation mixtures may be used to transform E.
coli, and successful transformants may be selected by known
techniques, such as ampicillin or tetracycline resistance as
described above. Plasmids from the transformants are then prepared,
analyzed by restriction endonuclease digestion, and/or sequenced to
confirm the presence of the desired construct.
[0122] Vectors that provide for the transient expression of DNA
encoding truncated GDNF in mammalian cells may also be used. In
general, transient expression involves the use of an expression
vector that is able to replicate efficiently in a host cell, such
that the host cell accumulates many copies of the expression vector
and, in turn, synthesizes high levels of the desired protein
encoded by the expression vector. Transient expression systems,
comprising a suitable expression vector and a host cell, allow for
the convenient positive identification of proteins encoded by
cloned DNAs, as well as for the rapid screening of such proteins
for desired biological or physiological properties. Thus, transient
expression systems are particularly useful in identifying variants
of the protein.
Selection and Transformation of Host Cells
[0123] Host cells (e.g., bacterial, mammalian, insect, yeast, or
plant cells) transformed with nucleic acid sequences for use in
expressing a recombinant truncated GDNF protein are also provided
by the present invention. The transformed host cell is cultured
under appropriate conditions permitting the expression of the
nucleic acid sequence. The selection of suitable host cells and
methods for transformation, culture, amplification, screening and
product production and purification are well known in the art. See
for example, Gething and Sambrook, Nature 293: 620-625 (1981), or
alternatively, Kaufman et al., Mol. Cell. Biol., 5 (7): 1750-1759
(1985) or Howley et al., U.S. Pat. No. 4,419,446. Truncated GDNF
may be expressed in E. coli in accordance with the description of
Lin et al. (U.S. patent application Ser. No. 07/855,413
(abandoned); Application No. PCT/US92/07888; WO 93/06116) which
involved the expression of mature GDNF. Other exemplary materials
and methods are discussed in further detail below. The transformed
host cell is cultured in a suitable medium, and the expressed
factor is then optionally recovered, isolated and purified from the
culture medium (or from the cell, if expressed intracellularly) by
an appropriate means known to those skilled in the art.
[0124] Suitable host cells for cloning or expressing the vectors
herein are the prokaryote, yeast, or higher eukaryote cells as
described above. Prokaryotic host cells include, but are not
limited to, eubacteria, such as Gram-negative or Gram-positive
organisms, for example, E. coli, Bacilli such as B. subtilis,
Pseudomonas species such as P. aeruginosa, Salmonella typhimurium,
or Serratia marcescans. Alternatively, in vitro methods of cloning,
e.g., PCR or other nucleic acid polymerase reactions, are
suitable.
[0125] In addition to prokaryotic host cells, eukaryotic microbes
such as filamentous fungi or yeast may be suitable hosts for the
expression of truncated GDNF proteins. Saccharomyces cerevisiae, or
common bakers yeast, is the most commonly used among lower
eukaryotic host microorganisms, but a number of other genera,
species, and strains are well known and commonly available.
[0126] Suitable host cells for the expression of glycosylated
truncated GDNF protein are derived from multicellular organisms.
Such host cells are capable of complex processing and glycosylation
activities. In principle, any higher eukaryotic cell culture might
be used, whether such culture involves vertebrate or invertebrate
cells, including plant and insect cells. Vertebrate cells are
generally used as the propagation of vertebrate cells in culture
(tissue culture) is a well known procedure. Examples of useful
mammalian host cell lines include, but are not limited to, monkey
kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture),
baby hamster kidney cells, and Chinese hamster ovary cells. Other
suitable mammalian cell lines include but are not limited to, HeLa,
mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH
mice, BHK or HaK hamster cell lines.
[0127] Similarly useful as host cells suitable for the present
invention are bacterial cells. For example, the various strains of
E. coli (e.g., HB101, DH5.alpha., DH10, and MC1061) are well-known
as host cells in the field of biotechnology. Various strains of
Streptomyces spp. and the like may also be employed. Presently
preferred host cells for producing truncated GDNF proteins are
bacterial cells (e.g., Escherichia coli) and mammalian cells (such
as Chinese hamster ovary cells, COS cells, etc.)
[0128] The host cells are transfected and preferably transformed
with the above-described expression or cloning vectors and cultured
in a conventional nutrient medium. The medium may be modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences. Transfection
and transformation are performed using standard techniques which
are well known to those skilled in the art and which are selected
as appropriate to the host cells involved. For example, for
mammalian cells without cell walls, the calcium phosphate
precipitation method may be used. Electroporation, micro injection
and other known techniques may also be used.
Culturing the Host Cells
[0129] Transformed cells used to produce truncated GDNF proteins of
the present invention are cultured in suitable media. The media may
be supplemented as necessary with hormones and/or other growth
factors (such as insulin, transferrin, or epidermal growth factor),
salts (such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleosides (such as adenosine and
thymidine), antibiotics (such as gentamicin), trace elements
(defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or other
energy source. Other supplements may also be included, at
appropriate concentrations, as will be appreciated by those skilled
in the art. Suitable culture conditions, such as temperature, pH,
and the like, are also well known to those skilled in the art for
use with the selected host cells.
[0130] It is also possible that truncated GDNF proteins may be
produced by homologous recombination, or with recombinant
production methods utilizing control elements introduced into cells
already containing DNA encoding GDNF. Homologous recombination is a
technique originally developed for targeting genes to induce or
correct mutations in transcriptionally active genes (Kucherlapati,
Prog. in Nucl. Acid Res. and Mol. Biol. 36:301 (1989)). The basic
technique was developed as a method for introducing specific
mutations into specific regions of the mammalian genome (Thomas et
al., Cell. 44:419-428, 1986; Thomas and Capecchi, Cell. 51:503-512,
1987; Doetschman et al., Proc. Natl. Acad. Sci. 85:8583-8587, 1988)
or to correct specific mutations within defective genes (Doetschman
et al., Nature. 330:576-578, 1987). Exemplary homologous
recombination techniques are described in U.S. Pat. No. 5,272,071
(EP 91 90 3051, EP Publication No. 505 500; PCT/US90/07642,
International Publication No. WO 91/09955) the disclosure of which
is hereby incorporated by reference.
[0131] Through homologous recombination, the DNA sequence to be
inserted into the genome can be directed to a specific region of
the gene of interest by attaching it to targeting DNA. The
targeting DNA is DNA that is complementary (homologous) to a region
of the genomic DNA. Small pieces of targeting DNA that are
complementary to a specific region of the genome are put in contact
with the parental strand during the DNA replication process. It is
a general property of DNA that has been inserted into a cell to
hybridize and therefore recombine with other pieces of endogenous
DNA through shared homologous regions. If this complementary strand
is attached to an oligonucleotide that contains a mutation or a
different sequence of DNA, it too is incorporated into the newly
synthesized strand as a result of the recombination. As a result of
the proofreading function, it is possible for the new sequence of
DNA to serve as the template. Thus, the transferred DNA is
incorporated into the genome.
[0132] If the sequence of a particular gene is known, such as the
nucleic acid sequence of GDNF, the pre-pro sequence or expression
control sequence, a piece of DNA that is complementary to a
selected region of the gene can be synthesized or otherwise
obtained, such as by appropriate restriction of the native DNA at
specific recognition sites bounding the region of interest. This
piece serves as a targeting sequence upon insertion into the cell
and will hybridize to its homologous region within the genome. If
this hybridization occurs during DNA replication, this piece of
DNA, and any additional sequence attached thereto, will act as an
Okazaki fragment and will be backstitched into the newly
synthesized daughter strand of DNA.
[0133] In the present invention, attached to these pieces of
targeting DNA are regions of DNA which may interact with the
expression of a GDNF protein. For example, a promoter/enhancer
element, a suppresser, or an exogenous transcription modulatory
element is inserted in the genome of the intended host cell in
proximity and orientation sufficient to influence the transcription
of DNA encoding the desired truncated GDNF. The control element
does not encode truncated GDNF, but instead controls a portion of
the DNA present in the host cell genome. Thus, the expression of
truncated GDNF proteins may be achieved not by transfection of DNA
that encodes the truncated GDNF gene itself, but rather by the use
of targeting DNA (containing regions of homology with the
endogenous gene of interest) coupled with DNA regulatory segments
that provide the endogenous gene sequence with recognizable signals
for transcription of a truncated GDNF protein.
[0134] In accordance with the present invention, homologous
recombination methods may also be used to modify a cell that
contains a normally transcriptionally silent GDNF gene to produce a
cell which expresses GDNF. The GDNF protein may then be processed
to form a truncated GDNF protein(s).
Truncated GDNF Pharmaceutical Compositions
[0135] Truncated GDNF protein product pharmaceutical compositions
typically include a therapeutically effective amount of a truncated
GDNF protein product in admixture with one or more pharmaceutically
and physiologically acceptable formulation materials. Suitable
formulation materials include, but are not limited to,
antioxidants, preservatives, coloring, flavoring and diluting
agents, emulsifying agents, suspending agents, solvents, fillers,
bulking agents, buffers, delivery vehicles, diluents, excipients
and/or pharmaceutical adjuvants. For example, a suitable vehicle
may be water for injection, physiological saline solution, or
artificial cerebrospinal fluid (CSF), possibly supplemented with
other materials common in compositions for parenteral
administration. Neutral buffered saline or saline mixed with serum
albumin are further exemplary vehicles.
[0136] The primary solvent in a vehicle may be either aqueous or
non-aqueous in nature. In addition, the vehicle may contain other
pharmaceutically-acceptable excipients for modifying or maintaining
the pH, osmolarity, viscosity, clarity, color, sterility,
stability, rate of dissolution, or odor of the formulation.
Similarly, the vehicle may contain still other
pharmaceutically-acceptable excipients for modifying or maintaining
the stability, rate of dissolution, or rate of release of truncated
GDNF protein product, or for promoting the absorption or
penetration of truncated GDNF protein product across the
blood-brain barrier. Such excipients are those substances usually
and customarily employed to formulate dosages for parenteral
administration in either unit dose or multi-dose form or for direct
infusion into the CSF by continuous or periodic infusion from an
implanted pump.
[0137] Once the therapeutic composition has been formulated, it may
be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or dehydrated or lyophilized powder. Such
formulations may be stored either in a ready to use form or in a
form, e.g., lyophilized, requiring reconstitution prior to
administration.
[0138] The optimal pharmaceutical formulation will be determined by
one skilled in the art depending upon the route of administration
and desired dosage. See for example, Remington's Pharmaceutical
Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042)
pages 1435-1712 the disclosure of which is hereby incorporated by
reference. The composition may also involve particulate
preparations of polymeric compounds such as polylactic acid,
polyglycolic acid, etc. or into liposomes. Hylauronic acid may also
be used, and this may have the effect of promoting sustained
duration in the circulation. Such compositions may influence the
physical state, stability, rate of in vivo release, and rate of in
vivo clearance of the present proteins and derivatives.
[0139] Other effective administration forms, such as parenteral
slow-release formulations, inhalant mists, orally active
formulations, or suppositories, are also envisioned. Current
truncated GDNF protein product pharmaceutical compositions are
formulated for parenteral administration, e.g.,
intracerebroventricular injection. Such parenterally administered
therapeutic compositions are typically in the form of a
pyrogen-free, parenterally acceptable aqueous solution comprising
truncated GDNF protein product in a pharmaceutically acceptable
vehicle. One preferred vehicle is physiological saline.
[0140] It is also contemplated that certain formulations containing
truncated GDNF protein product are to be administered orally.
Truncated GDNF protein product which is administered in this
fashion may be encapsulated and may be formulated with or without
those carriers customarily used in the compounding of solid dosage
forms. The capsule may designed to release the active portion of
the formulation at the point in the gastrointestinal tract when
bioavailability is maximized and pre-systemic degradation is
minimized. Additional excipients may be included to facilitate
absorption of truncated GDNF protein product. Diluents, flavorings,
low melting point waxes, vegetable oils, lubricants, suspending
agents, tablet disintegrating agents, and binders may also be
employed.
Administration of Truncated GDNF Protein Product
[0141] The truncated GDNF protein product may be administered
parenterally via a subcutaneous, intramuscular, intravenous,
transpulmonary, transdermal, intrathecal or intracerebral route.
Protein growth factors that do not cross the blood-brain barrier
may be given directly intracerebrally or otherwise in association
with other elements that will transport them across the barrier. It
is preferred that the truncated GDNF protein product is
administered intracerebroventricularly or into the brain or spinal
cord subarachnoid space. Truncated GDNF protein product may also be
administered intracerebrally directly into the brain parenchyma.
Slow-releasing implants in the brain containing the neurotrophic
factor embedded in a biodegradable polymer matrix can also deliver
truncated GDNF protein product. Truncated GDNF protein product may
be administered extracerebrally in a form that has been modified
chemically or packaged so that it passes the blood-brain barrier,
or it may be administered in connection with one or more agents
capable of promoting penetration of truncated GDNF protein product
across the barrier. For example, a conjugate of NGF and monoclonal
anti-transferrin receptor antibodies has been shown to be
transported to the brain via binding to transferrin receptors. To
achieve the desired dose of truncated GDNF protein product,
repeated daily or less frequent injections may be administered, or
truncated GDNF protein product may be infused continuously or
periodically from a constant- or programmable-flow implanted pump.
The frequency of dosing will depend on the pharmacokinetic
parameters of the truncated GDNF protein product as formulated, and
the route of administration.
[0142] Regardless of the manner of administration, the specific
dose is typically calculated according to body weight or body
surface area. For diseases involving the brain, the specific dose
is typically calculated according to the approximate brain weight
of the patient, which also may be estimated based on body weight or
body surface area. Further refinement of the calculations necessary
to determine the appropriate dosage for treatment involving each of
the above mentioned formulations is routinely made by those of
ordinary skill in the art, especially in light of the dosage
information and assays disclosed herein. Appropriate dosages may be
ascertained through use of the established assays for determining
dosages utilized in conjunction with appropriate dose-response
data. The final dosage regimen involved in a method of treating a
specific condition will be determined by the attending physician,
considering various factors which modify the action of drugs, e.g.,
the age, condition, body weight, sex and diet of the patient, the
severity of any infection, time of administration and other
clinical factors.
[0143] Truncated GDNF protein product of the present invention may
also be employed, alone or in combination with other growth factors
in the treatment of nerve disease. For example, truncated GDNF
protein product may be used in treating some forms of nerve disease
in combination with nerve growth factor. In addition, other factors
or other molecules, including chemical compositions, may be
employed together with truncated GDNF protein product. In the
treatment of Parkinson's Disease, it is contemplated that truncated
GDNF protein product be used by itself or in conjunction with the
administration of Levodopa, wherein the truncated GDNF would
enhance the production of endogenous dopamine and the neuronal
uptake of the increased concentration of dopamine.
[0144] As stated above, it is also contemplated that additional
neurotrophic or neuron nurturing factors will be useful or
necessary to treat some neuronal cell populations or some types of
injury or disease. Other factors that may be used in conjunction
with truncated GDNF include, but are not limited to: mitogens such
as insulin, insulin-like growth factors, epidermal growth factor,
vasoactive growth factor, pituitary adenylate cyclase activating
polypeptide, interferon and somatostatin; neurotrophic factors such
as brain derived neurotrophic factor, neurotrophin-3,
neurotrophin-4/5, neurotrophin-6, insulin-like growth factor,
ciliary neurotrophic factor, acidic and basic fibroblast growth
factors, fibroblast growth factor-5, transforming growth
factor-.beta., cocaine-amphetamine regulated transcript (CART) and
mature GDNF; and other growth factors such as epidermal growth
factor, leukemia inhibitory factor, interleukins, interferons, and
colony stimulating factors; as well as molecules and materials
which are the functional equivalents to these factors.
[0145] It is envisioned that the continuous administration or
sustained delivery of a truncated GDNF protein product may be
advantageous for a given treatment. While continuous administration
may be accomplished via a mechanical means, such as with an
infusion pump, it is contemplated that other modes of continuous or
near continuous administration may be practiced. For example,
chemical derivatization may result in sustained release forms of
the protein which have the effect of continuous presence in the
blood stream, in predictable amounts, based on a determined dosage
regimen. Thus, truncated GDNF protein products include truncated
GDNF protein derivatized to effectuate such continuous
administration.
[0146] Truncated GDNF protein cell therapy, e.g., intracerebral
implantation of cells producing truncated GDNF protein, is also
contemplated. This embodiment of the present invention may include
implanting into patients cells which are capable of synthesizing
and secreting a biologically active form of truncated GDNF protein.
Such truncated GDNF protein producing-cells may be cells which do
not normally produce a neurotrophic factor but have been modified
to produce truncated GDNF, or they could be cells whose ability to
produce GDNF has been augmented by transformation with a
polynucleotide suitable for the expression and secretion of
truncated GDNF protein. In order to minimize a potential
immunological reaction in patients from administering GDNF of a
foreign species, it is preferred that the cells be of human origin
and produce truncated human GDNF protein.
[0147] Implanted cells may be encapsulated to avoid infiltration of
the cells into brain tissue. Human or non-human animal cells may be
implanted in patients in biocompatible, semi-permeable polymeric
enclosures or membranes to allow release of a truncated GDNF
protein product, but that prevent destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissue. Alternatively, the patient's own cells,
transformed ex vivo to produce truncated GDNF, could be implanted
directly into the patient without such encapsulation.
[0148] The methodology for the membrane encapsulation of living
cells is familiar to those of ordinary skill in the art, and the
preparation of the encapsulated cells and their implantation in
patients may be accomplished. See for example, U.S. Pat. Nos.
4,892,538; 5,011,472; and 5,106,627, the disclosures of which are
hereby incorporated by reference. A system for encapsulating living
cells is also described in PCT Application WO 91/10425 of Aebischer
et al., specifically incorporated herein by reference. See also,
PCT Application WO 91/10470 of Aebischer et al.; Winn et al.,
Exper. Neurol., 113:322-329, 1991; Aebischer et al., Exper.
Neurol., 111:269-275, 1991; Tresco et al., ASAIO, 38:17-23, 1992,
the disclosures of which are hereby incorporated by reference.
[0149] Truncated GDNF protein gene therapy in vivo is also
envisioned, wherein a nucleic acid sequence encoding a truncated
GDNF protein is introduced directly into the patient. For example,
a nucleic acid sequence encoding a truncated GDNF protein is
introduced into target cells via local injection of a nucleic acid
construct with or without an appropriate delivery vector, such as
an adeno-associated virus vector. Alternative viral vectors
include, but are not limited to, retrovirus, adenovirus, herpes
simplex virus and papilloma virus vectors. Physical transfer may be
achieved in vivo by local injection of the desired nucleic acid
construct or other appropriate delivery vector containing the
desired nucleic acid sequence, liposome-mediated transfer, direct
injection (naked DNA), receptor-mediated transfer (ligand-DNA
complex), or microparticle bombardment (gene gun).
[0150] It should be noted that the truncated GDNF protein product
formulations described herein may be used for veterinary as well as
human applications and that the term "patient" should not be
construed in a limiting manner. In the case of veterinary
applications, the dosage ranges should be the same as specified
above.
[0151] As a means of further characterizing truncated GDNF proteins
of the present invention, antibodies can be developed which bind to
the truncated GDNF protein such as to epitopes within the
X-[Cys.sup.41-Cys.sup.133]-Y amino acid sequence. One of ordinary
skill in the art can use well-known, published procedures to obtain
monoclonal and polyclonal antibodies, or recombinant antibodies,
which specifically recognize and bind to the various proteins
encoded by the amino acid sequences of the present invention. Such
antibodies may then be used to purify and characterize truncated
GDNF protein. Alternatively, the antibodies may be used as
therapeutical inhibitors of the proteins to which they are
directed.
[0152] Other aspects and advantages of the present invention will
be understood upon consideration of the following illustrative
examples. Example 1 addresses the expression of mature GDNF in a
mammalian cell system and the preparation of truncated GDNF
protein. Example 2 addresses the expression of mature GDNF in a
bacterial cell system. Example 3 addresses the expression of
various truncated GDNF proteins in a bacterial cell system. Example
4 compares the biological activity of the mature GDNF protein and
truncated GDNF protein in an assay for dopaminergic neuron
neurotrophic activity.
EXAMPLES
Example 1
Expression of Mature Human GDNF in Cho Cells and the Purification
of Cho Cell-Derived Truncated GDNF Protein Materials
[0153] The following materials are used in the expression of human
GDNF in dihydrofolate reductase-deficient CHO cells (CHOd.sup.-
cells; for example, as described by Urlaub and Chasin, Proc. Natl.
Acad. Sci., USA 77(7): 4216-4220 (1980)).
[0154] CHOd.sup.- medium contained: Dulbecco's Modified Eagle's
Medium (DMEM)--high glucose (Gibco/BRL); 5% fetal bovine serum
(HyClone); MEM non-essential amino acids (1%) (Gibco/BRL);
hypoxanthine/thymidine (1%) (Gibco/BRL); and
glutamine/penicillin/streptomycin (1%) (Irvine Scientific).
[0155] Selective medium contained: DMEM (high glucose); 5% dialyzed
fetal bovine serum (HyClone); MEM non-essential amino acids; and
glutamine/penicillin/streptomycin.
[0156] 2.times.HEPES-buffered saline (HBS) contained: 280 mM NaCl;
10 mM KCl; 1.5 mM Na.sub.2HPO.sub.4; 12 mM dextrose; and 50 mM
HEPES.
[0157] Tris-buffered saline plus Tween (TBST) contained: 137 mM
NaCl; 20 mM Tris/HCl pH 7.5; and 0.1% Tween-20.
Methods
Transfection and Selection
[0158] CHOd.sup.- cells (passage 20) were seeded into 60 mm tissue
culture dishes (Falcon) at a density of 8.times.10.sup.5 cells per
dish in CHOd.sup.- growth medium. On the following day, about three
hours prior to transfection, the medium on the cells was replaced
with fresh medium.
[0159] Plasmid constructs containing the appropriate GDNF cDNA were
prepared using well known techniques. For example, the plasmid
construct pDSR.alpha.2 was prepared substantially in accordance
with the process described in the co-owned and copending U.S. Pat.
No. 501,904 filed Mar. 29, 1990 (abandoned) (also see, European
Patent Application No. 90305433, Publication No. EP 398 753, filed
May 18, 1990 and WO 90/14363 (1990), the disclosures of which are
hereby incorporated by reference. An exemplary plasmid map which
illustrates the structural organization of the vector is depicted
in FIG. 2. It will be appreciated by those skilled in the art that
a variety of nucleic acid sequences encoding the mature GDNF
protein, such as the sequences depicted in FIGS. 1, 3 and 4, may
also be used.
[0160] A HindIII-XbaI DNA fragment containing the human GDNF coding
sequences and the consensus Kozak sequences, CCACC(ATG) was
retrieved by restriction enzyme digestion from a pcDNA3 based
expression vector (Invitrogen, San Diego, Calif.). The DNA fragment
was directly cloned into HindIII/XbaI cut pDSR.alpha.2. The
resulting plasmid was called pSW5. The plasmid DNA of pSW5 was
linearized at the PuvI site prior to transfection.
[0161] pDSR.alpha.2 (FIG. 2) is a derivative of the plasmid pCD
(Okayama & Berg, Mol. Cell Biol. 3: 280-289, 1983) with three
main modifications: (i) the SV40 polyadenylation signal has been
replaced with the signal from the .alpha.-subunit of bovine
follicular stimulating hormone, .alpha.-bFSH (Goodwin et al.,
Nucleic Acids Res. 11: 6873-6882, 1983); (ii) a mouse dihydrofolate
reductase minigene (Gasser et al., Proc. Natl. Acad. Sci. 79:
6522-6526, 1982) has been inserted downstream from the expression
cassette to allow selection and amplification of the transformants;
and (iii) a 267 bp fragment containing the "R-element" and part of
the "U5" sequences of the long terminal repeat (LTR) of human
T-cell leukemia virus type I (HTLV-I) has been cloned and inserted
between the SV40 promoter and the splice signals as described
previously (Takebe et al., Mol. Cell. Biol. 8: 466-472, 1988).
[0162] Solutions of DNA were prepared containing a final
concentration of 3.0 .mu.g/dish GDNF-plasmid DNA, 7.0 .mu.g/dish
mouse kidney genomic carrier DNA (Clontech), 25 .mu.l/dish 2.5M
CaCl.sub.2, and sterile distilled water to a final volume of 250
.mu.l/dish. DNA solutions containing pDSR.alpha.2 vector DNA or
carrier DNA alone were similarly prepared as positive and negative
controls, respectively. The DNA solutions were added dropwise to an
equal volume of 2.times.HEPES-buffered saline while passing air
bubbles through the solution. The DNA/HBS solutions were incubated
at room temperature for 30 minutes.
[0163] The medium was removed from the CHOd.sup.- cell cultures,
and 500 .mu.l of the DNA solutions were added per dish. The dishes
were incubated at room temperature for 30 minutes, after which time
CHOd.sup.- medium (5.0 ml.) was added to each dish. The dishes were
then incubated at 37.degree. C. overnight.
[0164] On the following day the medium was replaced with fresh
CHOd.sup.- medium. The next day, when the cells had reached
confluence, the cultures were trypsinized and replated in 100 mm
dishes (Falcon) at a ratio of 1.times.60 mm dish to 8.times.100 mm
dishes. Cells were replated in selective medium. The cultures were
re-fed with fresh medium every two to three days.
[0165] After 15 days, colonies of transfected cells were isolated
using glass cloning cylinders, trypsinized, and replated into
24-well dishes (Falcon). A total of 40 colonies was isolated from
the GDNF/pSW5-transfected cells. The remaining cells on the dishes
were trypsinized, pooled, and replated into two 100 mm dishes (one
pool for each DNA construct).
Screening of Transfected Cells:
[0166] The 24-well and pool cultures were grown to confluence, at
which time the growth medium was removed and replaced with
serum-free medium (400 .mu.l/well or 4 ml/dish). Cells were
incubated for 48 hours, and the conditioned medium was harvested.
The conditioned medium samples were analyzed for GDNF protein
expression by western blot. Aliquots of conditioned medium (20
.mu.l or 40 .mu.l) were diluted with electrophoresis sample buffer
(with or without R-mercaptoethanol). Samples containing
.beta.-mercaptoethanol were boiled for three minutes (reducing
conditions). Both reduced and non-reduced samples were run on 16%
Tris-glycine gels (Novex). Gels were electroblotted onto
nitrocellulose filters (Schleicher and Schuell BA-83, 0.2.mu.). The
blots were rinsed with TBST and then incubated in a blocking
solution of 5% dried milk (Carnation) in TBST for 30 minutes at
room temperature. Blots were then treated with GDNF antiserum
(rabbit polyclonal antisera raised against E. coli-derived GDNF;
1:1000 in 5% milk/TBST) for one hour at room temperature. The blots
were then rinsed with TBST and washed 1.times.10 minutes and
2.times.5 minutes with 1% milk/TBST. They were then treated with
anti-rabbit Ig-horse radish peroxidase-conjugated secondary
antibody (1:15,000 in 1% milk/TBST) for 20 minutes. Blots were
rinsed and washed with TBST 1.times.20 minutes and 2.times.10
minutes, followed by treatment with ECL reagents (Amersham) for one
minute and exposure to Hyperfilm-ECL (Amersham).
[0167] The following process describes the purification of
CHO-expressed GDNF and a CHO-derived clipped GDNF homodimer, from
one liter of conditioned media. Because of significant protease
action in the CHO medium, clipping the chain at residue 31, the
procedure may include the use of a protease inhibitor during
purification.
Step 1. Bead Chromatography:
[0168] Serum free conditioned media was made 20 mM
2-[N-Morpholino]ethane sulfonate (MES), pH 6.0, by adding one
fiftieth volume of 1 M MES, pH 6.0. Twenty five milliliters of SP
Sepharose Big Bead resin (Pharmacia), equilibrated with 20 mM MES,
pH 6.0, was added and stirred at 4.degree. C. for one hour. The
resin was collected by allowing it to settle and decanting off the
conditioned media. The decanted media was filtered through a
fritted disc filter to recover any unsettled resin. The settled
resin and that recovered by filtration was resuspended and poured
into a 2.5 cm diameter column and washed with three column volumes
of 0.15 M NaCl, 20 mM MES, pH 6.0 (A buffer). Protein was eluted
with a 300 ml gradient from A buffer to 1.0 M NaCl, 20 mM MES, pH
6.0 (B buffer), at a flow rate of 0.2 column volumes/minute with
absorbance monitored at 280 nm. Fractions containing 1.1 column
volumes were collected. The presence of GDNF in the fractions was
detected by Western blotting analysis. Fractions containing GDNF
were pooled for further purification. GDNF eluted between 0.3 and
0.6 M NaCl.
Step 2. HPLC C4 Chromatography:
[0169] The pool from Step 1 was made 0.1% (v/v) trifluoroacetic
acid (TFA), vacuum filtered through a 0.45 micron filter, and
applied to a Vydac C4 column (0.46.times.25 cm) conditioned with
aqueous 10% acetonitrile, 0.1% TFA (A buffer). Protein was eluted
with a 2%/minute linear gradient over 50 minutes from A buffer to
aqueous 90% acetonitrile, 0.1% TFA (B buffer) with absorbance
measured at 280 nm. One milliliter fractions were collected, and
the presence of GDNF was detected by Western blotting analysis.
GDNF was eluted between 45% and 55% acetonitrile. Fractions were
taken to dryness in vacuum.
Step 3. High Performance S Chromatography:
[0170] Fractions containing GDNF from Step 2 were resolublized in
one milliliter of 0.15 M NaCl, 10 mM Tris, pH 8.0, and applied to a
0.75.times.7.5 cm TSK-Gel 5WP high performance S column (Toso
Haas). A linear gradient of 0.4%/minute was run from 0.15 M NaCl,
10 mM Tris, pH 8.0 (A buffer) to 1.0 M NaCl, 10 mM Tris, pH 8.0 (B
buffer) over 50 minutes at a flow rate of 1 ml/min. One minute
fractions were collected with absorbance measured at 280 nm. At 35%
B buffer, the gradient was changed to 6.5%/minute over 10 minutes.
Western blot analysis of the fractions showed four major GDNF
components. Three of the components eluted during the 0.4%/minute
gradient and the fourth was eluted during the 6.5%/minute gradient.
Appropriate pools were made of similar components and submitted for
sequencing. Sequencing analyses identified an approximately 29-36
kD pool as [Arg.sup.32-Ile.sup.134] truncated GDNF protein. The
component at approximately 38-40 kD was identified as an
[Arg.sup.32-Ile.sup.134] truncated GDNF/mature GDNF heterodimer.
Finally, the approximately 41-44 kD component which was isolated
during the latter portion of the gradient was identified by
sequencing as the mature GDNF homodimer.
Example 2
Mature Human GDNF Produced in E. coli
[0171] The bacterial expression of mature human GDNF may be
achieved in accordance with the process described in Lin et al.
(U.S. patent application Ser. No. 08/182,183 filed May 23, 1994,
now U.S. Pat. No. 7,226,758, issued Jun. 15, 2007, and its parent
applications; PCT/US92/07888 filed Sep. 17, 1992 (WO 93/06116); and
European Patent Application No. 92921022.7 (Publication No. EP 610
254); the disclosures of which are hereby incorporated by
reference. Based upon the description of the present invention,
those of ordinary skill in the art will appreciate that a variety
of materials and methods may readily be used or adapted for
suitable expression in E. coli and other bacteria. For example,
alternate polynucleotides, such as those depicted in FIGS. 1, 3 and
4, may be used in the expression process.
Refolding and Purification of Expressed Mature GDNF
[0172] The transformed cells were processed at 5.degree. C. (unless
otherwise noted) as follows: cell paste (30 gm) was suspended into
a final volume of 200 milliliters using 25 mM Tris, pH 8.5
containing 5 mM EDTA, to yield a final cell slurry of 15% (w/v).
The cells were thoroughly dispersed using a Biospec hand-held low
shear homogenizer. The slurry was passed twice through a
microfluidizer at 14,500 psi to break the cells and release
inclusion bodies. The resulting homogenate was then centrifuged for
30 minutes at 16,000.times.g. The pellet of inclusion bodies
resulting from the centrifugation was washed by resuspension in
chilled water to a final volume of 240 milliliters using the
Biospec homogenizer, as before, to form a slurry. A sample of this
slurry was kept for HPLC analysis of the GDNF expression level. The
remaining slurry was centrifuged for 30 minutes at 16,000.times.g.
The supernatant was discarded, and a small amount of cold water was
added to the centrifuge bottle and gently swirled to remove the
loosely formed membrane layer on top of the inclusion bodies
pellet. The pellet was resuspended with the Biospec homogenizer
using a sufficient volume of cold water to yield a concentration of
2 mg/ml of GDNF. The inclusion bodies were then solubilized by
mixing the final inclusion bodies suspension (25 ml) and 8M
guanidine HCl (25 ml) containing 180 mM cysteine HCl, and 50 mM
Tris HCl, pH 8.7. The solubilization mixture was stirred at
25.degree. C. for 60 to 90 minutes after which it was poured, with
mixing, into 2 M urea (450 ml, at 5.degree. C.) containing 20 mM
Tris HCl, pH 8.75 and 0.2 M guanidine HCl. This refold mixture was
slowly stirred at 5.degree. C. for 72 hours.
[0173] The refolded GDNF was partially purified as follows: 20 mM
sodium acetate buffer (250 ml, pH 5) at 5.degree. C. was added with
rapid stirring to the refold mixture, and the pH was adjusted to 5
with glacial acetic acid. The resulting precipitate was removed by
centrifugation at 13,600.times.g for 45 minutes at 5.degree. C. The
supernatant from this centrifugation was used as the load solution
for the next purification step involving cation exchange
chromatography with SP-big bead resin (Pharmacia). The column was
operated at 5.degree. C. using 20 mM sodium acetate (pH 5) as the
equilibration, rinsing, and elution buffer system. A bed of resin
(5 ml) was sanitized with 5 column volumes (CV) of 0.2N NaOH and
then equilibrated with the acetate buffer (5 CV). The load solution
(190 ml) was applied to the column at 0.5 CV/minute followed by a
10 CV rinse with acetate buffer at the same flow rate. The GDNF was
then eluted off the resin with a 20 CV linear gradient of NaCl from
0.3 M to 0.9M in the acetate buffer at a flow rate of 0.1
CV/minute. The column eluate was monitored by absorbance at 280 nm
and collected as fractions which were assayed by SDS-PAGE. The
fractions containing GDNF were pooled from the front of the GDNF
peak at 10% peak height through to the back of the peak to 10% peak
height. The protein in this pool was entirely GDNF and, depending
upon the production strain used, contained 32% to 12% contamination
as modified GDNF forms. The pool was then dialyzed against PBS or
other formulation buffers and, in some cases, concentrated by
ultrafiltration to 25 mg/ml. Both wild type and analogue forms of
GDNF purified by this procedure were characterized by reverse phase
HPLC, cation exchange HPLC, mass spectrometry, and endotoxin levels
in order to compare purities of the preparations in relation to the
corresponding production strains.
Example 3
Recombinant Production of Truncated GDNF in E. coli
[0174] Exemplary truncated GDNF proteins were produced
substantially in accordance with the techniques described in Lin et
al. (U.S. patent application Ser. No. 08/182,183 filed May 23,
1994, now U.S. Pat. No. 7,226,758, issued Jun. 15, 2007, supra).
Alternative bacterial expression materials and methods, as
described above, may also be used. The E. coli-expressed truncated
GDNF proteins included the [Pro.sup.23-Ile.sup.134],
[Arg.sup.32-Ile.sup.134], and [Gly.sup.33-Ile.sup.134] truncated
GDNF proteins as depicted in FIGS. 5, 6 and 7, respectively. The
polynucleotides encoding these exemplary truncated GDNF proteins
were constructed as depicted in FIGS. 5, 6 and 7, but corresponding
polynucleotides such as those depicted in FIGS. 1, 3 and 4 may also
be used. The polynucleotides were constructed by standard PCR
procedures as described in PCR Technology, Principles and
Applications for DNA Amplification, Henry A. Erlich, ed., Stockton
Press, NY, 1989 (Chapter 6, Using PCR to Engineer DNA) the
disclosure of which is hereby incorporated by reference.
Example 4
Bioassay for Dopaminergic Neuron Neurotrophic Activity
[0175] The E. coli-expressed [Pro.sup.23-Ile.sup.134],
[Arg.sup.32-Ile.sup.134], [Gly.sup.33-Ile.sup.134] and
[Lys.sup.37-Ile.sup.134] truncated GDNF proteins of Example 3 and
the CHO-derived [Arg.sup.32-Ile.sup.134] truncated GDNF protein of
Example 1 were assessed on a qualitative basis for their ability to
promote dopamine uptake by substantia nigra dopaminergic
neurons.
Materials
[0176] The following materials are used in an assay to assess the
survival of dopaminergic neurons in the presence of truncated GDNF
proteins:
Cell Culture Media
[0177] High glucose Dulbecco's Modified Eagle's Medium (DMEM;
catalog #11965-092), Ham's F12 medium (F12; #11765-021),
Leibovitz's L15 medium without sodium bicarbonate (#41300-039), B27
medium supplement (#17504-010), penicillin/streptomycin
(#15070-014), L-glutamine (#25030-016), Dulbecco's
phosphate-buffered saline (D-PBS; #14190-052), Hank's balanced salt
solution with calcium and magnesium salts (HBSS; #24020-026),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES;
#15630-015), mouse laminin (#23017-015), and bovine serum albumin
fraction V (#110-18-017) were all from GIBCO, Grand Island, N.Y.
Heat-inactivated horse serum was from HyClone, Logan, Utah.
Conalbumin (C-7786), poly-L-ornithine hydrobromide (P-3655), bovine
insulin (1-5500), human transferrin (T-2252), putrescine (P-6024),
progesterone (P-6149), sodium selenite (S-9133), metrizamide
(M-3383) were all from Sigma Chemical Company, Saint-Louis, Mo.
Papain, deoxyribonuclease I (DNAase) and ovalbumin (Papain
dissociation system) were from Worthington Biochemicals, Freehold,
N.J.
[0178] Falcon sterile 96-well microplates (#3072), tissue culture
plastic ware and polypropylene centrifuge tubes were from
Becton-Dickinson, Oxnard, Calif. Nunc Lab-Tek tissue culture
chamber coverglasses (#136439) were from Baxter, Irvine, Calif.; 20
.mu.m (#460) nylon mesh was from Tetko, Elmsford, N.Y.; and 4''
dissecting forceps and 4'' dissecting scissors were from Roboz
Surgical, Washington, D.C.
Antibodies and Related Reagents
[0179] Polyclonal rabbit anti-tyrosine hydroxylase antibodies
(TE101) were from Eugene Tech, Ridgefield Park, N.J.; polyclonal
rabbit anti-neuronal-specific enolase antibodies (NSE, AB951) were
from Chemicon, Temecula, Calif.; and biotinylated goat anti-rabbit
IgG and peroxidase-conjugated avidin/biotin complex (ABC Elite;
Vectastain kit PK-6100) were from Vector Laboratories, Burlingame,
Calif. 3',3'-diaminobenzidine was from Cappel Laboratories, West
Chester, Pa. Superblock blocking buffer in PBS (#37515) was from
Pierce Chemical Company, Rockford, Ill. Triton X-100 (X100),
Nonidet P-40 (N6507) and hydrogen peroxide (30%, v/v; H1009) were
from Sigma. GBR-12909 dopamine uptake inhibitor (D-052) was from
RBI, Natick, Mass. .sup.3H-dopamine (tritiated dopamine, NE-131; 21
Ci/mmol) was from New England Nuclear, Boston, Mass. Optiphase
Supermix scintillation cocktail was from Wallac, Turku, Finland.
White ViewPlate-96 microplates (#6005182) were from Packard
Instruments Corporation, Meriden, Conn. All other reagents were
obtained from Sigma Chemical Company, unless otherwise
specified.
Preparation of Media
[0180] The basal medium was prepared as a 1:1 mixture of DMEM and
F12 medium, and was supplemented with B27 medium supplement added
as a 50-fold concentrated stock solution. L-glutamine was added at
a final concentration of about 2 mM, penicillin at about 100 IU/I,
and streptomycin at about 100 mg/l. Heat-inactivated horse serum
was added to a final concentration of about 15 percent. After
mixing, the pH was adjusted to about 7.3, and the media were kept
at 4.degree. C. The media were prepared fresh just before use in
order to minimize inter-experimental variations. Plastic pipettes
and containers were used throughout to minimize protein
adsorption.
Culture Substratum
[0181] To encourage optimal attachment of substratum neurons and
neurite outgrowth, microtiter plate surfaces (the culture
substratum) were modified by sequential coatings with
poly-L-ornithine and laminin, as follows. The plate surfaces were
completely covered with a 0.1 mg/ml sterile solution of
poly-L-ornithine in 0.1 M boric acid (pH 8.4) for at least one hour
at room temperature, followed by a sterile wash with Super-Q water.
The water wash was then aspirated, and a 1.0 .mu.g/ml solution of
mouse laminin in PBS was added and incubated at 37.degree. C. for
two hours. These procedures were conducted just before using the
plates in order to ensure reproducibility of the results.
Preparation of Embryonic Rat Substantia Nigra Cultures
[0182] Embryonic rat brains were used as the source of substantia
nigra dopaminergic neurons. Timed-pregnant Sprague-Dawley rats at
embryonic day 15 were used. A maximum of 36 embryos (about three
litters) were processed per experiment. The pregnant rats were
killed by exposure to CO.sub.2, their abdominal cavities opened
with dissecting scissors, and the fetuses were removed from the
uteri. Fetal brains were then dissected and cleaned of blood and
meninges, and the ventral tegmental area containing the substantia
nigra was dissected using well-defined anatomical landmarks (Altman
and Bayer, Atlas of Prenatal Rat Brain Development, CRC Press, Boca
Raton, Fla., 1995). The tissues were collected in ice-cold D-PBS,
transferred into 10 milliliters of dissociation medium (120 units
papain and 2000 units DNAase in HBSS) and then incubated for 45
minutes at about 37.degree. C. on a rotary platform shaker at about
200 rpm. The cells were then dispersed by trituration through
fire-polished Pasteur pipettes, sieved through a 20 .mu.m Nitex
mesh to discard undissociated tissue, and centrifuged for five
minutes at 200.times.g using an IEC clinical centrifuge. The
resulting cell pellet was resuspended into HBSS containing
ovalbumin and about 500 units DNAase, layered on top of a 4%
ovalbumin solution (in HBSS) and centrifuged for about 10 minutes
at 500.times.g. The final pellet was resuspended in complete
culture medium (see above), adjusted to about 28,000 cells/ml, and
seeded in aliquots (90 .mu.l) into the 6 mm-wells of the 96-well
microplates previously coated with polyornithine and laminin.
Attachment of cells occurred rapidly, and the plating efficiency
was about 75 percent.
Immunohistochemistry of Dopaminergic Neurons
[0183] An indirect immunoperoxidase method described by Louis et
al. (J. Pharmacol. Exp. Therap., 262:1274-1283, 1992; Science,
259:689-692, 1993) was used with slight modifications, as follows,
to characterize the dopaminergic neurons in cultures of substantia
nigra. Cultures were fixed for about 30 minutes at room temperature
with 4% paraformaldehyde in D-PBS, pH 7.4, followed by three washes
in D-PBS (200 .mu.l per 6-mm well). The fixed cultures were then
incubated in Superblock blocking buffer in PBS, containing 1% NP-40
to increase the penetration of the antibodies. The primary rabbit
anti-tyrosine hydroxylase antibodies were then applied at a
dilution of about 1:2000 in the same buffer and incubated for one
hour at 37.degree. C. on a rotary shaker. After three washes with
D-PBS, the bound antibodies were detected using goat-anti-rabbit
biotinylated IgG at about a 1:500 dilution; these secondary
antibodies were incubated with the cells for about one hour at
37.degree. C. The cells were then washed three times with D-PBS,
and the secondary antibodies were detected with
avidin-biotin-peroxidase complex diluted at 1:500, and the cells
were incubated for about 45 minutes at 37.degree. C. After three
more washes with D-PBS, the cultures were reacted for 5-20 minutes
in a solution of 0.1 M Tris-HCl, pH 7.4, containing 0.04%
3',3'-diaminobenzidine-(HCl)4, 0.06 percent NiCl.sub.2 and 0.02
percent hydrogen peroxide.
Determining Neuronal Survival
[0184] Substantia nigra cultures were fixed and processed for
immunostaining as described above, and then examined with
bright-light optics at 200.times. magnification. The number of
neurons stained for tyrosine hydroxylase was counted in the entire
6-mm well of the 96-well microplates Viable neurons were
characterized as having a regularly-shaped cell body, with a major
axon-like process and several dendrite-like processes. Neurons
showing signs of degeneration, such as having irregular, vacuolated
perikarya or fragmented neurites, were excluded from the counts
(most of the degenerating neurons, however, detached from the
culture substratum). Dopaminergic neuron cell numbers were
expressed either as TH-positive neurons/6-mm well or as the
fold-change relative to control dopaminergic neuron density.
Determining Dopamine Uptake
[0185] Dopamine uptake was determined in cultures of 15-day-old
embryonic rat substantia nigra neurons that had been established in
white ViewPlate-96 microplates. The cultures were washed with
pre-warmed uptake buffer (about 100 .mu.l) which consists of a
modified Krebs-Ringer solution, pH 7.4, containing about 120 mM
NaCl, 4.7 mM KCl, 1.8 mM CaCl.sub.2, 1.2 mM MgSO.sub.4, 32 mM
NaHPO.sub.4, 1.3 mM EDTA, and 5.6 mM D-glucose. The uptake buffer
also contained 1 mM ascorbic acid and 50 .mu.M pargyline to prevent
the oxidation of dopamine. The cells were then preincubated at
37.degree. C. for about 10 minutes in uptake buffer. Tritiated
dopamine (.sup.3H-DA, 21 Ci/mmol) was then added to the substantia
nigra cultures at a concentration of about 50 nM in 75 .mu.l of
uptake buffer, and the cultures were incubated for about 60 minutes
at 37.degree. C. Non-specific dopamine uptake was determined by
incubating the cultures with uptake buffer containing the dopamine
uptake inhibitor GBR-12909 (1 .mu.M). Non-specific uptake
represented less than about one percent of total uptake. The uptake
assays were arrested by aspiration of the incubation medium
followed by three rapid washes with ice-cold uptake buffer (about
120 .mu.l). The cells were then lysed by addition of Optiphase
Supermix scintillation cocktail (200 .mu.l), and radioactivity was
determined by scintillation spectrometry using a Wallac
MicrobetaPlus 96-well microplate counter (i.e., dopamine uptake is
analyzed by scintillation counting of the retained tritium in the
cultures). The results are expressed either as dpm/6-mm well or as
the fold-change relative to control cultures.
Assays
Dopaminergic Neuron Survival and Morphological Development
[0186] Cultures of embryonic day 15 (E15) rat substantia nigra
enriched in dopaminergic neurons were used to demonstrate the
effect of truncated GDNF proteins on the survival of dopaminergic
neurons. The cultures were grown in polyornithine- and
laminin-coated 96-well microplates for up to six days alone or in
the presence of various concentrations (ranging from about 1
.mu.g/ml to about 10 ng/ml) of the following proteins: E.
coli-expressed mature hGDNF; E. coli-expressed
[Pro.sup.23-Ile.sup.134], [Arg.sup.32-Ile.sup.134],
[Gly.sup.33-Ile.sup.134] and [Lys.sup.37-Ile.sup.134] truncated
GDNF proteins; CHO cell-expressed mature hGDNF; and CHO
cell-derived [Arg.sup.32-Ile.sup.134] truncated GDNF protein. The
culture medium consisted of DMEM/F12 supplemented with 15%
heat-inactivated horse serum (E15 cultures) or 2.5%
heat-inactivated horse serum, D-glucose, HEPES, insulin and
transferrin (P6 cultures). Immunostaining for tyrosine hydroxylase
(TH), the rate-limiting enzyme in dopamine biosynthesis, was used
as a marker for dopaminergic neurons. Since noradrenergic neurons
in the rhombencephalon also stain positive for TH, great care was
taken to dissect an area restricted to the ventral tegmentum of the
mesencephalon and to avoid the more caudal regions containing the
noradrenergic cell bodies. After six days, the E15 cultures
typically consisted of about 70% neurons as identified by neuronal
specific enolase immunostaining (described above) and 30%
non-neuronal cells (which had a flattened, phase-dark appearance);
dopaminergic neurons represented about 10-15% of the neuron
population.
[0187] After six days, the cultures were fixed with
paraformaldehyde and immunostained for tyrosine hydroxylase, a
marker that identifies dopaminergic neurons in these cultures. All
the tyrosine-hydroxylase-positive neurons present in a 6-mm well
were counted under brightfield optics. Three to six different wells
were analyzed for each experimental condition. The results were
expressed as the percentages of the number of
tyrosine-hydroxylase-positive neurons found in control
cultures.
[0188] Cultures of E15 substantia nigra treated with 1.0 ng/ml of
GDNF, CHO cell-expressed GDNF or E. coli-expressed GDNF, contained
about 38% and 27% more TH-immunoreactive neurons, respectively,
than untreated control cultures, indicating that both GDNF species
promote the survival of dopaminergic neurons. Cultures of E15
substantia nigra treated with 1.0 ng/ml of truncated GDNF protein
showed a similar increase in the number of TH-positive neurons in
cultures after six days in vitro: 42% for CHO cell-derived
[Arg.sup.32-Ile.sup.134] truncated GDNF protein; and 26% and 17%
for E. coli-expressed [Arg.sup.32-Ile.sup.134] and
[Gly.sup.33-Ile.sup.134] truncated GDNF proteins, respectively.
[0189] Comparison of control cultures and cultures treated with
mature and truncated GDNF proteins also revealed pronounced effects
of all the GDNF proteins on the morphological differentiation of
dopaminergic neurons. The effects of the [Arg.sup.32-Ile.sup.134]
and [Gly.sup.33-Ile.sup.134] truncated GDNF proteins were identical
to their respective mature GDNF protein counterparts.
TH-immunoreactive neurons in all GDNF-treated cultures possessed
significantly more complex and extensive neuritic arborization, as
well as a higher degree of neurite branching and an overall larger
soma size, than did TH-positive neurons in control cultures.
Dopamine Uptake
[0190] Dopamine uptake measures the number and activity of high
affinity dopamine reuptake transporter sites and reflects the
functional differentiation of dopaminergic neurons. Dopamine uptake
was measured in cultures of E15 rat substantia nigra after six days
in vitro either with or without mature GDNF or truncated GDNF
proteins. In these cultures, dopamine uptake had the
pharmacological profile characteristic of dopaminergic neurons,
i.e., it was nearly completely blocked (more than 98 percent) by
1.0 .mu.M GBR-12909, a dopamine transporter inhibitor specific for
dopaminergic neurons (ID50=20 nM). This indicates that dopamine
uptake measurements do not reflect the presence of contaminating
noradrenergic neurons, which can take up dopamine through
norepinephrine transporters but are not sensitive to GBR-12909
inhibition. The effects of CHO-cell-expressed mature GDNF and the
CHO-derived [Arg.sup.32-Ile.sup.134] truncated GDNF protein were
identical: about 65% increase, with an ED50 of about 20 pg/ml. E.
coli-expressed [Pro.sup.23-Lys.sup.37.DELTA.Asn.sup.37-Ile.sup.134]
truncated GDNF protein, as depicted in FIG. 5, demonstrated a 65%
increase, with an ED50 of about 40 pg/ml. The effects on dopamine
uptake of the E. coli-expressed mature protein and the E.
coli-expressed [Arg.sup.32-Ile.sup.134], [Gly.sup.33-Ile.sup.134]
and [Lys.sup.37-Ile.sup.134] truncated GDNF proteins were the same:
about 50% increases, with ED50s of about 50 pg/ml.
[0191] These results indicate that the truncated GDNF proteins act
as potent survival-promoting and differentiation-inducing factors
for substantia nigra dopaminergic neurons. As such, they are
envisioned to be particularly useful in the treatment of
Parkinson's disease, a neurological disorder characterized by
decreased emotional acuity, slowing of both voluntary and
involuntary muscle movement, muscular rigidity, and tremor. Such
symptoms are attributed to the progressive degeneration of
dopamine-producing neurons located in the substantia nigra.
Degeneration of these neurons ("dopaminergic neurons") results in a
decrease of dopamine in an adjacent region of the brain called the
striatum.
Sequence CWU 1
1
531402DNAHomo sapiensCDS(1)..(402) 1tca cca gat aaa caa atg gca gtg
ctt cct aga aga gag cgg aat cgg 48Ser Pro Asp Lys Gln Met Ala Val
Leu Pro Arg Arg Glu Arg Asn Arg1 5 10 15cag gct gca gct gcc aac cca
gag aat tcc aga gga aaa ggt cgg aga 96Gln Ala Ala Ala Ala Asn Pro
Glu Asn Ser Arg Gly Lys Gly Arg Arg 20 25 30ggc cag agg ggc aaa aac
cgg ggt tgt gtc tta act gca ata cat tta 144Gly Gln Arg Gly Lys Asn
Arg Gly Cys Val Leu Thr Ala Ile His Leu 35 40 45aat gtc act gac ttg
ggt ctg ggc tat gaa acc aag gag gaa ctg att 192Asn Val Thr Asp Leu
Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu Ile 50 55 60ttt agg tac tgc
agc ggc tct tgc gat gca gct gag aca acg tac gac 240Phe Arg Tyr Cys
Ser Gly Ser Cys Asp Ala Ala Glu Thr Thr Tyr Asp65 70 75 80aaa ata
ttg aaa aac tta tcc aga aat aga agg ctg gtg act gac aaa 288Lys Ile
Leu Lys Asn Leu Ser Arg Asn Arg Arg Leu Val Thr Asp Lys 85 90 95gta
ggg cag gca tgt tgc aga ccc atc gcc ttt gat gat gac ctg tcg 336Val
Gly Gln Ala Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu Ser 100 105
110ttt tta gat gat aac ctg gtt tac cat att cta aga aag cat tcc gct
384Phe Leu Asp Asp Asn Leu Val Tyr His Ile Leu Arg Lys His Ser Ala
115 120 125aaa agg tgt gga tgt atc 402Lys Arg Cys Gly Cys Ile
1302134PRTHomo sapiens 2Ser Pro Asp Lys Gln Met Ala Val Leu Pro Arg
Arg Glu Arg Asn Arg1 5 10 15Gln Ala Ala Ala Ala Asn Pro Glu Asn Ser
Arg Gly Lys Gly Arg Arg 20 25 30Gly Gln Arg Gly Lys Asn Arg Gly Cys
Val Leu Thr Ala Ile His Leu 35 40 45Asn Val Thr Asp Leu Gly Leu Gly
Tyr Glu Thr Lys Glu Glu Leu Ile 50 55 60Phe Arg Tyr Cys Ser Gly Ser
Cys Asp Ala Ala Glu Thr Thr Tyr Asp65 70 75 80Lys Ile Leu Lys Asn
Leu Ser Arg Asn Arg Arg Leu Val Thr Asp Lys 85 90 95Val Gly Gln Ala
Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu Ser 100 105 110Phe Leu
Asp Asp Asn Leu Val Tyr His Ile Leu Arg Lys His Ser Ala 115 120
125Lys Arg Cys Gly Cys Ile 13034PRTHomo sapiens 3Lys Asn Arg
Gly145PRTHomo sapiens 4Gly Lys Asn Arg Gly1 556PRTHomo sapiens 5Arg
Gly Lys Asn Arg Gly1 567PRTHomo sapiens 6Gln Arg Gly Lys Asn Arg
Gly1 578PRTHomo sapiens 7Gly Gln Arg Gly Lys Asn Arg Gly1
589PRTHomo sapiens 8Arg Gly Gln Arg Gly Lys Asn Arg Gly1
5910PRTHomo sapiens 9Arg Arg Gly Gln Arg Gly Lys Asn Arg Gly1 5
101011PRTHomo sapiens 10Gly Arg Arg Gly Gln Arg Gly Lys Asn Arg
Gly1 5 101112PRTHomo sapiens 11Lys Gly Arg Arg Gly Gln Arg Gly Lys
Asn Arg Gly1 5 101213PRTHomo sapiens 12Gly Lys Gly Arg Arg Gly Gln
Arg Gly Lys Asn Arg Gly1 5 101314PRTHomo sapiens 13Arg Gly Lys Gly
Arg Arg Gly Gln Arg Gly Lys Asn Arg Gly1 5 101415PRTHomo sapiens
14Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg Gly Lys Asn Arg Gly1 5 10
151516PRTHomo sapiens 15Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg
Gly Lys Asn Arg Gly1 5 10 151617PRTHomo sapiens 16Glu Asn Ser Arg
Gly Lys Gly Arg Arg Gly Gln Arg Gly Lys Asn Arg1 5 10
15Gly1718PRTHomo sapiens 17Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg
Gly Gln Arg Gly Lys Asn1 5 10 15Arg Gly1819PRTHomo sapiens 18Asn
Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg Gly Lys1 5 10
15Asn Arg Gly1920PRTHomo sapiens 19Ala Asn Pro Glu Asn Ser Arg Gly
Lys Gly Arg Arg Gly Gln Arg Gly1 5 10 15Lys Asn Arg Gly
202021PRTHomo sapiens 20Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly
Arg Arg Gly Gln Arg1 5 10 15Gly Lys Asn Arg Gly 202122PRTHomo
sapiens 21Ala Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg
Gly Gln1 5 10 15Arg Gly Lys Asn Arg Gly 202223PRTHomo sapiens 22Ala
Ala Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly1 5 10
15Gln Arg Gly Lys Asn Arg Gly 202324PRTHomo sapiens 23Gln Ala Ala
Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg1 5 10 15Gly Gln
Arg Gly Lys Asn Arg Gly 202425PRTHomo sapiens 24Arg Gln Ala Ala Ala
Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg1 5 10 15Arg Gly Gln Arg
Gly Lys Asn Arg Gly 20 252526PRTHomo sapiens 25Asn Arg Gln Ala Ala
Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly1 5 10 15Arg Arg Gly Gln
Arg Gly Lys Asn Arg Gly 20 252627PRTHomo sapiens 26Arg Asn Arg Gln
Ala Ala Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys1 5 10 15Gly Arg Arg
Gly Gln Arg Gly Lys Asn Arg Gly 20 252728PRTHomo sapiens 27Glu Arg
Asn Arg Gln Ala Ala Ala Ala Asn Pro Glu Asn Ser Arg Gly1 5 10 15Lys
Gly Arg Arg Gly Gln Arg Gly Lys Asn Arg Gly 20 252829PRTHomo
sapiens 28Arg Glu Arg Asn Arg Gln Ala Ala Ala Ala Asn Pro Glu Asn
Ser Arg1 5 10 15Gly Lys Gly Arg Arg Gly Gln Arg Gly Lys Asn Arg Gly
20 252930PRTHomo sapiens 29Arg Arg Glu Arg Asn Arg Gln Ala Ala Ala
Ala Asn Pro Glu Asn Ser1 5 10 15Arg Gly Lys Gly Arg Arg Gly Gln Arg
Gly Lys Asn Arg Gly 20 25 303031PRTHomo sapiens 30Pro Arg Arg Glu
Arg Asn Arg Gln Ala Ala Ala Ala Asn Pro Glu Asn1 5 10 15Ser Arg Gly
Lys Gly Arg Arg Gly Gln Arg Gly Lys Asn Arg Gly 20 25 303132PRTHomo
sapiens 31Leu Pro Arg Arg Glu Arg Asn Arg Gln Ala Ala Ala Ala Asn
Pro Glu1 5 10 15Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg Gly Lys
Asn Arg Gly 20 25 303233PRTHomo sapiens 32Val Leu Pro Arg Arg Glu
Arg Asn Arg Gln Ala Ala Ala Ala Asn Pro1 5 10 15Glu Asn Ser Arg Gly
Lys Gly Arg Arg Gly Gln Arg Gly Lys Asn Arg 20 25 30Gly3334PRTHomo
sapiens 33Ala Val Leu Pro Arg Arg Glu Arg Asn Arg Gln Ala Ala Ala
Ala Asn1 5 10 15Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg
Gly Lys Asn 20 25 30Arg Gly3435PRTHomo sapiens 34Met Ala Val Leu
Pro Arg Arg Glu Arg Asn Arg Gln Ala Ala Ala Ala1 5 10 15Asn Pro Glu
Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg Gly Lys 20 25 30Asn Arg
Gly 353536PRTHomo sapiens 35Gln Met Ala Val Leu Pro Arg Arg Glu Arg
Asn Arg Gln Ala Ala Ala1 5 10 15Ala Asn Pro Glu Asn Ser Arg Gly Lys
Gly Arg Arg Gly Gln Arg Gly 20 25 30Lys Asn Arg Gly 35 3637PRTHomo
sapiens 36Lys Gln Met Ala Val Leu Pro Arg Arg Glu Arg Asn Arg Gln
Ala Ala1 5 10 15Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg
Gly Gln Arg 20 25 30Gly Lys Asn Arg Gly 353738PRTHomo sapiens 37Asp
Lys Gln Met Ala Val Leu Pro Arg Arg Glu Arg Asn Arg Gln Ala1 5 10
15Ala Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln
20 25 30Arg Gly Lys Asn Arg Gly 353839PRTHomo sapiens 38Pro Asp Lys
Gln Met Ala Val Leu Pro Arg Arg Glu Arg Asn Arg Gln1 5 10 15Ala Ala
Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly 20 25 30Gln
Arg Gly Lys Asn Arg Gly 3539417DNAHomo sapiens 39catatgtctc
cggataaaca aatggctgtt cttccacgtc gtgaacgtaa ccgtcaggcg 60gccgctgcta
acccggagaa ttcccgtggt aaaggtcgtc gtggtcagcg tggtaaaaac
120cgcggttgcg ttctgaccgc tatccacctg aacgttaccg acctgggtct
cggttacgaa 180accaaagaag aattaatctt ccgttactgc tccggttcct
gcgacgctgc tgaaaccacg 240tacgacaaaa tcctgaaaaa cctgtcccgt
aaccgtcgtc tggtttccga caaagttggt 300caagcttgct gccgtccgat
cgctttcgac gacgacctgt ccttcctgga cgacaacctg 360gtttaccaca
tcctgcgtaa acactccgct aagcgttgcg gttgcatcta aggatcc 41740417DNAHomo
sapiens 40catatgagcc cggacaaaca gatggcagta cttccacgtc gtgaacgtaa
tcgccaggca 60gcagctgcaa acccggaaaa ctcccgtggt aaaggtcgcc gtggccagcg
cggcaaaaac 120cgtggttgtg ttctgactgc aatccacctg aacgttactg
acctgggtct gggctacgaa 180accaaagaag aactgatctt ccgctactgc
agcggctctt gcgacgcagc tgaaaccact 240tacgacaaaa tcctgaaaaa
cctgtcccgt aaccgccgtc tggtaagcga caaagtaggt 300caggcatgct
gccgtccgat cgcattcgac gatgacctga gcttcctgga tgacaacctg
360gtttaccaca tcctgcgtaa acactccgct aaacgctgcg gttgcatcta aggatcc
41741345DNAHomo sapiensCDS(1)..(342) 41atg tcc cca gaa aat tct cgt
ggt aaa ggt cgt cgt ggt cag cgt ggt 48Met Ser Pro Glu Asn Ser Arg
Gly Lys Gly Arg Arg Gly Gln Arg Gly1 5 10 15aat aac cgc ggt tgc gtt
ctg acc gct atc cac ctg aac gtt acc gac 96Asn Asn Arg Gly Cys Val
Leu Thr Ala Ile His Leu Asn Val Thr Asp 20 25 30ctg ggt ctc ggt tac
gaa acc aaa gaa gaa tta atc ttc cgt tac tgc 144Leu Gly Leu Gly Tyr
Glu Thr Lys Glu Glu Leu Ile Phe Arg Tyr Cys 35 40 45tcc ggt tcc tgc
gac gct gct gaa acc acg tac gac aaa atc ctg aaa 192Ser Gly Ser Cys
Asp Ala Ala Glu Thr Thr Tyr Asp Lys Ile Leu Lys 50 55 60aac ctg tcc
cgt aac cgt cgt ctg gtt tcc gac aaa gtt ggt caa gct 240Asn Leu Ser
Arg Asn Arg Arg Leu Val Ser Asp Lys Val Gly Gln Ala65 70 75 80tgc
tgc cgt ccg atc gct ttc gac gac gac ctg tcc ttc ctg gac gac 288Cys
Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu Ser Phe Leu Asp Asp 85 90
95aac ctg gtt tac cac atc ctg cgt aaa cac tcc gct aag cgt tgc ggt
336Asn Leu Val Tyr His Ile Leu Arg Lys His Ser Ala Lys Arg Cys Gly
100 105 110tgc atc taa 345Cys Ile42114PRTHomo sapiens 42Met Ser Pro
Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg Gly1 5 10 15Asn Asn
Arg Gly Cys Val Leu Thr Ala Ile His Leu Asn Val Thr Asp 20 25 30Leu
Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu Ile Phe Arg Tyr Cys 35 40
45Ser Gly Ser Cys Asp Ala Ala Glu Thr Thr Tyr Asp Lys Ile Leu Lys
50 55 60Asn Leu Ser Arg Asn Arg Arg Leu Val Ser Asp Lys Val Gly Gln
Ala65 70 75 80Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu Ser Phe
Leu Asp Asp 85 90 95Asn Leu Val Tyr His Ile Leu Arg Lys His Ser Ala
Lys Arg Cys Gly 100 105 110Cys Ile43315DNAHomo sapiensCDS(1)..(312)
43atg cgt ggt caa cgt ggt aaa aac cgc ggt tgc gtt ctg act gca atc
48Met Arg Gly Gln Arg Gly Lys Asn Arg Gly Cys Val Leu Thr Ala Ile1
5 10 15cac ctg aac gtt act gac ctg ggt ctg ggc tac gaa acc aaa gaa
gaa 96His Leu Asn Val Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys Glu
Glu 20 25 30ctg atc ttc cgc tac tgc agc ggc tct tgc gac gca gct gaa
acc act 144Leu Ile Phe Arg Tyr Cys Ser Gly Ser Cys Asp Ala Ala Glu
Thr Thr 35 40 45tac gac aaa atc ctg aaa aac ctg tcc cgt aac cgc cgt
ctg gta agc 192Tyr Asp Lys Ile Leu Lys Asn Leu Ser Arg Asn Arg Arg
Leu Val Ser 50 55 60gac aaa gta ggt cag gca tgc tgc cgt ccg atc gca
ttc gac gat gac 240Asp Lys Val Gly Gln Ala Cys Cys Arg Pro Ile Ala
Phe Asp Asp Asp65 70 75 80ctg agc ttc ctg gat gac aac ctg gtt tac
cac atc ctg cgt aaa cac 288Leu Ser Phe Leu Asp Asp Asn Leu Val Tyr
His Ile Leu Arg Lys His 85 90 95tcc gct aaa cgc tgc ggt tgc atc taa
315Ser Ala Lys Arg Cys Gly Cys Ile 10044104PRTHomo sapiens 44Met
Arg Gly Gln Arg Gly Lys Asn Arg Gly Cys Val Leu Thr Ala Ile1 5 10
15His Leu Asn Val Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys Glu Glu
20 25 30Leu Ile Phe Arg Tyr Cys Ser Gly Ser Cys Asp Ala Ala Glu Thr
Thr 35 40 45Tyr Asp Lys Ile Leu Lys Asn Leu Ser Arg Asn Arg Arg Leu
Val Ser 50 55 60Asp Lys Val Gly Gln Ala Cys Cys Arg Pro Ile Ala Phe
Asp Asp Asp65 70 75 80Leu Ser Phe Leu Asp Asp Asn Leu Val Tyr His
Ile Leu Arg Lys His 85 90 95Ser Ala Lys Arg Cys Gly Cys Ile
10045312DNAHomo sapiensCDS(1)..(309) 45atg ggt caa cgt ggt aaa aac
cgt ggt tgt gtt ctg act gca atc cac 48Met Gly Gln Arg Gly Lys Asn
Arg Gly Cys Val Leu Thr Ala Ile His1 5 10 15ctg aac gtt act gac ctg
ggt ctg ggc tac gaa acc aaa gaa gaa ctg 96Leu Asn Val Thr Asp Leu
Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu 20 25 30atc ttc cgc tac tgc
agc ggc tct tgc gac gca gct gaa acc act tac 144Ile Phe Arg Tyr Cys
Ser Gly Ser Cys Asp Ala Ala Glu Thr Thr Tyr 35 40 45gac aaa atc ctg
aaa aac ctg tcc cgt aac cgc cgt ctg gta agc gac 192Asp Lys Ile Leu
Lys Asn Leu Ser Arg Asn Arg Arg Leu Val Ser Asp 50 55 60aaa gta ggt
cag gca tgc tgc cgt ccg atc gca ttc gac gat gac ctg 240Lys Val Gly
Gln Ala Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu65 70 75 80agc
ttc ctg gat gac aac ctg gtt tac cac atc ctg cgt aaa cac tcc 288Ser
Phe Leu Asp Asp Asn Leu Val Tyr His Ile Leu Arg Lys His Ser 85 90
95gct aaa cgc tgc ggt tgc atc taa 312Ala Lys Arg Cys Gly Cys Ile
10046103PRTHomo sapiens 46Met Gly Gln Arg Gly Lys Asn Arg Gly Cys
Val Leu Thr Ala Ile His1 5 10 15Leu Asn Val Thr Asp Leu Gly Leu Gly
Tyr Glu Thr Lys Glu Glu Leu 20 25 30Ile Phe Arg Tyr Cys Ser Gly Ser
Cys Asp Ala Ala Glu Thr Thr Tyr 35 40 45Asp Lys Ile Leu Lys Asn Leu
Ser Arg Asn Arg Arg Leu Val Ser Asp 50 55 60Lys Val Gly Gln Ala Cys
Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu65 70 75 80Ser Phe Leu Asp
Asp Asn Leu Val Tyr His Ile Leu Arg Lys His Ser 85 90 95Ala Lys Arg
Cys Gly Cys Ile 10047135PRTHomo sapiens 47Met Ser Pro Asp Lys Gln
Met Ala Val Leu Pro Arg Arg Glu Arg Asn1 5 10 15Arg Gln Ala Ala Ala
Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg 20 25 30Arg Gly Gln Arg
Gly Lys Asn Arg Gly Cys Val Leu Thr Ala Ile His 35 40 45Leu Asn Val
Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu 50 55 60Ile Phe
Arg Tyr Cys Ser Gly Ser Cys Asp Ala Ala Glu Thr Thr Tyr65 70 75
80Asp Lys Ile Leu Lys Asn Leu Ser Arg Asn Arg Arg Leu Val Ser Asp
85 90 95Lys Val Gly Gln Ala Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp
Leu 100 105 110Ser Phe Leu Asp Asp Asn Leu Val Tyr His Ile Leu Arg
Lys His Ser 115 120 125Ala Lys Arg Cys Gly Cys Ile 130
13548104PRTHomo sapiens 48Met Arg Gly Gln Arg Gly Lys Asn Arg Gly
Cys Val Leu Thr Ala Ile1 5 10 15His Leu Asn Val Thr Asp Leu Gly Leu
Gly Tyr Glu Thr Lys Glu Glu 20 25 30Leu Ile Phe Arg Tyr Cys Ser Gly
Ser Cys Asp Ala Ala Glu Thr Thr 35 40 45Tyr Asp Lys Ile Leu Lys Asn
Leu Ser Arg Asn Arg Arg Leu Val Ser 50 55 60Asp Lys Val Gly Gln Ala
Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp65 70 75 80Leu Ser Phe Leu
Asp Asp Asn Leu Val Tyr His Ile Leu Arg Lys His
85 90 95Ser Ala Lys Arg Cys Gly Cys Ile 10049103PRTHomo sapiens
49Met Gly Gln Arg Gly Lys Asn Arg Gly Cys Val Leu Thr Ala Ile His1
5 10 15Leu Asn Val Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys Glu Glu
Leu 20 25 30Ile Phe Arg Tyr Cys Ser Gly Ser Cys Asp Ala Ala Glu Thr
Thr Tyr 35 40 45Asp Lys Ile Leu Lys Asn Leu Ser Arg Asn Arg Arg Leu
Val Ser Asp 50 55 60Lys Val Gly Gln Ala Cys Cys Arg Pro Ile Ala Phe
Asp Asp Asp Leu65 70 75 80Ser Phe Leu Asp Asp Asn Leu Val Tyr His
Ile Leu Arg Lys His Ser 85 90 95Ala Lys Arg Cys Gly Cys Ile
10050114PRTHomo sapiens 50Met Ser Pro Glu Asn Ser Arg Gly Lys Gly
Arg Arg Gly Gln Arg Gly1 5 10 15Asn Asn Arg Gly Cys Val Leu Thr Ala
Ile His Leu Asn Val Thr Asp 20 25 30Leu Gly Leu Gly Tyr Glu Thr Lys
Glu Glu Leu Ile Phe Arg Tyr Cys 35 40 45Ser Gly Ser Cys Asp Ala Ala
Glu Thr Thr Tyr Asp Lys Ile Leu Lys 50 55 60Asn Leu Ser Arg Asn Arg
Arg Leu Val Ser Asp Lys Val Gly Gln Ala65 70 75 80Cys Cys Arg Pro
Ile Ala Phe Asp Asp Asp Leu Ser Phe Leu Asp Asp 85 90 95Asn Leu Val
Tyr His Ile Leu Arg Lys His Ser Ala Lys Arg Cys Gly 100 105 110Cys
Ile5119PRTHomo sapiens 51Ser Pro Glu Asn Ser Arg Gly Lys Gly Arg
Arg Gly Gln Arg Gly Lys1 5 10 15Asn Arg Gly526PRTHomo sapiens 52Arg
Gly Gln Arg Gly Lys1 5537PRTHomo sapiens 53Lys Asn Arg Gly Cys Val
Leu1 5
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