U.S. patent application number 09/859894 was filed with the patent office on 2002-10-17 for use of antibodies specific for growth differentiation factor-11.
This patent application is currently assigned to Johns Hopkins University School of Medicine. Invention is credited to Lee, Se-Jin, McPherron, Alexandra C..
Application Number | 20020150577 09/859894 |
Document ID | / |
Family ID | 23041174 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020150577 |
Kind Code |
A1 |
Lee, Se-Jin ; et
al. |
October 17, 2002 |
USE OF ANTIBODIES SPECIFIC FOR GROWTH DIFFERENTIATION FACTOR-11
Abstract
A transgenic non-human animal of the species selected from the
group consisting of avian, bovine, ovine and porcine having a
transgene which results in disrupting the production of and/or
activity of growth differentiation factor-11 (GDF-11) chromosomally
integrated into the germ cells of the animal is disclosed. Also
disclosed are methods for making such animals, and methods of
treating animals, including humans, with antibodies or antisense
directed to GDF-11. The animals so treated are characterized by
increased muscle tissue.
Inventors: |
Lee, Se-Jin; (Baltimore,
MD) ; McPherron, Alexandra C.; (Baltimore,
MD) |
Correspondence
Address: |
Lisa A. Haile, Ph.D.
Gray Cary Ware & Freidenrich LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
Johns Hopkins University School of
Medicine
|
Family ID: |
23041174 |
Appl. No.: |
09/859894 |
Filed: |
May 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09859894 |
May 16, 2001 |
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09019901 |
Feb 6, 1998 |
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09019901 |
Feb 6, 1998 |
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08795671 |
Feb 6, 1997 |
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6008434 |
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09019901 |
Feb 6, 1998 |
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08706958 |
Sep 3, 1996 |
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08706958 |
Sep 3, 1996 |
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08272763 |
Jul 8, 1994 |
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Current U.S.
Class: |
424/146.1 ;
435/320.1; 435/325; 435/69.4; 530/399; 536/23.5; 800/19 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 38/00 20130101; A61P 37/00 20180101; C07K 14/51 20130101; C07K
14/475 20130101 |
Class at
Publication: |
424/146.1 ;
800/19; 435/69.4; 435/320.1; 435/325; 530/399; 536/23.5 |
International
Class: |
A01K 067/027; C07H
021/04; A61K 039/395; C12P 021/02; C12N 005/06; C07K 014/475 |
Claims
1. A transgenic non-human animal having a transgene disrupting or
interfering with expression of growth differentiation factor-11
(GDF-11) chromosomally integrated into the germ cells of the
animal.
2. The transgenic animal of claim 1, wherein the animal is selected
from the group of species consisting of avian, bovine, ovine,
piscine, murine, and porcine.
3. The transgenic animal of claim 1 where the species is avian.
4. The transgenic animal of claim 1 where the species is
bovine.
5. The transgenic animal of claim 1 where the species is
porcine.
6. The transgenic animal of claim 1 where the species is ovine.
7. The transgenic animal of claim 1 where the species is
piscine.
8. The transgenic animal of claim 1, wherein the transgene
comprises GDF-11 antisense polynucleotide(s).
9. The transgenic animal of claim 1, wherein the trans gene
comprises a gene encoding a dominant negative GDF-11
polypeptide.
10. The transgenic animal of claim 1, wherein the animal is
homozygous or heterozygous for GDF-11 polynucleotide.
11. A chicken or turkey egg produced by the transgenic animal of
claim 3.
12. Beef obtained from the transgenic animal of claim 4.
13. Milk obtained from the transgenic animal of claim 4.
14. Pork obtained from the transgenic animal of claim 5.
15. Lamb obtained from the transgenic animal of claim 6.
16. Chicken or turkey meat produced by the transgenic animal of
claim 3.
17. A method of producing animal food products having increased
muscle mass comprising: a) introducing a transgene disrupting or
interfering with expression of growth differentiation factor-11
(GDF-11) into an embryo into germ cells of a pronuclear embryo of
the animal; b) implanting the embryo into the oviduct of a
pseudopregnant female thereby allowing the embryo to mature to full
term progeny; c) testing the progeny for presence of the transgene
to identify transgene-positive progeny; d) cross-breeding
transgene-positive progeny to obtain further transgene-positive
progeny; and e) processing the progeny to obtain foodstuff.
18. The method of claim 17, wherein the transgene comprises GDF-11
antisense polynucleotides.
19. The method of claim 17, wherein the transgene comprises a gene
encoding a dominant negative GDF-11 polypeptide.
20. A method of producing avian food products having reduced
cholesterol levels comprising: a) introducing a transgene
disrupting or interfering with expression of growth differentiation
factor-11(GDF-11) into an embryo of an avian animal; b) culturing
the embryo under conditions whereby progeny are hatched; c) testing
the progeny for presence of the transgene to identify
transgene-positive progeny; d) cross-breeding transgene-positive
progeny; and e) processing the progeny to obtain foodstuff.
21. The method of claim 20, wherein the transgene comprises GDF-11
antisense polynucleotides.
22. The method of claim 20, wherein the transgene comprises a gene
encoding a dominant negative GDF-11 polypeptide.
23. The transgenic animal of claim 20, wherein the transgene
comprises a polynucleotide encoding a truncated GDF-11
polypeptide.
24. A method for increasing the muscle mass in an animal comprising
administering to the animal an antibody, or fragment thereof, which
binds to GDF-11 polypeptide.
25. The method of claim 24, wherein anti-GDF-11 antibody is
administered to a domesticated animal.
26. The method of claim 24, wherein the antibody is a monoclonal
antibody or a polyclonal antibody.
27. The method of claim 24, wherein the anti-GDF-11 antibody is
administered by intravenous, intramuscular, multiple bolus, or
subcutaneous injections.
28. The method of claim 27, wherein the anti-GDF-11 antibody is
administered within a dose range of 0.1 .mu.g/kg to 100 mg/kg.
29. The method of claim 27, wherein the antibody is formulated in a
formulation suitable for administration by injection into an
animal.
30. A method of inhibiting the growth regulating actions of GDF-11
comprising contacting a GDF-11 agent with fetal or adult muscle
cells or progenitor cells.
31. The method of claim 30, wherein the agent is selected from the
group consisting of a monoclonal antibody, an antisense nucleic
acid and a dominant negative encoding nucleic acid sequence or
polypeptide.
32. The method of claim 31, wherein the antibody is a humanized
monoclonal antibody or a chimeric monoclonal antibody or fragment
thereof.
33. The method of claim 30, wherein the agent is administered to a
patient suffering from a disorder selected from the group
consisting of muscle wasting disease, neuromuscular disorder,
muscle atrophy and aging.
34. The method of claim 30, wherein the agent is administered to a
patient suffering from a disorder selected from the group
consisting of muscular dystrophy, spinal cord injury, traumatic
injury, congestive obstructive pulmonary disease (COPD), AIDS and
cachechia.
35. The method of claim 30, wherein the agent is administered to a
patient with muscle wasting disease or disorder by intravenous,
intramuscular or subcutaneous injection.
36. The method of claim 31, wherein the monoclonal antibody is
administered within a dose range between about 0.1/kg to about 100
mg/kg.
37. The method of claim 31, wherein the monoclonal antibody is
formulated in a formulation suitable for administration to a
patient.
38. A method for treating a muscle or adipose tissue disorder in a
subject, comprising administering a therapeutically effective
amount of a GDF-11 agent to the subject, thereby inhibiting
abnormal growth of muscle or adipose tissue.
39. The method as in claim 38, wherein the GDF-11 agent is selected
from the group consisting of an antisense polynucleotide, a
polynucleotide encoding a dominant negative GDF-11 polypeptide, a
GDF-11 antibody and a polynucleotide encoding a truncated GDF-11
polypeptide.
40. The method of claim 38, wherein the disorder is a cancer
selected from the group consisting of muscle, connective tissue, or
bone.
41. The method of claim 38, wherein the subject has an obesity
disorder.
42. A method of inhibiting the growth regulating actions of GDF-11
in a subject comprising administering to the subject, a GDF-11
agent that inhibits the action of GDF-11 in the subject.
43. The method as in claim 42, wherein the GDF-11 agent is selected
from the group consisting of an antisense polynucleotide, a
polynucleotide encoding a dominant negative GDF-1 polypeptide, a
GDF-11 antibody and a polynucleotide encoding a truncated GDF-11
polypeptide.
44. A method for identifying a compound that affects GDF-11
activity or gene expression comprising: a) incubating the compound
with GDF-11 polypeptide, or with a recombinant cell expressing
GDF-11 under conditions sufficient to allow the components to
interact; and b) determining the effect of the compound on GDF-11
activity or expression.
45. The method of claim 44, wherein the effect is inhibition of
GDF-11 activity or expression.
46. The method of claim 44, wherein the effect is stimulation of
GDF-11 activity or expression.
47. An isolated polynucleotide encoding a truncated GDF-11
polypeptide wherein the truncation is a loss of the C-terminal
active fragment of GDF-11.
48. The isolated polynucleotide of claim 47, wherein the
polynucleotide is set forth in FIG. 9.
Description
[0001] This application is a continuation-in-part application of
U.S. Application Serial No. 08/795,671, filed Feb. 6, 1997 and U.S.
application Ser. No. 08/706,958, filed Aug. 3, 1996, which is a
continuation of U.S. application Ser. No. 08/272,763,filed Jul. 8,
1994, now abandoned.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates generally to growth factors and
specifically to a new member of the transforming growth factor beta
(TGF-.beta.) superfamily, which is denoted, growth differentiation
factor-11 (GDF-11) and methods of use for modulating muscle cell
and adipose tissue growth.
[0004] 2. Description of Related Art
[0005] The transforming growth factor .beta.(TGF-.beta.)
superfamily encompasses a group of structurally-related proteins
which affect a wide range of differentiation processes during
embryonic development. The family includes, Mullerian inhibiting
substance (MIS), which is required for normal male sex development
(Behringer, et al., Nature, 345:167, 1990), Drosophila
decapentaplegic (DPP) gene product, which is required for
dorsal-ventral axis formation and morphogenesis of the imaginal
disks (Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Vg-1
gene product, which localizes to the vegetal pole of eggs ((Weeks,
et al., Cell, 51:861-867, 1987), the activins (Mason, et al.,
Biochem, Biophys. Res. Commun., 135:957-964, 1986), which can
induce the formation of mesoderm and anterior structures in Xenopus
embryos (Thomsen, et al., Cell, 63:485, 1990), and the bone
morphogenetic proteins (BMPs, osteogenin, OP-1) which can induce de
novo cartilage and bone formation (Sampath, et al., J Biol. Chem.,
265:13198, 1990). The TGF-.beta.s can influence a variety of
differentiation processes, including adipogenesis, myogenesis,
chondrogenesis, hematopolesis, and epithelial cell differentiation
(for review, see Massague, Cell 49:437, 1987).
[0006] The proteins of the TGF-.beta. family are initially
synthesized as a large precursor protein which subsequently
undergoes proteolytic cleavage at a cluster of basic residues
approximately 110-140 amino acids from the C-terminus. The
C-terminal regions, or mature regions, of the proteins are all
structurally related and the different family members can be
classified into distinct subgroups based on the extent of their
homology. Although the homologies within particular subgroups range
from 70% to 90% amino acid sequence identity, the homologies
between subgroups are significantly lower, generally ranging from
only 20% to 50%. In each case, the active species appears to be a
disulfide-linked dimer of C-terminal fragments. Studies have shown
that when the pro-region of a member of the TGF-.beta. family is
coexpressed with a mature region of another member of the
TGF-.beta. family, intracellular dimerization and secretion of
biologically active homodimers occur (Gray, A. et al., Science,
247:1328, 1990). Additional studies by Hammonds, et al., (Molec.
Endocrin. 5:149, 1991) showed that the use of the BMP-2 pro-region
combined with the BMP-4 mature region led to dramatically improved
expression of mature BMP-4. For most of the family members that
have been studied, the homodimeric species has been found to be
biologically active, but for other family members, like the
inhibins (Ling, et al., Nature, 321 :779, 1986) and the TGF-.beta.s
(Cheifetz, et al., Cell, 48:409, 1987), heterodimers have also been
detected, and these appear to have different biological properties
than the respective homodimers.
[0007] In addition, it is desirable to produce livestock and game
animals, such as cows, sheep, pigs, chicken and turkey, fish which
are relatively high in musculature and protein, and low in fat
content. Many drug and diet regimens exist which may help increase
muscle and protein content and lower undesirably high fat and/or
cholesterol levels, but such treatment is generally administered
after the fact, and is begun only after significant damage has
occurred to the vasculature. Accordingly, it would be desirable to
produce animals which are genetically predisposed to having higher
muscle content, without any ancillary increase in fat levels.
[0008] The food industry has put much effort into increasing the
amount of muscle and protein in foodstuffs. This quest is
relatively simple in the manufacture of synthetic foodstuffs, but
has been met with limited success in the preparation of animal
foodstuffs. Attempts have been made, for example, to lower
cholesterol levels in beef and poultry products by including
cholesterol-lowering drugs in animal feed (see e.g. Elkin and
Rogler, J. Agric. Food Chem. 1990, 38, 1635-1641). However, there
remains a need for more effective methods of increasing muscle and
reducing fat and cholesterol levels in animal food products.
SUMMARY OF THE INVENTION
[0009] The present invention provides a cell growth and
differentiation factor, GDF-11, a polynucleotide sequence which
encodes the factor, and antibodies which are immunoreactive with
the factor. This factor appears to relate to various cell
proliferative disorders, especially those involving muscle, nerve,
and adipose tissue.
[0010] In one embodiment, the invention provides a method for
detecting a cell proliferative disorder of muscle, nerve, or fat
origin and which is associated with GDF-11. In another embodiment,
the invention provides a method for treating a cell proliferative
disorder by suppressing or enhancing GDF-11 activity.
[0011] In another embodiment, the subject invention provides
non-human transgenic animals which are useful as a source of food
products with high muscle and protein content, and reduced fat and
cholesterol content. The animals have been altered chromosomally in
their germ cells and somatic cells so that the production of GDF-11
is produced in reduced amounts, or is completely disrupted,
resulting in animals with decreased levels of GDF-11 in their
system and higher than normal levels of muscle tissue, preferably
without increased fat and/or cholesterol levels. Accordingly, the
present invention also includes food products provided by the
animals. Such food products have increased nutritional value
because of the increase in muscle tissue. The transgenic non-human
animals of the invention include bovine, porcine, ovine and avian
animals, for example.
[0012] The subject invention also provides a method of producing
animal food products having increased muscle content. The method
includes modifying the genetic makeup of the germ cells of a
pronuclear embryo of the animal, implanting the embryo into the
oviduct of a pseudopregnant female thereby allowing the embryo to
mature to full term progeny, testing the progeny for presence of
the transgene to identify transgene-positive progeny,
cross-breeding transgene-positive progeny to obtain further
transgene-positive progeny and processing the progeny to obtain
foodstuff. The modification of the germ cell comprises altering the
genetic composition so as to disrupt or reduce the expression of
the naturally occurring gene encoding for production of GDF-11
protein. In a particular embodiment, the transgene comprises anti
sense polynucleotide sequences to the GDF-11 protein.
Alternatively, the transgene may comprise a non-functional sequence
which replaces or intervenes in the native GDF-11 gene.
[0013] The subject invention also provides a method of producing
avian food products having improved muscle content. The method
includes modifying the genetic makeup of the germ cells of a
pronuclear embryo of the avian animal, implanting the embryo into
the oviduct of a pseudopregnant female into an embryo of a chicken,
culturing the embryo under conditions whereby progeny are hatched,
testing the progeny for presence of the genetic alteration to
identify transgene-positive progeny, cross-breeding
transgene-positive progeny and processing the progeny to obtain
foodstuff.
[0014] The invention also provides a method for treating a muscle
or adipose tissue disorder in a subject. The method includes
administering a therapeutically effective amount of a GDF-11 agent
to the subject, thereby inhibiting abnormal growth of muscle or
adipose tissue. The GDF-11 agent may include an antibody, a GDF-11
antisense molecule or a dominant negative polypeptide, for example.
In one aspect, a method for inhibiting the growth regulating
actions of GDF-11 by contacting an anti-GDF-11 monoclonal antibody,
a GDF-11 antisense molecule or a dominant negative polypeptide (or
polynucleotide encoding a dominant negative polypeptide) with fetal
or adult muscle cells or progenitor cells is included. These agents
can be administered to a patient suffering from a disorder such as
muscle wasting disease, neuromuscular disorder, muscle atrophy,
obesity or other adipocyte cell disorders, and aging, for
example.
[0015] The invention also provides a method for a method for
identifying a compound that affects GDF-11 activity or gene
expression including incubating the compound with GDF-11
polypeptide, or with a recombinant cell expressing GDF-11 under
conditions sufficient to allow the compounds to interact and
determining the effect of the compound on GDF-11 activity or
expression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows the nucleotide and predicted amino acid
sequences of murine (FIG. 1a; SEQ ID NOS: 3 and 4, respectively)
and human (FIG. 1b; SEQ ID NOS: 1 and 2, respectively) GDF-11 The
putative proteolytic processing sites are shown by the shaded
boxes. In the human sequence, the potential N-linked glycosylation
signal is shown by the open box, and the consensus polyadenylation
signal is underlined; the poly A tail is not shown.
[0017] FIG. 2 shows Northern blots of RNA prepared from adult (FIG.
2a) or fetal and neonatal (FIG. 2b) tissues probed with a murine
GDF-11 probe.
[0018] FIG. 3 shows amino acid homologies among different members
of the TGF-.beta. superfamily. Numbers represent percent amino acid
identities between each pair calculated from the first conserved
cysteine to the C-terminus. Boxes represent homologies among
highly-related members within particular subgroups.
[0019] FIG. 4 shows an alignment of the predicted amino acid
sequences of human GDF-11 (top lines; SEQ ID NO: 2) with human
GDF-8 (bottom lines; SEQ ID NO: 5). Vertical lines indicate
identities. Dots represent gaps introduced in order to maximize the
alignment. Numbers represent amino acid positions relative to the
N-terminus. The putative proteolytic processing sites are shown by
the open box. The conserved cysteine residues on the C-terminal
region are shown by the shaded boxes.
[0020] FIG. 5 shows the expression of GDF-11 in mammalian cells.
Conditioned medium prepared from Chinese hamster ovary cells
transfected with a hybrid GDF-8/GDF-11 gene (see text) cloned into
the MSXND expression vector in either the antisense (lane 1) or
sense (lane 2) orientation was dialyzed, lyophilized, and subjected
to Western analysis using antibodies directed against the
C-terminal portion of GDF-8 protein. Arrows at right indicate the
putative unprocessed (pro-GDF-8/GDF-11) or processed GDF-11
proteins. Numbers at left indicate mobilities of molecular weight
standards.
[0021] FIG. 6 shows the chromosomal mapping of human GDF-11. DNA
samples prepared from human/rodent somatic cell lines were
subjected to PCR, electrophoresed on agarose gels, blotted, and
probed. The human chromosome contained in each of the hybrid cell
lines is identified at the top of each of the first 24 lanes (1-22,
X, and Y). In the lanes designated CHO, M, and H, the starting DNA
template was total genomic DNA from hamster, mouse, and human
sources, respectively. In the lane marked B1, no template DNA was
used. Numbers at left indicate the mobilities of DNA standards.
[0022] FIG. 7 shows the FISH localization of GDF-11. Metaphase
chromosomes derived from peripheral blood lymphocytes were
hybridized with digoxigenin-labelled human GDF-11 probe (a) or a
mixture of human GDF-11 genomic and chromosome 12-specific
centromere probes (b) and analyzed as described in the text. A
schematic showing the location of GDF-11 at position 12q13 is shown
in panel (c).
[0023] FIGS. 8a and 8b show the nucleotide sequence (SEQ ID NO: 10)
and deduced amino acid sequence (SEQ ID NO: 11) of murine
GDF-8.
[0024] FIG. 9 shows a map of the GDF-8 locus (top line) and
targeting construct (second line). The black and stippled boxes
represent coding sequences for the pro- and C-terminal regions,
respectively. The white boxes represent 5' and 3' untranslated
sequences. A probe derived from the region downstream of the 3'
homology fragment and upstream of the most distal HindIII site
shown hybridizes to an 11.2 kb HindIII fragment in the GDF-8 gene
and a 10.4 kb fragment in an homologously targeted gene.
Abbreviations: H, HindIII; X, Xba I.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides a growth and differentiation
factor, GDF-1, and a polynucleotide sequence encoding GDF-11.
GDF-11 is expressed at highest levels in muscle, brain, uterus,
spleen, and thymus and at lower levels in other tissues.
[0026] The TGF-.beta. superfamily consists of multifunctional
polypeptides that control proliferation, differentiation, and other
functions in many cell types. Many of the peptides have regulatory,
both positive and negative, effects on other peptide growth
factors. The structural homology between the GDF-11 protein of this
invention and the members of the TGF-.beta. family, indicates that
GDF-11 is a new member of the family of growth and differentiation
factors. Based on the known activities of many of the other
members, it can be expected that GDF-11 will also possess
biological activities that will make it useful as a diagnostic and
therapeutic reagent.
[0027] Certain members of this superfamily have expression patterns
or possess activities that relate to the function of the nervous
system. For example, one family member, namely GDNF, has been shown
to be a potent neurotrophic factor that can promote the survival of
dopaminergic neurons (Lin, et al., Science, 260:1130). Another
family member, namely dorsalin-1, is capable of promoting the
differentiation of neural crest cells (Basler, et al., Cell,
73:687, 1993). The inhibins and activins have been shown to be
expressed in the brain (Meunier, et al., Proc. Nat'l. Acad. Sci.,
USA, 85:247, 1988; Sawchenko, et al., Nature, 334:615, 1988), and
activin has been shown to be capable of functioning as a nerve cell
survival molecule (Schubert, et al., Nature, 344:868, 1990).
Another family member, namely GDF-1, is nervous system-specific in
its expression pattern (Lee, Proc. Nat'l. Acad. Sci., USA, 88:4250,
1991), and certain other family members, such as Vgr-1 (Lyons, et
al., Proc. Nat'l. Acad. Sci., USA, 86:4554, 1989; Jones, et al.,
Development, 111:581, 1991), OP-1 (Ozkaynak, et al., J. Biol.
Chem., 267:25220, 1992), and BMP-4 (Jones, et al., Development,
111:531, 1991), are also known to be expressed in the nervous
system. The expression of GDF-11 in brain and muscle suggests that
GDF-11 may also possess activities that relate to the function of
the nervous system. In particular, it is known, for example, that
skeletal muscle produces a factor or factors that promote the
survival of motor neurons (Brown, Trends Neurosci., 7:10, 1984).
The known neurotrophic activities of other members of this family
and the expression of GDF-11 in muscle suggest that one activity of
GDF-11 may be as a trophic factor for motor neurons; indeed, GDF-11
is highly related to GDF-8, which is virtually muscle-specific in
its expression pattern. Alternatively, GDF-11 may have neurotrophic
activities for other neuronal populations. Hence, GDF-11 may have
in vitro and in vivo applications in the treatment of
neurodegenerative diseases, such as amyotrophic lateral sclerosis,
or in maintaining cells or tissues in culture prior to
transplantation.
[0028] GDF-11 may also have applications in treating disease
processes involving muscle, such as in musculodegenerative diseases
or in tissue repair due to trauma. In this regard, many other
members of the TGF-.beta. family are also important mediators of
tissue repair. TGF-.beta. has been shown to have marked effects on
the formation of collagen and to cause a striking angiogenic
response in the newborn mouse (Roberts, et al., Proc. Natl. Acad.
Sci., USA 83:4167, 1986). TGF-.beta. has also been shown to inhibit
the differentiation of myoblasts in culture (Massague, et al.,
Proc. Natl. Acad. Sci., USA 83:8206, 1986). Moreover, because
myoblast cells may be used as a vehicle for delivering genes to
muscle for gene therapy, the properties of GDF-11 could be
exploited for maintaining cells prior to transplantation or for
enhancing the efficiency of the fusion process.
[0029] GDF-11 may also have applications in the treatment of
immunologic disorders. In particular, TGF-.beta. has been shown to
have a wide range of immunoregulatory activities, including potent
suppressive effects on B and T cell proliferation and function (for
review, see Palladino, et al., Ann. N.Y. Acad. Sci., 593:181,
1990). The expression of GDF-11 in spleen and thymus suggests that
GDF-11 may possess similar activities and therefore, may be used as
an anti-inflammatory agent or as a treatment for disorders related
to abnormal proliferation or function of lymphocytes.
[0030] The animals contemplated for use in the practice of the
subject invention are those animals generally regarded as useful
for the processing of food stuffs, i.e. avian such as meat bred and
egg laying chicken and turkey, ovine such as lamb, bovine such as
beef cattle and milk cows, piscine and porcine. For purposes of the
subject invention, these animals are referred to as "transgenic"
when such animal has had a heterologous DNA sequence, or one or
more additional DNA sequences normally endogenous to the animal
(collectively referred to herein as "transgenes") chromosomally
integrated into the germ cells of the animal. The transgenic animal
(including its progeny) will also have the transgene fortuitously
integrated into the chromosomes of somatic cells.
[0031] The TGF-P superfamily consists of multifunctional
polypeptides that control proliferation, differentiation, and other
functions in many cell types. Many of the peptides have regulatory,
both positive and negative, effects on other peptide growth
factors. The structural homology between the GDF-11 protein of this
invention and the members of the TGF-.beta. family, indicates that
GDF-11 is a new member of the family of growth and differentiation
factors. Based on the known activities of many of the other
members, it can be expected that GDF-11 will also possess
biological activities that will make it useful as a diagnostic and
therapeutic reagent.
[0032] In particular, certain members of this superfamily have
expression patterns or possess activities that relate to the
function of the nervous system. For example, the inhibins and
activins have been shown to be expressed in the brain (Meunier, et
al., Proc. Natl. Acad. Sci., USA, 85:247, 1988; Sawchenko, et al.,
Nature, 334:615, 1988), and activin has been shown to be capable of
functioning as a nerve cell survival molecule (Schubert, et al.,
Nature, 344:868, 1990). Another family member, namely, GDF-1, is
nervous system-specific in its expression pattern (Lee, S. J.,
Proc. Natl. Acad. Sci., USA, 88:4250, 1991), and certain other
family members, such as Vgr-1 (Lyons, et al., Proc. Natl. Acad.
Sci., USA, 86:4554, 1989; Jones, et al., Development, 111:531,
1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 267:25220, 1992),
and BMP-4 (Jones, et al., Development, 111:531, 1991), are also
known to be expressed in the nervous system. Because it is known
that skeletal muscle produces a factor or factors that promote the
survival of motor neurons (Brown, Trends Neurosci., 7:10, 1984),
the expression of GDF-11 in muscle suggests that one activity of
GDF-11 may be as a trophic factor for neurons. In this regard,
GDF-11 may have applications in the treatment of neurodegenerative
diseases, such as amyotrophic lateral sclerosis or muscular
dystrophy, or in maintaining cells or tissues in culture prior to
transplantation.
[0033] GDF-11may also have applications in treating disease
processes involving muscle, such as in musculodegenerative diseases
or in tissue repair due to trauma. In this regard, many other
members of the TGF-.beta. family are also important mediators of
tissue repair. TGF-.beta. has been shown to have marked effects on
the formation of collagen and to cause a striking angiogenic
response in the newborn mouse (Roberts, et al., Proc. Natl. Acad.
Sci., USA 83:4167, 1986). TGF-.beta. has also been shown to inhibit
the differentiation of myoblasts in culture (Massague, et al.,
Proc. Natl. Acad. Sci., USA 83:8206, 1986). Moreover, because
myoblast cells may be used as a vehicle for delivering genes to
muscle for gene therapy, the properties of GDF-11 could be
exploited for maintaining cells prior to transplantation or for
enhancing the efficiency of the fusion
[0034] The expression of GDF-11 in adipose tissue also raises the
possibility of applications for GDF-11 in the treatment of obesity
or of disorders related to abnormal proliferation of adipocytes. In
this regard, TGF-.beta. has been shown to be a potent inhibitor of
adipocyte differentiation in vitro (Ignotz and Massague, Proc.
Natl. Acad. Sci., USA 82:8530,1985).
Polypeptides, Polynucleotides, Vectors and Host Cells
[0035] The invention provides substantially pure GDF-8 polypeptide
and isolated polynucleotides that encode GDF-11. The term
"substantially pure" as used herein refers to GDF-11 which is
substantially free of other proteins, lipids, carbohydrates or
other materials with which it is naturally associated. One skilled
in the art can purify GDF-11 using standard techniques for protein
purification. The substantially pure polypeptide will yield a
single major band on a non-reducing polyacrylamide gel. The purity
of the GDF-11 polypeptide can also be determined by amino-terminal
amino acid sequence analysis. GDF-11 polypeptide includes
functional fragments of the polypeptide, as long as the activity of
GDF-11 remains. Smaller peptides containing the biological activity
of GDF-11 are included in the invention.
[0036] The invention provides polynucleotides encoding the GDF-11
protein. These polynucleotides include DNA, cDNA and RNA sequences
which encode GDF-11. It is understood that all polynucleotides
encoding all or a portion of GDF-11 are also included herein, as
long as they encode a polypeptide with GDF-11 activity. Such
polynucleotides include naturally occurring, synthetic, and
intentionally manipulated polynucleotides. For example, GDF-11
polynucleotide may be subjected to site-directed mutagenesis. The
polynucleotide sequence for GDF 11 also includes antisense
sequences. The polynucleotides of the invention include sequences
that are degenerate as a result of the genetic code. There are 20
natural amino acids, most of which are specified by more than one
codon. Therefore, all degenerate nucleotide sequences are included
in the invention as long as the amino acid sequence of GDF-11
polypeptide encoded by the nucleotide sequence is functionally
unchanged.
[0037] Specifically disclosed herein is a DNA sequence containing
the human GDF-11 gene. The sequence contains an open reading frame
encoding a polypeptide 407 amino acids in length. The sequence
contains a putative RXXR (SEQ ID NO: 6) proteolytic cleavage site
at amino acids 295-298. Cleavage of the precursor at this site
would generate an active C-terminal fragment 109 amino acids in
length with a predicted molecular weight of approximately 12,500
kD. Also disclosed herein is a partial murine genomic sequence.
Preferably, the human GDF-11 nucleotide sequence is SEQ ID NO: 1
and the mouse nucleotide sequence is SEQ ID NO:3.
[0038] The polynucleotide encoding GDF-11 includes SEQ ID NO:1 and
3, as well as nucleic acid sequences complementary to SEQ ID NO's:
1 and 3. A complementary sequence may include an antisense
nucleotide. When the sequence is RNA, the deoxynucleotides A, G, C,
and T of SEQ ID NO: 1 and 3 are replaced by ribonucleotides A, G.
C, and U, respectively. Also included in the invention are
fragments of the above-described nucleic acid sequences that are at
least 15 bases in length, which is sufficient to permit the
fragment to selectively hybridize to DNA that encodes the protein
of SEQ ID NO: 2 or 4 under physiological conditions (e.g., under
stringent conditions).
[0039] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0040] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined
empirically.
[0041] The C-terminal region of GDF-11 following the putative
proteolytic processing site shows significant homology to the known
members of the TGF-.beta. superfamily. The GDF-11 sequence contains
most of the residues that are highly conserved in other family
members (see FIG. 1). Like the TGF-.beta.s and inhibin .beta.s,
GDF-11 contains an extra pair of cysteine residues in addition to
the 7 cysteines found in virtually all other family members. Among
the known family members, GDF-11 is most homologous to GDF-8 (92%
sequence identity) (see FIG. 3).
[0042] Minor modifications of the recombinant GDF-11 primary amino
acid sequence may result in proteins which have substantially
equivalent activity as compared to the GDF-11 polypeptide described
herein. Such modifications may be deliberate, as by site-directed
mutagenesis, or may be spontaneous. All of the polypeptides
produced by these modifications are included herein as long as the
biological activity of GDF-11 still exists. Further, deletion of
one or more amino acids can also result in a modification of the
structure of the resultant molecule without significantly altering
its biological activity. This can lead to the development of a
smaller active molecule which would have broader utility. For
example, one can remove amino or carboxy terminal amino acids which
are not required for GDF-11 biological activity.
[0043] The nucleotide sequence encoding the GDF-11 polypeptide of
the invention includes the disclosed sequence and conservative
variations thereof. The term "conservative variation" as used
herein denotes the replacement of an amino acid residue by another,
biologically similar residue. Examples of conservative variations
include the substitution of one hydrophobic residue such as
isoleucine, valine, leucine or methionine for another, or the
substitution of one polar residue for another, such as the
substitution of arginine for lysine, glutamic for aspartic acid, or
glutamine for asparagine, and the like. The term "conservative
variation" also includes the use of a substituted amino acid in
place of an unsubstituted parent amino acid provided that
antibodies raised to the substituted polypeptide also immunoreact
with the unsubstituted polypeptide.
[0044] DNA sequences of the invention can be obtained by several
methods. For example, the DNA can be isolated using hybridization
techniques which are well known in the art. These include, but are
not limited to: 1) hybridization of genomic or cDNA libraries with
probes to detect homologous nucleotide sequences, 2) polymerase
chain reaction (PCR) on genomic DNA or cDNA using primers capable
of annealing to the DNA sequence of interest, and 3) antibody
screening of expression libraries to detect cloned DNA fragments
with shared structural features.
[0045] Preferably the GDF-11 polynucleotide of the invention is
derived from a mammalian organism, and most preferably from mouse,
rat, cow, pig, or human. GDF-11 polynucleotides from chicken, fish
and other species are also included herein. Screening procedures
which rely on nucleic acid hybridization make it possible to
isolate any gene sequence from any organism, provided the
appropriate probe is available. Oligonucleotide probes, which
correspond to a part of the sequence encoding the protein in
question, can be synthesized chemically. This requires that short,
oligopeptide stretches of amino acid sequence must be known. The
DNA sequence encoding the protein can be deduced from the genetic
code, however, the degeneracy of the code must be taken into
account. It is possible to perform a mixed addition reaction when
the sequence is degenerate. This includes a heterogeneous mixture
of denatured double-stranded DNA. For such screening, hybridization
is preferably performed on either single-stranded DNA or denatured
double-stranded DNA. Hybridization is particularly useful in the
detection of cDNA clones derived from sources where an extremely
low amount of mRNA sequences relating to the polypeptide of
interest are present. In other words, by using stringent
hybridization conditions directed to avoid non-specific binding, it
is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement (Wallace, et al., Nucl. Acid Res. 9:879,
1981).
[0046] The development of specific DNA sequences encoding GDF-11
can also be obtained by: 1 ) isolation of double-stranded DNA
sequences from the genomic DNA; 2) chemical manufacture of a DNA
sequence to provide the necessary codons for the polypeptide of
interest; and 3) in vitro synthesis of a doublestranded DNA
sequence by reverse transcription of mRNA isolated from a
eukaryotic donor cell. In the latter case, a double-stranded DNA
complement of mRNA is eventually formed which is generally referred
to as cDNA.
[0047] Of the three above-noted methods for developing specific DNA
sequences for use in recombinant procedures, the isolation of
genomic DNA isolates is the least common. This is especially true
when it is desirable to obtain the microbial expression of
mammalian polypeptides due to the presence of introns.
[0048] The synthesis of DNA sequences is frequently the method of
choice when the entire sequence of amino acid residues of the
desired polypeptide product is known. When the entire sequence of
amino acid residues of the desired polypeptide is not known, the
direct synthesis of DNA sequences is not possible and the method of
choice is the synthesis of cDNA sequences. Among the standard
procedures for isolating cDNA sequences of interest is the
formation of plasmid- or phage-carrying cDNA libraries which are
derived from reverse transcription of mRNA which is abundant in
donor cells that have a high level of genetic expression. When used
in combination with polymerase chain reaction technology, even rare
expression products can be cloned. In those cases where significant
portions of the amino acid sequence of the polypeptide are known,
the production of labeled single or double-stranded DNA or RNA
probe sequences duplicating a sequence putatively present in the
target cDNA may be employed in DNA/DNA hybridization procedures
which are carried out on cloned copies of the cDNA which have been
denatured into a single-stranded form (Jay, et al., Nucl. Acid
Res., 11:2325, 1983).
[0049] A cDNA expression library, such as lambda gt11, can be
screened indirectly for GDF-11 peptides having at least one
epitope, using antibodies specific for GDF-11. Such antibodies can
be either polyclonally or monoclonally derived and used to detect
expression product indicative of the presence of GDF-11 cDNA.
[0050] DNA sequences encoding GDF-11 can be expressed in vitro by
DNA transfer into a suitable host cell. "Host cells" are cells in
which a vector can be propagated and its DNA expressed. The term
also includes any progeny of the subject host cell. It is
understood that all progeny may not be identical to the parental
cell since there may be mutations that occur during replication.
However, such progeny are included when the term "host cell" is
used. Methods of stable transfer, meaning that the foreign DNA is
continuously maintained in the host, are known in the art.
[0051] In the present invention, the GDF-11 polynucleotide
sequences may be inserted into a recombinant expression vector. The
term "recombinant expression vector" refers to a plasmid, virus or
other vehicle known in the art that has been manipulated by
insertion or incorporation of the GDF-11 genetic sequences. Such
expression vectors contain a promoter sequence which facilitates
the efficient transcription of the inserted genetic sequence of the
host. The expression vector typically contains an origin of
replication, a promoter, as well as specific genes which allow
phenotypic selection of the transformed cells. Vectors suitable for
use in the present invention include, but are not limited to the
T7-based expression vector for expression in bacteria (Rosenberg,
et al., Gene, 56:125, 19117), the pMSXND expression vector for
expression in mammalian cells (Lee and Nathans, J. Biol. Chem.,
263:3521, 1988) and baculovirus-derived vectors for expression in
insect cells. The DNA segment can be present in the vector operably
linked to regulatory elements, for example, a promoter (e.g., T7,
metallothionein 1, or polyhedrin promoters).
[0052] Polynucleotide sequences encoding GDF-11 can be expressed in
either prokaryotes or eukaryotes. Hosts can include microbial,
yeast, insect and mammalian organisms. Methods of expressing DNA
sequences having eukaryotic or viral sequences in prokaryotes are
well known in the art. Biologically functional viral and plasmid
DNA vectors capable of expression and replication in a host are
known in the art. Such vectors are used to incorporate DNA
sequences of the invention. Preferably, the mature C-terminal
region of GDF-11 is expressed from a cDNA clone containing the
entire coding sequence of GDF-11. Alternatively, the C-terminal
portion of GDF-11 can be expressed as a fusion protein with the
pro- region of another member of the TGF-.beta. family or
co-expressed with another pro-region (see for example, Hammonds, et
al., Molec. Endocrin 5:149, 1991; Gray, A., and Mason, A., Science,
247:1328, 1990).
[0053] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method using procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired.
[0054] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used. Eukaryotic cells can also be cotransformed with DNA
sequences encoding the GDF-11 of the invention, and a second
foreign DNA molecule encoding a selectable phenotype, such as the
herpes simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein. (see for example, Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
[0055] Isolation and purification of microbial expressed
polypeptide, or fragments thereof, provided by the invention, may
be carried out by conventional means including preparative
chromatography and immunological separations involving monoclonal
or polyclonal antibodies. GDF-11 Antibodies and Methods of Use
[0056] The invention includes antibodies immunoreactive with GDF-11
polypeptide or functional fragments thereof. Antibody which
consists essentially of pooled monoclonal antibodies with different
epitopic specificities, as well as distinct monoclonal antibody
preparations are provided. Monoclonal antibodies are made from
antigen containing fragments of the protein by methods well known
to those skilled in the art (Kohler, et al., Nature, 256:495,
1975). The term antibody as used in this invention is meant to
include intact molecules as well as fragments thereof, such as Fab
and F(ab').sub.2, Fv and SCA fragments which are capable of binding
an epitopic determinant on GDF-11.
[0057] (1) An Fab fragment consists of a monovalent antigen-binding
fragment of an antibody molecule, and can be produced by digestion
of a whole antibody molecule with the enzyme papain, to yield a
fragment consisting of an intact light chain and a portion of a
heavy chain.
[0058] (2) An Fab' fragment of an antibody molecule can be obtained
by treating a whole antibody molecule with pepsin, followed by
reduction, to yield a molecule consisting of an intact light chain
and a portion of a heavy chain. Two Fab' fragments are obtained per
antibody molecule treated in this manner.
[0059] (3) An (Fab').sub.2 fragment of an antibody can be obtained
by treating a whole antibody molecule with the enzyme pepsin,
without subsequent reduction. A (Fab').sub.2 fragment is a dimer of
two Fab' fragments, held together by two disulfide bonds.
[0060] (4) An Fv fragment is defined as a genetically engineered
fragment containing the variable region of a light chain and the
variable region of a heavy chain expressed as two chains.
[0061] (5) A single chain antibody ("SCA") is a genetically
engineered single chain molecule containing the variable region of
a light chain and the variable region of a heavy chain, linked by a
suitable, flexible polypeptide linker.
[0062] As used in this invention, the term "epitope" refers to an
antigenic determinant on an antigen, such as a GDF-11 polypeptide,
to which the paratope of an antibody, such as an GDF-11-specific
antibody, binds. Antigenic determinants usually consist of
chemically active surface groupings of molecules, such as amino
acids or sugar side chains, and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics.
[0063] As is mentioned above, antigens that can be used in
producing GDF-11-specific antibodies include GDF-11 polypeptides or
GDF-11 polypeptide fragments. The polypeptide or peptide used to
immunize an animal can be obtained by standard recombinant,
chemical synthetic, or purification methods. As is well known in
the art, in order to increase immunogenicity, an antigen can be
conjugated to a carrier protein. Commonly used carriers include
keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum
albumin (BSA), and tetanus toxoid. The coupled peptide is then used
to immunize the animal (e.g., a mouse, a rat, or a rabbit). In
addition to such carriers, well known adjuvants can be administered
with the antigen to facilitate induction of a strong immune
response.
[0064] The term "cell-proliferative disorder" denotes malignant as
well as non-malignant cell populations which often appear to differ
from the surrounding tissue both morphologically and genotypically.
Malignant cells (i.e. cancer) develop as a result of a multistep
process. The GDF-11 polynucleotide that is an antisense molecule or
that encodes a dominant negative GDF-11 is usefull in treating
malignancies of the various organ systems, particularly, for
example, cells in muscle or adipose tissue. Essentially, any
disorder which is etiologically linked to altered expression of
GDF-11 could be considered susceptible to treatment with a GDF-11
agent (e.g., a suppressing or enhancing agent). One such disorder
is a malignant cell proliferative disorder, for example.
[0065] The invention provides a method for detecting a cell
proliferative disorder of muscle, uterine or neural tissue, for
example, which comprises contacting an anti-GDF-11 antibody with a
cell suspected of having a GDF-11 associated disorder and detecting
binding to the antibody. The antibody reactive with GDF-11 is
labeled with a compound which allows detection of binding to
GDF-11. For purposes of the invention, an antibody specific for
GDF-11 polypeptide may be used to detect the level of GDF-11 in
biological fluids and tissues. Any specimen containing a detectable
amount of antigen can be used. A preferred sample of this invention
is muscle, uterus, spleen, thymus, or neural tissue. The level of
GDF-11 in the suspect cell can be compared with the level in a
normal cell to determine whether the subject has a
GDF-11-associated cell proliferative disorder. Such methods of
detection are also useful using nucleic acid hybridization to
detect the level of GDF-11 mRNA in a sample or to detect an altered
GDF-11 gene. Preferably the subject is human.
[0066] The antibodies of the invention can be used in any subject
in which it is desirable to administer in vitro or in vivo
immunodiagnosis or immunotherapy. The antibodies of the invention
are suited for use, for example, in immunoassays in which they can
be utilized in liquid phase or bound to a solid phase carrier. In
addition, the antibodies in these immunoassays can be detectably
labeled in various ways. Examples of types of immunoassays which
can utilize antibodies of the invention are competitive and
non-competitive immunoassays in either a direct or indirect format.
Examples of such immunoassays are the radioimmunoassay (RIA) and
the sandwich (immunometric) assay. Detection of the antigens using
the antibodies of the invention can be done utilizing immunoassays
which are run in either the forward, reverse, or simultaneous
modes, including immunohistochemical assays on physiological
samples. Those of skill in the art will know, or can readily
discern, other immunoassay formats without undue
experimentation.
[0067] The antibodies of the invention can be bound to many
different carriers and used to detect the presence of an antigen
comprising the polypeptide of the invention. Examples of well-known
carriers include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon, amylases, natural and modified celluloses,
polyacrylamides, agaroses and magnetite. The nature of the carrier
can be either soluble or insoluble for purposes of the invention.
Those skilled in the art will know of other suitable carriers for
binding antibodies, or will be able to ascertain such, using
routine experimentation.
[0068] There are many different labels and methods of labeling
known to those of ordinary skill in the art. Examples of the types
of labels which can be used in the present invention include
enzymes, radioisotopes, fluorescent compounds, colloidal metals,
chemiluminescent compounds, phosphorescent compounds, and
bioluminescent compounds. Those of ordinary skill in the art will
know of other suitable labels for binding to the antibody, or will
be able to ascertain such, using routine experimentation.
[0069] Another technique which may also result in greater
sensitivity consists of coupling the antibodies to low molecular
weight haptens. These haptens can then be specifically detected by
means of a second reaction. For example, it is common to use such
haptens as biotin, which reacts with avidin, or dinitrophenyl,
puridoxal, and fluorescein, which can react with specific
antihapten antibodies.
[0070] In using the monoclonal antibodies of the invention for the
in vivo detection of antigen, the detectably labeled antibody is
given a dose which is diagnostically effective. The term
"diagnostically effective" means that the amount of detectably
labeled monoclonal antibody is administered in sufficient quantity
to enable detection of the site having the antigen comprising a
polypeptide of the invention for which the monoclonal antibodies
are specific.
[0071] The concentration of detectably labeled monoclonal antibody
which is administered should be sufficient such that the binding to
those cells having the polypeptide is detectable compared to the
background. Further, it is desirable that the detectably labeled
monoclonal antibody be rapidly cleared from the circulatory system
in order to give the best target-to-background signal ratio.
[0072] As a rule, the dosage of detectably labeled monoclonal
antibody for in vivo diagnosis will vary depending on such factors
as age, sex, and extent of disease of the individual. Such dosages
may vary, for example, depending on whether multiple injections are
given, antigenic burden, and other factors known to those of skill
in the art.
[0073] For in vivo diagnostic imaging, the type of detection
instrument available is a major factor in selecting a given
radioisotope. The radioisotope chosen must have a type of decay
which is detectable for a given type of instrument. Still another
important factor in selecting a radioisotope for in vivo diagnosis
is that deleterious radiation with respect to the host is
minimized. Ideally, a radioisotope used for in vivo imaging will
lack a particle emission, but produce a large number of photons in
the 140-250 keV range, which may readily be detected by
conventional gamma cameras.
[0074] For in vivo diagnosis radioisotopes may be bound to
immunoglobulin either directly or indirectly by using an
intermediate functional group. intermediate functional groups which
often are used to bind radioisotopes which exist as metallic ions
to immunoglobulins are the bifunctional chelating agents such as
diethylenetriaminepentacetic acid (DTPA) and
ethylenediaminetetraacetic acid (EDTA) and similar molecules.
Typical examples of metallic ions which can be bound to the
monoclonal antibodies of the invention are .sup.111In, .sup.97Ru,
.sup.67Ga, .sup.68Ga, .sup.72As , .sup.89Zr and .sup.201Tl.
[0075] The monoclonal antibodies of the invention can also be
labeled with a paramagnetic isotope for purposes of in vivo
diagnosis, as in magnetic resonance imaging (MRI) or electron spin
resonance (ESR). In general, any conventional method for
visualizing diagnostic imaging can be utilized. Usually gamma and
positron emitting radioisotopes are used for camera imaging and
paramagnetic isotopes for MRI. Elements which are particularly
useful in such techniques include .sup.157Gd, .sup.55Mn,
.sup.162Dy, .sup.52Cr, and .sup.56Fe.
[0076] The monoclonal antibodies of the invention can be used in
vitro and in vivo to monitor the course of amelioration of a
GDF-11-associated disease in a subject. Thus, for example, by
measuring the increase or decrease in the number of cells
expressing antigen comprising a polypeptide of the invention or
changes in the concentration of such antigen present in various
body fluids, it would be possible to determine whether a particular
therapeutic regimen aimed at ameliorating the GDF-11-associated
disease is effective. The term "ameliorate" denotes a lessening of
the detrimental effect of the GDF-11-associated disease in the
subject receiving therapy.
Additional Methods of Treatment and Diagnosis
[0077] The present invention identifies a nucleotide sequence that
can be expressed in an altered manner as compared to expression in
a normal cell, therefore it is possible to design appropriate
therapeutic or diagnostic techniques directed to this sequence.
Treatment includes administration of a reagent which modulates
activity. The term "modulate" envisions the suppression or
expression of GDF-11 when it is over-expressed, or augmentation of
GDF-11 expression when it is underexpressed. When a
muscle-associated disorder is associated with GDF-11
overexpression, such suppressive reagents as antisense GDF-11
polynucleotide sequence, dominant negative sequences or GDF-11
binding antibody can be introduced into a cell. In addition, an
anti-idiotype antibody which binds to a monoclonal antibody which
binds GDF-11 of the invention, or an epitope thereof, may also be
used in the therapeutic method of the invention. Alternatively,
when a cell proliferative disorder is associated with
underexpression or expression of a mutant GDF-11 polypeptide, a
sense polynucleotide sequence (the DNA coding strand) or GDF-11
polypeptide can be introduced into the cell. Such muscle-associated
disorders include cancer, muscular dystrophy, spinal cord injury,
traumatic injury, congestive obstructive pulmonary disease (COPD),
AIDS or cachecia. Neurodegenerative disorders are also envisioned
as treated by the method of the invention. In addition, the method
of the invention can be used in the treatment of obesity or of
disorders related to abnormal proliferation of adipocytes. One of
skill in the art can determine whether or not a particular
therapeutic course of treatment is successful by several methods
described herein (e.g., muscle fiber analysis or biopsy;
determination of fat content). The present examples demonstrate
that the methods of the invention are useful for decreasing fat
content, and therefore would be useful in the treatment of obesity
and related disorders (e.g., diabetes).
[0078] Thus, where a cell-proliferative disorder is associated with
the expression of GDF-11, nucleic acid sequences that interfere
with GDF-11 expression at the translational level can be used. This
approach utilizes, for example, antisense nucleic acid and
ribozymes to block translation of a specific GDF-11 mRNA, either by
masking that mRNA with an antisense nucleic acid or by cleaving it
with a ribozyme. Such disorders include neurodegenerative diseases,
for example. In addition, dominant-negative GDF-11 mutants would be
useful to actively interfere with function of "normal" GDF-11.
[0079] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub, Scientific American, 262:40, 1990). In the cell, the
antisense nucleic acids hybridize to the corresponding mRNA,
forming a double-stranded molecule. The antisense nucleic acids
interfere with the translation of the mRNA, since the cell will not
translate a mRNA that is double-stranded.
[0080] Antisense oligomers of about 15 nucleotides are preferred,
since they are easily synthesized and are less likely to cause
problems than larger molecules when introduced into the target
GDF-11-producing cell. The use of antisense methods to inhibit the
in vitro translation of genes is well known in the art
(Marcus-Sakura, Anal. Biochem., 172:289, 1988).
[0081] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030,
1988). A major advantage of this approach is that, because they are
sequence-specific, only mRNAs with particular sequences are
inactivated.
[0082] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-based
recognition sequences are preferable to shorter recognition
sequences.
[0083] In another embodiment of the present invention, a nucleotide
sequence encoding a GDF-11 dominant negative protein is provided.
For example, a genetic construct that contain such a dominant
negative encoding gene may be operably linked to a promoter, such
as a tissue-specific promoter. For example, a skeletal muscle
specific promoter (e.g., human skeletal muscle .alpha.-actin
promoter) or developmentally specific promoter (e.g., MyHC 3, which
is restricted in skeletal muscle to the embryonic period of
development, or an inducible promoter (e.g., the orphan nuclear
receptor TIS1).
[0084] Such constructs are useful in methods of modulating a
subject's skeletal mass. For example, a method include transforming
an organism, tissue, organ or cell with a genetic construct
encoding a dominant negative GDF-11 protein and suitable promoter
in operable linkage and expressing the dominant negative encoding
GDF-11 gene, thereby modulating muscle mass by interfering with
wild-type GDF-11 activity.
[0085] GDF-11 most likely forms dimers, homodimers or heterodimers
and may even form heterodimers with other GDF family members, such
as GDF-11 (see Example 4). Hence, while not wanting to be bound by
a particular theory, the dominant negative effect described herein
may involve the formation of non-functional homodimers or
heterodimers of dominant negative and wild-type GDF-11 monomers.
More specifically, it is possible that any non-functional homodimer
or any heterodimer formed by the dimerization of wild-type and
dominant negative GDF-11 monomers produces a dominant effect by: 1)
being synthesized but not processed or secreted; 2) inhibiting the
secretion of wild type GDF-11; 3) preventing normal proteolytic
cleavage of the preprotein thereby producing a nonfunctional GDF-11
molecule; 4) altering the affinity of the non-functional dimer
(e.g., homodimeric or heterodimeric GDF-11) to a receptor or
generating an antagonistic form of GDF-11 that binds a receptor
without activating it; or 5) inhibiting the intracellular
processing or secretion of GDF-11 related or TGF-.beta. family
proteins.
[0086] Non-functional GDF-1 can function to inhibit the growth
regulating actions of GDF-11 on muscle cells that include a
dominant negative GDF-11 gene. Deletion or missense dominant
negative forms of GDF-11 that retain the ability to form dimers
with wild- type GDF-11 protein but do not function as wild-type
GDF-11 proteins may be used to inhibit the biological activity of
endogenous wild- type GDF-11. For example, in one embodiment, the
proteolytic processing site of GDF-11 may be altered (e.g.,
deleted) resulting in a GDF-11 molecule able to undergo subsequent
dimerization with endogenous wild-type GDF-11 but unable to undergo
further processing into a mature GDF-11 form. Alternatively, a
non-functional GDF-11 can function as a monomeric species to
inhibit the growth regulating actions of GDF-11 on muscle
cells.
[0087] Any genetic recombinant method in the art may be used, for
example, recombinant viruses may be engineered to express a
dominant negative form of GDF-11 which may be used to inhibit the
activity of wild-type GDF-11. Such viruses may be used
therapeutically for treatment of diseases resulting from aberrant
over-expressions or activity of GDF-11 protein, such as in
denervation hypertrophy or as a means of controlling GDF-11
expression when treating disease conditions involving muscle, such
as in musculodegenerative diseases or in tissue repair due to
trauma or in modulating GDF-11 expression in animal husbandry
(e.g., transgenic animals for agricultural purposes).
[0088] The invention provides a method for treating a muscle or
adipose tissue disorder in a subject. The method includes
administering a therapeutically effective amount of a GDF-11 agent
to the subject, thereby inhibiting abnormal growth of muscle or
adipose tissue. The GDF-11 agent may include a GDF-11 antisense
molecule or a dominant negative polypeptide, for example. A
"therapeutically effective amount" of a GDF-11 agent is that amount
that ameliorates symptoms of the disorder or inhibits GDF-11
induced growth of muscle, for example, as compared with a normal
subject.
[0089] The present invention also provides gene therapy for the
treatment of cell proliferative or immunologic disorders which are
mediated by GDF-11 protein. Such therapy would achieve its
therapeutic effect by introduction of the GDF-11 antisense
polynucleotide or dominant negative encoding polynucleotide
sequences into cells having the proliferative disorder. Delivery of
antisense GDF-11 polynucleotide can be achieved using a recombinant
expression vector such as a chimeric virus or a colloidal
dispersion system. Especially preferred for therapeutic delivery of
antisense or dominant negative sequences is the use of targeted
liposomes. In contrast, when it is desirable to enhance GDF-11
production, a .quadrature. sense.quadrature. GDF-11 polynucleotide
is introduced into the appropriate cell(s).
[0090] Various viral vectors which can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A
number of additional retroviral vectors can incorporate multiple
genes. All of these vectors can transfer or incorporate a gene for
a selectable marker so that transduced cells can be identified and
generated. By inserting a GDF-11 sequence of interest into the
viral vector, along with another gene which encodes the ligand for
a receptor on a specific target cell, for example, the vector is
now target specific. Retroviral vectors can be made target specific
by attaching, for example, a sugar, a glycolipid, or a protein.
Preferred targeting is accomplished by using an antibody to target
the retroviral vector. Those of skill in the art will know of, or
can readily ascertain without undue experimentation, specific
polynucleotide sequences which can be inserted into the retroviral
genome or attached to a viral envelope to allow target specific
delivery of the retroviral vector containing the GDF-1 antisense
polynucleotide.
[0091] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence which enables
the packaging mechanism to recognize an RNA transcript for
encapsulation. Helper cell lines which have deletions of the
packaging signal include, but are not limited to .psi.2, PA317 and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
cells in which the packaging signal is intact, but the structural
genes are replaced by other genes of interest, the vector can be
packaged and vector virion produced.
[0092] Alternatively, NIH 3T3 or other tissue culture cells can be
directly transfected with plasmids encoding the retroviral
structural genes gag, pol and env, by conventional calcium
phosphate transfection. These cells are then transfected with the
vector plasmid containing the genes of interest. The resulting
cells release the retroviral vector into the culture medium.
[0093] Another targeted delivery system for GDF-11 antisense
polynucleotides is a colloidal dispersion system. Colloidal
dispersion systems include macromolecule complexes, nanocapsules,
microspheres, beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, and liposomes. The preferred
colloidal system of this invention is a liposome. Liposomes are
artificial membrane vesicles which are useful as delivery vehicles
in vitro and in vivo. It has been shown that large unilamellar
vesicles (LUV), which range in size from 0.2-4.0 82 m can
encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules. RNA, DNA and intact virions can be
encapsulated within the aqueous interior and be delivered to cells
in a biologically active form (Fraley, et al., Trends Biochem.
Sci., 6:77, 19111). In addition to mammalian cells, liposomes have
been used for delivery of polynucleotides in plant, yeast and
bacterial cells. in order for a liposome to be an efficient gene
transfer vehicle, the following characteristics should be present:
(1) encapsulation of the genes of interest at high efficiency while
not compromising their biological activity; (2) preferential and
substantial binding to a target cell in comparison to non-target
cells; (3) delivery of the aqueous contents of the vesicle to the
target cell cytoplasm at high efficiency; and (4) accurate and
effective expression of genetic information (Manning, et al.,
Biotechniques, 6:682, 1988).
[0094] The composition of the liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0095] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidyiglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0096] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0097] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various linking groups can
be used for joining the lipid chains to the targeting ligand.
[0098] Due to the expression of GDF-11 in muscle and adipose
tissue, there are a variety of applications using the polypeptide,
polynucleotide, and antibodies of the invention, related to these
tissues. Such applications include treatment of cell proliferative
disorders involving these and other tissues, such as neural tissue.
In addition, GDF-11 may be useful in various gene therapy
procedures. In embodiments where GDF-11 polypeptide is administered
to a subject, the dosage range is about 0.1 ug/kg to 100 mg/kg;
more preferably from about 1 ug/kg to 75 mg/kg and most preferably
from about 10 mg/kg to 50 mg/kg.
Chromosomal Location of GDF-11
[0099] The data in Example 6 shows that the human GDF-11 gene is
located on chromosome 2. By comparing the chromosomal location of
GDF-11 with the map positions of various human disorders, it should
be possible to determine whether mutations in the GDF-11 gene are
involved in the etiology of human diseases. For example, an
autosomal recessive form of juvenile amyotrophic lateral sclerosis
has been shown to map to chromosome 2 (Hentati, et al., Neurology,
42 [Suppl.3]:201, 1992). More precise mapping of GDF-11 and
analysis of DNA from these patients may indicate that GDF-11 is, in
fact, the gene affected in this disease. In addition, GDF-11 is
useful for distinguishing chromosome 2 from other chromosomes.
Transgenic Animals and Methods of Making the Same
[0100] Various methods to make the transgenic animals of the
subject invention can be employed. Generally speaking, three such
methods may be employed. In one such method, an embryo at the
pronuclear stage (a "one cell embryo") is harvested from a female
and the transgene is microinjected into the embryo, in which case
the transgene will be chromosomally integrated into both the germ
cells and somatic cells of the resulting mature animal. In another
such method, embryonic stem cells are isolated and the transgene
incorporated therein by electroporation, plasmid transfection or
microinjection, followed by reintroduction of the stem cells into
the embryo where they colonize and contribute to the germ line.
Methods for microinjection of mammalian species is described in
U.S. Pat. No. 4,873,191. In yet another such method, embryonic
cells are infected with a retrovirus containing the transgene
whereby the germ cells of the embryo have the transgene
chromosomally integrated therein. When the animals to be made
transgenic are avian, because avian fertilized ova generally go
through cell division for the first twenty hours in the oviduct,
microinjection into the pronucleus of the fertilized egg is
problematic due to the inaccessibility of the pronucleus.
Therefore, of the methods to make transgenic animals described
generally above, retrovirus infection is preferred for avian
species, for example as described in U.S. Pat. No. 5,162,215. If
microinjection is to be used with avian species, however, a
recently published procedure by Love et al., (Biotechnology, Jan.
12, 1994) can be utilized whereby the embryo is obtained from a
sacrificed hen approximately two and one-half hours after the
laying of the previous laid egg, the transgene is microinjected
into the cytoplasm of the germinal disc and the embryo is cultured
in a host shell until maturity. When the animals to be made
transgenic are bovine or porcine, microinjection can be hampered by
the opacity of the ova thereby making the nuclei difficult to
identify by traditional differential interference-contrast
microscopy. To overcome this problem, the ova can first be
centrifuged to segregate the pronuclei for better
visualization.
[0101] The "non-human animals" of the invention bovine, porcine,
ovine and avian animals (e.g., cow, pig, sheep, chicken). The
"transgenic non-human animals" of the invention are produced by
introducing "transgenes" into the germline of the non-human animal.
Embryonal target cells at various developmental stages can be used
to introduce transgenes. Different methods are used depending on
the stage of development of the embryonal target cell. The zygote
is the best target for micro-injection. The use of zygotes as a
target for gene transfer has a major advantage in that in most
cases the injected DNA will be incorporated into the host gene
before the first cleavage (Brinster et al., Proc. Natl. Acad. Sci.
USA 82:4438-4442, 1985). As a consequence, all cells of the
transgenic non-human animal will carry the incorporated transgene.
This will in general also be reflected in the efficient
transmission of the transgene to offspring of the founder since 50%
of the germ cells will harbor the transgene.
[0102] The term "transgenic" is used to describe an animal which
includes exogenous genetic material within all of its cells. A
"transgenic" animal can be produced by cross-breeding two chimeric
animals which include exogenous genetic material within cells used
in reproduction. Twenty-five percent of the resulting offspring
will be transgenic i.e., animals which include the exogenous
genetic material within all of their cells in both alleles. 50% of
the resulting animals will include the exogenous genetic material
within one allele and 25% will include no exogenous genetic
material.
[0103] In the microinjection method useful in the practice of the
subject invention, the transgene is digested and purified free from
any vector DNA e.g. by gel electrophoresis. It is preferred that
the transgene include an operatively associated promoter which
interacts with cellular proteins involved in transcription,
ultimately resulting in constitutive expression. Promoters useful
in this regard include those from cytomegalovirus (CMV), Moloney
leukemia virus (MLV), and herpes virus, as well as those from the
genes encoding metallothionin, skeletal actin, P-enolpyruvate
carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, and thymidine
kinase. Promoters for viral long terminal repeats (LTRs) such as
Rous Sarcoma Virus can also be employed. When the animals to be
made transgenic are avian, preferred promoters include those for
the chicken .beta.-globin gene, chicken lysozyme gene, and avian
leukosis virus. Constructs useful in plasmid transfection of
embryonic stem cells will employ additional regulatory elements
well known in the art such as enhancer elements to stimulate
transcription, splice acceptors, termination and polyadenylation
signals, and ribosome binding sites to permit translation.
[0104] Retroviral infection can also be used to introduce transgene
into a non-human animal, as described above. The developing
non-human embryo can be cultured in vitro to the blastocyst stage.
During this time, the blastomeres can be targets for retro viral
infection (Jaenich, R., Proc. Natl. Acad. Sci USA 73:1260-1264,
1976). Efficient infection of the blastomeres is obtained by
enzymatic treatment to remove the zona pellucida (Hogan, et al.
(1986) in Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). The viral vector
system used to introduce the transgene is typically a
replication-defective retro virus carrying the transgene (Jahner,
et al., Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der
Putten, et al., Proc. Natl. Acad. Sci USA 82:6148-6152, 1985).
Transfection is easily and efficiently obtained by culturing the
blastomeres on a monolayer of virus-producing cells (Van der
Putten, supra; Stewart, et al., EMBO J. 6:383-388, 1987).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele (D.
Jahner et al., Nature 2-98:623-628, 1982). Most of the founders
will be mosaic for the transgene since incorporation occurs only in
a subset of the cells which formed the transgenic nonhuman animal.
Further, the founder may contain various retro viral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line, albeit with low
efficiency, by intrauterine retroviral infection of the
midgestation embryo (D. Jahner et al., supra).
[0105] A third type of target cell for transgene introduction is
the embryonal stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(M. J. Evans et al. Nature 292:154-156, 1981; M. O. Bradley et al.,
Nature 309: 255-258, 1984; Gossler, et al., Proc. Natl. Acad. Sci
USA 83: 9065-9069, 1986; and Robertson et al., Nature 322:445-448,
1986). Transgenes can be efficiently introduced into the ES cells
by DNA transfection or by retro virus-mediated transduction. Such
transformed ES cells can thereafter be combined with blastocysts
from a nonhuman animal. The ES cells thereafter colonize the embryo
and contribute to the germ line of the resulting chimeric animal.
(For review see Jaenisch, R., Science 240: 1468-1474, 1988).
[0106] "Transformed" means a cell into which (or into an ancestor
of which) has been introduced, by means of recombinant nucleic acid
techniques, a heterologous nucleic acid molecule. "Heterologous"
refers to a nucleic acid sequence that either originates from
another species or is modified from either its original form or the
form primarily expressed in the cell.
[0107] "Transgene" means any piece of DNA which is inserted by
artifice into a cell, and becomes part of the genome of the
organism (i.e., either stably integrated or as a stable
extrachromosomal element) which develops from that cell. Such a
transgene may include a gene which is partly or entirely
heterologous (i.e., foreign) to the transgenic organism, or may
represent a gene homologous to an endogenous gene of the organism.
Included within this definition is a transgene created by the
providing of an RNA sequence which is transcribed into DNA and then
incorporated into the genome. The transgenes of the invention
include DNA sequences which encode GDF-11, and include GDF-sense
and antisense polynucleotides and dominant negative encoding
polynucleotides, which may be expressed in a transgenic non-human
animal. The term "transgenic" as used herein additionally includes
any organism whose genome has been altered by in vitro manipulation
of the early embryo or fertilized egg or by any transgenic
technology to induce a specific gene knockout. The term "gene
knockout" as used herein, refers to the targeted disruption of a
gene in vivo with complete loss of function that has been achieved
by any transgenic technology familiar to those in the art. In one
embodiment, transgenic animals having gene knockouts are those in
which the target gene has been rendered nonfunctional by an
insertion targeted to the gene to be rendered non-functional by
0homologous recombination. As used herein, the term "transgenic:
includes any transgenic technology familiar to those in the art
which can produce an organism carrying an introduced transgene or
one in which an endogenous gene has been rendered non-functional or
"knocked out." An example of a transgene used to "knockout" GDF-11
function in the present Examples is described in Example 6 and FIG.
9. Thus, in another embodiment, the invention provides a transgene
wherein the entire mature C-terminal region of GDF-11 is
deleted.
[0108] The transgene to be used in the practice of the subject
invention is a DNA sequence comprising a modified GDF-11 coding
sequence. In a preferred embodiment, the GDF-11 gene is disrupted
by homologous targeting in embryonic stem cells. For example, the
entire mature C-terminal region of the GDF-11 gene may be deleted
as described in the examples below. Optionally, the GDF-11
disruption or deletion may be accompanied by insertion of or
replacement with other DNA sequences, such as a non-functional
GDF-11 sequence. In other embodiments, the transgene comprises DNA
antisense to the coding sequence for GDF-11. In another embodiment,
the transgene comprises DNA encoding an antibody or receptor
peptide sequence which is able to bind to GDF-11. The DNA and
peptide sequences of GDF-11 are known in the art, the sequences,
localization and activity disclosed in W095/08543 and pending
United States patent application 08/706,958, filed on September 3,
1996, incorporated by reference in its entirety. The disclosure of
both of these applications are hereby incorporated herein by
reference. Where appropriate, DNA sequences that encode proteins
having GDF-11 activity but differ in nucleic acid sequence due to
the degeneracy of the genetic code may also be used herein, as may
truncated forms, allelic variants and interspecies homologues.
[0109] Therefore the invention also includes animals having
heterozygous mutations in GDF-11. A heterozygote would likely have
an intermediate increase in muscle mass as compared to the
homozygote.
[0110] After an embryo has been microinjected, colonized with
transfected embryonic stem cells or infected with a retrovirus
containing the transgene (except for practice of the subject
invention in avian species which is addressed elsewhere herein) the
embryo is implanted into the oviduct of a pseudopregnant female.
The consequent progeny are tested for incorporation of the
transgene by Southern blot analysis of blood samples using
transgene specific probes. PCR is particularly useful in this
regard. Positive progeny (G0) are crossbred to produce offspring
(G1) which are analyzed for transgene expression by Northern blot
analysis of tissue samples. To be able to distinguish expression of
like-species transgenes from expression of the animals endogenous
GDF-11 gene(s), a marker gene fragment can be included in the
construct in the 3' untranslated region of the transgene and the
Northern probe designed to probe for the marker gene fragment. The
serum levels of GDF-11 can also be measured in the transgenic
animal to establish appropriate expression. Expression of the
GDF-11 transgenes, thereby decreasing the GDF-11 in the tissue and
serum levels of the transgenic animals and consequently increasing
the muscle tissue content results in the foodstuffs from these
animals (i.e. eggs, beef, pork, poultry meat, milk, etc.) having
markedly increased muscle content, and preferably without
increased, and more preferably, reduced levels of fat and
cholesterol. By practice of the subject invention, a statistically
significant increase in muscle content, preferably at least a 2%
increase in muscle content (e.g., in chickens), more preferably a
25% increase in muscle content as a percentage of body weight, more
preferably greater than 40% increase in muscle content in these
foodstuffs can be obtained.
Additional Methods of Use
[0111] Thus, the present invention includes methods for increasing
muscle mass in domesticated animals, characterized by inactivation
or deletion of the gene encoding growth and differentiation
factor-11 (GDF-11). The domesticated animal is preferably selected
from the group consisting of ovine, bovine, porcine, piscine and
avian. The animal may be treated with an isolated polynucleotide
sequence encoding growth and differentiation factor-11 which
polynucleotide sequence is also from a domesticated animal selected
from the group consisting of ovine, bovine, porcine, piscine and
avian. The present invention includes methods for increasing the
muscle mass in domesticated animals characterized by administering
to a domesticated animal monoclonal antibodies directed to the
GDF-11 polypeptide. The antibody may be an anti-GDF-11, and may be
either a monoclonal antibody or a polyclonal antibody.
[0112] The invention includes methods comprising using an
anti-GDF-11 monoclonal antibody, antisense, or dominant negative
mutants as a therapeutic agent to inhibit the growth regulating
actions of GDF-11 on muscle cells. Muscle cells are defined to
include fetal or adult muscle cells, as well as progenitor cells
which are capable of differentiation into muscle. The monoclonal
antibody may be a humanized (e.g., either fully or a chimeric)
monoclonal antibody, of any species origin, such as murine, ovine,
bovine, porcine or avian. Methods of producing antibody molecules
with various combinations of "humanized" antibodies are well known
in the art and include combining murine variable regions with human
constant regions (Cabily, et al Proc. Natl. Acad. Sci. USA,
81:3273, 1984), or by grafting the murine-antibody complementary
determining regions (CDRs) onto the human framework (Richmann, et
al., Nature 332:323, 1988). Other general references which teach
methods for creating humanized antibodies include Morrison, et al,
Science, 229:1202, 1985; Jones, et al., Nature, 321:522, 1986;
Monroe, et al., Nature 312:779, 1985; Oi, et al., BioTechniques,
4:214, 1986; European Patent Application No. 302,620; and U.S. Pat.
No. 5,024,834. Therefore, by humanizing the monoclonal antibodies
of the invention for in vivo use, an immune response to the
antibodies would be greatly reduced.
[0113] The monoclonal antibody, GDF-11 polypeptide, or GDF-11
polynucleotide (all "GDF-11 agents") may have the effect of
increasing the development of skeletal muscles. In preferred
embodiments of the claimed methods, the GDF-11 monoclonal antibody,
polypeptide, or polynucleotide is administered to a patient
suffering from a disorder selected from the group consisting of
muscle wasting disease, neuromuscular disorder, muscle atrophy or
aging. The GDF-11 agent may also be administered to a patient
suffering from a disorder selected from the group consisting of
muscular dystrophy, spinal cord injury, traumatic injury,
congestive obstructive pulmonary disease (COPD), AIDS or
cachechia.
[0114] In a preferred embodiment, the GDF-11 agent is administered
to a patient with muscle wasting disease or disorder by
intravenous, intramuscular or subcutaneous injection; preferably, a
monoclonal antibody is administered within a dose range between
about 0.1 mg/kg to about 100 mg/kg; more preferably between about 1
ug/kg to 75 mg/kg; most preferably from about 10 mg/kg to 50 mg/kg.
The antibody may be administered, for example, by bolus injunction
or by slow infusion. Slow infusion over a period of 30 minutes to 2
hours is preferred. The GDF-11 agent may be formulated in a
formulation suitable for administration to a patient. Such
formulations are known in the art.
[0115] The dosage regimen will be determined by the attending
physician considering various factors which modify the action of
the GDF-11 protein, e.g. amount of tissue desired to be formed, the
site of tissue damage, the condition of the damaged tissue, the
size of a wound, type of damaged tissue, the patient's age, sex,
and diet, the severity of any infection, time of administration and
other clinical factors. The dosage may vary with the type of matrix
used in the reconstitution and the types of agent, such as
anti-GDF-11 antibodies, to be used in the composition. Generally,
systemic or injectable administration, such as intravenous (IV),
intramuscular (IM) or subcutaneous (Sub-Q) injection.
Administration will generally be initiated at a dose which is
minimally effective, and the dose will be increased over a
preselected time course until a positive effect is observed.
Subsequently, incremental increases in dosage will be made limiting
such incremental increases to such levels that produce a
corresponding increase in effect, while taking into account any
adverse affects that may appear. The addition of other known growth
factors, such as IGF I (insulin like growth factor I), human,
bovine, or chicken growth hormone which may aid in increasing
muscle mass, to the final composition, may also affect the dosage.
In the embodiment where an anti-GDF-11 antibody is administered,
the anti-GDF-11 antibody is generally administered within a dose
range of about 0.1 ug/kg to about 100 mg/kg.; more preferably
between about 10 mg/kg to 50 mg/kg.
[0116] Progress can be monitored by periodic assessment of tissue
growth and/or repair. The progress can be monitored, for example,
x-rays, histomorphometric determinations and tetracycline labeling.
Screening for GDF-11 Modulating Compounds
[0117] In another embodiment, the invention provides a method for
identifying a compound or molecule that modulates GDF-11 protein
activity or gene expression. The method includes incubating
components comprising the compound, GDF-11 polypeptide or with a
recombinant cell expressing GDF-11 polypeptide, under conditions
sufficient to allow the components to interact and determining the
effect of the compound on GDF-11 activity or expression. The effect
of the compound on GDF-11 activity can be measured by a number of
assays, and may include measurements before and after incubating in
the presence of the compound. Compounds that affect GDF-11 activity
or gene expression include peptides, peptidomimetics, polypeptides,
chemical compounds and biologic agents. Assays include Northern
blot analysis of GDF-11 mRNA (for gene expression), Western blot
analysis (for protein level) and muscle fiber analysis (for protein
activity).
[0118] The above screening assays may be used for detecting the
compounds or molecules that bind to the GDF-11 receptor or GDF-11
polypeptide, in isolating molecules that bind to the GDF-11 gene,
for measuring the amount of GDF-11 in a sample, either polypeptide
or RNA (mRNA), for identifying molecules that may act as agonists
or antagonists, and the like. For example, GDF-11 antagonists are
useful for treatment of muscular and adipose tissue disorders
(e.g., obesity).
[0119] Incubating includes conditions which allow contact between
the test compound and GDF-11 polypeptide or with a recombinant cell
expressing GDF-11 polypeptide. Contacting includes in solution and
in solid phase, or in a cell. The test compound may optionally be a
combinatorial library for screening a plurality of compounds.
Compounds identified in the method of the invention can be further
evaluated, detected, cloned, sequenced, and the like, either in
solution or after binding to a solid support, by any method usually
applied to the detection of a specific DNA sequence such as PCR,
oligomer restriction (Saiki, et al., Bio/Technology, 3:1008-1012,
1985), allele-specific oligonucleotide (ASO) probe analysis
(Conner, et al., Proc. Natl. Acad. Sci. USA, 80:278, 1983),
oligonucleotide Landegren, et al., Science, 241:1077, 1988), and
the like. Molecular techniques for DNA analysis have been reviewed
(Landegren, et al, Science, 242:229-237, 1988).
[0120] All references cited herein are hereby incorporated by
reference in their entirety.
[0121] The following examples are intended to illustrate but not
limit the invention. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
EXAMPLE 1
Identification and Isolation of a Novel TGF-.beta. Family
Member
[0122] To identify novel members of the TGF-.beta. superfamily, a
murine genomic library was screened at reduced stringency using a
murine GDF-8 probe (FIG. 8; nucleotides 865-1234) spanning the
region encoding the C-terminal portion of the GDF-8 precursor
protein. Hybridization was carried out as described (Lee, Mol.
Endocrinol., 4:1034, 1990) at 65.degree. C., and the final wash was
carried out at the same temperature in a buffer containing 0.5 M
NaCl. Among the hybridizing phage was one that could be
distinguished from GDF-8-containing phage on the basis of its
reduced hybridization intensity to the GDF-8 probe. Partial
nucleotide sequence analysis of the genomic insert present in this
weakly hybridizing phage showed that this clone contained a
sequence highly related to but distinct from murine GDF-8.
[0123] A partial nucleotide sequence of the genomic insert present
in this phage is shown in FIG. 1a. The sequence contained an open
reading frame extending from nucleotides 198 to 575 that showed
significant homology to the known members of the TGF-.beta.
superfamily (see below). Preceding this sequence was a 3' splice
consensus sequence at precisely the same position as in the GDF-8
gene. This new TGF-.beta. family member was given the designation
GDF-11 (growth/differentiation factor-11).
EXAMPLE 2
Expression of GDF-11
[0124] To determine the expression pattern of GDF-11, RNA samples
prepared from a variety of tissues were screened by Northern
analysis. RNA isolation and Northern analysis were carried out as
described previously (Lee, Mol. Endocrinol., 4:1034, 1990) except
that the hybridization was carried out in 5.times.SSPE, 10% dextran
sulfate, 50% formamide, 1% SDS, 200 .mu.g/ml salmon DNA, and 0.1%
each of bovine serum albumin, ficoll, and polyvinylpyrrolidone.
Five micrograms of twice poly A-selected RNA prepared from each
tissue (except for 2 day neonatal brain, for which only 3.3 .mu.g
RNA were used) were electrophoresed on formaldehyde gels, blotted,
and probed with GDF-11. As shown in FIG. 2, the GDF-11 probe
detected two RNA species, approximately 4.2 and 3.2 kb in length,
in adult thymus, brain, spleen, uterus, and muscle as well as in
whole embryos isolated at day 12.5 or 18.5 and in brain samples
taken at various stages of development. On longer exposures of
these blots, lower levels of GDF-11 RNA could also be detected in a
number of other tissues.
EXAMPLE 3
Isolation of cDNA Clones Encoding GDF-11
[0125] In order to isolate cDNA clones encoding GDF-11, a cDNA
library was prepared in the lambda ZAP II vector (Stratagene) using
RNA prepared from human adult spleen. From 5 .mu.g of twice poly
A-selected RNA prepared from human spleen, a cDNA library
consisting of 21 million recombinant phage was constructed
according to the instructions provided by Stratagene. The library
was screened without amplification. Library screening and
characterization of cDNA inserts were carried out as described
previously (Lee, Mol. Endocrinol., 4:1034, 1990). From this
library, 23 hybridizing phage were obtained.
[0126] The entire nucleotide sequence of the clone extending
furthest toward the 5' end of the gene was determined. The 1258
base pair sequence contained a single long open reading frame
beginning from the 5' end of the clone and extending to a TAA stop
codon. Because the open reading frame and the homology with GDF-8
(see below) extended to the very 5' end of the clone, it seemed
likely that this clone was missing the coding sequence
corresponding to the N-terminal portion of the GDF-11 precursor
protein. In order to obtain the remaining portion of the GDF-11
sequence, several genomic clones were isolated by screening a human
genomic library with the human GDF-11 cDNA probe. Partial sequence
analysis of one of these genomic clones showed that this clone
contained the GDF-11 gene. From this clone, the remaining GDF-11
coding sequence was obtained. FIGURE 1b shows the predicted
sequence of GDF-11 assembled from the genomic and cDNA sequences.
Nucleotides 136 to 1393 represent the extent of the sequence
obtained from a cDNA clone. Nucleotides 1 to 135 were obtained from
a genomic clone. The sequence has been arbitrarily numbered
beginning with a Sac II site present in the genomic clone, but the
location of the mRNA start site is not known. The sequence contains
a putative initiating methionine at nucleotide 54. Whether the
sequence upstream of this methionine codon is all present in the
mRNA is not known. Beginning with this methionine codon, the open
reading frame extends for 407 amino acids. The sequence contains
one potential N-linked glycosylation site at asparagine 94. The
sequence contains a predicted RXXR proteolytic cleavage site at
amino acids 295 to 298, and cleavage of the precursor at this site
would generate an active C-terminal fragment 109 amino acids in
length with a predicted molecular weight of approximately 12,500
kD. In this region, the predicted murine and human GDF-11 amino
acid sequences are 100% identical. The high degree of sequence
conservation across species suggests that GDF-11 plays an important
role in vivo.
[0127] The C-terminal region following the predicted cleavage site
contains all the hallmarks present in other TGF-.beta. family
members. GDF-11 contains most of the residues that are highly
conserved in other family members, including the seven cysteine
residues with their characteristic spacing. Like the TGF-.beta.'s,
the inhibin .beta.'s, and GDF-8, GDF-11 also contains two
additional cysteine residues. In the case of TGF-.beta.2, these
additional cysteine residues are known to form an intramolecular
disulfide bond (Daopin, et al., Science, 257:369, 1992; Schlunegger
and Grutter, Nature, 358:430, 1992). A tabulation of the amino acid
sequence homologies between GDF-11 and the other TGF-.beta. family
members is shown in FIG. 3. Numbers represent percent amino acid
identities between each pair calculated from the first conserved
cysteine to the C-terminus. Boxes represent homologies among
highly-related members within particular subgroups. In this region,
GDF-11 is most highly related to GDF-8 (92% sequence identity).
[0128] An alignment of GDF-8 (SEQ ID NO:5) and GDF-11 (SEQ ID NO:2)
amino acid sequences is shown in FIG. 4. The two sequences contain
potential N-linked glycosylation signals (NIS) and putative
proteolytic processing sites (RSRR) at analogous positions. The two
sequences are related not only in the C-terminal region following
the putative cleavage site (90% amino acid sequence identity), but
also in the pro-region of the molecules (45% amino acid sequence
identity).
EXAMPLE 4
Construction of a Hybrid GDF-8/GDF11 Gene
[0129] In order to express GDF-11 protein, a hybrid gene was
constructed in which the N-terminal region of GDF-11 was replaced
by the analogous region of GDF-8. Such hybrid constructs have been
used to produce biologically-active BMP-4 (Hammonds, et al., Mol.
Endocrinol., 5:149, 1991) and Vg-1 (Thomsen and Melton, Cell,
74:433, 1993). In order to ensure that the GDF-11 protein produced
from the hybrid construct would represent authentic GDF-11, the
hybrid gene was constructed in such a manner that the fusion of the
two gene fragments would occur precisely at the predicted cleavage
sites. In particular, an AvaII restriction site is present in both
sequences at the location corresponding to the predicted
proteolytic cleavage site. The N-terminal pro-region of GDF-8 up to
this AvaII site was obtained by partial digestion of the clone with
AvaII and fused to the C-terminal region of GDF-11 beginning at
this AvaII site. The resulting hybrid construct was then inserted
into the pMSXND mammalian expression vector (Lee and Nathans, J.
Biol. Chem., 263:3521) and transfected into Chinese hamster ovary
cells. As shown in FIG. 5, Western analysis of conditioned medium
from G41 8-resistant cells using antibodies raised against the
C-terminal portion of GDF-8 showed that these cells secreted GDF-11
protein into the medium and that at least some of the hybrid
protein was proteolytically processed. Furthermore, these studies
demonstrate that the antibodies directed against the C-terminal
portion of GDF-8 will also react with GDF-11 protein.
EXAMPLE 5
Chromosomal Localization of GDF-11
[0130] In order to map the chromosomal location of GDF-11, DNA
samples from human/rodent somatic cell hybrids (Drwinga, et al.,
Genomics, 16:311-313, 1993; Dubois and Naylor, Genomics,
16:315-319, 1993) were analyzed by polymerase chain reaction
followed by Southern blotting. Polymerase chain reaction was
carried out using primer #101, 5'-GAGTCCCGCTGCTGCCGATATCC-3', (SEQ
ID NO:7) and primer #102, 5'-TAGAGCATGTTGATTGGGGACAT-3', (SEQ ID
NO:8) for 35 cycles at 94.degree. C. for 2 minutes, 58.degree. C.
for 1 minutes, and 72.quadrature.C for 1 minute. These primers
correspond to nucleotides 981 to 1003 and the reverse complement of
nucleotides 1182 to 1204, respectively, in the human GDF-11
sequence. PCR products were electrophoresed on agarose gels,
blotted, and probed with oligonucleotide #104,
5'-AAATATCCGCATACCCATTT-3'- , (SEQ ID NO:9) which corresponds to a
sequence internal to the region flanked by primer #101 and #102.
Filters were hybridized in 6.times.SSC, 1.times.Denhardt's
solution, 100 .mu.g/ml yeast transfer RNA, and 0.05% sodium
pyrophosphate at 50.degree. C.
[0131] As shown in FIG. 6, the human-specific probe detected a band
of the predicted size (approximately 224 base pairs) in the
positive control sample (total human genomic DNA) and in a single
DNA sample from the human/rodent hybrid panel. This positive signal
corresponds to human chromosome 12. The human chromosome contained
in each of the hybrid cell lines is identified at the top of each
of the first 24 lanes (1-22, X, and Y). In the lanes designated
CHO, M, and H, the starting DNA template was total genomic DNA from
hamster, mouse, and human sources, respectively. In the lane marked
B1, no template DNA was used. Numbers at left indicate the
mobilities of DNA standards. These data show that the human GDF-11
gene is located on chromosome 12.
[0132] In order to determine the more precise location of GDF-11 on
chromosome 12, the GDF-11 gene was localized by florescence in situ
hybridization (FISH). These FISH localization studies were carried
out by contract to BIOS laboratories (New Haven, Connecticut).
Purified DNA from a human GDF-11 genomic clone was labelled with
digoxigenin dUTP by nick translation. Labelled probe was combined
with sheared human DNA and hybridized to normal metaphase
chromosomes derived from PHA stimulated peripheral blood
lymphocytes in a solution containing 50% formamide, 10% dextran
sulfate and 2.times.SSC. Specific hybridization signals were
detected by incubating the hybridized slides in
fluorescein-conjugated sheep antidigoxigenin antibodies. Slides
were then counterstained with propidium iodide and analyzed. As
shown in FIG. 7a, this experiment resulted in the specific
labelling of the proximal long arm of a group C chromosome, the
size and morphology of which were consistent with chromosome 12. In
order to confirm the identity of the specifically labelled
chromosome, a second experiment was conducted in which a chromosome
12- specific centromere probe was cohybridized with GDF-11. As
shown in FIG. 7b, this experiment clearly demonstrated that GDF-11
is located at a position which is 23% of the distance from the
centromere to the telomere of the long arm of chromosome 12, an
area which corresponds to band 12q3 (FIG. 7c). A total of 85
metaphase cells were analyzed and 80 exhibited specific
labelling.
EXAMPLE 6
GDF-11 Transgenic Knockout Mice
[0133] The GDF-11 gene is disrupted by homologous targeting in
embryonic stem cells. To ensure that the resulting mice are null
for GDF-11 function, the entire mature C-terminal region is deleted
and replaced by a neo cassette (example using GDF-8 sequences is
shown in FIG. 9). A murine 129 SV/J genomic library is prepared in
lambda FIX II according to the instructions provided by Stratagene
(La Jolla, Calif.). The structure of the GDF-11 gene was deduced
from restriction mapping and partial sequencing of phage clones
isolated from this library. Vectors for preparing the targeting
construct were kindly provided by Philip Soriano and Kirk Thomas
University. R1 ES cells are transfected with the targeting
construct, selected with gancyclovir (2.quadrature.M) and G418 (250
.mu.g/ml), and analyzed by Southern analysis. Homologously targeted
clones are injected into C57BL/6 blastocysts and transferred into
pseudopregnant females. Germline transmission of the targeted
allele is obtained in male chimeras from independently-derived ES
clones. Genomic Southern blots are hybridized at 42/C as described
above and washed in 0.2.times.SSC, 0.1% SDS at 42/C.
[0134] For whole leg analysis, legs of 14 week old mice are
skinned, treated with 0.2 M EDTA in PBS at 4/C for 4 weeks followed
by 0.5 M sucrose in PBS at 4/C. For fiber number and size analysis,
samples are directly mounted and frozen in isopentane as described
(Brumback and Leech, Color Atlas of Muscle Histochemistry, pp.
9-33, PSG Publishing Company, Littleton, Mass., 1984). Ten to 30
.mu.m sections are prepared using a cryostat and stained with
hematoxylin and eosin. Muscle fiber numbers are determined from
sections taken from the widest part of the tibialis cranialis
muscle. Muscle fiber sizes are measured from photographs of
sections of tibialis cranialis and gastrocnemius muscles. Fiber
type analysis is carried out using the mysosin ATPase assay after
pretreatment at pH 4.35 as described (Cumming, et al., Color Atlas
of Muscle Pathology, pp. 184-185, 1994) and by immunohistochemistry
using an antibody directed against type I myosin (MY32, Sigma) and
the Vectastain method (Vector Labs); in the immunohistochemical
experiments, no staining is seen when the primary antibodies were
left out. Carcasses are prepared from shaved mice by removing the
all of the internal organs and associated fat and connective
tissue. Fat content of carcasses from 4 month old males is
determined as described (Leshner, et al., Physiol. Behavior, 9:281,
1972).
[0135] For protein and DNA analysis, tissue is homogenized in 150
mM NaCl, 100 mM EDTA. Protein concentrations are determined using
the Biorad protein assay. DNA is isolated by adding SDS to 1%,
treating with 1 mg/ml proteinase K overnight at 55/C, extracting 3
times with phenol and twice with chloroform, and precipitating with
ammonium acetate and EtOH. DNA is digested with 2 mg/ml RNase for 1
hour at 37/C, and following proteinase K digestion and phenol and
chloroform extractions, the DNA is precipitated twice with ammonium
acetate and EtOH.
[0136] Homologous targeting of the GDF-11 gene is seen in
gancyclovir/G418 doubly-resistant ES cell clones. Following
injection of these targeted clones into blastocysts, chimeras are
obtained from independently-derived ES clones that produce
heterozygous pups when crossed to C57BL/6 females (FIG. 8b).
[0137] Homozygous mutants are viable and fertile when crossed to
C57BL/6 mice and to each other. Homozygous mutant animals, however,
are approximately 30% larger than their heterozygous and wild type
littermates. The difference between mutant and wild type body
weights is relatively constant irrespective of age and sex in adult
animals. Adult mutants display an abnormal body shape, with
pronounced shoulders and hips. When the skin is removed from
animals that had been sacrificed, it is apparent that the muscles
of the mutants were much larger than those of wild type animals.
The increase in skeletal muscle mass is widespread throughout the
body. Individual muscles isolated from homozygous mutant animals
weighed approximately 2-3 times more than those isolated from wild
type littermates. Although the magnitude of the weight increase
appears to roughly correlate with the level of GDF-11 expression in
the muscles examined. To determine whether the increased muscle
mass could account for the entire difference in total body weights
between wild type and mutant animals or whether many tissues were
generally larger in the mutants, the total body weights are
compared to carcass weights. The difference in carcass weights
between wild type and mutant animals is comparable to the
difference in total body weights. Moreover, because the fat content
of mutant and wild type animals is similar, these data are
consistent with all of the total body weight difference resulting
from an increase in skeletal muscle mass.
[0138] To determine whether the increase in skeletal muscle mass
resulted from hyperplasia or from hypertrophy, histologic analysis
of several different muscle groups is performed. The mutant muscle
appears grossly normal. No excess connective tissue or fat is seen
nor are there any obvious signs of degeneration, such as widely
varying fiber sizes or centrally-placed nuclei.
[0139] Finally, fiber type analysis of various muscles is carried
out to determine whether the number of both type I (slow) and type
II (fast) fibers is increased in the mutant animals.
[0140] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
11 1 1393 DNA Homo sapiens CDS (54)...(1274) Human GDF-11 1
ccgcgggact ccggcgtccc cgccccccag tcctccctcc cctcccctcc agc atg 56
Met 1 gtg ctc gcg gcc ccg ctg ctg ctg ggc ttc ctg ctc ctc gcc ctg
gag 104 Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu
Glu 5 10 15 ctg cgg ccc cgg ggg gag gcg gcc gag ggc ccc gcg gcg gcg
gcg gcg 152 Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala
Ala Ala 20 25 30 gcg gcg gcg gcg gcg gca gcg gcg ggg gtc ggg ggg
gag cgc tcc agc 200 Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly
Glu Arg Ser Ser 35 40 45 cgg cca gcc ccg tcc gtg gcg ccc gag ccg
gac ggc tgc ccc gtg tgc 248 Arg Pro Ala Pro Ser Val Ala Pro Glu Pro
Asp Gly Cys Pro Val Cys 50 55 60 65 gtt tgg cgg cag cac agc cgc gag
ctg cgc cta gag agc atc aag tcg 296 Val Trp Arg Gln His Ser Arg Glu
Leu Arg Leu Glu Ser Ile Lys Ser 70 75 80 cag atc ttg agc aaa ctg
cgg ctc aag gag gcg ccc aac atc agc cgc 344 Gln Ile Leu Ser Lys Leu
Arg Leu Lys Glu Ala Pro Asn Ile Ser Arg 85 90 95 gag gtg gtg aag
cag ctg ctg ccc aag gcg ccg ccg ctg cag cag atc 392 Glu Val Val Lys
Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln Ile 100 105 110 ctg gac
cta cac gac ttc cag ggc gac gcg ctg cag ccc gag gac ttc 440 Leu Asp
Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp Phe 115 120 125
ctg gag gag gac gag tac cac gcc acc acc gag acc gtc att agc atg 488
Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser Met 130
135 140 145 gcc cag gag acg gac cca gca gta cag aca gat ggc agc cct
ctc tgc 536 Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro
Leu Cys 150 155 160 tgc cat ttt cac ttc agc ccc aag gtg atg ttc aca
aag gta ctg aag 584 Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr
Lys Val Leu Lys 165 170 175 gcc cag ctg tgg gtg tac cta cgg cct gta
ccc cgc cca gcc aca gtc 632 Ala Gln Leu Trp Val Tyr Leu Arg Pro Val
Pro Arg Pro Ala Thr Val 180 185 190 tac ctg cag atc ttg cga cta aaa
ccc cta act ggg gaa ggg acc gca 680 Tyr Leu Gln Ile Leu Arg Leu Lys
Pro Leu Thr Gly Glu Gly Thr Ala 195 200 205 ggg gga ggg ggc gga ggc
cgg cgt cac atc cgt atc cgc tca ctg aag 728 Gly Gly Gly Gly Gly Gly
Arg Arg His Ile Arg Ile Arg Ser Leu Lys 210 215 220 225 att gag ctg
cac tca cgc tca ggc cat tgg cag agc atc gac ttc aag 776 Ile Glu Leu
His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe Lys 230 235 240 caa
gtg cta cac agc tgg ttc cgc cag cca cag agc aac tgg ggc atc 824 Gln
Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly Ile 245 250
255 gag atc aac gcc ttt gat ccc agt ggc aca gac ctg gct gtc acc tcc
872 Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser
260 265 270 ctg ggg ccg gga gcc gag ggg ctg cat cca ttc atg gag ctt
cga gtc 920 Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu
Arg Val 275 280 285 cta gag aac aca aaa cgt tcc cgg cgg aac ctg ggt
ctg gac tgc gac 968 Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly
Leu Asp Cys Asp 290 295 300 305 gag cac tca agc gag tcc cgc tgc tgc
cga tat ccc ctc aca gtg gac 1016 Glu His Ser Ser Glu Ser Arg Cys
Cys Arg Tyr Pro Leu Thr Val Asp 310 315 320 ttt gag gct ttc ggc tgg
gac tgg atc atc gca cct aag cgc tac aag 1064 Phe Glu Ala Phe Gly
Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys 325 330 335 gcc aac tac
tgc tcc ggc cag tgc gag tac atg ttc atg caa aaa tat 1112 Ala Asn
Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys Tyr 340 345 350
ccg cat acc cat ttg gtg cag cag gcc aat cca aga ggc tct gct ggg
1160 Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala
Gly 355 360 365 ccc tgt tgt acc ccc acc aag atg tcc cca atc aac atg
ctc tac ttc 1208 Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn
Met Leu Tyr Phe 370 375 380 385 aat gac aag cag cag att atc tac ggc
aag atc cct ggc atg gtg gtg 1256 Asn Asp Lys Gln Gln Ile Ile Tyr
Gly Lys Ile Pro Gly Met Val Val 390 395 400 gat cgc tgt ggc tgc tct
taagtgggtc actacaagct gctggagcaa 1304 Asp Arg Cys Gly Cys Ser 405
agacttggtg ggtgggtaac ttaacctctt cacagaggat aaaaaatgct tgtgagtatg
1364 acagaaggga ataaacaggc ttaaagggt 1393 2 407 PRT Homo sapiens 2
Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5
10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala
Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly
Glu Arg Ser 35 40 45 Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro
Asp Gly Cys Pro Val 50 55 60 Cys Val Trp Arg Gln His Ser Arg Glu
Leu Arg Leu Glu Ser Ile Lys 65 70 75 80 Ser Gln Ile Leu Ser Lys Leu
Arg Leu Lys Glu Ala Pro Asn Ile Ser 85 90 95 Arg Glu Val Val Lys
Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln 100 105 110 Ile Leu Asp
Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp 115 120 125 Phe
Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser 130 135
140 Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu
145 150 155 160 Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr
Lys Val Leu 165 170 175 Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val
Pro Arg Pro Ala Thr 180 185 190 Val Tyr Leu Gln Ile Leu Arg Leu Lys
Pro Leu Thr Gly Glu Gly Thr 195 200 205 Ala Gly Gly Gly Gly Gly Gly
Arg Arg His Ile Arg Ile Arg Ser Leu 210 215 220 Lys Ile Glu Leu His
Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe 225 230 235 240 Lys Gln
Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly 245 250 255
Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr 260
265 270 Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu
Arg 275 280 285 Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly
Leu Asp Cys 290 295 300 Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg
Tyr Pro Leu Thr Val 305 310 315 320 Asp Phe Glu Ala Phe Gly Trp Asp
Trp Ile Ile Ala Pro Lys Arg Tyr 325 330 335 Lys Ala Asn Tyr Cys Ser
Gly Gln Cys Glu Tyr Met Phe Met Gln Lys 340 345 350 Tyr Pro His Thr
His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala 355 360 365 Gly Pro
Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 370 375 380
Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val 385
390 395 400 Val Asp Arg Cys Gly Cys Ser 405 3 630 DNA Mus musculus
CDS (198)...(575) 3 tctagatgtc aagagaagtg gtcacaatgt ctgggtggga
gccgtaaaca agccaagagg 60 ttatggtttc tggtctgatg ctcctgttga
gatcaggaaa tgttcaggaa atcccctgtt 120 gagatgtagg aaagtaagag
gtaagagaca ttgttgaggg tcatgtcaca tctctttccc 180 ctctccctga ccctcag
cat cct ttc atg gag ctt cga gtc cta gag aac 230 His Pro Phe Met Glu
Leu Arg Val Leu Glu Asn 1 5 10 acg aaa agg tcc cgg cgg aac cta ggc
ctg gac tgc gat gaa cac tcg 278 Thr Lys Arg Ser Arg Arg Asn Leu Gly
Leu Asp Cys Asp Glu His Ser 15 20 25 agt gag tcc cgc tgc tgc cga
tat cct ctc aca gtg gac ttt gag gct 326 Ser Glu Ser Arg Cys Cys Arg
Tyr Pro Leu Thr Val Asp Phe Glu Ala 30 35 40 ttt ggc tgg gac tgg
atc atc gca cct aag cgc tac aag gcc aac tac 374 Phe Gly Trp Asp Trp
Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr 45 50 55 tgc tcc ggc
cag tgc gaa tac atg ttc atg caa aag tat cca cac acc 422 Cys Ser Gly
Gln Cys Glu Tyr Met Phe Met Gln Lys Tyr Pro His Thr 60 65 70 75 cac
ttg gtg caa cag gcc aac cca aga ggc tct gct ggg ccc tgc tgc 470 His
Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys 80 85
90 acc cct acc aag atg tcc cca atc aac atg ctc tac ttc aat gac aag
518 Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys
95 100 105 cag cag att atc tac ggc aag atc cct ggc atg gtg gtg gat
cga tgt 566 Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val Val Asp
Arg Cys 110 115 120 ggc tgc tcc taagttgtgg gctacagtgg atgcctccct
cagaccctac 615 Gly Cys Ser 125 cccaagaacc ccagc 630 4 126 PRT Mus
musculus 4 His Pro Phe Met Glu Leu Arg Val Leu Glu Asn Thr Lys Arg
Ser Arg 1 5 10 15 Arg Asn Leu Gly Leu Asp Cys Asp Glu His Ser Ser
Glu Ser Arg Cys 20 25 30 Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu
Ala Phe Gly Trp Asp Trp 35 40 45 Ile Ile Ala Pro Lys Arg Tyr Lys
Ala Asn Tyr Cys Ser Gly Gln Cys 50 55 60 Glu Tyr Met Phe Met Gln
Lys Tyr Pro His Thr His Leu Val Gln Gln 65 70 75 80 Ala Asn Pro Arg
Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met 85 90 95 Ser Pro
Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr 100 105 110
Gly Lys Ile Pro Gly Met Val Val Asp Arg Cys Gly Cys Ser 115 120 125
5 375 PRT Homo sapiens 5 Met Gln Lys Leu Gln Leu Cys Val Tyr Ile
Tyr Leu Phe Met Leu Ile 1 5 10 15 Val Ala Gly Pro Val Asp Leu Asn
Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30 Val Glu Lys Glu Gly Leu
Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 35 40 45 Lys Ser Ser Arg
Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 Arg Leu
Glu Thr Ala Pro Asn Ile Ser Lys Asp Val Ile Arg Gln Leu 65 70 75 80
Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 85
90 95 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr
His 100 105 110 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser
Asp Phe Leu 115 120 125 Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe
Phe Lys Phe Ser Ser 130 135 140 Lys Ile Gln Tyr Asn Lys Val Val Lys
Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160 Arg Pro Val Glu Thr Pro
Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170 175 Ile Lys Pro Met
Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 Lys Leu
Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205
Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210
215 220 Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val
Thr 225 230 235 240 Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe
Leu Glu Val Lys 245 250 255 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg
Asp Phe Gly Leu Asp Cys 260 265 270 Asp Glu His Ser Thr Glu Ser Arg
Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285 Asp Phe Glu Ala Phe Gly
Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295 300 Lys Ala Asn Tyr
Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 Tyr
Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325 330
335 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr
340 345 350 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala
Met Val 355 360 365 Val Asn Arg Cys Gly Cys Ser 370 375 6 4 PRT
Artificial Sequence putative proteolytic cleavage site 6 Arg Xaa
Xaa Arg 1 7 23 DNA Artificial Sequence oligonucleotide for PCR 7
gagtcccgct gctgccgata tcc 23 8 23 DNA Artificial Sequence
oligonucleotide for PCR 8 tagagcatgt tgattgggga cat 23 9 20 DNA
Artificial Sequence oligonucleotide for PCR 9 aaatatccgc atacccattt
20 10 2676 DNA Mus musculus CDS (104)...(1231) 10 gtctctcgga
cggtacatgc actaatattt cacttggcat tactcaaaag caaaaagaag 60
aaataagaac aagggaaaaa aaaagattgt gctgattttt aaa atg atg caa aaa 115
Met Met Gln Lys 1 ctg caa atg tat gtt tat att tac ctg ttc atg ctg
att gct gct ggc 163 Leu Gln Met Tyr Val Tyr Ile Tyr Leu Phe Met Leu
Ile Ala Ala Gly 5 10 15 20 cca gtg gat cta aat gag ggc agt gag aga
gaa gaa aat gtg gaa aaa 211 Pro Val Asp Leu Asn Glu Gly Ser Glu Arg
Glu Glu Asn Val Glu Lys 25 30 35 gag ggg ctg tgt aat gca tgt gcg
tgg aga caa aac acg agg tac tcc 259 Glu Gly Leu Cys Asn Ala Cys Ala
Trp Arg Gln Asn Thr Arg Tyr Ser 40 45 50 aga ata gaa gcc ata aaa
att caa atc ctc agt aag ctg cgc ctg gaa 307 Arg Ile Glu Ala Ile Lys
Ile Gln Ile Leu Ser Lys Leu Arg Leu Glu 55 60 65 aca gct cct aac
atc agc aaa gat gct ata aga caa ctt ctg cca aga 355 Thr Ala Pro Asn
Ile Ser Lys Asp Ala Ile Arg Gln Leu Leu Pro Arg 70 75 80 gcg cct
cca ctc cgg gaa ctg atc gat cag tac gac gtc cag agg gat 403 Ala Pro
Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val Gln Arg Asp 85 90 95
100 gac agc agt gat ggc tct ttg gaa gat gac gat tat cac gct acc acg
451 Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His Ala Thr Thr
105 110 115 gaa aca atc att acc atg cct aca gag tct gac ttt cta atg
caa gcg 499 Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu Met
Gln Ala 120 125 130 gat ggc aag ccc aaa tgt tgc ttt ttt aaa ttt agc
tct aaa ata cag 547 Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser
Ser Lys Ile Gln 135 140 145 tac aac aaa gta gta aaa gcc caa ctg tgg
ata tat ctc aga ccc gtc 595 Tyr Asn Lys Val Val Lys Ala Gln Leu Trp
Ile Tyr Leu Arg Pro Val 150 155 160 aag act cct aca aca gtg ttt gtg
caa atc ctg aga ctc atc aaa ccc 643 Lys Thr Pro Thr Thr Val Phe Val
Gln Ile Leu Arg Leu Ile Lys Pro 165 170 175 180 atg aaa gac ggt aca
agg tat act gga atc cga tct ctg aaa ctt gac 691 Met Lys Asp Gly Thr
Arg Tyr Thr Gly Ile Arg Ser Leu Lys Leu Asp 185 190 195 atg agc cca
ggc act ggt att tgg cag agt att gat gtg aag aca gtg 739 Met Ser Pro
Gly Thr Gly Ile Trp Gln Ser Ile Asp Val Lys Thr Val 200 205 210 ttg
caa aat tgg ctc aaa cag cct gaa tcc aac tta ggc att gaa atc 787 Leu
Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly Ile Glu Ile 215 220
225 aaa gct ttg gat gag aat ggc cat gat ctt gct gta acc ttc cca gga
835 Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr Phe Pro Gly
230 235 240 cca gga gaa gat ggg ctg aat ccc ttt tta gaa gtc aag gtg
aca gac 883 Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys Val
Thr Asp 245 250 255 260 aca ccc aag agg tcc cgg aga gac ttt ggg ctt
gac tgc gat gag cac 931 Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu
Asp Cys Asp Glu His 265 270 275 tcc acg gaa tcc cgg tgc tgc
cgc tac ccc ctc acg gtc gat ttt gaa 979 Ser Thr Glu Ser Arg Cys Cys
Arg Tyr Pro Leu Thr Val Asp Phe Glu 280 285 290 gcc ttt gga tgg gac
tgg att atc gca ccc aaa aga tat aag gcc aat 1027 Ala Phe Gly Trp
Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn 295 300 305 tac tgc
tca gga gag tgt gaa ttt gtg ttt tta caa aaa tat ccg cat 1075 Tyr
Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys Tyr Pro His 310 315
320 act cat ctt gtg cac caa gca aac ccc aga ggc tca gca ggc cct tgc
1123 Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro
Cys 325 330 335 340 tgc act ccg aca aaa atg tct ccc att aat atg cta
tat ttt aat ggc 1171 Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met
Leu Tyr Phe Asn Gly 345 350 355 aaa gaa caa ata ata tat ggg aaa att
cca gcc atg gta gta gac cgc 1219 Lys Glu Gln Ile Ile Tyr Gly Lys
Ile Pro Ala Met Val Val Asp Arg 360 365 370 tgt ggg tgc tca
tgagctttgc attaggttag aaacttccca agtcatggaa 1271 Cys Gly Cys Ser
375 ggtcttcccc tcaatttcga aactgtgaat tcaagcacca caggctgtag
gccttgagta 1331 tgctctagta acgtaagcac aagctacagt gtatgaacta
aaagagagaa tagatgcaat 1391 ggttggcatt caaccaccaa aataaaccat
actataggat gttgtatgat ttccagagtt 1451 tttgaaatag atggagatca
aattacattt atgtccatat atgtatatta caactacaat 1511 ctaggcaagg
aagtgagagc acatcttgtg gtctgctgag ttaggagggt atgattaaaa 1571
ggtaaagtct tatttcctaa cagtttcact taatatttac agaagaatct atatgtagcc
1631 tttgtaaagt gtaggattgt tatcatttaa aaacatcatg tacacttata
tttgtattgt 1691 atacttggta agataaaatt ccacaaagta ggaatggggc
ctcacataca cattgccatt 1751 cctattataa ttggacaatc caccacggtg
ctaatgcagt gctgaatggc tcctactgga 1811 cctctcgata gaacactcta
caaagtacga gtctctctct cccttccagg tgcatctcca 1871 cacacacagc
actaagtgtt caatgcattt tctttaagga aagaagaatc tttttttcta 1931
gaggtcaact ttcagtcaac tctagcacag cgggagtgac tgctgcatct taaaaggcag
1991 ccaaacagta ttcatttttt aatctaaatt tcaaaatcac tgtctgcctt
tatcacatgg 2051 caattttgtg gtaaaataat ggaaatgact ggttctatca
atattgtata aaagactctg 2111 aaacaattac atttatataa tatgtataca
atattgtttt gtaaataagt gtctcctttt 2171 atatttactt tggtatattt
ttacactaat gaaatttcaa atcattaaag tacaaagaca 2231 tgtcatgtat
cacaaaaaag gtgactgctt ctatttcaga gtgaattagc agattcaata 2291
gtggtcttaa aactctgtat gttaagatta gaaggttata ttacaatcaa tttatgtatt
2351 ttttacatta tcaacttatg gtttcatggt ggctgtatct atgaatgtgg
ctcccagtca 2411 aatttcaatg ccccaccatt ttaaaaatta caagcattac
taaacatacc aacatgtatc 2471 taaagaaata caaatatggt atctcaataa
cagctacttt tttattttat aatttgacaa 2531 tgaatacatt tcttttattt
acttcagttt tataaattgg aactttgttt atcaaatgta 2591 ttgtactcat
agctaaatga aattatttct tacataaaaa tgtgtagaaa ctataaatta 2651
aagtgttttc acatttttga aaggc 2676 11 376 PRT Mus musculus 11 Met Met
Gln Lys Leu Gln Met Tyr Val Tyr Ile Tyr Leu Phe Met Leu 1 5 10 15
Ile Ala Ala Gly Pro Val Asp Leu Asn Glu Gly Ser Glu Arg Glu Glu 20
25 30 Asn Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg Gln
Asn 35 40 45 Thr Arg Tyr Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile
Leu Ser Lys 50 55 60 Leu Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys
Asp Ala Ile Arg Gln 65 70 75 80 Leu Leu Pro Arg Ala Pro Pro Leu Arg
Glu Leu Ile Asp Gln Tyr Asp 85 90 95 Val Gln Arg Asp Asp Ser Ser
Asp Gly Ser Leu Glu Asp Asp Asp Tyr 100 105 110 His Ala Thr Thr Glu
Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe 115 120 125 Leu Met Gln
Ala Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser 130 135 140 Ser
Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr 145 150
155 160 Leu Arg Pro Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu
Arg 165 170 175 Leu Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly
Ile Arg Ser 180 185 190 Leu Lys Leu Asp Met Ser Pro Gly Thr Gly Ile
Trp Gln Ser Ile Asp 195 200 205 Val Lys Thr Val Leu Gln Asn Trp Leu
Lys Gln Pro Glu Ser Asn Leu 210 215 220 Gly Ile Glu Ile Lys Ala Leu
Asp Glu Asn Gly His Asp Leu Ala Val 225 230 235 240 Thr Phe Pro Gly
Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val 245 250 255 Lys Val
Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp 260 265 270
Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr 275
280 285 Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys
Arg 290 295 300 Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val
Phe Leu Gln 305 310 315 320 Lys Tyr Pro His Thr His Leu Val His Gln
Ala Asn Pro Arg Gly Ser 325 330 335 Ala Gly Pro Cys Cys Thr Pro Thr
Lys Met Ser Pro Ile Asn Met Leu 340 345 350 Tyr Phe Asn Gly Lys Glu
Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met 355 360 365 Val Val Asp Arg
Cys Gly Cys Ser 370 375
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