U.S. patent application number 09/871604 was filed with the patent office on 2003-09-04 for transgenic non-human animals expressing a gdf-11 dominant negative polypeptide, and methods of making and using same.
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 | 20030167492 09/871604 |
Document ID | / |
Family ID | 46279975 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030167492 |
Kind Code |
A1 |
Lee, Se-Jin ; et
al. |
September 4, 2003 |
TRANSGENIC NON-HUMAN ANIMALS EXPRESSING A GDF-11 DOMINANT NEGATIVE
POLYPEPTIDE, AND METHODS OF MAKING AND USING SAME
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 and bone 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 1600
4365 Executive Drive
San Diego
CA
92121-2189
US
|
Assignee: |
Johns Hopkins University School of
Medicine
|
Family ID: |
46279975 |
Appl. No.: |
09/871604 |
Filed: |
May 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09871604 |
May 31, 2001 |
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09123929 |
Jul 28, 1998 |
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09123929 |
Jul 28, 1998 |
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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: |
800/15 ; 800/17;
800/19 |
Current CPC
Class: |
C07K 2319/00 20130101;
A01K 67/0276 20130101; A61P 21/00 20180101; C12N 15/1136 20130101;
C12N 2310/11 20130101; C07K 14/51 20130101; C07K 14/475 20130101;
A01K 2217/075 20130101; C12N 2310/111 20130101; A61P 35/00
20180101; A01K 2207/05 20130101; A01K 2267/02 20130101; A61P 13/12
20180101; A01K 2227/105 20130101; C12N 15/8509 20130101 |
Class at
Publication: |
800/15 ; 800/17;
800/19 |
International
Class: |
A01K 067/027 |
Claims
1. A method of producing animal food products having an increased
number of ribs 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.
2. The method of claim 1, wherein the transgene comprises GDF-11
antisense polynucleotides.
3. The method of claim 1, wherein the transgene comprises a gene
encoding a dominant negative GDF-11 polypeptide.
4. A method of producing avian, porcine or bovine food products
having an increased number of ribs comprising: a) introducing a
transgene disrupting or interfering with expression of growth
differentiation factor-11 (GDF-11) into an embryo of an avian,
porcine or bovine 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.
5. The method of claim 4, wherein the transgene comprises GDF-11
antisense polynucleotides.
6. The method of claim 4, wherein the transgene comprises a gene
encoding a dominant negative GDF-11 polypeptide.
7. The transgenic animal of claim 4, wherein the transgene
comprises a polynucleotide encoding a truncated GDF-11
polypeptide.
8. A method of treating a chronic or acute renal disease in a
subject having such a disease, comprising: administering to the
subject, a reagent which affects GDF-11 activity or expression.
9. The method of claim 8, wherein the reagent is an agonist of
GDF-11.
10. The method of claim 8, wherein the reagent is an antagonist of
GDF-11.
11. The method of claim 10, wherein the antagonist is an antibody
to GDF-11.
12. The method of claim 10, wherein the antagonist is an antisense
polynucleotide to GDF-11.
Description
[0001] This application is a continuation-in-part application of
U.S. application Ser. No. 09/019,901, filed Feb. 6, 1998 which is a
continuation-in-part of U.S. application Ser. No. 08/795,671, filed
Feb. 6, 1997 and U.S. application Ser. No. 08//706,958, filed Sep.
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,
bone, kidney 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,
bone, kidney and adipose tissue.
[0010] In one embodiment, the invention provides a method for
detecting a cell proliferative disorder of muscle, nerve, bone,
kidney 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, bone 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 and bone tissue, such as ribs, 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 and bone tissue to which the muscle attaches. 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
antisense 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
animal food products having increased bone 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
antisense 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.
[0014] The subject invention also provides a method of producing
avian food products having improved muscle and/or bone 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.
[0015] The invention also provides a method for treating a muscle,
bone, kidney or adipose tissue disorder in a subject. The method
includes administering a therapeutically effective amount of a
GDF-11 agent to the subject, thereby affecting growth of muscle,
bone, kidney 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. In another aspect of the
invention, the agent may be an agonist of GDF-11 activity.
[0016] The invention also provides 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
[0017] FIG. 1 shows the nucleotide and predicted amino acid
sequences of murine (FIG. 1a) and human (FIG. 1b) 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.
[0018] 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.
[0019] 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.
[0020] FIG. 4a shows an alignment of the predicted amino acid
sequences of human GDF-11 (top lines) with human GDF-8 (bottom
lines). 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.
[0021] FIG. 4b shows the predicted amino acid sequences of murine
and human GDF-11 aligned with murine (McPherron et al., 1997) and
human (McPherron and Lee, 1997) myostatin (MSTN). Shaded boxes
represent amino acid homology with the murine and human GDF-11
sequences. Amino acids are numbered relative to the human GDF-11
sequence. The predicted proteolytic processing sites are located at
amino acids 295-298.
[0022] 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.
[0023] 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.
[0024] FIG. 7 shows the FISH localization of GDF-11. Metaphase
chromosomes derived from peripheral blood lymphocytes were
hybridized with digoxigenin-labeled 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).
[0025] FIG. 8 shows a genomic Southern analysis of DNA isolated
from different species.
[0026] FIG. 9 shows the construction of GDF-11 null mice by
homologous targeting. a) is a map of the GDF-11 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 targeting construct contains a total of 11 kb of
homology with the GDF-11 gene. A probe derived from the region
upstream of the 3' homology fragment and downstream of the first
EcoRI site shown hybridizes to a 6.5 kb EcoR1 fragment in the
GDF-11 gene and a 4.8 kb fragment in a homologously targeted gene.
Abbreviations: X, Xba1; E, EcoR1. b) Geneomic Southern of DNA
prepared from F1 heterozygous mutant mice (lanes 1 and 2) and
offspring derived from a mating of these mice (lanes 3-12).
[0027] FIG. 10 shows kidney abnormalities in GDF-11 knockout mice.
Kidneys of newborn animals were examined and classified according
to the number of normal sized or small kidneys as shown at the top.
Numbers in the table indicate number of animals falling into each
classification according to genotype.
[0028] FIG. 11 shows homeotic transformations in GDF-11 mutant
mice. a) Newborn pups with missing (first and second from left) and
normal looking tails. b-j) Skeleton preparations for newborn
wild-type (b, e, h), heterozygous (c, f, i) and homozygous (d, g,
j) mutant mice. Whole skeleton preparations (b-d), vertebral
columns (e-g), vertebrosternal ribs (h-j) showing transformations
and defects in homozygous and heterozygous mutant mice. Numbers
indicate thoracic segments.
[0029] FIG. 12 is a table summarizing the anterior transformations
in wild-type, heterozygous and homozygous GDF-11 mice.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides a growth and differentiation
factor, GDF-11, 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.
[0031] 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.
[0032] 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 BMP4 (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.
[0033] GDF-11 may also have applications in treating disease
processes involving the musculoskeletal system, such as in
musculodegenerative diseases, osteoporosis 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. GDF-11 may also have applications in treating disease
processes involving the kidney or in kidney repair due to
trauma.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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).
[0039] Polypeptides, Polynucleotides, Vectors and Host Cells
[0040] The invention provides substantially pure GDF-11 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.
[0041] 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 GDF11 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.
[0042] 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 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.
[0043] 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).
[0044] 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 immobilized, for example, on a
filter.
[0045] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about is 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.
[0046] 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).
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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).
[0051] 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.
[0052] 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.
[0053] 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).
[0054] 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.
[0055] 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.
[0056] 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).
[0057] 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).
[0058] 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.
[0059] 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).
[0060] 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.
[0061] GDF-11 Antibodies and Methods of Use
[0062] 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.
[0063] (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.
[0064] (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.
[0065] (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.
[0066] (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.
[0067] (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.
[0068] 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.
[0069] 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.
[0070] 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 useful in treating
malignancies of the various organ systems, particularly, for
example, cells in muscle, bone, kidney 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.
[0071] The invention provides a method for detecting a cell
proliferative disorder of muscle, bone, kidney, 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, bone, kidney, 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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 dinitrophenyi,
puridoxal, and fluorescein, which can react with specific
antihapten antibodies.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] Additional Methods of Treatment and Diagnosis
[0084] 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, musculoskeletal, and kidney
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).
[0085] 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.
[0086] 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.
[0087] 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).
[0088] 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.
[0089] 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.
[0090] 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).
[0091] 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, bone content and/or
kidney growth by interfering with wild-type GDF-11 activity.
[0092] 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 nonfumctional 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.
[0093] Non-functional GDF-11 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,
bone cells and kidney cells at any point in a tissue's or
organism's development.
[0094] 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-expression 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).
[0095] In addition, the expression of GDF-11 may be used, for
example to help in kidney development. The method includes
administering a therapeutically effective amount of a GDF-11 agent
to the subject, thereby promoting kidney cell growth and
differentiation in kidney tissue. The agent may be an antagonist or
agonist of GDF-11 activity. For example, the agent may include a
GDF-11 antisense molecule or a dominant negative polypeptide.
[0096] The invention provides a method for treating a muscle,
kidney (chronic or acute) 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 or stimulating growth
in kidney 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.
[0097] 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 "sense" GDF-11 polynucleotide is introduced into the
appropriate cell(s).
[0098] 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-11 antisense
polynucleotide.
[0099] 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.
[0100] 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.
[0101] 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 .mu.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).
[0102] 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.
[0103] 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
phosphatidylchloline, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] Chromosomal Location of GDF-11
[0108] 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.
[0109] Transgenic Animals and Methods of Making the Same
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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 298:623-628, 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).
[0115] 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).
[0116] "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.
[0117] "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
polynculeotides, 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
homologous 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.
[0118] 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 WO95/08543 and pending U.S.
patent application Ser. No. 08/706,958, filed on Sep. 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.
[0119] 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.
[0120] 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.
[0121] In addition decrease in GDF-11 in the tissue and serum
levels of the transgenic animals can be used to increase the bone
content of animals use as foodstuffs, for example the transgenic
animals having reduced GDF-11 can be provided to have an additional
number of ribs.
[0122] Additional Methods of Use
[0123] Thus, the present invention includes methods for increasing
muscle mass and/or rib content 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 or rib content 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.
[0124] 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.
[0125] 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 bone cells. bone cells are defined to include
fetal or adult bone cells, as well as progenitor cells which are
capable of differentiation into bone. 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.
[0126] 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 or skeletal bones.
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. In another preferred embodiment the
invention provides a method for treating bone degenerative
disorders, such as osteoporosis by administering to a patient
suffering from a disorder antibodies, polypeptides or
polynucleotides effecting GDF-11 activity. 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.
[0127] In a preferred embodiment, the GDF-11 agent is administered
to a patient with muscle or bone 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.
[0128] 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.
[0129] 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.
[0130] Screening for GDF-11 Modulating Compounds
[0131] 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).
[0132] 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).
[0133] 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).
[0134] All references cited herein are hereby incorporated by
reference in their entirety.
[0135] 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
[0136] 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.
[0137] 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
[0138] 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
[0139] 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.
[0140] 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. FIG. 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.
[0141] 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).
[0142] An alignment of GDF-8 (SEQ ID NO:5) and GDF-11 (SEQ ID NO:6)
amino acid sequences is shown in FIG. 4a. 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
[0143] 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 G418-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
[0144] 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.degree. 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.
[0145] 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.
[0146] 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, Conn.). 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 12q13 (FIG. 7c). A total of
85 metaphase cells were analyzed and 80 exhibited specific
labelling.
EXAMPLE 6
GDF-11 Homology in Mammalian Species
[0147] Like most other TGF-.beta. family member, GDF-11 also
appears to be highly conserved across species. By genomic Southern
analysis, homologous sequences were detected in all mammalian
species examined as well as in chickens and frogs (FIG. 4b). In
most species, the GDF-11 probe also detected a second, more faintly
hybridizing fragment corresponding to the myostatin gene (McPherron
et al., 1997).
EXAMPLE 7
GDF-11 Transgenic Knockout Mice
[0148] To determine the biological function of GDF-11, we disrupted
the GDF-11 gene by homologous targeting in embryonic stem cells. A
murine 129 SV/J genomic library was prepared in lambda FIXII
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 the library. Vectors for preparing the targeting construct
were kindly provided by Philip Soriano and Kirk Thomas. To ensure
that the resulting mice would be null for GDF-11 function, the
entire mature C-terminal region was deleted and replaced by a neo
cassette (FIGS. 9a,b). R1 ES cells were transfected with the
targeting construct, selected with gancyclovir (2 .mu.M) and G418
(250 .mu.g/ml), and analyzed by Southern analysis. Homologous
targeting of the GDF-11 gene was seen in 8/155 gancyclovir/G418
doubly resistant ES cell clones. Following injection of several
targeted clones into C57BL/6J blastocysts, we obtained chimeras
from one ES clone that produced heterozygous pups when crossed to
both C57BL/6J and 129/SvJ females. Crosses of C57BL/6J/129/SvJ
hybrid F1 heterozygotes produced 49 wild-type (34%), 94
heterozygous (66%) and no homozygous mutant adult offspring.
Similarly, there were no adult homozygous null animals seen in the
129/SvJ background (32 wild-type (36%) and 56 heterozygous mutant
(64%) animals).
[0149] To determine the age at which homozygous mutants were dying,
we genotyped litters of embryos isolated at various gestational
ages from heterozygous females that had been mated to heterozygous
males. At all embryonic stages examined, homozygous mutant embryos
were present at approximately the predicted frequency of 25%. Among
hybrid newborn mice, the different genotypes were also represented
at the expected Mendelian ratio of 1:2:1 (34 +/+ (28%), 61 +/-
(50%), and 28 -/- (23%)). Homozygous mutant mice were born alive
and were able to breath and nurse. All homozygous mutants died,
however, within the first 24 hours after birth. The precise cause
of death was unknown, but the lethality may have been related to
the fact that the kidneys in homozygous mutants were either
severely hypoplastic or completely absent. A summary of the kidney
abnormalities in these mice is shown in FIG. 10.
EXAMPLE 8
Anatomical Differences in Knockout Mice
[0150] Homozygous mutant animals were easily recognizable by their
severely shortened or absent tails (FIG. 11a). To further
characterize the tail defects in these homozygous mutant animals,
we examined their skeletons to determine the degree of disruption
of the caudal vertebrae. A comparison of wild-type and mutant
skeleton preparations of late stage embryos and newborn mice,
however, revealed differences not only in the caudal region of the
animals but in many other regions as well. In nearly every case
where differences were noted, the abnormalities appeared to
represent homeotic transformations of vertebral segments in which
particular segments appeared to have a morphology typical of more
anterior segments. These transformations, which are summarized in
FIG. 12, were evident throughout the axial skeleton extending from
the cervical region to the caudal region. Except for the defects
seen in the axial skeleton, the rest of the skeleton, such as the
cranium and limb bones, appeared normal.
[0151] Anterior transformations of the vertebrae in mutant newborn
animals were most readily apparent in the thoracic region, where
there was a dramatic increase in the number of thoracic (T)
segments. All wild-type mice examined showed the typical pattern of
13 thoracic vertebrae each with its associated pair of ribs (FIGS.
11(b,e)). In contrast, homozygous mutant mice showed a striking
increase in the number of thoracic vertebrae. All homozygous
mutants examined had 4 to 5 extra pairs of ribs for a total of 17
to 18 (FIGS. 11(d,g)) although in over 1/3 of these animals, the
18th rib appeared to be rudimentary. Hence, segments that would
normally correspond to lumbar (L) segments L1 to L4 or L5 appeared
to have been transformed into thoracic segments in mutant
animals.
[0152] Moreover, transformations within the thoracic region in
which one thoracic vertebra had a morphology characteristic of
another thoracic vertebra were also evident. For example, in
wild-type mice, the first 7 pairs of ribs attach to the sternum,
and the remaining 6 are unattached or free (FIGS. 11(e,h)). In
homozygous mutants, there was an increase in the number of both
attached and free pairs of ribs to 10-11 and 7-8, respectively
(FIGS. 11(g,j)). Therefore, thoracic segments T8, T9, T10, and in
some cases even T11, which all have free ribs in wild-type animals,
were transformed in mutant animals to have a characteristic typical
of more anterior thoracic segments, namely, the presence of ribs
attached to the sternum. Consistent with this finding, the
transitional spinous process and transitional articular processes
which are normally found on T10 in wild-type animals were instead
found on T13 in homozygous mutants (data not shown). Additional
transformations within the thoracic region were also noted in
certain mutant animals. For example, in wild-type mice, the ribs
derived from T1 normally touch the top of the sternum. However, in
{fraction (2/23)} hybrid and 2/3 129/SvJ homozygous mutant mice
examined, T2 appeared to have been transformed to have a morphology
resembling that of T1; that is, in these animals, the ribs derived
from T2 extended to touch the top of the sternum. In these cases,
the ribs derived from T1 appeared to fuse to the second pair of
ribs. Finally, in 82% of homozygous mutants, the long spinous
process normally present on T2 was shifted to the position of T3.
In certain other homozygous mutants, asymmetric fusion of a pair of
vertebrostemal ribs was seen at other thoracic levels.
[0153] The anterior transformations were not restricted to the
thoracic region. The anterior most transformation that we observed
was at the level of the 6th cervical vertebra (C6). In wild-type
mice, C6 is readily identifiable by the presence of two anterior
tuberculi on the ventral side. In several homozygous mutant mice,
although one of these two anterior tuberculi was present on C6, the
other was present at the position of C7 instead. Hence, in these
mice, C7 appeared to have been partially transformed to have a
morphology resembling that of C6. One other homozygous mutant had 2
anterior tuberculi on C7 but retained one on C6 for a complete C7
to C6 transformation but a partial C6 to C5 transformation.
[0154] Transformations of the axial skeleton also extended into the
lumbar region. Whereas wild-type animals normally have only 6
lumbar vertebrae, homozygous mutants had 8-9. At least 6 of the
lumbar vertebrae in the mutants must have derived from segments
that would normally have given rise to sacral and caudal vertebrae
as the data described above suggest that 4 to 5 lumbar segments
were transformed into thoracic segments. Hence, homozygous mutant
mice had a total of 33-34 presacral vertebrae compared to 26
presacral vertebrae normally present in wild-type mice. The most
common presacral vertebral patterns were C7/T18/L8 and C7/T18/L9
for mutant mice compared to C7/T13/L6 for wild-type mice. The
presence of additional presacral vertebrae in mutant animals was
obvious even without detailed examination of the skeletons as the
position of the hindlimbs relative to the forelimbs was displaced
posteriorly by 7-8 segments.
[0155] Although the sacral and caudal vertebrae were also affected
in homozygous mutant mice, the exact nature of each transformation
was not as readily identifiable. In wild-type mice, sacral segments
S1 and S2 typically have broad transverse processes compared to S3
and S4. In the mutants, there did not appear to be an identifiable
S1 or S2 vertebra. Instead, mutant animals had several vertebrae
that appeared to have morphology similar to S3. In addition, the
transverse processes of all 4 sacral vertebrae are normally fused
to each other although in newborns often only fusions of the first
3 vertebrae are seen. In homozygous mutants, however, the
transverse processes of the sacral vertebrae were usually unfused.
In the caudalmost region, all mutant animals also had severely
malformed vertebrae with extensive fusions of cartilage. Although
the severity of the fusions made it difficult to count the total
number of vertebrae in the caudal region, we were able to count up
to 15 transverse processes in several animals. We were unable to
determine whether these represented sacral or caudal vertebrae in
the mutants because we could not establish morphologic criteria for
distinguishing S4 from caudal vertebrae even in wild-type newborn
animals. Regardless of their identities, the total number of
vertebrae in this region was significantly reduced from the normal
number of approximately 30. Hence, although the mutants had
significantly more thoracic and lumber vertebrae than wild-type
mice, the total number of segments was reduced in the mutants due
to the truncation of the tails.
[0156] Heterozygous mice also showed abnormalities in the axial
skeleton although the phenotype was much milder than in homozygous
mice. The most obvious abnormality in heterozygous mice was the
presence of an additional thoracic segment with an associated pair
of ribs (FIGS. 11(c,f)). This transformation was present in every
heterozygous animal examined, and in every case, the additional
pair of ribs was attached to the sternum (FIG. 11(I)). Hence, T8,
whose associated rib normally does not touch the sternum, appeared
to have been transformed to a morphology characteristic of a more
anterior thoracic vertebra, and L1 appeared to have been
transformed to a morphology characteristic of a posterior thoracic
vertebra. Other abnormalities indicative of anterior
transformations were also seen to varying degrees in heterozygous
mice. These included a shift of the long spinous process
characteristic of T2 by one segment to T3, a shift of the articular
and spinous processes from T10 to T11, a shift of the anterior
tuberculus on C6 to C7, and transformation of T2 to T1 where the
rib associated with T2 touched the top of the sternum.
[0157] In order to understand the basis for the abnormalities in
axial patterning seen in GDF-11 mutant mice, we examined mutant
embryos isolated at various stages of development and compared them
to wild-type embryos. By gross morphological examination,
homozygous mutant embryos isolated up to day 9.5 of gestation were
not readily distinguishable from corresponding wild-type embryos.
In particular, the number of somites present at any given
developmental age was identical between mutant and wild-type
embryos, suggesting that the rate of somite formation was unaltered
in the mutants. By day 10.5-11.5 p.c., mutant embryos could be
easily distinguished from wild-type embryos by the posterior
displacement of the hindlimb by 7-8 somites. The abnormalities in
tail development were also readily apparent at this stage. Taken
together, these data suggest that the abnormalities observed in the
mutant skeletons represented true transformations of segment
identities rather than the insertion of additional segments, for
example, by an enhanced rate of somitogenesis.
[0158] Alterations in expression of homeobox containing genes are
known to cause transformations in Drosophila and in vertebrates. To
see if the expression patterns of Hox genes (the vertebrate
homeobox containing genes) were altered in GDF-11 null mutants we
determined the expression pattern of 3 representative Hox genes,
Hoxc-6, Hoxc-8 and Hoxc-11, in day 12.5 p.c. wild-type,
heterozygous and homozygous mutant embryos by whole mount in situ
hybridization. The expression pattern of Hoxc-6 in wild-type
embryos spanned prevertebrae 8-15 which correspond to thoracic
segments T1-T8. In homozygous mutants, however, the Hoxc-6
expression pattern was shifted posteriorly and expanded to
prevertebrae 9-18 (T2-T11). A similar shift was seen with the
Hoxc-8 probe. In wild-type embryos, Hoxc-8 was expressed in
prevertebrae 13-18 (T6-T11) but, in homozygous mutant embryos,
Hoxc-8 was expressed in prevertebrae 14-22 (T7-T15). Finally,
Hoxc-11 expression was also shifted posteriorly in that the
anterior boundary of expression changed from prevertebrae 28 tin
wild-type embryos to prevertebrae 36 in mutant embryos. (Note that
because the position of the hindlimb is also shifted posteriorly in
mutant embryos, the Hoxc-11 expression patterns in wild-type and
mutant appeared similar relative to the hindlimbs). These data
provide further evidence that the skeletal abnormalities seen in
mutant animals represent homeotic transformations.
[0159] The phenotype of GDF-11 mice suggested that GDF-11 acts
early during embryogenesis as a global regulator of axial
patterning. To begin to examine the mechanism by which GDF-11
exerts its effects, we determined the expression pattern of GDF-11
in early mouse embryos by whole mount in situ hybridization. At
these stages the primary sites of GDF-11 expression correlated
precisely with the known sites at which mesodermal cells are
generated. Expression of GDF-11 was first detected at day 8.25-8.5
p.c. (8-10 somites) in the primitive streak region, which is the
site at which ingressing cells form the mesoderm of the developing
embryo. Expression was maintained in the primitive streak at day
8.75, but by day 9.5 p.c., when the tail bud replaces the primitive
streak as the source of new mesodermal cells, expression of GDF-11
shifted to the tail bud. Hence at these early stages, GDF-11
appears to be synthesized in the region of the developing embryo
where new mesodermal cells arise and presumably acquire their
positional identity.
[0160] The phenotype of GDF-11 knockout mice in several respects
resembles the phenotype of mice carrying a deletion of a receptor
for some members of the TGF-.beta. superfamily, the activin type
IIB receptor (ActRIIB). As in the case of GDF-11 knockout mice, the
ActRIIB knockout mice have extra pairs of ribs and a spectrum of
kidney defects ranging from hypoplastic kidneys to complete absence
of kidneys. The similarity in the phenotypes of these mice raises
the possibility that ActRIIB may be a receptor for GDF-11. However,
Act RIIB cannot be the sole receptor for GDF-11 Ibecause the
phenotype of GDF-11 knockout mice is more severe than the phenotype
of ActRIIB mice. For example, whereas the GDF-11 knockout animals
have 4-5 extra pairs of ribs and show homeotic transformations
throughout the axial skeleton, the ActRIIB knockout animals have
only 3 extra pairs of ribs and do not show transformations at other
axial levels. In addition, the data indicate that the kidney
defects in the GDF-11 knockout mice are also more severe than those
in ActRIIB knockout mice. The ActRIIB knockout mice show defects in
left/right axis formation, such as lung isomerixm and a range of
heart defects that we have not yet observed in GDF-11 knockout
mice. ActRIIB can bind the activins and certain BMPs, although none
of the knockout mice generated for these ligands show defects in
left/right axis formation.
[0161] If GDF-11 does act directly on mesodermal cells to establish
positional identity, the data presented here would be consistent
with either short range or morphogen models for GDF-11 action. That
is, GDF-11 may act on mesodermal precursors to establish patterns
of Hox gene expression as these cells are being generated at the
site of GDF-11 expression, or alternatively, GDF-11 produced at the
posterior end of the embryo may diffuse to form a morphogen
gradient. Whatever the mechanism of action of GDF-11 may be, the
fact that gross anterior/posterior patterning still does occur in
GDF-11 knockout animals suggests that GDF-11 may not be the sole
regulator of anterior/posterior specification. Nevertheless, it is
clear that GDF-11 plays an important role as a global regulator of
axial patterning and that further study of this molecule will lead
to important new insights into how positional identity along the
anterior/posterior axis is established in the vertebrate
embryo.
[0162] 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
10 1 1393 DNA Human GDF-11 CDS (54)..(1274) 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 Human GDF-11 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 Mouse GDF-11 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 Mouse GDF-11 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 Human GDF-8 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 Asp
Arg Cys Gly Cys Ser 370 375 6 407 PRT Human GDF-11 6 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 7 23 DNA Artificial sequence Primer
for PCR 7 gagtcccgct gctgccgata tcc 23 8 23 DNA Artificial sequence
Primer for PCR 8 tagagcatgt tgattgggga cat 23 9 20 DNA Artificial
sequence Oligonucleotide probe for GDF-11 9 aaatatccgc atacccattt
20 10 376 PRT Mouse 10 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
* * * * *