Note: Descriptions are shown in the official language in which they were submitted.
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DORSAL TISSUE AFFECTING FACTOR AND COMPOSITIONS
Field of the Inventi
on
The invention generally relates to growth factors and neurotrophic factors,
and more particularly to a soluble growth factor with dorsal growth inducing
activity,
to complexes including the factor, and to DNA or RNA coding sequences for the
factor.
This invention was made, in part, with government support under Grant
Contract No. ROI-GM-42341, awarded by the National Institutes of Health. The
U.S. government has certain rights in this invention.
on
Backaround of the Inventi
Growth factors are substances, such as polypeptide hormones, which affect
the growth of defined populations of animal cells in vivo or in vitro, but
which are
not nutrient substances. Proteins involved in the growth and differentiation
of tissues
may promote or inhibit growth, and promote or inhibit differentiation, and
thus the
general term "growth factor" includes cytokines and trophic factors. Among
growth,
or neurotrophic factors presently known are those that can be classified into
the
insulin family [insulin, insulin-like growth)
A.
WO 94/05791 2143442 PCT/US93/08326
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factors (e.g., IGF-I, IGF-II), mammary stimulating factor (MSF), and
nerve growth factor (NGF)]; those classified into the epidermal
growth factor family [epidermal growth factor (EGF) and
transforming growth factors (TGFa, TGF(3, TGFy)]; those classified
into the platelet-derived growth factor family [platelet-derived
growth factor (PDGF), osteosarcoma-derived growth factor (ODGF),
and fibroblast growth factor (FGF)]; the neurotrophins [nerve growth
factor (NGF), brain derived neurotrophic factors (BDNF)
neurotrophins 3, 4, 5, (NT-3, NT-4, NT-5)]; and others [colony
stimulating factor (CSF), T-cell growth factor, tumor angiogenesis
factor (TAF), DNA synthesis promoting factor (DSF), tumor-derived
growth factors, fibroblast-derived growth factor (FDGF)].
Receptors that affect growth (that is, receptors for growth-
associated ligands) are proteins found associated with cell
surfaces that specifically bind their growth factors as ligands.
Growth factor receptors are utilized in various clinical and
diagnostic applications.
U.S. Patent 4,857,637, issued August 15, 1989, inventors
Hammonds et al., describes a method for immunizing an animal
against its growth hormone receptor through use of vaccinating with
antibodies in order to stimulate growth of the animals.
U.S. Patent 4,933,294, issued June 12, 1990, inventors
Waterfield et al., describes studies of structural alterations of the
human EGF receptor and its gene and a relationship in tumorigenesis
for assays and therapies involving the human EGF receptor. For
example, such assays can involve detection of structurally altered
or abnormally expressed growth factor receptor and the mRNA
transcripts and genes which encode them. EGF may have a role in cell 2
WO 94/05791 2143442 PCT/US93/08326
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proliferation and differentiation since it induces early eyelid
opening and incisor development in new born mice.
U.S. Patent 5,030,576, issued July 9, 1991, inventors Dull et
= al., describes the role of receptors, such as receptors for growth
factors, in designing drugs by the pharmaceutical industry, and
discloses use of a receptor hybrid for screening drug purposes, such
as in studies of EGF binding domains. U.S. Patent 5,087,616, issued
February 11, 1992, inventors Myers and Bichon, describes a method
for destroying tumor cells with a composition including a drug
conjugate. The conjugate has a growth factor as one moiety and a
polymeric carrier with a cytotoxic compound as another moiety.
Thus, compositions of the patent are described as binding
preferentially to tumor cells bearing EGF-binding receptors (when an
EGF growth factor, for example, is used as a first moiety).
U.S. Patent 5,098,833, issued March 24, 1992, inventors
Lasky, et al., describes a DNA isolant capable of hybridizing to the
epidermal growth factor domain. Expression systems for
recombinant production are said to be useful in therapeutic or
diagnostic compositions.
A good background review of a neurotrophic factor related to
NGF is provided by W092/05254, published April 2, 1992, which
also describes state of the art methods of: preparing amino acid
sequence variations, site-directed mutagenesis techniques,
ligation of coding DNA into a replicable vector for further cloning or
for expression, choice of promoters for expression vectors, suitable
host cells for expression, particularly mammalian cells, protein
purification upon recovery from culture medium as a secreted
protein, derivatization with bifunctional agents to cross-link
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protein to a support matrix for use with antibodies, entrapment in systems for
drug
delivery, preparation of therapeutic formulations, and methods of
administration. In
addition, preparation of polyclonal and monoclonal antibodies are described,
such as
are useful in diagnostic assays. These various aspects of isolation,
preparation, and
applications for. a novel neurotrophic factor, are illustrated by the WO
92/05254.
Thus, growth factors, their receptors, and DNA or RNA coding sequences,
therefore, and fragments thereof are useful in a number of therapeutic,
clinical,
research, diagnostic and drug design applications.
Brief Description of the Drawin~s
Figure 1. Nucleotide sequence (SEQ ID NO: 1) for the human noggin gene
and deduced amino acid sequence (SEQ ID NO: 2).
Figure 2(A). Experimental design: competent animal cap(AC) ectoderm was
dissected from staged embryos as shown. St10.5 dorsal and ventral AC and
ventral
marginal zones (VMXZ) also dissected as shown. Explants were washed once in
low
Ca/Mg Ringers (LCMR) solution and then placed in treatment medium containing
factor diluted in LCMR + 0.5%BSA. Explants cultured to late stages (St20+)
were
removed from treatment medium 6-16 hours after the start of treatment and
placed in
LCMR. When explants reached the desired stage they were either harvested for
RNA, or
WO 94/05791 214 3442 PCT/US93/08326
they were fixed for whole mount in situ hybridization or antibody
staining.
(B). Neural induction by noggin in the absence of muscle. Lanes
= 1-3 show specific fragments protected by N-CAM, 0-tubulin, and XIF-
3 probes respectively in whole St24 embryo RNA. Lanes 4-8 show
protection by the mixture of these three probes while lanes 9-13
show protection by an actin probe on tRNA(t), St24 embryo RNA (E),
and RNA collected from St9 AC treated with 5OpM activin (A), 25%
of 20 fold concentrated control CHO cell medium (C) or 25% of 20
fold concentrated noggin conditioned CHO cell medium (N).
Ubiquitously expressed cytoskeletal actin used as a loading control
shows that RNA levels in all treatments are comparable (lanes 11-
13).
Figure 3. 12% SDS-PAGE run under reducing conditions.
Proteins were visualized by silver staining. Lane 1 shows molecular
size standards. Lanes 2-7 show 0, 0.1, 0.2, 0.5, 1, and 2 g of
purified human noggin.
Figure 4 (A). Time course of animal caps treated with purified
noggin vs. activin; direct vs indirect neural induction. Animal caps
were dissected as shown in Fig. 2A and treated with LCMR + 0.5%
BSA (U), a 20% dilution of activin conditioned COS cell medium (A),
or 1 g/mi purified human noggin(N). RNA isolated from treated
animal caps (lanes 2-13) along with St22 whole embryo RNA (lanel)*
and tRNA (lane 14) was probed for N-CAM, 0-tubulin, muscle and
cytoskeletal actins, coliagen type II, and EF-la.
(B). Expression of early mesoderm markers in activin but not
noggin induced animal caps. Animal caps were dissected from St8
embryos, treated as described in (A), and harvested at St11. Lanes 1
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and 2 respectively show goosecoid and Xbra probe protection by
St10.5 whole embryo RNA. Lanes 3-6 show protection by, a mix of
these two probes. Relative RNA levels are demonstrated by separate
EF-la probe protection.
(C). Plasmid directed gastrula stage noggin expression directly
induces neural tissue. One cell stage embryos were injected with
20pg of pCSKAlacZ or pCSKAnoggin into the animal pole. Animal caps
from injected embryos were dissected at St8-9 and cultured until
St20, when they were harvested for analysis by RNase protection.
Figure 5. Responsiveness of dorsal and ventral animal caps to
neural induction by noggin. St 105 ventral and dorsal animal caps
were dissected as shown in Fig. 2. Dorsal and ventral animal caps
were treated with activin medium (DA,VA) or 1 g/ml human noggin
(DN, VN) and harvested at St26 for RNase protection analysis using N-
1 5 CAM, R-tubulin, and actin as probes.
Figure 6. Dose response of ventral marginal zones and animal
caps to human noggin protein. St 10.5 VMZs and St9 animal caps
were dissected as shown in Fig. 2A., and treated with 0, 1, 10, 50,
200, and 1000 ug/mI of human noggin (lanes 3-8 and 10-15
respectively). RNA from treated explants and control whole embryos
aged to St26 was then analyzed by RNase protection, using the
probes N-CAM, 0-tubulin, actin and collagen type II. In this
experiment, muscle induction at the dose of 1 ng/ml is stronger than
at 10ng/mI, and there is a low level of muscle actin expression in
the uninduced VMZs. This could be due to experimental variability
since in repeated experiments we saw muscle induction only at the doses of
50ng/mi and above (data not shown).
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Figure 7. In situ hybridization and antibody staining. Tailbud
embryos stained for NCAM showing side and dorsal views (a,b); NCAM
RNA is only detected in the neural tube, and not the somites. For
comparison, somites of a tailbud embryo stain for muscle actin,
dorsal view (c). Neural specific 6F11 antibody staining at St30 (d-f).
Some cement gland pigment remained in these embryos after
bleaching as seen in (d), however this pigment is distinct from
antibody staining. The inner mass of staining in the noggin treated
animal caps is due to the 6F11 antibody detection. Cement gland
specific XAG-1 transcripts detected at St23 (g-i), and anterior brain
otxA transcripts detected at St35 (j-1) in whole embryos at (d,g,j),
human noggin treated (1 g/mi) animal caps (e,h,k), and untreated
animal caps (f,i,l).
Figure 8. Reverse phase HPLC profile of two refolded isoforms
of noggin. The refolded noggin solution was applied onto a Brownlee
Aquapore AX-300, 0.46 x 22 cmHPLC column at a flow rate of 1
mi/min. The column was equilibrated with solvent A containing 0.1%
TFA in water. Solvent B was 0.1% TRA in acetonitrile. The column
was developed according to the following protocol: a) 2 min
isocratically at 95% of solvent A-5% of solvent B; 60 min linear
gradient to 65% of solvent B and 35% of solvent A. Correctly
refolded noggin elutes earlier at 44%-46% solvent B.
Figure 9. Reverse-phase HPLC chromatography characterization
of recombinant noggin refolded and purified from E. coli. Conditions
as in the legend to Fig. 8.
Figure 10. Recombinant noggin produced in E. coli and in insect
cells analyzed by 12.5% SDS-PAGE. Lanes H, L: High and low
molecular weight markers of the indicated size, respectively. Lanes
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1,2: Recombinant noggin produced in E. coli and in insect cells
respectively, treated with 2-mercaptoethanol before
electrophoresis. The slower mobility of noggin from insect cells
corresponds to the size increase that would occur due to N-linked
glycosylation at the single consensus site. Lanes 2,3: Recombinant
noggin produced in E. coli and in insect cells respectively, not
treated with 2-mercaptoethanol before electrophoresis.
Figure 11. Circular dichroism spectra of recombinant noggin
produced in E. coli (--), and in insect cells (-).
Figure 12. Ventral marginal zone assay showing induction of
muscle actin mRNA after expo'sure to human noggin (0.01, 0.05, 0.2
g/ml) produced in baculovirus, a mock transfected culture of
baculovirus (0.02, 1 g/ml) or human noggin produced in E.coli (0.1,
0.5, 2, or 10 g/ml).
Figure 13. Nucleotide sequence (SEQ ID NO:25) for the mouse
noggin gene and deduced amino acid sequence (SEQ ID NO:26).
Summary of the Invention
In one aspect of the present invention a peptide that can be in
substantially purified form is characterized by one or more of the
following, highly conserved amino acid sequences:
QMWLWSQTFCPVLY (SEQ ID NO:3);
RFWPRYVKVGSC (SEQ ID NO:4);
SKRSCSVPEGMVCK (SEQ ID NO:5);
LRWRCQRR (SEQ ID NO:6); and,
ISECKCSC (SEQ ID NO:7).
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Peptides of the invention induce dorsal growth in vertebrates
and can be prepared in soluble, physiologically active form for a
number of therapeutic, clinical, and diagnostic applications.
In a preferred embodiment, human noggin protein as set forth
in Figure 1 (SEQ ID NO: 2) is prepared for use in therapeutic, clinical
and diagnostic applications.
In another aspect of the present invention an oligonucleotide,
such as cDNA, is provided having substantial similarity to (or being
the same as) SEQ ID NO:8 (deduced amino acid sequence, SEQ ID NO:
9), SEQ ID NO: 10 (deduced amino acid sequence, SEQ ID NO: 11), or
SEQ ID NO:1. This oligonucleotide can be single or double stranded,
be formed of DNA or RNA bases, and can be in the antisense direction
with respect to SEQ ID NOS: 8, 10 or 1. SEQ ID NO: 8, SEQ ID NO: 10
and SEQ ID NO:1 each code for a functional polypeptide that we have
designated "noggin," which is capable of inducing dorsal
development in vertebrates when expressed.
Noggin or fragments thereof (which also may be synthesized by
in vitro methods) may be fused (by recombinant expression or in
vitro covalent methods) to an immunogenic polypeptide and this, in
turn, may be used to immunize an animal in order to raise antibodies
against a noggin epitope. Anti-noggin is recoverable from the serum
of immunized animals. Alternatively, monoclonal antibodies may be
prepared from cells to the immunized animal in conventional
fashion. Antibodies identified by routine screening will bind to
noggin but will not substantially cross-react with "wnt" or other
growth factors. Immobilized anti-noggin antibodies are useful
particularly in the diagnosis (in vitro or in vivo) or purification of
noggin.
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Substitutional, deletional, or insertional mutants of noggin
may be prepared by in vitro or recombinant methods and screened for
immuno-crossreactivity with noggin and for noggin antagonist or
agonist activity. =
Noggin also may be derivatized in vitro in order to prepare
immobilized noggin and labelled noggin, particularly for purposes of
. diagnosis of noggin or its antibodies, or for affinity purification of
noggin antibodies.
The present invention further provides for expression of
biologically active noggin molecules in prokaryotic and eukaryotic
expression systems.
The present invention further provides for the production of
noggin in quantities sufficient for therapeutic and diagnostic
applications. Likewise, anti-noggin antibodies may be utilized in
therapeutic and diagnostic applications. For most purposes, it is
preferable to use noggin genes or gene products from the same
species for therapeutic or diagnostic purposes, although cross-
species utility of noggin may be useful in specific embodiments of
the invention.
In additional embodiments, the noggin nucleic acids, proteins,
and peptides of the invention may be used to induce neural tissue
formation in mammals.
Description of the Preferred Embodiments
We have discovered a structurally unique growth factor that is
readily available in substantially pure, soluble form. We have
named the inventive polypeptide "noggin." This newly isolated
= neurotrophic factor induces dorsal deveiopment in vertebrates.
WO 94/05791 2143442 PGT/US93/08326
An earlier described family of proteins that also induces
dorsal development are the "wnt" proteins. These, however, in
contrast to noggin remain tenaciously bound to cell surfaces. Our
= initial work with noggin has been in Xenopus embryos; however,
noggin is highly conserved among vertebrates, as our work with
mouse noggin has demonstrated. The prior known FGF growth factor
family is also known to be involved in early embryonic induction,
but both the FGF proteins and their receptors are distinctly different
from noggin. Noggin modifies the actions of FGF (and also activin),
for example by potentiating growth, and is thus particularly
suggested in therapeutic compositions for use in combination with
other growth factors (as therapeutic adjuvants), such as to modify
or potentiate their effects.
We have cloned cDNA for noggin. The noggin cDNA contains a
single reading frame encoding a 26 kDa protein with a hydrophobic
amino-terminal sequence. Noggin is secreted. Noggin's cDNA encodes
the protein as a 26 kDa protein, but we have determined that noggin
is secreted in vivo, apparently as a dimeric glycoprotein with a
starting apparent molecular weight of about 33 kDa (as the wild-
type subunit). When not glycosylated, the monomeric unit has an
apparent molecular weight on SDS PAGE of about 25-30 kDa.
We have cloned the gene for human noggin (Figure 1; SEQ ID NO:
1). The sequence codes for a protein which has noggin activity (SEQ
ID NO: 2). The carboxy terminal region of noggin shows homology to a
Kunitz-type protease inhibitor, indicating that noggin protein, or
fragments thereof, may exhibit activities of a protease inhibitor.
We have been able to express biologically active noggin in
both eukaryotic and prokaryotic host cells. Two expression systems
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we have successfully used to express biologically active noggin have
been mammalian cell (ines (COS and mouse 293). A third expression
system is injection of synthetic mRNA into Xenopus oocytes. In
addition, we have successfully expressed biologically active human 5 noggin in
a prokaryotic system, E. coli, and in baculovirus.
Expression in these several different systems also illustrates
the high degree of conservation for noggin. We have found, for
example, substantial sequence similarity between frog noggin and
mouse noggin with a number of completely conserved stretches.
Thus, the following amino acid sequences represent completely
conserved portions as between frog noggin and mouse noggin:
QMWLWSQTFCPVLY (SEQ ID NO:3);
RFWPRYVKVGSC (SEQ ID NO:4);
SKRSCSVPEGMVCK (SEQ ID NO:5);
LRWRCQRR (SEQ ID NO:6); and,
ISECKCSC (SEQ ID NO:7).
There is about 87% overall conservation between the mouse and frog
sequences, and we have also observed a unique cysteine distribution
between the two.
Noggin nucleic acids, or oligonucleotides, encode a noggin
polypeptide or hybridize to such DNA and remain stably bound to it
under stringent conditions and are greater than about 10 bases in
length; provided, however, that such hybridizing nucleic acid is
novel and unobvious over any~ prior art nucleic acid including that
which encodes or is complementary to nucleic acid encoding other
growth factors.
By "stringent conditions" we mean are those which (1) employ
low ionic strength and high temperature for washing, for example,
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0.15 M NaCU0.015 M sodium citrate/0.1% NaDodSo4 at 50 C, or (2)
use during hybridization a denaturing agent such as formamide, for
example, 50% (voVvol) formamide with 0.1% bovine serum
albumin/0.1%F'IcX]IlT"'l0.1 ! poiyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCi, 75 mM sodium citrate
at 42 C.
By "substantial similarity," when we are referring to a
nucleotide sequence, is meant cross hybridization of sequences
under conditions of moderate stringency using a probe greater than
100 nucleotides long at 30 C in a standard buffer (Wahl et al.,
PNAS,76, 3683) and washes at 37 C in 300 mM NaCi, 30 mM sodium
citrate, 0.2% SDS at pH 7. Alternatively, one is able to isolate; by
polymerase chain reaction, a fragment of DNA coding for noggin or
noggin family members when using primers of degenerate sequence
that encode those SEQ ID NOS:3-7.
By "substantial similarity" when reference is made to
proteins is that noggin from different species, or noggin family
members within a species, will preserve the positions of cysteine
residues in at least 80% of positions throughout the protein. Like the
neurotrophin family, the sequence of the mature form of noggin and
noggin related polypeptides will be identical in at least 40% of
positions. Substantial similarity at the protein level includes an
ability of a subject protein to compete with noggin for binding to
receptors and some (but not all) monoclonal antibodies raised
against noggin epitopes.
The cloned cDNA for noggin (derived from frog) is designated
herein as SEQ ID NO: 8, partial sequence from mouse as SEQ ID NO:
10 or full sequence of mouse noggin as shown in Figure 13 (SEQ ID
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WO 94/05791 2143442"' PCT/US93/08326
NO: 25). The human sequence is designated herein as SEQ ID NO: 1. *
We have used RNA transcripts from the SEQ ID NO: 8 clone to rescue
embryos and return them to substantially normal development when
the noggin RNA is injected into ventralized embryos. In high doses
this results in excessive head development and it is for this reason
we named the protein "noggin." In northern blot analysis the noggin
cDNA hybridizes to two mRNAs that are expressed both maternally
and zygotically.
When using nucleotide sequences coding for part or all of
noggin in accordance with this invention, the length of the sequence
should be at least sufficient in size to be capable of hybridizing
with endogenous mRNA for the vertebrate's own noggin. Typically,
sufficient sequence size (for example, for use as diagnostic probes)
will be about 15 consecutive bases (DNA or RNA). In some diagnostic
and therapeutic applications, one may wish to use nucleotide noggin
coding sequences (analogous to all or a portion of SEQ ID NO: 8, SEQ
ID NO: 10, SEQ ID NO: 25 or SEQ ID NO:1) in the anti-sense direction
with respect to either SEQ ID NOS: 8, 10, 25, or 1.
We suggest as a few preferred primers for amplifying noggin
from other species (e.g. human):
5' Primer 1 SEQ ID NO: 12
CAA/GACNTTC/TTGC/TCCNGTN
5' Primer 2 SEQ ID NO: 13
TTC/TTGGCCNC/AGNTAC/TGTNAAA/G GTNGG
5'Primer3SEQIDNO:14
CCNGAA/GGGNATGGTNTG
= 3' Primer 1 SEQ ID NO: 15
C A N C/G T/A A/G C A C/T T T A/G C A C/T T C
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3' Primer 2 SEQ ID NO: 16
CANACCATNCCC/TTCNGG
3' Primer 3 SEQ ID NO: 17
CG/TNCG/TT/CTGG/ACANCG/TCCA
where N represents a mixture of all four nucleotides and mixtures of
two nucleotides are represented by alternates (e.g. A/G).
Although noggin transcript is not localized in the oocyte and
cleavage stage embryo, zygotic transcripts are initially restricted
to the presumptive dorsal mesoderm, and reach their highest levels
at the gastrula stage in the dorsal lip of the blastopore (Spemann's
organizer). In the neurula, noggin is transcribed in the notochord and
prechordal mesoderm.
Without being bound by theory, we have formulated hypotheses
about the embryological effects of noggin based on where it is
expressed, and on the effects of RNA injection in embryos. Since
noggin is , expressed in the Spemann organizer, we believe noggin to
be a mediator of the effects of the Spemann organizer, namely
neural induction and dorsalization of the mesoderm. We have shown
that noggin is able to directly induce neural tissue formation. Since
noggin is expressed in the notochord and head mesoderm, we believe
noggin to influence either the dorsal-ventral pattern or anterior-
posterior pattern of the neural plate. Since noggin is expressed in
the branchial arch neural crest, we believe it may therefore
influence whether neural crest cells deposit cartilage and also to
influence later branchial arch growth and remodelling. Noggin is
expressed in the tail fin neural crest, and since neural crest is
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required for growth of the fin, noggin may act as a growth factor for
epidermis or mesenchyme.
Although much of our experimental work has involved rescue of
embryonic development, because expression in the notochord
persists in the growing tail bud and a discontinuous line of stained
cells (indicating expression of noggin initiated at new sites) runs
the iength of the roof plate of the neural tube (and is also apparent
in the head mesoderm), we believe noggin is expressed as an adult
cell function also.
A number of applications for noggin are suggested from its
properties.
The noggin cDNA should be useful as a diagnostic tool (such as
through use of antibodies in assays for proteins in cell lines or use
of oligonucleotides as primers in a PCR test to amplify those with
sequence similarities to the oligonucleotide primer, and to see how
much noggin is present, e.g. primers such as 5' Primers 1-3 and 3'
Primers 1-3).
Because noggin has a pattern of expression that suggests it is
used to regulate cartilage production in the embryonic head, clinical
uses to regulate cartilage and bone growth are suggested for noggin
in therapeutic compositions and particularly in combination with
other growth factors due to a property of noggin to potentiate at
least some growth factors. Since neural crest cells are required for
the tadpole fin to grow, noggin seems to be a growth factor for the
tissue matrix and epidermis and should prove useful, for example, in
wound healing compositions.
Noggin, of course, provides the key to isolate its receptor
Since many receptors mutate to cellular oncogenes, the noggin
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receptor should prove useful as a diagnostic probe for certain tumor
types. Thus, when one views noggin as ligand in complexes, then
complexes in accordance with the invention include antibody bound
to noggin, antibody bound to peptides derived from noggin, noggin
bound to its receptor, or peptides derived from noggin bound to its
receptor. Mutant forms of noggin, which are either more potent
agonists or antagonists, are believed to be clinically useful. Such
complexes of noggin and its binding protein partners will find uses
in a number of applications.
Practice of this invention includes use of an oligonucleotide
construct comprising a sequence coding for noggin and for a
promoter sequence operatively linked to noggin in a mammalian,
bacterial or a viral expression vector. Expression and cloning
vectors contain a nucleotide sequence that enables the vector to
replicate in one or more selected host cells. Generally, in cloning
vectors this sequence is one that enables the vector to replicate
independently of the host chromosomes, and includes origins of
replication or autonomously replicating sequences. The well-known
plasmid pBR322 is suitable for most gram negative bacteria, the 2
plasmid origin for yeast and various .viral origins (SV40, polyoma,
adenovirus, VSV or BPV) are useful for cloning vectors in
mammalian cells.
Expression and cloning vectors should contain a selection
gene, also termed a selectable marker. Typically, this is a gene that
encodes a protein necessary for the survival or growth of a host
cell transformed with the vector. The presence of this gene ensures
that any host cell which deletes the vector will not obtain an
advantage in growth or reproduction over transformed hosts.
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Is
Typical selection genes encode proteins that (a) confer resistance
to antibiotics or other toxins, e.g. ampicillin, neomycin,
methotrexate or tetracycline, (b) complement auxotrophic
deficiencies.
Examples of suitable selectable markers for mammalian cells
are dihydrofolate reductase (DHFR) or thymidine kinase. Such
markers enable the identification of cells which were competent to
take up the noggin nucleic acid. The mammalian cell transformants
are placed under selection pressure in which only the transformants
are uniquely adapted to survive by virtue of having taken up the
marker. Selection pressure is imposed by culturing the
.transformants under conditions in which the concentration of
selection agent in the medium is successively changed.
Amplification is the process by which genes in greater demand for
the production of a protein critical for growth are reiterated in
tandem within the chromosomes of successive generations of
recombinant cells. Increased quantities of noggin can therefore be
synthesized from the amplified DNA.
For example, cells transformed, with the DHFR selection gene
are first identified by culturing all of the transformants in a culture
medium which contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell in this case is the
Chinese hamster ovary (CHO) cell line deficient in DHFR activity,
prepared and propagated as described by Urlaub and Chasin, Proc. Nat.
Acad. Sci., 77, 4216 (1980). The transformed cells then are exposed
to increased levels of Mtx. This leads to the synthesis of multiple
copies of the DHFR gene and, concomitantiy, muftipie copies of other
DNA comprising the expression vectors, 'such as the DNA encoding
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noggin. Alternatively, host cells transformed by an expression
vector comprising DNA sequences encoding noggin and
aminoglycoside 3' phosphotransferase (APH) protein can be selected
= by cell growth in medium containing an aminoglycosidic antibiotic
such as kanamycin or neomycin or G418. Because eukarotic cells do
not normally express an endogenous APH activity, genes encoding
APH protein, commonly referred to as neo resistant genes, may be
used as dominant selectable markers in a wide range of eukaryotic
host cells, by which cells transformed by the vector can readily be
identified.
Expression vectors, unlike cloning vectors, should contain a
promoter which is recognized by the host organism and is operably
linked to the noggin nucleic acid. Promoters are untranslated
sequences located upstream from the start codon of a structural
gene (generally within about 100 to 1000 bp) that control the
transcription and translation of nucleic acid under their control.
They typically fall into two classes, inducible and constitutive.
Inducible promoters are promoters that initiate increased levels of
transcription from DNA under their control in response to some
change in culture conditions, e.g. the presence or absence of a
nutrient or a change in temperature. At this time a large number of
promoters recognized by a variety of potential host cells are well
known. These promoters can be operably linked to noggin encoding
DNA by removing them from their gene of origin by restriction
enzyme digestion, followed by insertion 5' to the start codon for
noggin.
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L~ 0
Nucleic acid is operably linked when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
which participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects the
.transcription of the sequence; or a ribosome binding site is operably
linked to a coding sequence if it is positioned so as to facilitate
translation. Generally, operably linked means that the DNA
sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist then synthetic oligonucleotide adapters or linkers
are used in accord with conventional practice.
Transcription of noggin-encoding DNA in mammalian host
cells is controlled by promoters obtained from the genomes of
viruses such as polyoma, cytomegalovirus, adenovirus, retroviruses,
hepatitis-B virus, and most preferably Simian Virus 40 (SV40), or
from heterologous mammalian promoters, e.g. the actin promoter.
Of course, promoters from the host cell or related species also are
useful herein.
In particular embodiments of the invention expression of
noggin in E. coli is preferably performed using vectors which
comprise the following: a jaa UV5 promoter which may be controlled
by the lactose operon repressor; a strong ribosome binding site, for
example, the ribosome binding site of bacteriophage T7; a mutation
in the replication control region of the plasmid which may increase
2143442.
WO 94/05791 PCT/US93/08326
copy number; and a mutation which limits the expression of the
antibiotic resistance protein.
In a preferred embodiment, noggin is expressed in a high copy
number kanamycin resistant pBR322-derived plasmid under the
control of a j= UV5 promoter. In an additional preferred
embodiment, noggin is expressed in baculovirus under the control of
the polyhedrin promoter of Autrographa californica Multiple Nuclear
Polyhedrosis virus in insect host cells.
Noggin is believed to find use as an agent for enhancing the
survival or inducing the growth of nerve and muscle cells. It,
therefore, is useful in the therapy of congenital conditions or
degenerative disorders of the nervous system ("neurodegenerative
diseases"), including such diseases as Alzheimer's disease,
Parkinson's disease, Huntington's chorea, ALS, peripheral
neuropathies, and other conditions characterized by necrosis or loss
of neurons, whether central, peripheral, or motorneurons. In
addition, it may be useful for treating damaged nerve cells, e.g.,
nerves damaged by traumatic conditions such as burns and wounds,_
diabetes, kidney dysfunction, and the toxic effects of
chemotherapeutics used to treat cancer and AIDS. It also is useful
as a component of culture media for use in culturing nerve cells in
vitro.
The capacity of noggin to induce neural tissue may be useful in
diseases where neural tissue is formed improperly or incompletely
during development. Thus, noggin and the noggin gene are also useful
in treating congenital malformations such as anencephaly, or the
loss of cerebral hemispheres which results from failure of closure
of the anterior neural tube during development.
21
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2143442
Practice of this invention includes preparation and uses of a
diagnostic or therapeutic agent comprising a nucleotide sequence of
at least about 15 DNA or RNA bases analogous to all or a portion of
either SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID N0:25, or SEQ ID NO: 1 or of
the nucleic acid sequences contained in bacteriophages, hnoga,-9 or
hnog7,-10. That is, noggin preparations are useful as standards in
assays for noggin and in competitive-type receptor binding assays
when labelled with radioiodine, enzymes, fluorophores, spin labels,
and the like. Therapeutic formulations of noggin are prepared for
storage by mixing noggin having the desired degree of purity with
optional physiologically acceptable carriers, excipients or
stabilizers, in the form of lyophilized cake or aqueous solutions.
Acceptable carriers, excipients or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins. Other components can include
glycine, blutamine, asparagine, arginine, or lysine;
monosaccharides,disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar
alcohols such as mannitol or sorbitol; salt-forming counterions such
as sodium; and/or nonionic surfactants such as Tween, Pluronics or
PEG.
Noggin may be used according to the invention as described
supra. The concentration of the active ingredient used in the
formulation will depend upon the effective dose required and the
mode of administration used. The dose used should be sufficient to
22
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~
achieve circulating plasma concentrations of active ingredient that
are efficacious. Effective doses may be extrapolated from dose-
response curves derived from in vitro or animal model test systems.
By referring to noggin, the present invention also contemplates
the use of fragments, derivatives, agonists or antagonists of noggin
molecules.
Noggin may be administered in any pharmacologically
acceptable carrier. The administration route may be any mode of
administration known in the art, including but not limited to
intravenously, intrathecally, subcutaneously, by injection into
involved tissue, intraarterially, intranasally, orally, or via an
implanted device. The present invention provides for pharmaceutical
compositions comprising noggin in a pharmacologically acceptable
carrier.
Administration may result in the distribution of noggin
throughout the body or in a localized area. For example, in some
conditions which involve distant regions of the nervous system,
intravenous or intrathecal administration of noggin may be
desirable. Alternatively, and not by way of limitation, when
localized regions of the nervous system are involved, local
administration may be desirable. In such situations, an implant
containing noggin may be placed in or near the lesioned area.
Suitable implants include, but are not limited to, gelfoam, wax, or
microparticle-based implants.
Depending upon the mode of administration, the active
ingredient may be formulated in a liquid carrier such as saline,
incorporated into liposomes, microcapsuies, polymer or wax-based
23
WO 94/05791 A PC'T/US93/08326
4~~~ ~i ~ ~
and controlled release preparations, or formulated into tablet, pill
or capsule forms.
Inventive complexes comprise a ligand characterized by one or
more of the SEQ ID NOS:3-7. The ligand can be bound to a protein,
such as antibody. Such antibodies can be polyclonal or monoclonal.
Polyclonal antibodies to noggin generally are raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
noggin and an adjuvant. It may be useful to conjugate noggin or a
fragment containing the target amino acid sequence to a protein
which is immunogenic in the species to be immunized, e.g., keyhole
limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor using a bifunctional or derivatizing , agent, for
example, maleimidobenzoyl sulfosuccinimide ester (coniugation
through cysteine residues), N-hydroxy-succinimide (through lysine
residues), glutaraidehyde, succinic anhydride, SOC12, or R'N- C=
NR.
Animals can be immunized against the immunogenic conjugates
or derivatives by combining 1 mg or 1 g of conjugate (for rabbits or
mice, respectively) with 3 volumes of Freund's complete adjuvant
and injecting the solution intradermally in multiple sites. One
month later the animals are boosted with 1/5 to 1/10 the original
amount of conjugate in Fruend's complete adjuvant by subcutaneous
injection at multipie sites. Seven to 14 days later animals are bled
and the serum is assayed for anti-noggin titer. Animals are boosted
until the titer piateaus. Preferably, the animal is boosted with the
conjugate of the same noggin polypeptide, but conjugated to a
different protein and/or through a different cross-linking agent.
Conjugates also can be made in recombinant cell culture as protein
24
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~ R
fusions. Also, aggregating agents such as alum are used to enhance
the immune response.
Monoclonal antibodies are prepared by recovering spleen celis
from immunized animals and immortalizing the cells in conventional
fashion, e.g. by fusion with myeloma cells or by EB virus
transformation and screening for clones expressing the desired
antibody..
In a preferred embodiment, a rat mohoclonal antibody such as
RP57-16, prepared after immunization of a rat with recombinant
human noggin, reacts specifically with both Xenopus and human
noggin, but not with the neurotrophins BDNF, NT-3 and NT-4.
Noggin antibodies are useful in diagnostic assays for noggin or
its antibodies. In one embodiment of a receptor binding assay, an
antibody composition which binds to all of a selected plurality of
members of the noggin family is immobilized on an insoluble
matrix, the test sample is contacted with the immobilized antibody
composition in order to adsorb all noggin family members, and then
the immobilized family members are contacted with a plurality of
antibodies specific for each member, each of the antibodies being
individually identifiable as specific for a predetermined family
member, as by unique labels such as discrete fluorophores or the
like. By determining the presence and/or amount of each unique
label, the relative proportion and amount of each family member can
be determined.
Noggin antibodies also are useful for the affinity purification
of noggin from recombinant cell culture or natural sources. Noggin
antibodies that do not detectably cross-react with other growth
WO 94/05791 PCT/US93/08326
2143442
factors can be used to purify noggin free from these other family
members.
Aspects of the invention will now be illustrated by the
following examples.
EXPERIMENTAL PROCEDURES
Production of XenoRus embryos
Xenopus embryos were prepared by the protocol described by
Condie and Harland (Development, 101, 93-105, 1987). Embryos were
staged according to the table of Nieuwkoop and Faber ("Normal Table
of Xenopus laevis"(Daubin), Amsterdam: North Holland, 1967).
Ventralized embryos were produced by irradiation with a Statalinker
(Stratagene), and dorsalized embryos were produced by treatment
with LiCl as described by us in our paper on certain "wnt" proteins
(designated "Xwnt-8"), Smith and Harland, Cell, Vol. 67, pp. 753-765
(1991) (incorporated by reference and occasionally referred to
hereinafter as "S&H, supra").
EXAMPLE 1
Isolation and Sequencing of Noggin cDNA
The construction of the size-selected plasmid cDNA library
from stage 11 LiCI-treated embryos was as follows. Sixty
micrograms of poly(A) RNA from stage 11 LiCI-treated embryos was
size fractionated on a 10% to 30% sucrose gradient in the presence
of methylmercuric hydroxide. First strand cDNA was synthesized
from 2 g of the size-fractionated poly(A) RNAs primed with
26
CA 02143442 2004-10-06
oligo(dT) - oligonucleotide containing the recognition site for Noti.
After synthesis of the second strand, cDNAs were treated with
EcoRl methylase, ligated with EcoRl linkers, digested with EcoRl and
Notl, and finally ligated to 125 ng of modified pGEM-5Zf( )TM
(Promega). The pGEM-5Zf(-)TM used here was modified by the addition
of an oligonucleotide into the Nsil site to create an EcoRi site. The
vector was not treated with alkaline phosphatase, but the excised
polylinker sequence was removed on a sepharose 4BCL columnT"'. The
ligated products were used to transform XL-1 BIueTM cells
(Stratagene), and plated to give 100,000 colonies per
10 cm plate. Plasmid DNAs were isolated from plate cultures, by the
alkali ne-lysis/polyethylene glycol precipitation protocol.
Dorsalizing activity in the library was assayed by injecting
RNA transcripts made from pooled plasmid DNA. Single clones were
isolated by a process of sib selection. In this procedure the plasmid
{ibrary was replated on 12 plates with 10-fold fewer colonies per
plate. RNA was synthesized from pooled plasmid DNAs isolated from
each plate and tested for dorsalizing activity by injection into UV-
ventralized embryos. Those pools with dorsalizing activity were
replated and screened as described above. This process was
repeated until single clones were isolated.
In vitro RNA synthesis, injection assay for dorsal axis rescue
and sib-selections were also done, as described by us in S&H, supra.
The nucleotide sequence of both strands of the isolated noggin
cDNA clone was determined by the dideoxy termination method
using modified T7 DNA polymerase (US Biochem). Deletions were
prepared in sequencing templates by both restriction enzyme and
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~
exonuclease III digestion (Henikoff, Meth. Enzymol, 155, 156-165,
1987).
In vitro translation
One-half g of in vitro synthesized noggin, Xwnt-8, and
goosecoid mRNAs were translated in a nuclease treated rabbit
reticulocyte lysate (Promega) with added 35S-methionine according
to the manufacturer's instructions. The translation products were
visualized by SDS-polyacrylamide gel electrophoresis (12% gels)
followed by fluorography. Noggin protein had the molecular weight
predicted by the open reading frame.
RNA Isolation and Analysis
Total RNA was isolated from embryos and oocytes by a small
scale protocol as described by Condie and Harland, supra. Dorsal lips
were dissected from 30 unfixed stage 10.5 embryos and pooled for
total RNA preparation. Samples containing either the total RNA
equivalent of 2.5 embryos or approximately 2 g of poly A+ RNA
were analyzed by northern blotting. Random primed DNA probes were
prepared from a 1,323 bp fragment of noggin cDNA from the EcoRl
site at nucleotide -83 to an EcoRV site that lies in the vector
immediately 3' to the end of the cDNA.
RNAse protection assays were done using a protocol as
detailed by Melton et al. (Nuc. Acids Res., 12, 7035-7056, 1984)
with minor modifications (C. Kintner, Salk Institute, La Jolla,
California). A noggin cDNA exonuclease III deletion clone,
illustrated by SEQ ID NO: 8 but having a deletion from the 3' end to
nucleotide 383, was used as a template for synthesizing RNA
28
2143442 WO 94/05791 2143PCT/US93/08326
~
probes. The template DNA was linearized by EcoRl restriction
enzyme digestion and a 463 base antisense RNA incorporating 32P
was synthesized with T7 RNA polymerase. A 387 base antisense
EFla RNA probe was used as a control for amount of RNA per sample.
Probes were gel purified prior to use.
In situ hybridization
After fixation and storage, the embryos were checked to
ensure the blastocoel and archenteron were punctured. Care was
taken to puncture the residual blastocoel of neurulae and tadpoles
as well as the archenteron. Embryos were rewashed at room
temperature in 100% ethanol for two hours to remove residual lipid.
After hybridization, staining was allowed to develop overnight and
the embryos were then fixed in Bouin's. Newly stained embryos have
a high background of pink stain but most of this washes out, leaving
the specific stain. Following overnight fixation, the embryos were
washed well with 70% ethanol, 70% ethanol buffered with PBS and
methanol. Embryos were cleared in Murray's mix and photographed
with Kodak Ektar 25 film, using a Zeiss axioplan microscope (2.5 or
5x objective with 3x12B telescope to assist with focusing).
Lineaqe Tracing
Lineage tracing with mRNA that encodes nuclear localized B-
galactosidase was as we described in S&H, supra. Ventralized
embryos were coinjected at the 32 cell stage with 0.5 ng B-
galactosidase and 25 pg noggin05' mRNAs. Embryos were fixed and
stained with X-gal at approximately stage 22.
29
WO 94/05791 PCr/US93/08326
2143442
Results
Noggin cDNA Encodes a Novel Polype t{ide_
The 1833 nucleotide sequence of the selected clone is shown
by SEQ ID NO: 8 and sometimes also referred to as "clone A3." The
sequence contains a single long open reading frame encoding a 222
amino acid polypeptide with a predicted molecular weight of 26
kDa. At the amino terminus, the hydrophobic stretch of amino acids
suggests that the polypeptide enters the secretory pathway. There
is a single potential site for N-linked glycosylation at amino acid
61. Extensive untranslated regions are located both 5' and 3' to the
reading frame (593 and 573 bp, respectively). The 3' untranslated
region is particularly rich in repeated dA and dT nucleotides, and
contains, in addition to a polyadenylation signal sequence located 24
bp upstream from the start of the poly A tail, a second potential
polyadenylation sequence 147 bp further upstream.
Sense RNA synthesized from clone A3 with SP6 RNA
polymerase was translated in a rabbit reticulocyte lysate system.
The 3S-labeled products were fractionated on a 12% SDS-
polyacrylamide gel and visualized by fluorography. The major
protein product had the
expected molecular weight of approximately 26 kDa.
Comparison of the amino acid sequence of the predicted
polypeptide to the National Center for Biotechnology Information
BLAST network (non-redundant data base) did not identify any
similar sequence. Thus, this clone encodes the new type of protein
we have named "noggin" which is secreted, and which has dorsal
inducing activity in Xenopus.
WO 94/05791 Z143442 PCr/US93/08326
~
Noggin mRNA can Rescue a Complete Dorsal-Ventral Axis
Injection of noggin RNA into a single blastomere of a four cell
stage UV-ventralized embryo can restore the complete spectrum of
dorsal structures. The degree of axis rescue was dependent upon the
amount of RNA injected, with embryos receiving low doses having
only posterior dorsal structures, while embryos receiving higher
doses had excess dorsal-anterior tissue. RNA transcripts from two
noggin plasmids were tested. The first contained the full cDNA. The
second (pNogginA5') had a deletion removing the first 513
nucleotides of the 5' untranslated region up to the EcoRl site. The
resulting embryos from injection of RNA transcripts of these two
plasmids, as well as Xwnt-8 RNA for comparison, were scored
according to the dorsoanterior index (DAI) scale of Rao and Elinson
(Dev. Biol., 127, 64-77, 1988). In this scale, a completely
ventralized embryo is scored as zero, a normal embryo is scored as
5, and the most severely dorsoanteriorized embryos, those having
radial dorsoanterior structures, were scored as 10. RNA
synthesized from pNogginA5' (nogginA5' mRNA) repeatedly gave a
higher DAI than the equivalent amount of mRNA synthesized from the
complete cDNA. The dose-dependency of axis rescue by nogginA5'
mRNA was very similar to that of Xwnt-8 mRNA.
UV treated embryos were also injected with a higher doses
(1,000 pg) of the noggin mRNAs. Injection of this dose of noggin
mRNA into one blastomere at the four cell stage resulted in
embryos with very severe hyperdorsalization (DAI >7). However,
most of these embryos died at the late gastrula/early neurula stage.
Apparently excessively strong gastrulation movements resulted in
31
WO 94/05791 PCT/US93/08326
a
the thinning and rupture of the blastocoel roof. We have also
observed this effect with high doses of injected Xwnt-8 mRNA.
The rescue of dorsal development by both nogginA5' and Xwnt-
8 mRNAs followed a consistent pattern in which increasing amounts
of the mRNAs lead to progressively more anterior structures being
rescued. For example, embryos that received 1 pg of the RNAs had
primarily the posterior and trunk dorsal structures rescued, and for
the most part lacked head structures. Higher doses (10 or 100 pg) of
both of the RNAs resulted in embryos with more anterior
development, and many had either nearly normal or hyperdorsalized
phenotypes.
Noggin Injected Blastomeres Act as a Nieuwkooo Center
The effect of varying the site of noggin mRNA injection was
investigated. Thirty-two cell stage UV-treated embryos were
injected with either 0.5 ng of B-galactosidase mRNA alone or 0.5 ng
B-galactosidase mixed with 25 pg nogginA5' mRNA. Injection of
noggin mRNA into blastomeres of the vegetal tier gave the most
strongly dorsoanteriorized embryos. In both of the vegetal injected
embryos the nuclear X-Gal staining was found almost exclusively in
the endoderm (the mRNA encodes a B-galactosidase that
translocates to the nucleus, allowing distinction from the diffuse
background stain). One of the embryos shown was strongly
hyperdorsalized (DAI approximately 7) as a result of the noggin
mRNA injection, and had a severely truncated tail and enlarged head
structures. Embryos were also rescued by noggin mRNA injections
into the marginal zone.
In these embryos B-galactosidase staining was observed primarily
32
WO 94/05791 %14344Z PCT/US93/08326
~
in the axial and head mesoderm. Injection of noggin mRNA into the
animal pole had very little effect on axis formation. Likewise, B-
galactosidase mRNA alone was without effect.
Noggin mRNA is Exoressed Both Maternally and Zy otically-
Iri northern blot analysis of RNA from Xenopus embryos two
noggin mRNA species of approximate sizes 1.8 and 1.4 kb were
observed. A relatively low level of noggin mRNA was detected in
oocytes. By stage 11 the level of noggin mRNA was significantly
higher, reflecting zygotic transcription (as opposed to the
maternally deposited transcripts seen in oocytes). Noggin mRNA
remained at the elevated level up to the latest stage examined
(stage 45).
We expect that the primary dorsalizing RNA in our library to
be elevated in LiCI-treated embryos relative to normal or UV-
treated embryos. Lithium ion treatment resulted in a large increase
in the amount of noggin mRNA expressed, relative to untreated
embryos.
UV treatment had the opposite effect. Noggin mRNA expression was
essentially undetectable in total RNA samples from these embryos.
Thus, the abundance of noggin mRNA in manipulated embryos
parallels the rescuing activity.
We analyzed the distribution of noggin in oocytes and cleavage
stage embryos. Since the amount of maternally deposited noggin
RNA is too low for in situ hybridization to detect above background,
we used an RNAse protection assay. Oocytes were dissected into
animal and vegetal halves. No enrichment of noggin mRNA was seen
in either hemisphere relative to total oocyte RNA. Four-cell stage
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WO 94/05791 2143442 PCT/U593/08326
embryos were dissected into dorsal and ventral halves, as well as
animal and vegetal halves. Noggin transcripts were found to be
distributed evenly between dorsal and ventral hemispheres as well
as animal and vegetal hemispheres. The same result was obtained
with embryos that were tilted 900 immediately following
fertilization and then marked with a vital dye on their uppermost
side to indicate the future dorsal side. Older (32 cell stage)
blastula embryos were also dissected into dorsal-ventral and
animal-vegetal halves. No enrichment of noggin mRNA in any of the
hemispheres was seen relative to the total embryo. In addition,
treatment did not alter the abundance of maternally deposited
noggin RNA, indicating no preferential degradation in ventral
tissues. Samples with known amounts of in vitro synthesized noggin
mRNA were included in the RNAase protection assay. From these and
other data 'we estimate that there is approximately 0.1 pg of noggin
mRNA per blastula stage embryo and 1 pg per gastrula stage embryo.
The localization of noggin transcripts was investigated in
early gastrula stage embryos. Dorsal lips were dissected from stage
10.5 embryos. A northern blot of equai amounts of total RNA from
intact embryos, dissected dorsal lips, and from the remaining
embryo after dissection of the dorsal lip was hybridized with a
noggin probe and then re-hybridized with an EFla probe, as a control
for amount of RNA loaded per sample. The autoradiograph of the blot
showed that noggin mRNA at this stage is enriched in the dorsal lip.
In situ Hybridization: Zvaotic Expression of No aig, n in
the Spemann Organizer
The localization of noggin transcripts in developing embryos
was examined in greater detail using whole mount in situ
34
2,14 344 2
WO 94/05791 PCT/US93/08326
~
hybridization. Whole fixed embryos were hybridized with digoxigenin
containing RNA probes.
Hybridized RNA probe was then visualized with an alkaline
phosphatase-conjugated anti-digoxigenin antibody. The specificity
of hybridization seen with antisense noggin probes was tested both
by hybridizing embryos with sense noggin probes, and by using two
non-overlapping antisense probes. Due both to the low level of
expression, and to background staining, noggin mRNA could not be
detected unequivocally before the late blastula stage. The increased
level of noggin mRNA that was detected by northern blot following
activation of zygo'tic transcription was apparent in in situ
hybridization at stage 9 as a patch of stairiing cells on the dorsal
side of the embryo. Viewed from the vegetal pole, this patch of
cells was restricted to a sector of about 600. A side view of the
same embryo shows that the staining cells were located within the
marginal zone (i.e., between the animal and vegetal poles and within
the presumptive dorsal mesoderm forming region). Transcripts are
largely restricted to the nucleus at this stage.
A side view of an early gastrula stage embryo 30
(approximate stage 10.5) shows specific hybridization primarily in
the involuting mesoderm at the dorsal lip. A vegetal view of the
same embryo (blastopore (ip arrowed) shows that noggin mRNA is
most abundant on the dorsal side, but expression extends at the
lower level to the ventral side of the embryo. This method of in situ
hybridization does not detect transcripts in the most yolky,
endodermal region of embryos, therefore we cannot rule out
expression in more vegetal regions than those seen in the Figure.
Treatments which are known to affect the size of the dorsal lip
WO 94/05791 214314G y PCT/US93/08326
(LiCI treatment, UV irradiation) had a profound effect on the pattern
of noggin in situ hybridization. In LiCI treated embryos the
staining is intense throughout the marginal zone. UV treatment
reduced the hybridization signal to low levels. This result is
consistent with amounts of noggin mRNA seen by northern blot
analysis. The UV treated embryo also is a negative control for
specificity of hybridization.
As gastrulation proceeds, noggin mRNA staining follows the
involuting dorsal mesoderm, and is highest in the presumptive
notochord. By the late neurula stage (approximately 18) noggin
mRNA expressing, cells are clearest in the most dorsal mesoderm,
primarily in the notochord but also extending more anteriorly into
the pre-chordal mesoderm. The anterior tip of the notochord is
arrowed. During tailbud stages expression of noggin in the dorsal
mesoderm declines, through expression in the notochord persists in
the growing tailbud. Expression of noggin initiates at several new
sites, which become progressively clearer as the tadpole matures. A
discontinuous line of stained cells runs the length of the roof plate
of the neural tube. Staining is also apparent in the head mesoderm,
primarily in the mandibular and gill arches. We suspect that this
expression corresponds to skeletogenic neural crest cells.
Furthermore, subsets of these cells express homeobox genes that
mark different anterior-posterior levels of the head neural crest,
for example En-2 in the mandibular arch is seen by antibody
staining. Cells with steilate morphology stained from noggin mRNA
in the tail fin. These stellate cells are also likely to be derived
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WO 94/05791 Z143442, PCT/US93/08326
~
from the neural crest. None of these patterns were seen with the
sense probe, or with a number of other probes.
EXAMPLE 2
Noaain cDNA Transfected into COS Cells Produces Active
Qonditioned Medium
For COS cells the noggin cDNA was inserted into a COS cell
expression vector. COS cells were transfected, and medium
harvested after allowing expression of the introduced noggin genes.
This medium has been tested in an animal cap assay for mesoderm
inducing or dorsalizing activity. We have tested two transfection
protocols, a standard one, where cells recover and then are
transferred to serum-free medium, and an alternate where cells are
transferred to a defined medium lacking serum but containing
transferrin, insulin, and BSA. Cells remain healthy in the
supplemented medium and a cotransfected P-galactosidase gene
gives 100 fold more activity than in the unsupplemented medium.
The results of treating cells with these media is shown below in
Table 1. Animal caps were taken from ventralized animals, treated
and at the end of neurulation they were scored for elongation,
usually a sign that notochord or neural tissues have been induced.
Elongation is indicated in Table 1 by a "+" and even greater
elongation a "++." In addition, they are scored for a molecular
marker by Northern blotting.
As shown by the data of Table 1, the noggin cDNA has a large
effect on the COS cell conditioned medium. However, noggin is
probably interacting with something else in the medium, since COS-
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WO 94/05791 214 3.44+ 1 PCr/US93/08326
cell conditioned medium alone has some activity. It is possible that
noggin is causing the cells to secrete something that they normally
would not, but the experiments do indicate that noggin is secreted
and is responsible for some of the activity.
38
WO 94/05791 21A 3442 PCr/US93/08326
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TABLE 1
Cos Cell Conditioned Medium: Effects on Animal Caps
Elongation N-CAM
expression
Transferred to serum
free medium +
transferrin, BSA,
and insulin
1. Vector only +/- +
2. Noggin cDNA ++ ++
Transferred to serum
free medium without
supplements
1. Vector only - -
2. Noggin cDNA - -
Noqgin mRNA Injected into Oocytes Produces Active Secreted Noqgin
protein
A second approach to studying whether protein can be secreted
in active form is to inject oocytes with mRNA and take material
secreted by the oocyte. A particular advantage of this method is
that the injected mRNA is efficiently translated, and most of the
translation of the oocyte can be taken up by the injected mRNA. A
new protein, whose synthesis is directed by injected noggin mRNA
is secreted into the medium. Noggin clearly synergizes with activin
to produce elongated expl~tnts that express elevated levels of
muscle actin.
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WO 94/05791 PCT/U593/08326
~14 3 44 2
Biochemical Prooerties of Noaoin
Injected oocytes are injected with mRNA, and labelled with
35S methionine. Most of the radioactive protein secreted into the
medium is from the injected mRNA. The noggin protein, which is
almost isotopically pure, can then be analyzed. From this analysis
we have determined that noggin is a dimeric glycoprotein. When run
under reducing conditions, and treated with N-glycanase to remove
sugar residues, noggin migrates only slightly slower than its
predicted molecular weight of 26 kDa. The removal of sugar side
chains results in a loss of about 4 kDa from a starting apparent
molecular weight of 33 kDa. When run under non-reducing conditions
it migrates at double this value.
We do not yet know if the dimer of the protein is the active
species, or if there is a proteolytically processed form which is
active. In a control experiment with activin mRNA, oocytes produce
activin activity, but the bulk of the radiolabelled protein migrates
as the precursor form. Only a small amount of processed protein (15
kDa) was detected. It is possible that noggin injected oocytes
secrete predominantly unprocessed protein and a trace of extremely
active processed protein that we have not detected. Despite the
caveats, the main point from analysis of injected oocytes and
transfected COS cells is that active noggin can be obtained as a
freely soluble secreted polypeptide. This sets it apart from the
other group of genes with dorsalizing activity, the wnts. Wnt
proteins have not been available in soluble form and this has greatly
hampered the analysis of their biological activities, and of the
receptor that binds to them.
WO 94/05791 21.43442 PCf/US93/08326
0
EXAMPLE 3
Cloning of the Mouse Noggin Homoloa
It is currently impossible to eliminate zygotic noggin
transcription from developing Xenopus embryos. In contrast, it
should be possible to generate homozygous null mutations in the
mouse. We have cloned the mouse noggin cDNA (SEQ ID NO: 10). This
is useful to generate mutant mice. In addition to generating the
probes and tools to make mutant mice, a comparison of the noggin
1 0 sequences should be a useful predictor of conserved domains and
functions. The C-terminal 80 amino acids are 87% identical between
SEQ ID NOS: 8 and 10.
Mouse noggin was isolated from an embryonic
cDNA library by probing with a radiolabelled frog noggin
cDNA under conditions of moderate stringency (as defined
earlier). Subsequently a genomic clone was isolated by
probing a genomic library with the mouse noggin cDNA
15 under conditions of high stringency (as defined, but hybridized at
42 C and washed at 50 C in 15 mM NaCI, 1.5 mM sodium citrate).
The full nucleotide sequence of mouse noggin cDNA (SEQ ID NO: 25)
as well as the deduced amino acid sequence (SEQ ID NO: 26) are
shown in Figure 13. There are only two amino acid differences
between mouse noggin and human noggin.
41
CA 02143442 2004-10-06
EXAMPLE 4
Cloning of the Human Nogqin Homoloa
Materials and Methods
Probe reparation
Two oligonucleotides were synthesized based on the mouse
noggin sequence (supra). The sequence of the oligonucleotides is
n~ggjr~ 5': 5'-CAG ATG TGG CTG TGG TCA-3' (SEQ ID NO: 18)
corresponding to amino acids QMWLWS (SEQ ID NO: 19) and noggin 3'
5'-GCAGGAACACTTACACTC-3' (SEQ ID NO: 20) corresponding to amino
acids ECKCSC (SEQ ID NO: 21) of the mouse noggin protein .
The oligonucleotides were used for PCR amplification of a
segment of DNA of 260 nucleotides using as a template a mouse
cDNA clone prepared as set forth in Example 3. The amplified
fragment had a nucleotide sequence that corresponds to nucleotides
2 through 262 of the mouse sequence as set forth in SEQ ID NO: 10.
After amplification, the PCR reaction was electrophosed in agarose
gels, the DNA band of 260 nts purified by Magic PCRTM (Promega), and
used as template for the probe labeling reaction. The probe was
labeled using a standard PCR reaction (Perkin-Elmer) on 20 ng of
DNA template and 0.2 m Curie of alpha 32P-dCTP (Du Pont 3000
Ci/mmol) instead of dCTP. Unincorporated label was separated from
the probes on a G50 N1CK columnT"" (Pharmacia). The excluded volume
of the reaction contained a total of 1.8 x108 cpm.
In addition, one degenerated oligonucleotide, named nog ig n D,
corresponding to conserved mouse and Xenopus noggin sequences,
was synthesized as follows: Nogpin D: 5'- GARGGIATGGTITGYAARCC-
42
CA 02143442 2004-10-06
3 ' (SEQ lD NO: 22). Noggin D(SEQ ID NO: 22) was labeled by kinase
reaction using T4 polynucleotide kinase and gamma 32P ATP. The
labeled oligonucleotide was pur,i,fied by NAP5TM (Pharmacia) column
and used for library hybridization.
Library screening
A human placental genomic library (Clontech Cat#HL1067J,
average insert size 15 kb) in vector EMBL-3 was plated according to
manufacturer specifications in NM 538 E.coli. Approximately 3
million plaques were transferred to nitrocellulose filters (BA-85
Schleicher and Schuell) in three replicas (named A, B and C) and
screened according to Maniatis, et al.[Sambrook, et a., Molecular
cloning a laboratory manual, CSH Lab Press, New York (1989)]. The
replica filters A and C were hybridized in a buffer containing 0.5 M
sodium phosphate, pH 7.2, 7% sodium dodecyl sulphate, 1%
crystalline BSA, 1 mM EDTA, 40 m g/ml denaturated salmon sperm
DNA and about 1 x106 cpm/ml of the PCR probe (supra). After
hybridization for 12 h at 65 C, the filters were washed twice at
room temperature in 2x SSC (30 mM sodium citrate, 0.3 M NaCI),
0.1% SDS and then at 65 C in 2xSSC, 0.1% SDS for 20 min and
exposed to Kodak X-OMAT AR filmTM . The filter replica B were
hybridized with the labeled oligonucleotide Oogain D in 6xSSC , 0.1%
SDS at 51 C for 12 h followed by wash at 2xSCC, 0.1% SDS at room
temperature, and in 6xSSC, 0.1% SDS at 50 C and exposed to Kodak X-
OMAT AR film. Positive plaques from all replicas were isolated and
purified by re-screening as above. Purified positive plaques were
suspended in 500 l SM (100 mM NaCI, 10mM MgSO4 x 7H20, 50 mM
Tris HCI pH 7.5, 0.01% gelatin). 160 l of phage suspension was
43
r t r .
WO 94/05791 ,r~ ~ ~ ~ ~ ~ PCT/US93/08326
G 0
mixed with 0.5 mi saturated NM538 culture, incubated for 20 min at
37 C and then inoculated into 250 ml LB containing 10 mM Mg S04,
0.2% maltose. The cultures were incubated untii cell lysis (7-8 hr)
at 37 C. The phage lysates were used for phage DNA purification by
the Qiagen procedure according to the manufacturers
recommendations (Qiagen).
Seguencina
Sequencing was performed by using the Applied Biosystems
Model 373A automatic sequencer and Applied Biosystems Taq
DyeDeoxyTM Terminator Cycle Seque.ncing Kit.
Fiesults
Filters hybridized to the PCR mouse noggin probes (SEQ ID NOS:
18 and 20). showed two strong signals corresponding 'to phage
plaques named hnog?,-9 and hnogk-10. These plaques also hybridized
to degenerate oligonucleotide probe noaainD (SEQ ID NO: 22) revealed
that these clones correspond to the human noggin gene. In addition,
two other plaques named hnoga,-5 and hnog7,-7 produced slightly
weaker signals when hybridized to the PCR probes. These clones
correspond to either human noggin or related gene(s). All of the
human DNA inserts can be excised from the vectors using known
restriction sites as described in the literature regarding each
particular library.
A 1.6 kb Sacl fragment from clone hnogk-9 containing the
human noggin gene was subcloned and the nucieotide sequence
determined as set forth in Figure 1. The amino acid sequence for
human noggin, as deduced from the nucleotide sequence, is also set
44
WO 94/05791 '~~ ~ ~" ~ ~ ~ PC'T/US93/08326
=
forth in Figure 1. The gene or cDNA may be expressed in various
eukaryotic or prokaryotic expression systems to produce
biologically active human noggin protein. It is expected that the
human protein will exhibit neurotrophic activity similar to that
exhibited by Xenopus noggin protein.
EXAMPLE 5
Tissue Localization of inessacle for human noggin
Materials and Methods
Probe preparation
Probes were prepared as set forth in Example 4. The oligos
used are as follows:
SEQIDNO:23:
5' GAC.TCG.AGT.CGA.CAT.CGC.AGA.TGT.GGC.TGT.GGT.CAC
SEQ ID NO: 24:
5' CCA.AGC.TTC.TAG.AAT.TCG.CAG.GAA.CAC.TTA.CAC.TCG.G
(The underlined sequence represent mouse noggin sequence; the rest
of the sequence are tails containing restriction sites for cloning.)
A DNA fragment of approximately 300 bp was obtained by PCR
amplification of a mouse cDNA clone prepared as described in
Example 3.
CA 02143442 2004-10-06
RNA Pregaration and Northern Blots
Selected tissues were dissected from Sprague-Dawley rats
and immediately frozen in liquid nitrogen. RNAs were isolated by
homogenization of tissues in 3 M LiCI, 6 M urea, as described in
Bothwell, et al. 1990 (Methods of Cloning and Analysis of Eukaryotic
Genes, Boston, MS, Jones and Bartlett). RNAs (10 g) were
fractionated by electrophoresis through quadruplicate 1% agarose-
formaldehyde gels (Bothwell, et al., 1990, Methods of Cloning and
Analysis of Eukaryotic Genes, Boston, MS, Jones and Bartlett)
followed by capillary transfer to nylon membranes (MagnaGraph,
Micron Separations Inc.) with 10xSSC (pH7). RNAs were UV-cross-
linked to the membranes by exposure to ultraviolet light
( Stratal{nkerTM , Stratagen, Inc.) and hybridized at 68 C with
radiolabled probes in the presence of 0.5 M NaPO4 (pH 7), 1% bovine
serum albumin (fraction V, Sigma, Inc.) 7% SDS, 1 mM EDTA [Mahoudi,
et al., Biotechniques Z:331-333 (1989)], 100 g/mi sonicated,
denatured salmon sperm DNA. Filters were washed at 68 C with
3xSSC, 0.1% SDS and subjected to autoradiography for 1 day to 2
weeks with one or two intensifying screens (CronexTM; DuPont) and X-
ray film (AR-5, Kodak) at 70 C. Ethidium bromide staining of the
gels demonstrated that equivalent levels of totai RNA were being
assayed for the different samples [as in Maisonpierre, et al., Science
2AZ:1446-1451 (1990)].
46
WO 94/05791 U43,442 PCT/US93/08326
9 RNA was prepared from the following human cell lines:
Neuroblastoma Neuroepithelioma
CHP-134 SK-N-MC
LA-N-1 CHP-100
LA-N-5 IARC-EWI
I M R-32 SK-N-LO
SHSY5Y SK-ES
SKNSH DADY
SHEP
Hemato oetic Small Cell Luna Carcinoma Cervical Carcinoma
K562 Calu 3 HeLa
U937 SKLu
M1 NCI-H69
TF1 SKMES
BAF
B9
47
WO 94/05791 PCT/US93/08326
~
Sympathoadrenal Precursor Hepatoblastoma Medulloblastoma
MAH HEPG2 Madsen =
Med
U266
Pheochromocytoma
PC12
RESULTS
We have amplified a DNA fragment from the mouse noggin
plasmid, corresponding to the region conserved between Xenopus and
mouse noggin.
The amplified fragment of approximately 300bp was used as
probe to hybridize to northerns, with RNAs prepared from adult and
embryonic tissues, as well as from various cell lines. Noggin
transcript of about 2kb in size was detected in adult rat brain, and
in a cell line, SKMES, a small cell lung carcinoma.
Expression of noggin transcripts was examined in various
tissues from rat and mouse at different stages of development and
in adult. In the mouse, noggin transcripts can be detected in
embryos or head from E9 to E12, as well as in newborn brain and
adult brain. There was no detectable signal in peripheral tissues
examined except in skeletal muscle. Abundant level of expression
was also found in hippocampal astrocytes isolated from postnatal
mouse.
48
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WO 94/05791 PG'T/US93/08326
~
In the rat, noggin transcripts were detectable in embryos or
head from E9 to E18, as well as in brain from P1, P19 and adult
brain. In the cerebellum, expression of noggin appeared to be higher
in E18 and P1; in the spinal cord, expression of noggin mRNA peaked
at P1. Examination of noggin expression in all of the CNS regions,
especially the olfactory bulb, midbrain, hindbrain and cerebellum. In
the adult, noggin mRNA could be detected in all CNS regions,
especially the olfactory bulb and cerebellum. There also appeared to
be low levels in the skin.
EXAMPLE 6
Neural Induction by Noggjn
Materials and Methods
Preparation of Xeno us noggin CHO cell conditioned medium
Xenopus noggin CHO conditioned.medium was made by selecting
for stably transfected CHO cells. Dihydrofolate reductase (DHFR)
deficient CHO parental cells ( J. Papkoff, Syntex Research) were
transfected with a Xenopus noggin expression plasmid containing
noggin in tandem with the dihydrofolate reductase gene. Growth in
nucleoside free medium was used to select for successfully
transfected cells. Nine colonies of transfectants were picked and
grown up individually. The noggin gene in these cells was amplified
by slowly increasing the dose of methotrexate, an inhibitor of DHFR.
The presence of noggin transcripts was first tested by Northern
49
CA 02143442 2004-10-06
..analysis. Subsequently, two clones, B3 and C3, were shown to
secrete noggin protein, since conditioned. medium from these lines
was capable of dorsalizing ventral marginal zones. Furthermore, by
labeling B3 cellular proteins with 35S-methionine, noggin protein
could be identified as a band of about 30kD on reducing SDS-PAGE,
and a band of 60kD on non-reducing SDS-PAGE indicating it forms
the expected dimer. These properties matched those of the noggin
protein previously produced in Xenopus oocytes supra, (Smith et al.,
Nature 361, 547-49, 1993). B3 conditioned medium was collected in
a mixture of 1 part alpha MEM and 9 parts CHO-S-SFMII (Gibco-BRL).
The cells were allowed to condition the medium for 3 days. Control
medium from parental cells (CHO dhfr-) was collected identically.
Twenty fold concentrated medium was made using Centriprep 10T""
concentrators, where the 20 fold change is measured by volume.
Purification of human nog,qin from COS cells
Human noggin protein was purified by a cationic exchange
column. COS/M5 cells were transiently transfected with a human
noggin expression plasmid, pCAE11. Cells were allowed to condition
DMEM (Specialty Media) for two to three days, after which the
medium was removed. Particulates from the medium were removed
by a centrifugation step and subsequent passage through a 0.2um
cellulose acetate filter. This cleared medium was pumped onto a
MonoST"~ (Pharmacia) column which was washed with several volumes
40mM sodium phosphate (pH 7.3), 150mM NaCI, 1 mM EDTA. Proteins
were then eluted in a linear gradient with 40mM sodium phosphate
WO 94/05791 2143144Z PCT/US93/08326
~
(pH 8.5), 1.8M NaCi, 1 mM EDTA. Noggin protein elutes at 0.8M NaCI
and is 290% pure by SDS-PAGE.
Xenopus otx isolation
To isolate Xenopus Otx clones a tadpole head cDNA library
(Hemmati-Brivaniou, et al., Development 106, 611-617, 1989)
was screened with a mouse otx cDNA (S-L Ang and Rossant, Toronto)
at low stringency. The clones that were picked fell into two
classes. One class, which we have designated otxA, included
pXOT21.2, the probe used here. By in situ hybridization, transcripts
are first detected prior to gastrulation in the superficial layer on
the dorsal side. During neurulation a large anterior domain
expressed the gene, and includes both neural and non-neural tissues.
After a decline in expression in the tailbud tadpole, the gene is
reexpressed specifically in the brain and eyes.
Ventral marginal zone assay
Embryo preQaration
Xenopus laevis embryos are fertilized and de-jellied as
described (Condie and Harland, 1987. Development 101, 93-105),
routinely the evening before dissections. Embryos are cultured
overnight at 15 C. The vitelline membrane surrounding each
developing embryo is manually removed the following morning at the
late blastula stage. Until dissection, the embryos are maintained in
1/3x modified ringers in agarose coated dishes.
51
WO 94/05791 2~ ~ 3442 PCT/US93/08326
~
Ventral marginal zone dissection
Embryos are oriented with their yolky vegetal hemisphere up
so the dorsal side can be identified. The dorsal side of the early
gastrula is marked by the presence of a small arc of dense pigment
called the "dorsal lip" which marks the start of involution of dorsal
mesoderm. The ventral marginal zone (VMZ) is found directly
opposite the dorsal lip, and is dissected. Since the vitelline
membrane has been removed, the embryo tends to flatten. Using a
specially constructed knife made of an eyebrow, mounted onto a
glass pipet with wax, two cuts are made through the flattened
embryo from the top facing vegetal pole through to the animal pole.
The cuts are made such that they isolate approximately 30-60
degrees of the ventral side away from more lateral tissues. A third
cut which is perpendicular to the first two cuts completely isolates
the ventral marginal zone tissue away from the rest of the embryo.
This third cut is at the level of approximately two thirds of the
radius of the embryo from the center. Prior to treatment the VMZ is
washed lx in the culture medium.
Assay
Approximately between 5 to 10 VMZs are used per assay. The
washed VMZs are dropped gently (trying to minimize transfer of
liquid) into eppendorf tubes containing the desired treatment protein
medium for assay. The VMZs are allowed to develop to the late
neurula or early tailbud stage as assessed by control whole embryo
development. At this time RNA is isolated from the VMZs and control
whole embryos as described (Condie and Harland, ibid). The
expression of muscle actin in VMZs indicates a dorsalization event
52
WO 94/05791 U43442 PCT/US93/08326
~
(Lettice and Slack, 1933. Development, 117, 263-72). RNA from
each sample is run on a formaldehyde-agarose gel and blotted to
gene screen. The blot is then hybridized with a Xenopus muscle actin
probe (Dworkin-Rastl et al., 1986. J. Embryol. exp. Morph. 91, 153-
68) . Quantitation of dorsalization can be carried out by normalizing
muscle actin signal to that of the ubiquitously expressed EF-la
(Krieg et al., 1989. Devl. Biol. 133, 93-100) . Quantitation is done
using phosphor imaging.
RNase protection assay
RNase protection was carried out as described (D.A. Melton et
al., Nucleic Acids Res 12,7035-56, 1984), with the modification
that digestion was carried out at room temperature (22 C) using
RNase T1 only (Calbiochem 556785) at 10 units/mI. 20-30 animal
caps were harvested for each lane, of this 80% was used for neural
markers and 10% for muscle actin and coliagen type II. For goosecoid
and brachyury 20 caps were used. Exposures ranged from 12 hours to
5 days. In all cases, films were preflashed. In cases where a marker
was not expressed, the. result was confirmed with greater
sensitivity using phosphor imaging.
Results
The development of vertebrate embryos requires several
inductive interactions. Mesoderm, which eventually forms tissues
such as notochord, muscle, heart, mesenchyme and blood, is induced
in the equatorial region of the embryo (Nieuwkoop, Wilhelm Roux'
Arch. EntwMech. Org, 162, 341-373, 1969) . This inductive event is
well studied, and there are several candidates for the endogenous
53
ZM442
WO 94/05791 PC'T/US93/08326
inducer(s) including members of the fibroblast growth factor(FGF)
family and activin (Jessell and Melton, Cell 68, 257-70 1992; Sive,
Genes Dev 7, 1-12, 1993) and TGFb family (Asashima, et al.,
Roux's Arch. Dev. Biol. 198, 330-335, 1990; Asashima, et al.,
Naturwissenschaften 77, 8, 389-91, 1990; Green and Smith, Nature
347, 391-394, 1990; Smith, et al., Nature 345, 6277, 729-31, 1990;
Thomsen, et al., Cell 63, 485-493, 1990; van, et al., Nature 345,
6277, 732-4, 1990) . The use of dominant negative receptors for
both FGF (Amaya, et al., Cell 66, 257-270, 1991) and activin
(Hemmati-Brivaniou and Melton, Nature 359, 609-614, 1992) in
Xenopus embryos strongly suggests that the signaling pathways
activated by these molecules are essential for proper mesoderm
formation . Molecules such as wnts (Christian, et al., Development
111, 1045-1055, 1991; McMahon and Moon, Cell 58, 1075-84, 1989;
Smith and Harland, Cell 67, 753-765, 1991; Sokol, et al., Cell 67,
741-752, 1991) and noggin (Smith, et al., Nature 361, 547-49,
1993) modify the kinds of mesoderm made without inducing
mesoderm directly.
In a subsequent induction, the dorsal mesoderm of the Spemann
organizer signals nearby lateral mesoderm to take on a more dorsal
fate (Dale and Slack, Development 100, 2, 279-95, 1987; Lettice
and Slack, Development, 117, 263-271, 1993; Spemann and Mangold,
Arch. Mikrosk. Anat. EntwMech. 100, 599-638, 1924; Stewart and
Gerhart, Development 109, 363-372, 1990) . The only known
factor which is expressed in the organizer and can mimic its
dorsalizing activity is noggin.
Dorsal mesoderm of the Spemann organizer also signals nearby
ectoderm to become neural tissue. Neural induction by dorsal
54
*p 442 PC.T/US93/08326
WO 94/05791 Z14~3
mesoderm has been demonstrated in amphibians (Dixon and Kintner,
Development 106, 749-757, 1989; Doniach, et al., Science 257,
5069, 542-5, 1992; Hamburger, The Heritage of Experimental
Embryology: Hans Spemann and the Organizer, 1988; Kintner and
Melton, Development 99, 311-25, 1987; Spemann, Arch. mikrosk.
Anat. EntwMech. 100, 599-638, 1938) , birds (Kintner and Dodd,
Development 113, 1495-1506, 1991; Tsung, et al., Acta Biol exp
Sinica 10, 69-80, 1965) , and recently in mice (Ang and Rossant,
Development 118, 139-149, 1993) . Despite decades of effort,
little is known about the molecular nature of the factors responsible
for this induction. Among known inducers, activin can promote
formation of neural tissue, but this is due to a secondary induction
by the dorsal mesoderm that activin induces (Green, et al.,
Development 108, 1, 173-83, 1990; Green and Smith, Nature 347,
391-394, 1990; Kintner and Dodd, Development 113, 1495-1506,
1991) . Thus, activin cannot promote formation of neural tissue
when added to gastrula ectoderm; however, such ectoderm 'remains
competent to be neuralized by dorsal mesoderm until the end of
gastrulation (Sharpe and Gurdon, Development 109, 765-74,
1990)
Direct Neural fnduction by Noggin
Candidates for the endogenous inducer are expected to induce
neural tissue in the absence of dorsal mesoderm. Competent animal
cap ectoderm from late blastula stage embryos (St9) was used to
test noggin's neural inducing capacity. Xenopus noggin protein
conditioned medium was collected from stably transfected CHO cells
and twenty fold concentrated medium was used to treat St 9 animal
WO 94/05791 214344Z PCT/US93/08326
caps. Markers used in an RNase protection assay were N-
CAM (Jacobson and Rutishauser, Developmental Biology 116, 524-
31, 1986; Kintner and Melton, Development 99, 311-25, 1987) , a
neural cell adhesion molecule, a neural specific isoform of b-
tubulin (Good, et al. Nucleic Acids Res 17, 8000, 1989; Good, et al.,
Dev Biol 137, 414-8, 1990; Richter, et al., Proc Natl Acad Sci USA 85, 8086-
90, 1988) that is expressed in the hind brain and spinal
cord, and XIF3, a neurally expressed intermediate filament gene
(Sharpe, et al., Development 107, 701-14, 1989) to assay for
neural induction. AII these markers are restricted to neural tissue,
however, only NCAM is expressed throughout the nervous system. We
-found that Xenopus-noggin conditioned medium induces high levels of
N-CAM and XIF3 expression[Fig. 2.; lane8] in treated animal caps,
without inducing muscle actin(lane 13) (Dworkin-Rastl, et al., J.
Embryol. exp. Morph. 91, 153-168, 1986; Mohun, et al., Nature 311,
716-721, 1984) . Control CHO cell medium induces neither muscle
nor neural tissues (lanes 7,12). St 9 activin treated animal caps
express muscle actin(lanell) and all three neural markers(lane 6),
demonstrating activin's ability to generate neural tissue indirectly.
It is interesting to note that noggin induces very little, if any b-
tubulin expression, while inducing high levels of N-CAM, but activin
induction has nearly the converse effect.
To determine whether noggin protein is sufficient to induce
neural tissue, COS cells were transfected with pCAE11, a human
noggin expression plasmid, and the conditioned medium was purified
by cation exchange chromatography resulting in noggin preparations
that were 90% pure [Fig. 3.]. Such purified human noggin protein is
also able to induce neural tissue in animal caps [Fig. 4a., see below].
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WO 94/05791 ~ ~ 4344Z, PCT/US93/08326
We have shown that noggin does not induce muscle in late
blastula stage animal caps, however, it is possible that noggin
induces other types of dorsal mesoderm. To address this concern,
we asked whether noggin could induce the expression of the early
mesoderm markers goosecoid (Blumberg, et al., Science 253, 194-6,
1991; Cho, et al., Cell 67, 1111-20, 1991) , a marker of organizer
tissue and subsequently head mesoderm or X-brachyury (Smith, et
al., Cell 67, 79-87, 1991) , which appears to be expressed in all
mesodermal precursors early, and subsequently is expressed in
posterior mesoderm and notochord. Animal caps were treated at
stage 9 and collected at stage 11, when expression of goosecoid and
brachyury in the normal embryo is high. Neither marker is turned on
by purified human noggin (Fig. 4b. lane 5) at a dose with
demonstrated neural inducing activity (Fig 6 lanel5); in contrast
animal caps treated in the same fashion with activin show both
goosecoid and X-bra expression (Fig. 4b. lane 4) as expected for this
mesoderm inducing factor (Cho, et al., Cell 67, 1111-20, 1991;
Smith, et al., Cell 67, 79-87, 1991) . Untreated animal caps show
no expression of these mesodermal markers (lane 3), and RNA levels
in the collected animal caps are shown to be comparable using EF-la
levels (Krieg, et al., Dev Biol 133, 93-100, 1989).
Since purified human noggin is capable of driving neural
induction, no additional factors which may have been present in the
crude conditioned medium are..required. Furthermore, Xenopus and
human noggin, with 80% amino acid identity, can both act to induce
neural tissue in Xenopus, suggesting a conserved function for these
two proteins. However, for noggin to be a candidate endogenous
neural inducer it must be able to induce neural tissue at a stage
57
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0
when neural induction occurs in normal whole embryos. It is unclear
when the first instructive" signals are sent from dorsal mesoderm to
ectoderm in embryos. However, it is known that by early gastrula
stages, dorsal ectoderm has already been specified to become neural
tissue (Jones and Woodland, Development 107, 785-91, 1989) .
The neural inducing signal is therefore likely to start before this
stage. The latest stage at which animal caps have been shown to be
competent to respond to neural-inducing mesoderm is the early
neurula (St13-14) (Sharpe and Gurdon, Development 109, 765-74,
1990). Thus, a candidate endogenous neural inducer must be able
to induce neural tissue from gastrula stage competent ectoderm. -
Neural Induction at the Gastrula Stage
In order to assess the competence of ectoderm to respond to
noggin we treated animal caps taken from blastula (St8), late
blastula (St9), early gastrula (St10) and ventral animal caps from
mid-gastrula (StlO.5) stage embryos with purified human noggin[Fig.
2.]. We also treated similarly staged animal caps with activin to
demonstrate its mesoderm inducing and secondary neural inducing
activities, and to contrast activin's effects with those of noggin[Fig.
4a.]. Activin treated animal caps show neural induction only in
conjunction with induction of dorsal mesoderm, such as muscle and
notochord (lanes 3,6,9). In a number of experiments, we confirmed
that activin's ability to induce = dorsal mesoderm, and consequently
neural tissue, declines rapidly at the gastrula stage (lane
12) (Green, et al., Development 108, 173-83, 1990; Kintner and
Dodd, Development 113, 1495-1506, 1991). In the experiment
shown here a larger than usual dose of activin was given. Under
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these conditions, only a small amount of neural tissue is made,
perhaps because so much mesoderm is induced that there is not much
competent ectoderm left in the explant to be neuralized. In contrast
noggin can induce neural tissue in animal caps taken from all of
these stages without inducing the notochord and somite marker,
collagen type II (Amaya, et al., Development 118, 477-87, 1993;
Bieker and Yazdani-Buicky, J Histochem Cytochem 40, 1117-20,
1992), or muscle actin (lanes 4,7,10,13). This gives additional
support to the proposal that noggin is a direct neural inducer, since
it can act in the absence of both early.and late mesoderm markers.
Furthermore, we have shown that noggin can induce neural tissue in
competent ectoderm at a time when mesoderm inducers are inactive.
In some experiments, noggin addition to gastrula (but not
blastula) animal caps resulted in induction of muscle (data not
shown). This occurred at stages when activin could no longer induce
muscle. We interpret this as a result of a dorsalizing action by
noggin on tissues that have received a weak mesoderm-inducing
signal. The mesoderm-inducing signal which spreads into the
gastrula animal cap is not enough to induce mesoderm, but in the
presence of Xwnt-8 or noggin, muscle is formed. One interesting
corollary of the induction of muscle is that the kinds of neural
tissue seen in the explant are modified. Induction in explants that
contain no muscle usually yields N-CAM expression, but if muscle is
present, expression of both N-CAM and b-tubulin is seen. This
phenomenon is demonstrated in the secondary neural induction by
activin in St. 9 animal caps[Fig. 2.] and in the comparison of neural
tissue induced by noggin in ventral marginal zones versus animal
caps [Fig. 6.]. In the ventral marginal zones and animal caps in which
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muscle is present, both N-CAM and b-tubulin are expressed, whereas
induced animal caps without muscle, show only N-CAM expression.
Neural induction after injection of DNA coding for noggin
To confirm our conclusions using a different experimental
approach, we have directed noggin expression to gastrula stage
animal caps by injecting the plasmid pCSKA-noggin into the animal
pole of a one cell stage embryo. This plasmid, in which noggin is
under the control of the cytoskeletal actin promoter, turns on the
expression of noggin mRNA at the onset of gastrulation (Smith, et
al., Nature 361, 547-49, 1993) . At the blastula stage, the animal
caps are. dissected and then matured to tailbud stages for molecular
analysis. Animal caps injected with the noggin plasmid show
expression of N-CAM in the absence of muscle or notochord markers
(Fig. 4c. lane 2). A control plasmid directing the expression of lac Z
showed no neural or mesodermal induction as expected (lane 1). This
experiment demonstrates that ectopic noggin expression can
directly induce neural tissue in gastrula stage ectoderm, a stage
when neural induction is taking place in. whole embryos.
Differences in competence between dorsal and ventral animal caos.
Animal caps taken from the dorsal side of gastrula stage
embryos show greater competence to form neural tissue than ventral
animal caps (Otte and Moon, Cell 68, 1021-29, 1992; Sharpe, et al.,
Cell 50, 749-58, 1987) , when involuted anterior mesoderm is used
as the inducer. This type of mesoderm, however, has weaker
inducing capacity than the rest of the involuted mesoderm (Sive, et
al., Cell 58, 171-180, 1989) . Furthermore, the ventral side of an
WO 94/05791 Z443442 PGT/US93/08326
embryo can support the formation of a complete secondary axis when
the organizer is placed on that side (Gimlich and Cooke, Nature
306, 471-3, 1983; Smith and Slack; J. Embryol. Exp. Morph. 78, 299-
317, 1983; Spemann, Arch. Mikjrosk. Anat. EntwMech. 100, 599-638,
1938) , indicating that there is no qualitative difference in
competence. Thus, while a weak inducer might unmask slight
differences in competence of the ectoderm, it has been suggested
that a robust neural inducer would show little difference in its
effects on dorsal and ventral ectoderm (Servetnick and Grainger,
Development 112, 177-88, 1991) . Therefore we tested noggin's
effects on dorsal and ventral ectoderm from the early gastrula. No
difference in N-CAM expression is detected (Fig. 5, lanes 4,6), while
the ventral animal caps treated with noggin show a greater amount
of muscle actin expression (presumably through dorsalization of
tissues that received a low-grade mesoderm induction). Activin
treated dorsal caps show induction of roughly the same level of
muscle actin expression (lane 5) as the ventral noggin treated caps,
however, activin treatment did not induce detectable neural specific
transcripts (lanes 3,5). This indicates that muscle tissue induced at
this stage is not sufficient to secondarily induce neural tissue, and
that noggin must be present to induce neural tissue.
We conclude that there is no dorsal-ventral difference in
noggin mediated neural induction, suggesting that noggin behaves
like the robust neural inducing signal of the Spemann organizer, not
like the weaker signal from early anterior mesoderm.
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Dose DeRendenGSt
To determine what levels of noggin protein are required for
neural inducing activity, we carried out a dose response experiment.
In addition to determining the doses required for neural induction in
animal caps, we have also carried out a dose response of the
dorsalization of ventral marginal zones in order to compare the
doses required for these two types of inductions. Stage 9 animal
caps or St. 10.5 VMZ were treated with purified human noggin, and N-
CAM and [3-tubulin were used to assay neural induction, while muscle
actin was used as a marker of dorsal mesoderm. This experiment
shows that neural induction occurs at a dose of 1 g/ml, which is a
twenty fold higher dose than required for dorsalization of VMZ [Fig.
5]. There are several observations that may account for the
apparently high dose requirement. First, to get a maximal neural
response from dorsal mesoderm, the tissues must be left in contact
through most of neurulation (Sharpe and Gurdon, Development 109,
765-74, 1990) ; in contrast, the animal caps treated with noggin
close up rapidly, this inhibits factor access, and consequently they
receive only a brief effective dose. Second, it is likely that noggin
is not the only neural inducer active in the embryo; it has been
shown in a variety of amphibians that the somites (Hemmati-
Brivanlou, et al., Science 250, 800-802, 1990; Jones and Woodland,
Development 107, 785-91, 1989) and the neural plate have neurai inducing
activity (Hamburger, The Heritage of Experimental
Embryology: Hans Spemann and the Organizer, 1988; Servetnick and
Grainger, Dev Biol 147, 73-82, 1991) and noggin transcripts are
not detected there. Thus it is plausible that noggin is one of several
neural-inducing activities. In this connection it is worth noting that
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noggin is equally potent in inducing neural tissue in ventral marginal
zones as in dorsalizing them to generate muscle. Numerous other
experiments (see Fig. 5) show that induction of a similar amount of
muscle at this stage by activin does not result in neural induction.
Fourth, it may be that only a small fraction of the purified protein is
active, and that the experiment overestimates the amount of protein
needed for neural induction. Finally', it is possible that the
accessibility of exogenously added soluble noggin is significantly
lower than noggin protein being secreted endogenously.
Patterninq
Embryonic neural tissue develops an anteroposterior (A-P)
pattern, with various brain structures, eyes, and the spinal cord. It
is thought that A-P neural pattern requires the presence of dorsal
mesoderm, whether it be adjacent to the responding ectoderm in a
planar configuration (Dixon and Kintner, Development 106, 749-
757, 1989; Doniach, et al., Science 257, 542-5, 1992; Kintner and
Melton, Development 99, 311-25, 1987; Ruiz i Altaba, Development
108,595-604, 1990) , or directly beneath it in a vertical
interaction (Dixon and Kintner, Development 106, 749-757, 1989)
(Hemmati-Brivanlou, et al., Science 250, 800-802, 1990; Sharpe
and Gurdon, Development 109, 765-74, 1990; Sive, et al., Cell 58,
171-180, 1989) . Both of these types of interactions occur in
normal development, and' both probably contribute to the resulting
pattern. To determine if noggin induces patterned neural tissue, and
if so, what neural regions are represented, we used Xenopus otx as a
marker of forebrain and mid brain; En-2 (Hemmati-Brivaniou, et al.,
Development 111, 715-724, 1991) as a marker of the mid brain-
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hind brain boundary, and Krox-20 (Wilkinson, et al., Nature 337, 461-
4, 1989) as a marker of the third and fifth rhombomeres of the
hind brain in in situ hybridization (Harland, Methods in Cell Biology,
36, 675-685, 1991) . Antibodies directed against XlHbox
6 (Wright, et al., Development 109, 225-34, 1990) mark posterior
hind brain and spinal cord structures. Prior to the use of these
markers, we observed the formation of cement glands in noggin
treated animal caps. Since cement glands are induced organs of
ectodermal origin found anterior to the neural plate, this result
suggests that noggin induces anterior structures. In situ
.hybridization confirms this by 'showing the presence of a cement
gland specific transcript, XAG-1 (Sive, et al., Cell 58, 171-180,
1989) in noggin treated, animal caps, but not in control treated
animal caps[Fig. 7.]. In situ hybridization with the region specific
neural markers[Fig. 7.] show that noggin induces forebrain type
tissue as seen by the expression of otx in noggin treated animal
caps. We have not detected En-2, Krox20, or XlHbox, suggesting that
these more posterior markers are not induced by noggin.
ExRression of neural antigens
We have demonstrated that noggin directly induces the
expression of neural specific transcripts. A further demonstration
is to use antibodies against neural specific antigens to show that
the noggin induced tissue' is phenotypically neural. To this end, we
have treated animal cap tissue with noggin and cultured them to a
late stage (St 35) for antibody staining. We have used the 61711 anti-
N-CAM antibody, which stains the entire neural tube of a normal
embryo. Noggin treated animal caps express this antigen [Fig. 7.]
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-65-
while control untreated animal caps do not. This indicates that noggin can
induce the
production of neural specific proteins in treated animal caps. We have failed
to
detect the expression of numerous other antigens that are characteristic of
various
subclasses of differentiated neural cells. These included 2G9, which stains
most
neural tissue, including peripheral neurons, Tor 24.55, which stains sensory
neurons,
and Tor 23, which stains a variety of neurons including motor neurons.
EXAMPLE 7
Product of recombinant human nouzin from E. coli and baculovirus
Materials and Methods
Genetic Engineering and Cell Culture
A lactose inducible expression plasmid was constructed by replacing the
Swal/Bsml region of pRPN40 (Masiakowski et al, J. Neurochem. 57, 1003-1012,
1991) with the SwalBsml region of the human noggin gene obtained by PCR and
spanned by the same restriction sites, resulting in plasmid pRG301. pRG301 is
a
high copy number kanamycin resistant plasmid derived from pBR322 with the
human noggin gene under the control of the lacUV5 promoter. A plasmid
containing
the high copy number kanamycin resistant gene was deposited with the
Agricultural
Research Collection (NRRL), Peoria, Illinois, and bears accession number B-
18600.
This plasmid was described in United States Patent 5,256,568. E. coli
W31101aclq
cells transformed with pRG301 were grown at 37 C, induced with lactose,
harvested
by centrifugation,
CA 02143442 2004-10-06
washed once with 100 mM Tris-HCI, 50 mM EDTA pH 8 and. stored
frozen, essentially as described (Masiakowski et al, ibid. ).
ecove from inclusion bodies
E. coli cell paste (32 g) was suspended in ten volumes (v/w) of
50 mM TrisHCl-pH 8.0-5 mM EDTA, lysed in a French Press at 8,000
psi and 8 C and centrifuged at 8,000xg for 30 min at 4 C. The pellet
containing noggin was suspended in the original volume of 2 M urea-
20 mM TrisHCl, pH 8.0 and stirred for 30 min. The suspension was
centrifuged at 8,000xg at 4 C for 30 min and the pellet consisting
mostly of inclusion bodies (IB) was suspended in 20 volumes (v/w)
of 6 M guanidine HCI, 50 mM TrisHCl,1 mM EDTA, 50 mM DTT and
stirred for one hour at room temperature. After centrifugation at
8,000xg for 30 min, the supernatant containing 0.45-0.50 g
denatured and reduced noggin was diafiltered against 10 volumes of
6 M urea-50 mM sodium acetate pH 4.5-1 mM EDTA-0.1 mM 'DTT using
Omega 10,000 MW cut-off membranes. The diafiltrate containing 0.4-
0.44 g noggin was loaded at a flow rate of 30 mVmin onto a 2.6 x 10
cm column of S-SephamseT"" (Pharmacia), equilibrated in 6 M urea-50
mM , sodium acetate-1 mM EDTA-0.1 mM DTT pH 4.5 . The column was
first washed with the same buffer and then with a one liter gradient
(0-1 M NaCI) at a flow rate of 30 ml/min. Fractions containing
noggin were identified by gel electrophoresis and pooled. The yield
was 0.2-0.25 g noggin.
Refolding.
The denatured and reduced noggin solution was adjusted to .05-
.2 mg/ml protein concentration and brought to 1.5-2.5 M
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guanidineHCl-0.1 M TrisHCl pH 8.0-0.1 mM EDTA-0.2-2 mM reduced
glutathione-0.02-0.2 mM oxidized glutathione (preferably at a ratio
of 10:1 reduced to oxidized glutathione) at 4 C under slow stirring.
After 24-72 hours, two refolded noggin isoforms were identified by
RP-HPLC chromatography (Fig. 8). The refolded noggin solution was
diafiltered against 20 volumes of 0.05 M sodium acetate pH 4.5,
brought to 50 mM potassium phosphate pH 7.2 and stirred slowly at
4 C for 1 hour minimum. Misfolded noggin precipitated and was
removed by centrifugation for 30 min at 8,000 xg.
Reverse nhasP HPLC chromato r~ aohy.
Refolded noggin can be purified by chromatography on a 12 mm
C8, 1 x 25 cm DynamaxTM 300 A column equilibrated in solvent A (0.1%
TFA in water). After loading, the column was washed with solvent A
and was developed at a flow rate of 4 mi/min according to the
following protocol: (a) 10 min isocratically at 70% of solvent A, 30
% of solvent B (0.1% TFA in acetonitrile); 30 min linear gradient to
60 % solvent B and 40 % solvent A. Correctly refolded noggin elutes
earlier at 44%-46% solvent B. The yield was 0.07-0.1 g noggin.
Production of human noggin in Baculovirus cell culture.
The SF21 line of Spodoptera frugiperda was routinely
maintained as cell monolayers in Grace's Insect Cell medium
supplemented with lactalbumin hydrolysate and yeastolate (Gibco).
This medium completed with 10% v/v heat-inactivated fetal calf
serum (Irvine Scientific) is identified as TMNFH-10. Cells were also
cultured in serum-free medium (SF-900-II; Gibco) after adaptation.
Suspension cultures in either medium were raised in microcarrier
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culture flasks (Bellco) using a stirring speed of 80 rpm. All cultures
were maintained at >96 % viability, as judged by trypan blue
exclusion.
Construction of recombinant baculovirus.
Sequences corresponding to human noggin were excised as a 5'-
BamHl-Pstl-3' fragment from an expression plasmid containing the
human noggin gene. This fragment was inserted into BamHl-Pstl
digested pVL1393 (Invitrogen). The resulting plasmid, pTR 1009,
has the human noggin sequence immediately downstream of the
polyhedrin promoter of Autrographa californica Multiple Nuclear
Polyhedrosis Virus (AcMNPV), and this promoter-heterologous gene
fusion is flanked in turn by recombination targets derived from the
AcMNPV polyhedrin region. Recombinant plasmid DNA was purified
by alkaline lysis and CsCl centrifugation. SF21 cells were co-
transfected with plasmid and viral DNA by the following method:
Plasmid DNA (3 mg) was mixed with 0.5 mg linearized, deleted viral
DNA (Baculo GoidTM, Pharminigen), and precipitated with ethanol.
Dried DNA was then resuspended in water (50 mi), mixed with 1.5ml
Grace's medium, and 30 ml LipofectinTM cationic liposomes (BRL).
The DNA-liposome mixture was vortexed, allowed to stand at room
temperature for 15 minutes and added dropwise to a monolayer of
SF21 cells (2x106 cells/60 mm plate). After incubation at 27 C f or
four hours, 2 mi TMNFH-10 'was added and the culture returned to
incubation for 5 days. Tissue culture medium was harvested and
used as a source of virus for plaque isolation.
Recombinant virus was isolated by multiple rounds of plaque
purification on SF21 ceils. Diluted -virus (0.5 mi) was adsorbed to
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cell monolayers (2x106 cells/60 mm plate) for a period of one hour
at 27 C, aspirated, and virus plaques were allowed to develop with
an overlay of 0.5% agarose in TMNFH-10 medium for a period of 6
days. Virus plaques were picked after microscopic inspection, and
eluted into 2mi SF900-I1 medium. Virus stocks were amplified by
low multiplicity (0.1 pfu/cell) infection. Virus clones expressing
noggin were identified by metabolic labeling of infected cultures
with 35S-methionine and 35S-cysteine and analyzing total labeled
protein by polyacrylamide gel electrophoresis and autoradiography.
A labeled protein of the expected apparent Mr of 20,000-30,000 was
detected by this method in candidate clones but not in control
cultures.
Excression and purification of baculovirus-derived noagin
SF21 cells were cultured in suspension flasks to a density of
approximately 1.8x106/ml in SF900-11 medium. Cultures (500 mi)
were collected by centrifugation at 1000xg for 10 min .and
resuspended in 20m1 of growth medium containing 5-10 pfu/cell
recombinant virus. Virus was aliowed to adsorb for 1 hour at room
temperature with gentle mixing. Infected cells were then diluted to
their original volume with- fresh growth medium, and incubated at
27 C for 3 days. Cells and debris were subsequently clarified from
the growth medium by centrifugation at 1000x g for 20 min.
Cell supernatants were brought to pH 8.0, passed through a
0.45 mm Millipak6OTM filter and applied to a FastSTM column that had
been equilibrated in 25 mM HEPES pH 8Ø The column was washed
with the same buffer and developed with a linear NaCI gradient to
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0
remove other medium components. Noggin eluted from this column
at 1 M NaCi.
Results
Characterization of human no,qqin croduced in E. coli and i n
baculovirus
Reverse-phase HPLC chromatography shows that recombinant
noggin refolded and purified from E. coli elutes in a single sharp
peak, indicating the presence of one predominant isoform (Fig. 9).
Electrophoresis on 15 % polyacrylamide-SDS-reducing gels
shows that noggin from either E. coli or insect cells is better than
95 % pure and migrates in a single band corresponding to a protein of
20-30 kD. Noggin from insect cells shows slightly slower mobility,
apparently due to additional mass from N-linked glycosylation at the
single consensus site (Fig. 10). Treatment with Endo F converts the
mobility of insect-produced noggin to that of the bacterially
produced protein (data not shown).
In the absence of reducing agents, noggin produced either in
E.coli or in baculovirus behaves as a disulfide-Iinked oligomeric
pTotein (Fig. 10). However, by gel filtration analysis and mass
spectroscopy noggin is primarily a dimeric protein (data not shown).
Circular dichroism studies show that recombinant noggin
refolded and purified from E. coli as well as noggin purified from
insect cells have very similar conformations (Fig. 11). Secondary
structure determined by this method indicates that noggin consists
of 48% alpha-helix, 0 % beta-structure, and 52 % random coil.
WO 94/05791 2143444 PCT/US93/08326
Biological Activity of human noggin groduced in E. coli and i n
bacufovirus
Biological activity of human noggin produced in E. coli or in
baculovirus was determined by assay of muscle actin expression in
the ventral marginal zone assay, as described supra. Results shown
in Fig. 12 indicate a positive dose response for induction of muscle
actin mRNA in VMZ exposed to either bacterially produced human
noggin, or baculovirus produced human noggin.
EXAMPLE 8
Production and characterization of rat monocional_ antibody RP57-16
reactive with human noggin.
Materials and Methods
Production of antibody
RP57-16 rat monoclonal antibody reactive with recombinant
human and Xenopus noggin was produced by the immunization of a
female Lewis rat with four 35 g injections of purified
recombinant human noggin (produced in E. coli) over a two month
period. For the initial immunization, the protein was injected in the
rear foot pad in Freund's complete adjuvant. Subsequent injections
were given in the same foot pad in Freund's incomplete adjuvant.
The rat was euthanized 3 days after the fourth injection.
Lymph node cells from the immunized rat were mixed with
SP2/0-E.O. mouse myeloma cells at a ratio of 2:1. After
centrifugation, the cell mixture was resuspended in 0.25 ml of 42%
(w/v) PEG 3350 (Baker) in phosphate-buffer-saiine with 10% (v/v)
71
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dimethylsulfoxide (Sigma) for a total of 3 minutes in a 37 C water
bath. Cells were plated at a density of 5 X 104 lymphocytes per well
in 96-well plates (Falcon 3072) in DMEM/F-12 (Mediatech, Inc.)
containing 10% FBS (supplemented with streptomycin, penicillin,
pyruvate, and glutamine) and HMGT (1.6 x 10-3 M thymidine, 4.0 x 10 -
4 methotrexate, 1.3 x 10-3 sodium bicarbonate and 1.0 x 10-2
hypoxanthine). After 10 days in culture, supernatants were
harvested and assayed for antibody activity against recombinant
human noggin by indirect ELISA. Supernatant from COS-M5 cells
transfected with the plasmid containing the human noggin gene was
air dried overnight in ProbindTM 96-well assay plates (Falcon 3915).
Non-specific binding was eliminated by 2 hour incubation at ambient
temperature with PBS/1% BSA (Sigma). Plates were washed 2 times
with PBS/0.02 % Tween 20. Culture supernants were then added and
incubated at ambient temperature for 1 hour. Plates were washed 4
times with PBS/0.02 % Tween 20T"" . Secondary antibody, Goat anti-Rat
IgG (H+L) alkaline phosphatase conjugate(Caltag) diluted 1:2000 in
PBS/1% BSA was added to each wefl and the plates 'incubated at
ambient temperature for 1 hour. Plates were again washed 4 times
with PBS/0.02 % Tween20T"'. Antibody binding was visualized by 1
hour incubation at ambient temperature in the dark with pNPP (p-
nitrophenyl phosphate, Sigma) 1 mg/mI in diethanolamine buffer, pH
9.8. The reaction was stopped by the addition of an equal volume of
100 mM EDTA. Absorbance was read at 405 nm on a TherrnomaxTM
Microplate Reader (Molecular Devices). A reaction was considered
positive if the absorbance was 2 times that of the negative control
(diluent alone followed by secondary antibody and substrate).
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Positive clones were expanded and culture supernatant containing
monoclonal antibody was collected for specificity analysis.
RP57-16 was cloned in soft agar. Cloned hybrid cells were
expanded in DMEM/F-12 (Mediatech, Inc.) containing 10% FBS
(supplemented with streptomycin, penicillin, pyruvate, and
glutamine). Supernatant containing antibody was aliquoted and
stored at -70 C until use.
S ecificity Analxsis
ELISA 100 ng of purified recombinant human noggin, Xenopus noggin,
BDNF, NT-3, and NT4 protein was individually passively adsorbed to
Probind 96-well assay plates by overnight incubation at 4 C in 50
mM bicarbonate buffer, pH 9.6. BDNF, NT-3 and NT-4 were used to
assess non-specific binding of rat monoclonal antibody RP57-16.
Supernatants from COS-M5 cells transfected with either the plasmid
containing the human noggin gene or the plasmid containing the fig C-
terminal tagged Xenopus noggin gene were air dried to Probind 9 6-
well plates overnight. Non-specific binding was eliminated by 2
hour incubation at ambient temperature with PBS/1% BSA (Sigma).
Plates were washed 2 times with PBS/0.02 % Tween 20. Undiluted
RP57-16 was added and incubated at ambient temperature for 1
hour. Plates were washed 4 times with PBS/0.02 % Tween 20.
Secondary antibody, Goat anti-Rat IgG (H+L) alkaline phosphatase
conjugate(Caltag) diluted 1:2000 in PBS/1% BSA was added to each
well and the plates incubated at ambient temperature for 1 hour.
Plates were again washed 4 times with PBS/0.02 % Tween 20.
Antibody binding was visualized by 1 hour incubation at ambient
73
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temperature in the dark with pNPP (p-nitrophenyl phosphate, Sigma)
1 mg/ml in diethanolamine buffer, pH 9.8. The reaction was stopped
by the addition of an equal volume of 100 mM EDTA. Absorbance was
read at 405 nm on a Thermomax Microplate Reader (Molecular
Devices). A reaction was considered positive if the absorbance was
2 times that of the negative control (diluent alone followed by
secondary antibody and substrate).
El -c r ghoresis and Western Blotting
Rat monoclonal antibody RP57-16 was also analyzed by
Western blotting. 50 ng of recombinant human noggin, non-reduced
and reduced, were electrophoresed on 12.5% SDS-polyacrylamide
gels and electroblotted on nitrocellulose membranes. Membranes
were blocked with PBS/1% Casein/0.1 % Tween 20T"', and then
incubated for 2 hours with undiluted RP57-16 culture supernatant.
Following 4 washes in PBS/0.02% Tween 20T"", the membranes were
incubated with a 1:5000 dilution of Goat anti-Rat tgG (H+L)
horseradish peroxidase conjugate (Pelfreeze) in PBS/1% BSA/0.1 !0
Tween 20T"". Membranes were washed 4 times with PBS/0.02% Tween
20r Proteins were visualized with ECL Western Blotting ReagentsTM
(Amersham) according to the manufacturer's instructions.
Membranes were then exposed to XAR 5 Scientific Imaging film
(Kodak) for 5 seconds.
RESULTS
Rat monoclonaf antibody RP57-16 reacts with both
recombinant human and Xenopus noggin and with recombinant human
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noggin produced in E. coli, in insect cells, and in COS-M5 cells. The
antibody does not react with the neurotrophins BDNF, NT-3 and NT-4.
Western blotting showed that the antibody detects both reduced and
non-reduced protein.
DEPOSfT OF MICROORGANISMS
The following were deposited with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 under
the terms of the Budapest Treaty:
ATCC Accession Date of
No. Dgg2osit
phage hnog,%-5 75311 9-23-92
phage hnog7,-7 75309 9-23-92
phage hnogk-9 75310 9-23-92
phage hnoga,-10 75308 9-23-92
hybridoma RP57-16 CRL 11446 8-25-93
It is to be understood that while the invention has been
described above in conjunction with preferred specific
embodiments, the description and examples are intended to
illustrate and not limit the scope of the invention, which is defined
by the scope of the appended claims.
