THIS APPLICATION HAS BEEN DIVIDED OUT OF INDIAN
APPLICATION NO. 1583/KOLNP/2005
FIELD OF THE INVENTION
The invention is in the field of molecular biology. More specifically, the
invention pertains to methods and compositions for the diagnosis, prognosis,
prevention, treatment, and evaluation of therapies for bone-related disorders.
BACKGROUND OF THE INVENTION
The topic of bone-related disorders and diseases has gained considerable
attention over the past years. Bone-related disorders are characterized by bone loss
resulting from an imbalance between bone resorption and bone formation.
Throughout life, there is a constant remodeling of skeletal bone. In this remodeling
process, there is a delicate balance between bone resorption by osteoclasts and
subsequent restoration by osteoblasts. Osteoblasts, involved in both endochondral
and intramembranous ossification, are the specialized cells in bone tissue that make
matrix proteins resulting in the formation of new bone. Bone formation, i.e.
osteogenesis, is essential for the maintenance of bone mass in the skeleton. Unlike
osteoblasts, osteoclasts are associated with bone resorption and removal. In normal
bone, the balance between osteoblast-mediated bone formation and osteoclast-
mediated bone resorption is maintained through complex regulated interactions.
There are many deficiencies, diseases, and disorders associated with the
skeletal system. Examples of a few include, but are not limited to, osteoporosis,
bone cancer, arthritis, rickets, bone fracture, periodontal disease, bone segmental
defects, osteolytic bone disease, primary and secondary hyperparathyroidism,
Paget's disease, osteomalacia, hyperostosis, and osteopetrosis. Identification of the
mechanisms involved in osteogenic differentiation and the renewal processes are
crucial for the understanding of bone physiology and skeletal disorders, such as
osteoporosis. These disorders may involve deficient bone formation due to defective
maturation of putative osteoblastic progenitors.
There exists a need to develop methods of treating diseases or disorders
associated with bone growth, methods of hastening bone formation, methods of
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identifying agents that modulate (increase or decrease) bone formation, and
methods of identifying genes or their protein products associated with bone related
disorders.
Identification of the mechanisms involved in bone formation and bone
resorption are crucial for the understanding of bone physiology and skeletal
disorders, such as osteoporosis. The genes or their protein products which are
associated with bone related disorders may be used for the elucidation of the
molecular mechanisms of bone formation, bone resorption, for the screening and
development of new drugs, for diagnosis, prognosis, prevention, and treatment of
bone development and bone loss disorders, and evaluation of therapies for bone-
related disorders such as osteoporosis.
The present invention not only provides a method for modulating bone related
activity, it also provides a method by which the understanding of the mechanisms
involved in bone formation and bone resorption are furthered.
SUMMARY OF THE INVENTION
The present invention relates to an expression cassette comprising a
polynucleoticle encoding a Ror polypeptide or homologues or derivatives or
fragments or variants or mutants thereof wherein said polynucleotide is under the
control of a promoter operable in bone cells.
The present invention relates to a host cell comprising an expression cassette
comprising a polynucleotide encoding a Ror polypeptide or homologues or
derivatives or fragments or variants or mutants thereof, wherein said polynucleotide
is under the control of a promoter operable in eukaryotic cells, said promoter being
heterologous to said polynucleotide.
The present invention relates to a composition for modulating bone-related
activity comprising an effective amount of Ror molecule or homologues or derivatives
or fragments or variants or mutants thereof.
The present invention relates to a method of screening for agents, the method
comprising: (a) combining an agent with a Ror molecule; and (b) detecting an effect
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of said agent on Ror activity; wherein detection of a decrease or an increase in Ror
activity is indicative of an agent being a bone-related agent.
The present invention relates to a method of screening for agents, the method
comprising: (a) combining an agent with an isolated cell comprising a Ror promoter
sequence operatively linked to a reporter gene; and (b) detecting an effect of said
agent on reporter activity; wherein detection of a decrease or an increase in Ror
promoter activity as measured by the reporter activity is indicative of an agent being
a bone-related agent.
The present invention relates to a method of screening for agents that
modulate the binding of Ror polypeptide to a binding partner comprising: (a)
contacting Ror polypeptide with a Ror binding partner in the presence of an agent;
(b) contacting Ror polypeptide with a Ror binding partner in the presence of a control
or in the absence of the agent; and (c) selecting the agent that modulates Ror
polypeptide binding to Ror binding partner by comparing the binding of said Ror
polypeptide to the binding partner in step (a) to the binding of said Ror polypeptide to
a binding partner in step (b).
The present invention relates to a method of modulating bone-related activity
in a subject comprising administering to a subject an agent which modulates target
Ror molecule expression or activity.
The present invention relates to a method of modulating Wnt-1 and Wnt-3
activity in a subject comprising administering an agent which modulates target Ror2
molecule expression or activity in an amount effective to regulate Wnt-1 and Wnt-3
activity.
The present invention relates to a method of modulating Wnt-3 activity in a
subject comprising administering an agent which modulates target Ror1 polypeptide
expression or activity in an amount effective to regulate Wnt-3 activity.
The present invention relates to a method for identifying an agent for
modulating bone-related activity comprising: (a) expressing Ror molecule in a cell or
using endogenous Ror expression; (b) contacting the cell with the agent; and (c)
monitoring the expression or the activity of Ror molecule wherein an increase or
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decrease in the expression or activity of Ror molecule in the presence of the agent
identifies the agent as modulating bone-related activity.
The present invention relates to a method for identifying an agent for
modulating Wnt signaling pathway comprising: screening one or more agents for the
ability to modulate expression or activity of Ror molecule, wherein the agent that can
modulate expression or activity of Ror molecule is an agent that modulates Wnt
signaling pathway.
The present invention relates to a method of linking a bioactive molecule to a
cell expressing a Wnt polypeptide, said method comprising contacting said cell with a
Ror2 polypeptide that is bound to said bioactive molecule and allowing said Wnt
polypeptide and said Ror2 polypeptide to bind to one another, thereby linking said
bioactive molecule to said cell.
The present invention relates to a method for screening a subject for a bone-
related disorder comprising the steps of: measuring the expression of Ror molecule
in a subject and determining the relative expression of said Ror molecule in the
subject compared to its expression in normal subjects, or compared to its expression
in the same subject after being treated for bone-related disorders.
The present invention relates to a method for screening a subject for a bone-
related disorder comprising the steps of: measuring the activity of Ror polypeptide in
a subject and determining the relative activity of said Ror polypeptide in the subject
compared to its activity in normal subjects, or compared to its activity in the same
subject after being treated for bone-related disorders.
The present invention relates to a method of identifying genes that participate
in bone formation comprising: a) overexpressing Ror molecule in a cell, b) monitoring
the changes in gene expression profile and c) determining which genes are regulated
by Ror expression thereby identifying genes that participate in bone formation.
The present invention relates to a method of identifying genes that modulate
Wnt signaling pathway comprising: a) overexpressing Ror molecule in a cell, b)
monitoring the changes in gene expression profile and c) determining which genes
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are regulated by Ror expression thereby identifying genes that modulate Wnt
signaling pathway.
The present invention relates to a method for identifying proliferating human
pre-osteoblastic cells using Ror2 as a marker, comprising determining expression of
Ror2 gene in a human osteoblastic cell wherein the increased Ror2 expression
identifies the cell as being proliferating pre-osteoblastic cells.
The present invention relates to a method for identifying mouse osteoblastic
cells at the stage of matrix maturation using Ror2 as a marker, comprising
determining expression of Ror2 gene in a mouse osteoblastic cell wherein the
increased Ror2 expression identifies the cell as being an osteoblastic cell at the
stage of matrix maturation.
The present invention relates to a method of determining Ror 2 kinase activity
comprising: (a) obtaining Ror2 polypeptide; (b) labeling Ror2 polypeptide in presence
of 32P Y ATP; (c) determining Ror2 kinase activity by measuring the amount of
incorporated 32P wherein the amount of 32P indicates activity of Ror2 kinase.
Brief Description of the Drawings and Sequence Descriptions
The invention can be more fully understood from the following detailed
description and the accompanying drawings and Sequence Listing that form a part of
this application.
Figure 1 shows that the expression of Ror kinases decreases during late
stages of human osteoblast differentiation. Expression of Ror2 (Figure 1A) and Ror1
(Figure 1B) in cells representing different stages of osteoblast differentiation from
pre-osteoblasts to mature osteocytes was assessed by, gene chip analysis on
GIHumania chip (circles) and by real-time RT-PCR (bars). For both methods, the
relative mRNA expression in HOB-03-C5 was set at one. Real-time RT-PCR was
performed using probes and primers listed in Table 1 in Example 1. The levels of
mRNA were normalized to the expression of 18S rRNA in each sample. Means ±
Standard Errors (SE) of three RT-PCR reactions per cell line. Ror2 expression in
HOB-05-T1 cells was undetectable by RT-PCR. Cell lines: 03-C5 - HOB-03-C5,
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pre-osteoblasts, 03-CE6 - HOB-03-CE6, early osteoblasts; 02-C1 - HOB-02-C1,
mature osteoblasts; 01-C1 - HOB-01-C1, pre-osteocytes; 05-T1 - HOB-05-T1,
mature osteocytes (Figure 1 is referred to in Example 1).
Figure 2 shows that SFRP-1 suppresses Ror2 expression. Results of the
gene chip analysis of Ror2 (closed bars) and Ror1 (open bars) expression on
GIHumania chip using polyA(+)RNA from HOB-01-09 osteocytes stably
overexpressing SFRP-1 (01-09SFRP-1) or empty vector (01-09 vector) or from
calvarial bones of wild type or SFRP-1 -/- mice. Levels of Ror1 expression in wild
type and SFRP1-/- mice were below the chip detection limits. The relative mRNA
expression in the 01-09vector cells and in wild type mice was set at one (Figure 2 is
referred to in Example 1).
Figure 3 shows a predicted domain structure of Ror proteins. The domains
are: IG - immunoglobulin; FRZ - frizzled; kringle - triple loop structure linked by
three pairs of disulfide bonds originally found in prothrombin; M - transmembrane;
Tyr Kin - tyrosine kinase. B Localization of disulfide bonds in the frizzled module of
Ror1 (Roszmusz, et al., E., 2001, Journal of Biological Chemistry. 276:18485-90).
Numbers refer to the 10 conserved cysteines in the frizzled domain (Figure 3 is
referred to in Example 1).
Figure 4 shows that the expression of Ror2, but nor Ror1, kinase increases
during early stages of human osteoblast differentiation and through later stages of
mouse osteoblast differentiation. A Human MSC were incubated in human
osteogenic medium (0.1 mM dexamethasone, 0.05 mM ascorbic acid and 10 mM β-
glycerophosphate in growth medium) and total cellular RNA was collected at times
indicated and analyzed by RT-PCR for expression of Ror1 and Ror2 genes. B.
Murine MC3T3-E1 cells were incubated in mouse osteogenic medium (25 μg/ml
ascorbic acid and 10 mM β-glycerophosphate in growth medium) and total cellular
RNA was collected at times indicated and analyzed by RT-PCR for expression of
Ror1, Ror2, alkaline phosphatase (AP), and osteocalcin (OC) genes (Figure 3 is
referred to in Example 2).
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Figure 5 shows expression of Ror proteins in U2OS cells. A. Schematic
representations of the full-length Ror1 and Ror2 kinases and the Ror2 mutants.
FLAG - flag epitope tag; M - transmembrane domain; Tyr Kin - tyrosine kinase
domain. B. Western immunoblot for the flag epitope tag of the whole-cell protein
extracts (50 μg/lane) from U2OS cells transfected with the indicated Ror constructs.
Position of Ror1, Ror2 and Ror2KD is marked by an arrowhead and Ror2AC-flag, by
an arrow. C. The top panel shows an autoradiograph of the results of in vitro
autophosphorylation assay performed as described under General Methods using
Ror2-flag or Ror2KD-flag immunoprecipitated on flag affinity agarose. In the bottom
panel, ten percent of the flag immunoprecipitated proteins were separated by SDS-
PAGE and analyzed by Silver staining to assess kinase levels in the
autophosphorylation reactions (Figure 5 is referred to in Examples 3, 6, and 8).
Figure 6 shows that Ror2 kinase inhibits Wnt-3, but potentiates Wnt-1 activity.
U2OS cultures were transiently tranfected with a recombinant luciferase reporter
gene containing 16 copies of the TCF binding site cloned 5' to the thymidine kinase
promoter. In A, the promoter-reporter gene was co-transfected with pcDNA3.1(+)
(vector), Ror2-flag (R2), Wnt-3-HA (w3), or Wnt-3-HA plus the indicated amounts of
Ror2-flag (in ng per well of a 96-well plate) or SFRP-1 (S) or both. In B, the
promoter-reporter gene was co-transfected with pcDNA3.1(+), Wnt-1-HA (w1), or
Wnt-1-HA plus the indicated amounts of Ror2-flag or SFRP-1 or both. Luciferase
values measured after transfection of a reporter gene in presence of pcDNA3.1 (+)
have been arbitrarily given a value of 1. In A, the results are means ± SE of at least
two independent experiments with n≥16 and the asterisks indicate significant
decreases in luciferase activity below the level obtained with Wnt-3-HA alone (* - p <
0.05, ** - p < 0.0001). In B, the results are means ± SE of at least three independent
experiments with n≥24 and the asterisks indicate significant increases in luciferase
activity above the level in presence of Wnt-1-HA alone (* - p < 0.05, ** - p < 0.0001)
(Figure 6 is referred to in Example 4).
Figure 7 shows that Ror1 kinase inhibits Wnt-3, but has no effect on Wnt-1
activity. As in Figure 6 except Ror1-flag (R1) was used in place of Ror2-flag. Means
± SE, n≥8. In A, the asterisks indicate significant decreases in luciferase activity
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below the level obtained with Wnt-3-HA alone (* - p < 0.01, ** - p < 0.0001) (Figure 7
is referred to in Example 5).
Figure 8 shows that the tyrosine kinase activity of Ror2 is required for
potentiation of Wnt-1 and for most of inhibition of Wnt-3 activity. As in Figure 6
except Ror2KD-flag (R2KD) was used in place of Ror2-flag. In A and B, the results
are means ± SE of at least three independent experiments with n≥24. The asterisks
indicate significant decreases in luciferase activity below the level obtained with Wnt-
3-HA alone (** - p < 0.0001) (Figure 8 is referred to in Example 6).
Figure 9 shows that the cytoplasmic domain of Ror2 is required for
potentiation of Wnt-1 and for part of inhibition of Wnt-3 activity. As in Figure 6 except
Ror2AC-flag (R2d) was used in place of Ror2-flag. In A, the results are means + SE
of at least two independent experiments with n≥16. The asterisks indicate significant
decreases in luciferase activity below the level obtained with Wnt-3-HA alone (* - p <
0.05, **-p< 0.0001). In B, n≥8 (Figure 9 is referred to in Example 6).
Figure 10 shows that Ror2 and Ror2KD bind to Wnt-1 and Wnt-3. COS7
cells were transiently transfected with vector controls or the indicated combinations
of Ror2-flag and Wnts-HA. The total amount of DNA was kept constant by addition
of pcDNA3.1(+) or pUSEamp in place of Ror2 or Wnts, respectively. At 24 h, lysates
were analyzed by SDS-PAGE, directly (top) or after immunoprecipitation with anti-
flag antibody (bottom). Immunoblotting was performed with anti-HA antibody (Figure
10 is referred to in Example 7).
Figure 11 shows that Ror2 discriminates between different Wnts. COS7 cells
were transiently transfected with the indicated Wnts-HA and Ror2-Flag (+) or
pcDNA3.1. At 24 h, lysates were immunoprecipitated with anti-flag antibody and
analyzed for presence of Wnts by anti-HA antibody (top). The bottom panel shows
western blot analysis with anti-HA antibody of the COS7 extracts containing the
indicated Wnts and the Ror2-flag (control for equal loading in the
immunoprecipitation reactions) (Figure 11 is referred to in Example 7).
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Figure 12 shows that overexpression of Wnt-1 and Wnt-3 has no effect on the
extent of Ror2 autophosphorylation. A. The top panel shows an autoradiograph of
the results of in vitro autophosphorylation assay performed as described under
General Methods using Ror2-flag immunoprecipitated on flag affinity agarose out of
U2OS cells co-transfected with Ror2-flag and the indicated Wnts. The bottom panel
shows western immunoblotting of the same membrane with anti-flag antibody.
B. Autoradiographic signals were normalized to the total amount of immunoreactive
Ror2 protein in each reaction and the relative signal obtained in absence of Wnts
was set at one. Means ± SE of three independent experiments (Figure 12 is referred
to in Example 8).
Figure 13 shows that Ror2 inhibits Wnt-mediated stabilization of cytosolic p-
catenin. U2OS cultures were transiently transfected with the indicated combinations
of Ror2 (R2) and Wnts (W). The total amount of DNA was kept constant by addition
of pcDNA3.1(+) or pUSEamp in place of Ror2 or Wnts, respectively. At 24 h,
cytoplasmic proteins were analyzed by western immunoblotting with anti-β-catenin
antibody. The levels of p-catenin were normalized to the signal obtained in absence
of Wnts after equal loading was verified by staining with anti-p-actin antibody. The
numbers are means of three independent experiments (Figure 13 is referred to in
Example 9).
Figure 14 shows a proposed model for Ror2 activity whereby Ror2 binds both
Wnts, sequestering them away from Frizzled receptors and inhibiting their ability to
stabilize β-catenin. In addition, Wnt1 binding to the Ror2 receptor causes activation
of an unidentified signaling cascade that requires tyrosine kinase activity of the Ror2
receptor and results in potentiation of Wnt-responsive promoter activity. Wnt3
binding does not stimulate the same cascade, but instead activates other tyrosine
kinase-dependent events that lead to inhibition of Wnt-responsive promoters. FZ -
Frizzled receptor, GSK-3 ft ~ glycogen synthase kinase B, B-cat-β-catenin, Lef/Tcf -
lymphoid-enhancer binding factor/T-cell transcription factor (Figure 14 is referred to
in Example 10).
Figure 15 illustrates identification of the Ror2 binding partners in U2OS cells.
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Whole-cell extracts from U2OS cells transiently transfected with the constructs
identified on top were immunoprecipitated with anti-flag antibody and subjected to
SDS-PAGE analysis on 4-12% (A) or 7% (B) polyacrylamide gels. In parallel
experiments, the 4-12% gels were transblotted onto nitrocellulose membrane and
western immunoblotting was performed with anti-flag (C) or anti-phosphotyrosine (D)
antibody. Arrows point to bands that appear to be Ror2-dependent. M - molecular
weight in kDa. The prominent bands around 55 and 25 kDa are IgG subunits
dissociated from the flag affinity agarose (Figure 15 is referred to in Example 11).
Figure 16 shows that Ror2 binds to the intracellular domain of Notch2
(Notch2IC). U2OS cells were transiently transfected with vector controls or the
indicated combinations of Ror2-flag and Notch2IC-V5-his. The total amount of DNA
was kept constant by addition of pcDNA3.1(+). At 24 h, lysates were analyzed by
SDS-PAGE directly (top) or after immunoprecipitation with anti-flag antibody.
Immunoblotting was performed with anti-V5 (top) or anti-his (bottom) antibody
(Figure 16 is referred to in Example 12).
Figure 17 confirms generation of cell lines stably over-expressing Ror2 and
Ror1. A. Relative Ror2 and Ror1 mRNA expression in HOB-01-09 cells over-
expressing Ror2, Ror2-Flag, Ror1-Flag, or empty vector (pcDNA 3.1(+)). The
relative mRNA expression in HOB-01-09-pcDNA cells was set at one. Real-time RT-
PCR was performed using probes,and primers listed in Table 1 in Example 1. The
levels of mRNA were normalized to the expression of 18S rRNA in each sample. B.
Western immunoblot with the indicated antibodies of the whole-cell protein extracts
(50 ng/lane) from HOB-01-09 cells over-expressing Ror2, Ror2-Flag, Ror1-Flag, or
empty vector (Figure 17 is referred to in Example 13).
Table 1 shows primers and probes used in the real-time RT-PCR analysis of
human Ror mRNA (Table 1 is referred to in Example 1).
Table 2 shows primers and probes used in the real-time RT-PCR analysis of
mouse Ror mRNA (Table 2 is referred to in Example 2).
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Table 3 shows primers and probes used in the real-time RT-PCR analysis of
mouse alkaline phosphatase and osteocalcin mRNA (Table 3 is referred to in
Example 2).
Table 4 shows potential Ror2 interacting proteins (Table 4 is referred to in
Example 9).
The following 48 sequence descriptions and sequence listings attached
hereto comply with the rules governing nucleotide and/or amino acid sequence
disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
("Requirements for Patent Applications containing nucleotide sequences and/or
Amino Acid Sequence Disclosure-the Sequence Rules") and consistent with World
Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence
listing requirements of the EPO and PCT (Rules 5.2 and 4.95(a-bis) and Section 208
and Annex C of the Administrative Instructions). The Sequence Descriptions
contains the one letter code for nucleotide sequence characters and the three letter
codes for amino acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Res., 13, 3021-3030, (1985) and in the Biochemical J.,
219(2), 345-373, (1984) which are herein incorporated by reference. The symbols
and format used for nucleotide and amino acid sequence data comply with the rules
set forth in 37 C.F.R. §1.822.
SEQ ID NO:1 is the first nucleotide sequence containing the TCF DNA
binding sites originally identified in the TCR-alpha enhancer, the CD3-e enhancer,
and the consensus TCF DNA binding site.
SEQ ID NO:2 is the second nucleotide sequence containing the TCF DNA
binding sites originally identified in the TCR-alpha enhancer, the CD3-e enhancer,
and the consensus TCF DNA binding site.
SEQ ID NO:3 is the nucleotide sequence that codes for Ror1 protein.
SEQ ID NO:4 is the deduced amino acid sequence of Ror1 protein.
SEQ ID NO:5 is the nucleotide sequence that codes for Ror2 protein.
SEQ ID NO:6 is the deduced amino acid sequence of Ror2 protein.
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SEQ ID N0:7 is the nucleotide sequence that codes for Ror1-flag protein
SEQ ID NO:8 is the deduced amino acid sequence of Rori-flag protein .
SEQ ID NO:9 is the nucleotide sequence that codes for Ror2-flag protein
SEQ ID NO:10 is the deduced amino acid sequence of Ror2-flag protein.
SEQ ID NO:11 is the nucleotide sequence that codes for Ror2AC-flag
protein.
SEQ ID NO:12 is the deduced amino acid sequence of Ror2AC-flag protein.
SEQ ID NO: 13 is the nucleotide sequence of the top strand primer used to
construct Ror1-flag.
SEQ ID NO: 14 is the nucleotide sequence of the bottom strand primer used
to construct Ror1-flag.
SEQ ID NO:15 is the nucleotide sequence of the first top strand primer used
to construct Ror2-flag.
SEQ ID NO: 16 is the nucleotide sequence of the first bottom strand primer
used to construct Ror2-flag.
SEQ ID NO:17 is the nucleotide sequence of the second top strand primer
used to construct Ror2-flag.
SEQ ID NO:18 is the nucleotide sequence of the second bottom strand
primer used to construct Ror2-flag.
SEQ ID NO:19 is the nucleotide sequence of the third top strand primer used
to construct Ror2-flag.
SEQ ID NO:20 is the nucleotide sequence of the third bottom strand primer
used to construct Ror2-flag.
SEQ ID NO:21 is the nucleotide sequence of the top strand primer used to
construct Ror2KD-flag.
SEQ ID NO:22 is the nucleotide sequence of the bottom strand primer used
to construct Ror2KD-flag.
SEQ ID NO:23 is the nucleotide sequence of the top strand primer used to
construct Ror2AC-flag.
SEQ ID NO:24 is the nucleotide sequence of the bottom strand primer used
to construct Ror2AC-flag.
SEQ ID NO:25 is the nucleotide sequence of the forward primer to identify
human Ror1 (2993-3013). .
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SEQ ID NO:26 is the nucleotide sequence of the reverse primer to identify
human Ror1 (3049-3074).
SEQ ID NO:27 is the nucleotide sequence of the probe to identify human
Ror1 (3018-3044).
SEQ ID NO:28 is the nucleotide sequence of the forward primer to identify
human Ror2 (1149-1169).
SEQ ID NO:29 is the nucleotide sequence of the reverse primer to identify
human Ror2 (1239-1259).
SEQ ID NO:30 is the nucleotide sequence of the probe to identify human
Ror2 (1174-1198).
SEQ ID NO:31 is the nucleotide sequence of the forward primer to identify
mouse Ror1 (2350-2370).
SEQ ID NO:32 is the nucleotide sequence of the reverse primer to identify
mouse Ror1 (2402-2421).
SEQ ID NO:33 is the nucleotide sequence of the probe to identify mouse
Ror1 (2372-2395).
SEQ ID NO:34 is the nucleotide sequence of the forward primer to identify
mouse Ror2 (364-386).
SEQ ID NO:35 is the nucleotide sequence of the reverse primer to identify
mouse Ror2 (429-448).
SEQ ID NO:36 is the nucleotide sequence of the probe to identify mouse
Ror2 (400-424).
SEQ ID NO:37 is the nucleotide sequence of the forward primer to identify
mouse alkaline phosphatase (1354-1373).
SEQ ID NO:38 is the nucleotide sequence of the reverse primer to identify
mouse alkaline phosphatase (1445-1464).
SEQ ID NO:39 is the nucleotide sequence of the probe to identify mouse
alkaline phosphatase (1416-1442).
SEQ ID NO:40 is the nucleotide sequence of the forward primer to identify
mouse osteocalcin (78-96).
SEQ ID NO:41 is the nucleotide sequence of the reverse primer to identify
mouse osteocalcin (124-145).
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SEQ ID NO:42 is the nucleotide sequence of the probe to identify mouse
osteocalcin (98-121).
SEQ ID NO:43 is the nucleotide sequence of the 5' untranslated region of the
human Ror1 gene (-2000 to +1).
SEQ ID NO:44 is the nucleotide sequence of the 5' untranslated region of the
mouse Ror2 gene (-2000 to +1).
SEQ ID NO:45 is the nucleotide sequence of the top strand primer used to
construct the 5' portion of Notch2IC (1-782).
SEQ ID NO:46 is the nucleotide sequence of the bottom strand primer used
to construct the 5' portion of Notch2IC (1-782).
SEQ ID NO:47 is the nucleotide sequence of the top strand primer used to
construct the 3' portion of Notch2IC (783-2307).
SEQ ID NO:48 is the nucleotide sequence of the bottom strand primer used
to construct the 3' portion of Notch2IC (783-2307).
DETAILED DESCRIPTION OF THE INVENTION
Applicants have discovered that expression of genes coding for human
receptor tyrosine kinase-like orphan receptors 1 and 2 (Ror1 and Ror2) is
significantly down-regulated during human osteoblast differentiation. Applicants have
also provided evidence that Ror2 expression is inversely related to the expression of
secreted frizzled-related protein 1 (SFRP-1). SFRP-1 has been reported to be a
potential osteoporosis target that stimulates apoptosis of osteoblasts in vitro and in
vivo (WO 01/19855).
Moreover, Applicants discovered that in osteoblastic cells, Ror1 and Ror2
modulate Wnt signaling pathways that regulate survival of bone-forming osteoblasts.
In one embodiment, Ror2 kinase inhibits Wnt-3 but potentiates Wnt-1 activity.
Furthermore, Ror2 binds to both Wnt-1 and Wnt-3 proteins. In another embodiment,
the cytoplasmic domain of Ror2 is required for potentiation of Wnt-1 but not for
inhibition of Wnt-3 activity. In yet another embodiment, Ror1 kinase inhibits Wnt-3
but has no effect on Wnt-1 activity.
Several Ror2 binding partners have also been identified.
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The present invention provides an expression cassette comprising Ror
polypeptide under the control of a promoter operable in bone cells. The present
invention further provides a composition for modulating bone-related activity
comprising effective amount of Ror molecule or homologues or derivatives or
fragments or variants or mutants thereof.
The present invention provides for a method of screening for agents, the
method comprising: (a) combining a agent with a Ror molecule; and (b) detecting an
effect of said agent on Ror activity; wherein detection of a decrease or an increase in
Ror activity is indicative of an agent being a bone-related agent.
Moreover, the present invention provides for a method of screening for agents
that modulate the binding of Ror to a binding partner comprising: (a) contacting Ror
with the Ror binding partner in the presence of an agent; (b) contacting Ror with a
Ror binding partner in the presence of a control or in the absence of the agent and,
(c) selecting the agent that modulates Ror molecule by comparing the binding of said
Ror to the binding partner in step (a) to the binding of said Ror to the binding partner
in step (b).
The method can also include a method of screening for agents, the method
comprising: (a) combining an agent with an isolated cell comprising a Ror promoter
sequence operatively linked to a reporter gene; and (b) detecting an effect of said
agent on Ror activity; wherein detection of a decrease or an increase in Ror activity
as measured by the reporter is indicative of an agent being a bone-related agent.
The present invention provides methods of administering an agent identified
to modulate Ror expression or activity in the form of a pharmaceutical composition to
subjects to treat bone-related disorders. Moreover, the present invention provides
methods of administering an agent identified to modulate Ror expression to treat
subjects diagnosed with diseases or conditions associated with Wnt signaling
pathway.
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The present invention further provides for the use of an effective amount of an agent
which modulates target Ror molecule expression or activity in the preparation of a
composition for modulating bone-related activity in the treatment of bone related
disorders. Preferably the agent is identified by a screening method provided therein.
The agent which modulates target Ror molecule expression or activity used in
the preparation of the composition may be selected from the group consisting of an
antibody, a small molecule, a peptide, an oligopeptide and a polypeptide.
Preferably an agent for use in accordance with the invention comprises an
antisense nucleic acid or siRNA molecule specific for Ror gene, wherein said
antisense nucleic acid or siRNA molecule recognizes and binds to a nucleic acid
encoding one or more Ror polypeptides, homologues, derivatives, fragments,
variants or mutants thereof.
In an alternative embodiment an agent for use in accordance with the present
invention to modulate Ror molecule expression or activity is capable of binding to a
Ror binding partner.
Where the target Ror module is Ror2 the agent for use in the preparation of a
composition according to the invention may be selected from the group consisting of
ADP/ATP carrier protein, UDP-glucose ceramide glucosyltransferase-like 1, 14-3-3
protein beta/alpha, 14-3-3 protein gamma, ribophorin I, arginine N-methyltransferase
1, cellular apoptosis susceptibility protein, NOTCH2 protein, and human skeletal
muscle LIM-protein 3.
The present invention further provides a method of preparing a composition
for modulating bone-related activity wherein said method comprises:
(i) identifying a bone-related agent by combining an agent with a Ror
molecule and detecting an effect of said agent on Ror activity
(ii) combining an effective amount of the bone-related agent identified in step
(i) with a pharmaceutically acceptable carrier to form said composition.
The present invention also provides additional uses of the Ror molecules,
such as identifying agents that modulate bone formation and/or Wnt signaling,
identifying genes or proteins that participate in bone formation and/or Wnt signaling,
diagnostic uses, uses as pharmaceutical drug targets, evaluating the efficacy of
drugs, and generating host cells and transgenic animals.
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The present provides for a method of identifying proliferating human pre-
osteoblastic cells using Ror2 as a marker, comprising determining expression of
Ror2 gene in a human osteoblastic cell wherein the increased Ror2 expression
identifies the cell as being proliferating pre-osteoblastic ceils.
The present invention further provides for a method of identifying mouse
osteoblastic cells at the stage of matrix maturation using Ror2 as a marker,
comprising determining expression of Ror2 gene in a mouse osteoblastic cell
wherein the increased Ror2 expression identifies the cell as being an osteoblastic
cell at the stage of matrix maturation.
In addition, the present invention provides for a of determining Ror 2 kinase
activity comprising: (a) obtaining Ror2 polypeptide; (b) labeling Ror2 polypeptide in
presence of 32P γ ATP; (c) determining Ror2 kinase activity by measuring the amount
of incorporated 32P wherein the amount of 32P indicates activity of Ror2 kinase.
Definitions of Abbreviations and Terms:
The following definitions are provided for the full understanding of terms and
abbreviations used in this specification.
As used herein and in the appended claims, the singular forms "a", "an", and
"the" include the plural reference unless the context clearly indicates otherwise.
Thus, for example, a reference to "a host cell" includes a plurality of such host cells,
and a reference to "an antibody" is a reference to one or more antibodies and
equivalents thereof known to those skilled in the art, and so forth.
The abbreviations in the specification correspond to units of measure,
techniques, properties or compounds as follows: "g" means gram(s), "mg" means
milligram(s), "ng" means nanogram(s), "kDa" means kilodalton(s), "°C" means
degree(s) Celsius, "cm" means centimeter(s), "s" means second(s), "min" means
minute(s), "h" means hour(s), "I" means liter(s), "ml" means miHiliter(s), "ul" means
microliter(s), "pi" means picoliter(s), "M" means molar, "mM" means millimolar,
-17-

'mmole" means millimole(s), "kb" means kilobase(s), "bp" means base pair(s), and
"RT means room temperature.
"Dulbecco's-modified Eagle Medium" is abbreviated DMEM.
"High performance liquid chromatography" is abbreviated HPLC.
"High throughput screening" is abbreviated HTS.
"Open reading frame" is abbreviated ORF.
"Mass-spectroscopy" is abbreviated MS.
"Tandem mass-spectroscopy" is abbreviated MS/MS.
"Polyacrylamide gel electrophoresis" is abbreviated PAGE.
"Polymerase chain reaction" is abbreviated PCR.
"Reverse transcriptase polymerase chain reaction" is abbreviated RT-PCR.
"Sodium dodecyl sulfate" is abbreviated SDS.
"Sodium dodecyl sulfate-polyacrylamide gel electrophoresis" is abbreviated
SDS-PAGE.
"Human skeletal muscle LIM-protein 3" is abbreviated (SLIM 3).
"Adenine nucleotide translocator 2" is abbreviated ADP/ATP carrier protein.
"Bone Mineral Density" is abbreviated BMD.
"Ribosomal RNA" is abbreviated rRNA".
"Untranslated region" is abbreviated UTR.
"T-cell factor" is abbreviated TCF.
"Dithiothreitol" is abbreviated DTT.
In the context of this disclosure, a number of terms shall be utilized. As used
herein, the term Ror refers to a family of receptor tyrosine kinase-like orphan
receptors. "Ror molecule" refers to Ror polypeptides, Ror peptides, fragments,
variants, and mutants thereof as well as to nucleic acids that encode Ror
polypeptides, Ror peptides and fragments or variants or mutants thereof. "Ror
molecule" also refers to Ror polynucleotides, genes and variants and mutants
thereof. "Ror molecule" and "Ror" refer to both Ror1 and Ror2 molecules.
"Target Ror molecule" refers to a Ror molecule whose activity is modulated
by the agent of the present invention. The target Ror molecule can be Ror
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polypeptide, homologues, derivatives or fragments or variants or mutants thereof.
Ror molecule of interest can also be nucleic acid (oligonucleotide or polynucleotide
of RNA or DNA). For example, if transcripts of genes are the interest of an
experiment, the target Ror molecules would be the transcripts. It is to be understood
that the term target Ror molecule refers to both full-length molecules and to
fragments, variants, and mutants thereof, such as an epitope of a protein. The target
Ror molecule may be either Ror1 molecule or Ror2 molecule or both.
The term "Wnt" refers to a family of conserved, cysteine-rich, secreted
glycoproteins that are involved in critical aspects of early embryonic development.
Wnt genes are also implicated in cancer. "Wnt" as used herein specifically includes
Wnt genes of all human and non-human animal species, including, but not limited to,
mammals, such as human, mouse, rat and other rodents, etc. The term "Wnt"
includes native human Wnt genes and encoded polypeptides, including human Wnt-
1 (previously called int-1) (van Ooyen et al., EMBO J, 4, 2905-9, (1985)), Wnt-3
(Roelink et al., Genomics, 17, 790-792, (1993); Huguet et al., Cancer Res., 54, 2615-
2521, (1994)), and their variants, in particular amino acid sequence variants.
The "Wnt signaling pathway" refers to any pathway modulated by Wnt
proteins such as Wnt-1, Wnt-2, Wnt-3, and the like.
The "canonical Wnt signaling pathway" refers to activation by Wnt proteins of
the Disheveled protein which in turn inhibits glycogen synthetase kinase-3 from
phosphorylating β-catenin. Phosphorylated β-catenin is rapidly degraded following
ubiquitination. However, the unphosphorylated β-catenin accumulates and
translocates to the nucleus where it acts as a cofactor of the T-cell factor
transcription activator complex.
The term "secreted frizzled related protein" or "SFRP" relates to a secreted
receptor of the Wnt signaling pathway.
The term "nucleic acid molecule" refers to the phosphate ester form of
ribonucleotides (RNA molecules) or deoxyribonucleotides (DNA molecules), or any
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phosphodiester analogs, in either single-stranded form, or a double-stranded helix.
Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The
term nucleic acid molecule, and in particular DNA or RNA molecule, refers to the
primary and secondary structure of the molecule, and does not limit it to any
particular tertiary forms. Thus, this term includes double-stranded DNA found, inter
alia, in linear (e.g., restriction fragments) or circular DNA molecules, plasmids, and
chromosomes. In discussing the structure of particular double-stranded DNA
molecules, sequences may be described according to the normal convention of
giving only the sequence in the 5' to 3* direction along the nontranscribed strand of
DNA (i.e., the strand having a sequence homologous to the mRNA).
A "recombinant nucleic acid molecule" is a nucleic acid molecule that has
undergone a molecular biological manipulation, i.e., non-naturally occurring nucleic
acid molecule. Furthermore, the term "recombinant DNA molecule" refers to a
nucleic acid sequence which is not naturally occurring, or can be made by the
artificial combination of two otherwise separated segments of sequence, i.e., by
ligating together pieces of DNA that are not normally continuous. By "recombinantly
produced" is meant artificial combination often accomplished by either chemical
synthesis means, or by the artificial manipulation of isolated segments of nucleic
acids, e.g., by genetic engineering techniques using restriction enzymes, ligases,
and similar recombinant techniques as described by, for example, Sambrook et al.,
Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.;
(1989), or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols
(1989), and DNA Cloning: A Practical Approach, Volumes I and II (ed. D. N. Glover)
IREL Press, Oxford, (1985).
Such is usually done to replace a codon with a redundant codon encoding
the same or a conservative amino acid, while typically introducing or removing a
sequence recognition site. Alternatively, it may be performed to join together nucleic
acid segments of desired functions to generate a single genetic entity comprising a
desired combination of functions not found in the common natural forms. Restriction
enzyme recognition sites are often the target of such artificial manipulations, but
other site specific targets, e.g., promoters, DNA replication sites, regulation
-20-

sequences, control sequences, or other useful features may be incorporated by
design. Examples of recombinant nucleic acid molecule include recombinant
vectors, such as cloning or expression vectors which contain DNA sequences
encoding phi gene proteins which are in a 5' to 3' (sense) orientation or in a 31 to 51
(antisense) orientation.
The terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic
acid molecule", "nucleic acid sequence", "oligonucleotide", refer to a series of
nucleotide bases (also called "nucleotides") in DNA and RNA, and mean any chain
of two or more nucleotides. The polynucleotides can be chimeric mixtures or
derivatives or modified versions thereof, single-stranded or double-stranded. The
oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate
backbone, for example, to improve stability of the molecule, its hybridization
parameters, etc. The antisense oligonuculeotide may comprise a modified base
moiety which is selected from the group including but not limited to 5-fluorouracil, 5-
bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-
(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-
carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-
methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine,
7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-
methylthio-N6- isopentenyladenine, wybutoxosine, pseudouracil, queosine, 2-
thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil- 5-
oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2- thiouracil, 3-(3-
amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. A nucleotide sequence
typically carries genetic information, including the information used by cellular
machinery to make proteins and enzymes. These terms include double- or single-
stranded genomic and cDNA, RNA, any synthetic and genetically manipulated
polynucleotide, and both sense and antisense polynucleotides. This includes single-
and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids,
as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino
acid backbone. This also includes nucleic acids containing modified bases, for
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example thio-uracil, thio-guanine and fluoro-uracil, or containing carbohydrate, or
lipids.
Polynucleotides of the invention may be synthesized by standard methods
known in the art, e.g. by use of an automated DNA synthesizer (such as those that
are commercially available from Biosearch, Applied Biosystems, etc.). As examples,
phosphorothioate oligonucleotides may be synthesized by the method of Stein et al.,
Nucl. Acids Res., 16, 3209, (1988), methylphosphonate oligonucleotides can be
prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl.
Acad. Sci. U.S.A. 85, 7448-7451, (1988), etc. A number of methods have been
developed for delivering antisense DNA or RNA to cells, e.g., antisense molecules
can be injected directly into the tissue site, or modified antisense molecules,
designed to target the desired cells (antisense linked to peptides or antibodies that
specifically bind receptors or antigens expressed on the target cell surface) can be
administered systemically. Alternatively, RNA molecules may be generated by in
vitro and in vivo transcription of DNA sequences encoding the antisense RNA
molecule. Such DNA sequences may be incorporated into a wide variety of vectors
that incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters. Alternatively, antisense cDNA constructs that synthesize
antisense RNA constitutively or inducibly, depending on the promoter used, can be
introduced stably into cell lines. However, it is often difficult to achieve intracellular
concentrations of the antisense sufficient to suppress translation of endogenous
mRNAs. Therefore a preferred approach utilizes a recombinant DNA construct in
which the antisense oligonucleotide is placed under the control of a strong promoter.
The use of such a construct to transfect target cells in the patient will result in the
transcription of sufficient amounts of single stranded RNAs that will form
complementary base pairs with the endogenous target gene transcripts and thereby
prevent translation of the target gene mRNA. For example, a vector can be
introduced in vivo such that it is taken up by a cell and directs the transcription of an
antisense RNA. Such a vector can remain episomal or become chromosomally
integrated, as long as it can be transcribed to produce the desired antisense RNA.
Such vectors can be constructed by recombinant DNA technology methods standard
in the art. Vectors can be plasmid, viral, or others known in the art, used for
-22-

replication and expression in mammalian cells. Expression of the sequence encoding
the antisense RNA can be by any promoter known in the art to act in mammalian,
preferably human cells. Such promoters can be inducible or constitutive. Such
promoters include but are not limited to: the SV40 early promoter region (Bernoist
and Chambon, Nature, 290, 304-310, (1981), the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus, Yamamoto et al., Cell, 22, 787-797, (1980),
the herpes thymidine kinase promoter, Wagner et al., Proc. Natl. Acad. Sci. U.S.A.
78, 1441-1445, (1981), the regulatory sequences of the metallothionein gene
Brinster et al., Nature 296, 39-42, (1982), etc. Any type of plasmid, cosmid, yeast
artificial chromosome or viral vector can be used to prepare the recombinant DNA
construct that can be introduced directly into the tissue site. Alternatively, viral
vectors can be used which selectively infect the desired tissue, in which case
administration may be accomplished by another route (e.g., systemically).
Ribozymes are RNA molecules possessing the ability to specifically cleave
other single-stranded RNA in a manner analogous to DNA restriction endonucleases.
Through the modification of nucleotide sequences that encode these RNAs, it is
possible to engineer molecules that recognize specific nucleotide sequences in an
RNA molecule and cleave it, Cech, J. Amer. Med. Assn., 260, 3030, (1988). A major
advantage of this approach is that, because they are sequence-specific, only mRNAs
with particular sequences are inactivated.
The polynucleotides may be flanked by natural regulatory (expression
control) sequences, or may be associated with heterologous sequences, including
promoters, internal ribosome entry sites (IRES) and other ribosome binding site
sequences, enhancers, response elements, suppressors, signal sequences,
polyadenylation sequences, introns, 5'- and 3'-non-coding regions, and the like. The
nucleic acids may also be modified by many means known in the art. Non-limiting
examples of such modifications include methylation, "caps", substitution of one or
more of the naturally occurring nucleotides with an analog, and internucleotide
modifications such as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
-23-

Polynucleotides may contain one or more additional covalently linked moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-
lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals,
radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides
may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl
phosphoramidate linkage. Furthermore, the polynucleotides herein may also be
modified with a label capable of providing a detectable signal, either directly or
indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and
the like.
"RNA transcript" refers to the product resulting from RNA polymerase-
catalyzed transcription of a DNA sequence. When the RNA transcript is a
complementary copy of the DNA sequence, it is referred to as the primary transcript
or it may be an RNA sequence derived from post-transcriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger RNA (mRNA)"
refers to the RNA that is without introns and can be translated into polypeptides by
the cell. "cRNA" refers to complementary RNA, transcribed from a recombinant
cDNA template. "cDNA" refers to DNA that is complementary to and derived from an
mRNA template. The cDNA can be single-stranded or converted to double-stranded
form using, for example, the Klenow fragment of DNA polymerase I.
A sequence "complementary" to a portion of an RNA, refers to a sequence
having sufficient complementarity to be able to hybridize with the RNA, forming a
stable duplex; in the case of double-stranded antisense nucleic acids, a single strand
of the duplex DNA may thus be tested, or triplex formation may be assayed. The
ability to hybridize will depend on both the degree of complementarity and the length
of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the
more base mismatches with an RNA it may contain and still form a stable duplex (or
triplex, as the case may be). One skilled in the art can ascertain a tolerable degree
of mismatch by use of standard procedures to determine the melting point of the
hybridized complex.
-24-

An "anti-sense" copy of a particular polynucleotide refers to a complementary
sequence that is capable of hydrogen bonding to the polynucleotide and can therefor
be capable of modulating expression of the polynucleotide. These are DNA, RNA or
analogs thereof, including analogs having altered backbones, as described above.
The polynucleotide to which the anti-sense copy binds may be in single-stranded
form or in double-stranded form. A DNA sequence linked to a promoter in an "anti-
sense orientation" may be linked to the promoter such that an RNA molecule
complementary to the coding mRNA of the target gene is produced.
The antisense polynucleotide may comprise at least one modified sugar
moiety selected from the group including but not limited to arabinose, 2-fluoro-
arabinose, xylulose, and hexose. In one embodiment, the antisense oligonucleotide
may comprise at least one modified phosphate backbone selected from the group
consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate,
a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof.
The term "sense" refers to sequences of nucleic acids that are in the same
orientation as the coding mRNA nucleic acid sequence. A DNA sequence linked to a
promoter in a "sense orientation" is linked such that an RNA molecule which contains
sequences identical to an mRNA is transcribed. The produced RNA molecule,
however, need not be transcribed into a functional protein.
A "sense" strand and an "anti-sense" strand when used in the same context
refer to single-stranded polynucleotides that are complementary to each other. They
may be opposing strands of a double-stranded polynucleotide, or one strand may be
predicted from the other according to generally accepted base-pairing rules. Unless
otherwise specified or implied, the assignment of one or the other strand as "sense"
or "antisense" is arbitrary.
The terms "nucleic acid" or "nucleic acid sequence", "nucleic acid molecule",
"nucleic acid fragment" or "polynucleotide" may be used interchangeably with "gene",
"mRNA encoded by a gene" and "cDNA".
-25-

The term "polynucleotide encoding polypeptide" encompasses a
polynucleotide that may include only the coding sequence as well as a
polynucleotide that may include additional coding or non-coding sequence.
A nucleic acid molecule is "hybridizable" to another nucleic acid molecule,
such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic
acid molecule can anneal to the other nucleic acid molecule under the appropriate
conditions of temperature and solution ionic strength, Sambrook, J. et al. eds.,
Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor
Laboratory Press, NY. Vols. 1-3 (ISBN 0-87969-309-6). The conditions of
temperature and ionic strength determine the "stringency" of the hybridization. For
preliminary screening for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a Tm of 55°C, can be used, e.g., 5x SSC, 0.1% SDS,
0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS. Moderate
stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide,
with 5x or 6x SSC. High stringency hybridization conditions correspond to the
highest Tm, e.g., 50% formamide, 5x or 6x SSC. Hybridization requires that the two
nucleic acids contain complementary sequences, although depending on the
stringency of the hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the length of the
nucleic acids and the degree of complementation, variables well known in the art.
The greater the degree of similarity or homology between two nucleotide sequences,
the greater the value of Tm for hybrids of nucleic acids having those sequences. The
relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases
in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than
100 nucleotides in length, equations for calculating Tm have been derived, Sambrook
et al. eds., Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring
Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6), 9.50-9.51). For
hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the oligonucleotide
determines its specificity, Sambrook et al. eds., Molecular Cloning: A Laboratory
-26-

Manual (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-
87969-309-6, 11.7-11.8).
The term "complementary" is used to describe the relationship between
nucleotide bases that are capable to hybridizing to one another. For example, with
respect to DNA, adenosine is complementary to thymine and cytosine is
complementary to guanine.
"Identity" or "similarity", as known in the art, are relationships between two or
more polypeptide sequences or two or more polynucleotide sequences, as
determined by comparing the sequences. In the art, identity also means the degree
of sequence relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such sequences. Both
identity and similarity can be readily calculated by known methods such as those
described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University
Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology,
von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I,
Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and
Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,
New York, 1991. Methods commonly employed to determine identity or similarity
between sequences include, but are not limited to those disclosed in Carillo, H., and
Lipman, D., SIAM J Applied Math., 48:1073 (1988). Methods to determine identity
and similarity are codified in publicly available computer programs. Preferred
computer program methods to determine identity and similarity between two
sequences include, but are not limited to, GCG program package, Devereux, J., et
at., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA
Atschul, S. F. et al., J Molec. Biol., 215,403 (1990)).
"Homologous" refers to the degree of sequence similarity between two
polymers (i.e. polypeptide molecules or nucleic acid molecules). The homology
percentage figures referred to herein reflect the maximal homology possible between
-27-

the two polymers, i.e., the percent homology when the two polymers are so aligned
as to have the greatest number of matched (homologous) positions.
The term "percent homology" refers to the extent of amino acid sequence
identity between polypeptides. The homology between any two polypeptides is a
direct function of the total number of matching amino acids at a given position in
either sequence, e.g., if half of the total number of amino acids in either of the
sequences are the same then the two sequences are said to exhibit 50% homology.
The term "fragment", "analog", and "derivative" when referring to the
polypeptide of the present invention (e.g. SEQ ID NOs:4, 6, 8,10, and 12), refers to a
polypeptide which may retain essentially the same biological function or activity as
such polypeptide. Thus, an analog includes a precursor protein that can be activated
by cleavage of the precursor protein portion to produce an active mature polypeptide.
The fragment, analog, or derivative of the polypeptide of the present invention (e.g.
SEQ ID NOs: 4, 6, 8, 10, and 12) may be one in which one or more of the amino
acids are substituted with a conserved or non-conserved amino acid residues and
such amino acid residues may or may not be one encoded by the genetic code, or
one in which one or more of the amino acid residues includes a substituent group, or
one in which the polypeptide is fused with a compound such as polyethylene glycol to
increase the half-life of the polypeptide, or one in which additional amino acids are
fused to the polypeptide such as a signal peptide or a sequence such as polyhistidine
tag which is employed for the purification of the polypeptide or the precursor protein.
Such fragments, analogs, or derivatives are deemed to be within the scope of the
present invention.
"Conserved" residues of a polynucleotide sequence are those residues that
occur unaltered in the same position of two or more related sequences being
compared. Residues that are relatively conserved are those that are conserved
amongst more related sequences than residues appearing elsewhere in the
sequences.
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Related polynucleotides are polynucleotides that share a significant
proportion of identical residues.
Different poiynucleotides "correspond" to each other if one is ultimately
derived from another. For example, messenger RNA corresponds to the gene from
which it is transcribed. cDNA corresponds to the RNA from which it has been
produced, such as by a reverse transcription reaction, or by chemical synthesis of a
DNA based upon knowledge of the RNA sequence. cDNA also corresponds to the
gene that encodes the RNA. Polynucleotides also "correspond" to each other if they
serve a similar function, such as encoding a related polypeptide in different species,
strains or variants that are being compared.
An "analog" of a DNA, RNA or a polynucleotide, refers to a molecule
resembling naturally occurring polynucleotides in form and/or function (e.g. in the
ability to engage in sequence-specific hydrogen bonding to base pairs on a
complementary polynucleotide sequence) but which differs from DNA or RNA in, for
example, the possession of an unusual or non-natural base or an altered backbone.
See for example, Uhlmann et al., Chemical Reviews 90, 543-584, (1990).
A "coding sequence" or a sequence "encoding" an expression product, such
as an RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when
expressed, results in the production of that RNA, polypeptide, protein, or enzyme,
i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide,
protein, or enzyme.
"Codon degeneracy" refers to divergence in the genetic code permitting
variation of the polynucleotide sequence without affecting the amino acid sequence
of an encoded polypeptide. The skilled artisan is well aware of the "codon-bias"
exhibited by a specific host cell to use nucleotide codons to specify a given amino
acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is
desirable to design the gene such that its frequency of codon usage approaches the
frequency of preferred codon usage of the host cell.
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A "substantial portion" of an amino acid or nucleotide sequence is a portion
comprising enough of the amino acid sequence of a polypeptide or the nucleotide
sequence of a gene to putatively identify that polypeptide or gene, either by manual
evaluation of the sequence by one skilled in the art, or by computer automated
sequence comparison and identification using algorithms such as BLAST (Basic
Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215, 403-410,
(1993); see also www.ncbi.nlm.nih.gov/BLAST).
Accordingly, a "substantial portion" of a nucleotide sequence comprises
enough of the sequence to specifically identify and/or isolate a nucleic acid fragment
comprising the sequence. The skilled artisan, having the benefit of the sequences
as reported herein, may now use all or a substantial portion of the disclosed
sequences for purposes known to those skilled in this art.
"Synthetic genes" can be assembled from oligonucleotide building blocks that
are chemically synthesized using procedures known to those skilled in the art.
These building blocks are ligated and annealed to form gene segments that are then
enzymatically assembled to construct the entire gene. "Chemically synthesized", as
related to a sequence of DNA, means that the component nucleotides were
assembled in vitro. Manual chemical synthesis of DNA may be accomplished using
well-known procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly, the genes can be
tailored for optimal gene expression based on optimization of nucleotide sequence to
reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood
of successful gene expression if codon usage is biased towards those codons
favored by the host. Determining preferred codons can be based on a survey of
genes derived from the host cell where sequence information is available.
"Gene" refers to a nucleic acid fragment that expresses a specific protein,
including regulatory sequences preceding (51 non-coding sequences) and following
(3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as
found in nature with its own regulatory sequences. "Chimeric gene" or "chimeric
construct" refers to any gene or a construct, not a native gene, comprising regulatory
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and coding sequences that are not found together in nature. Accordingly, a chimeric
gene or chimeric construct may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory sequences and
coding sequences derived from the same source, but arranged in a manner different
than that found in nature. "Endogenous gene" refers to a native gene in its natural
location in the genome of an organism. A "foreign" gene refers to a gene not
normally found in the host organism, but which is introduced into the host organism
by gene transfer. Foreign genes can comprise native genes inserted into a non-
native organism, or chimeric genes. A "transgene" is a gene that has been
introduced into the genome by a transformation procedure.
"Regulatory sequences" refer to nucleotide sequences located upstream (5'
non-coding sequences), within, or downstream (31 non-coding sequences) of a
coding sequence, and which influence the transcription, RNA processing or stability,
or translation of the associated coding sequence. Regulatory sequences may
include promoters, translation leader sequences, introns, and polyadenylation
recognition sequences.
"Gene control sequence" refers to the DNA sequences required to initiate
gene transcription plus those required to regulate the rate at which initiation occurs.
Thus a gene control sequence may consist of the promoter, where the general
transcription factors and the polymerase assemble, plus all the regulatory sequences
to which gene regulatory proteins bind to control the rate of these assembly
processes at the promoter. For example, the control sequences that are suitable for
prokaryotes may include a promoter, optionally an operator sequence, and a
ribosome-binding site. Eukaryotic cells may utilize promoters, enhancers, and/or
polyadenylation signals.
"Promoter" refers to a nucleotide sequence capable of controlling the
expression of a coding sequence or functional RNA. In general, a coding sequence
is located 31 to a promoter sequence. The promoter sequence consists of proximal
and more distal upstream elements, the latter elements often referred to as
enhancers. Accordingly, an "enhancer" is a nucleotide sequence that can stimulate
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promoter activity and may be an innate element of the promoter or a heterologous
element inserted to enhance the level or tissue-specificity of a promoter. Promoters
may be derived in their entirety from a native gene, or be composed of different
elements derived from different promoters found in nature, or even comprise
synthetic nucleotide segments. It is understood by those skilled in the art that
different promoters may direct the expression of a gene in different tissues or cell
types, or at different stages of development, or in response to different environmental
conditions.
The "3' non-coding sequences" refer to nucleotide sequences located
downstream of a coding sequence and include polyadenylation recognition
sequences and other sequences encoding regulatory signals capable of affecting
mRNA processing or gene expression. The polyadenylation signal is usually
characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the
mRNA precursor.
The "translation leader sequence" refers to a nucleotide sequence located
between the promoter sequence of a gene and the coding sequence. The
translation leader sequence is present in the fully processed mRNA upstream of the
translation start sequence. The translation leader sequence may affect processing
of the primary transcript to mRNA, mRNA stability or translation efficiency.
The term "operatively linked" refers to the association of two or more nucleic
acid fragments on a single nucleic acid fragment so that the function of one is
affected by the other. For example, a promoter is operatively linked with a coding
sequence when it is capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of the promoter).
Coding sequences can be operatively linked to regulatory sequences in sense or
antisense orientation. A "promoter operable in bone cells" refer to a promoter that is
recognized by the RNA polymerase of the bone cell.
"RNA transcript" refers to the product resulting from RNA polymerase-
catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect
complementary copy of the DNA sequence, it is referred to as the primary transcript
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or it may be an RNA sequence derived from posttranscriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger RNA (mRNA)"
refers to the RNA that is without introns and that can be translated into polypeptide.
"cDNA" refers to a double-stranded DNA that is complementary to and derived from
mRNA. "Sense" RNA refers to an RNA transcript that includes the mRNA and so
can be translated into a polypeptide by the cell. "Antisense RNA" refers to an RNA
transcript that is complementary to all or part of a target primary transcript or mRNA
and that blocks the expression of a target gene (see U.S. Patent No. 5,107,065,
incorporated herein by reference). The complementarity of an antisense RNA may
be with any part of the specific nucleotide sequence, i.e., at the 5' non-coding
sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional
RNA" refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may
not be translated but yet has an effect on cellular processes.
The term "siRNA" or "RNAi" refers to small interfering RNAs, that are capable
of causing interference and can cause post-transcriptional silencing of specific genes
in cells, for example, mammalian cells (including human cells) and in the body, for
example, mammalian bodies (including humans). The phenomenon of RNA
interference is described and discussed in Bass, Nature, 411, 428-29, (2001);
Elbahir et al., Nature, 411, 494-98, (2001); and Fire et al., Nature, 391, 806-11,
(1998), where methods of making interfering RNA also are discussed. The siRNAs
based upon the sequence disclosed herein can be made by approaches known in
the art, including the use of complementary DNA strands or synthetic approaches.
Exemplary siRNAs could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps,
10 bps, 5 bps or any integer thereabout or therebetween.
The term "expression" refers to the transcription and stable accumulation of
sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the
invention. Expression may also refer to translation of mRNA into a polypeptide.
"Antisense inhibition" refers to the production of antisense RNA transcripts capable
of suppressing the expression of the target protein.
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"Overexpression" refers to the production of a gene product in an organism
that exceeds levels of production in normal or non-transformed organisms.
"Suppression" refers to suppressing the expression of foreign or endogenous genes
or RNA transcripts.
"Altered levels" refers to the production of gene product(s) in organisms in
amounts or proportions that differ from that of normal or non-transformed organisms.
Overexpression of the polypeptide of the present invention may be accomplished by
first constructing a chimeric gene or chimeric construct in which the coding region is
operatively linked to a promoter capable of directing expression of a gene or
construct in the desired tissues at the desired stage of development. For reasons of
convenience, the chimeric gene or chimeric construct may comprise promoter
sequences and translation leader sequences derived from the same genes. 3' Non-
coding sequences encoding transcription termination signals may also be provided.
The instant chimeric gene or chimeric construct may also comprise one or more
introns in order to facilitate gene expression. Plasmid vectors comprising the instant
chimeric gene or chimeric construct can then be constructed. The choice of plasmid
vector is dependent upon the method that will be used to transform host cells. The
skilled artisan is well aware of the genetic elements that must be present on the
plasmid vector in order to successfully transform, select and propagate host cells
containing the chimeric gene or chimeric construct. The skilled artisan will also
recognize that different independent transformation events will result in different
levels and patterns of expression, Jones et al., EMBO J., 4, 2411-2418, (1985);
De Almeida et al., Mol. Gen. Genetics, 218, 78-86, (1989), and thus that multiple
events must be screened in order to obtain lines displaying the desired expression
level and pattern. Such screening may be accomplished by southern analysis of
DNA, northern analysis of mRNA expression, western or immunnocytochemical
analysis of protein expression, or phenotypic analysis.
An "expression cassette" refers to a DNA coding sequence or segment of
DNA that codes for an expression product that can be inserted into a vector at
defined restriction sites. The cassette restriction sites are designed to ensure
insertion of the cassette in the proper reading frame. Generally, foreign DNA is
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inserted at one or more restriction sites of the vector DNA, and then is carried by the
vector into a host cell along with the transmissible vector DNA. A segment or
sequence of DNA having inserted or added DNA, such as an expression vector, can
also be called a "DNA construct."
The term "polypeptide" refers to a polymer of amino acids without regard to
the length of the polymer; thus, "peptides," "oligopeptides", and "proteins" are
included within the definition of polypeptide and used interchangeably herein. This
term also does not specify or exclude chemical or post-expression modifications of
the polypeptides of the invention, although chemical or post-expression modifications
of these polypeptides may be included or excluded as specific embodiments.
Therefore, for example, modifications to polypeptides that include the covalent
attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the
like are expressly encompassed by the term polypeptide. Further, polypeptides with
these modifications may be specified as individual species to be included or
excluded from the present invention. The natural or other chemical modifications,
such as those listed in examples above can occur anywhere in a polypeptide,
including the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. It will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given polypeptide. Also,
a given polypeptide may contain many types of modifications. Polypeptides may be
branched, for example, as a result of ubiquitination, and they may be cyclic, with or
without branching. Modifications include acetylation, acylation, ADP-ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of
a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation, formation of covalent cross-
links, formation of cysteine, formation of pyroglutamate, formylation, gamma-
carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination,
methylation, myristoylation, oxidation, pegylation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
(See, for instance, proteins--structure and molecular properties, 2nd Ed., T. E.
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Creighton, W. H. Freeman and Company, New York (1993); posttranslational
covalent modification of proteins, b. c. Johnson, Ed., Academic Press, New York,
pgs. 1-12, 1983; Seifter et al., Meth Enzymol 182:626-646, 1990; Rattan et al., Ann
NY Acad Sci 663:48-62, 1992). Also included within the definition are polypeptides
which contain one or more analogs of an amino acid (including, for example, non-
naturally occurring amino acids, amino acids which only occur naturally in an
unrelated biological system, modified amino acids from mammalian systems etc.),
polypeptides with substituted linkages, as well as other modifications known in the
art, both naturally occurring and non-naturally occurring. The term "polypeptide" may
also be used interchangeably with the term "protein"or "peptide".
The term "peptide" refers to any polymer of two or more amino acids, wherein
each amino acid is linked to one or two other amino acids via a peptide bond
(-CONH-) formed between the NH.sub.2 and the COOH groups of adjacent amino
acids. Preferably, the amino acids are naturally occurring amino acids, particularly
alpha.-amino acids of the L-enantiomeric form. However, other amino acids,
enantiomeric forms, and amino acid derivatives may be included in a peptide.
Peptides include "polypeptides," which, upon hydrolysis, yield more than two amino
acids. Polypeptides may include proteins, which typically comprise 50 or more
amino acids.
The terms "variant" or "variants" refer to variations of the nucleic acid or
amino acid sequences of Ror molecule. Encompassed within the term "variant(s)"
are nucleotide and amino acid substitutions, additions, or deletions of Ror molecules.
Also, encompassed within the term "variant(s)" are chemically modified natural and
synthetic Ror molecules. For example, variant may refer to polypeptides that differ
from a reference polypeptide. Generally, the differences between the polypeptide
that differs in amino acid sequence from reference polypeptide, and the reference
polypeptide are limited so that the amino acid sequences of the reference and the
variant are closely similar overall and, in some regions, identical. A variant and
reference polypeptide may differ in amino acid sequence by one or more
substitutions, deletions, additions, fusions and truncations that may be conservative
or non-conservative and may be present in any combination. For example, variants
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may be those in which several, for instance from 50 to 30, from 30 to 20, from 20 to
10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acids are inserted,
substituted, or deleted, in any combination. Additionally, a variant may be a
fragment of a polypeptide of the invention that differs from a reference polypeptide
sequence by being shorter than the reference sequence, such as by a terminal or
internal deletion. A variant of a polypeptide of the invention also includes a
polypeptide which retains essentially the same biological function or activity as such
polypeptide, e.g., precursor proteins which can be activated by cleavage of the
precursor portion to produce an active mature polypeptide. These variants may be
allelic variations characterized by differences in the nucleotide sequences of the
structural gene coding for the protein, or may involve differential splicing or post-
translational modification. Variants also include a related protein having substantially
the same biological activity, but obtained from a different species. The skilled artisan
can produce variants having single or multiple amino acid substitutions, deletions,
additions, or replacements. These variants may include, inter alia: (i) one in which
one or more of the amino acid residues are substituted with a conserved or non-
conserved amino acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the genetic code,
or (ii) one in which one or more amino acids are deleted from the peptide or protein,
or (iii) one in which one or more amino acids are added to the polypeptide or protein,
or (iv) one in which one or more of the amino acid residues include a substituent
group, or (v) one in which the mature polypeptide is fused with another compound,
such as a compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), or (vi) one in which the additional amino acids are fused to the
mature polypeptide such as a leader or secretory sequence or a sequence which is
employed for purification of the mature polypeptide or a precursor protein sequence.
A variant of the polypeptide may also be a naturally occurring variant such as a
naturally occurring allelic variant, or it may be a variant that is not known to occur
naturally. All such variants defined above are deemed to be within the scope of
teachings in the art.
The polypeptides and the polynucleotides of the present invention are
preferably provided in an isolated form, and may be purified to homogeneity.
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The term "isolated" means that the material is removed from its original or
native environment (e.g., the natural environment if it is naturally occurring).
Therefore, a naturally-occurring polynucleotide or polypeptide present in a living
animal is not isolated, but the same polynucleotide or polypeptide, separated by
human intervention from some or all of the coexisting materials in the natural system,
is isolated. For example, an "isolated nucleic acid fragment" is a polymer of RNA or
DNA that is single- or double-stranded, optionally containing synthetic, non-natural or
altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic DNA or
synthetic DNA and combined with carbohydrate, lipid, protein or other materials.
Such polynucleotides could be part of a vector and/or such polynucleotides or
polypeptides could be part of a composition, and still be isolated in that such vector
or composition is not part of the environment in which it is found in nature. Similarly,
the term "substantially purified" refers to a substance, which has been separated or
otherwise removed, through human intervention, from the immediate chemical
environment in which it occurs in Nature. Substantially purified polypeptides or
nucleic acids may be obtained or produced by any of a number of techniques and
procedures generally known in the field.
The term "purification" refers to increasing the specific activity or
concentration of a particular polypeptide or polypeptides in a sample. In one
embodiment, specific activity is expressed as the ratio between the activity of the
target polypeptide and the concentration of total polypeptide in the sample. In
another embodiment, specific activity is expressed as the ratio between the
concentration of the target polypeptide and the concentration of total polypeptide.
Purification methods include but are not limited to dialysis, centrifugation, and column
chromatography techniques, which are well-known procedures to those of skill in the
art. See, e.g., Young et al., 1997, "Production of biopharmaceutical proteins in the
milk of transgenic dairy animals," BioPharm 10(6): 34-38.
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The terms "substantially pure" and "isolated" are not intended to exclude
mixtures of polynucleotides or polypeptides with substances that are not associated
with the polynucleotides or polypeptides in nature.
The terms "cell," "cell line," and "cell culture" may be used interchangeably.
All of these terms also include their progeny, which is any and all subsequent
generations. It is understood that all progeny may not be identical due to deliberate
or inadvertent mutations. In the context of expressing a heterologous nucleic acid
sequence, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial cells
such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells,
fish cells, and insect cells), whether located in vitro or in vivo. For example, host and
may include any transformable organisms that are capable of replicating a cells may
be located in a transgenic animal. Host cell can be used as a recipient for vectors
vector and/or expressing a heterologous nucleic acid encoded by a vector.
General methods for expressing and recovering foreign protein produced by
a mammalian cell system are provided by, for example, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles
and Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996). Standard
techniques for recovering protein produced by a bacterial system is provided by, for
example, Grisshammer et al., "Purification of over-produced proteins from E. coli
cells," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Transformation of insect cells and
production of foreign polypeptides therein is disclosed by Guarino et al., US5162222
and WIPO publication WO94/06463. Methods for isolating recombinant proteins
from a baculovirus system are also described by Richardson (ed.), "Baculovirus
Expression Protocols" (The Humana Press, Inc. 1995). In one embodiment, the
polypeptides of the invention can be expressed using a baculovirus expression
system (see, Luckow et al., Bio/Technology, 1988, 6, 47, "Baculovirus Expression
Vectors: a Laboratory Manual", O'Rielly et al. (Eds.), W. H. Freeman and Company,
New York, 1992, US4,879,236, each of which is incorporated herein by reference in
its entirety). In addition, the MAXBAC.TM. complete baculovirus expression system
(Invitrogen) can, for example, be used for production in insect cells.
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The polypeptides of the present invention can also be isolated by exploitation
of particular properties. For example, immobilized metal ion adsorption (IMAC)
chromatography can be used to purify histidine-rich proteins, including those
comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to
form a chejate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-rich proteins
will be adsorbed to this matrix with differing affinities, depending upon the metal ion
used, and will be eluted by competitive elution, lowering the pH, or use of strong
chelating agents. Other methods of purification include purification of glycosylated
proteins by lectin affinity chromatography and ion exchange chromatography (M.
Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additional embodiments of
the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-
binding protein, an immunoglobulin domain) may be constructed to facilitate
purification.
Host cells of the invention can be used in methods for the large-scale
production of Ror polypeptides wherein the cells are grown in a suitable culture
medium and the desired polypeptide products are isolated from the cells, or from the
medium in which the cells are grown, by purification methods known in the art, e.g.,
conventional chromatographic methods including immunoaffinity chromatography,
receptor affinity chromatography, hydrophobic interaction chromatography, lectin
affinity chromatography, size exclusion filtration, cation or anion exchange
chromatography, high pressure liquid chromatography (HPLC), reverse phase
HPLC, and the like. Other methods of purification include those methods wherein
the desired protein is expressed and purified as a fusion protein having a specific
tag, label, or chelating moiety that is recognized by a specific binding partner or
agent. The purified protein can be cleaved to yield the desired protein, or can be left
as an intact fusion protein. Cleavage of the fusion component may produce a form
of the desired protein having additional amino acid residues as a result of the
cleavage process.
The term "in Situ:" refers to and includes the terms "in vivo," "ex vivo," and
"in vitro" as these terms are commonly recognized and understood by persons
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ordinarily skilled in the art. Furthermore, the phrase "in situ" is employed herein in its
broadest connotative and denotative contexts to identify an entity, cell or tissue as
found or in place, without regard to its source or origin, its condition or status or its
duration or longevity at that location or position.
The term "in vitro" refers to an artificial environment and to reactions or
processes that occur within an artificial environment. In vitro environments include,
but are not limited to, test tubes and cell cultures. The term "in vivo" refers to the
natural environment (e.g., an animal or a cell) and to processes or reaction that
occur within a natural environment.
The methods of the present invention may be performed in vitro using cells
(cultured cells) and cell lysates, including nuclear extracts. Examples of cells
contemplated for identifying agents that modulate bone formation include but are not
limited to calvarial cells, osteoblasts, osteoclasts, chondrocytes, and pluripotent
precursor cells, such as multipotent bone marrow stromal cells. Specific examples of
osteoblast and osteoblast precursor cell lines include MC3T3-E1, C2C12, MG-63
cells, U2OS cells, UMR106 cells, ROS 17/2.8 cells, SaOS-2 cells, and the like that
are provided in the catalog from the ATCC (WO 01/19855) as well as HOB cell lines
described in Bodine PV, Vernon SK, Komm BS., Endocrinology, 137, 4592-4604,
(1996), Bodine PVN, TrailSmith M, Komm BS., J Bone Min Res, 11, 806-819, (1996),
Bodine PV, Green J, Harris HA, Bhat RA, Stein GS, Lian JB, Komm BS., J Cell
Biochem, 65, 368-387, (1997), Bodine PV, Komm BS., Bone, 25, 535-43 (1999),
Bodine PVN, Harris HA, Komm BS., Endocrinology, 140, 2439-2451, (1999),
Prince M, Banerjee C, Javed A, Green J, Lian JB, Stein GS, Bodine PV, Komm BS, J
Cell Biochem, 80, 424-40, (2001).
The methods of the present invention may also be performed using a cell-free
system.
The term "expression system" refers to a host cell and compatible vector
under suitable conditions, e.g., for the expression of a protein coded for by foreign
DNA carried by the vector and introduced into the host cell. Common expression
systems include E. coli host cells and plasmid vectors, insect host cells and
Baculovirus vectors, and mammalian host cells and vectors.
-41 -

"Transformation" refers to the transfer of a nucleic acid fragment into the
genome of a host organism, resulting in genetically stable inheritance. Host
organisms containing the transformed nucleic acid fragments are referred to as
"transgenic" organisms.
"Clone" refers to a population of cells derived from a single cell or common
ancestor by mitosis. A "cell line" refers to a clone of a primary cell that is capable of
stable growth in vitro for several generations.
The term "differentiate" refers to having a different character or function from
the original type of tissues or cells. Thus, "differentiation" is the process or act of
differentiating.
The term "osteoblast differentiation" refers to the process in which a cell
develops specialized functions during maturation into an osteoblast cell. Osteoblast
differentiation may include pre-osteoblast, early and mature osteoblast, pre-
osteocyte and mature osteocyte stages (Bodine et al, Vitamins and Hormones 65,
101-151 (2002), Stein et al. Endocrine Reviews 14, 424-442 (1993), and Lian et al.
Vitamins and Hormones 55,443-509 (1999)).
The term "proliferation" refers to the growth and production of similar cells.
The term "phenotype" refers to the observable character of a cell or an
WE CLAIM:
1. An expression cassette comprising a polynucleoticle encoding a Ror
polypeptide or homologues or derivatives or fragments or variants or mutants thereof
wherein said polynucleotide is under the control of a promoter operable in bone cells.
2. The expression cassette of Claim 1, wherein Ror polypeptide is Ror1
polypeptide.
3. The expression cassette of Claim 1, wherein Ror polypeptide is Ror2
polypeptide.
4. The expression cassette of Claim 1, wherein said promoter is heterologous to
the coding sequence.
5. The expression cassette of Claim 1, wherein the promoter is a bone-specific
promoter.
6. The expression cassette of Claim 5, wherein the bone-specific promoter is a
rat 3.6 kb type I collagen or rat 1.7 kb osteocalcin promoter.
7. The expression cassette of any of Claims 1 to 6, wherein said promoter is an
inducible promoter.
8. A vector comprising the expression cassette of any of Claims 1 to 7.
9. The vector of Claim 8, wherein the vector is a viral vector.
10. The vector of Claim 9, wherein said viral vector is selected from the group
consisting of a retroviral vector, an adenoviral vector, an adeno-associated viral
vector, a vaccinia viral vector, and a herpes viral vector.
11. The expression cassette of Claim 1, wherein said expression cassette further
comprises a polyadenylation signal.

12. A host cell comprising an expression cassette comprising a polynucleotide
encoding a Ror polypeptide or homologues or derivatives or fragments or variants or
mutants thereof, wherein said polynucleotide is under the control of a promoter
operable in eukaryotic cells, said promoter being heterologous to said polynucleotide.
13. The host cell of Claim 12, wherein Ror polypeptide is Ror1 polypeptide.
14. The host cell of Claim 12, wherein Ror polypeptide is Ror2 polypeptide.
15. A composition for modulating bone-related activity comprising an effective
amount of Ror molecule or homologues or derivatives or fragments or variants or
mutants thereof.
16. The composition of Claim 15, wherein Ror molecule is Ror1 molecule.
17. The composition of Claim 15, wherein Ror molecule is Ror2 molecule.
18. The composition of Claim 15, further comprising a pharmaceutically
acceptable carrier.
19. The composition of Claim 15, wherein bone-related activity is osteoblast
differentiation, osteoclast differentiation, osteoblast survival, osteoclast survival,
osteoblast activity, or osteoclast activity.
20. A method of screening for agents, the method comprising: (a) combining an
agent with a Ror molecule; and (b) detecting an effect of said agent on Ror activity;
wherein detection of a decrease or an increase in Ror activity is indicative of an
agent being a bone-related agent.
21. The method of Claim 20, wherein a decrease or an increase in Ror activity is
detected by a decrease or an increase in Ror-induced inhibition of Wnt-3 signaling.
22. The method of Claims 20, wherein Ror molecule is Ror1 molecule and Ror
activity is Ror1 activity.
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23. The method of Claims 20, wherein Ror molecule is Ror2 molecule and Ror
activity is Ror2 activity.
24. The method of Claim 20, wherein Ror molecule is Ror2 molecule and a
decrease or an increase in Ror activity is detected by a decrease or an increase in
Ror2-induced activation of Wnt-1 signaling.
25. The method of Claim 20, wherein Ror molecule is a Ror polypeptide and a
decrease or an increase in Ror activity is detected by a decrease or an increase in
Ror autophosphorylation.
26. The method of Claim 25, wherein Ror polypeptide is Ror1 polypeptide, and
Ror activity is Ror1 activity.
27. The method of Claim 25, wherein Ror polypeptide is Ror2 polypeptide, and
Ror activity is Ror2 activity.
28. A method of screening for agents, the method comprising: (a) combining an
agent with an isolated cell comprising a Ror promoter sequence operatively linked to
a reporter gene; and (b) detecting an effect of said agent on reporter activity;
wherein detection of a decrease or an increase in Ror promoter activity as measured
by the reporter activity is indicative of an agent being a bone-related agent.
29. The method of Claim 28, wherein Ror promoter is Ror1 promoter.
30. The method of Claim 29, wherein Ror1 promoter is human Ror1 promoter.

31. The method of Claim 28, wherein Ror promoter is Ror2 promoter.
32. The method of Claim 31, wherein Ror2 promoter is mouse Ror2 promoter.
33. A method of screening for agents that modulate the binding of Ror
polypeptide to a binding partner comprising: (a) contacting Ror polypeptide with a
Ror binding partner in the presence of an agent; (b) contacting Ror polypeptide with
a Ror binding partner in the presence of a control or in the absence of the agent; and
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(c) selecting the agent that modulates Ror polypeptide binding to Ror binding partner
by comparing the binding of said Ror polypeptide to the binding partner in step (a) to
the binding of said Ror polypeptide to a binding partner in step (b).
34. The method of Claim 33, wherein Ror polypeptide is Ror2 polypeptide and
Ror binding partner is Ror2 binding partner.
35. The method of Claim 34, wherein Ror2 binding partner is selected from the
group consisting of ADP/ATP carrier protein, UDP-glucose ceramide
glucosyltransferase-like 1, 14-3-3 protein beta/alpha, 14-3-3 protein gamma,
ribophorin I, arginine N-methyltransferase 1, cellular apoptosis susceptibility protein,
NOTCH2 protein, and human skeletal muscle LIM-protein 3.
36. A method of modulating bone-related activity in a subject comprising
administering to a subject an agent which modulates target Ror molecule expression
or activity.
37. The method of Claim 36, wherein said agent comprises one or more of Ror
molecules or homologues or derivatives or fragments or variants or mutants thereof.
38. The method of Claim 36, wherein said agent comprises one or more of Ror
molecule binding partners or homologues or derivatives or fragments or variants or
mutants thereof.
39. The method of Claims 36, wherein target Ror molecule is Ror1 molecule.
40. The method of Claims 36, wherein target Ror molecule is Ror2 molecule.
41. The method of Claim 36, wherein target Ror molecule activity is tyrosine
kinase activity.
42. The method of Claim 36, wherein bone-related activity is osteoblast
differentiation, osteoclast differentiation, osteoblast survival, osteoclast survival,
osteoblast activity, or osteoclast activity.
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13. The method of Claim 36, wherein said agent is selected from the group
consisting of an antibody, a small molecule, a peptide, an oligopeptide, and a
polypeptide.
44. The method of Claim 36, wherein said agent comprises an antisense nucleic
acid or siRNA molecule specific for Ror gene and wherein antisense nucleic acid or
siRNA molecule recognize and bind to a nucleic acid encoding one or more Ror
polypetides or homologues or derivatives or fragments or variants or mutants thereof.
45. The method of Claim 44, wherein Ror gene is Ror1 gene and Ror polypeptide
is Ror1 polypeptide.
46. The method of Claim 44, wherein Ror gene is Ror2 gene and Ror polypeptide
is Ror2 polypeptide.
47. The method of Claim 36, wherein said agent modulates expression and/or
activity of target Ror molecules or homologues or derivatives or fragments or variants
or mutants thereof by binding to Ror binding partners.
48. The method of Claim 47, wherein said agent inhibits expression and/or
activity of target Ror molecules or homologues or derivatives or fragments or variants
or mutants thereof by binding to Ror binding partners.
49. The method of Claim 47, wherein said agent enhances expression and/or
activity of target Ror molecules or homologues or derivatives or fragments or variants
or mutants by binding to Ror binding partners.

50. The method as in any one of Claims 47-49, wherein target Ror molecule is
Ror2 molecule and Ror binding partner is Ror2 binding partner.
51. The method of Claim 50, wherein Ror2 binding partners are selected from the
group consisting of ADP/ATP carrier protein, UDP-glucose ceramide
glucosyltransferase-like 1, 14-3-3 protein beta/alpha, 14-3-3 protein gamma,
ribophorin I, arginine N-methyltransferase 1, cellular apoptosis susceptibility protein,
NOTCH2 protein, and human skeletal muscle LIM-protein 3.
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52. The method of Claim 36, wherein said agent modulates binding of Ror
molecules or homologues or derivatives or fragments or variants or mutants thereof
to Ror binding partners.
53. The method of Claim 52, wherein said agent enhances binding of Ror
molecules or homologues or derivatives or fragments or variants or mutants thereof
to Ror binding partners.
54. The method of Claim 52, wherein said agent inhibits binding of Ror moelcules
or homologues or derivatives or fragments or variants or mutants thereof to Ror
binding partners.
55. The method as in any one of claims 52, wherein Ror molecule is Ror2
molecule and Ror binding partner is Ror2 binding partner.
56. The method of Claim 55, wherein Ror2 binding partners are selected from the
group consisting of ADP/ATP carrier protein, UDP-glucose ceramide
glucosyltransferase-like 1, 14-3-3 protein beta/alpha, 14-3-3 protein gamma,
ribophorin I, arginine N-methyltransferase 1, cellular apoptosis susceptibility protein,
NOTCH2 protein, and human skeletal muscle LIM-protein 3.
57. The method of Claim 36, wherein the agent is administered to isolated cells in
culture.

58. The method of Claim 57, wherein the cells are primary osteoblasts,
immortalized cell lines of osteoblastic origin, or immortalized cell lines of non-
osteoblastic origin.
59. The method of Claim 58, wherein immortalized cell lines of osteoblastic origin
are selected from the group consisting of HOB, U2OS, and SaOS-2 cells.
60. The method of Claim 36, wherein the agent is administered to a non-human
transgenic animal.
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61. The method of Claim 60, wherein, the transgenic animal is a mouse.
62. The method of Claim 60, wherein the agent is administered to a non-human
knockout animal.
63. The method of Claim 36, wherein said subject is a vertebrate or an
invertebrate organism.
64. The method of Claim 36, wherein said subject is a mammal.
65. The method of Claim 64, wherein said mammal is a human.
66. A method of modulating Wnt-1 and Wnt-3 activity in a subject comprising
administering an agent which modulates target Ror2 molecule expression or activity
in an amount effective to regulate Wnt-1 and Wnt-3 activity.
67. A method for identifying an agent for modulating bone-related activity
comprising: (a) expressing Ror molecule in a cell or using endogenous Ror
expression; (b) contacting the cell with the agent; and (c) monitoring the expression
or the activity of Ror molecule wherein an increase or decrease in the expression or
activity of Ror molecule in the presence of the agent identifies the agent as
modulating bone-related activity.
68. The method of Claim 67, wherein Ror molecule is Ror1 molecule.
69. The method of Claim 67, wherein Ror molecule is Ror2 molecule.
70. The method of Claim 67, wherein Ror molecule activity is tyrosine kinase
activity.
71. The method of Claim 67, wherein bone-related activity is osteoblast
differentiation, osteoclast differentiation, osteoblast survival, osteoclast survival,
osteoblast activity, or osteoclast activity.
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72. The method of Claim 67, wherein said agent is selected from the group
consisting of antibody, small molecule, peptide, oligopeptide, ribozyme, and
polypeptide.
73. The method of Claim 67, wherein said agent comprises an antisense nucleic
acid or siRNA molecule specific for Ror gene and wherein antisense nucleic acid or
siRNA molecule recognize and bind to a nucleic acid encoding one or more Ror
polypetides or homologues or derivatives or fragments or variants or mutants thereof.
74. The method of Claim 73, wherein Ror gene is Ror1 gene and Ror polypeptide
is Ror1 polypeptide.
75. The method of Claim 73, wherein Ror gene is Ror2 gene and Ror polypeptide
is Ror2 polypeptide.
76. The method of Claim 67, wherein said agent modulates expression and/or
activity of Ror molecules or homologues or derivatives or fragments or variants or
mutants thereof by binding to Ror binding partners.
77. The method of Claims 76, wherein Ror molecule is Ror2 molecule and Ror
binding partner is Ror2 binding partner.
78. The method of Claim 77, wherein Ror2 binding partners are selected from the
group consisting of ADP/ATP carrier protein, UDP-glucose ceramide
glucosyltransferase-like 1, 14-3-3 protein beta/alpha, 14-3-3 protein gamma,
ribophorin I, arginine N-methyltransferase 1, cellular apoptosis susceptibility protein,
NOTCH2 protein, and human skeletal muscle LiM-protein 3.
79. The method of Claim 67, wherein said agent modulates binding of Ror
molecules or homologues or derivatives or fragments or variants or mutants thereof
to Ror binding partners.
80. The method of Claims 79, wherein Ror molecule is Ror2 molecule and Ror
binding partner is Ror2 binding partner.
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81. The method of Claim 67, wherein the cells are primary osteoblasts,
immortalized cell lines of osteoblastic origin, or immortalized cell lines of non-
osteoblastic origin
82. The method of Claim 81, wherein immortalized cell lines of osteoblastic origin
are selected from the group consisting of HOB, U2OS, and SaOS-2 cells.
83. The method of Claim 67, wherein the agent is further administered to a
vertebrate organism to monitor the expression or the activity of Ror molecule wherein
an increase or decrease in the expression or activity of Ror molecule in the presence
of the agent identifies the agent as modulating bone-related activity.
84. The method of Claim 83, wherein said vertebrate organism is a mammal.
85. The method of Claim 83, wherein said vertebrate organism is a non-human
transgenic animal.
86. A method for identifying an agent for modulating Wnt signaling pathway
comprising: screening one or more agents for the ability to modulate expression or
activity of Ror molecule, wherein the agent that can modulate expression or activity
of Ror molecule is an agent that modulates Wnt signaling pathway.
87. A method of linking a bioactive molecule to a cell expressing a Wnt
polypeptide, said method comprising contacting a cell with a Ror2 polypeptide that is
bound to a bioactive molecule and allowing a Wnt polypeptide and said Ror2
polypeptide to bind to one another, thereby linking said bioactive molecule to said
cell.
88. The method of Claim 87, wherein Wnt polypeptide is selected from the group
consisting of Wnt-1 and Wnt-3.
89. A method for screening a subject for a bone-related disorder comprising the
steps of: measuring the expression of Ror molecule in a subject and determining the
relative expression of said Ror molecule in the subject compared to its expression in
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normal subjects, or compared to its expression in the same subject after being
treated for bone-related disorders.
90. A method of identifying genes that participate in bone formation comprising:
a) overexpressing Ror molecule in a cell, b) monitoring the changes in gene
expression profile and c) determining which genes are regulated by Ror expression
thereby identifying genes that participate in bone formation.
91. A method of identifying genes that modulate Wnt signaling pathway
comprising: a) overexpressing Ror molecule in a cell, b) monitoring the changes in
gene expression profile and c) determining which genes are regulated by Ror
expression thereby identifying genes that modulate Wnt signaling pathway.
92. A method for identifying proliferating human pre-osteoblastic cells using Ror2
as a marker, comprising determining expression of Ror2 gene in a human
osteoblastic cell wherein the increased Ror2 expression identifies the cell as being
proliferating pre-osteoblastic cells.

This invention relates to modulating bone-related activity in a subject by modulating Ror molecules. The invention further relates to compositions and methods for the screening, diagnosis and development of therapies for bone-related disorders.