Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
[0001] COMPOSITIONS AND METHODS FOR THE TREATMENT OF
OSTEOPOROSIS.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and compositions for
the treatment of bone related disorders. In addition, the present invention
relates to
the field of recombinant DNA technology transgenic animals and signal
transduction.
BACKGROUND OF THE INVENTION
[0003] Osteoporosis is a debilitating bone disease that is associated
primarily with the ageing skeleton and characterized by reduced bone mass and
micro-architectural damage, which leads to increased bone fragility and
susceptibility to fracture. The amount and rate at which bone is lost is
determined
in large part by genetics, as well as alterations in the availability of
circulating
hormones and locally-derived factors (Ralston 2000, Curr Opin Pharmacol. 3:286-
290, Goltzman 2002, Nature Reviews: Drug Discovery 1:784-796). The bone
modeling and remodeling coordinated signaling by these hormones and growth
factors results in a balance between anabolic and catabolic activity. An
imbalance
that favors the anabolic activity of osteoblasts (Ducy et al. 2000, Science
299:1501-1504) over the catabolic activity of osteoclasts (Teitelbaum et al.
2003,
Nature reviews Genetics 4:638-649) results in a net gain in bone, such as that
seen in physiologic bone growth and in osteopetrotic disorders. The converse
is
known to result in osteoporosis (Harada et al. 2003, Nature 423:349-55).
[0004] Osteoporosis currently affects 1.4 million Canadians over the
age of 50 and more than 3 times that number are estimated to have a reduction
in
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bone mass sufficient to predisposes them to fracture. As the mean age of the
Canadian population increases over the next 20 years it is estimated that the
$1.5
billion in direct healthcare costs currently spent on osteoporosis and related
fractures will increase to more than $30 billion. The situation described
above for
the Canadian population occurs in all industrial countries worldwide. There is
therefore an urgent need to develop specific therapeutic strategies to prevent
bone
loss and promote bone growth in the ageing population.
[0005] The skeleton is continuously renewed throughout life in a
homeostatic process called remodeling that involves resorption of old bone by
osteoclasts and formation of new bone by osteoblasts. In "post -menopausal" or
estrogen-deficiency osteoporosis, this process has been attributed to
increased
osteoclast survival and increased osteoblast apoptosis, whereas "senile" or
age-
related osteoporosis, which affects both men and women, is a consequence of a
decrease in the number of pre-osteoblasts and an increase in osteoblast
apoptosis.
[0006] The major focus of drug development for osteoporosis has been
aimed at an inhibition of osteociast function to prevent excessive bone loss
in post-
menopausal women. These drugs are known as anti-resorptive drugs. Examples
include the bisphosphonates and pyrophosphate analogs that bind with high
affinity to bone mineral and induce osteoclasts apoptosis. Although these
compounds are potent inhibitors of bone resorption, patient compliance is
often
low due to gastric intolerance. They may also inhibit bone apposition by
osteoblasts, which would exacerbate the existing problem of low bone mass.
[0007] Estrogenic compounds act via the classic steroid hormone
receptor pathway to inhibit expression of cytokines that promote osteoclast
formation. Treatment with estrogen effectively prevents the rapid bone loss
that
accompanies the decline in ovarian function at menopause but may be harmful in
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the long term by increasing the risk of uterine and breast cancer, coronary
heart
disease and stroke. Selective estrogen receptor modulators (SERMs) are not as
effective as estrogen in inhibiting bone loss but have the advantage that they
do
not carry an increased risk for breast or uterine cancer. Their potential
cardiovascular risks or benefits have not been evaluated.
[0008] Another class of osteoporosis drugs includes anabolic agents
such as estrogen, PTH and growth factors: Estrogen and SERMS stimulate
osteoblast proliferation via the classic pathway that involves interaction
with the
transcriptional machinery. It has been proposed that they also inhibit
osteoblast
apoptosis via a non-classic pathway that involves interaction with the
Src/Ras/MAP
kinase pathway. Unlike estrogen and its receptors, parathyroid hormone (PTH)
and its analogs have both anabolic and catabolic functions in bone.
Intermittent
administration PTH increases bone mass by increasing the size of the pre-
osteoblast pool and by promoting osteoblast survival. For this reason it has
been
exploited by the pharmaceutical industry and, despite evidence that it caused
osteosarcomas when administered intermittently to rats, it was recently
approved
by the FDA as the first anabolic agent for the treatment of severe
osteoporosis.
However, PTH is also the major hormonal regulator of calcium homeostasis and
can promote bone resorption and hypercalcemia when present continuously at
high concentrations. This represents a serious risk when used for human
consumption. Nevertheless, short-term clinical studies have demonstrated that
PTH increases bone mass and reduces fracture incidence. Growth factors
represent the third major class of compounds that has been tested for anabolic
activity in bone. They include parathyroid hormone related protein (PTHrP),
insulin
like growth factor (IGF-1 ), fibroblast growth factors (FGFs), transforming
growth
factors (TGFs) and bone morphogenetic proteins (BMPs). All of these growth
factors signal through cell surface receptors linked to the Ras/MAP kinase
signaling pathway and promote osteoblast survival by inhibiting apoptosis.
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(0009] The identification of genetic and epi-genetic factors that regulate
bone growth and skeletal remodeling have relied on inbred and congenic strains
of
mice, as well as animal models harboring spontaneous and targeted mutations in
their genomes (Lazner et al. 1999, Human Mol Genetics 8:1839-1846, Huang et
al.
2003, Osteoporosis Intl 14:701-715). The Src tyrosine kinase was shown to play
a
role in bone remodeling when Src -/- mice died in the peri-natal period with
osteopetrosis (Soriano et al. 1991, Cell 64:693-702) caused by impaired
function
of mature osteoclasts (Horne et al. 1992, J Cell Biol 119:1003-1013, Amling et
al.
2000, Bone 27:603-610). Although Src kinase activity was originally thought to
be
dispensable for this function (Scwartzberg et al. 1997, Genes Dev 11:2835-
2844),
its requirement was recently confirmed (Miyazaki et al. 2004, J. Biol. Chem.
279:17660-6). The role of tyrosine phosphorylation in bone remodeling is
further
supported by the presence of bone remodeling defects in tyrosine phosphatase
SHIP and PTPepsilon-deficient mice (Takeshita et al. 2002, Nat. Med. 8:943-9,
Chiusaroli et al. 2004, Mol. Biol. Cell 15:234-44). Tyrosine phosphorylation
of c-Cbl
and Pyk2 by Src and complex formation is necessary for osteoclast-mediated
bone
resorption (Tanaka et al. 1996, Nature 383:528-531, Sanjay et al. 2001, J.
Cell
Biol. 152:181-195). The severity of the Src-/- phenotype (Soriano et al. 1991,
supra) suggests that bone defects could be observed in transgenic animal
models
of Src substrates. Furthermore, while c-Cbl mice do display an osteoclast
defect in
vitro the mice do not display an overt bone phenotype (Chiusaroli et al. 2003,
Dev
Biol 261:537-547), therefore, there is a lack of animal models of Src
substrates
that display bone-remodeling defects.
[0010] The Src-associated substrate during mitosis of 68 kDa (Sam68;
also known as p62) belongs to the heteronuclear ribonucleoprotein K homology
(KH) domain family of RNA-binding proteins (Wong et al. 1992, Cell 69:551-558,
Richard et al. 1995, Mol. Cell. Biol. 15:186-197). In human, Sam68 is a 443
amino
acid protein which contains several functional domains. Mouse Sam68 also
comprises 443 amino acids and is very well conserved with human Sam68 sharing
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more than 94% sequence identity. A natural splice variant in which the KH
domain
is absent also exists in human and mice and is called Sam68deIKH.
[0011] The KH domain is the second most prevalent RNA binding motif
in proteins. The KH domain of Sam68 is flanked by conserved N- and C-terminal
sequences which are required for RNA binding activity. The entire RNA binding
domain contains approximately 200 amino acids and is referred to as the GSG or
STAR domain. In addition, Sam68 has several proline-rich sequences that are
the
sites of protein-protein interaction with SH3 and WW domains as well as
arginine-
glycine rich regions that are methylated by protein arginine
methyltransferases.
Sam68 also has a tyrosine-rich domain at the C-terminus that is the site of
phosphorylation by tyrosine kinases and interaction with SH2 domain containing
polypeptides.
[0012] The tyrosine phosphorylation of Sam68 by Src and Sik/BRK
tyrosine kinases negatively regulate its RNA binding activity (Wang et al.
1995, J
Biol Chem 270:2010-2013, Derry et al. 2000, Mol Cell Biol 20:6114-6126). Hence
Sam68 and several other single KH domain containing proteins have been
referred
to as signal transduction and activation of RNA (STAR) proteins (Vernet et al.
1997, Trends Genet 13:479-484, Lukong and Richard 2003, Biochimica
Biophysics Acta 1653:73-86). Despite the extensive literature that
characterizes
the functional domains and biochemical interactions of STAR proteins, the
biological relevance of Sam68 and its physiological link with Src remain
undefined.
[0013] Thus, there remains a need to generate animal models of Src
substrates that display bone-remodeling defects.
[0014] There also remains a need to identify specific cellular targets
involved in bone metabolism.
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[0015] More particularly, there remains a need for the identification of
cellular targets involved in bone loss with ageing.
[0016] There also remains a need for the development of safe and
effective anabolic agents to treat the increasing number of people over the
age of
50 who have reduced bone mass which predispose them to fracture.
[0017] There remains a need for the development of specific
therapeutic strategies to prevent bone loss and to promote bone growth in an
ageing population.
[0018] The present invention seeks to meet these needs and other
needs and refers to a number of documents, the content of which is herein
incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
[0019] The present invention is based on the demonstration of the
importance of Sam68 in bone metabolism. This has been demonstrated by the
generation and characterization of Sam68 deficient mice which do not show bone
loss with ageing. Thus, the present invention relates to the identification of
Sam68
as a target for new therapeutic development in the field of bone metabolism
related
diseases. More particularly, the present invention relates to the
identification of
Sam68 as a therapeutic target for osteoporosis treatment.
[0020] In one embodiment, the present invention relates to Sam68
deficient animals for studying bone metabolism.
[0021] In another embodiment, the present invention relates to the use
of Sam68 knock out mice to produce an array of Sam68 specific antibodies and
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ligands, notably monoclonal antibodies.
[0022] In another embodiment, the present invention relates to
methods of treatment of disorders related to bone metabolism comprising a
modulation of the expression of Sam68 in a cell or organism. Such methods
include, in particular embodiments, the use of an antisense nucleic acid of
Sam68,
of Sam68 siRNAs or of a Sam68 specific ribozyme. Other agents, which decrease
the expression level andlor activity of Sam68 (e.g. antibodies (vaccines),
small
molecules, peptides) are also encompassed as agents useful in the treatment of
Sam68 related diseases involving bone loss, such as osteoporosis.
[0023] In a related aspect, the present invention relates to antisense
oligonucleotides hybridizing to a nucleic acid sequence encoding Sam68 protein
(e.g. SEQ ID NO: 1, SEQ 1D N0:2) thereby enabling the control of the
transcription
or translation of the Sam68 gene in cells. The antisense sequences of the
present
invention consist of all or part of nucleic acid sequences SEQ ID N0:1 or SEQ
ID
N0:2 in reverse orientation and variants thereof. The present invention
further
relates to small double stranded RNA molecules (siRNAs) derived from Sam68
nucleic acid sequence (SEQ ID N0:1, SEQ ID N0:2, and variants thereof) which
also decrease Sam68 protein cell expression. In a particular embodiment, the
present invention relates to antisense oligonucleotides and siRNAs that
specifically
inhibit the expression of the Sam68deItaKH splice variant (e.g. SEQ ID NO: 5).
The present invention also relates to methods utilizing siRNA or antisense RNA
to
reduce Sam68 mRNA and/or protein expression and therefore, to significantly
decrease bone loss which is dependent on Sam68 expression and biological
activity. In a particular embodiment, inhibition or reduction of Sam68
expression
significantly reduces osteoporosis in a human subject. The Sam68 complementary
sequences of the present invention can either be directly transcribed in
target cells
or synthetically produced and incorporated into cells by well-known methods.
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[0024] In one embodiment, the present invention features a method of
reducing Sam68 expression in a subject by administering thereto a dsRNA (e.g.,
siRNA), or vector producing same in an effective amount, to reduce Sam68
expression thereby decreasing bone loss and treating or preventing
osteoporosis
and related disorders. The dsRNA can be modified so as to be less susceptible
to
enzymatic degradation or to facilitate its delivery to a target cell (e.g., an
osteoblast). RNA interference (i.e., RNAi) toward a targeted DNA segment in a
cell
can be achieved by administering a double stranded RNA (e.g., siRNA) molecule
to the cell, wherein the ribonucleotide sequence of the double stranded RNA
molecule corresponds to the ribonucleotide sequence of the targeted DNA
segment. In one particular case where the siRNA is chemically modified or
contains point mutations, the antisense region of the siRNAs of the present
invention is still capable of hybridizing to the ribonucleotide sequence of
the
targeted gene (e.g., Sam68 mRNA) and to trigger RNAi.
[0025] In another embodiment, the present invention relates to
screening assays to identify modulators of Sam68 biological functions which
are
useful in the treatment of bone diseases.
[0026] In a further embodiment, the present invention relates to
screening assays to identify compounds (e.g. peptides or nucleic acids) that
completely or partially inhibit a functional activity of Sam68 associated with
bone
loss. Screening assays to identify compounds which stimulate Sam68 expression
or activity are also encompassed by the present invention. Such compounds may
be useful in the treatment of osteopetrosis (i.e. excess bone).
[0027] In one embodiment, the invention provides assays for screening
candidates or test compounds which interact with substrates of a Sam68 protein
or
biologically active portion thereof.
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[0028) In another embodiment, the invention provides assays for
screening candidates or test compounds, which bind to or modulate the activity
of
a Sam68 protein or polypeptide or biologically active portion thereof.
[0029] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a Sam68 protein or biologically active portion thereof,
either
natural or of recombinant in origin, is contacted with a test compound and the
ability of same to modulate Sam68 biological activity, e.g., modulation of
transcription of specific target genes, RNA binding activity, apoptosis, or
any other
measurable biological activity of Sam68 is determined. Determining the ability
of
same to modulate Sam68 activity can be accomplished by monitoring, for
example, the expression and/or activity of a specific gene modulated by Sam68
in
the presence of the test compound as compared to the expression and/or
activity
in the absence thereof.
[0030] In another embodiment, candidate compounds are tested for
their ability to inhibit Sam68 dependent cellular proliferation with the
incorporated
tritiated thymidine method.
[0031] In yet a further embodiment, modulators of Sam68 expression
are identified in a method wherein a cell is contacted with a candidate
compound
and the expression of Sam68 mRNA or protein in the cell is determined. The
level
of expression of Sam68 mRNA or protein in the presence of the candidate
compound is compared to the level of expression of Sam68 mRNA or protein in
the absence of the candidate compound. The candidate compound can then be
identified as a modulator of Sam68 expression based on this comparison. For
example, when expression of Sam68 mRNA or protein is greater (statistically
significantly greater) in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of Sam68 mRNA or
protein expression. Alternatively, when expression of Sam68 mRNA or protein is
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less (statistically significantly less) in the presence of the candidate
compound
than in its absence, the candidate compound is identified as an inhibitor of
Sam68
mRNA or protein expression. The level of Sam68 mRNA or protein expression in
the cells can be determined by methods described herein or other methods known
in the art for detecting Sam68 mRNA or protein.
[0032] In one embodiment, the screening assays of the present
invention comprise 1 ) contacting a Sam68 protein, or functional variant
thereof,
with a candidate compound; and 2) measuring a biological activity of Sam68, or
variant thereof, in the presence of the candidate compound, wherein a compound
that inhibits Sam68 function is selected when Sam68 biological activity is
significantly reduced in the presence of said candidate compound as compared
to
in the absence thereof.
[0033] Thus, the present invention also concerns inhibitors of Sam68
function associated with bone loss. Such inhibitors are useful in the
treatment of
diseases associated with bone loss, such as osteoporosis. Without being
limited to
a particular mechanism of action, Sam68 inhibitors of the present invention
may
decrease osteoclastic activity or increase osteoblastic activity. In addition,
Sam68
inhibitors of the present invention may also reduce osteoblast apoptosis,
thereby
shifting the imbalance in bone metabolism in order to reduce or completely
abrogate bone loss.
[0034] In one embodiment, the inhibitors of the present invention
reduce or completely abolish the RNA binding activity of Sam68. In a
particular
embodiment, the inhibitors of the present invention compete with natural
endogenous RNAs for binding with Sam68. In further embodiment, the inhibitors
of
the present invention interact with the KH or GSG domains of Sam68, thereby
blocking the access of endogenous substrates to the RNA binding domain.
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[0035] In another embodiment, the inhibitors of the present invention
inhibit Sam68 interaction with interacting proteins. Such an inhibition
reduces
Sam68 activity related to bone metabolism thereby reducing the rate of bone
loss.
For example, peptides or small molecules mimicking SH2, SH3 or WW domains
could be used to inhibit a Sam68 interaction with endogenous proteins.
[0036] In a related embodiment, the compounds of the present
invention specifically promote phosphorylation or inhibit dephosphorylation of
Sam68, thereby modulating its function associated with bone metabolism (e.g.
RNA binding activity).
[0037] In a related aspect, the present invention also relates to the use
of any compound capable of inhibiting Sam68 expression in a cell for the
preparation of a pharmaceutical composition intended for the treatment or
prevention of bone related disorders such as osteoporosis.
[0038] Since mice deficient in Sam68 do not show a reduction of bone
mineral density (BMD) with age (osteoporosis) the use of a vaccine against
Sam68
should be an alternative and/or complementary way to treat osteoporosis. In
the
specific case of a Sam68 vaccine, the Sam68 exogenous sequence may be linked
to other molecules including diphtheria toxin, other immunogenic toxin
peptides or
helper antigen peptides in order to improve its efficiency in eliciting the
desire
immunological response in vivo. Humanized mouse monoclonal antibodies or DNA
vaccines comprising a Sam68 nucleic acid sequence or fragment thereof (for an
example on DNA vaccines see US 6,472,375) may be used in accordance with the
present invention to prevent bone loss or treat osteoporosis and related
diseases.
[0039] The present invention further relates to cells expressing Sam68
useful to screen for agents that modulate a Sam68 biological function.
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[0040] The present invention also concerns the use of transgenic mice,
bearing at least one copy of a highly expressed Sam68 gene, as an animal model
for diseases involving Sam68 activity. Such animal model is also useful to
screen
for agents that reduces a Sam68 activity related to bone metabolism.
[0041] In a further embodiment, the present invention features
pharmaceutical composition comprising a compound of the present invention
(e.g.
antisense, siRNA, ribozyme, peptides, nucleic acids, small molecules,
antibodies
etc) which can be chemically modified, in a pharmaceutically acceptable
carrier or
diluent. In another embodiment, the present invention features a method for
treating or preventing a disease or condition in a subject (e.g.,
osteoporosis),
comprising administering to the subject a composition of the invention under
conditions suitable for the treatment or prevention of the disease or
condition in the
subject (e.g., osteoporosis), alone, or in conjunction with one or more
therapeutic
compounds.
[0042] In one embodiment, pharmaceutical compositions of the present
invention comprise a specific nucleic acid sequence (e.g., a mammalian Sam68
sequence, siRNA, antisense and the like) or fragment thereof in a vector,
under
the control of appropriate regulatory sequences to target its expression into
a
specific type of cell (e.g., bone cells such as osteoblasts) thereby reducing
or
preventing bone loss.
[0043] The methods of the present invention can be used for subjects
with preexisting condition (e.g. already suffering of osteoporosis), or
subject
predisposed to bone loss. Additionally, the methods of the present invention
can
be used to correct or compensate for cellular or physiological abnormalities
involved in conferring susceptibility to osteoporosis in patients and/or
alleviate
symptoms of bone loss or as a preventive measure in patients.
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[0044] The compounds of the present invention include lead
compounds and derivative compounds constructed so as to have the same or
similar molecular structure or shape, as the lead compounds, but may differ
from
the lead compounds either with respect to susceptibility to hydrolysis or
proteolysis
(e.g. bioavailability), or with respect to their biological properties (e.g.,
increased
affinity for Sam68). The present invention also relates to compounds and
compositions that are useful for the treatment or prevention of conditions,
diseases
or disorders associated with inappropriate Sam68 production or function.
[0045] In another embodiment, the present invention also relates to
pharmaceutical compositions comprising one or more of the compounds described
herein and a physiologically acceptable carrier. These pharmaceutical
compositions can be in a variety of forms including oral dosage forms, topic
creams, suppository, nasal spray and inhaler, as well as injectable and
infusible
solutions. Methods for preparing pharmaceutical composition are well known in
the art as reference can be made to Remington's Pharmaceutical Sciences, Mack
Publishing Company, Eaton, Pa., USA.
[0046] The method of treatment of the present invention may be
preventive and reduce the risk of developing a Sam68 associated disease or
condition, and may be used to alleviate or obviate the condition (e.g.
osteoporosis). The administration of the therapeutic agent can be in any
pharmaceutically acceptable form in a suitable carrier, and in therapeutically
acceptable dose.
[0047] The compounds can be used as competitive inhibitors in assays
to screen for, or to characterize similar new Sam68 antagonists. In such
assays,
the compounds of the present invention can be used without modification or
they
can be labeled (i.e., covalently or non-covalently linked to a moiety which
directly
or indirectly provide a detectable signal). Examples of labels include
radiolabels
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such as X251, 14G.' and 3H, enzymes such as alkaline phosphatase and
horseradish
peroxidase (US Pat. 3,645,090), ligands such as biotin, avidin, luminescent
compounds including bioluminescent, phosphorescent, chemiluminescent or
fluorescent labels (US Pat. 3,940,475).
[0048] The compounds of the present invention can be administered to
a subject to completely or partially inhibit the activity of Sam68 in vivo.
Thus the
methods of the present invention are useful in the therapeutic treatment of
Sam68
related disorders such as osteoporosis. For example, the compositions of the
present invention can be administered in a therapeutically effective amount to
treat
symptoms related to inappropriate function of Sam68.
[0049] In order to provide a clear and consistent understanding of
terms used in the specification and claims, including the scope to be given
such
terms, a number of definitions are provided herein below.
DEFINITIONS
[0050] Unless defined otherwise, the scientific and technological terms
and nomenclature used herein have the same meaning as commonly understood
by a person of ordinary skill to which this invention pertains. Commonly
understood
definitions of molecular biology terms can be found for example in Dictionary
of
Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John
Wiley &
Sons, New York, NY), The Harper Collins Dictionary of Biology (Hale & Marham,
1991, Harper Perennial, New York, NY), Rieger et al., Glossary of genetics:
Classical and molecular, 5'" edition, Springer-Verlag, New-York, 1991; Alberts
et
al., Molecular Biology of the Cell, 4t" edition, Garland science, New-York,
2002;
and, t_ewin, Genes VII, Oxford University Press, New-York, 2000. Generally,
the
methods traditionally used in molecular biology, such as preparative
extractions of
plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient,
agarose
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or acrylamide gel electrophoresis, purification of DNA fragments by
electroelution,
phenol or pheol-chloroform extraction of proteins, ethanol or isopropanol
precipitation of DNA in saline medium, transformation into bacteria or
transfection
into cells, procedure for cell culture, infection, methods and the like are
common
methods used in the art. Such standard techniques can be found in reference
manuals such as for example Sambrook et al. (2000, Molecular Cloning - A
Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories); and
Ausubel
et al. (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New-
York). In addition, methods and procedures to produce transgenic animals are
well-known in the art and described in details for example in: Hogan ef al.,
1994,
Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press; Nagy et
al., 2002, Manipulating the Mouse Embryo, 3rd edition, Cold Spring Harbor
Laboratory Press.
[0051] The use of the word "a" or "an" when used in conjunction with
the term "comprising" in the claims and/or the specification may mean "one"
but it
is also consistent with the meaning of "one or more", "at least one", and "one
or
more than one".
[0052) Throughout this application, the term "about" is used to indicate
that a value includes the standard deviation of error for the device or method
being
employed to determine the value. In general, the terminology "about" is meant
to
designate a possible variation of up to 10%. Therefore, a variation of 1, 2,
3, 4, 5,
6, 7, 8, 9 and 10 % of a value is included in the term about.
[0053] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and "comprises"),
"having" (and any form of having, such as "have" and "has"), "including" (and
any
form of including, such as "includes" and "include") or "containing" (and any
form of
containing, such as "contains" and "contain") are inclusive or open-ended and
do
CA 02494575 2004-12-24
16
not exclude additional, un-recited elements or method steps.
[0054] Nucleotide sequences are presented herein by single strand, in
the 5' to 3' direction, from left to right, using the one-letter nucleotide
symbols as
commonly used in the art and in accordance with the recommendations of the
IUPAC-IUB Biochemical Nomenclature Commission.
[0055] As used herein, "nucleic acid molecule" or "polynucleotides",
refers to a polymer of nucleotides. Non-limiting examples thereof include DNA
(e.g.
genomic DNA, cDNA), RNA molecules (e.g. mRNA) and chimeras thereof. The
nucleic acid molecule can be obtained by cloning techniques or synthesized.
DNA
can be double-stranded or single-stranded (coding strand or non-coding strand
[antisense]). Conventional ribonucleic acid (RNA) and deoxyribonucleic acid
(DNA)
are included in the term "nucleic acid" and polynucleotides as are analogs
thereof.
A nucleic acid backbone may comprise a variety of linkages known in the art,
including one or more of sugar-phosphodiester linkages, peptide-nucleic acid
bonds (referred to as "peptide nucleic acids" (PNA); Hydig-Hielsen et al., PCT
Int'I
Pub. No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages
or combinations thereof. Sugar moieties of the nucleic acid may be ribose or
deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy
substitutions (containing a 2'-O-methylribofuranosyl moiety; see PCT No. WO
98/02582) and/or 2' halide substitutions. Nitrogenous bases may be
conventional
bases (A, G, C, T, U), known analogs thereof (e.g., inosine or others; see The
Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11 th ed., 1992),
or
known derivatives of purine or pyrimidine bases (see, Cook, PCT Int'I Pub. No.
WO
93/13121 ) or "abasic" residues in which the backbone includes no nitrogenous
base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481 ). A
nucleic
acid may comprise only conventional sugars, bases and linkages, as found in
RNA
and DNA, or may include both conventional components and substitutions (e.g.,
conventional bases linked via a methoxy backbone, or a nucleic acid including
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conventional bases and one or more base analogs). The terminology 'Sam68
nucleic acid" or "Sam68 polynucleotide" refers to a native Sam68 nucleic acid
sequence. In one embodiment, the human Sam68 nucleic acid sequence has the
sequence set forth in SEQ ID NO: 1. In another embodiment, the mouse Sam68
nucleic acid sequence has the sequence as set forth in SEQ ID N0:2. In one
particular embodiment, the Sam68 nucleic acid encodes Sam68 protein (SEQ ID
NO: 3 (human) or SEQ ID NO 4 (mouse)). In another particular embodiment, the
Sam68 nucleic acid encodes a splice variant of the Sam68 gene (e.g.
Sam68deIKH, SEQ ID N0:5)
[0056] Isolated nucleic acid molecule. An °isolated nucleic acid
molecule", as is generally understood and used herein, refers to a polymer of
nucleotides, and includes but should not be limited to DNA and RNA. The
"isolated" nucleic acid molecule is purified from its natural in vivo state.
[0057] A nucleic acid segment. A DNA or RNA segment (or chimera),
as is generally understood and used herein, refers to a molecule comprising a
linear stretch of nucleotides wherein the nucleotides are present in a
sequence
that can encode, through the genetic code when applicable (not all segments
being coding sequences), a molecule comprising a linear sequence of amino acid
residues that is referred to as a protein, a protein fragment or a
polypeptide.
[0058] By "RNA" or "mRNA" is meant a molecule comprising at least
one ribonucleotide residue. By ribonucleotide is meant a nucleotide with a
hydroxyl
group at the 2' position of a p-D-ribo-furanose moiety. The term include
double
stranded RNA, single stranded RNA, isolated RNA such as partially purified
RNA,
essentially purified RNA, synthetic RNA, recombinantly produced RNA, as well
as
altered RNA that differs from naturally occurring RNA by the addition,
deletion,
substitution and/or alteration of one or more nucleotide. Such alterations can
include addition of non-nucleotide material, such as to the ends) of a siRNA
or
CA 02494575 2004-12-24
18
internally, for example at one or more nucleotides of the RNA molecule.
Nucleotides in the RNA molecules of the instant invention can also comprise
non-
standard nucleotides or chemically synthesized nucleotides or
deoxynucleotides.
These altered RNAs can be referred to as analogs or analogs of naturally
occurring RNA.
[0059] Complementary DNA (cDNA). Recombinant nucleic acid
molecules synthesized by reverse transcription of messenger RNA ("mRNA").
[0060] Gene. A DNA sequence related to a polypeptide chain or
protein, and as used herein includes the 5' and 3' untranslated ends. The
polypeptide can be encoded by a full-length sequence or any portion of the
coding
sequence, so long as the functional activity of the protein is retained.
[0061] Expression. By the term "expression" is meant the process by
which a gene or otherwise nucleic acid sequence produces a polypeptide. It
involves transcription of the gene into mRNA, and the translation of such mRNA
into polypeptide(s). When referring to a RNA nucleic acid, the term expression
relates to its translation into a polypeptide(s).
[0062] The term "vector" is commonly known in the art and defines a
plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA
vehicle into which nucleic acid of the present invention can be cloned.
Numerous
types of vectors exist and are well known in the art. One specific type of
vector is
called a targeting vector which may be used for homologous recombination with
an
endogenous target gene in a cell. Homologous recombination occurs between two
sequences (i.e. the targeting vector and endogenous gene sequences) that are
partially or fully complementary. homologous recombination may be used to
alter a
gene sequence in a cell (e.g. embryonic stem cells, (ES cells)) in order to
completely shut down protein expression or to introduce point mutations,
CA 02494575 2004-12-24
19
substitutions or deletions in the target gene sequence. Such method is used
for
example to generate transgenic animals and is well known in the art.
[0063] Expression Vector. A vector or vehicle similar to a cloning vector
but which is capable of expressing a gene which has been cloned into it, after
transformation into a host. The cloned gene (or nucleic acid sequence) is
usually
placed under the control of (i.e., operably linked to) certain control
sequences such
as promoter sequences which may be cell or tissue specific (e.g. bone).
[0064] Expression control sequences will vary depending on whether
the vector is designed to express the operably linked gene (or nucleic acid
sequence) in a prokaryotic and/or eukaryotic host and can additionally contain
transcriptional elements such as enhancer elements, termination sequences,
tissue-specificity elements, and/or translational initiation and termination
sites.
Vectors which can be used both in prokaryotic and eukaryotic cells are often
called
shuttle vectors.
[0065] A DNA construct can be a vector comprising a promoter that is
operably linked to an oligonucleotide sequence of the present invention, which
is in
turn, operably linked to a heterologous gene, such as the gene for the
luciferase
reporter molecule. "Promoter" refers to a DNA regulatory region capable of
binding
directly or indirectly to RNA polymerase in a cell and initiating
transcription of a
downstream (3' direction) coding sequence. For purposes of the present
invention,
the promoter is bound at its 3' terminus by the transcription initiation site
and
extends upstream (5' direction) to include the minimum number of bases or
elements necessary to initiate transcription at levels detectable above
background.
Within the promoter will be found a transcription initiation site
(conveniently defined
by mapping with S1 nuclease), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters
will often, but not always, contain "TATA" boxes and "CCAT" boxes. Prokaryotic
CA 02494575 2004-12-24
promoters contain Shine-Dalgarno sequences in addition to the -10 and -35
consensus sequences.
(0066] As used herein, the term "gene therapy" relates to the
introduction and expression in an animal (preferably a human) of an exogenous
sequence (e.g., a Sam68 gene or cDNA sequence, a Sam68 siRNA or antisense
nucleic acid) to supplement, replace or inhibit a target gene (i.e., Sam68
gene), or
to enable target cells to produce a protein (e.g., a Sam68 chimeric protein to
target
a specific molecule to bones) having a prophylactic or therapeutic effect
toward
osteoporosis and other Sam68 related diseases.
(0067] Agarose Gel Electrophoresis. The most commonly used
technique (though not the only one) for fractionating double strand DNA is
agarose
gel electrophoresis. The principle of this method is that DNA molecules
migrate
through the gel as though it were a sieve that retards the movement of the
largest
molecules to the greatest extent and the movement of the smallest molecules to
the least extent. Note that the smaller the DNA fragment, the greater the
mobility
under electrophoresis in the agarose gel.
(0068] The DNA fragments fractionated by agarose gel electrophoresis
can be visualized directly by a staining procedure (e.g. EtBr) if the number
of
fragments included in the pattern is small. In order to visualize a small
subset of
these fragments, a methodology referred to as the Southern hybridization
procedure can be applied.
(0069] Southern Transfer Procedure. The purpose of the Southern
transfer procedure (alsa referred to as blotting) is to physically transfer
DNA
fractionated by agarose gel electrophoresis onto a nitrocellulose filter paper
or
another appropriate surface or method, while retaining the relative positions
of
DNA fragments resulting from the fractionation procedure. The methodology used
CA 02494575 2004-12-24
21
to accomplish the transfer from agarose gel to nitrocellulose involves drawing
the
DNA from the gel onto the nitrocellulose paper by capillary action, or other
action.
[0070] Nucleic Acid Hybridization. Nucleic acid hybridization depends
on the principle that two single-stranded nucleic acid molecules that have
complementary base sequences will reform the thermodynamically favored
double-stranded structure if they are mixed under the proper conditions. The
double-stranded structure will be formed between two complementary single-
stranded nucleic acids even if one is immobilized on a nitrocellulose filter.
In the
Southern or Northern hybridization procedures, the latter situation occurs.
The
DNA/RNA of the individual to be tested may be digested with a restriction
endonuclease if applicable, prior to its fractionation by agarose gel
electrophoresis,
conversion to the single-stranded form, and transfer to nitrocellulose paper,
making it available for reannealing to the hybridization probe. Non-limiting
examples of hybridization conditions can be found in Ausubel, F.M. et al.,
Current
protocols in Molecular Biology, John Wiley & Sons, Inc., New York, NY (1994).
For
purposes of illustration, an example of moderately stringent conditions for
testing
the hybridization of a polynucleotide of the present invention with other
polynucleotides, include prewashing, in a solution of 5X SSC, 0.5% SDS, 1mM
EDTA (pH 8.0); hybridizing at 50 °C-60 °C, 5X SSC and 100
,ug/ml denatured
salmon sperm DNA overnight (12-16 hours); followed by washing twice at 60
°C for
15 minutes with each of 2X SSC, 0.5X SSC and 0.2X SSC containing 0.1 % SDS.
For example for highly stringent hybridization conditions, the hybridization
temperature is changed to 62, 63, 64, 65, 66, 67 or 68 °C. One skilled
in the art will
understand that the stringency of hybridization can be readily manipulated,
such as
by altering the salt and SDS concentration of the hybridizing and washing
solutions
and/or temperature at which the hybridization is performed. The temperature
and
salt concentration selected is determined based on the melting temperature
(Tm)
of the DNA hybrid. Other protocols or commercially available hybridization
kits
using different annealing and washing solutions can also be used as well known
in
CA 02494575 2004-12-24
22
the art. The use of formamide in different mixtures to lower the melting
temperature
may also be used and is well known in the art.
(0071] A "probe" is meant to include a nucleic acid oligomer that
hybridizes specifically to a target sequence in a nucleic acid or its
complement,
under conditions that promote hybridization, thereby allowing detection of the
target sequence or its amplified nucleic acid. Detection may either be direct
(i.e,
resulting from a probe hybridizing directly to the target or amplified
sequence) or
indirect (i.e., resulting from a probe hybridizing to an intermediate
molecular
structure that links the probe to the target or amplified sequence). A probe's
"target" generally refers to a sequence within an amplified nucleic acid
sequence
(i.e., a subset of the amplified sequence) that hybridizes specifically to at
least a
portion of the probe sequence by standard hydrogen bonding or "base pairing."
[0072] By "sufficiently complementary" is meant a contiguous nucleic
acid base sequence that is capable of hybridizing to another sequence by
hydrogen bonding between a series of complementary bases. Complementary
base sequences may be complementary at each position in sequence by using
standard base pairing (e.g., G:C, A:T or A:U pairing) non standard base
pairing
(e.g., I:C) or may contain one or more residues (including a basic residues)
that
are not complementary by using standard base pairing, but which allow the
entire
sequence to specifically hybridize with another base sequence in appropriate
hybridization conditions. Contiguous bases of an oligomer are preferably at
least
about 80% (81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98,
99, 100%), more preferably at least about 90% complementary to the sequence to
which the oligomer specifically hybridizes. In reference to more specific
nucleic
acid molecules of the present invention, the binding free energy for a nucleic
acid
molecule with its complementary sequence is sufficient to allow the relevant
function of the nucleic acid to proceed (e.g., RNAi activity). For example,
the
degree of complementarity between the sense and antisense region (or strand)
of
CA 02494575 2004-12-24
23
the siRNA construct can be the same or can be different from the degree of
complementarity between the antisense region of the siRNA and the target RNA
sequence (e.g., Sam68 RNA sequence). Complementarity to the target sequence
of less than 100% in the antisense strand of the siRNA duplex (including
deletions,
insertions and point mutations) is reported to be tolerated when these
differences
are located between the 5'-end and the middle of the antisense siRNA (Elbashir
et
al., 2001, Embo, 20(23):68-77-6888). Determination of binding free energies
for
nucleic acid molecules is well known in the art (e.g., see Turner et al.,
1987, J. Am.
Chem. Soc. 190:3783-3785; Frier ef al., 1986 Proc. Nat. Acad. Sci. USA, 83
:9373-
9377) "Perfectly complementary" means that all the contiguous residues of a
nucleic acid molecule will hydrogen bond with the same number of contiguous
residues in a second nucleic acid sequence. Appropriate hybridization
conditions
are well known to those skilled in the art, can be predicted readily based on
sequence composition and conditions, or can be determined empirically by using
routine testing (see Sambrook et al., (cf. Molecular Cloning: A Laboratory
Manual,
Third Edition, edited by Cold Spring Harbor Laboratory, 2000) at ~~ 1.90-1.91,
7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly at ~~ 9.50-9.51, 11.12-
11.13,
11.45-11.47 and 11.55-11.57). Sequences that are "sufficiently complementary"
allow stable hybridization of a probe sequence to a target sequence, even if
the
two sequences are not completely complementary.
[0073] Nucleic acid sequences may be detected by using hybridization
with a complementary sequence (e.g., oligonucleotide probes) (see U.S. Patent
Nos. 5,503,980 (Cantor), 5,202,231 (Drmanac et al.), 5,149,625 (Church ef
al.),
5,112,736 (Caldwell et al.), 5,068,176 (Vijg et al.), and 5,002,867
(Macevicz)).
Hybridization detection methods may use an array of probes (e.g., on a DNA
chip)
to provide sequence information about the target nucleic acid which
selectively
hybridizes to an exactly complementary probe sequence in a set of four related
probe sequences that differ by one nucleotide (see U.S. Patent Nos. 5,837,832
and 5,861,242 (Chee et al.).
CA 02494575 2004-12-24
24
[0074] A detection step may use any of a variety of known methods to
detect the presence of nucleic acid by hybridization to a probe
oligonucleotide.
One specific example of a detection step uses a homogeneous detection method
such as described in detail previously in Arnold et al. Clinical Chemistry
35:1588-
1594 (1989), and U.S. Patent Nos. 5,658,737 (Nelson et al.), and 5,118,801 and
5,312,728 (Lizardi et al.).
[0075] The types of detection methods in which probes can be used
include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and
Northern
blots (RNA detection). Labeled proteins could also be used to detect a
particular
nucleic acid sequence to which it binds (e.g protein detection by far western
technology: Guichet et al., 1997, Nature 385(6616): 548-552; and Schwartz et
al.,
2001, EMBO 20(3): 510-519). Other detection methods include kits containing
reagents of the present invention on a dipstick setup and the like. Of course,
it
might be preferable to use a detection method which is amenable to automation.
A
non-limiting example thereof includes a chip or other support comprising one
or
more (e.g. an array) different probes.
[0076] A "label" refers to a molecular moiety or compound that can be
detected or can lead to a detectable signal. A label is joined, directly or
indirectly,
to a nucleic acid probe or the nucleic acid to be detected (e.g., an amplified
sequence). Direct labeling can occur through bonds or interactions that link
the
label to the nucleic acid (e.g., covalent bonds or non-covalent interactions),
whereas indirect labeling can occur through the use of a "linker or bridging
moiety,
such as additional oligonucleotide(s), which is either directly or indirectly
labeled.
Bridging moieties may amplify a detectable signal. Labels can include any
detectable moiety (e.g., a radionuclide, ligand such as biotin or avidin,
enzyme or
enzyme substrate, reactive group, chromophore such as a dye or colored
particle,
luminescent compound including a bioluminescent, phosphorescent or
chemiluminescent compound, and fluorescent compound). In one particular
CA 02494575 2004-12-24
embodiment, the label on a labeled probe is detectable in a homogeneous assay
system, i.e., in a mixture, the bound label exhibits a detectable change
compared
to an unbound label.
[0077] Other methods of labeling nucleic acids are known whereby a
label is attached to a nucleic acid strand as it is fragmented, which is
useful for
labeling nucleic acids to be detected by hybridization to an array of
immobilized
DNA probes (e.g., see PCT No. PCT/IB99/02073).
[0078] A "homogeneous detectable label" refers. to a label whose
presence can be detected in a homogeneous fashion based upon whether the
labeled probe is hybridized to a target sequence. A homogeneous detectable
label
can be detected without physically removing hybridized from unhybridized forms
of
the labeled probe. Homogeneous detectable labels and methods of detecting
them have been described in detail elsewhere (e.g., see U.S. Pat. Nos.
5,283,174,
5,656,207 and 5,658,737).
[0079] As used herein, "oligonucleotides" or "oligos" define a molecule
having two or more nucleotides (ribo or deoxyribonucleotides). The size of the
oligo will be dictated by the particular situation and ultimately on the
particular use
thereof and adapted accordingly by the person of ordinary skill. An
oligonucleotide
can be synthesized chemically or derived by cloning according to well-known
methods. While they are usually in a single-stranded form, they can be in a
double-
stranded form and even contain a "regulatory region". They can contain
natural,
rare or synthetic nucleotides. They can be designed to enhance a chosen
criterion
like stability, for example. Chimeras of deoxyribonucleotides and
ribonucleotides
may also be within the scope of the present invention.
[0080] "Amplification" refers to any known in vitro procedure for
obtaining multiple copies ("amplicons") of a target nucleic acid sequence or
its
CA 02494575 2004-12-24
26
complement or fragments thereof. In vitro amplification refers to the
production of
an amplified nucleic acid that may contain less than the complete target
region
sequence or its complement. Known in vitro amplification methods include,
e.g.,
transcription-mediated amplification, replicase-mediated amplification,
polymerase
chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification,
nucleic acid sequence-based amplification (NASBA), and strand-displacement
amplification (SDA). Replicase-mediated amplification uses self-replicating
RNA
molecules, and a replicase such as Qf3-replicase (e.g., Kramer et al., U.S.
Pat. No.
4,786,600). PCR amplification is well known and uses DNA polymerase, primers
and thermal cycling to synthesize multiple copies of the two complementary
strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat. Nos. 4,683,195,
4,683,202,
and 4,800,159). LCR amplification uses at least four separate oligonucleotides
to
amplify a target and its complementary strand by using multiple cycles of
hybridization, ligation, and denaturation (e.g., EP Pat. App. Pub. No. 0 320
308).
SDA is a method in which a primer contains a recognition site for a
restriction
endonuclease that permits the endonuclease to nick one strand of a
hemimodified
DNA duplex that includes the target sequence, followed by amplification in a
series
of primer extension and strand displacement steps (e.g., Walker et al., U.S.
Pat.
No. 5,422,252). Another known strand-displacement amplification method does
not require endonuclease nicking (Dattagupta et al., U.S. Patent No.
6,087,133).
Transcription-mediated amplification (TMA) can also be used in the present
invention. In one embodiment, TMA and NASBA isothermic methods of nucleic
acid amplification are used. Those skilled in the art will understand that the
oligonucleotide primer sequences of the present invention may be readily used
in
any in vitro amplification method based on primer extension by a polymerase
(see
generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25 and (Kwoh et al.,
1989,
Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology
6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28;253-260; and Sambrook
et
al., (cf. Molecular Cloning: A Laboratory Manual, Third Edition, edited by
Cold
Spring Harbor Laboratory, 2000). As commonly known in the art, the oligos are
designed to bind to a complementary sequence under selected conditions.
CA 02494575 2004-12-24
27
(0081] As used herein, a "primer" defines an oligonucleotide which is
capable of annealing to a target sequence, thereby creating a double stranded
region which can serve as an initiation point for nucleic acid synthesis under
suitable conditions. Primers can be, for example, designed to be specific for
certain alleles so as to be used in an allele-specific amplification system.
The
primer's 5' region may be non-complementary to the target nucleic acid
sequence
and include additional bases, such as a promoter sequence (which is referred
to
as a "promoter primer"). Those skilled in the art will appreciate that any
oligomer
that can function as a primer can be modified to include a 5' promoter
sequence,
and thus function as a promoter primer. Similarly, any promoter primer can
serve
as a primer, independent of its functional promoter sequence. Of course the
design
of a primer from a known nucleic acid sequence is well known in the art. As
for the
oligos, it can comprise a number of types of different nucleotides.
[0082] Polymerise chain reaction (PCR). PCR is carried out in
accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195;
4,683,202; 4,800,159; and 4,965,188 (the disclosures of all three U.S. Patent
are
incorporated herein by reference). In general, PCR involves a treatment of a
nucleic acid sample (e.g., in the presence of a heat stable DNA polymerise)
under
hybridizing conditions, with one oligonucleotide primer for each strand of the
specific sequence to be detected. An extension product of each primer which is
synthesized is complementary to each of the two nucleic acid strands, with the
primers sufficiently complementary to each strand of the specific sequence to
hybridize therewith. The extension product synthesized from each primer can
also
serve as a template for further synthesis of extension products using the same
primers. Following a sufficient number of rounds of synthesis of extension
products, the sample is analyzed to assess whether the sequence or sequences
to
be detected are present. Detection of the amplified sequence may be carried
out
by visualization following like, for example, EtBr staining of the DNA
following gel
electrophoresis, or using a detectable label in accordance with known
techniques,
CA 02494575 2004-12-24
28
and the like. For a review on PCR techniques (see for example "PCR Protocols,
A
Guide to Methods and Amplifications", Michael et al. Eds, Acad. Press, 1990).
[0083] Ligase chain reaction (LCR). Another example of amplification
technique is LCR. It is carried out in accordance with known techniques
(Weiss,
1991, Science 254:1292). Adaptation of the protocol to meet the desired needs
can be carried out by a person of ordinary skill. Strand displacement
amplification
(SDA) is also carried out in accordance with known techniques or adaptations
thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad.
Sci.
USA 89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696).
[0084] Transcription-associated amplification. Amplifying a target
nucleic acid sequence by using at least two primers can be accomplished using
a
variety of known nucleic acid amplification methods, but preferably uses a
transcription-associated amplification reaction that is substantially
isothermal. By
using such an in vitro amplification method, many strands of nucleic acid are
produced from a singly copy of target nucleic acid, thus permitting detection
of the
target in the sample by specifically binding the amplified sequences to one or
more
detection probes. Transcription-associated amplification methods have been
described in detail elsewhere (e.g., U.S. Pat. Nos. 5,399,491 and 5,554,516).
Briefly, transcription-associated amplification uses two types of primers (one
being
a promoter primer because it contains a promoter sequence for an RNA
polymerase), two enzyme activities (a reverse transcriptase (RT) and an RNA
polymerase), substrates (deoxyribonucleoside triphosphates, ribonucleoside
triphosphates) and appropriate salts and buffers in solution to produce
multiple
RNA transcripts from a nucleic acid template. Initially, a promoter primer
hybridizes specifically to a target sequence (e.g., RNA) and reverse
transcriptase
creates a first complementary DNA strand (cDNA) by extension from the 3' end
of
the promoter primer. The cDNA is made available for hybridization with the
second primer by any of a variety of methods, such as, by denaturing the
target-
CA 02494575 2004-12-24
29
cDNA duplex or using RNase H activity supplied by the RT that degrades RNA in
a
DNA:RNA duplex. A second primer binds to the cDNA and a new strand of DNA is
synthesized from the end of the second primer using the RT activity to create
a
double-stranded DNA (dsDNA) having a functional promoter sequence at one end.
An RNA polymerise binds to the dsDNA promoter sequence and transcription
produces multiple transcripts ("amplicons"). Amplicons are used in subsequent
steps or cycles of the transcription-associated amplification process by
serving as
a new template for replication, thus generating many copies of amplified
nucleic
acid (i.e., about 100 to 3,000 copies of RNA are synthesized from each
template).
[0085] Nucleic acid fragments in accordance with the present invention
include epitope-encoding portions of the polypeptides of the invention. Such
portions can be identified by the person of ordinary skill using the nucleic
acid
sequences of the present invention in accordance with well-known methods. Such
epitopes are useful in raising antibodies that are specific to the
polypeptides of the
present invention. The invention also provides nucleic acid molecules which
comprise polynucleotide sequences capable of hybridizing under stringent
conditions to the polynucleotide sequences of the present invention or to
portions
thereof.
[0086] As used herein, the twenty natural amino acids and their
abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids)
such
as a,a-disubstituted amino acids, N-alkyl amino acids, lactic acid and other
unconventional amino acids may also be suitable components for the
polypeptides
of the present invention. Examples of unconventional amino acids include but
are
not limited to selenocysteine, citrulline, ornithine, norvaline, 4-(E)-butenyl-
4(R) -
methyl-N-methylthreonine (MeBmt), N-methyl-leucine (MeLeu), aminoisobutyric
acid, statine, N-methyl-alanine (MeAla).
[0087] As used herein, "protein" or "polypeptide" means any peptide-
CA 02494575 2004-12-24
linked chain of amino acids, regardless of postranslational modifications
(e.g.
phosphorylation, glycosylation, sulfatation, sumoylation, prenylation,
ubiquitination
etc). A " SAM68 protein" or a " Sam68 polypeptide" is an expression product of
Sam68 nucleic acid (e.g. Sam68 gene) such as native human Sam68 protein
(SEQ ID NO: 3), a Sam68 natural splice variant such as Sam68deIKH (SEQ ID
N0:5) or a Sam68 protein homolog (e.g. mouse Sam68, SEQ ID NO: 4) that
shares at least 60% (but preferably, at least 65, 70, 75, 80, 85, 86, 87, 88,
89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) amino acid sequence identity with
Sam68
and displays functional activity of native Sam68 protein. For the sake of
brevity, the
units (e.g. 66, 67...81, 82%...) have not been specifically recited but are
nevertheless considered within the scope of the present invention. A 'Sam68
interacting protein" refers to a protein which binds directly or indirectly
(e.g. via
RNA or another bridging protein or molecule) to Sam68 in order to modulate or
participate in a functional activity of Sam68. These proteins include kinases,
scaffolding proteins or any other proteins known to interact with Sam68 (see
below). An "isolated protein" or "isolated polypeptide" is purified from its
natural in
vivo state.
[0088] The terms "biological activity" or "functional activity" or
"function" are used interchangeably and refer to any detectable biological
activity
associated with a structural, biochemical or physiological activity of the
protein (i.e.
Sam68 protein). For instance, one non-limiting example of a functional
activity of
Sam68 protein includes ribonucleotide homopolymers binding activity (e.g.RNA
binding activity). The UAAA and UUUA sequences are among the specific
sequences that interact with Sam68. Other specific RNA substrates of Sam68
include mRNAs encoding: DAP3/IRCP, nucleolar protein-p40, hnRNP A2/B1
(UAAA), PAP/ANXS, PBP/PEA-BP, and ~-actin (UUUUU). Therefore, interaction of
Sam68 with any of these RNA substrates is considered a functional activity of
Sam68 protein. A Sam68 biological activity also include for example, simple
binding of Sam68 with compounds, substrates, interacting proteins and the
like.
CA 02494575 2004-12-24
31
Thus, oligomerization of Sam68 with specific proteins such as proteins
containing
SH2, SH3, and WW domains as well as with itself is also considered a
biological
activity of Sam68. Such interaction may be stable or transient. Another
example of
a Sam68 functional activity is its capacity to become phosphorylated by
several
kinases. Therefore, other biological activities of Sam68 include its
interaction with
p59~'", p60S'~, p56~~k, ZAP_70 and Sik/BRK. Other molecules such as insulin
and
leptin have also reported to induce Sam68 phosphorylation. Thus, in accordance
with the present invention, oligomerization and phosphorylation of Sam68 are
also
considered as functional or biological activities of Sam68. Interaction of
Sam68
with other known ligands (e.g. Grb2, Grap, Nck, PLC-y, P13K p85a and ItklTec
family of kinases) not explicitly listed in the present invention may also be
considered functional activities of Sam68. A complete review on Sam68
functional
activities may be found for example in Lukong and Richard (2003, Biochimica
Biophysica Acta 1653:73-86). Thus, in accordance with the present invention,
measuring the effect of a test compound on its ability to inhibit or increase
(e.g.,
modulate) Sam68 binding or interaction, level of expression as well as
phosphorylation status is considered herein as measuring a biological activity
of
Sam68. Broadly intra-or inter-molecular binding of Sam68 in the absence vs the
presence of the modulating compounds of the present invention is yet another
example of a biological activity according to the invention. As noted above,
Sam68
biological activity also includes any biochemical measurement of the protein,
conformational changes, phosphoryfation status (or any other posttransiational
modification e.g. ubiquitination, sumolylation, palmytoylation, prenylation
etc), any
downstream effect of Sam68's signaling such as protein phosphorylation in
signaling cascades, indirect gene expression modulation, or any other feature
of
the protein that can be measured with techniques known in the art. Finally,
Sam68
biological activities include a detectable change in cell architecture, cell
proliferation and apoptosis or other cell phenotype that is modulated by the
action
of Sam68.
CA 02494575 2004-12-24
32
[0089] Sam68 antibody. As used herein, the term "Sam68 antibody" or
"immunologically specific Sam68 antibody" refers to an antibody that
specifically
binds to (interacts with) a Sam68 protein and displays no substantial binding
to
other naturally occurring proteins other than the ones sharing the same
antigenic
determinants as the Sam68 protein. Sam68 antibodies include polyclonal,
monoclonal, humanized as well as chimeric antibodies.
[0090] The term animal is used herein to include all vertebrate animals
except humans. It also includes an individual animal at all stages of
development
including embryonic and fetal stages.
[0091] A "transgenic animal" is any animal containing one or more
cells bearing genetic information altered or received, directly or indirectly,
by
deliberate genetic manipulation at the subcellular level, such as targeted
recombination (homologous recombination) or microinjection or infection with
recombinant virus. The term transgenic animal is not meant to encompass
classical cross-breading or in vitro fertilization but rather is meant to
include
animals in which one or more cells are altered by or received a recombinant
DNA
molecule. This molecule may be targeted to a specific genetic locus, be
randomly
integrated within a chromosome or it may be extrachromosomally replicating
DNA.
[0092] The term "germ cell line transgenic animal" refers to a
transgenic animal in which the genetic alteration or genetic information was
introduced into a germ-line cell, thereby conferring the ability to transfer
the genetic
information to offspring. In the case where such offspring possess some or all
the
of that alteration or genetic information, then they too are also considered
transgenic animals.
[0093] As used herein, a "targeted gene" or "knock out" is a DNA
sequence introduced into a germline or a non-human animal by way of human
CA 02494575 2004-12-24
33
intervention, including, but not limited to, the methods described herein
(e.g.
homologous recombination, random integration...). The targeted genes of the
present invention include DNA sequences which are designed to alter cognate
endogenous alleles.
[0094) When referring to nucleic acid molecules, proteins or
polypeptides, the term native refers to a naturally occurring nucleic acid or
polypeptide. A homolog is a gene sequence encoding a polypeptide isolated from
an organism other than a human being. Similarly, a homolog of a native
polypeptide is an expression product of a gene homolog. Of course, the non-
coding portion of a gene can also find a homolog portion in another organism.
[0095) Polyacrylamide Gel Electrophoresis (PAGE). The most
commonly used technique (though not the only one) for achieving a
fractionation of
polypeptides on the basis of size is polyacrylamide gel electrophoresis. The
principle of this method is that polypeptide molecules migrate through the gel
as
though it were a sieve that retards the movement of the largest molecules to
the
greatest extent and the movement of the smallest molecules to the least
extent.
The smaller the polypeptide fragment, the greater the mobility under
electrophoresis in the polyacrylamide gel. Both before and during
electrophoresis,
the polypeptides typically are continuously exposed to the detergent sodium
dodecyl sulfate (SDS), under which conditions the polypeptides are denatured.
Native gels are run in the absence of SDS. The polypeptides fractionated by
polyacrylamide gel electrophoresis can be visualized directly by a staining
procedure if the number of polypeptide components is small.
[0096) Western blotting Procedure. The purpose of the Western
transfer procedure (also referred to as blotting) is to physically transfer
polypeptides fractionated by polyacrylamide gel electrophoresis onto a
nitrocellulose filter paper or another appropriate surface or method, while
retaining
CA 02494575 2004-12-24
34
the relative positions of polypeptides resulting from the fractionation
procedure.
The blot is then probed with an antibody that specifically binds to the
polypeptide
of interest.
[0100] As used herein, the designation "functional derivative" denotes,
in the context of a functional derivative of an amino acid sequence, a
molecule that
retains a biological activity (either function or structural) that is
substantially similar
to that of the original sequence. This functional derivative or equivalent may
be a
natural derivative or may be prepared synthetically. Such derivatives include
amino
acid sequences having substitutions, deletions, or additions of one or more
amino
acids, provided that the biological activity of the protein is conserved. The
substituting amino acid generally has chemico-physical properties, which are
similar to that of the substituted amino acid. The similar chemico-physical
properties include, similarities in charge, bulkiness, hydrophobicity,
hydrophylieity
and the like. The term "functional derivatives" is intended to include
"segments",
"variants", "analogs" or "chemical derivatives" of the subject matter of the
present
invention.
[0101] As used herein, "chemical derivatives" is meant to cover
additional chemical moieties not normally part of the subject matter of the
invention. Such moieties could affect the physico-chemical characteristic of
the
derivative (i.e. solubility, absorption, half life and the like, decrease of
toxicity).
Such moieties are exemplified in Remington: The Science and Practice of
Pharmacy by Alfonso R. Gennaro, 2003, 21t" edition, Mack Publishing Company.
Methods of coupling these chemical-physical moieties to a polypeptide are well
known in the art.
[0102] As commonly known, a "mutation" is a detectable change in the
genetic material which can be transmitted to a daughter cell. As well known, a
mutation can be, for example, a detectable change in one or more
CA 02494575 2004-12-24
deoxyribonucleotide. For example, nucleotides can be added, deleted,
substituted
for, inverted, or transposed to a new position. Spontaneous mutations and
experimentally induced mutations exist. The result of a mutation of nucleic
acid
molecule is a mutant nucleic acid molecule. A mutant polypeptide can be
encoded
from this mutant nucleic acid molecule.
[0103] The term "variant" refers herein to a protein, which is
substantially similar in structure and biological activity to the protein, or
nucleic acid
of the present invention to maintain at least one of its biological
activities. Thus,
provided that two molecules possess a common activity and can substitute for
each other, they are considered variants as that term is used herein, even if
the
composition, or secondary, tertiary or quaternary structure of one molecule is
not
identical to that found in the other, or if the amino acid sequence or
nucleotide
sequence is not identical. A homolog is a gene sequence encoding a polypeptide
isolated from an organism other than a human being. Similarly, a homolog of a
native polypeptide is an expression product of a gene homolog. Expression
vectors, regulatory sequences (e.g. promoters), leader sequences and method to
generate same and introduce them in cells are well known in the art.
[0104] In accordance with the present invention, it shall be understood
that the "in vivo" experimental model can also be used to carry out an "in
vitro"
assay. For example, cellular extracts from the indicator cells can be prepared
and
used in one of the aforementioned "in vitro" tests (such as in binding assays
or in
vitro translation assays).
[0105] The term "subject" or "patient" as used herein refers to an
animal, preferably a mammal, most preferably a human who is the object of
treatment, observation or experiment.
[0106] As used herein, the term "purified" refers to a molecule (e.g.
CA 02494575 2004-12-24
36
Sam68 polypeptides, antisense or RNAi molecule, RNA substrates of Sam68)
having been separated from a component of the composition in which it was
originally present. Thus, for example, a "purified Sam68 polypeptide or
polynucieotide" has been purified to a level not found in nature. A
"substantially
pure" molecule is a molecule that is lacking in most other components (e.g.,
30,
40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of
contaminants). By
opposition, the term "crude" means molecules that have not been separated from
the components of the original composition in which it was present. Therefore,
the
terms "separating" or "purifying" refers to methods by which one or more
components of the biological sample are removed from one or more other
components of the sample. Sample components include nucleic acids in a
generally aqueous solution that may include other components, such as
proteins,
carbohydrates, or lipids. A separating or purifying step preferably removes at
least
about 70% (e.g., 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100%), more
preferably at
least about 90% (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) and, even
more preferably, at least about 95% (e.g., 95, 96, 97, 98, 99, 100%) of the
other
components present in the sample from the desired component. For the sake of
brevity, the units (e.g. 66, 67...81, 82,...91, 92%....) have not
systematically been
recited but are considered, nevertheless, within the scope of the present
invention.
(0107] The terms °inhibiting," "reducing" or any variation of these
terms,
when used in the claims and/or the specification includes any measurable
decrease or complete inhibition of at least one biological activity of Sam68
to
achieve a desired result. For example, a compound is said to be inhibiting
Sam68
activity when a decrease in RNA binding is measured following a treatment with
the compounds of the present invention as compared to in the absence thereof.
[0108] As used herein, the terms "molecule", "compound", "agent" or
"ligand" are used interchangeably and broadly to refer to natural, synthetic
or semi-
synthetic molecules or compounds. The term "compounds therefore denotes for
CA 02494575 2004-12-24
37
example chemicals, macromolecules, cell or tissue extracts (from plants or
animals) and the like. Non-limiting examples of compounds include peptides,
antibodies, carbohydrates, nucleic acid molecules and pharmaceutical agents.
The compound can be selected and screened by a variety of means including
random screening, rational selection and by rational design using for example
protein or ligand (e.g. RNA) modeling methods such as computer modeling. The
terms "rationally selected" or "rationally designed" are meant to define
compounds
which have been chosen based on the configuration of interacting domains of
the
present invention. As will be understood by the person of ordinary skill,
macromolecules having non-naturally occurring modifications are also within
the
scope of the term "molecule". For example, the modulating compounds of the
present invention are modified to enhance their stability and their
bioavailability.
The compounds or molecules identified in accordance with the teachings of the
present invention have a therapeutic value in diseases or conditions in which
the
physiology or homeostasis of the cell and/or tissue is compromised by Sam68
production or response. For example, compounds of the present invention, by
acting on a biological activity of Sam68 (e.g. RNA binding) reduce bone loss
and
thereby treat osteoporosis.
[0109] As used herein "antagonists", "Sam68 antagonists" or "sam68
inhibitors" refer to any molecule or compound capable of inhibiting
(completely or
partially) a biological activity of Sam68.
[0110] In general, techniques for preparing antibodies (including
monoclonal antibodies and hybridomas) and for detecting antigens using
antibodies are well known in the art (Campbell, 1984, In "Monoclonal Antibody
Technology: Laboratory Techniques in Biochemistry and Molecular Biology",
Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al.,
1988 (in: Antibody - A Laboratory Manual, CSH Laboratories). The present
invention also provides polyclonal, monoclonal antibodies, or humanized
versions
CA 02494575 2004-12-24
38
thereof, chimeric antibodies and the like which inhibit or neutralize their
respective
interaction domains and/or are specific thereto.
[0111] Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It should be
understood, however, that the detailed description and the specific examples,
while indicating specific embodiments of the invention, are given by way of
illustration only, since various changes and modifications within the spirit
and
scope of the invention will become apparent to those skilled in the art from
this
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0112] Figure 1 shows the localization of Sam68 in primary mouse
osteoblasts and osteoclasts. (a-d) Primary mouse osteoblasts were released by
collagenase digest from the calvaria of 3 day-old C57BU6 mice and grown on
cover-slips in differentiation medium for 48 hours before fixing in 4%
paraformaldehyde and performing immunofluorescence staining with anti-Sam68
antibodies (AD-1, red) and for ALP activity with using the fluorescent
substrate
ELF97 (Green). The cells were visualized by phase contrast and fluorescence
microscopy. (e-h) Mouse osteoclasts were isolated from a crushed mouse femur
and the cells labeled for immunofluorescence using anti-Sam68 antibodies (red)
and for TRAP with the fluorescent substrate ELF97 (green). The cells were
visualized by phase contrast and fluorescence microscopy.
[0113] Figure 2 shows the generation of Sam68-deficient mice. (a) The
genomic organization of the wild-type and targeted sam68 alleles after
homologous recombination are depicted. The location of the DNA fragment used
as a probe for the Southern-blot analysis is shown, as well as the sizes of
the two
Bglll fragments detected for wild-type and targeted sam68 alleles. The
targeted
CA 02494575 2004-12-24
39
allele replaces exon 4 and part of exon 5 of sam68 with PGK-neomycin cassette.
(b) Southern-blot analysis of genomic DNA from wild-type (+/+), heterozygous
(+/-)
and homozygous (-/-) mice. DNA fragments corresponding to wild-type (4.5 kb)
and the targeted (5.5 kb) alleles are illustrated. (c) Western blot analysis
of Sam68
expression. Protein extracts from wild-type, heterozygous and homozygous cells
were immunoblotted with anti-Sam68 (AD-1 ) and anti-actin antibodies.
[0114] Figure 3 shows a FaxitronT"" X-ray of the femur and spine of
Sam68+/+ and Sam68-/- mice. Mice were given a lethal dose of anesthetic at the
indicated times and contact X-rays of the distal femora and lumbar vertebrae
obtained on a FaxitronT"" MX20 equipped with an FPX-2 Imaging system.
Representative X-rays of the distal femur (a-d) and lumbar spine (e-h) of
Sam68+/+ (+/+) and Sam68-/- (-/-) mice revealed comparable radio-opacity at 4
months (left panels). At 12 months (right panels), cortical thinning (b,
arrow) and
radio-lucency (b, asterisk) are apparent in the distal femur and lumbar spine
(f,
arrow) of +/+ mice but not -/- mice (d, h). The images are representative of
those
obtained from 6-7 animals in each group.
[0115] Figure 4 shows micro computed tomography of distal femur and
fourth lumbar vertebra. Bones were dissected free of soft tissue and fixed
overnight in 4% paraformaldehyde before scanning on a Skyscan 1072 static
instrument equipped with 3D creator analytical software. Representative 3
dimensional re-constructions and 2D cross-sectional scans demonstrated similar
architecture in the distal femur (a-d) and the fourth lumbar vertebra (e-h) of
Sam68+/+ (+/+) and Sam68-/- (-/-) mice. In keeping with the results from
Faxitron
X-ray, trabecular bone (b, asterisk) and cortical thickness (b, arrow) were
reduced
in the femur and vertebra (f, arrow) of 12 month-old +/+ mice compared with
age-
matched -/- mice (d, h) and 4 month-old mice (a, c, e, g). The images are
representative of those from 5-7 animals in each group.
CA 02494575 2004-12-24
[0116] Figure 5 shows the histological analysis of un-decalcified bone
from Sam68+/+ and Sam68-/- mice. Mice were injected with calcein and
tetracycline at 7 and 3 days prior to euthanization by exsanguination. Femora
were
fixed overnight in 4% paraformaldehyde and processed for embedding in
methylmethacrylate, for von Kossa staining of mineralized tissue (a, e, i, m)
and for
fluorescence microscopy to identify the mineralization fronts labeled with
calcein
(green) and tetracycline (yellow, b, f, j, n). After overnight fixation in 4%
paraformaldehyde the tibiae were processed for embedding in
methylmethacrylate:
glycolmethacrylate and sections stained for alkaline phosphatase (ALP)
activity to
identify osteoblasts (brown, c, g, k, o) and for tartrate resistant acid
phosphatase
(red, TRAP) to identify osteoclasts (d, h, I, p). Von kossa stained sections
were
counter-stained with toluidine blue and ALP and TRAP stained sections with
fast
green. Staining patterns were similar in 4 month-old Sam68+/+ (a-d), 4 month-
old
Sam68-/- (i-I) and 12 month-old Sam68-/- (m-p) mice. In contrast, the 12 month-
old Sam68+/+ mice had less bone (e), primarily a single fluorochrome label
(f), less
ALP-positive (g) and less TRAP-positive (h) cells. Magnification at source x
1.5 (a,
e, i, m); x 10 main panel and x 40 inset panel (b-d, f-h, j-I, n-p).
Representative
images for each group were selected from 6-7 von Kossa stained sections, 4-6
fluorochrome images; 4-6 ALP stained sections and 6-7 TRAP stained sections.
[0117] Figure 6 shows the quantitation of bone volume, trabecular
architecture and bone cell complement. (a) Histomorphometry was performed on
von Kossa and TRAP stained sections using a Leica DMR microscope equipped
with a Retiga 1300 camera and Bioquant Nova Prime image analysis software.
The panels represent the Mean t SD for 4 regions of interest on 6-7 mice in
each
group. Quantitative histomorphometry demonstrated significant reductions in
bone
volume per tissue volume (BV/TV), in osteoid volume (OV/TV), in osteoblasts
(OB/TV) and in TRAP-positive osteoclasts (OC/TV) in the 12 month-old Sam68+/+
mice (hatched black) compared with 4 month-old Sam68+/+ mice (solid black) and
with 12 month-old Sam68-/- mice (hatched grey) and 4 month-old Sam68-/- mice
CA 02494575 2004-12-24
41
(solid grey). (b, c) Quantitative micro-CT was performed using the 3D
CreatorT""
software supplied with the Skyscan instrument. The panels represent the Mean t
SD for 6-7 mice in each group. Differences in the percent bone (BV/'fV),
structure
model index (SMI) and trabecular separation (Tr Sp), as quantitated by micro-
CT,
were also apparent between 4 month-old and 12 month-old Sam68+/+ mice but
not Sam68-/- mice. * p < 0.01, ** p < 0.05..
(0118] Figure 7 shows an alignment between human and mouse
Sam68 nucleotide sequences. Alignment was performed using ClustalW.
(0119] Figure 8 shows an alignment between human and mouse
Sam68 amino acid sequences. The human (hSam68, hSam68del) and mouse
(mSam68) homologues were aligned using ClustalW. The functional domains of
hSam68 are highlighted. Proline-rich domains (blue) represent SH3-domain-
binding sites; the KH domain (lavender), an RNA-binding motif, occurs within
the
larger GSG domain (boxed); the C-terminal tyrosine residues (green) are
potential
sites of phosphorylation by Src family kinases; RG repeats (yellow) represents
potential arginine methylation sites.
(0120] The present invention is illustrated in further details by the
following non-limiting description.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
(0121] In a broad sense, the present invention relates to the
identification of Sam68 as an important player in bone metabolism. This has
been
demonstrated by the production and analysis of Sam68 deficient mice, which do
not show age-related bone loss. Thus the present invention has identified
Sam68
as an important target for new therapeutic development in the field of bone
related
diseases such as osteoporosis.
CA 02494575 2004-12-24
42
SAM68 SEQUENCES ARE CONSERVED THROUGHOUT THE EVOLUTION
[0122] To date, several mammalian Sam68 orthologs have been
identified. Human (Genbank accession number NM 006559, SEQ ID N0:1 )
mouse (Genbank accession number U17046), rat (Genbank accession number
NM_130405), and chicken (Genbank accession number AY057837) as well as
Sam68 orthologs in Torpedo caiifornica and D. melanogaster have been cloned.
The amino acid sequences within the functional domains are highly conserved
(Figure 7). Sam68 is located on chromosome 1 p32 (in a contig spanning 3834
base pairs) and contains nine exons encoding an open reading frame of 1437 bp.
The human and mouse genes are structurally similar with the same number and
size of exons. With more than 94% identity at the protein level, with all
functional
domains and related activities conserved (e.g. RNA binding activity,
interaction
with SH2, SH3 domains etc) mouse and human Sam68 are expected to share the
same activities in cells. Thus it is expected that the results presented
herein are
translatable to the human system.
[0123] Among the functional domains that are conserved between
Sam68 orthologs including mouse and human are: (1 ) the KH domain or RNA
binding domain; (2) the proline-rich SH3 and WW-domains binding sites; (3) the
tyrosine rich SH2 domain binding and phosphorylation sites; (4) the RG-rich
arginine methylation sites; and (5) the nuclear localization signal (see
figure 8).
Both the human and mouse Sam68 contain identical functional motifs and thus,
are likely to function in a similar manner in protecting against age-related
bone
loss(Lukong and Richard 2003, Biochimica Biophysics Acta 1653:73-86).
GSGIKH domain
[0124] The RNA binding domain of Sam68 corresponds to a tripartite
region containing the KH domain and its flanking homology sequences. The
CA 02494575 2004-12-24
43
flanking sequences of approximately 80 and 30 amino acids are referred to as
the
N-terminal of KH (NK) and the C-terminal of KH (CK) respectively. Via its RNA
binding domain, Sam68 binds to ribonucleotides homopolymers with higher
affinity
to polyU and polyA. The a/u rich sequences, more particularly UAAA and UUUA
were identified as high affinity RNA targets (Lin et al., J. Biol. Chem.,
1997,
272:27274-27280). In addition, several putative Sam68 RNA targets were
identified by differential display and cDNA representational differences
analysis
among which, ten of them were also KH containing proteins (Itoh et al.,
Nucleic
Acids Res., 2002, 30:5452-5464). Of these targets, 10 showed a KH dependent
binding to Sam68 in vivo; these mRNAs are encoding: DAP3/IRCP, nucleolar
protein-p40, hnRNP A2/B1, PAP/ANXS, PBP/PEA-BP and ~i-actin. All of these bind
Sam68 through their 3'UTR region. In addition, Sam68 self associates via its
GSG
domain (comprising the NK, KH and CK tripartite region), thereby forming homo-
oligomers which are disrupted upon tyrosine phosphorylation. Thus, it has been
postulated that Sam68 binds RNA in its unphosphorylated state.
Tyrosine-rich SH2 domain binding and phosphorylation site
[0125] The C-terminal end of Sam68 protein is characterized by the
presence of several tyrosine residues which are potential sites for
phosphorylation.
Sam68 has been demonstrated to be phosphorylated by several kinases including
p60sro,p59fyn,Sik/BRK, p56'~k and ZAP-70. Cell surface receptors such as
insulin,
leptin, and ligation of the CD16, CD32 and T-cell receptor have been observed
to
increase tyrosine-phosphorylation of Sam68. Phosphorylated Sam68 can than
interact with several SH2-containing protein such as but not limited to Src
family
kinases, Grap. Nck, PLCy-1,P13K p85a, Sik/BRK, Grb2, RasGap and Itk/Tec family
kinases(Lukong and Richard 2003, Biochimica Biophysics Acta 1653:73-86).
CA 02494575 2004-12-24
44
Proline-rich SH3 and WW-domain binding sites
[0126] Sam68 contain numerous proline-rich sequences that are the
binding sites of SH3 and WW domain containing proteins(Lukong and Richard
2003, Biochimica Biophysics Acta 1653:73-86). SH3 domain ligands consists of
short contiguous proline-rich amino acid sequences with core consensus
sequence PXXP. Ligands with a basic residue located N-terminal or C-terminal
to
the PXXP motif have been designated class I (RXXPXXP) or class II (PXXPXR)
ligands, respectively. The WW domain is a short conserved sequence of about 40
amino acid residues in single or tandem repeats with two signature tryptophan
residues spaced 22 or 23 residues apart and has an affinity for proline-rich
sequences. WW and SH3 domains share similar or overlapping proline-rich
sequences and it is conceivable that they may compete for the same ligands in
vivo. Sam68 has been shown to interact with the SH3-domain containing Src
kinases, Sik/BRK kinases, p85 PI-3K, PLCgamma-1, PRMT2, Grb-2, GRAP,
Itk/Tec/BTK, Nck and vav(Lukong and Richard 2003, Biochimica Biophysics Acta
1653:73-86).
RG-rich arginine methylation sites
[0127] Another prominent feature of STAR proteins as well as many
other proteins involved in RNA metabolism is the presence of RG-rich regions
and
RGG boxes, potential sites for protein arginine methylation (Cote et al.,
2003,Mo1.
Biol. Cell 14:274-287). Arginine methylation is a prevalent post-translational
covalent modification in eukaryotes that has been shown to modulate several
cellular processes including protein-protein interactions, transcription and
intracellular localization (Gary & Clarke, 1998. Prog. Nucleic Acid Res. Mol.
Biol.
61: 65-131 ). Protein-arginine N-methyltransferases (PRMTs) catalyze the
sequential transfer of methyl groups from S-adenosyl-L-methionine to the
guanidino nitrogen atoms of specific arginine residues within proteins. At
least 7
CA 02494575 2004-12-24
mammalian PRMTs, named simply PRMT1 through PRMT7, have been cloned to
date and are classified in two groups (type I and type II) based on substrate
and
reaction product specificity.
Nuclear localization signal
[0128] The predominantly nuclear localization of mammalian Sam68 is
dictated by a nonconventional nuclear localization signal (NLS) embedded in
the
last 24 amino acids (42°RPSLKAPPARPVKGAYREHPYGRY~3, SEQ ID N0:16) in
the C-terminal of the polypeptide (Ishidate et al., 1997, FEBS lett. 409: 237-
41 ).
SAM68 EXPRESSION IN PRIMARY MOUSE OSTEOBLASTS AND
OSTEOCLASTS
[0129] In order to determine if the Src substrate Sam68 could be
involved in bone metabolism, the first step was to investigate whether or not
Sam68 is expressed in primary osteoblasts and osteoclasts. Osteoblasts (Fig. 1
a-
d) were isolated from the calvaria of 3 day-old mice and osteoclasts (Fig. 1 e-
h)
from a femur of 14 day-old mice. Cells plated on glass coverslips were
immunostained with anti-Sam68 antibodies (Fig. 1b, 1f, red) and the identity
of
osteoblasts and osteoclasts were confirmed using fluorescent assays for
alkaline
phosphatase (ALP, Fig. 1 c-d) and tartrate-resistant acid phosphatase (TRAP,
Fig.
1g-h), respectively. Sam68 localized to the nuclei of osteoblasts (Fig. 1b)
and to
the nuclei and cytoplasm of osteoclasts (Fig. 1f and data not shown). These
data
illustrate that Sam68 is expressed in primary osteoblasts and osteoclasts.
GENERATION Of= MICE DEFICIENT IN SAM68
[0130] As a means to define the role that Sam68 plays in mammalian
CA 02494575 2004-12-24
46
physiology, mice that do not express Sam68 because of a targeted mutational
disruption in the Sam68 gene have been generated.
[0131] In accordance with the present invention, the altered Sam68
gene generally should not fully encode the same Sam68 protein native to the
host
animal and its expression product should be altered to a minor or great
degree, or
preferably, absent altogether. However, it is conceivable that a more modestly
modified Sam68 gene will fall within the scope of the present invention if it
is a
specific alteration that would inhibit a Sam68 biological function (e.g.
mutation in
the KH domain, mutations/deletions in specific interacting domains -e.g.
proline-
rich domain or arginine-glycine rich regions or mutations in specific tyrosine
residues that are normally phosphorylated and important for interaction with
SH2
containing proteins). Modifications and deletions render the naturally
occurring
gene non-functional, thereby producing a knock out animal. In addition,
dominant
negative mutations are also encompassed in the scope of the present invention.
[0132] The DNA used for altering a target gene may be obtained by a
wide variety of techniques that include, but are not limited to, isolation
from
genomic sources, preparation of cDNA from isolated mRNA templates, direct
synthesis or combination thereof.
[0133] A type of target cell for transgene introduction is embryonic stem
cell (ES). ES cells may be obtained from preimplantation embryos cultured in
vitro
by methods well known in the art. Methods to generate transgenic animals (e.g.
knock out mouse) are well known in the art and detailed methods may be found
for
example in Hogan et al., 1994, Manipulating the Mouse Embryo, Cold Spring
Harbor Laboratory Press; Nagy et al., 2002, Manipulating the Mouse Embryo, 3rd
edition, Cold Spring Harbor Laboratory Press.
[0134] In order to generate Sam68-deficient mice, a bacteriophage
CA 02494575 2004-12-24
47
clone encompassing Sam68 exons 3-9 was isolated from a 129/SvJ genomic
library using full-length Sam68 cDNA as a probe. Xbal digested Sam68 genomic
DNA fragments of 4kb (encompassing exon 4 and part of exon 5) and 3kb
(spanning part of exon 5 and exon 6) were subcloned in BluescriptT"" SK
resulting
in pBS4 and pBS3, respectively. A DNA fragment was amplified from pBS4 with
the following oligonucleotides (5'-AAT GTC TAG AAA CAA CTC ATA TAC AGA C-
3-SEQ ID NO: 6) and the Universal primer (5'-GGA AAC AGC TAT GAC CAT G-3'
SEQ ID NO: 15). The Xbal digested 1 kb DNA fragment was subcloned in the Xbal
site of pPNT (Andrew Karaplis, McGill University, Canada). The 3kb fragment
from
pBS3 was amplified by PCR with (5'-GGG ATG CGG CCG CTC TAG AAT TGT
CCT ACT TGA ACG G-3'-SEQ ID NO: 7) and (5'-CGG TGG CGG CCG CTG TCG
ACC TGA GTA ACA TTT CTT A-3'-SEQ ID NO: 8) and subcloned in the Notl site
of pPNT. The targeting vector pPNT-Sam68 replaces exon4 and part of exon 5
with a neomycin resistant gene cassette. A Sall site was introduced at the 3'
end of
the 3kb DNA fragment and was used to linearize the plasmid for electroporation
into embryonic stem (ES) cells. Approximately 1000 ES colonies were screened
and 2 clones were identified that contained the Sam68 mutant allele, as
determined by Southern blotting. Targeted ES cells were injected into 3.5-day-
old
BALB/c blastocysts and were transferred into CD-1 foster mothers, and animals
classified as chimeras by coat color were mated with BALB/c mice. Germ line
transmission was achieved and the mice were maintained on C57BU6
background.
Genotyping
[0135] All mouse procedures were performed in accordance with
McGil1 University guidelines, which are set by the Canadian Council on Animal
Care. Genomic DNA was isolated from tail biopsies and analyzed by Southern
blotting and genomic PCR analysis. The DNA fragment utilized as the probe for
the Southern blotting analysis was amplified with the following two
oligonucleotides
CA 02494575 2004-12-24
48
(5'-AAG CCT TTA CTG GTT GTG T-3'-SEQ ID NO: 9) and (5'- GAA ACG CAC
CGT AGG CT-3'-SEQ ID NO: 10). The wild-type sam68 allele was identified by
genomic PCR using the following oligonucleotides 5'-AAA TCC TAA CCC TCC
TCA GTC AG-3' (SEQ ID NO: 11 ) and 5'-GAT ATG ATG GAT GAT ATC TGT
CAG-3' (SEQ ID NO: 12). The Sam68 targeted allele was identified by genomic
PCR using the following oligonucleotides 5'-CTT GGG TGG AGA GGC TAT TCG-
3' (SEQ ID NO: 13) and 5'-GTC GGG CAT GCG CGC CTT GAG C-3' (SEQ ID
NO: 14).
SAM68 IS NOT ESSENTIAL FOR MOUSE DEVELOPMENT
[0136] Thus, to define the physiologic role of Sam68, Sam68-deficient
mice were generated by gene targeting. Sam68 exons (Karsenty 2003, Nature
423:316-318, Riggs et al. 2002, Endocrine Rev 23:279-302) which encode the
functional region of the KH domain were deleted (Fig. 2a). Mice heterozygous
for
the sam68 mutation were phenotypically normal and the genotypes of the
offspring
from heterozygote intercrosses exhibited a Mendelian segregation at embryonic
day18.5, but not at post-natal day 1 (Table 1 ). Actually, most of the Sam68-/-
pups
were killed by their mothers for unknown reasons. The integrity of the
targeted
allele was verified by Southern blot analysis (Fig. 2b) and by PCR of genomic
DNA
(data not shown). The Sam68-/- mice were devoid of Sam68 protein expression,
as analyzed by immunoblotting with several C-terminal Sam68 antibodies
including
AD-1 (Fig. 2c, ref. 31 ). Sam68 transcripts encoded by exons (Harada et al.
2003,
supra, Riggs et al. 2002, Endocrine Rev 23:279-302) were absent, as evidenced
by reverse transcription PCR (data not shown), confirming that indeed Sam68-
deficient mice were generated.
(0137] Despite evidence that Sam68 is ubiquitously expressed (Wong
et al. 1992, supra), the Sam68-/- mice that survived into adulthood lived a
normal
lifespan, did not develop tumors and showed no immunological or other major
CA 02494575 2004-12-24
49
illnesses. Sam68-/- mice did, however, have difficulty breeding due to male
infertility and the females rarely provided adequate care to their young.
SAM68 DEFICIENCY PROTECTS MICE FROM AGE-RELATED BONE LOSS
[0138] Cohorts of adult Sam68+/+ and Sam68-/- mice were euthanized
by exsanguination at 4 and 12 months of age for skeletal phenotyping. To
minimize differences in the bone phenotype that might arise secondary to
differences in sex or weight, age-matched female mice were selected for the
analysis. The female mice demonstrated similar increases in body weight, body
fat
content and bone length, in the axial and appendicular skeleton between 4 and
12
months of age (Table 2). FaxitronT"' X-ray (Fig. 3) revealed cortical thinning
(arrow)
in the distal femora of 12 month-old Sam68+/+ mice (Fig. 3b) compared with 4
month-old mice of either genotype (Fig. 3a, c) and with 12 month-old Sam68-/-
mice (Fig. 3d). Trabecular bone was significantly decreased in the 12 month-
old
Sam68+/+ mice, as evidenced by the radio-lucent appearance of the distal
femoral
metaphysis (Fig. 3b, asterisk) and the lumbar spine (Fig. 3f, arrow). In
contrast, the
radio-opaque appearance of the lumbar vertebra of 12 month-old Sam68-/- mice
(Fig. 3h) was indicative of increased trabecular bone compared with the young
mice (Fig. 3e, g) and with the 12 month-old Sam68+/+ mice (Fig. 3f). Total
body
bone mineral content (BMC), quantitated with a Pffximus~ densitometer,
increased
in both Sam68+/+ (427.5 to 482.5) and Sam68-/- (387.5 to 565.5) mice between 4
months and 12 months of age, although the increase was not statistically
significant in the wild-type mice (Table 2). However, bone mineral density
decreased over time at the level of the spine in Sam68+/+ mice (vertebra BMD,
63.87 to 57.57), whereas it increased in both the femur and the spine in Sam68-
/-
mice (Table 2). These data demonstrate that Sam68-/- mice continued to thrive
and accrue bone in the axial and appendicular skeleton for 12 months, in
contrast
to age-matched wild-type littermate controls.
CA 02494575 2004-12-24
THREE-DIMENSIONAL ARCHITECTURE OF BONE IS PRESERVED IN AGED
SAM68-/- MICE
[0139] To confirm the apparent differences in bone content of the femur
and lumbar vertebra in 12 month-old Sam68+1+ and Sam68-/- mice, quantitative
micro computed tomography (micro-CT) using a Skyscan 1072~ static imaging
instrument (Fig. 4) was performed. Three-dimensional reconstruction of the
distal
femur showed comparable architecture in 4 month-old Sam68+/+ (Fig. 4a), 4
month-old Sam68-/- (Fig. 4c) mice and in 12 month-old Sam68-/- (Fig. 4d) mice.
In
contrast, there was a significant decrease in metaphyseal bone (asterisk) and
cortical thinning in the diaphysis of 12 month-old Sam68+/+ mice (Fig. 4b).
These
are characteristic features of the skeletons of aged C57BL/6 mice and resemble
the clinical features of age-related bone loss in humans (Lazner et al. 1999,
Human Mol Genetics 8:1839-1846, Harada et al. 2003, Nature 423:349-355). A
similar loss of trabecular bone was seen in the 12 month-old Sam68+/+ vertebra
(Fig. 4f, arrow), whereas that of the 12 month-old Sam68-/- (Fig. 4h) mice was
in
fact more dense than that observed in the 4 month-ofd mice (Fig. 4e, g). These
observations confirmed the FaxitronT"" X-ray and BMD data (Fig. 3 and Table 2)
and showed that the absence of Sam68 expression protected mice from age-
related bone loss in the femur and in the vertebra.
MINERAL APPOSITION AND BONE REMODELING ARE PRESERVED IN
AGED SAM68-/- MICE
[0140] To determine the molecular mechanisms involved in the
preservation of bone mass in aged Sam68-!- mice, un-decalcified femora and
tibia
were embedded in plastic and sections from the mid-saggital region were
prepared
to identify mineralized bone and the mineralization fronts. Sections were
stained in
situ for ALP and TRAP activity to identify osteoblasts and osteoclasts,
respectively
(Fig. 5). In the 4 month-old mice, little difference was seen at low or high
(inset)
CA 02494575 2004-12-24
51
magnification in bones of either genotype stained for mineral with von Kossa
and
counter-stained with toluidine blue (Fig. 5a, 5i). Similar rates of bone
deposition
were also observed in the 4 month-old Sam68+/+ and Sam68-/- mice, as
demonstrated by the deposition of calcein and tetracycline at the
mineralization
fronts (Fig. 5b, 5j). These apparent similarities in bone metabolism in 4
month-old
wild-type and mutant mice were corroborated by in situ staining for ALP (Fig.
5c,
5k) and TRAP activity (Fig. 5d, 51), which identified osteoblasts and
osteoclasts,
respectively. At 12 months of age, the bones of Sam68-/- mice showed
remarkably
similar parameters of trabecular bone content (Fig. 5m), mineral apposition
(Fig.
5n), osteoblast activity (Fig. 50) and osteoclast activity (Fig. 5p) to the 4
month-old
mice. In sharp contrast, the 12 month-old Sam68+/+ exhibited the anticipated
age-
related bone loss characterized by decreased trabecular bone (Fig. 5e),
decreased
deposition of fluorochrome at the mineralization fronts (Fig. 5f) and
decreased ALP
(Fig. 5g) and TRAP (Fig. 5h) activity. Quantitation of the mean surface area t
standard deviation of bone labeled with fluorochrome shown no significant
difference between 4 month-old Sam68+/+ (5.85 ~ 1.41 ) and Sam68-/- mice (6.78
t 3.46), whereas there was significantly less labeled surface between 12 month-
old Sam68+/+ (0.71 t 0.87) and Sam68-/- (8.30 t 5.11 Sam68-/-, p < 0.01 )
mice.
Collectively, the histological data demonstrate an age-mediated reduction in
bone
remodeling in 12 month-old Sam68+/+ mice that was not observed in12 month-old
Sam68-/- mice, as the latter had active remodeling surfaces with abundant
levels
of osteoblasts and osteoclasts.
QUANTITATIVE MICRO-CT AND HISTOMORPHOMETRY CONFIRM BONE
PRESERVATION IN AGED SAM68-/- MICE
[0141 To quantify the observed alterations in bone volume, cellular
composition and trabecular architecture, traditional two-dimensional
histomorphometric techniques were used and quantitative three-dimensional
micro-CT analyses (Fig, 6) were performed. Histomorphometric analysis of the
CA 02494575 2004-12-24
52
tibia from wild-type 4 month-old (black bars) and 12 month-old mice (black
hatched
bars) demonstrates a greater than ~75% reduction in 1 ) bone volume per tissue
volume (BV/TV), 2) un-mineralized bone matrix, or osteoid (OV/TV), 3) the
number
of osteoblasts (OBITV), 4) the number of osteoclasts (OCITV) per tissue volume
(Fig. 6a). Four month-old Sam68-/- mice had slight increases in BV/TV, OV/TV,
OB/TV and OC/TV compared with wild-type 4 month-old mice (Fig. 6a, gray bars).
Remarkably, the histomorphometric parameters of the 12 month-ofd Sam68-/-
mice (gray hatched bars) were similar to the 4 month-old wild-type mice (Fig.
6a),
further demonstrating the fact that Sam68-/- mice maintain their bone mass.
[0142] Quantitative data from micro-CT analyses revealed a similar
reduction in BV/'fV in the 12 month-old Sam68+J+ mice in contrast to the 4 and
12
month-old Sam68-/- mice analyzed (Fig. 6b, BV/TV). The 12 month-old Sam68+l+
mice were associated with a significant increase in the structure model index
(SMI)
and trabecular separation (Tr Sp), but not in the trabecular thickness (Tr Th)
compared with age-matched Sam68-/- mice or 4 month-old mice of either
genotype (Fig. 6b). Quantitation of the trabecular separation distribution by
micro-
CT showed that the decrease in the mean Tr Sp was due to an increase in the
proportion of spaces in the 700-1400 micron range in the Sam68+/+ mice (Fig.
6c,
black dashed line). In effect, this meant that there were fewer trabeculae
rather
than equivalent numbers of thin trabeculae in 12 month-old Sam68+l+ mice
compared with any of the other groups of mice (Fig. 6c). The data presented
herein further demonstrate that loss of Sam68 expression protects against age-
related bone loss, as 12 month-old Sam68-/- mice had similar histomorphometric
and micro-CT parameters as 4 month-old mice.
DISCUSSION
[0143] Applicants report herein the generation of Sam68-deficient mice
using a traditional approach where part of the Sam68 RNA binding domain (KH
CA 02494575 2004-12-24
53
domain) was replaced with a neomycin resistance gene cassette. Cohorts of
Sam68+/+ and Sam68-/- mice were euthanized at 4 months and 12 months for
skeletal phenotyping to determine if the Src substrate Sam68 influenced bone
remodeling. Results obtained from FaxitronTM X-ray and micro-CT analysis
showed
that the bone mass was preserved in 12 month-old Sam68-/- mice. This was in
sharp contrast to 12 month-old wild-type mice in which bone mass was decreased
up to ~75% with ageing. In fact, the BV/TV ratio of the 12 month-old Sam68-/-
mice
was virtually indistinguishable from that of 4 month-old wild-type and Sam68-/-
mice. Histological analyses of the femur and vertebra showed that preservation
of
bone in the 12 month-old Sam68-/- mice was accompanied by equivalent numbers
of osteoblasts and osteoclasts as observed in the young adult mice. These data
demonstrate that bone cell activity and bone mass were maintained in 12 month-
old Sam68-/- mice and did not decline with age.
[0144] The quantitative analysis of bone density and architecture
demonstrated that 12 month-old Sam68-/- mice maintained the bone mass
observed in 4 month-old Sam68-/- mice. This phenotype was significantly
different
from that of the Src-/- mice, which exhibited severe osteopetrosis and failure
of
tooth eruption at birth and odontomas by 4 months of age (Soriano et al. 1991,
supra, Amling et al. 2000, supra). The developmental defect in the Src-/- mice
has
been attributed primarily to a deficiency in osteoclast bone resorption (Horne
et al.
1992, supra, Lowe ef al. 1993, Proc Natl Acad Sci (USA) 90:4485-4489). it was
also noted that decreased Src expression enhanced osteoblast differentiation
(Marzia et al. 2000, J. Cell Biol. 151:311-320), which could have contributed
to the
continued post-natal increase in bone mass (Amling et al. 2000, supra). As
visualized by in vivo labeling of the mineralization fronts and quantitative
histomorphometry, osteoblasts from 12 month-old Sam68-/- were actively forming
bone, as were those in the Src-l- mice (Amling et al. 2000, supra), suggesting
a
genetic link between Src and Sam68. Thus, the present invention provides the
first
genetic link between Src and aSrc substrate affecting bone metabolism.
CA 02494575 2004-12-24
54
[0145] Although the precise alteration in bone cell function in Sam68-/-
mice remains to be identified, the continued presence of numerous osteoblasts
suggests that these cells might be resistant to age-related apoptosis. Given
the
documented role of STAR proteins in the induction of apoptosis (Pilotte et al.
2001,
Genes Dev 15:845-858, Taylor et al. 2004, BMC Cell Biol. 5:1-12) and the
identification of Sam68 as a mitotic substrate of Src (Fumagalli et al. 1994,
Nature
368:871-874, Taylor et al. 1994, Nature 368:867-871, Pawson 1995, Nature 373:
573-580), it is quite possible that the osteoblasts from Sam68-/- mice have
both a
differentiation and survival advantage.
[0146] An alternative explanation for the maintenance of bone mass in
the Sam68-/- mice could be a mild impairment of osteoclast function. The
presence of Sam68 in membrane spreading initiation centers (deHoog et al.
2004,
Cell 117:649-662) and the whole cell distribution of Sam68 in osteoclasts
(Fig. 1 )
make this a viable possibility. However, a similar osteoblast to osteoclast
ratio was
observed in wild-type and Sam68-/- mice, suggesting that osteoblast/osteoclast-
coupled bone remodeling is normal. Preliminary results of serum levels of CTX,
a
type I collagen breakdown product and ALP, which is an index of osteoblast
activity, revealed no difference behnreen the wild-type and Sam68-/- mice
(data not
shown). In vitro functional assays are under way to further delineate the
precise
role of Sam68 in the differentiation program and activity of cells of the
osteoblast
and osteoclast lineage.
[0147] There are a number of mouse models of osteopetrosis and
osteoporosis that resemble to some extent their human counterparts (Lazner et
al.
1999, supra, Huang et al. 2003, supra, Chaloub ef al. 2003, Nat Med 9:399-406,
Klein et al. 2004, Science 303:229-232). The data presented herein showing the
involvement of Sam68 in bone remodeling is the first to demonstrate a
physiological role for Sam68 and the first report of an overt bone phenotype
resulting from the targeting of an Src substrate. The phenotype observed with
the
CA 02494575 2004-12-24
Sam68-/- deficient mice implies that inhibitors of Sam68 could prevent age-
related
bone loss. These results suggest that Sam68 expression levels, hypomorphism
and mutations may influence the susceptibility of individuals to osteoporosis.
Thus,
the present invention identifies Sam68-/- mice as a unique animal model to
investigate bone remodeling and identify the STAR protein Sam68 as a
therapeutic
target for age-related bone loss.
[0148] The present invention is further illustrated by the following
specific examples. The examples are provided for illustration only and should
not
be construed.as limiting the scope of the invention.
EXAMPLE 1
ISOLATION AND IMMUNOFLUORESCENT STAINING OF PRIMARY MOUSE
OSTEOBLASTS AND OSTEOCLASTS
[0149] Multi-nucleate osteoclasts were obtained by mincing the femora
and tibia of 14 day-old mice as described (Miyazaki ef al. 2004, supra) and
the
cells were plated on glass coverslips for microscopy. Cultures were maintained
for
48 hrs, fixed and prepared for immunofluorescence as described (Laroque et al.
2002, Neuron 36:815-829). The established criteria of multi-nucleation
(minimum
of 3) and expression of TRAP40 were used to identify osteoclasts. Cultures
were
immunostained with a polyclonal antiserum raised against Sam68 (AD, Harada et
al. 2003, supra, Chen et al. 1999, Mol Cell Biol 10:3015-3033) and positive
cells
localized using a secondary antibody conjugated to rhodamine. The cells were
counterstained with 3 mg/ml 4, 6-diamidino-2-phenylindole (DAPI) and multi-
nucleate cells visualized by fluorescence microscopy. Osteoblasts were
released
with collagenase from the calvaria of 3 day-old mice and analyzed as described
above except that the identity of the osteoblasts was confirmed by the
presence of
ALP.
CA 02494575 2004-12-24
56
EXAMPLE 2
RADIOLOGIC ASSESSMENT OF 4 AND 12 MONTH-OLD MICE
[0150) X-ray, bone mineral density (BMD) and micro computed
tomography (micro-CT) were performed essentially as described previously
(Valverde et al. 2004, Human Mol Genetics 13:271-284). Mice were administered
a lethal dose of anesthetic at the indicated times, exsanguinated and X-rayed
on a
Faxitron MX20 equipped with an FPX-2 Imaging system (balsa Medoptics,
Waterloo, Ontario ). BMD was determined using a Lunar PixiMUS 1.46 (GE-Lunar,
Madison, Wisconsin). Morphometric parameters were determined on anesthetized
mice at the time of sacrifice by direct measurement or from the X-ray.
[0151] Micro-CT was performed on the left femur and 4th lumbar
vertebra after removal of soft tissues and overnight fixation in 4%
paraformaldehyde. The distal metaphysis was scanned with a Skyscan 1072
micro-CT instrument (Skyscan, Antwerp, Belgium). Image acquisition was
performed at 100kV and 98NA, with a 0.9° rotation between frames. 2D
images
were used to generate 3D reconstructions and to quantitate parameters with the
3D Creator software supplied with the instrument.
EXAMPLE 3
HISTOLOGIC, HISTOCHEMICAL AND HISTOMORPHOMETRIC ANALYSES
[0152] All histologic and histomorphometric analyses were performed
essentially as described previously (Valverde et al. 2004, supra, Miao ef al.
2003,
Exptl Cell Res 294:210-222). Mice were given intra-peritoneal injections of 30
mg/kg tetracycline or 30 mg/kg calcein at 7 days and 2 days prior to sacrifice
to
label active mineralization surfaces (Valverde et al. 2004, supra). After
overnight
fixation in 4% paraformaldehyde the left femur was embedded in
pofymethylmethacrylate (MMA) and the left tibia in a mixture of 50% MMA and
CA 02494575 2004-12-24
57
50% glycolmethacrylate (GMA) and 2 m sections cut on a modified Leica RM 2155
rotary microtome (Leica Microsystems, Richmond Hill, Ontario). Fluorescence
images were captured using a Leica DMR microscope equipped with a Retiga
1300 camera (Qimaging, Burnaby, British Columbia) and histomorphometric data
obtained using Bioquant Nova Prime image analysis software (Bioquant Image
Analysis Corp, Nashville, Tennessee).
[0153] Sections of MMA-embedded bone were stained with von Kossa
and counterstained with tofuidine blue to show mineralized and un-mineralized
tissue respectively. Adjacent sections of MMA:GMA embedded bones were
stained for TRAP and ALP as described (Valverde et al. 2004, supra).
EXAMPLE 4
THERAPEUTIC NUCLEIC ACID MOLECULES
[0154] The present invention, has identified Sam68 as a target for the
treatment of osteoporosis and related bone disorders. Thus, in one embodiment,
the present invention generally relates to Sam68 expression modulation and the
use of Sam68 expression modulation (i.e. Sam68 overexpression, and Sam68
expression inhibition) to treat or prevent bone loss (e.g. osteoporosis).
[0155) The present invention further relates to RNA interference (RNAi)
to decrease Sam68 expression in target cells. "RNA interference" refers to the
process of sequence specific suppression of gene expression mediated by small
interfering RNA (siRNA) without generalized suppression of protein synthesis.
While the invention is not limited to a particular mode of action, RNAi may
involve
degradation of messenger RNA (e.g., Sam68 mRNA) by an RNA induced silencing
complex (RISC), preventing translation of the transcribed targeted mRNA.
Alternatively, it may involve methylation of genomic DNA, which shuts down
transcription of a targeted gene. The suppression of gene expression caused by
CA 02494575 2004-12-24
58
RNAi may be transient or it may be more stable, even permanent.
[0156) RNA interference is triggered by the presence of short
interfering RNAs of about 20-25 nucleotides in length which comprise about 19
base pair duplexes. These siRNAs can be of synthetic origin or they can be
derived from a ribonucfease III activity (e.g., dicer ribonuclease) found in
cells. The
RNAi response also features an endonuclease complex containing siRNA,
commonly referred to as an RNA-induced silencing complex (RISC), which
mediates the cleavage of single stranded RNA having a sequence complementary
to the antisense region of the siRNA duplex. Cleavage of the target RNA (e.g.,
Sam68 mRNA) takes place in the middle of the region complementary to the
antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev.,
15:188).
[0157] "Small interfering RNA" of the present invention refers to any
nucleic acid molecule capable of mediating RNA interference "RNAi" or gene
silencing (see for example, Bass, 2001, Nature, 411:428-429; Elbashir et al.,
2001,
Nature, 411:494-498; Kreutzer et al., International PCT publication No. WO
00/44895; Zernicka-Goetz et al., International PCT publication No. WO
01/36646;
Fire, International PCT publication No. W099/32619; Mello and Fire,
International
PCT publication No. W001/29058; Deschamps-Depaillette, International PCT
publication No. W099/07409; Han et al., International PCT puplication No. WO
2004/011647; Tuschl et al., International PCT publication No. WO 02/44321; and
Li et al., International PCT publication No. WO 00/44914). For example, siRNA
of
the present invention are double stranded RNA molecules from about ten to
about
30 nucleotides long that are named for their ability to specifically interfere
with
protein expression. In one embodiment, siRNA of the present invention are 12-
28
nucleotides long, more preferably 15-25 nucleotides long, even more preferably
19-23 nucleotides long and most preferably 21-23 nucleotides long. Therefore
preferred siRNA of the present invention are 12, 13, 14, 15, 16, 17, 18, 19,
20, 21,
22, 23, 24, 25, 26, 27, 28 nucleotides in length. As used herein, siRNA
molecules
CA 02494575 2004-12-24
59
need not to be limited to those molecules containing only RNA, but further
encompasses chemically modified nucleotides and non-nucleotides.
[0158] The length of one strand designates the length of an siRNA
molecule. For example, a siRNA that is described as a 23 ribonucleotides long
(a
23 mer) could comprise two opposite strands of RNA that anneal together for 21
contiguous base pairing. The two remaining ribonucleotides on each strand
would
form what is called an "overhang". In a particular embodiment, the siRNA of
the
present invention contains two strands of different lengths. In this case, the
longer
strand designates the length of the siRNA. For example, a dsRNA containing one
strand that is 20 nucleotides long and a second strand that is 19 nucleotides
long
is considered a 20 mer.
[0159] siRNAs that comprises an overhang are desirable. The
overhang may be at the 3' or 5' end. Preferably, the overhangs are at the 3'
end of
an RNA strand. The length of an overhang may vary but preferably is about 1 to
5
nucleotides long. Generally, 21 nucleotides siRNA with two nucleotides 3'-
overhang are the most active siRNAs.
[0160] siRNA of the present invention are designed to decrease Sam68
expression in a target cell by RNA interference. siRNA of the present
invention
comprise a sense region and an antisense region wherein the antisense region
comprises a sequence complementary to a Sam68 mRNA sequence (e.g., SEQ ID
NO: 1, or SEQ ID NO: 2) and the sense region comprises a sequence
complementary to the antisense sequence of Sam68 mRNA. A siRNA molecule
can be assembled from two nucleic acid fragments wherein one fragment
comprises the sense region and the second fragment comprises the antisense
region of siRNA molecule. The sense region and antisense region can also be
covalently connected via a linker molecule. The linker molecule can be a
polynucleotide linker or a non polynucleotide linker.
CA 02494575 2004-12-24
[0161] In one embodiment, the present invention features a siRNA
molecule having RNAi activity against Sam68 RNA, wherein the siRNA molecule
comprises a sequence complementary to any RNA having a Sam68 encoding
sequence. A siRNA molecule of the present invention can comprise any
contiguous Sam68 sequence (e.g. 19-23 contiguous nucleotides present in a
Sam68 sequence such as SEQ ID NO: 1, SEQ ID NO: 2). In the particular case
where alternate splicing produces a family of transcripts that are
distinguished by
specific exons, the present invention can be used to inhibit gene expression
of a
particular gene family member through the targeting of the appropriate exon(s)
(e.g., to specifically knock down the expression of the Sam68deItaKH
transcript
(Barlat et al., 1997, J. Biol. Chem. 272: p3129-32) or of the full length
transcript
(SEQ ID NO: 1 and 2).
[0162] siRNA of the present invention comprises a ribonucleotide
sequence that is at least 80% identical to a Sam68 ribonucleotide sequence.
Preferably, the siRNA molecule is at least 90%, at least 95% (e.g., 95, 96,
97, 99,
99, 100%), at least 98% (e.g., 98, 99, 100%) or at least 99% (e.g., 99, 100%)
identical to the ribonucleotide sequence of the target gene (e.g., Sam68 RNA).
siRNA molecule with insertion, deletions, or single point mutations relative
to the
target may also be effective. Mutations that are not in the center of the
siRNA
molecule are more tolerated. Tools to assist siRNA design are well known in
the
art and readily available to the public. For example, a computer-based siRNA
design tool is available on the Internet at www.dharmacon.com or are available
on
the web site of several companies that offer the synthesis of siRNA molecules.
[0163] In one embodiment, the siRNA molecules of the present
invention are chemically modified to confer increased stability against
nuclease
degradation but retain the ability to bind to the target nucleic acid that is
present in
a cell. Modified siRNAs of the present invention comprise modified
ribonucleotides,
and are resistant to enzymatic degradation such as RNAse degradation, yet they
CA 02494575 2004-12-24
61
retain their ability to reduce Sam68 expression in a target cell. The siRNA
may be
modified at any position of the molecule so long as the modified siRNA is
still
capable of binding to the target sequence and is more resistant to enzymatic
degradation. Modifications in the siRNA may be in the nucleotide base (i.e.,
purine
or pyrimidine), the ribose or phosphate.
[0164] More specifically, the siRNA may be modified in at least one
purine, in at least one pyrimidine or a combination thereof. Generally, all
purines
(adenosine or guanine) or all pyrimidine (cytosine or uracyl) or a combination
of all
purines and all pyrimidines of the siRNA are modified. Ribonucleotides on
either
one or both strands of the siRNA may be modified.
[0165] Non-limiting examples of chemical modification that can be
included in an siRNA molecule include phosphorothioate internucleotide
linkages
(see US 2003/0175950), 2'-O-methyl ribonucleotides, 2'-O-methyl modified
ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro
modified
pyrimidines nucleotides, 5-C-methyl nucleotides and deoxyabasic residue
incorporation. The ribonucleotides containing pyrimidine bases can be modified
at
the 2' position of the ribose residue. A preferable modification is the
addition of a
molecule from the halide chemical group such as fluorine. Other chemical
moieties
such as methyl, methoxymethyl and propyl may also be added as modifications
(see International PCT publication No. W02004/011647). These chemical
modifications, when used in various siRNA constructs, are shown to preserve
RNAi activity in cells while at the same time, dramatically increasing their
stability
in cells or serum. Chemical modifications of the siRNA of the present
invention can
also be used to improve the stability of the interaction with the target RNA
sequence.
[0166] siRNA of the present invention may also be modified by the
attachment of at least one receptor binding ligand to the siRNA. Receptor
binding
CA 02494575 2004-12-24
62
ligand can be any ligand or molecule that directs the siRNA of the present
invention to a specific target cell (e.g., osteoblasts and/or osteoclasts).
Such
ligands are useful to direct delivery of siRNA to a target cell in a body
system,
organ or tissue of a subject such as bone cells. Receptor binding ligand may
be
attached to one or more siRNA ends, including any combination of 5' or 3'
ends.
The selection of an appropriate ligand for delivering siRNAs depends on the
cells,
tissues or organs that are targeted and is considered to be within the
ordinary skill
of the art. For example, to target a siRNA to hepatocytes, cholesterol may be
attached at one or more ends, including 3' and 5' ends. As another example,
siRNA molecules can be targeted to bones by attaching at the 3' end or 5' end
of a
siRNA molecules an acidic moiety which will specifically interact with bone
matrix.
Other conjugates such as other ligands for cellular receptors (e.g., peptides
derived from naturally occurring protein ligands), protein localization
sequences
(e.g., ZIP code sequences), antibodies, nucleic acid aptamers, vitamins and
other
cofactors such as N-acetylgalactosamine and folate, polymers such as
polyethyleneglycol (PEG), polyamines (e.g., spermine or spermidine) and
phospholipids can be linked (directly or indirectly) to the siRNA molecule for
improving its bioavailability. Bisphosphonates examples of bone seeking
compounds that could be conjugated to the siRNAs of the present invention. The
bond between the siRNA and the biphosphonate may be made susceptible to
degradation by one or more degradation enzymes such as Phex (Cameos et al.,
Biochem J. 2003 Jul 1;373(Pt 1):271-9) which is very abundant in the bone
micro-
environment). Another approach that could be used in accordance with the
present
invention would be to conjugate the siRNAs to Tat or PTHrP nuclear targeting
sequences, which are endocytosed and transported to the nuclei of target
cells. In
yet another embodiment, the siRNAs of the present invention could be
transduced
in autogenous bone marrow ex vivo and then re-introduce into the host as a
bone
marrow transplant. This approach has been previously used with sucess in kids
with osteogenesis imperfecta (OI).
CA 02494575 2004-12-24
63
[0167] siRNAs can be prepared in a number of ways well known in the
art, such as by chemical synthesis, T7 polymerise transcription, or by
treating long
double stranded RNA (dsRNA) prepared by one of the two previous methods with
Dicer enzyme. Dicer enzyme create mixed population of dsRNA from about 21 to
23 base pairs in length from double stranded RNA that is about 500 base pairs
to
about 1000 base pairs in size. Dicer can effectively cleave modified strands
of
dsRNA, such as 2'-fluoromodified dsRNA (see W02004/011647).
[0168] In one embodiment, vectors are employed for producing siRNAs
by recombinant techniques. Thus, for example, a DNA segment encoding a siRNA
derived from a Sam68 sequence (e.g., SEQ ID N0:1, SEQ ID N0:2) may be
included in anyone of a variety of expression vectors for expressing any DNA
sequence derived from a Sam68 sequence. Such vectors include synthetic DNA
sequences (e.g., derivatives of SV40, bacterial plasmids, baculovirus, yeast
plamids, viral DNA such as vaccinia, fowl pox virus, adenovirus, lentivirus,
retrovirus, adeno-associated virus, alphavirus etc), chromosomal, and non
chromosomal vectors. Any vector may be used in accordance with the present
invention as long as it is replicable and viable in the desired host. The DNA
segment in the expression vector is operatebly linked to an appropriate
expression
control sequences) (e.g., promoter) to direct siRNA synthesis. Preferably, the
promoters of the present invention are from the type III class of RNA
polymerise III
promoters (e.g., U6 and H1~ promoters). The promoters of the present invention
may also be inducible, in that the expression may be turned on or turned off
(e.g.,
tetracycline-regulatable system employing the U6 promotor to control the
production of siRNA targeted to Sam68).
[0169] In a particular embodiment, the present invention utilizes a
vector wherein a DNA segment encoding the sense strand of the RNA
polynucleotide is operatebly linked to a first promoter and the antisense
strand of
the RNA polynucleotide is operably linked to a second promoter (i.e., each
strand
CA 02494575 2004-12-24
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of the RNA polynucleotide is independently expressed).
[0170] In another embodiment, the DNA segment encoding both
strands of the RNA polynucleotide are under the control of a single promoter.
In a
particular embodiment, the DNA segment encoding each strand are arranged on
the vector with a loop region connecting the two DNA segments (e.g., sense and
antisense sequences), where the transcription of the DNA segments and loop
region creates one RNA transcript. When transcribed, the siRNA folds back on
itself to form a short hairpin capable of inducing RNAi. The loop of the
hairpin
structure is preferably from about 4 to 6 nucleotides in length. The short
hairpin is
processed in cells by endoribonucleases wick removes the loop thus forming a
siRNA molecule. In this particular embodiment, siRNAs of the present invention
comprising a hairpin or circular structures are about 35 to about 65
nucleotides in
length (e.g., 35, 36, 37, 38, 49, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 65 nucleotides in length),
preferably
between 40 and 64 nucleotides in length comprising for example about 18, 19,
20,
21, 22, or 23, 24,25 base pairs.
[0171 In yet a further embodiment, the vector of the present invention
comprises opposing promoters. For example, the vector may comprise two RNA
polymerase III promoters on either side of the DNA segment (e.g., a specific
Sam68 DNA segment) encoding the sense strand of the RNA polynucleotide and
placed in opposing orientations, with or without a transcription terminator
placed
between the two opposing promoters.
[0172] Non-limiting examples of expression vectors used for siRNA
expression are described in Lee et al., 2002, Nature Biotechnol., 19:505;
Miyagishi
and Taira, 2002, Nature Biotechnol., 19:497; Pau et al., 2002, Nature
Biotechnol.,
19:500 and Novina et al., 2002, Nature Medecine, July 8(7):681-686).
CA 02494575 2004-12-24
(0173 The present invention also relates to antisense nucleic acid
molecules which can be used for example to decrease or abrogate the expression
of Sam68. An antisense nucleic acid molecule according to the present
invention
refers to a molecule capable of forming a stable duplex or triplex with a
portion of
its targeted nucleic acid sequence (DNA or RNA). The use of antisense nucleic
acid molecules and the design and modification of such molecules is well known
in
the art as described for example in WO 96/32966, WO 96/11266, WO 94/15646,
WO 93/08845, and USP 5,593,974. Antisense nucleic acid molecules according to
the present invention can be derived from the nucleic acid sequences and
modified
in accordance to well known methods. For example, some antisense molecules
can be designed to be more resistant to degradation to increase their affinity
to
their targeted sequence, to affect their transport to chosen cell types or
cell
compartments, and/or to enhance their lipid solubility by using nucleotide
analogs
and/or substituting chosen chemical fragments thereof, as commonly known in
the
art.
j0174) In one embodiment, antisense approach of the present invention
involves the design of oligonucleotides (either DNA or RNA) that are
complementary to Sam68 mRNA. The antisense oligonucleotides bind to Sam68
mRNA and prevent its translation. Absolute complementarity, although
preferred,
is not absolutely a prerequisite. One skilled in the art can identify a
certain
tolerable degree of mismatch by use of standard methods to determine the
melting
point of the hybridized antisense complex. In general, oligonucleotides that
are
complementary to the 5'untranslated region (up to the first AUG initiator
codon) of
Sam68 mRNA should work more efficiently at inhibiting translation and
production
of Sam68 protein. However, oligonucleotides that are targeted to a coding
portion
of the sequence may produce inactive truncated protein or diminish the
efficiency
of translation thereby lowering the overall expression of Sam68 protein in a
cell.
Antisense oligonucleotides targeted to the 3' untranslated region of messages
have also proven to be efficient in inhibiting translation of targeted mRNAs
CA 02494575 2004-12-24
66
(Wagner, R. (1994), Nature, 372:333-335). The Sam68 antisense oligonucleotides
of the present invention are less than 100 nucleotides in length,
particularly, less
than 50 nucleotides in length and more particularly less 30 nucleotides in
length.
Generally, effective antisense oligonucleotides are at least 15 or more
oligonucleotides in length.
[0175] The antisense oligonucleotides of the present invention can be
DNA, RNA, Chimeric DNA-RNA analogue, and derivatives thereof (see Inoue et al.
(1987), Nucl. Acids. Res. 15: 6131-6148; Inoue et al. (1987), FEBS lett. 215:
327-
330; Gauthier at al. (1987), Nucl. Acids, Res. 15: 6625-6641.). As mentioned
above, antisense oligonucleotides of the present invention may include
modified
bases or sugar moiety. Examples of modified bases include xanthine,
hypoxanthine, 2-methyladenine, N6-isopentenyladenine, 2-methylguanine, 3-
methylcytosine, 5-methylcytosine, N6-adenine, 7-methyguanine, 5-fluorouracil,
5-
chlorouracil, 5-bromouracil, 5-iodouracyl, 5-carboxymethylaminomethyluracil, 5-
methoxycarboxymethyluracil, queosine, 4-thiouracil and 2,6-diaminopurine.
Examples of modified sugar moieties include hexose, xylulose, arabinose and 2-
fluoroarabinose. The antisense oligonucleotides of the present invention may
also
include modified phosphate backbone such as methylphosphonate,
phosphoramidate, phosphoramidothioates, phosphordiamidate and alkyl
phosphotriesters. The synthesis of modified oligonucleotides can be done
according to methods well known in the art.
[0176] Once an antisense oligonucleotide or siRNA is designed, its
effectiveness can be appreciated by conducting in vitro studies that assess
the
ability of the antisense to inhibit gene expression (e.g., Sam68 protein
expression).
Such studies ultimately compare the level of Sam68 RNA or protein with the
level
of a control experiment (e.g., an oligonucleotide which is the same has that
of
antisense experiment but being a sense oligonucleotide or an oligonucleotide
of
the same size as the antisense oligonucleotide but that does not bind to a
specific
CA 02494575 2004-12-24
67
Sam68 sequence).
[0177] Non virus-based and virus-based vectors (e.g., adenovirus- and
lentivirus-based vectors) for insertion of exogenous nucleic acid sequences
into
eukaryotic cells are well known in the art and may be used in accordance with
the
present invention. Virus-based vectors (and their different variations) for
use in
gene therapy have already been described in details (see). In virus-based
vectors,
parts of a viral gene are replaced by the desired exogenous sequence so that a
viral vector is produced. Viral vectors are no longer able to replicate due to
DNA
manipulations.
[0178] In one specific embodiment, lentivirus derived vectors are used
to target a Sam68 sequence (e.g., siRNA, antisense, nucleic acid encoding a
partial or complete Sam68 protein) into specific target cells (e.g., bone
cells such
as osteoblasts). These vectors have the advantage of infecting quiescent cells
(for
example see US 6,656,706; Amado et al., 1999, Science 285: 674-676).
[0179] In addition to a Sam68 nucleic acid sequence, siRNA or
antisense, the vectors of the present invention may contain a gene that acts
as a
marker by encoding a detectable product.
[0180] One way of performing gene therapy is to extract cells from a
patient, infect the extracted cells with a viral vector and reintroduce the
cells back
into the patient. A selectable marker may or may not be included to provide a
means for enriching for infected or transduced cells. Alternatively, vectors
for gene
therapy that are specially formulated to reach and enter target cells may be
directly
administered to a patient (e.g., intravenously, orally etc.).
[0181] The exogenous sequences (e.g., antisense RNA, siRNA or a
Sam68 targeting vector for homologous recombination) may be delivered into
cells
CA 02494575 2004-12-24
68
that express Sam68 according to well known methods. Apart from infection with
virus-based vectors, examples of methods to deliver nucleic acid into cells
include
DEAE dextran lipid formulations, liposome-mediated transfection, CaCl2-
mediated
transfection, electroporation or using a gene gun. Synthetic cationic
amphiphilic
substances, such as dioleoyloxypropylmethylammonium bromide (DOTMA) in a
mixture with dioleoylphosphatidylethanolamine (DOPE), or lipopolyamine (Behr,
Bioconjugate Chem., 1994 5:382), have gained considerable importance in
charged gene transfer. Due to an excess of cationic charge, the substance
mixture
complexes with negatively charged genes and binds to the anionic cell surface.
Other methods include linking the exogenous oligonucleotide sequence (e.g.,
siRNA, antisense, Sam68 targeting vector for homologous recombination) to
peptides or antibodies that especially binds to receptors or antigens at the
surface
of a target cell. US 6,358,524 describe target cell-specific non-viral vectors
for
inserting at least one gene into cells of an organism. The method described
the
uses of non-viral carriers that are cationized to enable them to complex with
the
negatively charged DNA. Moreover, the method also includes the use of a ligand
(e.g., a monoclonal antibody or fragment thereof that is specific for membrane
antigen present on the surface of bone cells (e.g. PTH receptors, receptors
for
hormone and growth factors etc) can specifically bind to the desired target
cell in
order to enter it.
(0182] To achieve high cellular concentration of the Sam68 antisense
nucleic acid or small inhibitor RNAs of the present invention an effective
method
utilizes a recombinant DNA construct in which the nucleic acid sequence is
placed
under a strong promoter and the entire construct is targeted into the cell.
Such
promoter may constitutively or inducibly produce Sam68 antisense RNA or siRNA
of the present invention.
CA 02494575 2004-12-24
69
EXAMPLE 5
IDENTIFICATION OF SAM 68 AS A TARGET TO PREVENT BONE LOSS
(0183] Sam68 is a ubiquitously expressed binding protein that belongs
to a novel class of apoptotic inducers (Pilotte et al., 2001 Genes & Dev.,
15:845-
858). To define the physiologic role of Sam68, mice homozygous for targeted
disruption of the Sam68 gene were generated. Despite evidence that Sam68 is
ubiquitously expressed, Sam68-/- mice live a normal lifespan, do not develop
tumors, show no immunological or motor abnormalities or other major illnesses.
The only phenotypes are male sterility and preservation of bone mass in old
mice.
Over the past 6 months the skeletal phenotype of young and old female Sam68-/-
mice was characterized using advanced instrumentation and expertise. Using a
combination of quantitative imaging and histologic techniques it was
demonstrated
that the absence of Sam68 prevented the age-related bone loss and micro-
architectural damage seen in intact old female mice.
[0184] Thus, the present invention reports for the first time the
generation of Sam68-deficient mice as well as the analysis of their skeletal
phenotype. The absence of Sam68 confers resistance to age-related bone loss in
mice. These observations identify the Sam68-deficient mouse as a unique animal
model to study bone metabolism in ageing mice and validate Sam68 as a new
molecular target for the prevention and treatment of osteoporosis
[0185] Peak bone mass, which is achieved by the age of 30 in men and
women, has been identified as a major determinant of resistance or
susceptibility
to osteoporosis. It has been estimated that greater than 70% of the variance
in
peak bone mass is genetically determined and that genetic factors also
determine
the rate at which bone is lost from the ageing skeleton .For these reasons the
mouse has become the genetic model of choice to investigate disorders of bone
development and skeletal metabolism (Q-Y Huang, R R Recker and H-W Deng,
CA 02494575 2004-12-24
Osteoporosis International 2003 v14 pp701-715 provides many references for
mouse genes that have been associated with osteoporosis).
[0186] The location and activity of the basic multicelluiar units (BMUs)
that constantly remodel and renew bone is tightly regulated by signals arising
from
numerous systemic hormones, from locally derived growth factors, from the bone
matrix and from the cells themselves. A net loss in bone mass results from an
imbalance that favours osteoclast over osteoblast activity. As discussed this
can
result in part from decreased osteoclast apoptosis or increased osteoblast
apoptosis. These observations suggest that apoptosis plays a significant role
in the
pathogenesis of age-related bone loss and that therapeutic intervention to
promote
osteoclast apoptosis (bisphosphonates, estrogen) or inhibit osteoblast
apoptosis
(estrogen, PTH) is a useful strategy to prevent age-related bone loss.
[0187] Src kinases function at the plasma membrane to phosphorylate
the focal adhesion kinase (FAK), the ubiquitin ligase (Cbl) and other
signaling
proteins during the formation of focal adhesion contacts. In bone, this
function is
critically important in osteoclasts to enable their attachment to the bone
surface
and form a sealing zone that localizes bone dissolution to the immediate area.
Src's role in bone was clearly demonstrated more than a decade ago when Src-/-
mice developed severe osteopetrosis (too much bone) caused by defective
osteoclastic bone resorption.
[0188] When the Sam68 mouse cDNA was cloned in 1995 it was
proposed that it functions as a multi-functional effector protein linking Src
to
downstream effectors such as Ras (Richard ef al., 1995). In molecular
biochemical
studies it was also shown that Sam68 binds to itself, to other proteins and to
RNA
and that these interactions are regulated by post-translational modifications
including phosphorylation and methylation (Lukong and Richard 2003, Biochimica
Biophysics Acta 1653:73-86). Despite this apparent promiscuity and
multiplicity of
CA 02494575 2004-12-24
71
function, it was determined that the biological function of Sam68 is regulated
through its interaction with a very selective UAAA motif in uridine-rich RNA.
This
specific interaction enables Sam68 and related proteins to act as an inducer
of
programmed cell death.
[0189] The neo-natal osteopetrotic phenotype of the Src knock-out has
led to a search for Src inhibitors that can be targeted to bone for the
treatment of
osteoporosis. Given its promiscuity and multiple mechanisms of action it is
not
surprising that these efforts have had little success. As is true with other
broad
spectrum kinases that have been identified as potential drug targets, for
example
BCR-ABL and Raf kinase (FDA CDER), rigorous pre-clinical testing is required
to
allow identification of unanticipated, deleterious activity. The age-related
preservation of bone mass in Sam68 null mice provides the first genetic and
physiologic evidence that Sam68 lies in the Src signaling pathway. The
specificity
of the interaction between Sam68 and RNA that mediates its biological activity
most probably explains the less severe phenotype of Sam68 knock-out mice
compared with Src-null mice. This specificity also identifies Sam68 as a
valuable
target for drug discovery. Thus, by having identified a high bone mass
phenotype
in old Sam68'~' females, the present invention has identified a new target for
pharmacological intervention to treat osteoporosis and related disorders.
EXAMPLE 6
CHARACTERIZATION OF THE FUNCTIONAL PHENOTYPE OF SAM68'~'
OSTEOBLASTS
[0190] The most striking feature of the Sam68-/- phenotype is the
preservation of trabecular bone in aged female mice, which is accompanied by
maintenance of the osteoblast population. It is well recognized that increased
osteoblast apoptosis makes a significant contribution to the decline in bone
mass
with age. Sam68 family members induce apoptosis by a mechanism linked to RNA
CA 02494575 2004-12-24
72
binding (Taylor et al., 2004 BMC Cell Biol., 5: 1-12). The goal of these
experiments
is to determine if the absence of Sam68 promotes osteoblast survival.
[0191] Bone marrow will be flushed from the tibia and femora of 2
month old female wild type and Sam68'~' mice and cultured as described (HMG).
Cultures will be screened for viability using Hoechst stain to identify
condensed
nuclei, and for differentiation using ALP and von Kossa stain to identify
differentiated and mineralizing nodules respectively. The results will be
confirmed
using a combination AnnexinV/TUNEL kit (Intergen Corp) to evaluate apoptosis
and immunochemistry to identify differences in common markers of osteoblast
differentiation, such as type I collagen, osteocalcin and matrix MMP13. We
anticipate that Sam68-~- cultures will show fewer apoptotic cells at all
stages of
differentiation, which will yield more mineralized nodules.
EXAMPLE 7
CHARACTERIZATION OF THE FUNCTIONAL PHENOTYPE OF SAM68-/-
OSTEOCLASTS
[0192] Although the skeletal phenotype of Sam68-/- mice resembles
that of Src-/- mice in having mare bone, it differs in degree, age of onset
and
apparent mechanism. Src-/- mice exhibit osteopetrosis, impaired in tooth
eruption
and defective osteoclast activity at birth whereas Sam68-/- mice resemble
their
wild type littermates except for preservation of bone in old age.
[0193] The characterization of the functional phenotype of Sam68-/-
osteoclasts should rule-out a major contribution from osteoclasts and will
thus
distinguish Sam68 from Src as a molecular target. The fact that the Sam68 null
mice are normal at 4 months attests to the normal activity of osteoclasts.
Although
our data demonstrate that the phenotype is mainly osteoblastic, we cannot rule
out
an effect with age of the osteoclasts.
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73
(0194] Small numbers of osteoclasts for preliminary morphological,
histochemical and immunochemical analyses will be obtained by mincing the
femora and tibia of 2-4 day old mice and plating the cells directly on cover
slips as
described (Miyazaki ef al., 2004 J. Biol. Chem. 279: 17660-6). The established
criteria of multi-nucleation (minimum of 3) and expression of tartrate
resistant acid
phosphatase (TRAP) to identify osteoclasts and immunochemistry to co-localize
Sam68 within these cells will be used. A well characterized co-culture system
of
marrow cells and osteoblasts derived from neonatal mouse calvaria will be used
to
obtain large numbers of osteoclasts for functional studies. (Miyazaki et al.,
2004 J.
Biol. Chem. 279: 17660-6). The capacity of wild type and Sam68-/- osteoclasts
to
resorb bone can be assessed using commercially available OsteoAssay plates
(Cambrex Bio Science) that contain particles of bone. The number of
osteoclasts
can be evaluated using TRAP staining and resorption activity can be measured
as
the amount of type I collagen breakdown down product, measured by a
commercial ELISA, released into the culture medium. It is predicted that in
Sam68-
l- cultures there will be more osteoclasts and that their resorption activity
will be
only mildly impaired.
EXAMPLE 8
VALIDATION OF THE KH DOMAIN OF SAM68 AS A TARGET FOR DRUG
DISCOVERY IN OSTEOBLASTS
[0195] The KH domain in Sam68 is a phylogenetically conserved
region that allows STAR proteins to bind RNA (chen 1997). Although the
cellular
RNA targets for Sam68 have not yet been identified, it has been established
that a
single glycine 178 to aspartate (Sam68:G178D) substitution abrogates binding
of
Sam68 to RNA. The hypothesis that the RNA binding function of Sam68 is
necessary to mediate its pro-apoptotic activity in cells of the osteoblast
lineage will
be tested by the following approach.
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74
[0196] Cultures of wild type and Sam68-/- osteoblasts will be prepared
by flushing bone marrow from the tibia and femora of 2 month old female wild
type
and Sam68'~' mice and cultured as described (HMG). The cells will be
transduced
with adenoviruses expressing either wild-type Sam68 or Sam68:G178D carrying
the dominant negative mutation. Inhibition of the basal level of apoptosis and
in
similar numbers of mineralized nodules as seen in Sam68-/- cultures are
anticipated by the expression of Sam68:G178D in wild type cells. Conversely,
expression of wild-type Sam68 in Sam68-/- cultures should promote cell death
and
lead to fewer bone nodules. Further corroboration will be sought in parallel
experiments where Sam68 expression will be knocked-down using RNA
interference and anti-sense RNA. Proof-of-principle for this approach comes
from
anti-sense studies performed in primary cultures of Src-/- osteoblasts (Marzia
et al,
2000). These experiments will validate our strategy to target the KH domain of
Sam68 and inactivate its apoptotic function in osteoblasts in vivo.
EXAMPLE 9
ASSAYS TO IDENTIFY MODULATORS OF SAM68.
[019.7] In order to identify inhibitors of Sam68 activity in bone
metabolism, several screening assays aiming at reducing or inhibiting a
functional
activity of Sam68 in bone can be designed in accordance with the present
invention. One possible way is by screening libraries of candidate compounds
for
inhibitors of the RNA binding activity of Sam68. Inhibitors of other Sam68
functional activities may also be identified in accordance with the present
invention, as long as such functional activities are related to Sam68 function
in
bone metabolism. In addition, screening assays and compounds which directly or
indirectly modulate (e.g. decrease) Sam68 expression in cells are encompassed
by the present invention.
[0198] For example, combinatorial library methods known in the art,
CA 02494575 2004-12-24
including: biological libraries; spatially addressable parallel solid phase or
solution
phase libraries; synthetic library methods requiring deconvolution; the 'one-
bead
one-compound' library method; and synthetic library methods using affinity
chromatography selection may be used in order to identify modulators of Sam68
biological activity. The biological library approach is limited to peptide
libraries,
while the other four approaches are applicable to peptide, non-peptide
oligomer or
small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145,
1997).
Examples of methods for the synthesis of molecular libraries can be found in
the
art, for example in: DeWitt ef al. (1993) Proc. Natl. Acad. Sci. USA. 90:6909;
Erb et
al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994), J.
Med.
Chem. 37:2678; Cho ef al. (1993) Science 261 :1303; Carrell et al. (1994)
Angew.
Chem, Int. Ed Engl. 33:2059; and ibid 2061; and in Gallop et al. (1994). Med
Chem. 37:1233. Libraries of compounds may be presented in solution (e.g..
Houghten (1992) Biotechniques 13:412-421 ) or on beads (Lam (1991 ) Nature
354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria or spores
(Ladner
USP 5,223,409), plasmids (Cull et al.(1992) Proc Natl Acad Sci USA 89:1865-
1869) or on phage (Scott and Smith (1990); Science 249:386-390). Examples of
methods for the synthesis of molecular libraries can be found in the art, for
example in: DeWitt et al. (1993) supra; Erb et al. (1994) supra; Zuckermann et
al.
(1994) supra; Cho et al. (1993) supra; Carrell et al. (1994) supra, or
luciferase, and
the enzymatic label detected by determination of conversion of an appropriate
substrate to product. The choice of a particular combinatorial library depends
on
the specific Sam68 activity which needs to be modulated. Without being limited
to
this particular example, nucleic acids libraries, nucleic acid derivatives or
peptide
nucleic acid (PNA) combinatorial libraries may be used in order to screen for
compounds that inhibit the RNA binding activity of Sam68.
[0199] The RNA-binding KH domain has been identified as one of the
functional region of Sam68 (Lukong and Richard 2003, Biochimica Biophysics
Acts 1653:73-86). Thus, to inactivate Sam68, thus the present invention
relates to
CA 02494575 2004-12-24
76
the development of assays that will permit rapid identification of small
molecules to
inhibit Sam68 RNA binding activity or other Sam68 functional activities
related to
bone metabolism. An existing Sam68/RNA binding assay can readily be adapted
to an ELISA format for rapid screening purposes (Chen et al., 2001, J. Biol.
Chem.
276: 30803-11 ).
[0200] Sam68 has a preference for uridine-rich RNA ligands (Chen et
al., 1997) and has a high-affinity for the UAAA motif (Lin et al., 1997). RNA
binding
activity was often observed by gel mobility shift assay or in vitro 'pull-
downs' (Chen
et al., 2001 ). Thus one non limiting example of a screening assay that may be
used in accordance with the present invention utilizes biotinylated RNA bound
to
Streptavidin-coated 96-well plates and assesses the ability of recombinant
Sam68
to bind the plated RNA in the presence versus the absence of candidate
compounds by ELISA using anti-Sam68 antibodies. To confirm specificity,
competitor or control RNA is pre-incubated with Sam68 and the competitor RNA
should prevent Sam68 binding to the RNA bound to the plate. In addition, the
inability of Sam68:G178D to bind the immobilized RNA can be used as a negative
control. Synthetic RNA can be purchased from Dharmacon Inc. This assay could
readily be adapted to a 384 well format and the procedure automated to a high-
throughput assay for the development of small molecule inhibitors of Sam68.
[0201] Thus, all methods and assays of the present invention may be
developed for low-throughput, high-throughput, or ultra-high throughput
screening
formats. Of course, methods and assays of the present invention are amenable
to
automation. Automation and low-throughput, high-throughput, or ultra-high
throughput screening formats is possible for the screening of agents which
modulates the level and/or activity of Sam68.
[0202] Generally, high throughput screens for Sam68 modulators i.e.
candidate or test compounds or agents (e.g., peptides, peptidomimetics, small
CA 02494575 2004-12-24
77
molecules, antisense RNA, Ribozyme, or other drugs) may be based on assays
which measure biological activity of Sam68. The invention therefore provides a
method (also referred to herein as a "screening assay") for identifying
modulators,
which have an inhibitory effect on, for example, Sam68 biological activity or
expression, or which bind to or interact with Sam68 proteins, or which have a
stimulatory or inhibitory effect on, for example, the expression or activity
of Sam68
interacting proteins (targets) or substrates (e.g. specific mRNAs).
[0203] The assays described above may be used as initial or primary
screens to detect promising lead compounds for further development. Often,
lead
compounds will be further assessed in additional, different screens.
Therefore,
this invention also includes secondary Sam68 screens which may involve assays
utilizing mammalian cell lines expressing Sam68.
[0204] Tertiary screens may involve the study of the identified
modulators in rat and mouse models for bone disorders (e.g. osteoporosis).
Accordingly, it is within the scope of this invention to further use an agent
identified
as described herein in an appropriate animal model. For example, a test
compound identified as described herein (e.g., a Sam68 modulating agent, an
antisense Sam68 nucleic acid molecule, a Sam68 siRNA, a Sam68 antibody or a
Sam68-binding partner etc.) can be used in an animal model to determine the
efficacy, toxicity, or side effects of treatment with such an agent.
Alternatively, an
agent identified as described herein can be used in an animal model to
determine
the mechanism of action of such an agent. Furthermore, this invention pertains
to
uses of novel agents identified by the above-described screening assays for
treatment of bone diseases, as described herein.
CA 02494575 2004-12-24
78
EXAMPLE 10
FUNCTIONAL ASSAYS DESIGNED TO ASSESS THE EFFECT OF SAM68
MODULATORS ON BONE METABOLISM
[0205] The following assays may be used in accordance with the
present invention to determine the effects of Sam68 modulators on bone
metabolism. All assays could be performed in the presence and absence of a
candidate compound and the results compared in order to determine the effect
on
bone metabolism.
Analysis of the differentiation properties of pre-osteoblasts
[0206] Osteoporosis can result from a decrease in the capacity of bone
marrow stromal cells to differentiate into bone-forming osteoblasts. This
capacity
can be tested in vitro using a bone nodule assay. The long bones of 6-8-week
old
mice are removed asceptically, the ends removed with scissors and the bone
marrow cavity flushed with culture medium formulated to support osteoblast
differentiation from bone marrow stromal cells. Marrow cells are plated at
high
density and fed every 3 days for 18 days. Clusters of cells that stain
positive for
alkaline phosphatase (ALP) activity appear between 4-6 days and mineralized
nodules, identified with von Kossa stain, between 15-18 days. Quantitation of
ALP
activity and V Kossa stained nodules provides an index of differentiation
capacity,
which is enhanced in the absence of Sam68.
Analysis of the differentiation properties of pre-osteoclasts
[0207] Osteoporosis can also result from an increase in the capacity of
pre-osteoclasts to differentiate into bone-resorbing osteoclasts. This
capacity can
be tested in vitro by harvesting and plating bone marrow in an identical
manner as
described above. Cultures are then maintained in medium formulated to support
CA 02494575 2004-12-24
79
osteoclast differentiation from hematopoietic precursor cells. Cultures are
stained
between 4-6 days for tartrate resistant acid phosphatase (TRAP) activity, to
identify Multi-nucleate, osteoclast-like cells, and for ALP activity, to
identify the
adjacent ALP-positive clusters.
Analysis of the functional properties of mature osteoblasts (bone formation)
[0208] Osteoporosis can result from decreased activity of mature bone-
forming osteoblasts. This can be evaluated in a similar manner to that
described
above using a bone nodule assay. Osteoblasts are harvested asceptically from
the
calvariae (skull) of 8 week old mice, trimmed to remove soft tissue and
sutures and
cut into small fragments, which are subjected to sequential enzyme digest. The
digested bone fragments are cultured in medium formulated to support
osteoblast
differentiation and the outgrowth of cells continued for 11-15 days.
Trypsinized
cells are re-plated, expanded in culture and used for functional or molecular
analyses.
Analysis of the functional properties of mature osteoclasts (bone resorption)
(0209] Osteoporosis can result from an increase in osteoclast activity
relative to osteoblast activity, which leads to net bone loss. The long bones
of 6-8-
week old mice are removed asceptically, trimmed to remove soft tissue and
chopped finely before suspending in culture medium. The suspension is
aspirated
several times through a wide bore pipet, allowed to settle, and the
supernatant
transferred into a 96 well plate containing adherent particles of bone powder.
Culture medium is changed every 2 days and is formulated to support osteoclast
differentiation and activity. The resorptive capacity of TRAP-positive
osteoclasts is
quantitated by measuring the release of type I collagen fragments into the
culture
medium between days 5-8.
CA 02494575 2004-12-24
[0210] All of the above assays are well known in the art and are
described in more details in "Bone Research Protocols, Ed. M.H. Helfrich and
S.H.
Ralston. Humana Press 2003" the content of which is incorporated herein in its
entirety.
EXAMPLE 11
USE OF SAM68 KNOCK OUT MICE FOR THE GENERATION OF SAM68
SPECIFIC MONOCLONAL ANTIBODIES
(0211] As mentioned in the previous examples, Sam68 is a widely
expressed protein that has been conserved through-out evolution. The extensive
similarity of Sam68 protein among species can present certain problems for the
generation of Sam68-specific monoclonal antibodies. Antibodies are produced
following exposure to foreign antigens and must be recognized as non-self
before
an immune response is built-up. Thus, the Sam68 knock out mice of the present
invention may be used to produce more efficiently Sam68 antibodies since these
animals do not express Sam68. Utilization of knock out mice for this purpose
ensures that that the immunizing protein antigen will be recognized as non-
self and
therefore invoke a powerful immune response.
[0212] Potential applications for the antibodies of the present invention
include their use to reduce Sam68 activity in cells and thus may be useful to
treat
disorders associated with Sam68 activities (e.g. osteoporosis). For example,
loss
of bone in osteoporosis appears to require the presence of Sam68 in bone.
Therefore, a monoclonal antibody immunospecific for a determinant critical for
activity of Sam68 in bone may prevent Sam68 from stimulating bone resorption
that occurs during osteoporosis. Sam68 antibodies may also be useful in assays
to determine if a particular epitope has been modified. For example, the Sam68
antibodies may be used to assess post-translational modifications associated
with
a particular disease related to Sam68 activity (e.g. phosphorylation).
Finally, the
CA 02494575 2004-12-24
81
Sam68 antibodies of the present invention may also be used to quantify various
species of Sam68, for example in ELISA, radioimmunoassay, diffusion based
Ouchterlony, immunoprecipitation, western blot or rocket immunofluorescent
assays. These assays can readily be adapted to detect the Sam68 proteins of
the
present invention. Examples of such assays can be found in Chard, An
Introduction to Radioimmunoassay and Related Techniques, Elsevier Science
Publishers, Amsterdam, The Netherlands (1986); Bullock et al., Techniques in
Immunocytochemistry, Academic Press, Orlando, FL (1997); Tijssen, Practice and
Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and
Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands
(1985); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, New-York (1997). It ~is likely that Sam68 levels in cells
are
associated with disease state. Thus the ability to accurately and easily
quantify
Sam68 levels would be clinically useful.
[0213] Polyclonal antibodies can be raised by administration of Sam68
protein (or fragment thereof) to the knock out mice using well known
immunization
procedures.
[0214] Thus, techniques for preparing antibodies (including monoclonal
antibodies and hybridomas) and for detecting antigens using antibodies are
well
known in the art (Howard et Bethell, Basic methods in Antibody production and
characterization, InterpharmiCRC press, Boca Raton FL, (2000)), Harlow and
Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
New York (1997). In the methods of the present invention, polyclonal,
monoclonal
antibodies, or humanized versions thereof, chimeric antibodies and the like
may be
used. The invention further includes single chain antibodies. Antibody
fragments
which contain the idiotype of the molecule can be generated by known
techniques.
For example, such fragments include but are not limited to: the F(ab')2
fragment;
the Fab' fragments, Fab fragments, and Fv fragments.
CA 02494575 2004-12-24
82
j0215] For polyclonal antibodies, antisera containing antibody is
isolated from the immunized animal and is screened for the presence of
antibodies
with the desired specificity using one of the above-described procedures.
[0216] Humanized antibodies can be produced, for example by
replacing an immunogenic portion of an antibody with a corresponding, but non-
immunogenic portion (i.e. chimeric antibodies) (Robinson, R.R. et al.,
International
Patent Publication PCT/US86/02269; Akira, K. et al., European Patent
Application
184,187; Taniguchi, M., European Patent Application 171,496; Morrison, S.L.
et al., European Patent Application 173,494; Neuberger, M.S. et al., PCT
Application WO 86/01533; Cabilly, S. et al., European Patent Application
125,023;
Better, M. et al., Science 240:1041-1043 (1988); Liu, A.Y. et al., Proc. Natl.
Acad.
Sci. USA 84:3439-3443 (1987); Liu, A.Y. et al., J. Immunol. 139:3521-3526
(1987);
Sun, L.K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y,
et al.,
Canc. Res. 47:999-1005 (1987); Wood, C.R. et aL, Nature 314:446-449 (1985));
Shaw et al., J. Natl.Cancer Insf. 80:1553-1559 (1988). General reviews of
"humanized" chimeric antibodies are provided by Morrison, S.L. (Science,
229:1202-1207 (1985)) and by Oi, V.T. et al., 8ioTechniques 4:214 (1986)).
Suitable "humanized" antibodies can be alternatively produced by CDR or CEA
substitution (Jones, P.T. et al., Nature 321:552-525 (1986); Verhoeyan et al.,
Science 239:1534 (1988); Beidler, C,B. et al., J. Immunol. 141:4053-4060
(1988)).
[0217] Methods for immunization are well known in the art. Such
methods include subcutaneous or interperitoneal injection of the polypeptide.
One
skilled in the art will recognize that the amount of polypeptide used for
immunization will vary based on the animal which is immunized, the
antigenicity of
the polypeptide and the site of injection.
j0218] The polypeptide can be modified or administered in an adjuvant
in order to increase the peptide antigenicity. Methods of increasing the
antigenicity
CA 02494575 2004-12-24
83
of a polypeptide are well known in the art. Such procedures include coupling
the
antigen with a heterologous protein (such as globulin or ~3-galactosidase) or
through the inclusion of an adjuvant during immunization.
[0219] Unlike preparation of polyclonal antisera, the choice of animal
for monoclonal antibody production depends on the availability of appropriate
immortal lines capable of fusing with lymphocytes thereof. Mouse and rat have
been the animals of choice in hybridoma technology and are preferably used. A
number of cell lines suitable for fusion have been developed, and the choice
of any
particular cell line for hybridization protocols is directed by anyone of a
number of
criteria such as speed, uniformity and growth characteristics, absence of
immunoglobulin production and secretion by nonfused cell line, potential for
good
fusion frequency and deficiency of metabolism for a component of the growth
medium. In general, intraspecies hybrids, particularly between like strains
work
better than interspecies fusions.
[0220] For monoclonal antibodies, spleen cells from the immunized
animals are removed, fused with myeloma cells, and allowed to become
monoclonal antibody producing hybridoma cells. Any one of a number of methods
well known in the art can be used to identify the hybridoma cell which
produces an
antibody with the desired characteristics. These include screening the
hybridomas
with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al.,
Exp.
Cell Res. 175:109-124 (1988)).
[0221] Hybridomas secreting the desired antibodies are cloned and the
class and subclass is determined using procedures known in the art (Campbell,
Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and
Molecular Biology, supra (1984)).
[0222] Furthermore, one skilled in the art can readily adapt currently
CA 02494575 2004-12-24
84
available procedures, as well as the techniques, methods and kits disclosed
above
with regard to antibodies, to generate peptides capable of binding to a
specific
peptide sequence in order to generate rationally designed antipeptide
peptides, for
example see Hurby et al., "Application of Synthetic Peptides: Antisense
Peptides",
In Synthetic Peptides, A User's Guide, W.H. Freeman, NY, pp. 289-307 (1992),
and Kaspczak et al., Biochemistry 28:9230-8 (1989).
[0223] Although the present invention has been described hereinabove
by way of preferred embodiments thereof, it can be modified, without departing
from the spirit and nature of the subject invention as defined in the appended
claims.
CA 02494575 2004-12-24
SEQUENCE LISTING
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CA 02494575 2004-12-24
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CA 02494575 2004-12-24
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CA 02494575 2004-12-24
$$
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CA 02494575 2004-12-24
89
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CA 02494575 2004-12-24
35 40 45
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180 185 190
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Cys Gln Glu Gln Phe Leu Glu Leu Ser Tyr Leu Asn Gly Val Pro Glu
225 230 235 240
Pro Ser Arg Gly Arg Gly Val Pro Val Arg Gly Arg Gly Ala Ala Pro
245 250 255
CA 02494575 2004-12-24
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Pro Pro Pro Pro Val Pro Arg Gly Arg Gly VaI Gly Pro Pro Arg Gly
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Ala Leu Val Arg Gly Thr Pro Val Arg Gly Ala Ile Thr Arg Gly Ala
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CA 02494575 2004-12-24
92
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CA 02494575 2004-12-24
93
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Tyr Ala Leu Met Ala His Ala Met Glu Glu Val Lys Lys Phe Leu Val
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Pro Asp Met Met Asp Asp Ile Cys Gln Glu Gln Phe Leu Glu Leu Ser
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Arg Gly Arg Gly Ala Ala Pro Pro Pro Pro Pro Val Pro Arg Gly Arg
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Gly Val Gly Pro Pro Arg Gly Ala Leu Val Arg Gly Thr Pro Val Arg
305 310 315 320
Gly Ser Ile Thr Arg Gly Ala Thr Val Thr Arg Gly Val Pro Pro Pro
325 330 335
Pro Thr Val Arg Gly Ala Pro Thr Pro Arg Ala Arg Thr Ala Gly Ile
340 345 350
Gln Arg Ile Pro Leu Pro Pro Thr Pro Ala Pro Glu Thr Tyr Glu Asp
355 360 365
Tyr Gly Tyr Asp Asp Thr Tyr Ala Glu Gln Ser Tyr Glu Gly Tyr Glu
370 375 380
Gly Tyr Tyr Ser Gln Ser Gln Gly Glu Ser Glu Tyr Tyr Asp Tyr Gly
385 390 395 400
His Gly Glu Leu Gln Asp Ser Tyr Glu Ala Tyr Gly Gln Asp Asp Trp
405 410 415
Asn Gly Thr Arg Pro Ser Leu Lys Ala Pro Pro Ala Arg Pro Val Lys
420 425 430
Gly Ala Tyr Arg Glu His Pro Tyr Gly Arg Tyr
435 440
CA 02494575 2004-12-24
94
<210> 5
<211> 404
<212> PRT
<213> Homo Sapiens
<400> 5
Met Gln Arg Arg Asp Asp Pro Ala Ala Arg Met Ser Arg Ser Ser Gly
1 5 10 15
Arg Ser Gly Ser Met Asp Pro Ser Gly AIa His Pro Ser Val Arg Gln
20 25 30
Thr Pro Ser Arg Gln Pro Pro Leu Pro His Arg Ser Arg Gly Gly Gly
35 40 45
Gly Gly Ser Arg Gly Gly Ala Arg Ala Ser Pro Ala Thr Gln Pro Pro
50 55 60
Pro Leu Leu Pro Pro Ser Ala Thr Gly Pro Asp Ala Thr Val Gly Gly
65 70 75 80
Pro Ala Pro Thr Pro Leu Leu Pro Pro Ser Ala Thr Ala Ser Val Lys
85 90 95
Met Glu Pro Glu Asn Lys Tyr Leu Pro Glu Leu Met Ala Glu Lys Asp
100 105 110
Ser Leu Asp Pro Ser Phe Thr His Ala Met Gln Leu Leu Thr Ala Glu
115 120 125
Ile Glu Lys Ile Gln Lys Gly Asp Ser Lys Lys Asp Asp Glu Glu Asn
130 135 140
Tyr Leu Asp Leu Phe Ser His Lys Asn Met Lys Leu Lys Glu Arg Val
145 150 155 160
Leu Ile Pro Val Lys Gln Tyr Pro Lys Glu Glu Glu Leu Arg Lys Gly
165 170 175
CA 02494575 2004-12-24
Gly Asp Pro Lys Tyr Ala His Leu Asn Met Asp Leu His Val Phe Ile
180 185 190
Glu Val Phe Gly Pro Pro Cys Glu Ala Tyr Ala Leu Met Ala His Ala
195 200 205
Met Glu Glu Val Lys Lys Phe Leu Val Pro Asp Met Met Asp Asp Ile
210 215 220
Cys Gln Glu Gln Phe Leu Glu Leu Ser Tyr Leu Asn Gly Val Pro Glu
225 230 235 240
Pro Ser Arg Gly Arg Gly Val Pro Val Arg Gly Arg Gly Ala Ala Pro
245 250 255
Pro Pro Pro Pro Val Pro Arg Gly Arg Gly Val Gly Pro Pro Arg Gly
260 265 270
Ala Leu Val Arg Gly Thr Pro Val Arg Gly Ala Ile Thr Arg Gly Ala
275 280 285
Thr Val Thr Arg Gly Val Pro Pro Pro Pro Thr Val Arg Gly Ala Pro
290 295 300
Ala Pro Arg Ala Arg Thr Ala Gly Ile Gln Arg Ile Pro Leu Pro Pro
305 310 315 320
Pro Pro Ala Pro Glu Thr Tyr Glu Glu Tyr Gly Tyr Asp Asp Thr Tyr
325 330 335
Ala Glu Gln Ser Tyr Glu Gly Tyr Glu Gly Tyr Tyr Ser Gln Ser Gln
340 345 350
Gly Asp Ser Glu Tyr Tyr Asp Tyr Gly His Gly Glu Val Gln Asp Ser
355 360 365
Tyr Glu Ala Tyr Gly Gln Asp Asp Trp Asn Gly Thr Arg Pro Ser Leu
370 375 380
Lys Ala Pro Pro Ala Arg Pro Val Lys Gly Ala Tyr Arg Glu His Pro
CA 02494575 2004-12-24
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385 390 395 400
Tyr Gly Arg Tyr
<210> 6
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223> Synthetic construct
<400> 6
aatgtctaga aacaactcat atacagac
28
<210> 7
<211> 37
<212> DNA
<213> Artificial sequence
<220>
<223> Synthetic construct
<400> 7
gggatgcggc cgctctagaa ttgtcctact tgaacgg
37
<210> 8
<211> 37
<212> DNA
<213> Artificial sequence
<220>
<223> Synthetic construct
<400> 8
cggtggcggc cgctgtcgac ctgagtaaca tttctta
37
<210> 9
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Synthetic construct
CA 02494575 2004-12-24
97
<400> 9
aagcctttac tggttgtgt
19
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Synthetic construct
<400> 10
cttgaaacgc accgtaggct
<210> 11
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> Synthetic construct
<400> 11
aaatcctaac cctcctcagt cag
23
<210> 12
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Synthetic construct
<400> 12
gatatgatgg atgatatctg tcag
24
<210> 13
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Synthetic construct
CA 02494575 2004-12-24
98
<400> 13
cttgggtgga gaggctattc g
21
<210> 14
<211> 22
<212> DNA
c213> Artificial sequence
<220>
<223> Synthetic construct
<400> 14
gtcgggcatg cgcgccttga gc
22
<210> 15
<211> 19
c212> DNA
<213> Artificial sequence
<220>
c223> Synthetic construct
<400> 15
ggaaacagct atgaccatg
19
c210> 16
<211> 24
c212> PRT
<213> Homo sapiens
c400> 16
Arg Pro Ser Leu Lys Ala Pro Pro Ala Arg Pro Val Lys Gly Ala Tyr
1 5 10 15
Arg Glu His Pro Tyr Gly Arg Tyr
