CA2494575A1 - Compositions and methods for the treatment of osteoporosis - Google Patents

Abstract

The present invention relates to the generation and characterization of Sam68-deficient mice and to the recognition of Sam68 as a target for bone related disorders including osteoporosis. Methods to screen for agents that affect Sam68 activity or expression and methods of treatment of osteoporosis are also described.

Description

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 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 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.

(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 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.

[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 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.

[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.

[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 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.

[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.

[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.

[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 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 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 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 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 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, 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 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 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 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 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.).

[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 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 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.

(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, 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-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-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.

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.

[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 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 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 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.

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 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 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 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.

[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 (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.

[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 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).

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 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 ).

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 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 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 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 (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).

[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 illnesses. Sam68-/- mice did, however, have difficulty breeding due to male infertility and the females rarely provided adequate care to their young.

[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 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.

THREE-DIMENSIONAL ARCHITECTURE OF BONE IS PRESERVED IN AGED

[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

[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) 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

[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 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 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

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.

[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 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.

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.

[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.

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 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).

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 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-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 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 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.

[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 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 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).

[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 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).

(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 (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:

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, 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 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 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.

(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, 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 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.

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

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.

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.

(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.

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.

[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.

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, 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 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 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.

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 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.

[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.

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 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.

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 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 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.

SEQUENCE LISTING
<110> McGill University <120> COMPOSITIONS AND METHODS FOR THE TREATMENT OF OSTEOPOROSIS
<130> 11168.244 <160> 16 <170> PatentIn version 3.2 <210> 1 <211> 2685 <212> DNA
<213> Homo sapiens <400> 1 ggcttcggtc gctaccgctc ccgctctgcc acccccgcca accgccgctc gggcctccgt cgctgccgcg tcgctttctc gctccttgga tcgcacatcc tcccagatgc agcgccggga cgaccccgcc gcgcgcatga gccggtcttc gggccgtagc ggctccatgg acccctccgg tgcccacccc tcggtgcgtc agacgccgtc tcggcagccg ccgctgcctc accggtcccg gggaggcgga gggggatccc gcgggggcgc ccgggcctcg cccgccacgc agccgccacc gctgctgccg ccctcggcca cgggtcccga cgcgacagtg ggcgggccag cgccgacccc gctgctgccc ccctcggcca cagcctcggt caagatggag ccagagaaca agtacctgcc cgaactcatg gccgagaagg actcgctcga cccgtccttc actcacgcca tgcagctgct gacggcagaa attgagaaga ttcagaaagg agactcaaaa aaggatgatg aggagaatta cttggattta ttttctcata agaacatgaa actgaaagag cgagtgctga tacctgtcaa gcagtatccc aagttcaatt ttgtggggaa gattcttgga ccacaaggga atacaatcaa aagactgcag gaagagactg gtgcaaagat ctctgtattg ggaaagggct caatgagaga ..

caaagccaag gaggaagagc tgcgcaaagg tggagacccc aaatatgccc acttgaatat ggatctgcat gtcttcattg aagtctttgg acccccatgt gaggcttatg ctcttatggc ccatgccatg gaggaagtca agaaatttct agtaccggat atgatggatg atatctgtca ggagcaattt ctagagctgt cctacttgaa tggagtacct gaaccctctc gtggacgtgg ggtgccagtg agaggccggg gagctgcacc tcctccacca cctgttccca ggggccgtgg tgttggacca cctcgggggg ctttggtacg tggtacacca gtaaggggag ccatcaccag aggtgccact gtgactcgag gcgtgccacc cccacctact gtgaggggtg ctccagcacc aagagcacgg acagcgggca tccagaggat acctttgcct ccacctcctg caccagaaac atatgaagaa tatggatatg atgatacata cgcagaacaa agttacgaag gctacgaagg ctattacagc cagagtcaag gggactcaga atattatgac tatggacatg gggaggttca agattcttat gaagcttatg gccaggacga ctggaatggg accaggccgt cgctgaaggc ccctcctgct aggccagtga agggagcata cagagagcac ccatatggac gttattaaaa acaaacatga ggggaaaata tcagttatga gcaaagttgt tactgatttc ttgtatctcc caggattcct gttgctttac ccacaacaga caagtaattg tctaagtgtt tttcttcgtg gtccccttct tctccccacc ttattccatt cttaactctg cattctggct tctgtatgta gtattttaaa atgagttaaa atagatttag gaatattgaa ttaatttttt aagtgtgtag atgctttttt ctttgttgtt taaatataaa cagaagtgta ccttttataa taaaaaaaag aagttgagta aaaaaaaaaa acacacaaac ctgttagttt caaaaatgac attgcttgct taaaggttct gaagtaaagg cttgttaagt ttctcttagt tttgatttga ggcatcccgt aaagttgtag ttgcagaatc ccaaactagg ctacatttca aaattcaggg ctgtttaaga tttaaaatca caaacattaa cggcagtagg caccaccatg taaaagtgag ctcagacgtc tctaaaaaat gtttccttta taaaagcaca tggcggttga atcttaaggt taaattttaa tatgaaagat cctcatgaat taaatagttg atgcaatttt taacgttaat tgatataaaa aaaaaaacaa caaaattagg cttgtaaaac tgactttttc attacgtggg ttttgaaatc tagccccaga catactgtgt tgagagatac ttagagggag ggagtaggtt ttgaagaggt tgatggtggt ggggagggaa ggcctcctga attgagtttg atgcagagct ttttagccat gaagaatctt tcagtcatag tactaataat taaattttca gtatttaaaa agacaaagta ttttgtccat ttgagattct gcactccatg aaaagttcac ttggacgctg gggccaaaag ctgttgattt tcttaagttg acggttgtca atatatcgaa ctgttcccaa gttagtcaag tatgtctcaa cactagcatg atataaaaag ggacactgca gctgaatgaa aaaggaatca aaatccactt tgtacataag ttaaagtcct aattggattt gtaccgtcct cccattttgt tctcggaaga ttaaatgcta catgtgtaag tctgcctaaa taggtagctt aaacttatgt caaaatgtct gcagcagttt gtcaataaag tttagtcctt tttta <210> 2 <211> 1709 <212> DNA
<213> Mus musculus $$
<400> 2 cgctgtcgct cctgttcttc cacccccgcc aaccgccgct cgggcctcag ctgccgccgc gtcgcttcct cgctcgttcg ctcgccaccc atcatccccg atgcagcgcc gggacgatcc tgcctcgcgc ctcacccggt cctcgggccg cagctgctcc aaggacccgt caggtgccca cccctcggtg cgtctgaccc cgtctcggcc gtcgccgctt cctcaccggc cccggggagg gggaggtggg cccagaggag gcgctcgggc ctcgcccgcc acccagccgc cgccgctgct gcctccctcc acccctggtc ccgacgcgac ggtggtgggt tccgcgccga ccccgctgct gcccccgtca gccacagccg cggtcaagat ggagccggag aataagtacc cgcctgaact catggccgag aaggactcgc tcgacccgtc cttcactcac gccatgcagc tgctgtccgt agaaattgag aagattcaga agggagagtc aaaaaaagat gacgaggaga attatttgga tttattttct cataagaaca tgaagctgaa agaacgcgtg ctgatacctg tcaagcagta tccaaagttc aattttgtgg ggaagattct tggaccacaa ggaaatacaa tcaaaagact ccaggaagag actggtgcaa agatctctgt cttggggaag ggttcaatga gagacaaagc caaggaggaa gagttgcgca agggtggaga ccccaaatat gcccatttaa atatggatct gcatgtcttc attgaagtct ttggaccccc gtgtgaagct tatgctctta tggcccatgc tatggaagaa gtcaagaagt tcctagtacc agatatgatg gatgatatct gtcaggagca gtttctagaa ttgtcctact tgaacggagt acctgaaccc tctcgtggtc gtggggtatc tgtgagagga cgaggagctg cccctcctcc tccacctgtt cccagaggac gtggtgttgg accacctaga ggagctttgg ttcgtggaac cccagtgaga ggctccatca ccagaggtgc cactgtgact cgaggagtgc cacccccacc tactgtgagg ggtgctccaa caccaagagc tcggacagct gggattcaga gaataccttt gcctcccaca cctgcaccag aaacatacga agattatgga tatgatgata catacgcaga acagagttac gaaggctatg aaggctatta cagccagagt caaggggagt cagagtatta tgactatgga catggggagc tccaagattc ttacgaagcc tacggacaag atgactggaa tgggaccagg ccatcactga aggctcctcc agctaggcca gtgaagggag catacagaga gcatccatat ggacgttatt aaaaacaaac aggagggaaa aatatcagtt atgagcaaag ttgttactga tttcttgtat cccaggattc ctgttgcttt acccacaaca gacaagtaat tgtctaagtg tttttcttcg tggtcccttc tttccccact tcctccattc ttaactctcg attctggctt ctgtaatgta gtattttaaa atgagttaaa atagatttag gaatatcgaa ttaacccccc aagtgtgtaa gatgcttttt tttctttgtt gtttaaatat aaacagtgt <210> 3 <211> 404 <212> PRT
<213> Homo Sapiens <400> 3 Met Gln Arg Arg Asp Asp Pro Ala Ala Arg Met Ser Arg Ser Ser Gly Arg Ser Gly Ser Met Asp Pro Ser Gly Ala His Pro Ser Val Arg Gln Thr Pro Ser Arg Gln Pro Pro Leu Pro His Arg Ser Arg Gly Gly Gly Gly Gly Ser Arg Gly Gly Ala Arg Ala Ser Pro Ala Thr Gln Pro Pro Pro Leu Leu Pro Pro Ser Ala Thr Gly Pro Asp Ala Thr Val Gly Gly Pro Ala Pro Thr Pro Leu Leu Pro Pro Ser Ala Thr Ala Ser Val Lys Met Glu Pro Glu Asn Lys Tyr Leu Pro Glu Leu Met Ala Glu Lys Asp Ser Leu Asp Pro Ser Phe Thr His Ala Met Gln Leu Leu Thr Ala Glu Ile Glu Lys Ile Gln Lys Gly Asp Ser Lys Lys Asp Asp Glu Glu Asn Tyr Leu Asp Leu Phe Ser His Lys Asn Met Lys Leu Lys Glu Arg Val Leu Ile Pro Val Lys Gln Tyr Pro Lys Glu Glu Glu Leu Arg Lys Gly Gly Asp Pro Lys Tyr Ala His Leu Asn Met Asp Leu His Val Phe Ile Glu Val Phe Gly Pro Pro Cys Glu Ala Tyr Ala Leu Met Ala His Ala Met Glu Glu Val Lys Lys Phe Leu Val Pro Asp Met Met Asp Asp Ile Cys Gln Glu Gln Phe Leu Glu Leu Ser Tyr Leu Asn Gly Val Pro Glu Pro Ser Arg Gly Arg Gly Val Pro Val Arg Gly Arg Gly Ala Ala Pro Pro Pro Pro Pro Val Pro Arg Gly Arg Gly VaI Gly Pro Pro Arg Gly Ala Leu Val Arg Gly Thr Pro Val Arg Gly Ala Ile Thr Arg Gly Ala Thr Val Thr Arg Gly Val Pro Pro Pro Pro Thr Val Arg Gly Ala Pro Ala Pro Arg Ala Arg Thr Ala Gly Ile Gln Arg Ile Pro Leu Pro Pro Pro Pro Ala Pro Glu Thr Tyr Glu Glu Tyr Gly Tyr Asp Asp Thr Tyr Ala Glu Gln Ser Tyr Glu Gly Tyr Glu Gly Tyr Tyr Ser Gln Ser Gln Gly Asp Ser Glu Tyr Tyr Asp Tyr Gly His Gly Glu Val Gln Asp Ser Tyr Glu Ala Tyr Gly Gln Asp Asp Trp Asn Gly Thr Arg Pro Ser Leu Lys Ala Pro Pro Ala Arg Pro Val Lys Gly Ala Tyr Arg Glu His Pro Tyr Gly Arg Tyr <220> 4 <211> 443 <212> PRT
<213> Mus musculus <400> 4 Met Gln Arg Arg Asp Asp Pro Ala Ser Arg Leu Thr Arg Ser Ser Gly Arg Ser Cys Ser Lys Asp Pro Ser Gly Ala His Pro Ser Val Arg Leu Thr Pro Ser Arg Pro Ser Pro Leu Pro His Arg Pro Arg Gly Gly Gly Gly Gly Pro Arg Gly GIy Ala Arg Ala Ser Pro Ala Thr Gln Pro Pro Pro Leu Leu Pro Pro Ser Thr Pro Gly Pro Asp Ala Thr Val Val Gly Ser Ala Pro Thr Pro Leu Leu Pro Pro Ser Ala Thr Ala Ala Val Lys Met Glu Pro Glu Asn Lys Tyr Pro Pro Glu Leu Met Ala Glu Lys Asp Ser Leu Asp Pro Ser Phe Thr His Ala Met Gln Leu Leu Ser Val Glu Ile Glu Lys Ile Gln Lys Gly Glu Ser Lys Lys Asp Asp Glu GIu Asn Tyr Leu Asp Leu Phe Ser His Lys Asn Met Lys Leu Lys Glu Arg Val Leu Ile Pro Val Lys Gln Tyr Pro Lys Phe Asn Phe Val Gly Lys Ile Leu Gly Pro Gln Gly Asn Thr Ile Lys Arg Leu Gln Glu Glu Thr Gly Ala Lys Ile Ser Val Leu Gly Lys Gly Ser Met Arg Asp Lys Ala Lys Glu Glu Glu Leu Arg Lys Gly Gly Asp Pro Lys Tyr Ala His Leu Asn Met Asp Leu His Val Phe Ile Glu Val Phe Gly Pro Pro Cys Glu Ala Tyr Ala Leu Met Ala His Ala Met Glu Glu Val Lys Lys Phe Leu Val Pro Asp Met Met Asp Asp Ile Cys Gln Glu Gln Phe Leu Glu Leu Ser Tyr Leu Asn Gly Val Pro Glu Pro Ser Arg Gly Arg Gly Val Ser Val Arg Gly Arg Gly Ala Ala Pro Pro Pro Pro Pro Val Pro Arg Gly Arg Gly Val Gly Pro Pro Arg Gly Ala Leu Val Arg Gly Thr Pro Val Arg Gly Ser Ile Thr Arg Gly Ala Thr Val Thr Arg Gly Val Pro Pro Pro Pro Thr Val Arg Gly Ala Pro Thr Pro Arg Ala Arg Thr Ala Gly Ile Gln Arg Ile Pro Leu Pro Pro Thr Pro Ala Pro Glu Thr Tyr Glu Asp Tyr Gly Tyr Asp Asp Thr Tyr Ala Glu Gln Ser Tyr Glu Gly Tyr Glu Gly Tyr Tyr Ser Gln Ser Gln Gly Glu Ser Glu Tyr Tyr Asp Tyr Gly His Gly Glu Leu Gln Asp Ser Tyr Glu Ala Tyr Gly Gln Asp Asp Trp Asn Gly Thr Arg Pro Ser Leu Lys Ala Pro Pro Ala Arg Pro Val Lys Gly Ala Tyr Arg Glu His Pro Tyr Gly Arg Tyr <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 Arg Ser Gly Ser Met Asp Pro Ser Gly AIa His Pro Ser Val Arg Gln Thr Pro Ser Arg Gln Pro Pro Leu Pro His Arg Ser Arg Gly Gly Gly Gly Gly Ser Arg Gly Gly Ala Arg Ala Ser Pro Ala Thr Gln Pro Pro Pro Leu Leu Pro Pro Ser Ala Thr Gly Pro Asp Ala Thr Val Gly Gly Pro Ala Pro Thr Pro Leu Leu Pro Pro Ser Ala Thr Ala Ser Val Lys Met Glu Pro Glu Asn Lys Tyr Leu Pro Glu Leu Met Ala Glu Lys Asp Ser Leu Asp Pro Ser Phe Thr His Ala Met Gln Leu Leu Thr Ala Glu Ile Glu Lys Ile Gln Lys Gly Asp Ser Lys Lys Asp Asp Glu Glu Asn Tyr Leu Asp Leu Phe Ser His Lys Asn Met Lys Leu Lys Glu Arg Val Leu Ile Pro Val Lys Gln Tyr Pro Lys Glu Glu Glu Leu Arg Lys Gly Gly Asp Pro Lys Tyr Ala His Leu Asn Met Asp Leu His Val Phe Ile Glu Val Phe Gly Pro Pro Cys Glu Ala Tyr Ala Leu Met Ala His Ala Met Glu Glu Val Lys Lys Phe Leu Val Pro Asp Met Met Asp Asp Ile Cys Gln Glu Gln Phe Leu Glu Leu Ser Tyr Leu Asn Gly Val Pro Glu Pro Ser Arg Gly Arg Gly Val Pro Val Arg Gly Arg Gly Ala Ala Pro Pro Pro Pro Pro Val Pro Arg Gly Arg Gly Val Gly Pro Pro Arg Gly Ala Leu Val Arg Gly Thr Pro Val Arg Gly Ala Ile Thr Arg Gly Ala Thr Val Thr Arg Gly Val Pro Pro Pro Pro Thr Val Arg Gly Ala Pro Ala Pro Arg Ala Arg Thr Ala Gly Ile Gln Arg Ile Pro Leu Pro Pro Pro Pro Ala Pro Glu Thr Tyr Glu Glu Tyr Gly Tyr Asp Asp Thr Tyr Ala Glu Gln Ser Tyr Glu Gly Tyr Glu Gly Tyr Tyr Ser Gln Ser Gln Gly Asp Ser Glu Tyr Tyr Asp Tyr Gly His Gly Glu Val Gln Asp Ser Tyr Glu Ala Tyr Gly Gln Asp Asp Trp Asn Gly Thr Arg Pro Ser Leu Lys Ala Pro Pro Ala Arg Pro Val Lys Gly Ala Tyr Arg Glu His Pro Tyr Gly Arg Tyr <210> 6 <211> 28 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic construct <400> 6 aatgtctaga aacaactcat atacagac <210> 7 <211> 37 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic construct <400> 7 gggatgcggc cgctctagaa ttgtcctact tgaacgg <210> 8 <211> 37 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic construct <400> 8 cggtggcggc cgctgtcgac ctgagtaaca tttctta <210> 9 <211> 19 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic construct <400> 9 aagcctttac tggttgtgt <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 <210> 12 <211> 24 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic construct <400> 12 gatatgatgg atgatatctg tcag <210> 13 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic construct <400> 13 cttgggtgga gaggctattc g <210> 14 <211> 22 <212> DNA
c213> Artificial sequence <220>
<223> Synthetic construct <400> 14 gtcgggcatg cgcgccttga gc <210> 15 <211> 19 c212> DNA
<213> Artificial sequence <220>
c223> Synthetic construct <400> 15 ggaaacagct atgaccatg 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 Arg Glu His Pro Tyr Gly Arg Tyr

Claims (15)

1. A transgenic mouse whose germ cell comprises a homozygous null mutation in the endogenous nucleic acid sequence encoding Sam68 such that said mouse does not express a functional mouse Sam68 protein.

2. The transgenic mouse of claim 1, wherein said mouse is protected against age-related bone loss and transmits said null mutation to its offspring.

3. The transgenic mouse of claim 1, wherein said mouse is a male and is infertile.

4. The transgenic mouse of claim 1, wherein said mutation is created by insertion of a neomycin resistant gene cassette in order to replace exon4 and part of exon5 of Sam68 gene.

5. The transgenic mouse of claim 1, wherein said null mutation has been introduced into an ancestor of said mouse at an embryonic stage following introduction of embryonic stem cells bearing said Sam68 mutation into a blastocyst.

6. A method to identify potentially therapeutic agents which inhibit Sam68 activity useful for the treatment and prevention of osteoporosis, comprising:
i) contacting said agent with cells expressing Sam68; and ii) assessing said cells for an alteration in a Sam68 biological function, said biological function being related to bone remodeling;
wherein a potentially therapeutic agent useful for the treatment of osteoporosis is identified when said biological function related to bone remodeling is decreased in the presence of a candidate agent as compared to in the absence thereof.

7. A short interfering RNA (siRNA) molecule, useful for the treatment of osteoporosis, that down regulates the expression of Sam68 gene by RNA
interference comprising a sense region and an antisense region, wherein said antisense region comprises a sequence complementary to a Sam68 RNA
sequence and the sense region comprises a sequence complementary to the antisense of said Sam68 RNA sequence, and wherein the sense region of said siRNA is at least 95% identical to a portion of Sam68 nucleic acid selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:2.

8. The siRNA of claim 7, wherein said siRNA molecule is assembled from two nucleic acid fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of said siRNA
molecule.

9. The siRNA of claim 8, wherein said sense region and said antisense region are covalently connected via a linker molecule.

10. The siRNA of claim 9, wherein said linker molecule is a polynucleotide linker molecule.

11. The siRNA molecule of claims 7, wherein said sense region comprises a 3'-terminal overhang of 1 to 5 nucleotides in length and said antisense region comprises a 3'-terminal overhang of 1 to 5 nucleotides in length.

12. The siRNA molecule of claims 7, wherein said said sense and antisense regions comprise at least one nucleotide that is chemically modified in at least one of sugar, base, or backbone moiety.

13. The siRNA molecule of claims 7, comprising a double stranded region of about 10 to 28 nucleotides in length.

14. The siRNA molecule of claim 13, wherein said siRNA molecule is linked to at least one receptor binding ligand.

15. The siRNA molecule of claim 14, wherein said receptor binding ligand is attached to the 5'-end, the 3'end or both ends of the sense or antisense region of the siRNA molecule.