WO2005021757A1

WO2005021757A1 - Polypeptides and polynucleotides for use as a medicament

Info

Publication number: WO2005021757A1
Application number: PCT/EP2003/010086
Authority: WO
Inventors: Peter Herwig Maria Tomme; Luc Van Rompaey
Original assignee: Galapagos Genomics N.V.
Priority date: 2003-09-01
Filing date: 2003-09-01
Publication date: 2005-03-10
Also published as: AU2003266376A1

Abstract

The present invention relates to polypeptides and polynucleotides for use as a medicament. The invention in particular relates to the use of the polypeptides and polynucleotides for the manufacture of a medicament for the treatment of disorders involving a systemic or local decrease or increase in mean bone density.

Description

Polypeptides and polynucleotides for use as a medicament

The present invention relates to polypeptides and polynucleotides for use as a medicament. The invention in particular relates to the use of the polypeptides and polynucleotides for the manufacture of a medicament for the treatment of disorders involving a systemic or local decrease in mean bone density. Bone is not a static tissue. It is subject to constant breakdown and resynthesis in a complex process mediated by osteoblasts, which produce new bone, and osteoclasts, which destroy bone. The activities of these cells are regulated by a large number of cytokines and growth factors, many of which have now been identified and cloned. A number of diseases are caused by a disturbance of the fine-tuned balance between bone resorption and bone build-up. More precisely, increases or decreases of osteoclast activity or increases or decreases of osteoblast proliferation and differentiation result in a variety of diseases. The single most important bone disease is osteoporosis, but a number of other diseases affect a large number of patients such as hypercalce ia of malignancy, Paget's disease, inflammatory bone diseases like rheumatoid arthritis and osteo-arthritis and periodontal disease, focal osteogenesis occurring during skeletal metastases, Crouzon' s syndrome, rickets, opsismodysplasia, pycnodysostosis/Toulouse-Lautrec disease, osteogenesis imperfecta. As mentioned above, the cells which are responsible for forming bone, are osteoblasts. As osteoblasts differentiate from precursors to mature bone-forming cells, they express and secrete a number of enzymes and structural proteins of the bone matrix, including Type-1 collagen, osteocalcin, osteopontin and alkaline phosphatase. In addition, the bone morphogenetic proteins (BMPs) are expressed by cultured osteoblasts as they proliferate and differentiate. There is a plethora of conditions, which are characterized by the need to enhance bone formation: osteoporosis, hypercalcemia of malignancy, multiple myelomatosis, hyperparathyroidism, and hyperthyroidism. In addition, in the case of bone fractures, it would be desirable to stimulate bone growth and to hasten and complete bone repair. Agents that enhance bone formation would also be useful in facial reconstruction procedures. Other bone deficit conditions include bone segmental defects, periodontal disease, metastatic bone disease, osteolytic bone disease and conditions where connective tissue repair would be beneficial, such as healing or regeneration of cartilage defects or injury. Also of great significance are the chronic conditions of rheumatoid arthritis and osteo-arthritis and osteoporosis, including age-related osteoporosis and osteoporosis associated with post-menopausal hormone status. Other conditions characterized by the need for bone growth include disuse osteoporosis, diabetes-related osteoporosis, and glucocorticoid-related osteoporosis. Bone remodeling relies on an equilibrium between an anabolic - osteogenic - and a catabolic - bone resorption - process. After bone fractures, bone remodeling processes are required to heal the fracture. However, in many instances, patients are encountered with poorly healing fractures. A surgical intervention is often required to accelerate the recovery. Prostheses can be implanted with or without bone grafting procedures. In some cases where the bone is too porous or where previous implants failed to be incorporated into the bone, current medical practices can offer little or no help. There are currently no satisfactory pharmaceutical approaches to managing any of these conditions. Bone fractures are still treated exclusively using casts, braces, anchoring devices and other strictly mechanical means. Further bone deterioration associated with post-menopausal osteoporosis has been decreased or prevented with estrogens or bisphosphonates . Current therapies for the disease focus, however on stopping or slowing bone loss and stabilizing a person's existing bone mass, and not on building new bone to replace bone that has already deteriorated. A very limited number of compounds have been identified that are able to induce osteoblast differentiation in vitro, e.g. dexamethasone or recombinant human secreted proteins, such as BMP-2 or BMP-7. BMPs are potent stimulators of bone formation in vitro and in vivo . There are, however disadvantages to their use as therapeutic agents to enhance bone healing. Receptors for the bone morphogenetic proteins have been identified in many tissues, and the BMPs themselves are expressed in a large variety of tissues in specific temporal and spatial patterns. This suggests that BMPs may have effects on many tissues other than bone, potentially limiting their usefulness as therapeutic agents when administered systemically. There are also conditions which are characterized by increased bone mass such as osteosclerosis, osteosclerotic myeloma, pyknodysostosis, ca uratie-engelmand disease, osteopoikilosis, melorheostosis, and osteopetrosis . Current treatment for these condition primarily aim at increasing bone resorption by, for example, administering high doses of 1, 25-dihydroxyVitD/calcitriol and recombinant human interferon gamma, but also radiotherapy and prednisolone therapy are used. These therapies partially alleviate the diseases but do not cure them and some of them have severe side effects. It is important that factors are found that can induce osteoblast differentiation in vitro, starting from the pluripotent bone marrow mesenchymal progenitor cells (MPCs) or even from totipotent stem cells. These factors can be used for inducing bone formation. Inhibition of these factors can be beneficial for diseases with increased bone mass. Accordingly, there is a large interest in finding new human targets that are involved in the specific modulation of differentiation of osteoblasts or of other cell types involved in bone homeostasis. For example, human secreted proteins that specifically induce differentiation of osteoblasts can be used as biopharmaceuticals . But also proteins involved in the pathway of osteoblast induction can be used as targets to find drugs that modulate osteoblast differentiation. The present invention relates to polypeptides that are new drug targets for inducing the differentiation of osteoblasts. According to a first aspect, the present invention thus relates to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62, or a derivative, or a fragment thereof, for use as a medicament. Overexpression of these polypeptides in undifferentiated mammalian cells causes these cells to differentiate into osteoblasts. As osteoblasts differentiate from precursor- to mature bone-forming cells, they express and secrete a number of enzymes and structural proteins of the bone matrix, including Type-1 collagen, osteocalcin, osteopontin and alkaline phosphatase. Overexpression of the polypeptides of the present invention induces alkaline phosphatase levels by at least 3-fold as compared to non-differentiated precursor cells. According to the present invention overexpression refers to either expression of a polypeptide which normally is not expressed in the cell, or enhancing the expression of the polypeptide in the cell compared to endogenous expression. The term "polypeptides" may refer to peptides, oligopeptides, proteins and enzymes. Derivatives of a polypeptide are those peptides, oligopeptides, polypeptides, proteins and enzymes that comprise at least about 10 contiguous amino acid residues of the polypeptide and that retain the biological activity of the protein, e.g. polypeptides that have amino acid mutations compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may further comprise additional naturally-occurring, altered, glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally- occurring form of the polypeptide. It may also contain one or more non-amino acid substituents compared to the amino acid sequence of a naturally-occurring form of the polypeptide, for example a reporter molecule or other ligand, covalently or non- covalently bound to the amino acid sequence. In the present invention, fragment of polypeptides are peptides, oligopeptides, polypeptides, proteins and enzymes that comprise at least about 5 contiguous amino acid residues, preferably at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 contiguous amino acid residues, and exhibit substantially a similar, but not necessarily identical, activity as the complete sequence. According to a second aspect, the present invention relates to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for use as a medicament. The nucleic acid sequences encode the polypeptides of the present invention. The polynucleotides may be introduced into cells so that the polypeptides encoded by the nucleic acid sequences are expressed in the cells. The polynucleotides of the present invention can be introduced into cells by any method known in the art such as electroporation, injection, transfection with liposomes, DEAE- dextran or calcium phosphate and biolistic particle delivery. The nucleic acid sequence can be naked or in the form of a plasmid. The polynucleotides can also be introduced into cells by viral delivery. Expression of the polypeptide is achieved by methods known in the art such as placing the polynucleotide under control of a promoter such as CMV, HSV, TK, SV40, or elongation factor, but also bone-specific promoters such as osteocalcin and collagen type I promoter. Derivatives of a polynucleotide of the present invention may be DNA- and RNA- molecules, and oligonucleotides that comprise at least about 10 contiguous nucleic acid residues of the polynucleotide, e.g. polynucleotides that have nucleic acid mutations compared to the nucleic acid sequence of a naturally- occurring form of the polynucleotide. A derivative may further comprise nucleic acids with modified backbones such as peptide nucleic acid (PNA), polysiloxane, and 2 ' -0- (2-methoxy) ethyl, phosphorothioate, non-naturally occurring nucleic acid residues, or one or more nuclei acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection. Fragment of polynucleotide relate to oligonucleotides that comprise at least about 5 contiguous nucleic acid residues, preferably at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 contiguous nucleic acid residues, and exhibit substantially a similar, but not necessarily identical, activity as the complete sequence. In addition, the present invention relates to a vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for use as a medicament. Vectors, including plasmids and viruses, can be used to introduce nucleic acids into cells, such that the polypeptides encoded by the nucleic acid sequence are expressed in the cells. In the vector, the nucleic acid sequence may be placed under the control of a promoter, such as CMV, HSV, TK, SV40, or, the elongation factor. Preferably the vector is an adenoviral, retroviral, adeno- associated viral, lentiviral or a sendaiviral vector. Recombinant viruses are commonly used for gene transfer. To date, the three most commonly used viruses for gene transfer are adenovirus, retrovirus and adeno-associated virus. More recently lentiviruses, a subgroup of the retroviruses, and sendaivirus are being used. Adenoviruses are able to transduce both dividing and non-dividing cells and can be produced at high viral titres. Retroviruses can infect dividing cells only and integrate their genome into the host chromosome.

Integration achieves long-term gene expression. Lentiviruses, a sub-family of retroviruses, share all the standard properties of retroviruses but in addition they have the capacity to transduce non-dividing cells. Adeno-associated virus (AAV) is a small, non-pathogenic, single-stranded DNA virus. It requires co-infection with a helper virus (adenovirus or herpes virus) in order to undergo productive infection. In the absence of helper virus, wild-type AAV integrates site-specifically, into the host genome. Similarly to retrovirus, integration facilitates longer gene expression. AAV can infect both non- dividing and dividing cells. Sendai virus is a member of the paramyxoviridae, and is a single-stranded RNA virus that is able to transfect both dividing and non-dividing cells. Its method of entering cells involves sialic acid and cholesterol that are common to many cell types. Sendai viral gene expression and replication are localized in the cytoplasm, in contrast to most viruses that need to enter the nucleus. The present invention further relates to the use of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local decrease in mean bone density. The present invention further relates to the use of a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local decrease in mean bone density. In addition, the present invention relates to the use of a vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local decrease in mean bone density. Preferably the disorder is selected from the group consisting of osteoporosis, hypercalcemia of malignancy, multiple myelomatosis, hyperparathyroidis , and hyperthyroidism. In a further aspect, the present invention relates to a method for inducing the differentiation of an undifferentiated mammalian cell into an osteoblast comprising (a) introducing a polynucleotide, or a vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, into the cell; and (b) overexpressing a polypeptide, or a derivative, or a fragment thereof, encoded by the nucleic acid sequence, or a derivative, or a fragment thereof, in the cell; such that the level of bone alkaline phosphatase is increased compared to cells without the polynucleotide or vector. Preferably, the polypeptide encoded by the polynucleotide is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62, Undifferentiated cells are pluripotent cells which are in an early stage of specialization, i . e . , which do not yet have their final function and can be induced to form almost any given cell type. In particular, these are cells which have not yet differentiated to e . g. , osteoblasts or osteoclasts. Such cells are especially blood cells and cells present in bone marrow, as well as cells derived from adipose tissue. In addition, cells which still can be differentiated into mesenchymal precursor cells are contemplated in the present invention, such as, for example, totipotent stem cells such as embryonic stem cells. According to another aspect the present invention relates to a method for identifying a compound that induces differentiation of undifferentiated mammalian cells into osteoblasts comprising: (a) contacting one or more compounds with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62, or a derivative, or a fragment thereof (b) determining the binding affinity of the compound to the polypeptide, (c) contacting a population of undifferentiated mammalian cells with the compound that exhibits a binding affinity of at least 10 μM, and (d) identifying the compound that induces the differentiation of the cells. The present invention further relates to a method for identifying a compound that induces differentiation of undifferentiated mammalian cells into osteoblasts comprising: (a) contacting one or more compounds with a polynucleotide sequence or a vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, (b) determining the binding affinity of the compound to the polynucleotide or to the vector, (c) contacting a population of undifferentiated mammalian cells with the compound that exhibits a binding affinity of at least 10 μM, and (d) identifying the compound that induces the differentiation of the cells. Osteoblast differentiation can be measured by measuring the level of enzymes that are induced during the differentiation process, such as alkaline phosphatase, type-1 collagen, osteocalcin and osteopontin. The alkaline phosphatase activity can be measured by adding methylu belliferyl heptaphosphate (MUP) solution (Sigma) to the cells. The fluorescence generated upon cleavage of the MUP substrate by the AP activity is measured on a fluorescence plate reader (Fluostar, BMG) . The polypeptides or the polynucleotides of the present invention employed in the methods described above may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. To perform the methods it is feasible to immobilize either the polypeptide of the present invention or the compound to facilitate separation of complexes from uncomplexed forms of the polypeptide, as well as to accommodate automation of the assay. Interaction (e.g., binding of) of the polypeptide of the present invention with a compound can be accomplished in any vessel suitable for containing the reactants . Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the polypeptide to be bound to a matrix. For example, the polypeptide of the present invention can be "His" tagged, and subsequently adsorbed onto Ni-NTA microtitre plates, or ProtA fusions with the polypeptides of the present invention can be adsorbed to IgG, which are then combined with the cell lysates (e.g., (35) ^s- labelled) and the candidate compound, and the mixture incubated under conditions favorable for complex formation (e.g., at physiological conditions for salt and pH) . Following incubation, the plates are washed to remove any unbound label, and the matrix is immobilized. The amount of radioactivity can be determined directly, or in the supernatant after dissociation of the complexes. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of the protein binding to the protein of the present invention quantitated from the gel using standard electrophoretic techniques. Other techniques for immobilizing protein on matrices can also be used in the method of identifying compounds. For example, either the polypeptide of the present invention or the compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated protein molecules of the present invention can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical) . Alternatively, antibodies reactive with the polypeptides of the present invention but which do not interfere with binding of the polypeptide to the compound can be derivatized to the wells of the plate, and the polypeptide of the present invention can be trapped in the wells by antibody conjugation. As described above, preparations of a labeled candidate compound are incubated in the wells of the plate presenting the polypeptide of the present invention, and the amount of complex trapped in the well can be quantitated. The binding affinity of the compound with the polypeptide or polynucleotide can be measured by methods known in the art, such as using surface plasmon resonance biosensors (Biacore) , by saturation binding analysis with a labeled compound (e.g. Scatchard and Lind o analysis) , via displacement reactions, by differential UV spectrophotometer, fluorescence polarisation assay, Fluorometric Imaging Plate Reader (FLIPR^®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer. The binding affinity of compounds can also be expressed in a dissociation constant (Kd) or as IC50 or EC50. The IC50 represents the concentration of a compound that is required for 50% inhibition of binding of another ligand to the polypetide. The EC50 represents the concentration required for obtaining 50% of the maximum effect in vitro. The dissociation constant, Kd, is a measure of how well a ligand binds to the polypeptide, and is equivalent to the ligand concentration required to saturate exactly half of the binding-sites on the polypeptide. Compounds with a high affinity binding have low Kd, IC50 and

EC50 values, i.e. in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e. in the micromolar range. For high-throughput purposes, libraries of compounds can be used such as peptide libraries (e.g. LOPAP™, Sigma Aldrich) , lipid libraries (BioMol) , synthetic compound libraries (e.g. LOPAC™, Sigma Aldrich) or natural compound libraries (Specs, TimTec) . Preferably the compounds are low molecular weight compounds. Low molecular weight compounds, i.e. with a molecular weight of 500 Dalton or less, are likely to have good absorption and permeation in biological systems and are consequently more likely to be successful drug candidates than compounds with a molecular weight above 500 Dalton (Lipinski et al. (1997) ) . According to a preferred embodiment the compounds are peptides. Peptides can be excellent drug candidates and there are multiple examples of commercially valuable peptides such as fertility hormones and platelet aggregation inhibitors. According to another preferred embodiment the compounds are natural compounds . Natural compounds are compounds that have been extracted from natural sources, e.g. plants. Using natural compounds in screens has the advantage that more diverse molecules are screened. Natural compounds have an enormous variety of different molecules. Synthetic compounds do not exhibit such variety of different molecules. According to another aspect, the present invention relates to a method for in vitro production of bone tissue, comprising the steps of: (a) applying undifferentiated mammalian cells on a substrate to form a cellular substrate, (b) contacting the cells with a polypeptide having an amino acid sequence selected from the group of SEQ ID NO: 32- 62, or a derivative, or a fragment thereof, for a time sufficient to differentiate the undi ferentiated cells into osteoblasts, thereby producing a continuous bone matrix. Acoording to another preferred embodiment the present invention relates to a method for in vitro production of bone tissue, comprising the steps of: (a) applying undifferentiated mammalian cells on a substrate to form a cellular substrate, (b) introducing a polynucleotide sequence, or a vector comprising a nucleic acid sequence selected from the group of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for a time sufficient to differentiate the undifferentiated cells into osteoblasts, thereby producing a continuous bone matrix. Preferably the continuous bone matrix comprises a thickness of at least 0.5 μm on the surface of the substrate. The invention thus provides a method for producing a substrate with a matrix grown thereon, which can be used for the provision of load-bearing implant, including joint prostheses, such as artificial hip joints, knee joints and finger joints, and axillofacial implants, such as dental implants. It can also be used for special surgery devices, such as spacers, or bone fillers, and for use in augmentation, obliteration or reconstitution of bone defects and damaged or lost bone. Bone formation can be optimized by variation in mineralization, both by inductive and by conductive processes. A combination of the provision of a load-bearing implant (preferably coated with a matrix as described above) with a bone filler comprising a matrix as described, constitutes a significant advantage of to the present invention. The method of the invention is also very suitable in relation to revision surgery, i.e., when previous surgical devices have to be replaced. Suitable undifferentiated cells are bone marrow cells, including haematopoietic cells and in particular stromal cells. The marrow cells, and especially the stromal cells are found to be very effective in the bone producing process when taken from their original environment. The undifferentiated cells can be directly applied on the substrate or they can advantageously be multiplied in the absence of the substrate before being applied on the substrate. In the latter mode, the cells are still largely undif erentiated after multiplication and, for the purpose of the invention, they are still referred to as undifferentiated. Subsequently, the cells are allowed to differentiate.

Differentiation can be induced or enhanced by the presence of suitable inductors, such as glucocorticoids, and dexamethasone. Especially suitable inductors of differentiation are the polynucleotides and polypeptides of the present invention. The use of undifferentiated cells provides several advantages. Firstly, their lower differentiation implies a higher proliferation rate and allows the eventual functionality to be better directed and controlled. Moreover, culturing these cells not only produces the required bone matrix containing organic and inorganic components, but also results in the presence, in the culture medium and in the matrix, of several factors which are essential for growth of the tissue and for adaptation to existing living tissue. Also, the culture medium can be a source of active factors such as growth factors, to be used in connection with the implanting process. Furthermore, such undifferentiated cells are often available in large quantities and more conveniently than e . g. , mature bone cells, and exhibit a lower morbidity during recovery. Moreover, the undifferentiated cells can be obtained from the patient for whom the implant is intended. The bone resulting from these cells is autologous to the patient and thus no immune response will be induced. Matrices as thick as 100 μm can be produced as a result of the use of undifferentiated cells. The substrate on which the undifferentiated cells can be applied and cultured can be a metal, such as titanium, cobalt/chromium alloy or stainless steel, a bioactive surface such as a calcium phosphate, polymer surfaces such as polyethylene, and the like. Although less preferred, siliceous material such as glass ceramics, can also be used as a substrate. Most preferred are metals, such as titanium, and calcium phosphates, even though calcium phosphate is not an indispensable component of the substrate. The substrate may be porous or non-porous. The cells can be applied at a rate of e . g. , 10³ -10⁶ per cm², in particular 10⁴ -2X10⁵ cells per cm². The culture medium to be used in the method according to the invention can be a commonly known culture medium such as MEM (minimum essential medium) . Advantageously, the medium can be a conditioned medium. In this context, a conditioned medium is understood to be a medium wherein similar cells have previously been incubated, causing the medium to contain factors such as polypeptides, secreted by the cells which are important for cell growth and cell differentiation. The cells are cultured for a time to produce a sufficient matrix layer, e . g. , a matrix layer having a thickness of at least 0,5 μm, in particular from 1 up to 100 μm, more in particular of 10-50 μm. The cells may be contacted with the culture medium for e . g. , 2-15 weeks, in particular 4-10 weeks. The production of the matrix, when applied on a substrate, results in a continuous or quasi-continuous coating covering the substrate for at least 50%, in particular at least 80% of its surface area. The present invention further relates to a method for diagnosing a pathological condition involving a systemic or local decrease in mean bone density or a susceptibility to the condition in a subject, comprising: (a) obtaining a sample of the subject's RNA corresponding to a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 or a sample of the subject's genomic DNA corresponding to a genomic sequence of a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 (b) determining the nucleic acid sequence of the subject's mRNA or genomic DNA; (c) comparing the nucleic acid sequence of the subject's mRNA or genomic DNA with a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 or with a genomic sequence encoding a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 obtained from a database; and (d) identifying any difference (s) between the nucleic acid sequence of the subject's mRNA or genomic DNA and the nucleic acid selected from the group consisting of SEQ ID NO: 1-31 or the genomic sequence encoding a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 obtained from a database . In addition, the present invention relates to a method for diagnosing a pathological condition, involving a systemic or local decrease in mean bone density, or a susceptibility to the condition in a subject, comprising determining the amount of polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62 in a biological sample, and comparing the amount with the amount of the polypeptide in healthy subjects, wherein a decrease of the amount of polypeptide compared to the healthy subjects is indicative of the condition. Preferably the condition is selected from the group consisting of osteoporosis, hypercalcemia of malignancy, multiple myelomatosis, hyperparathyroidism, and hyperthyroidism. A subject's mRNA corresponds to a nucleic acid of the present invention when the subject's mRNA is transcribed from the same gene as the nucleic acid of the present invention. Furthermore, a subject's genomic DNA corresponds to a genomic sequence encoding a nucleic acid of the present invention when the subject's genomic DNA encodes for the same gene as the nucleic acid of the present invention. It is well understood in the art that databases such as GenBank, can be searched to identify genomic sequences that contain regions of identity (exons) to a nucleic acid. Such genomic sequences are thus said to encode for the nucleic acid. Another aspect of the invention relates to polynucleotides comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 63-175, or a derivative, or a fragment thereof, for use as a medicament. In addition the present invention involves a vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 63-175, or a derivative, or a fragment thereof, for use as a medicament. Preferably the vector is an adenoviral, retroviral, adeno- associated viral, lentiviral or a sendaiviral vector. According to another embodiment the present invention relates to the use of a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 63- 175, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local increase in mean bone density. Furthermore, the present invention relates to the use of a vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 63-175, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local increase in mean bone density. Preferably the disorder is selected from the group consisting of osteosclerosis, osteosclerotic myeloma, pyknodysostosis, Camuratie-Engelmand disease, osteopoikilosis, melorheostosis, and osteopetrosis . According to the present invention the polynucleotides comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 63-175, preferably are expression- inhibitory agents that inhibit the translation in the cell of a polyribonucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62. The expression-inhibitory agents can be helpful with disorders with increased bone density as they reduce the differentiation of cells into osteoblast. When at the same time the amount of osteoclasts remain the same, the reduction of the amount of osteoblasts will result in a decrease of bone mass. The expression-inhibitory agents of the present invention include molecules comprising nucleic acid sequences designed to bind in a complementary manner to a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 1-31 expressed in a cell, and include antisense oligonucleotides, genetic antisense constructs, ribozymes, and interfering RNAs (RNAi) . The term complementary to a nucleotide sequence in the context of antisense oligonucleotides and methods therefore means sufficiently complementary to such a sequence as to allow hybridization to that sequence in a cell, i . e . , under physiological conditions. One type of expression-inhibitory agents contemplated by the invention is a nucleic acid that is antisense to a nucleic acid comprising SEQ ID NO: 1-31. For example, an antisense nucleic acid (e.g. DNA) may be introduced into cells in vitro, or administered to a subject in vivo, as gene therapy to inhibit cellular expression of nucleic acids comprising SEQ ID NO: 1-31. Anti-sense nucleic acid is intended to mean an oligonucleotide that has a nucleotide sequence that interacts through base pairing with a specific complementary nucleic acid sequence involved in the expression of the target such, that the expression of the gene is reduced. Preferably, the specific nucleic acid sequence involved in the expression of the gene is a genomic DNA molecule or mRNA molecule that encodes the gene. This genomic DNA molecule can comprise regulatory regions of the gene, or the coding sequence for the mature gene. Antisense oligonucleotides preferably comprise a sequence containing from about 17 to about 100 nucleotides and more preferably the antisense oligonucleotides comprise from about 18 to about 30 nucleotides. Antisense nucleic acids of the invention are preferably nucleic acid fragments capable of specifically hybridizing with all or part of a nucleic acid comprising SEQ ID NO: 1-31 or the corresponding messenger RNA, or they may be DNA sequences whose expression in the cell produces RNA complementary to all or part of the mRNA comprising SEQ ID NO: 1-31. Antisense nucleic acids may be prepared by expression of all or part of a sequence selected from the group consisting of SEQ ID NO: 1-31, in the opposite orientation. Preferably the antisense nucleic acid is prepared by expression of a sequence selected from the group consisting of SEQ ID NO: 63-175, in the opposite orientation. Preferably, the antisense sequence is at- least about 17 nucleotides in length. Antisense oligonucleotides can also contain a variety of modifications that confer resistance to nucleolytic degradation such as, for example, modified internucleoside linkages, modified nucleic acid bases and/or modified sugars and the like. The antisense oligonucleotides of the invention can also be modified by chemically linking the oligonucleotide to one or more moieties or conjugates to enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide. Such moieties or conjugates include lipids such as cholesterol, cholic acid, thioether, aliphatic chains, phospholipids, polyamines, polyethylene glycol (PEG), or palmityl moieties. Genetic antisense refers to the incorporation of antisense constructs that are complementary to sequences of genes into the genome of a cell. Such incorporation allows for the continued synthesis of the antisense molecule. Another type of expression-inhibitory agent contemplated by the invention is a nucleic acid that is able to catalyze cleavage of RNA molecules. The expression "ribozymes" as used herein relates to catalytic RNA molecules capable of cleaving other RNA molecules at phosphodiester bonds in a manner specific to the sequence. The hydrolysis of the target sequence to be cleaved is initiated by the formation of a catalytically active complex consisting of ribozyme and substrate RNA. All ribozymes capable of cleaving phosphodiester bonds in trans, that is to say intramolecularly, are suitable for the purposes of the invention. Apart from ribonuclease P the known naturally occurring ribozymes (hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme, Neurospora mitochondrial VS ribozyme, group I and group II introns) are catalysts, which cleave or splice themselves and which act in cis (intramolecularly) . Yet another method of expression-inhibition is RNA interference (RNAi) . RNAi is the post-transcriptional process of gene silencing mediated by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA and is observed in animals and plants. A self-complementing single stranded siRNA molecule polynucleotide according to the present invention comprises a first guide sequence, a second sequence capable of forming a stem-loop structure within said second sequence, and a third sequence, which complements the first guide sequence and is covalently linked to the distal end of the second sequence. All nucleotides in the first and third sequences base pair, or alternatively there may be mismatches between the first and third sequences, and are preferably between about 17 and 23 nts in length. The first or third sequence is complementary to a portion of SEQ ID NO: 1-31. Preferably the first and third sequence comprise a nucleic acid sequence consisting of SEQ ID NO: 63-175. Preferably, the second sequence is 4-30 nucleotides long, more preferably 5-15 nucleotides long and most preferably 8 nucleotides long. In a most preferred embodiment the linker sequence is UUGCUAUA (SEQ ID NO: 180) . The term "compound" refers to proteins, peptides, organic compounds, inorganic compounds, lipids, nucleotides, peptides, oligonucleotides, polynucleotides and natural compounds. The term "expression" comprises both endogenous expression and overexpression by transduction. The term "overexpression" relates to expression of a polypeptide of polynucleotide in a cell that does not normally express the polypeptide or polynucleotide, or enhancement of the normal expression of the polypeptide or polynucleotide. The term "osteoblast differentiation" means the process of differentiation of undifferentiated cells (progenitor cells or precursor cells) into osteoblasts and/or preosteoblasts . The term "osteogenesis" is used as a synonym in this context.

"Abnormal osteoblast differentiation" means a situation where either too much or too little osteoblast differentiation is occurring. The invention is further illustrated by the following non- limiting Examples and Figures. Figure 1: Principle of the osteoblast differentiation assay. Mesenchymal stem cells derived from bone marrow are infected with the placenta PhenoSelect™ cDNA library viruses. Six days post infection bone alkaline phosphatase activity is measured following addition of 4-methylumbelliferylphosphate (MUP) substrate. Figure 2: Proof of principle. Control plates in a 96 well plate format are prepared containing different control viruses transducing the following transgenes: BMP2, eGFP, PPAR-gamma, empty virus and luciferase. All cells infected with virus containing BMP2 had an increased BAP value, none of the negative controls (eGFP, PPAR-gamma, empty and luciferase) scored. Figure 3: Construction of knock-down constructs. This figure is a schematic representation of the cloning strategy for construction of adenoviral knock-down constructs showing utilization of Sapl sites and an E. coli death gene. Figure 4 : A schematic representation of the cloning strategy for construction of adenoviral knock-down constructs showing the structure of the 56 nt knock-down constructs coding inserts . Figure 5: A schematic representation of the knock-down vector pKD122.

Table 1: Hits from the AP screen inducing osteoblast differentation.

Table 2: Known osteogenic factors picked up in the AP assay ccession Official Description symbol

Table 3: Target sequences that can be used for the expression-inhibitory agents accession sequence ■aaaiaaw

EXAMPLES Example 1: Screening of the PhenoSelect library for up- regulation of bone alkaline phosphatase Adenovirus serotypes There are at least 49 human adenovirus serotypes which have been divided into six subgenera (A to F) with distinctly different tropsim. Most adenovirus vector gene transfer systems are derived from adenovirus serotype 2 and 5 (Ad2 and Ad5) . The PhenoSelect™ library used for the screen is produced in Ad5 adenovirus. Adenoviral infection is initiated by the formation of complexes between the globular knob domain of the adenoviral fiber protein and a host cell receptor. The cellular receptor for the fiber protein of Ad5 (and the other members of Adeno subgroups A, C, D, E and F) has been identified as the Coxsackievirus and Adenovirus Receptor (CAR) . Thus, cells that do not carry the CAR receptor or that express the receptor at very low basal levels are difficult to infect efficiently with any of these adenoviral vectors, including Ad5 viruses. Mesenchymal progenitor cells (MPCs) are difficult to transduce with Ad5 virus. One way to infect these cells more efficiently is to use an Ad virus from group B or to use a chimeric adenovirus containing the Ad5 capsid and fiber proteins from a adenovirus with different serotypes ( e . g. , Ad5fib35 or Ad5fib51, See also WO0224933) that can enter the cell through a different receptor. Although this approach has proven to be quite successful, it is not feasible for the existing placental PhenoSelect™ library. Therefore an alternative strategy is devised wherein the cells are first infected with Ad5 or an Ad5 fiber variant expressing hCAR (human receptor) prior to infection with the placental PhenoSelect™ cDNA library viruses. The cells infected with this virus will express hCAR receptors on their surfaces and therefore can be easily infected with the PhenoSelect™ library. Although the transduction efficiency of the hCAR containing Ad5 viruses (or variants) may be modest, sufficient hCAR receptor is expressed on the surface of the MPCs after adenoviral mediated transfer to subsequently allow efficient infection with the library viruses. All the viruses described in this patent application have the Ad5 genome backbone with the E1A, E1B and E2A genes deleted and fiber variant 5 (See US 6,340,595 and US 6,413,776). The viruses Ad5fib35-hCAR and Ad5fib51-hCAR, described in this paragraph, have other fiber proteins and do not have the E2A gene deleted in their genome. When no fiber protein is mentioned the serotype 5 fiber is used. Description of the assay Mesenchymal precursor cells (MPCs) are determined to differentiate into osteoblasts in the presence of appropriate factors ( e . g. , BMP2, see Figure 1) . An assay to screen for such factors monitors activity of alkaline phosphatase (AP) enzyme, an early marker in the osteoblast differentiation program. MPCs are seeded in 384 well plates and simultaneously co-infected one day later with Ad5fib5-hCAR and individual PhenoSelect™ viruses. Six days post infection (dpi), cellular alkaline phosphatase activity is determined as follows: the conditioned medium is removed from the cells and the cells are washed once with PBS before the addition of 10 μl of a methylumbelliferyl heptaphosphate (MUP) solution (Sigma cat no M3168) to each well. The plates are then incubated at 37°C for 30 minutes and the enzymatic reaction is stopped by the addition of 5 μL of 1M Na₂C0₃ solution to each well. The fluorescence generated upon cleavage of the MUP substrate by the AP activity is measured on a fluorescence plate reader (Fluostar, BMG) . (See also PCT/EP02/09530) . High Throughput AP assay MPCs isolated from bone marrow of patients that have undergone prosthetic implantation/replacement surgery, are obtained from Isotis. The MPCs are seeded in black 384 well plates with clear bottom (Costar or Nunc) at a density of 1000 cells per well. One day after seeding, the cells are co- infected with Ad5-hCAR (MOI 100) and the PhenoSelect™ library (MOI 4000) . Morphogenic protein 2 virus (Ad5-BMP2) is used as a positive control and Ad5-eGFP or Ad5-empty virus are used as negative controls to evaluate background effects and the effects of the viral infection on the cells, respectively. Six days post infection all conditioned medium is aspirated before 15 μl MUP is added to each well. The plates are incubated for 15 min at 37°C and monitored for AP activity using a fluorescence plate reader (Fluostar, BMG) . The data obtained from the FluoStar are analyzed as follows: for control plates, the mean of the fluorescence units obtained for all the viruses except Ad5-BMP2 virus is calculated. The standard deviation for these data points is also calculated. The cut-off value for hit calling is then pre-set at the mean plus 3.4 times the standard deviation. Values above this cut-off value have more than a 99% statistical chance of scoring truly above background. All the signals obtained for BMP2 viruses are clearly above this cutoff value (Figure 2) , showing the validity of this approach. After screening 112 000 PhenoSelect adenoviruses in this assay, the polypeptides listed in Table 1 are identified as new inducers of alkaline phosphatase levels. Also known osteoblast inducers were picked up in the screen (see Table 2) validating the assay used. The viruses encoding the identified polypeptides in the AP screen are re-propagated from the original viral stocks. In addition new viruses are derived from the pAdapt plasmids present in the glycerol stocks used to make the PhenoSelect™ library. These viruses are re-screened. All viruses again induce up-regulation of AP. The virusses that induced upregulation of AP are sequenced. Two of the genes indentified in the screen were known osteogenic factors (Table 2) : activating transcription factor 2 (ATF2) and bone morphogenetic protein receptor, type II (BMPR2) . Disruption of the gene encoding ATF-2 in mice causes reduced proliferation of chondrocytes and chondrodysplasia. ATF- 2 is a member of the ATF/cAMP response element-binding protein (CREB) family of transcription factors. Members of this family can bind to the cAMP response element (CRE) found in many mammalian gene promoters. They all contain a basic region- leucine zipper motif and can bind to DNA as homodi ers and heterodimers in various combinations. In addition to associating with other members of the ATF/CREB family, ATF-2 has been reported to form heterodimers with c-Jun. The protein is also a histone acetyltransferase (HAT) that specifically acetylates histones H2B and H4 in vitro; thus it may represent a class of sequence-specific factors that activate transcription by direct e ects on chromatin components . BMPR2 is a member of the bone morphogenetic protein (BMP) receptor family of transmembrane serine/threonine kinases. The ligands of this receptor are BMPs, which are members of the TGF-beta superfamily. BMPs are involved in endochondral bone formation and embryogenesis . These proteins transduce their signals through the formation of heteromeric complexes of 2 different types of serine (threonine) kinase receptors: type I receptors of about 50-55 kD and type II receptors of about 70- 80 kD. Type II receptors bind ligands in the absence of type I receptors, but they require their respective type I receptors for signaling, whereas type I receptors require their respective type II receptors for ligand binding. Twenty seven other sequences have been identified in the screen that before have not been recognized as inducers of bone formation, see table 1. Three of these sequences had thus far unknown functions . Short description of hits LEGUMAIN Legumain is a human cysteine protease, also known as PRSC1, with 40% amino acid identity to hemoglobinase of the schistosome parasite and 36% identity to a vacuolar processing enzyme of the soybean. The observed size of the protein is approximately 34 kD, the enzyme is glycosylated and acts as a cysteine peptidase with specificity for asparaginyl bonds. It is inhibited by lodoacetamide and maleimides, but unaffected by compound E64 (trans-epoxysuccinyl-L-leucylamido- (4- guanidino) butane) . It is also inhibited by ovocystatin (cystatin from chicken egg white) and human cystatin C with Ki values < 5 nM. Northern-blot analysis revealed the strongest expression of this gene in kidney, among the human tissues examined. UBIQUITIN-SPECIFIC PROTEASE 8 Ubiquitin, a highly conserved protein involved in the regulation of intracellular protein breakdown, cell cycle regulation, and stress response, is released from degraded proteins by disassembly of the polyubiquitin chains. The disassembly process is mediated by ubiquitin-specific proteases (USPs) Deubiquitinating enzymes (DUBs) are capable of producing a free ubiquitin moiety from ubiquitin-fused precursors and ubiquitinylated proteins in cells. DUBs possess conserved catalytic regions known as the ' cys domain' and the 'his domain.' There are 2 distinct families of DUBs: ubiquitin- specific processing proteases (UBPs, or USPs) and ubiquitin C- terminal hydrolases (UCHs) . Ubiquitin-specific protease 8 (USP8), also known as deubiquitinating enzyme HUMORF8 and ubiquitin isopeptidase, UBPY, is able to cleave linear or isopeptide-linked ubiquitin chains. It accumulates upon growth stimulation of starved human fibroblasts, and its levels decrease in response to growth arrest induced by cell-cell contact. Inhibition of USP8 accumulation prevents fibroblasts from entering S-phase in response to serum stimulation. MITOGEN-ACTI ATED PROTEIN KINASE KINASE KINASE 4 (MAP3K4) In mammalian cells, a variety of extracellular stimuli generate intracellular signals that converge on a limited number of so-called mitogen-activated protein (MAP) kinase pathways. The central core of each MAP kinase (MAPK) pathway is a conserved cascade of 3 protein kinases: an activated MAPK kinase kinase (MAPKKK) phosphorylates and activates a specific MAPK kinase (MAPKK) , which then activates a specific MAPK. While the ERK MAPKs are activated by mitogenic stimulation, the CSBP2 and JNK MAPKs are activated by environmental stresses such as osmotic shock, UV irradiation, wound stress, and inflammatory factors. MAP3K4, also called map/erk kinase kinase 4 (MEKK4), map three kinase 1 (MTK1), is the human homolog of the S. cerevisiae Ssk2 and Ssk22 MAPKKKs . The 1, 607-amino acid protein, contains a protein kinase catalytic domain at the C terminus that is 98%, 42%, 36%, and 41% identical to the kinase domains of mouse MEKK4, Ssk2, Ssk22, and S. pombe Wikl, respectively. Northern blot analysis detected an approximately 6-kb transcript in various human tissues, with highest levels in heart, placenta, skeletal muscle, and pancreas. RAFl, MURINE LEUKEMIA VIRAL ONCOGENE HOMOLOG 1 (V-RAF-1. Raf 1 is also known as oncogene rafl, transforming replication-defective urine retrovirus 3611-msv, oncogene MIL. The viral genome bears close similarities to the Moloney murine leukemia virus. The cellular homolog, c-raf, is present in 1 or 2 copies per haploid genome in mouse and human DNA. RAFl lies on chromosome 3 and was assigned to 3p25 by in situ hybridization. This suggests that RAFl may be involved in mixed parotid gland tumors with the t(3;8) (p25;q21) translocation . Rafl has been associated with small cell cancer of the lung, familial renal cell carcinoma, and stomach cancer (Japan) . It was shown that RAFl can be targeted to the mitochondria by BCL2, a regulator of apoptotic cell death. Active RAFl improved BCL2-mediated resistance to apoptosis. It was also showed that RAFl phosphorylates BAD. ZAK: STERILE ALPHA MOTIF (SAM) AND LEUCINE ZIPPER (LC) CONTAINING KINASE AZK ZAK is also known as MLK-like mitogen-activated protein triple kinase (MLTK) , mlklak, and MLK-related kinase (MRK) , MLK stands for mixed lineage kinase. The cDNA has 2456 bp and encodes a protein of 800 amino acids that contains a kinase catalytic domain, a leucine-zipper and a sterile alpha motif (SAM) . The molecular weight of this protein is 91kDa. Northern blot analysis revealed that the expression of this ZAK gene is found in various parts of human tissues. It was also found that ZAK proteins form homodimers or oligomers in mammalian cells. The leucine-zipper (LZ) and sterile-alpha motif (SAM) kinase (ZAK) belongs to the MAP kinase kinase kinase (MAP3K) when upon over-expression in mammalian cells activates the JNK/SAPK pathway. Overexpression of the ZAK gene induces apoptosis of a hepatoma cell line. Expression of wild-type ZAK increases the cell population in the G(2)/M phase of the cell cycle, which may indicate G(2) arrest. Western blot analysis shows that the decreased cyclin E level correlated strongly with the low proliferative capacity of ZAK-expressed cells. BC017083 The BC017083 sequence is similar to serine threonine kinase pim3 from Rattus norvegicus. The gene has a protein product with unknown function and has homology to serine threonine kinase pim3 [Rattus norvegicus] (84% identity in aligned region, p=9e-26) and PIM-1, a human proto-oncogene and serine/threonine-protein kinase (51% identity in aligned region, p=le-07) and the mouse PIM-1 Proto-oncogene serine/threonine-protein kinase (69% identity in aligned region, p=3e-08) The human proto-oncogene PIM-1 encodes a protein kinase which is upregulated in prostate cancer. The 33-kD product of the PIM gene is highly expressed in the liver and spleen during fetal hematopoiesis . It was overexpressed in hematopoietic malignancies, particularly in myeloid and lymphoid acute leukemias. DUAL-SPECIFICITY PHOSPHATASE 6 (DUSP6) Dual-specificity phosphatase 6, is also known as MAP kinase phosphatase 3 (MKP3), and PYST. Members of the mitogen- activated protein (MAP) kinase family play a pivotal role in cellular signal transduction. The dual-specificity phosphatases can reverse MAP kinase activation by dephosphorylating critical phosphotyrosine and phosphothreonine residues. When expressed in mammalian cells, MKP3 blocked both the phosphorylation and enzymatic activation of the MAP kinase ERK2 by mitogens . DUSP6, a predicted 381-amino acid protein is 36% identical to MKP-1 and contains the characteristic extended active-site sequence motif VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) as well as two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase. It is localized to the cytoplasm and is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens . HUMAN GROWTH FACTOR-REGULATED TYROSINE KINASE SUBSTRATE (HGS) HGS is also known as hepatocyte growth factor-regulated tyrosine kinase substrate, hrs or hgf-regulated tyrosine kinase substrate. Activation of tyrosine kinases is an initial biochemical event in intracellular signal transduction from cytokine receptors after their binding with ligands. Several families of tyrosine kinases are known to be associated with the cytoplasmic domains of cytokine receptors. Upon activation of tyrosine kinases, cytokine receptor subunits are phosphorylated on tyrosine residues, which results in association of the receptor subunits with signal transducers and activators of transcription (STATs) . The cDNA encodes a deduced 777-amino acid protein. The 110-kD phosphotyrosine protein is inducible by stimulation with interleukin-2 and is 93% homologous to mouse Hrs . HGS contains double zinc finger (FYVE) motifs, a putative coiled-coil sequence, and nucleotide- binding sites. MYELOID DIFFERENTIATION PRIMARY RESPONSE GENE 88 (MYD88) Human MYD88 cDNA encodes a 296-amino acid polypeptide with a predicted mass of 33 kD. MYD88 shares 81% amino acid identity with murine MyD88. The 150-amino acid C-terminal region has significant homology to the type I interleukin-1 receptor cytoplasmic domain. Northern blotting revealed that human MYD88 is expressed as 2 MYD88 hybridizing 1.6- and 3-kb mRNAs in a variety of tissues and cell lines. The MYD88 gene is encoded by 5 exons and overexpression of MYD88 causes an increase in the level of transcription from the interleukin-8 promoter. MyD88 acts as adapter protein involved in the toll-like receptor and IL-1 receptor signaling pathway in the innate immune response, acts via irakl, irak2 and trafβ, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. It increases IL-8 transcription and may be involved in myeloid differentiation. ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM(AMSH) AMSH, a molecule that associates with STAM1, is involved in the in vitro cell growth signaling mediated by interleukin 2 and granulocyte-macrophage colony-stimulating factor. The deduced 424-amino acid protein contains 2 potential SH3-binding domains (PxxP motifs), a JAB1 subdomain homologous (JSH) region, and a putative bipartite nuclear localization signal. VAV3 ONCOGENE (VAV3) VAV3, a third representative of the Vav family, participates in signal transduction processes. It is also involved in the activation of several members of the Rho family. This activity, when deregulated, leads to marked cytoskeletal changes and to alterations in the process of cell division. Sequence analysis predicted that the 847-amino acid protein, which shares approximately 69% and 66% sequence similarity with VAV1 and VAV2, respectively, contains Dbl homology (DH) and pleckstrin homology domains, common in Rho and Rac GEFs, as well as a calponin homology (CH) region, an acidic motif, a zinc finger (ZF) domain, 2 SH3 regions, and 1 SH2 domain, like the other VAVs . DNA METHYLTRANSFERASE 3A (DNMT3A) The Dnmt3A cDNA is 4,192 bp in length, encoding a protein of 909 amino acids. The human DNMT3A and DNMT3B cDNA are highly homologous to the mouse genes. Dnmt3a and Dnmt3b transcripts are abundantly expressed in undifferentiated embryonic stem cells. Dnmt3a and Dnmt3b encode de novo DNA methyltransferases . De novo methylation of genomic DNA is a developmentally regulated process that appears to play a pivotal role in regulation of genomic imprinting and X-chromosome inactivation in mammals. Aberrant de novo methylation of growth regulatory genes is associated with tumor genesis in humans. dUTP PYROPHOSPHATASE (DUT) DUT is also known as deoxyuridine triphosphate nucleotidohydrolase, deoxyuridine triphosphatase, and dUTPase. DUT hydrolyzes dUTP to dUMP and pyrophosphate . It produces dUMP, the immediate precursor of thymidine nucleotides and it decreases the intracellular concentration of dUTP so that uracil cannot be incorporated into DNA. PLASMINOGEN ACTIVATOR, UROKINASE RECEPTOR (PLAUR) PLAUR is also known as UPA receptor (UPAR) and CD87 antigen. The urokinase-type plasminogen activator receptor is a key molecule in the regulation of cell-surface plasminogen activation and, as such, plays an important role in many normal as well as pathologic processes. The complete coding sequence of the cDNA was found to encode 335 amino acids including a predicted signal peptide of 22 residues and a hydrophobic C-terminal portion and is highly glycosylated. PLAUR is directly associated with the carbohydrate-binding domain of SELL in the membrane of neutrophils, an association analogous to that between PLAUR and beta-2 integrins . UPAR mRNA levels correlate with the invasive potential of endometrial carcinomas. Furthermore, the increase in UPAR mRNA levels correlate linearly with the progression of disease stage. PROTEIN C RECEPTOR (PROCR) PROCR is also known as endothelial protein c receptor (EPCR) , cell cycle, centrosome-associated protein (CCCA) , and CCD41. Protein C, a vitamin K-dependent serine protease zymogen, plays a major role in blood coagulation and may also prevent the lethal effects of gram-negative sepsis. Deficiency of protein C leads to life-threatening thrombophilia . Protein C is activated when thrombin, the terminal enzyme of the coagulation system, binds to an endothelial cell surface protein, thrombomodulin (THBD) The receptor for protein C is a 238-amino acid type 1 transmembrane protein and has a 15-amino acid N-terminal signal sequence; an extracellular domain with 4 potential N- glycosylation sites and 4 cys residues; a C-terminal 25-amino acid transmembrane region; and a short cytoplasmic tail containing only 3 amino acids. Northern blot analysis detected high levels of a 1.3-kb EPCR transcript only in endothelial cell lines. PROCR is expressed strongly in the endothelial cells of arteries and veins in heart and lung, less intensely in capillaries in the lung and skin, and not at all in the endothelium of small vessels of the liver and kidney. Immunoblot analysis showed that EPCR is expressed as a 49-kD protein that is reduced to 25 kD by deglycosylation. RETIN01C ACID RECEPTOR ALPHA (RARA) RARA protein binds retinoic acid with high affinity, and was found to be homologous to the receptors for steroid hormones, thyroid hormones, and vitamin D3, and appeared to be a retinoic acid-inducible transacting enhancer factor. The genes for all the steroid/thyroid receptors show a common pattern of structure, with 4 regions: A/B, C, D, and E. The function of region A/B is unknown; C encodes the DNA-binding domain; D is believed to be a hinge region; and E encodes the ligand-binding domain. In general, the DNA-binding domain is most highly conserved, both within and between the 2 groups of receptors (steroid and thyroid) ; the ligand-binding domains show less homology. There are 3 retinoic acid receptors, RAR- alpha, RAR-beta, and RAR-gamma. The alpha and beta forms of RAR were found to be more homologous to the 2 closely related thyroid hormone receptors alpha and beta, than to any other members of the nuclear receptor family. Retinoic acid receptor alpha is implicated in leukemogenesis and Acute promyelocytic leukemia (APL, also known as acute myeloid leukemia-3, AML3, or M3) . Bone is a target tissue for action of retinoids though their precise role remains unclear. Some reports describe the promotion of osteoblastic differentation upon supplementing with retinoic acid (RA) . However most studies show an inhibition of osteogenesis and a decrease in osteoblastic marker. In one study the number of large differentiated osteoblast cells decreased in RA-treated cultures. The production of bone specific markers, alkaline phosphatase, was also reduced in the RA-treated cultures. Human osteoblastic cell line (SV-HFO) are mineralized by treatment with dexamethasone (Dex) . RA inhibited the mineralization of these cells, coincident with the inhibition of alkaline phosphatase (ALP) . A specific inhibitor of the RAR alpha, Ro 41-5253, completely blocks the induction of ALP triggered by AM580. These results suggest that, at physiological concentrations, RA suppresses the differentiation of osteoprogenitor cells and regulates osteoblastic functions. Therefore it is surprising that up-regulation of RARA induces osteoblast formation. CHROMODOMAIN HELICASE DNA-BINDING PROTEIN 3 (CHD3) CHD3 is also known as Mi2-alpha and zinc-finger helicase (Snf2- like) . The protein has 1944 amino acids, and a molecular weight of 220 kDa . The protein is characterized by the presence of chromo (chromatin organization modifier) domains, 2 phd-type zinc fingers and SNF2-related helicase/ATPase domains. Patients with dermatomyositis develop antibodies against the nuclear antigen chromodomain helicase DNA binding protein 3. It is a central component of the nucleosome remodelling and histone deacetylase (nurd) repressive complex. NUCLEAR RECEPTOR COACTIVATOR 3 (NCOA3) NCOA3 is also known as AMPLIFIED IN BREAST CANCER 1 (AIB1), ACTR, and THYROID HORMONE RECEPTOR ACTIVATOR MOLECULE 1 (TRAMl) . NCOA3 has 1424 amino acids, and has a molecular weight of 155 kDa. It is a nuclear receptor coactivator that directly binds nuclear receptors and stimulates the transcriptional activities in hormone-dependent fashion. NCOA3 recruits 2 other nuclear factors, CBP and PCAF and thus plays a central role in creating a multisubunit coactivator complex. NCOA3 NCOA3 is frequently amplified or ovexpressed in breast and ovarian cancers . SWI/SNF-RELATED MATRIX-ASSOCIATED ACTIN-DEPENDENT REGULATOR OF CHROMATIN, SUBFAMILY A, MEMBER 5 (SMARCA5) SMARCA5 is also known as homolog of yeast sucrose nonfermenting, sucrose nonfermenting-like 5, HSNF2H, SNF2H, and WCRF135. SMARCA5 contains 1052 amino acids and has a molecular weight: of 122 kDa. SMARCA5 is a member of the SWI/SNF family of proteins. Members of this family have helicase and ATPase activities and are thought to regulate transcription of certain genes by altering the chromatin structure around those genes. It is a component of the chromatin remodeling and spacing factor RSF, a facilitator of the transcription of class II genes by RNA polymerase II. RAS p21 PROTEIN ACTIVATOR 1 (RASA1) RASA 1 is also known as guanosine triphosphatase- activating protein, gtpase-activating protein (GAP) . The protein contains 1044 amino acids and has a molecular weight of 116 kDa. It is an inhibitory regulator of the RAS-cyclic amp pathway. It stimulates the GTPase of normal but not oncogenic RAS P21. The RAS gene family encodes membrane-associated, guanine nucleotide-binding proteins (p21) that are involved in the control of cellular proliferation and differentiation. Similar to other guanine-binding proteins (such as the heterotrimeric G proteins), the RAS proteins cycle between an active guanosine-triphosphate (GTP) bound form and an inactive, guanosine-diphosphate (GDP) bound form. The weak intrinsic GTPase activity of RAS proteins is greatly enhanced by the action of GTPase-activating proteins (GAPs) . HEPATOMA-DERIVED GROWTH FACTOR (HDGF) HDGF is also known as High-mobility group protein 1- like 2 (HMG-1L2) . This protein contains 240 amino acids and has a molecular weight of 27 kDa. It is a heparin-binding protein, with mitogenic activity for fibroblasts. It contains a well- conserved N-terminal amino acid sequence (homologous to the amino terminus of HDGF; hath) and nuclear localization signals (NLSs) in gene-specific regions other than the hath region. The primary sequence shares homology with the high mobility group-1 protein, 23.4% amino acid identity and 35.6% amino acid similarity. FORKHEAD BOX P4 (FOXP4) FOXP4 is also known as fork head-related protein like A and winged-helix repressor FOXP . It is a transcription activator for a number of liver genes such as afp, albumin, tyrosine aminotransferase, pepck, and it interacts with the cis-acting regulatory regions of these genes . It contains a 1 fork-head domain. V-SKI, AVIAN SARCOMA VIRAL ONCOGENE HOMOLOG (SKI); Ski is also known as C-ski, chicken viral oncogene sk, sk oncogene, SKV, Ski oncogene . It may play a role in neural tube development and terminal differentiation of skeletal muscle cells but not in the determination of cells to the myogenic lineage. The nuclear protooncogene protein SKI associates with SMAD3 in response to the activation of TGFBl signaling leading to repression of TGF-beta, activin and bone morphogenetic protein responses. Association with SKI repressed transcriptional activation by SMAD3, and overexpression of SKI rendered cells resistant to the growth-inhibitory effects of TGFBl. SKI is a novel component of the TGFBl signaling pathway and shed light on the mechanism of action of the SKI oncoprotein. SKI disrupts the formation of a functional complex between the comediator SMADs (Co-SMADs) and receptor SMADs (R- SMADs) , explaining how it could lead to repression of TGF-beta, activin and bone morphogenetic protein responses. The structure of the SKI fragment, stabilized by a bound zinc atom, resembled the SAND domain found in transcription factors and other nuclear proteins, in which the corresponding I loop is responsible for DNA binding. HOMO SAPIENS HYPOTHETICAL PROTEIN FLJ23751. It is a gene with protein product, however the function is unknown. The gene codes for a predicted 480 amino acid protein with a molecular weight of 55 kD. It propably has 1 transmembrane domain and a histidine acid phosphatase domain. It is similar to mouse hypothetical histidine acid phosphatase containing protein (EMBL database: AK036973; BAC29653) , 89% identity. HUMAN HYPOTHETICAL PROTEIN FLJ22169. It is a gene with protein product however the function is unknown. The gene gives a predicted protein of 668 amino acids with a molecular weight of 75 kDa. It has 2 possible transmembrane domains. The sequence is slightly similar to autophagy protein 9 of Dictyostelium discoideum (Slime mold) ,32% identical. In yeast, 15 Apg proteins coordinate the formation of autophagosomes . Autophagy is a bulk degradation process induced by starvation in eukaryotic cells. Apg9 plays a direct role in the formation of the cytoplasm to vacuole targeting and autophagic vesicles, possibly serving as a marker for a specialised compartment essential for these vesicle- mediated alternative targeting pathways MULTIPLE COAGULATION FACTOR DEFICIENCY 2 (MCFD2) MCFD2 is also known as multiple coagulation factor deficiency protein 2, neural stem cell derived neuronal survival protein, F5F8D, SDNSF, and LMAN1IP MCFD2 is a 175 amino acid protein with a molecular weight of 20 kDa. Notable features include a predicted signal peptide at the N terminus and 2 calmodulin-like EF hands for putative calcium binding at the C terminus. Northern blot analysis showed that MCFD2 is expressed in multiple tissues.

Example 2: Validation of identified hits for osteoblast differentiation: Mineralization study The process of osteogenesis consists of several successive events. During the initial phases of osteogenesis, bone alkaline phosphatase (BAP) becomes upregulated. As there are 4 alkaline phosphatases and only one is bone specific, alkaline phosphatase activity per se is not a fail-safe osteogenic marker. It is therefore important to look at more specific events occurring in later stages of osteogenesis such as mineralization. Bone tissue consists of cells embedded in a matrix of organic materials (e.g., collagen) and inorganic materials (e.g. Ca²⁺ and phosphate) . Bone mineralization is shown in vitro by staining differentiated bone cells for the matrix they deposited. The Von Kossa and Alizarin RedS stains allow the visualization of deposited phosphate and calcium, respectively. On day one, primary human MPCs are seeded in a 6 well plate (Costar or Nunc) at a density of 50,000 to 100,000 cells per well. MPCs are co-infected one day later with Ad5fib35-hCAR (MOI 2500) and Ad5-control (eGFP or BMP2) or hit-virus (Ad5) (at MOIs of 500, 1500, or 4500) . Medium (DMEM, 10% heat- inactivated fetal bovine serum, Penicillin, Streptomycin) , supplemented with 100 μg/ml L-ascorbate and 10 mM beta- glycerophosphate, is refreshed 3 times a week. 20 to 30 days after the start of the experiment, cells are stained with Von Kossa stain (Lecanda et al 2000) or with Alizarin RedS stain (Kale et al . , 2000) . The Alizarin RedS staining is carried out as follows: cells are washed twice with PBS, fixed with 10% paraformaldehyde for 45 minutes at 4°C, and washed 3 times with PBS. Cells are incubated with 40 mM aqueous Alizarin RedS solution, pH 4.1-4.3 for 10 minutes followed by 5 washes with distilled water. Staining is evaluated and photographed using white light For the Von Kossa staining, cells are first washed 3 times with PBS then fixed with 4% paraformaldehyde for 4 hours at 4°C and washed again 3 times with distilled water. After addition of 5% silver nitrate solution, the plates are exposed to UV light for 1 hour. The cells are rinsed 3 times with distilled water before and after the residual silver nitrate is neutralized with 2.5% sodium thiosulphate . Phosphate deposits become visible as black clusters and are easily photographed under white light microscopy.

Example 3: Expression of hits Total RNA is isolated from MPCs and subjected to DNAse digestion and then used to make cDNA. cDNA synthesis is performed using 'Taqitian reverse transcription reagents' (Applied Biosystems) in the presence or absence of reverse transcriptase . Parts of both reaction mixtures are then used to perform real time PCR. Different PCR primer sets are used on both samples: pri ersets for housekeeping genes (18S rRNA and human β-actin taq an primer/probe mix, Applied Biosystems) and for the hits (SYBR green PCR master mix, Applied Biosystems) are used. Depending on the amounts of mRNA present in the total RNA, linear ranges of PCR amplificied products are coming up at a specific amplification cycles. The linear ranges are then used to calculate Ct values. The more mRNA is present, the lower the Ct value.

Example 4. Validation of the hits for osteoblast differentiation: calvarial skull model Technique for Neonatal Mouse Calvaria Assay (In vitro) This assay is described in US6413998. Briefly, four days after birth, the front and parietal bones of ICR Swiss white mouse pups are removed by microdissection and split along the sagittal suture. The bones are incubated in BGJb medium plus

0.02% (or lower concentration) beta-methylcyclodextrin, wherein the medium also contains test or control substances or recombinant adenoviruses encoding control cDNAs (eGFP and BMP2) or the SPINT-1 cDNA, at 37 °C in a humidified atmosphere of 5% C0₂ and 95% air for 96 hours. Following this, the bones are removed from the incubation media and fixed in 10% buffered formalin for 24-48 hours, decalcified in 14% EDTA for 1 week, processed through graded alcohols; and embedded in paraffin wax. Three μm sections of the calvaria are prepared. Representative sections are selected for histomorphometric assessment of bone formation and bone resorption. Bone changes are measured on sections cut 200 μm apart. Osteoblasts and osteoclasts are identified by their distinctive morphology. In vivo assay of effects of compounds and recombinant adenoviruses on murine calvarial bone growth Male ICR Swiss white mice, aged 4-6 weeks and weighing 13- 26 grams, are employed, using 4-5 mice per group. The calvarial bone growth assay is performed as described in WO 9524211.

Briefly, the SPINT-1 adenoviruses or a compound and appropriate control vehicle is injected into the subcutaneous tissue over the right calvaria of normal mice. Typically, the control vehicle is the vehicle in which the compound is solubilzed, in this case empty virus, eGFP, or LacZ virus in PBS containing 5% DMSO or is PBS containing Tween (2 μl/10 ml) . The animals are sacrificed on day 14 and bone growth measured by histomorphometry. Bone samples for quantitation are cleaned from adjacent tissues and fixed in 10% buffered formalin for 24-48 hours, decalcified in 14% EDTA for 1-3 weeks, processed through graded alcohols, and embedded in paraffin wax. Three to five μm sections of the calvaria are prepared, and representative sections are selected for histomorphometric assessment of the effects on bone formation and bone resorption. Sections are measured by using a camera lucida attachment to trace directly the microscopic image onto a digitizing plate. Bone changes are measured on sections cut 200 μm apart, over 4 adjacent 1X1 mm fields on both the injected and non-injected sides of the calvaria. New bone is identified by its characteristic woven structure, and osteoclasts and osteoblasts are identified by their distinctive morphology. Histomorphometry software (such as BIOQUANT NOVA PRIME 6.5, Imagepro from or MediaCybernetics, OsteoMetrics Software or any other histomorphometry software which is known to persons skilled in the art) is used to process digitizer input to determine cell counts and measure areas or perimeters . Example 5: Screening for compounds that bind to the polypeptides of the present invention Introduction Compounds are screened for binding to the polypeptides of the present invention. The affinity of the compounds to the polypeptides is determined in a displacement reaction. In brief, the polypeptides of the present invention are incubated with a labeled ligand that is known to bind to the polypeptide and then treated with an unlabeled compound. The freeing of the labeled ligand is measured and plotted to calculate IC50 or EC50 values that reflect the binding affinity of the compound to its target, i.e. the polypeptides of the present invention. Strong binders have an IC50 or EC50 in the nanomolar and even picomolar range. Compounds that have an IC50 or EC50 of at least 10 micromol or better (nmol to p ol) are applied in the AP assay to check for osteogenic function (Example 1) Preparation of the polypeptides of the present invention The polypeptides of the present invention can be prepared in a number of ways depending on whether the assay will be run on cells, cell fractions or biochemically, on purified proteins. If the polypeptide of the present invention is a receptor anchored in a membrane (BMPR2), the polypeptide of the present invention will be overexpressed in cells. If the target binds the ligand extracellularly, entire cells are used to study binding affinities. Alternatively, or for intracellular receptors (RARA) , membrane preparations are prepared and used to study binding affinities. Reaction for IC50 or EC50 determination A. Extracellular receptor - binding study on cell surface The receptor is expressed in Per.Cδ cells by adenovirally transducing the cells (see US 6,340,595). The cells are incubated with radiolabeled ligand (iodinated, tritiated) and the unlabeled compound at 2, 10, 50, 250, 1250 fold molar excess (3 hours at 4° C: 25 mM HEPES, 140 mM NaCl, 1 mM CaCl₂, 5 mM MgCl₂ and 0.2% BSA, adjusted to pH 7.1) . Reactions mixtures are aspirated onto PEI-treated GF/B glass filters using a cell harvester (Packard) . The filters are washed twice (25 mM HEPES, 500 mM NaCl, 1 mM CaCl₂, 5 mM MgCl₂, adjusted to pH 7.1) . Scintillant (MicroScint-10; 35 μl) is added to dried filters and the filters counted in a Packard Topcount scintillation counter. Data are analyzed and plotted using Prism software (GraphPad Software, San Diego, Calif.). Competition curves are analyzed and IC50 values calculated. If 1 or more datapoints do not fall within the linear range of the competition curve or close to the linear range (in case the linear range is steep; typically seen for high affinity compounds) the assay is repeated and concentrations of labeled ligand and unlabeled compound adapted to have more datapoints close to or in the linear range. B. Receptor binding studies on membrane preparations Membranes preparations are isolated from Per.C6 cells overexpressing the receptor as follows: Medium is aspirated from the transduced cells and cells are harvested in 1 x PBS by gentle scraping. Cells are pelleted (2500 rp 5 min) and resuspended in 50 mM Tris pH 7.4 (10 x 10E6 cells/ml). The cell pellet is homogenized by sonicating 3 x 5 sec (UP50H; sonotrode MSl; max amplitude: 140 μm; max Sonic Power Density: 125W/cnr) . Membrane fractions are prepared by centrifuging 20 min at maximal speed (13000 rpm -15 000 to 20 OOOg or rcf) . The resulting pellet is resuspended in 500 μl 50 mM Tris pH 7.4 and sonicated again for 3 x 5 sec. The membrane fraction is isolated by centrifugation and finally resuspended in PBS/1% Triton. Binding competition and derivation of IC50 values are determined as desicribed above with the difference that a detergent (e.g. 1% Triton) is included in the assay buffer. Biochemical assays - use of (semi-) purified protein targets (Semi-) purified proteins are obtained by synthesizing them in vitro by e.g. using the pSG5-expression vectors and the TNT^® T7 Quick Coupled Transcription/Translation System (Promega) . Reaction mixtures containing the translated proteins are snap- frozen in 45-μl aliquots at -70 °C until required. [³⁵S]Methionine-radiolabeled translation products (translated in parallel) are separated on 10% SDS-polyacrylamide gels in order to check the integrity of the ER translation products. Translation products are diluted 20-fold in ligand binding buffer (20 mM Hepes, pH 7.4, 1.5 mM EDTA, 0.1% bovine serum albumin, 0.25 mM dithiothreitol, 10% glycerol) and kept on ice. Aliquots (45 μl) of 20-fold diluted receptor preparation are saturated with radiolabeled ligand and incubated in the presence, or absence, of various concentrations of compounds for at least 16 hours at 4 C. The final incubation volume is 50 μl . Free and bound radioligand are separated by adding 50 μl of ice- cold DCC (0.1 g of dextran T70, 1.0 g of activated charcoal, 4.0 ml of 1 M Tris, pH 7.4, 0.8 ml of 0.5 M EDTA, made up to a final volume of 400 ml in distilled water) to each reaction tube. The tubes are mixed briefly, incubated on ice for 5 min, and centrifuged for 5 min at 4 C to pellet the charcoal. 80 μl of supernatant is removed and added directly to beta-vials (Pony Vial™, Packard^®) containing scintillant and measured. Data are analyzed and plotted using Prism software (GraphPad Software, San Diego, Calif.). Competition curves are analyzed and IC50 values calculated. If one or more datapoints do not fall within the linear range of the competition curve or close to the linear range (in case the linear range is steep; typically seen for high affinity compounds) the assay is repeated and concentrations of labeled ligand and unlabeled compound are adapted to have more datapoints close to or in the linear range. Alternatively, (semi-) urified proteins are prepared from E. coli (BL21 (DE3)pLysS, Novagen) producing GST-fusions with the protein target. E. coli expressing the fusion proteins are harvested by centrifugation and subjected to freeze thawing in buffer containing 50 mM Tris-HCl pH 7.9, 250 mM KC1, 10% glycerol, 1% Triton X-100, 10 mM DTT, 1 mM PMSF, 10 μg/mL DNase and 10 mM MgCl . Competitive ligand binding assays are performed on immobilized GST-fusion proteins from crude extracts incubated with glutathione-Sepharose 4B (Amersham Pharmacia Biotech) . Following immobilization, the slurry is washed three times in binding buffer containing 50 mM Tris-HCL, pH 7.9, 50 mM KC1, 0.1% Triton-XlOO, 10 mM DTT, 2 mM EDTA, dispensed in 96-well filter plates (MHVB N45, Millipore) and incubated with a fixed amount labeled ligand and different concentrations of cold competing compounds. Equilibrium binding is reached after incubation for 2 hours at room temperature on a plate shaker. The plates are then washed 3 times in binding buffer, dried overnight at room temperature followed by scintillation counting after the addition of 25 μl of scintillant per well (MicroScint-10) . Data are analyzed and plotted using Prism software (GraphPad Software, San Diego, Calif.). Competition curves are analyzed and IC50 values calculated. If one or more datapoints do not fall within the linear range of the competition curve or close to the linear range (in case the linear range is steep; typically seen for high affinity compounds) the assay is repeated and concentrations of labeled ligands and unlabeled compound are adapted to have more datapoints close to or in the linear range.

Example 6: siRNA and siRNA expression constructs Inhibition of an identified protease induces the differentiation of MPCs into osteoblasts. One way of inhibiting the protease is by RNA interference. 21 nt dsRNA targeted against the hit is used (see Table 3) . In addition, siRNA expression constructs, both viral and non-viral are synthesized. In Table 3 target sequence are given for siRNA molecules designed against the polypeptides of the present inveniton from Table 1. For each polypeptide, 3 different siRNA molecules are given. siRNA expression constructs The construction of the siRNA expression constructs is depicted in Figures 3 and 4. In short, oligos containing knockdown target sequences as depicted in Table 3 are cloned in the knock-down vector, pKD122 (Figure 5) . These DNA expression plasmids are used using DNA transfer methods known in the art, such as lipofectamine or PEL The individual knockdown constructs for each gene are pooled or used separately. Adenoviral siRNA expression constructs The viruses are made in an arrayed format, if preferred. The arrayed viruses mediate expression of the siRNA constructs; each well contains a unique recombinant virus carrying a siRNA expression construct targeted against a gene, i.e., one target gene per well. Further details about the concept of arrayed adenoviral vectors can be found in W09964582, US 6,340,595 and US 6,413,776 . In addition to the knock-down vector pKD122, two other materials are needed for the generation of recombinant adenovirus particles: a helper cosmid and a packaging cell line (see also W09964582. US 6,340,595). The cosmid (pWE/Ad.AfIII- rITRΔE2A) contains the main part of the adenovirus serotype 5 genome (bp 3534-35953) from which the E2A gene is deleted. The Per.C6/E2A packaging cell line is derived from human embryonic retina cells (HER) transfected with plasmids mediating the expression of the El and E2A genes. The adenoviral genes that are integrated into the genome of the PER.C6/E2A cell line share no homology with the adenoviral sequences on the knock- down plasmid and the cosmid. Consequently, vector stocks free of replication competent adenoviruses (RCAs) are prepared. To obtain viruses, the knockdown plasmid is co-transfected with the helper cosmid into a packaging cell line PER.C6/E2A. Once these plasmids are transfected into the PER.C6/E2A cell line, the complete Ad5 genome is reconstituted by homologous recombination. The helper and knock-down plasmids contain homologous sequences (bp 3535-6093) , which are a substrate for this recombination event. Design of oligos Oligonucleotides are designed to target specific mRNAs . These sequences are used for the construction of knock-down adenoviral expression clones. Specific pairs of forward (F) and reverse (R) oligonucleotides are annealed together resulting in a duplexed structure that is used for cloning into the knock-down vector. The 56 nt oligos targeted against knock-down target sequences have the following structure: Forward oligonucleotide: 5' -ACC-G-N18*-GTTTGCTATAAC-Nl 8-CTTT Reverse oligonucleotide: 3'-C-N18*-CAAACGATATTG- N18 -GAAAAAT-5' Where N18 are 18 nucleotides that are homologous to the target sequences in the mRNA encoding the polypeptides of the present invention; i.e. the nucleotides in bold in Table 3. N18* are 18 nucleotides that are complementary to the mRNA encoding the polypeptides of the present invention; i.e. the antisense sequence of the nucleotides in bold in Table 3. The single stranded oligonucleotide components are synthesized and annealed in 96 or 384 well plates to generate double stranded oligonucleotides at a final concentration of 50 p ol/μl, 100 μl total volume per well (Sigma) . 2 μl annealing buffer (NEBuffer 2, lOx concentrated, New England Biolabs), in a 96 well PCR plate, is added to 18 μl oligos. The plates are spun down briefly and subsequently sealed. The plates are incubated in the PCR machine for 5 minutes at 95°C and slowly cooled. The annealed oligos are diluted 1000 fold. Design of pKD122 The pIPspAdAptδ plasmids contain the 5' part (bp 1-454 and bp 3511-6093 of the adenovirus serotype 5 genome in which the El gene is deleted and a promoter is introduced. In contrast to the plasmid pIPspAdAptδ, the siRNA expression vectors lack the CMV promoter and the SV40 polyadenylation site and the larger part of the polylinker. pKD122 further contains U6 promoter, Sap I recognition sites and the E. Coli lethal gene, ccdB. Sap I cuts adjacent to its recognition sites (GCTCTTC (N) χ_/4) creating a 3' overhang. This has the advantage in that it cuts any sequence and that the recognition sequence is not present in the final construct since it is present on the excised fragment. Therefore it is used for the construction of expression plasmids, without disturbing the sequence. The ccdB is included in the fragment to be excised. When the restriction fragment is not correctly excised and the ccdB gene remains in the plasmid, no E. coli colonies are formed after transfection. Only E. coli containing correct expression plasmids (with the two unique Sapl overhangs and without the ccdB gene) will form colonies. pIPspAdapt6 is grown in the methylase negative E. coli strain DM1 to prevent methylation of the second Xba I-site. The DNA is isolated and digested with Xba I, thereby excising a 142 bp fragment containing the poly A signal. The religated vector is called pIPspAdapt6-deltaPolyA. The polylinker is removed from pIPspAdapt6-deltaPolyA by digestion with EcoRI and BamHI, blunted with Klenow, religated and digested with Ascl to reduce background. This religated vector is called pIPspAdapt6-deltaPolyA delta-polylinker . ) pIPspAdapt6-deltaPolyA-delta polylinker is digested with Avrll and HindiII to remove the CMV promoter, purified on a 1% agarose TAE gel, and isolated using the Qiaquick gel extraction kit (Qiagen) . The ccdB gene is cut from pIPspAdaptlOZeoDestA (W09964582) with BamHI and Sal I. The 676 bp ccdB fragment is purified on a 0.8% agarose TAE gel and isolated using the Qiaquick gel extraction kit (Qiagen) . The human genomic U6 gene (Accession number M14486) is cloned by a PCR based strategy using human genomic DNA. The region to be cloned starts at nucleotide -265 upstream of the transcription start site ends at nucleotide +198 downstream of the transcription start site. The primers used are: 5'-GcacgTTCTAGAAGGTCGGGCAGGAAGAGGGCCT-3' (SEQ ID NO: 176) 5'-ccgtgcAAGCTTTGGTAAACCGTGCACCGGCGTA-3' (SEQ ID NO: 177)

The PCR product is cloned into the Xba I and Hind III sites of pIPspAdapt6-deltaPolyA. The resulting vector is hU6(+l)pIPspAdapt6-deltaPolyA Two U6 Sap I PCR fragments (a left (L) and a right (R) ) containing the U6 promoter sequences together with the Sapl recognition sequences are made with the following primers: 5'-CGACCATGCGCGGATCCGCTCTTCTGGTGTTTCGTCCTT-3' (SEQ ID NO: 178) 5'-CGGATCCGCGCATGGTCGACGCTCTTCATTACATCAGGTTGTTT-3' (SEQ ID NO: 179) SEQ ID NO: 178 with SEQ ID NO: 176 is used to give the L fragment and SEQ ID NO: 179 with SEQ ID NO: 177 is used to give the R fragment. The hU6(+l) pIPspAdapt6-dpA delta polylinker is used as template. The PCR fragments are purified on a 1% agarose TAE gel and isolated using the Qiaquick gel extraction kit (Qiagen) . The R-fragment is digested with Xbal and BamHI, and the L fragment is digested with Sail and Hindlll. pIPspAdapt6- deltaPolyA-deltapolylinker is digested with Avrll and Hindlll. The digested R- and L-fragments together with the digested pIPspAdapt6-deltaPolyA-deltapolylinker and the ccdB fragment are ligated using T4 ligase in ligase buffer (about 30 ng of each fragment in the ligation) and transformed in DB3.1 cells (from Invitrogen) . The ccdB protein is not lethal for the DB3.1 cells . A colony PCR with primers SEQ ID NO: 176 and 177 is performed to check the sequences. A 1000 bp fragment should be generated. Positive clones are digested with Hindi and Bglll. The correct clones give fragments of 3800, 1400, 538, 402 and 134 bp in size. Clones that give these fragments are sequenced. The resulting vector is pKD122 (Figure 5) . Cloning of the oligos The knockdown vector (Fig 5) is digested by Sap I and gel- purified. The digestion mix is 30 μl Neb 4 (New England BioLabs), 10 μl Sap I in 300 μl total volume for 9 μg of knockdown vector, and is incubated at 37 °C over night. The gel is 1 % agarose in lx TAE and 5 μl of digestion mix with 2 μl lOx loading bufferis loaded. The digested vector is isolated from gel with QIAquick gel extraction kit (Qiagen) . Ligation of the annealed oligos in the knock-down vector 0.5 μl digested knock-down vector (40 ng/μl) , 1 μl T4 DNA ligase buffer (lOx concentrated, Biolabs) , 0.5 μl T4 DNA ligase (Biolabs) and 7 μl illiQ H₂0 are added per well. Added to this mixture is 1 μl of the diluted annealed oligos. The plates are incubated over night at room temperature. Transformation 5 μl of each ligation mix is put into a new PCR plate and put on ice. 25 μl competent DH5α-cells (Subcloning efficiency, Invitrogen) is added and incubated on ice for 30 minutes. The bacteria are heat shocked for 40 seconds at 37 °C and put on ice for 2 minutes. 170 μl RT SOC-medium (Invitrogen) is added to each well. The bacteria are recovered by shaking for 1 hour at 37°C and 100-150 rp . Cells are spun down at 1700 g for 1 min. 100 μl supernatant is taken and the bacteria are resuspended in the remainder of the supernatant (100 μl) . 50 μl of the cell suspension (50%) is plated out in 1 well of a 6-wells plate (filled with 3 ml LB agar+100 μg/ml ampicillin /well) . The plates are incubated over night at 37 °C Colony picking 3 colonies of each construct are picked and inoculated as agar-stab (LB agar with 100 μg/ml ampicillin) and liquid culture (LB medium with 100 μg/ml ampicillin) . The clones in the agar-stab are sequenced. Clones with the correct sequence are transferred to a new 96 well plate. The knock-down vector with the annealed oligos is digested with PI-PspI. PI-PspI digestion mix (lx) contains 0.5 μg annealed knockdown vector, 2.5 μl PI-PspI enzyme (lU/μl New England Biolabs), 2.5 μl PI-PspI NEBuffer (lOx concentrated New England Biolabs), and 0.25 μl BSA (lOOx concentrated, New England Biolabs) in a total volume of 25 μl (end concentration = ~20 ng/μl) . The mixture is incubated over night at 65°C in a humidified box. The digestion is checked on a gel: 10 μl PI-PspI digestion mix is added to 2 μl loading buffer (lOx concentrated) and put loaded onto a 1% agarose gel in lx TAE buffer + ethidium bromide. Transfection Each clone is co-transfected into PER.C6/E2A cells together with the cosmid pWE/Ad.AflII-rITRΔE2A (W09964582; US 6,340,595). CPE is scored 14 days after transfection. After the final scoring the plates are stored at -80°C until further propagation of the viruses. Virus propagation The final virus propagation step is aimed at obtaining a higher percentage of wells showing CPE and more homogenous virus titers. Viruses are propagated according to the following procedure. The transfection plates stored at -80°C are thawed at room temperature for about 1 hour. By means of a 96 channel Hydra dispenser (Robbins), 20 μl of the supernatant is transferred onto PER.C6/E2A cells seeded in 96 well plates at a density of 2.25xl0⁴ cells/well in 180 μl of DMEM + 10% FBS. After transfering one series of 96 viruses, the needles of the dispenser are disinfected and sterilized by pipetting up 60 μl of 5% bleach three times. The traces of bleach present in the needles are removed by three successive washes with 70 μl of sterile water. Cells are incubated at 34°C, 10% C0₂ for approximately 10 days and the number of wells showing CPE is scored. In general, the number of wells showing CPE is increased after propagation. The plates are then stored at - 80°C. In addition, modifications to the viral coat proteins can be introduced to obtain a different or improved tropism (WO02/24933) . The individual knockdown adenoviruses can be used as arrays but also can be pooled to various degrees, i.e., sets of pools or one large pool.

Claims

1. Polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62, or a derivative, or a fragment thereof, for use as a medicament.

2. Polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for use as a medicament.

3. Vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for use as a medicament.

4. Vector according to claim 3, wherein said vector is an adenoviral, retroviral, adeno-associated viral, lentiviral or a sendaiviral vector.

5. Use of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local decrease in mean bone density.

6. Use of a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local decrease in mean bone density.

7. Use of a vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local decrease in mean bone density.

8. Use according to any of claims 5-7 wherein the disorder is selected from the group consisting of osteoporosis, hypercalce ia of malignancy, multiple myelomatosis, hyperparathyroidism, and hyperthyroidism.

9. Method for inducing the differentiation of undifferentiated mammalian cells into osteoblasts comprising (a) introducing a polynucleotide or a vector comprising a nucleic acid sequence selected from the group consisting of SEQ

ID NO: 1-31, or a derivative, or a fragment thereof, into the cell; and (b) overexpressing a polypeptide, or a derivative, or a fragment thereof, encoded by the nucleic acid sequence, or a derivative, or a fragment thereof, in the cell; such that the level of bone alkaline phosphatase is increased compared to cells without the introduced polynucleotide or vector.

10. Method for identifying a compound that induces differentiation of undifferentiated mammalian cells into osteoblasts comprising: (a) contacting one or more compounds with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62 or a derivative or a fragment thereof (b) determining the binding affinity of the compound to the polypeptide, (c) contacting a population of undifferentiated mammalian cells with the compound that exhibits binding affinity of at least 10 micromolar, and (d) identifying the compound that induces the differentiation of the cells.

11. Method for identifying a compound that induces differentiation of undifferentiated mammalian cells into osteoblasts comprising: (a) contacting one or more compounds with a polynucleotide sequence or a vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-31, or a derivative, or a fragment thereof, (b) determining the binding affinity of the compound to the polynucleotide or to the vector, (c) contacting a population of undifferentiated mammalian cells with the compound that exhibits binding affinity of at least 10 micromolar, and (d) identifying the compound that induces the differentiation of the cells.

12. Method according to claim 10 or 11 wherein the compounds are low molecular weight compounds.

13. Method according to claims 10 or 11 wherein the compounds are peptides.

14. Method according to claims 10 or 11 wherein the compounds are natural compounds .

15 Method for in vitro production of bone tissue, comprising the steps of: (a) applying undifferentiated mammalian cells on a substrate to form a cellular substrate, (b) contacting the cells with a polypeptide having an amino acid sequence selected from the group of SEQ ID NO: 32- 62, or a derivative, or a fragment thereof, for a time sufficient to differentiate the undifferentiated cells into osteoblasts, thereby producing a continuous bone matrix.

16. Method for in vitro production of bone tissue, comprising the steps of: (a) applying undifferentiated mammalian cells on a substrate to form a cellular substrate, (b) introducing a polynucleotide sequence or a vector comprising a nucleic acid sequence selected from the group of

SEQ ID NO: 1-31, or a derivative, or a fragment thereof, for a time sufficient to differentiate the undifferentiated cells into osteoblasts, thereby producing a continuous bone matrix.

17. Method according to claim 15 or 16, wherein the continuous bone matrix comprises a thickness of at least 0.5 μm on the surface of the substrate.

18. Method for diagnosing a pathological condition involving a systemic or local decrease in mean bone density or a susceptibility to the condition in a subject, comprising: (a) obtaining a sample of the subject's mRNA corresponding to a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 or a sample of the subject's genomic DNA corresponding to a genomic sequence of a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 (b) determining the nucleic acid sequence of the subject's mRNA or genomic DNA; (c) comparing the nucleic acid sequence of the subject's mRNA or genomic DNA with a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 or with a genomic sequence encoding a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 obtained from a database; and (d) identifying any difference (s) between the nucleic acid sequence of the subject's mRNA or genomic DNA and the nucleic acid selected from the group consisting of SEQ ID NO: 1-31 or the genomic sequence encoding a nucleic acid selected from the group consisting of SEQ ID NO: 1-31 obtained from a database .

19. Method for diagnosing a pathological condition involving a systemic or local decrease in mean bone density or a susceptibility to the condition in a subject comprising determining the amount of polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 32-62 in a biological sample, and comparing the amount with an amount of the polypeptide in healthy subjects, whereby a decrease of the amount of polypeptide compared to the healthy subjects is indicative of the condition.

20. Method according to any of claims 18 or 19 wherein the condition is selected from the group consisting of osteoporosis, hypercalcemia of malignancy, multiple myelomatosis, hyperparathyroidism, and hyperthyroidism.

21. Polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 63-175, or a derivative, or a fragment thereof, for use as a medicament.

22. Vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 63-175, or a derivative, or a fragment thereof, for use as a medicament.

23. Vector according to claim 22, wherein said vector is an adenoviral, retroviral, adeno-associated viral, lentiviral or a sendaiviral vector.

24. Use of a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 63- 175, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local increase in mean bone density.

25. Use of a vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 63-175, or a derivative, or a fragment thereof, for the manufacture of a medicament for treatment of a disorder involving a systemic or local increase in mean bone density.

26. Use according to claims 24 or 25 wherein the disorder is selected from the group consisting of osteosclerosis, osteosclerotic myeloma, pyknodysostosis, Camuratie-Engelmand disease, osteopoikilosis, melorheostosis, and osteopetrosis .