WO2019038420A1 - Pharmaceutical compositions for the treatment of osteoclast diseases - Google Patents

Abstract

The invention relates to methods and pharmaceutical compositions for the treatment of osteoclast diseases in a subject in need thereof. The inventors investigated osteoclastogenesis and osteoclast differentiation and demonstrated that CSF-1R expression and RANK expression are down-regulated by HCMV infection during osteoclastogenesis and CSF-1R stimulation induces RANK expression and osteoclast differentiation. The inventors also demonstrated that HCMV infection inhibits cell proliferation and differentiation during osteoclastogenesis. The inventors also demonstrated that HCMV-mediated osteoclastogenesis inhibition is mediated by QK15 upregulation during infection and that expression of the regulatory protein QK15 inhibits osteoclastogenesis by inducing CSF-1R mRNA degradation and RANK inhibition. The inventors also investigated articular bone erosion and demonstrate that HCMV seropositivity has a protective effect in a cohort of 487 Rheumatoid Arthritis (RA) patients. Thus, the invention relates to a QK15 polypeptide and/or an agent for QK15 polypeptide expression for use in the treatment of osteoclast disease in a subject in need thereof.

Description

PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF OSTEOCLAST DISEASES

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of osteoclast diseases in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Osteoclasts (OCs) are large multinucleated highly specialised cells capable of breaking down both the inorganic (hydroxyapatite) and organic (collagen and multiple-non-collagenous proteins) matrix of bone, dentine and mineralised cartilage and are unique in their ability to resorb mineralised bone matrix (Helfrich et al., 2003). They form as a result of the fusion of mononuclear precursors derived from the monocyte/macrophage lineage, and this differentiation is controlled by interactions between osteoblasts and/or stromal cells and preosteoclasts (Crockett et al., 2011).

In addition to their central role in inflammation, monocytes/macrophages are at the origin of pathological bone erosion in RA due to their excessive differentiation into OCs, which are the only cells specialized in bone resorption (Thurlings et al., 2009). Their differentiation is mediated by two major cytokines, M-CSF and RANKL (receptor activator of nuclear factor kB ligand). M-CSF binds to its receptor c-FMS, (cellular-feline McDonough strain sarcoma virus oncogene homologue, or CSF-1 receptor, or CD115), which in turn induces the expression of RANK on monocytes (Davignon et al., 2013).

Inflammatory diseases such as rheumatoid arthritis, spondylitis ankylosing, psoriatic disease are characterized by bone erosion mediated by OCs.

Osteoporosis is due to imbalance between bone resorption and bone deposition. For example, menopause is increasing the risk of osteoporosis due accelerated bone resorption.

Bone resorption in cancer metastases is also due to OCs hyperactivity.

There are several osteoclast diseases, which are the result of overactivity of OCs and deregulation of osteoclastogenesis and OC differentiation such as Paget's disease of bone (PDB), Juvenile Paget's disease (JPD), early onset PDB, expansile skeletal hyperphosphatasia (ESH) and familiar expansile osteolysis (FEO). The clinical phenotype of these diseases is one of uncontrolled bone remodelling, with presence of many, often enlarged, osteoclasts accompanied by areas of active bone formation (Helfrich et al., 2005). Accordingly, there is a need to develop new approaches and drugs that will be suitable for effective and efficient treatment of osteoclast diseases. In this way, it has been suggested that characterization of new therapeutic compounds in osteoclast diseases may be highly desirable.

There is no disclosure in the art of QKI5 effects in osteoclast diseases, the inhibition of osteoclastogenesis by QKI5 and the use of QKI5 polypeptide and agent for QKI5 polypeptide expression in the treatment of osteoclast diseases.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of osteoclast diseases in a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors investigated osteoclastogenesis and OC differentiation and demonstrated that CSF-1R expression and RANK expression are down-regulated by HCMV infection during osteoclastogenesis. The inventors also demonstrated that HCMV infection inhibits cell proliferation and differentiation during osteoclastogenesis. The inventors also demonstrated that HCMV-mediated osteoclastogenesis inhibition is mediated by QKI5 upregulation during infection and that experimental induction of QKI5 expression inhibits osteoclastogenesis by inducing CSF-1R mRNA degradation and RANK inhibition. The inventors also investigated articular bone erosion and demonstrate that HCMV seropositivity has a protective effect in a cohort of 487 Rheumatoid Arthritis (RA) patients.

Accordingly, the present invention relates to a QKI5 polypeptide and/or an agent for QKI5 polypeptide expression for use in the treatment of osteoclast disease in a subject in need thereof.

As used herein, the term "subject" denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with osteoclast disease. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with bone erosion diseases, osteoporosis, rheumatoid arthritis, chronic inflammatory rheumatism, bone cancer or bone cancer metastasis.

As used herein, the term "osteoclast disease" has its general meaning in the art and includes, but is not limited to, bone erosion diseases and bone cancer. Examples of osteoclast disease that may be treated by methods and compositions of the present invention include, but are not limited to, bone erosion diseases, osteoporosis, arthritis, rheumatoid arthritis, chronic inflammatory arthritis, rheumatism, chronic inflammatory rheumatism, bone cancer, osteosarcoma, bone cancer metastasis, Paget' s disease of bone (PDB), Juvenile Paget' s disease, periodontal disease, Familial Expanile Osteolysis (FEO), Expansile Skeletal Hyperphosphatasia (ESH) and Camurati Engelmann Disease (CED).

In some embodiments, the subject suffers from bone erosion diseases, osteoporosis, rheumatoid arthritis, chronic inflammatory rheumatism, bone cancer, osteosarcoma or bone cancer metastasis.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). The term "QKI5" has its general meaning in the art and refers to quaking (QK) 1-5 protein. The term "QKI5" refers to the RNA-binding protein belonging to the evolutionarily conserved signal transduction and activator of RNA (STAR) family implicated in embryogenesis and central nervous system development. The QKI (quaking, Qk) locus encodes a diverse set of proteins created by alternative splicing, and the three QKI proteins QKI-5, QKI- 6, and QKI-7 different by their carboxyl tails (Wu et al, 1999; Fu et al, 2012). The term "QKI5" also refers to QKI5 polypeptide deposited in the National Center for Biotechnology Information, U.S. National Library of Medicine under the NCBI accession number: NP 001288014.1 (SEQ ID NO: 1). The term "QKI5" also refers to QKI5 polypeptide encoded by the nucleic acid sequence deposited in the NCBI under the NCBI accession number NM 001301085.1 (SEQ ID NO: 2).

In some embodiments, the QKI5 polypeptide of the invention is an isolated, synthetic or recombinant QKI5 polypeptide.

In some embodiments, said QKI5 polypeptide comprises a sequence as set forth by SEQ ID NO: l.

SEQ ID NO: 1

In some embodiments, the polypeptide of the present invention comprises or consists of an amino acid sequence having at least 70% of identity with SEQ ID NO: 1.

According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99, or 100%) of identity with the second amino acid sequence. Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990). In particular the polypeptide of the invention is a functional conservative variant of the polypeptide according to the invention. As used herein the term "function-conservative variant" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Accordingly, a "function-conservative variant" also includes a polypeptide which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent polypeptide to which it is compared. Functional properties of the polypeptide of the invention could typically be assessed in any functional assay as described in the EXAMPLE.

A further aspect of the present invention relates to a fusion protein comprising the polypeptide according to the invention that is fused to at least one heterologous polypeptide.

The term "fusion protein" refers to the polypeptide according to the invention that is fused directly or via a spacer to at least one heterologous polypeptide.

According to the invention, the fusion protein comprises the polypeptide according to the invention that is fused either directly or via a spacer at its C-terminal end to the N-terminal end of the heterologous polypeptide, or at its N-terminal end to the C-terminal end of the heterologous polypeptide.

As used herein, the term "directly" means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the polypeptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide.

In other words, in this embodiment, the last amino acid of the C-terminal end of said polypeptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said polypeptide is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous polypeptide.

As used herein, the term "spacer" refers to a sequence of at least one amino acid that links the polypeptide of the invention to the heterologous polypeptide. Such a spacer may be useful to prevent steric hindrances.

In some embodiments, the heterologous polypeptide is a cell-penetrating peptide, a

Transactivator of Transcription (TAT) cell penetrating sequence, a cell permeable peptide or a membranous penetrating sequence.

The term "cell-penetrating peptides" are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

In some embodiment, the heterologous polypeptide is an osteoclast targeting agent.

Osteoclast targeting agent include but are not limited to ligands or antibodies directed against the integrin ανβ3, calcitonin receptors, the vitronectin receptor (VNR), vacuolar ATPases (V- ATPases), the transcription factors PU.l, c-Fos, and NFKB I and 2, the growth factors M-CSF (macrophage colony stimulating factor) and RANKL (TNFRSF11), the receptor RANK ((receptor activator of NFKB), member 11 A of the tumour necrosis factor (TNF) receptor superfamily), the cathepsin K (CTSK), the tartrate-resistant acid phosphatase (TrAP), ClC-7 chloride channel, or antibodies or agents interacting with osteoclast membrane-bound and intracellular targets.

In another embodiment, the heterologous polypeptide is an osteoclast disease therapeutic polypeptide.

The term "osteoclast disease therapeutic polypeptide" refers to any polypeptide that has anti-osteoclast disease activities such as described below. Several such polypeptides are known in the art such as described in Sun et al, 2008; Araujo et al, 2009; Tang et al., 2010; Broadhead et al, 2011; Kim and Moon, 2013.

The polypeptides or fusion proteins of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides or fusion proteins, by standard techniques for production of amino acid sequences. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the polypeptides or fusion proteins of the invention can be synthesized by recombinant DNA techniques as is now well- known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (polypeptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

Polypeptides or fusion proteins of the invention can be used in an isolated (e.g., purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).

In specific embodiments, it is contemplated that polypeptides or fusion proteins according to the invention may be modified in order to improve their therapeutic efficacy and their stability using well-known techniques. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. A strategy for improving drug stability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

For example, Pegylation is a well-established and validated approach for the modification of a range of polypeptides (Chapman, 2002). The benefits include among others: (a) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and/or through evasion of cellular clearance mechanisms; (b) reduced antigenicity and immunogenicity of the molecule to which PEG is attached; (c) improved pharmacokinetics; (d) enhanced proteolytic resistance of the conjugated protein (Cunningham- Rundles et.al, 1992); and (e) improved thermal and mechanical stability of the PEGylated polypeptide.

Therefore, advantageously, the polypeptides of the invention may be covalently linked with one or more polyethylene glycol (PEG) group(s). One skilled in the art can select a suitable molecular mass for PEG, based on how the pegylated polypeptide will be used therapeutically by considering different factors including desired dosage, circulation time, resistance to proteolysis, immunogenicity, etc.

In one embodiment, the PEG of the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CFb ("methoxy PEG"). In addition, such a PEG can consist of one or more PEG side-chains which are linked together. PEGs with more than one PEG chain are called branched PEGs. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide. One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et al., 1995).

To effect covalent attachment of PEG groups to the polypeptide, the hydroxyl end groups of the polymer molecule must be provided in activated form, i. e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate (SPA), succinimidyl carboxymethylate (SCM),benzotriazole carbonate (BTC), N- hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)). Suitable activated polymer molecules are commercially available, e. g. from Shearwater Polymers, Inc., Huntsville, AL, USA, or from PolyMASC Pharmaceuticals pic, UK. Alternatively, the polymer molecules can be activated by conventional methods known in the art, e. g. as disclosed in WO 90/13540. Specific examples of activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEGs : NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM- PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS.

The conjugation of the polypeptides or fusion proteins and the activated polymer molecules is conducted by use of any conventional method. Conventional methods are known to the skilled artisan. The skilled person will be aware that the activation method and/or conjugation chemistry to be used depends on the attachment group(s) of the polypeptides as well as the functional groups of the PEG molecule (e.g., being amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate).

In one embodiment, polypeptides are conjugated with PEGs at amino acid D and E (for COOH), T, Y and S (for OH), K (for NH₂), C (for SH if at least one cysteine is conserved) or/and Q and N (for the amide function).

In one embodiment, additional sites for PEGylation can be introduced by site-directed mutagenesis by introducing one or more lysine residues. For instance, one or more arginine residues may be mutated to a lysine residue. In another embodiment, additional PEGylation sites are chemically introduced by modifying amino acids on polypeptides of the invention.

In one embodiment, PEGs are conjugated to the polypeptides or fusion proteins through a linker. Suitable linkers are well known to the skilled person. A preferred example is cyanuric chloride ((Abuchowski et al, 1977); US 4,179, 337).

Conventional separation and purification techniques known in the art can be used to purify pegylated polypeptides of the invention, such as size exclusion (e.g. gel filtration) and ion exchange chromatography. Products may also be separated using SDS-PAGE.

In one embodiment, the pegylated polypeptides provided by the invention have a serum half- life in vivo at least 50%, 75%, 100%, 150% or 200% greater than that of an unmodified polypeptide. In some embodiments, the agent for QKI5 polypeptide expression of the invention is selected from the group consisting of an isolated, synthetic or recombinant nucleic acid encoding for QKI5 polypeptide, a nucleic acid sequence encoding for the fusion protein, a nucleic acid encoding a fragment of a QKI5 polypeptide, a cell expressing QKI5 polypeptide, and agent inducing QKI5 gene and polypeptide expression and their combinations.

In some embodiments, said nucleic acid encoding for QKI5 polypeptide comprises a sequence as set forth by SEQ ID NO: 2.

SEQ ID NO: 2

In some embodiments, the nucleic acid encoding for QKI5 polypeptide for example comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 2, preferably at least 85% identical to sequence SEQ ID NO: 2, more preferably at least 90% identical to sequence SEQ ID NO: 2, still more preferably at least 95%, at least 96%>, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 2.

As used herein, a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

These nucleic acid sequences can be obtained by conventional methods well known to those skilled in the art. Typically, said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

So, a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule encoding for a polypeptide or a fusion protein of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted. These recombinant vectors may, for example, be cloning vectors, or expression vectors.

As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

Any expression vector for animal cell can be used. Examples of suitable vectors include pAGE107 (Miyaji et al, 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al,

1984) , pKCR (O'Hare et al, 1981), pSGl beta d2-4 (Miyaji et al, 1990) and the like.

Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.

Other examples of viral vectors include adenoviral, lentiviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.

Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami and Itoh, 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana et al, 1987), promoter (Mason et al,

1985) and enhancer (Gillies et al., 1983) of immunoglobulin H chain and the like.

A further aspect of the invention relates to a host cell comprising a nucleic acid molecule encoding for a polypeptide or a fusion protein according to the invention or a vector according to the invention. In particular, a subject of the present invention is a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule or vector according to the invention.

The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed". In a particular embodiment, for expressing and producing polypeptides or fusion proteins of the invention, prokaryotic cells, in particular E. coli cells, will be chosen. Actually, according to the invention, it is not mandatory to produce the polypeptide or the fusion protein of the invention in a eukaryotic context that will favour post-translational modifications (e.g. glycosylation). Furthermore, prokaryotic cells have the advantages to produce protein in large amounts. If a eukaryotic context is needed, yeasts (e.g. saccharomyces strains) may be particularly suitable since they allow production of large amounts of proteins. Otherwise, typical eukaryotic cell lines such as CHO, BHK-21 , COS-7, C127, PER.C6, YB2/0, HEK293, mononuclear macrophage/monocyte-lineage hematopoietic precursors, Haematopoietic stem cells, Mononuclear precursor cells, osteoblast or inactive osteoclast could be used, for their ability to process to the right post-translational modifications of the fusion protein of the invention.

The construction of expression vectors in accordance with the invention, and the transformation of the host cells can be carried out using conventional molecular biology techniques. The polypeptide or the fusion protein of the invention, can, for example, be obtained by culturing genetically transformed cells in accordance with the invention and recovering the polypeptide or the fusion protein expressed by said cell, from the culture. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractional precipitation, in particular ammonium sulfate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins may be used for producing the proteins in accordance with the invention.

A further aspect of the invention relates to a method for producing a polypeptide or a fusion protein of the invention comprising the step consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said polypeptide or fusion protein; and (ii) recovering the expressed polypeptide or fusion protein.

In some embodiments, the agent for QKI5 polypeptide expression of the invention is an agent inducing QKI5 gene and polypeptide expression selected from the group consisting of, but not limited to, Human Cytomegalovirus (HCMV), VHL/E HCMV strain, and TB40/E HCMV strain.

In a further aspect, the present invention relates to the QKI5 polypeptide and/orthe agent for QKI5 polypeptide expression according to the invention in combination with one or more anti-osteoclast compound for use in the treatment of osteoclast disease in a subject in need thereof. The term "anti-osteoclast compound" has its general meaning in the art and refers to compounds and therapeutic active agent used in anti-osteoclast therapy such as Dendrimer; Calcium; vitamin D; antioxidant; Bisphosphonates such as ibandronate, aminobisphosphonates (e.g., risedronate, alendronate and zoledronate) and non-aminobisphosphonates (e.g., clodronate, tiludronate and etidronate); Calcitonin; Teriparatide; 1-34 parathyroid hormone (PTH 1-34); Raloxifene (selective estrogen receptor modulator (SERM)); Strontium ranelate; Disease-modifying antirheumatic drugs (DMARDs) such as adalimumab, etanercept, infliximab, agents that inhibit TNF-a; Nonsteroidal anti-inflammatory drugs; selective cyclooxygenase-2 inhibitors; agent targeting the RANK/RANKL/OPG signaling pathway such as Estrogen, SERMs such as ospemifene, lasofoxifene, bazedoxifene and arzoxifene; agents targeting RANKL such as IgG2 monoclonal antibody denosumab; (Fc) - OPG, a genetically engineering OPG fusion protein (Fc-fragment of IgGl); Inhibitors of integrin ανβ3 such as humanized monoclonal antibody LM-609, L-000845704; Inhibitors of vacuolar proton- pumping-ATPase such as SB-242784 , FR-177995, FR-202126, FR-167356; Inhibitors of carbonic anhydrase II; Inhibitors of the CLC-7 chloride channel such as NS-3696, NS-3736 and NS-5818; Inhibitors of Cathepsin K such as peptidyl aldehyde Cbz-(Leu)₃ CHO, AAE-581, MIV-701, odanacatib; Inhibitors of matrix metalloproteases (such as MMP-9) by inhibiting JAK and p38; Agents targeting the Wnt signaling pathway such as sclerostin; agents targeting signal transduction by protein kinases including inhibitors of p38 kinase, JAK, c-frns tyrosine kinase, ERK, and Jun N-terminal kinase and inhibitors of tyrosine kinase c-Src such as Dasatinib; soluble RANK and Recombinant Murine RANK Protein and compounds described in Davignon et al., 2013; Sun et al., 2008 ; Araujo et al., 2009 ; Tang et al., 2010 ; Broadhead et al, 2011; Kim and Moon, 2013.

The term "anti-osteoclast compound" also refers to osteoclast differentiation inhibitors such as autophagy-activating compounds such as mammalian target of rapamycin (mTOR)- independent autophagy inducers including spermidine and its derivatives, and promethazine and its derivatives, e.g., trifluoperazine, and mTOR-dependent autophagy inducers, such as rapamycin and everolimus; Ceramide glucosyltransferase (CGT) inhibitors and glucosidase inhibitors such as miglustat (iminosugar N-butyl-deoxynojirimycin, also referred to as NB- DNJ); calcitonin (CT) peptide and bisphosphonate-linked calcitonin; Indenone derivatives such as 6-(2-morpholinoethoxy)-3-phenyl-2-(pyridin-3-yl)-lH-inden-l-one hydrochloride salt; Cystatin C-mimetic compounds; Sulfonamide compounds such as ABD-295, ABD-455, ABD- 456, ABD-599, ABD-781, ABD-789; Benzamidine derivatives such as DW-1350 and their combination with bisphosphonates; Fatty acid amides such as N-oleoyl-L-serine HU-639; Pyrazole derivatives; Furo [3,2-b]pyrol derivatives; D-pinitol; Epothilones; Diphyllin; Anti- RANKL Nanobody, ALX-0141; TNF receptor loop-mimic peptide, WP9QY; anti-voltage dependent anion channel (VDAC) antibody; Anti-Siglec-15; Cilengitide, targeting integrin ανβ3 and ανβ5 such as EMD 121974, NSC 707544; RANK loop 3-mimic peptide L3-3; Anti- DC-STAMP (Dendritic cell- specific transmembrane protein); Anti-TGF-b antibody 1D1 1; Fusion protein RIG; Peptides with RANK cytoplasmic tail domain sequence; and compounds described in Kim and Moon, 2013.

In some embodiments, the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent.

In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

Typically the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression according to the invention as described above are administered to the subject in a therapeutically effective amount.

By a "therapeutically effective amount" of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention as above described is meant a sufficient amount of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression for treating osteoclast disease at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific QKI5 polypeptide and/or the agent for QKI5 polypeptide expression employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific QKI5 polypeptide and/or the agent for QKI5 polypeptide expression employed; the duration of the treatment; drugs used in combination or coincidental with the specific QKI5 polypeptide and/or the agent for QKI5 polypeptide expression employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention, preferably from 1 mg to about 100 mg of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

In a particular embodiment, the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression according to the invention may be used in a concentration between 0.01 μΜ and 20 μΜ, particularly, the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 μΜ.

According to the invention, the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention is administered to the subject in the form of a pharmaceutical composition. Typically, the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the present invention plus any additional desired ingredient from a previously sterile- filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of osteoclast disease in a subject in need thereof.

In a further aspect, the present invention relates to a method for treating osteoclast disease in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the invention. The invention also provides kits comprising the QKI5 polypeptide and/or the agent for QKI5 polypeptide expression of the invention. Kits containing the QKI5 polypeptide and/orthe agent for QKI5 polypeptide expression of the invention find use in therapeutic methods. The invention will be further illustrated by the following figures and examples.

However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: HCMV infection inhibits CSF-IR expression in a replication-dependent manner. Purified monocytes were incubated with recombinant M-CSF and RANKL (both at 50 ng/mL) and after 24 hours, infected or not (Ni) with infectious (VHL/E) or UV-inactivated HCMV (VHL/E UV). Before infection (0) or after 24 hours of infection (24h), cells were harvested to assess CSF-IR mRNA expression by qPCR. RT-qPCR Experiments are presented as average of 5 different donors normalized on GAPDH expression (n=5, *** p<0.001, error bars as s.d.).

Figure 2: HCMV infection inhibits RANK expression in a replicative dependent manner. Purified monocytes were incubated with recombinant M-CSF and RANKL (both at 50 ng/mL) and after 24 hours, infected or not (Ni) with infectious (VHL/E) or UV-inactivated HCMV (VHL/E UV). Before infection (0) or after 24 hours of infection (24h), cells were harvested to assess RANK mRNA expression by qPCR. Scale bar represents 50 μιη. RT-qPCR Experiments are presented as average of 5 different donors normalized on GAPDH expression (n=5, *** pO.001, error bars as s.d.).

Figure 3: HCMV infection inhibits monocytes proliferation and differentiation during osteoclastogenesis in a replication-dependent manner. Purified monocytes were incubated with recombinant M-CSF and RANKL (both at 50 ng/mL) and after 24 hours, infected or not (Ni) with UV-inactivated HCMV or not (VHL/E or VHL/E UV). (A) Cell proliferation was determined using CellTiter 96® Aq_ueous One Solution Cell Proliferation Assay at days 0, 2, 6, 8 and 10 after infection. (B) After 12 days of differentiation, osteoclasts were stained using Leukocyte Acid Phosphatase TRAP Kit and counted. Scale bar in white represents 50 μιη. Experiments are presented as an average of 5 different donors (n=5, *** p<0.001, error bars as s.d.).

Figure 4: HCMV inhibitis osteoclastogenesis through a downregulation of CSF-IR mediated by QKI5 upregulation. Purified monocytes were incubated with recombinant M- CSF and RANKL (both at 50 ng/mL) and after 24 hours, infected (VHL/E) or not (Ni) with HCMV. Cells were harvested at TO, 2, 4, 8, 16 and 24 hours after infection to assess CSF-1R, QKI5, RANK and UL122 mRNA expression by qPCR. Scale bar represents 50 μιη. RT-qPCR Experiments are presented as average of 5 different donors normalized on GAPDH expression (n=5, * p<0.05, ** p<0.01, *** pO.001, error bars as s.d.).

Figure 5: Over-expression of QKI5 decrease CSF-1R and RANK expression and inhibitis osteoclastogenesis. Purified monocytes were transfected by Amaxa or not (NT) with a plasmid inducing QKI5 overexpression (QKI5) or a control plasmid (empty) and then incubated with recombinant M-CSF and RANKL (both at 50 ng/mL). After 2 days of differentiation, cells were harvested and QKI5, CSF1-R and RANK mRNA and protein expression was analyzed by RT-qPCR (A). (B) After 12 days of differentiation, Osteoclasts were stained using Leukocyte Acid Phosphatase TRAP Kit and counted. Scale bar in white represents 50 μιη. Experiments are presented as an average of 5 different donors (n=5, *** pO.001, error bars as s.d.).

Figure 6: HCMV seropositivity protects against bone erosion during rheumatoid arthritis, study of ESPOIR cohort. Delta Erosion Sharp score evolution (0-1 year) is represented for 214 HCMV- and 273 HCMV+ RA patients from the ESPOIR cohort, (n = 487, * p<0.05, error bars as s.d.).

Figure 7: Control of arthritis by lentivirus expressing qki5. Lentivirus encoding for QKI5 was injected ( 2* 107 PFU, 10 μΐ) in the left knee joint of mice (n=7). Controls consisted of left knees injected with empty virus (2* 107 PFU, 10 μΐ, n=7) and non injected left knees (n=7). Mice were injected intraperitoneally with K/BxN serum to induce arthritis. Arthritic score was evaluated on the rear left paws. Table 1: Demographic and disease characteristics of patients with RA at inclusion.

Based on the report of the hands and feet radiographs, the documented disease stage was determined in all patients according to Sharp Score. RA, rheumatoid arthritis; ACPA+, anti- citrullinated peptide antibody presence; RF+, rheumatoid factor presence; CMV+, HCMV seropositivity; DAS28, disease Activity Score on 28 joints; n, number of patients; OR, odds ratio (n=487, * p<0.05, *** p<0.001).

Table 2: Demographic and disease characteristics of patients with RA at 1 year after inclusion. Based on the report of the hands and feet radiographs, the documented disease stage was determined in all patients according to Sharp Score. Sharp score was separated in two different observations: Erosion score and Joint space narrowing score. RA, rheumatoid arthritis; ACPA+, anti-citrullinated peptide antibody presence; n, number of patients; OR, odds ratio (n=487, * p<0.05).

EXAMPLE:

Material & Methods

Cells Viruses and Reagents

MRC5 fibroblasts, HCT116 and Hek-293T cells were propagated in DMEM medium (InVitrogen) containing 10% fetal calf serum and 5% penicillin/streptomycin. Monocytes were purified from HCMV seropositive and negative donors buffy coat, obtained at the EFS (Etablissement Francais du Sang, France), layered on Pancoll gradient (PanBiotech, Aidenbach, Germany), and purified by adherence 30 min in cell cultures plates and PBS washes to discard lymphocytes. Purified monocytes were cultures for osteoclast differentiation.

The VHL/E HCMV strain (gift from Christian Sinzger, Germany) was used in this study. Virus stocks were generated in MRC5 or HUVEC cells, collecting particles when cytopathic effects were >90%. Supematants were clarified of cell debris by centrifugation at l,500xg for 10 min, ultracentrifuged at 100,000xg for 30 min at 4°C, pellets were resuspended and virus stocks were stored at -80°C until use. Virus titers were determined by plaque assay on MRC5 cells using standard methods (MEM2X diluted two fold with solution of 1.6% agarose). UV irradiation of HCMV was performed with a Spectroline irradiator (EF-140/F) for 20 min. Monocytes were infected the day after purification and start of culture at an m.o.i. of 3.

Recombinant Human M-CSF was purchased from Biolegend (San Diego, CA, USA) and Recombinant Human RANKL from Peprotech (Rocky Hill, NJ, USA). Anti- Cytomegalovirus IE1 and IE2 (ab53495) and anti-Histone H3 (abl791) antibodies were purchased from Abeam (Cambridge, UK), anti-RANK monoclonal antibody (MA5-16153) from ThermoFisher Scientific (Waltham, MA, USA), anti-QKI5 polyclonal antibody (AB9904) from Millipore (Temecula, CA, USA), anti-CSF-lR antibody (sc-692) from Santa Cruz (Dallas, TX, USA) and anti-FOXPl monoclonal antibody (3210-S) from EPITOMICS (Burlingame, CA, USA).

Osteoclasts Differentiation

Purified monocytes were incubated with M-CSF and RANKL (both at 50 ng/ml) and cultured for 12 days at 37°C, 5% C02 in RPMI supplemented with 10% FCS (Invitrogen). Culture medium was replaced with fresh one every 4 days with M-CSF and RANKL (both at 50 ng/ml). Osteoclast formation was evaluated by Leukocyte Acid Phosphatase TRAP Kit (Sigma, Saint-Louis, MO, USA) staining and manual counting of the number of multinucleated TRAP+ cells.

Cells transfection

5 Millions Human monocytes freshly enriched by adherence from peripheral blood mononuclear cells were transfected with 2.5 μg of plasmids vectors using the Amaxa Human Monocyte Nucleofector®Kit (Lonza, Basel, Switzerland).

Lentiviral vectors

Lentiviral vectors pseudotyped with the vesicular stomatitis G protein were generated by transfection into 293T cells of a packaging construct, pCMVR8.74 (Addgene #22036), a plasmid producing the vesicular stomatitis G protein envelope (pMD2.G, Addgene #12259); and the vector itself. pRRL empty backbone (pRRL. empty, Addgene #12252) was complemented with PGK promoter controlling QKI gene (pRRL.QKI). pLKO vectors coding an shQKI5 were constructed by replacing shKAPl in previously described experiments. Culture medium was collected at 36 and 48 h, pooled, 0.2mm filtered, concentrated by ultracentrifugation, aliquoted and stored at -80°C, until used. Viral titers (infectious particles) were determined by transductions of HCT1 16 cells with serial dilutions of the vector preparation in a 12-well plate. 72 h later, cells were analyzed for eGFP expression by FACS and the infectious particles titer was determined (IP.ml-1).

Quantitative Real-Time PCR

RNA was extracted by using High Pure RNA Isolation Kit (Roche, and reverse transcribed with RevertAid Reverse Transcriptase (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. All qPCR were performed with SYBR green mix (Roche, Switzerland).

Cell Proliferation Assay

The number of viable cells in proliferation was determined using CellTiter 96®

Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Cells were cultured in 96 wells plate at a concentration a 104 cells per wells in presence of M-CSF and RANKL (both at 50ng/ml) and infected or not with HCMV (m.o.i. of 3). Cell proliferation was measured at day 3, day 6, day 8 and day 10, by adding 20 μΐ of reagent per wells and incubated 2 hours at 37°C. Absorbance was recorded at 490 nm using a 96-well plate reader.

Western Blot Analysis

Cells lysates were subjected to SDS/PAGE on 4-12% polyacrylamide gels (ThermoFisher scientific, Walham, MA, USA). After transferring on 0.22 μιη nitrocellulose membrane, proteins were revealed using specific antibodies and anti-rabbit or anti-mouse HRP- linked polyclonal antibodies (Cell Signaling, Danvers, MA, USA).

Flow Cytometry Analysis

Cells used for cell surface staining were washed with PBS-EDTA 5 mM, PBS-SVF 5% and stained with monoclonal anti-CSFIR Fluorescein-conjugated antibodies (R&D systems, Minneapolis, MN, USA). Flow cytometry analyses were performed witha FACSCalibur cytometer (BD Biosciences). Data were analyzed with Flow Jo software.

Microscopy Analysis

Cells were fixed and permeabilised with Methanol during 5 min at -20°C, and saturated with PBS 5% FCS during 2 hr at room temperature (RT). Primary staining with specific antibodies was performed in PBS 5% FCS at 4°C overnight or 3 hr at RT, before addition of secondary antibodies (Alexa-488 or -565) in PBS 5% FCS during 1 hr at RT. Dapi bath was performed during 10 min at RT, and slides were mounted with Fluoromount-G (Southern Biotech, Birmingham, AL).

Rheumatoid arthritis cohort (ESPOIR)

This work is derived from a large national, multicentre, longitudinal, prospective cohort of 813 French patients with early arthritis, the ESPOIR (Etude et Suivi des POlyarthrites Indifferenciees Recentes) cohort. The characteristics of the cohort have been described previously elsewhere. Briefly, 813 patients with early arthritis recruited in 14 centers in France with arthritis duration <6 months and no prior treatment with disease-modifying antirheumatic drugs were included between 2002 and 2005. Patients underwent clinical, biological and radiological assessments at baseline and at each subsequent visit. For the present study, we selected 487 individuals who fulfilled the 2010 American College of Rheumatology (ACR)/ EUropean League Against Rheumatisms (EULAR) criteria for RA. Local institutional review boards approved the study, and written informed consent was obtained from all participants in the study. One biological resources centre (Joelle Benessiano, Paris-Bichat) was in charge of centralising and managing biological data collection. C. Lukas for expert x-ray reading, S. Martin (Paris Bichat) who did all the central dosages of CRP, IgA and IgM rheumatoid factor and anti-CCP antibodies HCMV Serology for these patients was determined using Architect CMV IgG assay (Abbott, Chicago, IL, USA). 214 patients were detected seronegative and 273 seropositive for HCMV.

Statistics

All experiments were performed more than three times. The values are expressed as the mean ± standard deviation (SD). The t test was used to assess the significance of differences between two conditions. All p values are two-sided, and p values equal to or below 0.05 were considered significant.

Results

CSF-IR expression is down-regulated during HCMV infection. It has already been shown that infection of macrophages with HCMV clinical strain TB40/E down regulates cell surface expression of CSF-IR (CD115, MCSF-R) (1). Consequently, we asked if HCMV infection could also interfere with CSF-IR cell-surface expression during osteoclastogenesis by infecting monocytes in osteoclast differentiation (stimulated with recombinant M-CSF and RANKL, both at 50 ng/mL) with an HCMV clinical strain (VHL/E) at an m.o.i. of 3. Figure 1 shows that HCMV infection lead to a rapid inhibition of CSF-IR mRNA transcription (Fig.1) and protein expression (data not shown) implicating a strong decrease of cell surface expression (data not shown). This inhibition was also observed by fluorescence microscopy in VHL/E infected cells in comparison with non- infected ones (data not shown). Interestingly, viral gene expression was required for this inhibition as UV-inactivated virus showed no effect on CSF- IR expression. These results demonstrate that HCMV infection during osteoclastogenesis inhibits CSF-IR expression. As CSF-IR stimulation induces RANK expression and osteoclast differentiation, we further analyzed the impact of infection on these particular mechanisms.

Inhibition of CSF-IR is accompanied with RANK down regulation during HCMV infection. As M-CSF induces RANK up-regulation during osteoclastogenesis (2), we investigated the effect of HCMV infection on RANK expression during this phenomenon. We observed that, as expected, after 24h of VHL/E infection, RANK mRNA expression was dramatically decreased in a viral replicative dependent manner (Fig.2). We confirmed this observation with protein expression analysis by Western Blot. We could clearly observed that in infected cells, expressing IE1 and IE2 proteins, the noticed decrease of CSF-IR protein expression is followed by an inhibition of RANK expression (data not shown) (cell surface expression by FACS is ongoing). These results demonstrate that HCMV infection, in addition of CSF-IR inhibition during osteoclastogenesis, inhibits RANK expression.

HCMV infection inhibits cell proliferation and differentiation during osteoclastogenesis. Since M-CSF and RANKL signalization are essential to osteoclastogenesis, we then examined the effect of HCMV infection on osteoclast differentiation. As expected, HCMV infection impaired cell proliferation and in a viral gene expression-dependent mechanism (Fig.3 A). After 12 days of differentiation, osteoclasts were labelled using a TRAP assay and counted. We clearly observed that HCMV infection completely blocked OCs differentiation in a replicative dependent manner (Fig. 3B) whereas in non-infected control wells, large multinucleated osteoclastic cells had clearly developed (data not shown). New experiments using with TRAP staining are ongoing. These results demonstrated for the first time that HCMV infection of monocytes interfere with osteoclast differentiation.

HCMV-mediated osteoclastogenesis inhibition is mediated by QKI5 upregulation during infection. We then sought to identify the protein responsible for CSF-1R down- regulation. We first investigated the principal known regulators of CSF-1R such as foxpl and RUNX but no significant modulation by HCMV was observed (data not shown). It has been recently demonstrated that the RNA-Binding protein QKI5 could facilitate the degradation of CSF-1R mRNA3. We thus investigated this possibility and followed the effect of HCMV infection during 24 hours on CSF-1R, RANK and QKI5 expression during osteoclastogenesis. As expected, 2 hours after infection UL122 viral gene was expressed showing a replicative infection in our model. We could observe an inhibition of CSF-1R mRNA expression after 16 hours just before RANK inhibition. Remarkably, QKI5 mRNA was increased as of 8 hours of infection meaning that QKI5 upregulation occured before CSF-1R inhibition and could be responsible of this mechanism (Fig.4). Protein expression analysis by western blots confirmed these observations and showed an upregulation of QKI5 in infected cells (i.e. expressing IE1) followed by an inhibition of CSF-1R expression andRANK (data not shown). This upregulation of QKI5 protein expression was confirmed by fluorescence microscopy (data not shown). Multiple stainings with anti-QKI5 and anti-IE proteins are ongoing. These results, taken together, demonstrate the possibility that HCMV infection increases the expression of the regulatory protein QKI5 leading to CSF-1R mRNA degradation and RANK inhibition to finally block osteoclastogenesis.

Does Knock down of QKI5 restore osteoclastogenesis in HCMV infected monocytes? To confirm the hypothesis that QKI5 is the key mediator of osteoclastogenesis inhibition during HCMV infection, knock down experiments are in progress, using lentiviral vectors to transduce monocytes and silence QKI5 in cells. After 24 hours of HCMV infection, we will analyze CSF-1R, RANK, QKI5 and UL122 mRNA and protein expression by RT- qPCR and Western blot. A TRAP staining will be performed to test for the restoration of OCs differentiation when QKI5 is knocked down, (data not shown).

Over-expression of QKI5 decreases CSF-1R and RANK expression and inhibits osteoclastogenesis. We further investigated the ability of QKI5 to inhibit osteoclastogenesis by inducing its expression in monocytes during OCs differentiation. Freshly purified monocytes were transfected or not (NT) by Amaxa® with a control (empty) or a QKI5 coding plasmid under the control of a CMV promoter (QKI5) and incubated during osteoclast differentiation with recombinant M-CSF and RANKL. After 48 hours, cells were harvested and mRNA expression analyzed. We could observe that QKI5 was upregulated in transfected cells in comparison with non-transduced ones (NT) or empty control (Fig.5A). This QKI5 over- expression lead to a strong decrease of CSF-1R and RANK mRNA expression (Fig.5A). When we compared expression between empty and QKI5, we could clearly observe this strong inhibition of CSF-1R and RANK in QKI5 over-expressing cells. Next experiments will be repeated and performed with NT corresponding to Amaxa® treated cells without plasmid DNA. Complementary experiments are in progress to study protein expression (data not shown). As expected, we could clearly observe that over-expression of QKI5 inhibits completely OCs differentiation (Fig.5B). These experiments, already performed twice, show the potential of QKI5 over-expression to inhibit osteoclast differentiation and use it as a target to control this phenomenon in diseases implicating OCs.

HCMV seropositivity has a protective effect on articular bone erosion in

Rheumatoid Arthritis (RA). Sera from 487 patients with RA were analyzed for the presence of anti-CMV IgG antibodies. 273 patients were tested as HCMV seropositive (56.1 %) whereas 214 were HCMV seronegative (43.9%) (Table 1). No significant differences were detected at the inclusion except an older population for anti-HCMV positive patients in comparison with seronegative ones (median age of 52.9 years and 47.8 years respectively; p<0.0001), a smaller proportion of ACPA+ in HCMV positive patients (49.8% for 58.9% in HCMV negative patients, p<0.0465) and a DAS28 higher with HCMV seropositivity (mean of 5.55 +/- 1.24 for 5.20 +/- 1.14, p<0.0013). In 2012, another laboratory published a study demonstrating the association of HCMV seropositivity with most severe joint destruction and surgery in rheumatoid arthritis 4. The Steinbrocker test used not specific for bone erosion and was thus not adapted for this kind of study and the article was rapidly refuted 5. To refine our study, we chose to use Sharp's Score that can distinguish between bone erosion and Joint Space Narrowing. Although there was no difference at inclusion, HCMV seropositive patients displayed less severe bone erosion evolution in comparison with HCMV negative ones after one year of disease (Table 2). Indeed, 17.2% of HCMVpos patients had a delta sharp total score > 1 compared to 26.3 % of HCMVneg patients (p<0.0151). When bone erosion sharp score was specifically assessed, it was clearly observed that only bone erosion was negatively impacted by HCMV seropositivity (only 16.1% of HCMV+ patients have an score evolution superior to 1 in comparison with 25.2% of HCMV- patients, 0.0128) whereas joint space narrowing was not significantly different. When we looked at the evolution of bone erosion between HCMV+ and HCMV- patients, it confirmed that bone erosion was limited to the HCMV+ ones (Fig.6). These study of ESPOIR cohort demonstrates a protective effect of HCMV infection from bone erosion during inflammatory diseases as RA.

Is HCMV reactivated during osteoclastogenesis? To understand the link between HCMV seropositivity and protection to bone erosion during RA, many hypotheses are available. HCMV could reactivate from latently infected monocytes or hematopoietic stem cells when they differentiate to macrophages 6 and this phenomenon could occur during articular inflammation, inducing infection of osteoclastic progenitors and inhibiting their differentiation. The other possibility is that differentiation of monocytes into osteoclast could also reactivate HCMV from latency and directly induce a viral release during erosion and better target this articular erosion. To answer to this question, we are now performing differentiation experiments, taking freshly purified monocytes from seropositive donors and differentiating them to macrophage in presence or not of recombinant LPS as a positive control for HCMV reactivation. In parallel we will differentiate monocytes into osteoclast and check if reactivation occurs assessed by viral DNA expression detection and mRNA expression, followed by new viral particles production.

In vivo data (figure 7):

Arthritis score was found to be lower in the left paws of mice injected in the left knees with lentivirus expressing QKI5. This suggests that the expression of QKI5 in situ is able to inhibit inflammation due to K/BxN serum-induced arthritis.

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Claims

CLAIMS:

1. A QKI5 polypeptide and/or an agent for QKI5 polypeptide expression for use in the treatment of osteoclast disease in a subject in need thereof.

2. The QKI5 polypeptide and/or the agent for QKI5 polypeptide expression for use according to claim 1 wherein the subject is afflicted with or susceptible to be afflicted with osteoclast disease selected from bone erosion diseases, osteoporosis, arthritis, rheumatoid arthritis, chronic inflammatory arthritis, rheumatism, chronic inflammatory rheumatism, bone cancer, osteosarcoma, bone cancer metastasis, Paget's disease of bone (PDB), Juvenile Paget's disease, periodontal disease, Familial Expanile Osteolysis (FEO), Expansile Skeletal Hyperphosphatasia (ESH) and Camurati Engelmann Disease (CED).

3. The QKI5 polypeptide and/or the agent for QKI5 polypeptide expression for use according to claim 1 wherein the QKI5 polypeptide comprises a sequence as set forth by SEQ ID NO: 1.

4. The QKI5 polypeptide and/or the agent for QKI5 polypeptide expression for use according to claim 1 wherein the agent for QKI5 polypeptide expression is selected from the group consisting of an isolated, synthetic or recombinant nucleic acid encoding for QKI5 polypeptide, a nucleic acid encoding a fragment of a QKI5 polypeptide, a cell expressing QKI5 polypeptide, and agent inducing QKI5 gene and polypeptide expression and their combinations.

5. The QKI5 polypeptide and/or the agent for QKI5 polypeptide expression for use according to claim 4 wherein the said nucleic acid encoding for QKI5 polypeptide comprises a sequence as set forth by SEQ ID NO: 2.

6. The QKI5 polypeptide and/or the agent for QKI5 polypeptide expression for use according to claim 4 wherein the agent inducing QKI5 gene and polypeptide expression is selected from the group consisting of Human Cytomegalovirus (HCMV), VHL/E HCMV strain and TB40/E HCMV strain.

7. The QKI5 polypeptide and/or the agent for QKI5 polypeptide expression for use according to any of claims 1 to 6 in combination with one or more anti-osteoclast compound.

8. A method for treating osteoclast disease in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of a QKI5 polypeptide and/or an agent for QKI5 polypeptide expression.