WO2006024096A1 - Procédé servant à moduler l'ostéoclastogénèse - Google Patents

Procédé servant à moduler l'ostéoclastogénèse Download PDF

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WO2006024096A1
WO2006024096A1 PCT/AU2005/001322 AU2005001322W WO2006024096A1 WO 2006024096 A1 WO2006024096 A1 WO 2006024096A1 AU 2005001322 W AU2005001322 W AU 2005001322W WO 2006024096 A1 WO2006024096 A1 WO 2006024096A1
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trx
tbp
activity
ratio
osteoclast
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Gregory Royce Collier
Geoffrey Charles Nicholson
Nigel Alexander Morrison
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Chemgenex Pharmaceuticals Limited
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to a method for modulating the process of osteoclastogenesis in an animal subject. More particularly, the present invention contemplates a method of modulating the process of osteoclastogenesis by manipulating the ratio of Thioredoxin-1 (Trx-1) to Thioredoxin Binding Protein-2 (TBP-2) in osteoclast progenitor cells.
  • the present invention further identifies a range of agents, including expression constructs, gene silencing constructs and antioxidants, which may be used to effect changes in the Trx-l:TBP-2 ratio in the osteoclast progenitor cells and thereby modulate the process of osteoclastogenesis.
  • the present invention further extends to methods of treating and diagnosing diseases associated with excessive or unregulated osteoclastogenesis in a subject.
  • ROS cytotoxic reactive oxygen species
  • organisms have developed redox systems that are also critically important in the regulation of transcription factors (Droge, Physiol Rev 82(l):47-95, 2002, Holmgren, J Biol Chem 264(24):13963-13969, 1989 and Holmgren, Antioxid Redox Signal 2(4):U ⁇ - 820, 2000).
  • Redox signaling regulates many cellular functions including proliferation, activation, growth inhibition and apoptosis (Haddad, Cell Signal 14(ll):%79-%97, 2002).
  • Oxidative stress Alteration of the redox status of cells by exposure to oxidants (“oxidative stress”) has been shown to play important roles in ageing and almost all common diseases including malignancy, diabetes mellitus, neurodegenerative diseases, atherosclerosis, ischemia, autoimmunity, and HIV infection (Droge 2002 supra).
  • the role of free radicals in these conditions has been intensely researched, particularly in the last decade, but relatively little attention has been paid to their involvement in the pathogenesis of osteoporosis (Droge 2002 supra), arguably the most common degenerative disease of all, or in osteoclast biology (Boyle et al, Nature 423 (6937):337 -342, 2003).
  • Osteoclastic bone resorption (Bax et al, Biochem Biophys Res Commun 753(3 ⁇ :1143-1158, 1992, Garrett et al, J Clin Invest 85(3):632-639, 1990 and Fraser et al, Bone I9(3):223-226, 1996) are stimulated by ROS whereas free radical scavengers are inhibitory (Hall et al, Biochem Biophys Res Commun 207(%) :280-287, 1995 and Rao et al, J Med Food 6(2):69-78, 2003).
  • 17- ⁇ estradiol stimulated glutathione reductase and Trx reductase levels, and osteoclast generation and NF- ⁇ B activity were increased by L- buthionine-(S,R)-sulphoximine or hydrogen peroxide, but inhibited by NAC.
  • 17- ⁇ estradiol did not modulate glutathione or glutathione reductase activity in three different cultured osteoblastic cell types. The investigators concluded that excess bone resorption associated with estrogen deficiency might be attributed to reduced antioxidant defences in osteoclasts and/or other cells in the bone marrow.
  • hM-CSF human macrophage-colony stimulating factor
  • sRANKL soluble RANKL
  • Trx-1 activity is required for osteoclast differentiation.
  • TBP-2 thioredoxin binding protein-2
  • Trx-1 a key endogenous modulator of cellular redox and signaling.
  • TBP-2 expression is reduced during human osteoclast differentiation compared to macrophage differentiation. It is further determined that in the presence of sRANKL, expression of TBP-2 is decreased whereas Trx-1 expression is increased.
  • the antioxidant ⁇ -acetylcysteine reversed this pattern and markedly inhibited osteoclastogenesis.
  • Adenoviral over-expression of human TBP-2 in precursors also inhibited osteoclastogenesis and Trx-1 expression, inhibited sRANKL-induced DNA- binding of AP-I, but enhanced sRANKL-induced DNA binding of NF- ⁇ B.
  • the data generated in accordance with the present invention identify a role for TBP-2 and the Trx system in osteoclast differentiation mediated by redox regulation of AP-I transcription.
  • Modulators of the Trx system such as antioxidants have been identified as having potential as anti-resorptive therapies.
  • one aspect of the present invention contemplates a method of modulating osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an agent which modulates the Trx-1 :TBP-2 ratio in one or more osteoclast progenitor cells of said subject.
  • the present invention provides a method of modulating osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an agent which alters the Trx-l:TBP-2 ratio in one or more osteoclast progenitor cells of said subject wherein lowering or elevating the Trx-1 :TBP-2 ratio inhibits and promotes osteoclastogenesis, osteoclast survival and/or osteoclast activity, respectively.
  • the present invention provides, therefore, agents including genetic agents which lower the Trx-1 :TBP-2 ratio by elevating levels or activity of TBP-2 or reducing levels or activity of Trx-1 and agents which elevate the Trx-l:TBP-2 ratio by increasing levels or activity of Trx-1 or reduce levels of TBP-2.
  • the agent for up-regulating protein activity or protein levels is an expression construct comprising a nucleic acid sequence encoding one or more peptides, polypeptides or proteins operably linked to a promoter.
  • the agent is a TBP-2 expression construct.
  • the present invention provides a method of inhibiting osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject a TBP-2 expression construct which decreases the Trx- l:TBP-2 ratio in one or more cells of said subject, wherein said reduction in the Trx- l:TBP-2 ratio inhibits one or more of osteoclastogenesis, osteoclast survival and/or osteoclast activity in said subject.
  • the present invention includes, inter alia, a genetic approach to down-regulating expression of Trx-1 and/or TBP-2.
  • a genetic approach uses nucleic acid molecules or molecules having a genetic component to induce pre- or post-transcriptional gene silencing.
  • the present invention further extends to a method of modulating osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an agent which indirectly modulates the Trx-1 :TBP-2 ratio in one or more osteoclast progenitor cells of said subject.
  • the present invention provides a method of inhibiting osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an agent which inhibits RANKL signalling and thereby indirectly reduces the Trx-1 :TBP-2 ratio in one or more osteoclast progenitor cells of said subject, wherein said reduction in Trx-1 :TBP-2 ratio inhibits osteoclastogenesis in said subject.
  • the present invention provides a method of inhibiting osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an antioxidant, wherein said antioxidant directly or indirectly reduces the Trx-l:TBP-2 ratio in one or more osteoclast progenitor cells of said subject, wherein said reduction in Trx-l:TBP-2 ratio inhibits osteoclastogenesis in said subject.
  • the present invention further provides methods for the treatment and/or diagnosis of conditions associated with excessive or unregulated osteoclastic activity.
  • the present invention provides a method of treating or preventing a condition associated with excessive or unregulated osteoclastic activity in a subject, said method comprising administering to said subject an agent which effects an decrease in the Trx-1 :TBP-2 ratio in one or more osteoclast progenitor cells in said subject, wherein said decrease in the Trx-1 :TBP-2 ratio inhibits osteoclastic activity.
  • the "subject” is a human.
  • Figure 1 is a graphical representation showing decreased TBP-2 protein expression and increased Trx-1 protein expression during osteoclast, compared to macrophage, differentiation. Protein expression was assessed by western blot in 4d and 7d colony forming unit-granulocyte-macrophage (CFU-GM) cultures treated with hM-CSF (25 ng/niL) alone or together with sRANKL (62.5 ng/mL). At 4d, TBP-2 and Trx-1 expression was similar in both treatment groups. However, at 7d, TBP-2 expression was lower and Trx-1 expression higher in cells treated with sRANKL. Treatments are shown in boxes.
  • CFU-GM colony forming unit-granulocyte-macrophage
  • Figure 2 is a graphical representation illustrating that antioxidants block RANKL-induced modulation of TBP-2/Trx-l protein expression and inhibit osteoclast differentiation,
  • the antioxidants NAC, selenium and ebselen potently inhibit osteoclast differentiation.
  • Figure 3 is a graphical representation showing that adenoviral infection of CFU-GM osteoclast precursors with TBP-2 construct (AdTBP -2) inhibits osteoclast differentiation, up-regulates TBP-2 and down-regulates Trx-1 protein expression, (a) Infection of CFU-GM osteoclast precursors with TBP-2 construct (AdTBP -2)
  • GM cultures incubated in the presence of hM-CSF (25 ng/ml) and sRANKL (62.5 ng/ml) for 14d with increasing MOI of AdTBP-2 dose-responsively inhibited osteoclast formation
  • Figure 4 is a graphical representation demonstrating that Adenoviral over-expression of TBP-2 inhibits sRANKL-induced AP-I DNA binding, but enhances NF- ⁇ B DNA binding.
  • FIG. 5 is a graphical representation showing that retroviral infection of CFU-GM osteoclast precursors with the antiTBP-2 shRNA construct promotes osteoclast differentiation and resorption.
  • CFU-GM cultures were incubated in the presence of hM- CSF (25 ng/ml), sRANKL (125 ng/ml) and GM-CSF (10 ng/ml) for 24 hours in the presence of 1 moi of shRNA retrovirus and then for 13d with M-CSF and RANKL alone.
  • an agent includes a single agent, as well as two or more agents and so forth.
  • the present invention is predicated in part on the finding that the process of osteoclastogenesis in animal cells is modulated by the ratio of Trx-1 and TBP -2 in osteoclast progenitor cells in a subject.
  • TBP-2 negatively regulates Trx-1, a key endogenous modulator of cellular redox and signaling.
  • TBP-2 is a 46 kDa protein that is expressed ubiquitously but its biological functions are not well known. It was originally cloned as vitamin D-up-regulated protein 1 (VDUPl) in HL-60 promyelocyte leukemia cells treated with 1,25-dihydroxy- vitamin D (Chen and DeLuca, Biochim Biophys Acta 1219(l):26-32, 1994).
  • TBP-2 acts as an endogenous negative regulator of Trx-1: It inhibits Trx-1 reducing activity by binding to it at the redox-active site; interferes with JNK and arabidopsis Skpl-like gene 1 (ASKl) signaling; inhibits Trx-1 nuclear translocation; and inhibits Trx-1 protein expression.
  • TBP-2 TBP-2
  • Trx-1 Trx-1 expression is increased (Lincoln et al, Anticancer Res 23 (35j:2425-2433, 2003).
  • aspect of the present invention contemplates a method of modulating osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an agent which directly or indirectly modulates the Trx-1 :TBP-2 ratio in one or more osteoclast progenitor cells of said subject.
  • the present invention provides a method of modulating osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an agent which alters the Trx-1 :TBP-2 ratio in one or more osteoclast progenitor cells of said subject wherein lowering or elevating the Trx-1 :TBP-2 ratio inhibits and promotes osteoclastogenesis, osteoclast survival and/or osteoclast activity, respectively.
  • the term "subject" refers to any animal comprising a internal skeleton. Accordingly, animals contemplated herein include, inter alia, mammals, marsupials, monotremes, reptiles, fish and avians.
  • the subject is a mammalian animal such as livestock animals (e.g. sheep, cows, pigs, horses, donkeys and goats), companion animals (e.g. cats, dogs, rabbits), laboratory animals (e.g. mice, rats, rabbits, guinea pigs, hamsters), human and non-human primates, avian species (e.g. poultry birds, aviary birds), reptiles and amphibians.
  • livestock animals e.g. sheep, cows, pigs, horses, donkeys and goats
  • companion animals e.g. cats, dogs, rabbits
  • laboratory animals e.g. mice, rats, rabbits, guinea pigs, hamsters
  • human and non-human primates avian species (e.
  • osteoclastogenesis should be understood to encompass all aspects of the proliferation, surivial and activity of osteoclasts in a subject.
  • modulation of osteoclastogenesis, osteoclast survival and/or osteoclast activity should be understood to extend to modulation of osteoclast activity in a subject including osteoclastic bone resorption.
  • the present invention should be understood to extend to, inter alia, a method of modulating osteoclastic bone resorption.
  • the agents for use in accordance with the present methods include any agents which can modulate the Trx-1 :TBP-2 ratio in one or more osteoclast progenitor cells in a subject.
  • modulation of the Trx-1 :TBP-2 ratio should be understood to encompass changes in the amounts of the proteins and/or changes in the relative activity of the proteins which may or may not be associated with a change in the amounts thereof.
  • the ratio may be altered by increasing or decreasing Trx-1 and/or increasing or decreasing TBP-2.
  • the increasing or decreasing relates to protein levels and/or protein activity. Modulation may be at the level of the proteins or via expression of the genes encoding one or both of Trx-1 or TBP-2.
  • the agent is an agent which directly modulates the Trx-1 :TBP-2 ratio.
  • agents contemplated herein include those which modulate the Trx-l :TBP-2 ratio by up-regulation of the activity of a protein, such as Trx-1 or TBP-2.
  • agents include, but are not limited to:
  • agents which increase transcription of an endogenous gene encoding the subject protein (i) agents which increase transcription of an endogenous gene encoding the subject protein
  • Trx-1 adenoviral over- expression of TBP-2 is associated with decreased Trx-1 levels and impaired osteoclastogenesis. While it is known that TBP-2 down-regulates Trx-1 (Nishiyama et al, J Biol Chem 274(37j:21645-21650, 1999 and Junn et al, J Immunol 164(12) -.6287-6295, 2000), there is no evidence that Trx-1 regulates TBP-2. Moreover, although Trx-1 has multiple effects on intracellular signaling, TBP-2 is not known to directly influence signaling. Taken together, these results indicate that maintenance of Trx-1 expression is required for osteoclastogenesis. Without wishing to limit the present invention to any one theory or mode of action, it is proposed that maintenance of Trx-1 expression and osteoclastogenesis is likely to be via facilitation of RANKL signaling events, such as AP-I activation.
  • Trx-1 has been shown to have opposing effects on NF -KB activation depending on whether it is over-expressed in the cytosol or nucleus (Hirota et al, J Biol Chem 274(39):27891-27897, 1999). In the cytosol, Trx-1 blocks the dissociation of I- ⁇ B from NF -KB, inhibiting nuclear translocation and DNA binding, whereas in the nucleus, Trx-1 facilitates DNA binding of NF- ⁇ B.
  • the agent for up-regulating protein activity is an expression construct comprising a nucleic acid sequence encoding one or more peptides, polypeptides or proteins operably linked to a promoter.
  • the agent is a TBP -2 expression construct.
  • TBP -2 expression construct should be understood to refer to a nucleic acid construct comprising a nucleotide sequence encoding TBP-2 or a homolog or derivative thereof in addition to any other nucleic acid sequences which effect expression of the nucleotide sequence encoding TBP-2 or a homolog or derivative thereof in a host cell.
  • the "TBP-2 expression construct” comprises a nucleotide sequence encoding TBP-2 or a homolog or derivative thereof operably linked to a promoter.
  • promoter includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • a promoter is usually, but not necessarily, positioned upstream or 5' of a structural gene region, the expression of which it regulates.
  • the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
  • promoter is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell.
  • Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the sense molecule and/or to alter the spatial expression and/or temporal expression of said sense molecule.
  • regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense molecule, thereby conferring copper inducibility on the expression of said molecules. Placing a nucleic acid molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence.
  • heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e. the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e. the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
  • the promoter may regulate the expression constitutively, or differentially with respect to the cell, tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or pathogens, or metal ions, amongst others.
  • the vectors contemplated by the present invention include vectors operable in any prokaryotic or eukaryotic cell type that would be suitable for the expression of a nucleic acid molecule in a host cell.
  • Constitutive or inducuble promoters are particularly useful for the purposes of the present invention, as the present invention specifically contemplates the use of the vector described herein as an expression vector.
  • the terms "in operable connection with” or “operably under the control” or similar shall be taken to indicate that expression of the nucleic acid molecule is under the control of the promoter sequence with which it is spatially connected in a cell, tissue, organ or whole plant.
  • the construct preferably contains additional regulatory elements for efficient transcription, for example, a transcription termination sequence.
  • Terminator refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3 '-non-translated DNA sequences generally containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3 '-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants or synthesized de novo.
  • the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used.
  • Exemplary terminators which are compatible with the present invention include the SV40 polyadenylation signal or the HSV TK polyadenylation signal which are operable in animal cells, tissues and organs.
  • constructs described herein are capable of being modified further, for example, by the inclusion of marker nucleotide sequences encoding a detectable marker enzyme or a functional analogue or derivative thereof, to facilitate detection of the synthetic gene in a cell, tissue or organ in which it is expressed.
  • the marker nucleotide sequences will be present in a translatable format and expressed.
  • a nucleic acid is inserted into a suitable vector or episome molecule, such as a bacteriophage vector, viral vector or a plasmid, cosmid or artificial chromosome vector which is capable of being maintained and/or replicated and/or expressed in the host cell, tissue or organ into which it is subsequently introduced.
  • a suitable vector or episome molecule such as a bacteriophage vector, viral vector or a plasmid, cosmid or artificial chromosome vector which is capable of being maintained and/or replicated and/or expressed in the host cell, tissue or organ into which it is subsequently introduced.
  • an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the genetic construct which are intended to be expressed or transferred to an animal cell, or integrated into the genome of an animal cell.
  • selectable marker gene includes any gene which confers a phenotype on a cell on which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.
  • Suitable selectable marker genes contemplated herein include the ampicillin-resistance gene (Amp r ), tetracycline-resistance gene (Tc 1 ), bacterial kanamycin-resistance gene
  • Kan 1 is the zeocin resistance gene (Zeocin is a drug of the bleomycin family which is trade mark of InVitrogen Corporation), the A URI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gen (npt ⁇ ), hygromycin-resistance gene, ⁇ -glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein-encoding gene or the luciferase gene, amongst others.
  • Zeocin is a drug of the bleomycin family which is trade mark of InVitrogen Corporation
  • a URI-C gene which confers resistance to the antibiotic aureobasidin A
  • phosphinothricin-resistance gene neomycin phosphotransferase gen (npt ⁇ )
  • the selectable marker gene is the nptll gene or Kan r gene or green fluorescent protein (GFP)-encoding gene.
  • GFP green fluorescent protein
  • Standard methods may be used to introduce the constructs into the cell, tissue or organ, for example, liposome-mediated transfection or transformation, transformation of cells with attenuated virus particles or bacterial cells, cell mating, transformation or transfection procedures known to those skilled in the art.
  • the present invention provides a method of inhibiting osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject a TBP-2 expression construct which decreases the Trx-l:TBP-2 ratio in one or more cells of said subject, wherein said reduction in the Trx- l:TBP-2 ratio inhibits one or more of osteoclastogenesis, osteoclast survival and/or osteoclast activity in said subject.
  • agents contemplated herein include those which modulate the Trx-l:TBP-2 ratio by downregulation of the amount or activity of a protein, such as Trx-1 or TBP-2 or both.
  • agents contemplated herein include, but are not limited to:
  • agents which directly or indirectly inhibit transcription of the endogenous target gene encoding the protein into the corresponding mRNA agents which directly or indirectly inhibit transcription of the endogenous target gene encoding the protein into the corresponding mRNA
  • agents which effect post-transcriptional inhibition including RNAi agents and antisense nucleic acids;
  • the present invention contemplates a genetic approach to down- regulating expression of Trx-1 and/or TBP-2.
  • Such an approach uses nucleic acid molecules or molecules having a genetic component to induce pre- or post-transcriptional gene silencing.
  • Anti-sense, sense, RNAi and ribozyme methods of gene regulation are all contemplated by the present invention.
  • nucleic acids include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g.
  • phosphorothioates phosphorodithioates, etc.
  • pendent moieties e.g. polypeptides
  • intercalators e.g. acridine, psoralen, etc.
  • chelators e.g. acridine, psoralen, etc.
  • alkylators e.g. ⁇ -anomeric nucleic acids, etc.
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • Antisense polynucleotide sequences are useful in silencing transcripts encoding Trx-1 and/or TBP-2.
  • polynucleotide vectors containing all or a portion of a gene locus may be placed under the control of a promoter in either the sense or antisense orientation and introduced into a cell. Expression of such a sense or antisense construct within a cell interferes with target transcription and/or translation.
  • co-suppression i.e. using sense-suppresion
  • mechanisms to induce RNAi or siRNA may also be employed.
  • antisense or sense molecules may be directly administered.
  • the antisense or sense molecules may be formulated in a composition and then administered by any number of means to target cells.
  • the present invention employs compounds such as oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding Trx-1 and/or TBP-2, ie. the oligonucleotides induce transcriptional or post- transcriptional gene silencing. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding the inhibitor. The oligonucleotides may be provided directly to a cell or generated within the cell.
  • target nucleic acid encompasses DNA encoding the Trx-1 or TBP-2, RNA (including pre-mRNA and/or mRNA or portions thereof) transcribed from such DNA, and cDNA derived from such RNA.
  • the hybridization of a compound of the subject invention with its target nucleic acid is generally referred to as "antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.”
  • antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
  • the functions of DNA to be interfered with can include replication and transcription.
  • Replication and transcription for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise.
  • the functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA.
  • hybridization means the pairing of complementary strands of oligomeric compounds.
  • the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
  • nucleobases complementary nucleoside or nucleotide bases
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Hybridization can occur under varying circumstances.
  • An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired.
  • oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.
  • compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (e.gS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid.
  • these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops.
  • the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.
  • RNAse H a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
  • antisense compound is a single-stranded antisense oligonucleotide
  • dsRNA double-stranded RNA
  • siRNA short-interfering RNA molecule
  • siRNA's are double stranded, partially double-stranded or hairpin-forming RNA molecules which typically comprise a double stranded region of between 10-30 nucleotides in length.
  • siRNAs comprising double stranded regions of between 18 and 25 nucleotides in length have shown strong activity, with 22 nucleotide double stranded regions having particularly strong gene silencing activity.
  • RNAi RNA-mediated gene-silencing phenomenon
  • dsRNA Double- stranded RNA
  • PTGS post-transcriptional gene silencing
  • RNAi The central process of RNAi is the cleavage of dsRNA into smaller pieces of a defined length by the enzyme Dicer.
  • Dicer cleaves dsRNA into two smaller RNA molecules, referred to herein as "siRNAs” — that are around 21-23 nucleotides in length.
  • Dicer delivers these Guide RNAs to a group of proteins called the RNA-induced silencing complex (RISC), which uses the antisense strand of the Guide RNA to bind to and degrade the corresponding mRNA, resulting in gene silencing.
  • RISC is an enzyme and catalyses multiple rounds of RNAi, perhaps hundreds or thousands in vivo, which makes RNAi particularly efficient.
  • RNAi also exhibits substantial persistence in some organisms, such as worms, which suggests there may also be an amplification response.
  • RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense or sense compounds which are "DNA-like" elicit RNAse H . Activation of RNase H 5 therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
  • siRNAs have an integral role in the phenomenon of RNA interference (RNAi).
  • RNAi RNA interference
  • dsRNAs introduced into certain organisms or cells are degraded into approximately 22 nucleotide fragments. These 22 nucleotide siRNA molecules then bind to the complementary portion of their target mRNA and tag it for degradation.
  • the "antisense compounds" contemplated by the present invention may include, inter alia, siRNAs and precursors thereof including hairpin RNAs, double stranded RNAs and the like.
  • oligomeric compound refers to a polymer or oligomer comprising a plurality of monomeric units.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly.
  • modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
  • the nucleic acid agents described supra may be targeted to the open reading frame (ORF) or "coding region" of the target gene which is known in the art to refer to the region between the translation initiation codon and the translation termination codon.
  • ORF open reading frame
  • coding region of the target gene which is known in the art to refer to the region between the translation initiation codon and the translation termination codon.
  • one region is the intragenic region encompassing the translation initiation or termination codon of the ORF of a gene.
  • target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene).
  • 5'UTR 5' untranslated region
  • 3'UTR 3' untranslated region
  • the 5' cap site of an mRNA comprises an N7 -methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage.
  • the 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5' cap region.
  • nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred.
  • linear compounds may have internal nucleobase complementarity and may, therefore, fold in a manner as to produce a fully or partially double-stranded compound.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2
  • Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts, mixed salts and free acid forms are also included.
  • the present invention contemplates a method of inhibiting osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject a nucleic acid agent which reduces the transcription or translation of Trx-1 in one or more osteoclast progenitor cells in said subject.
  • the "nucleic acid agent” may be an antisense molecule, an RNAi inducing molecule (including siRNA molecules) or a knockout construct.
  • the present invention further extends to a method of modulating osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an agent which indirectly modulates the Trx-1 :TBP-2 ratio in one or more osteoclast progenitor cells of said subject.
  • hM-CSF human macrophage-colony stimulating factor
  • sRANKL soluble RANKL
  • Osteoclast differentiation, survival and activity are dependent on the presence of both M-CSF and RANKL, which signals through multiple pathways.
  • NF- ⁇ B and AP-I Signaling through NF- ⁇ B and AP-I are both essential, as mutation of the p50/p52 component of NF- ⁇ B, or the c-Fos component of AP-I, result in failure of osteoclastogenesis and osteopetrosis (Boyle et al, 2003 supra).
  • Trx-1 and TBP-2 protein were detected in freshly isolated CFU-GM cells (colony forming unit-granulocyte- macrophage) and, in early time-course experiments (0-48h), we did not observe any significant temporal change in cells treated with hM-CSF alone nor any regulation by sRANKL was observed.
  • Cells treated with hM-CSF and sRANKL are entirely mononuclear at 4d but predominately multinuclear at 7d (Hodge et al, 2004 supra), suggesting a relationship between TBP-2/Trx-l expression and cell fusion.
  • the present invention provides a method of inhibiting osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an agent which inhibits RANKL signalling and thereby indirectly reduces the Trx-l:TBP-2 ratio in one or more osteoclast progenitor cells of said subject, wherein said reduction in Trx-l:TBP-2 ratio inhibits osteoclastogenesis in said subject.
  • Osteoclast formation is stimulated by hydrogen peroxide, indicating that ROS induce osteoclast differentiation.
  • Oxidative stress causes Trx-1 translocation to the nucleus, which can be blocked by NAC (Hirota et al, 1999 supra).
  • Reduced nuclear Trx-1 mediates DNA binding of AP-I, NF- ⁇ B, and other transcription factors, via Ref-1 (Nishinaka et al, Redox Rep ⁇ 5(5j:289-295, 2001).
  • NAC treatment of osteoclast precursors inhibits their osteoclastic differentiation by inhibiting nuclear translocation of Trx-1 and subsequent Trx-1 -dependent activation of AP-I and NF- ⁇ B.
  • NAC may directly modulate expression of Trx-1 and TBP -2, although only a small change was found in their mRNA expression after 7 days of NAC treatment was observed in accordance with the present invention.
  • inducers of ROS reduce expression of TBP -2 that is blocked by NAC, but they do not effect expression of Trx-1 (Schulze et al, Circ Res 91 fS):689-695, 2002).
  • the lowering of TBP-2 is associated with increased Trx-1 reducing activity.
  • redox signals can indirectly activate Trx-1 via suppression of TBP-2 expression.
  • cardiac myocytes biomechanical or oxidative stress down-regulates TBP-2 expression and increases Trx-1 activity, while over-expression of TBP-2 inhibits Trx-1 activity and induces apoptosis (Wang et al, J Biol Chem 277 (29) -.26496-26500, 2002).
  • the relative under-expression of TBP-2 protein and over-expression of Trx-1 protein seen in sRANKL-treated cells was completely reversed by co-treatment with 10 mM NAC, a thiol ROS scavenger.
  • NAC a thiol ROS scavenger.
  • expression of TBP-2 mRNA was increased Trx-1 mRNA decreased 2.0-fold.
  • NAC dose-dependently inhibited osteoclast formation.
  • the present invention provides a method of inhibiting osteoclastogenesis, osteoclast survival and/or osteoclast activity in a subject, said method comprising administering to said subject an antioxidant, wherein said antioxidant directly or indirectly reduces the Trx-l:TBP-2 ratio in one or more osteoclast progenitor cells of said subject, wherein said reduction in Trx-l:TBP-2 ratio inhibits osteoclastogenesis in said subject.
  • antioxidant encompasses any natural or synthetic substance that prevents or impedes destructive oxidation reactions and includes compounds which reacts directly with free radicals and/or other chemical substances which release free radicals that would otherwise attack molecules in the body, and abnormally oxidize them.
  • the antioxidant by reacting with the oxidant, protects these important molecules from being damaged.
  • the term “antioxidant” should also be understood to specifically include compounds which react with ROS or oxygen free radicals, which are highly reactive compounds created during normal cell metabolism.
  • Antioxidants scavenge free radicals, convert them to harmless substances, absorb them or attach to them before the free radicals can, inter alia, directly or indirectly increase the Trx-l :TBP-2 ratio and thereby initiate or promote the process of osteoclastogenesis.
  • antioxidants include antioxidants that exist as vitamins, minerals and other compounds in foods; the food antioxidants BHT and BHA; lycopenes; carotenoids; vitamins vitamins A, C, E, B-I, B-5, B-6; the minerals selenium, germanium, magnesium, copper and zinc; the enzymes catalase and superoxide dismutase (SOD); coenzyme Q-IO; amino acids such as cysteine.
  • the antioxidant comprises NAC.
  • the present invention further provides methods for the treatment and/or diagnosis of conditions associated with excessive or unregulated osteoclastic activity.
  • Exemplary diseases to which the methods of the present invention may be adapted include bone loss associated conditions including post-menopausal osteoporosis, Paget's disease of bone, hyperparathyroidism, inflammatory arthritis, peridontal disease, multiple myeloma, and bone metastases.
  • the present invention provides a method of treating or preventing a condition associated with excessive or unregulated osteoclastic activity in a subject, said method comprising administering to said subject an agent which effects an decrease in the Trx-1 :TBP-2 ratio in one or more osteoclast progenitor cells in said subject, wherein said decrease in the Trx-1 :TBP-2 ratio inhibits osteoclastic activity.
  • Osteoclasts were generated from adult human CD14 + cells as previously described (Nicholson et al, Clin Sci (Lond) (99) (2) :133-140, 2000). Briefly, CD14 + derived from peripheral blood mononuclear cells were cultured for 3 wk in MEM with hM-CSF (25 ng/nil) alone or with sRANKL (62.5 ng/ml) to produce macrophages or osteoclasts, respectively. Osteoclasts were also generated from human CFU-GM as previously described (Hodge et al, 2004 supra).
  • CFU-GM or colony forming unit- granulocyte-macrophage is a precursor cell in the granulocytic series that can grow into a myeloblast in the presence of appropriate stimulators in vitro.
  • CFU-GM derived from umbilical cord blood were incubated in MEM/10% FBS in either 6-well plates (4-8 x 10 5 in 2 mL/well) or 96-well plates (4 x 10 4 in 200 ⁇ L /well) containing 4 x 4 mm sperm whale dentine slices with hM-CSF (25 ng/mL) and sRANKL (62.5 ng/mL).
  • Osteoclast formation and resorption were quantified using MCID image analysis as previously described (Hodge et al, 2004 supra).
  • the MCID was calibrated so that only TRAP + cells with plan area >500 ⁇ m 2 (equivalent to at least 2 nuclei/cell) were counted.
  • Hybridisation was for 16h in a traditional hybridisation fluid (6xSSPE, 50% v/v formamide, Ix Denhardt's solution). Filters were washed in a standard series of decreasing ionic strength at 55C, exposed simultaneously to X-ray film and images matched manually for genes that showed differential signals.
  • TBP-2 primers were based on the 3' end of the gene as revealed by the BLAT server (http://genome.ucsc.edu), to amplify a short fragment in cDNA and to skip a short exon in genomic DNA.
  • Trx-1 primers were obtained from Applied Biosystems (product number 4331182). The method of comparison was based on reference to 18S or ⁇ -actin mRNA as an internal control for RNA (delta-delta method). For each treatment/time-point mRNA was extracted separately from 3 wells of a 6-well plate and the Real-time PCR was done in duplicate. Statistical differences were assessed using the 2-sample t-test.
  • the recombinant replication deficient adenovirus vectors were constructed using the Adeno-X (trade mark) Expression System (Clontech, Palo Alto, CA, USA).
  • the LacZ adenovirus expressing E. coli ⁇ -galactosidase (AdLacZ) was used to optimise infection conditions and as the normal control.
  • TBP-2 adenovirus (AdTBP-2) was constructed using full-length human cDNA.
  • Recombinant virus was produced in HEK 293 cells and assayed using the Adeno-X (trade mark) Rapid Titre Kit (Clontech).
  • TBP-2 protein was probed using rabbit anti-human TBP-2 antibody and detected using anti-rabbit IgG peroxidase conjugate.
  • Trx-1 protein was probed using a monoclonal anti- human Trx-1 antibody (abl754) and detected using anti-mouse IgG peroxidase conjugate.
  • ⁇ -Actin protein was probed using a monoclonal anti- ⁇ -actin antibody and detected using anti-mouse IgG peroxidase conjugate. TBP-2, Trx-1 and ⁇ -actin bands were detected by enhanced chemiluminescence (ECL plus) using Hyperfilm (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).
  • CFU-GM cultured in hM-CSF and sRANKL for 1Od were infected with AdlacZ or AdTBP-2 and cultured for a further 4d.
  • the cells were starved of sRANKL for 2-16h and then stimulated with sRANKL (62.5 ng/ml) or vehicle for Ih before harvesting.
  • Nuclear extracts prepared using the Nuclear Extraction Kit (Panomics, Redwood City, CA, USA) were run on 6% w/v acrylamide 0.5XTBE gels and transferred onto Biodyne B 0.45 ⁇ filters (PALL Life Sciences).
  • Filters were processed using EMSA "Gel-Shift” Kits (Panomics) according to the manufacturers instructions and bands visualised using Hyperfilm (trade mark) ECL (Amersham Pharmacia Biotech). Biotin labelled or 'cold' NF- ⁇ B or AP-I oligonucleotides were obtained from Panomics. NF -KB activity was also assessed using the EZ-Detect (trade mark) NF- ⁇ B p65 Transcription Factor Kit (Pierce, Rockford, USA). Cells were prepared and stimulated with sRANKL as above and whole cell protein extracts were prepared using M-PER Mammalian Protein Extraction Reagent (Pierce).
  • Human CD14 + or CFU-GM precursors treated with hM-CSF and sRANKL generate TRAP + multinucleate cells from about 7d but substantial substrate resorption is not seen until 14-21d (Hodge et al, 2004 supra and Nicholson et al, 2000 supra). In the presence of hM-CSF only, no resorption occurs and the cells formed are mononuclear, TRAP" 68 , non ⁇ specific esterase "1" , and CDlIb + , consistent with macrophage phenotype (Hodge et al, 2004 supra).
  • TBP-2 and Trx-1 protein (Figure 1) in CFU-GM osteoclastogenesis cultures was examined.
  • protein expression of both genes was similar in cells treated with hM-CSF alone compared to cells treated with hM-CSF and sRANKL.
  • expression of TBP-2 protein was substantially less and Trx-1 protein substantially more in cells exposed to sRANKL.
  • the net effect between 4d and 7d was a substantial up- regulation of TBP-2 protein and suppression of Trx-1 protein in cells treated with hM-CSF alone, whereas in the presence of sRANKL, TBP-2 protein expression remained low and Trx-1 protein expression was sustained.
  • Trx-1 and TBP-2 protein were both detected in freshly isolated CFU-GM cells and, in early time-course experiments (0-48h), no significant temporal change in cells treated was observed hM-CSF alone nor any regulation by sRANKL.
  • Cells treated with hM-CSF and sRANKL are entirely mononuclear at 4d but predominately multinuclear at 7d (Hodge et al, 2004 supra), suggesting a relationship between TBP-2/Trx-l expression and cell fusion.
  • TBP-2 and Trx-1 mRNA was quantified at early time-points with Real ⁇ time PCR using ⁇ -actin mRNA as an internal control for RNA. The results are shown in Table I. In cultures treated with sRANKL, TBP-2 mRNA was significantly reduced at 6 and 7d and Trx-1 mRNA was significantly increased at 5 and 6d. No differences in mRNA expression were seen at 4d. No changes in mitochondrial Trx (Trx-2) mRNA expression were seen at either time-point. EXAMPLE 3
  • Antioxidants block RANKL-induced modulation of TBP-2/Trx-l expression and inhibit osteoclast differentiation.
  • Trx reductase contains selenocysteine and its activity is enhanced by selenium (Mustachich and Powis, Biochem J 346 Pt /:l-8, 2000).
  • Ebselen is a substrate for Trx reductase and a superfast Trx-1 oxidant and it is considered that its antioxidant properties are mediated by the Trx system (Zhao et al, Proc Natl Acad Sci USA 9P ⁇ 73J:8579-8584, 2002).
  • Adenoviral over-expression of TBP-2 inhibits osteoclast formation, reduces osteoclast size and reduces Trx-1 protein expression.
  • AdTBP-2-infected cultures showed minimal increase in size, suggesting an effect of TBP-2 over-expression on cell fusion.
  • TRAP + mononuclear cells were quantified at 14d (when the greatest difference in osteoclast numbers between the treatment groups exists).
  • TBP-2 Over-expression of TBP-2 inhibits RANKL-induced AP-I activation, but enhances NF-
  • osteoclasts were prepared in the normal way by culturing CFU-GM infected with either AdLacZ or AdTBP-2 in the presence of sRANKL and hMCSF.
  • sRANKL was removed from the cultures for 2-16h prior to re-stimulation.
  • AdLacZ cultures activation of AP-I, as assessed by EMSA, was stimulated by treatment with sRANKL for Ih.
  • AdTBP-2 infection had no apparent effect on basal AP-I activation but blocked the response to sRANKL ( Figure 4a).
  • Double stranded oligonucleotides containing a target gene sense and anti-sense sequence were cloned into a retroviral vector (RNAi-Ready pSIREN-RetroQ Vector).
  • RNAi-Ready pSIREN-RetroQ Vector Double stranded oligonucleotides containing a target gene sense and anti-sense sequence
  • antiTBP-2 expresses a short hairpin RNA (shRNA) that is processed in vivo into siRNA- like molecules capable of carrying out gene-specific silencing.
  • shRNA short hairpin RNA
  • the construct was transfected into a packaging cell line (PT 67) and stably transfected cells were selected with puromycin. These cells produce replication-incompetent infective retroviruses that were then used to infect mitotically active CFU-GM cells to express shRNA. Results are shown in Figure 5 and Table 2.
  • the shRNA antiTBP-2 down-regulates TBP-2 RNA consequently decreasing expression of TBP-2 protein in the cell.
  • TBP-2 binds and inhibits the reduced form of TRX there is more TRX available in the cell in the presence of the antiTBP-2 shRNA.
  • An increase in TRX has been shown to promote osteoclast differentiation leading to increased osteoclast numbers and resorption. It is likely to do this by promoting the activity of transcription factors important in osteoclastogenesis such as NFkB and AP-I.

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Abstract

La présente invention concerne de façon générale un procédé servant à moduler le processus d'ostéoclastogénèse chez un sujet animal. Plus particulièrement, la présente invention concerne un procédé de modulation du processus d'ostéoclastogénèse en manipulant le rapport de la thiorédoxine-1 (Trx-1) sur la protéine liant la thiorédoxine-2 (TBP-2) dans des cellules progénitrices d'ostéoclastes. La présente invention identifie en plus une gamme d'agents, dont des constructions d'expression, des constructions d'extinction de gènes et des antioxydants, qu'on peut utiliser pour modifier le rapport Trx-1:TBP-2 dans les cellules progénitrices d'ostéoclastes et pour moduler de cette manière le processus d'ostéoclastogénèse. En adaptant les procédés décrits ici, la présente invention s'étend en plus à des procédés de traitement et de diagnostic de maladies associées à une ostéoclastogénèse excessive ou non régulée chez un sujet.
PCT/AU2005/001322 2004-08-31 2005-08-31 Procédé servant à moduler l'ostéoclastogénèse WO2006024096A1 (fr)

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