La protein as a novel regulator of osteoclast generation

Abstract

The present invention provides a method for regulating osteoclast fusion, a pharmaceutical composition for use in the method, and a kit. [Solution] In some embodiments, these methods include administering an effective amount of lupus autoantigen (La) protein or an agent that modulates the expression or activity of the La protein to a subject in need, thereby modulating osteoclast fusion in the subject. In some embodiments, the method increases osteoclast fusion and bone resorption. In other embodiments, the method decreases osteoclast fusion and bone resorption.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63 / 155,896, filed on 3 March 2021, which is incorporated herein by reference.

[0002] Statement of government support This invention was made with government support from the National Institutes of Health and the National Institute of Child Health and Human Development. The U.S. government has certain rights to this invention.

[0003] Field of Invention The present invention relates to the field of osteoclast fusion, and more specifically to the use of an effective amount of lupus autoantigen (La) protein or an agent that modulates the expression or activity of the La protein for regulating osteoclast fusion, for example, for regulating bone resorption. [Background technology]

[0004] background Osteoclasts are multinucleated bone-eating cells that are essential for lifelong skeletal remodeling, and dysfunction of these cells is a major cause of several bone diseases, including osteoporosis, fibrous dysplasia, Paget's disease, and marble bone disease. These conditions, which affect more than 10 million Americans, all exhibit disturbances in osteoclast-mediated bone resorption. Multinucleated osteoclasts are formed by the sequential fusion of mononuclear precursor cells. The number of nuclei per syncytial osteoclast, and thus the number of fusion events that generated that cell, is directly correlated with the cell's bone resorption propensity, and the number of osteoclasts and / or the number of fusion events that give rise to osteoclasts is significantly altered in many bone diseases. Despite the fundamental role of fusion in osteoclast formation and skeletal remodeling, an understanding of how osteoclasts control their fusion to reach an "appropriate size" in order to perform their biological functions is currently lacking. A better understanding of how osteoclasts fuse may help elucidate the pathology underlying these bone diseases and provide new treatments for skeletal disorders. SUMMARY OF THE INVENTION

[0005] SUMMARY OF THE DISCLOSURE Methods for modulating osteoclast fusion are disclosed herein. In some embodiments, these methods include administering to a subject in need thereof an effective amount of a La protein or an agent that modulates the expression or activity of a La protein, thereby modulating osteoclast fusion in the subject. Methods for modulating bone resorption are also disclosed.

[0006] In some embodiments, the method increases osteoclast fusion, and the active agent modulating the expression or activity of La protein is either an active agent that increases the expression or activity of La protein, or La protein (or a fragment thereof). In further embodiments, these methods include administering an effective amount of La protein (or a fragment thereof) or an active agent that increases the expression or activity of La protein to a subject in need, thereby increasing osteoclast fusion in the subject. In further embodiments, the subject has a disease including reduced bone resorption, and the method increases bone resorption. In some non-limiting examples, La protein (or a fragment thereof) is administered to the subject.

[0007] In another embodiment, the method reduces osteoclast fusion in a subject, and the active agent reduces the expression or activity of La protein. In a further embodiment, these methods include administering an active agent that reduces the expression or activity of La protein to a subject in need thereof, thereby reducing osteoclast fusion in the subject. In a further embodiment, the subject has a disease including increased bone resorption. In some non-limiting examples, the active agent is an inhibitory RNA or an antagonist antibody that specifically binds to La. In another non-limiting example, the active agent is an inhibitory La peptide.

[0008] A pharmaceutical composition is disclosed that comprises an effective amount of La protein (or a fragment thereof) or an active agent (e.g., a nucleic acid molecule, vector, or antibody) that modulates the expression or activity of La protein, and is useful in any of the disclosed methods.

[0009] The features of this disclosure described above and other features will become even more apparent from the detailed description of several embodiments set forth below with reference to the accompanying drawings. [Brief explanation of the drawing]

[0010] [Figure 1A]Figures 1A-1G: Osteoclastic differentiation involves dramatic changes in the steady-state levels and localization of La molecular isoforms. (A) Representative images of the stages of osteoclast induction in human monocytes after M-CSF (6 days) and M-CSF + RANKL (5 days), respectively (staining = phalloidin-Alexa488, nuclear dots = Hoechst). (B) Quantification of the number of fusion events normalized to the total number of nuclei observed over time after RANKL addition (n=3). Each dot represents the average of scored >7,500 nuclei. (C) Representative Bis-Tris PAGE separation and silver staining of total protein lysates from the osteoclastic stage illustrated in A. Maximum separation of proteins of this molecular weight was achieved by electrophoresis of the lysates until the 50kDa marker almost completely leached out of the 4-12% polyacrylamide gel, resulting in a potentially misleadingly heavy band of interest. < represents the band of interest as evaluated by mass spectrometry after being excised from both lanes. (D) Representative Tris-glycine Western blots evaluating La expression in primary human monocytes and total protein lysates from the osteoclast generation stage shown in A. (Anti-La antibody (α-La), Abcam 75927). (E) Representative Tris-glycine Western blots evaluating the time course of La expression after RANKL addition. (α-La, Abcam 75927) (α-GAPDH loading control). (F) Representative digital immunofluorescence images of La in M-CSF-induced osteoclast precursors and 3 days after RANKL application. (α-La, Abcam). (G) Representative digital immunofluorescence images of La in osteoclasts using La antibodies that specifically recognize low molecular weight (LMW) La (α-LMW La) and full-length (FL) La (α-FL La). Cells were fixed 4 days after RANKL addition, which is rich in both LMW La and FL La. [Figure 1B] Please refer to the explanation in Figure 1A. [Figure 1C] Please refer to the explanation in Figure 1A. [Figure 1D] Please refer to the explanation in Figure 1A. [Figure 1E] Please refer to the explanation in Figure 1A. [Figure 1F] Please refer to the explanation in Figure 1A. [Figure 1G] Please refer to the explanation in Figure 1A. [Figure 2A] Figures 2A-2I: Osteoclast formation depends on truncated La, but function of the La domain is not required. (A) qPCR evaluation of SSBs after siRNA treatment of human osteoclast precursors. (n=4) (P=0.0043). (B) Representative phase-contrast images of untargeted and SSB-targeted human osteoclasts stained with TRAP, as shown in a. (C) Quantification of the number of fusion events in the formation of syncytia with 3+ nuclei in an experiment like one in B. (n=3) (P=0.0306). (D) Representative immunofluorescence images of La in RAW 264.7 before mRANKL addition (control), 3 days after mRANKL addition, and 5 days after mRANKL addition, where clumpy multinucleated osteoclasts were consistently observed, as imaged here. (E) Representative tris-glycine Western blot of whole cell lysates taken from mouse RAW 264.7 treated as in D. Mouse RANKL (mRANKL)-treated cells were enriched into mononuclear (mononuclear) or multinuclear (multinuclear) populations as described in the Methods section (cyclophyllin B (α-Cyclo B) loading control). (F) Topological diagrams of LMW La 1-375, "uncleavable" La D371A, D374A, and "RNAΔ" La 1-375 Q20A_Y24A_D33I. (G) Quantification of the number of fusion events in the formation of syncytia with 3+ nuclei in RAW 264.7 cells transfected with empty expression plasmids, La 1-375 expression plasmids, or La 1-375 Q20A_Y24A_D33I expression plasmids. (n=3) (P=0.0213 and 0.0173, respectively). (H) Representative fluorescence images of human monocyte-derived osteoclasts transfected with an empty expression plasmid, a La 1-375 expression plasmid, or a La D371A, D374A expression plasmid. (I) Quantification of the number of fusion events in syncytia with 3+ nuclei in H. (n=4) (P=0.0205 and 0.325, respectively). Statistical significance was assessed by paired t-tests. *=P<0.05. **=P<0.001. Error bars = SEM. [Figure 2B] Please refer to the explanation in Figure 2A. [Figure 2C] Please refer to the explanation in Figure 2A. [Figure 2D] Please refer to the explanation in Figure 2A. [Figure 2E] Please refer to the explanation in Figure 2A. [Figure 2F] Please refer to the explanation in Figure 2A. [Figure 2G] Please refer to the explanation in Figure 2A. [Figure 2H] Please refer to the explanation in Figure 2A. [Figure 2I] Please refer to the explanation in Figure 2A. [Figure 3A]Figures 3A-3G: La associates with the membrane, migrates to the surface, and controls osteoclast membrane fusion. (A) Western blot comparing cytosolic fraction and membrane-bound protein fraction from human osteoclasts. (B) Representative digital immunofluorescence images comparing surface staining of α-Fish / TKS5 (osteoclast superficial membrane protein used as a negative control) or α-La antibody in human osteoclasts under impermembrane treatment conditions (top) and DIC (bottom). (C) Representative digital immunofluorescence images comparing surface staining of isotype control or α-La antibody in RAW 264.7-derived osteoclasts under impermembrane treatment conditions. (D) Representative digital immunofluorescence images of La in forming osteoclasts 2-5 days after RANKL application (α-La, Abcam). Cells were stained with La at the indicated time points without membrane permeabilization. (E) Schematic diagram illustrating the sequential process of multinucleated osteoclast formation (top) and an approach to isolate the membrane fusion stage from the pre-fusion stage of osteoclast differentiation (bottom). When the half-fusion inhibitor lysophosphatidylcholine (LPC) is applied 48 hours after RANKL-induced osteoclast generation, the pre-fusion differentiation stage is permitted, but half-fusion is inhibited, resulting in cell synchronization. By removing LPC, it becomes possible to specifically search for membrane fusion between osteoclasts. (F) Quantification of human osteoclast fusions isolated from the pre-fusion stage and synchronized, as illustrated in E. Fusion in the presence of α-La and fusion without antibody addition (washing) were normalized to α-RANK control. (n=3) (P=0.0086 and 0.1330, respectively). (G) Effect of α-FL La application or α-LMW La application on synchronized human osteoclast fusions. Data normalized to data after application of isotype control (IgG). (n=2) (P=0.03 and 0.4581, respectively). (E, F) "LPC" - Fusion observed without removing LPC. Statistical significance was assessed by paired t-tests. *=P<0.05. **=P<0.001. Error bars = SEM. [Figure 3B] Please refer to the explanation in Figure 3A. [Figure 3C] Please refer to the explanation in Figure 3A. [Figure 3D] Please refer to the explanation in Figure 3A. [Figure 3E]Please refer to the explanation in Figure 3A. [Figure 3F] Please refer to the explanation in Figure 3A. [Figure 3G] Please refer to the explanation in Figure 3A. [Figure 4A] Figures 4A-4E: Recombinant La promotes osteoclast fusion. (A) Representative fluorescence images of human osteoclasts 3 days after RANKL addition with or without overnight (end of day 2 after RANKL) addition of recombinant thermoinactivated La 1-408, La 1-408, La 1-375, or La 1-375 Q20A / Y24A / D33I. Recombinant protein was added at approximately 40 nM at the end of day 2 after RANKL addition, and cells were fixed the following morning. (B) Quantification of A. (Inactivated n=2; Others n=3) (P=0.1232, 0.0015, 0.0035, and 0.0491, respectively). (C) Fractions of nuclei in fusion cells present in syncytia of various sizes from A. (n=3). (D) Quantification of the number of fusion events with or without the addition of La 1-187 or La 188-375. Recombinant protein was added at approximately 40 nM at the end of day 2 after RANKL addition, and cells were fixed the following morning. (E) Quantification of synchronized fusion events (illustrated in Figure 3E) with and without the addition of recombinant La species (washed). "LPC" indicates that the half-fusion inhibitor remained until fixation. (n=3) (P=0.001 and 0.03, respectively). Statistical significance was assessed by paired t-tests. In (B, D, E), data were normalized to the control data (no protein addition in B and D, and washing and no protein addition in E). Error bars = SEM. [Figure 4B] Please refer to the explanation in Figure 4A. [Figure 4C] Please refer to the explanation in Figure 4A. [Figure 4D] Please refer to the explanation in Figure 4A. [Figure 4E] Please refer to the explanation in Figure 4A. [Figure 5A]Figures 5A-5E: La interference affects bone resorption by human osteoclasts. (A) Illustration illustrating the use of fluorescein amine-labeled chondroitin sulfate captured on a calcium phosphate-coated plate for assaying bone resorption. (B) Bone resorption in osteoclasts overexpressing uncleavable La (D371A, D374A) or truncated La (1-375), normalized to values ​​for osteoclasts transfected with empty plasmids. (n=3) (P=0.0564 and 0.0471). (C) Bone resorption in osteoclasts transfected with siRNA targeting La transcripts, normalized to values ​​for untargeted siRNA. (n=5) (P=0.0045). (D) Bone resorption in osteoclasts exposed to different concentrations of α-La, normalized to values ​​for cells treated with 7.5 μg / ml isotype control antibody. (n=3)(P=0.0723, 0.0098, and 0.004). (E) Bone resorption in osteoclasts treated with 40nM recombinant La 1-375, normalized to control after the addition of the same amount of PBS. (n=3). Statistical significance was assessed by paired t-tests. Error bars = SEM. [Figure 5B] Please refer to the explanation in Figure 5A. [Figure 5C] Please refer to the explanation in Figure 5A. [Figure 5D] Please refer to the explanation in Figure 5A. [Figure 5E] Please refer to the explanation in Figure 5A. [Figure 6A]Figures 6A-6C: Osteoclast formation in human osteoblast / osteoclast precursor coculture is dependent on the La protein. (A) Schematic diagram of the multiwell configuration of the human osteoblast / osteoclast coculture system used: 3 regions for osteoblasts (shown in dark gray) and 1 region for osteoclast precursors (light gray). After M-CSF induction of human monocytes, the partitions in the multiwell were removed and the osteoclast precursors and primary osteoblast medium were mixed. (B) Representative immunofluorescence images comparing osteoclast fusion using control IgG or α-La antibody. Arrowheads represent syncytia with ≥3 nuclei. (C) Quantification of osteoclast fusion in coculture in the presence of α-La, normalized to values ​​in the presence of control IgG. (n=4) (P=0.0331). Statistical significance was evaluated by paired t-tests. Error bars = SEM. [Figure 6B] Please refer to the explanation in Figure 6A. [Figure 6C] Please refer to the explanation in Figure 6A. [Figure 7A]Figures 7A-7G: α-La treatment suppresses ectopic osteoclast formation in fibrous dysplasia (FD) explants, confirming that La regulates osteoclasts in both healthy and diseased conditions. (A) Illustration of the ex vivo bone marrow culture system based on the tetracycline-inducible model of FD. (B) TRAP staining of bone marrow explants from homozygous Gαs R201C mice (FD) and wild-type littermates (WT). Explants were cultured with M-CSF alone or with M-CSF and Doxy. (C) ELISA quantification of mRANKL produced in FD and WT cultures after Doxy treatment. (n=4)(P=0.0005)(D) Representative images of Doxy-activated FD explants treated with isotype control or α-La antibody. (E) Quantification of the number of fusions producing osteoclast syncytia with ≥3 nuclei. (n=4)(P=0.0054). (F) Quantification of the number of osteoclast syncytia with ≥3 nuclei from D. (n=4)(P=0.0178). In B and D, arrowheads represent multinucleated osteoclasts, and arrows represent fibrillary cell aggregates specific to FD after Doxy addition. Statistical significance was assessed by unpaired t-tests in c and paired t-tests in E and F. Error bars = SEM. (G) Illustration of the La protein in the process of osteoclast formation. La is an essential ubiquitous RNA-binding protein. In the nuclei of all eukaryotic cells, La performs its normative, ancient function. While not theoretically bound, La has further specialized functions in the formation of multinucleated osteoclasts. During osteoclast formation, La dissipates as circulating monocytes become osteoclast precursor cells. Once osteoclast restraint is initiated by RANKL, La returns but is rapidly cleaved by proteases and shuttled to the surface of the fusing osteoclast. On the surface of the fusing osteoclast, La performs a novel function as a membrane fusion control factor. When the osteoclast reaches the "appropriate size" for its biological function, the surface La disappears and is replaced by normative non-cleaved La, which returns to the nucleus of the mature osteoclast. [Figure 7B] Please refer to the explanation in Figure 7A. [Figure 7C] Please refer to the explanation in Figure 7A. [Figure 7D] Please refer to the explanation in Figure 7A. [Figure 7E] Please refer to the explanation in Figure 7A. [Figure 7F] Please refer to the explanation in Figure 7A. [Figure 7G] Please refer to the explanation in Figure 7A. [Figure 8A] Figures 8A-8G: Characterization of monocyte-derived osteoclasts and identification of La protein in osteoclasts. (A) qPCR evaluation of the osteoclast differentiation marker CTSK from the conditions shown in Figure 1a. (n=4) (P=0.0079). (B) Quantification of fluorescent bone resorption in osteoclast precursors (M-CSF) and differentiated osteoclasts (RANKL) (n=3) (P=0.0066) (5 days post-RANKL). (C) Nuclear fraction in syncytial osteoclasts of various sizes (n=4) (5 days post-RANKL). (D) Mass spectrometry data from six excised bands separated in six separate lanes electrophoresed on a single gel, as seen in Figure 1b. Each lane corresponded to a separate cell lysate. Cells were collected from two healthy donors (1 and 2), differentiated in M-CSF for 6 days, and further differentiated in M-CSF or M-CSF + RANKL for 3 days. Cells from donor 2 were differentiated in three independent technical replicates (2a-2c). Vimentin was also detected in each sample. Although vimentin has a molecular weight similar to La, vimentin levels were nearly equivalent between the M-CSF and RANKL samples. This table shows the total number of distinctly different peptide sequences identified in the protein group (peptides) and peptide spectrum match (PSM). (E) qPCR evaluation of SSB (La gene) from the osteoclast generation stage shown in A (n=3) (P=0.027). (F) Representative tris-glycine Western blots evaluating the dominant La species 2 and 5 days after RANKL addition. (G) Representative immunofluorescence images of La in forming osteoclasts 2-5 days after RANKL application (α-La, Abcam). Cells were stained with La at the indicated time points after membrane permeabilization. Statistical significance was evaluated by paired t-tests. **=P<0.001. Error bars = SEM. [Figure 8B] Please refer to the explanation in Figure 8A. [Figure 8C] Please refer to the explanation in Figure 8A. [Figure 8D] Please refer to the explanation in Figure 8A. [Figure 8E] Please refer to the explanation in Figure 8A. [Figure 8F] Please refer to the explanation in Figure 8A. [Figure 8G] Please refer to the explanation in Figure 8A. [Figure 9] Exogenous La constructs are expressed at similar levels in human osteoclasts. ΔCq values ​​of La signaling in human osteoclasts transfected with empty mammalian expression plasmids, La D371A, D374A mammalian expression plasmids, La 1-375 mammalian expression plasmids, and "RNAΔ" La 1-375 Q20A_Y24A_D33I mammalian expression plasmids. GAPDH was used as a housekeeping transcript control. (n=2). Error bars = SEM. [Figure 10A] Figures 10A-10D: Multinucleated osteoclast formation depends on La cleavage. (A) Western blot demonstrating that the caspase inhibitor z-VAD-fmk can inhibit the production of cleaved La in human osteoclasts (3-4 days after RANKL). (B) Representative immunofluorescence images of α-FL La staining in primary human osteoclasts during active fusion (day 3) and when fusion is almost complete (day 5). (C) Representative immunofluorescence images of α-FL La staining in primary human osteoclasts (day 3) during active fusion, under control conditions (vehicle) and after inhibition of La cleavage by the application of the pancaspase inhibitor z-VAD-fmk. (D) Quantification of the number of fusion events in cells with 3+ nuclei from c. (n=3) (P=0.0455). Statistical significance was evaluated by paired t-tests. *=P<0.05. Error bars = SEM. [Figure 10B] Please refer to the explanation in Figure 10A. [Figure 10C] Please refer to the explanation in Figure 10A. [Figure 10D] Please refer to the explanation in Figure 10A. [Figure 11] RNAi repression of La does not alter the steady-state transcript levels of proteins involved in osteoclastogenesis differentiation or osteoclast fusion. qPCR evaluation of two osteoclast differentiation markers, NFATc1 and CTSK, as well as osteoclast fusion-related transcripts, syncytin 1 (SYN1), annexin A5 (ANXA5), S100A4, and ANO6, after siRNA treatment as described in Figure 2A (n=4). Statistical significance was assessed by paired t-tests (P=0.4051, 0.4679, 0.45650, 0.6172, 0.7899, and 0.1129). Error bars = SEM. [Figure 12A] Figures 12A-12D: La interacts with Anx A5. (A) Immunoprecipitation of osteoclast lysates 3 days after RANKL addition, with and without DTSSP surface crosslinking. La supramolecular complexes were captured on immunomagnetic beads via mouse α-La or isotype control, and the complexes were blotted with rabbit antibodies induced against targets of interest. (B) Immunoprecipitation of osteoclast lysates with DTSSP surface crosslinking. La (left) or Anx A5 (right) supramolecular complexes were captured on immunomagnetic beads via mouse α-La or α-Anx A5, and the complexes were blotted with rabbit antibodies induced against targets of interest. (C) Schematic illustration of an approach to identify membrane affinity by separating liposome-bound proteins (lower fraction) from soluble proteins (upper fraction). (D) Quantification of recombinant La and Anx A5 in liposome-bound fractions as illustrated in C. (n=2). Error bars = SEM. [Figure 12B] Please refer to the explanation in Figure 12A. [Figure 12C] Please refer to the explanation in Figure 12A. [Figure 12D] Please refer to the explanation in Figure 12A. [Figure 13]Figures 13A-13B: α-La suppresses osteoclast-dependent bone loss in fibrous dysplasia (FD). (A) Representative X-ray images of the hind limbs of FD mice before and 21 days after doxy. addition to the diet. The numbers indicate a score quantifying the progression of FD for each bone, which is then averaged to give the FD score presented in b. (B) Time course of FD progression after doxy. addition to the diet, scored on X-ray images like a, with antibody injection. [Figure 14A] Figures 14A-14C: Identification of La osteoclast fusion domains and peptide inhibitors. (A) Linear diagram of La 1-375 protein motifs. The lines at the bottom of the figure represent 12 overlapping peptides designed to represent protein motifs in La 188-375. (B) Quantification of fusion event counts with and without the addition of La 1-187 or La 188-375. Recombinant proteins were added overnight at approximately 40 nM between days 2 and 3 post-RANKL. (C) Same as A, using La 188-375 (+ control) or the 12 peptides illustrated in B. Peptides 2 and 9 inhibit fusion and multinucleated osteoclast formation. [Figure 14B] Please refer to the explanation in Figure 14A. [Figure 14C] Please refer to the explanation in Figure 14A. [Figure 15A] Figures 15A-15C: La directly interacts with the osteoclast fusion apparatus annexin A5 (Anx A5). (A) La supramolecular protein complex immunoprecipitated by immunomagnetic beads from osteoclast lysates 3 days after RANKL. (B) Quantification of recombinant La and Anx A5 concentrated on the membranes of PC:PS liposomes, PC:PS liposomes containing 5 mM Ca2+, or PC liposomes containing 5 mM Ca2+. (C) Magnetic streptavidin pull-down of biotin-Anx A5. Lane 1 = 6 × His-La only, Lane 2 = sample before pull-down, and Lane 3 = after pull-down and 4 washes. [Figure 15B] Please refer to the explanation in Figure 15A. [Figure 15C] Please refer to the explanation in Figure 15A. [Figure 16A] Figures 16A-16C: siRNA inhibition of sorting nexin 10 (SNX10) increases the La content on the osteoclast surface, leading to osteoclast hyperfusion, a phenotype specific to infant osteopetrosis associated with SNX10 deficiency. (A) Quantification of α-La surface staining after 48 hours of siRNA treatment. (B) Quantification of nuclei per syncytium after 48 hours of siRNA treatment. Normalized for untargeting. +IgG / +α-La indicates the addition of 6 μg / ml antibody. (C) Schematic representation of how La migrates to the osteoclast surface, is removed to halt osteoclast fusion, how SNX10 loss in osteopetrosis interferes with La surface removal, and how inhibition of excess surface La rescues the multinucleation of disrupted osteoclasts. [Figure 16B] Please refer to the explanation in Figure 16A. [Figure 16C] Please refer to the explanation in Figure 16A. [Modes for carrying out the invention]

[0011] Sequence List The nucleic acid and amino acid sequences listed are indicated using standard letter abbreviations for nucleotide bases and three (or one) letter symbols for amino acids, as specified in 37C.FR1.822. Only one strand of each sequence is shown, but references to the shown strands are understood to include the complementary strand. This sequence listing was submitted as an ASCII text file [Sequence_Listing, March 2, 2022, 12.7KB], which is incorporated herein by reference. The attached sequence listing is as follows: SEQ ID NO: 1 is an exemplary amino acid sequence of the full-length human La protein, including the nuclear localization sequence NLS. SEQ ID NO: 2 is an exemplary amino acid sequence of amino acids 1-375 of the human La protein lacking NLS. SEQ ID NO: 3 is an exemplary nucleic acid sequence encoding the human La protein. SEQ ID NO: 4 and SEQ ID NO: 5 are nucleic acid sequences of exemplary antisense RNA specific to La. SEQ ID NO: 6 is an exemplary amino acid sequence of the caspase's active site. SEQ ID NO: 7 is an exemplary amino acid sequence of amino acids 188-375 of the human La protein. SEQ ID NO: 8 and SEQ ID NO: 9 are amino acid sequences of exemplary inhibitory peptide fragments of the La protein. SEQ ID NO: 10 and SEQ ID NO: 11 are exemplary nucleic acid sequences encoding peptide 2 and peptide 9, respectively.

[0012] Detailed description of several aspects Osteoclasts are induced from circulating monocytes through the sequential production of macrophage precursors in response to macrophage colony-stimulating factor (M-CSF) and further osteogenic differentiation mediated by nuclear factor kappa B-activating receptor ligand (RANKL) (Feng et al., Bone Res. 2013;1(1):11-26. Epub 2013 / 03 / 01. doi:10.4248 / BR201301003. PubMed PMID:26273491; PMCID:PMC4472093). While investigating proteomic changes during osteoclast fusion, it was found that lupus autoantigen (called La protein), encoded by the SSB gene, is involved in osteoclast generation. La is an abundant, seemingly ubiquitous RNA chaperone, observed almost exclusively in the nucleus of all human cell types and tissues (Wolin et al., Annu Rev). Biochem.2002;71:375-403.Epub 2002 / 06 / 05.doi:10.1146 / annurev.biochem.71.090501.15000). Intranuclear retention of La is due to the normative nuclear localization sequence (NLS) of its C-terminus (Rosenblum et al., J Cell Biol.1998;143(4):887-99.Epub 1998 / 11 / 17.doi:10.1083 / jcb.143.4.887.PubMed PMID:9817748).In a few specific biological processes (e.g., apoptosis, viral infection, serum starvation), La is reported to be cleaved by caspases and transported to the extracellular surface of the plasma membrane (PM) (Bachmann et al., Autoimmunity.1992;12(1):37-45.Epub 1992 / 01 / 01.doi:10.3109 / 08916939209146128;Bachmann et al., Autoimmunity.1991;9(2):99-107.Epub 1991 / 01 / 01.doi:10.3109 / 08916939109006745;Rutjes et al., Cell Death Differ.1999;6(10):976-86.Epub 1999 / 11 / 11.doi:10.1038 / sj.cdd.4400571.5;Shiroki et al., J Virol.1999;73(3):2193-200.Epub 1999 / 02 / 11).

[0013] As disclosed herein, in osteoclast generation, La is neither "ubiquitous" nor nuclear, but bidirectionally controls osteoclast membrane fusion and bone resorption. Therefore, suppressing La inhibits osteoclast membrane fusion and bone resorption, while increasing La increases both. La is abundant in primary human monocytes but disappears upon differentiation into osteoclast precursors. After activation of osteoclast generation, La reappears as a low molecular weight species detected on the surface of fusion cells. After osteoclast fusion, the low molecular weight La is degraded and replaced by the full-length La species, which returns to the nucleus of syncytial osteoclasts. Reduction of La, inhibition of its cleavage, or blocking of La on the osteoclast surface with specific antibodies inhibits osteoclast fusion. Addition of recombinant La 1-375, which corresponds to the NLS-deficient La protein and therefore to the low molecular weight cleavage form of human La protein, or overexpression of La 1-375, vigorously promotes large-scale osteoclast fusion. The addition of recombinant full-length La also promotes osteoclast fusion. However, overexpression of "non-cleavable" La mutants is ineffective because they are not cleaved and cannot reach the osteoclast surface. By disrupting osteoclast size by targeting La on the osteoclast surface, the bone resorption tendency of primary human osteoclasts can be regulated bidirectionally.

[0014] This specification discloses that the La protein (or its fragments, e.g., SEQ ID NO: 7), or agents that increase the activity or function of the La protein, e.g., nucleic acid molecules encoding La, can be used to increase osteoclast fusion in vivo and in vitro. These methods increase bone resorption in subjects where increasing bone resorption is required. In contrast, this specification discloses that agents that decrease the activity or function of the La protein can be used to decrease osteoclast fusion in vivo and in vitro. These methods decrease bone resorption in subjects where decreasing bone resorption is required.

[0015] I. Terminology Unless otherwise noted, technical terms are used according to their conventional usage. Definitions of terms commonly used in molecular biology can be found in Krebs et al., Lewin's Genes XII, Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the” refer to both singular and plural unless the context indicates otherwise. For example, the term “a protein” encompasses one or more proteins and can be considered equivalent to the phrase “at least one protein.” As used herein, the term “comprises” means “includes.” Unless otherwise indicated, “approximately” means within 5%. Furthermore, any base size or amino acid size and any molecular weight or molecular mass values ​​given to nucleic acids or polypeptides are approximate and provided for illustrative purposes only, unless otherwise indicated. Appropriate methods and materials are specifically described below, but many similar or equivalent methods and materials can be used. In case of any inconsistency, this specification, including the definitions of terms, shall prevail. Furthermore, the materials, methods, and examples are merely illustrative and not intended to be limiting. To facilitate consideration of various embodiments, a definition of the terms is provided below.

[0016] Administration: Introduction of a composition (e.g., one containing an active ingredient that increases or decreases La activity) into a subject via a selected route. Administration can be topical or systemic. For example, if the route is intravenous, the composition is administered by introducing the composition into the vein of the subject. Similarly, if the route is intramuscular, the composition is administered by introducing the composition into the muscle of the subject. If the selected route is oral, the composition is administered by ingesting the composition. Useful exemplary routes of administration in the methods disclosed herein include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intraosseous, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes. Administration can also be topical, such as administration into the bone of the subject.

[0017] Animals: Living multicellular vertebrates; this category includes, for example, mammals and birds. The term mammal includes both humans and non-human mammals. Similarly, the term "subject" includes both human subjects and veterinary subjects.

[0018] Amino acid substitution: The replacement of one or more different amino acids in a polypeptide (e.g., La, e.g., SEQ ID NO: 1 or SEQ ID NO: 2) with one or more different amino acids. In relation to protein sequences, amino acid substitutions are also called mutations.

[0019] Antibody: A polypeptide comprising at least a light-chain immunoglobulin variable region or a heavy-chain immunoglobulin variable region that specifically recognizes and binds to an epitope (an antigen such as the La protein or a fragment thereof). This includes intact immunoglobulins, as well as their variants and portions known in the art, e.g., Fab' fragments, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which the light-chain variable region and the heavy-chain variable region of an immunoglobulin are linked by a linker, while dsFv proteins have mutations added to their chains that introduce disulfide bonds to stabilize the association of those chains. This term also includes genetically modified forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Co., Rockford, Illinois), and Kuby, J., Immunology, 3rd edition, WHFreeman & Co., New York, 1997.

[0020] Typically, immunoglobulins have a heavy chain and a light chain. The heavy and light chains each contain a constant region and a variable region (these regions are also known as “domains”). The heavy and light chain variable regions work together to specifically bind to an antigen. The light and heavy chain variable regions contain a “framework” region, separated by three hypervariable regions also called “complementarity-determining regions” or “CDRs.” The extent of the framework regions and CDRs is defined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; this document is incorporated herein by reference). The Kabat database is currently maintained online. The sequences of different light or heavy chain framework regions are relatively conserved within species. The antibody framework region is a composite framework region of its constituent light and heavy chains, which helps to position and align the CDRs in three-dimensional space.

[0021] The binding of the antigen to the epitope is primarily carried out by the CDRs. The CDRs on each chain are typically numbered sequentially from the N-terminus and called CDR1, CDR2, and CDR3, and are also typically identified by the chain on which that particular CDR is located. Therefore, V H CDR3 is located in the variable domain of the antibody heavy chain where it is found, while V L CDR1 is the CDR1 found in the variable domain of the light chain of the antibody.

[0022] "V H References to "VH" or "VH" refer to the variable region of the immunoglobulin heavy chain, including those of Fv, scFv, dsFv, or Fab. L "VL" refers to the variable region of an immunoglobulin light chain, including Fv, scFv, dsFv, or Fab.

[0023] A "monoclonal antibody" is an antibody produced by a single clone of a B lymphocyte or by cells transfected with the light chain and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, such as creating hybrid antibody-forming cells by fusing myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0024] A “humanized” immunoglobulin is an immunoglobulin that comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the “donor,” and the human immunoglobulin providing the framework is called the “acceptor.” In one embodiment, in a humanized immunoglobulin, all CDRs are derived from the donor immunoglobulin. A constant region is not required, but if one is present, it must be substantially identical to the human immunoglobulin constant region, i.e., at least about 85-90% identical, e.g., about 95% or more identical. Thus, all parts of a humanized immunoglobulin are substantially identical to the corresponding parts of the natural human immunoglobulin sequence, except perhaps the CDRs. A “humanized antibody” is an antibody that comprises a humanized light chain immunoglobulin and a humanized heavy chain immunoglobulin. The humanized antibody binds to the same antigen as the donor antibody providing the CDRs. The acceptor framework of a humanized immunoglobulin or humanized antibody may have a limited number of substitutions by amino acids adopted from the donor framework. Humanized monoclonal antibodies and other monoclonal antibodies may have further conserved amino acid substitutions that have no substantial effect on antigen-binding function or other immunoglobulin function. Humanized immunoglobulins can be constructed using genetic engineering (see, for example, U.S. Patent No. 5,585,089).

[0025] An "antagonist antibody" or "inhibitory antibody" is an antibody that interferes with one of the biological activities of its target polypeptide, such as the La protein.

[0026] Bone disease: encompasses any disease, defect, or disorder that affects the strength, function, and / or integrity of bone, for example, by reducing bone tensile strength and modulus. Examples of bone disease include, but are not limited to, bone fragility disorders such as osteoporosis, and genetic disorders that result in abnormal bone formation, such as McCune-Albright syndrome (MAS) and osteogenesis imperfecta. Other examples of bone disease include malignant diseases and / or cancers of the bone, such as sarcomas, such as osteosarcoma. Other bone diseases include Paget's disease of bone, fibrous dysplasia, rheumatoid arthritis, osteomyelitis, and osteopetrosis.

[0027] Bone Healing and Fracture Healing: Bone heals (fused) in a unique way compared to other connective tissues. Bone has the ability to completely regenerate itself rather than developing scar tissue. The majority of fractures heal through secondary fracture healing, which involves a combination of intramembranous ossification and endochondral ossification. While not bound by theory, it is generally believed that five distinct healing stages are involved in the fracture healing pathway. These include three sequential stages: an initial stage where hematoma is formed and inflammation occurs; a subsequent stage where cartilage formation begins and angiogenesis takes place; then, cartilage calcification, cartilage resorption and bone deposition; and finally, a longer-term bone remodeling stage. Generally, the process of fracture healing involves constrained bone progenitor cells and unconstrained, undifferentiated mesenchymal stem cells. Bone formed by intramembranous ossification is initially found distant from the fracture site, leading to the formation of callus and directly forming bone without first forming cartilage. Generally, cell proliferation decreases two weeks after a fracture, and hypertrophic chondrocytes become the dominant cell type in the callus. The resulting endochondral bone forms adjacent to the fracture site.

[0028] Bone resorption: Osteoclasts break down bone tissue, releasing minerals. Osteoclasts are generally located on the outer layer of bone, just below the periosteum. This process begins when osteoclasts attach to bone cells. Next, the osteoclasts induce invagination of their cell membranes, forming an isolated acidic microenvironment between themselves and the bone surface, and secrete collagenase, cathepsin K, and other enzymes that are important in the resorption process. As osteoclasts burrow through the calcified bone, high levels of calcium, magnesium, phosphate, and collagen products are released into the extracellular fluid.

[0029] Bisphosphonates are a class of drugs that work by slowing bone loss. Structurally, bisphosphonates are chemically stable derivatives of inorganic pyrophosphate (PPi), a naturally occurring compound in which two phosphate groups are linked by esterification. Bisphosphonates have a very high affinity for bone mineral deposits because they bind to hydroxyapatite crystals. Therefore, the intraskeletal storage of bisphosphonates depends on the availability of hydroxyapatite binding sites. Bisphosphonates are preferentially incorporated into sites of active bone remodeling, as is often seen in conditions characterized by accelerated skeletal turnover. Bisphosphonates inhibit bone resorption by attaching to hydroxyapatite binding sites on the bone surface and by shortening the lifespan of osteoclasts and / or preventing osteoclast precursors from associating with bone. For more information on bisphosphonates, see, for example, Drake et al., Mayo Clin. Proc. 83:1032-1045, 2008. In some cases, bisphosphonates are used in combination with the methods described herein.

[0030] Cas9: An RNA-guided DNA endonuclease enzyme capable of cleaving DNA. Cas9 has two active cleavage sites (HNH and RuvC), one for each strand of the double helix. This disclosure also includes catalytically inactive (inactivated) Cas9 (dCas9). In some examples, dCas9 contains one or more of the following point mutations: D10A, H840A, and N863A.

[0031] The nucleic acid and protein sequences of Cas9 are publicly available. For example, nucleotides 796693..800799 in GenBank® accession number CP012045.1 and nucleotides 1100046..1104152 in CP014139.1 disclose the Cas9 nucleic acid, while GenBank® accession numbers AMA70685.1 and AKP81606.1 disclose the Cas9 protein. In some cases, Cas9 is an inactivated form of Cas9 (dCas9), such as a nuclease-deficient form (e.g., shown in GenBank® accession numbers AKA60242.1 and KR011748.1). In certain cases, Cas9 maintains DNA cleavage ability while possessing at least 80% sequence identity to such sequences, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0032] Caspases: Enzymes that are cysteine-aspartate proteases, cysteine ​​aspartases, or cysteine-dependent aspartate-targeting proteases. Caspases are a family of protease enzymes that play a crucial role in programmed cell death. They are called caspases because of their specific cysteine ​​protease activity, in which the cysteine ​​in their active site nucleophilically attacks and cleaves target proteins only after the aspartate residue.

[0033] cDNA (complementary DNA): A piece of DNA lacking internal non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

[0034] Conservative Variants: A “conservative” amino acid substitution is one that substantially does not affect, or reduces, the activity of a polypeptide, such as the La protein’s ability to regulate osteoclast fusion. Non-exclusive examples of conservative substitutions include the following: TIFF2026053494000002.tif121137

[0035] The term "conservative variation" includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the polypeptide binds with the same affinity as the unsubstituted (parent) polypeptide. Non-conservative substitutions are those that reduce the polypeptide's ability to bind.

[0036] ~essentially consisting of and comprising: A polypeptide comprising an amino acid sequence that is essentially consisting of a specified amino acid sequence contains no additional amino acid residues. However, residues in the polypeptide can be modified to include non-peptide components, such as labels (e.g., fluorescent labels, radioactive labels, or solid particle labels), sugars, or lipids, and the N-terminus or C-terminus of the polypeptide can be joined to a linker for conjugation chemistry (e.g., by a peptide bond). A polypeptide comprising or essentially consisting of a specified amino acid sequence may be glycosylated or have amide modifications. A polypeptide comprising a specified amino acid sequence contains no additional amino acid residues, no additional biological components, such as nucleic acids, lipids, sugars, or labels.

[0037] The N-terminus or C-terminus of a polypeptide can be conjugated (e.g., by peptide bonds) to heterologous amino acids, such as peptide tags, or cysteine ​​(or other) residues, in relation to linkers for conjugation chemistry. A polypeptide consisting of or essentially derived from a specific amino acid sequence can be conjugated to an independent, distinct heterologous polypeptide via its N-terminus or C-terminus or linker, for example, in the case of a fusion protein containing a first polypeptide consisting of a first sequence linked (via peptide bonds) to a heterologous polypeptide consisting of a second sequence. In another example, the N-terminus or C-terminus of a polypeptide consisting of or essentially derived from a specific amino acid sequence can be conjugated (via peptide bonds) to a peptide linker further conjugated to one or more additional heterologous polypeptides. In yet another example, the N-terminus or C-terminus of a polypeptide consisting of or essentially derived from a specific amino acid sequence can be conjugated to one or more amino acid residues, such as a histidine tag, to facilitate further modification or manipulation of the polypeptide.

[0038] Control: Reference standard. In some embodiments, the control is a negative control sample obtained from healthy patients (e.g., patients without bone disease), or subjects treated with a carrier, untargeted nucleic acid sequence, scrambled nucleic acid / amino acid sequence, or untreated cells from healthy patients. In another embodiment, the control is a positive control sample obtained from patients treated with the active agent. In yet another embodiment, the control is a historical control or standard reference value or standard reference range (e.g., a previously tested control sample, e.g., a group of patients with known prognosis or outcome, or a group of samples representing baseline or normal values).

[0039] The difference between the test sample and the control may be an increase or a decrease. The difference may be qualitative or quantitative, such as statistically significant. In some cases, the difference is an increase or decrease of at least about 5%, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or more than 500% compared to the control.

[0040] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) editing system: A bacterial-based engineered nuclease system used for genome manipulation. It is partly based on the adaptive immune responses of many bacteria and archaea. Using such a method, it becomes possible to add, remove, or modify genetic material at specific locations in, for example, a target DNA sequence or target RNA sequence (e.g., La nucleic acid sequence). Thus, the CRISPR / Cas system can be used for nucleic acid targeting (e.g., DNA or RNA) to, for example, detect target DNA or target RNA, modify target DNA or target RNA at any desired location, or cleave target DNA or target RNA at any desired location. Thus, such a method can be used to modify the expression of the La protein, for example, by introducing mutations to silence expression, for example, by knocking out the La gene.

[0041] In one example, this method edits DNA, such as genomes, using the Cas9 nuclease. The Cas9 nuclease cleaves DNA, generating blunt ends at double-strand breaks at sites specified by the 20-nucleotide complementary strand sequence contained within the crRNA transcript. Thus, the CRISPR / Cas system can be manipulated to create double-strand breaks at desired targets in the cell's genome and to repair the induced breaks by homologous recombination repair (HDR) or non-homologous end ligation (NHEJ) using mechanisms inherent in the cell. In another example, this method edits RNA, such as La RNA, using the Cas13d nuclease. The Cas13d nuclease cleaves RNA (see, for example, WO 2019 / 040664).

[0042] Non-restrictive examples of Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas13d, Cpf1, C2c3, C2c2 and C2c1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc 2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and their homologs are included.

[0043] Degenerate variant: A polynucleotide encoding a peptide that contains a degenerate sequence as a result of the genetic code. There are 20 natural amino acids, most of which are specified by two or more codons. Therefore, all degenerate nucleotide sequences are included in this disclosure, insofar as the amino acid sequence of the polypeptide encoded by that nucleotide sequence remains unchanged.

[0044] Regulatory Sequence: A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which it is functionally ligated. A regulatory sequence is functionally ligated to a nucleic acid sequence if it controls and regulates the transcription and, where appropriate, translation of the nucleic acid sequence. Thus, a regulatory sequence may include appropriate promoters, enhancers, transcriptional terminators, start codons (i.e., ATGs) before protein-coding genes, splicing signals for introns, maintenance of the correct gene reading frame to enable proper translation of mRNA, and stop codons. The term "regulatory sequence" includes at least a component whose presence can influence expression, and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences. A regulatory sequence may include a promoter.

[0045] A promoter is the smallest sequence sufficient to direct transcription. It also includes promoter elements sufficient to control promoter-dependent gene expression in a cell-type specific, tissue-specific, or induced manner by external signals or agents, and such elements may be located in the 5' or 3' region of the gene. Both constitutive and inducible promoters are included (see, e.g., Bitter et al., 1987, Methods in Enzymology 153, 516-544). For example, when cloning in bacterial systems, inducible promoters such as the bacteriophage lambda pL, plac, ptrp, and ptac (ptrp-lac hybrid promoter) can be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., retroviral long-terminal repeats; adenovirus late promoter; vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA techniques or synthetic techniques can also be used to produce transcription of nucleic acid sequences.

[0046] Fibrous dysplasia of bone is a disorder characterized by bone loss due to the ectopic formation of osteoclasts that are excessively numerous and large in size, pathologically eroding the bone. Normal bone and bone marrow are then replaced by fibrous tissue, resulting in the formation of weak, swollen bone. Consequently, most complications result in fractures, deformities, functional impairment, and pain. Fibrous dysplasia of bone can affect one bone (monostotic), multiple bones (polyostotic), or all bones (panostotic), and can occur alone or in conjunction with café-au-lait spots and hyperactive endocrine disorders, known as McCune-Albright syndrome. Fibrous dysplasia of bone is a mosaic disorder resulting from post-junctional activating mutations in the GNAS gene locus, located at 20q13.2-q13.3 and encoding the α-subunit of the Gs G-coupled protein receptor. On X-rays, fibrous dysplasia of bone presents as foamy, soluble lesions or a frosted-glass appearance in the bone.

[0047] Heterogeneous: These components originate from different genetic sources and are therefore not found together in nature. These components can be host cells, genes, or regulatory regions such as promoters. While heterogeneous components are not found together in nature, they can function together, for example, when a promoter heterogeneous to a particular gene is ligated to that gene.

[0048] Host cell: A cell in which a vector can grow and express its DNA. The cell can be a prokaryotic or eukaryotic cell. The cell can be a mammalian cell, such as a human cell. This term also includes any offspring of the target host cell. It is understood that not all offspring are identical to the parent cell because mutations can occur during replication. However, when the term "host cell" is used, such offspring are included.

[0049] To inhibit or treat a disease: Treating a disease, for example, but not limited to, osteoporosis or osteopetrosis, means inhibiting the complete progression of the disease. In some cases, inhibiting a disease means reducing the symptoms of that particular disease. "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or a pathological condition associated with that disease. Treatment can be measured using any objective or subjective parameters, such as success or signs of success in reducing or improving an injury, condition, or state, including improvement, remission, reduction of symptoms or making the condition tolerable for the patient, slowing the rate of degeneration or decline, or reducing the debilitating nature of the final stage of degeneration, or improving the physical condition of the subject. Treatment can be evaluated by objective or subjective parameters, such as the results of physical examinations or biological tests.

[0050] Inhibitory nucleic acid molecules include inhibitory RNA molecules and inhibitory DNA molecules such as antisense oligonucleotides, siRNAs, microRNAs (miRNAs), shRNAs, or ribozymes. The use of any type of antisense compound that specifically targets and controls the expression of the nucleic acid encoding La is envisioned. La antisense compounds are compounds that specifically hybridize with the La nucleic acid molecule to regulate its expression. These compounds can be introduced as single-stranded, double-stranded, cyclic, branched, or hairpin compounds and may contain structural elements such as internal or terminal bulges or loops. Double-stranded antisense compounds can be two strands forming a double-stranded compound, or a single strand with sufficient self-complementarity to allow hybridization and the formation of a fully or partially double-stranded compound. In some examples, an antisense oligonucleotide is a single-stranded antisense compound such that the antisense oligonucleotide hybridizes with the mRNA encoding the La protein, and the resulting double helix is ​​recognized by RNaseH, causing mRNA cleavage. In some cases, miRNAs are single-stranded RNA molecules, e.g., about 21–23 nucleotides long, at least partially complementary to mRNA molecules, that regulate gene expression via the RNAi pathway. In further cases, shRNAs are RNA oligonucleotides that are cleaved into siRNAs, forming a tight hairpin. siRNA molecules are generally about 15–40 nucleotides long, e.g., 20–25 nucleotides long, and may have 0–5 nucleotide overhangs at the 3' or 5' ends, or they may be blunt-ended. Generally, one strand of the siRNA is at least partially complementary to the nucleic acid molecule encoding the La protein. Antisense compounds that specifically target the La gene can be prepared by designing compounds complementary to the target nucleotide sequence, such as the mRNA sequence. Antisense compounds can specifically hybridize to the target and regulate its expression even if they are not 100% complementary to the nucleic acid molecule encoding the La protein.For example, if an antisense compound or a double-stranded compound is used, the antisense strand of the compound can be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% complementary to the nucleic acid molecule encoding the La protein (e.g., SEQ ID NO: 3). Methods for screening antisense compounds with respect to specificity are known (see, for example, U.S. Patent Application Publication No. 2003-0228689).

[0051] Isolated: An “isolated” biological component (e.g., a nucleic acid molecule, protein, or organelle) is substantially separated or purified from other biological components in the organism’s cells that naturally contain it, namely other chromosomes and extrachromosomal DNA and RNA, proteins, and organelles. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. This term also encompasses nucleic acids and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids and proteins.

[0052] Lupus autoantigen (La) protein: In vivo, the La protein is a 47 kDa polypeptide that often behaves as an autoantigen in patients with systemic lupus erythematosus and Sjögren's syndrome. The La protein is primarily located in the nucleus of cells. In the nucleus, the La protein acts as an RNA polymerase III (RNAP III) transcription factor by participating in tRNA production and binding to the U-rich 3'UTR of nascent transcripts to assist their folding and maturation. In the cytoplasm, the La protein acts as a translation factor by facilitating the translation of specific mRNAs. As an RNA-binding protein (RBP), La associates with a subset of mRNAs containing the 5'-terminal oligopyrimidine (5'TOP) motif, which is known to control protein synthesis. The binding of La to specific RNA molecular classes controls their downstream processes, protects them from endonuclease digestion, and coordinates their export from the nucleus. This specification discloses that the La protein regulates osteoclast fusion.

[0053] Exemplary La protein sequences and the nucleic acid sequences encoding these protein sequences are disclosed herein and also disclosed in GENBANK® accession numbers NP_003133, February 15, 2021, NM_003142, February 15, 2021, and NM_001294145, February 15, 2021, all of which are incorporated herein by reference.

[0054] Label: A detectable compound or composition that is directly or indirectly conjugated to another molecule to facilitate its detection. Non-exclusive examples of labels include fluorescent tags, enzyme linkage, and radioisotopes.

[0055] Mammals: This term encompasses both humans and non-human mammals. Similarly, the term "subject" includes both human subjects and veterinary subjects.

[0056] Adjustment: To modify something to make it statistically significant. Adjustment can be an increase or a decrease. Those skilled in the art can identify appropriate assays for determining a significant increase or decrease in a parameter. These include, but are not limited to, Student's t-test or the paired ratio t-test. Exemplary methods are described in the Examples section.

[0057] Osteoblasts: Mononuclear cells responsible for bone formation. Osteoblasts produce osteoid, which is mainly composed of type I collagen. Osteoblasts are also responsible for the calcification of the osteoid matrix. Bone is a dynamic tissue that is constantly being rebuilt by osteoblasts, which create bone, and osteoclasts, which absorb bone. Osteoblasts arise from bone progenitor cells located in the periosteum and bone marrow. Bone progenitor cells are immature progenitor cells that express the master regulatory transcription factor Cbfa1 / Runx2. Once bone progenitor cells begin to differentiate into osteoblasts, they begin to express a range of markers, including osterix, type I collagen, alkaline phosphatase, osteocalcin, osteopontin, and osteonectin.

[0058] Osteoclasts: A type of osteocyte that removes bone tissue by removing the calcified matrix of bone tissue through the process of bone resorption and the breakdown of the organic phase of osteoid. Osteoclasts are formed by the fusion of cells from the monocyte-macrophage cell lineage. Osteoclast formation consists of several steps, including precursor survival, differentiation into mononuclear preosteoclasts, and fusion into multinucleated mature osteoclasts. Classically, osteoclast fusion involves four basic stages: (1) attraction / migration, (2) recognition, (3) cell-cell adhesion, and (4) membrane fusion. Osteoclasts are characterized by high expression of tartrate-resistant acid phosphatases and cathepsin K, and the process of osteoclast formation is evaluated by the detection of specific osteoclast-generating transcription factors (e.g., nuclear factor of activated T-cells, cytoplasmic 1). A single osteoclast can contain 3 to 100 nuclei, and their diameter varies from 10 to 300 μM. In humans, mature osteoclasts typically have 4 to 8 nuclei per cell.

[0059] Osteocytes: Mature, non-dividing osteocytes housed in their own foveae (small lumens within the bone). Osteocytes originate from osteoblasts and represent the final stage of maturation in the osteocyte lineage. They are less active than osteoblasts and do not contribute to the net increase of bone matrix, but they are essential for matrix maintenance, normal turnover, and play a crucial role in osteoclast production. The slender cytoplasmic processes of osteocytes maintain attachment to each other and to osteoblasts via tubules (small channels in the bone).

[0060] Osteomyelitis: Inflammation of the bone. Symptoms may include redness of the surface, fever, and pain in certain bones accompanied by weakness. The cause is usually a bacterial infection, but it can also be a fungal infection. Osteomyelitis is accompanied by the formation and increased activity of osteoclasts, which absorb bone, as well as bone loss.

[0061] Osteopetrosis: A rare genetic disorder characterized by hardening and densification of bone, also known as marble bone disease or Albers-Schoenberg disease. Osteopetrosis can cause bone resorption and fracture. Human osteopetrosis is a heterogeneous disorder encompassing various molecular lesions and a range of clinical features, but all forms share a single pathogenic nexus to osteoclasts. Individuals with osteopetrosis experience insufficient bone resorption, resulting in too little bone being absorbed and too much bone being created. Osteopetrosis is classified into two groups of disorders: autosomal dominant osteopetrosis and autosomal recessive osteopetrosis (ARO), which is more severe and sometimes fatal and is also known as infantile malignant osteopetrosis (see Sobacchi et al., Nat Rev Endocrinol.9, 522-536, and Penna et al. Autosomal recessive osteopetrosis: mechanisms and treatments. Dis Model Mech.2021 May 1;14(5):dmm048940.doi:10.1242 / dmm.048940 PMCID:PMC8188884;PMID:33970241). ARO disorder has an incidence of 1 in 250,000 births and arises from mutations in various genes involved in osteoclast formation, including loss-of-function mutations in the genes encoding TNFSF11 or TNFRSF11A, which impair the expression of RANKL or its receptor RANK, respectively (osteoclast-poor osteopetrosis). Another type of osteoclast-rich osteopetrosis disorder, called osteoclast-rich osteopetrosis, arises from defects in osteoclast function caused by mutations in the TCIRG1, CLCN7, or OSTM1 genes, which encode type V proton ATPase or chloride ion voltage-gated channel 7 or osteoclastogenesis-associated transmembrane protein 1, respectively.Most cases of osteopetrosis (ARO) are caused by the presence of inactive osteoclasts ("hypoosteoclastic ARO"), including 5% of ARO cases associated with mutations in SNX10, which encodes the protein sortin nexin-10 ("hypoosteoclastic ARO"). (See Sobacchi et al., op. cit., Penn et al., op. cit.) Low osteopetrosis has been disclosed, for example, by Penn et al., op. cit.

[0062] Osteoporosis: A systemic skeletal disorder characterized by low bone mass, deterioration of the microstructure of bone tissue leading to bone fragility, and a resulting increased risk of fracture. In older adults, this is the most common reason for bone breakage. The main consequence of osteoporosis is an increased risk of fracture. Osteoporotic fractures occur in situations where healthy people would not normally fracture, and therefore they are considered fragility fractures. Typical fragility fractures occur in the spine, ribs, hips, and wrists. The World Health Organization (WHO) defines osteoporosis as bone mineral density 2.5 standard deviations lower than the reference bone mineral density (i.e., generally healthy young adults around 30 years of age). "Osteopenia" refers to a decrease in bone mineral density that is not as severe as osteoporosis, and is detected by appropriate diagnostic procedures such as radiography, regardless of whether osteoporosis is present. The WHO defines osteopenia as bone mineral density 1 to 2.5 standard deviations lower than the reference bone mineral density mentioned above.

[0063] Open Reading Frame (ORF): A sequence of nucleotide triplets (codons) that encode amino acids without any internal stop codons. These sequences are typically translated into proteins.

[0064] Functionally linked: A first nucleic acid sequence is functionally linked to a second nucleic acid sequence if the first nucleic acid sequence is positioned in a functional relationship with it. For example, if a promoter affects the transcription or expression of a coding sequence, such as a polypeptide sequence, then the promoter is functionally linked to that coding sequence. In general, functionally linked DNA sequences are contiguous and, if two protein-coding regions need to be joined, they are in the same reading frame.

[0065] Paget's disease of bone: A chronic bone disorder typically found in the spine, pelvis, long bones of the limbs, and skull, characterized by excessive bone destruction and regrowth in the affected area. This is because the bone in these areas is excessively eroded by ectopic formation of osteoclasts that are too numerous and too large in size. Subsequently, the bone undergoes excessively rapid regrowth, resulting in bone that is larger and softer than normal, may be deformed, and easily fractured. These areas can be treated with bisphosphonates and / or calcitonin in combination with methods provided herein, for example. The first stage of this disorder is characterized by increased bone resorption in the lesion area, and osteolytic lesions are commonly detected on radiographic examinations. The osteoclasts are larger and have more nuclei than normal adult osteoclasts. Following the excessive bone resorption, increased bone formation occurs, which is characterized by an increase in the number of osteoblasts that appear normal. However, the rapidly deposited bone has a structurally disordered appearance, exhibiting soft and porous characteristics, which explains the increased risk of skeletal deformity and fracture. Reflecting the increased rate of bone remodeling, there are elevated serum alkaline phosphatase levels and elevated urinary excretion levels of hydroxypurine and pyridinoline.

[0066] Nucleic acid molecules: Polymers composed of nucleotide units (ribonucleotides, deoxyribonucleotides, their associated native structural variants, and synthetic non-natural analogs) linked by phosphodiester bonds, their associated native structural variants, and synthetic non-natural analogs. Therefore, this term includes nucleotide polymers in which the nucleotides and the links between them are unnatural synthetic analogs, such as, but are not limited to, phosphorothioates, phosphoramides, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). Such polynucleotides can be synthesized, for example, using automated DNA synthesizers. The term "oligonucleotide" typically refers to short polynucleotides, generally of about 50 nucleotides or less. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes RNA sequences (i.e., A, U, G, C) in which "T" is replaced by "U".

[0067] This specification uses conventional annotations to describe nucleotide sequences. That is, the left end of a single-stranded nucleotide is the 5' end, and the left-handed direction of a double-stranded nucleotide sequence is called the 5' direction. The direction of 5'→3' nucleotide addition to a nascent RNA transcript is called the transcription direction. The DNA strand having the same sequence as mRNA is called the "coding strand". The sequence located 5' to the sequence of interest on a nucleic acid sequence is called the "upstream sequence", and the nucleotide sequence located 3' to the sequence of interest is called the "downstream sequence".

[0068] "Code" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, that it serves as a template for synthesizing other polymers and macromolecules that have either a given nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a given amino acid sequence, along with the biological properties it imposes, in a biological process. Therefore, if the transcription and translation of mRNA produced by a gene in a cell or other biological system produce a protein, then that gene codes for a protein. Both the coding strand, i.e., the nucleotide sequence identical to the mRNA sequence and usually listed in sequence listings, and the non-coding strand used as a template for the transcription of a gene or cDNA, can be said to code for a protein or other products of that gene or cDNA. Unless otherwise explicitly stated, "nucleotide sequences that code for an amino acid sequence" encompass all nucleotide sequences that are degenerate of each other and code for the same amino acid sequence. Nucleotide sequences that code for proteins and RNA may contain introns.

[0069] "Recombinant nucleic acids" refer to nucleic acids that have nucleotide sequences that, compared to, for example, wild-type genes, are not naturally joined together. This includes nucleic acid vectors containing amplified or assembled nucleic acids that can be used to transform suitable host cells. In one example, recombinant nucleic acids have sequences that are not naturally occurring, or sequences created by the artificial combination of two sequence segments that would normally be separated. This artificial combination is often achieved by chemical synthesis or by artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques. Host cells containing recombinant nucleic acids are called "recombinant host cells." Recombinant nucleic acids may also provide non-coding functions (such as promoters, origins of replication, and ribosome binding sites).

[0070] If a polynucleotide having the first sequence specifically hybridizes with a polynucleotide having the second sequence, then the first sequence is "antisense" to the second sequence. Therefore, these two sequences are complementary.

[0071] Pharmacopoeia-acceptable carriers: Useful pharmaceutically acceptable carriers are conventional. EW Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 15th edition (1975) describes compositions and formulations suitable for the pharmaceutically active substance disclosed herein (e.g., La protein, or substances that modulate the function or activity of La protein).

[0072] Generally, the properties of the carrier will depend on the specific administration method used. For example, parenteral formulations typically contain an injectable fluid that includes a pharmaceutically and physiologically acceptable fluid as a vehicle, such as water, saline, equilibrium salt solutions, dextrose aqueous solution, or glycerol. In the case of solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers include, for example, pharmaceutically-grade mannitol, lactose, starch, or magnesium stearate. In addition to the biologically neutral carrier, the administered pharmaceutical composition may also contain trace amounts of non-toxic auxiliary substances, such as sodium acetate or sorbitan monolaurate, such as wetting or emulsifying agents, preservatives, and pH buffers.

[0073] The "therapeutic dose" is the amount of composition or cells necessary to achieve the desired effect in the subject being treated. For example, this could be the amount required to influence osteoclast fusion. When administered to a subject, the dosage used is generally such that a target tissue concentration, which has been shown to achieve an in vitro effect, is achieved.

[0074] Polypeptide: Any chain of amino acids, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). With respect to polypeptides and proteins, the word "approximately" indicates an integer quantity. Therefore, in one example, a polypeptide with "approximately" 29 amino acids is 28-30 amino acids long. Thus, a polypeptide having "approximately" a specified number of residues may be one amino acid shorter or one amino acid longer than that specified number. A fusion polypeptide contains the amino acid sequences of a first polypeptide and a second different polypeptide (e.g., a heterologous polypeptide) and can be synthesized as a single amino acid sequence. Recombinant polypeptides have amino acid sequences that do not exist naturally, or amino acid sequences made up of two segments of an amino acid sequence that would normally be separated.

[0075] Promoter: A series of nucleic acid regulatory sequences that direct the transcription of a nucleic acid. A promoter includes essential nucleic acid sequences close to the transcription start site, such as the TATA element in the case of a polymerase type II promoter. Optionally, a promoter may also include distal enhancer or repressor elements, which may be located thousands of base pairs away from the transcription start site.

[0076] Purified: The polypeptides and polynucleotides disclosed herein can be purified (and / or synthesized) by any means known in the art (see, for example, Guide to Protein Purification, edited by Deutscher, Meth.Enzymol.185, Academic Press, San Diego, 1990, and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982). Substantially purified means purification from other proteins, nucleic acid molecules, or cellular components. A substantially purified protein is at least about 60%, 70%, 80%, 90%, 95%, 98%, or 99% pure. Thus, in one non-limiting specific example, a substantially purified protein contains 90% less other proteins or cellular components. Thus, the term purified is a relative term, not requiring absolute purity.

[0077] Similarly, purified nucleic acids are those in which the nucleic acid is more concentrated than it would be in its natural environment within a cell.

[0078] In some cases, the purified populations of nucleic acids, proteins, or cells are approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, or free from other nucleic acids, proteins, or cells, respectively.

[0079] Nuclear factor kappa B-activated receptor ligand (RANKL): A protein encoded by the TNFSF11 gene in humans. RANKL binds to RANK on cells in the myeloid cell lineage and functions in osteoclast differentiation and activation. RANKL can also bind to osteoprotegerin, a protein secreted primarily by cells in the osteoblast lineage. Osteoprotegerin is a potent inhibitor of osteoclast formation by preventing RANKL from binding to RANK. RANKL also has a function in the immune system, expressed by T helper cells and thought to be involved in dendritic cell maturation. It is a dendritic cell survival factor and helps regulate the T cell-dependent immune response. T cell activation induces RANKL expression, which can lead to increased osteoclast formation and bone loss. Exemplary amino acid and nucleic acid sequences of human RANKL can be found in GENBANK accession number NM_003701.4, February 16, 2021, incorporated herein by reference.

[0080] Rheumatoid arthritis is a chronic autoimmune disorder that affects the joints, causing inflammation of the synovial membrane. The joints become swollen, tender, warm to the touch, and stiff, limiting their movement. Over time, multiple joints may be affected (polyarthritis). Most commonly, the small joints of the hands, feet, and cervical spine are affected, but larger joints such as the shoulders and knees may also be affected. Bone damage in rheumatoid arthritis is mainly caused by increased differentiation and activity of osteoclasts.

[0081] Sequence Identity: The similarity between amino acid sequences is expressed by sequence similarity, also known as sequence identity. Sequence identity is often measured by an identity (or similarity or homology) percentage, with a higher percentage indicating greater similarity between the two sequences. Homologs or variants of polypeptides will exhibit a relatively high degree of sequence identity when aligned using standard methods.

[0082] Methods for aligning sequences for comparison are well known. Various programs and alignment algorithms are described in Smith and Waterman, Adv.Appl.Math.2:482, 1981; Needleman and Wunsch, 1970, J Mol Biol 48, 443-453; Higgins and Sharp, 1988, Gene 73, 237-244; Higgins and Sharp, 1989, CABIOS 5, 151-153; Corpet et al., 1988, Nucleic Acids Research 16, 10881-10890; and Pearson and Lipman, 1988, Proc Natl Acad Sci USA 85, 2444-2448. Altschul et al., 1994, Nature Genet 6, 119-129, provides a detailed discussion of sequence alignment methods and homology calculations.

[0083] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990, J Mol Biol 215, 403-410) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Maryland), and can be accessed via the Internet for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to determine sequence identity using this program can be found on the NCBI website.

[0084] Polypeptide homologs and variants typically retain at least 75%, or even at least 80%, of sequence identity when counted in full-length alignment with the polypeptide amino acid sequence using NCBI Blast 2.0, gapped blastp with default parameters. For comparing amino acid sequences longer than approximately 30 amino acids, use the default BLOSUM62 matrix and the Blast 2 sequences function with default parameters (gap existence cost of 11 and per residue gap cost of 1). For aligning shorter peptides (less than approximately 30 amino acids), the PAM30 matrix should be used and the Blast 2 sequences function should be used with default parameters (open gap penalty of 9, extension gap penalty of 1). Proteins with greater similarity to the reference sequence will show increased identity percentages when evaluated by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When the sequence being compared for sequence identity is shorter than the entire sequence, homologs and variants typically have at least 80% sequence identity within a short window of 10–20 amino acids, and can have at least 85%, 90%, or 95% sequence identity depending on their respective similarity to the reference sequence. Methods for determining sequence identity within such short windows are available on the NCBI website. It will be understood by those skilled in the art that these sequence identity ranges are provided only as guidelines, and there is a good chance that extremely important homologs will be obtained that do not fall within these ranges.

[0085] Therefore, in some examples, polypeptide or nucleic acid sequence variants typically retain at least about 75%, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, counted by full-length alignment with the amino acid or nucleotide sequence of interest. Sequences with greater similarity to the reference sequence will show increased identity percentages when evaluated in this manner, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When the sequence being compared for sequence identity is shorter than the entire sequence, homologs and variants typically have at least 80% sequence identity in a short window of 10–20 amino acids (or 30–60 nucleotides), and may have at least 85%, or at least 90%, or 95%, sequence identity depending on their respective similarity to the reference sequence. Methods for determining sequence identity within such a short window are available on the NCBI website.

[0086] In this specification, when we refer to "at least 90% identity," it (or a similar expression) means "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" with respect to a given reference sequence. Therefore, a La protein (or fragment thereof) having at least 90% sequence identity with SEQ ID NO: 1, 2, 7, 8, or 9 is considered to have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity with respect to SEQ ID NO: 1 or 2. Similarly, an La-coded sequence having at least 90% sequence identity to SEQ ID NO: 3 is one that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identity to SEQ ID NO: 3.

[0087] Subjects: Living multicellular vertebrates, this category includes humans and non-human mammals, such as non-human primates, rats, mice, dogs, cats, horses, cattle, and pigs. In one example, the subject is a human. In a further example, subjects that require regulation of osteoclast fusion are selected. For example, a subject may require an increase or decrease in osteoclast fusion, or an increase or decrease in bone resorption.

[0088] A vector is a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may contain nucleic acid sequences, such as an origin of replication, that enable it to replicate in the host cell. A vector may also contain one or more selectable marker genes and other genetic elements known in the art. Vectors include plasmid vectors, such as plasmids for expression in Gram-negative and Gram-positive bacterial cells. Exemplary vectors include vectors for expression in Escherichia coli (E. coli) and Salmonella. Vectors also include viral vectors, such as, but are not limited to, retroviruses, lentiviruses, adeno-associated viruses (AAV), orthopoxviruses, tripoxviruses, fowlpox viruses, capripoxviruses, porcineviruses, adenoviruses, herpesviruses, alphaviruses, baculoviruses, Sindbisviruses, vaccinia viruses, and polioviruses. Vectors also include vectors for expression in yeast cells or mammalian cells.

[0089] Virus: A microscopic infectious organism that replicates within living cells. Viruses essentially consist of a core of single nucleic acid surrounded by a protein coat and possess the ability to replicate only within living cells. "Viral replication" refers to the production of a new virus through the occurrence of at least one viral life cycle.

[0090] II. La protein and agents that increase the function and / or activity of La protein for use in increasing osteoclast fusion Methods for increasing osteoclast fusion are disclosed herein. These methods can be used to increase bone resorption in a subject. These methods may utilize La protein, or agents that increase the expression or activity of La protein, such as nucleic acid molecules encoding La protein. Exemplary agents useful in these methods are disclosed below.

[0091] A. La Protein This specification discloses that the addition of "cleaved" recombinant La, which corresponds to the amino acid sequence of the La protein but lacks the carboxyl-terminal nuclear localization sequence, or the overexpression of "cleaved" La, vigorously promotes osteoclast fusion. Any form of the La protein that functions to promote osteoclast fusion is useful in the disclosed method.

[0092] An example human La protein sequence is shown below. Regarding the positional numbers in TIFF2026053494000003.tif54128, the N-terminal amino acid is assigned position 1, and the remaining positions are assigned sequential numbers to the amino acids.

[0093] In some embodiments, the useful La protein contains or consists of amino acids 1-375 of SEQ ID NO: 1. In a further embodiment, the useful La protein contains or consists of amino acids 188-375 of SEQ ID NO: 1. In a non-limiting specific example, the La protein contains, essentially consists of, or comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7.

[0094] Any fragment or variant of the La protein is useful in the methods disclosed herein, insofar as it functions to promote osteoclast fusion. In some embodiments, the La protein useful in the disclosed methods contains a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, and promotes osteoclast fusion. The La protein may contain up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative substitution in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. In some examples, the La protein is SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, containing 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative substitution. The La protein useful in the methods disclosed herein is either naturally occurring or recombinant.

[0095] In some embodiments, the La protein contains, essentially consists of, or is composed of, an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2, and this La protein is at most 375 amino acids long and does not contain amino acids 376-408 of SEQ ID NO: 1. In further embodiments, the La protein contains the amino acid sequence of SEQ ID NO: 2, is at least 95% identical to SEQ ID NO: 1, and the substitutions are located only within amino acids 376-408 of SEQ ID NO: 2. In some embodiments, the substitutions are conservative substitutions.

[0096] In a further embodiment, the La protein contains, essentially consists of, or is composed of, an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7, and this La protein does not contain amino acids 1-187 and 376-408 of SEQ ID NO: 1. In a further embodiment, the La protein contains the amino acid sequence of SEQ ID NO: 7, is at least 95% identical to SEQ ID NO: 1, and the substitutions are present only within amino acids 1-187 and 376-408 of SEQ ID NO: 1. In some embodiments, the substitutions are conserved substitutions.

[0097] In a further embodiment, the La protein contains, is essentially, or consists of, amino acids 300-375 and / or amino acids 6-242 of SEQ ID NO: 1, and this La protein does not contain amino acids 376-408 of SEQ ID NO: 1. In a further embodiment, the La protein contains, is essentially, or consists of, amino acids 300-375 and / or amino acids 6-242 of SEQ ID NO: 1, and includes a substitution within amino acids 376-408 of SEQ ID NO: 1. In some embodiments, the substitution is a conservative substitution.

[0098] In the disclosed method, La protein fragments are also useful, in which case the fragments increase osteoclast fusion. The La protein can contain deletions of up to 5 amino acids, e.g., 1, 2, 3, 4, or 5 amino acids. In some embodiments, the La protein can contain deletions of up to 3 amino acids, e.g., 1, 2, or 3 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. In some embodiments, the deletion is within amino acids 376-408 of SEQ ID NO: 1. In a non-limiting example, the fragment is between amino acids 6-242 of SEQ ID NO: 1.

[0099] Useful La proteins can be prepared using recombinant methods, such as expression in host cells. Exemplary nucleic acid molecules can be prepared by cloning techniques (see below). Examples of appropriate cloning and sequencing techniques, and instructions sufficient to guide those skilled in the art who have performed many cloning operations, are publicly available (see, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, up to supplement 104, 2013)).

[0100] Suitable hosts include microorganisms, yeasts, insects, and mammals. Methods for expressing DNA sequences containing eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archaea, insects, fungi (e.g., yeast), plant, and animal cells (e.g., mammalian cells, e.g., human cells). Useful exemplary cells include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, HEK293 cells, Neurospora, and immortalized myeloid and lymphoid cell lines of mammals. Techniques for growing mammalian cells in culture are well known (see, e.g., Helgason and Miller, eds., 2012, Basic Cell Culture Protocols (Methods in Molecular Biology), 4th edition, Humana Press). Commonly used mammalian host cell lines include VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, but cell lines such as those designed to obtain higher expression levels, desired glycosylation patterns, or other characteristics may also be used. In some embodiments, the host cell may be HEK293 cells or their derivatives, e.g., GnTI - / - This includes cells (ATCC® number CRL-3022) or HEK-293F cells.

[0101] Transformation of host cells with recombinant DNA can be carried out by conventional techniques. In some embodiments where the host is a prokaryotic host (but not limited to, such as E. coli), competent cells with DNA uptake ability can be prepared from cells harvested after the exponential growth phase and treated with CaCl2. Alternatively, heat shock, MgCl2, or RbCl can be used. Transformation can be carried out after protoplast formation in the host cells, if desired, or by electroporation.

[0102] When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical procedures such as microinjection, electroporation, insertion of liposome-encapsulated plasmids, or viral vectors can be used. Eukaryotic cells can also be co-transformed with a polynucleotide sequence encoding the antigen of this disclosure and a second exogenous DNA molecule encoding a selectable phenotype, such as a herpesthymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells with eukaryotic viral vectors, e.g., Simian virus 40 (SV40) or bovine papillomavirus, to express the protein (see, e.g., Viral Expression Vectors, Springer press, Muzyczka, ed., 2011). Suitable expression systems, such as plasmids and vectors, that are useful for producing proteins in cells, including higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0103] Nucleic acid molecules and vectors encoding B. La Nucleic acid molecules encoding La proteins, variants, or fragments are also useful in the disclosed method. By introducing nucleic acids encoding La proteins, variants, or fragments, the amount of La protein increases, and therefore the activity of the La protein also increases.

[0104] Nucleic acid molecules can be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustained sequence replication systems (3SR). A wide variety of cloning methods, host cell and in vitro amplification methods are well known to those skilled in the art. RNA molecules are also useful.

[0105] Polynucleotides encoding La proteins, variants, or fragments can include recombinant DNA that is incorporated into a vector (e.g., an expression vector), incorporated into an autonomously replicating plasmid or virus, incorporated into the genomic DNA of a prokaryotic or eukaryotic organism, or recombinant DNA that exists as a separate molecule independent of other sequences (e.g., cDNA). Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. This term encompasses both single-stranded and double-stranded DNA. An exemplary nucleic acid molecule encoding SEQ ID NO: 1 is listed below. The following is an example nucleic acid sequence encoding peptide 2 (see below) in TIFF2026053494000006.tif172154. The following is an example nucleic acid sequence encoding peptide 2 (see below) in TIFF2026053494000007.tif10140. TIFF2026053494000008.tif10153

[0106] Polynucleotides encoding La proteins, their variants, or fragments useful in the disclosed methods include DNA, cDNA, and RNA sequences encoding La proteins. Silent mutations in coding sequences are due to the degeneracy (i.e., redundancy) of the genetic code, where two or more codons can code for the same amino acid residue. For example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT, or AAC; aspartic acid can be encoded by GAT, or GAC; cysteine ​​can be encoded by TGT, or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA, or CAG; tyrosine can be encoded by TAT, or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (e.g., L. Stryer, 1988, Biochemistry, 3rd edition, WH5 Freeman and Co., NY). Degenerate variants are also useful in the methods disclosed herein.

[0107] Those skilled in the art can easily use the amino acid sequences and genetic code provided herein to construct further nucleic acid molecules encoding La proteins, variants, or fragments. Nucleic acid sequences encoding La proteins, variants, or fragments can be synthesized by any suitable method, for example, by cloning of a suitable sequence, or by direct chemical synthesis using methods such as the phosphotryster method described in Narang et al., Meth.Enzymol.68:90-99, 1979, the phosphodiester method described in Brown et al., Meth.Enzymol.68:109-151, 1979, the diethylphosphoramidite method described in Beaucage et al., Tetra.Lett.22:1859-1862, 1981, or the solid-phase phosphoramidite triester method described in Beaucage & Caruthers, Tetra.Letts.22(20):1859-1862, 1988, for example, Needham-VanDevanter et al., Nucl.Acids Res.12:6159-6168. These can be prepared using automated synthesis apparatuses as described in 1984, etc., and the solid support method described in U.S. Patent No. 4,458,066. Chemical synthesis produces single-stranded (ss) oligonucleotides, which can be converted to double-stranded (ds) DNA by hybridization with a complementary sequence or by polymerization using a DNA polymerase that uses the single strand as a template. Exemplary nucleic acids containing sequences encoding La proteins, variants, or fragments can be prepared by cloning techniques.

[0108] Nucleic acid molecules encoding La proteins, variants, or fragments can be cloned or amplified by in vitro methods such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification (TAS), self-sustaining sequence replication (3SR), and Qβ replicase amplification (QB). For example, polynucleotides encoding La proteins, variants, or fragments can be isolated by polymerase chain reaction of cDNA using primers based on the molecule's DNA sequence. A wide variety of cloning and in vitro amplification methods can be used. The PCR method is described, for example, in U.S. Patent No. 4,683,195, Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987, and Erlich (ed.), PCR Technology (Stockton Press, NY, 1989). Polynucleotides can also be isolated by screening a genomic library or cDNA library with probes selected from the sequence of the desired polynucleotide under stringent hybridization conditions.

[0109] Typically, a polynucleotide sequence encoding a La protein, variant, or fragment is functionally linked to a transcriptional regulatory sequence, such as a promoter and a polyadenylation signal. Any promoter, which is a polynucleotide sequence recognized by the host cell's transcriptional mechanism (or introduced synthetic mechanism) involved in transcription initiation, can be used. A polyadenylation signal is a polynucleotide sequence that instructs the addition of a series of nucleotides to the end of an mRNA transcript for proper processing and translation-ready transport from the nucleus to the cytoplasm.

[0110] Exemplary promoters include viral promoters, such as the cytomegalovirus pre-early gene promoter ("CMV"), herpes simplex virus thymidine kinase ("tk"), SV40 early transcription unit, polyomas, retroviruses, papillomaviruses, hepatitis B virus, and human and monkey immunodeficiency viruses. Other promoters include promoters isolated from mammalian genes, such as immunoglobulin heavy chain, immunoglobulin light chain, T cell receptor, HLA DQα and DQβ, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II, HLA-DRα, β-actin, muscle creatine kinase, prealbumin (transthyretin), elastase I, metallothionein, collagenase, albumin, fetoprotein, β-globin, c-fos, c-HA-ras, neuronal adhesion molecules (NCAM), α1-antitrypsin, H2B (TH2B) histone, type I collagen, glucose regulatory proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TNI), platelet-derived growth factor, and dystrophin, as well as osteocyte-specific promoters.

[0111] Promoters can be inductive or constitutive. Inductive promoters are those that are inactive or exhibit low activity unless an inducer is present. Further examples of promoters include, but are not limited to, the promoters of MT II, ​​MMTV, collagenase, stromelysin, SV40, the mouse MX gene, α-2-macroglobulin, the MHC class I gene h-2kb, HSP70, proliferin, tetracycline-inducible, tumor necrosis factor, or the thyroid-stimulating hormone gene. An example of an inductive promoter is the interferon-inducible ISG54 promoter (see Bluyssen et al., Proc. Natl Acad. Sci. 92:5645-5649, 1995, incorporated herein by reference). In some embodiments, promoters are constitutive promoters that, once introduced into host cells, result in high levels of transcription even without the presence of additional factors. In some embodiments, the promoter is a bone-specific promoter such as the osteocalcin promoter, osteopontin promoter, or osteonectin promoter (see also Grienberg and Benayahu, BMC Genomics, 6, 46(2005), doi.org / 10.1186 / 1471-2164-6-46 005, which is incorporated herein by reference in its entirety). Furthermore, in some embodiments, the promoter is an osteoclast or osteoclast precursor-specific promoter, such as the cathepsin K promoter, tartrate-resistant acid phosphatase promoter, lysozyme M promoter, nuclear factor κB-activated receptor promoter, macrophage surface antigen 1 promoter, or macrophage colony-stimulating factor receptor promoter. See the internet ncbi.nlm.nih.gov / pmc / articles / PMC6397767 / .

[0112] Optionally, the transcriptional regulatory sequence may contain one or more enhancer elements. These are binding recognition sites for one or more transcription factors that increase transcription beyond what is observed with minimal promoter alone, and these are also functionally ligated to the polynucleotide encoding the nucleic acid molecule encoding the La protein. For the nucleic acid molecule encoding the La protein, introns that help stabilize the mRNA and increase its expression level may also be included.

[0113] To achieve proper termination and polyadenylation of the La gene transcript, it may be desirable to include a polyadenylation signal. Exemplary polyadenylation signals have been isolated from betaglobin, bovine growth hormone, SV40, and herpes simplex virus thymidine kinase genes.

[0114] Nucleic acid molecules encoding La proteins, variants, or fragments can be included in a viral vector for purposes such as the expression of a protomer to produce the corresponding La protein, variant, or fragment in a host cell, or for administration to a subject disclosed herein. Typically, such a viral vector contains a nucleic acid molecule encoding a La protein, variant, or fragment. In some examples, the viral vector encoding a La protein, variant, or fragment can be replication-competent. For example, the viral vector may have mutations in its viral genome that attenuate but do not completely block viral replication in a host cell (e.g., insertion of a nucleic acid encoding a protomer).

[0115] Various viral vectors that can be used in nucleic acid-based therapies as taught herein include adenoviruses or adeno-associated viruses, herpesviruses, vaccinia, or RNA viruses, such as retroviruses (including HVJ; see Kotani et al., Curr. Gene Ther. 4:183-194, 2004). In one embodiment, the retroviral vector is a derivative of a mouse or tri-retrovirus or a human or primate lentivirus. Examples of retroviral vectors into which foreign genes can be inserted include, but are not limited to, Moloney's mouse leukemia virus (MoMLV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), and Rous sarcoma virus (RSV). In one embodiment, when the target is human, viruses such as gibbon leukemia virus (GaLV) can be used. Pseudotyped retroviral vectors containing heterologous envelope genes can be used.

[0116] Some of the further retroviral vectors can incorporate multiple genes. Any of these vectors can incorporate genes for selectable markers so that transduced cells can be identified and generated. The vector becomes target-specific when a nucleic acid encoding the La protein, variant, or fragment is inserted into the viral vector, along with another gene that can function, for example, as a viral envelope protein and also encode a ligand for a receptor on a specific target cell. Retroviral vectors can also be made target-specific by modifying the envelope protein, for example, by attaching sugars, glycolipids, or proteins. In one non-limiting example, targeting is achieved by directing the retroviral vector to a target using an antibody.

[0117] Recombinant retroviruses are non-replicating by design and therefore require assistance to produce infectious vector particles. This assistance can be provided, for example, by using a helper cell line containing a plasmid that encodes all of the retrovirus's structural genes under the control of regulatory sequences within terminal repeats (LTRs). These plasmids lack nucleotide sequences that would allow the packaging mechanism to recognize the RNA transcript for capsid formation. Helper cell lines lacking the packaging signal include, but are not limited to, ψ2, PA317, and PA12. These cell lines produce empty virions because the genome is not packaged. If a retroviral vector with an intact packaging signal, but in which the structural genes are replaced with genes of other interest, is introduced into such cells, the vector can be packaged and a vector virion can be produced.

[0118] Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol, and env using conventional transfection methods. These cells are then transfected with a vector plasmid containing the gene of interest. The resulting cells release the retroviral vector into the culture medium.

[0119] Adenovirus vectors are also useful. Adenovirus vectors include replicable, replication-deficient, and gutless forms. Deficient viruses that completely or nearly completely lack viral genes, such as adenovirus vectors or adeno-associated virus (AAV) vectors, can be used. The use of defective viral vectors allows for administration to specific cells without the concern that the vector may infect other cells. Useful AAV vectors are replication-deficient. Although not bound by theory, adenovirus vectors are known to exhibit strong expression, excellent titers, and the ability to transduce dividing and non-dividing cells in vivo (Hitt et al., Adv in Virus Res 55:479-505, 2000). When used in vivo, these vectors result in strong, but transient, gene expression due to an immune response induced against the vector backbone. In some non-specific cases, useful vectors include attenuated adenovirus vectors, such as those described by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630 1992, La Salle et al., Science 259:988-990, 1993), or deficient AAV vectors (Samulski et al., J. Virol., 61:3096-3101, 1987, Samulski et al., J. Virol., 63:3822-3828, 1989, Lebkowski et al., Mol. Cell. Biol., 8:3988-3996, 1988).

[0120] Recombinant AAV vectors are characterized by their ability to direct the expression and production of selected transgenic products in targeted cells. Therefore, these recombinant vectors contain at least all AAV sequences essential for the physical structure necessary for capsid formation and infection of target cells.

[0121] AAV belongs to the family Parvoviridae and the genus Dependovirus. AAV is a small, non-enveloped virus that packages a linear single-stranded DNA genome. The sense and antisense strands of AAV DNA are packaged in the AAV capsid at equal frequency. In some embodiments, the AAV DNA comprises nucleic acid containing a promoter functionally linked to a nucleic acid molecule encoding a La protein, variant, or fragment. Recombinant vectors, such as recombinant adenovirus vectors and recombinant adeno-associated virus (rAAV) vectors containing the nucleic acid molecules disclosed herein, are further provided. In some embodiments, the AAV is rAAV8 and / or AAV2. However, the AAV serotype can be any other suitable AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11 or AAV12, or a hybrid of two or more AAV serotypes.

[0122] La expression is influenced by polyomas, namely SV40 (Madzak et al., 1992, J.Gen.Virol., 73:15331536), herpesviruses such as HSV, EBV, and CMV (Margolskee, 1992, Curr.Top.Microbiol.Immunol., 158:67-90, Johnson et al., 1992, J.Virol., 66:29522965, Fink et al., 1992, Hum.Gene Ther.3:11-19, Breakfield et al., 1987, Mol.Neurobiol., 1:337-371, Fresse et al., 1990, Biochem.Pharmacol., 40:2189-2199), and Sindbisvirus (H.Herweijer et al., 1995, Human Gene Therapy 6:1161-1167, U.S. Patent Nos. 5,091,309 and 5,2217,879, alphavirus (S. Schlesinger, 1993, Trends Biotechnol. 11:18-22, I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377), as well as birds (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754, Petropouplos et al., 1992, J. Virol., 66:3391-3397), mice (Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24, Miller et al., 1985, Mol.Cell Biol., 5:431-437, Sorge et al., 1984, Mol.Cell Biol., 4:1730-1737, Mann et al., 1985, J.Virol., 54:401-407) and humans (Page et al., 1990, J. Virol., 64:5370-5276, Buchschalcher et al., 1992, J. Virol.Further viral vectors, such as retroviruses derived from (66:2731-2739), can also be used. Baculovirus (Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors are also known in the art and are available from commercial sources (e.g., PharMingen (San Diego, California), Protein Sciences Corp. (Meriden, Connecticut), Stratagene (La Jolla, California)).

[0123] Another targeted delivery system for polynucleotides encoding La proteins, variants, or fragments is a colloidal dispersion system. These systems are also useful in the disclosed methods.

[0124] Colloidal dispersion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. One colloidal dispersion system is the liposome. Liposomes are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUVs) in the size range of approximately 0.2–4 microns can encapsulate a significant proportion of aqueous buffer containing large polymers. RNA, DNA, and intact virions can be encapsulated within the aqueous solution and delivered to cells in a biologically active form (Fraley et al., Trends Biochem. Sci. 6:77, 1981). Liposomes have been used for the delivery of polynucleotides not only in mammalian cells but also in plant, yeast, and bacterial cells. For liposomes to be an efficient gene delivery vehicle, they should possess the following characteristics: (1) highly efficient encapsulation of nucleic acids of interest without impairing their biological activity; (2) preferential and substantial binding to target cells compared to non-target cells; (3) highly efficient delivery of the aqueous contents of the vesicle to the cytoplasm of the target cell; and (4) accurate and effective expression of genetic information (Mannino et al., Biotechniques 6:682, 1988).

[0125] The composition of liposomes is typically a combination of phospholipids, especially those with high phase transition temperatures, usually combined with steroids, particularly cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0126] Examples of lipids useful for liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Diacylphosphatidylglycerol, in which the lipid portion contains 14 to 18 carbon atoms, particularly 16 to 18 carbon atoms and is saturated, is especially useful. Exemplary phospholipids include phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

[0127] Liposome targeting can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, such as organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based on whether it is passive or active. Passive targeting utilizes the liposome's natural tendency to distribute to cells in the reticular system (RES) within organs, including sinusoidal capillaries. Active targeting, on the other hand, involves modifying liposomes by coupling them to specific ligands, such as monoclonal antibodies, sugars, glycolipids, or proteins, or by altering the composition or size of the liposomes, in order to achieve targeting to organs and cell types other than their natural localization sites.

[0128] Another targeted delivery system is the use of biodegradable and biocompatible polymer scaffolds for use in bone (Jang et al., Expert Rev. Medical Devices 1:127-138, 2004). These scaffolds typically contain a mixture of one or more biodegradable polymers, such as, but not limited to, saturated aliphatic polyesters, e.g., poly(lactic acid: PLA), poly(glycolic acid), or poly(lactic-co-glycolide: PLGA) copolymers, unsaturated linear polyesters, e.g., polypropylene fumarate (PPF), or aliphatic polyesters produced by microorganisms, e.g., polyhydroxyalkanoate (PHA) (see Rezwan et al., Biomaterials 27:3413-3431, 2006; Laurencin et al., Clin. Orthopaed. Rel. Res. 447:221-236). By varying the ratios of these various components, polymer scaffolds with different mechanical properties can be obtained. Commonly used scaffolds have a PLA to PGA ratio of 75:25, but this ratio can vary depending on the specific application. Other commonly used scaffolds include surface bioeroding polymers, such as poly(anhydrous), e.g., trimellitilimidoglycine (TMA-gly) or pyromelitylimidoalanine (PMA-ala), or poly(phosphazenes), e.g., high molecular weight poly(organophasphazenes) (P[PHOS]), and bioactive ceramics. The gradual biodegradation of these scaffolds allows for the gradual release of drugs or genes from the scaffold. Therefore, the advantage of these polymer carriers is that they correspond not only to scaffolds but also to drug or gene delivery systems.This system can be applied to plasmid DNA delivery, as well as to viral vectors such as AAV or retroviral vectors, and transposon-based vectors.

[0129] The surface of targeted delivery systems can be modified in various ways. In the case of liposome-targeted delivery systems, lipid groups can be incorporated into the liposome's lipid bilayer to stably associate the targeting ligand with the liposome bilayer. Various linking groups can be used to attach the lipid chain to the targeting ligand.

[0130] C. Chemical compounds and low molecular weight agonists La protein agonists include molecules identified from large libraries of natural products or synthetic (or semi-synthetic) extracts, or molecules identified from chemical libraries. Screening methods that detect increased La activity, such as by measuring osteoclast fusion, are useful for identifying compounds by activity from diverse sources. Initial screening can be performed using diverse libraries of compounds, various other compounds, and compound libraries. In this way, molecules that increase La protein activity can be identified. These small molecules can be identified from combinatorial libraries, natural product libraries, or other small molecule libraries. In addition, La agonists can also be identified as compounds from commercial sources and as commercially available analogs of identified inhibitors.

[0131] The source of the test extract or compound itself is not important for the identification of La protein agonists. Therefore, La protein agonists can be identified from virtually countless chemical extracts or compounds. Examples of such extracts or compounds include, but are not limited to, extracts based on plants, fungi, prokaryotes, or animals, fermentation broths, and synthetic compounds, as well as modifications of existing compounds. Numerous methods can also be used to perform random or targeted synthesis (e.g., semi-synthesis or total synthesis) of many chemical compounds, for example, but not limited to compounds based on sugars, lipids, peptides, and nucleic acids. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, New Hampshire) and Aldrich Chemical (Milwaukee, Wisconsin). La protein agonists can be identified from synthetic compound libraries commercially available from several companies, including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, New Jersey), Brandon Associates (Merrimack, New Hampshire), and Microsource (New Milford, Connecticut). La protein agonists can be identified from rare chemical libraries, such as those available from Aldrich (Milwaukee, Wisconsin). La protein agonists can also be identified in libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts, commercially available from several suppliers, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Fort Pierce, Florida), and PharmaMar, USA (Cambridge, Massachusetts). Natural and synthetically produced libraries and compounds are readily modified by conventional chemical, physical, and biochemical means.

[0132] Useful compounds can be found within numerous compound classes. However, they are typically organic compounds, including low molecular weight organic compounds. The methods disclosed herein can utilize low molecular weight organic compounds having molecular weights greater than 50 daltons but less than about 2,500 daltons, for example, less than about 750 daltons or less than about 350 daltons. Exemplary classes include heterocyclic compounds, peptides, sugars, and steroids. Compounds can be modified to enhance potency, stability, pharmaceutically acceptable properties, etc.

[0133] III. Substances that reduce the function and / or activity of La protein Methods for reducing osteoclast fusion in a subject are disclosed herein. These methods can reduce bone resorption. These methods use agents that reduce the expression or activity of La protein in the subject. Exemplary agents are disclosed below.

[0134] A. Antibodies and their antigen-binding fragments The substance that reduces the activity of the La protein can be an antagonist antibody that specifically binds to the La protein, such as, but is not limited to, a monoclonal antibody. Antibodies that specifically bind to the La protein are commercially available. In some embodiments, the antibody binds to the La protein and reduces osteoclast fusion. Exemplary antibodies are disclosed in Example 8.

[0135] Antibodies that specifically bind to and substantially reduce or inhibit the activity of the La protein (e.g., reduction of at least 20%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, and even 100%) are useful in the methods disclosed herein. Antibodies include monoclonal antibodies, human antibodies, humanized antibodies, deimmunized antibodies, and immunoglobulin (Ig) fusion proteins. Fully human antibodies and humanized antibodies that bind to the La protein can also be prepared using methods known to those skilled in the art.

[0136] Polyclonal antagonist antibodies can be prepared, for example, by immunizing a suitable subject (e.g., a human or veterinary subject) with La protein. Anti-La protein antibody titers in the immunized subject can be monitored over time, for example, using an enzyme-linked immunosorbent assay (ELISA) with immobilized La protein or its epitope. In one example, antibody molecules that specifically bind to La protein can be isolated from mammals (e.g., from serum) and further purified, for example, by using protein A chromatography to isolate IgG antibodies. In some embodiments, antibodies can also be selected using functional assays, such as detecting inhibition of osteoclast fusion.

[0137] Antibody-producing cells can be obtained from subjects such as immunized subjects and can be used to prepare monoclonal antibodies (Kohler and Milstein Nature 256:495 49, 1995; Brown et al., J.Immunol.127:539 46, 1981; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77 96, 1985; Gefter, M. Let al.(1977) Somatic Cell Genet.3:231 36; Kenneth, RH, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, NY (1980); Kozbor et al. Immunol.Today 4:72, 1983; Lerner, EA(1981) Yale (See J.Biol.Med.54:387 402, Yeh et al., Proc.Natl.Acad.Sci.76:2927 31, 1976). In one example, an immortalized cell line (typically myeloma) is fused with lymphocytes (typically spleen cells) from a mammal immunized with La protein, and the resulting hybridoma cell culture supernatant is screened to identify hybridomas that specifically bind to a polypeptide of interest and produce monoclonal antibodies that inhibit the function of that polypeptide.

[0138] In one embodiment, to produce hybridomas, the immortalized cell line (such as a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, mouse hybridomas can be produced by fusing lymphocytes from mice immunized with the La protein or its epitope with an immortalized mouse cell line. In one example, a mouse myeloma cell line sensitive to a culture medium containing hypoxanthine, aminopterin, and thymidine ("HAT medium") is used. Any of the many myeloma cell lines available from the American Type Culture Collection (ATCC) in Rockville, Maryland, including the P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653, or Sp2 / O-Ag14 myeloma cell lines, can be used as fusion partners. HAT-sensitive mouse myeloma cells can be fused with mouse splenocytes using polyethylene glycol ("PEG"). The hybridoma cells obtained by fusion are then selected using HAT medium, which kills unfused (and unproductively fused) myeloma cells. Hybridoma cells that produce monoclonal antibodies of interest can be detected, for example, by screening the hybridoma culture supernatant for the production of antibodies that bind to La polypeptide, using immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)).

[0139] As an alternative method for preparing monoclonal antibody-secreting hybridomas, monoclonal antibodies that specifically bind to the La protein can also be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the La protein or its epitope, and isolating immunoglobulin library members that specifically bind to that polypeptide. Library members with specific activity, such as activity that binds to the La protein, or activity that inhibits osteoclast fusion in an in vitro assay, can be selected. Kits for creating and screening phage display libraries are commercially available (not limited to, but e.g., Pharmacia and Stratagene). Examples of methods and reagents particularly suitable for use in the preparation and screening of antibody display libraries can be found, for example, in U.S. Patent No. 5,223,409, PCT Publication Nos. WO 90 / 02809, WO 91 / 17271, WO 92 / 18619, WO 92 / 20791, WO 92 / 15679, WO 92 / 01047, WO 93 / 01288, WO 92 / 09690, Barbas et al., Proc.Natl.Acad.Sci.USA 88:7978 7982, 1991, and Hoogenboom et al., Nucleic Acids Res. 19:4133 4137, 1991. Suitable assays for monitoring osteoclast fusion are disclosed, for example, in the Examples section.

[0140] In one example, the sequence of the specificity determining region of each CDR is determined. Residues outside the SDR (specificity determining region, e.g., non-ligand contact site) are substituted. For example, up to one, two, or three amino acids can be substituted in any of the CDR sequences. The creation of chimeric antibodies, which include a framework region from one antibody and a CDR from a different antibody, is known in the art. For example, humanized antibodies can be created. The antibody or antibody fragment may be a humanized immunoglobulin having a CDR from a donor monoclonal antibody that binds to the La protein or its epitope, and an immunoglobulin from a human acceptor immunoglobulin heavy chain and light chain framework, as well as a heavy chain and light chain variable region framework.

[0141] Humanized monoclonal antibodies can be produced by transferring the CDR from the variable heavy and light chains of donor mouse immunoglobulin (which specifically binds to the La protein) to the human variable domain, and then substituting human residues in the framework region if necessary to maintain affinity. The use of antibody components derived from humanized monoclonal antibodies eliminates potential challenges related to the immunogenicity of the constant region of donor antibodies. Techniques for producing humanized monoclonal antibodies are described, for example, in Jones et al., Nature 321:522, 1986, Riechmann et al., Nature 332:323, 1988, Verhoeyen et al., Science 239:1534, 1988, Carter et al., Proc.Natl.Acad.Sci.USA89:4285, 1992, Sandhu, Crit.Rev.Biotech.12:437, 1992, and Singer et al., J.Immunol.150:2844, 1993. The antibody may be any isotype, but in some embodiments, the antibody is IgG, including, but not limited to, IgG1, IgG2, IgG3, and IgG4.

[0142] In one aspect, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. The human framework regions and mutations that can be added in the humanized antibody framework regions are known in the art (see, e.g., U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety).

[0143] Exemplary human antibodies are LEN and 21 / 28CL. Sequences of many human heavy and light chain frameworks are known. Generally, antibodies, such as human antibodies or humanized antibodies, specifically bind to the La protein and / or its epitope with an affinity constant of at least 10 7 M -1 , e.g., at least 10 8 M -1 , at least 5×10 8 M -1 or at least 10 9 M -1 . In some instances, the antibody specifically binds to the La protein or its epitope with an affinity constant of at least 10 8 M -1 , at least 5×10 8 M -1 or at least 10 9 M -1 . These antibodies can inhibit osteoclast fusion as compared to a control, e.g., compared to osteoclast fusion in the absence of the antibody or when using a control isotype-matched antibody. The antibody can be a fully human antibody.

[0144] Antibodies, such as mouse monoclonal antibodies, chimeric antibodies, and humanized antibodies, contain not only full-length molecules but also fragments such as Fab, F(ab')2, and Fv, which contain variable regions in the heavy and light chains and can bind to specific epitopes. These antibody fragments retain some ability to selectively bind to their antigens or receptors. These fragments include the following: (1) Fab is a fragment containing a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digesting the entire enzyme with the enzyme papain to obtain an intact light chain and a portion of one heavy chain. (2) Fab' is a fragment of an antibody molecule, which can be obtained by treating the entire antibody with pepsin, then reducing it to obtain an intact light chain and a portion of the heavy chain, yielding two Fab' fragments per antibody molecule. (3)(Fab')2, an antibody fragment that can be obtained by treating the entire antibody with the enzyme pepsin and then not performing reduction. F(ab')2 is a dimer of two Fab' fragments joined together by two disulfide bonds. (4) Fv, a genetically engineered fragment containing a variable region of the light chain and a variable region of the heavy chain expressed as two strands, and (5) Single-chain antibodies (such as scFv) are defined as genetically engineered molecules containing a light chain variable region and a heavy chain variable region, linked as a gene-fused single-chain molecule by an appropriate polypeptide linker.

[0145] Methods for constructing these fragments are publicly known (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). In some examples, the variable region includes a variable region of the light chain and a variable region of the heavy chain, which are expressed as separate polypeptides. Fv antibodies are typically about 25 kDa and contain a complete antigen-binding site with three CDRs in each heavy chain and each light chain. To construct these antibodies, two separate nucleic acid constructs are used in host cells, and V H and V L It can express V H and V L When Fv is expressed discontinuously, the Fv antibody chains are typically bound together by non-covalent interactions. However, these chains tend to dissociate when diluted, so methods have been developed to crosslink them using glutaraldehyde, intermolecular disulfides, or peptide linkers. Thus, in one example, Fv can be a disulfide-stabilized Fv (dsFv) in which the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.

[0146] In another example, an Fv fragment linked by a peptide linker is V H Chain and V L These single-chain antigen-binding proteins (scFv) are linked by an oligonucleotide. H Domain and V LIt is prepared by constructing a structural gene containing the DNA sequence encoding the domains. This structural gene is inserted into an expression vector and then introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFv are publicly known (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol.2, p. 97, 1991; Bird et al., Science 242:423, 1988, U.S. Patent No. 4,946,778; Pack et al., Bio / Technology 11:1271, 1993; and Sandhu, op. cit.).

[0147] Antibody fragments can be prepared by proteolytic hydrolysis of antibodies or by the expression of DNA encoding the fragment in E. coli. Antibody fragments can also be obtained by pepsin digestion or papain digestion of the whole antibody using conventional methods. For example, an antibody fragment can be prepared by enzymatically cleaving an antibody with pepsin to obtain a 5S fragment called F(ab')2. A 3,5S Fab' monovalent fragment can be prepared by further cleaving this fragment with a thiol reducing agent and optionally a protecting group for the sulfhydryl group resulting from the cleavage of the disulfide bond. Alternatively, pepsin-based enzymatic cleavage directly generates two monovalent fragments: a Fab' fragment and an Fc fragment (see U.S. Patent Nos. 4,036,945 and 4,331,647 and the references contained therein, Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960, Porter, Biochem. J. 73:119, 1959, Edelman et al., Methods in Enzymology, Vol. 1, p. 422, Academic Press, 1967, and sections 2.8.1-2.8.10 and 2.10.1-2.10.4 of Coligan et al.).

[0148] Other methods for cleaving antibodies, such as the formation of monovalent light-heavy chain fragments by separating the heavy chain, further cleavage of the fragment, or other enzymatic, chemical, or genetic techniques, may also be used, as long as the fragment binds to an antigen recognized by the intact antibody. Any of the antigen-binding fragments described herein are useful.

[0149] Conservative variants of antibodies can be produced. Such conservative variants, used in antibody fragments such as dsFv fragments or scFv fragments, are produced with correct folding and V H Region and V L It will preserve the critical amino acid residues necessary for stabilization between regions and retain the residue charge characteristics so as not to lose the molecule's low pI and low toxicity. H Region and V L Amino acid substitutions (e.g., at most one, two, three, four, or five amino acid substitutions) can be made in the region. In some embodiments, these substitutions are made in the framework region and not in the CDR. A table of conserved amino acid substitutions is provided above. Those skilled in the art can easily examine the amino acid sequence of an antibody of interest, find one or more amino acids in the above simplified table, identify the conserved substitutions, and construct a conserved variant using molecular techniques.

[0150] Effector molecules, such as therapeutic, diagnostic, or detection molecules, can be linked to antagonist antibodies that specifically bind to the La protein using several methods. Both covalent and non-covalent attachments can be used. The procedure for attaching effector molecules to antibodies varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which can be used in reactions with appropriate functional groups on the antibody to result in the attachment of the effector molecule. Alternatively, antibodies can be derivatized to expose or attach additional reactive functional groups. Derivatization may involve the attachment of one of several linker molecules, such as those available from Pierce Chemical Company in Rockford, Illinois. The linker can be any molecule used to conjugate the antibody to the effector molecule. The linker can form both covalent bonds to the antibody and covalent bonds to the effector molecule. Suitable linkers include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and effector molecule are polypeptides, the linker can be attached to the constituent amino acids via their side groups (e.g., to cysteine ​​via disulfide bonds) or to the alpha-carbon amino and alpha-carbon carboxyl groups of the terminal amino acids.

[0151] The nucleic acid sequence encoding the antibody can be synthesized by any suitable method, for example, by cloning a suitable sequence, or by direct chemical synthesis using methods such as the phosphotryster method described in Narang et al., Meth.Enzymol.68:90-99, 1979, the phosphodiester method described in Brown et al., Meth.Enzymol.68:109-151, 1979, the diethylphosphoramidite method described in Beaucage et al., Tetra.Lett.22:1859-1862, 1981, or the solid-phase phosphoramidite triester method described in Beaucage & Caruthers, Tetra.Letts.22(20):1859-1862, 1988, for example, Needham-VanDevanter et al., Nucl.Acids Res.12:6159-6168. It can be prepared using automated synthesis equipment described in 1984, etc., and the solid support method described in U.S. Patent No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides. These can be converted to double-stranded DNA oligonucleotides by hybridization with complementary sequences or by polymerization using DNA polymerase with the single strand as a template. Longer sequences can also be obtained by ligation of the short sequences produced by chemical synthesis.

[0152] Exemplary nucleic acids encoding sequences that encode antagonist antibodies specifically binding to the La protein can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to guide those skilled in the art who have performed many cloning operations, can be found in Sambrook et al., op. cit., Berger and Kimmel, eds., op. cit., and Ausubel, op. cit. Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (St. Louis, Missouri), R&D Systems (Minneapolis, Minnesota), Pharmacia Amersham (Piscataway, New Jersey), CLONTECH Laboratories, Inc. (Palo Alto, California), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wisconsin), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaythersburg, Maryland), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Swiss Books), Invitrogen (San Diego, California), and Applied Biosystems (Foster City, California), as well as other commercial sources.

[0153] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification (TAS), and self-sustaining sequence replication (3SR). A wide variety of cloning methods, host cell and in vitro amplification methods are known.

[0154] In one example, a useful antibody is prepared by inserting a cDNA encoding a variable region from an antagonist antibody that specifically binds to the La protein into a vector containing a cDNA encoding an effector molecule (EM). This insertion is performed so that the variable region and the EM are read in the same reading frame, producing a single continuous polypeptide. Thus, the encoded polypeptide contains a functional Fv region and a functional EM region. In one embodiment, a cDNA encoding a detectable marker (e.g., an enzyme) is ligated to an scFv such that the marker is positioned at the carboxyl end of the scFv. In another example, the detectable marker is positioned at the amino terminus of the scFv. In yet another example, a cDNA encoding a detectable marker is ligated to the heavy chain variable region of an antagonist antibody that specifically binds to the La protein such that the marker is positioned at the carboxyl end of the heavy chain variable region. The heavy chain variable region can then be ligated to the light chain variable region of an antibody that specifically binds to the La protein using a disulfide bond. In yet another example, a cDNA encoding a marker is ligated to the light chain variable region of an antagonist antibody that binds to the La protein, such that the marker is positioned at the carboxyl end of the light chain variable region. The light chain variable region can then be ligated to the heavy chain variable region of an antagonist antibody that specifically binds to the La protein using disulfide bonds.

[0155] Once a nucleic acid encoding a La antagonist antibody or its functional fragment is isolated and cloned, the protein can be expressed in recombinant cells such as bacterial cells, plant cells, yeast cells, insect cells, and mammalian cells. One or more DNA sequences encoding the antibody or its functional fragment can be expressed in vitro by DNA introduction into a suitable host cell. The cell can be prokaryotic or eukaryotic. This term also includes any offspring of the target host cell. It is understood that not all offspring will be identical to the parent cell, as mutations may occur during replication. Stable introduction methods, meaning that the foreign DNA is continuously maintained in the host, are known.

[0156] Polynucleotide sequences encoding an antibody or its functional fragment (e.g., scFV) can be functionally ligated to an expression regulatory sequence. The expression regulatory sequence functionally ligated to the coding sequence is ligated so that expression of the coding sequence is achieved under conditions compatible with the expression regulatory sequence. Expression regulatory sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, pre-protein coding gene start codons (i.e., ATGs), splicing signals for introns, maintenance of the correct reading frame of the gene to enable proper translation of mRNA, and stop codons. RNA encoding the disclosed antibody is also useful.

[0157] Polynucleotide sequences encoding antibodies or their functional fragments can be manipulated to allow for insertion or incorporation into expression vectors, for example, but not limited to, plasmids, viruses, or other vehicles that can be expressed in either prokaryotes or eukaryotes. Examples of hosts include microorganisms, yeasts, insects, and mammals. Methods for expressing DNA sequences containing eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral DNA vectors and plasmid DNA vectors that can be expressed and replicated in hosts are known.

[0158] Transformation of host cells with recombinant DNA can be performed using conventional techniques. When the host is a prokaryotic host such as E. coli, competent cells capable of DNA uptake can be prepared from cells harvested after the exponential growth phase and then treated with the CaCl2 method. Alternatively, MgCl2 can be used. Transformation can be performed, if desired, after the formation of host cell protoplasts, or by electroporation.

[0159] When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, mechanical procedures such as microinjection, electroporation, insertion of liposome-encapsulated plasmids, or viral vectors may be used. Eukaryotic cells can also be co-transformed with a polynucleotide sequence encoding an antibody or a functional fragment thereof, and a second exogenous DNA molecule encoding a selectable phenotype, such as a herpesthymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells with eukaryotic viral vectors, such as Simian virus 40 (SV40) or bovine papillomavirus, to express proteins (see, e.g., Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman, ed., 1982). Those skilled in the art can readily use expression systems such as plasmids and vectors that are useful for producing proteins in cells, including higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0160] The isolation and purification of recombinantly expressed polypeptides can be carried out by conventional means, including preparative chromatography and immunological separation. Once recombinant antibodies have been expressed, they can be purified by standard procedures in the art, including ammonium sulfate precipitation, affinity columns, and column chromatography (see R. Scopes, Protein Purification, Springer-Verlag, NY, 1982 for general information). This specification discloses substantially pure compositions having at least about 90–95% homogeneity, and for pharmaceutical purposes, homogeneity of 98–99% or higher may be used. When used therapeutically, once partially or homogeneously purified as desired, the polypeptides should be substantially free of endotoxins.

[0161] Methods for expressing single-chain antibodies from bacteria such as Escherichia coli and / or for refolding single-chain antibodies into suitable active forms have been described and are applicable to the antibodies disclosed herein. See Buchner et al., Anal. Biochem. 205:263-270, 1992, Pluckthun, Biotechnology 9:545, 1991, Huse et al., Science 246:1275, 1989, and Ward et al., Nature 341:544, 1989. All of these publications are incorporated herein by reference.

[0162] Functional heterologous proteins from E. coli or other bacteria are often isolated from inclusion bodies and require solubilization with strong denaturants followed by refolding. During the solubilization process, a reducing agent must be present to separate the disulfide bonds. An exemplary buffer containing a reducing agent is 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, and 0.3 M DTE (dithioerythritol). The redox reaction of disulfide bonds can occur in the presence of reduced and oxidized low molecular weight thiol reagents, as described in Saxena et al., Biochemistry 9:5015-5021, 1970, which is incorporated herein by reference, and especially as described by Buchner et al., cited above.

[0163] Restoration is typically achieved by diluting the denatured and reduced protein in a refolding buffer (e.g., 100-fold dilution). An example buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

[0164] As a modification of the double-chain antibody purification protocol, the heavy and light chain regions are solubilized and reduced separately, and then mixed in a refolding solution. Exemplary yields are obtained by mixing these two proteins in a molar ratio such that one protein does not exceed a 5-fold molar excess of the other. After the redox shuffling is complete, it is desirable to add an excess amount of oxidized glutathione or other oxidized low molecular weight compounds to the refolding solution.

[0165] In addition to recombinant methods, the antagonist antibodies and their functional fragments disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid-phase synthesis of polypeptides less than approximately 50 amino acids in length can be achieved by attaching the C-terminal amino acid of the sequence to an insoluble support and then sequentially adding the remaining amino acids in the sequence. Techniques for solid-phase synthesis are described in Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol.2: Special Methods in Peptide Synthesis, Part A, pp. 3-284; Merrifield et al., J.Am.Chem.Soc.85:2149-2156, 1963; and Stewart et al., Solid Phase Peptide Synthesis, 2nd edition, Pierce Chem.Co., Rockford, Illinois, 1984. Longer proteins can be synthesized by condensation of the amino-terminus and carboxy-terminus of shorter fragments. Methods for forming peptide bonds by activating the carboxyl terminus (for example, by using the coupling reagent N,N'-dicyclohexylcarbodiimide) are well known.

[0166] B. Inhibitory nucleic acid molecules The methods disclosed herein may also utilize inhibitory nucleic acids that reduce the expression and / or activity of the La protein. In some examples, such inhibitory nucleic acid molecules reduce the expression or activity of the La protein by at least 20%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, and even 100%. One embodiment is RNA interference (RNAi), such as, but not limited to, small inhibitory RNA (siRNA) or short hairpin RNA, which can be used to interfere with or inhibit the expression of a target. RNAs that specifically target the La protein are commercially available, for example, from Santa Cruz Biotechnology, Inc., ThermoFisher Scientific, and Sigma Aldrich. Exemplary commercially available La-specific RNAi sequences that can be used in the disclosed methods include: sense TIFF2026053494000009.tif4128; and antisense This includes TIFF2026053494000010.tif4128.

[0167] Generally, siRNA is produced by the cleavage of relatively long double-stranded RNA molecules with dicers or DCL enzymes (Zamore, Science, 296:1265-1269, 2002; Bernstein et al., Nature, 409:363-366, 2001). In animals and plants, siRNA is incorporated into RISC and guides the sequence-specific ribonucleotide cleavage activity of RISC, resulting in the cleavage of mRNA or other RNA targets in the cytoplasm. In the nucleus, siRNA guides the methylation of heterochromatin-associated histones and DNA, which results in transcriptional silencing of individual genes or large chromatin domains.

[0168] This disclosure provides RNA suitable for interfering with or inhibiting the expression of La protein, comprising a double-stranded RNA of about 19 to about 40 nucleotides having a sequence substantially identical to a portion of the mRNA or transcript of a target gene, such as the La protein, whose expression is to be interfered with or inhibited. With respect to this disclosure, the difference between the RNA sequence that is "substantially identical" to a specific portion of the mRNA or transcript of the target gene whose expression is to be interfered with or inhibited, and that specific portion of the mRNA or transcript of the target gene, is about 30% or less, and in some embodiments, about 10% or less or 5% or less. In certain embodiments, the RNA sequence is strictly identical to a specific portion of the mRNA or transcript of the target gene (e.g., the La protein transcript).

[0169] Accordingly, the siRNAs disclosed herein comprise a double-stranded RNA approximately 15 to 40 nucleotides long and a 3' or 5' overhang on each strand having a length of 0 to 5 nucleotides, wherein the sequence of the double-stranded RNA is substantially identical (see above) to a portion of the mRNA or transcript of the nucleic acid encoding the La protein. In certain examples, the double-stranded RNA contains approximately 19 to 25 nucleotides, e.g., 20, 21, or 22 nucleotides, substantially identical to the nucleic acid encoding the La protein. In further examples, the double-stranded RNA contains approximately 19 to 25 nucleotides, 100% identical to the nucleic acid encoding the La protein. It should be noted that in this context, "approximately" refers only to integer quantities. In one example, "approximately" 20 nucleotides refers to nucleotides 19 to 21 nucleotides long.

[0170] With respect to overhangs on double-stranded RNA, the length of the overhang is independent between the two strands in that the length of one overhang does not depend on the length of the overhang on the other strand. In specific examples, the length of the 3' or 5' overhang is 0 nucleotides on at least one strand, and in some examples, it is 0 nucleotides on both strands (and therefore blunt-ended dsRNA). In other examples, the length of the 3' or 5' overhang is 1 to 5 nucleotides on at least one strand. More specifically, in some examples, the length of the 3' or 5' overhang is 2 nucleotides on at least one strand, or 2 nucleotides on both strands. In a specific example, a dsRNA molecule has a 3' overhang of 2 nucleotides on both strands.

[0171] Therefore, in one of the specific RNA embodiments provided, the double-stranded RNA contains 20, 21, or 22 nucleotides, and the 3' overhang length is 2 nucleotides in both strands. In the RNA embodiments provided herein, the double-stranded RNA contains approximately 40–60% adenine + uracil (AU) and approximately 60–40% guanine + cytosine (GC). More specifically, in certain examples, the double-stranded RNA contains approximately 50% AU and approximately 50% GC.

[0172] This specification also discloses RNAs, for example, double-stranded RNA, further comprising at least one modified nucleotide in the sense strand. In certain examples, the modified ribonucleotide is located in the 3' overhang of at least one strand, more specifically in the 3' overhang of the sense strand. Examples of modified ribonucleotides may include ribonucleotides with detectable labels (e.g., rhodamine or FITC fluorophores), thiophosphate nucleotide analogs, deoxynucleotides (considered modified because the base molecule is ribonucleic acid), 2'-fluorouracil, 2'-aminouracil, 2'-aminocytidine, 4-thiouracil, 5-bromouracil, 5-iodouracil, 5-(3-aminoallyl)-uracil, inosine, or 2'O-Me-nucleotide analogs.

[0173] Antisense and ribozyme molecules for the La protein are useful in the methods disclosed herein. An antisense nucleic acid is a DNA or RNA molecule complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990). In cells, the antisense nucleic acid hybridizes to the corresponding mRNA to form a double-stranded molecule. Since cells will not translate double-stranded mRNA, the antisense nucleic acid interferes with mRNA translation. Antisense oligomers of about 15 nucleotides can be used because they are easy to synthesize and less likely to cause problems than longer molecules when introduced into target cells producing the La protein. The use of antisense methods to inhibit in vitro translation of genes is well known (see, e.g., Marcus-Sakura, Anal. Biochem. 172:289, 1988).

[0174] Antisense oligonucleotides are, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides long. Antisense nucleic acids can be constructed using chemosynthesis and enzymatic ligation reactions. For example, antisense nucleic acid molecules can be chemosynthesized using native nucleotides, or various modified nucleotides, such as phosphorothioate derivatives and acridine-substituted nucleotides, can be used to increase the biological stability of the molecule or to increase the physical stability of the double helix formed between the antisense nucleic acid and the sense nucleic acid. Examples of modified nucleotides that can be used to produce antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueusin, and inosine.

[0175] The use of oligonucleotides to halt transcription is known as the triplex strategy, in which case the oligonucleotide wraps around the double-helix DNA to form a triple helix. These triplex compounds can therefore be designed to recognize unique sites on selected genes (Maher, et al., Antisense Res. and Dev. 1(3):227, 1991; Helene, C., Anticancer Drug Design 6(6):569), 1991). This type of inhibitory oligonucleotide is also useful in the methods disclosed herein.

[0176] Ribozymes, which are RNA molecules that have the ability to specifically cleave other single-stranded RNAs in a manner similar to DNA restriction endonucleases, are also useful. By modifying the nucleotide sequences that encode these RNAs, it is possible to engineer molecules that recognize and cleave specific nucleotide sequences within RNA molecules (Cech, J.Amer.Med.Assn.260:3030, 1988). The advantage of this approach is that, because they are sequence-specific, only mRNA with a specific sequence is inactivated.

[0177] There are two basic types of ribozymes: the tetrahymena type (Hasselhoff, Nature 334:585, 1988) and the "hammerhead" type. Tetrahymena ribozymes recognize sequences of 4 nucleotides in length, while "hammerhead" ribozymes recognize sequences of 11 to 18 nucleotides in length. The longer the recognition sequence, the higher the probability that the sequence is present exclusively in the target mRNA species. Therefore, hammerhead ribozymes are preferred over tetrahymena ribozymes for inactivating specific mRNA species, and 18-nucleotide recognition sequences are preferred over shorter recognition sequences.

[0178] Various delivery systems are known and can be used to administer siRNA and other inhibitory nucleic acid molecules as therapeutic agents. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, nanoparticles, recombinant cells capable of expressing therapeutic molecules (see, e.g., Wu et al., J. Biol. Chem. 262, 4429, 1987), and the construction of therapeutic nucleic acids as part of retroviral vectors or other vectors.

[0179] C. Chemical compounds, small molecules, and caspase inhibitors La protein inhibitors include molecules identified from large libraries of natural products or synthetic (or semi-synthetic) extracts, or molecules identified from chemical libraries. Screening methods for detecting a decrease in La protein activity are useful for identifying compounds by activity from diverse sources. Initial screening can be performed using diverse libraries of compounds, various other compounds, and compound libraries. Thus, molecules that bind to La protein, molecules that inhibit La protein expression, and molecules that inhibit La protein activity can be identified. These small molecules can be identified from combinatorial libraries, natural product libraries, or other small molecule libraries. In addition, La antagonists can also be identified as compounds from commercial sources and as commercially available analogs of identified inhibitors. La antagonists can be tested, for example, in assays to confirm that they reduce osteoclast fusion.

[0180] The source of the test extract or test compound itself is not important for the identification of La protein small molecule antagonists. Therefore, La protein inhibitors can be identified from virtually countless chemical extracts or chemical compounds. Examples of such extracts or compounds that can be La protein inhibitors include, but are not limited to, extracts based on plants, fungi, prokaryotes, or animals, fermentation broths, and synthetic compounds, as well as modifications of existing compounds. Numerous methods can also be used to perform random or targeted synthesis (e.g., semi-synthesis or total synthesis) of many chemical compounds, for example, but not limited to compounds based on sugars, lipids, peptides, and nucleic acids. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, New Hampshire) and Aldrich Chemical (Milwaukee, Wisconsin). La inhibitors can be identified from synthetic compound libraries commercially available from several companies, including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, New Jersey), Brandon Associates (Merrimack, New Hampshire), and Microsource (New Milford, Connecticut). La protein inhibitors can be identified from rare chemical libraries, such as those available from Aldrich (Milwaukee, Wisconsin). La protein inhibitors can also be identified in libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts, commercially available from several suppliers, including Biotics (Sussex, UK), Xenova (Slaugh, UK), Harbor Branch Oceangraphics Institute (Fort Pierce, Florida), and PharmaMar, USA (Cambridge, Massachusetts). Natural and synthetically produced libraries and compounds are readily modified by conventional chemical, physical, and biochemical means.

[0181] Useful compounds that function as inhibitors can be found within numerous compound classes. However, they are typically organic compounds, including low molecular weight organic compounds. The methods disclosed herein can utilize low molecular weight organic compounds having molecular weights greater than 50 daltons but less than about 2,500 daltons, for example, less than about 750 daltons or less than about 350 daltons. Exemplary classes include heterocyclic compounds, peptides, sugars, and steroids. Compounds can be modified to enhance potency, stability, pharmaceutically acceptable properties, etc.

[0182] In some embodiments, caspase inhibitors are useful in the methods disclosed herein. Caspases are a family of intracellular endoproteases that use cysteine ​​residues to initiate the cleavage of peptide substrates. The enzymatic properties of caspases are governed by the presence of a catalytic dyad (cysteine, histidine), where cysteine ​​acts as a nucleophile to initiate the cleavage of peptide bonds. The active sites of caspases are highly conserved and consist of catalytic cysteine ​​contained in the peptide sequence QACXG (SEQ ID NO: 6) (where X is arginine (R), glutamine (Q), or glycine (G)), and a basic subsite SI that gives them the specificity of substrate cleavage after an aspartic acid residue, which is unique among mammalian proteases except for serine proteases and granzyme B. Generally, caspases recognize tetrapeptide motifs P1-P4 located on the N-terminal side of cleavable bonds, which are recognized by the enzyme's subsites S1-S4, respectively. Downstream aspartates (P'1 and P'2) are also involved in caspase recognition and specificity. See PCT application publication number WO2017162674A1, incorporated herein by reference.

[0183] Caspases are classified into three groups according to their preferred or primarily recognized amino acid sequence. One group of caspases, including caspases 1, 4, and 5, has been shown to prefer hydrophobic aromatic amino acids at position 4 on the N-terminal side of the cleavage site. Another group, including caspases 2, 3, and 7, recognizes aspartyl residues, preferably Asp-Glu-X-Asp, at both positions 1 and 4 on the N-terminal side of the cleavage site. A third group, including caspases 6, 8, 9, and 10, accepts many amino acids in the primary recognition sequence but appears to prefer residues with branched aliphatic side chains, such as valine and leucine, at position 4. Further information is provided, for example, in PCT application publication number WO2001010383A2, which is incorporated herein by reference in its entirety. Caspase inhibitors are also disclosed in Lee et al., Expert Opinion on Therapeutic Patents 28(1), DOI:10.1080 / 13543776.2017.1378426, incorporated herein by reference. Caspase inhibitors can be pancaspase inhibitors, such as, but not limited to, Q z-VAD-fmk, Q-VD-OPH or Z-VKD-FMK, Emricasane or IDN-6556. In some embodiments, the caspase inhibitor is a pancaspase inhibitor such as the caspase inhibitor z-VAD-fmk. Exemplary caspase inhibitors also include Z-DEVD-FMK, Ac-DMPD-CMK and Ac-DMLD-CMK, Ac-ATS010-KE, rosmarinic acid and curcumin, Ac-DNLD-CHO, nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, naproxen, ketorolac, IDN-6556, emricasane, GS 9450, and VRT-043198 / VX-765 (vernacasan). In some non-limiting examples, caspase inhibitors may be caspase-3, caspase-7, or caspase-8 inhibitors.

[0184] D. Inhibitory La peptide This specification discloses peptide inhibitors of La. These peptides bind to La or to target molecules that specifically bind to full-length La protein and / or low-molecular-weight cleaved La, thereby inhibiting the interaction between full-length La protein and its target. The peptide inhibitors can reduce osteoclast fusion and / or bone resorption in the target. These peptide inhibitors reduce La activity in the control.

[0185] In some embodiments, these peptide inhibitors bind to annexin A5 and inhibit the interaction between full-length La protein and annexin A5. In further embodiments, the peptide inhibitors reduce osteoclast fusion compared to controls such as no treatment or treatment with vehicle alone.

[0186] In some embodiments, the peptide inhibitor contains at least 15 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, but does not contain the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 7. The peptide inhibitor may contain at least 15, 20, 25, 30, 35, or 40 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, but does not contain the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 7. In a further embodiment, the peptide inhibitor comprises 15 to 40 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, for example, 20 to 40, 25 to 35, 25 to 40, or 30 to 40, 15 to 35, 15 to 30, or 15 to 25 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. In a further embodiment, the peptide inhibitor comprises 15, 20, 25, 30, 35, or 40 amino acids or less from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. In yet another embodiment, the peptide inhibitor comprises 15, 20, 25, 30, 35, or 40 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. The peptide inhibitor inhibits osteoclast fusion induced by the La protein. A suitable assay is disclosed, for example, in the Examples section.

[0187] In some embodiments, the peptide inhibitor comprises at least 15 amino acids from (a) an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 and promotes osteoclast fusion, or (b) an amino acid sequence that has at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved substitution in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. The peptide inhibitor contains at least 15, 20, 25, 30, 35, or 40 amino acids from (a) an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 and promotes osteoclast fusion, or (b) an amino acid sequence that has at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved substitution in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. In a further embodiment, the peptide inhibitor comprises 15 to 40 amino acids from (a) an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 and promotes osteoclast fusion, or (b) an amino acid sequence that has at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved substitution in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7.In a further embodiment, the peptide inhibitor is (a) an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 and promotes osteoclast fusion, or (b) an amino acid sequence of 15, 20, 25, 30, 35, or 40 amino acids or less that has at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved substitution in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. In a further embodiment, the peptide inhibitor is (a) an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 and promotes osteoclast fusion, or (b) 15 to 40 amino acids from an amino acid sequence having at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative substitution in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, for example, (a) an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 and promotes osteoclast fusion, or (b) an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 contains 20-40, 25-40, or 30-40, 15-35, 15-30, or 15-25 amino acids from an amino acid sequence having at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative substitution.In a further embodiment, the peptide inhibitor is (a) an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 and promotes osteoclast fusion, or (b) an amino acid sequence of 15, 20, 25, 30, 35, or 40 amino acids or less that has at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved substitution in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. In yet another embodiment, the peptide inhibitor is (a) an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 and promotes osteoclast fusion, or (b) 15, 20, 25, 30, 35, or 40 amino acids from an amino acid sequence that has at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved substitution in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. These peptide inhibitors inhibit osteoclast fusion induced by the La protein. Suitable assays are disclosed, for example, in the Examples section.

[0188] In non-specific examples, peptide inhibitors are, Peptide 2: TIFF2026053494000011.tif4128 or Peptide 9: Contains or consists of TIFF2026053494000012.tif4128.

[0189] In a further embodiment, the peptide contains at least one amino acid substitution in either SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the peptide contains only one amino acid substitution in relation to SEQ ID NO: 8 or SEQ ID NO: 9. In another example, the peptide contains two, three, four, five or more amino acid substitutions, for example, two, three, four, five or more amino acid substitutions in relation to SEQ ID NO: 8 or SEQ ID NO: 9. In a further embodiment, the amino acid sequence of the peptide is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8 or SEQ ID NO: 9. These peptide inhibitors inhibit osteoclast fusion induced by the La protein. Suitable assays are disclosed, for example, in the Examples section. In one embodiment, the peptide is at most 35 amino acids long. In some embodiments, the peptide is 25, 26, 27, 28, 29, or 30 amino acids long. In one non-limiting example, the peptide is 30 amino acids long. In a non-limiting specific example, the inhibitory peptide is (a) consisting of SEQ ID NO: 8 or SEQ ID NO: 9, or (b) containing SEQ ID NO: 8 or SEQ ID NO: 9 and having a maximum length of 35 amino acids, or (c) consisting of SEQ ID NO: 8 or SEQ ID NO: 9 with 1, 2, 3, 4, or 5 conservative substitutions, or (d) containing SEQ ID NO: 8 or SEQ ID NO: 9 with 1, 2, 3, 4, or 5 conservative substitutions and having a maximum length of 35 amino acids.

[0190] Useful La peptide inhibitors can be prepared using recombinant methods, such as expression in host cells. Exemplary nucleic acid molecules can be prepared by cloning techniques, as disclosed above for the La protein. Nucleic acid molecules and vectors encoding La peptide inhibitors are also useful in the disclosed methods, as disclosed above for the La protein. By introducing nucleic acids and / or vectors encoding La peptide inhibitors, the activity of La in the target is reduced. In this regard, as disclosed above with respect to the use of the La protein, the same expression regulators, vectors, and host cells can be used.

[0191] E. CRISR / Cas9 This disclosure includes methods for site-specific modification of nucleic acid molecules (e.g., genomes, RNA) in cells. These modifications may include, but are not limited to, site-specific mutations, deletions, insertions, and substitutions of nucleotides. These modifications can be made anywhere within the genome, particularly within genomic elements, including coding sequences, regulatory elements, and non-coding DNA sequences. Any number of such modifications can be made, and they can be made in any order or combination, for example, all at once or one by one. Such methods can be used to modify the expression of genes such as La. Techniques for making such modifications by genome editing include the use of CRISPR-Cas systems, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs).

[0192] A typical CRISPR system consists of two components: CRISPR-associated nuclease 9 (Cas9) and one or more guide RNAs (gRNAs), each containing CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA). Simple gene disruption can be caused by cleavage of the target site, followed by nucleic acid changes, such as deletion, and repair via the non-homologous end junction (NHEJ) pathway. Target recognition by crRNA occurs through complementary base pairing with the target DNA, which directs the Cas protein to cleave the foreign sequence. In some embodiments, DNA recognition by guide RNA and the resulting endonuclease cleavage require complementary base pairing with protospacer-adjacent motifs (PAMs) (e.g., 5'-NGG-3') and protospacer regions in the target (Jinek et al., Science. 337:816-821, 2012). The PAM motif recognized by Cas9 differs depending on the Cas9 protein. Any Cas9 protein can be used in the systems and methods disclosed herein. In another embodiment of the systems and methods disclosed herein, the promoter is functionally linked to the nucleic acid encoding Cas9. In one non-limiting example, the bone-specific promoter is Runx2. [Please select a suitable promoter. Although this promoter is bone-specific, we suspect it may not be suitable for the correct cells.]

[0193] As described above, the Cas9 RNA guide system includes a mature crRNA that base-pairs with a transactivating crRNA (tracrRNA) to form a two-RNA structure that directs Cas9 to the desired double-strand (ds) cleavage site locus in the target DNA, i.e., the gene encoding La. In some embodiments, the base-paired tracrRNA:crRNA combination is engineered to create a single RNA chimera, thereby producing a guide sequence (e.g., gRNA) that retains the ability to direct sequence-specific Cas9 dsDNA cleavage (see Jinek et al., Science. 337:816-821, 2012). In some embodiments, the Cas9-guide sequence complex results in cleavage of one or both strands at the target sequence within the La gene. Thus, Cas9 endonucleases (Jinek et al., Science. 337:816-821, 2012; Mali et al., Nat Methods. 2013 Oct;10(10):1028-1034) and gRNA molecules are used for sequence-specific target recognition, cleavage, and genome editing of the La gene. In one embodiment, the cleavage site is at a specific nucleotide, for example, at the 16th, 17th, or 18th nucleotide of a 20-nucleotide (nt) target, though not limited to these. In one non-limiting example, the cleavage site is at the 17th nucleotide of the 20nt target sequence. The cleavage can be a double-strand break.

[0194] In some embodiments, the gRNA molecule is selected so that the target genome holds a protospacer adjacent motif (PAM). In some embodiments, DNA recognition by the guide RNA and the resulting endonuclease cleavage require the presence of a protospacer adjacent motif (PAM) (e.g., 5'-NGG-3') immediately after the target. The PAM is present in the targeted nucleic acid sequence but not in the crRNA constructed to target it. In some embodiments, the protospacer adjacent motif (PAM) corresponds to 2-5 nucleotides beginning immediately or near the protospacer at the distal end of the leader. The PAM motif may also be NNAGAA, NAG, NGGNG, AWG, CC, CC, CCN, TCN, or TTC.

[0195] In some embodiments, the cleavage occurs approximately 3 base pairs upstream of the PAM. In some embodiments, the Cas9 nuclease cleaves the double-stranded nucleic acid sequence.

[0196] In some embodiments, guide sequences are selected to reduce the degree of secondary structure within the sequence. Secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. One example of such an algorithm is mFold (Zuker and Stiegler, Nucleic Acids Res. 9(1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, which uses a centroid structure prediction algorithm (see, e.g., Gruber et al., 2008, Cell 106(1):23-24 and Can and Church, 2009, Nature Biotechnology 27(12):1151-62). Guide sequences can be designed using the MIT CRISPR design tool found at crispr.mit.edu, the Harvard and University of Bergen CHOPCHOP web tool found at chophop.cbu.uib.no, or the E-CRISP tool found at www.e-crisp.org / E-CRISP. Further tools for designing tracrRNA and guide sequences are described in Naito et al., Bioinformatics. 2014 Nov 20 and Ma et al. BioMed Research International, Volume 2013 (2013), Article ID 270805. CrRNA can be 18–48 nucleotides long. CrRNA can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In one example, the crRNA is 20 nucleotides long.

[0197] The system disclosed herein introduces double-strand DNA breaks into the La gene so that the La target is cleaved by Cas9. This prevents the production of a functional La protein. In some embodiments, two or more DNA breaks can be introduced by using two or more gRNAs. For example, two gRNAs can be used so that two breaks are achieved. When two or more gRNAs are used to locate two or more cleavage events, in one embodiment, two or more cleavage events in the target nucleic acid may be carried out by the same or different Cas9 proteins. For example, when two gRNAs are used to locate two double-strand breaks, only one Cas9 nuclease can be used to create both double-strand breaks.

[0198] In some embodiments, the disclosed method involves the use of one or more vectors comprising (a) a bone-specific promoter such as Runx2 functionally ligated to a nucleotide sequence encoding a type II Cas9 nuclease, and (b) a promoter such as a U6 promoter functionally ligated to one or more nucleotide sequences encoding one or more CRISPR-Cas guide RNAs that hybridize with the La gene in target cells such as human cells. Components (a) and (b) may be located on the same vector or different vectors, the one or more guide RNAs target the La gene in the target cell, and the Cas9 protein cleaves the La gene. In non-limiting examples, the one or more vectors are viral vectors such as lentiviral vectors. In another non-limiting example, the viral vectors are adenovirus vectors, adeno-associated virus vectors, or retroviral vectors.

[0199] IV. Treatment Methods and Pharmaceutical Compositions This specification discloses methods for modulating osteoclast fusion. These methods can be performed in vivo or in vitro. In some embodiments, the disclosed methods increase osteoclast fusion. In other embodiments, the disclosed methods decrease osteoclast fusion. When performed in vivo, the disclosed methods can modulate bone resorption. In some embodiments, the disclosed methods increase bone resorption. In other embodiments, the disclosed methods decrease bone resorption. These methods include administering an effective amount of La protein, a nucleic acid molecule encoding La protein, or other active ingredients that modulate the expression or activity of La protein to the target.

[0200] The subjects may be human or veterinary subjects. The subjects may be mammals. The disclosed methods will typically be used to treat human subjects, but may also be used to treat similar or the same diseases in other vertebrates, such as other primates, dogs, cats, horses, and cattle.

[0201] Administration can be systemic or topical. Examples of methods for administering the composition to mammals include, but are not limited to, oral, subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous administration. Other routes, such as inhalation and rectal administration, are also possible. Topical administration includes administration to the bone or joint of the target. Generally, an effective dose modulates osteoclast fusion compared to the target, for example, compared to no treatment or treatment with a carrier.

[0202] In one embodiment, administration is local, for example, by administration to the periosteum. In another embodiment, administration is local, for example by intramedullary injection. Intramedullary administration can be achieved by direct injection into the medullary cavity at the fracture site without injecting into the periosteum or bone cortex. Intramedullary administration can be achieved by direct injection into the bone marrow or by insertion of a K-wire through the intramedullary canal.

[0203] Administration can be systemic for subjects requiring systemic administration. In some embodiments, an effective dose may increase osteoclast fusion in the subject. An effective dose may increase bone resorption in the subject. In other embodiments, an effective dose may decrease osteoclast fusion in the subject. An effective dose may decrease bone resorption in the subject.

[0204] The administration may be a local dose, for example, an administration to the bone of the subject requiring it. In some embodiments, the effective dose may increase osteoclast fusion in the subject. The effective dose may increase bone resorption in the subject. In other embodiments, the effective dose may decrease osteoclast fusion in the subject. The effective dose may decrease bone resorption in the subject.

[0205] La protein, a nucleic agent encoding La protein, or another agent that modulates the expression or activity of La protein may be administered as a single or multiple doses, depending on the dosage and frequency required and tolerated by the subject. In any case, the composition should be administered in an amount sufficient to effectively treat the subject, such as by modulating osteoclast fusion in the patient. The composition may be administered as a single dose, but may be applied periodically until the therapeutic outcome is achieved or until it is appropriate to discontinue treatment due to side effects. In one example, the dose is infused over time. In one example, a continuous infusion may be administered over approximately 1 to 10 days, for example, over approximately 2 to 5 days, for example, over 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In another example, the dose of the agent is administered as a single or multiple bolus.

[0206] The subject may be treated at regular intervals, for example, daily, twice a week, weekly, twice a month, or monthly, until the desired therapeutic outcome is achieved. Generally, the dose is sufficient to treat or improve the symptoms or signs of the disease without causing unacceptable toxicity to the patient. The effective dose for this use will depend on the activity of the active ingredient, the severity of the disease, and the patient's general health condition. The effective dose will result in a subjective relief of symptoms or an objectively identifiable improvement that can be noticed by a clinician or other qualified observer. Concomitant use of active ingredients is also possible. Administration can be initiated whenever the suppression or prevention of the disease is desired, for example, at a certain age in the subject or prior to environmental exposure. In some embodiments, the method can regulate bone resorption in the subject before the appearance of one or more symptoms of the disease. In other embodiments, the method can regulate bone resorption in the subject after the onset of one or more symptoms of the disease.

[0207] The appropriate dosage for the treatment will vary depending on the activity of the active ingredient, the method of administration, the nature and severity of the disorder, and the patient's age and weight. However, under certain circumstances, increasing or decreasing the dosage may be appropriate. Dosage can be administered in the form of individual dose units or in several subdivided dose units as a single dose, or by administering subdivided doses multiple times at specific intervals. An experienced clinician can easily determine the effective dose.

[0208] For administration to a target, an effective amount of La protein, a nucleic acid molecule encoding La protein, or another active agent that modulates the expression or activity of La protein can be included in a pharmaceutically acceptable carrier. These pharmaceutical compositions can be prepared and administered in dose units. Solid dose units include tablets, capsules, single injections, and even suppositories. The appropriate dosage format should best be determined individually by the healthcare professional for each target. Various pharmaceutically acceptable carriers and their formulations are described in standard pharmaceutical textbooks, e.g., E.W. Martin's Remington's Pharmaceutical Sciences. See also Wang, Y.Jand Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988. The dosage form of the pharmaceutical composition will depend on the chosen mode of administration. Generally, pharmaceutical compositions contain an effective amount of La protein, or an active agent that modulates the function or activity of La protein.

[0209] Suitable forms of solid or liquid pharmaceutical preparations include, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops, or ampoule-type injectable solutions, and preparations in which the active compound is released sustainably. In these preparations, excipients and additives and / or auxiliaries, such as disintegrants, binders, coatings, swelling agents, lubricants, flavorings, sweeteners, solubilizers, or scaffolds, are typically used as described above. Pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief overview of current methods for drug delivery, see Langer, Science 249:1527-1533, 1990.

[0210] The parenteral formulation for controlled release of the present composition can be made as an implant, an oily injection, or a particle system. To provide a broad overview of protein delivery systems, see Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA (1995), which is incorporated herein by reference. Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein such as a cytotoxin or drug as a core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Since capillaries have a diameter of approximately 5 μm, only nanoparticles are administered intravenously. Microparticles typically have a diameter of around 100 μm and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, edited by J. Kreuter, Marcel Dekker, Inc., New York, NY, pages 219 - 342, 1994, and Tice & Tabibi, Treatise on Controlled Drug Delivery, edited by A. Kydonieus, Marcel Dekker, Inc., New York, NY, pages 315 - 339, 1992. Both of these references are incorporated herein by reference.

[0211] To extend the time during which an agent is available, the therapeutic agent can be provided as an implant, an oily injection, or a particle system. The particle system can be microparticles, microcapsules, microspheres, nanocapsules, or similar particles.

[0212] Polymers can be used for the ion-controlled release of the compositions disclosed herein. In the art, various degradable and non-degradable polymer matrices are known for use in drug delivery control (Langer, Accounts Chem.Res.26: 537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous but fluid liquid at low temperatures, but forms a semi-solid gel at body temperature. It has been shown to be an effective medium for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm.Res.9:425-434, 1992 and Pec et al., J.Parent.Sci.Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the control of protein release (Ijntema et al., Int.J.Pharm.112:215-224, 1994). In another context, liposomes are used for release control as well as drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, Pennsylvania, 1993). Numerous other systems for controlling the delivery of therapeutic proteins are also known. See, for example, U.S. Patents 5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,028, 4,957,735, 5,019,369, 5,055,303, 5,514,670, 5,413,797, 5,268,164, 5,004,697, 4,902,505, 5,506,206, 5,271,961, 5,254,342, and 5,534,496. Each of these U.S. patents is incorporated herein by reference.

[0213] In some embodiments, scaffolds comprising, for example, a combination of polylactic acid and glycolic acid are utilized for local administration to bone. By varying the ratio of these two components, polymers with different mechanical properties can be obtained. Thus, in some embodiments, the ratio of polylactic acid:glycolic acid is about 1:1, about 2:1, about 3:1 or about 4:1. In one example, the scaffold material comprises about 75% polylactic acid and about 25% glycolic acid.

[0214] In another embodiment, the scaffold is porous. For example, in the case of non-weight bearing tissue, etc., the porosity of the scaffold can be about 85%, about 90%, about 95%, about 98%. In a further example, in the case of weight bearing tissue, etc., the porosity of the scaffold is about 5%, about 10%, about 15% or about 20%. The porosity can be determined by, for example, the fusion of microspheres by CO2 treatment. In this process, commercially available polymer pellets are converted into microspheres of a desired size and they are fused to form a porous structure. By changing the micropore size, scaffolds with different microporosities can be obtained. These scaffolds can be used, for example, with plasmid DNA, AAV viral vectors, transposon vectors and MLV vectors. Other scaffolds are described above.

[0215] A. Additional description of methods for increasing osteoclast fusion In some embodiments, these methods increase osteoclast fusion in subjects. These methods utilize the La protein, nucleic acids encoding the La protein, or La protein agonists disclosed above. In some embodiments, subjects have diseases including reduced bone resorption. These include, but are not limited to, fractures and hypoosteoclastic osteopetrosis and genetic conditions that reduce osteoclastic bone resorption. These methods increase bone resorption and can be used to treat disorders in which increased bone resorption is beneficial to the subject. These subjects include, but are not limited to, subjects with fractures and / or osteopetrosis. These methods can also affect spinal fusion.

[0216] Methods are provided to promote fracture healing. Fractures can occur in any bone, for example, but are not limited to: the skull, e.g., the frontal bone, parietal bone, temporal bone, occipital bone, sphenoid bone, ethmoid bone; facial bones, e.g., the zygomatic bone, superior and inferior maxilla, nasal bone, mandible, palatine bone, lacrimal bone, vomer, inferior concha; ear bones, e.g., malleus, incus, stapes; hyoid bone; shoulder bones, e.g., clavicle or scapula; rib cage bones, e.g., sternum or ribs; spinal bones, e.g., cervical, lumbar and thoracic vertebrae; arm bones, e.g., humerus, ulna and radius; hand bones, e.g., scaphoid, lunate, triquetrum, pisiform, trapezium, trapezium, capitate Fractures can include those of the bones (e.g., the hamate bone), the bones of the palm (e.g., the metacarpals), the bones of the fingers (e.g., the proximal, middle, and distal phalanges), the bones of the pelvis (e.g., the ilium, sacrum, and coccyx), the bones of the leg (e.g., the femur, tibia, patella, and fibula), the bones of the foot (e.g., the calcaneus, talus, navicular bone, medial cuneiform, intermediate cuneiform, lateral cuneiform, cuboid, metatarsal, proximal, middle, and distal phalanges), and the pelvic bones. In some cases, the fracture is repaired without extraosseous ossification, for example, without ossification in soft tissue.

[0217] Methods for promoting spinal fixation using the vectors described herein are also provided. Spinal fixation can be induced in any of the vertebrae, including but not limited to the cervical, lumbar, and thoracic vertebrae. In one example, spinal fixation occurs without extraosseous ossification, for example, without ossification in soft tissue.

[0218] In further embodiments, the methods disclosed herein can be used to treat subjects whose bones are damaged by some disease, defect, or disorder that affects bone strength, function, and / or integrity, for example, by reducing bone tensile strength and modulus. Examples of bone diseases include, but are not limited to, bone fragility disorders such as osteoporosis.

[0219] In some embodiments, the therapeutically effective dose is the amount required to induce bone growth, increase prostaglandin expression, or heal a fracture. Administration of La protein, nucleic acids encoding La protein, or other agents that increase the activity and / or expression of La protein and increase osteoclast fusion can suppress symptoms of fracture or spinal disorder in a subject, such as pain and its complications. The amount effective for this use will, of course, depend on the severity of the pain as well as the patient's weight and general condition. Typically, the dosage used in vitro can serve as a useful guide for the amount useful for in situ administration of the pharmaceutical composition, and animal models can be used to determine the effective dosage for treating a particular disorder. Various considerations are presented, for example, in Gilman et al., eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th edition, Pergamon Press, 1990, and in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, Pennsylvania, 1990, and these references are incorporated herein by reference, respectively.

[0220] Exemplary assays for determining whether a bone defect can be treated by a certain method include radiography (Lehmann et al., Bone 35:1247-1255, 2004, Rundle et al., Bone 32:591-601, 2003, Nakamura et al., J.Bone Miner.Res.13:942-949, 1998); microcomputed tomography (μCT) (Nakamura et al., J.Bone Miner.Res.13:942-949, 1998, Lehmann et al., Bone 35:1247-1255, 2004, Tamasi et al., J.Bone Miner.Res.18:1605-1611, 2003, Shefelbine et al., Bone 36:480-488, 2005), peripheral quantitative computed tomography (Rundle et al., Bone 32:591-601, 2003, Tamasi et al., J.Bone Miner.Res.18:1605-1611, 2003), dual-energy X-ray absorptiometry (Holzer et al., Clin.Orthop.Rel.Res.366:258-263, 1999, Nakamura et al., J.Bone Miner.Res.13:42-949, 1998), histomorphometry (Lehmann et al., Bone 35:247-1255, 2004, Tamasi et al., J.Bone Miner.Res.18:1605-1611, 2003, Li et al., J.Bone Miner.Res.17:791-799, 2002, Schmidmaier et al., Bone 30:816-822;2002, Nakamura et al., J.Bone Miner.Res.13:942-949, 1998, Sheng et al., Bone 30:486-491, 2002), Masson's trichrome staining of collagen (Rundle et al., Bone 32:591-601, 2003), Goldner staining of collagen (Holzer et al., Clin.Orthop.Rel.Res.Examples include von Kossa silver staining of bone (366:258-263;1999), safranin orange staining of collagen (Schmidmaier et al., Bone 30:816-822, 2002), and immunohistochemistry (Rundle et al., Bone 32:591-601, 2003, Li et al., J.Bone Miner.Res.17:791-799, 2002, Safadi et al., J.Cell Physiol.196:51-62, 2003, Iwaki et al., J.Bone Miner.Res.12:96-102, 1997).

[0221] Treatment of bone defects involves stimulating bone formation to at least partially fill the gap or structural discontinuity at the site of the bone defect. Treatment of bone defects does not require a complete healing process or a treatment that is 100% effective in restoring the defect to its pre-defect state. Successful treatment of bone defects involves partial repair or healing, for example, filling of the bone defect with a new amount of bone material to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0222] If using a viral vector, at least 10 5 , at least 10 6 , or at least 10 7 Plaque-forming units / mg in mammals, for example, about 10 5 ~about 10 10 It may be desirable to administer to the recipient the dosage of each recombinant virus in the composition, in terms of plaque-forming units / mg per mammal. However, lower or higher doses may also be administered. Recombinant viral vector compositions can be introduced into the target.

[0223] Generally, the amount of recombinant viral vector carrying the nucleic acid sequence of the polypeptide to be administered is based on the titer of the viral particles. An exemplary range of the administered virus is 10 per mammal, such as humans. 5 ~10 10 These are virus particles.

[0224] In some embodiments, La protein, nucleic acids encoding La protein, or agents that increase the activity and / or function of La protein can be administered together with additional therapeutic agents. For example, for the treatment of fractures, LMP-1, FGF-2, BMP, or related proteins, or nucleic acids encoding one or more of these proteins, may be administered. For example, LMP-1 is a transcription factor that has been shown to induce osteomorphogenesis by recruiting multiple bone morphogenetic proteins (BMPs) (see Liu et al., Bone 35:673-681, 2004). While not theoretically bound, LMP-1 expression causes cells to produce osteoinducible paracrine factors such as prostaglandins and BMPs, which further enhance osteoblast differentiation in surrounding cells. In another embodiment, bone morphogenetic proteins, such as BMP-2, BMP-4, BMP-7, and / or BMP-2 / 4 hybrids, or nucleic acids encoding bone morphogenetic proteins, are administered. In another embodiment, growth factors such as FGF-2 are administered to further enhance fracture repair.

[0225] Other active agents, such as chemical compounds, may also be administered. In one embodiment, an anti-inflammatory agent, such as a nonsteroidal anti-inflammatory agent, is administered to the subject. In another embodiment, an antibiotic, antifungal agent, or antiviral agent is administered to the subject. Thus, in the disclosed method, other therapeutic agents may also be used to promote fracture healing and / or spinal fixation.

[0226] If the subject has osteoporosis, the method may further include the step of administering an effective amount of bisphosphonate or calcitonin. In a further embodiment, the method includes the step of administering an effective amount of bisphosphonate, an antibody that specifically binds to nuclear factor kappa B activating receptor ligand (RANKL), and / or teriparatide. In one non-limiting example, the antibody that specifically binds to RANKL is denosumab. Examples of bisphosphonates include, but are not limited to, zoledronic acid, pamidronate, ibandronate, alendronate, and risedronate.

[0227] B. Additional explanation of methods to reduce osteoclast fusion In several embodiments, methods for reducing osteoclast fusion in a subject are disclosed. The method comprises administering to the subject an effective amount of an agent that reduces the activity or expression of La protein in the subject. The method can reduce bone resorption in the subject. In further embodiments, the subject has a disease including increased bone resorption. These methods reduce bone resorption and can be used to treat disorders in which increased bone resorption is observed in the subject. In some embodiments, the subject has osteoporosis, Paget's disease of bone, fibrous dysplasia, rheumatoid arthritis, osteomyelitis, or metastatic bone disease. In one non-limiting example, the subject has fibrous dysplasia. In another embodiment, the subject has hyperosteoclastic osteopetrosis.

[0228] In some embodiments, the active agent is an inhibitory nucleic acid molecule. In one non-limiting specific example, a therapeutically effective dose of polynucleotides is administered to a subject to treat a disease involving increased bone resorption.

[0229] In a further embodiment, the active agent is an inhibitory La peptide, a nucleic acid molecule encoding the inhibitory La peptide, or a vector containing the nucleic acid molecule. Administration of an effective amount of the inhibitory La peptide, a nucleic acid molecule encoding the inhibitory La peptide, or a vector containing the nucleic acid molecule reduces osteoclast fusion in a subject.

[0230] The administration of nucleic acid constructs is taught, for example, in U.S. Patents 5,643,578, 5,593,972, 5,817,637, and 5,880,103. This method involves liposome delivery of nucleic acids (or La proteins).

[0231] One approach for nucleic acid administration is the direct administration of plasmid DNA, such as mammalian expression plasmids. As mentioned above, the nucleotide sequence encoding the polypeptide can be placed under the control of a promoter to increase the expression of the molecule. CRISPR / Cas systems are also useful in the methods disclosed herein.

[0232] Inhibitory nucleic acid molecules can also be expressed by attenuated viral hosts or vectors, or bacterial vectors. Recombinant vaccinia viruses, adeno-associated viruses (AAVs), herpesviruses, retroviruses, or other viral vectors can be used to express peptides or proteins. For example, vaccinia vectors and methods of administration are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) is another vector for expressing peptides (see Stover, Nature 351:456-460, 1991).

[0233] If using a viral vector, at least 10 5 , at least 10 6 , or at least 10 7 Plaque-forming units / mg in mammals, for example, about 10 5 ~about 10 10It may be desirable to administer to a recipient a dosage of each recombinant virus in the composition per plaque-forming unit / mg of mammal. However, lower or higher dosages can also be administered. The composition of the recombinant virus vector can be introduced to a subject. Generally, the amount of recombinant virus vector administered is based on the titer of virus particles. An exemplary range of virus administered is 10 5 ~10 10 virus particles per mammal such as a human.

[0234] In some embodiments, an effective amount of an antagonist antibody or an antigen-binding fragment thereof is administered to a subject. The antagonist antibody and its antigen-binding fragment are provided as a sterile solution of known concentration, but can also be provided as a lyophilized product and rehydrated with sterile water prior to administration. Next, the antibody solution is added to an infusion bag containing 0.9% sodium chloride USP and administered typically at a dosage of 0.5 to 15 mg / kg body weight. Since the approval of RITUXAN® in 1997, the extensive experience in the administration of antibody drugs sold in the United States can be utilized in the art. Antibody drugs can be administered by slow infusion rather than by IV push or IV bolus. In one example, a higher initial loading dose is administered followed by a maintenance dose at a lower level. For example, an initial loading dose of at least 0.5 mg / kg, such as at least 1 mg / kg, such as 4 mg / kg, is infused over approximately 90 minutes, and then, if the tolerance to the previous dose is high, a maintenance dose of at least 0.5 mg / kg, such as at least 1 mg / kg, such as 2 mg / kg, is infused over 30 minutes once a week for 4 to 8 weeks.

[0235] In a non-limiting specific example, a pharmaceutical composition for intravenous administration contains from about 0.1 μg to 10 mg of an antagonist antibody or an antigen-binding fragment thereof per patient per day. When the antagonist antibody or an antigen-binding fragment thereof is administered into a body cavity or the lumen of an organ, a dosage of 0.1 mg to up to about 100 mg per subject per day can be used in particular.

[0236] In some embodiments, additional active ingredients are administered to the subject. In some embodiments, the subject has osteoporosis. In further embodiments, the method includes administering an effective amount of a bisphosphonate, an antibody that specifically binds to nuclear factor kappa B-activated receptor ligand (RANKL), and / or teriparatide. In one non-limiting specific example, the antibody that specifically binds to RANKL is denosumab. Examples of bisphosphonates include, but are not limited to, zoledronic acid, pamidronate, ibandronate, alendronate, and risedronate.

[0237] In some embodiments, the subjects have Paget's disease of bone. The method may include a step of administering an effective amount of bisphosphonate or calcitonin. Examples of bisphosphonates include, but are not limited to, zoledronic acid, pamidronate, ibandronate, alendronate, and risedronate.

[0238] In a further embodiment, the subjects have fibrous dysplasia. The method may include a step of administering denosumab (Boyce et al., J Bone Miner Res. 2012 Jul;27(7):1462-1470). The method may also include a step of administering an effective dose of bisphosphonate. Examples of bisphosphonates include, but are not limited to, zoledronic acid, pamidronate, ibandronate, alendronate, and risedronate.

[0239] In a further embodiment, the subject has rheumatoid arthritis. The method may include the step of administering an effective amount of a nonsteroidal anti-inflammatory agent, a steroid, methotrexate, leflunomide, hydroxychloroquine, sulfasalazine, tofacitinib, abatacept, adalimumab, anakinara, baricitinib, certolizumab, entanercept, golimumab, infliximab, rituximab, sarilumab, and / or tocilzumab.

[0240] In a further embodiment, the subject has osteomyelitis. The method may include a step of administering an effective dose of antibiotic. Suitable antibiotics include, but are not limited to, amoxicillin-clavulanate, ciprofloxacin + clindamycin, levofloxacin + clindamycin, or moxifloxacin. The method may also include a step of administering an antifungal agent, for example, but not limited to, itraconazole, fluconazole, ketoconazole, terbinafine, and voriconazole. The method may also include a step of administering denosumab and / or teriparatide.

[0241] In further embodiments, the subject has metastatic bone disease. In further embodiments, the subject has osteosarcoma. The method may also include a step of treating the subject with a chemotherapeutic agent, immunotherapy, or radiation. The primary cancer may be, for example, a mammary gland tumor, lung tumor, thyroid tumor, kidney tumor, or prostate tumor. The method may include a step of administering an effective dose of bisphosphonate or calcitonin. Examples of bisphosphonates include, but are not limited to, zoledronic acid, pamidronate, ibandronate, alendronate, and risedronate. The method may also include a step of administering denosumab.

[0242] V. Kit Kits are also provided. In some embodiments, the kit may include the La protein, or a fragment thereof. The kit may also include nucleic acids or vectors encoding the La protein. In another embodiment, the kit may include an agent that reduces the expression or activity of the La protein in a subject.

[0243] In some embodiments, the kit comprises (a) an La protein or an effective fragment thereof, or a nucleic acid molecule or vector encoding an La protein or an effective fragment thereof, and optionally, (b) one or more proteins that interact with La and modulate its activity, or an effective fragment thereof, or a nucleic acid molecule or vector encoding these proteins or their effective fragments, and (c) a nucleic acid encoding LMP-1, FGF-2, BMP, or related proteins, or one or more of these proteins. In some embodiments, the kit comprises elements (a) and (c).

[0244] In a further embodiment, the kit comprises (a) an activator that reduces the expression or activity of La protein in a target, and (b) a bisphosphonate, an antibody that specifically binds to RANKL, and / or teriparatide. In a further embodiment, the activator that reduces the expression or activity of La protein in a target is i) an inhibitory nucleic acid molecule, ii) an antagonist antibody that specifically binds to La protein, iii) a caspase inhibitor, or iv) an inhibitory peptide or a nucleic acid molecule encoding an inhibitory peptide. In some non-limiting examples, the inhibitory peptide comprises or consists of SEQ ID NO: 8 or SEQ ID NO: 9, or comprises or consists of SEQ ID NO: 8 or SEQ ID NO: 9 with 1, 2, 3, 4, or 5 conserved substitutions. In a further embodiment, the kit comprises a CRISPR nuclease or a nucleic acid molecule encoding a CRISPR nuclease.

[0245] The kit may also include additional components to facilitate the specific application for which it was designed. For example, the kit may further include buffers and other reagents commonly used to carry out a particular method.

[0246] The kit may include a container and labels or accompanying documents on or attached to the container. Suitable containers include, for example, bottles, vials, and syringes. Containers may be made from a variety of materials, such as glass or plastic. In some embodiments, the container may have an access port to allow a specific amount of the active substance to be withdrawn (for example, the container may be an intravenous bag or a vial with a stopper that can be punctured with a subcutaneous needle).

[0247] Labels or accompanying documents indicate the intended use of the composition. Accompanying documents typically include instructions usually found on the product's market packaging, containing information on the product's dosage, contraindications, and / or precautions for use. Educational materials may be in written form, electronic format (e.g., computer diskette or compact disc), or visual format (e.g., video files).

[0248] The present disclosure is illustrated below by non-limiting embodiments. [Examples]

[0249] We hereby disclose that osteoclast formation involves and depends on drastic changes in the steady-state levels, molecular species, and intracellular localization of the La protein. La is shown to function as a regulator of osteoclast fusion and to influence the bone resorption capacity of osteoclasts. Surprisingly, La present in primary human monocytes is almost completely absent in M-CSF-induced osteoclast precursors. RANKL-induced constraint on osteoclast generation promotes the reappearance of cleaved La protein on the surface of osteoclasts during constrained fusion. As osteoclast fusion reaches a plateau, cleaved La disappears, and high molecular weight full-length protein (FL-La) is observed in the nucleus of mature multinucleated osteoclasts. Disruption of La expression, cleavage, or surface function inhibits osteoclast fusion, while exogenous surface La promotes fusion. Furthermore, the mechanism by which La promotes osteoclast fusion does not depend on La's ability to interact with RNA via its highly conserved La domain. In fact, the C-terminal portion of La (SEQ ID NO: 7) lacks the La domain and RNA recognition motif 1 (RRM1), yet it is sufficient to promote fusion between human osteoclasts. This finding indicates that the La protein is adapted in mammals to function as an osteoclast fusion regulator. Therefore, La is a target for treating bone diseases caused by disruption of bone turnover.

[0250] Example 1 La protein is involved in the formation of multinucleated osteoclasts. Osteoclast formation has been shown to involve dramatic changes in the levels, molecular species, and location of La within the osteoclasts during fusion. Severed non-nuclear La species promote osteoclast formation, and as the cells reach mature size, LMW La is replaced by FL La, which is detected in the nuclei of syncytial osteoclasts.

[0251] Human osteoclast generation was modeled by inducing mononuclear osteoclast precursors by treating primary monocytes with M-CSF, and then adding recombinant RANKL to obtain multinucleated osteoclasts that readily resorb bone (Figures 1A, 1B, 8A-8C) (Verma et al., J Biol Chem 293, 254-270, doi:10.1074 / jbc.M117.809681(2018)). The osteoclast precursors began to fuse approximately 2 days after RANKL addition, and reached the size characteristic of mature multinucleated osteoclasts (approximately 5-10 nuclei / cell) after about 5 days. 10, 35, 36 Reaching this stage (Figure 1B, Figure 8C) (Moller et al., Int J Mol Sci 21, doi:10.3390 / ijms21176368(2020), Abdallah et al., Front Immunol 9, 632, doi:10.3389 / fimmu.2018.00632(2018), Stattin et al., Sci Rep 7, 3012, doi:10.1038 / s41598-017-02533-2(2017)).

[0252] While evaluating proteomic changes associated with osteoclast generation, a distinctly different protein was discovered that was almost absent in M-CSF-inducible precursors but abundantly expressed in osteoclasts after approximately 3 days of RANKL-stimulated osteoclast generation, during which cells rapidly fuse (Figure 1C, arrow). Using mass spectrometry, this protein was identified as La (Figure 8D). The low level of La in M-CSF-inducible macrophages was unexpected, as La is generally considered to be an abundant and ubiquitous protein (see, for example, Wolin and Cedervall, Annu Rev Biochem 71, 375-403, doi:10.1146 / annurev.biochem.71.090501.150003 (2002)).

[0253] Western blot analysis confirmed that La is sufficiently expressed in monocytes, significantly reduced in M-CSF-induced osteoclast precursors, and returns to a high steady-state level of La during RANKL-induced osteoclast formation (Figure 1D). This data suggests that strict regulation of La during osteoclast generation occurs post-translationally, as M-CSF-induced precursors contained even higher levels of La transcripts (gene SSBs) than those after RANKL application (Figure 8E). During osteoclast generation, La appeared as two distinctly different, temporally separated molecular species (Figure 1E). The low molecular weight (LMW La) species detected at the initiation of fusion and during strong fusion is replaced by a higher molecular weight species corresponding to full-length La (FL La) as fusion slows and osteoclasts reach mature size.

[0254] In addition to changes in molecular weight, osteoclast-generating differentiation of human monocytes involves dramatic changes in the intracellular location of La. Normatively, La exhibits strong nuclear staining, as exemplified in HeLa cells (Wolin and Cedervall, Annu Rev Biochem 71, 375-403, doi:10.1146 / annurev.biochem.71.090501.150003(2002)). In contrast, M-CSF-induced osteoclast precursors exhibit only slight La staining (Figure 1F), which is consistent with biochemical analysis (Figure 1D). The addition of RANKL produced abundant La signaling in constrained, fusing osteoclasts, but in contrast to other human cell types and tissues (Wolin and Cedervall, Annu Rev Biochem 71, 375-403, doi:10.1146 / annurev.biochem.71.090501.150003(2002), Maraia et al., Wiley Interdiscip Rev RNA 8, doi:10.1002 / wrna.1430(2017)), La appeared throughout fusing osteoclasts as distinct, primarily non-nuclear plaques during the early stages of osteoclast fusion (Figure 1F).

[0255] The anti-La antibody (α-La) used in Figure 1D recognizes both LMW La and FL La species. To determine whether different molecular weight species of La exhibit different localization during osteoclast formation, several commercially available antibodies were analyzed to select an antibody that preferentially recognizes LMW La species (see also α-LMW La, Figure 8F, and the table below).

[0256] (Table) Antibodies used to detect La molecular species in human osteoclasts TIFF2026053494000013.tif23164

[0257] FL La is largely phosphorylated at Ser366 and localized to the nucleus (Intine et al., Mol Cell 12, 1301-1307, doi:10.1016 / s1097-2765(03)00429-5(2003)). Previous studies have shown that LMW La is produced by cleavage of FL La, however, that FL La must be dephosphorylated at Ser366 to become cleavable. 33 Furthermore, previous reports have shown that antibodies specific to phosphoSer366 La do not recognize LMW La (Rutjes et al., Cell Death Differ 6, 976-986, doi:10.1038 / sj.cdd.4400571(1999)). These experiments used an α-phosphoSer366 La antibody to preferentially stain FL La (α-FL La). At intermediate time points where both La isoforms are present, LMW La was detected primarily extranuclear and throughout the cell, while FL La was observed exclusively within the nucleus of fused cells (Figure 1G). The shift in La distribution during osteoclast formation was further confirmed by α-La staining at various days after RANKL application (Figure 8G).

[0258] Osteoclastic differentiation was found to be accompanied by drastic changes in motivated La expression and localization. We determined whether La is functionally involved in osteoclast formation. La expression was found to tightly regulate the formation of multinucleated osteoclasts. RNAi-mediated reduction of La transcripts (SSBs) severely inhibited osteoclast fusion (Figure 2A-2C). Cytosolic localization of La during fusion was also observed during osteoclastic differentiation and fusion of mouse osteoclast precursors derived from RAW 264.7 (Figure 2D). Furthermore, Western blot analysis of cell lysates collected separately from predominantly mononuclear and predominantly multinucleated cells showed that strong fusion on day 3 after RANKL was accompanied by a drastic increase in steady-state levels of La (Figure 2E). These findings suggest that La dependence in osteoclast formation is conserved between humans and mice. It should be noted that, unlike human cells, in the case of RAW 264.7 cells, after the active fusion phase, the La level returned to the low pre-fusion level by day 5.

[0259] To identify functional La morphologies associated with osteoclast generation, we determined the relationship between the appearance of LMW La and osteoclast fusion. During apoptosis, human La is cleaved at Glu-375 by caspases, and the NLS of La is removed (Rutjes et al., Cell Death Differ 6, 976-986, doi:10.1038 / sj.cdd.4400571(1999), Ayukawa et al., J Biol Chem 275, 34465-34470, doi:10.1074 / jbc.M003673200(2000)). Overexpression of La 1-375, which mimics this cleaved species, was found to significantly promote fusion in both mouse osteoclasts derived from RAW 264.7 and human osteoclasts derived from monocytes (Figure 2F~2I). In contrast, the non-cleavable mutants of FL La, D371A and D374A La (point mutations disrupting the expected caspase cleavage site of La), did not have any effect on osteoclast fusion despite being expressed at similar levels, suggesting that multinucleated osteoclast formation is dependent on LMW La (Figures 2H, 2I, and 9). The pancaspase inhibitor z-VAD was also found to inhibit LMW La production in differentiating osteoclasts (Figure 10A). Inhibition of caspase-dependent production of LMW La leads to premature tethering of FL La in the nuclei of unfused osteoclasts (Figure 10C vs. Figure 10B), significantly disrupting the ability of osteoclasts to form multinucleated syncytia (Figure 10D). See also Szymczyk et al., J Cell Physiol 209, 836-844, doi:10.1002 / jcp.20770 (2006). This data further supports the role of caspase-cleaved LMW La in the formation of multinucleated osteoclasts.

[0260] Example 2 Cell surface-bound La controls the cell fusion stage of osteoclast formation. Although La alone does not possess fusion activity, it has been demonstrated that it promotes membrane fusion on the surface of differentiating osteoclasts. This promotion does not require the La domain, RRM1, or NLS domain of La, and involves a complex formed by La together with Anx A5, one of the components of the osteoclast fusion mechanism.

[0261] In characterizing the role of La in osteoclast formation, we first investigated whether La indirectly exerts its function in osteoclast formation by altering the expression of factors related to osteoclast differentiation or osteoclast fusion. While La expression in many cell types affects the steady-state levels of many transcripts / proteins (Sommer et al., Oncogene 30, 434-444, doi:10.1038 / onc.2010.425(2011)), La RNAi repression did not alter the steady-state transcript levels of the essential osteoclast factors NFATc1 and CTSK, nor the transcripts encoding the fusion-related proteins syncytin 1, Anx A5, S100A4, or the lipid scramblase anoctamin 6 / TMEM16F (Figure 11) (Verma et al., J Biol Chem 293, 254-270, doi:10.1074 / jbc.M117.809681(2018)). Therefore, while knockdown of La inhibited osteoclast syncytial formation, it did not significantly affect osteoclast differentiation or other mechanisms critically important for intercellular fusion. To further investigate the mechanism by which La affects osteoclast formation but not their differentiation, we evaluated whether the formation of multinucleated osteoclasts depends on La's highly characterized RNA-binding function. This highly conserved function is based on a high-affinity interaction between the La domain and its high-affinity oligo(U)-3' binding site common to RNA polymerase III transcripts. To evaluate the requirements for high-affinity interactions between the La domain and transcripts in osteoclast generation, we overexpressed a mutant La 1-375 (La 1-375 RNAΔ) containing three point mutations Q20A / Y24A / D33I (Bayfield et al., Nat Struct Mol Biol 16, 430-437, doi:10.1038 / nsmb.1573 (2009), Vinayak et al., Nucleic Acids Res 46, 4228-4240, doi:10.1093 / nar / gky090 (2018)) that are known to functionally impair La domain function.La 1-375 RNAΔ was found to promote multinucleated osteoclast formation as strongly as wild-type La 1-375, indicating that high affinity of the La domain to RNA polymerase III transcripts is not necessary for La's role in osteoclast formation (Figure 2F, Figure 3G).

[0262] A second piece of evidence suggesting that La's role in multinucleated osteoclast formation is not due to its normative role in RNA metabolism was the demonstration that La's function in osteoclasts is on the cell surface, rather than in the nucleus or cytoplasm. As previously mentioned, in differentiating osteoclasts, La loses its NLS and appears throughout the cell, in the mottled structure. Proteins from RANKL-constrained osteoclasts at the time of active cell fusion were concentrated in either a soluble cytosolic protein fraction or a membrane-bound protein fraction. As expected, actin was found mostly in the cytosolic fraction, the transmembrane RANK receptor in the membrane fraction, and the superficial membrane protein Anx A5 in both fractions (Figure 3A). Although La proteins are presumed to be soluble in differentiating osteoclasts, the presence of La in both the cytosolic and membrane-bound fractions suggests that La unexpectedly associates with the membrane during osteoclast formation (Figure 3A).

[0263] Previous reports indicated that La cleavage in apoptotic cells was accompanied by the detection of La on the cell surface (Rutjes et al., Cell Death Differ 6, 976-986, doi:10.1038 / sj.cdd.4400571(1999), Ayukawa et al., J Biol Chem 275, 34465-34470, doi:10.1074 / jbc.M003673200(2000)), but it was previously unknown whether this surface La performed any cellular function or simply acted as a surface antigen. To evaluate whether osteoclast La is transported to the cell surface following cleavage, fusion osteoclasts were stained with α-La antibody under impermeable conditions (Figures 3B and 3C). The osteoclast superficial membrane protein Fish (also known as TSK5) (Oikawa et al., J Cell Biol 197, 553-568, doi:10.1083 / jcb.201111116(2012)) is concentrated during osteoclast fusion and binds to the cytoplasmic leaflet of the plasma membrane (PM) (Oikawa et al., J Cell Biol 197, 553-568, doi:10.1083 / jcb.201111116(2012)). In contrast, La extensively modified the surface of human osteoclasts during fusion (Figure 3B). Furthermore, this surface pooling of La is not limited to human osteoclasts. Significant La surface staining was observed in mouse osteoclasts derived from RAW 264.7 (Figure 3C), suggesting that surface La is a common feature of fusion-promoting osteoclasts in mammals.

[0264] Surface staining with α-La at various time points after RANKL application revealed a transient increase in La at the point of strong fusion (Figure 3D vs. Figure 1B), further linking La to fusion.

[0265] Next, we evaluated whether La on the surface of human osteoclasts functions in the cell fusion stage of osteoclast formation. All intercellular fusion events in development and tissue maintenance proceed by a slow (daily) asynchronous differentiation process that prepares fusion-competent cells (Brukman et al., J Cell Biol 218, 1436-1451, doi:10.1083 / jcb.201901017(2019)). Then, PM fusion occurs by a rapid (minute-by-minute) progression from the formation of the initial semi-fusion junction to the fusion pore that combines the volumes of the two cells (Figure 3D, top). We used the hemifusion inhibitor lysophosphatidylcholine (LPC) to detach these steps in the formation of multinucleated syncytia (Verma et al., J Biol Chem 293, 254-270, doi:10.1074 / jbc.M117.809681(2018)). The inverted conical shape of LPC does not contribute to the concave shape of the hemifusion stalk, so ready-to-fuse cells are captured upstream of hemifusion. After removal of LPC, cells undergo synchronized fusion relatively quickly (within 90 minutes), making it possible to specifically evaluate protein function at the membrane fusion stage of osteoclast formation, detached from the upstream differentiation process (Figure 3D, bottom). In the presence of LPC, ready-to-fuse cells constrained by RANKL accumulate, and this hemifusion inhibition is then released by washing away the LPC. Application of α-La antibody at the time of LPC removal significantly inhibited synchronized osteoclast membrane fusion (Figure 3E). In contrast, antibodies targeting the PM receptor RANK on the osteoclast surface had no effect (Figure 3F). RANKL-RANK signaling triggers upstream osteoclastogenesis, but inhibition of RANK after semi-fusion synchronization cannot inhibit membrane fusion. This is because fusion itself does not depend on RANK activity (Verma et al., J Biol Chem 293, 254-270, doi:10.1074 / jbc.M117.809681(2018)).Furthermore, the α-LMW La antibody completely blocked synchronized osteoclast membrane fusion, while the α-FL La antibody had no effect (Figure 3G).

[0266] In contrast to the fusion-inhibiting effect of antibodies targeting surface La, the application of recombinant La dramatically promoted osteoclast fusion. FL La (La 1-408), truncated La (La 1-375), or truncated RNA-binding mutant La 1-375 RNAΔ outside of fusion osteoclasts significantly promoted multinucleated syncytia formation (Figures 4A-4C). This promotion was not observed when recombinant La was thermally inactivated (Figure 4B). Since recombinant La 1-375 RNAΔ promoted fusion similarly to La 1-408 and La 1-375, it was confirmed that high-affinity interaction of La with RNA polymerase III transcripts is not required for La's role in regulating osteoclast fusion (Figures 4A-4C). Furthermore, the ability of FL La to promote osteoclast fusion suggests that FL La itself does not lack fusion ability, but rather that proteolytic processing and dephosphorylation of La are important for its delivery to the cell surface.

[0267] To further analyze the contributions of the La domain, La and RRM1 domain, which are critically important for RNA binding (Wolin et al., Annu Rev Biochem 71, 375-403, doi:10.1146 / annurev.biochem.71.090501.150003 (2002), Maraia et al., Wiley Interdiscip Rev RNA 8, doi:10.1002 / wrna.1430 (2017), Bayfield et al., Nat Struct Mol Biol 16, 430-437, doi:10.1038 / nsmb.1573 (2009), Vinayak et al., Nucleic Acids Res 46, 4228-4240, doi:10.1093 / nar / gky090 (2018)), La La 1-375 was divided into La 1-187 and La 188-375. La 188-375 significantly promoted multinucleated osteoclast formation, while La 1-187 had no effect (Figure 4D). These data demonstrate that the La domain, RRM1, and the C-terminal 33AA of La are not necessary for La's role in osteoclast formation (Figure 4D). Importantly, La promoted osteoclast formation at the membrane fusion stage, rather than at any pre-fusion stage of differentiation. Up to this point, the application of recombinant La to LPC-synchronized osteoclasts dramatically promoted osteoclast membrane fusion (Figure 4E).

[0268] All of these data indicate that La functions to promote the formation of large, multinucleated osteoclasts on the cell surface during the membrane fusion phase of osteoclast formation. Proteins involved in membrane fusion can be classified into fusion factors, which are sufficient to generate semi-fusion intermediates and open fusion pores, and proteins that regulate the activity of fusion factors (Bruckman et al., J Cell Biol 218, 1436-1451, doi:10.1083 / jcb.201901017(2019)). To investigate whether cell surface La can independently fuse membranes and function as an active protein fusion factor, we evaluated the ability of La to promote fusion between 3T3 fibroblasts stably expressing HA0 (non-cleaving influenza fusion factor hemagglutinin (HA)) and erythrocytes (RBCs) labeled with lipid probes and content probes (Leikina et al., Dev Cell 46, 767-780 e767, doi:10.1016 / j.devcel.2018.08.006(2018)). Although HA0 does not possess fusion ability, it establishes extremely close contact between HA0-expressing fibroblasts and RBCs. Of the 872 HA0-cell-bound RBCs analyzed, none underwent lipid probe exchange (an indicator of partial fusion) or cytoplasmic probe exchange (an indicator of fusion pores) in response to the application of 40 nM recombinant La. According to Wilson's method (Ludbrook et al., ANZ J Surg 79, 565-570, doi:10.1111 / j.1445-2197.2009.04994.x(2009)), the probability of La-mediated fibroblast-RBC fusion does not exceed 0.0044 per cell contact, and under these conditions, it exhibits less than 1 / 100th the activity of HA activated with a true fusion factor (approximately 0.5 per contact). These data suggest that La does not possess detectable intercellular fusion activity itself, but rather that La likely controls some larger fusion mechanism specific to osteoclasts.

[0269] The lack of direct fusion activity of La suggested that cell surface La interacts with some other protein involved in fusion. To test this hypothesis, we evaluated whether La interacts with Anx A5, a superficial membrane protein similarly involved in the membrane fusion stage of osteoclasts (Verma et al., J Biol Chem 293, 254-270, doi:10.1074 / jbc.M117.809681(2018), Whitlock and Chernomordik, J Biol Chem, 100411, doi:10.1016 / j.jbc.2021.100411(2021)) and upregulated at a similar time in osteoclast formation. When La and La-containing protein complexes were immunoprecipitated from fused human osteoclasts onto magnetic beads containing α-La Ab, it was found that the La protein complex contained Anx A5 (Figure 12A). The specificity of the association between La and Anx A5 was demonstrated by the fact that neither of the La supramolecular complexes from fusing osteoclasts contained Anx A1 or Anx A4, which are abundant in fusing osteoclasts (Figure 12B). Furthermore, immunoprecipitation of Anx A5 supramolecular complexes isolated using α-Anx A5 Ab from fusing human osteoclasts contained La (Figure 12B).

[0270] Further evidence of the La-Anx A5 interaction was obtained from experiments that showed that direct binding of La to Anx A5 immobilizes La on phosphatidylserine (PS)-containing phospholipid liposomes. Anx A5 then binds Ca to the PS-containing membrane. 2+ It binds in a dependent manner. Recombinant La alone or together with recombinant Anx A5 was introduced into PS-containing liposomes, and the liposomes were pelleted by centrifugation to evaluate whether La was concentrated with the liposomes or in the supernatant (Figure 12C). La alone was not pelleted well with the liposomes because it does not itself possess a membrane-binding domain. In contrast, with both La and Anx A5, Ca 2+In response, it was pelleted together with liposomes (Figure 12D). La membrane association was Anx A5, Ca 2+ And PS was required. Neither La nor Anx A5 pelleted with liposomes lacking PS (Figure 12D). These findings suggest that a direct interaction between La and extracellular Anx A5 bound to PS transiently exposed on the surface of osteoclasts during fusion (Verma et al., J Biol Chem 293, 254-270, doi:10.1074 / jbc.M117.809681(2018)) facilitates the association of La with the surface of differentiating osteoclast precursors.

[0271] Example 3 La is a potential target for influencing osteoclast formation and function. Cell surface lamina (La) has been shown to regulate the formation of multinucleated osteoclasts in humans and mice, triggered by biologically meaningful interactions between osteoclast precursors and osteogenic cells. Targeting La modulates the fusion of osteoclast precursors, which in turn alters the resulting osteoclast bone resorption tendency. Furthermore, La has been found to be involved in osteoclast formation in an ex vivo FD model. This demonstrates that targeting the function of La on the surface of developing osteoclasts is an effective therapeutic intervention in FD and other resorbable bone diseases caused by excessive osteoclast activity.

[0272] There is a relationship between osteoclast fusion and bone resorption (Makris and Saffar, Arch Oral Biol 27, 965-969, doi:10.1016 / 0003-9969(82)90104-2(1982), Piper et al., Anat Embryol(Berl)186, 291-299, doi:10.1007 / BF00185977(1992), Moller et al., Int J Mol Sci 21, doi:10.3390 / ijms21176368(2020)). La hypothesized that bone resorption is controlled by controlling osteoclast size. This hypothesis was evaluated by differentiating osteoclasts on fluoresceinated calcium phosphate, a biomimetic of bone, and osteoclast-dependent bone resorption was assessed by the release of fluorescein into the culture medium (Figure 5A). Monocyte-derived precursors (M-CSF only) released only a small amount of captured fluorescein, but the addition of RANKL resulted in the formation of multinucleated osteoclasts that readily absorbed calcium phosphate and released fluorescein (Figure 8B). Overexpression of La 1-375 promoted bone resorption, while the cleavage-inhibiting La mutants D371 and 374A had no effect (Figure 5B). Furthermore, reduction of La by RNAi reduced bone resorption by approximately 40% compared to untargeted controls (Figure 5C). An α-La antibody that inhibits fusion (Figure 3E) also dramatically reduced osteoclast-dependent bone resorption in a dose-dependent manner (Figure 5D). Finally, extracellular addition of recombinant La 1-375 to fusion osteoclasts dramatically increased osteoclast bone resorption (Figure 5E). Based on these data, we concluded that targeting cell surface La bidirectionally regulates both osteoclast fusion and subsequent bone resorption.

[0273] In biologically meaningful contexts, osteoclast formation occurs against the backdrop of interactions between osteoblasts / osteocytes and other cell types, resulting in much lower concentrations of RANKL and many other osteoclast regulatory factors (Kitura et al., Int J Mol Sci 21, doi:10.3390 / ijms21145169(2020)). To investigate whether La is involved in osteoblast-induced osteoclast formation, primary human osteoblasts were isolated from cancellous bone and co-cultured with human osteoclast precursors obtained by M-CSF induction of primary human monocytes. Osteoblasts and osteoclast precursors were cultured isolated from each other using well inserts (Figure 6A). No fusion was observed between osteoclast precursors unless the well inserts were removed. Upon removal of the well inserts, the media from the osteoblast / osteoclast wells were mixed, and the co-cultured osteoclast precursors rapidly fused to produce multinucleated osteoclasts. The addition of α-La antibodies inhibited fusion between osteoclasts in such co-cultures by nearly 75% (Figure 6B, 6C), confirming the involvement of La in osteoclast formation in a biologically meaningful model of bone remodeling lesions.

[0274] To investigate whether La plays a role in bone pathology, the experiment focused on fibrous dysplasia (FD), an osteoclast-dependent bone disease (de Castro et al., J Bone Miner Res 34, 290-294, doi:10.1002 / jbmr.3602(2019)). FD is caused by gain-of-function mutations in Gαs that lead to constitutively increased cAMP signaling and upregulation of cAMP / RANKL-dependent osteoclast generation (Boyce and Collins, Endocr Rev 41, doi:10.1210 / endrev / bnz011(2020)). In a conditionally tetracycline-inducible mouse model, FD-like bone lesions develop in adult mice within two weeks of doxycycline (Doxy) administration (Xao et al., Proc Natl Acad Sci USA 115, E428-E437, doi:10.1073 / pnas.1713710115(2018)). The formation of these lesions is attributed to inducible gain-of-function mutant Gα, specifically in skeletal stem cell lineage cells, which are responsible for the excessive RANKL production observed in FD. s R201C It is driven by the activation of RANKL. This excessive RANKL production leads to the ectopic formation of numerous large osteoclasts that excessively erode healthy bone. Using bone marrow explants from these FD mice, we established a robust ex vivo model of ectopic osteoclast formation observed in FD (Figure 7A). As illustrated in Figure 7B, culturing these FD explants in the presence of M-CSF alone resulted in numerous adherent cells, but multinucleated TRAP + Osteoclasts were not produced. In contrast, the addition of Doxy produced inducible Gα s R201C Fibrous cell aggregates (arrows) and numerous multinucleated TRAPs are not observed in explants from wild-type littermates lacking certain elements. +This resulted in rapid development of osteoclasts (arrowheads). Doxy-induced osteoclast generation was accompanied by an approximately 17-fold increase in mRANKL produced by the explant (Figure 7C). Importantly, the α-La antibody inhibited approximately 60% of osteoclast fusion induced by the addition of Doxy to the FD explant and reduced the number of observed multinucleated osteoclasts by approximately 40% (Figures 7D-7F).

[0275] The differentiation of mouse and human monocytes into multinucleated osteoclasts depends on strictly programmed changes in the steady-state level, post-translational modification, and intracellular localization of La (Figure 7G). At the initiation of osteoclast formation, M-CSF-inducible precursors exhibit a dramatic loss of La protein, suggesting that this differentiation process may require coordinated downregulation of a specific La regulatory pool of mRNA triggered by the loss of steady-state La. In the subsequent RANKL-inducible stage of osteoclast formation, La reappears as an unphosphorylated, proteolytically cleaved species in the cytoplasm and on the surface of the osteoclast precursors during fusion. As osteoclast growth slows, in the later stages of fusion, La is observed at its conventional size and nuclear localization. The rate of formation, the size of the multinucleated syncytium, and the subsequent bone resorption activity of osteoclasts are regulated by the cell surface La protein. In fact, cell surface La regulates osteoclast function by modulating the membrane fusion stage of osteoclast formation, rather than the upstream differentiation process. Reducing the amount of La by suppressing the steady-state level of its transcript, blocking the proteolytic processing required for its transport to the cell surface, or inhibiting its activity with an antibody inhibits fusion. Conversely, increasing the steady-state concentration of La by either overexpression or application of recombinant protein promotes fusion. In summary, these data demonstrate that La, a key protein in eukaryotic RNA biology, is present on the surface of osteoclasts, where it acts as a master regulator of osteoclast membrane fusion.

[0276] The data presented herein demonstrate that the role of La in regulating osteoclast fusion and bone resorption is distinct from the detailed normative role of La and represents a novel function of the La protein. First, the ability to inhibit or promote synchronized osteoclast membrane fusion with non-cell-permeable reagents (e.g., antibodies, recombinant La) indicates that the regulation of osteoclast fusion is dependent on surface La.

[0277] La regulation of osteoclast formation is independent of the highly conserved La domain and the interaction between RRM1 and RNA. This conclusion is supported by the finding that mutations in critically important residues within the La domain, as well as deletion of the entire N-terminal half of the La protein (containing both the La domain and RRM1), did not result in the loss of recombinant La's ability to promote osteoclast fusion.

[0278] Since La alone does not initiate either partial fusion or fusion between bound membranes, it is unlikely that La directly catalyzes and / or drives membrane fusion. It is more likely that La recruits or stimulates other components of the osteoclast fusion complex. The latter scenario is supported by these findings, which shed light on the association of La with the fusion regulator Anx A5. Recombinant La and Anx A5 interact directly, and it was found that Anx A5 facilitates the association of La with PS-containing membranes in a Ca2+-dependent manner. These observations, along with previously reported dependence of osteoclast fusion on cell surface PS and Anx A5 (Verma et al., J Biol Chem 293, 254-270, doi:10.1074 / jbc.M117.809681(2018)), provide clues to how osteoclasts utilize PS to trigger the assembly of fusion complexes between constrained precursors.

[0279] The imbalance between bone formation and bone resorption in many skeletal diseases is associated with either excessive activity of osteoclasts (e.g., osteoporosis, Paget's disease, and FD) or insufficient activity (e.g., osteopetrosis). It is disclosed herein that the formation of multinucleated human osteoclasts can be inhibited or promoted by treatment targeting the La protein on the surface of osteoclast precursors. Importantly, α-La antibodies inhibit fusion and bone resorption by osteoclasts derived from RANKL-activated monocytes. α-La antibody treatment also inhibited the formation of multinucleated osteoclasts in human osteoclast precursor / osteoblast cocultures modeling bone remodeling lesions in which osteoclast-generating factors are produced by osteoblasts within the lesion (Ikebuchi et al., Nature 561, 195-200, doi:10.1038 / s41586-018-0482-7(2018)). Furthermore, the hypothesis that cell surface La plays a crucial role in osteoclast formation under biologically significant circumstances was validated by experiments using an ex vivo model of FD. The progression of FD is characterized by a sharp increase in serum levels of RANKL and other osteoclast-generating factors, and the ectopic formation of numerous multinucleated osteoclasts near bone lesions. As expected, induction of the FD phenotype in bone marrow exgrafts resulted in high concentrations of RANKL and ectopic osteoclast formation. The finding that α-La inhibits multinucleated osteoclast formation in both size and number confirms the importance of La as a novel target in bone pathology and highlights its potential as a target for future therapeutic development.

[0280] Some proteins involved in the early stages of osteoclastogenesis have already been tested as potential therapeutic targets in animal and / or clinical studies (Boyce et al., J Bone Miner Res 28, 711-722, doi:10.1002 / jbmr.1885(2013)). The α-RANKL antibody denosumab is an FDA-approved drug for treating osteoporosis (Sordillo et al., Cancer 97, 802-812, doi:10.1002 / cncr.11134(2003)). La-dependent osteoclast fusion downstream of the RANKL / RANK / osteoprotegerin signaling pathway corresponds to therapeutic targets at different mechanistic stages of bone remodeling. Considering that mononuclear osteoclasts resorb bone, inhibiting La-dependent osteoclast fusion may have a more subtle and selective effect on bone resorption than inhibiting upstream osteoclast precursor formation with α-RANKL antibodies (see, e.g., Miyamoto et al., J Bone Miner Res 27, 1289-1297, doi:10.1002 / jbmr.1575(2012)). Cell-impermeable drugs, such as RANKL, can access cell surface La. In some clinical situations, the more subtle effects of La-targeting treatments may be advantageous. Furthermore, unlike RANKL, which regulates not only osteoclast generation but also the immune response (Ono et al., Inflamm Regen 40, 2, doi:10.1186 / s41232-019-0111-3(2020)), the only known function of cell surface La is its role in regulating osteoclast fusion, as disclosed here. Therefore, due to the specificity of surface La, off-target effects can be minimized. Finally, it is known that osteoclasts release factors that regulate osteoblast activity (Sims et al., Curr Osteoporos Rep 10, 109-117, doi:10.1007 / s11914-012-0096-1(2012)). Completely inhibiting osteoclast generation by targeting RANKL is likely to inhibit osteoclast-osteoblast signaling.By targeting La, it is possible to suppress the fusion stage of osteoclast formation while maintaining the differentiation potential of osteoclasts, thereby preserving this osteoclast-osteoblast crosstalk within the lesion, which may be important for bone remodeling in diseases where it is lost. In summary, the disclosed data demonstrate the function of the La protein as a key regulatory element of osteoclast formation, in which its site of action, mechanism of action, and partner protein (Anx A5) play a significantly different role from its function as a well-known RNA chaperone, and provide therapeutic measures to modulate osteoclast function.

[0281] Example 4 Use of La antibodies in a model of fibrous dysplasia (FD) Fibrillar dysplasia (FD) is an osteolytic bone disease in which an imbalance in osteoclast-osteoblast (OC-OB) signaling and coordination is central to its pathophysiology. Postjunctional mosaicism Gα occurs only in osteoblasts. s The mutation disrupts OB-OC signaling, leading to overproduction of osteoclast-generating signals and altering osteoclast formation and activity. We used an inducible mouse model of FD in which the addition of doxycytrin to the diet induced the progression of FD in approximately three weeks. The progression of FD was detected by visually observing the decrease in bone mass in the mouse hind limbs over time using X-rays, and the progression of FD was scored by observing the development of radiolucent resorption pits and woven bone deposition along calcified bone. Each bone was assigned a score from 1 to 6 representing the degree of bone loss, and an FD progression score was given to each mouse by averaging these scores (Figure 13A). In contrast to animals treated with isotype controls, administration of α-La antibody was found to suppress the continued progression of FD in the mouse model (Figure 13B).

[0282] Example 5 Inhibitory domain of La protein La is a normative nuclear regulator of RNA metabolism in eukaryotes. A second function of La was detected: it is cleaved, exposed on the PM, and controls the multinucleation and activity of osteoclasts. The first 187AA of La (e.g., aa 1-187 in SEQ ID NO: 2), which contains the domain essential for normative La-RNA binding, is not required for La's ability to promote osteoclast multinucleation (Figure 14B). We hypothesized that the uncharacterized region of AA188-375 contains the La domain that controls osteoclast multinucleation. To identify the domain within AA188-375 responsible for regulating osteoclast multinucleation, we synthesized a peptide library consisting of overlapping peptides (30AA length (12 total) (Figure 14A)) spanning AA188-375. Screening these peptides identified two peptides (2, SEQ ID NO: 8, and 9, SEQ ID NO: 9) that specifically inhibit osteoclast multinucleation (Figure 14C). In this figure, the control is the untreated group. The + indicates the addition of LA 188-375, which promoted fusion. Peptide 2: TIFF2026053494000014.tif4128peptide 9: TIFF2026053494000015.tif4128

[0283] Example 6 Interaction between La and annexin A5 (Anx A5) Anx A5 associates with the surface of fusion-promoting osteoclasts by binding to phosphatidylserine (PS) in a Ca2+-dependent manner. Transient plasma membrane (PM) PS exposure and Anx A5 binding promote osteoclast fusion. Data demonstrate that native La interacts with native Anx A5 in fusion-promoting osteoclasts, and that recombinant Anx A5 enriches recombinant La on the membrane in a PS-dependent and Ca2+-dependent manner (Figure 15A, Figure 15B). This indicates that at the time and location of osteoclast fusion, Anx A5 enriches La on osteoclast PMs that are transiently exposed to PS. We hypothesize that a direct La-Anx A5 protein interaction promotes osteoclast multinucleation by enriching La on the surface of fusion-promoting osteoclasts. An assay was developed to evaluate direct La-Anx A5 binding by incubating recombinant 6×his-La and biotin-Anx A5 in solution, pulling down the biotin-Anx A5, and evaluating the coprecipitation of 6×his-La (Figure 15C). The La peptide library described in Example 5 is used to identify peptides that reduce La coprecipitation and to further evaluate La-Anx A5 binding in osteoclast formation.

[0284] Example 7 Treatment of osteopetrosis by inhibiting osteoclast fusion Changes in osteoclast size alter bone resorption and underlie bone diseases such as Paget's disease and osteopetrosis. Loss of Snx10 leads to the production of extremely large osteoclasts that no longer properly attach to bone, resulting in resorption defects and osteopetrosis (M. Barnea-Zoha et al., 2021., An SNX10-dependent mechanism downregulates fusion between mature osteoclasts, J Cell Sci. 134(9):jcs254979. PMCID:PMC8182410;PMID:33975343). Data showed that La significantly concentrates on the surface of osteoclasts following Snx10 loss (Figure 16A). Inhibiting this excess surface La partially rescued the dysregulation of osteoclast multinucleation observed after Snx10 loss (Figure 16B). Data show that SNX10 plays a role in breaking down surface lamina (La) and halting osteoclast formation, allowing osteoclasts to resorb bone in a physiologically required manner. In this model, loss of SNX10, which leads to osteopetrosis, causes defects in osteoclast formation / function because it leaves lamina on the surface of the osteoclasts, thus allowing the osteoclasts to continue growing and resulting in defects in attachment and function. In hyperosteoclastic osteopetrosis, inhibiting surface lamina can rescue osteoclasts from defective occlusion formation and restore proper osteoclast function.

[0285] Example 8 material and method Reagents: Human M-CSF and RANKL, as well as mouse M-CSF and RANKL, were purchased from Cell Sciences (catalog numbers #CRM146B, #CRR100B, #CRM735B, and CRR101D, respectively). LPC (1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine, #855475), PC (1,2-dioleoyl-sn-glycero-3-phosphocholine, #850375C), PS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine, #840035C), and lysamin rhodamine phosphatidylethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lysamin rhodamine B sulfonyl, #810150C) were purchased from Avanti Polar Lipids. The bone resorption assay kit was purchased from Cosmo Bio Co. (catalog number CSR-BRA-24KIT) and used according to the manufacturer's instructions. Hoechst 33342 and phalloidin-Alexa555 were purchased from Invitrogen (#H3570 and A30106, respectively). TRAP staining reagent was purchased from Cosmo Bio Co. (#PMC-AK04F-COS). Fluorescent lipid PKH26 (PKH26GL-1KT) and carboxyfluorescein CF (5-(and-6)-carboxyfluorescein, isomer mixture, #C368) were purchased from Sigma and Invitrogen, respectively.

[0286] Animals: Hyperfunctional Gα in cells of the osteogenic lineage s R201C Bone marrow exgrafts were obtained using a mouse model of fibrous dysplasia with inducible expression of (Zhao, X. et al., Proc Natl Acad Sci USA 115, E428-E437, doi:10.1073 / pnas.1713710115(2018), Boyce et al., Endocr Rev 41, doi:10.1210 / endrev / bnz011(2020)) (described below). Female mice aged 12-18 weeks were used in this study.

[0287] Mouse bone marrow explant culture: Tibia and femur were excised from a previously described inducible mouse model of fibrous dysplasia (Boyce et al., Endocr Rev 41, doi:10.1210 / endrev / bnz011(2020)) or from wild-type littermates. Holes were made in the epiphyses of each bone using a 22-gauge subcutaneous needle, and bone marrow was washed into a culture dish using alpha-MEM. These bone marrow isolates were further dissociated using a new 22-gauge subcutaneous needle to obtain single-cell suspensions, which were cultured in T-75 culture flasks with alpha-MEM + 20% FBS, 1× pen / strep, and 1× Normocin (InvivoGen, #Ant-nr-1) for 7 days. Cells attached to the flask were washed three times with PBS, subcultured using 0.05% trypsin and a cell scraper, and cultured for up to three subcultures in alpha-MEM + 20% FBS and 1×pen / strep. Gα in bone marrow stromal cell subsets of explants. s R201C For expression induction, cells were plated to near confluence in 6-well plates and treated with 1 μM doxycycline (Sigma, #D9891-5G). The culture medium was changed daily during induction. For antibody treatment, when initial cell fusion was observed (typically after about 4 days of doxycycline treatment), 6 μg / ml of antibody was added overnight.

[0288] Cell culture: Osteoclasts: Elutriated monocytes were obtained from healthy donors using the elutriation method. For imaging, cells were placed in 35 mm dishes with polymer coverslip bottoms (Ibidi #81156), and for biochemical experiments, cells were placed in 35 mm or 10 cm dishes in complete medium [α-MEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin-L-glutamine (Gibco Invitrogene #12571063, #26140079, and #10378016, respectively)] in 1 cm³ of a culture medium. 2 Approximately 2.9 x 10 5The cells were plated at a density of [number] cells. Monocytes were differentiated into M2 macrophages for 6 days in the presence of 100 ng / ml M-CSF, and then differentiated for 3 days in 100 ng / ml M-CSF and 100 ng / ml RANKL, unless otherwise indicated. RAW 264.7 cells (ATCC, Manassas, Virginia, #TIB-71) were maintained in DMEM supplemented with 10% FBS for up to 8 passages. RAW 264.7 cells were differentiated into osteoclasts for 5 days in the presence of 100 ng / ml mouse RANKL. To separate unfused mononuclear RAW 264.7 cells from fused multinuclear RAW 264.7 cells into separate fractions, the fact that multinuclear cells adhere much more strongly to the culture dish plastic was utilized. After washing in PBS, the mixed RAW culture (after RANKL differentiation) was treated with Ca 2+ and Mg 2+ The cells were left in untreated PBS at room temperature for 10 minutes. Next, the culture dish was tapped on the workbench to release most of the unfused mononuclear raw cells, which were then collected by centrifugation. This process was repeated 2-4 times until a population of mainly fused multinuclear syncytia remained in the dish. The mononuclear and multinuclear cell fractions were then prepared for the biochemical or imaging experiments described below.

[0289] Human osteoblast / osteoclast co-culture: Osteoblasts isolated from the cancellous bone of healthy individuals were obtained from PromoCell (#C12720) and cultured according to the manufacturer's instructions. Osteoclast precursors were obtained from primary human monocytes by culturing in M-CSF for 6 days as described above. Osteoblasts and osteoclast precursors were cultured in 35 mm dishes using 4-well culture inserts in a 3:1 well ratio (ibidi, #81156). 48 hours prior to co-culture mixing, osteoblasts were switched to serum-free alpha-MEM containing 1 × pen / strep (Gibco). Following serum starvation, the 4-well culture inserts were removed, and the cells were cultured overnight in their respective prepared media, with or without treatment. The following morning, the cells were fixed with 4% paraformaldehyde.

[0290] HA0-expressing cells and RBCs: NIH 3T3 mouse fibroblasts from 15 clone cell lines stably expressing influenza were used. These HA0-expressing cells were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS and antibiotics. To maintain the HA in a non-fusion-capable form, these cells were used without trypsin pretreatment. Human erythrocytes (RBCs) were isolated from anonymous healthy donors. As described in (77), RBCs were labeled with the fluorescent dye PKH26 and loaded with the water-soluble fluorescent dye CF.

[0291] Constructs, Recombinant Proteins, and Transfection: Recombinant La 1-408, 1-375, 1-375 Q20A_Y24A_D33I, 1-187, and 188-375 were amplified using primers designed with overlap sequences and inserted between NdeI / HindIII in a V78 pET28A E. coli expression vector, each tagged with an N-terminal 6×his affinity tag. La 1-408 was expressed as a recombinant protein in E. coli by SD Biosciences (San Diego, California) and purified using an IMAC column. The remaining constructs were transformed into BL2 (DE3) chemically competent E. coli (Thermo Fisher Scientific), and protein expression was induced using IPTG. Cells were lysed with Bugbuster (Sigma) according to the manufacturer's instructions, and the 6×his-La protein was affinity-purified using a HisPur Cobalt Spin column (Thermo Fisher Scientific). Endotoxin contaminants were depleted from affinity-purified 6×his-La protein using a Pierce high-volume endotoxin removal column (Thermo Fisher Scientific) according to the manufacturer's instructions. The protein was then sterile filtered, aliquot, and stored at -80°C.

[0292] Plasmids were introduced into primary osteoclasts by jetPRIME (Polyplus Transfection) on day 2 after RANKL stimulation. FLAG-La 1-408, FLAG-La 1-375, and FLAG-La 1-375 Q20A_Y24A_D33I plasmids were provided by Maraia Lab (NICHD). Briefly, an SSB (UniProt P05455) was inserted between HindIII and BamHI in the pFLAG-CMV2 vector (Sigma). The "uncleavable" La was created by employing the FLAG-La 1-408 plasmid and introducing two point mutations at amino acids D371A and D374A that caused the loss of the caspase cleavage site in the C-terminal region of this protein (Emory Integrated Genomics Core). siRNAs were introduced into primary osteoclasts by Lipofectamine RNAiMAX (Thermo Fisher Scientific) one day after RANKL stimulation. Non-targeted siRNA (catalog number 4390843) and SSB-targeted siRNA (catalog number 4392420_ID:s13469) were introduced at a concentration of 5 ng / ml (Silencer Select, Ambion).

[0293] Antibodies: α-Cyclophyllin B (CST, D1VdJ), α-GAPDH (CST, D16H11), α-Tubulin (Abcam, 7750), α-RANK (Abcam, 13918), α-Anx A5 (Abcam, 54775), control rabbit polyclonal IgG (Abcam, 27478), α-La (Abcam, 75927), IgG2a (Abcam, 18415) used as an isotype control for α-Anx A5 (Abcam, 54775), IgG1 (Abcam, 170190; used as an isotype control for α-Anx A5 (Abcam, 54775)), α-Anx A1 (Abcam, 47661), α-Anx A4 (Abcam, 65846), α-6×His (Abcam, 18184), and α-FISH (Abcam, 118575).

[0294] Where indicated, the following anti-La antibodies were used for immunoblotting, immunostaining, and immunoinhibition: Abcam, 75927 (referred to as α-La); anti-SSB antibody Invitrogen, PA5-29763 (referred to as α-LMW La); anti-La phospho Ser366 Abcam, 61800 (referred to as α-FL La). The La antibodies used in this study were characterized to identify the La molecular species found in human osteoclasts derived from monocytes. See the table above. To evaluate the selectivity of these antibodies against the two La species observed during osteoclast formation, lysates from differentiating human osteoclasts were probed at a point in time when only one of the two La species described was predominantly present (i.e., almost entirely LMW La or almost entirely FL La). Abcam 75927 antibody ("α-La") recognized both La species in Western blots. Invitrogen PA5-29763 ("α-LMW La") preferentially recognized LMW La. Since LMW La is a cleavage product of FL La, FL La contains all the residues in LMW La. The finding that α-LMW La preferentially recognizes LMW La suggests that this antibody must recognize a posttranslational or conformational epitope that is different between LMW La and FL La, rather than simply a primary amino acid sequence common to both. To specifically recognize FL La, we used the Abcam 61800 antibody against phosphorylated human La (phospho Ser366). This α-FL La antibody did not function in Western blotting in these experiments, while immunofluorescence staining showed that this antibody recognized FL La but not LMW La. In differentiating human osteoclasts at an intermediate point (day 4 after RANKL addition) when Western blot analysis with α-La recognized both osteoclast La isoforms, α-FL La exclusively recognized nuclear La, while α-LMW primarily recognized cytoplasmic La.

[0295] Biochemical approach: Cells were lysed on ice by pulsed sonication and rotated end-over-end at 4°C for 45 minutes in the presence of a protease inhibitor (cOmplete, Sigma, #118361700010). Steady-state protein levels were assessed by SDS-PAGE followed by immunoblotting. Bulk proteins were assessed by SDS-PAGE followed by silver staining (SilverQuest, Thermo Fisher Scientific). Bands of interest were excised from the silver-stained gel, destained, and evaluated by liquid chromatography connected to tandem mass spectrometry (Proteomics Core, NHLBI). Selective enrichment of cytosolic and membrane-bound protein fractions was performed using the MEM-PER® Plus membrane protein extraction kit (Thermo Fisher Scientific, catalog no. 89842) according to the manufacturer's instructions.

[0296] Immunoprecipitation was performed as previously described (78). Briefly, multiprotein complexes were crosslinked quasi-stoichiometrically using a non-membrane-permeable, 12 Å-long cleavable crosslinking agent, 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), according to the manufacturer's instructions (Thermo Fisher Scientific). Supramolecular complexes were immunoprecipitated using sheep α-Ms IgG magnetic Dynabeads (Invitrogen) modified with Ms antibodies targeting the substance of interest (α-La, Abcam, 75927; α-Anx A5, Abcam, 54775; IgG2a, Abcam, 18415; or IgG1, Abcam, 170190). The supramolecular complexes were denatured, the crosslinks were cleaved by the addition of a reducing agent (BME, BioRad), and the proteins within these complexes were separated by PAGE. Proteins were transcribed, and the target protein was probed using immunoblotting (as described above). For the target protein, the membrane was probed using Rb antibodies (α-La PA5-29763, α-Anx A5 14196, α-Anx A1 47661, or α-Anx A4 65846).

[0297] Transcript Analysis: For real-time PCR, total RNA was collected from cell lysates using the PURELINK® RNA kit according to the manufacturer's instructions (Invitrogen #12183018A). cDNA was generated from total RNA by reverse transcription using the High-Capacity RNA-to-cDNA kit according to the manufacturer's instructions (Applied Biosystems, #4387406). The cDNA was then amplified using IQ®SYBR®Green Supermix (Biorad). All primers were pre-designed KICQSTART®SYBR®Green primers with the highest rank score specific to the gene of interest or the GAPDH control, and were used according to the manufacturer's instructions (Sigma). All real-time PCR reactions were performed and analyzed using the CFX96 Real-Time System (Biorad) with GAPDH as the internal control. The ΔΔCt method was used to determine the fold change in gene expression. Three to four independent experiments were performed, each analyzed in duplicate.

[0298] Fusion assay: Osteoclast fusion in cultured cells was evaluated by fluorescence microscopy (Verma et al., J Biol Chem 293, 254-270, doi:10.1074 / jbc.M117.809681(2018)). Briefly, cells were fixed with 4% paraformaldehyde at the time point of interest, permeabilized with 0.1% TRITON® X-100, and blocked with 5% FBS. Next, the cells were stained with phallodin-ALEXAFLURO® 488 and Hoechst to label the actin cytoskeleton and nucleus, respectively. Sixteen randomly selected fields were imaged using a Lionheart FX microscope with a 10× / 0.3 NA Plan Fluorite WD objective lens (BioTek) and Gen3.10 software (BioTek), using ALEXAFLURO® 488, Hoechst, and phase-contrast fitted filter set (BioTek). Osteoclast fusion efficiency was assessed as the number of intercellular fusion events in these images, as previously described. Regardless of the order of fusion events, the number of intercellular fusion events required to produce a syncytium with N nuclei is always equal to N-1, so Σ(N) i -1)=N total -N syn The fusion number index was calculated as follows: Here, N i = The number of nuclei in each syncytium, N syn = Total number of syncytia. To account for small variations in cell density between dishes, the number of fusion events was normalized to the total number of nuclei (including unfused cells). In contrast to traditional fusion index measurements, this approach equally considers fusions between two mononuclear cells, between one mononuclear cell and one multinuclear cell, and between two multinuclear cells. In traditional fusion index calculations, fusions between two multinuclear cells do not change the percentage of nuclei in a syncytia. If the number of syncytia is counted instead, fusion events between two multinuclear cells are not only missed but also decrease the number of syncytia. In contrast, the fusion index includes all fusion events.

[0299] Synchronization of osteoclast membrane fusion: Osteoclast fusion was synchronized as described in (Verma, J Biol Chem 293, 254-270, doi:10.1074 / jbc.M117.809681(2018)). Briefly, 72 hours after RANKL treatment, osteoclast medium was supplemented with 100 ng / ml M-CSF, 100 ng / ml RANKL, and 350 μm lauroyl-LPC. After 16 hours, LPC was removed by washing five times with fresh medium, and cells were fused for 90 minutes in or without antibody treatment or recombinant La.

[0300] HA0-RBC Fusion Assay: To investigate whether La can mediate fusion, the protein was applied to HA0-expressing cells in which RBCs were strongly bound by an interaction between the sialic acid receptor on the surface of RBCs and the HA1 subunit of HA0 (82). HA0, being an uncleaved form of HA, bound but did not mediate fusion. HA cells were washed twice with PBS and incubated with 1 ml of RBC suspension (0.05% hematocrit) for 10 minutes. HA cells with 0-2 RBCs bound per cell were washed with PBS to remove unbound RBCs. Cells were then exposed to 40 nM FL La. Fusion activity (content mixture and / or lipid mixture) was assayed by fluorescence microscopy 1 hour after La application.

[0301] Liposome Binding Assay: Multilamellar liposomes were formed from pure PC or a 9:1 (w / w) mixture of PC and PS. Both lipid compositions were supplemented with 0.5 mol% lysamine rhodamine phosphatidylethanolamine. To prepare the liposomes, the lipid stock solution in benzene / methanol (95:5) was frozen in liquid nitrogen and lyophilized overnight using SPEEDVAC® (Savant). The dried lipids were resuspended in aqueous buffer (100 mM NaCl, 10 mM Hepes, pH 7.0) at a total lipid concentration of 1 mM and vortexed. Protein and CaCl2 were added to the liposomes, and the mixture was incubated on ice for 30 minutes. To pelletize, the liposomes were centrifuged at 15,000 g for 20 minutes, and based on rhodamine fluorescence, approximately 95% of the liposomes were pelletized. Next, the centrifuged sample was fractionated into an upper liposome-depleted fraction and a lower fraction containing liposomes and liposome-binding proteins. The fractions were then solubilized by adding Laemmli buffer (Bio Rad) and separated by SDS-PAGE as described above. Recombinant La and recombinant Anx A5 were detected using α-6×His antibody (Abcam) based on their respective n-terminal 6×His tags, and the signals of the soluble protein fraction and the liposome-binding protein fraction were evaluated by densitometry. The data were expressed as the percentage of liposome-bound protein signals, as shown in the following formula. TIFF2026053494000016.tif6128

[0302] Fluorescence microscopy imaging: In immunofluorescence experiments, cells were washed with PBS and fixed at 37°C using warm, freshly prepared 4% formaldehyde (Sigma, F1268) in PBS. The cells were washed three times with PBS. To permeabilize the cells, they were incubated for 5 minutes in 0.1% TRITON® X100 in PBS. The cells were washed three times again with PBS and placed in PBS containing 10% FBS at room temperature for 10 minutes to suppress nonspecific binding. The cells were then incubated with the primary antibody in PBS containing 10% FBS for 1 hour. After washing five times in PBS, the cells were placed in PBS containing 10% FBS with secondary antibodies (anti-rabbit IgG Fab2 ALEXAFLUOR® or anti-mouse IgG Fab2 ALEXAFLUOR® 488, both from Cell Signaling Technology, catalog numbers #647 4414S and #4408S respectively, 1:500 dilution) at room temperature for 1 hour, and then the cells were washed five more times in PBS.

[0303] For experiments requiring immunostaining of impermeable cells (Figures 3B and 3C), cells were fixed as described above and then incubated with primary antibody (10 mg / ml) at 37°C for 10 minutes. After washing twice with complete medium and three times with PBS, the cells were fixed as described above. After fixation, the cells were washed three times with PBS and then placed in PBS containing 10% FBS for 10 minutes at room temperature to suppress nonspecific binding. Next, the cells were incubated with secondary antibody as described above (in PBS containing 10% FBS at room temperature for 1 hour), and finally washed five times with PBS. Images were captured using a Zeiss LSM 800 airyscan confocal microscope with a C-Apochromat 63x / 1.2 water immersion objective lens.

[0304] Bone resorption: Bone resorption was evaluated using the Cosmo Bio USA bone resorption assay kit, following the manufacturer's instructions. Briefly, 24-well calcium phosphate coated plates were labeled with fluorescein amine-labeled chondroitin sulfate. Human monocyte-derived osteoclasts were differentiated using alpha-MEM without phenol red as described above. The culture medium was collected 4-5 days after RANKL addition, and the fluorescence intensity in the medium was evaluated as recommended by the manufacturer.

[0305] Statistical Analysis: Each graph represents data from three independent biological replicas repeated on independent occasions, unless otherwise stated in the caption. Data were assembled and analyzed using GraphPad Prism 8.0. For each experiment, cells from the same passage number, donor, or animal were paired across the various conditions described. All reported functional dependencies were observed in each independent experiment. However, as is well known for human monocyte-derived osteoclasts (Moller et al., Int J Mol Sci 21, doi:10.3390 / ijms21176368(2020)), the time course of osteoclastogenic differentiation and the degree of fusion at baseline varied considerably among monocytes from different donors. Statistical significance was analyzed using paired ratio t-tests, where raw values ​​were logarithmically transformed before evaluation. In the analysis of the HA0-RBC experiment, confidence intervals for binomial ratios were calculated using the Wilson method in R (v.4.1.1) using the binconf function from the Hmisc package (v.4.5.0).

[0306] In view of the numerous conceivable embodiments to which the principles of our invention may be applied, it should be recognized that the exemplary embodiments are merely examples of the present invention and should not be considered limitations on the scope of the invention. Rather, the scope of the invention is defined by the following claims. Therefore, we assert that our invention includes everything contained within the scope and gist of these claims.

Claims

1. A process of administering an effective amount of lupus autoantigen (La) protein or a substance that modulates the expression or activity of La protein to a subject in need, thereby modulating osteoclast fusion in the subject. A method for regulating osteoclast fusion, including

2. The method according to claim 1, wherein the subject is a human.

3. The method according to claim 1 or 2, wherein the administration comprises systemically administering an effective amount of La protein or an active substance that modulates the expression or activity of La protein to a subject.

4. A method according to any one of claims 1 to 3, which increases osteoclast fusion and bone resorption in a subject.

5. The method according to claim 4, wherein the active substance that regulates the expression or activity of the La protein is an active substance that increases the expression or activity of the La protein.

6. The method according to claim 4 or 5, wherein the subject has a disease including reduction of bone resorption.

7. The method according to claim 6, wherein the disease is osteopetrosis.

8. The method according to claim 4 or claim 5, wherein the subject has a fracture.

9. The method according to any one of claims 4 or 6 to 8, comprising the step of administering La protein to a subject.

10. La Protein (a) SEQ ID NO: 1 amino acids 300-375, (b) SEQ ID NO: 1 Amino acids 6-242 including and / or The La protein does not contain amino acids 376-408 of SEQ ID NO:

1. The method according to claim 9.

11. La Protein (a) An amino acid sequence that is at least 95% identical to SEQ ID NO: 2, wherein the La protein is at most 375 amino acids long and does not contain amino acids 376-408 of SEQ ID NO:

1. (b) Amino acid sequence that is at least 95% identical to SEQ ID NO: 2, (c) Amino acid sequence of SEQ ID NO: 2 [Amino acids 1-375 of La] (d) Amino acid sequence of SEQ ID NO: 1, (e) Amino acid sequence that is at least 95% identical to SEQ ID NO: 7, (f) An amino acid sequence that is at least 95% identical to SEQ ID NO: 7, wherein the La protein does not contain amino acids 1-187 and 376-408 of SEQ ID NO: 1, or (g) SEQ ID NO: 7 The method according to any one of claims 1 to 9, comprising or consisting of the following.

12. The method according to any one of claims 4 to 8, wherein the La protein or the substance that modulates its activity is a nucleic acid molecule that encodes the La protein.

13. La Protein (a) Amino acids 300–375 of SEQ ID NO: 1, and / or (b) SEQ ID NO: 1 Amino acids 6-242 Includes, The La protein does not contain amino acids 376-408 of SEQ ID NO:

1. The method according to claim 12.

14. La Protein (a) An amino acid sequence that is at least 95% identical to SEQ ID NO: 2, wherein the La protein is at most 375 amino acids long and does not contain amino acids 376-408 of SEQ ID NO:

1. (b) Amino acid sequence that is at least 95% identical to SEQ ID NO: 2, (c) Amino acid sequence of SEQ ID NO: 2 [Amino acids 1-375 of La] (d) Amino acid sequence of SEQ ID NO: 1, (e) Amino acid sequence that is at least 95% identical to SEQ ID NO: 7, (f) An amino acid sequence that is at least 95% identical to SEQ ID NO: 7, wherein the La protein does not contain amino acids 1-187 and 376-408 of SEQ ID NO: 1, or (g) SEQ ID NO: 7 The method according to claim 12, comprising or consisting of.

15. The method according to claim 12, wherein the nucleic acid molecule contains SEQ ID NO:

3.

16. The method according to any one of claims 12 to 15, comprising the step of administering an expression vector containing a nucleic acid molecule encoding the La protein to a target.

17. The method according to claim 16, wherein the vector is an adenovirus vector, a lentivirus vector, or an adeno-associated virus vector.

18. The method according to any one of claims 1 to 3, wherein the method reduces osteoclast fusion and bone resorption in the subject, and the active substance reduces the expression or activity of La protein in the subject.

19. The method according to claim 18, wherein the subject has a disease including increased bone resorption.

20. The method according to claim 19, wherein the disease is osteoporosis, Paget's disease of bone, fibrous dysplasia, rheumatoid arthritis, osteopetrosis (high osteoclast-rich osteopetrosis), osteomyelitis, or metastatic bone disease.

21. The method according to any one of claims 18 to 20, wherein the active substance is an inhibitory nucleic acid molecule, a nucleic acid molecule encoding an inhibitory La peptide, or a CRISPR / Cas system.

22. The method according to claim 21, wherein the inhibitory nucleic acid molecule is a small inhibitory (SiO)RNA, antisense RNA, or ribozyme.

23. The method according to claim 21 or 22, wherein the active substance is an siRNA comprising or consisting of SEQ ID NO: 4 or SEQ ID NO:

5.

24. The method according to any one of claims 18 to 20, wherein the active substance is an antagonist antibody that specifically binds to the La protein.

25. The method according to any one of claims 18 to 20, wherein the active substance is a caspase inhibitor.

26. The method according to any one of claims 18 to 20, wherein the active substance is an inhibitory peptide, a nucleic acid molecule encoding an inhibitory peptide, or a vector containing a nucleic acid molecule encoding an inhibitory peptide.

27. The inhibitory peptide is (a) SEQ ID NO: 8 or SEQ ID NO: 9 (b) containing SEQ ID NO: 8 or SEQ ID NO: 9, and having a maximum length of 35 amino acids, (c) SEQ ID NO: 8 or SEQ ID NO: 9 with 1, 2, 3, 4 or 5 conservative substitutions, (d) containing SEQ ID NO: 8 or SEQ ID NO: 9 with 1, 2, 3, 4 or 5 conservative substitutions, and having a maximum length of 35 amino acids, The method according to claim 26.

28. The method according to claim 26, wherein the active substance is a nucleic acid molecule encoding an inhibitory peptide, and the nucleic acid molecule comprises SEQ ID NO: 10 or SEQ ID NO:

11.

29. If the subject has osteoporosis, the above method is The process of administering to the subject one or more of the following in an effective amount: a bisphosphonate, an antibody that specifically binds to nuclear factor kappa B activating receptor ligand (RANKL), or teriparatide. The method according to any one of claims 19 to 28, further comprising:

30. The method according to claim 29, wherein the antibody that specifically binds to RANKL is denosumab.

31. A pharmaceutical composition comprising an effective amount of La protein or an active agent that modulates the expression or activity of La protein, for use in the method according to any one of claims 1 to 30.

32. (a) A substance that reduces the expression or activity of La protein in the target, and (b) Bisphosphonate, antibody that specifically binds to RANKL, or teriparatide A kit that includes this.

33. A substance that reduces the expression or activity of La protein in the target is (a) Inhibitory nucleic acid molecules, (b) CRISPR / Cas system, (c) Antagonist antibody that specifically binds to La protein, (d) Caspase inhibitors, (e) inhibitory peptide, or (f) Nucleic acid molecules encoding inhibitory peptides The kit according to claim 32.

34. The substance that reduces the expression or activity of the La protein is an inhibitory peptide, and the said inhibitory peptide is (a) SEQ ID NO: 8 or SEQ ID NO: 9 (b) containing SEQ ID NO: 8 or SEQ ID NO: 9, and having a maximum length of 35 amino acids, (c) SEQ ID NO: 8 or SEQ ID NO: 9 with 1, 2, 3, 4 or 5 conservative substitutions, (d) containing SEQ ID NO: 8 or SEQ ID NO: 9 with 1, 2, 3, 4 or 5 conservative substitutions, and having a maximum length of 35 amino acids, The kit according to claim 32.

35. (a) La protein, its effective fragment, or a nucleic acid molecule or vector encoding the La protein or its effective fragment, and (b) Latent membrane protein (LMP)-1, fibroblast growth factor (FGF)-2, or bone morphogenetic protein (BMP), or nucleic acids encoding LMP-1, FGF-2, or BMP A kit that includes this.

Images