Engineering of a safe bone anabolic gene therapy to treat alveolar bone loss in osteoporosis

Abstract

Aspects of the disclosure relate to compositions and methods for modulating bone growth, for example by increasing osteogenesis and / or decreasing osteoclastogenesis. The disclosure is based, in part, on recombinant adeno-associated virus (rAAV) vectors encoding microRNAs or miRNA inhibitors that inhibit expression or activity of gene products in the WNT pathway, for example Schnurri-3 (SHN3), sclerostin (SOST), and / or dickkopf-1 (DKK-1). In some embodiments, compositions described by the disclosure are useful for treating certain bone diseases, disorders, or injuries, such as alveolar bone loss and bone fractures.

Description

[0001]ENGINEERING OF A SAFE BONE ANABOLIC GENE THERAPY TO TREAT ALVEOLAR BONE LOSS IN OSTEOPOROSIS RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 553,103, entitled “ENGINEERING OF A SAFE BONE ANABOLIC GENE THERAPY TO TREAT ALVEOLAR BONE LOSS IN OSTEOPOROSIS” filed on February 13, 2024, the entire content of which is incorporated by reference herein. BACKGROUND Alveolar bone loss that increases a risk in tooth loss, gum disease susceptibility, and facial deformity is highly prevalent in aged populations. However, there are limited treatment options to restore alveolar bone loss. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (U012070199WO00-SEQ-SCC.xml; Size: 25,774 bytes; and Date of Creation: February 11, 2025) is herein incorporated by reference in its entirety. SUMMARY Aspects of the disclosure relate to compositions and methods for modulating bone growth. In some embodiments, compositions and methods described herein are useful for treatment of certain diseases, disorders, or injuries associated with bone, for example alveolar bone loss (e.g., due to osteoporosis) or bone fractures. The disclosure is based, in part, on inhibitors of certain gene products of the Wnt pathway (e.g., SHN3, SOST, DKK-1, etc.) that are administered locally (e.g., to a jaw of a subject or bone of a subject). In some embodiments, the inhibitors are interfering nucleic acids. In some embodiments, the inhibitors are delivered by viral vectors, such as recombinant adeno-associated virus (rAAV) vectors. Accordingly, in some aspects, the disclosure provides a method for treating alveolar bone loss in a subject in need thereof, the method comprising administering to the jaw of a subject in need thereof an isolated nucleic acid comprising a nucleotide sequence encoding an inhibitor of a gene selected from Schnurri-3 (SHN3), sclerostin (SOST), and dickkopf-1 (DKK- 1). 12075767.1 In some embodiments, an inhibitor is selected from the group consisting of dsRNA, siRNA, shRNA, miRNA, and artificial miRNA (amiRNA). In some embodiments, an amiRNA comprises a miR-33 scaffold or a mir-155 scaffold; and a nucleic acid sequence having a region of complementarity with Schnurri-3 (SHN3), sclerostin (SOST), or dickkopf-1 (DKK-1). In some embodiments, an amiRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-14. In some embodiments, an isolated nucleic acid further comprises a promoter. In some embodiments, a promoter is an inducible promoter operably linked to the nucleotide sequence encoding the inhibitor. In some embodiments, a promoter is induced by inflammation in a subject or by mechanical stress (e.g., vibration). In some embodiments, a promoter comprises a NF-kB promoter (pNF-kB), a bone morphogenic protein promoter (pBRE), or an Lef / Tcf promoter. In some embodiments, a promoter comprises the sequence set forth in any one of SEQ ID NOs: 16-18. In some embodiments, an isolated nucleic acid further comprises one or more miRNA binding sites. In some embodiments, one or more miRNA binding sites are miR-122 binding sites, miR208a binding sites, or a combination thereof. In some embodiments, one or more miRNA binding sites comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, an isolated nucleic acid further comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs) flanking a nucleotide sequence encoding a promoter. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, an isolated nucleic acid is administered to the subject as a recombinant adeno-associated virus (rAAV). In some embodiments, an rAAV comprises an AAV capsid protein selected from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 capsid protein, or a variant thereof. In some embodiments, a capsid protein comprises the sequence set forth in any one of SEQ ID NOs: 19-21. In some embodiments, administering to a jaw of a subject comprises injection. In some embodiments, injection comprises direct injection to the jaw of the subject. In some embodiments, a subject is a mammal. In some embodiments, a subject is a human or a mouse. In some embodiments, a subject has or is suspected of having osteoporosis. 12075767.1 In some aspects, the disclosure provides an artificial miRNA (amiRNA) comprising a miR-33 scaffold or a mir-155 scaffold; and a nucleic acid sequence having a region of complementarity with human or mouse dickkopf-1 (DKK-1). In some embodiments, an amiRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 4-10. In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising: an isolated nucleic acid comprising a transgene encoding an amiRNA as described herein, flanked by AAV inverted terminal repeats (ITRs); and one or more capsid proteins. In some embodiments, a transgene further comprises a promoter. In some embodiments, a promoter is an inducible promoter operably linked to a nucleotide sequence encoding an inhibitor. In some embodiments, a promoter is induced by inflammation in a subject or by mechanical stress (e.g., vibration). In some embodiments, a promoter comprises a NF-kB promoter (pNF-kB), a bone morphogenic protein promoter (pBRE), or an Lef / Tcf promoter. In some embodiments, a transgene further comprises one or more miRNA binding sites. In some embodiments, one or more miRNA binding sites are miR-122 binding sites, miR208a binding sites, or a combination thereof. In some embodiments, one or more miRNA binding sites comprises the sequence set forth in SEQ ID NO: 15. In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising an rAAV vector as described herein; and one or more AAV capsid proteins. In some embodiments, one or more AAV capsid proteins are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 capsid proteins, or a variant thereof. BRIEF DESCRIPTION OF DRAWINGS FIGs. 1A-1K show SHN3 deletion increases alveolar bone and tooth root dentin mass. (A, C) Immunohistochemistry showing expression of SOST and DKK1 (A) and SHN3 and β - Catenin (C) in the mandible of 2-month-old mice. AB: alveolar bone, PDL: periodontal ligament, BM: bone marrow, DP: dental pulp, ob, osteoblasts, od: odontoblasts, ocy: osteocytes. (B, D) Human SOST, DKK1, and SHN3 mRNA expression in the mandible of young vs. aged patients (n = 4 / group). (E) Mouse Shn3 mRNA expression in the mandible of 2-month-old vs. 24-month-old mice (n =5 / group, left) and 6-month-old mice with sham control vs ovariectomy (OVX) surgery (n = 4–6 / group, right). (F, G, H) MicroCT and histologic analyses showing 12075767.1 alveolar bone mass in the first molar of 1-month-old and 7-month-old Shn3- / - and Shn3+ / + (WT) mandibles. MicroCT images showing sagittal and cross sections of the first molar (F, left and middle; H, left). H&E (G, left and middle; H, middle) and Masson’s trichrome staining of sagittal sectioned molar (G, right). MicroCT quantification of alveolar bone mass in the first molar (n = 5 / group, F, left). AB.BV / TV: alveolar bone volume per tissue volume, AB.Th: alveolar bone thickness (F, right). ( J, K) MicroCT analysis showing root dentin and enamel volume of the first molar of 1- and 7-month-old Shn3- / - and Shn3+ / + mandible. 3D- reconstruction images(J, K-bottom) and relative quantification (n = 5 / group) are displayed (J, K- top). The representative image of the methods used to quantitatively measure the parameters listed in (I). The area highlighted in green marks the crown dentin region of interest (ROI), the yellow area marks the enamel ROI, the pink area marks the root dentin ROI, and the gray area marks the root dentin.MR: mesial root , DR: distal root. Scale bars: 50 um (A, C), 1 mm (F- left and middle, H-left), 100 um (G, H-middle, J-bottom, K-bottom). A two-tailed unpaired Student’s t-test for comparing two groups (B, D, E, F, H, J, K; error bars, data represent mean ± SD). Representative images of three replicates are displayed (A, C, G, H, J, K). FIGs. 2A-2K show mesenchyme-specific deletion of SHN3 increases alveolar bone and tooth root dentin mass. (A) Fluorescence microscopy was performed on cryo-sectioned mandibles from 2-month-old Pdgfrα-GFP reporter mice to visualize GFP-expressing Pdgfrα- lineage cells in the alveolar bone and tooth. (B, C) Alveolar bone marrow mesenchymal stromal cells (ABMSCs) were isolated from the mandible of 4-week-old Shn3Pdgfrα and Shn3fl / fl mice and cultured under osteogenic conditions. mRNA expression of Shn3, Bglap, and Dmp1 (B) and mineralization activity (C) were assessed by RT-PCR and alizarin red staining, respectively (n = 4 / group). (D, E) MicroCT and histologic analyses showing alveolar bone mass in the first molar of 1- and 7-month-old Shn3Pdgfrα and Shn3fl / fl mandible. MicroCT images showing sagittal and cross sections of the mandibular first molar (left). H&E staining of sagittal sectioned molar (D-right, E-middle). MicroCT quantification of alveolar bone mass in the mandibular first molar (n =5 / group, D-middle, E-right). (F) MicroCT analysis showing root dentin and enamel volume of the first molar of 1- or 7- month-old Shn3Pdgfrα and Shn3fl / fl mandibles. 3D- reconstruction images and relative quantification (n = 5 / group) are displayed. (G) Masson’s trichome staining (left) and scanning electron microscopy (SEM, right) were performed in sagittal sectioned tooth dentin of the first molar of 1-month-old Shn3Pdgfrα and Shn3fl / fl mandible, demonstrating normal microstructure of tooth dentin. (H) Fluorescence microscopy 12075767.1 was performed on cryo-sectioned mandibles from 2-month-old Prx1-GFP (left) and Dmp1- cre;Rosa26mTmG (right) reporter mice to visualize GFP-expressing Prx1- or Dmp1-lineage cells in the alveolar bone and tooth. (I) MicroCT and histologic analyses showing alveolar bone mass in the first molar of 1-month-old Shn3Dmp1, Shn3Prx1, and Shn3fl / fl mandibles. MicroCT images (left), H&E staining (middle), and microCT quantification of alveolar bone mass in the molar (right) are displayed (n = 5 / group). (J, K) MicroCT analysis showing root dentin and enamel volume of the first molar of 1-month-old Shn3Prx1, Shn3Dmp1, and Shn3fl / fl mandibles. 3D-reconstruction images and relative quantification (n = 5 / group) are displayed. Scale bars: 50 um (A, G-left, H), 1 mm (D and E-left, I-left), 100 um (D-right and E- middle, F-right, I-middle, J-right, K-right), 30 um (G, right). A two-tailed unpaired Student’s t- test for comparing two groups (B, D, E, F, I, J, K; error bars, data represent the mean ± SD). Representative images of three replicates are displayed (A, C, D, E-K). FIGs. 3A-3K show AAV-mediated silencing of human SHN3 increases bone and collagen formation. (A) The AAV vector genome containing the CMV enhancer / chicken β-actin promoter (CBA), hs-amiR-ctrl, hs-amiR-hSHN3, an Egfp reporter gene (EGFP), β-globin polyA sequence (PA), and inverted terminal repeat (ITR) was packaged into the rAAV9 capsid. (B, C) Human periodontal ligament stem cells (hPDLCs) and bone marrow-derived stromal cells (hBMSCs) were treated with rAAV9 carrying hs-amiR-ctrl or hs-amiR-hShn3 and cultured under osteogenic conditions for 4 days. rAAV9’s transduction efficiency was examined by fluorescence microscopy using EGFP expression (B). mRNA expression of SHN3 and the osteogenic marker IBSP were assessed by RT-PCR (n = 4 / group, C). (D-K) Diagram of the study and treatment methods. Human BMSC-seeded hydroxyapatite (HA)-scaffold was implanted into the interscapular fat pads of immunodeficient SCID mice and one week later, PBS or rAAV9 carrying hs-amiR-ctrl or hs-amiR-hSHN3 was injected into the implantation site (D). Four weeks later, the implanted scaffold was visualized by radiography (E), and EGFP expression in the scaffold was assessed by IVIS optical imaging system (F). mRNA expression of SHN3, BGLAP, AXIN2, and COL1 in the scaffold was assessed by RT-PCR (n = 4 / group, G). Bone accrual and collagen production of the scaffold were assessed by microCT (H, I), histology (J), and SEM (K). 2D microCT images (H) and relative quantification showing mineral density distribution (I) are displayed. Masson’s trichome staining shows a significant increase in collagen production in the hs-amiR-hSHN3 treated mice (blue, J) while an increase in collagen fibers, calcified matrix, and number of osteoblast-like cells was observed by SEM 12075767.1 analysis (n = 3 / group, K, left and middle). Scale bars: 200 um (B), 1 mm (H), 100 um (J), 30 um (K, left and middle), 2 um (K, right). A two-tailed unpaired Student’s t-test for comparing two groups (C, G; error bars, data represent mean ± SD). Representative images of three replicates are displayed (B, E, F, H, J, K). FIGs. 4A-4L show AAV-mediated silencing of SHN3 reverses alveolar bone loss in osteoporotic mice. (A–C) 2-month-old mice were treated with PBS or rAAV9.egfp via intravenous (IV) injection or periodontal ligament (PL) injection into the mandibular first molar. 10 days later, EGFP expression in individual tissues was monitored by IVIS-100 optical imaging (A) and RT-PCR (n = 4–5 / group, B, C). The y-axis in A shows radiant efficiency (p / s / cm2 / sr / μW / cm2). (D) 2-month-old Pdgfrα-GFP reporter mice were treated with PBS or dss.rAAV9.mCherry via PL injection to visualize AAV-transduced Pdgfrα-lineage cells in the alveolar bone (n = 3 / group). 10 days later, mCherry expression in mandibles was monitored by IVIS-100 optical imaging (top) and fluorescence microscopy on cryo-sectioned mandibles (bottom). PDL: periodontal ligament, BM: bone marrow, DP: dental pulp, ob: osteoblast, ocy: osteocyte. Scale bar: 50 um. (E–I) Diagram of the study and treatment methods. Sham or OVX surgery was performed on 3-month-old female mice and 4 weeks later, mice were treated with dss.rAAV9 carrying amiR-ctrl or amiR-SHN3 via IV injection or PL injection (E). 8 weeks later, Shn3 mRNA expression in the mandible was assessed by RT-PCR (n = 6 / group, F). 2D microCT images (G, top and middle) and relative quantification (H) and H&E staining (G, bottom), showing an increase in alveolar bone mass (n = 6 / group) in the amiR-SHN3 treated OVX mice. MicroCT analysis showing root dentin and enamel volume of mandibular first molar (n = 6 / group, I). (J, K) 20-month-old male mice were treated with dss.rAAV9 carrying amiR-ctrl or amiR-SHN3 via PL injection and 8 weeks later, Shn3 mRNA expression in the mandible was assessed by RT-PCR (n = 7 / group, J). 2D microCT images (K, left and middle) and relative quantification (L) and H&E staining (K, right), showing an increase in alveolar bone mass (n = 7 / group) in the amiR-SHN3 treated mice. Scale bars: 100 um (D, G-bottom, K-right), 1 mm (G- top and middle, K-left and middle). A two-tailed unpaired Student’s t-test for comparing two groups (J, L) or ordinary one-way ANOVA with Dunnett’s multiple comparisons test (B, C, F, H, I). B, C, F, H–K; data represent mean ± SD. Representative images are displayed (A, D, G, K). FIGs. 5A-5K show liver / heart-detargeting AAV-mediated silencing of SHN3 reverses bone loss in osteoporotic mice. (A) Diagram of the liver and heart-detargeting AAV vector 12075767.1 construct containing an egfp, amiR-ctrl, or amiR-SHN3. Target sequences of the liver-abundant miR-122 and the heart-abundant miR-208a were inserted into the 3’-UTR of egfp to repress transgene expression. (B, C) 2-month-old mice were treated with PBS, dss.rAAV9.egfp, or dss.rAAV9.egfp.MIR-TS via IV injection and 10 days later, EGFP expression in individual tissues was assessed by IVIS-100 optical imaging (B) and RT-PCR (C). (D–F) Sham or OVX surgery was performed on 3-month-old female mice and 4 weeks later, mice were treated with dss.rAAV9 carrying amiR-ctrl, amiR-SHN3, or amiR-shn3-MIR-TS via IV injection. 8 weeks later, Shn3 mRNA expression in the tibia was assessed by RT-PCR (n = 5–6 / group, D). Femoral bone mass was assessed by microCT. 3D microCT images (E) and relative quantification (F) are displayed (n = 5 / group). Tb.BV / TV: trabecular bone volume per tissue volume, Tb.N: trabecular number, Tb.Sp: trabecular space. (G, H) 2-month-old mice were treated with PBS, dss.rAAV9.egfp, or dss.rAAV9.egfp.MIR-TS via PL injection into the mandibular first molar. 10 days later, EGFP expression in the mandible was assessed by IVIS-100 optical imaging (G), RT-PCR (n = 6 / group, H, left), and fluorescence microscopy on cryo-sectioned mandible (H, right). (I-K) Sham or OVX surgery was performed on 3-month-old female mice and 4 weeks later, mice were treated with dss.rAAV9 carrying amiR-ctrl, amiR-SHN3, or amiR-SHN3.MIR- TS via PL injection. 8 weeks later, Shn3 mRNA expression in the mandible was assessed by RT-PCR (n = 5 / group, I). 2D microCT images (J, top) and relative quantification (n = 5–7 / group, K) and H&E staining (J, bottom) show a reversal of alveolar bone loss in OVX mandible. Scale bars: top; 1 mm (E, J-top), 100 µm (H, J-bottom). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (C, D, F, H, I, K; data represent mean ± SD). Representative images of five (B, E-left) or six (G, H-right, J) replicates are displayed. FIGs. 6A-6J show development of a bone-targeting AAV with mechanical stress- inducible expression. (A) Fluorescence microscopy was performed on cryo-sectioned mandibles of 2-month-old TCF / Lef1-HIST1H2BB / EGFP reporter mice to locate cells with WNT signaling activation in the alveolar bone. (B) mRNA expression of WNT-responsive genes, Axin2 and Lef1, in the calvaria, mandible, and tibia of 2-month-old mice (n = 8–10 / group). (C) The AAV vector genome containing the LEF / TCF promoter (pLef / Tcf) and egfp reporter gene was packaged into the dss.rAAV9 capsid. (D, E) Calvarial osteoblasts (COB) were transduced with dss.rAAV9.pLef / Tcf-egfp and stimulated with recombinant WNT3a or flow stress. EGFP expression was assessed by fluorescence microscopy (D) and RT-PCR (n = 4 / group, E). WNT signaling activity was assessed by measuring mRNA expression of Axin2 and Lef1 (n = 12075767.1 4 / group, E). (F–J) Diagram of the study and treatment methods. 2-month-old mice were treated with dss.rAAV9.pLef / Tcf-egfp via PL injection into the mandibular first molar and 2 days later, the mandible was stimulated daily with HFV treatment for up to 15 days (F). RNA was isolated from the treated mandible at day 0, 5, 10, and 15 post-treatment. mRNA expression of egfp and Axin2 was assessed by RT-PCR (n = 5–7 / group, G). AAV-treated mandible was stimulated daily with or without HFV treatment for 10 days and WNT signaling activity was assessed by mRNA expression of egfp, Axin2, and Lef1 (n = 4–6 / group, H), IVIS-100 optical imaging (I), and fluorescence microscopy on cryo-sectioned mandible (J). AAV-treated mandible was stained with β-Catenin, demonstrating that EGFP expression in β-Catenin high cells was upregulated by HFV treatment (J). Scale bar: 50 um (A, J), 100 μm (D). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (B, E, G, H; data represent mean ± SD). Representative images of three (A, J), four (D), or six (I) are displayed (A, D, I, J). FIGs. 7A-7K show vibration-inducible AAV-mediated silencing of SHN3 / SOST reverses alveolar bone loss in osteoporotic mice. Sham or OVX surgery was performed on 3- month-old female mice and 4 weeks later, mice were treated with dss.rAAV9.pLef / Tcf carrying amiR-ctrl, amiR-SHN3, or amiR-SOST / SHN3 via PL injection to the mandibular first molar. 2 days later, AAV-treated mandibles were stimulated daily with HFV treatment for 10 days. 7 weeks later, mRNA expression of Shn3 and Sost in the mandible was assessed by RT-PCR (n = 6–10 / group, A, C). WNT signaling activity was assessed by measuring mRNA expression of Axin2 and Lef1 (n = 6 / group, B, D). Calcein / alizarin red labeling images and relative histomorphometric quantification of BFR / BS and MAR are displayed (n = 5 / group, E). BFR / BS, bone formation rate / bone surface; MAR, mineral apposition rate. 2D microCT images (F, top and middle) and relative quantification (n = 6–12 / group, G) and H&E staining (F, bottom) of alveolar bone are displayed. amiR-SOST / SHN3 upregulated mRNA expression of Osteoprotegerin (Opg) in HFV-treated mandibles (H). Osteoclast development in AAV-treated mandibles was assessed by measuring Trap mRNA expression (I) and numbers of TRAP- positive osteoclasts on the surface of alveolar bone (n = 5 / group, J, K). Oc.S / BS: osteoclast surface / bone surface; N.Oc / B.pm: osteoclast number / Bone perimeter. Scale bars: 200 μm (E), 1 mm (F-top and middle), 100 um (F-bottom). A two-tailed unpaired Student’s t-test for comparing two groups (B, C, D) or ordinary one-way ANOVA with Dunnett’s multiple comparisons test (A, E, G, H, I, K). A–E, G, H, I, K; data represent mean ± SD. Representative images are displayed (E, F, I). 12075767.1 FIGs. 8A-8C show osteoblast-specific deletion of Shn3 increases bone mass in the long bones. MicroCT analysis shows a significant increase in trabecular bone mass and cortical thickness of one-month-old Shn3Pdgfra (A), Shn3Prx1 (B), Shn3Dmp1 (C) femurs compared to Shn3fl / fl femurs (n = 5 / group). A two-tailed unpaired Student’s t-test for comparing two groups (A–C; error bars, data represent mean ± SD). FIGs. 9A-9D show biodistribution of locally or systemically injected rAAV9 in mice. (A) The AAV vector genome containing the CMV enhancer / chicken b-actin promoter (CBA), an Egfp reporter gene (EGFP), amiR-ctrl, amiR-SHN3, b-globin polyA sequence (PA), and inverted terminal repeat (ITR) was packaged into rAAV9 or dss.AAV9 capsid. (B) Diagram of the study and treatment methods for FIGs. 4A-4D. 2-month-old mice were treated with EGFP- expressing rAAV9 (rAAV9.egfp) or dss.rAAV9 (dss.rAAV9.egfp) via intravenous (IV) injection or periodontal ligament (PL) injection to the mandibular first molar. 10 days later, EGFP expression in individual tissues was assessed by fluorescence microscopy in cryosectioned tissues (C, D). PDL: periodontal ligament, ob: osteoblast, ocy: osteocyte. Scale bar, 100 μm, C; 50 μm, D. Representative images of three replicates are displayed (C, D). FIGs. 10A-10C show characterization of the liver / heart-detargeting AAV. (A) Expression of heart-abundant miR-208a and liver-abundant miR-122 in the mandible, liver, and heart was measured by RT-PCR analysis (n =3 / group). (B) 2-month-old mice were injected IV with PBS, dss.AAV9.egfp, or dss.rAAV9.egfp.MIR-TS and ten days later, EGFP expression in the liver and heart was assessed by fluorescence microscopy (n = 3 / group, scale bar: 200 μm). (C) Diagram of the study and treatment methods for FIGs. 5G-5K. Sham or ovariectomy (OVX) surgery was performed on 3-month-old female mice, and four weeks later, mice were treated with PBS or dss.rAAV9 carrying egfp, amiR-ctrl, amiR-SHN3, or amiR-SHN3-MIR-TS via PL injection. Eight weeks after injection, egfp and Shn3 expression and alveolar bone mass were assessed (n =5 / group). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (A; data represent mean ± SD). Representative images of three replicates are displayed (B). FIGs. 11A-11D show vibration treatment does not affect the skull, mandible, and tooth structure. (A) Diagram of the study and treatment methods. Calvarial osteoblasts (COB) were incubated for two days and then transduced with dss.rAAV9.pLef / Tcf-egfp for two days and cultured in osteoblast differentiation medium (DM) for two days. AAV-treated cells were stimulated with recombinant WNT3a or flow stress for 72 hours. EGFP expression was assessed by fluorescence microscopy and RT-PCR. (B-D) 2-month-old mice were treated with 12075767.1 dss.rAAV9.pLef / Tcf-egfp via PL injection into the mandibular first molar and ten days later, the mandible was stimulated daily with high-frequency vibration (HFV) for up to fifteen days. Representative photographs show that the molar structure of AAV-treated mandibles is grossly normal (B). Representative 2D microCT images and relative quantification show little to no effects of high-frequency vibration (HFV) treatment on skull length, intercondylar distance, and mandible length (C, D). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (D; data represent mean ± SD). Representative images of three replicates are displayed (B, C). FIGs. 12A-12E show vibration-inducible AAV-mediated silencing of SHN3 or SHN3 / SOST does not affect tooth dentin formation in osteoporotic mice. (A) Correlation analysis of mRNA expression of egfp and WNT-responsive genes, Axin2 and Lef1. The experiments were performed in Figure 6E. (B) Diagram of the mechanical stress-responsive AAV containing amiR-ctrl, amiR-SHN3, or amiR-SHN3 / SOST. The CBA promoter was replaced with the LEF / TCF promoter (pLef / Tcf). (C) Diagram of the study and treatment methods for FIG. 7. Sham or OVX surgery was performed on 3-month-old female mice and four weeks later, mice were treated with dss.rAAV9.pLef / Tcf carrying amiR-ctrl, amiR-SHN3, or amiR-SOST / SHN3 via PL injection to the mandibular first molar. Ten days later, AAV-treated mandibles were stimulated daily with HFV for ten days. Seven weeks later, mice were treated with calcein and alizarin red via intraperitoneal injection at six-day intervals and then euthanized. (D) Representative calcein / alizarin red labeling images and relative histomorphometric quantification of dentin deposition rates in the mandibular incisor are displayed (E). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (E; data represent mean ± SD). Representative images of five replicates are displayed (D). FIGs. 13A-13K show that SHN3 deletion increased alveolar bone and tooth root dentin mass. FIG. 13A and FIG. 13C show immunohistochemistry showing expression of SOST and DKK1 (FIG. 13A) and SHN3 and β-Catenin (FIG. 13C) in the mandible of 2-month-old mice. AB: alveolar bone, PDL: periodontal ligament, BM: bone marrow, DP: dental pulp, ob: osteoblasts, od: odontoblasts, ocy: osteocytes. FIG. 13B and FIG. 13D show human SOST, DKK1, and SHN3 mRNA expression in the mandible of young vs. aged patients (n = 4 / group). FIG. 13E shows Mouse Shn3 mRNA expression in the mandible of 2-month-old vs. 24-month- old mice (n =5 / group, left) and 6-month-old mice with sham control vs ovariectomy (OVX) surgery (n = 4–6 / group, right). FIG. 13F, FIG. 13G, and FIG. 13H show MicroCT and histologic analyses showing alveolar bone mass in the first molar of 1- and 7-month-old Shn3- / -and Shn3+ / +12075767.1 (WT) mandibles. MicroCT images showing sagittal and cross sections of the first molar (FIG. 13F, left and middle; FIG. 13H, left). H&E (FIG. 13G, left and middle; FIG. 13H, middle) and Masson’s trichrome staining of sagittal sectioned molar (FIG. 13G, right). MicroCT quantification of alveolar bone mass in the first molar (n = 5 / group, FIG. 13F, left). AB.BV / TV: alveolar bone volume per tissue volume, AB.Th: alveolar bone thickness (FIG. 13F, right). FIG. 13J and FIG. 13K show MicroCT analysis showing root dentin and enamel volume of the first molar of 1- and 7-month-old Shn3- / -and Shn3+ / +mandible. 3D-reconstruction images (FIG. 13J, FIG. 13K-bottom) and relative quantification (n = 5 / group) are displayed (FIG. 13J, FIG. 13K- top). The representative image of the methods used to quantitatively measure the parameters listed in (FIG. 13I). The area highlighted second from top marks the crown dentin region of interest (ROI), the top area marks the enamel ROI, the second from the bottom area marks the root dentin ROI, and the bottom area marks the root dentin. MR: mesial root, DR: distal root. Scale bars: 50 µm (FIG. 13A, FIG. 13C), 1 mm (FIG. 13F- left and middle, FIG. 13H-left), 100 µm (FIG. 13G, FIG. 13H-middle, FIG. 13J-bottom, FIG. 13K-bottom). A two-tailed unpaired Student’s t-test for comparing two groups (FIG. 13B, FIG. 13D, FIG. 13E, FIG. 13F, FIG. 13H, FIG. 13J, FIG. 13K; error bars, data represent mean ± SD). Representative images of three replicates are displayed (FIG. 13A, FIG. 13C, FIG. 13G, FIG. 13H, FIG. 13J, FIG. 13K). FIGs. 14A-14K show mesenchyme-specific deletion of SHN3 increased alveolar bone and tooth root dentin mass. FIG. 14A shows fluorescence microscopy was performed on cryo- sectioned mandibles from 2-month-old Pdgfrα-GFP reporter mice to visualize GFP-expressing Pdgfrα-lineage cells in the alveolar bone and tooth. FIG. 14B and FIG. 14C show that alveolar bone marrow mesenchymal stromal cells (ABMSCs) were isolated from the mandible of 4- week-old Shn3Pdgfrαand Shn3fl / flmice and cultured under osteogenic conditions. mRNA expression of Shn3, Bglap, and Dmp1 (FIG. 14B) and mineralization activity (FIG. 14C) were assessed by RT-PCR and alizarin red staining, respectively (n = 4 / group). FIG. 14D and FIG. 14E show MicroCT and histologic analyses showing alveolar bone mass in the first molar of 1- and 7-month-old Shn3Pdgfrαand Shn3fl / flmandibles. MicroCT images showing sagittal and cross sections of the mandibular first molar (left). H&E staining of sagittal sectioned molar (FIG. 14D-right, FIG. 14E-middle). MicroCT quantification of alveolar bone mass in the mandibular first molar (n =5 / group, FIG. 14D-middle, FIG. 14E-right). FIG. 14F shows MicroCT analysis showing root dentin and enamel volume of the first molar of 1- or 7- month-old Shn3Pdgfrαand Shn3fl / flmandibles. 3D-reconstruction images and relative quantification (n = 5 / group) are 12075767.1 displayed. FIG. 14G shows Masson’s trichome staining (left) and scanning electron microscopy (SEM, right) were performed in sagittal sectioned tooth dentin of the first molar of 1-month-old Shn3Pdgfrαand Shn3fl / flmandibles, demonstrating normal microstructure of tooth dentin. FIG. 14H shows fluorescence microscopy was performed on cryo-sectioned mandibles from 2- month-old Prx1-GFP (left) and Dmp1-cre;Rosa26mTmG(right) reporter mice to visualize GFP- expressing Prx1- or Dmp1-lineage cells in the alveolar bone and tooth. FIG. 14I shows MicroCT and histologic analyses showing alveolar bone mass in the first molar of 1-month-old Shn3Dmp1, Shn3Prx1, and Shn3fl / flmandibles. MicroCT images (left), H&E staining (middle), and microCT quantification of alveolar bone mass in the molar (right) are displayed (n = 5 / group). FIG. 14J and FIG. 14K show MicroCT analysis showing root dentin and enamel volume of the first molar of 1-month-old Shn3Prx1, Shn3Dmp1, and Shn3fl / flmandibles. 3D-reconstruction images and relative quantification (n = 5 / group) are displayed. Scale bars: 50 µm (FIG. 14A, FIG. 14G-left, FIG. 14H), 1 mm (FIG. 14D, FIG. 14E-left, FIG. 14I-left), 100 µm (FIG. 14D-right, FIG. 14E- middle, FIG. 14F-right, FIG. 14I-middle, FIG. 14J-right, FIG. 14K-right), 30 µm (G, right). A two-tailed unpaired Student’s t-test for comparing two groups (FIG. 14B, FIG. 14D, FIG. 14E, FIG. 14F, FIG. 14I, FIG. 14J, FIG. 14K; error bars, data represent the mean ± SD). Representative images of three replicates are displayed (FIG. 14A, FIG. 14C, FIG. 14D, FIGs. 14E-14K). FIGs. 15A-15K show AAV-mediated silencing of human SHN3 increased bone and collagen formation. FIG. 15A shows the AAV vector genome containing the CMV enhancer / chicken β-actin promoter (CBA), hs-amiR-ctrl, hs-amiR-hSHN3, an Egfp reporter gene (EGFP), β-globin polyA sequence (PA), and inverted terminal repeat (ITR) was packaged into the rAAV9 capsid. FIG. 15B and FIG. 15C show human periodontal ligament stem cells (hPDLCs) and bone marrow-derived stromal cells (hBMSCs) were treated with rAAV9 carrying hs-amiR-ctrl or hs-amiR-hShn3 (4 x 106vg / cell) and cultured under osteogenic conditions for 4 days. rAAV9’s transduction efficiency was examined by fluorescence microscopy using EGFP expression (FIG. 15B). mRNA expression of SHN3 and the osteogenic marker IBSP were assessed by RT-PCR (n = 4 / group, FIG. 15C). FIGs. 15D-15K show a diagram of the study and treatment methods. Human BMSC-seeded hydroxyapatite (HA)-scaffold was implanted into the interscapular fat pads of immunodeficient SCID mice and one week later, PBS or rAAV9 carrying hs-amiR-ctrl or hs-amiR-hSHN3 (2.5 x 1012vg / kg) was injected into the implantation site (FIG. 15D). Four weeks later, the implanted scaffold was visualized by radiography (FIG. 12075767.1 15E), and EGFP expression in the scaffold was assessed by IVIS-100 optical imaging system (FIG. 15F). mRNA expression of SHN3, BGLAP, AXIN2, and COL1 in the scaffold was assessed by RT-PCR (n = 4 / group, FIG. 15G). Bone accrual and collagen production of the scaffold were assessed by microCT (FIG. 15H, FIG. 15I), histology (FIG. 15J), and SEM (FIG. 15K). 2D microCT images (FIG. 15H) and relative quantification showing mineral density distribution (FIG. 15I) are displayed. Masson’s trichome staining shows a significant increase in collagen production in the hs-amiR-hSHN3 treated mice (dark [blue], FIG. 15J) while an increase in collagen fibers, calcified matrix, and number of osteoblast-like cells was observed by SEM analysis (n = 3 / group, FIG. 15K, left and middle). Scale bars: 200 µm (FIG. 15B), 1 mm (FIG. 15H), 100 µm (FIG. 15J), 30 µm (FIG. 15K, left and middle), 2 µm (FIG. 15K, right). A two- tailed unpaired Student’s t-test for comparing two groups (FIG. 15C, FIG. 15G; error bars, data represent mean ± SD). Representative images of three replicates are displayed (FIG. 15B, FIG. 15E, FIG. 15F, FIG. 15H, FIG. 15J, FIG. 15K). FIGs. 16A-16L show AAV-mediated silencing of SHN3 reversed alveolar bone loss in osteoporotic mice. FIGs. 16A–16C show 2-month-old mice were treated with PBS or rAAV9.egfp via intravenous (IV, 2.5 x 1013vg / kg) injection or periodontal ligament (PL, 2.5 x 1012vg / kg) injection into the mandibular first molar. 10 days later, EGFP expression in individual tissues was monitored by IVIS-100 optical imaging (FIG. 16A) and RT-PCR (n = 4– 5 / group, FIG. 16B, FIG. 16C). FIG. 16D shows 2-month-old Pdgfrα-GFP reporter mice were treated with PBS or dss.rAAV9.mCherry (2.5 x 1012vg / kg) via PL injection to visualize AAV- transduced Pdgfrα-lineage cells in the alveolar bone (n = 3 / group). 10 days later, mCherry expression in mandibles was monitored by IVIS-100 optical imaging (top) and fluorescence microscopy on cryo-sectioned mandibles (bottom). PDL: periodontal ligament, BM: bone marrow, DP: dental pulp, ob: osteoblast, ocy: osteocyte. Scale bar: 50 µm. FIG. 16E– FIG. 16I shows a diagram of the study and treatment methods. Sham or OVX surgery was performed on 3-month-old female mice and 4 weeks later, mice were treated with dss.rAAV9 carrying amiR- ctrl or amiR-SHN3 via IV (2.5 x 1013vg / kg) injection or PL (2.5 x 1012vg / kg) injection (FIG. 16E). 8 weeks later, Shn3 mRNA expression in the mandible was assessed by RT-PCR (n = 6 / group, FIG. 16F). 2D microCT images (FIG. 16G, top and middle) and relative quantification (FIG. 16H) and H&E staining (FIG. 16G, bottom), showing an increase in alveolar bone mass (n = 6 / group) in the amiR-SHN3 treated OVX mice. MicroCT analysis showing root dentin and enamel volume of mandibular first molar (n = 6 / group, FIG. 16I). FIG. 12075767.1 16J and FIG. 16K show 20-month-old male mice were treated with dss.rAAV9 carrying amiR- ctrl or amiR-SHN3 (2.5 x 1012vg / kg) via PL injection and 8 weeks later, Shn3 mRNA expression in the mandible was assessed by RT-PCR (n = 7 / group, FIG. 16J). 2D microCT images (FIG. 16K, left and middle) and relative quantification (FIG. 16L) and H&E staining (FIG. 16K, right), showing an increase in alveolar bone mass (n = 7 / group) in the amiR-SHN3 treated mice. Scale bars: 100 µm (FIG. 16D, FIG. 16G-bottom, FIG. 16K-right), 1 mm (FIG. 16G- top and middle, FIG.16K-left and middle). A two-tailed unpaired Student’s t-test for comparing two groups (FIG. 16J, FIG. 16L) or ordinary one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 16B, FIG. 16C, FIG. 16F, FIG. 16H, FIG. 16I). FIG. 16B, FIG. 16C, FIG. 16F, FIG. 16H– FIG. 16K; data represent mean ± SD. Representative images are displayed (FIG. 16A, FIG. 16D, FIG. 16G, FIG. 16K). FIGs. 17A-17K show liver / heart-detargeting AAV-mediated silencing of SHN3 reversed bone loss in osteoporotic mice. FIG. 17A shows a diagram of the liver and heart-detargeting AAV vector construct containing an egfp, amiR-ctrl, or amiR-SHN3. Target sequences of the liver-abundant miR-122 and the heart-abundant miR-208a were inserted into the 3’-UTR of egfp to repress transgene expression. FIG. 17B and FIG. 17C show 2-month-old mice were treated with PBS, dss.rAAV9.egfp, or dss.rAAV9.egfp.MIR-TS (2.5 x 1013vg / kg) via IV injection and 10 days later, EGFP expression in individual tissues was assessed by IVIS-100 optical imaging (FIG. 17B) and RT-PCR (FIG. 17C). FIG. 17D– FIG. 17F show Sham or OVX surgery was performed on 3-month-old female mice and 4 weeks later, mice were treated with dss.rAAV9 carrying amiR-ctrl, amiR-SHN3, or amiR-shn3-MIR-TS (2.5 x 1013vg / kg) via IV injection. 8 weeks later, Shn3 mRNA expression in the tibia was assessed by RT-PCR (n = 5–6 / group, FIG. 17D). Femoral bone mass was assessed by microCT. 3D microCT images (FIG. 17E) and relative quantification (FIG. 17F) are displayed (n = 5 / group). Tb.BV / TV: trabecular bone volume per tissue volume, Tb.N: trabecular number, Tb.Sp: trabecular space. FIG. 17G and FIG. 17H show 2-month-old mice were treated with PBS, dss.rAAV9.egfp, or dss.rAAV9.egfp.MIR- TS (2.5 x 1012vg / kg) via PL injection into the mandibular first molar. 10 days later, EGFP expression in the mandible was assessed by IVIS-100 optical imaging (FIG. 17G), RT-PCR (n = 6 / group, FIG. 17H, left), and fluorescence microscopy on cryo-sectioned mandible (FIG. 17H, right). FIG. 17I- FIG. 17K show Sham or OVX surgery was performed on 3-month-old female mice and 4 weeks later, mice were treated with dss.rAAV9 carrying amiR-ctrl, amiR-SHN3, or amiR-SHN3.MIR-TS (2.5 x 1012vg / kg) via PL injection. 8 weeks later, Shn3 mRNA expression 12075767.1 in the mandible was assessed by RT-PCR (n = 5 / group, FIG. 17I). 2D microCT images (FIG. 17J, top) and relative quantification (n = 5–7 / group, FIG. 17K) and H&E staining (FIG. 17J, bottom) show a reversal of alveolar bone loss in OVX mandible. Scale bars: top; 1 mm (FIG. 17E, FIG. 17J-top), 100 µm (FIG. 17H, FIG. 17J-bottom). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 17C, FIG. 17D, FIG. 17F, FIG. 17H, FIG. 17I, FIG. 17K; data represent mean ± SD). Representative images of five (FIG. 17B, FIG. 17E-left) or six (FIG. 17G, FIG. 17H-right, FIG. 17J) replicates are displayed. FIGs. 18A-18J show development of a bone-targeting AAV with vibration-inducible expression. FIG. 18A shows fluorescence microscopy was performed on cryo-sectioned mandibles of 2-month-old TCF / Lef1-HIST1:H2BB / EGFP reporter mice to locate cells with WNT signaling activation in the alveolar bone. FIG. 18B shows mRNA expression of WNT- responsive genes, Axin2 and Lef1, in the calvaria, mandible, and tibia of 2-month-old mice (n = 8–10 / group). FIG. 18C shows the AAV vector genome containing the LEF / TCF promoter (pLef / Tcf) and egfp reporter gene was packaged into the dss.rAAV9 capsid. FIG. 18D, FIG. 18E show Calvarial osteoblasts (COB) were transduced with dss.rAAV9.pLef / Tcf-egfp (4 x 106vg / cell) and stimulated with a recombinant WNT3a or flow stress. EGFP expression was assessed by fluorescence microscopy (FIG. 18D) and RT-PCR (n = 4 / group, FIG. 18E). WNT signaling activity was assessed by measuring mRNA expression of Axin2 and Lef1 (n = 4 / group, FIG. 18E). FIG. 18F– FIG. 18J show a diagram of the study and treatment methods. 2-month- old mice were treated with dss.rAAV9.pLef / Tcf-egfp (2.5 x 1012vg / kg) via PL injection into the mandibular first molar and 2 days later, the mandible was stimulated daily with HFV treatment for up to 15 days (FIG. 18F). RNA was isolated from the treated mandible at day 0, 5, 10, and 15 post-treatment. mRNA expression of egfp and Axin2 was assessed by RT-PCR (n = 5–7 / group, FIG. 18G). AAV-treated mandible was stimulated daily with or without HFV treatment for 10 days and WNT signaling activity was assessed by mRNA expression of egfp, Axin2, and Lef1 (n = 4–6 / group, FIG. 18H), IVIS-100 optical imaging (FIG. 18I), and fluorescence microscopy on cryo-sectioned mandible (FIG. 18J). AAV-treated mandible was stained with β-Catenin, demonstrating that EGFP expression in β-Cateninhighcells was upregulated by HFV treatment (FIG. 18J). Scale bar: 50 µm (FIG. 18A, FIG. 18J), 100 μm (FIG. 18D). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 18B, FIG. 18E, FIG. 18G, FIG. 18H; data represent mean ± SD). Representative images of three (FIG. 18A, FIG. 18J), four (FIG. 18D), or six (FIG. 18I) are displayed (FIG. 18A, FIG. 18D, FIG. 18I, FIG. 18J). 12075767.1 FIGs. 19A-19K show vibration-inducible AAV-mediated silencing of SHN3 / SOST reversed alveolar bone loss in osteoporotic mice. Sham or OVX surgery was performed on 3- month-old female mice and 4 weeks later, mice were treated with dss.rAAV9.pLef / Tcf carrying amiR-ctrl, amiR-SHN3, or amiR-SOST / SHN3 (2.5 x 1012vg / kg) via PL injection to the mandibular first molar. 2 days later, AAV-treated mandibles were stimulated daily with HFV treatment for 10 days. 7 weeks later, mRNA expression of Shn3 and Sost in the mandible was assessed by RT-PCR (n = 6–10 / group, FIG. 19A, FIG.19C). WNT signaling activity was assessed by measuring mRNA expression of Axin2 and Lef1 (n = 6 / group, FIG. 19B, FIG. 19D). Calcein / alizarin red labeling images and relative histomorphometric quantification of BFR / BS and MAR are displayed (n = 5 / group, FIG. 19E). BFR / BS, bone formation rate / bone surface; MAR, mineral apposition rate. 2D microCT images (FIG. 19F, top and middle) and relative quantification (n = 6–12 / group, FIG. 19G) and H&E staining (FIG. 19F, bottom) of alveolar bone are displayed. amiR-SOST / SHN3 treatment upregulated mRNA expression of Osteoprotegerin (Opg) in HFV-treated mandibles (FIG. 19H). Osteoclast development in AAV- treated mandibles was assessed by measuring Trap mRNA expression (FIG. 19I) and numbers of TRAP-positive osteoclasts on the surface of alveolar bone (n = 5 / group, FIG. 19J, FIG. 19K). Oc.S / BS: osteoclast surface / bone surface; N.Oc / B.pm: osteoclast number / Bone perimeter. Scale bars: 200 μm (FIG. 19E), 1 mm (FIG. 19F-top and middle), 100 µm (FIG. 19F-bottom). A two-tailed unpaired Student’s t-test for comparing two groups (FIG. 19B, FIG. 19C, FIG. 19D) or ordinary one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 19A, FIG. 19E, FIG. 19G, FIG. 19H, FIG. 19I, FIG. 19K). FIG. 19A– FIG. 19E, FIG. 19G, FIG. 19H, FIG. 19I, FIG. 19K; data represent mean ± SD. Representative images are displayed (FIG. 19E, FIG. 19F, FIG. 19I). FIGs. 20A-20C show osteoblast-specific deletion of Shn3 increased bone mass in the long bones. MicroCT analysis showed a significant increase in trabecular bone mass and cortical thickness of one-month-old Shn3Pdgfrα(FIG. 20A), Shn3Prx1(FIG. 20B), Shn3Dmp1(FIG. 20C) femurs compared to Shn3fl / flfemurs (n = 5 / group). A two-tailed unpaired Student’s t-test for comparing two groups (FIG. 20A– FIG. 20C; error bars, data represent mean ± SD). FIGs. 21A-21F show biodistribution of locally or systemically injected rAAV9 in mice. FIG. 21A shows the AAV vector genome containing the CMV enhancer / chicken β-actin promoter (CBA), an Egfp reporter gene (EGFP), amiR-ctrl, amiR-SHN3, β-globin polyA sequence (PA), and inverted terminal repeat (ITR) was packaged into rAAV9 or dss.AAV9 12075767.1 capsid. FIG. 21B shows a diagram of the study and treatment methods for FIGs. 16A-16D. 2- month-old mice were treated with EGFP-expressing rAAV9 (rAAV9.egfp) or dss.rAAV9 (dss.rAAV9.egfp) via intravenous (IV, 2.5 x 1013vg / kg) injection or periodontal ligament (PL, 2.5 x 1012vg / kg) injection to the mandibular first molar. 10 days later, EGFP expression in individual tissues was assessed by fluorescence microscopy in cryosectioned tissues (FIG. 21C, FIG. 21D). Sham or OVX surgery was performed on 3-month-old female mice and 4 weeks later, mice were injected IV with dss.rAAV9 carrying amiR-ctrl or amiR-SHN3 (2.5 x 1013vg / kg). 8 weeks later, mRNA levels of Shn3, MyoD, and Myogenin in the skeletal muscle were measured by RT-PCR (n = 6 - 8 / group, FIG. 21E). Alternatively, the skeletal muscle was stained with H&E (FIG. 21F), suggesting that SHN3 is dispensable of muscle homeostasis. PDL: periodontal ligament, ob: osteoblast, ocy: osteocyte. Scale bar, 100 µm, FIG. 21C, FIG. 21F; 50 µm, FIG. 21D. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 21E; data represent mean ± SD). Representative images of three replicates are displayed (FIG. 21C, FIG. 21D, FIG. 21F). FIGs. 22A-22C show characterization of the liver / heart-detargeting AAV. FIG. 22A shows expression of heart-abundant miR-208a and liver-abundant miR-122 in the mandible, liver, and heart was measured by RT-PCR analysis (n =3 / group). FIG. 22B shows 2-month-old mice were injected IV with PBS, dss.AAV9.egfp, or dss.rAAV9.egfp.MIR-TS (2.5 x 1013vg / kg) and ten days later, EGFP expression in the liver and heart was assessed by fluorescence microscopy (n = 3 / group, scale bar: 200 µm). FIG. 22C shows a diagram of the study and treatment methods for Figure 5G-K. Sham or ovariectomy (OVX) surgery was performed on 3- month-old female mice, and four weeks later, mice were treated with PBS or dss.rAAV9 carrying egfp, amiR-ctrl, amiR-SHN3, or amiR-SHN3-MIR-TS (2.5 x 1012vg / kg) via PL injection. Eight weeks after injection, egfp and Shn3 expression and alveolar bone mass were assessed (n = 5 / group). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 22A; data represent mean ± SD). Representative images of three replicates are displayed (FIG. 22B). FIGs. 23A-23D show vibration treatment did not affect the skull, mandible, and tooth structure. FIG. 23A shows a diagram of the study and treatment methods. Calvarial osteoblasts (COB) were incubated for two days and then transduced with dss.rAAV9.pLef / Tcf-egfp (4 x 106vg / cell) for two days and cultured in osteoblast differentiation medium (DM) for two days. AAV- treated cells were stimulated with recombinant WNT3a or flow stress for 72 hours. EGFP 12075767.1 expression was assessed by fluorescence microscopy and RT-PCR. FIG. 23B- FIG. 23D show 2- month-old mice were treated with dss.rAAV9.pLef / Tcf-egfp (2.5 x 1012vg / kg) via PL injection into the mandibular first molar and two days later, the mandible was stimulated daily with high- frequency vibration (HFV) for up to fifteen days. Representative photographs show that the molar structure of AAV-treated mandibles is grossly normal (FIG. 23B). Representative 2D microCT images and relative quantification show little to no effects of high-frequency vibration (HFV) treatment on skull length, intercondylar distance, and mandible length (FIG. 23C, FIG. 23D). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 23D; data represent mean ± SD). Representative images of three replicates are displayed (FIG. 23B, FIG. 23C). FIGs. 24A-24B show effects of HFV treatment on AAV’s biodistribution in mice. 2- month-old mice were treated with dss.rAAV9.pLef / Tcf-egfp (2.5 x 1012vg / kg) via PL injection into the mandibular first molar and two days later, the mandible was stimulated daily with HFV treatment for 10 days. EGFP expression in individual tissues was assessed by IVIS optical imaging system (FIG. 24A) and RT-PCR (n = 4 / group, FIG. 24B). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 24B; data represent mean ± SD). Representative images of three replicates are displayed (FIG. 24A). FIGs. 25A-25E show vibration-inducible AAV-mediated silencing of SHN3 or SHN3 / SOST did not affect tooth dentin formation in osteoporotic mice. FIG. 25A shows a correlation analysis of mRNA expression of egfp and WNT-responsive genes, Axin2 and Lef1. The experiments were performed in FIG. 18E. FIG. 25B shows a diagram of the mechanical stress-responsive AAV containing amiR-ctrl, amiR-SHN3, or amiR-SHN3 / SOST. The CBA promoter was replaced with the LEF / TCF promoter (pLef / Tcf). FIG. 25C shows a diagram of the study and treatment methods for FIG. 19A-FIG. 19K. Sham or OVX surgery was performed on 3-month-old female mice and four weeks later, mice were treated with dss.rAAV9.pLef / Tcf carrying amiR-ctrl, amiR-SHN3, or amiR-SOST / SHN3 (2.5 x 1012vg / kg) via PL injection to the mandibular first molar. Two days later, AAV-treated mandibles were stimulated daily with HFV for ten days. Seven weeks later, mice were treated with calcein and alizarin red via intraperitoneal injection at six-day intervals and then euthanized. FIG. 25D shows representative calcein / alizarin red labeling images and relative histomorphometric quantification of dentin deposition rates in the mandibular incisor are displayed (FIG. 25E). Ordinary one-way ANOVA 12075767.1 with Dunnett’s multiple comparisons test (FIG. 25E, FIG. 25F; data represent mean ± SD). Representative images of five replicates are displayed (FIG. 25D). FIG. 26 shows a comparison between constitutive and vibration-inducible silencing efficiency of Shn3 in alveolar bone using the CBA and pLEF-1 / TCF promoter, respectively. 2- month-old mice were treated with dss.rAAV9.pCBA carrying amiR-ctrl or amiR-SHN3 or dss.rAAV9.pLef / Tcf carrying amiR-SHN3 (2.5 x 1012vg / kg) via PL injection into the mandibular first molar and two days later, the mandible was stimulated daily with HFV treatment for 10 days. Egfp, Shn3 and Alp mRNA expression was measured by RT-PCR (n = 5– 7 / group). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (data represent mean ± SD). DETAILED DESCRIPTION Aspects of the disclosure relate to compositions and methods for modulating bone growth, for example by increasing osteogenesis and / or decreasing osteoclastogenesis. The disclosure is based, in part, on recombinant adeno-associated virus (rAAV) vectors encoding microRNAs or miRNA inhibitors that inhibit expression or activity of gene products in the WNT pathway, for example Schnurri-3 (SHN3), sclerostin (SOST), and / or dickkopf-1 (DKK-1). In some embodiments, compositions described by the disclosure are useful for treating certain bone diseases, disorders, or injuries, such as alveolar bone loss and bone fractures. Accordingly, in some aspects, the disclosure provides a method for treating alveolar bone loss in a subject in need thereof, the method comprising administering to the jaw of a subject in need thereof an isolated nucleic acid comprising a nucleotide sequence encoding an inhibitor of a gene selected from Schnurri-3 (SHN3), sclerostin (SOST), and dickkopf-1 (DKK- 1). Isolated Nucleic Acids Compositions and methods for delivering a transgene (e.g. an inhibitory RNA, such as an shRNA, miRNA, etc.) to a subject are provided in the disclosure. The compositions typically comprise an isolated nucleic acid encoding a transgene (e.g., a protein, an inhibitory nucleic acid, etc.) capable of modulating bone metabolism. For example, in some embodiments, a transgene reduces expression of a target protein, such as a target protein associated with promoting or inhibiting bone formation. 12075767.1 “Bone metabolism” generally refers to a biological process involving bone formation and / or bone resorption. In some embodiments, bone metabolism involves the formation of new bone as produced by osteoblasts (OBs) and differentiated osteocytes, and / or mature bone tissue being resorbed by osteoclasts (OCs). OBs arise from the bone marrow derived mesenchymal cells that ultimately differentiate terminally into osteocytes. OB (and osteocyte) functions or activities include but are not limited to bone formation, bone mineralization, and regulation of OC activity. Decreased bone mass has been observed to result from inhibition of OB and / or osteocyte function or activity. Increased bone mass has been observed to result from increased OB and / or osteocyte function or activity. OCs arise from bone marrow-derived monocytes and in some embodiments have been observed to be controlled by signals from OBs. OC functions include bone resorption. In some embodiments, decreased bone mass has been observed to result from increased OC activity. In some embodiments, increased bone mass has been observed to result from inhibition of OC activity. In some embodiments, an isolated nucleic acid or an rAAV as described by the disclosure comprises a transgene encoding at least one bone metabolism modulating agent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more bone metabolism modulating agents). As used herein, a “bone metabolism modulating agent” refers to a molecule (a nucleic acid or protein encoded by a nucleic acid, e.g., a transgene) that either induces or inhibits bone formation or deposition, for example by increasing or decreasing expression, activity, and / or function of proteins, cells, etc., that are involved in bone formation or bone resorption. Generally, a bone metabolism modulating agent can be a peptide, protein, or an interfering nucleic acid (e.g., dsRNA, siRNA, shRNA, miRNA, artificial miRNA, etc.). In some embodiments, a bone metabolism modulating agent is a bone formation inducing agent. In some embodiments, a bone metabolism modulating agent is a bone formation inhibiting agent. A “bone formation inducing agent” refers to a molecule that promotes bone synthesis either by promoting OB and / or osteocyte (OCY) differentiation or activity and / or by inhibiting OC activity. In some embodiments, a bone formation inducing agent is a nucleic acid (e.g., RNAi oligonucleotide or miRNA oligonucleotide or antisense oligonucleotide) or protein encoded by a nucleic acid (e.g., a transgene) that promotes OB and / or osteocyte function or activity (e.g., bone formation, mineralization, regulation of osteoclast activity or function, etc.). In some embodiments, a bone formation inducing agent is an inhibitory nucleic acid that inhibits 12075767.1 OC differentiation or activity, such as an inhibitory nucleic acid that targets sclerostin (SOST), schnurri-3 (SHN3), dickkopf-1 (DKK-1), etc. A “bone formation inhibiting agent” refers to a molecule that inhibits OB and / or osteocyte differentiation or activity and / or increases OC differentiation , activity or function. In some embodiments, a bone formation inhibiting agent is a nucleic acid (e.g., RNAi oligonucleotide or miRNA oligonucleotide or antisense oligonucleotide) or protein encoded by a nucleic acid (e.g., a transgene) that inhibits OB and / or osteocyte differentiation or activity. In some embodiments, examples of bone formation inhibiting agents that inhibit OB and / or osteocyte activity or function include but are not limited to a MAPK inhibitor, and pro- inflammatory cytokines (e.g., tumor necrosis factor alpha (TNF-α), etc. In some embodiments, a bone formation inhibiting agent is an inhibitory nucleic acid that inhibits OB and / or osteocyte differentiation or activity. In some embodiments, an isolated nucleic acid encodes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inhibitory nucleic acids, for example dsRNA, siRNA, shRNA, miRNA, artificial microRNA (ami-RNA), etc.). Generally, an inhibitory nucleic acid specifically binds to (e.g., hybridizes with) at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) continuous bases of a gene encoding a gene product (e.g., a protein) associated with bone metabolism (e.g., SOST, SHN3, DKK-1, etc.). As used herein “continuous bases” refers to two or more nucleotide bases that are covalently bound (e.g., by one or more phosphodiester bond, etc.) to each other (e.g. as part of a nucleic acid molecule). In some embodiments, the at least one inhibitory nucleic acid is about 50%, about 60% about 70% about 80% about 90%, about 95%, about 99% or about 100% identical to the two or more ( e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) continuous nucleotide bases of a gene encoding a gene product (e.g., a protein) associated with bone metabolism (e.g., SOST, SHN3, DKK-1, etc.). A “microRNA” or “miRNA” is a small non-coding RNA molecule capable of mediating transcriptional or post-translational gene silencing. Typically, miRNA is transcribed as a hairpin or stem-loop (e.g., having a self-complementarity, single-stranded backbone) duplex structure, referred to as a primary miRNA (pri-miRNA), which is enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) into a pre-miRNA. The length of a pri-miRNA can vary. In some embodiments, a pri-miRNA ranges from about 100 to about 5000 base pairs (e.g., about 100, about 200, about 500, about 1000, about 1200, about 1500, about 1800, or about 2000 base 12075767.1 pairs) in length. In some embodiments, a pri-miRNA is greater than 200 base pairs in length (e.g., 2500, 5000, 7000, 9000, or more base pairs in length. Pre-miRNA, which is also characterized by a hairpin or stem-loop duplex structure, can also vary in length. In some embodiments, pre-miRNA ranges in size from about 40 base pairs in length to about 500 base pairs in length. In some embodiments, pre-miRNA ranges in size from about 50 to 100 base pairs in length. In some embodiments, pre-miRNA ranges in size from about 50 to about 90 base pairs in length (e.g., about 50, about 52, about 54, about 56, about 58, about 60, about 62, about 64, about 66, about 68, about 70, about 72, about 74, about 76, about 78, about 80, about 82, about 84, about 86, about 88, or about 90 base pairs in length). Generally, pre-miRNA is exported into the cytoplasm, and enzymatically processed by Dicer to first produce an imperfect miRNA / miRNA* duplex and then a single-stranded mature miRNA molecule, which is subsequently loaded into the RNA-induced silencing complex (RISC). Typically, a mature miRNA molecule ranges in size from about 19 to about 30 base pairs in length. In some embodiments, a mature miRNA molecule is about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or 30 base pairs in length. In some aspects, the disclosure provides isolated nucleic acids and vectors (e.g., rAAV vectors) that encode one or more artificial miRNAs. As used herein “artificial miRNA” or “amiRNA” refers to an endogenous pri-miRNA or pre-miRNA (e.g., a miRNA backbone, which is a precursor miRNA capable of producing a functional mature miRNA), in which the miRNA and miRNA* (e.g., passenger strand of the miRNA duplex) sequences have been replaced with corresponding amiRNA / amiRNA* sequences that direct highly efficient RNA silencing of the targeted gene, for example as described by Eamens et al. (2014), Methods Mol. Biol. 1062:211- 224. For example, in some embodiments an artificial miRNA comprises a miR-155 pri-miRNA backbone into which a sequence encoding a bone metabolism modulating (e.g., bone formation inhibiting agent) miRNA has been inserted in place of the endogenous miR-155 mature miRNA- encoding sequence. In some embodiments, miRNA (e.g., an artificial miRNA) as described by the disclosure comprises a miR-155 backbone sequence, a miR-30 backbone sequence, a mir-64 backbone sequence, or a miR-122 backbone sequence. In some embodiments an artificial miRNA comprises a miR-33 pri-miRNA backbone into which a sequence encoding a bone metabolism modulating (e.g., bone formation inhibiting agent) miRNA has been inserted in place of the endogenous miR-33 mature miRNA-encoding sequence. In some embodiments, 12075767.1 miRNA (e.g., an artificial miRNA) as described by the disclosure comprises a miR-33 backbone sequence. In some embodiments, the present disclosure provides an isolated nucleic acid comprising a transgene encoding an artificial microRNA targeting the SHN3 gene (GeneID: 59269), which encodes the Schnurri-3 protein. The Schnurri-3 (SHN3) protein is a transcription factor that regulates NK-κβ protein expression and immunoglobulin and T-cell receptor antibody recombination. In some embodiments, the SHN3 gene is represented by the NCBI Accession Number NM_001127714.2 or NM_024503.5. In some embodiments, the SHN3 protein is represented by the NCBI Accession Number NP_001121186.1 or NP_078779.2. In some embodiments, an artificial microRNA targets (e.g., binds to, or comprises a region of complementarity with) at least 6 continuous nucleotides of a SHN3 gene. In some embodiments, an artificial microRNA targets (e.g., binds to, or comprises a region of complementarity with) between 6 and 30 continuous nucleotides of a SHN3 gene. In some embodiments, an artificial microRNA targets between 12-24 continuous nucleotides of a SHN3 gene. In some embodiments, an artificial microRNA targets between 9-27 continuous nucleotides of the SHN3 gene. In some embodiments, an artificial microRNA targets at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 continuous nucleotides of a SHN3 gene. In some embodiments, the present disclosure provides an isolated nucleic acid comprising a transgene encoding an artificial microRNA targeting the SOST gene (GeneID: 50964), which encodes the sclerostin protein. The sclerostin (SOST) protein is a secreted glycoprotein with a C-terminal cysteine knot-like (CTCK) domain and sequence similarity to the DAN (differential screening-selected gene aberrative in neuroblastoma) family of bone morphogenetic protein (BMP) antagonists. In some embodiments, the SOST gene is represented by the NCBI Accession Number NM_025237.3. In some embodiments, the SOST protein is represented by the NCBI Accession Number NP_079513.1. In some embodiments, an artificial microRNA targets (e.g., binds to, or comprises a region of complementarity with) at least 6 continuous nucleotides of a SOST gene. In some embodiments, an artificial microRNA targets (e.g., binds to, or comprises a region of complementarity with) between 6 and 30 continuous nucleotides of a SOST gene. In some embodiments, an artificial microRNA targets between 12-24 continuous nucleotides of a SOST gene. In some embodiments, an artificial microRNA targets between 9-27 continuous 12075767.1 nucleotides of the SOST gene. In some embodiments, an artificial microRNA targets at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 continuous nucleotides of a SOST gene. In some embodiments, the present disclosure provides an isolated nucleic acid comprising a transgene encoding an artificial microRNA targeting the DKK-1 gene (GeneID: 22943), which encodes the dikkopf WNT signaling pathway inhibitor 1 (DKK-1) protein. The DKK-1 protein is a secreted protein that binds to the LRP6 co-receptor and inhibits beta-catenin- dependent Wnt signaling. In some embodiments, the DKK-1 gene is represented by the NCBI Accession Number NM_012242.4. In some embodiments, the DKK-1 protein is represented by the NCBI Accession Number NP_036374.1. In some embodiments, an artificial microRNA targets (e.g., binds to, or comprises a region of complementarity with) at least 6 continuous nucleotides of a DKK-1 gene. In some embodiments, an artificial microRNA targets (e.g., binds to, or comprises a region of complementarity with) between 6 and 30 continuous nucleotides of a DKK-1 gene. In some embodiments, an artificial microRNA targets between 12-24 continuous nucleotides of a DKK-1 gene. In some embodiments, an artificial microRNA targets between 9-27 continuous nucleotides of the DKK-1 gene. In some embodiments, an artificial microRNA targets at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 continuous nucleotides of a DKK-1 gene. In some embodiments, an artificial microRNA is between 6-50 nucleotides in length. In some embodiments, an artificial microRNA is between 8-24 nucleotides in length. In some embodiments, an artificial microRNA is between 12-36 nucleotides in length. In some embodiments, an artificial microRNA is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, an isolated inhibitory nucleic acid decreases expression of a target gene by between 50% and 99% (e.g., any integer between 50% and 99%, inclusive). In some aspects, an isolated inhibitory nucleic acid decreases expression of a target gene by between 75% and 90%. In some aspects, an isolated inhibitory nucleic acid decreases expression of a target gene by between 80% and 99%. In some embodiments, an isolated inhibitory nucleic acid decreases expression of a SOST, SHN3, or DKK-1 gene by between 50% and 99% (e.g., any integer between 50% and 99%, inclusive). In some embodiments, an isolated inhibitory nucleic 12075767.1 acid decreases expression of a SOST, SHN3, or DKK-1 gene by between 75% and 90%. In some aspects, an isolated inhibitory nucleic acid decreases expression of a SOST, SHN3, or DKK-1 gene by between 80% and 99%. A region comprising a transgene (e.g., a second region, third region, fourth region, etc.) may be positioned at any suitable location of the isolated nucleic acid. The region may be positioned in any untranslated portion of the nucleic acid, including, for example, an intron, a 5’ or 3’ untranslated region, etc. In some cases, it may be desirable to position the region (e.g., the second region, third region, fourth region, etc.) upstream of the first codon of a nucleic acid sequence encoding a protein (e.g., a protein coding sequence). For example, the region may be positioned between the first codon of a protein coding sequence) and 2000 nucleotides upstream of the first codon. The region may be positioned between the first codon of a protein coding sequence and 1000 nucleotides upstream of the first codon. The region may be positioned between the first codon of a protein coding sequence and 500 nucleotides upstream of the first codon. The region may be positioned between the first codon of a protein coding sequence and 250 nucleotides upstream of the first codon. The region may be positioned between the first codon of a protein coding sequence and 150 nucleotides upstream of the first codon. In some cases (e.g., when a transgene lacks a protein coding sequence), it may be desirable to position the region (e.g., the second region, third region, fourth region, etc.) upstream of the poly-A tail of a transgene. For example, the region may be positioned between the first base of the poly-A tail and 2000 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 1000 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 500 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 250 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 150 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 100 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 50 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 20 nucleotides upstream of the first base. In some embodiments, the region is positioned between the last nucleotide base of a promoter sequence and the first nucleotide base of a poly-A tail sequence. 12075767.1 In some cases, the region may be positioned downstream of the last base of the poly-A tail of a transgene. The region may be between the last base of the poly-A tail and a position 2000 nucleotides downstream of the last base. The region may be between the last base of the poly-A tail and a position 1000 nucleotides downstream of the last base. The region may be between the last base of the poly-A tail and a position 500 nucleotides downstream of the last base. The region may be between the last base of the poly-A tail and a position 250 nucleotides downstream of the last base. The region may be between the last base of the poly-A tail and a position 150 nucleotides downstream of the last base. It should be appreciated that in cases where a transgene encodes more than one miRNA, each miRNA may be positioned in any suitable location within the transgene. For example, a nucleic acid encoding a first miRNA may be positioned in an intron of the transgene and a nucleic acid sequence encoding a second miRNA may be positioned in another untranslated region (e.g., between the last codon of a protein coding sequence and the first base of the poly-A tail of the transgene). In some embodiments, the transgene further comprises a nucleic acid sequence encoding one or more expression control sequences (e.g., a promoter, etc.). Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. For nucleic acids encoding proteins, a polyadenylation sequence generally is inserted following the transgene sequences and before the 3' AAV ITR sequence. A rAAV construct useful in the present disclosure may also contain an intron, desirably located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from 12075767.1 SV-40, and is referred to as the SV-40 T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES sequence would be used to produce a protein that contain more than one polypeptide chains. Selection of these and other common vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al., and references cited therein at, for example, pages 3.183.26 and 16.1716.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, a Foot and Mouth Disease Virus 2A sequence is included in polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11: 1921-1931.; and Klump, H et al., Gene Therapy, 2001; 8: 811-817). Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter [Invitrogen]. In some embodiments, a promoter is an enhanced chicken β-actin promoter. In some embodiments, a promoter is a U6 promoter. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible 12075767.1 sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only. Aspects of the disclosure relate to isolated nucleic acid and rAAVs comprising inducible promoters. In some embodiments the inducible promoter is responsive to inflammation in a subject. Examples of inflammation-responsive promoters include but are not limited to promoters containing endogenous BMP-responsive elements (BRE, EndolD1-BRE) and NF-kB- binding sites (pNF-kB), for example as set forth in SEQ ID NOs: 17 and 18. In some embodiments, an inducible promoter is responsive to mechanical stress, for example vibration (e.g., high frequency vibration, HFV). In some embodiments, a promoter responsive to mechanical stress comprises sequences derived from WNT signaling pathway genes such as WNT-responsive genes, Tcf and Lef-1. In some embodiments, a mechanical stress inducible promoter comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, a tissue-specific promoter is a bone tissue-specific promoter. Examples of bone tissue-specific promoters include but are not limited to promoters of osterix, osteocalcin, type 1 collagen α1, DMP1, cathepsin K, Rank, etc. Aspects of the disclosure relate to an isolated nucleic acid comprising more than one promoter (e.g., 2, 3, 4, 5, or more promoters). For example, in the context of a construct having a transgene comprising a first region encoding a protein and an second region encoding an inhibitory RNA (e.g., miRNA), it may be desirable to drive expression of the protein coding region using a first promoter sequence (e.g., a first promoter sequence operably linked to the protein coding region), and to drive expression of the inhibitory RNA encoding region with a second promoter sequence (e.g., a second promoter sequence operably linked to the inhibitory RNA encoding region). Generally, the first promoter sequence and the second promoter 12075767.1 sequence can be the same promoter sequence or different promoter sequences. In some embodiments, the first promoter sequence (e.g., the promoter driving expression of the protein coding region) is an RNA polymerase III (polIII) promoter sequence. Non-limiting examples of polIII promoter sequences include U6 and H1 promoter sequences. In some embodiments, the second promoter sequence (e.g., the promoter sequence driving expression of the inhibitory RNA) is an RNA polymerase II (polII) promoter sequence. Non-limiting examples of polII promoter sequences include T7, T3, SP6, RSV, and cytomegalovirus promoter sequences. In some embodiments, a polIII promoter sequence drives expression of an inhibitory RNA (e.g., miRNA) encoding region. In some embodiments, a polII promoter sequence drives expression of a protein coding region. Following systemic delivery, rAAV vectors can target additional tissues such as liver, lungs, heart, and skeletal muscle, which may cause adverse effects. The skilled artisan will appreciate that binding sites may be selected to control the expression of a transgene in a tissue specific manner. Therefore, the present disclosure provides tissue-specific, endogenous miRNAs to repress transgene expression in liver (e.g., miR-122) and / or heart (e.g., miR-208a), by engineering perfectly complementary miRNA-binding sites into the AAV vector genome. In some embodiments the rAAV comprises at least one tissue specific endogenous miRNA. In some embodiments, the tissue specific endogenous miRNA is a miR-122. In some embodiments, the tissue specific endogenous miRNA is a miR-208a. The target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region. Typically, the target site is in the 3’ UTR of the mRNA. Furthermore, the transgene may be designed such that multiple miRNAs regulate the mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites may result in the cooperative action of multiple RISCs and provide highly efficient inhibition of expression. The target site sequence may comprise a total of 5-100, 10-60, or more nucleotides. The target site sequence may comprise at least 5 nucleotides of the sequence of a target gene binding site. Recombinant AAVs (rAAVs) The isolated nucleic acids of the disclosure may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). In some embodiments, an isolated nucleic acid as described by the disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof. The isolated nucleic acid (e.g., the 12075767.1 recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and / or delivered to a selected target cell. “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may comprise, as disclosed elsewhere herein, one or more regions that encode one or more proteins and / or inhibitory nucleic acids (e.g., shRNA, miRNAs, etc.) comprising a nucleic acid that targets an endogenous mRNA of a subject. The transgene may also comprise a region encoding, for example, a protein and / or an expression control sequence (e.g., a poly-A tail), as described elsewhere in the disclosure. Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520532 (1996)). An example of such a molecule employed in the present invention is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, the isolated nucleic acid (e.g., the rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAV2 / 2-66, AAV2 / 2-84, AAV2 / 2-125, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region (e.g., a first region) encoding an AAV2 ITR. In some embodiments, the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR. In some embodiments, the second AAV ITR has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAV2 / 2-66, AAV2 / 2-84, AAV2 / 2-125, and variants thereof. In some embodiments, the second ITR is a mutant ITR that lacks a functional terminal resolution site (TRS). The term “lacking a terminal resolution site” can refer to an AAV ITR that comprises a mutation (e.g., a sense mutation such as a non-synonymous mutation, or missense mutation) that abrogates the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., a ΔTRS ITR). Without wishing to be bound by 12075767.1 any particular theory, a rAAV vector comprising an ITR lacking a functional TRS produces a self-complementary rAAV vector, for example as described by McCarthy (2008) Molecular Therapy 16(10):1648-1656. As used herein, the term “self-complementary AAV vector” (scAAV) refers to a vector containing a double-stranded vector genome generated by the absence of a terminal resolution site (TR) from one of the ITRs of the AAV. The absence of a TR prevents the initiation of replication at the vector terminus where the TR is not present. In general, scAAV vectors generate single-stranded, inverted repeat genomes, with a wild-type (wt) AAV TR at each end and a mutated TR (mTR) in the middle. The instant invention is based, in part, on the recognition that DNA fragments encoding RNA hairpin structures (e.g. shRNA, miRNA, and AmiRNA) can serve a function similar to a mutant inverted terminal repeat (mTR) during viral genome replication, generating self-complementary AAV vector genomes. For example, in some embodiments, the disclosure provides rAAV (e.g. self-complementary AAV; scAAV) vectors comprising a single-stranded self-complementary nucleic acid with inverted terminal repeats (ITRs) at each of two ends and a central portion comprising a promoter operably linked with a sequence encoding a hairpin-forming RNA (e.g., shRNA, miRNA, ami-RNA, etc.). In some embodiments, the sequence encoding a hairpin-forming RNA (e.g., shRNA, miRNA, ami- RNA, etc.) is substituted at a position of the self-complementary nucleic acid normally occupied by a mutant ITR. “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and / or expression in a cell of a target tissue. The instant disclosure provides a vector comprising a single, cis-acting wild-type ITR. In some embodiments, the ITR is a 5’ ITR. In some embodiments, the ITR is a 3’ ITR Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITR(s) is used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify ITR sequences is within 12075767.1 the skill of the art. (See, e.g., texts such as Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520532 (1996)). For example, an ITR may be mutated at its terminal resolution site (TR), which inhibits replication at the vector terminus where the TR has been mutated and results in the formation of a self-complementary AAV. Another example of such a molecule employed in the present disclosure is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' AAV ITR sequence and a 3’ hairpin-forming RNA sequence. AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, an ITR sequence is an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, and / or AAVrh10 ITR sequence. In some embodiments, the rAAVs of the disclosure are pseudotyped rAAVs. For example, a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with the proteins of Y will be designated as AAVX / Y (e.g. AAV2 / 1 has the ITRs of AAV2 and the capsid of AAV1). In some embodiments, pseudotyped rAAVs may be useful for combining the tissue-specific targeting capabilities of a capsid protein from one AAV serotype with the viral DNA from another AAV serotype, thereby allowing targeted delivery of a transgene to a target tissue. Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003 / 0138772), the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins. In some embodiments, capsid proteins are structural proteins encoded by the cap gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact 12075767.1 with the host. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner. In some embodiments, an AAV capsid protein is of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAVrh10, AAVrh39, and AAVrh43. In some embodiments, an AAV capsid protein is of a serotype derived from a non-human primate, for example scAAV.rh8, AAV.rh39, or AAV.rh43 serotype. In some embodiments, an AAV capsid protein is of an AAV9 serotype (e.g., as set forth in SEQ ID NO: 20). In some embodiments, an AAV capsid protein is of an AAV6 serotype (e.g., as set forth in SEQ ID NO: 19). The disclosure is based, in part, on rAAVs comprising capsid proteins that have increased tropism for bone tissue. In some embodiments, the capsid proteins are grafted to a bone-targeting peptide. A heterologous bone-targeting peptide may target OCs (e.g., specifically, or preferentially targets OCs relative to OBs) or OBs (e.g., specifically, or preferentially targets OBs relative to OCs). In some embodiments, a bone-targeting peptide is an (AspSerSer)6 peptide, which may also be referred to as a DSS6 peptide (e.g. SEQ ID NO: 21). Further examples of bone-targeting peptides include but are not limited to those described by Ouyang et al. (2009) Lett. Organic Chem 6(4):272-277. As used herein, “grafting” refers to joining or uniting of one molecule with another molecule. In some embodiments, the term grafting refers to joining or uniting of at least two molecules such that one of the at least two molecules is inserted within another of at least two molecules. In some embodiments, the term grafting refers to joining or uniting of at least two polymeric molecules such that one of at least two molecules is appended to another of at least two molecules. In some embodiments, the term grafting refers to joining or uniting of one polymeric molecule (e.g., a nucleic acid, a polypeptide) with another polymeric molecule (e.g., a nucleic acid, a polypeptide). In some embodiments, the term grafting refers to joining or uniting of at least two nucleic acid molecules such that one of at least two molecules is appended to another of at least two nucleic acid molecules. In some embodiments, the term grafting refers to joining or uniting of at least two nucleic acid molecules such that one of the at least two nucleic acid molecules is inserted within another of the at least two nucleic acid molecules. For example, it has been observed that targeting peptides may be grafted to certain loci of a nucleic acid encoding a VP2 AAV capsid protein. In some embodiments, a targeting peptide (e.g. a bone-targeting peptide) is inserted at a 12075767.1 position corresponding to the position between the codons encoding Q588 and A589 and / or N587 and R588 of an AAV2 or AAV9 VP2 capsid protein. In some embodiments, a targeting peptide is inserted at a position between the codons encoding N587 and R588 of an VP3 capsid protein (or a position corresponding to such amino acid positions in AAV2 or AAV9). In some embodiments, a targeting peptide is inserted at a position between the codons encoding S452 and G453 of an VP1 capsid protein. Other potential positions may be N587 and R588. In some embodiments, a nucleic acid formed through grafting (a grafted nucleic acid) encodes a chimeric protein. In some embodiments, a grafted nucleic acid encodes a chimeric protein, such that one polypeptide is effectively inserted into another polypeptide (e.g. not directly conjugated before the N-terminus or after the C-terminus), thereby creating a contiguous fusion of two polypeptides. In some embodiments, a grafted nucleic acid encodes a chimeric protein, such that one polypeptide is effectively appended to another polypeptide (e.g. directly conjugated before the N-terminus or after the C-terminus), thereby creating a contiguous fusion of two polypeptides. In some embodiments, the term grafting refers to joining or uniting of at least two polypeptides, or fragments thereof, such that one of the at least two polypeptides or fragments thereof is inserted within another of the at least two polypeptides or fragments thereof. In some embodiments, the term grafting refers to joining or uniting of at least two polypeptides or fragments thereof such that one of the at least two polypeptides or fragments thereof is appended to another of the at least two polypeptides or fragments thereof. In some embodiments, the disclosure relates to an adeno-associated virus (AAV) capsid protein that is conjugated to one or more bone-targeting moieties. A “bone-targeting moiety” generally refers to a small molecule, peptide, nucleic acid, etc., that facilitates trafficking of an rAAV to bone or bone tissue. For example, in some embodiments, a bone-targeting moiety is a peptide or small molecule that binds to a receptor on a bone cell (e.g., OB, OC, osteocyte, etc.). Examples of bone-targeting moieties include but are not limited to alendronate (ALE), polypeptides such as cyclic arginine-glycine-aspartic acid-tyrosine-lysine (cRGCyk), Asp-Asp- Asp-Asp-Asp-Asp-Asp-Asp (D-Asp8), and aptamers such as CH6. A bone-targeting moiety may be conjugated directly to a capsid protein or conjugated to a capsid protein via a linker molecule (e.g., an amino acid linker, a PEG linker, etc.). In some embodiments, a linker is a glycine-rich linker. In some embodiments, a linker comprises at least two glycine residues. In some embodiments, a linker comprises GGGGS. In some embodiments, the linker comprises a formula selected from the group consisting of: [G]n, 12075767.1 [G]nS, [GS]n, and [GGSG]n, wherein G is glycine and wherein n is an integer greater than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, n is an integer in a range of 2 to 10, 2 to 20, 5 to 10, 5 to 15, or 5 to 25. Accordingly, in some embodiments, a heterologous targeting peptide is conjugated to a linker. In some embodiments, a capsid protein comprises one or more azide-bearing unnatural amino acids which are capable of reacting with an ADIBO-tagged bone-targeting moiety (e.g., via “click chemistry” to form a capsid protein-bone-targeting moiety conjugate. Capsid proteins comprising unnatural azide-bearing amino acids are described, for example by Zhang et al. (2016) Biomaterials 80:134-145, and use of ADIBO-based click chemistry for peptide conjugation is described, for example by Prim et al. (2013) Molecules 18(8):9833-49. The components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contain the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art. The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold 12075767.1 Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745. In some embodiments, recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the "AAV helper function" sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the entirety of both incorporated by reference herein. The accessory function vector encodes nucleotide sequences for non-AAV derived viral and / or cellular functions upon which AAV is dependent for replication (i.e., "accessory functions"). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus. In some aspects, the disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells. A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. In some embodiments, a host cell is a bacterial 12075767.1 cell, yeast cell, insect cell (Sf9), or a mammalian (e.g., human, rodent, non-human primate, etc.) cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants. As used herein, the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced. In some aspects, the present disclosure provides a recombinant AAV comprising a capsid protein and an isolated nucleic acid comprising a first region encoding an AAV ITR and a second region comprising a transgene, wherein the transgene encodes an artificial microRNA. The artificial microRNA may decrease the expression of a target gene in a cell (e.g. osteoblasts, osteoclasts, osteocytes, chondrocytes) or a subject. In some embodiments, the rAAV comprises an artificial microRNA that decreases the expression of SHN3, SOST, and / or DKK-1 in a cell or a subject. The foregoing methods for packaging recombinant vectors in desired AAV capsids to produce the rAAVs of the disclosure are not meant to be limiting and other suitable methods will be apparent to the skilled artisan. Modes of Administration and Compositions 12075767.1 The rAAVs of the disclosure may be delivered to a subject in compositions according to any appropriate methods known in the art. For example, an rAAV, preferably suspended in a physiologically compatible carrier (e.g., in a composition), may be administered to a subject, e.g., host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque). In some embodiments a host animal does not include a human. Delivery of the rAAVs to a mammalian subject may be by, for example, intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In some embodiments, the rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially facilitating the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer the virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. Moreover, in certain instances, it may be desirable to deliver the virions to the bone (e.g., bone tissue) of a subject. By “bone tissue” is meant all cells and tissue of the bone and / or joint (e.g., cartilage, axial and appendicular bone, etc.) of a vertebrate. Thus, the term includes, but is not limited to, osteoblasts, osteocytes, osteoclasts, chondrocytes, and the like. Recombinant AAVs may be delivered directly to the bone by injection into, e.g., directly into the bone, via intrasynovial injection, knee injection, femoral intramedullary injection, etc., with a needle, catheter or related device, using surgical techniques known in the art. In some embodiments, rAAV as described in the disclosure are administered by intravenous injection. In some embodiments, the rAAV are administered by intramuscular injection. Aspects of the instant disclosure relate to compositions comprising a recombinant AAV comprising a capsid protein and a nucleic acid encoding a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more bone metabolism modulating agents. In some embodiments, the nucleic acid further comprises one or more AAV ITRs. In some embodiments, the rAAV comprises an rAAV vector comprising the sequence set forth in any one of SEQ ID NO: 1-14 (or the complementary sequence thereof), or a portion thereof. In some embodiments, a composition further comprises a pharmaceutically acceptable carrier. In some embodiments, an rAAV vector encoding one or more miRNAs and / or one or more miRNA 12075767.1 binding sites does not comprise a reporter protein (e.g., nucleic acid sequence encoding luciferase, EGFP, etc.). In some embodiments, an rAAV vector lacks protein coding nucleic acid sequences. In some embodiments, compositions comprise a recombinant AAV comprising a capsid protein and a nucleic acid comprising a first region encoding an AAV ITR and a second region comprising a transgene, wherein the transgene encodes one or more miRNAs that inhibit SHN3, SOST, and / or DKK-1. In some embodiments, the recombinant AAV comprises a sequence as set forth in any one of SEQ ID NO: 1-14. In some embodiments, the capsid protein is an AAV9 capsid protein or AAV6 capsid protein. In some embodiments, the capsid protein further comprises a heterologous bone-targeting peptide. Aspects of the disclosure provide a method of decreasing target gene (e.g., SHN3, SOST, and / or DKK-1) expression or activity in a cell. A cell may be a single cell or a population of cells (e.g., culture). A cell may be in vivo (e.g., in a subject) or in vitro (e.g., in culture). A subject may be a mammal, optionally a human, a mouse, a rat, a non-human primate, a pig, a dog, a cat, a chicken, or a cow. Expression or activity of the target gene(s) in a cell or subject may be decreased by between 50% and 99% (e.g., any integer between 50% and 99%, inclusive) using isolated nucleic acids, rAAVs, or compositions of the present disclosure. Expression or activity of target gene(s) in a cell or subject may be decreased by between 75% and 90% using isolated nucleic acids, rAAVs, or compositions of the present disclosure. Expression or activity of target gene(s) in a cell or subject may be decreased by between 80% and 99% using isolated nucleic acids, rAAVs, or compositions of the present disclosure. The compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure. 12075767.1 Optionally, the compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin. The rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired. The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., the units of dose in genome copies / per kilogram of body weight (GC / kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art. An “effective amount” of an rAAV is an amount sufficient to target infect an animal, target a desired tissue (e.g., bone tissue). The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 109to 1016genome copies. In some cases, a dosage between about 1011to 1013rAAV genome copies is appropriate. In certain embodiments, 1012or 1013rAAV genome copies is effective to target bone tissue. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose of rAAV is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than bi- weekly (e.g., once in a two-calendar week period). In some embodiments, a dose of rAAV is 12075767.1 administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than once per six calendar months. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year). In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ~1013GC / ml or more). Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171–178, the contents of which are incorporated herein by reference.) Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. In certain circumstances it will be desirable to deliver the rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intraopancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, femoral intramedullary, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver rAAVs. In some embodiments, a preferred mode of administration is by direct injection into the jaw of a subject. In some embodiments, a preferred mode of administration is by direct administration to a site of a bone fracture of a subject. 12075767.1 The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host. Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating 12075767.1 the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein. The formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). 12075767.1 Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587). Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 µm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å, containing an aqueous solution in the core. Alternatively, nanocapsule formulations of the rAAV may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 µm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use. In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the rAAV compositions to a host. Sonophoresis (i.e., ultrasound) has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback- controlled delivery (U.S. Pat. No. 5,697,899). Aspects of the disclosure relate to the surprising discovery that combining administration of isolated nucleic acids and rAAVs as described herein directly to the jaw or bone fracture site of a subject with high frequency vibration (HFV) results in improved gene expression and promotion of bone growth in the subject. Administration of HFV to subjects is known, for 12075767.1 example as described by Shipley et al., J Orthod Sci. 2019; 8: 15, the entire contents of which are incorporated herein by reference in their entirety. Therapeutic Methods Methods for delivering an effective amount of a transgene (e.g., a nucleic acid or rAAV encoding one or more miRNAs as described herein) to a subject are provided by the disclosure. In some embodiments, the methods comprise the step of administering to a subject an effective amount of an isolated nucleic acid encoding an interfering RNA capable of inhibiting bone loss (e.g., bone loss due to bone fracture, osteoporosis). In some embodiments, the methods comprise the step of administering to a subject an effective amount of an isolated nucleic acid encoding an interfering RNA capable of reversing bone loss. Thus, in some embodiments, isolated nucleic acids, rAAVs, and compositions described herein are useful for treating a subject having or suspected of having a disease or disorder associated with bone loss. As used herein, a “disease or disorder associated with dysregulated bone metabolism” refers to a condition characterized by an imbalance between bone deposition and bone resorption resulting in either 1) abnormally increased bone deposition (e.g., formation) relative to a healthy individual (e.g., a subject not having a disease characterized by imbalance between bone deposition and bone resorption), or 2) abnormally decreased bone deposition (e.g., formation) relative to a healthy individual (e.g., a subject not having a disease characterized by an imbalance between bone deposition and bone resorption), or 3) abnormally increased bone resorption (e.g., breakdown) relative to a healthy individual (e.g., a subject not having a disease characterized by imbalance between bone deposition and bone resorption), or 4) abnormally decreased bone resorption (e.g., breakdown) relative to a healthy individual (e.g., a subject not having a disease characterized by imbalance between bone deposition and bone resorption). A “disease associated with reduced bone density” refers to a condition characterized by increased bone porosity resulting from either 1) abnormally decreased bone deposition (e.g., formation) relative to a healthy individual (e.g., a subject not having a disease characterized by decreased bone density), or 2) abnormally increased bone resorption (e.g., breakdown) relative to a healthy individual (e.g., a subject not having a disease characterized by decreased bone density). A disease associated with increased bone porosity may arise from either 1) abnormally decreased OB and / or osteocyte differentiation, function, or activity relative to a healthy individual (e.g., a subject not having a disease characterized by decreased bone density) and / or 12075767.1 2) abnormally increased OC differentiation , function, or activity relative to a healthy individual (e.g., a subject not having a disease characterized by decreased bone density). “Porosity” generally refers to the volume of fraction of bone not occupied by bone tissue. A “disease associated with increased bone density” refers to a condition characterized by decreased bone porosity resulting from either 1) abnormally increased bone deposition (e.g., formation) relative to a healthy individual (e.g., a subject not having a disease characterized by increased bone density), or 2) abnormally decreased bone resorption (e.g., breakdown) relative to a healthy individual (e.g., a subject not having a disease characterized by increased bone density). A disease associated with decreased bone porosity may arise from either 1) abnormally increased OB and / or osteocyte differentiation, function, or activity relative to a healthy individual (e.g., a subject not having a disease characterized by increased bone density) and / or 2) abnormally decreased OC differentiation, function, or activity relative to a healthy individual (e.g., a subject not having a disease characterized by increased bone density). Aspects of the present disclosure provide methods of treating a disease or disorder associated with dysregulated bone metabolism. Dysregulated bone metabolism may be diseases associated with reduced bone density (e.g., osteoporosis, critical sized-bone defects, a mechanical disorder resulting from disuse or injury). Dysregulated bone metabolism may be diseases associated with increased bone density (e.g., osteopetrosis, pycnodysostosis, sclerosteosis, acromegaly, fluorosis, myelofibrosis, hepatitis C-associated osteosclerosis, heterotrophic ossification). In some embodiments, the disease is alveolar bone loss (e.g., bone loss in the jaw of a subject). In some embodiments, the alveolar bone loss is caused by osteoporosis in the subject. In some embodiments, administering the nucleic acid, the rAAV, the vector, the bone graft substitute improves bone formation and / or bone healing in a subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, administering the nucleic acid, the rAAV, the vector, the bone graft substitute stimulate bone regeneration and / or reversing bone loss in a subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5- fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. 12075767.1 As used herein, the improvement or stimulation is relative to a control. The control can be in a state that is prior to the administration of the isolated nucleic acid, the rAAV, the vector, and the bone graft substitute. The improvement or stimulation is relative to a subject that has not been administered the isolated nucleic acid, the rAAV, the vector, and the bone graft substitute. As used herein, a “normal, healthy subject” refers to a subject who does not have, is not suspected of, or is at risk of developing a disease or disorder. In some embodiments, the disease or disorder is an inflammatory disease. In some embodiments, the disease or disorder is associated with bone metabolism. In some embodiments, a normal, healthy subject can be a control described herein. As used herein, the term “treating” refers to the application or administration of a composition, isolated nucleic acid, vector, or rAAV as described herein to a subject having bone loss or a predisposition toward a bone loss condition, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the inflammatory condition. "Development" or "progression" of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of inflammatory diseases includes initial onset and / or recurrence. In some embodiments, methods of treating osteoporosis comprise administering to a subject in need thereof a recombinant AAV (rAAV) comprising a transgene. A rAAV may comprise a modification that promotes its targeting to bone cells (e.g., osteoclasts and osteoblasts). Non-limiting modifications of rAAVs that promote its targeting to bone cells include modification of capsid proteins with heterologous bone-targeting peptides, modification of rAAV vectors with bone-specific promoters, and use of AAV serotypes with increased targeting to bone relative to other tissues. In some embodiments, an “effective amount” or “amount effective of a substance” in the context of a composition or dose for administration to a subject refers to an amount sufficient to produce one or more desired effects (e.g., to preserve bone tissue or reverse bone loss). In some embodiments, an effective amount of a nucleic acid is an amount sufficient to transfect (or infect 12075767.1 in the context of rAAV-mediated delivery) a sufficient number of target cells of a target tissue of a subject. In some embodiments, a target tissue is bone tissue (e.g., bone and bone tissue cells, such as OBs, OCs, osteocytes, chondrocytes, etc.). In some embodiments, an effective amount of a nucleic acid (e.g., which may be delivered via an rAAV) may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to increase activity or function of OBs and / or osteocytes, to inhibit activity of OBs and / or osteocytes, to increase activity of function of OCs, to inhibit activity or function of OCs, etc. In some embodiments, an effective amount of an isolated nucleic acid disclosed herein may partially or fully rescue bone losses. In some embodiments, an effective amount of an isolated nucleic acid disclosed herein may partially or fully alleviate the effects of the genes that cause bone losses. An effective amount can also involve delaying the occurrence of an undesired response. The effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, the severity of a condition, the tissue to be targeted, the specific route of administration and like factors, and may thus vary among subject and tissue as described elsewhere in the disclosure. Exemplary embodiments of the invention will be described in more detail by the following examples. These embodiments are exemplary of the invention, which one skilled in the art will recognize is not limited to the exemplary embodiments. EXAMPLES Example 1 Alveolar bone is the portion of the mandibular and maxillary bones that not only supports the tooth sockets and muscles of mastication but also protects nerves, vessels, and glands. It undergoes rapid bone remodeling during tooth eruption due to the forces of mastication and positional adaptation of the teeth. Normal alveolar bone remodeling is controlled by bone-forming osteoblasts and bone-resorbing osteoclasts, but disruption of that balance leads to alveolar bone atrophy. Osteoporosis frequently develops atrophic alveolar bone and hampers proper chewing and speaking, heightens vulnerability to gum disease, and disrupts tooth alignment associated with facial deformity. Additionally, reduced alveolar bone density makes dental implant procedures challenging, as they require sufficient bone mass to achieve dental implantation. Therefore, preserving alveolar bone health is crucial for maintaining optimal oral health, function, and aesthetics. 12075767.1 Restoration of alveolar bone loss in osteoporosis poses a significant challenge in dental practice. Current treatments include anti-inflammatory agents and the transplantation of natural or synthetic bone grafts and resorbable or non-resorbable collagen membranes along with supplementary bioactive products. Additionally, osteoporosis drugs, such as bisphosphonates, anti-RANKL antibody, parathyroid hormone analogues, and estrogen replacement, are used for treatment. However, these treatments show limited success in the clinic due to a lack of long- term preservation, rehabilitation, and functional needs of alveolar bone. Moreover, while oral administration of bisphosphonates do not show significant improvement in preventing alveolar bone loss, they can cause oral bony lesions such as bisphosphonate-related osteonecrosis of the jaw. Surgical transplantation often fails and frequently leads to complications. For example, vertical bone augmentation is hindered by brittleness and inadequate toughness in newly formed bones, resulting in frequent fractures during the implantation process. The use of slow-resorbing bone biomaterials also can compromise the quantity and quality of newly formed bone in augmentation sites. Materials and Methods Cell culture and reagents: Human bone marrow-derived mesenchymal stromal cells (BMSCs, #7500) were purchased from ScienCell Research Laboratories and cultured according to the manufacturer’s manuals. Human Periodontal Ligament Stem Cells (PDLSCs, 36085-01) were purchased from Celprogen and cultured in the Human Periodontal Ligament Stem Cells Complete Growth Media with Serum (M36085-01S) for maintenance or cultured in the Human Periodontal Ligament Stem Cell Culture Differentiation Media with Serum (M36085-01DS) for osteogenic differentiation. Mouse alveolar bone-marrow-derived MSCs were isolated from 1- month-old Shn3Pdgfrαand Shn3fl / flmandibles as described previously. Briefly, mandibular bones were collected and processed by removing attached soft tissues and teeth, including molars and incisors. Cells were then obtained using a digestion solution that contains collagenase type I (Worthington Biochem, LS004196) and dispase II (Roche,10165859001). The resulting cell suspensions were filtered (40 µm) and washed using PBS and maintained in α-MEM medium (Gibco). Mouse COBs were isolated from the calvaria of wildtype neonates at postnatal day 3 (C57BL / 6J). Cells were maintained in α-MEM medium (Gibco) containing 10% FBS (Corning), 2 mM L-glutamine (Corning), 1% penicillin / streptomycin (Corning), and 1% nonessential amino acids (Corning) while they were differentiated into mature osteoblasts under osteogenic 12075767.1 medium containing ascorbic acid (200 µM, Sigma, #A8960) and β-glycerophosphate (10 mM, Sigma, #G9422). Recombinant WNT3a was purchased from R&D systems (#1324-WN). rAAV vector design and production: Bone-targeting AAV9 capsid (rAAV9.DSS) was generated as described in previous studies. Mouse or human miR-33 scaffold design rules were applied to generate optimized artificial miRNA (amiR) cassettes and amiR target sequences were designed using a custom Excel macro. Plasmids were constructed by Gibson assembly and standard molecular biology methods. Validated DNA sequences for amiR-ctrl, amiR-SHN3, hs- amiR-SHN3, and amiR-SOST / SHN3 were synthesized as gBlocksTM, cloned into the intronic region of the pAAVsc-CB6-Egfp plasmid at the restriction enzyme sites (PstI and BglII). To generate a liver and heart-detargeting AAV (dss.AAV9.egfp.MIR-TS), endogenous complementary sequences for miR-122 and miR-208a were synthesized as gBlocksTMand inserted into the 3′ untranslated region (UTR) of the egfp reporter gene in the pAAVsc-CB6- Egfp plasmid and packaged into the dss.AAV9 capsid. To generate a mechanical stress- inducible AAV (dss.AAV9.pLef / Tcf-egfp), DNA sequences for the TCF / LEF promoter (pTcf / Lef) adapted from the M50 super 8x TopFlash reporter gene (Addgene, #12456) were synthesized as gBlocksTMand then replaced the CB6 promoter of the pAAVsc-CB6-Egfp plasmid. Constructs were verified by sequencing and then packaged into the dss.AAV9 capsid. rAAV production was executed by transient transfection in HEK293 cells, purified by CsCl sedimentation, and quantified by droplet digital PCR (ddPCR) on a QX200 ddPCR system (Bio- Rad) using the Egfp or mCherry prime / probe set. Quantitative RT-PCR analysis: Total RNA was purified from cells using QIAzol (QIAGEN) and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems. Quantitative RT-PCR was performed using SYBR® Green PCR Master Mix (Bio-Rad) with the CFX connect RT-PCR detection system (Bio-Rad). To measure Shn3 mRNA levels in bone tissues, after removal of bone marrow, soft tissue and teeth, including molars and incisors, mandibles or tibias were snap-frozen in liquid nitrogen for 30 s and in turn, homogenized in 1 ml of QIAzol for 1 min. Alternatively, a TaqMan microRNA assay kit (Applied Biosystems) was used to measure the expression of miR-122 and miR-208a. miRNAs were isolated from the mandible, liver, or heart using the mirVana miRNA isolation kit (Ambion), followed by cDNA synthesis using the TaqMan miRNA reverse transcription kit 12075767.1 (Applied Biosystems). The cDNA was used for RT-PCR using a TaqMan miRNA assay kit (Applied Biosystems) according to the manufacturer’s protocol: miR-122-5p (assay ID: 002245), miR-208a-3p (assay ID: 000511). Human subjects and analysis: Four de-identified alveolar bone samples were obtained from young (20–25-year-old) and aged (older than 60-year-old) patients. Total RNAs were extracted from alveolar bone areas and mRNA expression of SOST, DKK1, and SHN3 was examined by RT-PCR. Mice: Mice were housed in a constant environment (up to 5 mice per cage), ambient temperature of 21 ± 2 °C, circulating air, and constant humidity of 50 ± 10%, in a 12 h light, 12 h dark cycle. Mice were given a standard mouse chow diet and monitored every three days for the amount of their food and water intake and signs of distress. For signs of severe distress, including general malaise, severe cachexia, or more than 20% loss of body weight, humane euthanasia was performed in consultation with veterinary staff. Mice were euthanized in a carbon dioxide chamber, followed by cervical dislocation. Neonates were euthanized by decapitation. Cre deleter mice (C57BL / 6J) that express Cre recombinase under the control of the Prrx1 promoter (Prx1-cre), the Pdgfrα promoter (Pdgfrα-cre), and the Dmp1 promoter (Dmp1- cre) were purchased from Jackson Laboratory. Shn3− / −and Shn3fl / flmice were previously generated and maintained on a BALB / c and C57BL / 6J background, respectively. Wild-type (C57BL / 6J) mice, Pdgfrα-GFP, and Rosa26mT / mGreporter mice, severe combined immunodeficiency (SCID) mice, and TCF / Lef1-HIST1H2BB / EGFP reporter mice were purchased from The Jackson Laboratory. Mouse genotypes were determined by PCR on tail genomic DNA; primer sequences are available upon request. High-frequency vibration (HFV): A portable high-frequency wand was applied to the mandible of both cheeks to produce high-frequency mechanical vibration in alveolar bone, teeth, and periodontal tissues within the mandible. All HFV treatment was applied to cheek skin around the buccal side of the mandible molar for 30 seconds per day, according to the experiment’s purpose and timeline. The portable high-frequency wand delivers a micro- vibration frequency at 200Hz with 10,000 / minute vibration. Working voltage & power: 1.5V, 0.1W. 12075767.1 MicroCT and radiography: MicroCT (µCT35; SCANCO Medical AG; Bruttisellen, Switzerland) was used for qualitative and quantitative assessment of bone mass and microarchitecture of alveolar bones and femurs, which was performed by an investigator blinded to the genotypes of the animals under analysis. MicroCT scanning was performed at 55 kVp and 114 mA energy intensity with 300-ms integration time. A specific voxel size 12 µm was used to measure the mandibular body, molars, and incisors. All images were reconstructed using image matrices of 1024 × 1024 pixels. For trabecular bone analysis of the distal femur, an upper 2.1 mm region beginning 280 μm proximal to the growth plate was contoured. Additionally, a mid-shaft region of 0.6 mm in length was used to analyze cortical bone. For mandibular bone analysis, the area between the mesial and distal root of the first molar was measured volumetrically through 100 serial images (120 μm span). Specifically, contour lines were drawn between the mesial and distal root of the mandibular first molar to define the alveolar bone region of interest (ROI) in micro-CT 2D sagittal sections: Shn3- / -, Shn3Prx1, Shn3Pdgfrα, Shn3Dmp1, wildtype mice, and AAV-treated OVX mice (100 serial images) or 20-month-old mice (140 serial images). For the assessment of mineral density and thickness of root dentin or cementum, 40 slices centered on the cut-through of the mesial root in the first molar were measured. The first slice in the sequence was below the pulp chamber when two roots separate to form two distinct channels. The last slice was at the apex of the tooth. For crown dentin and enamel, molar enamel and dentin volume and density were calculated and measured from the cementum- enamel junction to the highest cusp tip. Customized filtering and thresholding were applied to quantify the targeted area within the volume of interest (VOI). For the assessment of enamel mass, the Inventors set the fixed value of 1000 as a high threshold and increased the low threshold up to 900 to highlight dense segmentation within VOI. For the assessment of dentin mass, the Inventors set the fixed value of 900 as a high threshold and increased the low threshold up to 320 to highlight dense segmentation within VOI. 3D reconstruction images were obtained from contoured 2D images by methods based on the distance transformation of the binarized images. Alternatively, the Inveon multimodality 3D visualization program was used to generate fused 3D viewing of multiple static or dynamic volumes of microCT modalities (Siemens Medical Solutions USA, Inc.). All images presented are representative of the respective genotypes (n > 5). The Trident Specimen Radiography system (Hologic, USA) was used to generate detailed radiographic images of the whole mouse body after euthanasia. The X- 12075767.1 ray beam intensity was 1 mA 28–30 kV with AEC (automatic exposure control) for consistent image acquisition. Histology and histomorphometry: For histological analysis, alveolar bones were dissected from the mice, fixed in 10% neutral buffered formalin for 2 days, and decalcified by 14% tetrasodium EDTA for 2–4 weeks. Tissues were dehydrated by passage through an ethanol series, cleared twice in xylene, embedded in paraffin, and sectioned at a thickness of 6 μm along the coronal plate from anterior to posterior. Decalcified femoral sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, or Tartrate-Resistant Acid Phosphatase (TRAP). For dynamic histomorphometric analysis, 25 mg / kg calcein (Sigma, C0875) and 50 mg / kg alizarin-3-methyliminodiacetic acid (Sigma, A3882) dissolved in 2% sodium bicarbonate solution were subcutaneously injected into mice at 6-day intervals. After fixing in 10% neutral buffered formalin for two days, undecalcified mandibular bone samples were embedded in methyl methacrylate, and alveolar bone was sectioned longitudinally (5 μm) and stained with McNeal’s trichrome for osteoid assessment and TRAP for osteoclasts. A region of interest is defined under the mandibular first molar in the metaphysis and BFR / bone surface (BS), MAR, BS, Ob.S / BS, and osteoclast surface (Oc.S / BS) are measured using the Osteometrics. Measurements were taken on two sections / sample (separated by ~25 μm) and summed prior to normalization to obtain a single measure / sample in accordance with ASBMR standards. This methodology has undergone extensive quality control and validation, and the results were assessed by two different researchers in a blinded fashion. MAR was calculated by measuring the distance between the calcein-labeled bone and the alizarin red-labeled bone; this measurement was made at defined sites around the mandibular incisors in 5 separate mice. The same analyses were performed on the lower incisor for dentin apposition rate. Immunohistochemistry and immunofluorescence: For immunohistochemistry analysis, paraffin sections were dewaxed and stained according to the manufacturer’s directions, using the Discovery XT automated IHC stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). CC1 standard buffer (pH 8.4 buffer containing Tris / Borate / EDTA) and inhibitor D (3% H2O2, Endogenous peroxidase) were used for antigen retrieval and blocking, respectively. Sections were incubated with antibodies specific to SHN3 (PA5-52194, Thermo Fisher, 1:200), SOST (AF1589-SP, R&D systems, 1:100) or DKK1 (21112-1-AP, Proteintech, 1:100) for 40 min at 12075767.1 37 °C, and a secondary antibody for 20 min at 37 °C. Subsequently, they were incubated in SA- HRP D for 16 min at 37 °C and then DAB + H2O2 substrate for 8 min, followed by hematoxylin and bluing reagent counterstain at 37 °C. Reaction buffer (pH 7.6 Tris buffer) was used as a washing solution. Stained samples were visualized using an Aperio virtual microscope (Leica Microsystems, USA), and images of the sample were analyzed by the Aperio image scope program (ver. 12.3.2.8013, Leica Microsystems, USA). For immunofluorescence analysis, fresh alveolar bone dissected from Shn3- / -, Shn3Prx1, Shn3Pdgfrα, Shn3Dmp1, and wildtype mice and rAAV-treated mice were collected and immediately fixed in ice-cold 4% paraformaldehyde solution for 2 days. Semi-decalcification was carried out for 5 days in 0.5 M EDTA pH 7.4 at 4 °C with constant shaking (age ≥ 1 week), and infiltration was followed with a mixture of 20% sucrose phosphate buffer for 1 day and with 25% sucrose phosphate buffer the next day. All samples were embedded in a 50 / 50 mixture of 25% sucrose solution and OCT compound (Sakura) and cut into 12-μm-thick sagittal sections using a cryostat (Leica). Immunofluorescence staining and analysis was performed as described previously. Briefly, after treatment with 0.2% Triton X-100 for 10 min, sections were blocked with 5% donkey serum at room temperature for 30 min and incubated overnight at 4 °C with anti-b-CATENIN antibody (Thermo Fisher, PA5-77934, 1:100). Primary antibodies were visualized with donkey anti-rat IgG Alexa-594 (1:500, Molecular Probes). Nuclei were counterstained with 4-6,diamidino-2- phenylindole (DAPI). An Olympus IX81 confocal microscope or Leica TCS SP5 II Zeiss LSM- 880 confocal microscope was used to image samples. Scanned Electron Microscopy (SEM): Mandibular bones were isolated from one-month-old Shn3Pdgfraand Shn3fl / flmice. They were fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde in cacodylate buffer (pH7.2) overnight a 4℃ and dehydrated through an ethanol gradient (70%–100%) and 100% Acetone, followed by embedding in Spur’s epoxy resin and polymerized at 68℃ of 48 hours. The embedded mandibles were sectioned at the first molar using a slow-speed diamond saw and finally polished using Pol Metal Polish DKK100 (Pol, Denmark). The polished surfaces were coated with 8 nm of Gold-Palladium and imaged using a ThermoFisher, Quanta 200 FESEM (Hillsborough, OR) 10 Kv accelerating voltage. AAV treatment in a mouse model of postmenopausal osteoporosis: Mouse models of postmenopausal osteoporosis were generated by anesthetizing and bilaterally ovariectomizing 12075767.1 (OVX) 3-month-old female mice (Jackson Laboratory, C57BL / 6J). Four weeks after the surgery, sham or OVX mice were IV (2.5 x 1013vector genomes [vg] / kg) or PL (2.5 x 1012vg / kg) injected with rAAV9 or dss.rAAV9 carrying egfp, amiR-ctrl, amiR-SHN3, or amiR- SOST / hs-amiR-SHN3. Seven weeks after the injection, mice were subcutaneously injected with calcein and alizarin-3-methyliminodiacetic acid at six-day intervals for dynamic histomorphometric analysis. Non-labeled mice were used to monitor EGFP expression using the IVIS-100 optical imaging on frozen sections. AAV treatment of human skeletal organoid in xenograft mice: Human BMSCs were seeded on hydroxyapatite (HA)-scaffold (Osteogene Tech), cultured under osteogenic conditions for two days, and implanted into the interscapular fat pads of 3-month-old immunodeficient SCID mice. One week later, rAAV9 (2.5 x 1012vg / kg) carrying hs-amiR-ctrl or hs-amiR-hSHN3 was injected into the implantation site (FIG. 3D). Four weeks later, the implanted HA-scaffold was visualized by radiography and then, EGFP expression in the scaffold was assessed by the IVIS optical imaging system. Bone and collagen formation was assessed by microCT, histology, and SEM analyses. SHN3 deletion increases alveolar bone and tooth dentin mass The WNT pathway is a key regulator of bone formation by enhancing osteoblast development, and WNT antagonists, such as sclerostin (SOST), dickkopf-1 (DKK-1), and schnurri-3 (SHN3), are promising targets to promote bone formation in osteoporosis. While SOST and DKK-1 proteins are all present in the alveolar bone in young mice (FIG. 1A), mRNA expression of SOST and DKK-1 was substantially decreased in older human mandibles relative to young mandibles (FIG.1B). Accordingly, treatment of aged osteoporotic rats with anti-SOST antibody or anti-DKK-1 antibodies showed minimal effects on alveolar bone regeneration. SHN3 expression was also detected in the alveolar bone of young mice (FIG. 1C), but unlike SOST and DKK1, its expression was further upregulated in human and mouse aged mandibles (FIG. 1D). Similarly, postmenopausal osteoporosis in female mice also increased SHN3 expression in mandibles (FIG. 1E). SHN3 controls β-Catenin stability downstream of WNT signaling and that deleting SHN3 enhances WNT signaling, osteoblast development, and bone formation. SHN3 plays a role in alveolar bone development by regulating WNT signaling. To explore potential regulatory 12075767.1 interactions between SHN3 and WNT signaling within the alveolar bone, an immunohistochemistry (IHC) analysis was performed, demonstrating co-localization of SHN3 and β-Catenin in multiple cell populations within alveolar bone, including periodontal ligament cells (PDL), odontoblasts (OD), osteoblasts (OB), and osteocytes (OCY, FIG.1C). MicroCT analysis of alveolar bone demonstrated greater bone volume per tissue volume (BV / TV) and trabecular bone thickness (Tb.Th) in 1-month-old Shn3- / -mice than littermate controls (FIG.1F). This is consistent with a histologic analysis showing an increase in trabecular bone mass in the bone marrow of Shn3- / -mandibles (FIG. 1G). 7-month-old Shn3- / -mice also showed an increase in alveolar bone mass, but this increase was slightly compromised as trabecular bone mass became saturated (FIG. 1H). These results indicate that SHN3 acts as a key regulator of alveolar bone mass during skeletal development and homeostasis. SHN3 expression in dentin-producing odontoblasts within mandibles prompted testing of its potential role in dental development. MicroCT analysis of the mandibular first molar demonstrated a significant increase in root dentin volume in 1-month-old Shn3- / -mice relative to littermate controls while enamel volume, produced by dental ameloblasts, was comparable between Shn3- / -and control mice (FIGs. 1I and 1J). Notably, the increase in root dentin volume of Shn3- / -mice was halted as dentin production was saturated at age of 7 months (FIG. 1K). These results indicate that SHN3 in odontoblasts functions as a suppressor of dentin production at early developmental stages while its role in ameloblasts is dispensable for enamel production. Thus, SHN3 is crucial for alveolar bone and tooth dentin formation, which makes SHN3 an attractive therapeutic target to promote regeneration of alveolar bone and tooth dentin. Osteoblast-specific deletion of SHN3 increases alveolar bone and tooth dentin mass Platelet-derived growth factor receptors (PDGFRs) have been reported to express in a broad range of mesenchyme-lineage cells at multiple anatomical locations in the skeleton. Fluorescence microscopy of Pdgfrα-GFP reporter mice demonstrated GFP expression in periodontal ligament cells, osteoblast-lineage cells, and dental-lineage cells within mandibles (FIG. 2A). Shn3 was conditionally deleted in these cells by crossing mice with a Shn3-floxed allele with Pdgfrα-Cre mice (Shn3Pdgfrα). Reduced expression of SHN3 was confirmed in alveolar bone-derived mesenchymal stromal cells (ABMSCs) isolated from Shn3Pdgfrαmandibles (FIG. 2B). These cells showed an increase in expression of osteogenic marker genes, including osteocalcin (Bglap) and dentin matrix acidic phosphoprotein 1 (Dmp1, FIG. 2B), and 12075767.1 extracellular mineralization (alizarin red staining, FIG. 2C), indicating enhanced osteogenic activity. As seen in Shn3- / -mice, alveolar bone mass was markedly increased in Shn3Pdgfrαmice relative to littermate controls (Shn3fl / fl) at ages 1 and 7 months old, as shown by increased BV / TV and Tb.Th. This is consistent with a histologic analysis showing an increase in trabecular bone mass in the bone marrow of Shn3Pdgfrαmandibles (FIGs. 2D and 2E). Similarly, trabecular bone mass and cortical thickness in the long bones were both markedly increased in these mice (FIG. 8A). These results highlight the role of SHN3 in Pdgfrα+cells as a key regulator of alveolar and long bone mass during skeletal development and homeostasis. Since Pdgfrα-GFP expression was also detected in odontoblasts within mandibles (Figure 2A), the tooth dentin volume in the first molar of Shn3Pdgfrαmandibles was assessed using microCT. Similar to Shn3- / -mandibles, 1-month-old Shn3Pdgfrαmandibles displayed a significant increase in root dentin volume without any alteration in enamel volume and the increased root dentin volume was halted as dentin production was saturated at age of 7 months (FIG. 2F). Notably, Masson’s trichrome staining and scanning electron microscopy (SEM) of Shn3Pdgfrαtooth showed normal dentin matrix and tubule structure, showing that SHN3 deletion in Pdgfrα+cells increases the production of healthy dentin (FIG. 2G). Next, SHN3 expression was deleted at early and late stages of osteoblast differentiation by crossing Shn3fl / flmice with Prx1-Cre mice (Shn3Prx1) and Dmp1-Cre mice (Shn3Dmp1), respectively. Prx1-Cre mice and Dmp1-Cre mice were also crossed with Rosa26mT / mGreporter mice where Cre-recombinase uses GFP expression to visualize Cre-expressing cells within mandibles. Unlike Prx1-GFP proteins primarily expressed in osteoblasts on the alveolar bone surface, Dmp1-GFP expression was detected in osteocytes embedded in the alveolar bone matrix. Prx1- and Dmp1-GFP proteins were both expressed in odontoblasts on the dentin surface (FIG. 2H). Deletion of Shn3 in Prx1+skeletal progenitors and Dmp1+osteocytes resulted in a significant increase in alveolar bone mass (FIG. 2I) and femoral bone mass (FIGs. 8B and 8C). These results show that SHN3 acts in both early and late stages of osteoblast differentiation to produce alveolar bone and long bone. 1-month-old Shn3Prx1mandibles displayed an increase in root dentin volume of the first molar without any change in enamel volume (FIG. 2J). However, there was little to no increase in root dentin volume in Shn3Dmp1molar (FIGs. 2J and 2K), showing that SHN3 in skeletal progenitors, but not in osteocytes, drives dentin production 12075767.1 during skeletal development. Thus, inhibition of SHN3 in skeletal progenitors promotes formation of long bone, alveolar bone, and tooth dentin. AAV-mediated silencing of human SHN3 promotes bone formation in xenograft mice To test whether SHN3-deficiency in human osteoblasts can promote bone formation, human artificial miRNA (hs-amiR) was developed by embedding the guide strand of a small silencing RNA that targets human SHN3 into a human miR-33-derived miRNA scaffold (hs- amiR-hSHN3) and then packaged into the AAV9 capsid. In this design, the hs-amiR is inserted intronically between the chicken β-actin (CBA) promoter and the Egfp reporter gene (FIG. 3A), which allows for visual tracking of positively transduced cells or tissues. The AAV9’s ability to transduce human periodontal ligament stromal cells (PDLSCs) or bone marrow-derived mesenchymal stromal cells (BMSCs) was confirmed by GFP expression using fluorescence microscopy (FIG. 3B). rAAV9 carrying hs-amiR-hSHN3 effectively silenced SHN3 expression in these cells and increased osteogenic differentiation (FIG. 3C). Next, whether AAV-mediated silencing of human SHN3 promotes bone formation in vivo was examined using a xenograft mouse model implanted with a human skeletal organoid (FIG. 3D). The human skeletal organoid was generated by culturing human BMSCs in hydroxyapatite (HA)-scaffold under osteogenic conditions and then implanted into interscapular fat pads of immunodeficient mice (FIG. 3E). A single dose of rAAV9 carrying hs-amiR-ctrl or hs-amiR-hSHN3 was injected to the implanted site. Four weeks later, AAV’s ability to transduce the human skeletal organoid and to knockdown Shn3 expression were confirmed by GFP expression using the IVIS optical imaging system (FIG. 3F) and RT-PCR analysis (FIG. 3G), respectively. Compared to hs-amiR-ctrl, hs- amiR-hSHN3-treatment markedly increased osteogenic gene expression, osteocalcin (BGLAP) and type 1 collagen (COL1), and WNT-induced gene expression, AXIN2 (FIG. 3G). Likewise, this organoid showed a significant increase in mineral density distribution (microCT) and newly formed collagen and bone (Masson’s trichrome staining, FIGs. 3H-3J). This is consistent with SEM showing increased production of collagen fibers and calcified matrix in the human skeletal organoid at molecular levels (FIG. 3K). Thus, AAV-mediated silencing of SHN3 is effective in transducing human osteoblast lineage cells, enhancing osteogenic differentiation and WNT signaling and promoting bone formation in human skeletal organoids. AAV-mediated silencing of Shn3 reverses alveolar bone loss in osteoporosis 12075767.1 rAAV9’s ability to transduce alveolar bone was examined using GFP expression after intravenous (IV) injection or periodontal ligament (PL) injection to the mandibular first molar (FIGs. 9A, top and 9B). Individual organ imaging of IV-injected mice showed robust GFP expression in liver and muscle and a modest expression in the mandible. By contrast, PL- injected mice showed a modest expression in the liver and mandible with little to no expression in muscle, brain, heart, lung, spleen, and kidney (FIG. 4A). This is consistent with RT-PCR analysis and fluorescence microscopy on cryo-sectioned tissues showing a significant decrease in GFP expression in the liver, heart, and muscle when treated via PL injection relative to IV injection (FIGs. 4B and 9C). However, GFP expression in the PL-injected mandibles was increased 3-fold (FIG. 4C) when rAAV9 was transduced into periodontal ligament cells, odontoblasts, and osteoblast-lineage cells (FIG. 9D). Thus, PL injection of rAAV9 is an effective gene delivery strategy to transduce alveolar bone while limiting AAV’s expression in other tissues. To further improve the bone-specific tropism of the rAAV9 capsid, a bone-homing peptide motif (AspSerSer)6 was grafted onto an AAV9-VP2 capsid protein (dss.rAAV9). 8- week-old Pdgfrα-GFP reporter mice were treated with mCherry-expressing dss.rAAV9 (dss.rAAV9.mCherry) via PL injection into the mandibular first molar and mCherry expression in Pdgfrα-GFP+cells was assessed by fluorescence microscopy. Fluorescence microscopy showed mCherry expression in a subset of Pdgfrα+cells in alveolar bone, including periodontal ligament cells, osteoblast-lineage cells, and odontoblasts (FIG. 4D). Thus, PL injection of dss.rAAV9 effectively transduces alveolar Pdgfrα+bone-residing cells. Postmenopausal osteoporosis results in alveolar bone loss and deterioration of bone structure, increasing the risk of tooth loss, gum disease susceptibility, and facial deformity. This process can be modeled in mice; ovariectomy (OVX) surgery in female mice leads to estrogen deficiency-induced bone loss, recapitulating postmenopausal osteoporosis. To test whether AAV-mediated silencing of Shn3 can reverse alveolar bone loss in postmenopausal osteoporosis, an AAV-compatible artificial miRNA that silences mouse Shn3 expression by embedding Shn3-targeting sequences into a mouse miR-33-derived miRNA scaffold (amiR- SHN3, FIG. 9A, bottom) was examined. Sham control or OVX surgery was performed in 12- week-old female mice and four weeks later, a single dose of dss.rAAV9 carrying amiR-ctrl or amiR-SHN3 was administered via IV or PL injection (FIG. 4E). Eight weeks after injection, knockdown efficiency of Shn3 in AAV-treated OVX mandibles was confirmed by RT-PCR 12075767.1 (FIG. 4F). While amiR-ctrl-treated OVX mice showed a significant reduction in alveolar bone mass compared to sham control mice, this bone loss was reversed by both IV- and PL-injection of amiR-SHN3, as shown by increased trabecular BV / TV, thickness, and number and by decreased trabecular space (FIGs. 4G and 4H). This is consistent with a histological analysis showing a reversal of trabecular bone loss in the bone marrow of amiR-SHN3-treated mandible with OVX surgery (FIG. 4G, bottom). PL-injection of dss.rAAV9.amiR-SHN3 showed greater increase in alveolar bone mass than IV injection, demonstrating that local delivery is more effective for AAV transduction and alveolar bone formation than systemic delivery. Notably, unlike genetic deletion of Shn3, amiR-SHN3-treatment did not increase root dentin volume (FIG. 4I). This discrepancy may result from AAV’s low silencing efficiency in odontoblasts or AAV injection in older mice that may be too late to promote tooth dentin production. Next, AAV’s ability to restore alveolar bone loss in aging-associated osteoporosis was examined. 20-month-old male mice were treated with dss.rAAV9 carrying amiR-ctrl or amiR- SHN3 via PL injection into the mandibular first molar. Two months later, knockdown efficiency of Shn3 in AAV-treated mandibles was confirmed by RT-PCR (FIG. 4J). Compared to amiR- ctrl-treated mice, amiR-SHN3-treated mice showed a significant increase in alveolar bone mass, as shown by greater BV / TV and Tb.Th (FIGs. 4K and 4L). These results demonstrated that bone-targeting AAV-mediated silencing of Shn3 via PL injection into mandibles is effective in restoring alveolar bone loss in both aging-associated and postmenopausal osteoporosis. Taken together, the approach of local delivery of a SHN3 silencer to mandibles via a bone-targeted AAV may successfully treat alveolar bone loss in osteoporosis. Liver / heart-“detargeting” AAV-mediated silencing of Shn3 reverses osteoporosis Newly developed agents to treat bone loss, such as anti-sclerostin antibody and the small molecule inhibitor of Cathepsin K, have off-target cardiovascular and cerebrovascular events in clinical trials, respectively. Thus, AAV’s expression in non-skeletal tissues should be suppressed. Since dss.rAAV9 can transduce liver and heart, AAV’s expression in liver and heart was suppressed using liver- and heart-specific miRNA-mediated degradation. The specificity of miR-122 and miR-208a expression in liver and heart was validated by RT-PCR (FIG. 10A). Three tandem complementary sites for liver-specific miR-122 and heart-specific miR-208a were inserted into the 3′ untranslated region (UTR) of the egfp reporter gene in the vector genome and then, packaged into dss.rAAV9 capsid (dss.rAAV9.egfp.MIR-TS; FIG. 5A, top). 8-week-old 12075767.1 mice were IV injected with PBS, dss.rAAV9.egfp, or dss.rAAV9.egfp.MIR-TS and the tissue distribution of AAVs was assessed by EGFP expression using the IVIS optical imaging system (FIG. 5B) and fluorescence microscopy on cryo-sectioned tissues (FIG. 10B). dss.rAAV9.egfp- treated mice showed robust GFP expression in liver, but the expression in dss.rAAV9.egfp.MIR- TS-treated liver was markedly reduced. This is consistent with RT-PCR analysis showing a significant decrease in GFP expression in heart and liver treated with dss.rAAV9.egfp.MIR-TS relative to dss.rAAV9.egfp while the expression was comparable between tibia and muscle (FIG. 5C). These results indicated that systemic delivery of dss.rAAV9.egfp.MIR-TS facilitated GFP expression in the bone, but not in heart, liver, lung, kidney, spleen, and brain. To examine whether systemic delivery of dss.rAAV9.amiR-Shn3.MIR-TS can reverse femoral bone loss in postmenopausal osteoporosis, a single dose of dss.rAAV9 carrying amiR- ctrl, amiR-SHN3 or amiR-SHN3.MIR-TS was IV injected into 12-week-old female mice four weeks after sham control or OVX surgery. Eight weeks later, knockdown efficiency of Shn3 in AAV-treated OVX tibia was confirmed (FIG. 5D). While amiR-ctrl-treated OVX mice showed significant reductions in femoral bone mass compared to sham control mice, this bone loss was reversed by both amiR-SHN3 and amiR-SHN3.MIR-TS, as shown by increased trabecular BV / TV, thickness, and number and decreased trabecular space (FIGs. 5E and 5F). Thus, miR- 122- and miR-208a-mediated repression effectively “detargets” AAV expression in liver and heart without affecting anabolic bone increase by amiR-SHN3. Next, EGFP expression in the alveolar bone treated with dss.rAAV9.egfp and dss.rAAV9.egfp.MIR-TS via local delivery was compared. 8-week-old mice were treated with dss.rAAV9.egfp or dss.rAAV9.egfp.MIR-TS via PL injection into the mandibular first molar. Ten days later, EGFP expression in the mandible was assessed by IVIS optical imaging, RT- PCR, and fluorescence microscopy (FIGs. 5G and 5H), demonstrating equivalent expression levels in AAV-treated mandibles. Similar to dss.rAAV9.egfp, dss.rAAV9.egfp.MIR-TS effectively transduced a subset of periodontal ligament cells and osteoblast-lineage cells within alveolar bone. Thus, PL injection of dss.rAAV9.MIR-TS to the mandible may be a safe and effective strategy to target alveolar bone. To compare the therapeutic efficacy of dss.rAAV9.amiR-SHN3 vs. dss.rAAV9.amiR- SHN3.MIR-TS in osteoporotic alveolar bone, 12-week-old female mice were treated with the AAVs via PL injection to the mandibular first molar four weeks after sham control or OVX surgery (FIG. 10C). Eight weeks later, knockdown efficiency of Shn3 in AAV-treated OVX 12075767.1 mandible was confirmed by RT-PCR (FIG. 5I). While amiR-ctrl-treated OVX mice showed a significant reduction in alveolar bone mass compared to sham control mice, this bone loss was reversed by both amiR-SHN3.MIR-TS and amiR-SHN3, as shown by increased trabecular BV / TV and thickness (FIGs. 5J and 5K). Thus, PL injection of dss.rAAV9.amiR-SHN3.MIR-TS effectively restores alveolar bone loss in postmenopausal osteoporosis while securing AAV’s expression in mandibles. Vibration-inducible expression of bone-targeted AAV in mandibles Alveolar bone continuously receives mechanical stimuli, such as chewing, biting, and speaking, which is crucial to maintain alveolar bone mass. Given that WNT signaling is a major mechano-sensing pathway in osteoblast lineage cells to promote bone formation, activation of WNT signaling in the alveolar bone was examined using transgenic TCF / Lef1-GFP reporter mice that express a fused protein of histone 2B and GFP in response to WNT stimulation. Fluorescence microscopy showed GFP expression in multiple cell populations within alveolar bone, including periodontal ligament cells, odontoblasts, osteoblasts, and osteocytes (FIG. 6A). These results indicate robust WNT signaling in alveolar bone-residing cells. Notably, compared to skull bone requiring low mechanical demands, mandible and tibia that require high mechanical demands showed elevated expression of WNT-responsive genes, Axin2 and Lef-1 (FIG. 6B), indicating that WNT signaling in mandibles is activated in response to mechanical stress. Upon WNT stimulation, the key transcription factor β-Catenin is released from the degradation complex and forms a protein complex with other transcription factors, TCF and LEF-1, in the nucleus, resulting in transcriptional activation. To assess mechanical stress- induced activation of WNT signaling in alveolar bone, the dss.rAAV9 that expresses GFP protein under the control of the LEF-1 / TCF-responsive promoter (dss.rAAV9.pLef / Tcf-egfp, FIG. 6C) was produced. Calvarial osteoblasts (COBs) were treated with dss.rAAV9.pLef / Tcf- egfp and then stimulated with a recombinant Wnt3a (rWnt3a) ligand or flow stress. GFP expression (fluorescence microscopy, FIG. 6D) and mRNA expression of egfp, Axin2, and Lef-1 (RT-PCR, FIGs. 6E and 11A) in AAV-treated COBs were markedly upregulated in response to rWnt3a or flow stress. These results demonstrate the ability of dss.rAAV9.pLef / Tcf-egfp to induce GFP expression in response to mechanical stress via WNT signaling activation. 12075767.1 Since high frequency vibration (HFV) activates WNT signaling as a mechanical stress, whether HFV treatment can induce GFP expression in dss.rAAV9.pLef / Tcf-egfp-treated mandibles via WNT signaling activation was examined. Seven days after PL injection into the mandibular first molar, 8-week-old mice were treated daily with HFV (200 Hz, 30 sec) on the mandible (FIG. 6F). Of note, HFV did not induce any adverse effects on tooth, gum, skull, and mandible structures (FIGs. 11B–11D). Expression of egfp and Axin2 in the mandible was assessed by RT-PCR at different time points, demonstrating that their expression in the mandible peaked at both 10 and 5 days of daily HFV stimulation, respectively (FIG. 6G). Compared to no HFV or PBS treatment, ten days of HFV treatment significantly increased the expression of egfp, Axin2, and Lef1 in the mandible (FIG. 6H). Notably, HFV-induced expression of egfp in AAV-treated mandible corresponds to WNT-responsive expression of Axin2 and Lef1 (FIG. 12A). The IVIS optical imaging system also confirmed that GFP expression in AAV-treated mandible was detected only in the presence of HFV stimulation (FIG. 6I). Within alveolar bone, a subset of β-Catenin-expressing cells, including periodontal ligaments, osteoblasts, osteocytes, and odontoblasts expressed GFP proteins (FIG. 6J). These results indicated that PL injection of dss.rAAV9.pLef / Tcf to mandibles facilitated control of transgene expression in alveolar bone in response to HFV stimulation. Vibration-inducible AAV gene therapy reverses alveolar bone loss in osteoporosis A bone-targeted AAV that confers HFV-induced single silencing of Shn3 as a moderate WNT signaling activator (dss.rAAV9.pLef / Tcf.amiR-SHN3) and dual silencing of Shn3 and Sost as a strong WNT signaling activator (dss.rAAV9.pLef / Tcf.amiR-SHN3 / SOST) (FIG. 12B) was produced. 4 weeks after sham control or OVX surgery, 12-week-old female mice were treated with dss.rAAV9.pLef / Tcf carrying amiR-ctrl, amiR-SHN3, or amiR-SHN3 / SOST via PL injection to mandibular first molar and then stimulated daily with HFV for ten days (FIG. 12C). HFV stimulation of amiR-SHN3-treated mandibles led to a decrease in Shn3 expression and an increase in Lef-1 and Axin2 expression and these mandibles did not show any decrease in Shn3 expression in the absence of HFV stimulation (FIGs. 7A and 7B). In amiR-SHN3 / SOST-treated mandibles, HFV stimulation further upregulated Lef1 and Axin2 expression by silencing both Shn3 and Sost expression (FIGs. 7C and 7D). These results demonstrate that HFV stimulation of AAV-treated mandibles effectively silenced the expression of Shn3 and / or Sost and activated WNT signaling. 12075767.1 Regardless of HFV stimulation, amiR-ctrl-treated OVX mice showed a significant decrease in bone formation rate (BFR) and mineral apposition rate (MAR) in the alveolar bone, demonstrating reduced osteoblast activity in vivo (FIG. 7E). By contrast, HFV stimulation of amiR-SHN3 / SOST-treated mandibles led to nearly complete reversal of the BFR and MAR while only the modest increase was seen in amiR-SHN3-treated mandibles (FIG. 7E). Similarly, microCT analysis of amiR-SHN3 / SOST-treated mandibles showed an increase in trabecular BV / TV and thickness and a decrease in trabecular space when treated with HFV. amiR-SHN3- treated mandibles also showed a modest increase in alveolar bone mass after HFV treatment (FIGs. 7F and 7G). These results indicate that HFV-induced dual silencing of Shn3 and Sost is more effective than single silencing of Shn3 in enhancing WNT signaling and osteoblast activity and promoting alveolar bone formation in postmenopausal osteoporosis. Notably, amiR- SHN3 / SOST-treated mandibles did not show any change in dentin deposition rates after HFV stimulation, demonstrating that HFV-induced dual silencing of Shn3 and Sost in the mandible does not affect tooth dentin production (FIGs. 12D and 12E). HFV stimulation of amiR-SHN3 / SOST-treated mandibles upregulated Opg expression while the expression was not affected by amiR-SHN3 treatment (FIG. 7H). Compared to sham control mandibles, amiR-ctrl-treated OVX mandibles showed a significant increase in osteoclast development, as shown by an increase in tartrate-resistant acid phosphatase (Trap, Acp5) mRNA expression and TRAP-positive osteoclast numbers on alveolar bone surface. This increase was reversed by amiR-SHN3 / SOST treatment, but not by amiR-SHN3 treatment, in the presence of HFV stimulation (FIGs. 7I-7K). These results show that HFV-induced dual silencing of Shn3 and Sost in the mandible hyperactivates WNT / β-Catenin signaling in osteoblasts and inhibits osteoclast-mediated bone resorption via upregulation of OPG expression. However, HFV- induced single silencing of Shn3 shows a modest increase on WNT signaling without affecting OPG expression and osteoclast development. Example 2 Alveolar bone loss in elderly populations is highly prevalent and increases the risk of tooth loss, gum disease susceptibility, and facial deformity. Unfortunately, there are very limited treatment options available. Here, the Inventors developed a bone-targeted gene therapy that reversed alveolar bone loss in patients with osteoporosis by targeting the adaptor protein Schnurri-3 (SHN3). SHN3 is a promising therapeutic target for alveolar bone regeneration, 12075767.1 because SHN3 expression is elevated in human and mouse mandible tissues with osteoporosis while deletion of SHN3 in mice greatly increased alveolar bone and tooth dentin mass. The following was used: a bone-targeted recombinant adeno-associated virus (rAAV) carrying an artificial microRNA (miRNA) that silences SHN3 expression to restore alveolar bone loss in mouse models of both postmenopausal and senile osteoporosis by enhancing WNT signaling and osteoblast function. Additionally, rAAV-mediated silencing of SHN3 enhanced bone formation and collagen production of human skeletal organoids in xenograft mice. Finally, rAAV expression in the mandible was tightly controlled via liver- and heart-specific miRNA-mediated repression or via a vibration-inducible mechanism. Collectively, these results demonstrate that AAV-based bone anabolic gene therapy is a promising strategy to treat alveolar bone loss in osteoporosis. Alveolar bone is the portion of the mandibular and maxillary bones that not only supports the tooth sockets and muscles of mastication but also protects nerves, vessels, and glands. It undergoes rapid bone remodeling during tooth eruption due to the forces of mastication and positional adaptation of the teeth. Normal alveolar bone remodeling is controlled by bone-forming osteoblasts and bone-resorbing osteoclasts, but disruption of that balance leads to alveolar bone atrophy. Osteoporosis frequently develops atrophic alveolar bone and hampers proper chewing and speaking, heightens vulnerability to gum disease, and disrupts tooth alignment associated with facial deformity. Additionally, reduced alveolar bone density makes dental implant procedures challenging, as they require sufficient bone mass to achieve dental implantation. Therefore, preserving alveolar bone health is crucial for maintaining optimal oral health, function, and aesthetics. Restoration of alveolar bone loss in osteoporosis poses a significant challenge in dental practice. Current treatments include anti-inflammatory agents and the transplantation of natural or synthetic bone grafts and resorbable or non-resorbable collagen membranes along with supplementary bioactive products. Additionally, osteoporosis drugs, such as bisphosphonates, anti-RANKL antibody, parathyroid hormone analogues, and estrogen replacement, are used for treatment. However, these treatments have shown limited success in the clinic due to a lack of long-term preservation, rehabilitation, and functional needs of alveolar bone. Moreover, while oral administration of bisphosphonates have not shown significant improvement in preventing alveolar bone loss, they can cause oral bony lesions such as bisphosphonate-related osteonecrosis of the jaw. Surgical transplantation has often failed and frequently led to 12075767.1 complications. For example, vertical bone augmentation has been hindered by brittleness and inadequate toughness in newly formed bones, resulting in frequent fractures during the implantation process. The use of slow-resorbing bone biomaterials also can compromise the quantity and quality of newly formed bone in augmentation sites. Hence, the development of new therapeutics to restore alveolar bone loss while limiting worrisome side effects remains an unmet need. Recombinant adeno-associated viruses (rAAV) have demonstrated long-term durability of gene expression, lack of post-immunogenicity, and good safety profiles in clinical studies. Although rAAVs have targeted various organs, such as brain, eye, heart, liver, and skeletal muscle, rAAV-mediated gene therapy for craniofacial diseases have not been evaluated. These Inventors have demonstrated rAAV9 as a highly effective serotype for transducing osteoblast- lineage cells in the long bones and vertebrae of mice. Here, the following was tested: the ability of rAAV9 to promote alveolar bone regeneration by targeting Schnurri-3 (SHN3), an intracellular adaptor protein involved in WNT signaling that inhibits bone formation. It is herein demonstrated that rAAV-mediated gene therapy targeting SHN3 is an alternative approach to traditional osteoporosis drugs to reverse alveolar bone loss in osteoporotic patients. A bone- targeted AAV effectively delivered a SHN3 silencer to osteoblast-lineage cells that reside in alveolar bone, enhanced WNT signaling and osteoblast function, and promoted alveolar bone formation in osteoporotic mice. Moreover, rAAV expression in non-skeletal organs, heart and liver, was repressed via organ-specific miRNA-mediated degradation while the expression was tightly controlled in a vibration-inducible manner. Thus, the bone-trophic AAV targeting SHN3 is a promising bone anabolic agent that restored alveolar bone loss in osteoporosis while limiting potential side effects. Results SHN3 deletion increased alveolar bone and tooth dentin mass The WNT pathway is a key regulator of bone formation by enhancing osteoblast development, and WNT antagonists, such as sclerostin (SOST), dickkopf-1 (DKK-1), and schnurri-3 (SHN3), are promising targets to promote bone formation in osteoporosis. While SOST and DKK-1 proteins are all present in the alveolar bone in young mice (FIG. 13A), mRNA expression of SOST and DKK-1 was substantially decreased in older human mandibles relative to young mandibles (FIG. 13B). Accordingly, treatment of aged osteoporotic rats with anti-SOST 12075767.1 antibody or anti-DKK-1 antibodies showed minimal effects on alveolar bone regeneration. SHN3 expression was also detected in the alveolar bone of young mice (FIG. 13C), but unlike SOST and DKK1, its expression was further upregulated in human and mouse aged mandibles (FIG. 13D). Similarly, postmenopausal osteoporosis in female mice also increased SHN3 expression in mandibles (FIG. 13E). Thus, SHN3 is likely to be a more attractive target than SOST and DKK1 for alveolar bone regeneration in osteoporotic settings. SHN3 controls β-Catenin stability downstream of WNT signaling and that deleting SHN3 enhances WNT signaling, osteoblast development, and bone formation. Thus, it was herein hypothesized that SHN3 may play a role in alveolar bone development by regulating WNT signaling. To explore potential regulatory interactions between SHN3 and WNT signaling within the alveolar bone, an immunohistochemistry (IHC) analysis was performed, demonstrating co-localization of SHN3 and β-Catenin in multiple cell populations within alveolar bone, including periodontal ligament cells (PDL), odontoblasts (OD), osteoblasts (OB), and osteocytes (OCY, FIG. 13C). MicroCT analysis of alveolar bone demonstrated greater bone volume per tissue volume (BV / TV) and trabecular bone thickness (Tb.Th) in 1-month-old Shn3- / -mice than littermate controls (FIG. 13F). This was consistent with a histologic analysis showing an increase in trabecular bone mass in the bone marrow of Shn3- / -mandibles (FIG. 13G). 7-month-old Shn3- / -mice also showed an increase in alveolar bone mass, but this increase was slightly compromised as trabecular bone mass became saturated (FIG. 13H). These results suggest that SHN3 acts as a key regulator of alveolar bone mass during skeletal development and homeostasis. SHN3 expression in dentin-producing odontoblasts within mandibles prompted the Inventors to test its potential role in dental development. MicroCT analysis of the mandibular first molar demonstrated a significant increase in root dentin volume in 1-month-old Shn3- / -mice relative to littermate controls while enamel volume, produced by dental ameloblasts, was comparable between Shn3- / -and littermate control mice (FIG. 13I and FIG. 13J). Notably, the increase in root dentin volume of Shn3- / -mice was halted as dentin production was saturated at age of 7 months (FIG. 13K). These results suggest that SHN3 in odontoblasts functioned as a suppressor of dentin production at early developmental stages while its role in ameloblasts was dispensable for enamel production. Thus, SHN3 was crucial for alveolar bone and tooth dentin formation, which makes SHN3 an attractive therapeutic target to promote regeneration of alveolar bone and tooth dentin. 12075767.1 SHN3 deletion in mesenchyme-lineage cells increased alveolar bone and tooth dentin mass Platelet-derived growth factor receptors (PDGFRs) have been reported to express in a broad range of mesenchyme-lineage cells at multiple anatomical locations in the skeleton. Fluorescence microscopy of Pdgfrα-GFP reporter mice demonstrated GFP expression in periodontal ligament cells, osteoblast-lineage cells, and dental-lineage cells within mandibles (FIG. 14A). Shn3 was conditionally deleted in these cells by crossing mice with a Shn3-floxed allele (Shn3fl / fl) with Pdgfrα-Cre mice (Shn3Pdgfrα). Reduced expression of Shn3 was confirmed in alveolar bone-derived mesenchymal stromal cells (ABMSCs) isolated from Shn3Pdgfrαmandibles (FIG. 14B). These cells showed an increase in expression of osteogenic marker genes, including osteocalcin (Bglap) and dentin matrix acidic phosphoprotein 1 (Dmp1, FIG. 14B), and extracellular mineralization (alizarin red staining, FIG. 14C), indicating enhanced osteogenic activity. As seen in Shn3- / -mice, alveolar bone mass was markedly increased in Shn3Pdgfrαmice relative to littermate controls (Shn3fl / fl) at ages 1 and 7 months old, as shown by increased BV / TV and Tb.Th. This is consistent with a histologic analysis showing an increase in trabecular bone mass in the bone marrow of Shn3Pdgfrαmandibles (FIG. 14D and FIG. 14E). Similarly, trabecular bone mass and cortical thickness in the long bones were both markedly increased in these mice (FIG. 20A). These results highlight the role of SHN3 in Pdgfrα+cells as a key regulator of alveolar and long bone mass during skeletal development and homeostasis. Since Pdgfrα-GFP expression was also detected in odontoblasts within mandibles (FIG. 14A), the following was assessed: the tooth dentin volume in the first molar of Shn3Pdgfrαmandibles using microCT. Similar to Shn3- / -mandibles, 1-month-old Shn3Pdgfrαmandibles displayed a significant increase in root dentin volume without any alteration in enamel volume and the increased root dentin volume was halted as dentin production was saturated at age of 7 months (FIG. 14F). Notably, Masson’s trichrome staining and scanning electron microscopy (SEM) of Shn3Pdgfrαtooth showed normal dentin matrix and tubular structure, showing that Shn3 deletion in Pdgfrα+cells led to an increase in a good quality of dentin mass (FIG. 14G). Next, SHN3 expression was deleted at early and late stages of osteoblast differentiation by crossing Shn3fl / flmice with Prx1-Cre mice (Shn3Prx1) and Dmp1-Cre mice (Shn3Dmp1), respectively. Prx1-Cre mice and Dmp1-Cre mice were also crossed with Rosa26mT / mGreporter mice38where Cre-recombinase uses GFP expression to visualize Cre-expressing cells within 12075767.1 mandibles. Unlike Prx1-GFP proteins primarily expressed in osteoblasts on the alveolar bone surface, Dmp1-GFP expression was detected in osteocytes embedded in the alveolar bone matrix. Prx1- and Dmp1-GFP proteins were both expressed in odontoblasts on the dentin surface (FIG. 14H). Deletion of Shn3 in Prx1+skeletal progenitors and Dmp1+osteocytes resulted in a significant increase in alveolar bone mass (FIG. 14I) and femoral bone mass (FIG. 20B and FIG. 20C). These results show that SHN3 acted in both early and late stages of osteoblast differentiation to produce alveolar bone and long bone. 1-month-old Shn3Prx1mandibles displayed an increase in root dentin volume of the first molar without any change in enamel volume (FIG. 14J). However, there was little to no increase in root dentin volume in Shn3Dmp1molar (FIG. 14J and FIG. 14K), showing that SHN3 in skeletal progenitors, but not in osteocytes, drove dentin production during skeletal development. Thus, inhibition of SHN3 in skeletal progenitors promoted formation of long bone, alveolar bone, and tooth dentin. AAV-mediated silencing of human SHN3 promoted bone formation in xenograft mice To test whether SHN3-deficiency in human osteoblasts can promote bone formation, human artificial miRNA (hs-amiR) was developed by embedding the guide strand of a small silencing RNA that targets human SHN3 into a human miR-33-derived miRNA scaffold (hs-amiR- hSHN3), and then packaged into the AAV9 capsid. In this design, the hs-amiR was inserted intronically between the chicken β-actin (CBA) promoter and the Egfp reporter gene (FIG. 15A), which allowed for visual tracking of positively transduced cells or tissues. The AAV9’s ability to transduce human periodontal ligament stromal cells (PDLSCs) or bone marrow-derived mesenchymal stromal cells (BMSCs) was confirmed by GFP expression using fluorescence microscopy (FIG. 15B). rAAV9 carrying hs-amiR-hSHN3 effectively silenced SHN3 expression in these cells and increased osteogenic differentiation (FIG. 15C). Next, the following was examined: whether AAV-mediated silencing of human SHN3 promoted bone formation in vivo using a xenograft mouse model implanted with a human skeletal organoid (FIG. 15D). The human skeletal organoid was generated by culturing human BMSCs in hydroxyapatite (HA)- scaffold under osteogenic conditions and then implanted into interscapular fat pads of immunodeficient mice (FIG. 15E). A single dose of rAAV9 carrying hs-amiR-ctrl or hs-amiR- hSHN3 was injected to the implanted site. Four weeks later, AAV’s ability to transduce the human skeletal organoid and to knockdown Shn3 expression were confirmed by GFP expression using the IVIS optical imaging system (FIG. 15F) and RT-PCR analysis (FIG. 15G), 12075767.1 respectively. Compared to hs-amiR-ctrl, hs-amiR-hSHN3-treatment markedly increased osteogenic gene expression, osteocalcin (BGLAP) and type 1 collagen (COL1), and WNT- induced gene expression, AXIN2 (FIG. 15G). Likewise, this organoid showed a significant increase in mineral density distribution (microCT) and newly formed collagen and mineral (Masson’s trichrome staining, FIG. 15H- FIG. 15J). This was consistent with SEM showing increased production of collagen fibers and calcified matrix in the human skeletal organoid at molecular levels (FIG. 15K). Thus, AAV-mediated silencing of SHN3 was effective in transducing human osteoblast lineage cells, enhancing osteogenic differentiation and WNT signaling and promoting bone formation in human skeletal organoids, suggesting the clinical potential to human skeletal diseases with low bone mass. AAV-mediated silencing of Shn3 reversed alveolar bone loss in osteoporosis Since rAAV9 has been reported as a highly effective serotype that transduces osteoblast-lineage cells in the long bone and vertebrae in mice, rAAV9’s ability to transduce alveolar bone was examined using GFP expression after intravenous (IV) injection or periodontal ligament (PL) injection to the mandibular first molar (FIG. 21A, top and FIG. 21B). Individual organ imaging of IV-injected mice showed robust GFP expression in liver and muscle and a modest expression in the mandible. By contrast, PL-injected mice showed a modest expression in the liver and mandible with little to no expression in muscle, brain, heart, lung, spleen, and kidney (FIG. 16A). This was consistent with RT-PCR analysis and fluorescence microscopy on cryo-sectioned tissues showing a significant decrease in GFP expression in the liver, heart, and muscle when treated via PL injection relative to IV injection (FIG. 16B and FIG. 21C). However, GFP expression in the PL-injected mandibles was increased 3-fold (FIG. 16C) when rAAV9 was transduced into periodontal ligament cells, odontoblasts, and osteoblast-lineage cells (FIG. 21D). Thus, PL injection of rAAV9 was an effective gene delivery strategy to transduce alveolar bone while limiting AAV’s expression in other tissues. To further improve the bone-specific tropism of the rAAV9 capsid, a bone-homing peptide motif (AspSerSer)6 was grafted onto an AAV9-VP2 capsid protein (dss.rAAV9). 8- week-old Pdgfrα-GFP reporter mice were treated with mCherry-expressing dss.rAAV9 (dss.rAAV9.mCherry) via PL injection into the mandibular first molar and mCherry expression in Pdgfrα-GFP+cells was assessed by fluorescence microscopy. Fluorescence microscopy showed mCherry expression in a subset of Pdgfrα+cells in alveolar bone, including periodontal 12075767.1 ligament cells, osteoblast-lineage cells, and odontoblasts (FIG. 16D). Thus, PL injection of dss.rAAV9 effectively transduced Pdgfrα+alveolar bone-residing cells. Postmenopausal osteoporosis results in alveolar bone loss and deterioration of bone structure, increasing the risk of tooth loss, gum disease susceptibility, and facial deformity. This process can be modeled in mice; ovariectomy (OVX) surgery in female mice led to estrogen deficiency-induced bone loss, recapitulating postmenopausal osteoporosis. To test whether AAV- mediated silencing of Shn3 could reverse alveolar bone loss in postmenopausal osteoporosis, the following was generated: an AAV-compatible artificial miRNA that silenced mouse Shn3 expression by embedding Shn3-targeting sequences into a mouse miR-33-derived miRNA scaffold (amiR-SHN3, FIG. 21A, bottom). Sham control or OVX surgery was performed in 12- week-old female mice and four weeks later, a single dose of dss.rAAV9 carrying amiR-ctrl or amiR-SHN3 was administered via IV or PL injection (FIG. 16E). Eight weeks after injection, knockdown efficiency of Shn3 in AAV-treated OVX mandibles was confirmed by RT-PCR (FIG. 16F). While amiR-ctrl-treated OVX mice showed a significant reduction in alveolar bone mass compared to sham control mice, this bone loss was reversed by both IV- and PL-injection of amiR-SHN3, as shown by increased trabecular BV / TV, thickness, and number and by decreased trabecular space (FIG. 16G and FIG. 16H). This was consistent with a histological analysis showing a reversal of trabecular bone loss in the bone marrow of amiR-SHN3-treated mandible with OVX surgery (FIG. 16G, bottom). However, amiR-SHN3-treated muscle did not show any changes in myogenic gene expression and microstructures of muscle fibers, suggesting that SHN3 was dispensable of muscle homeostasis (FIG. 21E and FIG. 21F). PL-injection of dss.rAAV9.amiR-SHN3 showed greater increase in alveolar bone mass than IV injection, demonstrating that local delivery was more effective for AAV transduction and alveolar bone formation than systemic delivery. Notably, unlike genetic deletion of Shn3, amiR- SHN3-treatment did not increase root dentin volume (FIG. 16I). This discrepancy may have resulted from AAV’s low silencing efficiency in odontoblasts and osteoblast-lineage cells or AAV injection in older mice that may be too late to promote tooth dentin production. Next, the following was examined: AAV’s ability to restore alveolar bone loss in aging- associated osteoporosis. 20-month-old male mice were treated with dss.rAAV9 carrying amiR- ctrl or amiR-SHN3 via PL injection into the mandibular first molar. Two months later, knockdown efficiency of Shn3 in AAV-treated mandibles was confirmed by RT-PCR (FIG. 16J). Compared to amiR-ctrl-treated mice, amiR-SHN3-treated mice showed a significant increase in 12075767.1 alveolar bone mass, as shown by greater BV / TV and Tb.Th (FIG. 16K and FIG. 16L). These results demonstrated that bone-targeting AAV-mediated silencing of Shn3 via PL injection into mandibles was effective in restoring alveolar bone loss in both aging-associated and postmenopausal osteoporosis. Taken together, the approach of local delivery of a SHN3 silencer to mandibles via a bone-targeted AAV may successfully treat alveolar bone loss in osteoporosis. Liver / heart-“detargeting” AAV-mediated silencing of Shn3 reverses osteoporosis Newly developed agents to treat bone loss, such as anti-sclerostin antibody and the small molecule inhibitor of Cathepsin K, have off-target cardiovascular and cerebrovascular events in clinical trials, respectively. Thus, in some circumstances AAV’s expression in non-skeletal tissues needs to be suppressed. Since dss.rAAV9 can transduce liver and heart, the following was repressed: AAV’s expression in liver and heart using liver- and heart-specific miRNA- mediated degradation. The specificity of miR-122 and miR-208a expression in liver and heart was validated by RT-PCR (FIG. 22A). Three tandem complementary sites for liver-specific miR- 122 and heart-specific miR-208a were inserted into the 3′ untranslated region (UTR) of the egfp reporter gene in the vector genome and then, packaged into dss.rAAV9 capsid (dss.rAAV9.egfp.MIR-TS; FIG. 17A, top). 8-week-old mice were IV injected with PBS, dss.rAAV9.egfp, or dss.rAAV9.egfp.MIR-TS and the tissue distribution of AAVs was assessed by EGFP expression using the IVIS optical imaging system (FIG. 17B) and fluorescence microscopy on cryo-sectioned tissues (FIG. 22B). dss.rAAV9.egfp-treated mice showed robust GFP expression in liver, but the expression in dss.rAAV9.egfp.MIR-TS-treated liver was markedly reduced. This was consistent with RT-PCR analysis showing a significant decrease in GFP expression in heart and liver treated with dss.rAAV9.egfp.MIR-TS relative to dss.rAAV9.egfp while the expression was comparable between tibia and muscle (FIG. 17C). These results indicated that systemic delivery of dss.rAAV9.egfp.MIR-TS facilitated GFP expression in the bone, but not in heart, liver, lung, kidney, spleen, and brain. To examine whether systemic delivery of dss.rAAV9.amiR-Shn3.MIR-TS could reverse femoral bone loss in postmenopausal osteoporosis, a single dose of dss.rAAV9 carrying amiR- ctrl, amiR-SHN3 or amiR-SHN3.MIR-TS was IV injected into 12-week-old female mice four weeks after sham control or OVX surgery. Eight weeks later, knockdown efficiency of Shn3 in AAV-treated OVX tibia was confirmed (FIG. 17D). While amiR-ctrl-treated OVX mice showed significant reductions in femoral bone mass compared to sham control mice, this bone loss was 12075767.1 reversed by both amiR-SHN3 and amiR-SHN3.MIR-TS, as shown by increased trabecular BV / TV, thickness, and number and decreased trabecular space (FIG. 17E and FIG. 17F). Thus, miR-122- and miR-208a-mediated repression effectively “detargeted” AAV expression in liver and heart without affecting anabolic bone increase by amiR-SHN3. Next, the following was compared: EGFP expression in the alveolar bone treated with dss.rAAV9.egfp and dss.rAAV9.egfp.MIR-TS via local delivery. 8-week-old mice were treated with dss.rAAV9.egfp or dss.rAAV9.egfp.MIR-TS via PL injection into the mandibular first molar. Ten days later, EGFP expression in the mandible was assessed by IVIS optical imaging, RT-PCR, and fluorescence microscopy (FIG. 17G and FIG. 17H), demonstrating equivalent expression levels in AAV-treated mandibles. Similar to dss.rAAV9.egfp, dss.rAAV9.egfp.MIR-TS effectively transduced a subset of periodontal ligament cells and osteoblast-lineage cells within alveolar bone. Thus, PL injection of dss.rAAV9.MIR-TS to the mandible may be a safe and effective strategy to target alveolar bone. To compare the therapeutic efficacy of dss.rAAV9.amiR-SHN3 vs. dss.rAAV9.amiR- SHN3.MIR-TS in osteoporotic alveolar bone, 12-week-old female mice were treated with the AAVs via PL injection to the mandibular first molar four weeks after sham control or OVX surgery (FIG. 22C). Eight weeks later, knockdown efficiency of Shn3 in AAV-treated OVX mandible was confirmed by RT-PCR (FIG. 17I). While amiR-ctrl-treated OVX mice showed a significant reduction in alveolar bone mass compared to sham control mice, this bone loss was reversed by both amiR-SHN3.MIR-TS and amiR-SHN3, as shown by increased trabecular BV / TV and thickness (FIG. 17J and FIG. 17K). Thus, PL injection of dss.rAAV9.amiR- SHN3.MIR-TS effectively restored alveolar bone loss in postmenopausal osteoporosis while securing AAV’s expression in mandibles. The Inventors concluded that silencing of Shn3 in mandibles via a bone-targeted AAV with bone-specific tropism (dss.rAAV9 capsid) and liver- and heart-detargeting capability (miR-122 / miR-208a-mediated repression) was a targeted and safe approach to restore alveolar bone loss in osteoporosis. Vibration-inducible expression of bone-targeted AAV in mandibles Alveolar bone continuously receives mechanical stimuli, such as chewing, biting, and speaking, which is crucial to maintain alveolar bone mass. Given that WNT signaling is a major mechano- sensing pathway in osteoblast lineage cells to promote bone formation, activation of WNT signaling in the alveolar bone was examined using transgenic TCF / Lef1-GFP reporter mice that 12075767.1 express a fused protein of histone 2B and GFP in response to WNT stimulation. Fluorescence microscopy showed GFP expression in multiple cell populations within alveolar bone, including periodontal ligament cells, odontoblasts, osteoblasts, and osteocytes (FIG. 18A). These results indicate robust WNT signaling in alveolar bone-residing cells. Notably, compared to skull bone requiring low mechanical demands, mandible and tibia that require high mechanical demands showed elevated expression of WNT-responsive genes, Axin2 and Lef-1 (FIG. 18B), suggesting that WNT signaling in mandibles was activated in response to mechanical stress. Upon WNT stimulation, the key transcription factor β-Catenin is released from the degradation complex and forms a protein complex with other transcription factors, TCF and LEF-1, in the nucleus, resulting in transcriptional activation. To assess mechanical stress- induced activation of WNT signaling in alveolar bone, the following was generated: the dss.rAAV9 that expressed GFP protein under the control of the LEF-1 / TCF-responsive promoter (dss.rAAV9.pLef / Tcf-egfp, FIG. 18C). Calvarial osteoblasts (COBs) were treated with dss.rAAV9.pLef / Tcf-egfp and then stimulated with a recombinant Wnt3a (rWnt3a) ligand or flow stress. GFP expression (fluorescence microscopy, FIG. 18D) and mRNA expression of egfp, Axin2, and Lef-1 (RT-PCR, FIG. 18E and FIG. 23A) in AAV-treated COBs were markedly upregulated in response to rWnt3a or flow stress. These results demonstrated the ability of dss.rAAV9.pLef / Tcf-egfp to induce GFP expression in response to mechanical stress via WNT signaling activation. High-frequency vibration (HFV) treatment has shown promise in dental practice to enhance alveolar bone density and fibroblast stimulation within the periodontal ligament. Since HFV activates WNT signaling as a mechanical stress, the following was tested: whether HFV treatment could induce GFP expression in dss.rAAV9.pLef / Tcf-egfp-treated mandibles via WNT signaling activation. Two days after PL injection into the mandibular first molar, 8-week-old mice were treated daily with HFV (200 Hz, 30 sec) on the mandible (FIG. 18F). Of note, HFV did not induce any adverse effects on tooth, gum, skull, and mandible structures (FIG. 23B–FIG. 23D). Expression of egfp and Axin2 in the mandible was assessed by RT-PCR at different time points, demonstrating that their expression in the mandible peaked at both 10 and 5 days of daily HFV stimulation, respectively (FIG. 18G). Compared to no HFV or PBS treatment, ten days of HFV treatment significantly increased the expression of egfp, Axin2, and Lef1 in the mandible (FIG. 18H). Notably, HFV-induced expression of egfp in AAV-treated mandible corresponded to WNT-responsive expression of Axin2 and Lef1 (FIG. 25A). The IVIS optical imaging system 12075767.1 confirmed that GFP expression in AAV-treated mandible was detected only in the presence of HFV stimulation (FIG. 18I). AAV-treated liver also showed modest expression of GFP proteins, but the expression was markedly reduced upon HFV stimulation. There was little to no expression in brain, heart, lung, kidney, and spleen regardless of HFV stimulation (FIG. 24A- FIG. 24B). Within alveolar bone, a subset of β-Catenin-expressing cells, including periodontal ligaments, osteoblasts, osteocytes, and odontoblasts expressed GFP proteins (FIG. 18J). These results suggested that PL injection of dss.rAAV9.pLef / Tcf to mandibles facilitated control of transgene expression in alveolar bone in response to HFV stimulation. Vibration-inducible AAV gene therapy reversed alveolar bone loss in osteoporosis SHN3 deficiency has upregulated Sost expression among known WNT antagonists and inhibition of both factors further increased WNT / β-catenin signaling in osteoblast-lineage cells, suggesting that expression of SHN3 and SOST is connected via a negative feedback mechanism. Thus, the following was generated: a bone-targeted AAV that conferred HFV-induced single silencing of Shn3 as a moderate WNT signaling activator (dss.rAAV9.pLef / Tcf.amiR-SHN3) and dual silencing of Shn3 and Sost as a strong WNT signaling activator (dss.rAAV9.pLef / Tcf.amiR- SHN3 / SOST) (FIG. 25B). 4 weeks after sham control or OVX surgery, 12-week-old female mice were treated with dss.rAAV9.pLef / Tcf carrying amiR-ctrl, amiR-SHN3, or amiR-SHN3 / SOST via PL injection to mandibular first molar and then stimulated daily with HFV for ten days (FIG. 25C). HFV stimulation of amiR-SHN3-treated mandibles led to a decrease in Shn3 expression and an increase in Lef-1 and Axin2 expression and these mandibles did not show any decrease in Shn3 expression in the absence of HFV stimulation (FIG. 19A and FIG. 19B). In amiR- SHN3 / SOST-treated mandibles, HFV stimulation further upregulated Lef1 and Axin2 expression by silencing both Shn3 and Sost expression (FIG. 19C and FIG. 19D). These results demonstrated that HFV stimulation of AAV-treated mandibles effectively silenced the expression of Shn3 and / or Sost and activated WNT signaling. Regardless of HFV stimulation, amiR-ctrl-treated OVX mice showed a significant decrease in bone formation rate (BFR) and mineral apposition rate (MAR) in the alveolar bone, demonstrating reduced osteoblast activity in vivo (FIG. 19E). By contrast, HFV stimulation of amiR-SHN3 / SOST-treated mandibles led to nearly complete reversal of the BFR and MAR while only the modest increase was seen in amiR-SHN3-treated mandibles (FIG. 19E). MicroCT analysis of amiR-SHN3 / SOST-treated mandibles also showed an increase in trabecular BV / TV 12075767.1 and thickness and a decrease in trabecular space when treated with HFV. Unlike the CBA promoter-driven silencing of Shn3 showing a significant increase in alveolar bone mass, only a modest increase in bone mass was observed under the LEF-1 / TCF promoter-driven silencing after HFV treatment (FIG. 19F and FIG. 19G). This was consistent with the RT-PCR data showing that compared to control treatment, the CBA promoter-driven silencing of Shn3 resulted in high GFP expression, robust knockdown of Shn3, and increased expression of the osteogenic gene alkaline phosphatase (Alp) regardless of HFV stimulation. However, HFV- induced silencing of Shn3 under the LEF-1 / TCF promoter led to a modest increase in Egfp and Alp expression along with a modest knockdown of Shn3 (FIG. 26), resulting in weak osteogenesis. These results suggested that HFV-induced dual silencing of Shn3 and Sost was more effective than single silencing of Shn3 in enhancing WNT signaling and osteoblast activity and promoting alveolar bone formation in postmenopausal osteoporosis. Notably, amiR- SHN3 / SOST-treated mandibles did not show any change in dentin deposition rates after HFV stimulation, demonstrating that HFV-induced dual silencing of Shn3 and Sost in the mandible did not affect tooth dentin production (FIG. 25D and FIG. 25E). Hyperactivation of WNT / β-Catenin signaling in osteoblasts has inhibited osteoclast development via upregulated production of osteoprotegerin (OPG). Accordingly, HFV stimulation of amiR-SHN3 / SOST-treated mandibles upregulated Opg expression while the expression was not affected by amiR-SHN3 treatment (FIG. 19H). Compared to sham control mandibles, amiR-ctrl-treated OVX mandibles showed a significant increase in osteoclast development, as shown by an increase in tartrate-resistant acid phosphatase (Trap, Acp5) mRNA expression and TRAP-positive osteoclast numbers on alveolar bone surface. This increase was reversed by amiR-SHN3 / SOST treatment, but not by amiR-SHN3 treatment, in the presence of HFV stimulation (FIG. 19I-FIG. 19K). These results showed that HFV-induced dual silencing of Shn3 and Sost in the mandible hyperactivated WNT / β-Catenin signaling in osteoblasts and inhibited osteoclast-mediated bone resorption via upregulation of OPG expression. However, HFV-induced single silencing of Shn3 showed a modest increase on WNT signaling without affecting OPG expression and osteoclast development. Taken together, activation of WNT signaling in mandibles via SHN3 and / or SOST knockdown using the bone-targeted AAV with liver / heart-detargeting capability or HFV-induced expression could be a promising, safe, and targeted therapeutic option to restore alveolar bone loss in osteoporosis. 12075767.1 Materials and Methods Cell culture and reagents: Human bone marrow-derived mesenchymal stromal cells (BMSCs, #7500) were purchased from ScienCell Research Laboratories and cultured according to the manufacturer’s manuals. Human Periodontal Ligament Stem Cells (PDLSCs, 36085-01) were purchased from Celprogen and cultured in the Human Periodontal Ligament Stem Cells Complete Growth Media with Serum (M36085-01S) for maintenance or cultured in the Human Periodontal Ligament Stem Cell Culture Differentiation Media with Serum (M36085-01DS) for osteogenic differentiation. Mouse alveolar bone-marrow-derived MSCs were isolated from 1- month-old Shn3Pdgfrαand Shn3fl / flmandibles as described previously.70Briefly, mandibular bones were collected and processed by removing attached soft tissues and teeth, including molars and incisors. Cells were then obtained using a digestion solution that contains collagenase type I (Worthington Biochem, LS004196) and dispase II (Roche,10165859001). The resulting cell suspensions were filtered (40 µm) and washed using PBS and maintained in α-MEM medium (Gibco). Mouse COBs were isolated from the calvaria of wildtype neonates at postnatal day 3 (C57BL / 6J). Cells were maintained in α-MEM medium (Gibco) containing 10% FBS (Corning), 2 mM L-glutamine (Corning), 1% penicillin / streptomycin (Corning), and 1% nonessential amino acids (Corning) while they were differentiated into mature osteoblasts under osteogenic medium containing ascorbic acid (200 µM, Sigma, #A8960) and β-glycerophosphate (10 mM, Sigma, #G9422). Recombinant WNT3a was purchased from R&D systems (#1324-WN). rAAV vector design and production: Bone-targeting AAV9 capsid (dss.rAAV9) was generated as described in previous studies. Mouse or human miR-33 scaffold design rules were applied to generate optimized artificial miRNA (amiR) cassettes and amiR target sequences were designed using a custom Excel macro and cloned into a self-complementary AAV vector genome. Plasmids were constructed by Gibson assembly and standard molecular biology methods. Validated DNA sequences for amiR-ctrl, amiR-SHN3, hs-amiR-SHN3, and amiR-SOST / SHN3 were synthesized as gBlocksTM, cloned into the intronic region of the pAAVsc-CB6-Egfp plasmid at the restriction enzyme sites (PstI and BglII). To generate a liver and heart-detargeting AAV (dss.rAAV9.egfp.MIR-TS), endogenous complementary sequences for miR-122 and miR- 208a were synthesized as gBlocksTMand inserted into the 3′ untranslated region (UTR) of the egfp reporter gene in the pAAVsc-CB6-Egfp plasmid and packaged into the dss.AAV9 capsid. To generate a mechanical stress-inducible AAV (dss.rAAV9.pLef / Tcf-egfp), DNA sequences for the TCF / LEF promoter (pTcf / Lef) adapted from the M50 super 8x TopFlash reporter gene 12075767.1 (Addgene, #12456) were synthesized as gBlocksTMand then replaced the CB6 promoter of the pAAVsc-CB6-Egfp plasmid. Constructs were verified by sequencing and then packaged into the dss.rAAV9 capsid. rAAV9 production was executed by transient transfection in HEK293 cells, purified by CsCl sedimentation, and quantified by droplet digital PCR (ddPCR) on a QX200 ddPCR system (Bio-Rad) using the Egfp or mCherry prime / probe set. Quantitative RT-PCR analysis: Total RNA was purified from cells using QIAzol (QIAGEN) and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems. Quantitative RT-PCR was performed using SYBR® Green PCR Master Mix (Bio-Rad) with the CFX connect RT-PCR detection system (Bio-Rad). To measure Shn3 mRNA levels in bone tissues, after removal of bone marrow, soft tissue and teeth, including molars and incisors, mandibles or tibias were snap-frozen in liquid nitrogen for 30 s and in turn, homogenized in 1 ml of QIAzol for 1 min. Alternatively, a TaqMan microRNA assay kit (Applied Biosystems) was used to measure the expression of miR-122 and miR-208a. miRNAs were isolated from the mandible, liver, or heart using the mirVana miRNA isolation kit (Ambion), followed by cDNA synthesis using the TaqMan miRNA reverse transcription kit (Applied Biosystems). The cDNA was used for RT-PCR using a TaqMan miRNA assay kit (Applied Biosystems) according to the manufacturer’s protocol: miR-122-5p (assay ID: 002245), miR-208a-3p (assay ID: 000511). Human subjects and analysis: Four de-identified alveolar bone samples were obtained from young (20-25 year-old) and aged (older than 60 year-old) patients at the School of Stomatology Wuhan University during dental implant surgery. Total RNAs were extracted from alveolar bone areas and mRNA expression of SOST, DKK1, and SHN3 was examined by RT-PCR. These were obtained under Institutional Review Board approval (Protocol #: 2022D03). Mice: Mice were housed in a constant environment (up to 5 mice per cage), ambient temperature of 21 ± 2 °C, circulating air, and constant humidity of 50 ± 10%, in a 12 h light, 12 h dark cycle. Mice were given a standard mouse chow diet and monitored every three days for the amount of their food and water intake and signs of distress. For signs of severe distress, including general malaise, severe cachexia, or more than 20% loss of body weight, humane euthanasia was performed in consultation with veterinary staff. Mice were euthanized in a carbon dioxide chamber, followed by cervical dislocation. Neonates were euthanized by decapitation. Cre deleter mice (C57BL / 6J) that express Cre recombinase under the control of the 12075767.1 Prrx1 promoter (Prx1-cre), the Pdgfrα promoter (Pdgfrα-cre), and the Dmp1 promoter (Dmp1- cre) were purchased from Jackson Laboratory. Shn3− / −31and Shn3fl / fl 73mice were previously generated and maintained on a BALB / c and C57BL / 6J background, respectively. Wild-type (C57BL / 6J) mice, Pdgfrα-GFP, and Rosa26mT / mGreporter mice, severe combined immunodeficiency (SCID) mice, and TCF / Lef1-HIST1:H2BB / EGFP reporter mice were purchased from The Jackson Laboratory. Mouse genotypes were determined by PCR on tail genomic DNA; primer sequences are available upon request. All animals were used in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were handled according to protocols approved by the University of Massachusetts Chan Medical School Institutional Animal Care and Use Committee (Protocol#: A-202200036). High-frequency vibration (HFV): A portable high-frequency wand was applied to the mandible of both cheeks to produce high-frequency mechanical vibration in alveolar bone, teeth, and periodontal tissues within the mandible. All HFV treatment was applied to cheek skin around the buccal side of the mandible molar for 30 seconds per day, according to the experiment’s purpose and timeline. The portable high-frequency wand delivers a micro-vibration frequency at 200Hz with 10,000 / minute vibration. Working voltage & power: 1.5V, 0.1W. MicroCT and radiography: MicroCT (µCT35; SCANCO Medical AG; Bruttisellen, Switzerland) was used for qualitative and quantitative assessment of bone mass and microarchitecture of alveolar bones and femurs, which was performed by an investigator blinded to the genotypes of the animals under analysis. MicroCT scanning was performed at 55 kVp and 114 mA energy intensity with 300-ms integration time. A specific voxel size 12 µm was used to measure the mandibular body, molars, and incisors. All images were reconstructed using image matrices of 1024 × 1024 pixels. For trabecular bone analysis of the distal femur, an upper 2.1 mm region beginning 280 μm proximal to the growth plate was contoured. Additionally, a mid-shaft region of 0.6 mm in length was used to analyze cortical bone. For mandibular bone analysis, the area between the mesial and distal root of the first molar was measured volumetrically through 100 serial images (120 μm span). Specifically, contour lines were drawn between the mesial and distal root of the mandibular first molar to define the alveolar bone region of interest (ROI) in micro-CT 2D sagittal sections: Shn3- / -, Shn3Prx1, Shn3Pdgfrα, Shn3Dmp1, wildtype mice, and AAV-treated OVX 12075767.1 mice (100 serial images) or 20-month-old mice (140 serial images). For the assessment of mineral density and thickness of root dentin or cementum, 40 slices centered on the cut-through of the mesial root in the first molar were measured. The first slice in the sequence was below the pulp chamber when two roots separated to form two distinct channels. The last slice was at the apex of the tooth. For crown dentin and enamel, molar enamel and dentin volume and density were calculated and measured from the cementum-enamel junction to the highest cusp tip. Customized filtering and thresholding were applied to quantify the targeted area within the volume of interest (VOI). For the assessment of enamel mass, the Inventors set the fixed value of 1000 as a high threshold and increased the low threshold up to 900 to highlight dense segmentation within VOI. For the assessment of dentin mass, the Inventors set the fixed value of 900 as a high threshold and increased the low threshold up to 320 to highlight dense segmentation within VOI. 3D reconstruction images were obtained from contoured 2D images by methods based on the distance transformation of the binarized images. Alternatively, the Inveon multimodality 3D visualization program was used to generate fused 3D viewing of multiple static or dynamic volumes of microCT modalities (Siemens Medical Solutions USA, Inc.). All images presented were representative of the respective genotypes (n > 5). The Trident Specimen Radiography system (Hologic, USA) was used to generate detailed radiographic images of the whole mouse body after euthanasia. The X-ray beam intensity was 1 mA 28–30 kV with AEC (automatic exposure control) for consistent image acquisition. Histology and histomorphometry: For histological analysis, alveolar bones were dissected from the mice, fixed in 10% neutral buffered formalin for 2 days, and decalcified by 14% tetrasodium EDTA for 2–4 weeks. Tissues were dehydrated by passage through an ethanol series, cleared twice in xylene, embedded in paraffin, and sectioned at a thickness of 6 μm along the coronal plate from anterior to posterior. Decalcified femoral sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, or Tartrate-Resistant Acid Phosphatase (TRAP). For dynamic histomorphometric analysis, 25 mg / kg calcein (Sigma, C0875) and 50 mg / kg alizarin-3-methyliminodiacetic acid (Sigma, A3882) dissolved in 2% sodium bicarbonate solution were subcutaneously injected into mice at 6-day intervals. After fixing in 12075767.1 10% neutral buffered formalin for two days, undecalcified mandibular bone samples were embedded in methyl methacrylate, and alveolar bone was sectioned longitudinally (5 μm) and stained with McNeal’s trichrome for osteoid assessment and TRAP for osteoclasts. A region of interest was defined under the mandibular first molar in the metaphysis and BFR / bone surface (BS), MAR, BS, Ob.S / BS, and osteoclast surface (Oc.S / BS) were measured using the Osteometrics. Measurements were taken on two sections / sample (separated by ~25 μm) and summed prior to normalization to obtain a single measure / sample in accordance with ASBMR standards. This methodology has undergone extensive quality control and validation, and the results were assessed by two different researchers in a blinded fashion. MAR was calculated by measuring the distance between the calcein-labeled bone ([green] right line, FIG. 25D) and the alizarin red-labeled bone ([red] left line, FIG. 25D); this measurement was made at defined sites around the mandibular incisors in 5 separate mice. The same analyses were performed on the lower incisor for dentin apposition rate. Immunohistochemistry and immunofluorescence: For immunohistochemistry analysis, paraffin sections were dewaxed and stained according to the manufacturer’s directions, using the Discovery XT automated IHC stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). CC1 standard buffer (pH 8.4 buffer containing Tris / Borate / EDTA) and inhibitor D (3% H2O2, Endogenous peroxidase) were used for antigen retrieval and blocking, respectively. Sections were incubated with antibodies specific to SHN3 (PA5-52194, Thermo Fisher, 1:200), SOST (AF1589-SP, R&D systems, 1:100) or DKK1 (21112-1-AP, Proteintech, 1:100) for 40 min at 37 °C, and a secondary antibody for 20 min at 37 °C. Subsequently, they were incubated in SA- HRP D for 16 min at 37 °C and then DAB + H2O2substrate for 8 min, followed by hematoxylin and bluing reagent counterstain at 37 °C. Reaction buffer (pH 7.6 Tris buffer) was used as a washing solution. Stained samples were visualized using an Aperio virtual microscope (Leica Microsystems, USA), and images of the sample were analyzed by the Aperio image scope program (ver. 12.3.2.8013, Leica Microsystems, USA). For immunofluorescence analysis, fresh alveolar bone dissected from Shn3- / -, Shn3Prx1, Shn3Pdgfrα, Shn3Dmp1, and wildtype mice and rAAV-treated mice were collected and immediately fixed in ice-cold 4% paraformaldehyde solution for 2 days. Semi-decalcification was carried out for 5 days in 0.5 M EDTA pH 7.4 at 4 °C with constant shaking (age ≥ 1 week), and infiltration was followed with a mixture of 20% sucrose phosphate buffer for 1 day and with 25% sucrose 12075767.1 phosphate buffer the next day. All samples were embedded in a 50 / 50 mixture of 25% sucrose solution and OCT compound (Sakura) and cut into 12-μm-thick sagittal sections using a cryostat (Leica). Immunofluorescence staining and analysis was performed as described previously. Briefly, after treatment with 0.2% Triton X-100 for 10 min, sections were blocked with 5% donkey serum at room temperature for 30 min and incubated overnight at 4 °C with anti-b- CATENIN antibody (Thermo Fisher, PA5-77934, 1:100). Primary antibodies were visualized with donkey anti-rat IgG Alexa-594 (1:500, Molecular Probes). Nuclei were counterstained with 4-6,diamidino-2-phenylindole (DAPI). An Olympus IX81 confocal microscope or Leica TCS SP5 II Zeiss LSM-880 confocal microscope was used to image samples. Scanned Electron Microscopy (SEM): Mandibular bones were isolated from one-month-old Shn3Pdgfraand Shn3fl / flmice. They were fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde in cacodylate buffer (pH 7.2) overnight at 4℃ and dehydrated through an ethanol gradient (70%–100%) and 100% Acetone, followed by embedding in Spur’s epoxy resin and polymerized at 68℃ of 48 hours. The embedded mandibles were sectioned at the first molar using a slow-speed diamond saw and finally polished using Pol Metal Polish DKK100 (Pol, Denmark). The polished surfaces were coated with 8 nm of Gold-Palladium and imaged using a ThermoFisher, Quanta 200 FESEM (Hillsborough, OR) 10 kV accelerating voltage. AAV treatment in a mouse model of postmenopausal osteoporosis: Mouse models of postmenopausal osteoporosis were generated by anesthetizing and bilaterally ovariectomizing (OVX) 3-month-old female mice (Jackson Laboratory, C57BL / 6J). Four weeks after the surgery, sham or OVX mice were IV (2.5 x 1013vector genomes [vg] / kg) or PL (2.5 x 1012vg / kg) injected with rAAV9 or dss.rAAV9 carrying egfp, amiR-ctrl, amiR-SHN3, or amiR-SOST / hs- amiR-SHN3. Seven weeks after the injection, mice were subcutaneously injected with calcein and alizarin-3-methyliminodiacetic acid at six-day intervals for dynamic histomorphometric analysis. Non-labeled mice were used to monitor EGFP expression using the IVIS-100 optical imaging on frozen sections. AAV treatment of human skeletal organoid in xenograft mice: Human BMSCs were seeded on hydroxyapatite (HA)-scaffold (Osteogene Tech), cultured under osteogenic conditions for two days, and implanted into the interscapular fat pads of 3-month-old immunodeficient SCID 12075767.1 mice. One week later, rAAV9 (2.5 x 1012vg / kg) carrying hs-amiR-ctrl or hs-amiR-hSHN3 was injected into the implantation site (FIG. 15D). Four weeks later, the implanted HA-scaffold was visualized by radiography and then, EGFP expression in the scaffold was assessed by the IVIS optical imaging system. Bone and collagen formation was assessed by microCT, histology, and SEM analyses. REPRESENTATIVE SEQUENCES >amiR-human SHN3 (SEQ ID NO 1) ggcagccttggagtgggttcctgccccctcgggcacacaaacagagctgaagaccaccctgggcacctccttggctggccgcatacctc ctggcgggcagctgtgtttccatggtaagttcaaggctgttctggtggtacccagccttgaagatgccatggaaacacagaggcctgcctg gccctcgagagactgccctgactgaaggccctatcaggtgggggaggggatcctgatagagggcactgctgccactgttggggcccaa g >amiR-mouse SHN3 (SEQ ID NO: 2) tttgtcttttatttcaggtcccagatctagggctctgcgtttgctccaggtagtccgctgctcccttgggcctgggcccactgacagccctggtg cctctggccggctgcacacctcctggcgggcagctgtgtacaaactacttgagagcaggtgttctggcaatacctgcctgctctgtaatagtt tgtacacggaggcctgccctgactgcccacggtgccgtggccaaagaggatctaagggcaccgctgagggcctacctaaccatcgtggg gaataaggacagtgtcacccctgcaggggatccggtggtggtgcaaatca >amiR-mouse SOST+human amiR-33-mouse SHN3 (SEQ ID NO: 3) gatctagggctctgcgtttgctccaggtagtccgctgctcccttgggcctgggcccactgacagccctggtgcctctggccggctgcacac ctcctggcgggcagctgtgtgacctctgtggcatcattcctgttctggcaatacctgggaatgatcgcgcagaggtcacacggaggcctgc cctgactgcccacggtgccgtggccaaagaggatctaagggcaccgctgagggcctacctaaccatcgtggggaataaggacagtgtca cccctgcaggggatccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttct gctctaaaagctgcggaattgtacccgcggccgatccaccggtcgccaccatggggcagccttggagtgggttcctgccccctcgggca cacaaacagagctgaagaccaccctgggcacctccttggctggccgcatacctcctggcgggcagctgtgtacaaactacttgagagcag gtgttctggtggtacccacctgctctgtaatagtttgtacacagaggcctgcctggccctcgagagactgccctgactgaaggccctatcag gtgggggaggggatcctgatagagggcactgctgccactgttggggcccaagaagct >amiR-human DKK1-1 (SEQ ID NO: 4) AGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGAC AGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGcaatggtctggtact tattccTGTTCTGGCAATACCTGGGAATAAGATCTAGACCATTGCACGGAGGCCTGCCC TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC CTAACCATCGTGGGGAATAAGGACAGTGTCACCC >amiR-human DKK1-2 (SEQ ID NO: 5) AGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGAC AGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGacatagcgtgacgc atgcagcTGTTCTGGCAATACCTGGCTGCATGGCTTACGCTATGTCACGGAGGCCTGCC 12075767.1 CTGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTA CCTAACCATCGTGGGGAATAAGGACAGTGTCACCC >amiR-human DKK1-3 (SEQ ID NO: 6) AGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGAC AGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGatatttctagtccatg agagcTGTTCTGGCAATACCTGGCTCTCATCCATTAGAAATATCACGGAGGCCTGCCCT GACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTACC TAACCATCGTGGGGAATAAGGACAGTGTCACCC >amiR-mouse Dkk1-1 (SEQ ID NO: 7) AGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGAC AGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGtttcaatgatgctttc ctcaaTGTTCTGGCAATACCTGTTGAGGAATCCGTCATTGAAACACGGAGGCCTGCCCT GACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTACC TAACCATCGTGGGGAATAAGGACAGTGTCACCC >amiR-mouse Dkk1-2 (SEQ ID NO: 8) AGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGAC AGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGatgctttcctcaattt cccctTGTTCTGGCAATACCTGAGGGGAAAAAGGGGAAAGCATCACGGAGGCCTGCCC TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC CTAACCATCGTGGGGAATAAGGACAGTGTCACCC >amiR-mouse Dkk1-3 (SEQ ID NO: 9) AGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGAC AGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGttccgtttgtgcttgg tgcacTGTTCTGGCAATACCTGGTGCACCATCCGCAAACGGAACACGGAGGCCTGCCC TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC CTAACCATCGTGGGGAATAAGGACAGTGTCACCC >amiR-mouse Dkk1-4 (SEQ ID NO: 10) AGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGAC AGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGtttaaggacaggttt acagatTGTTCTGGCAATACCTGATCTGTAATGCCGTCCTTAAACACGGAGGCCTGCCC TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC CTAACCATCGTGGGGAATAAGGACAGTGTCACCC >amiR-33-mSOSTi-1 (SEQ ID NO: 11) tttgtcttttatttcaggtcccAGATCTAGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCC TTGGGCCTGGGCCCACTGACAGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGC GGGCAGCTGTGAcaagtaggcagatgaggcacTGTTCTGGCAATACCTGGTGCCTCAAG TACCTACTTGTCACGGAGGCCTGCCCTGACTGCCCACGGTGCCGTGGCCAAAGAG GATCTAAGGGCACCGCTGAGGGCCTACCTAACCATCGTGGGGAATAAGGACAGTGT CACCCCTGCAGgggatccggtggtggtgcaaatca 12075767.1 >amiR-33-mSOSTi-2 (SEQ ID NO: 12) tttgtcttttatttcaggtcccAGATCTAGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCC TTGGGCCTGGGCCCACTGACAGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGC GGGCAGCTGTGtgacctctgtggcatcattccTGTTCTGGCAATACCTGGGAATGATCGCG CAGAGGTCACACGGAGGCCTGCCCTGACTGCCCACGGTGCCGTGGCCAAAGAGG ATCTAAGGGCACCGCTGAGGGCCTACCTAACCATCGTGGGGAATAAGGACAGTGTC ACCCCTGCAGgggatccggtggtggtgcaaatca >amiR-33-hSosT-1 (SEQ ID NO: 13) gtcttttatttcaggtcccagatcttAGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGC CTGGGCCCACTGACAGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGCGGGCA GCTGTGatggtcttgttgttctccagcTGTTCTGGCAATACCTGGCTGGAGATGAGCAAGACCAT CACGGAGGCCTGCCCTGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCA CCGCTGAGGGCCTACCTAACCATCGTGGGGAATAAGGACAGTGTCACCCccctgcagggg atccggtggtggtgcaaat >amiR-33-hSosT-2 (SEQ ID NO: 14) gtcttttatttcaggtcccagatcttAGGGCTCTGCGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGC CTGGGCCCACTGACAGCCCTGGTGCCTCTGGCCGGCTGCACACCTCCTGGCGGGCA GCTGTGACGtCtttGGtCtCAAAGGGGTGTTCTGGCAATACCTGCCCCTTTGTCATCAAA GACGTCACGGAGGCCTGCCCTGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAA GGGCACCGCTGAGGGCCTACCTAACCATCGTGGGGAATAAGGACAGTGTCACCCccc tgcaggggatccggtggtggtgcaaat >miR-122 / miR-208a-TS (SEQ ID NO: 15) acaagctttttgctcgtcttatacaagctttttgctcgtcttatacaagctttttgctcgtcttatacaaacaccattgtcacactccaacaaacacc attgtcacactccaacaaacaccattgtcacactcca >Lef / Tcf promoter (SEQ ID NO: 16) cgagctcttacgcgagatcaaagggggtaagatcaaagggggtaagatcaaaggggcgcgagatcaaagggggtaagatcaaagggg gtaagatcaaagggggtaagatcaaaggggcgcgcccgcgtgctagcccgggctcgagatctagactctagagggtatataatggaagc tcgaattccagcttggcattccggtactgttggtaaaaagcttggcattccggtactgttggtaaa >PB2 (NF-kB promoter) (SEQ ID NO: 17) TGAGCTCACAGAGGGGACTTTCCGAGAGATCTACAGAGGGGACTTTCCGAGAGCGA GCTTGGGCTGCAGGTCGACCGTCCATCCATTCACAGCGCTTCTATAAAGGCGCCAGC TGAGGCGCCTACTACTCCAACCGCGACTGCAGCGAGCAACTGAGAAGACTGGATAG AGCCGGCGGTTCCGCGAACGAGCAGTGACCGCGCTCCCACCCAGCTCTGCTCTGCA GCTCCACCAGTGTCTCTCTAGA >pBRE promoter (SEQ ID NO: 18) GCTTCGCGCCCTAAGTCTGCAGGTGACGGGCTCAGGGGCGGGGGCTGGGTGGGGGG GAGCGGAGAATGCTCCAGCCCAGTTTGCCGTCTCCATGGCGACCGCCCGCGCGGCG 12075767.1 CCAGCCTGACAGCCCGTCCGGGTTTTATGAATGGGTGACGTCACGGGCCTGGCGTCT AACGGTCTGAGCCGCTTGTTCAGACGCTGACACAGACCAGCCCGGGAAAGG > AAV6 CAPSID (SEQ ID NO: 19) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGG NLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRL NFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNW HCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFS DSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQML RTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLF SRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPG TAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGT VAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMG GFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNP EVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL > AAV9 CAPSID (SEQ ID NO: 20) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKR LNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNW HCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQV FTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQ MLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLK FSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMN PGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYG QVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPL MGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL >DSSx6 peptide (SEQ ID NO: 21) DSSDSSDSSDSSDSSDSS 12075767.1

Claims

CLAIMS What is claimed is:

1. A method for treating alveolar bone loss in a subject in need thereof, the method comprising administering to the jaw of a subject in need thereof an isolated nucleic acid comprising a nucleotide sequence encoding an inhibitor of a gene selected from Schnurri-3 (SHN3), sclerostin (SOST), and dickkopf-1 (DKK-1).

2. The method of claim 1, wherein the inhibitor is selected from the group consisting of dsRNA, siRNA, shRNA, miRNA, and artificial miRNA (amiRNA).

3. The method of claim 2, wherein the amiRNA comprises: (i) a miR-33 scaffold or a mir-155 scaffold; and (ii) a nucleic acid sequence having a region of complementarity with Schnurri-3 (SHN3), sclerostin (SOST), or dickkopf-1 (DKK-1).

4. The method of any one of claims 1 to 3, wherein the amiRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-14.

5. The method of any one of claims 1 to 4, wherein the isolated nucleic acid further comprises a promoter.

6. The method of claim 5, wherein the promoter is an inducible promoter operably linked to the nucleotide sequence encoding the inhibitor.

7. The method of claim 5 or 6, wherein the promoter is induced by inflammation in a subject or by mechanical stress (e.g., vibration).

8. The method of any one of claims 5 to 7, wherein the promoter comprises a NF-kB promoter (pNF-kB), a bone morphogenic protein promoter (pBRE), or an Lef / Tcf promoter.

9. The method of any one of claims 1 to 8, wherein the isolated nucleic acid further comprises one or more miRNA binding sites. 12075767.

110. The method of claim 9, wherein the one or more miRNA binding sites are miR-122 binding sites, miR208a binding sites, or a combination thereof.

11. The method of any one of claims 1 to 10, wherein the isolated nucleic acid further comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs) flanking the nucleotide sequence encoding the promoter.

12. The method of any one of claims 1 to 11, wherein the isolated nucleic acid is administered to the subject as a recombinant adeno-associated virus (rAAV).

13. The method of claim 12, wherein the rAAV comprises an AAV capsid protein selected from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 capsid protein, or a variant thereof.

14. The method of any one of claims 1 to 13, wherein the administering to the jaw of the subject comprises injection, optionally direct injection to the jaw of the subject.

15. The method of any one of claims 1 to 14, wherein the subject is a mammal, optionally a human or a mouse.

16. The method of any one of claims 1 to 15, wherein the subject has or is suspected of having osteoporosis.

17. An artificial miRNA (amiRNA) comprising: (i) a miR-33 scaffold or a mir-155 scaffold; and (ii) a nucleic acid sequence having a region of complementarity with human or mouse dickkopf-1 (DKK-1).

18. The amiRNA of claim 17, wherein the nucleic acid sequence is set forth in any one of SEQ ID NOs: 4-10. 12075767.

119. A recombinant adeno-associated virus (rAAV) vector comprising: (i) an isolated nucleic acid comprising a transgene encoding the miRNA of claim 17 or 18, flanked by AAV inverted terminal repeats (ITRs); and (ii) one or more capsid proteins.

20. The rAAV vector of claim 19, wherein the transgene further comprises a promoter.

21. The rAAV vector of claim 20, wherein the promoter is an inducible promoter operably linked to the nucleotide sequence encoding the inhibitor.

22. The rAAV vector of claim 20 or 21, wherein the promoter is induced by inflammation in a subject or by mechanical stress (e.g., vibration).

23. The rAAV vector of any one of claims 20 to 22, wherein the promoter comprises a NF- kB promoter (pNF-kB), a bone morphogenic protein promoter (pBRE), or an Lef / Tcf promoter.

24. The rAAV vector of any one of claims 19 to 23, wherein the transgene further comprises one or more miRNA binding sites.

25. The rAAV vector of claim 24, wherein the one or more miRNA binding sites are miR- 122 binding sites, miR208a binding sites, or a combination thereof.

26. A recombinant adeno-associated virus (rAAV) vector comprising: (i) the rAAV vector of any one of claims 19 to 25; and (ii) one or more AAV capsid proteins.

27. The rAAV of claim 26, wherein the one or more AAV capsid proteins are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 capsid proteins, or a variant thereof. 12075767.1

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