Antibodies and methods for treating bone loss

Abstract

The present disclosure is directed to methods of treating or rescuing bone loss related to age, disease or injury in a subject.

Description

[0001] Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0002] ANTIBODIES AND METHODS FOR TREATING BONE LOSS

[0003] This application is an International Application, which claims the benefit of priority from U.S. provisional patent application no. 63 / 712,951, filed on October 28, 2024, the entire contents of each which are incorporated herein by reference in their entireties.

[0004] For countries that permit incorporation by reference, all patents, patent applications and publications cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted being prior art.

[0005] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

[0006] GOVERNMENT INTERESTS

[0007] This invention was made with government support under AG066963 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] FIELD OF THE INVENTION

[0009] The present disclosure is directed to methods of treating or rescuing a bone disorder or condition associated with bone loss related to age, disease or injury in a subject.

[0010] BACKGROUND

[0011] Musculoskeletal diseases are skyrocketing with the increasingly aging society. Osteoporosis poses a major public health threat for over 54 million Americans as it is interrelated with high fracture rates. Osteoporosis- related hip fractures are invariably associated with significant morbidity and strikingly, a 58% mortality rate in the elderly within the first year of injury. This problem is compounded by a lack of efficient preventive and medical therapies for age-related bone disease free of major side effects. Available bone anabolic and anti-bone Docket No.: 2974214-000002-W01

[0012] Date of filing: October 28, 2025 resorptive therapies to treat or prevent osteoporosis-related bone loss come with critical limitations and known side-effects. It was found that aging, steroid use and disease lead to a pathological shift in the cellular fates of human skeletal stem cells (SSCs) impairing regeneration and decreasing bone health. The aged SSCs have lower bone-forming potential due to their skewed lineage trajectory towards fibrostromal tissues and / or maintain an undifferentiated state with altered secretory profiles. Under these conditions, individuals are prone to diseases such as osteoporosis, osteonecrosis, sustain fractures and regenerate poorly.

[0013] Recent studies have revealed adult stem cell populations within bone were targeted as a regenerative source to maintain and restore skeletal health. However, breakthroughs in stem cell based- regenerative strategies have been hampered by the inability to isolate bona fide stem cell populations. What is needed are new effective methods that leverage stem cell biology to treat or rescue a bone disorder or condition associated with bone loss related to age, disease or injury in a subject.

[0014] SUMMARY

[0015] Disclosed herein are methods of treating a bone loss disorder or condition or methods of rescuing bone loss related to age, disease or injury in a subject by targeting basigin / CD147. Also disclosed herein are methods of screening for anti-basigin / CD147 agents.

[0016] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, and / or ameliorating a bone loss disease or disorder (including, but not limited to osteoporosis; osteopenia; lactation; traumatic bone injury; pathologic bone injury; periprosthetic bone loss; osteolysis; osteonecrosis; osteomalacia; Paget’s disease of bone; osteogenesis imperfecta; rheumatoid arthritis; bone loss resulting from menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, hyperthyroidism, hyperparathyroidism, cancer (including, but not limited to multiple myeloma, bone cancer, bone metastasis); bone loss resulting from a craniofacial disorder; bone loss resulting from oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy; lactation- induced bone loss disorder, menopause-induced bone loss disorder, skeletal stem cell (SSC)- dysfunction-induced-bone loss, systemic autoimmune disease (including but not limited to systemic lupus erythematous or antineutrophil cytoplasmic antibody (ANCA)- associated vasculitis (such as, for example, granulomatosis with polyangiitis (GPA), microscopic Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA)),and steroid- induced-bone loss disorder (including but not limited to glucocorticoid-induced osteoporosis (GIOP) or glucocorticoid-induced osteonecrosis (GION) and / or bone injury) in a subject, comprising administering to the subject an agent (such as, for example, a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody (such as, for example, Gavilimomab, Meplazumab), or an antibody fragment) that inhibits Basigin / CD147 or administering to the subject a gene-editing system to a subject in need thereof, wherein the gene-editing system comprises an endonuclease (such as, for example, Cas9 enzyme) and a guide RNA (gRNA), wherein the gRNA binds a CD147 gene comprising an Osteocalcin (OCN) promoter. In some aspects, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts. In some aspects, the agent (such as, for example an anti-Basigin / CD147 antibody or antibody fragment thereof) is administered to the subject via a systemic injection. In some aspects, the method increases BMD and / or bone vascularization in the subject compared to an untreated control.

[0017] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, and / or ameliorating a bone loss disease of any preceding aspect wherein the gene-editing system further comprises an adeno-associated virus (AAV) vector (such as, for example, AAV9). In some embodiments, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts) to the subject. In some embodiments, the gene-editing system is administered to the subject via a systemic injection. In some embodiments, the method increases BMD and / or bone vascularization in the subject compared to an untreated control.

[0018] In one aspect, disclosed herein are methods of rescuing age-related skeletal degeneration in a subject, comprising measuring bone mineral density (BMD) (using methods, such as, for example, dual-energy X-ray absorptiometry (DEXA) of the subject compared to a standardized level of BMD from age-matched normal subjects; wherein the decrease in BMD compared to the standardized level of BMD from the age-matched normal subject denotes age-related skeletal Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 degeneration; and administering to the subject an agent (such as, for example, a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, a mesenchymal stem cell (MSC)-derived product (including, but not limited to, cytokines, MSC- derived osteoblasts or MSC- cell-conditioned media), an antibody (such as, for example, Gavilimomab, Meplazumab) or an antibody fragment) that inhibits Basigin / CD147 or administering to the subject a gene-editing system to a subject in need thereof, wherein the geneediting system comprises an endonuclease (such as, for example, Cas9 enzyme) and a guide RNA (gRNA), wherein the gRNA binds a CD 147 gene comprising an Osteocalcin (OCN) promoter, when a decrease in BMD is detected in the subject. In some aspects, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)- derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts. . In some embodiments, the agent (such as, for example an anti-Basigin / CD147 antibody or antibody fragment thereof) is administered to the subject via a systemic injection. In some embodiments, the method increases BMD and / or bone vascularization in the subject compared to an untreated control.

[0019] Also, disclosed herein are methods of rescuing age-related skeletal degeneration of any preceding aspect wherein the gene-editing system further comprises an adeno-associated virus (AAV) vector (such as, for example, AAV9). In some aspects, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts) to the subject. In some aspects, the geneediting system is administered to the subject via a systemic injection. In some aspects, the method increases BMD and / or bone vascularization in the subject compared to an untreated control.

[0020] In one aspect, disclosed herein are methods of increasing vascularization in a subject comprising administering to the subject an agent (such as, for example, a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody (such as, for example, Gavilimomab, Meplazumab), or an antibody fragment) that inhibits Basigin / CD147 to the subject. In some embodiments, the agent comprises an anti- Basigin / CD147 antibody or antibody fragment. In some embodiments, the method of treating Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts) to the subject. In some aspects, the gene-editing system is administered to the subject via a systemic injection. In some aspects, the method increases BMD in the subject compared to an untreated control.

[0021] Also disclosed herein are methods of engineering a bone graft, comprising contacting a stem cell (such as, for example, a skeletal stem cell (SSC), an induced pluripotent stem cell (iPSC), or an MSC) with an agent (such as, for example, a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, a mesenchymal stem cell (MSC)- derived product (including, but not limited to, cytokines, MSC-derived osteoblasts or MSC-cell- conditioned media), an antibody (such as, for example, Gavilimomab, Meplazumab), or an antibody fragment) that targets Basigin / CD147. In some aspects, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts).

[0022] In one aspect disclosed herein are methods of screening to detect an anti-basigin / CD147 antibody, comprising, contacting a skeletal stem cell (SSC) with one or more agent(s) (including, but not limited to a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, a mesenchymal stem cell (MSC)-derived product (including, but not limited to, cytokines, MSC-derived osteoblasts or MSC-cell-conditioned media), an antibody (such as, for example, Gavilimomab, Meplazumab), or an antibody fragment) that targets Basigin / CD147 and measuring fibroblast colony forming unit (CFU-F) ability of the SSC, basigin / CD147 signaling, mineralization in vitro, and / or vascularization, wherein an increase in fibroblast CFU-F ability (such as, for example, colony number and / or colony size); a decrease in basigin / CD147 signaling, increase in mineralization in vitro and / or increase in vascularization compared to a control denotes an increase in anti-basigin activity.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

[0025] FIG. 1 shows a GC-mediated anti-osteogenic, pro-endothelial, pro-myeloid shift is reversed by PTH. (A) Experimental schematic for exposing mice to continuous glucocorticoids through subcutaneous Methylprednisolone treatment at two timepoints and varying interventions. Created in BioRender. Ambrosi, T. (2025) https: / / BioRender.com / 8pmjxe4. (B) Mechanical strength test of femurs by 3 -point bending. (C) Quantification of trabecular bone volume per total volume (Tb.BV / TV) shown as percentage change compared to day-0 age-matched controls. Analysis of femur bones from n=8 biologically independent mice for Placebo and GC removal groups, n=7 for GC d-28, n=6 for GC d-56 and n=9 for GC+hPTH. (D) Representative microCT images of distal femur trabecular bone at day-28 and day-56. Data shown as mean ± SEM. Statistical testing between Placebo and other group by two-sided unpaired student t-test. *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001. (E) UMAP of femur derived single cells from experimental groups and their distinct clustering by Leiden. (F) Specific markers of Leiden clusters determining cellular identity. (G) UMAP plot showing cellular clustering labeled by experimental group. (H) Bar graphs showing relative abundance of each cell type captured for each experimental group. (I) Dotplot showing selected osteochondrogenic gene expression in mesenchymal cell subsets. (J) Global pathway enrichment analysis of top 200 expressed genes in mesenchymal cells of GC group using EnrichR. (K) Dotplots of endothelial, and (L) myeloid gene expression in vascular and hematopoietic cell types, respectively, between different experimental groups.

[0026] FIG. 2 shows GCs drive impaired skeletal stem and progenitor function and altered bone marrow blood vessel characteristics. (A) Flow cytometric based quantification of skeletal stem cell (SSC, CD45-Terl l9-Tie2-CD90-6c3-CD105-CD51+) in femurs of experimental groups. n=8 biologically independent mice for Placebo d-28 group, n=7 for GC d-28 group, n=5 for Placebo d- 56 group, n=3 for GC d-56 group, n=6 for GC removal and GC+hPTH groups. (B) In vitro osteogenesis (Alizarin Red S stain) and (C) chondrogenesis (Alcian Blue stain) assays on purified, primary SSCs. Spectrophotometric quantification of staining (right). Cells from n=3 biologically independent mice per group. (D) Flow cytometric based quantification of endothelial cell populations (CD31+) in femurs of experimental groups. n=4 biologically independent mice for Placebo d-28, n=3 for GC d-28 and d-56 groups, n=5 for Placebo d-56 and GC+hPTH groups, n=6 Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 for GC removal group. (E) Immunohistochemistry staining for Endomucin (Emcn) in bone marrow of day-56 placebo and GC treated mice as well as in 24-month-old wild type mice. Arrow heads: sinusoids. (F) Representative immunohistochemistry staining for Endomucin (Emcn) in bone marrow of day-56 experimental groups and quantification of blood vessel area (right top) and size of sinusoid lumen (right bottom) based on immunohistochemistry staining for Endomucin. n=4. Data shown as mean ± SEM. Statistical testing between Placebo and other group by two- sided unpaired student t-test. *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001. Scale bars, 20pm.

[0027] FIG. 3 shows GC alters de novo in vivo bone formation by SSCs and the niches they generate. (A) Experimental schematic of renal capsule transplants of freshly purified wild type, GFP-labeled SSCs in mice exposed to placebo, GC or GC+human PTH (hPTH 1-34). Created in BioRender. Ambrosi, T. (2025) https: / / BioRender.com / r8dp0jv. (B) Light microscopic images (top) and GFP signal of grafts formed beneath the renal capsule of transplanted mice. (C) Single cell transcriptomic analysis of formed tissue grafts displayed as UMAP of single cells clustered by Leiden for cell type association and by group (top left). SSPC: Skeletal stem and progenitor cells. (D) Tracksplot of specific markers of Leiden clusters determining cellular identity. (E) Bar graphs showing relative abundance of each cell type captured for each experimental group. (F) Dotplot showing selected osteogchondrogenic (left) genes in SSPC cluster between different experimental groups. (G) Dotplot showing SASP (senescence associated secretory phenotype) / cellular stress related gene expression in cells between different experimental groups. (H) Dotplot showing pro- endothelial and oxidative stress related gene expression between different experimental groups.

[0028] FIG. 4 shows Basigin overexpression alters human skeletal lineage dynamics that impair endothelial function. (A) Single cell transcriptomic analysis of tissue grafts from mouse SSCs exposed to placebo, GC, human PTH or GC+human PTH (hPTH 1-34) displayed as UMAP showing high expression of Basigin in SSPCs. SSPC: Skeletal stem and progenitor cells. Left bottom: Violin plot showing increased expression of Bsg in GC group across cells. (B) Representative image of tube formation assay by VeraVec endothelial cells exposed to control supernatant or supernatant of human SSCs overexpressing Basigin. (C) Quantification of ImageJ- based tube formation analysis. n=6 independent samples from two independent experiments. (D) Representative brightfield images of endothelial scratch assay (left) and its quantification (right). n=6 independent samples from three independent experiments. (E) Measurement of reactive oxygen species in cultured endothelial cells after 24h supernatant exposure. Left: representative Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 fluorescence images. Right: Quantification of fluorescence signal. n=6. Experiments with supernatant from at least two donors and two independent experiments. (F) Colony forming unit ability of primary human SSCs overexpressing control vector or Basigin. CFU-Fs stained by Crystal violet. n=6 independent replicates from cells of two biologically independent donors. (G) In vitro osteogenesis (Alizarin Red S stain) by hSSCs of the same groups. n=3 independent replicates from cells of one biological donor. (H) In vitro chondrogenesis (Alcian Blue stain) assays of the same groups. n=6 independent replicates from cells of two biologically independent donors. (I) Schematic of subcutaneous transplant approach. Created in BioRender. Ambrosi, T. (2025) https: / / BioRender.com / 4wl26h3. (J) Immunohistochemistry of Basigin (green) expression in SSC-generated grafts with corresponding quantification. n=3 biologically independent donor cells in three biologically independent mice per group. (K) Representative Alizarin Red S staining of sectioned grafts and quantification of mineralized tissue in grafts containing transplanted human SSCs as assessed by Alizarin Red S staining. n=6 sections from three biologically independent grafts. (L) Representative TRAP staining of sectioned grafts and quantification of TRAP-positive area of grafts. n=6 sections from three biologically independent grafts. (M) Representative immunohistochemistry staining for Endomucin and DAPI of sectioned graft. Small insert shows Endomucin staining without DAPI. Data shown as mean ± SEM. Statistical testing between Placebo and other group by two-sided unpaired student t-test. *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001. Scale bars, 50 pm.

[0029] FIG. 5 shows antibody blockade of Basigin rescues GC-induced endothelial and skeletal impairments in vitro. (A) Tube formation assay of human VeraVec endothelial cells treated with supernatant from different experimental groups. n=6 independent replicates per group from two independent experiments. (B) Representative brightfield images of endothelial scratch assay (top) and its quantification displayed as boxplots (bottom) after 12h. Endothelial cells were treated with supernatant of cultured hSSCs treated as shown. n=6 independent replicates from two independent experiments for Ctrl+aBsg and Bsg-OE+aBsg groups. n=12 independent replicates from three independent experiments for Bsg-OE group. Box plots with box and whiskers Min to Max. (C) In vitro osteogenesis of patient-derived human SSCs. Left: Boxplots showing quantification of Alizarin Red S staining. Right: Representative images of Alizarin Red S staining. Ctrl: control media only; Ctrl+PTH: control media with PTH 1-34 treatment 6h before media change; Ctrl+aBsg: control media with Basigin antibody treatment. Bsg-OE: Lentivirally Basigin Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 overexpressing human SSCs with control media. n=10 independent replicates of cells from two independent donors. Data shown as mean ± SEM. Statistical testing in a,c by one-way ANOVA with Fisher-LSD test, in b by Wilcoxon signed-rank test. *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001. Scale bars, 100 pm

[0030] FIG. 6 shows pharmacological blockade and genetic knockout of Basigin reverses GC- induced skeletal changes. (A) Schematic of experimental setup. Created with BioRender.com. (B) Representative immunohistochemistry images and quantification of Basigin expression in the different experimental groups. n=3 biologically independent mice per group. (C) Representative H&E stain of distal femurs of experimental groups with quantification of trabecular bone area below the growth plate region. n=5 biologically independent mice per group. (D) TRAP staining and quantification of femur bones from experimental groups. n=3 biologically independent mice per group. (E) Representative images of Endomucin-positive endothelium in the bone marrow environment. Arrows: small sinusoids. (F) Flow cytometric analysis of femur bones of experimental groups for skeletal stem cells (SSCs). n=5 biologically independent mice per group. (G) In vitro osteogenic differentiation of bone marrow stromal cells derived from experimental groups stained with Alizarin Red. n=3 biologically independent mice per group. (H) Flow cytometric analysis of blood from mice of different experimental groups showing lymphoid to myeloid ratio. (I) Flow cytometric analysis of bone marrow (BM) common myeloid progenitor cells (CMPs) from mice of different experimental groups. n=5 biologically independent mice per group. (J) Experimental schematic for GC experiments in heterozygous Basigin knockout mice. Control (Ctrl) and tamoxifen treated conditional knockout (cKO) mice were compared for their skeletal response to GC exposure. Created in BioRender. Ambrosi, T. (2025) https: / / BioRender.com / 35tbvyl. (K) Co-staining of Basigin (green) and endothelial marker CD31 (red) in bones of experimental groups after GC exposure. (L) Micro-CT analysis of trabecular bone parameters at endpoint. n=3 mice per group. (M) TRAP staining for osteoclast activity at endpoint. Average from n=3 biologically independent mice per group. Data shown as mean ± SEM. Statistical testing by one-way ANOVA with Fisher-LSD test for pharmacological approach (B-I) or by two-sided unpaired student t-test for comparison of genetic knockout groups (K-M). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars, 50 pm

[0031] FIG. 7 shows antibody blockade of Basigin in aging mice reinvigorates skeletal remodeling improving bone mass. (A) IHC of Basigin expression in tibias of 24-month-old mice. Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0032] Right: Histology -based quantification Basigin-expression cells per analyzed area. n=3 biologically independent mice per sex and group. (B) H&E staining of proximal tibia regions below the growth plate. Right: Histology-based quantification of tibial trabecular BV / TV. n=12 biologically independent female mice per group. n=9 biologically independent male mice per group. (C) DEXA bone mineral density (BMD) measurements of vertebral L5 at treatment starting day-0 and at day- 14 of treatment. (D) Micro-CT measurement of vertebral L5 BV / TV at 4-week of treatment. n=5 biologically independent mice per sex and group. (E) Representative Alizarin Red S staining of in vitro osteogenesis. Cells from n=5 independent mice per group. (F) TRAP quantification in tibia sections of experimental groups. N.Oc / BS: number of osteoclasts per bone surface. Analysis of n=4 biologically independent female mice per group. n=3 biologically independent male mice per group. (G) Graphical summary of experimental findings. Created in BioRender. Ambrosi, T. (2025) https: / / BioRender.com / 03daips. Data shown as mean ± SEM. Statistical testing between IgG control and other group by two-sided unpaired student t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars, 50 pm.

[0033] FIG. 8 shows micro-CT bone parameters of experimental groups. (A) Representative H&E staining images of metaphyseal regions of distal femurs of different experimental groups. (B) Quantification of trabecular bone thickness (Tb.Th), number (Tb.N) and spacing (Tb.Sp) shown as percentage change compared to day 0. Analysis of femur bones from n=8 biologically independent mice for Placebo and GC removal groups, n=7 for GC d-28, n=6 for GC d-56 and n=9 for GC+hPTH. (C) Representative microCT images of cortical bone at day-28 and day-56. (D) Quantification of femoral cortical thickness (Cort.Th) and cortical area (Cort.Ar.). Analysis of femur bones from n=8 biologically independent mice for Placebo d-28 and GC+hPTH groups, n=7 for GC d-28, Placebo d-56 and GC removal groups, n=6 for GC d-56. Statistical testing between Placebo and other group by two-sided unpaired student t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars, 50 pm.

[0034] FIG. 9 shows bone parameters of experimental groups. (A) Representative Calcein double labeling for each experimental group. (B) Quantification of mineral apposition rate (MAR) and bone formation rate (BFR) based on Calcein double labeling. n=4 biologically independent mice per group. (C) Quantification of osteoclast surface per bone surface (OC.s / BS) by TRAP labeling. n=3 biologically independent mice per group. (D) Quantification of bone marrow derived osteoclast formation from bones harvested at day-56 (left) and representative images thereof Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 (right). Bone marrow-derived cells from n=5 biologically independent mice per group. Data shown as mean ± SEM. Statistical testing between Placebo and other group by two-sided unpaired student t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar, 50pm.

[0035] FIG. 10 shows flow cytometric gating strategies. (A) Representative gating strategy for skeletal stem cell (SSC) and bone-cartilage-stromal-progenitor cell (BCSP) populations. SSCs are defined as CD45-Terl 19-Tie2-CD90-6c3-CD105-CD51+[CD200+], CD200 was not used in this panel since SSCs (CD45-Terl 19-Tie2-CD90-6c3-CD105-CD51+) in adult mice are CD200+ as shown in our work related to reference (17). (B) Flow cytometric based quantification of transient BCSP (CD45-Terl 19-Tie2-CD90-6c3-CD105+CD51+) in femurs of experimental groups. n=8 biologically independent mice for Placebo d-28 group, n=7 for GC d-28 group, n=5 for Placebo d- 56 group, n=3 for GC d-56 group, n=6 for GC removal and GC+hPTH groups. (C) Representative gating strategy for CD31+ endothelial cell populations. (D) Representative immunohistochemistry staining for Endomucin (Emcn) in bone marrow of day-28 experimental groups. (E) Expression of the glucocorticoid receptor gene Nr3cl in SSC-graft derived tissue that underwent single cell RNA-sequencing. (F) Matrixplot based on single cell transcriptomic readouts of tissue grafts from mouse SSCs exposed to placebo, GC, human PTH or GC+human PTH (hPTH 1-34) displaying expression of selected genes for SSPC cluster. Plotted data shown as mean ± SEM. Statistical testing between Placebo and other group by two-sided unpaired student t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars, 20pm.

[0036] FIG. 11 shows cellular composition of bone marrow and blood in anti-Basigin treated mice. (A) Representative gating strategy for hematopoietic stem and progenitor cells. CLP: common lymphoid progenitor, GMP: granulo-monocyte progenitor, CMP: common myeloid progenitor, MEP: myelo-erythroid progenitor, LSK:LinlowScal+cKit+ hematopoietic stem and progenitor cells, MPPa: multipotent progenitor a, MPPb: multipotent progenitor b, MPPc: multipotent progenitor c, pHSC:phenotypic hematopoietic stem cell, myHSC: myeloid skewed HSC, balHSC: balanced HSC. (B) Frequency of adipogenic (APC, Lin-Scal+CD24-) progenitor cells in bone marrow. n=5 biologically independent mice per group. (C) Myeloid cell fraction in mouse blood samples. n=5 biologically independent mice per group. (D) Flow cytometric analysis of hematopoietic stem and progenitor cells in bone marrow. n=5 biologically independent mice per group. (E) Frequency of SSCs and APCs in conditional knockout mice exposed to GC. n=3 biologically independent mice per group. (F) Myeloid cell fraction in blood samples of same mice. Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 n=3 biologically independent mice per group. (G) Flow cytometric analysis of hematopoietic stem and progenitor cells in bone marrow of same mice. n=3 biologically independent mice per group. Data shown as mean ± SEM. Statistical testing by one-way ANOVA with Fisher-LSD test for pharmacological approach (b-d) or by two-sided unpaired student t-test for comparison of genetic knockout groups (e-g). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0037] FIG. 12 shows cellular composition of bone marrow in anti-Basigin treated aged mice. (A) Representative immunohistochemistry of endothelial Endomucin expression (green) bone marrow of aged female and male mice treated with control IgG or anti-Basigin. (B) Flow cytometric analysis of skeletal cell populations in female and male mice. n=5 biologically independent mice per group. (C) Flow cytometric analysis of myeloid and lymphoid cell frequency in blood samples. n=5 biologically independent mice for female and male control groups. n=6 for female and male aBSG groups. (D) Flow cytometric analysis of hematopoietic stem and progenitor cell populations in bone marrow. n=5 biologically independent mice for female groups and male aBSG group. n=4 for male control group. Data shown as mean ± SEM. Statistical testing between Placebo and other group by two-sided unpaired student t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar, 100pm.

[0038] FIG. 13 provides data showing 15-week-old Col2a-Cre ERT2 x Basiging flox / x (Heterozygous) mice 5 weeks after first Tamoxifen injection (lx per day for first 5 days) at 10 weeks of age. Trabecular bone volume per total volume (BV / TV) and cortical bone volume (BV) in femurs as well as spine BV / TV (top to bottom) showing sex specific values for control (fl / fl) and heterozygous (Cre fl / +) mice. Represented images of trabecular bone and cortical bone in control and heterozygous (Mutant Het) groups are shown on continued page. n=l-2 per sex and group.

[0039] DETAILED DESCRIPTION

[0040] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0041] A. Definitions

[0042] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0043] Ranges can be expressed herein as from “about” one value, and / or to “about” another value. When such a range is expressed, another embodiment includes from the one value and / or to the other value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a data point “10” and a data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0044] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0045] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0046] An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0047] A “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0048] “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0049] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e g., tumor growth). This can be in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0050] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a event or characteristic, to stabilize or delay the development or progression of a p event or characteristic, or to minimize the chances that a event or characteristic will occur. Prevent does not require comparison to a control as it is more absolute than, for example, reduce. As used herein, something can be reduced but not prevented, but something that is reduced can also be prevented. Likewise, something can be prevented but not reduced, but something that is prevented can also be reduced. Where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0051] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e g., physician.

[0052] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0053] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0054] “Comprising” is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. “Consisting essentially of ’ when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein cannot exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of ’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0055] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be “positive” or “negative.” Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0056] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA occurs.

[0057] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0058] A “protein”, “polypeptide”, or “peptide” each refer to a polymer of amino acids and does not imply a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide. This term also includes polypeptides with post-expression modification, such as glycosylation (e.g., the addition of a saccharide), acetylation, phosphorylation, and the like.

[0059] The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides.

[0060] The term “nucleobase” refers to the part of a nucleotide that bears the Watson / Crick basepairing functionality. The most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.

[0061] The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

[0062] The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.

[0063] As used throughout, by a “subject” (or a “host”) is meant an individual. Thus, the “subject” can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non- human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject can be a mammal such as a primate or a human. Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0064] Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject.

[0065] As used herein, a “therapeutic regimen” refers to a structured treatment plan or strategy designed to improve and maintain health. A therapeutic regimen will be designed, prescribed, and / or administered by a licensed medical practitioner. The therapeutic regimen specifies the treatment dosage, the treatment scheduling, and the duration of the treatment. In some embodiments, the therapeutic regimen comprises one or more therapeutic compositions. In some embodiments, the therapeutic regimen comprises one or more therapeutic agents. In some embodiments, the therapeutic regimen comprises any combination of therapeutic compositions and therapeutic agents, such as for example the combination of an inhibitor and an antibody. In some embodiments, a therapeutic regimen comprises modifying, continuing, and / or initiating at least one therapeutic agent and / or therapeutic composition. In some embodiments, a therapeutic regimen comprises treating and / or preventing a disease, disorder, and / or condition.

[0066] “Vascularization” is the process that regulates endothelial morphology and function including vascular supply. Vascularization involves the growth of blood vessels into a tissue or organ to improve the supply of nutrients and oxygen.

[0067] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0068] B. Methods of treating bone loss and / or age-related skeletal degeneration

[0069] Available bone anabolic and anti -bone resorptive therapies to treat or prevent osteoporosis- related bone loss come with critical limitations and known side-effects. It was found that aging, steroid use and disease lead to a pathological shift in the cellular fates of human skeletal stem cells (SSCs) impairing regeneration and decreasing bone health. Skeletal stem cells (SSCs) are a type of stem cell that can self-renew and differentiate into mature bone, cartilage, and stromal cells. SSCs are found in the bone marrow, periosteum, and growth plate reserve zone. SSCs are responsible for the growth, maintenance, and repair of the skeleton. Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0070] The aged SSCs have lower bone-forming potential due to their skewed lineage trajectory towards fibrostromal tissues and / or maintain an undifferentiated state with altered secretory profiles. Under these conditions, individuals are prone to diseases such as osteoporosis, osteonecrosis, sustain fractures and regenerate poorly. Accordingly, in one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, and / or ameliorating a bone loss disease (including, but not limited to osteoporosis; osteopenia; lactation; traumatic bone injury; pathologic bone injury; periprosthetic bone loss; osteolysis; osteonecrosis; osteomalacia; Paget’s disease of bone; osteogenesis imperfecta; rheumatoid arthritis; bone loss resulting from menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, hyperthyroidism, hyperparathyroidism, cancer (including, but not limited to multiple myeloma, bone cancer, bone metastasis); bone loss resulting from a craniofacial disorder; bone loss resulting from oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy; lactation-induced bone loss disorder, menopause-induced bone loss disorder, skeletal stem cell (SSC)-dysfuncti on-induced-bone loss, systemic autoimmune disease (including but not limited to systemic lupus erythematous or antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (such as, for example, granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA)), and steroid-induced- bone loss disorder (including but not limited to glucocorticoid- induced osteoporosis (GIOP) or glucocorticoid-induced osteonecrosis (GION) and / or bone injury) in a subject comprising administering to the subject an agent (such as, for example, a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody (such as, for example, Gavilimomab, Meplazumab), or an antibody fragment that targets Basigin / CD147) that inhibits Basigin / CD147. Basigin, also called CD147 or EMMPRIN, is a multifunctional transmembrane glycoprotein that belongs to the immunoglobulin superfamily. BSG is located on chromosome 19 at pl3.3 and consists of ten exons spanning approximately 12 kb. In some embodiments, the agent comprises an anti-Basigin / CD147 antibody (such as, for example, Gavilimomab, Meplazumab) or antibody fragment. In some embodiments, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts. Some other Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 exemplary osteogenic agents are dexamethasone, ascorbic acid, P-glycerophosphate, growth factors (such as, for example, fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and platelet derived growth factors (PDGFs)) or oxysterols. In some embodiments, the methods further comprises administration of other agents used for treating bone related disorders, including but not limited to bone forming (anabolic) agents, antiresorptive agent (such as, for example, estrogen, bisphosphonates, denosumab), anti-inflammatory drugs, and steroids. In some embodiments, the agent (such as, for example an anti-Basigin / CD147 antibody (such as, for example, Gavilimomab, Meplazumab) or antibody fragment thereof) is administered to the subject via a systemic injection.

[0071] In some embodiments, the agent is encapsulated within a hydrogel matrix and administered to the subject. In some embodiments, the hydrogel matrix encapsulating the agent is administered to the subject via localized implantation. In some embodiments, the hydrogel matrix comprises a biomaterial and a crosslinker. In some embodiments, the hydrogel matrix is functionalized. Some exemplary biomaterials are polyethylene glycol (PEG), hyaluronic acid (HA), alginate, chitosan, gelatin, collagen, fibrin or cellulose. In some embodiments, the crosslinker is glutaraldehyde, epichlorohydrin, glyoxal, GPA peptide, VPM peptide, imidoester crosslinker dimethyl suberimidate, the N-Hydroxysuccinimide-ester crosslinker BS3 or formaldehyde. In some embodiments, the agent- loaded hydrogels show similar rheology properties compared with the blank hydrogels, indicating that the formation of the hydrogel was not affected by the agent encapsulation.

[0072] In some embodiments, the method increases vascularization compared to an untreated control. Some other exemplary disorders that can be treated using the methods of any of the preceding aspects are bone disorders and conditions associated with bone degeneration including but not limited to, periprosthetic bone loss, osteolysis, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, multiple myeloma, a craniofacial disorder, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0073] In some embodiments, the agent and / or the osteogenic agent are administered at a therapeutically effective dosage. In some embodiments, the agent and / or the osteogenic agent are is administered 1, 2, 3, 4, 5, 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, 50, 51, Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more times until no longer indicated as being needed. In some embodiments, the agent and / or the osteogenic agent are administered 1, 2, or 3 times a day until no longer indicated as being needed. In some embodiments, the agent and / or the osteogenic agent are administered daily. In some embodiments, the agent and / or the osteogenic agent are administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some embodiments, the agent and / or the osteogenic agent are administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some embodiments, the agent and / or the osteogenic agent are administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some embodiments, the agent and / or the osteogenic agent are administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more.

[0074] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, and / or ameliorating a bone loss disease (including, but not limited to osteoporosis; osteopenia; lactation; traumatic bone injury; pathologic bone injury; periprosthetic bone loss; osteolysis; osteonecrosis; osteomalacia; Paget’s disease of bone; osteogenesis imperfecta; rheumatoid arthritis; bone loss resulting from menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, hyperthyroidism, hyperparathyroidism, cancer (including, but not limited to multiple myeloma, bone cancer, bone metastasis); bone loss resulting from a craniofacial disorder; bone loss resulting from oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy; lactation-induced bone loss disorder, menopause-induced bone loss disorder, skeletal stem cell (SSC)-dysfuncti on- induced- bone loss, systemic autoimmune disease (including but not limited to systemic lupus erythematous or antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (such as, for example, granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA)),and steroid-induced-bone loss disorder (including but not limited to glucocorticoid-induced osteoporosis (GIOP) or glucocorticoid- induced osteonecrosis (GION) and / or bone injury) in a subject or systemic , comprising administering a gene-editing system to a subject in need thereof, wherein the gene-editing system comprises an endonuclease (such as, for example, Cas9 enzyme) and a guide RNA (gRNA), Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 wherein the gRNA binds a CD 147 gene comprising an Osteocalcin (OCN) promoter. Some other exemplary Cas endonucleases are Cas3, Cas9, CaslO, Cast 2a, and Cast 3. In some embodiments, the gene-editing system further comprises an adeno-associated virus (AAV) vector (such as, for example, AAV9). In some embodiments, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts.

[0075] Some other exemplary osteogenic agents are dexamethasone, ascorbic acid, - glycerophosphate, growth factors (such as, for example, fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and platelet derived growth factors (PDGFs)) or oxysterols. In some embodiments, the methods further comprises administration of other agents used for treating bone related disorders, including but not limited to bone forming (anabolic) agents, antiresorptive agent (such as, for example, estrogen, bisphosphonates, denosumab), anti-inflammatory drugs, and steroids. In some embodiments, the gene-editing system is administered to the subject via a systemic injection. In some embodiments, the gene-editing system and / or the osteogenic agents administered at a therapeutically effective dosage. In some embodiments, the gene-editing system and / or the osteogenic agents are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

[0076] 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,

[0077] 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,

[0078] 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,

[0079] 96, 97, 98, 99, 100, or more times until no longer indicated as being needed. In some embodiments, the gene-editing system and / or the osteogenic agents are administered 1, 2, or 3 times a day until no longer indicated as being needed. In some embodiments, the gene-editing system and / or the osteogenic agents are administered daily. In some embodiments, the gene-editing system and / or the osteogenic agents are administered every day, every 2 days, every 3 days, every 4 days, every

[0080] 5 days, every 6 days, every 7 days, or more. In some embodiments, the gene-editing system and / or the osteogenic agents are administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some embodiments, the gene-editing system and / or the osteogenic agents are administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every

[0081] 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some embodiments, the gene-editing system and / or the osteogenic Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 agents are administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more. In some embodiments, the method increases BMD and / or vascularization compared to an untreated control. Some other exemplary disorders that can be treated using the methods of any of the preceding aspects are bone disorders and conditions associated with bone degeneration including but not limited to, periprosthetic bone loss, osteolysis, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, multiple myeloma, a craniofacial disorder, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0082] In one aspect, disclosed herein are methods of rescuing age-related skeletal degeneration in a subject, comprising measuring bone mineral density (BMD) (using methods, such as, for example, dual-energy X-ray absorptiometry (DEXA) of the subject compared to a standardized level of BMD from age-matched normal subjects; wherein a decrease in BMD relative to a standardized level of BMD from age-matched normal subjects denotes age-related skeletal degeneration; and administering to the subject an agent (such as, for example, a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody (such as, for example, Gavilimomab, Meplazumab), a mesenchymal stem cell (MSC)- derived product (including, but not limited to, cytokines, MSC-derived osteoblasts or MSC-cell- conditioned media) or an antibody fragment that targets Basigin / CD147) that inhibits Basigin / CD147, when a decrease in BMD is detected in the subject. In some embodiments, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts. Some other exemplary osteogenic agents are dexamethasone, ascorbic acid, P-glycerophosphate, growth factors (such as, for example, fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and platelet derived growth factors (PDGFs)) or oxysterols. In some embodiments, the methods further comprises administration of other agents used for treating bone related disorders, including but not limited to bone forming (anabolic) agents, antiresorptive agent (such as, for example, estrogen, bisphosphonates, denosumab), anti- inflammatory drugs, and steroids. In some embodiments the Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 agent (such as, for example an anti- Basigin / CD147 antibody (such as, for example, Gavilimomab, Meplazumab) or antibody fragment thereof) is administered to the subject via a systemic injection. In some embodiments, the agent and / or the osteogenic agent are administered at a therapeutically effective dosage. In some embodiments, the agent and / or the osteogenic agent are administered 1, 2, 3, 4, 5, 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,

[0083] 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,

[0084] 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

[0085] 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more times until no longer indicated as being needed. In some embodiments, the agent and / or the osteogenic agent are administered 1, 2, or 3 times a day until no longer indicated as being needed. In some embodiments, the agent and / or the osteogenic agent are administered daily. In some embodiments, the agent and / or the osteogenic agent are administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some embodiments, the agent and / or the osteogenic agent are administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some embodiments, the agent and / or the osteogenic agent are administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some embodiments, the agent and / or the osteogenic agent are administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more. In some embodiments, the method increases BMD and / or vascularization compared to an untreated control. Some other exemplary disorders that can be treated using the methods of any of the preceding aspects are bone disorders and conditions associated with bone degeneration including but not limited to, periprosthetic bone loss, osteolysis, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, multiple myeloma, a craniofacial disorder, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0086] In one aspect, disclosed herein are methods of rescuing age-related skeletal degeneration in a subject, comprising measuring bone mineral density (BMD) (using methods, such as, for example, dual-energy X-ray absorptiometry (DEXA) of the subject compared to a standardized level of BMD from age-matched normal subjects; wherein a decrease in BMD relative to a standardized level of BMD from age-matched normal subjects denotes age-related skeletal Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 degeneration; and administering a gene-editing system to a subject in need thereof, wherein the gene-editing system comprises an endonuclease (such as, for example, Cas9 enzyme) and a guide RNA (gRNA), wherein the gRNA binds a CD147 gene comprising an Osteocalcin (OCN) promoter. Some other exemplary Cas endonucleases are Cas3, Cas9, CaslO, Casl2a, and Casl3. In some embodiments, the gene- editing system further comprises an adeno-associated virus (AAV) vector (such as, for example, AAV9). comprising an endonuclease and a guide RNA (gRNA), when a decrease in BMD is detected in the subject, wherein the gRNA binds a CD 147 gene comprising an Osteocalcin (OCN) promoter. In some aspects, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)- derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts. Some other exemplary osteogenic agents are dexamethasone, ascorbic acid, 0- glycerophosphate, growth factors (such as, for example, fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and platelet derived growth factors (PDGFs)) or oxysterols. In some embodiments, the methods further comprises administration of other agents used for treating bone related disorders, including but not limited to bone forming (anabolic) agents, antiresorptive agent (such as, for example, estrogen, bisphosphonates, denosumab), anti-inflammatory drugs, and steroids. In some embodiments, the gene-editing system is administered to the subject via a systemic injection. In some embodiments, the gene-editing system and / or the osteogenic agents administered at a therapeutically effective dosage. In some embodiments, the gene-editing system and / or the osteogenic agents are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

[0087] 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,

[0088] 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,

[0089] 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more times until no longer indicated as being needed. In some embodiments, the gene-editing system and / or the osteogenic agents are administered 1, 2, or 3 times a day until no longer indicated as being needed. In some embodiments, the gene-editing system and / or the osteogenic agents are administered daily. In some embodiments, the gene-editing system and / or the osteogenic agents are administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some embodiments, the gene-editing system and / or the osteogenic agents are administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 embodiments, the gene-editing system and / or the osteogenic agents are administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some embodiments, the gene-editing system and / or the osteogenic agents are administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more. In some embodiments, the method increases BMD and / or vascularization compared to an untreated control. In some embodiments, the method increases BMD and / or vascularization compared to an untreated control. Some other exemplary disorders that can be treated using the methods of any of the preceding aspects are bone disorders and conditions associated with bone degeneration including but not limited to, periprosthetic bone loss, osteolysis, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, multiple myeloma, a craniofacial disorder, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0090] In one aspect, disclosed herein are methods of increasing vascularization in a subject, comprising administering an agent (such as, for example, a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody (such as, for example, Gavilimomab, Meplazumab), or an antibody fragment) that inhibits Basigin / CD147 to the subject. In some embodiments, the method of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)- derived osteoblasts. Some other exemplary osteogenic agents are dexamethasone, ascorbic acid, 0- glycerophosphate, growth factors (such as, for example, fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and platelet derived growth factors (PDGFs)) or oxysterols. In some embodiments, the methods further comprises administration of other agents used for treating bone related disorders, including but not limited to bone forming (anabolic) agents, antiresorptive agent (such as, for example, estrogen, bisphosphonates, denosumab), anti-inflammatory drugs, and steroids. In some embodiments, the agent (such as, for example an anti-Basigin / CD147 antibody (such as, for example, Gavilimomab, Meplazumab) or antibody fragment thereof) is administered to the subject via a systemic injection. In some embodiments, the agent and / or the osteogenic agent are administered at a therapeutically Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 effective dosage. Tn some embodiments, the agent and / or the osteogenic agent are administered 1, 2, 3, 4, 5, 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,

[0091] 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,

[0092] 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

[0093] 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more times until no longer indicated as being needed. In some embodiments, the agent and / or the osteogenic agent are administered 1, 2, or 3 times a day until no longer indicated as being needed. In some embodiments, the agent and / or the osteogenic agent are administered daily. In some embodiments, the agent and / or the osteogenic agent are administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some embodiments, the agent and / or the osteogenic agent are administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some embodiments, the agent and / or the osteogenic agent are administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some embodiments, the agent and / or the osteogenic agent are administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more. In some embodiments, the method increases BMD in the subject compared to an untreated control. Some other exemplary disorders that can be treated using the methods of any of the preceding aspects are bone disorders and conditions associated with bone degeneration including but not limited to, periprosthetic bone loss, osteolysis, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, multiple myeloma, a craniofacial disorder, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0094] Also disclosed herein are methods of engineering a bone graft, comprising contacting a stem cell (such as, for example, a skeletal stem cell (SSC), an induced pluripotent stem cell (iPSC), or an MSC) with an agent (such as, for example, a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody (such as, for example, Gavilimomab, Meplazumab), a mesenchymal stem cell (MSC)-derived product (including, but not limited to, cytokines, MSC-derived osteoblasts or MSC-cell-conditioned media), or an antibody fragment that targets Basigin / CD147). In some embodiments, the method Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 of treating further comprises administration of an osteogenic agent (such as, for example, Parathyroid Hormone (PTH) (1-34), mesenchymal stem cell (MSC)-derived osteoblasts, skeletal stem cells (SSC), or induced pluripotent stem cell (iPSC)-derived osteoblasts. Some other exemplary osteogenic agents are dexamethasone, ascorbic acid, P-glycerophosphate, growth factors (such as, for example, fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and platelet derived growth factors (PDGFs)) or oxysterols. In some embodiments, the methods further comprises administration of other agents used for treating bone related disorders, including but not limited to bone forming (anabolic) agents, antiresorptive agent (such as, for example, estrogen, bisphosphonates, denosumab), anti-inflammatory drugs, and steroids.

[0095] Also disclosed herein are methods of screening to detect an anti-basigin / CD147 antibody (such as, for example, Gavilimomab, Meplazumab), comprising, contacting a skeletal stem cell (SSC) with one or more agent(s) (including, but not limited to a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody (such as, for example, Gavilimomab, Meplazumab), a mesenchymal stem cell (MSC)-derived product (including, but not limited to, cytokines, MSC-derived osteoblasts or MSC-cell-conditioned media), an antibody (such as, for example, Gavilimomab, Meplazumab)or an antibody fragment) that targets Basigin / CD147) and measuring fibroblast colony forming unit (CFU-F) ability of the SSC, basigin / CD147 signaling, mineralization in vitro, and / or support of vascularization, wherein an increase in fibroblast CFU-F ability (such as, for example, colony number and / or colony size); a decrease in basigin / CD147 signaling, increase in mineralization in vitro and / or increase in vascularization support compared to a control denotes an increase in anti-basigin activity.

[0096] Antibodies

[0097] The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with CD 147. The antibodies can be tested for their activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and / or prophylactic activities are tested according to known clinical testing methods. There are five major Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 classes of human immunoglobulins: IgA, IgD, IgE, TgG and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2.

[0098] One skilled in the art can recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0099] The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that can be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a species or belonging to a antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the antagonistic activity.

[0100] The disclosed monoclonal antibodies can be made using any procedure which produces monoclonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal can be immunized with an immunizing agent to elicit lymphocytes that produce or can produce antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0101] The monoclonal antibodies can also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can bind specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

[0102] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, such as Fab fragments, can be accomplished using routine Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94 / 29348 published Dec. 22, 1994, and U.S. Pat. No. 4,342,566. Papain digestion of antibodies can produce two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of crosslinking antigen.

[0103] As used herein, the term “antibody or fragments thereof’ encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, sFv, scFv, bispecific T cell engager (BiTE), nanobodies, diabodies, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain CD 147 binding activity are included within the meaning of the term “antibody or fragment thereof.” Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory ManuaX. Cold Spring Harbor Publications, New York, (1988)).

[0104] Also included within the meaning of “antibody or fragments thereof’ are conjugates of antibody fragments and antigen binding proteins (single chain antibodies).

[0105] The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove / add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment can be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992). Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0106] As used herein, the term “antibody” or “antibodies” can also refer to a human antibody and / or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

[0107] Human antibodies

[0108] The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the activity are selected using Env-CD4-co-receptor complexes as described herein.

[0109] Humanized antibodies

[0110] Antibody humanization techniques involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.

[0111] To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies can also contain residues which are found neither in Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 the recipient antibody nor in the imported CDR or framework sequences. A humanized antibody has one or more amino acid residues introduced into it from a source which is non-human.

[0112] In practice, humanized antibodies can be human antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0113] Humanized antibodies contain at least a portion of an antibody constant region (Fc), such as that of a human antibody (Jones et al., Nature, 321 :522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

[0114] Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S. Patent No. 5,721,367 (Kay et al.), U.S. Patent No. 5,837,243 (Deo et al.), U.S. PatentNo. 5, 939,598 (Kucherlapati et al.), U.S. Patent No. 6,130,364 (Jakobovits et al.), and U.S. Patent No. 6,180,377 (Morgan et al.).

[0115] Administration of antibodies

[0116] Administration of the antibodies can be done as disclosed herein. Nucleic acid approaches for antibody delivery also exist. The anti-Basigin / CD147 antibodies (such as, for example, Gavilimomab, Meplazumab) and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient’s or subject’s own cells, that express the CD147 gene under the control of the Osteocalcin (OCN) promoter, take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment. In some embodiments, the anti-Basigin / CD147 antibody (such as, for example, Gavilimomab, Meplazumab) is administered to the subject via a systemic injection.

[0117] C. Examples

[0118] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations can be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0119] Example 1: Basigin Links Altered Skeletal Stem Cell Lineage Dynamics with Glucocorticoid- induced Bone Loss and Impaired Angiogenesis

[0120] Glucocorticoid (GC) induced osteoporosis (GIOP) and osteonecrosis remain a significant health issue with few approved therapies. Here, we investigate the cellular and molecular processes by which GCs affect osteogenesis and angiogenesis. We find that GC treatment reduces bone mass through decreased bone formation by skeletal stem cells (SSCs). Concomitantly, endothelial cells increase in numbers but display distorted phenotypical features. Transplantation studies of SSCs combined with molecular analysis by single cell RNA-sequencing and functional testing of primary human cells tie GC-induced skeletal changes to altered stem cell differentiation dynamics. This in turn perpetuates reduced osteogenesis and vascular malformation through direct SSC- endothelial crosstalk mediated at least in part by Basigin. The genetic deletion of Basigin in the skeletal lineage as well as antibody -mediated blockade of Basigin during GC treatment prevents bone loss. Intriguingly, when administered to 2-year-old mice, anti-Basigin therapy reinstates bone remodeling to significantly improve bone mass. These findings provide new therapeutic vantage points for GIOP and potentially other conditions associated with bone loss.

[0121] Introduction: Glucocorticoids (GCs) are potent anti-inflammatory compounds, however, continued exposure results in bone loss and osteonecrosis1. Since SSCs are crucial for maintaining skeletal homeostasis, it is tempting to speculate that GCs can affect their function. Recently, detrimental changes to angiogenesis have also been implicated in GC-induced bone loss2,3. Nevertheless, the mechanism by which GCs alter vascularity remains unknown. Our study aimed to evaluate GC-effects on both osteogenesis and angiogenesis, as well as if treatment with parathyroid hormone (hPTH 1-34) modifies the effects in a mouse model of GC induced bone loss.

[0122] GCs are frequently prescribed for both acute and chronic medical conditions to reduce inflammation and immune system activity. GCs are known to alter bone remodeling by reducing osteoblast activity, stimulating osteoclast maturation and activity and reducing gonadal hormone production which indirectly activates osteoclastogenesis1. Bone loss induced by GCs tends to have two phases, with an initial rapid loss of bone mass, such as trabecular, followed by a sustained Docket No.: 2974214-000002-W01

[0123] Date of filing: October 28, 2025 gradual reduction that results in the loss of cortical bone. Independent of the GC-induced loss of bone mass, subjects treated with GCs often rapidly reduce mechanical strength of bone such that many subjects experience fractures while treated4.

[0124] After the initial stages of GC-induced bone loss, a continued suppression of bone formation has been reported. This is mainly due to reduced activity of osteoblasts, the differentiated cells that are responsible for bone formation derived from a pool of skeletal stem cells (SSCs). hPTH 1-34, also known as teriparatide, is FDA-approved to treat GC-induced osteoporosis as it stimulates osteoblasts to generate bone in the presence of GCs5,6, decreases sclerostin production by osteocytes and prevents osteoclast maturation in the presence of GCs7.

[0125] GC exposure also can induce osteonecrosis, the result of damaged skeletal vasculature that provides essential nutrients for normal cell function and tissue homeostasis. This adverse event can be associated with higher cumulative doses and longer treatment courses of systemic GCs, however, it has also been described after intra-articular injections, topical administration or low- dose, short-term oral steroids3. No major gender differences have been identified. While the exact etiology of osteonecrosis is not clear, reduction in the vascular supply to the proximal femur is frequently observed. Angiogenesis is required for the osteogenic process, as endothelial cell invasion is needed to transition hypertrophic chondrocytes to osteoblasts at the growth plate, and angiogenesis through macrophage initiated signaling is needed for the formation of a bone remodeling unit. However, the influence of GCs on angiogenesis is unclear8.

[0126] Therefore, given the observation that GCs reduce bone formation and can alter vascular supply directly or indirectly to bone, the purpose of this study was to increase our understanding of how GCs affect the cellular and molecular components of the bone marrow environment. We performed a detailed analysis of the cellular composition, SSC activity and single cell gene expression in mice treated with GCs, after recovery from GCs and GC with hPTH 1-34 treatment. We determined that bone loss through GC exposure is initially associated with a reduced number of SSCs and vascular progenitor cells, while with continued GC exposure skeletal precursor cells accumulated but lost their ability to differentiate into osteogenic and chondrogenic cell types. Interestingly, these SSCs secrete specific factors, including Basigin, that alter endothelial morphology and function through direct crosstalk. Concomitant hPTH 1-34 during GC exposure or genetic and antibody-mediated Basigin blockade abrogated that detrimental signaling axis and reversed the GC-mediated phenotype. Excitingly, anti-Basigin treatment also improved bone Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 parameters in aged mice indicating a potential broader clinical utility. Altogether, these findings reveal a previously unappreciated connection between stem and endothelial cell interaction that can be targeted to prevent GC -induced bone loss.

[0127] Results:

[0128] GC-induced bone loss is reversed by concurrent hPTH treatment. To assess the cellular and molecular changes of continuous GC exposure on bone tissue, we subcutaneously transplanted 4-month-old, male Balb / cJ mice with 5 mg Methylprednisolone (GC) or placebo release pellets. Randomly grouped mice were housed for 28 days before analysis. To assess the consequences of prolonged GC treatment, additional animals were kept for another 28 days and compared to two separate groups having GC pellets removed after 28 days or animals receiving hPTH 1-34 at 40 pg / kg, 5 days / week during GC exposure throughout the experiment (FIG.1A). As expected from our previous study using a similar model9, micro-CT analyses and mechanical testing showed that 28- and 56-day GC exposure significantly reduced femoral trabecular bone parameters and bone strength, respectively, compared to placebo control (FIG.1B-D & FIG. 8A-B). Interestingly, removing GC pellets after 28 days did not reverse trabecular bone loss after an additional 28 days, while hPTH 1-34 in the presence of GC exposure normalized trabecular parameters to placebo levels. Cortical thickness and area were significantly reduced upon acute GC exposure but were unaltered in groups monitored over a 56-day period (FIG. 8C-D). To test how these observations were tied to changes in bone remodeling, we conducted dynamic histomorphometry and found that bone formation and osteoclast activity were inversely controlled by GCs. Mineral apposition and bone formation rate were significantly reduced by GC treatment at both investigated timepoints and only hPTH 1-34 treatment improved those parameters to placebo control levels (FIG. 9A-C). In contrast, osteoclast activity was strongly increased upon GC exposure as measured by TRAP staining and bone marrow-derived in vitro osteoclastogenesis; however, GC removal alone was sufficient to reduce bone resorption activity to control levels (FIG. 9C-D). Osteoclast surface per bone surface was elevated in GC mice that received hPTH 1-34 indicating stimulation of increased bone remodeling with high bone formation rates outweighing increased bone resorption. Altogether, these data provide insight into the complex effects of GC and hPTH 1-34 actions on bone parameters of our mouse model. Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0129] GC-exposure drives distinct cellular and molecular changes in hones : Having establi shed our model of GC-induced bone loss, we next sought to derive a more detailed view of the cellular changes of bone tissue. We conducted 10X Chromium single cell RNA-sequencing (scRNAseq) of dissected femurs from the five experimental groups on day 56. Given the low representation of non-hematopoietic cells in single cell preparations of bone tissue, we sorted equal numbers of CD45-positive and CD45-negative cell population into the same collection tube for each group. Unbiased Leiden clustering analysis of stringently quality filtered single cells established the cellular composition of captured cells for each experimental group (FIG.l E-F). This approach covered the broad heterogeneous composition of the bone / bone marrow (BM) composition including hematopoietic, mesenchymal and endothelial cell types. When we investigated specific differences in the cellular make up of each group, we observed that GCs increased stromal cell populations in the BM, including CXCL12 expressing reticular (CAR) cells (FIG. 1G-H). There were also slight changes to committed bone forming cell types, indicating potential alterations to mesenchymal lineage allocation commitment. Specifically, expression of genes associated with osteogenesis and chondrogenesis were reduced upon GC exposure but showed strong improvement to placebo levels if mice were simultaneously treated with hPTH 1-34. Conducting global pathway enrichment analysis with the top 200 differentially expressed genes for each group, revealed that GC exposure led to increases in angiogenesis-related signaling in the BM environment (FIG. 1J). Specific gene expression patterns of endothelial genes showed that GC removal and hPTH 1-34 treatment normalized the expression to placebo levels (FIG. IK). Similarly, while the immune cell compartment composition was not strongly altered by GC exposure, we observed an increase in pro-myeloid and pro-osteoclastic signaling that was partially reversed by GC removal and hPTH 1-34 treatment (FIG. IL). In sum, scRNAseq of the bone tissue from the different experimental groups revealed distinct cellular and molecular changes of the BM compartment supportive of alterations at the tissue level. Strikingly, the strongest changes were associated with alterations in angiogenic signaling.

[0130] GCs alter skeletal stem and progenitor function and blood vessel characteristics : Since we observed bone loss and reduced expression of osteochondrogenic genes in mice exposed to GC, we wondered if skeletal stem and progenitor activity can be altered. Therefore, we analyzed the frequency of phenotypic SSCs (CD45-Terl 19-Tie2-CD90-6c3-CD105-CD51+[CD200+]) and the directly downstream transient multipotent bone-cartilage-stromal progenitors (BCSPs; CD45- Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 Teri 19-Tie2-CD90-6c3-CD105+CD51+) using flow cytometry10. Results showed that GCs initially (day 28) decreased SSC and BCSP numbers, while extended exposure (day 56) drove an increase in SSCs (FIG. 2A & FIG. 10A-B). However, GC removal led to a significant accumulation of SSCs and BCSP compared to placebo controls. Again, only GC with concomitant hPTH 1-34 normalized skeletal progenitor abundance to control levels. To further assess functional properties of SSCs, we freshly purified them from bone tissue of each experimental group on day 56 and seeded them for standard in vitro osteogenic and chondrogenic assays. In alignment with micro-CT and transcriptomic data, GCs significantly reduced osteogenic and chondrogenic potential of skeletal progenitors compared to placebo controls which was also not altered upon GC removal assessed after 28 days (FIG. 2B). SSCs from bones of mice exposed to GC but treated with hPTH 1-34 injections showed strong osteochondrogenic activity. These results indicate that skeletal stem and progenitor function is negatively affected by GCs, directly associating it to negative structural and mechanical changes observed for bones.

[0131] Based on scRNAseq results, we also wondered how endothelial numbers and composition maybe altered. Flow cytometric analysis revealed a similar pattern as seen with SSCs. While after 28 days, endothelial cell numbers were reduced with GCs compared to the placebo group, longer GC exposure drove an accumulation of endothelial cell presence that was only normalized to placebo levels when mice received hPTH 1-34 with GCs (FIG. 2D & FIG. 10C). When we stained histological sections of femur bones after 56 days of GC exposure with the endothelial marker Endomucin, we observed an increase of blood vessel abundance as well as distinct morphological changes that resembled the endothelial compartment of aged (24-month-old) mice (FIG. 2E). Previous work has reported that cellular stress in the BM environment is connected to increased numbers of sinusoids with dilated lumina11. Quantitative analyses showed that at day 56 of GC exposure there were significantly more Endomucin-positive sinusoidal blood vessels with increased lumen area in the BM compared to placebo controls (FIG. 2F & FIG. 10D). The blood vessel area and dilation of lumens remained at placebo levels when mice were concomitantly treated with hPTH 1-34. In summary, GCs mediate specific changes to bone-forming lineage cells and angiogenic processes that can be reversed by co-stimulation with hPTH 1-34 treatment, indicating a direct connection between SSCs and endothelial cells. Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0132] GCs alter SSC-mediated ossicle formation. To determine if GC-mediated alterations were connected through changes observed in SSC and endothelial cell activity, we assessed SSC- specific bone formation characteristics via renal capsule transplantation12. To that end, we freshly FACS purified equal numbers of SSCs from ubiquitous GFP-reporter mice and transplanted them beneath the renal capsule of wild type C57BL / 6 mice. We randomly assigned mice to four groups, i.e., a control group receiving placebo pellets at the time of surgery, a group with hPTH 1-34 treatments but no GCs as well as two groups receiving GC release pellets. One of the GC groups received 5x weekly hPTH 1-34 over the 21 days of the experiment (FIG. 3A). At that time, we isolated tissue grafts and processed them for scRNAseq. Generated ossicles of groups contained mesenchymal, endothelial and hematopoietic cell types recognized by distinct single cell clustering based on overall gene expression profiles (FIG. 3B-E). Given limitations of scRNAseq approaches, including transcriptomic coverage, preventing faithful separation of rare, homogeneous SSC populations using unbiased clustering analysis, we identified the SSC-enriched cluster by known but not unique marker expression (e.g., Sox9, Col2al, Pthlh) within a broader pool of skeletal stem and progenitor cells (SSPCs). These SSPCs also showed high expression of the gene Nr3cl encoding the glucocorticoid receptor compared to other mesenchymal lineage cells (FIG. 10E) Mice treated with GCs alone presented with a higher number of undifferentiated SSPCs than placebo treated mice. The expression of osteogenic and chondrogenic gene programs also decreased with GC treatment alone but was rescued by hPTH 1-34 treatment (FIG. 3F). Analyzing gene expression patterns of cells revealed that GC exposure led to high expression of genes related to cellular stress including those associated with the senescence-associated secretory phenotype (SASP) and oxidative stress, supporting the idea that GCs appear to drive an aging-like and pro-inflammatory microenvironment (FIG. 3G). Interestingly, genes involved with blood vessel recruitment and formation were highly increased in the presence of GCs which also was associated with increased oxidative stress gene expression (FIG. 3H). Once again, hPTH 1-34 added to the GC treatment was able to reverse gene expression patterns towards placebo control levels. Of note, higher circulating levels of hPTH 1-34 elevated expression of SASP / cellular stress related genes compared to the placebo group. Slight increases in those markers can be the reflection of a more activated state of hPTH 1-34 exposed cells rather than increased inflammation. Since SSC derived ossicle formation strongly depends on host endothelial blood vessel recruitment, these Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 results support a co-regulation of SSC and endothelial lineages during GC exposures and provided a list of molecular interaction partners.

[0133] GC-induced SSC derived Basigin impairs bone forming and vascular network properties. Among the multiple pro-endothelial genes upregulated upon GC exposure and rescued by hPTH 1-34 treatment (Kdr / Vegfr, Vegfa, Pdgfa, Wnkl), we identified Basigin (Bsg) as well as its known intracellular interaction partners of the monocarboxylate transporter (Mctl-3) family to be highly expressed in accumulated SSPCs (FIG. IK, FIG. 3H, FIG. 4A & FIG. 10F)13. We therefore hypothesized that Basigin, a known activator of cell proliferation14 13, can drive GC- mediated aberrations in the BM environment through a distinct SSC-endothelial signaling axis. To further explore this concept and its translational relevance, we conducted functional tests in primary human SSCs (hSSCs) and in the human VeraVec HUVEC endothelial cell line. We collected 48h supernatant from hSSCs lentivirally overexpressing Basigin or vector controlled to directly tie SSPC derived Basigin expression to changes in endothelial cell activity. Then, we assessed the effect of the supernatants of both groups during in vitro tube formation and wound scratch assays of VeraVec endothelial cells. Strikingly, in the presence of high levels of Basigin in the supernatants, blood vessel architecture was significantly impaired, as indicated by reduced mesh, tube and node numbers compared to the control (FIG. 4B-C). We also found that Basigin impaired endothelial cell migration in a wound scratch assay (FIG. 4D). In addition, elevated Basigin levels increased ROS generation in cultured endothelial cells (FIG. 4E), confirming scRNAseq readouts (FIG. 3G,H). Furthermore, overexpression of Basigin in hSSCs drove increased colony forming ability, is a measure of proliferative activity, while it impaired in vitro osteogenic and chondrogenic differentiation compared to controls (FIG. 4F-H). Finally, we subcutaneously transplanted equal numbers of hSSCs overexpressing Basigin or control into immunodeficient NOD scid gamma (NSG) mice to determine if ossicles formed displayed differences in osteogenesis and angiogenesis in vivo (FIG. 4I-J). In line with a recent report on the effect of GCs on fracture healing16, we observed reduced bone remodeling dynamics in grafts derived from Basigin-overexpressing cells that were less mineralized and showed low bone resorption activity by host-derived osteoclasts (FIG. 4K-L). Interestingly, the shortened morphology of recruited blood vessels of the Basigin-SSC grafts mirrored the GC-induced phenotype observed in femurs (FIG. 4M). These results establish a direct connection between Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 SSC-derived Basigin and detrimental effects of skeletogenesis and blood vessel architecture caused by GCs that are transferable to human cells.

[0134] Antibody-mediated blockade of hSSC-derived Basigin reverses impaired endothelial function and bone formation in vitro. Next, we investigated whether the negative effects of Basigin-overexpression in human SSCs can be pharmacologically reversed. To that end, we treated human SSCs with a monoclonal antibody against Basigin (aBSG) and collected supernatant to test its effect on vascular modeling. Indeed, the detrimental paracrine effect on human endothelial tube formation in the presence of supernatant from Basigin-overexpressing SSCs was mostly returned to control levels with antibody treatment (FIG. 5A). Similarly, VeraVec cells exposed to Basigin and treated with aBSG performed similarly to controls in wound scratch assays (FIG. 5B). Strikingly, the Basigin induced osteogenesis-impairing effects due to overexpression in SSCs were rescued when we exposed the cells to hPTH 1-34 or aBSG (FIG. 5C), supporting Basigin antibody blockade as a new strategy to prevent or reverse GC-induced SSC dysfunction mediated bone loss.

[0135] Pharmacological and genetic ablation of Basigin in vivo prevents detrimental bone loss induced by GCs To confirm our in vitro findings, we next tested whether antibody blockade of Basigin can prevent GC-induced bone loss in mice. We compared placebo treated control mice with mice that were exposed to GCs for 28 days. Of these GC-treated mice we looked at mice receiving no additional therapy or treated with hPTH 1-34 or aBSG throughout that time period (FIG. 6A). Histological analyses of femoral bone sections at day 28 showed that hPTH 1-34 and aBSG treatments reduced Basigin expression seen in the GC only group (FIG. 6B). Also, the reduction in Basigin levels in these treatment groups correlated with trabecular bone volume, osteoclast numbers and bone marrow endothelial morphology of placebo controls (FIG.6 C-E), prevented the GC-induced short-term reduction in SSC frequency (FIG. 2A) and maintained their in vitro osteogenic potential (FIG. 6F-G). While we observed a GC-driven shift towards an increase in circulating myeloid cell types in blood which was reversed by aBSG treatment, we did not detect any significant differences on the hematopoietic stem and progenitor compartment in the bone marrow (FIG. 6H-I & FIG. 12).

[0136] To strengthen our findings and tie GC-induced bone loss specifically to skeletal stem and progenitor cell derived Basigin we crossed Collagen 2al (Col2al)-CreERT2 with Basiginflox / flox (Bsgfl / fl) mice to generate conditional knockout mice lacking Basigin expression in SSPCs. Our Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 previous studies17,18and current results (FIG. 3D) indicated SSPCs, but not endothelial or immune cell types express, Col2al. Female Col2al-CreERT2 x Bsgfl / - transgenic mice were tamoxifen- induced when they reached 8 weeks of age and implanted with 4-week GC release pellets one week later (FIG. 6J). Compared to vehicle treated (com oil) control mice, heterozygous SSPC- specific knockout mice presented with significantly reduced Basigin levels upon GC exposure (FIG. 6K). This reduction was accompanied by significantly higher trabecular bone parameters and reduced osteoclast numbers indicating that a reduction in expression of Basigin from SSPCs was sufficient to prevent GC induced bone loss (FIG. 6L-M & FIG. 12E-G). Thus, administration of aBSG during GC treatment or conditional genetic ablation specifically in the skeletal stem and progenitor lineage prior to GC exposure prevents skeletal maladaptation.

[0137] Basigin antibody therapy improves bone mass in aged mice independent of sex. Since Basigin has been reported to be a therapeutic target for a number of pro-inflammatory and pro- fibrotic conditions, we asked whether aBSG can also reverse age-related bone loss, i.e., osteoporosis19,20. Indeed, immunohistochemistry analysis showed higher expression of Basigin in bone marrow of old mice, in particular in females (FIG. 7A). When we treated 2-year-old female and male mice with aBSG three times / week at 1 mg / kg for four weeks we found a significant improvement in volumetric trabecular bone of long bones compared to IgG control treated mice (FIG. 7B-C). Vertebral L5 bone mineral density at 14 days and BV / TV at 28 days of treatment measured by DEXA and micro-CT, respectively, demonstrated an anabolic effect on the skeleton by aBSG independent of sex (FIG. 7C-D). Interestingly, SSCs of the same mice showed higher in vitro osteogenic potential whereas histological analysis indicated an increase in bone resorptive activity with aBSG (FIG. 7E-F). This was accompanied by endothelial restoration but no detectable changes in the frequency of skeletal and hematopoietic stem and progenitor cells (FIG. 12). Collectively, these results indicated that aBSG treatment boosts bone remodeling in aged mice in a sex -independent manner leading to a net gain in bone mass despite elevated osteoclast activity through higher bone-forming activity.

[0138] Discussion: In this study, we identified a previously unknown connection between SSC function and angiogenesis in GIOP. While it has been established that GCs increase osteoclastogenesis and alter osteogenesis and angiogenesis, a mechanism related to vascular changes has not been appreciated. Our findings demonstrate that continuous treatment with GCs promotes SSC proliferation leading to their accumulation at the expense of differentiation required Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 for bone formation. We uncovered a new signaling axis by which Basigin, a transmembrane protein, specifically expressed by skeletal stem and progenitor cells when exposed to GCs, alters endothelial function and remodels BM vasculature driving cellular stress and promoting bone loss through reduced stem cell-based bone formation and increased resorption.

[0139] Basigin has been considered an innovative marker in the context of cancer stem cells21, but little is known about Basigin in the realm of skeletal biology. Two studies report that high levels of Basigin promote osteoclastogenesis through NFATcl signaling22and its involvement in alveolar bone remodeling and soft tissue degradation23. It also acts as a potent stimulator of Interleukin-6 secretion in multiple cell lines that include monocytes24, altogether supporting our observation of increased pro-osteoclastic signaling and bone resorption activity in GC treated animals with high stem and progenitor cell specific Basigin expression. In line with SSC accumulation as well as their altered cytokine and elevated extracellular matrix factor expression upon GC exposure, Basigin has also been found to contribute to tumor progression by stimulation of proliferation, elevated growth factor secretion and its matrix metalloprotease activity25. More specifically, previous studies have shown that Basigin acts as a chaperone for the shuttling of monocarboxylate transporters (MCTs) to the cell surface where MCTs facilitate the movement of short-chain fatty acids such as lactate. Lactate can directly regulate the cell cycle to promote cellular proliferation26, for example by modifying histones through lactylation changing epigenetic states27. Indeed, in Basigin-expressing SSPCs we found concomitant upregulation of Mctl-3 needed for lactate influx but downregulation of Mct4 used for lactate efflux indicating that the altered metabolic state of SSPCs with high intracellular lactate can contribute to the functional changes observed. Follow-up studies will have to experimentally address this and potential other mechanisms, such as altered mechano-sensing of SSPCs due to Basigin mediated ECM remodeling28.

[0140] At the endothelial cell level, we found that GC induced vascular impairments are closely tied to SSC-derived Basigin. Supporting our findings connecting Basigin to blood vessel formation is its known role as a coreceptor for vascular endothelial growth factor receptor 2 (Kdr / Vegfr2) in endothelial cells enhancing their Vegfr mediated activation and downstream signaling29. Continuous GC treatment in mice increased endothelial cell number and blood vessel area. However, Basigin exposure to endothelial cells resulted in irregularities in the connectivity and thickness of the nascent blood vessels. Flow cytometric and histological readouts, single cell gene Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 expression data and functional experiments connect these vascular changes to enhanced Vegfr signaling with a pathological blood vessel phenotype and increased oxidative stress. This is corroborated by studies that have shown that overactivation of Vegfr signaling is a driver of cellular stress in part through the stimulation of cell damaging and inflammatory reactive oxygen species that negatively affect formation, migration and permeability of blood vessels30 31. One of the most perplexing questions in medicine is how glucocorticoids induce osteonecrosis (ON). While epidemiologic studies find GCs increase the risk of ON, the pathology of the ON lesion is that of reduced blood vessel density and increased adipose tissue within the bone marrow8. Therefore, our observations can be relevant to GC-induced ON, as the altered morphology of these nascent blood vessels can reduce the ability to deliver hemoglobin / oxygen, and other crucial nutrients, to the bones. In certain areas of specific skeletal compartments like the femoral head, this can lead to increased cell death. Potentially connected to this, low hemoglobin is independently associated with increased fracture risk in non-vertebral bones of older adults32. Altogether, it will be worthwhile to investigate if the pronounced changes in angiogenic signaling mediated by Basigin can initiate the development of GC induced ON and higher fracture risk.

[0141] Excitingly, the observation that this newly discovered pathological signaling mechanism is reversable presents new therapeutic vantage points to counter GC-induced bone loss and maybe ON. Concomitant administration of hPTH or aBSG prevented bone loss and other pathological skeletal phenotypes observed with GC exposure. Counterintuitively to the known antiinflammatory actions of GCs, one of the bone marrow specific changes we observed with GC exposure was an increase in myeloid signaling. This result is consistent with the bone cell activity changes observed in GC treated subjects as GC treatment reduces bone formation and increases the number and activity of osteoclasts8'9. The locally bone restricted pro-inflammatory marker upregulation often also seen with cellular stress and SASP are indirect and related to changes in bone formation dynamics that favor increased osteoclastogenesis and pathological remodeling of blood vessel architecture that drive disruption in oxygen supply favoring pro-necrotic states at least in part through Basigin mediated mechanisms. It remains to be determined if strategies preventing GC-induced bone loss have any impact on the intended clinical effects of GC therapy.

[0142] We observed that the withdrawal of GCs on day 28 did not restore bone mass to baseline levels by day 56. Elevated GCs result in a reduction in the release of GCs from the hypothalamus33, and our 28-day GC treatment can have sufficiently suppressed the hypothalamic-pituitary-adrenal Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0143] (HPA) axis such that the 28-day treatment period was insufficient to see recovery of the skeletal tissues. In support of this observation, both osteogenesis and angiogenesis measurements for this group were similar to those of the GC only group at 56 days. New studies will need to investigate the HPA axis recovery in this treatment group to contextualize our current findings. Clinical studies have reported that GIOP can be treated with hPTH 1-34 leading to an increase in trabecular bone mass and biochemical markers of increased bone formation. Furthermore, meta-analyses of multiple studies have reported improved bone strength6’34. However, the effect of hPTH 1-34 on angiogenesis in the presence of GCs has not been investigated. Our study demonstrated that hPTH 1-34 treatment during GC exposure reduced Basigin expression, restored blood vessel number and morphology, as well as osteochondrogenic differentiation of SSCs. The role of hPTH 1-34 in angiogenesis is complex and can be further affected by GC exposure. While hPTH 1-34 has previously been shown to be osteoanabolic and to direct blood vessels toward the bone forming surface, it has not been found to be pro-angiogenic35. In contrast, direct beneficial actions of hPTH 1-34 on endothelial cells have been reported in fracture regeneration settings36,37. Even though GCs and hPTH 1-34 act on multiple skeletal lineage cell populations, our results provide evidence for their crucial role in regulating SSC activity.

[0144] Intriguingly, we can show that the antibody treatment against Basigin and heterozygous conditional deletion of SSPC derived Basigin mitigate both its detrimental autocrine and paracrine effects on skeletogenesis and vascular modeling, respectively. Recognizing that many phenotypic changes in response to GC treatment resembled age-associated skeletal changes, i.e., dysfunctional SSCs, pro-osteoclastic signaling and loss of bone marrow blood vessel integrity, we deduced that GC-induced bone loss can underlie overlapping mechanistic processes17,38. Fittingly, we found Basigin to be highly abundant in bone marrow of 2-year-old mice. When we tested treatment with aBSG in aged male and female mice, bone parameters significantly improved indicating antibody treatment against Basigin can also serve as an anti-osteoporotic drug independent of sex. Additional work with variations in dosing and longer treatment regimens are needed to validate this. Similar to hPTH 1-34, aBsg can restore proper SSC function thereby preventing elevated Basigin levels and its negative downstream effects. Future work will have to inquire if aBSG elicits overlapping molecular pathways in SSCs or if it can be a superior therapeutic approach to hPTH 1-34 to prevent GC-mediated bone loss. Given that GC treatment is often prescribed to patients for many years and hPTH 1-34 can only be administered for a few years to restore bone mass, Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 aBSG can become a valuable alternative. Tn summary, our work is the first to report the detrimental role of Basigin signaling in osteo-angiogenic coupling of bones and provides a new therapeutic target to prevent or potentially reverse skeletal health decline. Future studies are needed to determine the specific autocrine and paracrine signaling cascades Basigin elicits on skeletal and endothelial cell populations in more detail and if its blockade can also be able to reverse established GIOP and prevent GIOP induced ON.

[0145] Materials & Methods:

[0146] Animals: Mouse experiments complied with relevant ethical regulations and were conducted under approved protocols. Mice were maintained at animal facilities in accordance with institutional guidelines. Mice were given food and water ad libitum and housed in temperature-, moisture- and light-controlled (12-h light-dark cycle) micro-insulators. Unless otherwise specified, experiments were conducted using 3-month-old Balb / cJ male mice, purchased from Jackson Laboratories (Strain#:000651). To study the effects of glucocorticoids on bone biology, Methylprednisolone 5 mg 60-day release pellets or vehicle (cat#SG-241, Innovative Research of America, Sarasota, FL, USA) were implanted under the skin near the lumbar spine at day 0, and pellets were maintained for 56 days or removed after 28 days as indicated. Human parathyroid hormone 1-34 (hPTH 1-34) was purchased from Sigma (cat#:P3796), and the treatment was administered subcutaneously at 40 pg / kg, 5x / week for four consecutive weeks. For 28-day studies with concomitant hPTH (40 pg / kg / day, subcutaneously 5x / week) or aBSG (Img / kg, i.v. 3x / week) treatment mice were implanted with Methylprednisolone 2.5 mg 21 -day release pellets or vehicle control pellets. Aged (24-months) male and female C57BL / 6 from were ordered from NIA and treated i.v. with aBSG or IgG controls (Img / kg, 3x / week for four weeks; ThermoFisher, cat#: 16- 1471-82 & 16-4321-82). For renal capsule transplantation experiments cells from three-month old male GFP reporter mice (C57BL / 6-Tg(CAG-EGFP)10sb / J; JAX: 003291) were transplanted into male B6 mice (C57BL / Ka-Thyl. l-CD45.1; JAX: 000406). At the same day mice were transplanted with GC pellets (2.5 mg 21 -day release pellets or vehicle) and hPTH treatment was initiated (40 pg / kg / day, 5x / week for 3 weeks). Three-month old immunodeficient NSG mice (NOD.Cg-Prkdcscid 112rgtmlWjl / SzJ; JAX: 005557) were used for human cell transplantation studies. Bsgflox / flox mice were obtained as previously published39. Tamoxifen inducible Col2al- CreERT2 mice were crossed with Bsgflox / flox mice to generate Col2al-CreERT2 x Bsgflox / + Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 (conditional heterozygous Basigin knockout, cKO) female offsprings. At 8-weeks of age induction of the Cre-recombinase was achieved by daily oral gavage of tamoxifen (75 mg / kg) for 5 consecutive days. For the control group transgenic mice were injected with vehicle (com oil). One week after the first dose mice were implanted with 21 -day Methylprednisolone release pellets.

[0147] Human primary cells and cell lines: Human skeletal stem cells (hSSCs) were obtained from fracture callus (day 3-5 after injury) tissues during open reduction and internal fixation procedures. Procurement and handling were in accordance with the guidelines. Informed consent was not required as samples were considered biological waste. No restrictions were made regarding the race, gender, or age of the specimen’s donor. Following excision, specimens were placed on ice, and hSSCs were isolated as described herein. Early passage (3-5) VeraVec HUVEC cells were used for endothelial assays as further explained herein.

[0148] Micro-computed tomography and dual-energy x-ray absorptiometry (DEXA) analysis: Soft tissue-free femurs were scanned within 2 h of dissection using a Bruker Skyscan 1276 (Bruker Preclinical Imaging) with a source voltage of 85 kV, a source current of 200 pA, a filter setting of Al 1 mm, and pixel size of 17.5pm at 2016 * 1344. Reconstructed samples were analyzed using CT Analyser (CTan) v.1.17.7.2 and CTvox v.3.3.0 software (Bruker). Anatomical landmarks as per ASBMR guidelines40were used to set region of interest for analyzing trabecular and (200 consecutive sections) cortical (100 consecutive sections) bone parameters. Subsequent mechanical strength testing was conducted using 3-point bending. The maximum load (N) sustained prior to fracture was recorded41. Whole-body DEXA imaging was performed at baseline (day of surgery) and 14 days post-surgery to determine lumbar spine (L5) BMD. Mice were anesthetized with isoflurane and placed in a cabinet x-ray system (Mozart®, Kubtec Medical Imaging) for analysis.

[0149] Histology: Soft-tissue-free specimens were fixed in 4% PFA at 4 °C overnight. Samples were decalcified in 400 mM EDTA (EDTA) in PBS (pH 7.2) at 4 °C for 2 weeks with a change of EDTA every other day. The specimens were then dehydrated in 30% sucrose at 4 °C overnight. Specimens were embedded in optimal cutting temperature compound (OCT) and sectioned at 5 pm. Representative sections were stained with freshly prepared hematoxylin and eosin (H&E) or Tartrate-resistant acid phosphatase (TRAP). Immunofluorescence on sections of cryopreserved long bone and ectopic bone specimens were incubated with 3% Bovine Serum Albumin in Tris Buffered Saline (TBS) for Ih. Then, samples were probed with primary antibody (Endomucin 1 :200, cat# sc-65495, Santa Cruz; CD31 1 :200: cat# AF3628, Thermofisher; Basigin 1 :200, cat# Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 16-1471-82, Thermofisher & cat# NB500-430, Novus Biologicals) diluted in 1% BSA / PBS and incubated in a humidified chamber at 4°C overnight. The specimens were washed with PBS three times. Secondary antibody (AF488 dk anti-rat 1 :500: Donkey anti-Rat IgG (H+L) Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor™ 488, cat# A21208; Donkey anti-Goat IgG (H+L) 1 :500 Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647, cat# A21447, both Thermofisher) was applied for 15 minutes at room temperature in the dark. Specimens were also incubated with 1 pg / ml of DAPI for 10 mins and then washed twice. The specimens were then mounted with a coverslip using Fluoromount-G and imaged. ImageJ (http: / / wsr.imagej.net / distros / osx / ij l52-osx- java8.zip; RRID:SCR_003070) was used to quantify Endomucin-positive blood vessel / sinusoid lumen area. H&E stains were analyzed using image deconvolution according to Landini et al.42. The bone marrow area spanning 1 mm below the growth plate between cortical bone region was selected and the image deconvolution plugin, Colour Deconvolution 2, was ran on the selected region. The H&E 2 filter within the plug-in was applied. The color threshold on panel “Colour_2” was adjusted to represent the Eosin-stained bone regions. To measure the BV / TV, the partial (bone) area and total area and were analyzed.

[0150] Bone Histo morphometry Analysis : Mice were injected with 20 mg / kg of Calcein (Sigma- Aldrich, St. Louis, MO, USA) 8 and 2 days before euthanasia. Histological sections of bones were prepared as described herein. A standard sampling site in the secondary spongiosa of the distal metaphysis was established. Mineral apposition rate (MAR), bone formation rate (BFR) and osteoclast number / bone surface (Oc / BS) were calculated.

[0151] Flow cytometric isolation of skeletal progenitor cells. SSC lineage populations were isolated using cell-surface-marker profiles as previously described10 12. In brief, femurs were dissected, cleaned of soft tissue and crushed using mortar and pestle. Then, the tissue was digested in M199 (cat#l 1150067, Thermo Fisher Scientific) with 2.2 mg / ml collagenase II buffer (cat#C6885, Sigma-Aldrich) at 37 °C for 60 min. Dissociated cells were strained through a 100- pm nylon filter, washed in staining medium (10% fetal bovine serum (FBS) in PBS) and pelleted at 200 g at 4°C. The cell pellet was resuspended in staining medium and red blood cells were depleted via ACK lysis for 5 min. The cells were washed again in staining medium and pelleted at 200 g at 4°C. Then, the cells were prepared for flow cytometry with fluorochrome-conjugated antibodies. For mouse SSC lineages: CD90.1 (1 :200, Thermo Fisher, 47-0900), CD90.2 (1 :200, Thermo Fisher, 47-0902), CD105 (1 :200, Thermo Fisher, 13-1051), CD51 (1 :200, BD Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0152] Biosciences, 551187), CD45 (1 :400, BioLegend, 103110), Teri 19 (1 :400, Thermo Fisher, 15- 5921), Tie2 (1 :100, Thermo Fisher, 14-5987), 6C3 (1 :200, BioLegend, 108312), streptavidin PE- Cy7 (1 :400, Thermo Fisher, 25-4317), Sca-1 (1 :200, Thermo Fisher, 56-5981), CD45 (1 :400, Thermo Fisher, 11-0451), CD31 (1 :200, Thermo Fisher, 12-0311) and CD24 (1 :200, Thermo Fisher, 47-0242). For human SSC lineages: CD45 (1:200, BioLegend, 304029), CD235a (1 :200, BioLegend, 306612), CD31 (1 :200, Thermo Fisher Scientific, 13-0319), CD202b (TIE-2) (1 : 100, BioLegend, 334204), streptavidin APC-AlexaFlour750 (1 :400, Thermo Fisher, SA1027), CD146 (1:200, BioLegend, 342010), PDPN (1 :200, Thermo Fisher Scientific, 17-9381), CD164 (1 :200, BioLegend, 324808) and CD73 (1 :200, BioLegend, 344016). Flow cytometry was conducted on a FACS Aria II Instrument (BD BioSciences) using a 70-pm nozzle in the Shared FACS Facility in the Lokey Stem Cell Institute (Stanford). The skeletal stem-cell lineage gating strategy was determined using appropriate isotype and fluorescence-minus-one controls. Propidium iodide staining was used to determine cell viability. Cells were sorted for purity. Flow cytometric analysis was conducted using FlowJo (FLOWJ LLC, vlO.10). Blood panels and bone marrow analysis was conducted on a CYTEK Aurora Sorter. Blood was stained with Teri 19-PE-Cy5 (1 :400, 116210, BioLegend), CD45-FITC (1 :400, 11-0451, Invitrogen), B220-APC-Cy7 (1:200, 103224, BioLegend), CDl lb-PE-Cy7 (1 :200, 101216, BioLegend), CD3-APC (1 :200, 100236, BioLegend), Grl-BV711 (1 :200, 108443, BioLegend). CDl lb+ and Grl+ cells within CD45+ were considered myeloid lineage, while CD45+CD1 lb-Gr-CD3+ and B220+ were marked as lymphoid lineage. HSC lineage populations were stained with Lineage cocktail-Pacific Blue (1 :50, 133310, BioLegend), CD127-BV711 (1 :200, 135035, BioLegend), CD117-APC-Cy7 (1 :200, 105826, BioLegend), Scal-APC (1 :200, 160904, BioLegend), CD16 / 32-PE (1 :200, 156606, BioLegend), CD34-BV786 (1 : 100, 742971, BD Biosciences), CD135-PE-Cy5 (1 :200, 135312, BioLegend), CD150-BV510 (1 :200, 115929, BioLegend). DAPI was used as live stain.

[0153] Mouse cell cultur . Cells were cultured in a-MEM with 10% FBS and 1% penicillinstreptomycin (Thermo Fisher Scientific; 15140-122). For in vitro osteogenic and chondrogenic differentiation assays, 12,000 FACS-purified mouse SSCs were cultured in expansion in wells of a 24-well tissue culture plate. Cells were washed in PBS, trypsinized and transferred to osteogenic differentiation medium containing 10% FBS, 100 pg / ml ascorbic acid and 10 mM P- glycerophosphate in a-MEM for 14 days. Alternatively, for chondrogenic differentiation micromass cultures were generated by a 5-pl droplet of cell suspension with approximately 1.5 x Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025

[0154] 107 cells per ml pipetted in the center of a 24-well plate and cultured for 2 h in the incubator before adding warm chondrogenic medium consisting of a-MEM (high glucose) with 10% FBS, 100 nM dexamethasone, 1 pM L-ascorbic acid-2-phosphate and 10 ng / ml TGF-01 (Invitrogen, cat#PHG9204). The micromass was maintained for 21 days with media changes every other day. At the end of differentiation cells were washed in PBS, fixed in 4% PFA and stained with Alizarin Red S (cat#A5533-25G, Sigma-Aldrich) solution (osteogenesis) or Alcian Blue (cat#A3157, Sigma-Aldrich) solution (chondrogenesis). For osteogenic potential Alizarin Red S stain was dissolved in 300 pl of 20% Methanol / 10% Acetic Acid solution. After complete liberation of staining, 80 pl of each well was transferred into a new 96-well plate in duplicates and absorbance was measured at 450 nm. Chondrogenic potential was assessed by spectrophotometrically measuring absorption at 595 nm. For osteoclastogenesis assays flushed bone marrow cells from long bones were plated in 24-well plates at a density of 200,000 cells per well with a-MEM without phenol red, 1% GlutaMAX supplement (cat#35050061, Gibco), 10% FBS, 1% Penicillin- Streptomycin 10,000 U / ml, 1 pM prostaglandin E2 (cat#P0409, Sigma), and 10 ng / ml Csfl recombinant murine protein (cat#315-02, Peprotech) for 3 days. Starting on day three, the media was changed daily to also include 10 ng / ml recombinant mouse RANKL (cat#315-l 1, Peprotech). Osteoclast culture continued for 10 days until large, multinucleated osteoclasts appeared. Plates were stained for osteoclasts using the TRAP kit (cat#387A, Sigma-Aldrich).

[0155] Human SSC (hSSC) culture Freshly sorted, primary hSSCs were cultured in a-MEM with 10% human platelet derived lysate (HPL; cat#06962, STEMCELLtechnologies), 1% Penicillin-Streptomycin, 0.01% heparin and maintained at 37°C with 5% CO2. For in vitro differentiation assays hSSCs were treated and supplemented with the same osteogenic and chondrogenic differentiation protocols as described for mouse cells herein. For viral overexpression experiments the coding region of Basigin (269aa) was cloned into a pCCLc backbone for manufacture of second generation lentivirus using Lenti-X 293T as packaging cells as previously described43. The viral titer was determined functionally, based on amount of virus necessary to reach 90-95% eGFP expression. Cells were transduced using Lipofectamin3000 (cat#L3000001, ThermoFisher). For rescue experiments media was supplemented with hPTH 1- 34 by adding a final concentration of 2.5 nM 6h before each media change (to resemble intermittent exposure) or with 1 pg / mL of monoclonal Basigin antibody (cat# NB55-430, Novus Biologicals) Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 during media change. Reactive oxygen species measurement was performed according to manufacturer’s instructions (cat# abl 13851, Abeam).

[0156] Cell line experiments: Low passage VeraVec endothelial cells were thawed in a 10 cm dish with 10 mL of EGM-2 medium (cat#CC-3156, Lonza Bioscience). VeraVecs were grown at 37°C with 5% CO2 until confluent with media changes every 3 days. Before the assays, VeraVec culture was starved with low serum medium. For migration (wound scratch assay) 44, VeraVecs were lifted with 0.05% trypsin, spun down and resuspended in supernatant from 48h cultured SSCs. They were then plated at 1.5x105 cells / well in a 24-well plate. To induce a gap for the migration, a scratch assay was generated using a 1000 pL micropipette tip, scratching vertically from one side of the well to the other. The gap was imaged, and area measured at times Oh and 12h. The area healed was calculated by (area at Oh - area at 12h) / (area at Oh) x 100%. For tube formation a matrix (cat#A1413201, Geltrex, ThermoFisher,) was deposited in wells of 48-well plate at an amount of 50 uL / cm2, for a total of 50 pL per well. As for migration, VeraVecs were lifted and resuspended in supernatant from SSCs. VeraVecs were plated at 1.5x104 cells / well. Cells were imaged at 12h.

[0157] Subcutaneous xenografts: Primary hSSCs were transplanted into the dorsum of 3-month- old immunodeficient NSG mice (NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ; JAX: 005557) as described previously45. Briefly, freshly sorted patient-derived hSSCs were sorted, expanded to confluency and virally transduced to overexpress Basigin or control. 1x106 cells were mixed with 5 pl Matrigel and seeded on macroporous composite scaffolds formed of hydroxyapatite (HA) and poly(lactide-co-glycolide) (PLG) (HA-PLG) on ice. Scaffolds were fabricated using a gas foaming / particulate leaching method as previously described46. Microspheres composed of PLG (85: 15; DLG 7E; Lakeshore Biomaterials, Birmingham, AL) were prepared using a doubleemulsion process and lyophilized. Lyophilized microspheres (7.1 mg) were combined with 17.8 mg of synthetic HA (particle size, <200 nm; Aldrich Chemistry, St. Louis, MO) and 134.9 mg of NaCl (300 to 500 pm in diameter) to yield a 2.5: 1 :19 mass ratio of ceramic:polymer:salt. The powdered mixture was compressed under 2 metric tons for 1 min to form solid disks (final dimensions, 8 mm in diameter and 1.5 mm in height) using a Carver Press (Carver Inc., Wabash, IN). Compressed disks were exposed to high-pressure CO2 gas (5.5 MPa) for at least 24 hours, followed by rapid pressure release to prompt polymer fusion. Salt particles were leached from scaffolds in distilled H2O for 24 h to generate HA / PLG composite scaffolds. HA / PLG composite Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 scaffolds were cut with a biopsy punch to produce scaffolds with final dimensions of 4 mm in diameter and 1.5 mm in height. Scaffolds were sterilized in 70% ethanol in 24 well plates for 20 minutes, followed by two rinses in sterile PBS. Sterile scaffolds were dried and kept until use. To promote cell adhesion, scaffolds were incubated in culture media at 37°C with 5% CO2 for 30 min directly before cell seeding. A small skin incision was made in the dorsum of anesthetized NSG mice and the cell containing scaffold was slid under the skin. Interrupted sutures were applied to close the incision and transplants were allowed to engraft and grow for 6 weeks.

[0158] Renal capsule transplantation. Renal capsule transplantations were conducted as previously described10,12. Briefly, in the anaesthetized mouse a 5-mm dorsal incision was made, and the kidney was exposed manually. Then, a 2-mm incision was created in the renal capsule using a needle bevel, and 5,000 FACS-purified mouse SSCs resuspended in 2 pl of Matrigel were transplanted beneath the capsule. The kidney was re-approximated manually and incisions were closed using sutures and staples. At time of surgery, mice were randomly divided into three groups. Mice received subcutaneous placebo or Methylprednisolone pellets as described herein. One group additionally was treated with hPTH (see herein). Grafts were collected after 21 days.

[0159] Single cell RNA-sequencing Femurs were collected from the treatment groups, and each were processed, digested and prepared for FACS as described herein. Single cell solutions of each treatment group were then pooled (n = 5 per group) and 1 x 106 PI-Terl 19-CD45+ and 1x106 PI-Terl 19-CD45- cells were sorted into one collection tube containing FACS buffer for each experimental group. Cells were then processed with 10X Chromium Next GEM Single Cell 3’ GEM kit (10X Genomics, v.3.1) according to the manufacturer’s instruction to target 5,000 cells per group. Barcoded samples were demultiplexed, aligned to the mouse genome (GRCm39.104), and UMI-collapsed with the Cellranger toolkit with standard settings (v.7.1.0, 10X Genomics), and sequenced on a partial lane Illumina NovaSeq platform. We used the Scanpy package (v.1.9.1.) to explore the data. First, quality filtering to exclude multiplets and cells of poor quality was conducted by only keeping cells with a gene count of more than 250 and fewer than 3,000, with less than 15% mitochondrial and 15% ribosomal gene content, leaving 8,614 cells. Genes expressed in fewer than three cells across cells were also removed from downstream analysis. Data were log-normalized, cell cycle-regressed and scaled for analysis. Dimensionality reduction and Leiden clustering as well as subclustering were conducted choosing parameters based on PCA elbow plots. For single cell RNA-sequencing of SSC-derived grafts the tissue the tissue was Docket No.: 2974214-000002-W01 Date of filing: October 28, 2025 dissected out and processed as described for femur bone processing to isolate SSCs. Single cell solution was then stained with PI and Teri 19, and living (Pl-negative), non-red blood cells (Teri 19-negative) were sorted into FACS buffer for each group. Based on yield, cells were then processed with 10X Chromium Next GEM Single Cell 3’ GEM kit (10X Genomics, v.3.1) according to the manufacturer’s instruction to target 2,000 cells for Placebo and GC+PTH groups as well as 500 cells for GC group. Other steps were conducted as described herein. A total of 595 graft-derived cells across groups passed stringent quality filtering. Data are available under GEO accession number GSE253044.

[0160] Statistical analysis: Statistical significance between placebo and treatment groups was determined using two-tailed, unpaired Student’s t-test or One-way ANOVA for multiple groups comparison with LSD Fisher test unless stated otherwise in the figure legend (GraphPad Prism; version 10). Statistical significance was defined as p < 0.05. Data points refer to biological replicates and are presented as mean ± standard error of the mean (SEM) unless otherwise stated in figure legend.

[0161] D. References Cited in this Example

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[0190] 28. Curtin, K. D., Meinertzhagen, I. A. & Wyman, R. J. Basigin (EMMPRIN / CD147) interacts with integrin to affect cellular architecture. J Cell Sci 118, 2649-2660 (2005).

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[0201] 39. Li, K., Li, Q., Bashir, S. T., Bany, B. M. & Nowak, R. A. Loss of basigin expression in uterine cells leads to subfertility in female micef. Biol Reprod 105, 859-875 (2021).

[0202] 40. Bouxsein, M. L. et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res 25, 1468-1486 (2010).

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Claims

Docket No.: 2974214-000002-W01Date of filing: October 28, 2025CLAIMSWhat is claimed is:

1. A method of treating a bone loss disorder in a subject, comprising administering to the subject, an agent that inhibits Basigin / CD147.

2. The method of treating of claim 1, wherein the agent comprises an antibody or antibody fragment thereof that targets Basigin / CD147, a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody, or a mesenchymal stem cell (MSC)-derived product.

3. The method of treating of any one of claims 1-2, wherein the agent comprises an anti- Basigin / CD147 antibody or antibody fragment thereof.

4. The method of treating of claim 2, wherein the MSC-derived product is a cytokine, an MSC- derived osteoblast or an MSC-cell-conditioned medium.

5. The method of treating of any of claims 1-4, further comprising administration of an osteogenic agent.

6. The method of treating of claim 5, wherein the osteogenic agent is Parathyroid Hormone (PTH) (1-34).

7. The method of treating of any of claims 5-6, wherein the osteogenic agent is a mesenchymal stem cell (MSC)-derived osteoblast, a skeletal stem cell (SSC), or an induced pluripotent stem cell (iPSC)-derived osteoblast.

8. The method of treating of any one of claims 1-7, wherein the agent is administered to the subject via a systemic injection.

9. The method of treating of any one of claims 1-8, wherein the bone loss disorder is selected from a group consisting of osteoporosis; osteopenia; lactation; traumatic bone injury; pathologic bone injury; periprosthetic bone loss; osteolysis; osteonecrosis; osteomalacia; Paget’s disease ofDocket No.: 2974214-000002-W01 Date of filing: October 28, 2025 bone; osteogenesis imperfecta; rheumatoid arthritis; bone loss resulting from menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, hyperthyroidism, hyperparathyroidism, cancer; bone loss resulting from a craniofacial disorder; bone loss resulting from oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy; lactation-induced bone loss disorder, menopause- induced bone loss disorder, skeletal stem cell (SSC)-dysfuncti on-induced-bone loss, systemic autoimmune disease (including but not limited to systemic lupus erythematous or antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (such as, for example, granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA)), and steroid-induced-bone loss disorder (including but not limited to glucocorticoid-induced osteoporosis (GIOP) or glucocorticoid-induced osteonecrosis (GION) and / or bone injury.

10. The method of treating of any one of claims 1-9, wherein the bone loss disorder is a steroid- induced-bone loss disorder.

11. The method of treating of any one of claims 9-10, wherein the steroid-induced-bone loss disorder is glucocorticoid-induced osteoporosis (GIOP) or glucocorticoid-induced osteonecrosis (GION).

12. The method of treating of any one of claims 1-11, wherein the method increases BMD and / or bone vascularization in the subject compared to an untreated control.

13. A method of treating a bone loss disorder in a subject comprising, administering a geneediting system to a subject in need thereof, wherein the gene-editing system comprises an endonuclease and a guide RNA (gRNA), wherein the gRNA binds a CD147 gene comprising an Osteocalcin (OCN) promoter.

14. The method of treating of claim 13, wherein the gene-editing system further comprises an adeno-associated virus (AAV) vector.

15. The method of treating of claim 14, wherein the AAV vector is AAV9.Docket No.: 2974214-000002-W01 Date of filing: October 28, 202516. The method of treating of any one of claims 13-15, wherein the endonuclease is a Cas9 enzyme.

17. The method of treating of any of claims 13-16, further comprising administration of an osteogenic agent.

18. The method of treating of claim 17, wherein the osteogenic agent is Parathyroid Hormone (PTH) (1-34).

19. The method of treating of any of claims 17-18, wherein the osteogenic agent is a mesenchymal stem cell (MSC)-derived osteoblast, a skeletal stem cell (SSC), or an induced pluripotent stem cell (iPSC)-derived osteoblast.

20. The method of treating of any one of claims 13-19, wherein the gene-editing system is administered to the subject via a systemic injection.

21. The method of treating of any one of claims 13-20, wherein the bone loss disorder is selected from a group consisting of osteoporosis; osteopenia; lactation; traumatic bone injury; pathologic bone injury; periprosthetic bone loss; osteolysis; osteonecrosis; osteomalacia; Paget’s disease of bone; osteogenesis imperfecta; rheumatoid arthritis; bone loss resulting from menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, hyperthyroidism, hyperparathyroidism, cancer; bone loss resulting from a craniofacial disorder; bone loss resulting from oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy; lactation-induced bone loss disorder, menopause- induced bone loss disorder, skeletal stem cell (SSC)-dysfuncti on- induced-bone loss, systemic autoimmune disease (including but not limited to systemic lupus erythematous or antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (such as, for example, granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA)), and steroid-induced-bone loss disorder (including but not limited to glucocorticoid-induced osteoporosis (GIOP) or glucocorticoid- induced osteonecrosis (GION) and / or bone injury.

22. The method of treating of any one of claims 13-21, wherein the bone loss disorder is a steroid-induced-bone loss disorder.Docket No.: 2974214-000002-W01 Date of filing: October 28, 202523. The method of treating of any one of claims 21 -22, wherein the steroid-induced-bone loss disorder is glucocorticoid-induced osteoporosis (GIOP) or glucocorticoid-induced osteonecrosis (GION).

24. The method of treating of any one of claims 13-23, wherein the method increases BMD and / or bone vascularization in the subject compared to an untreated control.

25. A method of rescuing age-related skeletal degeneration in a subject, comprising, a. measuring bone mineral density (BMD) of the subject compared to a standardized level of BMD from age-matched normal subjects; wherein a decrease in BMD relative to a standardized level of BMD from age-matched normal subjects denotes age-related skeletal degeneration and b. administering to the subject an agent that inhibits Basigin / CD147, when a decrease in BMD is detected in the subject.

26. The method of rescuing of claim 25, wherein measuring BMD comprises dual-energy X- ray absorptiometry (DEXA).

27. The method of rescuing of any of claims 25-26, wherein the agent comprises a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody, a mesenchymal stem cell (MSC)-derived product or an antibody fragment that targets Basigin / CD147.

28. The method of rescuing of any one of claims 25-27, wherein the agent comprises an anti- Basigin / CD147 antibody or antibody fragment thereof.

29. The method of rescuing of claim 27, wherein the MSC-derived product is a cytokine, an MSC-derived osteoblast or an MSC-cell-conditioned medium.

30. The method of rescuing of any of claims 25-29, further comprising administration of an osteogenic agent.Docket No.: 2974214-000002-W01 Date of filing: October 28, 202531 . The method of rescuing of claim 30, wherein the osteogenic agent is Parathyroid Hormone (PTH) (1-34).

32. The method of rescuing of any of claims 30-31, wherein the osteogenic agent is a mesenchymal stem cell (MSC)-derived osteoblast, a skeletal stem cell (SSC), or an induced pluripotent stem cell (iPSC)-derived osteoblast.

33. The method of rescuing of any of claims 25-32, wherein the agent is administered to the subject via a systemic injection.

34. The method of rescuing of any one of claims 25-33, wherein the method increases BMD and / or bone vascularization in the subject compared to an untreated control.

35. A method of rescuing age-related skeletal degeneration in a subject, comprising, a. measuring bone mineral density (BMD) of the subject compared to a standardized level of BMD from age-matched normal subjects; wherein a decrease in BMD relative to a standardized level of BMD from age-matched normal subjects denotes age-related skeletal degeneration; and b. administering a gene-editing system to a subject in need thereof, wherein the geneediting system comprises an endonuclease and a guide RNA (gRNA), when a decrease in BMD is detected in the subject, wherein the gRNA binds a CD147 gene comprising an Osteocalcin (OCN) promoter.

36. The method of rescuing of claim 35, wherein measuring BMD comprises dual-energy X- ray absorptiometry (DEXA).

37. The method of rescuing of any one of claims 35-36, wherein the gene-editing system further comprises an adeno-associated virus (AAV) vector.

38. The method of rescuing of claim 37, wherein the AAV vector is AAV9.

39. The method of rescuing of any one of claims 35-38, wherein the endonuclease is a Cas9 enzyme.Docket No.: 2974214-000002-W01 Date of filing: October 28, 202540. The method of rescuing of any of claims 35-39, further comprising administration of an osteogenic agent.

41. The method of rescuing of claim 40, wherein the osteogenic agent is Parathyroid Hormone (PTH) (1-34).

42. The method of rescuing of any of claims 40-41, wherein the osteogenic agent is a mesenchymal stem cell (MSC)-derived osteoblast, a skeletal stem cell (SSC), or an induced pluripotent stem cell (iPSC)-derived osteoblast.

43. The method of rescuing of any one of claims 35-42, wherein the gene-editing system is administered to the subject via a systemic injection.

44. The method of rescuing of any one of claims 35-43, wherein the method increases BMD and / or bone vascularization in the subject compared to an untreated control.

45. A method of increasing vascularization in a subject, comprising administering an agent that inhibits Basigin / CD147 to the subject.

46. The method of increasing vascularization of claim 45, wherein the agent comprises a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody, a mesenchymal stem cell (MSC)-derived product, or an antibody fragment that targets Basigin / CD147.

47. The method of increasing vascularization of any one of claims 45-46, wherein the agent comprises an anti-Basigin / CD147 antibody or antibody fragment thereof.

48. The method of increasing vascularization of claim 46, wherein the MSC-derived product is a cytokine, an MSC-derived osteoblast or an MSC-cell-conditioned medium.

49. The method of increasing vascularization of any of claims 45-48, further comprising administration of an osteogenic agent.Docket No.: 2974214-000002-W01 Date of filing: October 28, 202550. The method of increasing vascularization of claim 49, wherein the osteogenic agent is Parathyroid Hormone (PTH) (1-34).

51. The method of increasing vascularization of any of claims 49-50, wherein the osteogenic agent is a mesenchymal stem cell (MSC)-derived osteoblast, a skeletal stem cell (SSC), or an induced pluripotent stem cell (iPSC)-derived osteoblast.

52. The method of increasing vascularization of any one of claims 45-51, wherein the agent is administered to the subject via a systemic injection.

53. The method of increasing vascularization of any one of claims 45-52, wherein the method further increases BMD in the subject compared to an untreated control.

54. A method of engineering a bone graft, comprising contacting a stem cell with an agent that inhibits Basigin / CD147.

55. The method of engineering of claim 54, wherein the stem cell comprises a skeletal stem cell (SSC), an induced pluripotent stem cell (iPSC), or an MSC.

56. The method of engineering of any one of claims 54-55, wherein the agent comprises a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody, a mesenchymal stem cell (MSC)-derived product, or an antibody fragment that targets Basigin / CD147.

57. The method of engineering of any one of claims 54-56, wherein the agent comprises an anti- Basigin / CD147 antibody or antibody fragment thereof.

58. The method of engineering of claim 56, wherein the MSC-derived product is a cytokine, an MSC-derived osteoblast or an MSC-cell-conditioned medium.

59. The method of engineering of any of claims 54-58, further comprising administration of an osteogenic agent.Docket No.: 2974214-000002-W01 Date of filing: October 28, 202560. The method of engineering of claim 59, wherein the osteogenic agent is Parathyroid Hormone (PTH) (1-34).

61. The method of engineering of any of claims 59-60, wherein the osteogenic agent is a mesenchymal stem cell (MSC)-derived osteoblast, a skeletal stem cell (SSC), or an induced pluripotent stem cell (iPSC)-derived osteoblast.

62. A method of screening to detect an anti-basigin / CD147 antibody, comprising, a. contacting a skeletal stem cell (SSC) with one or more agent(s); and b. measuring fibroblast colony forming unit (CFU-F) ability of the SSC, basigin / CD147 signaling, mineralization in vitro, and / or vascularization, wherein an increase in fibroblast CFU-F ability; a decrease in basigin / CD147 signaling, increase in mineralization in vitro and / or increase in vascularization compared to a control denotes an increase in anti-basigin activity.

63. The method of screening of claim 62, wherein fibroblast CFU-F ability comprises colony number and / or colony size.

64. The method of screening of any one of claims 62-63, wherein the one or more agent(s) comprise a small molecule, a small interfering (si) ribonucleic acid (RNA) (siRNA), a microRNA (miRNA), a long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, a peptide, a protein, an antibody, a mesenchymal stem cell (MSC)-derived product, or an antibody fragment that targets Basigin / CD147.

65. The method of screening of claim 64, wherein the MSC-derived product is a cytokine, anMSC-derived osteoblast or an MSC-cell-conditioned medium.

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