Methods of using the brain-derived osteogenic factor ccn3 for treatment of bone and cartilage degeneration

Abstract

Methods of using cellular communication network factor 3 (CCN3) for treating bone or cartilage disorders are provided. Methods of gene therapy are also provided, including methods of expressing CCN3 in vivo in effective amounts sufficient to promote new bone or cartilage growth. In addition, methods of screening for agonists, mimics, and analogues of CCN3 are also provided.

Description

Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 METHODS OF USING THE BRAIN-DERIVED OSTEOGENIC FACTOR CCN3 FOR TREATMENT OF BONE AND CARTILAGE DEGENERATION CROSS-REFERENCE TORELATEDAPPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(e) of provisional application 63 / 578,795, filed August 25, 2023, which application is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. R01 AG062331 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION BYREFERENCE OFSEQUENCELISTING

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML file, “UCSF- 749WO” created on August 15, 2024 and having a size of 24,285 bytes. The contents of the Sequence Listing XML file are incorporated by reference herein in their entireties. INTRODUCTION

[0004] Musculoskeletal disorders are a significant global health burden. Osteoporosis significantly impacts healthy aging and is commonly experienced by more women than men. Females leverage estradiol (E2) as an anabolic hormone to increase energy expenditure (Ingraham et al., Annu Rev Physiol 84, 59-85 (2022)) and preserve bone mass by regulating bone remodeling (Khosla et al., Trends Endocrinol Metab 23, 576-581 (2012)) via osteocytes (Doolittle et al., J Bone Miner Res 37, 1750-1760 (2022)), osteoblasts (Almeida et al., J Clin Invest 123, 394-404 (2013)), and osteochondral skeletal stem cells (ocSSCs) 6, which are fated for bone and cartilage (Chan et al., Cell 160, 285-298 (2015); Chan et al., Cell 175, 43-56 e21 (2018)). For women, natural or drug-induced estrogen depletion during menopause or anti-hormone therapies slowly degrades bone mass, underscoring the anabolic features of estrogen on bone metabolism. However, the intimate association between estrogen and bone is oddly uncoupled during lactation when ovarian estrogens shut down, and bone formation and resorption rise sharply to supply the high calcium demand by progeny (reviewed in Kovacs, J Bone Miner Res 32, 676-680 (2017)). Parathyroid hormone-related protein (PTHrP), a close orthologue of parathyroid hormone (PTH) from mammary glands, is the main driver for stripping calcium from maternal bones for milkAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 (Ardawi et al., Eur J Endocrinol 137, 402-409 (1997); Ardeshirpour et al., Bone 38, 787-793 (2006)). The relentless demand for calcium by newborns eventually leads to significant bone loss in mothers, dropping nearly 30% in rodents due to large litter sizes (VanHouten & Wysolmerski, Endocrinology 144, 5521-5529 (2003)) and 10% in humans (Kalkwarf & Specker, Obstet Gynecol 86, 26-32 (1995); Bjornerem et al., J Bone Miner Res 32, 681-687 (2017)); these losses mostly normalize post-lactation (VanHouten & Wysolmerski, supra; Bjornerem et al., supra). Presumably, the maternal skeleton (and that of pups) would be severely compromised without this lactational-anabolic phase, as inferred by the increased bone mass in lactating mothers after conditional knock-out of PTHrP (VanHouten et al., J Clin Invest 112, 1429-1436 (2003); Karaplis et al., Genes Dev 8, 277-289 (1994)). Absent the underlying mechanism driving new bone formation during lactation; this notion remains to be proved.

[0005] Currently 52.2M Americans are diagnosed with arthritis and by 2040 this is estimated to rise to 78.4M. Of these disorders, the most common type is osteoarthritis (OA) which has a lifetime risk of 40%. With no effective treatments yet approved to prevent OA disease progression, symptomatic relief and eventual joint replacement are the standards of care. In an attempt to regenerate cartilage in OA, surgeons perform microfracture (MF) surgery, a technique developed in the 1950's and widely used today. During MF surgery, the surgeon drills into the debrided chondral bone until the marrow cavity is accessed. A hematoma forms at the MF site that is resorbed and replaced with fibrous tissues. The resulting “fibrocartilage” provides some symptomatic relief but has significantly reduced mechanical properties compared to normal articular cartilage6. Little is known about the mechanism through which MF causes fibrocartilage formation, the effect of this technique on resident stem cell populations, or how this can be leveraged for tissue regeneration.

[0006] The limitations of current approaches for addressing OA, including MF, have led to widespread interest in the potential of stem cell therapy for regenerating cartilage. Numerous clinical trials explore the use of autologous stem cell transplantation as alternative therapies for OA of the hip, knee, and thumb. The majority of stem cell-specific trials treating OA of the hip and knee are pilot or feasibility studies investigating the use of plastic-adherent, culture-expanded “mesenchymal stem cells” (MSC). These MSC are derived from bone marrow or adipose tissue. However, these MSC do not constitute validated stem cell populations and it is difficult to ascertain the degree of engraftment by the transplanted cells and their contribution to changes in functional outcomes. The trials also did not reveal specific mechanisms of action behind cases of apparent symptomatic improvement.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0007] More recently, several groups have made progress in identifying skeletal stem cells (SSC) in mice (mSSC) and humans (hSSC). Purified SSC are defined by their ability to self-renew and their multi-lineage contribution to bone, cartilage, and stroma, but not fat. However, it is important to consider that SSCs using different sorting strategies may be similar but not necessarily the same, for instance, in their ability to make adipocytes. Furthermore, SSC in the bones of mice and humans are stimulated to proliferate following injury and injury-activated progenitor populations also demonstrate enhanced skeletogenic potential relative to homeostatic populations. However, whether activation of resident SSC can be utilized to regenerate cartilage as a means to treat OA has yet to be determined.

[0008] There remains a need for better methods of treating and preventing bone and cartilage degeneration. SUMMARY

[0009] Methods of using cellular communication network factor 3 (CCN3) for treating bone or cartilage disorders are provided. Methods of gene therapy are also provided, including methods of expressing CCN3 in vivo in effective amounts sufficient to promote new bone or cartilage growth. In addition, methods of screening for agonists, mimics, and analogues of CCN3 are also provided.

[0010] In one aspect, a method of treating a bone disorder or condition associated with bone degeneration in a subject is provided, the method comprising administering a therapeutically effective amount of CCN3 to the subject.

[0011] By “therapeutically effective dose or amount” of a CCN3 protein is intended an amount that, when administered, as described herein, brings about a positive therapeutic response, such as improved recovery from a bone disorder or condition associated with bone degeneration. Improved recovery may include increased bone density, increased bone mass, increased bone strength, increased bone mineral density, and / or decreased fatty bone marrow. Additionally, a therapeutically effective dose or amount may stimulate osteochondral skeletal stem cells to produce bone.

[0012] Bone disorders and conditions associated with bone degeneration include any disease or condition resulting in decreased bone density, reduced bone mass, bone fragility, low bone mineral density, and / or increased fatty bone marrow such as, but not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0013] In certain embodiments, the CCN3 protein comprises an amino acid sequence having at least about 80-100% sequence identity to the sequence of SEQ ID NO:1, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto.

[0014] In certain embodiments, the treatment increases bone mass of a bone of the subject compared to the bone mass before the treatment.

[0015] In certain embodiments, the CCN3 is administered intravenously or intraperitoneally.

[0016] In certain embodiments, the CCN3 is administered locally to a bone of the subject.

[0017] In certain embodiments, multiple therapeutically effective doses of CCN3 are administered to the subject. In some embodiments, the CCN3 is administered according to a daily dosing regimen or intermittently.

[0018] In certain embodiments, the method further comprises administering an osteoanabolic agent or anti-resorptive agent to the subject. Exemplary osteoanabolic agents include, without limitation, parathyroid hormone, teriparatide, abaloparatide, and romosozumab. Exemplary anti-resorptive agents include, without limitation, estrogen, an estrogen agonist, a bisphosphonate, and denosumab.

[0019] In certain embodiments, the subject is human.

[0020] In another embodiment, a method of decreasing or preventing bone degeneration in a female subject during lactation is provided, the method comprising administering a therapeutically effective amount of CCN3 to the subject.

[0021] In another aspect, a composition comprising CCN3 for use in a method of treating a bone disorder or condition associated with bone degeneration is provided. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.

[0022] In another aspect, a method of treating a bone disorder or condition associated with bone degeneration in a subject is provided, the method comprising administering a vector comprising an expression cassette comprising a coding sequence encoding CCN3 to the subject.

[0023] In certain embodiments, the expression cassette comprises a promoter operably linked to the coding sequence encoding the CCN3.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0024] In certain embodiments, the coding sequence encoding the CCN3 is integrated into a chromosomal locus in the genome of the subject. In some embodiments, an endogenous promoter is operably linked to the integrated coding sequence encoding the CCN3 at the chromosomal locus.

[0025] In certain embodiments, the vector is a plasmid or viral vector. Exemplary viral vectors include, without limitation, adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, and retroviral vectors.

[0026] In certain embodiments, the vector is administered intravenously or intraperitoneally.

[0027] In certain embodiments, the vector is administered via a portal vein.

[0028] In certain embodiments, the vector is administered locally to a bone.

[0029] In another aspect, a method of providing a subject with CCN3 to promote bone growth in the subject is provided, the method comprising introducing a vector comprising a promoter operably linked to a coding sequence encoding the CCN3 into a cell, wherein the cell expresses the CCN3 in vivo in the subject in an effective amount sufficient to promote bone growth in the subject.

[0030] In certain embodiments, the vector is introduced into the cell ex vivo or in vivo.

[0031] In certain embodiments, the cell is a hepatocyte or an osteochondral skeletal stem cell.

[0032] In certain embodiments, the subject has a bone disorder or condition associated with bone degeneration such as, but not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0033] In another aspect, a method of stimulating an osteochondral skeletal stem cell to produce bone is provided, the method comprising contacting the osteochondral skeletal stem cell with an effective amount of CCN3, wherein bone is produced by the osteochondral skeletal stem cell.

[0034] In another aspect, a method of screening for an agonist of CCN3 that increases bone growth is provided, the method comprising: contacting a cell with the CCN3 and a candidate agent, wherein the cell is an osteochondral skeletal stem cell, a preosteoblast cell, an osteoblast cell, an osteoprogenitor cell, or an osteosarcoma cell; and measuring bone production by the cell,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 wherein an increased amount of bone production in the presence of the candidate agent compared to a reference value range for the amount of bone production in a control cell, wherein the candidate agent is absent, indicates that the candidate agent is an agonist of CCN3.

[0035] In another aspect, a method of screening for a mimic or an analogue of CCN3 that stimulates bone growth is provided, the method comprising: contacting a cell with a candidate agent, wherein the cell is an osteochondral skeletal stem cell, a preosteoblast cell, an osteoblast cell, an osteoprogenitor cell, or an osteosarcoma cell; and measuring bone mineralization by the cell, wherein an increased amount of bone mineralization in presence of the candidate agent compared to a reference value range for the amount of bone mineralization in a control cell, wherein the candidate agent is absent, indicates that the candidate agent is a mimic or an analogue of the CCN3.

[0036] In certain embodiments, the cell, used in a method of screening, is derived from a stem cell. In some embodiments, the stem cell is an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell. In some embodiments, the adult stem cell is a mesenchymal stem cell. In some embodiments, the stem cell is from a patient who has a bone disorder or condition associated with bone degeneration. In some embodiments, the cell is immortalized. In some embodiments, the cell is from a cell line. Exemplary cell lines include, without limitation, an MC3T3-E1 preosteoblast cell line, an SaOs2 osteosarcoma cell line, an MG-63 osteosarcoma cell line, a hFOB osteoblast cell line, an ASC52telo immortalized adipose derived mesenchymal stem cell line, a human telomerase reverse transcriptase-immortalized bone marrow mesenchymal stromal cell (hTERT-BMSC) line, and an ATDC5 chondrogenic mouse teratocarcinoma cell line.

[0037] In another aspect, a method of producing a bone graft is provided, the method comprising culturing osteochondral skeletal stem cells in the presence of CCN3 under suitable conditions, wherein the osteochondral skeletal stem cells produce bone for the bone graft.

[0038] In another aspect, a bone graft is provided, wherein the bone graft is produced by culturing osteochondral skeletal stem cells in the presence of CCN3 using a method described herein.

[0039] In another aspect, a method of transplanting a bone graft into a subject is provided, the method comprising transplanting a bone graft, produced as described herein, into a transplantation site in the subject.

[0040] In certain embodiments, the osteochondral skeletal stem cells, used to produce the bone graft, are autologous, allogeneic, or xenogeneic.

[0041] In certain embodiments, the bone graft replaces missing bone to repair a bone fracture at the transplantation site. In some embodiments, the bone fracture is a compound fracture.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0042] In certain embodiments, the bone graft provides new bone to repair a congenital bone defect at the transplantation site.

[0043] In certain embodiments, the method further comprises administering CCN3 locally at the transplantation site to stimulate new bone growth at the transplantation site.

[0044] In another aspect, a method of monitoring a lactating female subject to determine risk of bone degeneration from breast feeding is provided, the method comprising: obtaining a biological sample from the lactating female subject; measuring a level of CCN3 in the biological sample; comparing the level of the CCN3 in the biological sample to reference value ranges for CCN3 from a control subject, wherein levels of CCN3 below a threshold value indicate that the subject is at risk of bone degeneration from breast feeding, and wherein levels of CCN3 equal to or greater than a threshold value indicate that the female subject may continue breast feeding without substantial risk of bone degeneration.

[0045] In certain embodiments, the female subject discontinues breast feeding if the level of CCN3 indicates the female subject is at risk of bone degeneration from breast feeding.

[0046] In certain embodiments, the method further comprises administering a therapeutically effective amount of the CCN3 to the female subject to lower the risk of bone degeneration from breast feeding.

[0047] In certain embodiments, the biological sample is blood or plasma.

[0048] In certain embodiments, measuring the level of CCN3 comprises performing an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, an immunofluorescent assay, immunohistochemistry, fluorescence-activated cell sorting (FACS), a Western Blot, mass spectrometry, tandem mass spectrometry, a biochemical assay, liquid chromatography, or NMR.

[0049] In another aspect, CCN3 for use as a biomarker in monitoring a lactating female subject to determine risk of bone loss from breast feeding is provided.

[0050] In another aspect, a method for regenerating mammalian cartilage in a subject is provided, the method comprising: activating skeletal stem cells with a mechanical stimulus; and administering a therapeutically effective amount of cellular communication network factor 3 (CCN3) in combination with a therapeutically effective amount of a vascular endothelial growth factor (VEGF) inhibitor to the subject.

[0051] In certain embodiments, the CCN3 protein comprises an amino acid sequence having at least about 80-100% sequence identity to the sequence of SEQ ID NO:1, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto.

[0052] In certain embodiments, the VEGF inhibitor is cabozantinib.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0053] In certain embodiments, the cartilage is articular cartilage.

[0054] In certain embodiments, the mechanical stimulus is an acute local injury.

[0055] In certain embodiments, the acute local injury is a surgically performed microfracture procedure to bone tissue at a desired site for cartilage regeneration.

[0056] In certain embodiments, the VEGF inhibitor and the CCN3 are administered locally to the acute local injury.

[0057] In certain embodiments, the VEGF inhibitor and the CCN3 are administered to the subject immediately after said activating the skeletal stem cells with the mechanical stimulus.

[0058] In certain embodiments, the VEGF inhibitor and the CCN3 are administered to the subject within three days after said activating the skeletal stem cells with the mechanical stimulus.

[0059] In certain embodiments, the VEGF inhibitor and the CCN3 are encapsulated in a hydrogel.

[0060] In certain embodiments, the hydrogel comprises alginate. In some embodiments, the alginate is ionically cross-linked. In some embodiments, the alginate is ionically cross-linked by divalent cations, including, without limitation, divalent calcium cations. In some embodiments, the alginate concentration in the hydrogel ranges from 2 to 10 percentage by weight (wt%).

[0061] In certain embodiments, the VEGF inhibitor and the CCN3 are administered using a drug delivery device. In some embodiments, the drug delivery device provides sustained delivery of the VEGF inhibitor and the CCN3. In some embodiments, the drug delivery device is a reservoir implant or a monolithic implant. In some embodiments, the drug delivery device is a non- biodegradable implant. In some embodiments, the drug delivery device is a biodegradable implant. In some embodiments, the drug delivery device is implanted at the site of the local acute injury.

[0062] In certain embodiments, the subject has a cartilage disorder. Exemplary cartilage disorders include, without limitation, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, and malignancy.

[0063] In certain embodiments, the method further comprises administering a therapeutically effective amount of skeletal stem cells to the subject.

[0064] In another aspect, a composition comprising CCN3 and a VEGF inhibitor for use in a method of regenerating mammalian cartilage is provided.

[0065] In certain embodiments, the VEGF inhibitor in the composition is cabozantinib.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0066] In certain embodiments, the CCN3 and the VEGF inhibitor are encapsulated in a hydrogel. In some embodiments, the hydrogel comprises alginate. In some embodiments, the alginate is ionically cross-linked. In some embodiments, the alginate is ionically cross-linked by divalent cations, including, without limitation, divalent calcium cations. In some embodiments, the alginate concentration in the hydrogel ranges from 2 to 10 wt%.

[0067] In another aspect, a method of screening for an agonist of CCN3 that increases production of cartilage is provided, the method comprising: activating a skeletal stem cell with a mechanical stimulus; contacting the skeletal stem cell with CCN3, a VEGF inhibitor, and a candidate agent; and measuring cartilage production by the skeletal stem cell, wherein an increased amount of cartilage production in the presence of the candidate agent compared to a reference value range for the amount of cartilage production in a control skeletal stem cell, wherein the candidate agent is absent, indicates that the candidate agent is an agonist of CCN3.

[0068] In another aspect, a method of screening for a mimic or an analogue of CCN3 that stimulates production of cartilage is provided, the method comprising: activating a skeletal stem cell with a mechanical stimulus; contacting the skeletal stem cell with a VEGF inhibitor and a candidate agent; and measuring cartilage production by the skeletal stem cell, wherein an increased amount of cartilage production in presence of the candidate agent compared to a reference value range for the amount of cartilage production in a control skeletal stem cell, wherein the candidate agent is absent, indicates that the candidate agent is a mimic or an analogue of the CCN3. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIGS. 1A-1I. A Brain-Dependent Circulatory Factor Functions as an Osteoanabolic Hormone. FIG. 1A, Schematic showing stereotaxic deletion of ERα in the ARC using AAV2-Cre vector with representative µCT scans of the distal femur from females injected with AAV2 control virus (ERα-ControlARC) and ERα-KOARCfemale, as previously reported1. FIG. 1B, µCT images obtained from Esr1Nkx2.1-Cre, Esr1Kiss-Cre,and Esr1Pdyn-Cre4.5-5-week-old mice with %BV / TV indicated lower righthand corner. FIG.1C, Timeline of in vivo µCT imaging post-surgical pairing of Esr1fl / fl(WT) and Esr1Nkx2.1-Cre(Mutant) female mice. FIG. 1D, Representative in vivo µCT imaging of distal femur at baseline (Wk 0) and 3 weeks later (Wk 3) with %BV / TV indicated. FIG.1E, Bar graph shows the percent change in %BV / TV at Wks 3, 6, and 17 compared to Wk 0. FIG. 1F, Absolute %BV / TV graphed for Esr1fl / flfemales in the WT:WT (black) and WT:Mutant pairs (red) showing values for each animal at baseline (0) and 17 weeks later (17), (N = 5WT:5WT, 5WT:5Mut). FIG. 1G, Schematic of wildtype female andAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 male bone transplants into Esr1fl / fl(WT) and Esr1Nkx2.1-Cre(Mutant) female mice for 6 weeks. FIG. 1H, Representative image of µCT scan for control femur transplanted into WT (WT:WT) or mutant females (WT:Mutant). FIG.1I, Fractional bone volume of excised bones transplanted into Esr1fl / flfemales (black bars) or female (red bar) or male (blue bar) bones transplanted into Esr1Nkx2.1-Crefemales (N = 4-6). Two-way ANOVA in left panel e with repeated measures, and One-way ANOVA in right panel f (Šidák’s multiple-comparisons test). Unpaired Student’s T-test, 2-tailed in panel i. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Error Bars ± SEM.

[0070] FIGS. 2A-2K. Brain-Dependent Bone Factor Increases Osteogenic Capacity of ocSSCs. FIG. 2A, Schematic of FACS-isolation and the fate of ocSSCs (left panel) and pvSSCs (right panel) using listed cell-surface markers. FIG. 2B, Schematic of wild-type female ocSSCs (~15,000 live cells) transplanted into kidney capsule of Esr1fl / fland Esr1Nkx2.1-Crefemale mice. FIG. 2C, Images of outlined graft region with host-derived hematopoietic features (top panels, white arrows). Volumetric bone density images (left panels) and sections of graph region stained for mineralized bone (yellow), cartilage (blue), and marrow (red) in right panels). FIG. 2D, Fractional areas for marrow, cartilage, and bone quantified from stained sections in individual kidney grafts (N = 6, 5), and FIG. 2E, bone density for a separate cohort (N = 4, 4). FIG. 2F, Schematic of stereotaxic bilateral delivery of FACS-purified control ocSSCs (~550 live cells) from Esr1fl / fl-CAG-Luc,-GFPinto the MBH of Esr1fl / fland Esr1Nkx2.1-Crefemale mice. FIG. 2G, Representative images of Pentachrome (top panel) with ossicles from imaged brains (lower left) and anti-GFP (lower right) of stained brain sections, six weeks post-injection. FIG.2H. Measured volumetric bone density of ossicles in MBH of Esr1fl / fl(black) and Esr1Nkx2.1-Cre(red) female mice. FIG. 2I, Percent of FACS-purified ocSSCs, pvSSCs, APCs described in Methods isolated from Esr1fl / fland Esr1Nkx2.1-Cre3 weeks old (N = 3, 3) and 10-week-old females (N = 5, 5) and males (5, 3) with the legend for bar graphs above. FIG. 2J, Isolated ocSSCs pooled from 3- and 10-week- old females differentiated in defined media and stained with Alizarin Red (left panel) or Alcian Blue (right panel) with brightfield images from representative wells (FIG.2K) including staining with Oil Red O (n = 3, 3 per group). One-way ANOVA in FIGS.2D, 2I, and 2J (Šidák’s multiple- comparisons test). Unpaired Student’s T-test, 2-tailed for FIGS. 2E, 2H, and 2I (3 Wks). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error Bars ± SEM.

[0071] FIGS. 3A-3G. Identification of CCN3 as a Candidate Brain-Derived Osteoanabolic Factor. FIG.3A, Fractional bone volume, mechanical strength, and BMAT levels in long bones of Esr1fl / fland Esr1Nkx2.1-Crefemales fed SD or HFD for 17 weeks (N = 4-6 per group). FIG. 3B, Representative images of tibia stained for TRAP (top row), labeled for Calcein / Alizarin Red (middle row) and osmium stained (bottom row); lipid droplets (yellowAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 arrows). FIG. 3C, Normalized reads for candidate genes; Penk in the pituitary (N = 2-4). FIG. 3D, Heatmap of top DEGs changed in Esr1Nkx2.1-Crefemales at 12 weeks of age (adapted from RE), and at 27 weeks of age fed SD or HFD; Scale is Log2 fold. FIG. 3E, Transcript levels of Ccn3 and Penk in mutant female ARC by qPCR. FIG.3F, Staining for ERα (pink) and CCN3 (green) in brain sections from posterior ARC and SCN regions of Esr1fl / flfemale (10 wks) and Esr1Nkx2.1-Cre female and male (12 wks), Scale bar = 100 and 200 µm. FIG.3G, Merge of CCN3 and KISS1 in Esr1Nkx2.1-Crefemale ARC. One-way ANOVA in FIGS. 3A and 3C (Šidák’s multiple- comparisons test). Unpaired Student’s T-test, 2-tailed for FIG. 3E. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error Bars ± SEM. Abbreviations: ME median eminence, ARC arcuate nucleus, 3V third ventricle, SCN suprachiasmatic nucleus, oc optic chiasm.

[0072] FIGS. 4A-4J. CCN3 Independent of Sex- and Age Promotes New Bone Formation Through Enhanced Osteogenesis of ocSSCs in Mice and Humans. FIG.4A, Fractional bone volume for control Esr1fl / flfemale (red circles) and male (blue circles) femurs cultured ex vivo and treated daily for 5 days with plasma isolated from Esr1Nkx2.1-Cremutant females (6-12 wks); contralateral femurs were treated with control plasma (white circles), (N = 19, 10). FIG. 4B, Data from panel a, showing percent change in bone volume of contralateral female (red bar) or male (blue bar) femurs after adding mutant versus control plasma. FIG. 4C, Representative µCT images scanned from female and male femurs treated with plasma with corresponding %BV / TV. FIG. 4D, Fractional bone volume of female (N =11) and male (N=8) control femurs treated daily with recombinant mouse CCN3 (mCCN3, 3.0 nM) compared to untreated baseline control (Baseline). FIG.4E, Percent change in bone volume of female (red bar) or male (blue bar) femurs treated with either CCN3 or saline and then compared to the baseline value of freshly isolated and stored contralateral femur (N =5-10). FIG.4F, Representative images of H&E-stained femurs at baseline and after culturing with CCN3. FIG. 4G, Percent change in %BV / TV and trabecular thickness in Esr1fl / flfemales and males following daily CCN3 injections (i.p.7.5 µg / kg) or saline for 21 days. All data were normalized to the average for control females injected with only saline (N = 6,8 for females and 7,6 for males). FIG.4H, Representative µCT images scanned from female and male treated femurs with corresponding %BV / TV. FIG. 4I, Osteogenic differentiation assays of purified mouse ocSSCs treated with recombinant mouse (m)CCN3 protein or the Penk-encoded met-ENK and Bam22P peptides. Plotted values are normalized to Alizarin staining of control wells in defined media alone, set at 100% with brightfield images of wells below without or with mCCN3 (n = 3 per condition). FIG. 4J, Osteogenic differentiation assays of purified human ocSSCs treated with recombinant human (h)CCN3 protein. Plotted values are normalized to Alizarin staining of control wells in definedAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 media alone set at 100%, and representative images of wells treated without or with mCCN3 below (n = 3 per condition). One-way ANOVA in panels e, k, and i (Šidák’s multiple-comparisons test). Paired Student’s T-test, 2-tailed for FIGS.4A, 4D and Unpaired Student’s T-test for FIGS. 4B, 4G. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error Bars ± SEM.

[0073] FIGS.5A-5I. Brain-Derived CCN3 Drives Higher Bone Mass and Is Activated in Lactating Females When Estrogen Plummets. FIG. 5A, Bone volume and dynamic histomorphometry measurements after Calcein and Alizarin red double labeling (5 days apart) of female mice transduced with the lowest dose of AAVdj-CAG-Ccn3 viral vector compared to control vector (black) obtained in femurs from Esr1fl / flcontrol females. FIG.5B, Bone formation rate and mineral apposition rate (MAR) from dynamic histomorphometry measurements with representative images of femur sections described above from aged Esr1fl / flfemales (20-23 months of age) injected with AAVdj-CAG-CCN3. FIG. 5C, BV / TV (%) from µCT and bone formation rate (BFR) plotted as determined by dynamic histomorphometry analyses and assessed by Calcein (green) and Alizarin Red (red). Low dose (N = 4,6, High Dose N = 7,8). FIG. 5D, Injection of siRNA directed against mCcn3 into ARC of mutant Esr1Nkx2.1-Cremutant females. FIG.5E, Ccn3 expression in posterior ARC shown for scramble control oligos, a unilateral hit, and the best level of knockdown. Scale bar = 100 µm. Lower panels show corresponding µCT scans of sagittal and longitudinal views of distal femur; complete misses were added to the control group (N = 4, 4). FIG. 5F, XY plot of the number of CCN3-positive neurons in the posterior ARC versus the fractional bone volume. FIG. 5G, Representative images of coronal brain sections of females stained for ERα (pink) and CCN3 (green) in the posterior ARC region during pregnancy or different postpartum stages as indicated; higher resolution images are shown in the lower row (N ≥ 2). Scale bar = 100 µm upper row, 50 µm lower row. FIG.5H, Ccn3 transcripts quantified from microdissected ARC tissue obtained from Esr1fl / flvirgin, Esr1Nkx2.1-Crevirgin, and Esr1fl / fllactating (7DPP) females (N = 5, 3, 3). FIG. 5I, Schematic of brain-derived CCN3 as an osteoanabolic hormone that counteracts the catabolic actions of mammary-gland PTHrP to maintain adequate calcium and preserve the maternal skeleton during lactation when E2 has vanished. One-Way ANOVA for panel g (Šidák’s multiple-comparisons test), Unpaired Student’s T-test for FIGS.5B. *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant. Error Bars ± SEM. Abbreviations: ME median eminence, ARC arcuate nucleus, 3V third ventricle.

[0074] FIGS. 6A-6E. Sex-Dependent High Bone Mass in Genetic Models that Target KNDy ARC Neurons Occurs Before Puberty. FIG. 6A, Representative images µCT imaging in female and male distal femurs st 4.5 and 6.5 weeks. FIG.6B, Box and whisker plots of structuralAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 bone parameters of control Esr1fl / fland mutant Esr1Pdyn-Cre(mutant) females (red) and males (blue); legend on top. FIG. 6C, Time course of high bone mass in Esr1fl / fl(black) or Esr1Nkx2.1-Cre(red) females beginning at 1 week of age, data for the 4.5-week time point are taken from (RE) and re- graphed, (N = 4 for all groups except N = 2 for mutant at 4 wks), legend on top. FIG. 6D, µCT imaging of females at 3 and 4 weeks of age. FIG.6E, Modified Pentachrome staining of sections from control and mutant distal femur 4 wks of age, with Modified Movat’s stained sections showing enhanced mineralized bone (red) in the mutant femur. One-Way ANOVA for panel g (Šidák’s multiple-comparisons test), Unpaired Student’s T-test for FIG.6B. ****p < 0.0001, ns = not significant. Error Bars ± SEM.

[0075] FIGS.7A-7E. Increases in Trabecular Bone in WT:MUT and MUT:WT Parabiosis Without Weight Changes in Whole Body or Other Tissues. FIG. 7A, Bar graph of Evan’s Blue concentration in blood injected into Control or Mutant female mice 14 days post-surgery to assess pairing. FIG. 7B, Box and whisker plots of percent changes in structural bone parameters of the Esr1fl / fldistal femur after WT:MUT pairing for time indicated as determined by in vivo µCT imaging. FIG.7C, Body weights and other tissue measurements obtained 17 weeks post-joining after euthanasia, (N = 6, 4). FIG. 7D, Percent change (left panel) and fractional bone volume (%BV / TV. right panel) in Esr1Nkx2.1Crefemurs (N = 5) in MUT:WT parabionts, as determined by in vivo µCT scans. FIG. 7E, Representative µCT images of Esr1Nkx2.1Credistal femur from MUT:WT pairings. Legend is shown at top. Unpaired Student T-Test for panel a. Two- and One- Way ANOVA for repeated measures for panels in b, d, respectively (Šidák’s multiple- comparisons test). Ratio Paired Student T-Test for panel d (right). *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Error Bars ± SEM.

[0076] FIGS. 8A-8C. Higher bone mass of Wild-type Femurs Transplanted into Mutant Esr1Nkx2.1-CreFemales. FIG.8A, Images of wild-type female bones 6-weeks post-implantation into control or mutant females. FIG.8B, Box and whisker plots of µCT structural parameters of wild- type female femurs into Esr1fl / fl(black) or Esr1Nkx2.1-Cre(red, N = 5,6) females 6-weeks post- implantation. FIG. 8C, Box and whisker plots of µCT structural parameters of wild-type male bones into Esr1fl / fl(black) or Esr1Nkx2.1-Cre(blue, N = 4,4) females 6-weeks post-implantation. Unaired Student T-Test for all panels in b and c. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Legends to plots on top.

[0077] FIGS.9A-9E. Increased SSCs from Control Bones After Sharing Circulation with Mutant Esr1Nkx2.1-CreFemales, who Exhibit Youthful ocSSCs and Bone Mass. FIG. 9A, Box and whisker plots of live cells obtained following FACS-purification as described in Methods isolated from control femurs obtained from WT:WT or WT:MUT parabionts (N = 6, 4) or from controlAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 female (red, N = 6, 7) or male (blue, N = 4, 4) femurs transplanted into Esr1Nkx2.1-Crefemales as indicted by legend on top. FIG. 9B, Bar graphs of CFU-F from ocSSCs purified from control or mutant female long bones at age indicated, (N = 3-6). FIG. 9C, Differentiation of ocSSCs in defined media and stained with dye as indicated at top of representative images of culture wells (n = 3-4 replicates); AR (Alizarin Red) and AB (Alcian Blue). FIG.9D, Fractional bone volume of trabecular and cortical bone as well as other parameters obtained from µCT scanning. FIG.9E, Representative images of µCT-scans of femur from aged females (≥ 52 weeks of age) of distal and midshaft regions (N = 3, 3). Unaired Student T-Test for all panels in b and c. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error Bars ± SEM.

[0078] FIGS. 10A-10G. ScRNA-Sequencing of Prospectively Isolated Mouse OcSSCs Are Biased For Osteogenic Fate. FIG. 10A, UMAP plot SmartSeq2 scRNA sequencing of unbiased Leiden clustering of 264 high-quality filtered single ocSSCs (122 control and 142 mutant cells) obtained from 7-week-old female mice. FIG. 10B, Dot plot of cluster-specific markers. FIG. 10C, Heatmap of top fifty upregulated genes per cluster. FIG. 10D, UMAP with cell annotation of control Esr1fl / fl(122 cells) and mutant Esr1Nkx2.1Cre(142 cells). FIG. 10E, Distribution of genotypes within cluster populations. FIG. 10F, Dot plot showing higher expression of anti-inflammatory and pro-osteogenic markers within mutant Esr1Nkx2.1CreocSSCs compared to control ocSSCs. FIG.10G, BioPlanet 2019 pathway enrichment (top three bars) and GO Biological Process 2023 ontology (bottom three bars) displaying overexpression in mutants versus wild type based on top200 DEGs.

[0079] FIGS. 11A-11G. Dietary Challenge but Not High Glucose Degrades Bone Only in Mutant Females Without Altering Other Metabolic Parameters. FIG. 11A, XY plot of body weights versus age for Esr1fl / fland Esr1Nkx2.1-Creage-matched female littermates maintained on standard breeder chow (SD) or high-fat diet (HFD) for 17 weeks starting at 10 weeks of age (N ≥ 4 per group). Blood glucose levels after GTT (i.p.), area under the curve (AUC), fat mass, and serum triglycerides plotted for control and mutant females fed HFD for 15 weeks; legend on top. FIG. 11B, Trabecular and cortical bone parameters obtained after µCT scans for four experimental female cohorts. FIG.11C, Representative images of sections of TRAP-stained and double-labeled with Calcein green (green) and Alizarin red (red) femurs for Esr1fl / fland Esr1Nkx2.1-Cre cohorts; area in yellow squares shown in Main FIG. 3B. FIG. 11D, Dynamic histomorphometry obtained from tibias for four different experimental cohorts; osteocytes per bone surface (Oc / BS), bone formation rate / bone surface (BFR / BS), and mineralized surface / bone surface (MS / BS), N = 4 per group. FIG.11E, XY plot of body weights versus age for Esr1fl / fland Esr1Nkx2.1-Creage-matched male experimental cohorts, (N = 4 per group). FIG. 11F, TrabecularAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 and cortical bone parameters obtained by µCT imaging for four male experimental cohorts. Legend on top. FIG. 11G, Blood glucose and structural bone parameters obtained by µCT imaging for Esr1fl / fland Esr1Nkx2.1-Crefemale cohorts treated with vehicle (N = 3, 6) or S961 (N = 5, 5) delivered by implanted osmotic pumps filled with 40 nM Naloxone and delivered over a period of 8 weeks, (N = 5-6 per group). Two-Way ANOVA for repeated measures for panels in a and e (BW curve and GTT), respectively (Šidák’s multiple-comparisons test). One-Way ANOVA for FIGS. 11B, 11D, 11F, and 11G (Šidák’s multiple-comparisons test), Unpaired Student’s T- test for panels in a. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001, ns = not significant. Error Bars ± SEM.

[0080] FIGS. 12A-12C. A Cluster of ARC Genes Correlates with Changing Bone Mass in Female Esr1Nkx2.1-CreMutants. FIG. 12A, Relative expression of transcripts as listed in the female hypothalamus at 2.5 weeks of age shown in bar graphs of individual points (N = 4-8). FIG. 12B, Relative expression of transcripts in microdissected ARC harvested from control Esr1fl / fland mutant Esr1Nkx2.1-Creage-matched females (red) or males (blue), (N = 2-5). FIG.12C, Heatmaps of top 50 DEGs listed to the right following analyses of bulk RNA-Seq datasets of Esr1fl / fland Esr1Nkx2.1-Creage-matched female littermates maintained on standard breeder chow (SD) or high- fat diet (HFD) for 17 weeks starting at 10 weeks of age; samples include microdissected ARC (left panel), whole pituitary glands (middle panel) or liver tissue (right panel). The cluster of secreted proteins / peptides for the ARC attenuated by HFD are highlighted in red text. Legend for each heatmap shows relative Z-Scores. Unpaired Student’s T-test for FIGS. 12A and 12B. ns = not significant. Error Bars ± SEM.

[0081] FIGS. 13A-13E. Low Dose CCN3 Increases Bone Mass in Control Bones Cultured in a Dish and in Mice After Daily Injections. FIG.13A, Bar plots of change in fractional bone volume from whole femurs harvested from control females and then cultured with isolated plasma from Esr1fl / fland Esr1Nkx2.1-Creage-matched female littermates. Plasma (15 µl) was added daily for 1-7 days of culture as described in Methods. FIG.13B, Plots of fractional bone volume were determined after culturing the right tibia and right femur in media treated with 0.9 % NS (Saline). Baseline values were obtained for freshly isolated left tibia or left femur from the same mouse immediately fixed in 4% PFA for analysis, without culturing (Baseline). FIG.13C, H&E staining of sectioned contralateral left and right femurs from same female and male mouse at Baseline or Saline. FIG. 13D, Box and whiskers plots of bone parameters after saline (black) or CCN3 (red) daily treatments of control females. FIG. 13E, Effects of chronic perfusion of Naloxone over 28 days with fractional bone volume plotted for control Esr1fl / fland mutant Esr1Nkx2.1-Creage-matched females, 10-12 weeks of age at the beginning of treatment with vehicleAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 or Naloxone delivered via an implanted mini-osmotic pump (0.5 mg / 24 hrs) over 28 days. Legend above bar graph (N = 4 per group). Paired Student’s T-test for FIG. 13B and Unpaired Student’s T-test for FIG. 13D. One-Way ANOVA for FIG. 13E (Šidák’s multiple-comparisons test), *p < 0.05, **p < 0.01, ns = not significant., ns = not significant. Error Bars ± SEM.

[0082] FIGS. 14A-14D. Low Dose CCN3 Increases Osteogenesis in Young and Older Human ocSSCs Harvested from Either Males or Females. FIG.14A, Normalized Alizarin staining of cultured human ocSSCs harvested from a 14-year-old male growth plate and treated with either recombinant protein or purified peptides, with doses provided and differentiation assays described in Methods. Data shown for CCN3 are replotted from the left panels of Main FIG. 4j for comparison. The dotted line is 100% value for cells cultured in osteogenic media alone. FIG.14B, Same as FIG.14A, except human ocSSCs were harvested from a 15-year-old male fracture. FIG. 14C, Representative images of duplicate wells of Alizarin staining in Control media, osteogenic media minus or plus different doses of human CCN3 with magnified images of one well in far- right images of each panel. FIG. 14D, Representative images of duplicate wells of Alizarin staining in culture wells with osteogenic defined media minus or plus human CCN3. One-Way ANOVA for FIGS. 14A, 14B (Šidák’s multiple-comparisons test to control wells, black), *p < 0.05, ***p < 0.001, ****p< 0.0001, ns = not significant. Error Bars ± SEM.

[0083] FIGS.15A-15C. Ectopic Hepatic mCCN3 Expression in Control Esr1fl / flFemales Increases Bone Formation. FIG. 15A, Expression of mCCN3 protein in female liver transduced with a lower dose of AAVdj-CAG-Ccn3 2 weeks post-injection; panels to the right represent digitally magnified images of individual positive cells. Scale bar = 100 µm. FIG. 15B, Relative levels of Ccn3 transcripts in liver tissue 5 weeks post-injection after transduction of low dose (5*109) or high dose (3*1010) GC / mouse of AAVdj-CAG-Ccn3 viral vector (green bars) and control AAVdj-empty vector (black bars) into Esr1fl / flfemale littermates, (N = 4, 6 for lower titer, 6-8 months of age, and N = 7, 8 for higher titer, 3 months of age). FIG. 15C, Dynamic histomorphometry measurements of bone parameters using double Calcein and Alizarin red labeling in femurs from 6-8-month-old Esr1fl / flcontrol females. Unpaired Student’s T test for low and high dose groups in panels b and c. *p < 0.05, **p < 0.01, ****p < 0.0001. Error Bars ± SEM.

[0084] FIG. 16. Bone regeneration after bone fracture in mice. CCN3 was administered to 24-month-old male mice (NIA C57BL / 6) in a slow-release gel at a bone fracture site. Images obtained at 21 days post-fracture show about a 400% increase in bone at the fracture site for 24- month-old mice administered 2 µg CCN3.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0085] FIGS. 17A-17B. Cartilage regeneration through growth-factor enhanced microfracture (GEM). FIG. 17A, Safranin O staining of distal femur section from uninjured and GEM-treated mice 4-weeks after surgery. The mice were treated with cabozantinib, CCN3, or a combination of cabozantinib and CCN3 and compared to mice administered a PBS control. The injury site is outlined with dashed black line. FIGS. 17B, Quantification of regenerated area showing percentage of area staining for cartilage. Data shown as mean + / - SEM. N=3 per group. One-way ANOVA with Fisher LSD posthoc test. DETAILED DESCRIPTION

[0086] Methods of using CCN3 for treating bone or cartilage disorders are provided. Methods of gene therapy are also provided, including methods of expressing CCN3 in vivo in effective amounts sufficient to promote new bone or cartilage growth. In addition, methods of screening for agonists, mimics, and analogues of CCN3 are also provided.

[0087] Before exemplary embodiments of the present invention are described, it is to be understood that this invention is not limited to particular embodiments described, 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, since the scope of the present invention will be limited only by the appended claims.

[0088] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0089] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials may now be described. Any and all publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection withAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0090] It must be noted that as used herein and in 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 cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof, e.g., polypeptides and peptides, known to those skilled in the art, and so forth.

[0091] It is further noted that the claims may be drafted to exclude any element which may be optional. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[0092] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent such publications may set out definitions of a term that conflicts with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls.

[0093] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. DEFINITIONS

[0094] The term “bone disorder or condition associated with bone degeneration” is used herein to refer to any disease or condition resulting in decreased bone density, reduced bone mass, bone fragility, low bone mineral density, and / or increased fatty bone marrow. Bone disorders and conditions associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0095] The term “cartilage disorder” is used herein to refer to any disease or condition resulting in cartilage damage or loss. Cartilage disorders include, but are not limited to, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, and malignancy.

[0096] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already inflicted (e.g., those with a bone or cartilage disorder or condition associated with bone or cartilage degeneration) as well as those in which prevention is desired (e.g., those with increased susceptibility or a genetic predisposition to developing a bone or cartilage disorder or condition associated with bone or cartilage degeneration).

[0097] A therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming inflicted.

[0098] The terms “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to vertebrates including, but not limited to, mammals, including human and non- human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows; amphibians such as frogs, salamanders, and caecilians; bony fish such as ray-fins and lobe-fins; reptiles such as turtles, crocodilians, snakes, amphisbaenians, lizards, and tuatara; and birds such as domestic, wild and game birds, including chickens, turkeys and other gallinaceous birds, ducks,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 and geese. In some cases, the subject methods find use in experimental animals, in veterinary applications, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, primates, and transgenic animals.

[0099] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0100] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0101] By “therapeutically effective dose or amount” of a CCN3 protein or a recombinant polynucleotide comprising a coding sequence encoding a CCN3 protein is intended an amount that, when administered, as described herein, brings about a positive therapeutic response, such as improved recovery from a bone or cartilage disorder or condition associated with bone or cartilage degeneration. Improved recovery from a bone disorder may include increased bone density, increased bone mass, increased bone strength, increased bone mineral density, and / or decreased fatty bone marrow. Additionally, a therapeutically effective dose or amount may stimulate osteochondral skeletal stem cells to produce bone.

[0102] By "therapeutically effective dose or amount" of CCN3 and a VEGF inhibitor is intended an amount that, when administered in combination with a mechanical stimulus, as described herein, brings about a positive therapeutic response, such as improved recovery from a cartilage disorder. Improved recovery may include generation of new cartilage at a treatment site (e.g., a damaged joint). For example, a therapeutically effective dose or amount could be used to treat cartilage damage or loss resulting from a traumatic injury or a degenerative disease, such as arthritis or other disease involving cartilage degeneration. Preferably, a therapeutically effective amount restores function and / or relieves pain and inflammation associated with cartilage damage or loss.

[0103] In some embodiments, an effective amount of a CCN3 protein or a recombinant polynucleotide comprising a coding sequence encoding a CCN3 protein, when administered byAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 itself or in combination with a VEGF inhibitor, and optionally other factors, will increase bone or cartilage mass by at least about 5%, at least about 10%, at least about 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to an appropriate control, the control typically being a subject not treated with the composition.

[0104] The term “microfracture” refers to a surgical technique using an implement such as a sharp pick, awl, or drill to create small fracture holes in a bone. The microfracture surgical technique was developed to treat chondral defects, which are damaged areas of articular cartilage of the knee. It is commonly used to treat patients with full thickness damage to the articular cartilage that goes all the way down to the bone. Microfracture has been used in joints including, without limitation, the shoulder, hip, ankle, and knee joints.

[0105] Microfracture is typically performed as part of arthroscopic surgery. The area undergoing microfracture is generally prepared by removing any loose or damaged cartilage. Ideally, the area undergoing microfracture will be less than about 2 centimeters in diameter and have good, healthy surrounding cartilage. A small, sharp pick (awl) or drill is used to create the small microfracture holes in the bone. The number of microfractures created depends on the size of the joint that is being treated. Most patients with a 1- to 2-centimeter area of damage require five to 15 small microfracture holes in the bone. The penetration of the outer layers of bone allows blood and stem cells to form a clot in the area of the cartilage defect.

[0106] “Articular cartilage” is the highly specialized connective tissue of diarthrodial joints. Its main function is to provide a smooth, lubricated surface for articulation and to facilitate the transmission of loads with a low frictional coefficient. Articular cartilage is void of blood vessels, lymphatics, and nerves and is subject to a harsh biomechanical environment. Articular cartilage has a limited capacity for intrinsic healing and repair. In this regard, the preservation and health of articular cartilage are paramount to joint health.

[0107] The surfaces of articulating bones in mammalian joints are covered with articular cartilage. The articulating cartilage prevents direct contact of the opposing bone surfaces and permits the near frictionless movement of the articulating bones relative to one another. Two types of articular cartilage defects are commonly observed in mammals and include full-thickness and partial thickness defects. The two-types of defects differ not only in the extent of physical damage but also in the nature of repair response each type of lesion elicits.

[0108] Full-thickness articular cartilage defects include damage to the articular cartilage, the underlying subchondral bone tissue, and the calcified layer of cartilage located between the articular cartilage and the subchondral bone. Full-thickness defects typically arise during severeAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 trauma of the joint or during the late stages of degenerative joint diseases, for example, during osteoarthritis. Since the subchondral bone tissue is both innervated and vascularized, damage to this tissue is often painful. The repair reaction induced by damage to the subchondral bone usually results in the formation of fibrocartilage at the site of the full-thickness defect. Fibrocartilage, however, lacks the biomechanical properties of articular cartilage and fails to persist in the joint on a long-term basis.

[0109] Partial-thickness articular cartilage defects are restricted to the cartilage tissue itself. These defects usually include fissures or clefts in the articulating surface of the cartilage. Partial thickness defects are caused by mechanical arrangements of the joint which in turn induce wearing of the cartilage tissue within the joint. In the absence of innervation and vasculature, partial-thickness defects do not elicit repair responses and therefore tend not to heal. Although painless, partial-thickness defects often degenerate into full-thickness defects.

[0110] “Articular cartilage” is hyaline cartilage and is typically 2 to 4 mm thick. It is composed of a dense extracellular matrix (ECM) with a sparse distribution of chondrocytes. The ECM is mainly composed of water, collagen, and proteoglycans, with other noncollagenous proteins and glycoproteins present in lesser amounts. Along with collagen fiber ultrastructure and ECM, chondrocytes contribute to the various zones of articular cartilage — the superficial zone, the middle zone, the deep zone, and the calcified zone. Within each zone, 3 regions can be identified — the pericellular region, the territorial region, and the interterritorial region.

[0111] The thin superficial (tangential) zone protects deeper layers from shear stresses and makes up approximately 10% to 20% of articular cartilage thickness. The collagen fibers of this zone (primarily, type II and IX collagen) are packed tightly and aligned parallel to the articular surface. The superficial layer contains a relatively high number of flattened chondrocytes, and the integrity of this layer is imperative in the protection and maintenance of deeper layers. This zone is in contact with synovial fluid and is responsible for most of the tensile properties of cartilage, which enable it to resist the sheer, tensile, and compressive forces imposed by joint.

[0112] Immediately deep to the superficial zone is the middle (transitional) zone, which provides an anatomic and functional bridge between the superficial and deep zones. The middle zone represents 40% to 60% of the total cartilage volume, and it contains proteoglycans and thicker collagen fibrils. In this layer, the collagen is organized obliquely, and the chondrocytes are spherical and at low density. Functionally, the middle zone is the first line of resistance to compressive forces.

[0113] The deep zone is responsible for providing the greatest resistance to compressive forces, given that collagen fibrils are arranged perpendicular to the articular surface. The deepAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 zone contains the largest diameter collagen fibrils in a radial arrangement, the highest proteoglycan content, and the lowest water concentration. The chondrocytes are typically arranged in columnar orientation, parallel to the collagen fibers and perpendicular to the joint line. The deep zone represents approximately 30% of articular cartilage volume.

[0114] The tide mark distinguishes the deep zone from the calcified cartilage. The deep zone is responsible for providing the greatest amount of resistance to compressive forces, given the high proteoglycan content. Of note, the collagen fibrils are arranged perpendicular to the articular cartilage. The calcified layer plays an integral role in securing the cartilage to bone, by anchoring the collagen fibrils of the deep zone to subchondral bone. In this zone, the cell population is scarce and chondrocytes are hypertrophic.

[0115] Collagen is the most abundant structural macromolecule in the ECM, and it makes up about 60% of the dry weight of cartilage. Type II collagen represents 90% to 95% of the collagen in ECM and forms fibrils and fibers intertwined with proteoglycan aggregates. Collagen types I, IV, V, VI, IX, and XI are also present but contribute only a minor proportion. The minor collagens help to form and stabilize the type II collagen fibril network.

[0116] Proteoglycans account for 10% to 15% of the wet weight of cartilage. Articular cartilage contains a variety of proteoglycans that are essential for normal function, including aggrecan, decorin, biglycan, and fibromodulin. The largest in size and the most abundant by weight is aggrecan.

[0117] Chondrocytes are the resident cell type in articular cartilage. Chondrocytes are highly specialized, metabolically active cells that play a unique role in the development, maintenance, and repair of the ECM. Chondrocytes have limited potential for replication, a factor that contributes to the limited intrinsic healing capacity of cartilage in response to injury. Chondrocyte survival depends on an optimal chemical and mechanical environment. Biochemical markers of chondrocytes, include without limitation, collagen type II, chondroitin sulfate, keratin sulfate and characteristic morphologic markers of smooth muscle, including but not limited to the rounded morphology observed in culture, and able to secrete collagen type II, including but not limited to the generation of tissue or matrices with hemodynamic properties of cartilage in vitro.

[0118] Fibrocartilage is formed by acute local injury at a bone site in the absence of biochemical factors to direct cartilage formation. The mechanical properties are inferior to articular cartilage. For example, indicia of fibrocartilage include proteoglycan-producing chondrocytes and fibrotic cells, which stain positive for collagen (COL) 1 and matrix metalloproteinase (MMP) 13 and negative for COL 2.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0119] By "proliferate" it is meant to divide by mitosis, i.e. undergo mitosis. An "expanded population" is a population of cells that has proliferated, i.e. undergone mitosis, such that the expanded population has an increase in cell number, that is, a greater number of cells, than the population at the outset.

[0120] The term "tissue" refers to a group or layer of similarly specialized cells which together perform certain special functions.

[0121] The term "organ" refers to two or more adjacent layers of tissue, which layers of tissue maintain some form of cell-cell and / or cell-matrix interaction to form a microarchitecture.

[0122] The terms "protein," "peptide" and "polypeptide" refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and / or imino molecules. No particular size is implied by use of the terms "protein," "peptide" or "polypeptide" and these terms are used interchangeably. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic). Thus, synthetic oligopeptides, dimers, multimers (e.g., tandem repeats, linearly-linked peptides), cyclized, branched molecules and the like, are included within the definition. The terms also include molecules comprising one or more peptoids (e.g., N-substituted glycine residues) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos.5,831,005; 5,877,278; and 5,977,301; Nguyen et al. (2000) Chem Biol.7(7):463-473; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89(20):9367-9371 for descriptions of peptoids). Non-limiting lengths of peptides suitable for use in the present invention includes peptides of 3 to 5 residues in length, 6 to 10 residues in length (or any integer therebetween), 11 to 20 residues in length (or any integer therebetween), 21 to 75 residues in length (or any integer therebetween), 75 to 100 (or any integer therebetween), or polypeptides of greater than 100 residues in length. Typically, polypeptides useful in this invention can have a maximum length suitable for the intended application. Preferably, the polypeptide is between about 3 and 100 residues in length. Generally, one skilled in art can easily select the maximum length in view of the teachings herein. Further, proteins, peptides, and polypeptides, as described herein, for example synthetic peptides, may include additional molecules such as labels or other chemical moieties.

[0123] Thus, references to proteins, polypeptides, or peptides also include derivatives of the amino acid sequences of the invention including one or more non-naturally occurring amino acids. A first polypeptide or peptide is "derived from" a second polypeptide or peptide if it is (i)Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 encoded by a first polynucleotide derived from a second polynucleotide encoding the second polypeptide or peptide, or (ii) displays sequence identity to the second polypeptide or peptide as described herein. Sequence (or percent) identity can be determined as described below. Preferably, derivatives exhibit at least about 50% percent identity, more preferably at least about 80%, and even more preferably between about 85% and 99% (or any value therebetween) to the sequence from which they were derived. Such derivatives can include postexpression modifications of the polypeptide or peptide, for example, glycosylation, acetylation, phosphorylation, and the like.

[0124] Amino acid derivatives can also include modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the protein (or fragment thereof) maintains the desired activity (e.g., CCN3 biological activity, ability to increase bone density, bone mass, bone strength, and / or rate of bone formation, and / or increase cartilage growth, rate of cartilage formation, and / or regeneration of cartilage). These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification. Furthermore, modifications may be made that have one or more of the following effects: increasing ability to increase bone density, bone mass, bone strength, and / or rate of bone formation and / or increasing ability to increase cartilage growth, rate of cartilage formation, and / or regeneration of cartilage, or facilitating purification, delivery, or cell processing. Proteins or biologically active fragments thereof can be made recombinantly, synthetically, or in tissue culture.

[0125] The term “cellular communication network factor 3” or “CCN3” as used herein encompasses all forms of CCN3 and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain biological activity (e.g., ability to increase bone density, bone mass, bone strength, and / or rate of bone formation, and / or increase cartilage growth, rate of cartilage formation, and / or regeneration of cartilage).

[0126] A CCN3 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism but may be synthetically or recombinantly produced. A number of CCN3 nucleic acid and protein sequences are known. Representative sequences of a human CCN3 protein (SEQ ID NO:1), genomic sequence of a CCN3 gene (SEQ ID NO:2), and CCN3 mRNA (SEQ ID NO:3) are presented in the Sequence Listing. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM_002514, NG_009779, NM_010930.5, NM_002514, NM_030868, NM_205268, XM_058698906, XM_004580701, XM_046465005,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 XM_038555394, XM_057736139, XM_057791994, NP_002505, NP_035060, NP_110495, NP_990599, XP_03737995, XP_026919801, XP_032981561, XP_025306624, NP_001253825, XP_036292740, XP_010970891, XP_005564036, XP_004743541, XP_004000129, XP_037748011, XP_043457574, XP_038246527, XP_038540833, and XP_532317; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the variant CCN3 protein retains CCN3 biological activity (i.e., the ability to increase bone density, bone mass, bone strength, and / or rate of bone formation, and / or ability to regenerate cartilage, increase cartilage mass, and / or increase rate of cartilage formation), can be used to produce a CCN3 protein or recombinant polynucleotide comprising a coding sequence encoding a CCN3 protein for use in the methods described herein.

[0127] By "fragment" is intended a molecule consisting of only a part of the intact full- length sequence and structure. The fragment can include a C-terminal deletion an N-terminal deletion, and / or an internal deletion of the polypeptide. Active fragments of a particular protein or polypeptide will generally include at least about 5-14 contiguous amino acid residues of the full length molecule, but may include at least about 15-25 contiguous amino acid residues of the full length molecule, and can include at least about 20-50 or more contiguous amino acid residues of the full length molecule, or any integer between 5 amino acids and the full length sequence, provided that the fragment in question retains biological activity (e.g., CCN3 biological activity, ability to increase bone density, bone mass, bone strength, and / or rate of bone formation, and / or ability to regenerate cartilage, increase cartilage mass, and / or increase rate of cartilage formation).

[0128] "Substantially purified" generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0129] By "isolated" is meant, when referring to a protein, polypeptide, or peptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid moleculeAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

[0130] The term “derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

[0131] The terms “variant,” “analog” and “mutein” refer to biologically active derivatives of the reference molecule that retain desired activity, such as CCN3 biological activity (e.g., the ability to increase bone density, bone mass, bone strength, and / or rate of bone formation, and / or ability to regenerate cartilage, increase cartilage mass, and / or increase rate of cartilage formation), as described herein. In general, the terms “variant” and “analog” refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and / or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity, and which are “substantially homologous” to the reference molecule as defined below. In general, the amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned. Often, the analogs will include the same number of amino acids but will include substitutions, as explained herein. The term “mutein” further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and / or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like. The term also includes molecules comprising one or more N- substituted glycine residues (a “peptoid”) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al., Chem Biol. (2000) 7:463- 473; and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for descriptions of peptoids). Preferably, the analog or mutein has at least the same biological activity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below.

[0132] As explained above, analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic --Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 aspartate and glutamate; (2) basic -- lysine, arginine, histidine; (3) non-polar -- alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar -- glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or non- conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5-25, so long as the desired function of the molecule remains intact. One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp / Woods and Kyte-Doolittle plots, well known in the art.

[0133] By “derivative” is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, as long as the desired biological activity of the native polypeptide is retained. Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.

[0134] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

[0135] In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl.3:353358, National biomedical Research Foundation, Washington, DC, which adapts the local homologyAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 algorithm of Smith and Waterman Advances in Appl. Math.2:482489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0136] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

[0137] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al. (2001) Molecular Cloning, a laboratory manual (3rdedition, Cold Spring Harbor Laboratories, New York); DNA Cloning, supra; Nucleic Acid Hybridization, supra.

[0138] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptideAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0139] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included. The exogenous polynucleotide may be maintained as a non- integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0140] "Recombinant host cells," "host cells," "cells", "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.

[0141] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0142] Typical "control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.

[0143] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0144] "Encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequenceAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.

[0145] "Expression cassette" or "expression construct" refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. In certain embodiments, the expression cassette described herein may be contained within a vector construct (e.g., plasmid or viral vector). In addition to the components of the expression cassette, the vector may also include, one or more selectable markers, a signal which allows the vector to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0146] "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0147] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al., supra, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[0148] A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0149] "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expressionAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, adenoviruses, lentiviruses, alphaviruses, pox viruses, and vaccinia viruses.

[0150] A polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

[0151] As used herein, the term “determining” refers to both quantitative and qualitative determinations and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like.

[0152] An "effective amount" of a CCN3 agonist (e.g., small molecule, drug, receptor ligand, protein, polypeptide, peptide, peptoid, aptamer) is an amount sufficient to increase the biological activity of CCN3 or increase bone growth or cartilage growth. An effective amount can be administered in one or more administrations, applications, or dosages.

[0153] The term "sample" as used herein relates to a material or mixture of materials, typically, although not necessarily, in liquid form, containing one or more analytes of interest.

[0154] As used herein, a “biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents, including but not limited to, conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.

[0155] The term “CCN3 sample” with respect to an individual encompasses biological samples (e.g., blood or plasma samples) comprising CCN3 obtained from the individual. The CCN3 samples can be obtained by any suitable method such as by venipuncture or biopsy. TheAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, washed, centrifuged, or enriched for particular types of molecules (e.g., CCN3).

[0156] The terms "quantity", "amount", and "level" are used interchangeably herein and may refer to an absolute quantification of a molecule or an analyte in a sample, or to a relative quantification of a molecule or analyte in a sample, i.e., relative to another value such as relative to a reference value as taught herein, or to a range of values for the biomarker. These values or ranges can be obtained from a single patient or from a group of patients.

[0157] The term “assaying” is used herein to include the physical steps of manipulating a sample to generate data related to the sample. As will be readily understood by one of ordinary skill in the art, a sample must be “obtained” prior to assaying the sample. Thus, the term “assaying” implies that the sample has been obtained. The terms “obtained” or “obtaining” as used herein encompass the act of receiving an extracted or isolated sample. For example, a testing facility can “obtain” a sample in the mail (or via delivery, etc.) prior to assaying the sample. In some such cases, the sample was “extracted” or “isolated” from an individual by another party prior to mailing (i.e., delivery, transfer, etc.), and then “obtained” by the testing facility upon arrival of the sample. Thus, a testing facility can obtain the sample and then assay the sample, thereby producing data related to the sample.

[0158] The terms “obtained” or “obtaining” as used herein can also include the physical extraction or isolation of a sample from a subject. Accordingly, a sample can be isolated from a subject (and thus “obtained”) by the same person or same entity that subsequently assays the sample. When a sample is “extracted” or “isolated” from a first party or entity and then transferred (e.g., delivered, mailed, etc.) to a second party, the sample was “obtained” by the first party (and also “isolated” by the first party), and then subsequently “obtained” (but not “isolated”) by the second party. Accordingly, in some embodiments, the step of obtaining does not comprise the step of isolating a sample.

[0159] In some embodiments, the step of obtaining comprises the step of isolating a sample. Methods and protocols for isolating various samples (e.g., a blood sample, a serum sample, a plasma sample, a biopsy sample, an aspirate, etc.) will be known to one of ordinary skill in the art and any convenient method may be used to isolate a sample.

[0160] It will be understood by one of ordinary skill in the art that in some cases, it is convenient to wait until multiple samples have been obtained prior to assaying the samples. Accordingly, in some cases an isolated is stored until all appropriate samples have been obtained. One of ordinary skill in the art will understand how to appropriately store a variety of differentAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 types of samples and any convenient method of storage may be used (e.g., refrigeration) that is appropriate for the particular sample. In some embodiments, a pre-treatment sample is assayed prior to obtaining a post-treatment sample. In some cases, a pre-treatment sample and a post- treatment sample are assayed in parallel. In some cases, multiple different post-treatment samples and / or a pre-treatment sample are assayed in parallel. In some cases, samples are processed immediately or as soon as possible after they are obtained.

[0161] A "reference level" or "reference value" of a biomarker (e.g., CCN3) means a level of the biomarker that is indicative of a predisposition to developing a bone disorder or condition associated with bone degeneration (e.g., loss of bone density or bone mass, bone fragility, osteoporosis, or osteopenia). A "reference level" of a biomarker may be an absolute or relative amount or concentration of the biomarker, a presence or absence of the biomarker, a range of amount or concentration of the biomarker, a minimum and / or maximum amount or concentration of the biomarker, a mean amount or concentration of the biomarker, and / or a median amount or concentration of the biomarker. Reference levels may be tailored to specific techniques that are used to measure levels of biomarkers in samples (e.g., ELISA, mass spectrometry, Western blot), where the levels of biomarkers may differ based on the specific technique that is used.

[0162] The terms “determining”, “measuring”, “evaluating”, “assessing,” “assaying,” and “analyzing” are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and / or qualitative determinations. Assaying may be relative or absolute. For example, “assaying” can be determining whether the level is less than or “greater than or equal to” a particular threshold, (the threshold can be pre-determined or can be determined by assaying a control sample). On the other hand, “assaying to determine the level” can mean determining a quantitative value (using any convenient metric) that represents the level. The level can be expressed in arbitrary units associated with a particular assay (e.g., fluorescence units, e.g., mean fluorescence intensity (MFI)), or can be expressed as an absolute value with defined units (e.g., number of molecules or concentration). Additionally, the level can be compared to the level of another biomarker or standard to derive a normalized value that represents a normalized level. The specific metric (or units) chosen is not crucial as long as the same units are used (or conversion to the same units is performed) when evaluating multiple samples from the same individual (e.g., samples taken at different points in time from the same individual). This is because the units cancel when calculating a fold-change (i.e., determining a ratio) in the level from one sample to the next (e.g., samples taken at different points in time from the same individual).Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0163] For measuring protein levels, the amount or level of a protein in the sample is determined. In some cases, the protein comprises a post-translational modification (e.g., phosphorylation, glycosylation) associated with regulation of activity of the protein such as by a signaling cascade, wherein the modified protein is the biomarker, and the amount of the modified protein is therefore measured. In some embodiments, an extracellular protein level is measured. For example, in some cases, the protein (i.e., polypeptide) being measured is a secreted protein and the concentration can therefore be measured in blood or plasma. In some embodiments, concentration is a relative value measured by comparing the level of one protein relative to another protein. In other embodiments the concentration is an absolute measurement of weight / volume or weight / weight.

[0164] In some instances, the concentration of one or more additional proteins may also be measured, and biomarker concentration compared to the level of the one or more additional proteins to provide a normalized value for the biomarker concentration. Any convenient protocol for evaluating protein levels may be employed wherein the level of one or more proteins in the assayed sample is determined.

[0165] While a variety of different manners of assaying for protein levels are known to one of ordinary skill in the art and any convenient method may be used, one representative and convenient type of protocol for assaying protein levels is ELISA, an antibody-based method. In ELISA and ELISA-based assays, one or more antibodies specific for the proteins of interest may be immobilized onto a selected solid surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the assay plate wells are coated with a non-specific “blocking” protein that is known to be antigenically neutral with regard to the test sample such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of non-specific adsorption sites on the immobilizing surface, thereby reducing the background caused by non-specific binding of antigen onto the surface. After washing to remove unbound blocking protein, the immobilizing surface is contacted with the sample to be tested under conditions that are conducive to immune complex (antigen / antibody) formation. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. The occurrence and amount of immunocomplex formation may then be determined by subjecting the bound immunocomplexes to a second antibody having specificity for the target that differs from the first antibody and detecting binding of the second antibody. In certain embodiments, the second antibody will have an associated enzyme, e.g. urease, peroxidase, or alkaline phosphatase, which will generate a color precipitate upon incubating with an appropriate chromogenic substrate. AfterAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 such incubation with the second antibody and washing to remove unbound material, the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6- sulfonic acid (ABTS) and H2O2, in the case of a peroxidase label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.

[0166] The preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody. The solid substrate upon which the antibody or antibodies are immobilized can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate may be chosen to maximize signal to noise ratios, to minimize background binding, as well as for ease of separation and cost. Washes may be effected in a manner most appropriate for the substrate being used, for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, or rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent.

[0167] Alternatively, non-ELISA based-methods for measuring the levels of one or more proteins in a sample may be employed. Representative exemplary methods include but are not limited to antibody-based methods (e.g., immunofluorescence assay, radioimmunoassay, immunoprecipitation, Western blotting, proteomic arrays, xMAP microsphere technology (e.g., Luminex technology), immunohistochemistry, flow cytometry, and the like) as well as non- antibody-based methods (e.g., mass spectrometry or tandem mass spectrometry).

[0168] “Providing an analysis” is used herein to refer to the delivery of an oral or written analysis (i.e., a document, a report, etc.). A written analysis can be a printed or electronic document. A suitable analysis (e.g., an oral or written report) provides any or all of the following information: identifying information of the subject (name, age, etc.), a description of what type of sample(s) was used and / or how it was used, the technique used to assay the sample, the results of the assay (e.g., the level of the CCN3 measured and / or the fold-change of the level of CCN3 over time), the assessment as to whether the individual is at risk of bone loss from continued breast feeding, a recommendation to discontinue breast feeding if the level of CCN3 indicates that the subject is at risk of bone loss if breast feeding is continued, etc. The report can be in any format including, but not limited to printed information on a suitable medium or substrate (e.g., paper); or electronic format. If in electronic format, the report can be in any computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has beenAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 recorded. In addition, the report may be present as a website address which may be used via the internet to access the information at a remote site.

[0169] “Hydrogel” refers to a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel. Biocompatible hydrogel refers to a polymer that forms a gel which is not toxic to living cells, and allows sufficient diffusion of oxygen and nutrients to the entrapped cells to maintain viability.

[0170] The term "stem cell" refers to a cell that retains the ability to renew itself through mitotic cell division and that can differentiate into a diverse range of specialized cell types. Mammalian stem cells can be divided into three broad categories: embryonic stem cells, which are derived from blastocysts, adult stem cells, which are found in adult tissues, and cord blood stem cells, which are found in the umbilical cord. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body by replenishing specialized cells. Adult stem cells include, without limitation, mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells, and neural stem cells. Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a closely related family of cells (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state. Induced pluripotent stem cells can be derived, for example, from adult somatic cells such as peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes.

[0171] As used herein, “reprogramming factors” refers to one or more, i.e., a cocktail, of biologically active factors that act on a cell to alter transcription, thereby reprogramming a cell to multipotency or to pluripotency. Reprogramming factors may be provided individually or as a single composition, that is, as a premixed composition, of reprogramming factors to the cells, e.g., somatic cells from an individual with a family history or genetic make-up of interest, such as a patient who has a neurological disorder or a neurodegenerative disease. The factors may beAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 provided at the same molar ratio or at different molar ratios. The factors may be provided once or multiple times in the course of culturing the cells of the subject invention. In some embodiments the reprogramming factor is a transcription factor, including without limitation, Oct3 / 4; Sox2; Klf4; c-Myc; Nanog; and Lin-28.

[0172] The somatic cells may include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes, etc., which are contacted with reprogramming factors, as defined above, in a combination and quantity sufficient to reprogram the cell to pluripotency. Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. In some embodiments the reprogramming factors are provided as a plurality of coding sequences on a vector.

[0173] The term “skeletal stem cell” refers to a multipotent and self-renewing cell capable of generating bone marrow stromal cells, skeletal cells, and chondrogenic cells. By self-renewing, it is meant that when a skeletal stem cell undergoes mitosis, the cell produces at least one daughter cell that is a skeletal stem cell. By multipotent, it is meant that a skeletal stem cell is capable of giving rise to progenitor cells (skeletal progenitors) that give rise to all cell types of the skeletal system, but not capable of giving rise to cells of other organs in vivo.

[0174] Skeletal stem cells can be derived from non-skeletal cells such as embryonic stem cells, adult stem cells, and induced pluripotent stem cells. For example, skeletal stem cells can be generated by reprogramming mesenchymal stem cells, and adipose tissue containing such cells, such as human adipose stem cells (hAASC). Induced skeletal cells have characteristics of functional SSCs derived from nature; that is, they can give rise to the same lineages.

[0175] Human SSC cell populations may be characterized by their cell surface markers, though it will be understood by one of skill in the art that endogenous populations of SSC need not be characterized for effective stimulation. Human SSC are negative for expression of CD45, CD235, Tie2, and CD31; and express positively podoplanin (PDPN). A population of cells, e.g., cells isolated from bone tissue, having this combination of markers may be referred to as [PDPN+ / 146] cells. The [PDPN+ / 146] population can be further subdivided into three populations: a unipotent subset capable of chondrogenesis [PDPN+CD146 CD73 CD164], a unipotent cellular subpopulation capable of osteogenesis [PDPN+CD146 CD73 CD164+] and a multipotent [PDPN+CD146 CD73+CD164+] cell capable of endochondral (bone and cartilage) ossification. A population of cells of interest for use in the methods of the invention may be isolated from bone with respect to CD45, CD235, Tie2, and CD31 and PDPN. Other cell populations of interest areAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 [PDPN+CD146 CD73 CD164] cells; [PDPN+CD146 CD73 CD164+] cells; and [PDPN+CD146 CD73+CD164+] cells. The mouse skeletal lineage is characterized as CD45-, Ter119-, Tie2-, ocv integrin+. The SSC is further characterized as Thy1- 6C3- CD105- CD200+.

[0176] “Adipose-derived stem cells” or "adipose-derived stromal cells" refer to cells that originate from adipose tissue. By "adipose" is meant any fat tissue. The adipose tissue may be brown or white adipose tissue, derived from subcutaneous, omental / visceral, mammary, gonadal, or other adipose tissue site. Preferably, the adipose is subcutaneous white adipose tissue. Such cells may be provided as a primary cell culture or an immortalized cell line. The adipose tissue may be from any organism having fat tissue. Preferably, the adipose tissue is mammalian, most preferably the adipose tissue is human. A convenient source of adipose tissue is from liposuction surgery, however, the source of adipose tissue or the method of isolation of adipose tissue is not critical to the invention.

[0177] Adipose tissue is abundant and accessible to harvest methods with minimal risk to the patient. It is estimated that there are more than 104stem cells per gram of adipose tissue (Sen et al 2001, Journal of Cellular Biochemistry 81:312-319), which cells can be used immediately or cryopreserved for future autologous or allogeneic applications.

[0178] Methods for the isolation, expansion, and differentiation of human adipose tissue- derived cells have been reported. See for example, Burris et al 1999, Mol Endocrinol 13:410-7; Erickson et al 2002, Biochem Biophys Res Commun. Jan.18, 2002; 290(2):763-9; Gronthos et al 2001, Journal of Cellular Physiology, 189:54-63; Halvorsen et al 2001, Metabolism 50:407-413; Halvorsen et al 2001, Tissue Eng.7(6):729-41; Harp et al 2001, Biochem Biophys Res Commun 281:907-912; Saladin et al 1999, Cell Growth & Diff 10:43-48; Sen et al 2001, Journal of Cellular Biochemistry 81:312-319; Zhou et al 1999, Biotechnol. Techniques 13:513-517. Adipose tissue- derived stromal cells may be obtained from thinned human adipose tissue by collagenase digestion and differential centrifugation [Halvorsen et al 2001, Metabolism 50:407-413; Hauner et al 1989, J Clin Invest 84:1663-1670; Rodbell et al 1966. J Biol Chem 241:130-139].

[0179] Adipose tissue derived stem cells have been reported to express markers including: CD13, CD29, CD44, CD63, CD73, CD90, CD166, aldehyde dehydrogenase (ALDH), and ABCG2. The adipose tissue derived stem cells may be a population of purified mononuclear cells extracted from adipose tissue capable of proliferating in culture for more than 1 month.

[0180] For isolation of cells from tissue, an appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g., normal saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.

[0181] The cell population can be used immediately. Alternatively, the cell population may be frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused. In such cases, the cells will usually be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.

[0182] The adipose cells can be cultured in vitro under various culture conditions. Culture medium may be liquid or semi-solid, e.g., containing agar, methylcellulose, etc. The cell population may be conveniently suspended in an appropriate nutrient medium, such as Iscove's modified DMEM or RPMI-1640, normally supplemented with fetal calf serum (about 5-10%), L- glutamine, a thiol, particularly 2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin. In one embodiment of the invention, the adipose cells are maintained in culture in the absence of feeder layer cells, i.e. in the absence of serum, etc. The culture may contain growth factors to which the cells are responsive. Growth factors, as defined herein, are molecules capable of promoting survival, growth and / or differentiation of cells, either in culture or in the intact tissue, through specific effects on a transmembrane receptor. Growth factors include polypeptides and non-polypeptide factors.

[0183] The terms “efficiency of reprogramming”, “reprogramming efficiency”, “efficiency of conversion”, or “conversion efficiency” are used interchangeably herein to refer to the ability of cells of one cell lineage to give rise to an induced cell of another cell lineage when contacted with the appropriate reprogramming system, for example, the ability of adipose tissue cells to give rise to iSSC when contacted with high doses of BMP2. In other words, the cells produce about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold, about 20-fold, about 30-fold, about 50-fold, about 100-fold, about 200-fold the number of induced cells (e.g. iSSC) as the uncontacted population, or more. Promoting Bone and Cartilage Regeneration with CCN3

[0184] Compositions and methods are provided using CCN3 for regenerating or replacing bone or cartilage. In certain embodiments, CCN3 is used as an osteogenic factor to stimulate osteochondral skeletal stem cells to produce bone. CCN3 can be used to increase bone density, bone mass, bone strength, and / or the rate of bone formation, decrease fatty bone marrow in bone, and / or reverse bone loss. In some embodiments, CCN3 is used to treat a bone disorder or conditionAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 associated with bone degeneration. Bone disorders and conditions associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0185] In other embodiments, CCN3 is used as a chondrogenic factor to stimulate skeletal cells to produce cartilage. CCN3 can be used, for example, to treat a cartilage lesion or injury in a subject. Such lesions include any condition involving cartilaginous tissue which is inadequate for physiological or cosmetic purposes. Such defects include those that are congenital, including developmental malformations, and defects resulting from disease, trauma, or surgical or other medical procedures. Cartilage defects include those brought about by cartilage disorders including, but not limited to, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury (e.g., such as caused by surgery, accidental injury, sports injury), infection, or malignancy.

[0186] Various sites for articular cartilage regeneration can be treated, including without limitation a knee joint, elbow joint, joints in the phalanges and phalanxes, shoulder joints, hip joints, wrist joints, ankle joints, etc. The individual may be an adult, e.g., past adolescence, and may be an aged adult, e.g., a human over 55 years of age, over 65 years of age, over 70 years of age, etc.

[0187] An individual in need of cartilage regeneration can be treated with the methods described herein. For treatment of a cartilage disorder, a therapeutically effective amount of CCN3 is administered in combination with a therapeutically effective amount of a VEGF inhibitor and a mechanical stimulus.

[0188] The VEGF inhibitor can be any substance that decreases signaling by the VEGF- VEGFR pathway. VEGF inhibitors include, to name just a few examples, small molecules, peptides, polypeptides, proteins, including more specifically antibodies, including anti-VEGF antibodies, anti-VEGFR antibodies, intrabodies, maxibodies, minibodies, diabodies, Fc fusion proteins such as peptibodies, receptibodies, soluble VEGF receptor proteins and fragments, and a variety of others. Many VEGF inhibitors work by binding to VEGF or to a VEGF receptor. Others work more indirectly by binding to factors that bind to VEGF or to a VEGF receptor or to other components of the VEGF signaling pathway. Still other VEGF inhibitors act by altering regulatoryAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 posttranslational modifications that modulate VEGF pathway signaling. VEGF inhibitors also may act through more indirect mechanisms. Whatever the mechanism involved, as used herein, a VEGF inhibitor decreases the effective activity of the VEGF signaling pathway in a given circumstance over what it would be in the same circumstance in the absence of the inhibitor.

[0189] In some embodiments, a dose of a VEGF inhibitor is provided in an implant, e.g., a matrix or scaffold for localized delivery of the factor. The effective dose may be determined based on the specific tissue, rate of release from the implant, size of the implant, and the like and may be empirically determined by one of skill in the art. The dose may provide for biological activity equivalent to 1 mg soluble VEGF receptor, 10 mg, 100 mg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 250 mg, 500 mg, 750 mg , 1 g of soluble VEGF receptor. The dose may be administered at a single time point, e.g., as a single implant; or may be fractionated, e.g., delivered in a microneedle configuration. The dose may be administered, once, two, three times, 4 times, 5 times, 10 times, or more as required to achieve the desired effect, and administration may be daily, every 2 days, every 3 days, every 4 days, weekly, bi-weekly, monthly, or more.

[0190] A great many VEGF inhibitors have been described in the literature. For a description of VEGF inhibitors, see, e.g., the following patent documents: US 2003 / 0105091, US2006 / 0241115, US 5,521,184, US 5,770,599, US 5,990,141, US 6,235,764, US 6,258,812, US 6, 515.004, US 6,630,500, US 6,713,485, WO 02005 / 070891, WO 01 / 32651, WO 02 / 68406, WO 02 / 66470, WO 02 / 55501, WO 04 / 05279, WO 04 / 07481, WO 04 / 07458, WO 04 / 09784, WO 02 / 59110, WO 99 / 450029, WO 00 / 59509, WO 99 / 61422, WO 00 / 12089, WO 00 / 02871, and WO 01 / 37820, particularly parts relevant to VEGF inhibitors; herein incorporate by reference.

[0191] Exemplary VEGF inhibitors include, without limitation, ABT-869 (Abbott) including formulations for oral administration and closely related VEGF inhibitors; AEE-788 (Novartis) (also called AE-788 and NVP-AEE-788, among others) including formulations for oral administration and closely related VEGF inhibitors; AG-13736 (Pfizer) (also called AG-013736) including formulations for oral administration and closely related VEGF inhibitors; AG-028262 (Pfizer) and closely related VEGF inhibitors; Angiostatin (EntreMed) (also called CAS Registry Number 86090-08-6, K1-4, and rhuAngiostatin, among others) and closely related inhibitors as described in, among others, US Patent Nos.5,792,825 and 6,025,688, particularly in parts relating to Angiostatin and closely related VEGF inhibitors, their structures and properties, and methods for making and using them; Avastin™ (Genentech) (also called bevacizumab, R-435, rhuMAB- VEGF, and CAS Registry Number 216974-75-3, among others) and closely related VEGF inhibitors; AVE-8062 (Ajinomoto Co. and Sanofi-aventis) (also called AC-7700 and combretastatin A4 analog, among others), and closely related VEGF inhibitors; AZD-2171Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 (AstraZeneca) and closely related VEGF inhibitors; Nexavar® (Bayer AG and Onyx) (also called CAS Registry Number 284461-73-0, BAY-43-9006, raf kinase inhibitor, sorafenib, sorafenib analogs, and IDDBCP150446, among others) and closely related VEGF inhibitors; BMS-387032 (Sunesis and Bristol-Myers Squibb) (also called SNS-032 and CAS Registry Number 345627-80- 7, among others) and closely related VEGF inhibitors; CEP-7055 (Cephalon and Sanofi-aventis) (also called CEP-11981 and SSR-106462, among others) and closely related VEGF inhibitors; CHIR-258 (Chiron) (also called CAS Registry Number 405169-16-6, GFKI, and GFKI-258, among others) and closely related VEGF inhibitors; CP-547632 (OSI Pharmaceuticals and Pfizer) (also called CAS Registry Number 252003-65-9, among others) and closely related VEGF inhibitors such as, for instance, CP-564959; E-7080 (Eisai Co.) (also called CAS Registry Number 417716-92-8 and ER-203492-00, among others) and closely related VEGF inhibitors; 786034 (GlaxoSmithKline) and closely related VEGF inhibitors; GW-654652 (GlaxoSmithKline) and closely related indazolylpyrimidine Kdr inhibitors; IMC-1C11 (ImClone) (also called DC-101 and c-p1C11, among others) and closely related VEGF inhibitors; KRN-951 (Kirin Brewery Co.) and other closely related quinoline-urea VEGF inhibitors; PKC-412 (Novartis) (also called CAS Registry Number 120685-11-2, benzoylstaurosporine, CGP-41251, midostaurin, and STI-412, among others) and closely related VEGF inhibitors; PTK-787 (Novartis and Schering) (also called CAS Registry Numbers 212141-54-3 and 212142-18-2, PTK / ZK, PTK-787 / ZK-222584, ZK- 22584, VEGF-TKI, VEGF-RKI, PTK-787A, DE-00268, CGP-79787, CGP-79787D, vatalanib, ZK-222584, among others) and closely related anilinophthalazine derivative VEGF inhibitors; SU11248 (Sugen and Pfizer) (also called SU-11248, SU-011248, SU-11248J, Sutent®, and sunitinib malate, among others) and closely related VEGF inhibitors; SU-5416 (Sugen and Pfizer / Pharmacia) (also called CAS Registry Number 194413-58-6, semaxanib, 204005-46-9, among others) and closely related VEGF inhibitors; SU-6668 (Sugen and Taiho) (also called CAS Registry Number 252916-29-3, SU-006668, and TSU-68, among others) and closely related VEGF inhibitors as described in, among others, WO-09948868, WO- 09961422, and WO- 00038519, particularly in parts relating to SU-6668 and closely related VEGF inhibitors, their structures and properties, and methods for making and using them; VEGF Trap (Regeneron and Sanofi-aventis) (also called AVE-0005 and Systemic VEGF Trap, among others) and closely related VEGF inhibitors as described in, among others, WO-2004110490, particularly in parts relating to VEGF Trap and closely related VEGF inhibitors, their structures and properties, and methods for making and using them; Thalidomide (Celgene) (also called CAS Registry Number 50-35-1, Synovir, Thalidomide Pharmion, and Thalomid, among others) and closely related VEGF inhibitors; XL-647 (Exelixis) (also called EXEL-7647, among others) and closely relatedAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 VEGF inhibitors; XL-999 (Exelixis) (also called EXEL-0999, among others) and closely related VEGF inhibitors; XL-880 (Exelixis) (also called EXEL-2880, among others) and closely related VEGF inhibitors; ZD-6474 (AstraZeneca) (also called CAS Registry Number 443913-73-3, Zactima, and AZD-6474, among others) and closely related anilinoquinazoline VEGF inhibitors; and ZK-304709 (Schering) (also called CDK inhibitors (indirubin derivatives), ZK-CDK, MTGI, and multi-target tumor growth inhibitor, among others) and other closely related compounds including the indirubin derivative VEGF inhibitors described in WO-00234717 , WO-02074742, WO-02100401, WO-00244148, WO-02096888, WO-03029223, WO-02092079, and WO- 02094814, particularly in parts relevant to these and closely related VEGF inhibitors, their structures and properties, and methods for making and using them. VEGF inhibitors may be delivered in a manner appropriate to the nature of the inhibitor, e.g., as a protein, small molecule, nucleic acid, etc., including without limitation appropriate vehicles and vectors as required.

[0192] In some embodiments, the mechanical stimulus is an acute local injury. In some embodiments, an individual presents with an acute local injury, e.g., from an accident or sports injury, etc., which may be treated with CCN3 in combination with a VEGF inhibitor in order to reduce development of fibrocartilage and enhance regeneration of articular cartilage.

[0193] In other embodiments, the acute local injury is surgically performed through a microfracture process performed with an awl, drill, or the like. Microfracture refers to a surgical technique that produces “microfractures” in subchondral bone perpendicular to the surface. This technique may use various angled awls or “picks.”, or small drills. A rough, raw surface that could hold the clot may also be formed. A pick can be used, for example, to produce fracture fragments that attract and hold the clot. In order for tissue to regenerate, cells must be present. In this procedure, the controlled “microfractures” through the subchondral bone allow access to marrow- based progenitor cells and growth factors. A marrow clot forms at the base of a prepared chondral lesion, and pluripotent cells proliferate and differentiate.

[0194] General indications for microfracture include full-thickness defects, unstable cartilage that overlies the subchondral bone, and a partial-thickness lesion that, when probed, the cartilage simply scrapes off down to bone. Patient age is not a specific contraindication. While patients under 35 years of age have greater improvement, older patients still show improvement. The size of the lesion is also not a contraindication for microfracture. Lesions may less than 400 mm2or more than 400 mm2. The height of the cartilage rim surrounding the lesion may be adequate to hold the clot in place.

[0195] MRI can be used to assess the thickness of the cartilage and determine other associated injuries. The MRI enables imaging of morphological changes such as chondralAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 fibrillation, fissuring, focal defects and corresponding fragments, and more diffuse thinning and wear, all manifesting as changes of the chondral thickness and surface at the cartilage interface to joint fluid and synovium. Earlier chondral degenerative changes, such as softening or blistering, to later fibrotic change can also be visible as intrasubstance areas of MRI signal change and heterogeneity, although such evaluation is still qualitative in standard clinical practice.

[0196] A thorough diagnostic arthroscopic examination of the joint can be performed through 3 portals (inflow cannula, arthroscope, and working instruments). Particular attention is paid to anterior interval scarring, plicae, and the lateral retinaculum, which have the potential to increase compression between cartilage surfaces. Microfracture is the final intra-articular procedure performed. This allows the initial clot in the microfracture site to be preserved. This can also prevent loss of visualization with blood and fat droplets entering the knee from the microfracture.

[0197] After identification of the full-thickness articular cartilage lesion, all remaining unstable cartilage is removed. A hand-held curved curette and a full radius resector can be used to remove the loose or marginally attached cartilage back to a stable rim of cartilage. The calcified cartilage layer that remains as a cap to many lesions is removed, preferably by using a curette. The integrity of the subchondral plate should be maintained. It is important that the defect is debrided deep enough to remove calcified cartilage layer but not so deep that the subchondral plate is damaged. This prepared lesion, with a stable perpendicular edge of healthy well-attached viable cartilage surrounding the defect provides a pool that helps hold the marrow clot as it forms.

[0198] Arthroscopic awls are used to make multiple holes, or “microfractures.” An angled awl, typically 30° or 45°, allows the tip to be perpendicular to the bone as it is advanced. A 90° awl is used for the patella or other soft bone; however, it should only be advanced manually, not with a mallet. Starting at the periphery, microfracture holes are made, ending with holes toward the center of the defect. These are made far enough apart so they do not break into each other, and the subchondral plate between them is protected. Fat droplets from the marrow cavity are seen when the appropriate depth (approximately 2 to 4 mm) has been reached. When completed, the irrigation fluid pump pressure is reduced to observe the release of marrow fat droplets and blood from the microfracture holes. During microfracture, a rough surface has been created in the defect. This surface should not be debrided or shaved further to make it smooth. This rough surface allows for the marrow clot to adhere more easily, yet the integrity of the subchondral plate is maintained for joint surface shape.

[0199] At the time of microfracture, or shortly after a local acute injury, CCN3 is administered in combination with a VEGF inhibitor to treat a cartilage disorder. In some cases, aAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 drug delivery device is implanted or otherwise positioned to provide an effective dose of CCN3 and a VEGF inhibitor. The CCN3 and VEGF inhibitor may be provided in separate compositions or in a single composition, that is, as a premixed composition of factors. The CCN3 and a VEGF inhibitor may be provided at the same molar ratio or at different molar ratios, and may be provided once or multiple times during the course of treatment. For example, an implant including both CCN3 and a VEGF inhibitor may be provided to an individual, as described further below. Production of CCN3

[0200] A CCN3 protein or biologically active variants or fragments thereof can be prepared in any suitable manner (e.g., recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g., native, fusions, labeled, lipidated, amidated, acetylated, PEGylated, etc.). The CCN3 protein may include naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing proteins are well understood in the art. Proteins are preferably prepared in substantially pure form (i.e. substantially free from other host cell or non-host cell proteins).

[0201] CCN3 nucleic acid and protein sequences may be derived from any source. A number of CCN3 nucleic acid and protein sequences are known. Representative sequences of a human CCN3 protein (SEQ ID NO:1), a genomic sequence of a CCN3 gene (SEQ ID NO:2), and a CCN3 mRNA (SEQ ID NO:3) are presented in the Sequence Listing. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM_002514, NG_009779, NM_010930.5, NM_002514, NM_030868, NM_205268, XM_058698906, XM_004580701, XM_046465005, XM_038555394, XM_057736139, XM_057791994, NP_002505, NP_035060, NP_110495, NP_990599, XP_03737995, XP_026919801, XP_032981561, XP_025306624, NP_001253825, XP_036292740, XP_010970891, XP_005564036, XP_004743541, XP_004000129, XP_037748011, XP_043457574, XP_038246527, XP_038540833, and XP_532317; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the variant CCN3 protein retains CCN3 biological activity (i.e., the ability to increase bone density, bone mass, bone strength, and / or rate of bone formation, and / or ability to regenerate cartilage, increase cartilage mass, and / or increase rate of cartilage formation), can be used toAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 produce a CCN3 protein or recombinant polynucleotide comprising a coding sequence encoding a CCN3 protein for use in the methods described herein.

[0202] In one embodiment, a CCN3 protein is generated using recombinant techniques. One of skill in the art can readily determine nucleotide sequences that encode CCN3 using standard methodology and the teachings herein. Oligonucleotide probes can be devised based on the known sequences and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques and, e.g., restriction enzymes employed to truncate the gene at desired portions of the full-length sequence. Similarly, sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA.

[0203] The sequences encoding CCN3 can also be produced synthetically, for example, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Sci- ence 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311; Stemmer et al. (1995) Gene 164:49-53.

[0204] Recombinant techniques are readily used to clone sequences encoding CCN3 that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid. Such a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes. Alternatively, the mutations can be effected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See, e.g., Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409.

[0205] Once coding sequences have been isolated and / or synthesized, they can be cloned into any suitable vector or replicon for expression. (See, also, Examples). As will be apparent from the teachings herein, a wide variety of vectors encoding modified polypeptides can beAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 generated by creating expression constructs which operably link, in various combinations, polynucleotides encoding polypeptides having deletions or mutations therein.

[0206] Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra; Sambrook et al., supra; Perbal et al. A Practical Guide to Molecular Cloning (Wiley-Liss; 2ndedition, 1988).

[0207] Insect cell expression systems, such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit).

[0208] Plant expression systems can also be used to produce the CCN3. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems, see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; and Hackland et al., Arch. Virol. (1994) 139:1-22.

[0209] Viral systems, such as a vaccinia-based infection / transfection system, as described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74:1103-1113, will also find use with the present invention. In this system, cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).

[0210] The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequenceAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 may or may not contain a signal peptide or leader sequence. With the present invention, both the naturally occurring signal peptides or heterologous sequences can be used. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397. Such sequences include, but are not limited to, the TPA leader, as well as the honeybee mellitin signal sequence.

[0211] Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

[0212] The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

[0213] In some cases, it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and / or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et al. (2001) Molecular Cloning, a laboratory manual (3rdedition, Cold Spring Harbor Laboratories, New York); DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

[0214] The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Vero293 cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirusAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0215] Depending on the expression system and host selected, the CCN3 protein is produced by growing host cells transformed by an expression vector described above under conditions whereby the protein is expressed. The selection of the appropriate growth conditions is within the skill of the art.

[0216] In one embodiment, the transformed cells secrete the CCN3 protein product into the surrounding media. Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example using a tissue plasminogen activator (TPA) leader sequence, an interferon (γ or α) signal sequence or other signal peptide sequences from known secretory proteins. The secreted CCN3 protein product can then be isolated by various techniques described herein, for example, using standard purification techniques such as but not limited to, hydroxyapatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0217] Alternatively, the transformed cells are disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the recombinant peptides or polypeptides substantially intact. Intracellular proteins can also be obtained by removing components from the cell wall or membrane, e.g., by the use of detergents or organic solvents, such that leakage of the polypeptides occurs. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001).

[0218] For example, methods of disrupting cells for use with the present invention include but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freeze-thaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS. The particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, culture conditions and any pre- treatment used.

[0219] Following disruption of the cells, cellular debris is removed, generally by centrifugation, and the intracellularly produced peptides or polypeptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0220] For example, one method for obtaining the intracellular polypeptides involves affinity purification, such as by immunoaffinity chromatography using antibodies (e.g., previously generated antibodies), or by lectin affinity chromatography. Particularly preferred lectin resins are those that recognize mannose moieties such as but not limited to resins derived from Galanthus nivalis agglutinin (GNA), Lens culinaris agglutinin (LCA or lentil lectin), Pisum sativum agglutinin (PSA or pea lectin), Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinum agglutinin (AUA). The choice of a suitable affinity resin is within the skill in the art. After affinity purification, the peptides or polypeptides can be further purified using conventional techniques well known in the art, such as by any of the techniques described above.

[0221] The CCN3 protein can be conveniently synthesized chemically, for example by any of several techniques that are known to those skilled in the peptide art. See, e.g., Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W. C. Chan and Peter D. White eds., Oxford University Press, 1stedition, 2000) ; N. Leo Benoiton, Chemistry of Peptide Synthesis (CRC Press; 1stedition, 2005); Peptide Synthesis and Applications (Methods in Molecular Biology, John Howl ed., Humana Press, 1sted., 2005); and Pharmaceutical Formulation Development of Peptides and Proteins (The Taylor & Francis Series in Pharmaceutical Sciences, Lars Hovgaard, Sven Frokjaer, and Marco van de Weert eds., CRC Press; 1stedition, 1999); herein incorporated by reference.

[0222] In general, these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage. The protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support, if solid phase synthesis techniques are used) are removed sequentially or concurrently, to render the final peptide or polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., Rockford, IL 1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles ofAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 Peptide Synthesis, (Springer-Verlag, Berlin 1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol.1, for classical solution synthesis. These methods are typically used for relatively small polypeptides, i.e., up to about 50-100 amino acids in length, but are also applicable to larger polypeptides.

[0223] Typical protecting groups include t-butyloxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4- dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o- nitrophenylsulfonyl and the like.

[0224] Typical solid supports are cross-linked polymeric supports. These can include divinylbenzene cross-linked-styrene-based polymers, for example, divinylbenzene- hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene-benzhydrylaminopolystyrene copolymers.

[0225] The CCN3 protein can also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985) 82:5131-5135; U.S. Patent No.4,631,211. Nucleic Acids Encoding CCN3

[0226] Nucleic acids encoding CCN3 can be used in gene therapy applications to treat a bone or cartilage disorder or condition associated with bone or cartilage degeneration. Nucleic acids described herein can be inserted into an expression vector to create an expression cassette capable of producing the CCN3 in a suitable host cell. The ability of constructs to produce the CCN3 can be empirically determined.

[0227] Expression cassettes typically include control elements operably linked to the coding sequence, which allow for the expression of the gene in vivo in the subject species. For example, typical promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others. Other nonviral promoters, such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression. Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon. Preferably, a sequence for optimization of initiation of translation, located 5' to the coding sequence, is also present. Examples of transcription terminator / polyadenylation signals includeAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence.

[0228] Enhancer elements may also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMPO J. (1985) 4:761, the enhancer / promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence.

[0229] Once complete, the constructs encoding CCN3 can be administered to a subject using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos.5,399,346, 5,580,859, 5,589,466. Genes can be delivered either directly to a subject or, alternatively, delivered ex vivo, to cells derived from the subject and the cells reimplanted in the subject.

[0230] A number of viral based systems have been developed for gene transfer into mammalian cells. These include adenoviruses, retroviruses (γ-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses (see e.g., Warnock et al. (2011) Methods Mol. Biol.737:1-25; Walther et al. (2000) Drugs 60(2):249-271; and Lundstrom (2003) Trends Biotechnol.21(3):117-122; herein incorporated by reference).

[0231] For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No.5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (2011) Curr Pharm Des.17(24):2516-2527). Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2):132-159; herein incorporated by reference).

[0232] A number of adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene TherapyAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51- 58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). Additionally, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 (published 23 January 1992) and WO 93 / 03769 (published 4 March 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

[0233] Another vector system useful for delivering the polynucleotides encoding CCN3 is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct.14, 1997, herein incorporated by reference).

[0234] Additional viral vectors which will find use for delivering nucleic acid molecules encoding CCN3 include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the CCN3 can be constructed as follows. The DNA encoding the particular CCN3 coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome. The resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0235] Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with. respect to the production of vaccinia viruses. See, e.g., WO 91 / 12882; WO 89 / 03429; and WO 92 / 03545.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0236] Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

[0237] Members of the Alphavirus genus, such as, but not limited to, vectors derived from the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the polynucleotides of the present invention. For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol.70:508-519; and International Publication Nos. WO 95 / 07995, WO 96 / 17072; as well as Dubensky, Jr., T. W., et al., U.S. Pat. No.5,843,723, issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S. Patent No. 5,789,245, issued Aug. 4, 1998, both herein incorporated by reference. Particularly preferred are chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02 / 099035, WO 02 / 080982, WO 01 / 81609, and WO 00 / 61772; herein incorporated by reference in their entireties.

[0238] A vaccinia-based infection / transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest (for example, a CCN3 expression cassette) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

[0239] As an alternative approach to infection with vaccinia or avipox virus recombinants, or to the delivery of genes using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can beAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 introduced into cells along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For a further discussion of T7 systems and their use for transforming cells, see, e.g., International Publication No. WO 94 / 26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201- 1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No.5,135,855.

[0240] The synthetic expression cassette of interest can also be delivered without a viral vector. For example, the synthetic expression cassette can be packaged as DNA or RNA in liposomes prior to delivery to the subject or to cells derived therefrom. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed DNA to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991.) 1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol.101, pp.512-527.

[0241] Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192), in functional form.

[0242] Cationic liposomes are readily available. For example, N[1-2,3- dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include (DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; PCT Publication No. WO 90 / 11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

[0243] Similarly, anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed withAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0244] The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al., in Methods of Immunology (1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

[0245] The DNA and / or peptide(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta (1975) 394:483- 491. See, also, U.S. Pat. Nos.4,663,161 and 4,871,488.

[0246] The expression cassette of interest may also be encapsulated, adsorbed to, or associated with, particulate carriers. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362- 368; McGee J. P., et al., J Microencapsul. 14(2):197-210, 1997; O'Hagan D. T., et al., Vaccine 11(2):149-54, 1993.

[0247] Furthermore, other particulate systems and polymers can be used for the in vivo or ex vivo delivery of the nucleic acid of interest. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest. Similarly, DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods. See, e.g., Felgner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for a review of delivery systems useful for gene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No.5,831,005, issued Nov.3, 1998, herein incorporated by reference) may also be used for delivery of a construct of the present invention.

[0248] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering synthetic expression cassettes encoding CCN3.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 The particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun." For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744. Also, needle- less injection systems can be used (Davis, H. L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

[0249] Recombinant vectors carrying a synthetic expression cassette encoding CCN3 are formulated into compositions for delivery to a vertebrate subject. These compositions may either be prophylactic (to prevent bone loss) or therapeutic (to treat a bone or cartilage disorder or condition associated with bone or cartilage degeneration). The compositions will comprise a "therapeutically effective amount" of the nucleic acid of interest such that an amount of the CCN3 protein (or a biologically active fragment thereof) can be produced in vivo to stimulate bone or cartilage regeneration and repair (e.g., increase bone density, increase bone mass, increase bone strength and / or decrease fatty bone marrow, and / or increase cartilage growth, increase cartilage mass, regenerate cartilage) in the individual to which it is administered. The exact amount necessary will vary depending on the subject being treated; the age and general condition of the subject to be treated; the degree of protection desired; the severity of the condition being treated; the particular CCN3 protein produced and its mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. Thus, a "therapeutically effective amount" will fall in a relatively broad range that can be determined through routine trials.

[0250] The compositions will generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, surfactants and the like, may be present in such vehicles. Certain facilitators of nucleic acid uptake and / or expression can also be included in the compositions or coadministered.

[0251] Once formulated, the compositions can be administered directly to the subject (e.g., as described above) or, alternatively, delivered ex vivo, to cells derived from the subject, using methods such as those described above. For example, methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and can include, e.g., dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0252] Direct delivery of synthetic expression cassette compositions in vivo will generally be accomplished with or without viral vectors, as described above, by injection using either a conventional syringe, needless devices such as Bioject™ or a gene gun, such as the Accell™ gene delivery system (PowderMed Ltd, Oxford, England). Pharmaceutical Compositions

[0253] A CCN3 protein or a recombinant polynucleotide comprising a coding sequence encoding a CCN3 protein can be formulated into pharmaceutical compositions optionally comprising one or more pharmaceutically acceptable excipients. For treating a cartilage disorder, additionally, a VEGF inhibitor may be provided in the same pharmaceutical composition as the CCN3 protein or in separate pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0254] Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0255] A composition can also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0256] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the CCN3, VEGF inhibitor, or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0257] A surfactant can be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.

[0258] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

[0259] The amount of the CCN3 and / or VEGF inhibitor (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial). A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.

[0260] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred. These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19thed., Williams & Williams, (1995), the "Physician’s DeskAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 Reference", 52nded., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[0261] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional preferred compositions include those for oral, ocular, or localized delivery.

[0262] The pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use. Preferably, the compositions comprising a CCN3 and / or VEGF inhibitor are in unit dosage form, meaning an amount of a conjugate or composition appropriate for a single dose, in a premeasured or pre-packaged form.

[0263] The compositions herein may optionally include one or more additional agents, such as other drugs for treating a bone or cartilage disorder or condition associated with bone or cartilage degeneration, or other medications used to treat a subject for a condition or disease. Compounded preparations may include CCN3 and / or a VEGF inhibitor and one or more drugs for treating a bone or cartilage disorder or condition associated with bone or cartilage degeneration, such as osteoanabolic or bone-forming agents including, but not limited to, parathyroid hormone, teriparatide, abaloparatide, and romosozumab; anti-resorptive agents including, but not limited to, estrogen and estrogen agonists, bisphosphonates, and denosumab; analgesics including, but not limited to, acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs) including, but not limited to, aspirin, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, pelubiprofen, zaltoprofen, fenbufen, tiaprofenic acid, carprofen, indomethacin, acemetacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, fenclofenac, aceclofenac, bromfenac, fentiazac, nabumetone, piroxicam, ampiroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and etofenamate; and steroids including, but not limited to, hydrocortisone, triamcinolone, prednisone, prednisolone, methylprednisolone, and dexamethasone, or other medications. Alternatively, such agents can be contained in a separateAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 composition from the composition comprising CCN3 and / or VEGF inhibitor and co-administered concurrently, before, or after the composition comprising the CCN3 and / or VEGF inhibitor.

[0264] In some embodiments, a dose of CCN3 is provided in an implant, e.g., a matrix or scaffold for localized delivery of CCN3, where the CCN3 is provided as a CCN3 protein or biologically active variant or fragment thereof. The effective dose may be determined based on the specific tissue, rate of release from the implant, size of the implant, and the like and may be empirically determined by one of skill in the art. The dose may provide for biological activity equivalent to 1 mg CCN3 protein, 10 mg, 100 mg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 250 mg, 500 mg, 750 mg, 1 g of CCN3 protein. The dose may be administered at a single time point, e.g., as a single implant; or may be fractionated, e.g., delivered in a microneedle configuration. The dose may be administered, once, two, three times, 4 times, 5 times, 10 times, or more as required to achieve the desired effect, and administration may be daily, every 2 days, every 3 days, every 4 days, weekly, bi-weekly, monthly, or more.

[0265] Drug delivery devices include structures that can be implanted and that release the active agents, e.g. CCN3 for treatment of a bone disorder, or CCN3 in combination with a VEGF inhibitor for treatment of a cartilage disorder at the targeted site (e.g., site of bone or cartilage defect or injury). Implantable drug delivery devices can be broadly classified into two main groups: passive implants and active implants. The first group includes two main types of implants: biodegradable and non-biodegradable implants. Active systems rely on energy dependent methods that provide the driving force to control drug release. The second group includes devices such as osmotic pressure gradients and electromechanical drives.

[0266] Passive polymeric implants are normally relatively simple devices with no moving parts, they rely on passive diffusion for drug release. They are generally made of drugs packed within a biocompatible polymer molecule. Several parameters such as: drug type / concentration, polymer type, implant design and surface properties can be modified to control the release profile. Passive implants can be classified into two main categories: non-biodegradable and biodegradable systems.

[0267] Non-biodegradable implants are commonly prepared using polymers such as silicones, poly(urethanes), poly(acrylates) or copolymers such as poly(ethyelene vinyl acetate). Poly(ethylene-vinyl acetate) (PEVA) is a thermoplastic copolymer of ethylene and vinyl acetate. Poly(siloxanes) or silicones are organosilicon polymeric materials composed of silicon and oxygen atoms. Lateral groups can be methyl, vinyl or phenyl groups. These groups will influence the properties of the polymer. Poly(siloxanes) have been used extensively in medicine due to the unique combination of thermal stability, biocompatibility, chemical inertness and elastomericAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 properties. The silicones commonly used for medical devices are vulcanized at room temperature. They are prepared using a two-component poly(dimethylsiloxanes) (PDMS) in the presence of a catalyst (platinum based compound). The final material is formed via an addition hydrosilation reaction. An alternative method to obtain silicones for medical applications is the using linear PDMS with hydroxyl terminal groups. This linear polymer is cross-linked with low molecular weight tetra(alkyloxysilane) using stannous octoate catalyst.

[0268] This type of device can be monolithic or reservoir type implant. Monolithic type implants are made from a polymer matrix in which the drug is homogeneously dispersed. On the other hand, reservoir-type implants contain a compact drug core covered by a permeable non- biodegradable membrane. The membrane thickness and the permeability of the drug through the membrane will govern the release kinetics.

[0269] Biodegradable implants are made using polymers or block copolymers that can be broken down into smaller fragments that will be subsequently excreted or absorbed by the body. Normally they are made using polymers such as collagen, PEG, chitin, poly(caprolactone) (PCL), poly(lactic acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA). Numerous other biodegradable polymers for drug delivery exist including: poly(amides), poly(anhydrides), poly(phosphazenes) and poly(dioxanone). Poly(anhydrides) have a low hydrolytic stability resulting in rapid degradation rates, making them suitable for use in short-term controlled delivery systems. Poly(phosphazenes) have a degradation rate that can be finely tuned by appropriate substitution with specific chemical groups and use of these polymers has been investigated for skeletal tissue regeneration and drug delivery. Poly(dioxanone), like PCL, is a polylactone that has been used for purposes such as drug delivery, and tissue engineering They do not need to be extracted after implantation, as they will be degraded by the body of the patient. They can be manufactured as monolithic implants and reservoir-type implants. In addition to the biopolymers, such as the above-mentioned PLA, there are a few natural polymers which also represent a promising class of materials with a wide range of applications, including use in implantable devices. These natural polymers include, collagen, hyaluronic acid, cellulose, chitosan, silk and others naturally derived proteins, as well as collagen, gelatin, albumin, elastin and milk proteins. These materials present certain advantages compared to the traditional materials (metals and ceramics) or synthetic polymers, such as biocompatibility, biodegradation and non-cytotoxicity, which make them ideal to be used in implantable drug delivery devices.

[0270] Dynamic or active polymeric implants have a positive driving force to control the release of drugs from the device. The majority of the implants in this category are electronic systems made of metallic materials. Dynamic drug delivery implants are mainly pump typeAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 implants. The main type of polymeric active implants are osmotic pumps. This type of device is formed mainly by a semipermeable membrane that surrounds a drug reservoir. The membrane should have an orifice that will allow drug release. Osmotic gradients will allow a steady inflow of fluid within the implant. This process will lead to an increase in the pressure within the implant that will force drug release through the orifice. This design allows constant drug release (zero order kinetics). This type of device allows a favorable release rate but the drug loading is limited.

[0271] In some embodiments, the CCN3 and / or VEGF inhibitor are prepared as an injectable paste. The paste can be injected into the implant site. In some embodiments, the paste can be prepared prior to implantation and / or store the paste in the syringe at sub-ambient temperatures until needed. In some embodiments, application of the composite by injection can resemble a bone cement that can be used to join and hold bone fragments in place or to improve adhesion of, for example, a hip prosthesis, for replacement of damaged cartilage in joints, and the like. Implantation in a non-open surgical setting can also be performed.

[0272] In other embodiments the CCN3 and / or VEGF inhibitor are prepared as a formable putty. The hydrated graft putty can be prepared and molded to approximate any implant shape. The putty can then be pressed into place to fill a void in the cartilage, bone, tooth socket or other site. In some embodiments, graft putty can be used to repair defects in non-union bone or in other situations where the fracture, hole or void to be filled is large and requires a degree of mechanical integrity in the implant material to both fill the gap and retain its shape.

[0273] In some embodiments, additional factors and / or cells are provided during the course of treatment. While in many cases endogenous SSCs are sufficient for regeneration of bone or cartilage, exogenous cells may be provided at the site of a local acute injury. The cells may be SSCs, or non-SSCs, such as stem cells, e.g., adipose stem cells. The cells may be autologous or allogenic. The cells may be provided concomitant with the provision of CCN3 or CCN3 in combination with a VEGF inhibitor, e.g., simultaneously, shortly before, shortly, after, etc. and may be in a single implant with the CCN3 or the CCN3 in combination with a VEGF inhibitor, as a separate implant or injection, etc.

[0274] A system for pharmaceutical use, i.e., a drug delivery device with CCN3 and / or a VEGF inhibitor, and optionally other factors, can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the NR pharmaceutical composition orAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.

[0275] Toxicity and therapeutic efficacy of an active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determining the LD50 (the lethal dose to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are preferred.

[0276] The data obtained from cell culture and / or animal studies can be used in formulating a range of dosages for humans. The dosage of the active ingredient typically lines within a range of circulating concentrations that include the ED50 with low toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of utilization administration.

[0277] The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade) . Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxin, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

[0278] The effective amount of a therapeutic composition to be given to a particular patient will depend on a variety of factors, several of which will differ from patient to patient. A competent clinician will be able to determine an effective amount of a therapeutic agent to administer to a patient to halt or reverse the progression of the disease condition as required. Utilizing LD50 animal data, and other information available for the agent, a clinician can determine the maximum safe dose for an individual, depending on the route of administration. For instance, an intravenously administered dose may be more than an intrathecally administered dose, given the greater body of fluid into which the therapeutic composition is being administered. Similarly, compositions which are rapidly cleared from the body may be administered at higher doses, or in repeated doses, in order to maintain a therapeutic concentration. Utilizing ordinaryAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 skill, the competent clinician will be able to optimize the dosage of a particular therapeutic in the course of routine clinical trials. Administration of CCN3 for Treatment of a Bone Disorder

[0279] At least one therapeutically effective cycle of treatment with a CCN3 protein or a vector comprising a coding sequence encoding CCN3 will be administered to a subject for treatment of a bone disorder or condition associated with bone degeneration. Bone disorders and conditions associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury (e.g., bone fracture caused by a fall, road traffic accident, fight, or surgery), pathologic bone fracture, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

[0280] By “therapeutically effective dose or amount” of a CCN3 protein or a vector comprising a coding sequence encoding a CCN3 protein is intended an amount that, when administered, as described herein, brings about a positive therapeutic response, such as improved recovery from a bone disorder or condition associated with bone degeneration. Improved recovery may include increased bone density, increased bone mass, increased bone strength, increased bone mineral density, and / or decreased fatty bone marrow. Additionally, a therapeutically effective dose or amount may stimulate osteochondral skeletal stem cells to produce bone.

[0281] In certain embodiments, multiple therapeutically effective doses of compositions comprising a CCN3 protein, or a vector comprising a coding sequence encoding CCN3, and / or one or more other therapeutic agents, such as other drugs for treating a bone disorder or condition associated with bone degeneration, or other medications will be administered. The compositions of the present invention are typically, although not necessarily, administered orally, via injection (subcutaneously, intravenously, intramuscularly, or intraperitoneally), by infusion, or locally. Additional modes of administration are also contemplated, such as intrahepatic, intraosseous, pulmonary, rectal, transdermal, transmucosal, intrathecal, pericardial, intra-arterial, and so forth.

[0282] The preparations are also suitable for local treatment. In a particular embodiment, a composition is used for localized delivery of a CCN3 protein or a vector comprising a coding sequence encoding CCN3 for the treatment of a bone disorder or condition associated with bone degeneration. For example, compositions may be administered locally in the vicinity of a bone defect or injury in need of repair or regeneration. The particular preparation and appropriateAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 method of administration are chosen to target the CCN3 protein or vector expressing the CCN3 protein to the site where new bone growth or bone repair is needed.

[0283] The pharmaceutical preparation can be in the form of a liquid solution or suspension immediately prior to administration, but may also take another form such as a syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, or the like. The pharmaceutical compositions comprising a CCN3 protein and / or other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art.

[0284] In another embodiment, the pharmaceutical compositions comprising a CCN3 protein or a vector comprising a coding sequence encoding a CCN3 protein, and / or other agents are administered prophylactically, e.g., to prevent bone loss. Such prophylactic uses will be of particular value for female subjects during lactation or subjects who have a genetic predisposition or a disease or condition that increases the risk of developing osteoporosis or osteopenia (e.g. menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy). In another embodiment, the pharmaceutical compositions comprising a CCN3 protein or a vector comprising a coding sequence encoding a CCN3 protein and / or other agents are administered therapeutically to subjects with osteoporosis or osteopenia.

[0285] In another embodiment, the pharmaceutical compositions comprising a CCN3 protein or a vector comprising a coding sequence encoding a CCN3 protein and / or other agents are in a sustained-release formulation, or a formulation that is administered using a sustained- release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[0286] The disclosure also provides a method for administering a conjugate comprising a CCN3 protein to a patient suffering from a bone disorder or condition associated with bone degeneration or condition that is responsive to treatment with a CCN3 protein contained in the conjugate or composition. The method comprises administering, via any of the herein described modes, a therapeutically effective amount of the conjugate or drug delivery system, preferably provided as part of a pharmaceutical composition. The method of administering may be used to treat any condition that is responsive to treatment with a CCN3 protein.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0287] Those of ordinary skill in the art will appreciate which conditions a specific a CCN3 protein can effectively treat. The actual dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the particular bone disorder being treated, the severity of the condition being treated, the judgment of the health care professional, and the particular CCN3 protein or conjugate being administered. Therapeutically effective amounts can be determined by those skilled in the art, and will be adjusted to the particular requirements of each particular case.

[0288] In certain embodiments, multiple therapeutically effective doses of a CCN3 protein will be administered according to a daily dosing regimen or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By “intermittent” administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, every other week, and so forth. For example, in some embodiments, a composition comprising a CCN3 protein will be administered once-weekly, twice-weekly or thrice-weekly for an extended period of time, such as for 1, 2, 3, 4, 5, 6, 7, 8...10...15…24 weeks, and so forth. By “twice-weekly” or “two times per week” is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By “thrice weekly” or “three times per week” is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present disclosure, this type of dosing is referred to as “intermittent” therapy. In accordance with the methods described herein, a subject can receive intermittent therapy (i.e., once-weekly, twice-weekly or thrice-weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below. The amount administered will depend on the potency of the specific CCN3 protein, the particular bone or cartilage disorder that is treated, the magnitude of the effect desired, and the route of administration.

[0289] A purified CCN3 protein or a vector comprising a coding sequence encoding a CCN3 protein (again, preferably provided as part of a pharmaceutical preparation) can be administered alone or in combination with one or more other therapeutic agents such as osteoanabolic or bone-forming agents including, but not limited to, parathyroid hormone, teriparatide, abaloparatide, and romosozumab; anti-resorptive agents including, but not limited to,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 estrogen and estrogen agonists, bisphosphonates, and denosumab; analgesics including, but not limited to, acetaminophen, and non-steroidal anti-inflammatory drugs (NSAIDs) including, but not limited to, aspirin, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, pelubiprofen, zaltoprofen, fenbufen, tiaprofenic acid, carprofen, indomethacin, acemetacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, fenclofenac, aceclofenac, bromfenac, fentiazac, nabumetone, piroxicam, ampiroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and etofenamate; steroids including, but not limited to, hydrocortisone, triamcinolone, prednisone, prednisolone, methylprednisolone, and dexamethasone; or other medications used to treat a particular condition or disease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Preferred compositions are those requiring dosing no more than once a day.

[0290] A CCN3 protein or a vector comprising a coding sequence encoding a CCN3 protein can be administered prior to, concurrent with, or subsequent to other agents. If provided at the same time as other agents, the CCN3 protein or vector comprising a coding sequence encoding the CCN3 protein can be provided in the same or in a different composition. Thus, CCN3 protein or vector comprising a coding sequence encoding the CCN3 protein and / or other agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising a CCN3 protein or a vector comprising a coding sequence encoding a CCN3 protein, and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating a bone disorder or condition associated with bone degeneration, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen. Similarly, a CCN3 protein or a vector comprising a coding sequence encoding a CCN3 protein and one or more other therapeutic agents can be administered in at least one therapeutic dose. Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0291] The methods described herein can be used for treating a human subject for a bone disorder or condition associated with bone degeneration. The methods described herein will also find use in veterinary applications for treatment of such bone disorders or conditions, for example, in domestic animals including, without limitation, pets, such as dogs and cats, and farm animals, such as sheep, goats, pigs, horses and cattle. Bone Grafts

[0292] Also provided herein is a method of producing a bone graft by culturing osteochondral skeletal stem cells in the presence of CCN3 under suitable conditions for the osteochondral skeletal stem cells to produce bone. The osteochondral skeletal stem cells are, in some cases, encapsulated within a biocompatible scaffold such that the cells remain in the scaffold when the tissue graft is transplanted at a transplantation site (e.g., a site in need of bone replacement or repair) of an individual. In some embodiments, the scaffold provides a support for generating bone from the osteochondral skeletal stem cells and has a defined geometry mimicking the shape of a bone or a portion of a bone that needs replacement or repair. Preferably, the scaffold has similar mechanical properties to bone.

[0293] By way of example, a biocompatible scaffold may comprise any material that will allow the CCN3 to be incorporated and compatible with the addition of osteochondral skeletal stem cells. The carrier, matrix, or scaffold can be predominantly non-immunogenic and biodegradable. Examples of biodegradable materials include, but are not limited to, alginate, polyglycolic acid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suture material, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin, cotton, or other naturally occurring biodegradable materials. In some embodiments, the matrix is biodegradable over a time period of less than a year, more preferably less than six months, most preferably over two to ten weeks. The polymer composition, as well as method of manufacture, can be used to determine the rate of degradation. It may be preferable to sterilize the matrix or scaffold material prior to administration or implantation, e.g., by treating it with ethylene oxide or by gamma irradiation or irradiation with an electron beam. In addition, a number of other materials may be used to form the scaffold or framework structure, including but not limited to: hydroxyapatite, nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE, teflon), thermanox (TPX), polymers of hydroxy acids, such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes, and a variety of polyhydroxyalkanoates, and combinations thereof.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0294] Suitable matrices include a mesh, sponge, or a polymeric hydrogel. A hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure, which entraps water molecules to form a gel. In general, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions that have charged side groups, or a monovalent ionic salt thereof. Exemplary hydrogel polymers include, without limitation, natural polymers such as polysaccharides, including hyaluronic acid, chitosan, heparin, alginate, cellulose, dextran, and agarose, and proteins, including fibrin, fibrinogen, collagen, elastin, gelatin, silk, laminin, fibronectin, albumin, thrombin, and keratin; modified natural polymers, including hydroxymethylcellulose, hydroxyethylcellulose, gelatin methacrylate, polyanionic N-carboxymethyl chitosan, and polycationic N-trimethyl chitosan; and synthetic polymers, including polyvinyl alcohol, N-vinylpyrrolidone, polyethylene glycol, poly(ethylene glycol) diacrylate, polyacrylamide, poly(N-isopropylacrylamide), sodium polyacrylate, acrylate polymers and copolymers such as hydroxyethyl methacrylate, ethyl methacrylate, propylene glycol methacrylate, ethylene glycol di-methyl acrylate, methyl methacrylate, glycidyl methacrylate, and glycol methacrylate, poly(N-isopropylacrylamide-co- acrylic acid), polyesters, polyurethanes, nylon, synthetic polyamino acids, prolamines; and combinations thereof, and other such molecules, including recombinant versions of such polymers. In some embodiments, the hydrogel comprises polyacrylamide.

[0295] Methods of preparing hydrogels are well known in the art. See, e.g., Barbucci Hydrogels Biological Properties and Applications, Springer, 2009; Hydrogels in Cell-Based Therapies, edited by Connon and Hamley, Royal Society of Chemistry, 2014; Tunable Hydrogels, edited by Lavrentieva, Pepelanov, and Seliktar, Springer Nature Switzerland AG, 2020; herein incorporated by reference. In certain instances, a hydrogel is crosslinked by chemical crosslinking. Exemplary chemical crosslinking agents include, without limitation, N,N,N’,N’- tetramethylethylenediamine (TEMED), ammonium persulfate, glutaraldehyde, formaldehyde, epoxy compounds, dialdehyde, N,N′-methylenebis(acrylamide) (MBA), ethylene glycol diacrylate (EGDA), ethylene glycol dimethylacrylate (EGDMA), PEG diacrylate (PEGDA), glyoxal, epichlorohydrin, and sodium borate / boric acid. The crosslink density may vary depending on the type and the concentration of the chemical crosslinking agent employed. Alternatively, the hydrogel may be photo-crosslinked by exposure to light. UV light-sensitive photoinitiators (i.e., initiate polymerization with exposure to UV light in the range of 200-400 nm) or visible light- sensitive photoinitiators (i.e., initiate polymerization with exposure to visible light in the range of 400-800 nm) may be used. A photoinitiator may be used to induce photopolymerization.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 Photopolymerization reactions may involve a free-radical-initiated chain polymerization or bio- orthogonal click reactions. Exemplary photoinitiators include, without limitation, (1-[4-(2- hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one), lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), and Eosin-Y. The crosslink density may vary depending on the intensity of the electromagnetic radiation applied to promote photopolymerization of the hydrogel as well as the duration of irradiation. In some embodiments, the crosslink density of the crosslinked hydrogel ranges from 1×10-15moles / cm3to 1×10-3moles / cm3.

[0296] The compressive modulus (i.e., material stiffness, also referred to herein as "modulus") of a hydrogel can be in the range of from1 kPa to 70 kPa, including any stiffness within this range such as 1 kPa, 2 kPa, 3 kPa, 4 kPa, 5 kPa, 6 kPa, 7 kPa, 8 kPa, 9 kPa, 10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, 15 kPa, 16 kPa, 17 kPa, 18 kPa, 19 kPa, 20 kPa, 21 kPa, 22 kPa, 23 kPa, 24 kPa, 25 kPa, 26 kPa, 27 kPa, 28 kPa, 29 kPa, 30 kPa, 31 kPa, 32 kPa, 33 kPa, 34 kPa, 35 kPa, 36 kPa, 37 kPa, 38 kPa, 39 kPa, 40 kPa, 45 kPa, 50 kPa, 55 kPa, 60 kPa, 65 kPa, or 70 kPa.

[0297] The scaffold may have any suitable lateral dimensions (e.g., width and / or length, or diameter). In some cases, the polymeric scaffold has a lateral dimension of about 1.0 centimeter (cm) or more, e.g., about 2.0 cm or more, about 3.0 cm or more, about 4.0 cm or more, including 5 cm or more, and in some cases has a lateral dimension of about 10 cm or less, e.g., about 9.0 cm or less, about 8.0 cm or less, about 7.0 cm or less, about 6.0 cm or less, including about 5.0 cm or less. In some embodiments, the polymeric scaffold has a lateral dimension in the range of about 1.0 cm to about 10 cm, e.g., about 1.0 cm to about 9.0 cm, about 2.0 cm to about 8.0 cm, including about 3.0 cm to about 7.0 cm.

[0298] In certain embodiments, the composition is injected at or adjacent to the site of a bone fracture or defect. Any type of bone may be treated by the subject methods including, but not limited to, bones of the arms, hands, legs, feet, neck, head, or spine.

[0299] In some embodiments, the subject being treated has a bone disorder or condition associated with bone degeneration. Bone disorders and conditions associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury (e.g., bone fracture caused by a fall, road traffic accident, fight, or surgery), pathologic fracture, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy. In some embodiments, the bone graft provides new bone for craniofacial surgery (e.g., to repair craniofacial clefts, facial fractures, or congenitalAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 anomalies), oral and maxillofacial surgery, plastic surgery, or reconstructive surgery, or for a dental implant (e.g., to repair or replace bone in the skull or jaw).

[0300] The bone graft may comprise any suitable amount of the osteochondral skeletal stem cells. The amount of cells may depend on a variety of factors, such as the size of the bone graft, the length of time the bone graft is to be implanted, the condition to be treated by the bone graft and / or the desired therapeutic outcome. In some cases, the bone graft includes at least 105cells, e.g., at least 106cells, at least 107cells, at least 108cells, at least 109cells, at least 1010cells, or more cells. Administration of CCN3 and a VEGF Inhibitor for Treatment of a Cartilage Disorder

[0301] At least one therapeutically effective dose of CCN3 (or a vector comprising a coding sequence encoding CCN3) in combination with a therapeutically effective dose of a VEGF inhibitor and a mechanical stimulus are administered. By "therapeutically effective dose or amount" of CCN3 (or a vector comprising a coding sequence encoding CCN3) and a VEGF inhibitor is intended an amount that, when administered in combination with a mechanical stimulus (e.g., microfracture surgical procedure or accidental acute local injury), as described herein, brings about a positive therapeutic response, such as improved recovery from a cartilage disorder. Improved recovery may include generation of new cartilage at a treatment site (e.g., a damaged joint). For example, a therapeutically effective dose or amount could be used to treat cartilage damage or loss resulting from a traumatic injury or a degenerative disease, such as arthritis or other disease involving cartilage degeneration. Preferably, a therapeutically effective amount restores function and / or relieves pain and inflammation associated with cartilage damage or loss.

[0302] Cartilage disorders that can be treated by the methods described herein include, but are not limited to, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, and malignancy. In some embodiments, the subject methods are used to treat an injury to hyaline cartilage, elastic cartilage, or fibrocartilage. In some embodiments, a joint having damaged cartilage is treated by the subject methods. For example, the damaged cartilage may be in a joint of a knee, hip, elbow, shoulder, hand, ankle, or spine.

[0303] In certain embodiments, multiple therapeutically effective doses of each of the CCN3 and the VEGF inhibitor will be administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 two days a week, three days a week, four days a week, or five days a week, and so forth. By “intermittent” administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, and so forth. For example, in some embodiments, the CCN3 and the VEGF inhibitor will be administered twice-weekly or thrice- weekly for an extended period of time, such as for 1, 2, 3, 4, 5, 6, 7, 8...10...15…24 weeks, and so forth. By “twice-weekly” or “two times per week” is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By “thrice weekly” or “three times per week” is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as “intermittent” therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy (i.e., twice-weekly or thrice-weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below.

[0304] The CCN3 can be administered prior to, concurrent with, or subsequent to the VEGF inhibitor. If provided at the same time as the VEGF inhibitor, the CCN3 can be provided in the same or in a different composition. Thus, the two agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a human subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering at least one therapeutically effective dose of a pharmaceutical composition comprising CCN3 and at least one therapeutically effective dose of a pharmaceutical composition comprising a VEGF inhibitor according to a particular dosing regimen. Administration of the separate pharmaceutical compositions can be at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day, or on different days), so long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.

[0305] In other embodiments, the pharmaceutical compositions comprising the agents, such as the CCN3 and / or the VEGF inhibitor is a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0306] The pharmaceutical compositions comprising the CCN3 and / or the VEGF inhibitor may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art. Suitable routes of administration include parenteral administration, such as subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous (IV), or infusion, oral and pulmonary, nasal, topical, transdermal, and suppositories. Where the composition is administered via pulmonary delivery, the therapeutically effective dose is adjusted such that the soluble level of the agent, such as the CCN3 and the VEGF inhibitor in the bloodstream, is equivalent to that obtained with a therapeutically effective dose that is administered parenterally, for example SC, IP, IM, or IV. In some embodiments, the pharmaceutical composition comprising the CCN3 and / or the VEGF inhibitor is administered by IM or SC injection. In some embodiments, the CCN3 and the VEGF inhibitor are administered topically as drops, on a patch, or in a gel. In some embodiments, the CCN3 and the VEGF inhibitor are administered locally to a site of defective or damaged cartilage (e.g., joint).

[0307] Factors influencing the respective amount of the various compositions to be administered include, but are not limited to, the mode of administration, the frequency of administration (i.e., daily, or intermittent administration, such as twice- or thrice-weekly), the particular type of cartilage disorder undergoing therapy, the location of cartilage damage, the severity of the disease, the history of the disease, whether the individual is undergoing concurrent therapy with another therapeutic agent, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Generally, a higher dosage of an agent is preferred with increasing weight of the subject undergoing therapy. Kits

[0308] Also provided are kits containing any of the compositions described herein for treating a patient for a bone or cartilage disorder or condition associated with bone or cartilage degeneration. The CCN3 protein or a vector comprising a coding sequence encoding the CCN3 protein and optionally other therapeutic agents may be contained in separate compositions or in the same composition. In some embodiments, the kit further comprises a VEGF inhibitor (e.g., cabozantinib). In some embodiments, the kit further comprises an implement for performing microfracture such as an awl or drill.

[0309] In certain embodiments, the kit comprises a CCN3 protein comprising or consisting of the amino acid sequence of SEQ ID NO:1, or a variant comprising a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identityAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 thereto, or a biologically active fragment thereof, wherein the variant or fragment retains the ability to increase bone density, bone mass, bone strength, and / or rate of bone formation, and / or ability to regenerate cartilage, increase cartilage mass, and / or increase rate of cartilage formation. A subject kit may include at least one container comprising a solution comprising a unit dose of the CCN3 and / or VEGF inhibitor, and a pharmaceutically acceptable excipient; and instructions to administer a unit dose according to a desired regimen or exemplary regimen dependent upon the particular bone or cartilage disorder being treated, age, weight, and the like.

[0310] Kits may include unit doses of the formulations comprising the CCN3 and / or VEGF inhibitor suitable for use in the treatment methods described herein, e.g., in tablets or injectable dose(s). In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the treatment for a bone disorder or condition associated with bone degeneration. The kit can include, for example, a dosing regimen for the CCN3 protein and / or VEGF inhibitor.

[0311] Formulations suitable for intravenous or intraperitoneal administration are of particular interest, and in such embodiments the kit may further include a syringe or other device to accomplish such administration, which syringe or device may be pre-filled with the CCN3 protein. The instructions can be printed on a label affixed to the container or can be a package insert that accompanies the container.

[0312] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, Blu-ray, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site. Screening

[0313] The inventors have discovered that CCN3 is a brain-derived osteogenic factor that promotes bone and cartilage growth (see Examples). Therefore, agonists, mimics, and analogues of CCN3 may be useful in treating bone or cartilage disorders and conditions associated with boneAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 or cartilage degeneration. Accordingly, screening methods for identifying candidate agents that increase CCN3 activity or mimic CCN3 activity are provided.

[0314] A variety of assays may be used for this purpose, and in many embodiments, a candidate agent will be tested in different assays to confirm activity in stimulating bone or cartilage growth as well as efficacy in treating a bone or cartilage disorder. For example, cell- based assays may determine the effects of an agent on biological activity of CCN3 (e.g., ability to stimulate bone or cartilage production by osteochondral skeletal stem cells and / or increase rate of bone or cartilage formation). In some embodiments, the ability of an CCN3 mimic or analogue to stimulate osteochondral skeletal stem cells to produce bone or cartilage is assayed. Any convenient format may be used for the assay, e.g., wells, plates, flasks, etc., preferably a high throughput format, such as multi-well plates. A test agent of interest is added to the reaction mixture with or without the CCN3 protein, and the effect of the agent on bone or cartilage growth is determined.

[0315] For example, a cellular assay to identify agonists that increase bone production may be performed by contacting a cell with CCN3 and a candidate agent; and measuring bone mineralization by the cell, wherein an increased amount of bone mineralization in the presence of the candidate agent compared to a reference value range for the amount of bone mineralization in a control cell (i.e., cell not contacted with the candidate agent, cell where the candidate agent is absent) indicates that the candidate agent is an agonist of CCN3.

[0316] In another example, a cellular assay to identify a mimic or an analogue of CCN3 that stimulates bone growth may be performed by contacting a cell with a candidate agent, wherein the cell is an osteochondral skeletal stem cell, a preosteoblast cell, an osteoblast cell, an osteoprogenitor cell, or an osteosarcoma cell; and measuring bone mineralization by the cell, wherein an increased amount of bone mineralization in the presence of the candidate agent compared to a reference value range for the amount of bone mineralization in a control cell indicates that the candidate agent is a mimic or an analogue of the CCN3.

[0317] In a further example, a cellular assay to identify agonists that increase cartilage production may be performed by activating a skeletal stem cell with a mechanical stimulus; contacting the skeletal stem cell with CCN3, a VEGF inhibitor, and a candidate agent; and measuring cartilage production by the skeletal stem cell, wherein an increased amount of cartilage production in the presence of the candidate agent compared to a reference value range for the amount of cartilage production in a control skeletal stem cell indicates that the candidate agent is an agonist of CCN3.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0318] In yet another example, a cellular assay to identify a mimic or an analogue of CCN3 that stimulates production of cartilage may be performed by activating a skeletal stem cell with a mechanical stimulus; contacting the skeletal stem cell with a vascular endothelial growth factor (VEGF) inhibitor and a candidate agent; and measuring cartilage production by the skeletal stem cell, wherein an increased amount of cartilage production in presence of the candidate agent compared to a reference value range for the amount of cartilage production in a control skeletal stem cell indicates that the candidate agent is a mimic or an analogue of the CCN3.

[0319] A CCN3 agonist, mimic, or analogue can be any type of molecule including, without limitation, a small molecule, drug, receptor ligand, protein, polypeptide, peptide, fusion protein, nucleic acid, oligonucleotide, peptide nucleic acid, aptamer that increases bone and / or cartilage growth. In some embodiments, an agonist increases the activity of CCN3 or increases CCN3 mRNA or protein levels by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold or more, or any amount in between as compared to native or control levels. In some embodiments, an agonist or mimic increases bone mineralization by a cell by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, or more, or any amount in between as compared to native or control levels.

[0320] Cellular assays can be performed, for example, on osteochondral skeletal stem cells, preosteoblast cells, osteoblast cells, osteoprogenitor cells, or osteosarcoma cells. In some embodiments, the cellular assays are performed with skeletal stem cells derived from other types of stem cells such as adult stem cells (e.g., mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells, or neural stem cells), embryonic stem cells, or induced pluripotent stem cells. In some embodiments, the skeletal stem cell or stem cell, from which a skeletal stem cell is derived, is from a patient who has a bone or cartilage disorder or condition associated with bone or cartilage degeneration.

[0321] In some embodiments, cellular assays are performed using cells from a cell line. Exemplary cell lines that can be used in the subject screening methods include, without limitation, a MC3T3-E1 preosteoblast cell line, an SaOs2 osteosarcoma cell line, an MG-63 osteosarcoma cell line, a hFOB osteoblast cell line, an ASC52telo immortalized adipose derived mesenchymal stem cell line, a human telomerase reverse transcriptase-immortalized bone marrow mesenchymal stromal cell (hTERT-BMSC) line, and an ATDC5 chondrogenic mouse teratocarcinoma cell line.

[0322] Assays may further include suitable controls (e.g., a sample in the absence of the test agent). Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one ofAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection).

[0323] A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc., including agents that are used to facilitate optimal binding activity and / or reduce non-specific or background activity. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti- microbial agents, etc. may be used. The components of the assay mixture are added in any order that provides for the requisite activity. Incubations are performed at any suitable temperature, typically between 4°C and 40°C. Incubation periods are selected for optimum activity but may also be optimized to facilitate rapid high-throughput screening. In some embodiments, between 0.1 hour and 1 hour, between 1 hour and 2 hours, or between 2 hours and 4 hours, will be sufficient.

[0324] A variety of different test agents may be screened. Candidate agents encompass numerous chemical classes, e.g., small organic compounds having a molecular weight of more than 50 daltons and less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons. Test agents can comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, or at least two of the functional chemical groups. The test agents can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Test agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0325] Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Moreover, screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0

[0326] In some embodiments, test agents are synthetic compounds. A number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94 / 24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods.

[0327] In another embodiment, the test agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.

[0328] In some embodiments, the test agents are organic moieties. In this embodiment, test agents are synthesized from a series of substrates that can be chemically modified. “Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions may be done on the moieties to form new substrates or candidate agents which can then be tested using the present invention.

[0329] In some embodiments test agents are assessed for any cytotoxic activity it may exhibit toward a living eukaryotic cell, using well-known assays, such as trypan blue dye exclusion, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) assay, and the like. Agents that do not exhibit significant cytotoxic activity are considered candidate agents. Monitoring Risk of Bone Degeneration from Breast Feeding

[0330] Levels of CCN3 can be used to monitor a lactating female subject to determine the risk of bone degeneration from breast feeding and provide guidance on whether to continue or discontinue breast feeding. During lactation, mothers may have significant bone loss because of the high demand for calcium (Ca2+) during nursing of a baby. The brain-derived osteogenic factor, CCN3, which is secreted from neurons of the arcuate nucleus (ARC), counteracts the loss ofAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 calcium by promoting bone production in lactating females. The level of CCN3 produced by ARC neurons declines over time after birth of a baby. Therefore, continued breast feeding after the level of CCN3 falls below a critical threshold level puts a mother at risk of bone degeneration if breast feeding is continued. Accordingly, levels of CCN3 can be monitored in a lactating female subject to determine if the levels of CCN3 remain above the critical threshold level to avoid bone degeneration or have fallen below the critical threshold level which indicates the lactating female subject is at risk of bone degeneration if breast feeding is continued.

[0331] A sample can be obtained from a lactating female subject to measure the level of CCN3 present. The sample is typically a blood or plasma sample comprising CCN3 taken from the subject. A blood sample can be obtained from a subject by conventional techniques. For example, blood samples can be obtained by venipuncture according to methods well known in the art.

[0332] Levels of CCN3 can be compared to reference value ranges to determine if the level of CCN3 is indicative of risk of bone degeneration (e.g., loss of bone density or bone mass, bone fragility, osteoporosis, or osteopenia). For example, assaying can be used to determine whether the level of CCN3 is less than or “greater than or equal to” a particular threshold, (the threshold can be pre-determined or can be determined by assaying a control sample). In some embodiments, the level of CCN3 in a sample obtained from a female lactating subject is compared to reference value ranges representing the levels of CCN3 in control samples from one or more female lactating subjects who do not have a substantial risk of bone degeneration (i.e., having CCN3 levels at or above the critical threshold level). That is, levels of CCN3 at or above the threshold value indicate that the female lactating subject may continue breast feeding without substantial risk of bone degeneration, whereas levels of CCN3 below the critical threshold value indicate that the female lactating subject is at risk of bone degeneration if breast feeding is continued. The level of CCN3 may be monitored over time during the period the lactating female subject would like to continue breast feeding in order to determine if the level of CCN3 remains at or above the critical threshold level or has declined to a level below the critical threshold level to determine whether or not it remains safe to continue breast feeding without substantial risk of bone degeneration.

[0333] The determination that a female lactating subject is at risk or not at risk of bone degeneration from breast feeding is an active clinical application of the correlation between levels of CCN3 and the risk of bone degeneration. For example, “determining” requires the active steps of measuring CCN3 levels, reviewing the data, which is produced during the active assaying step(s), and resolving whether an individual does or does not have a substantial risk of boneAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 degeneration from continued breast feeding. Additionally, in some cases, a decision is made to continue or not continue breast feeding. In some cases, the subject methods include the step of a medical practitioner (e.g., nurse or physician) advising a female lactating subject on the risk of bone degeneration if breast feeding is continued, e.g., if CCN3 levels have declined below the critical threshold level.

[0334] In some embodiments, the subject methods include providing an analysis indicating whether or not a lactating female subject is determined to be at substantial risk of bone degeneration if breast feeding is continued. A suitable analysis (e.g., an oral or written report) provides any or all of the following information: identifying information of the subject (name, age, etc.), a description of what type of sample(s) was used and / or how it was used, the technique used to assay the sample, the results of the assay (e.g., the level of the CCN3 measured and / or the fold-change of the level of CCN3 over time), the assessment as to whether the individual is at risk of bone degeneration from continued breast feeding, a recommendation to discontinue breast feeding if the level of CCN3 indicates that the subject is at risk of bone degeneration if breast feeding is continued, etc. The analysis can be provided to the subject, to the subject’s physician, to a testing facility, etc. The analysis can also be accessible as a website address via the internet. In some such cases, the analysis can be accessible by multiple different entities (e.g., the subject, the subject’s physician, a testing facility, etc.). Detecting CCN3

[0335] It is understood that CCN3 in a sample can be measured by any suitable method known in the art. In some embodiments, antibody-based methods are used to measure the level of CCN3 using an antibody that specifically binds to CCN3. The terms "specifically binds" or "specific binding" as used herein refer to preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides or epitopes). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a Kd (dissociation constant) of 10-5M or less (e.g., 10-6M or less, 10-7M or less, 10-8M or less, 10-9M or less, 10-10M or less, 10-11M or less, 10-12M or less, 10-13M or less, 10- 14 M or less, 10-15M or less, or 10-16M or less). By "affinity" it is meant the strength of binding, increased binding affinity being correlated with a lower Kd.

[0336] While a variety of different manners of assaying for protein levels are known in the art, one representative and convenient type of protocol for assaying protein levels is the enzyme-linked immunosorbent assay (ELISA). In ELISA and ELISA-based assays, one or moreAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 antibodies specific for the proteins of interest may be immobilized onto a selected solid surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the assay plate wells are coated with a non-specific "blocking" protein that is known to be antigenically neutral with regard to the test sample such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of non-specific adsorption sites on the immobilizing surface, thereby reducing the background caused by non-specific binding of antigen onto the surface. After washing to remove unbound blocking protein, the immobilizing surface is contacted with the sample to be tested under conditions that are conducive to immune complex (antigen / antibody) formation. Such conditions include diluting the sample with diluents such as BSA or bovine gamma globulin (BGG) in phosphate buffered saline (PBS) / Tween or PBS / Triton-X 100, which also tend to assist in the reduction of nonspecific background, and allowing the sample to incubate for about 2-4 hours at temperatures on the order of about 25°-27° C. (although other temperatures may be used). Following incubation, the antisera-contacted surface is washed so as to remove non- immunocomplexed material. An exemplary washing procedure includes washing with a solution such as PBS / Tween, PBS / Triton-X 100, or borate buffer. The occurrence and amount of immunocomplex formation may then be determined by subjecting the bound immunocomplexes to a second antibody having specificity for the target that differs from the first antibody and detecting binding of the second antibody. In certain embodiments, the second antibody will have an associated enzyme, e.g. urease, peroxidase, or alkaline phosphatase, which will generate a color precipitate upon incubating with an appropriate chromogenic substrate. For example, a urease or peroxidase-conjugated anti-human IgG may be employed, for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS / Tween). After such incubation with the second antibody and washing to remove unbound material, the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6- sulfonic acid (ABTS) and H2O2, in the case of a peroxidase label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer. The preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody.

[0337] The solid substrate upon which the antibody or antibodies are immobilized can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate,Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 microbead, dipstick, resin particle, etc. The substrate may be chosen to maximize signal to noise ratios, to minimize background binding, as well as for ease of separation and cost. Washes may be effected in a manner most appropriate for the substrate being used, for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, or rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent.

[0338] Alternatively, non-ELISA based-methods for measuring the levels of CCN3 in a sample may be employed, and any convenient method may be used. Representative examples known to one of ordinary skill in the art include, but are not limited to, other immunoassay techniques such as radioimmunoassays (RIA), sandwich immunoassays, fluorescent immunoassays, enzyme multiplied immunoassay technique (EMIT), capillary electrophoresis immunoassays (CEIA), and immunoprecipitation assays; mass spectrometry, or tandem mass spectrometry, proteomic arrays, xMAP microsphere technology, western blotting, immunohistochemistry, flow cytometry, cytometry by time-of-flight (CyTOF), multiplexed ion beam imaging (MIBI), and detection in body fluid by electrochemical sensor.

[0339] As another example, electrochemical sensors may be employed. In such methods, a capture aptamer or an antibody that is specific for CCN3 is immobilized on an electrode. A second aptamer or antibody, also specific for the CCN3, is labeled with, for example, pyrroquinoline quinone glucose dehydrogenase ((PQQ)GDH). The sample of body fluid (e.g., blood or plasma) is introduced to the sensor either by submerging the electrodes in the body fluid or by adding the sample fluid to a sample chamber, and the CCN3 analyte allowed to interact with the labeled aptamer / antibody and the immobilized capture aptamer / antibody. Glucose is then provided to the sample, and the electric current generated by (PQQ)GDH is observed, where the amount of electric current passing through the electrochemical cell is directly related to the amount of analyte captured at the electrode.

[0340] For measuring protein activity levels, the amount or level of CCN3 activity in the sample is determined. For example, the ability of CCN3 in the sample to stimulate bone production by osteochondral skeletal stem cells can be assayed (see, e.g., Examples).

[0341] In other embodiments, the amount or level in the sample of the CCN3 protein is determined. Any convenient method for measuring protein levels in a sample may be used, e.g., antibody-based methods, e.g., immunoassays, e.g., enzyme-linked immunosorbent assays (ELISAs), immunohistochemistry, and mass spectrometry.

[0342] The resultant data provides information regarding the amount and / or activity of the CCN3 that have been measured, wherein the information is in terms of whether or not the CCN3 is present and at what level, including guidance on whether the level is greater than or equal to theAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 critical threshold level to avoid substantial bone degeneration, or below the critical threshold level, and wherein the data may be both qualitative and quantitative. UTILITY

[0343] The compositions and methods of the present disclosure find use in a variety of different applications, including the treatment of bone and cartilage disorders and conditions associated with bone or cartilage degeneration. Bone disorders and conditions associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury (e.g., bone fracture caused by a fall, road traffic accident, fight, or surgery), pathologic bone fracture, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy. The compositions and methods of the present disclosure also find use in treating cartilage disorders, including, but not limited to, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, and malignancy.

[0344] CCN3 may be used to stimulate osteochondral skeletal stem cells to produce new bone or cartilage in vivo, ex vivo, or in vitro. Treatment with CCN3 or a recombinant polynucleotide comprising a coding sequence encoding CCN3 may increase bone density, bone mass, and bone strength, decrease fatty bone marrow, increase cartilage mass, increase rate of bone or cartilage production, or regenerate, replace, or repair bone or cartilage. Additionally, CCN3 may be used for production of bone for bone grafts to repair bone fractures or bone defects or cartilage for cartilage grafts to repair cartilage defects or damage. CCN3 may also be used as a biomarker to determine the risk of bone degeneration of lactating females during breast feeding. Examples of Non-Limiting Aspects of the Disclosure

[0345] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-125 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support forAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 all such combinations of aspects and is not limited to combinations of aspects explicitly provided below: 1. A method of treating a bone disorder or condition associated with bone degeneration in a subject, the method comprising administering a therapeutically effective amount of cellular communication network factor 3 (CCN3) to the subject. 2. The method of aspect 1, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. 3. The method of aspect 1 or 2, wherein the bone disorder or condition associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy. 4. The method of any one of aspects 1-3, wherein the treatment increases bone mass of a bone of the subject compared to the bone mass before the treatment. 5. The method of any one of aspects 1-4, wherein the treatment increases bone density of a bone of the subject compared to the bone density before the treatment. 6. The method of any one of aspects 1-5, wherein the treatment decreases an amount of fatty bone marrow in a bone of the subject compared to the amount of fatty bone marrow before the treatment. 7. The method of any one of aspects 1-6, wherein the CCN3 is administered intravenously or intraperitoneally. 8. The method of any one of aspects 1-6, wherein the CCN3 is administered locally to a bone of the subject.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 9. The method of any one of aspects 1-8, wherein multiple therapeutically effective doses of CCN3 are administered to the subject. 10. The method of aspect 9, wherein the CCN3 is administered according to a daily dosing regimen or intermittently. 11. The method of any one of aspects 1-10, further comprising administering an osteoanabolic agent or anti-resorptive agent to the subject. 12. The method of aspect 11, wherein the osteoanabolic agent is parathyroid hormone, teriparatide, abaloparatide, or romosozumab. 13. The method of aspect 11, wherein the anti-resorptive agent is estrogen, an estrogen agonist, a bisphosphonate, or denosumab. 14. The method of any one of aspects 1-13, wherein the subject is human. 15. The method of any one of aspects 1-14, wherein the CCN3 is encapsulated in a hydrogel. 16. The method of aspect 15, wherein the hydrogel comprises alginate. 17. The method of aspect 16, wherein the alginate is ionically cross-linked. 18. The method of aspect 17, wherein the alginate is ionically cross-linked by divalent calcium cations. 19. The method of any one of aspects 16-18, wherein the alginate concentration in the hydrogel ranges from 2 to 10 percentage by weight (wt%). 20. The method of any one of aspects 1-19, wherein the CCN3 is administered using a drug delivery device.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 21. The method of aspect 20, wherein the drug delivery device provides sustained delivery of the CCN3. 22. The method of aspect 20, wherein the drug delivery device is a reservoir implant or a monolithic implant. 23. The method of aspect 20, wherein the drug delivery device is a non- biodegradable implant. 24. The method of aspect 20, wherein the drug delivery device is a biodegradable implant. 25. The method of any one of aspects 1-21, further comprising administering a therapeutically effective amount of osteochondral skeletal stem cells to the subject. 26. A method of decreasing or preventing bone degeneration in a female subject during lactation, the method comprising administering a therapeutically effective amount of cellular communication network factor 3 (CCN3) to the subject. 27. The method of aspect 26, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. 28. The method of aspect 26 or 27, wherein the CCN3 is administered intravenously or intraperitoneally. 29. The method of aspect 26 or 27, wherein the CCN3 is administered locally to a bone of the subject. 30. The method of any one of aspects 26-29, wherein the CCN3 is administered according to a daily dosing regimen or intermittently. 31. A composition comprising CCN3 for use in a method of treating a bone disorder or condition associated with bone degeneration.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 32. The composition of aspect 31, further comprising a pharmaceutically acceptable excipient. 33. The composition of aspect 31 or 32, further comprising a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment. 34. The composition of any one of aspects 31-33, wherein the bone disorder or condition associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy. 35. A method of treating a bone disorder or condition associated with bone degeneration in a subject, the method comprising administering a vector comprising an expression cassette comprising a coding sequence encoding cellular communication network factor 3 (CCN3) to the subject. 36. The method of aspect 35, wherein the expression cassette comprises a promoter operably linked to the coding sequence encoding the CCN3. 37. The method of aspect 35, wherein the coding sequence encoding the CCN3 is integrated into a chromosomal locus in the genome of the subject. 38. The method of aspect 37, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the CCN3 at the chromosomal locus. 39. The method of any one of aspects 35-38, wherein the vector is a plasmid or viral vector.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 40. The method of aspect 39, wherein the viral vector is an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, or a retroviral vector. 41. The method of any one of aspects 35-40, wherein the vector is administered intravenously or intraperitoneally. 42. The method of aspect 41, wherein the vector is administered via a portal vein. 43. The method of any one of aspects 35-40, wherein the vector is administered locally to a bone. 44. The method of any one of aspects 35-43, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. 45. The method of any one of aspects 35-44, wherein the bone disorder or condition associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy. 46. The method of any one of aspects 35-45, wherein the treatment increases bone mass of a bone of the subject compared to the bone mass before the treatment. 47. The method of any one of aspects 35-46, wherein the treatment increases bone density of a bone of the subject compared to the bone density before the treatment. 48. The method of any one of aspects 35-47, wherein the treatment decreases an amount of fatty bone marrow in a bone of the subject compared to the amount of fatty bone marrow before the treatment.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 49. A method of providing a subject with cellular communication network factor 3 (CCN3) to promote bone growth in the subject, the method comprising introducing a vector comprising a promoter operably linked to a coding sequence encoding the CCN3 into a cell, wherein the cell expresses the CCN3 in vivo in the subject in an effective amount sufficient to promote bone growth in the subject. 50. The method of aspect 49, wherein the vector is introduced into the cell ex vivo or in vivo. 51. The method of aspect 49 or 50, wherein the cell is a hepatocyte or an osteochondral skeletal stem cell. 52. The method of any one of aspects 49-51, wherein the vector is a plasmid or viral vector. 53. The method of aspect 52, wherein the viral vector is an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, or a retroviral vector. 54. The method of any one of aspects 49-53, wherein the subject has a bone disorder or condition associated with bone degeneration. 55. The method of aspect 54, wherein the bone disorder or condition associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy. 56. A method of stimulating an osteochondral skeletal stem cell to produce bone, the method comprising contacting the osteochondral skeletal stem cell with an effective amount of cellular communication network factor 3 (CCN3), wherein bone is produced by the osteochondral skeletal stem cell.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 57. The method of aspect 56, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. 58. A method of screening for an agonist of cellular communication network factor 3 (CCN3) that increases bone growth, the method comprising: contacting a cell with the CCN3 and a candidate agent, wherein the cell is an osteochondral skeletal stem cell, a preosteoblast cell, an osteoblast cell, an osteoprogenitor cell, or an osteosarcoma cell; and measuring bone production by the cell, wherein an increased amount of bone production in presence of the candidate agent compared to a reference value range for the amount of bone production in a control cell, wherein the candidate agent is absent, indicates that the candidate agent is an agonist of CCN3. 59. A method of screening for a mimic or an analogue of cellular communication network factor 3 (CCN3) that stimulates bone growth, the method comprising: contacting a cell with a candidate agent, wherein the cell is an osteochondral skeletal stem cell, a preosteoblast cell, an osteoblast cell, an osteoprogenitor cell, or an osteosarcoma cell; and measuring bone mineralization by the cell, wherein an increased amount of bone mineralization in presence of the candidate agent compared to a reference value range for the amount of bone mineralization in a control cell, wherein the candidate agent is absent, indicates that the candidate agent is a mimic or an analogue of the CCN3. 60. The method of aspect 58 or 59, wherein the cell is derived from a stem cell. 61. The method of aspect 60, wherein the stem cell is an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell. 62. The method of aspect 61, wherein the adult stem cell is a mesenchymal stem cell. 63. The method of aspect 60, wherein the stem cell is from a patient who has a bone disorder or condition associated with bone degeneration.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 64. The method of aspect 59, wherein the cell is immortalized. 65. The method of aspect 59, wherein the cell is from a MC3T3-E1 preosteoblast cell line, an SaOs2 osteosarcoma cell line, an MG-63 osteosarcoma cell line, a hFOB osteoblast cell line, an ASC52telo immortalized adipose derived mesenchymal stem cell line, a human telomerase reverse transcriptase-immortalized bone marrow mesenchymal stromal cell (hTERT- BMSC) line, or an ATDC5 chondrogenic mouse teratocarcinoma cell line. 66. A method of producing a bone graft, the method comprising culturing an osteochondral skeletal stem cell in presence of cellular communication network factor 3 (CCN3) under suitable conditions, wherein the osteochondral skeletal stem cell produces bone for the bone graft. 67. A bone graft produced by the method of aspect 66. 68. The bone graft of aspect 67, wherein the osteochondral skeletal stem cell is derived from an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell. 69. The bone graft of aspect 68, wherein the adult stem cell is a mesenchymal stem cell. 70. A method of transplanting a bone graft into a subject, the method comprising transplanting the bone graft of aspect 69 into a transplantation site in the subject. 71. The method of aspect 70, wherein the osteochondral skeletal stem cell is autologous, allogeneic, or xenogeneic. 72. The method of aspect 70 or 71, wherein the osteochondral skeletal stem cell is derived from an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell. 73. The method of aspect 72, wherein the adult stem cell is a mesenchymal stem cell. 74. The method of any one of aspects 70-73, wherein the bone graft replaces missing bone to repair a bone fracture at the transplantation site.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 75. The method of aspect 74, wherein the bone fracture is a compound fracture. 76. The method of any one of aspects 70-73, wherein the bone graft provides new bone to repair a congenital bone defect at the transplantation site. 77. The method of any one of aspects 70-73, wherein the bone graft provides new bone for a craniofacial surgery, oral and maxillofacial surgery, plastic surgery, or reconstructive surgery. 78. The method of any one of aspects 70-73, wherein the bone graft provides new bone for a dental implant. 79. The method of any one of aspects 70-78, further comprising administering cellular communication network factor 3 (CCN3) locally at the transplantation site to stimulate new bone growth at the transplantation site. 80. A method of monitoring a lactating female subject to determine risk of bone degeneration from breast feeding, the method comprising: obtaining a biological sample from the lactating female subject; measuring a level of communication network factor 3 (CCN3) in the biological sample; and comparing the level of the CCN3 in the biological sample to reference value ranges for CCN3 from a control subject, wherein levels of CCN3 below a threshold value indicate that the subject is at risk of bone degeneration from breast feeding, and wherein levels of CCN3 equal to or greater than a threshold value indicate that the female subject may continue breast feeding without substantial risk of bone degeneration. 81. The method of aspect 80, wherein the female subject discontinues breast feeding if the level of CCN3 indicates the female subject is at risk of bone degeneration from breast feeding.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 82. The method of aspect 80 or 81, further comprising administering a therapeutically effective amount of the CCN3 to the female subject to lower the risk of bone degeneration from breast feeding. 83. The method of any one of aspects 80-82, wherein the biological sample is blood or plasma. 84. The method of any one of aspects 80-83, wherein said measuring the level of CCN3 comprises performing an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, an immunofluorescent assay, immunohistochemistry, fluorescence-activated cell sorting (FACS), a Western Blot, mass spectrometry, tandem mass spectrometry, a biochemical assay, liquid chromatography, or NMR. 85. A communication network factor 3 (CCN3) for use as a biomarker in monitoring a lactating female subject to determine risk of bone loss from breast feeding. 86. A method for regenerating mammalian cartilage in a subject, the method comprising: activating skeletal stem cells with a mechanical stimulus; and administering a therapeutically effective amount of cellular communication network factor 3 (CCN3) in combination with a therapeutically effective amount of a vascular endothelial growth factor (VEGF) inhibitor to the subject. 87. The method of aspect 86, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. 88. The method of aspect 86 or 87, wherein the VEGF inhibitor is cabozantinib. 89. The method of any one of aspects 86-88, wherein the cartilage is articular cartilage. 90. The method of any one of aspects 86-89, wherein the mechanical stimulus is an acute local injury.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 91. The method of aspect 90, wherein the acute local injury is a surgically performed microfracture procedure to bone tissue at a desired site for cartilage regeneration. 92. The method of aspect 90 or 91, wherein the VEGF inhibitor and the CCN3 are administered locally to the acute local injury. 93. The method of any of aspects 86-92, wherein the VEGF inhibitor and the CCN3 are administered to the subject immediately after said activating the skeletal stem cells with the mechanical stimulus. 94. The method of any of aspects 86-92, wherein the VEGF inhibitor and the CCN3 are administered to the subject within three days after said activating the skeletal stem cells with the mechanical stimulus. 95. The method of any of aspects 86-94, wherein the VEGF inhibitor and the CCN3 are encapsulated in a hydrogel. 96. The method of aspect 95, wherein the hydrogel comprises alginate. 97. The method of aspect 96, wherein the alginate is ionically cross-linked. 98. The method of aspect 97, wherein the alginate is ionically cross-linked by divalent calcium cations. 99. The method of any one of aspects 96-98, wherein the alginate concentration in the hydrogel ranges from 2 to 10 percentage by weight (wt%). 100. The method of any of aspects 86-99, wherein the VEGF inhibitor and the CCN3 are administered using a drug delivery device. 101. The method of aspect 100, wherein the drug delivery device provides sustained delivery of the VEGF inhibitor and the CCN3.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 102. The method of aspect 100 or 101, wherein the drug delivery device is a reservoir implant or a monolithic implant. 103. The method of any of aspects 100-102, wherein the drug delivery device is a non- biodegradable implant. 104. The method of any of aspects 100-102, wherein the drug delivery device is a biodegradable implant. 105. The method of any of aspects 100-104, wherein the drug delivery device is implanted at the site of the local acute injury. 106. The method of any of aspects 86-105, wherein the subject has a cartilage disorder. 107. The method of aspect 106, wherein the cartilage disorder is osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, or malignancy. 108. The method of any of aspects 86-107, further comprising administering a therapeutically effective amount of skeletal stem cells to the subject. 109. The method of any of aspects 86-108, wherein the CCN3 is provided by a vector comprising an expression cassette comprising a coding sequence encoding the CCN3. 110. The method of aspect 109, wherein the expression cassette comprises a promoter operably linked to the coding sequence encoding the CCN3. 111. The method of aspect 109 wherein the coding sequence encoding the CCN3 is integrated into a chromosomal locus in the genome of the subject. 112. The method of aspect 111, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the CCN3 at the chromosomal locus.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 113. The method of any one of aspects 109-112, wherein the vector is a plasmid or viral vector. 114. The method of aspect 113, wherein the viral vector is an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, or a retroviral vector. 115. A composition comprising cellular communication network factor 3 (CCN3) and a vascular endothelial growth factor (VEGF) inhibitor for use in a method of regenerating mammalian cartilage. 116. The composition of aspect 115, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. 117. The composition of aspect 115 or 116, wherein the VEGF inhibitor is cabozantinib. 118. The composition of any one of aspects 115-117, wherein the cartilage is articular cartilage. 119. The composition of any one of aspects 115-118, wherein the CCN3 and the VEGF inhibitor are encapsulated in a hydrogel. 120. The composition of aspect 119, wherein the hydrogel comprises alginate. 121. The composition of aspect 120, wherein the alginate is ionically cross-linked. 122. The composition of aspect 121, wherein the alginate is ionically cross-linked by divalent calcium cations. 123. The composition of any one of aspects 120-122 wherein the alginate concentration in the hydrogel ranges from 2 to 10 percentage by weight (wt%).Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 124. A method of screening for an agonist of cellular communication network factor 3 (CCN3) that increases production of cartilage, the method comprising: activating a skeletal stem cell with a mechanical stimulus; contacting the skeletal stem cell with CCN3, a vascular endothelial growth factor (VEGF) inhibitor, and a candidate agent; and measuring cartilage production by the skeletal stem cell, wherein an increased amount of cartilage production in presence of the candidate agent compared to a reference value range for the amount of cartilage production in a control skeletal stem cell, wherein the candidate agent is absent, indicates that the candidate agent is an agonist of CCN3. 125. A method of screening for a mimic or an analogue of cellular communication network factor 3 (CCN3) that stimulates production of cartilage, the method comprising: activating a skeletal stem cell with a mechanical stimulus; contacting the skeletal stem cell with a vascular endothelial growth factor (VEGF) inhibitor and a candidate agent; and measuring cartilage production by the skeletal stem cell, wherein an increased amount of cartilage production in presence of the candidate agent compared to a reference value range for the amount of cartilage production in a control skeletal stem cell, wherein the candidate agent is absent, indicates that the candidate agent is a mimic or an analogue of the CCN3. EXAMPLES

[0346] As can be appreciated from the disclosure provided above, the present disclosure has a wide variety of applications. Accordingly, 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 to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, dimensions, etc.) but some experimental errors and deviations should be accounted for. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 EXAMPLE1: BRAIN-DERIVEDCCN3,ANANABOLICHORMONE TOBUILDBONE INLACTATINGFEMALES

[0347] We and others have shown that central estrogen signaling exerts a sex-dependent restraint on bone formation, alongside its role in promoting spontaneous activity and thermogenesis17-20. High bone mass in females results following the deletion of neuronal ERα21in the arcuate nucleus of the medial basal hypothalamus (MBH)1 22. Viral and genetic mouse models designed to eliminate ERα in the ARC, specifically in ARCKiss1neurons, resulted in high trabecular bone mass in the spine and long bones, independent of high E2 levels, confirming the central origins of this extraordinary bone phenotype1.

[0348] Here, using a combination of question-driven and discovery-based approaches, we set out to identify the osteogenic hormone-like factor responsible for the high bone mass in mutant females after first showing that this factor circulates in the blood. Cellular communication network factor 3 (CCN3 / Nov) emerged as the best candidate, fulfilling all anticipated criteria; it is secreted, its appearance in the ARC coincides with the onset and then loss of the bone phenotype after a dietary challenge, it causes new bone formation when ectopically delivered ex vivo to bones or in vivo to mice, and finally, bone mass degrades in mutant females after knockdown of Ccn3 transcripts in the ARC. We propose that CCN3 functions as a brain-derived osteoanabolic hormone in a newly-identified brain-bone axis evolved to sustain skeletal health in mammalian mothers and offspring. A Humoral Factor Mediates the High Bone Mass in Mutant Females

[0349] Our prior genetic and viral deletions of ERα in the ARC strongly suggested that a subset of KNDy neurons in the ARC regulates bone mass and bone strength in females but not males (FIGS. 1A, 1B)1. That KNDy neurons participate in this brain-bone axis was further supported after deleting ERα with the Prodynorphin-Cre driver (FIG.1B and FIGS.6A, 6B). To identify the molecular origins of the high bone mass phenotype, we relied exclusively on the Esr1Nkx2-1Crefemale mouse model, which exhibits this unusual phenotype by four weeks of age (FIGS. 6C-6E). Given the privileged position of the ARC as one of the few circumventricular organs of the brain just dorsal to the median eminence, we asked if this high bone mass in mutant females might originate from a circulatory factor.

[0350] Using classical parabiosis coupled with in vivo µCT imaging (FIG.1C), two female groups were surgically joined, generating control pairs (WT-WT) and controls joined to mutant females (WT-MUT). Shortly after surgery (2 wks), baseline bone microarchitecture parametersAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 on the contralateral femur opposite the surgical side were established for each animal in the pairings. Females in the WT-WT paring exhibited a net decrease in bone mass that was readily observed beginning at six weeks post-surgery (FIG.1E); this decline normalized by Wk 17, rising an average of ~37%. In the WT-MUT pairings, higher fractional bone volume (%BV / TV) was observed at all time points in control females, increasing ~152% by 17 Wks (FIG.1F and FIGS. 7A, 7B). Uterine weights, as well as other parameters, were unchanged between WT-WT and WT- MUT pairings, consistent with the notion that higher estrogen levels are not at play in generating high bone mass in mutant females (FIG.7C). We noted that the already high bone mass in mutant females increased further in some MUT-WT pairings (FIGS.7D, 7E).

[0351] Bone transplant studies confirmed that a humoral factor accounts for the high bone mass in mutant females. Female and male femurs from 4-week-old control donors were implanted subcutaneously into 8-week-old wild-type or mutant females (FIG.1G and FIG.8A). Significant increases in fractional bone mass were detected in both female and male femurs six weeks post- implantation into mutant females (FIGS. 1H, 1I and FIGS. 8B, 8C), arguing strongly that this brain-dependent, female-specific osteoanabolic hormone will function in both sexes. A Brain-Dependent Factor Alters Skeletal Stem Cell Dynamics and Bone Anabolic Activity.

[0352] Skeletal homeostasis is tightly regulated by skeletal stem cell (SSC)-based bone formation and osteoclast-based bone resorption. Chan, Ambrosi, and colleagues demonstrated that stem cells with distinct lineage hierarchies and cellular contributions facilitate new bone formation23. In particular, osteochondral SSCs (ocSSCs) form bone and cartilage (and fibro / stromal lineage cell populations but not bone marrow adipocytes) and are present in the growth plate and periosteum of bones8,24(FIG. 2A), whereas perivascular SSCs (pvSSCs) give rise to unilateral committed adipogenic progenitor cells (APCs) that generate all bone marrow adipose tissue (BMAT) in bones23,25. As such, we reasoned that the brain-dependent anabolic hormone might alter ocSSC activity, given the increased bone formation in mutant females1. OcSSCs from female wild-type mice were isolated by flow cytometry and transplanted beneath the renal capsule of control or mutant females (FIG. 2bB). As expected, wild-type ocSSCs transplanted into control Esr1fl / flfemale littermates formed an ectopic bone graft with a host- derived hematopoietic compartment over six weeks (FIGS.2C, 2D). However, wild-type ocSSCs grafted into Esr1Nkx2-1Crefemales exhibited significantly higher mineralization and little hematopoietic marrow (FIGS. 2C-2E), suggesting that the osteoanabolic hormone present in mutant females alters the ocSSC lineage to promote bone formation (FIG. 2E). Consistent with this hypothesis, the higher fractional bone mass observed in the WT-MUT parabiosis or wholeAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 bone transplants studies correlated with increased ocSSCs frequency (FIG. 9A). The robustness of this circulatory osteoanabolic hormone was further verified by stereotaxic delivery of GFP- positive wild-type ocSSCs to the vicinity of the ARC (FIG. 2F). Remarkably, µCT imaging of mutant Esr1Nkx2-1Crehypothalami revealed mineralized ossicles overlapping with transplanted GFP+ cells six weeks post-injection; no ossicles were detected in wild-type brains (FIGS. 2G, 2H). These data further establish the existence of a circulatory anabolic bone factor in mutant females, perhaps originating from the ARC or surrounding hypothalamic regions.

[0353] That a circulating factor might promote wild-type ocSSC activity prompted us to compare the differentiation capacity of mutant and control ocSSCs. Flow cytometric analysis revealed a sex-dependent increased frequency of ocSSCs in both pre-pubertal (3Wks) and young adult mutant (10Wks) females (FIG. 2I). This alteration was limited to ocSSCs, as pvSSCs and their progeny adipogenic progenitor cells (APCs) fated for BMAT23,25were equivalent in controls and mutants, except for the lower frequency of APCs in younger mutants (FIG.2I). Differentiation assays revealed that while ocSSCs from both genotypes showed no differences in colony-forming ability (CFU-F) (FIG.9B), mutant ocSSCs exhibit a high intrinsic potential for bone and cartilage formation (FIG. 2J). Enhanced ocSSCs activity was also observed in older Esr1Nkx2-1Crefemales, consistent with their attenuated bone loss compared to age-matched control littermates and the known linkage between ocSSC dysfunction and age-related bone loss (FIGS.9C-9E)26. Identification of CCN3 as a Candidate Brain-Derived Osteogenic Factor

[0354] Despite the expansion and enhanced osteogenic capacity of mutant ocSSCs, single- cell RNA sequencing (sc-RNAseq) data implied that while mutant ocSSCs differentiation dynamics were primed towards bone formation, only modest overall transcriptomic differences were detected; thus, unfortunately, providing few hints regarding the identity of the osteoanabolic hormone in Esr1Nkx2-1Crefemales (FIG. 10). However, the first helpful clue in our hunt for this anabolic bone factor arose after finding that a chronic high-fat dietary (HFD) challenge dramatically disrupts the sex-dependent brain-bone axis. While body weights, fat mass, blood triglycerides, and glucose homeostasis remained unchanged with HFD (FIG.11A), the high bone mass phenotype in Esr1Nkx2-1Crefemales completely reversed with this dietary challenge (FIG.3A). Reduction in trabecular and cortical bone mass was accompanied by expected structural changes (FIG. 11B) and reduced bone strength and secondary spongiosa (FIGS. 3A, 3B). Histomorphometry showed that osteoclast number, bone formation, and mineralized surface remained proportional to bone surface in mutant females fed HFD (FIG.3B and FIGS.11C, 11D). However, levels of BMAT, quantified by osmium staining27, were significantly lower in mutantAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 females on either SD or HFD compared to control littermates (FIGS.3A, 3B). Nevertheless, while dense bones in Esr1Nkx2-1Crefemales readily degrade with HFD, they fail to accumulate BMAT to a similar extent as control females, thus defying the normal linkage between BMAT expansion and bone loss28. Bone parameters in Esr1Nkx2-1Cremales remained unchanged from their control littermates (FIGS. 11E, 11F). Moreover, chronic hyperglycemia induced by the insulin receptor antagonist S96129, failed to degrade mutant female bone mass, demonstrating the specificity of HFD-induced bone loss in mutant females (FIG.11G).

[0355] We then exploited the dynamic changes in bone following HFD by profiling gene changes in microdissected ARC from Esr1Nkx2-1Crefemales fed SD or HFD. Bulk RNA-seq revealed a small set of upregulated differentially expressed genes (DEGs) encoding neuropeptides or secreted proteins in the ARC, including Ccn3, Fst, Grp, and Penk – all dropped significantly after HFD but were elevated on SD in young and older females (FIGS. 3C, 3D and FIG. 12A). Importantly, Ccn3 and Penk expression correlated well with the onset of the high bone mass at around four weeks of age (FIG. 3E) but were unchanged at earlier time points or in Esr1Nkx2-1Cremales (FIGS.12B, 12C). Except for Penk in the mutant pituitary, few notable candidates emerged after profiling the pituitary and liver, two common tissue sources of secreted proteins, (FIG. 3C and FIG.12A). CCN3 / Ccn3 expression was nearly absent in control females or males but readily detected in the basal region of the mutant female ARC colocalizing with KISS1, a known marker of KNDy neurons (FIGS. 3F, 3G); expression levels easily surpassed those detected in the suprachiasmatic nucleus (SCN)30. CCN3 Functions as an Osteoanabolic Hormone.

[0356] High expression of CCN3 in mutant ARC neurons that are KISS1-positive and ERα−negative, prompted us to test this founding member of the CCN family31. This secreted protein is postulated to antagonize CCN2 to inhibit osteogenesis32,33, although a single report suggests the opposite34. Nevertheless, the anabolic potential of CCN3 was evaluated in three distinct assays. First, ex vivo whole long bone cultures were treated with CCN3 after initially finding that plasma collected from Esr1Nkx2-1Crefemales elevated bone mass of freshly dissected control femurs after five days, rising an average of 37% and 50% for female and male femurs, respectively (FIGS. 4A, 4B and FIG. 13A). Importantly, long bones of both sexes showed substantial degradation from their baseline values when cultured in media alone (FIGS.13B, 13C). Using this simple but effective assay, we found that low doses of mouse (m)CCN3 (3.0 nM) induced an upward dynamic shift (60%) in bone mass compared to saline (FIGS.4D-4F and FIG. 13B). Second, adult wild type mice were injected with mCCN3 (i.p.) or saline daily over threeAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 weeks. Within this short time and at this low dose (7.5 µg / kg), a significant increase in bone mass was noted for both female and male mice treated with mCCN3 (FIGS. 4G, 4H and FIG. 13D). Finally, culturing primary ocSSC isolated from newborn wild-type mice treated daily with mCCN3 increased mineralization by ~200% (FIG. 4I). Met-ENK and BAMP-22, two major peptides encoded by Penk, failed to elicit any changes; these negative results are consistent with the fact that the competitive antagonist of the µ-opioid receptor, Naloxone, was unable to degrade the high bone mass in mutant female mice (FIG.14A). Additionally, low concentrations of human CCN3 increased osteogenesis in primary human ocSSCs from pubertal and aged / geriatric patients independent of sex; higher levels had no beneficial effect on osteogenesis and were inhibitory at the highest level (FIG.4J and FIGS.14B-14E). The pro-osteogenic effect on young and old human ocSSCs appears to be exclusive to CCN3 (FIGS. 14B, 14C). Taken together, these data suggest that at low doses, CCN3 is an osteoanabolic hormone in mice and humans. Brain-Derived CCN3 Is Used During Lactation to Preserve Bone Mass

[0357] To establish the linkage between CCN3 and increased bone density in Esr1Nkx2-1females, bone parameters were measured following overexpression or knockdown of CCN3 in control or mutant females, respectively. We leveraged the secretory capacity of hepatocytes to increase circulating CCN3 in Esr1fl / flcontrol females after systemic delivery of the AAV-dj-CCN3 vector with high liver tropism35. Ectopic CCN3 expression in liver hepatocytes was detected as early as two weeks post-injection (FIG. 5A and FIG. 15A). Even at exceedingly low levels of hepatic CCN3 expression, modest increases in bone formation rate were observed (Extended Data FIGS. 10A, 10B). At 10-fold higher levels of hepatic CCN3 expression, fractional bone volume was enhanced by nearly 80% (FIGS.5B, 5C and FIG.15B). Conversely, transient knockdown of Ccn3 expression in Esr1Nkx2-1female ARC neurons by stereotaxic delivery of siRNAs attenuated the dense bone phenotype with levels of Ccn3 / CCN3 tracking well with %BV / TV (FIGS.5D-5F). Together, these data show that elevating or lowering CCN3 levels in control and mutant females, respectively, affects bone mass and that maintenance of the high bone mass in Esr1Nkx2-1Crefemales relies on sufficient CCN3 in ARCERα-CCN3neurons.

[0358] To expand our findings beyond an artificial genetic phenomenon, we asked if CCN3 appears at any life stage in the ARC of control females, focusing on the postpartum period when maternal BFR escalates to maintain the skeletal calcium reservoir12,36, in the face of exceedingly low circulating E29. Similar to virgin controls, CCN3-expressing ARCERαneurons are non-existent in early and late-stage pregnancy (FIGS. 5G, 5H). However, by seven days postpartum (DPP), CCN3 is abundantly expressed in a subset of ARCERαneurons of lactatingAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 dams (FIG. 5G), reaching near equivalent levels as found in Esr1Nkx2-1Cremutant females (FIG. 5H). Forced weaning reduced CCN3 in ARCERαneurons when examined 3 or 7 days after removal of pups (10 or14 DPP, respectively), suggesting that the need for bone-promoting CCN3 lessens at cessation of lactation when calcium demand diminishes (FIG.5G). These data imply that brain- derived CCN3 in ARCERα-KISS1-CCN3neurons sustains sufficient bone formation to support inter- generational resource transfer when ovarian-derived estrogens fade away (FIG.5I). DISCUSSION

[0359] The role of ARCERα-KISS1neurons as the gatekeeper of female reproduction is well- established – these neurons control multiple facets of physiology, including regulating pubertal onset and the hypothalamic-pituitary-gonadal axis. Here, we discover yet another crucial function for ARCERα-KISS1neurons in females – controlling bone homeostasis during lactation via the brain- derived osteoanabolic hormone CCN3. Shutting down ovarian estradiol production and energy expenditure during lactation37poses a serious problem - how do mineralized bone surfaces keep up while being “plundered” for calcium during lactation9, especially in the trabecular-rich spine, which is particularly susceptible to lactational-associated osteoporosis13as well as the loss of ERα signaling in bone4. Through CCN3, ARCERα-KISS1-CCN3neurons solve this problem by lifting the usual restraints on energetically costly bone formation, thereby increasing trabecular bone in the spine and long bones1, which degrades during lactation. How does CCN3 become upregulated in the female ARC during this unique life stage? While Ccn3 could be coordinately upregulated along with Kiss1 due to the sharp rise in prolactin signaling38, we note that prolactin levels are only modestly elevated in mutant females1. In addition to bone homeostasis, it will be of interest to determine if the medial basal hypothalamus also directly or indirectly controls other adaptive responses, including the dramatic increases in intestinal length and nutrient absorption in lactating mothers39.

[0360] Our data are at odds with the proposed function of CCN3 as an inhibitor of osteogenesis, as reviewed in40. However, most prior studies relied on high doses and high overexpression of CCN3, making it likely that compensatory cellular responses or non-specific receptor activation in cellular bone niches account for the adverse effects on osteogenesis. We, too, observed inhibitory effects in both mouse and human ocSSC differentiation assays at CCN3 doses exceeding the sub-nanomolar range. Based on the presence of anti-parallel β-strands and the C-terminal Cystine knot (CTCK) domain that mediates disulfide-linked dimerization41, we predict that CCN3 circulates at low doses as a tightly held homodimer, binding its cognate receptor with high affinity, similar to other growth factors such as NGF. Identifying the molecular targetAtty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 of CCN3 in ocSSCs and possibly other cellular populations, including osteocytes that reversibly remodel their perilacunar / canalicular matrix during lactation42, will help resolve these discrepancies. We speculate that permanently deleting ERα from ARCKISS1neurons leads to continuous secretion of low levels of CCN3, recapitulating the anabolic phase of healthy, postpartum bone remodeli...

Claims

Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 CLAIMS WHAT IS CLAIMED IS:

1. A method of treating a bone disorder or condition associated with bone degeneration in a subject, the method comprising administering a therapeutically effective amount of cellular communication network factor 3 (CCN3) to the subject.

2. The method of claim 1, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

3. The method of claim 1 or 2, wherein the bone disorder or condition associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

4. The method of any one of claims 1-3, wherein the treatment increases bone mass of a bone of the subject compared to the bone mass before the treatment.

5. The method of any one of claims 1-4, wherein the treatment increases bone density of a bone of the subject compared to the bone density before the treatment.

6. The method of any one of claims 1-5, wherein the treatment decreases an amount of fatty bone marrow in a bone of the subject compared to the amount of fatty bone marrow before the treatment.

7. The method of any one of claims 1-6, wherein the CCN3 is administered intravenously or intraperitoneally.

8. The method of any one of claims 1-6, wherein the CCN3 is administered locally to a bone of the subject.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 9. The method of any one of claims 1-8, wherein multiple therapeutically effective doses of CCN3 are administered to the subject.

10. The method of claim 9, wherein the CCN3 is administered according to a daily dosing regimen or intermittently.

11. The method of any one of claims 1-10, further comprising administering an osteoanabolic agent or anti-resorptive agent to the subject.

12. The method of claim 11, wherein the osteoanabolic agent is parathyroid hormone, teriparatide, abaloparatide, or romosozumab.

13. The method of claim 11, wherein the anti-resorptive agent is estrogen, an estrogen agonist, a bisphosphonate, or denosumab.

14. The method of any one of claims 1-13, wherein the subject is human.

15. The method of any one of claims 1-14, wherein the CCN3 is encapsulated in a hydrogel.

16. The method of claim 15, wherein the hydrogel comprises alginate.

17. The method of claim 16, wherein the alginate is ionically cross-linked.

18. The method of claim 17, wherein the alginate is ionically cross-linked by divalent calcium cations.

19. The method of any one of claims 16-18, wherein the alginate concentration in the hydrogel ranges from 2 to 10 percentage by weight (wt%).

20. The method of any one of claims 1-19, wherein the CCN3 is administered using a drug delivery device.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 21. The method of claim 20, wherein the drug delivery device provides sustained delivery of the CCN3.

22. The method of claim 20, wherein the drug delivery device is a reservoir implant or a monolithic implant.

23. The method of claim 20, wherein the drug delivery device is a non-biodegradable implant.

24. The method of claim 20, wherein the drug delivery device is a biodegradable implant.

25. The method of any one of claims 1-21, further comprising administering a therapeutically effective amount of osteochondral skeletal stem cells to the subject.

26. A method of decreasing or preventing bone degeneration in a female subject during lactation, the method comprising administering a therapeutically effective amount of cellular communication network factor 3 (CCN3) to the subject.

27. The method of claim 26, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

28. The method of claim 26 or 27, wherein the CCN3 is administered intravenously or intraperitoneally.

29. The method of claim 26 or 27, wherein the CCN3 is administered locally to a bone of the subject.

30. The method of any one of claims 26-29, wherein the CCN3 is administered according to a daily dosing regimen or intermittently.

31. A composition comprising CCN3 for use in a method of treating a bone disorder or condition associated with bone degeneration.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 32. The composition of claim 31, further comprising a pharmaceutically acceptable excipient.

33. The composition of claim 31 or 32, further comprising a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.

34. The composition of any one of claims 31-33, wherein the bone disorder or condition associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

35. A method of treating a bone disorder or condition associated with bone degeneration in a subject, the method comprising administering a vector comprising an expression cassette comprising a coding sequence encoding cellular communication network factor 3 (CCN3) to the subject.

36. The method of claim 35, wherein the expression cassette comprises a promoter operably linked to the coding sequence encoding the CCN3.

37. The method of claim 35, wherein the coding sequence encoding the CCN3 is integrated into a chromosomal locus in the genome of the subject.

38. The method of claim 37, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the CCN3 at the chromosomal locus.

39. The method of any one of claims 35-38, wherein the vector is a plasmid or viral vector.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 40. The method of claim 39, wherein the viral vector is an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

41. The method of any one of claims 35-40, wherein the vector is administered intravenously or intraperitoneally.

42. The method of claim 41, wherein the vector is administered via a portal vein.

43. The method of any one of claims 35-40, wherein the vector is administered locally to a bone.

44. The method of any one of claims 35-43, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

45. The method of any one of claims 35-44, wherein the bone disorder or condition associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

46. The method of any one of claims 35-45, wherein the treatment increases bone mass of a bone of the subject compared to the bone mass before the treatment.

47. The method of any one of claims 35-46, wherein the treatment increases bone density of a bone of the subject compared to the bone density before the treatment.

48. The method of any one of claims 35-47, wherein the treatment decreases an amount of fatty bone marrow in a bone of the subject compared to the amount of fatty bone marrow before the treatment.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 49. A method of providing a subject with cellular communication network factor 3 (CCN3) to promote bone growth in the subject, the method comprising introducing a vector comprising a promoter operably linked to a coding sequence encoding the CCN3 into a cell, wherein the cell expresses the CCN3 in vivo in the subject in an effective amount sufficient to promote bone growth in the subject.

50. The method of claim 49, wherein the vector is introduced into the cell ex vivo or in vivo.

51. The method of claim 49 or 50, wherein the cell is a hepatocyte or an osteochondral skeletal stem cell.

52. The method of any one of claims 49-51, wherein the vector is a plasmid or viral vector.

53. The method of claim 52, wherein the viral vector is an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

54. The method of any one of claims 49-53, wherein the subject has a bone disorder or condition associated with bone degeneration.

55. The method of claim 54, wherein the bone disorder or condition associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathologic bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, a craniofacial disorder, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or ovariectomy.

56. A method of stimulating an osteochondral skeletal stem cell to produce bone, the method comprising contacting the osteochondral skeletal stem cell with an effective amount of cellular communication network factor 3 (CCN3), wherein bone is produced by the osteochondral skeletal stem cell.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 57. The method of claim 56, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

58. A method of screening for an agonist of cellular communication network factor 3 (CCN3) that increases bone growth, the method comprising: contacting a cell with the CCN3 and a candidate agent, wherein the cell is an osteochondral skeletal stem cell, a preosteoblast cell, an osteoblast cell, an osteoprogenitor cell, or an osteosarcoma cell; and measuring bone production by the cell, wherein an increased amount of bone production in presence of the candidate agent compared to a reference value range for the amount of bone production in a control cell, wherein the candidate agent is absent, indicates that the candidate agent is an agonist of CCN3.

59. A method of screening for a mimic or an analogue of cellular communication network factor 3 (CCN3) that stimulates bone growth, the method comprising: contacting a cell with a candidate agent, wherein the cell is an osteochondral skeletal stem cell, a preosteoblast cell, an osteoblast cell, an osteoprogenitor cell, or an osteosarcoma cell; and measuring bone mineralization by the cell, wherein an increased amount of bone mineralization in presence of the candidate agent compared to a reference value range for the amount of bone mineralization in a control cell, wherein the candidate agent is absent, indicates that the candidate agent is a mimic or an analogue of the CCN3.

60. The method of claim 58 or 59, wherein the cell is derived from a stem cell.

61. The method of claim 60, wherein the stem cell is an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell.

62. The method of claim 61, wherein the adult stem cell is a mesenchymal stem cell.

63. The method of claim 60, wherein the stem cell is from a patient who has a bone disorder or condition associated with bone degeneration.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 64. The method of claim 59, wherein the cell is immortalized.

65. The method of claim 59, wherein the cell is from a MC3T3-E1 preosteoblast cell line, an SaOs2 osteosarcoma cell line, an MG-63 osteosarcoma cell line, a hFOB osteoblast cell line, an ASC52telo immortalized adipose derived mesenchymal stem cell line, a human telomerase reverse transcriptase-immortalized bone marrow mesenchymal stromal cell (hTERT- BMSC) line, or an ATDC5 chondrogenic mouse teratocarcinoma cell line.

66. A method of producing a bone graft, the method comprising culturing an osteochondral skeletal stem cell in presence of cellular communication network factor 3 (CCN3) under suitable conditions, wherein the osteochondral skeletal stem cell produces bone for the bone graft.

67. A bone graft produced by the method of claim 66.

68. The bone graft of claim 67, wherein the osteochondral skeletal stem cell is derived from an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell.

69. The bone graft of claim 68, wherein the adult stem cell is a mesenchymal stem cell.

70. A method of transplanting a bone graft into a subject, the method comprising transplanting the bone graft of claim 69 into a transplantation site in the subject.

71. The method of claim 70, wherein the osteochondral skeletal stem cell is autologous, allogeneic, or xenogeneic.

72. The method of claim 70 or 71, wherein the osteochondral skeletal stem cell is derived from an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell.

73. The method of claim 72, wherein the adult stem cell is a mesenchymal stem cell.

74. The method of any one of claims 70-73, wherein the bone graft replaces missing bone to repair a bone fracture at the transplantation site.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 75. The method of claim 74, wherein the bone fracture is a compound fracture.

76. The method of any one of claims 70-73, wherein the bone graft provides new bone to repair a congenital bone defect at the transplantation site.

77. The method of any one of claims 70-73, wherein the bone graft provides new bone for a craniofacial surgery, oral and maxillofacial surgery, plastic surgery, or reconstructive surgery.

78. The method of any one of claims 70-73, wherein the bone graft provides new bone for a dental implant.

79. The method of any one of claims 70-78, further comprising administering cellular communication network factor 3 (CCN3) locally at the transplantation site to stimulate new bone growth at the transplantation site.

80. A method of monitoring a lactating female subject to determine risk of bone degeneration from breast feeding, the method comprising: obtaining a biological sample from the lactating female subject; measuring a level of communication network factor 3 (CCN3) in the biological sample; and comparing the level of the CCN3 in the biological sample to reference value ranges for CCN3 from a control subject, wherein levels of CCN3 below a threshold value indicate that the subject is at risk of bone degeneration from breast feeding, and wherein levels of CCN3 equal to or greater than a threshold value indicate that the female subject may continue breast feeding without substantial risk of bone degeneration.

81. The method of claim 80, wherein the female subject discontinues breast feeding if the level of CCN3 indicates the female subject is at risk of bone degeneration from breast feeding.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 82. The method of claim 80 or 81, further comprising administering a therapeutically effective amount of the CCN3 to the female subject to lower the risk of bone degeneration from breast feeding.

83. The method of any one of claims 80-82, wherein the biological sample is blood or plasma.

84. The method of any one of claims 80-83, wherein said measuring the level of CCN3 comprises performing an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, an immunofluorescent assay, immunohistochemistry, fluorescence-activated cell sorting (FACS), a Western Blot, mass spectrometry, tandem mass spectrometry, a biochemical assay, liquid chromatography, or NMR.

85. A communication network factor 3 (CCN3) for use as a biomarker in monitoring a lactating female subject to determine risk of bone loss from breast feeding.

86. A method for regenerating mammalian cartilage in a subject, the method comprising: activating skeletal stem cells with a mechanical stimulus; and administering a therapeutically effective amount of cellular communication network factor 3 (CCN3) in combination with a therapeutically effective amount of a vascular endothelial growth factor (VEGF) inhibitor to the subject.

87. The method of claim 86, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

88. The method of claim 86 or 87, wherein the VEGF inhibitor is cabozantinib.

89. The method of any one of claims 86-88, wherein the cartilage is articular cartilage.

90. The method of any one of claims 86-89, wherein the mechanical stimulus is an acute local injury.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 91. The method of claim 90, wherein the acute local injury is a surgically performed microfracture procedure to bone tissue at a desired site for cartilage regeneration.

92. The method of claim 90 or 91, wherein the VEGF inhibitor and the CCN3 are administered locally to the acute local injury.

93. The method of any of claims 86-92, wherein the VEGF inhibitor and the CCN3 are administered to the subject immediately after said activating the skeletal stem cells with the mechanical stimulus.

94. The method of any of claims 86-92, wherein the VEGF inhibitor and the CCN3 are administered to the subject within three days after said activating the skeletal stem cells with the mechanical stimulus.

95. The method of any of claims 86-94, wherein the VEGF inhibitor and the CCN3 are encapsulated in a hydrogel.

96. The method of claim 95, wherein the hydrogel comprises alginate.

97. The method of claim 96, wherein the alginate is ionically cross-linked.

98. The method of claim 97, wherein the alginate is ionically cross-linked by divalent calcium cations.

99. The method of any one of claims 96-98, wherein the alginate concentration in the hydrogel ranges from 2 to 10 percentage by weight (wt%).

100. The method of any of claims 86-99, wherein the VEGF inhibitor and the CCN3 are administered using a drug delivery device.

101. The method of claim 100, wherein the drug delivery device provides sustained delivery of the VEGF inhibitor and the CCN3.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 102. The method of claim 100 or 101, wherein the drug delivery device is a reservoir implant or a monolithic implant.

103. The method of any of claims 100-102, wherein the drug delivery device is a non- biodegradable implant.

104. The method of any of claims 100-102, wherein the drug delivery device is a biodegradable implant.

105. The method of any of claims 100-104, wherein the drug delivery device is implanted at the site of the local acute injury.

106. The method of any of claims 86-105, wherein the subject has a cartilage disorder.

107. The method of claim 106, wherein the cartilage disorder is osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, or malignancy.

108. The method of any of claims 86-107, further comprising administering a therapeutically effective amount of skeletal stem cells to the subject.

109. The method of any of claims 86-108, wherein the CCN3 is provided by a vector comprising an expression cassette comprising a coding sequence encoding the CCN3.

110. The method of claim 109, wherein the expression cassette comprises a promoter operably linked to the coding sequence encoding the CCN3.

111. The method of claim 109 wherein the coding sequence encoding the CCN3 is integrated into a chromosomal locus in the genome of the subject.

112. The method of claim 111, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the CCN3 at the chromosomal locus.Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 113. The method of any one of claims 109-112, wherein the vector is a plasmid or viral vector.

114. The method of claim 113, wherein the viral vector is an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

115. A composition comprising cellular communication network factor 3 (CCN3) and a vascular endothelial growth factor (VEGF) inhibitor for use in a method of regenerating mammalian cartilage.

116. The composition of claim 115, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

117. The composition of claim 115 or 116, wherein the VEGF inhibitor is cabozantinib.

118. The composition of any one of claims 115-117, wherein the cartilage is articular cartilage.

119. The composition of any one of claims 115-118, wherein the CCN3 and the VEGF inhibitor are encapsulated in a hydrogel.

120. The composition of claim 119, wherein the hydrogel comprises alginate.

121. The composition of claim 120, wherein the alginate is ionically cross-linked.

122. The composition of claim 121, wherein the alginate is ionically cross-linked by divalent calcium cations.

123. The composition of any one of claims 120-122 wherein the alginate concentration in the hydrogel ranges from 2 to 10 percentage by weight (wt%).Atty. Dckt.: UCSF-749WO Client Ref.: SF-2024-018-2-PCT-0 124. A method of screening for an agonist of cellular communication network factor 3 (CCN3) that increases production of cartilage, the method comprising: activating a skeletal stem cell with a mechanical stimulus; contacting the skeletal stem cell with CCN3, a vascular endothelial growth factor (VEGF) inhibitor, and a candidate agent; and measuring cartilage production by the skeletal stem cell, wherein an increased amount of cartilage production in presence of the candidate agent compared to a reference value range for the amount of cartilage production in a control skeletal stem cell, wherein the candidate agent is absent, indicates that the candidate agent is an agonist of CCN3.

125. A method of screening for a mimic or an analogue of cellular communication network factor 3 (CCN3) that stimulates production of cartilage, the method comprising: activating a skeletal stem cell with a mechanical stimulus; contacting the skeletal stem cell with a vascular endothelial growth factor (VEGF) inhibitor and a candidate agent; and measuring cartilage production by the skeletal stem cell, wherein an increased amount of cartilage production in presence of the candidate agent compared to a reference value range for the amount of cartilage production in a control skeletal stem cell, wherein the candidate agent is absent, indicates that the candidate agent is a mimic or an analogue of the CCN3.

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