Composition of matter for promoting bone fracture healing and methods of manufacture and use thereof

Abstract

A recombinant proteoglycan 4 protein used for treating bone fracture is provided. Also provided herein is a method for treating bone fracture that includes administering to a subject a composition comprising a therapeutically effective amount of a recombinant proteoglycan 4 protein or a nucleic acid construct encoding such recombinant proteoglycan 4 protein. Further provided is a composition including a therapeutically effective amount of a recombinant proteoglycan 4 protein or a nucleic acid construct encoding such recombinant proteoglycan 4 protein.

Description

Attorney Docket No. 0073605-001029COMPOSITION OF MATTER FOR PROMOTING BONE FRACTURE HEALING AND METHODS OF MANUFACTURE AND USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application Serial No. 63 / 700,185, filed on September 27, 2024, and entitled “APPARATUS, MATERIAL ANDPROCESS TO PROMOTE BONE FRACTURE HEALING,” the entirety of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. DK121327 awarded by the United States Department of Agriculture and Grant No. R01 AR085071 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention.FIELD OF THE INVENTION

[0003] Embodiments generally relate to therapeutics and orthopedics. Some embodiments are directed to a composition of matter for promoting bone fracture healing and methods manufacture and use thereof.REFERENCE TO SEQUENCE LISTING

[0004] This specification includes a sequence listing submitted herewith, which includes the file entitled 0073605-001029.xml having the following size: 9,017 bytes which was created September 4, 2025, the contents of which are incorporated by reference herein.BACKGROUND

[0005] Surgical management of bone fractures has advanced with the incorporation of advanced techniques. However, delayed bone healing remains a clinical challenge, with the prevalence of long bone nonunion that ranges from 10 to 15% among patients. The rate of nonunion also increases with comorbidities. For example, diabetes can increase the risk of nonunion by a factor of more than seven.

[0006] Growth factors can be used to promote bone healing by stimulating cellular processes including proliferation, migration, and differentiation. To address their often-limited stability and short half-life and to maintain local delivery, growth factors are typically delivered using sophisticated biomaterial-based systems. Growth factors are typically associated with high cost and often require multiple administrations, which can lead to both financial burden and physical discomfort for patients seeking treatment. Further, the use of growth factors can potentially increaseAttorney Docket No. 0073605-001029 the risk of ectopic bone formation, potential tumor formation, as well as various side effects associated with supraphy si ologi cal dosages.

[0007] Effective and safe clinical solutions for promoting bone fracture healing and bone regeneration remain elusive.SUMMARY OF THE DISCLOSURE

[0008] The present disclosure describes a splice variant of the extracellular proteoglycan 4 protein (Prg4). This splice variant was discovered using a murine model of tibial fracture. This variant is expressed only in the callus during bone fracture healing, has not been reported or detected in other musculoskeletal tissue, and is the only variant expressed in the mouse callus. The variant is relatively short in length, lacks 3 internal exons, and plays an essential for the healing process by promoting / regulating substantially all the core steps in fracture healing. Further, the human analogue of this murine short variant has never been identified or reported in any musculoskeletal tissue, nor is it listed in any database of the human transcriptome for musculoskeletal tissues. A recombinant human Prg4 protein that mimics the murine splice variant detected in callus was accordingly engineered. The more than 90% homology between human and mouse Prg4 proteins in regions that constitute the mouse splice variant facilitated the engineering of such human analogue. The engineered human variant exhibited strong osteogenic effects, while other human variants (found in other tissues) did not show such osteogenic effects. This engineered human Prg4 recombinant protein can be used in regenerative medicine for promoting and rescuing bone healing, especially in diseases with high rates of nonunion.

[0009] The technology described in the present disclosure can be used to address current clinical challenges in nonunion and delayed healing. As a regenerative medicine, Prg4 is safer than growth factors and is expressed at high levels locally in the callus. Importantly, an eight-fold decrease in Prg4 expression level was observed in the callus of obese / diabetic mice. This decrease in Prg4 expression level provides further support for the use of Prg4 to induce healing in diabetic patients and reduce the incidence of nonunion.

[0010] An aspect of the present disclosure is a method for treating bone fracture or promoting bone growth, bone regeneration, or bone fracture healing in a subject. In some embodiments, the method can include administering to the subject a composition including a therapeutically effective amount of a recombinant proteoglycan 4 protein, the recombinant proteoglycan 4 protein having an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1. In some embodiments, the method can include administering to the subject a composition including aAttorney Docket No. 0073605-001029 therapeutically effective amount of a recombinant proteoglycan 4 protein, the recombinant proteoglycan 4 protein having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the method can include administering to the subject a composition including a therapeutically effective amount of a nucleic acid construct, the nucleic acid construct having a nucleic acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 2. In some embodiments, the method can include administering to the subject a composition including a therapeutically effective amount of a nucleic acid construct, the nucleic acid construct having a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 2.

[0011] In some embodiments, the nucleic acid construct can include a ribonucleic acid (RNA). In some embodiments, the nucleic acid construct can encode a recombinant proteoglycan 4 protein. In some embodiments, the recombinant proteoglycan 4 protein encoded by the nucleic acid construct can have an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1. In some embodiments, the recombinant proteoglycan 4 protein encoded by the nucleic acid construct can have an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.

[0012] In some embodiments, the recombinant proteoglycan 4 protein can be produced or expressed in the subject as an extracellular protein or secreted protein. In some embodiments, the recombinant proteoglycan 4 protein can be produced or expressed inside a callus of the subject.

[0013] In some embodiments, the method can include administering the composition locally, e.g., to an articular joint or a callus of the subject.

[0014] In some embodiments, the subject can be diagnosed with, or expresses symptoms of, diabetes or obesity. In some embodiments, the subject can be associated with a higher risk of nonunion.

[0015] In some embodiments, the method described herein can further include inducing or promoting osteogenic or chondrogenic differentiation of mesenchymal stem cells in the subject, e.g., using the composition, recombinant protein, or nucleic acid construct described herein.

[0016] In some embodiments, the method described herein can further include maintaining or restoring chondrocyte homeostasis or osteoblast homeostasis in the subject.

[0017] Another aspect of the present disclosure is a recombinant proteoglycan 4 protein. In some embodiments, the recombinant proteoglycan 4 protein can have an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1. In some embodiments, the recombinant proteoglycan 4 protein can have an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.Attorney Docket No. 0073605-001029

[0018] In some embodiments, the recombinant proteoglycan 4 protein described herein can be used for treating bone fracture. Accordingly, in some embodiments, the recombinant proteoglycan 4 protein described herein can be used in the manufacture of a medicament for the treatment of bone fracture.

[0019] In some embodiments, the recombinant proteoglycan 4 protein described herein can be used for promoting bone growth, bone regeneration, or bone fracture healing. Accordingly, in some embodiments, the recombinant proteoglycan 4 protein described herein can be used in the manufacture of a medicament for promoting bone growth, bone regeneration, or bone fracture healing.

[0020] In some embodiments, the recombinant proteoglycan 4 protein described herein can be used in combination with a full-length proteoglycan 4 protein or a proteoglycan 4 protein having an amino acid sequence longer than that of the recombinant proteoglycan 4 protein for treating osteoarthritis. Accordingly, in some embodiments, the recombinant proteoglycan 4 protein described herein can be used in the manufacture of a medicament for the treatment of osteoarthritis.

[0021] In some embodiments, the recombinant proteoglycan 4 protein can be used in combination with a stem cell therapy for treating bone fracture. Accordingly, in some embodiments, the recombinant proteoglycan 4 protein described herein can be used in combination with a stem cell therapy in the manufacture of a medicament for the treatment of bone fracture. Further, in come embodiments, the recombinant proteoglycan 4 protein described herein can be used in combination with a stem cell therapy in the manufacture of a medicament for promoting bone growth, bone regeneration, or bone fracture healing.

[0022] In some embodiments, the recombinant proteoglycan 4 protein can be used for treating cartilage degeneration, as well as diseases associated with bone loss.

[0023] In some embodiments, the recombinant proteoglycan 4 protein or the short Prg4 isoform described herein can be used for treating conditions where longer Prg4 isoforms are typically applied, including without limitation wound healing, skin repair, and / or ophthalmological diseases (e g., dry eye syndrome).

[0024] Another aspect of the present disclosure is a composition for treatment of bone fracture or promoting bone growth, bone regeneration, or bone fracture healing. In some embodiments, the composition can include a therapeutically effective amount of a recombinant proteoglycan 4 protein having an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1 and a delivery vehicle, wherein the therapeutically effective amount of the recombinant proteoglycan 4Attorney Docket No. 0073605-001029 protein is stored within a matrix or cavity of the delivery vehicle. In some embodiments, the composition can include a delivery vehicle and a therapeutically effective amount of a recombinant proteoglycan 4 protein having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, at least 90% identical to SEQ ID NO: 1, at least 95% identical to SEQ ID NO: 1, at least 99% identical to SEQ ID NO: 1, or identical to SEQ ID NO: 1, wherein the therapeutically effective amount of the recombinant proteoglycan 4 is stored within a matrix or cavity of the delivery vehicle.

[0025] In some embodiments, the therapeutically effective amount of the recombinant proteoglycan 4 protein can be released from the delivery vehicle over a period of at least seven days and no greater than six months.

[0026] In some embodiments, the composition can include a therapeutically effective amount of a nucleic acid construct having a nucleic acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 2 and a delivery vehicle, wherein the therapeutically effective amount of the nucleic acid construct is stored within a matrix or cavity of the delivery vehicle.

[0027] In some embodiments, the composition can include a delivery vehicle and a therapeutically effective amount of a nucleic acid construct having a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 2, at least 90% identical to SEQ ID NO: 2, at least 95% identical to SEQ ID NO: 2, at least 99% identical to SEQ ID NO: 2, or identical to SEQ ID NO: 2 , wherein the therapeutically effective amount of the nucleic acid construct is stored within a matrix or cavity of the delivery vehicle.

[0028] In some embodiments, the composition described herein can be used in the treatment of bone fracture. Accordingly, in some embodiments, the composition described herein can be used in the manufacture of a medicament for the treatment of bone fracture.

[0029] In some embodiments, the composition can be used in promoting bone growth, bone regeneration, or bone fracture healing. Accordingly, in some embodiments, the composition described herein can be used in the manufacture of a medicament for promoting bone growth, bone regeneration, or bone fracture healing.

[0030] In some embodiments, the composition described herein can further include a therapeutically effective amount of a full-length proteoglycan 4 protein or therapeutically effective amount of a proteoglycan 4 protein having an amino acid sequence longer than that of the recombinant proteoglycan 4 protein. In some embodiments, the composition described herein can further include a therapeutically effective amount of a nucleic acid encoding the full-length proteoglycan 4 protein or therapeutically effective amount of a nucleic acid encoding a proteoglycanAttorney Docket No. 0073605-0010294 protein having an amino acid sequence longer than that of the recombinant proteoglycan 4 protein. Accordingly, in some embodiments, such composition can be used in the treatment of osteoarthritis or in the manufacture of a medicament for the treatment of osteoarthritis.

[0031] In some embodiments, the composition described herein can be used in combination with a stem cell therapy (e.g., as an adjuvant) for treating bone fracture or promoting bone growth, bone regeneration, or bone fracture healing. In some embodiments, the composition described herein can be used in combination with a stem cell therapy in the manufacture of a medicament for the treatment of bone fracture or for promoting bone growth, bone regeneration, or bone fracture healing.

[0032] In some embodiments, aspects of the present disclosure can be used, e.g., as therapeutics, to promote bone healing. In some embodiments, aspects of the present disclosure can be used to treat jaw and / or dental diseases or defects. In some embodiments, aspects of the present disclosure can be used in periodontology.

[0033] These and other aspects and features of nonlimiting embodiments of the present invention will become apparent to those skilled in the art upon review of the following description of specific nonlimiting embodiments of the invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] For the purpose of illustrating the invention, the drawings show aspects of one or more embodiments of the invention. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities shown in the drawings.

[0035] FIG. 1 depicts an exemplary schematic showing various phases of fracture healing. The inflammatory phase is initiated as a result of blood vessel rupture at the fracture area and develops into a fracture hematoma rich in inflammatory cells, inflammatory cytokines, and angiogenic factors. Within the fracture hematoma, fibrin-rich granulation tissue begins to develop. Mesenchymal progenitor cells recruited at the fracture area undergo chondrogenic differentiation leading to the replacement of granulation by a collagen-rich fibrocartilaginous callus. As the fracture healing progresses, the fibrocartilaginous callus is invaded by endothelial cells leading to neovascularization at the fracture area. During this stage of fracture repair, the fibrocartilaginous callus begins to undergo endochondral ossification resulting in a callus that bridges bone fragments. This step initiates fibrocartilaginous callus resorption and calcification. Neovascularization within the callus initiates another round of osteoprogenitor cell recruitment to initiate osteogenesis and the deposition of a hard calcified callus of immature bone. In the later stage of the fracture repair, with continuousAttorney Docket No. 0073605-001029 migration of the osteoblasts and osteoclasts, the hard callus undergoes coupled remodeling and finally replaced by mature compact bone.

[0036] FIG. 2A depicts a schematic of a proteoglycan.

[0037] FIG. 2B depicts a schematic showing the structure of proteoglycan and its role within a cartilage matrix.

[0038] FIG. 2C depicts a schematic showing the structure of proteoglycan 4 (Prg4).

[0039] FIG. 3A depicts an exemplary timeline of the healing course showing the healing phases and harvest timepoints used for bulk RNA-seq.

[0040] FIG. 3B depicts an exemplary analysis of RNA-seq data showing levels of Prg4 expression at the indicated timepoints (N = 3).

[0041] FIG. 3C depicts exemplary cell clusters identified using scRNA-seq on d5 and dlO combined. Three mice were combined each day, and 22,854 cells were sequenced (2207 median gene # per cell).

[0042] FIG. 3D depicts exemplary scRNA-sew violin plots showing Prg4 expression in the 32 cell clusters shown in FIG. 3C.

[0043] FIG. 3E depicts exemplary scRNA-seq violin plots showing the expression of the specified genes in the indicated clusters.

[0044] FIG. 3F depicts exemplary data showing the three stem cells / progenitors and stem-celllike clusters identified on days 5 and 10, respectively.

[0045] FIGS. 3G-H depict additional exemplary scRNA-seq violin plots showing the expression of the specified genes in the indicated clusters.

[0046] FIG. 4A depicts exemplary results based on an RT-qPCR analysis of Prg4 mRNA in PSC vs. other cells following sorting of callus cells using FACS. The level of Prg4 mRNA is normalized to that of P-actin mRNA, and the normalized level in PSC is defined as 100%. N = 4. (***) P < 0.001 using unpaired Student’s t-test. Bar graphs represent average ± SEM.

[0047] FIG. 4B depicts similar exemplary results shown in FIG. 4A except that Prg4 mRNA level was measured in the indicated tissues, and the normalized level in BM is defined as 1. Pdgfra+Scal+populations were sorted. Prg4 expression is substantially higher in the callus PSCs than in BMSCs or contralateral bone PSCs. These data suggest that Prg4 expression is highly induced, and its splicing is reprogrammed in the callus PSCs. (****) p < 0.0001 using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Bar graphs represent average ± SEM.Attorney Docket No. 0073605-001029

[0048] FIGS. 4C-D depict exemplary exon-intron structure of human Prg4 pre-mRNA (FIG. 4C) and mouse Prg4 pre-mRNA (FIG. 4D). Solid boxes indicate exons. Different splice variants are shown.

[0049] FIG. 4E depicts exemplary results based on an analysis of Prg4 splice variants using bulk RNA-seq data and rMAT (Top). The exon-intron structure of murine Prg4-L and Prg4-S variants are shown for comparison (Middle). Analysis of splicing around exon 7 (bottom) shows prevalence of events where exon-7 is skipped.

[0050] FIG. 4F depicts an illustration of the locations where Prg4 primer pairs 1 and 2 anneal (top) and a comparison between Prg4-1 and Prg4-S splice variants (bottom).

[0051] FIG. 4G depicts exemplary results based on PCR of callus RNA using Prg4 primer pair 1 (left) or 2 (right). The expected size of Prg4-S and Prg4-L PCR products are shown.

[0052] FIG. 4H depicts exemplary results as shown in FIG. 4G except that PCR was performed using RNA isolated from PSC that were sorted from d5 callus or intact bone. Primer pair 1 was used. These results suggest that Prg4 splicing is reprogramed when PSCs are mobilized from the periosteum to the callus to favor the production of Prg4-S. All gel images are representative of N = 3.

[0053] FIG. 5 A depicts an exemplary analysis of the sequence of Prg4-L protein, predicting the N-terminal 24 amino acids as a signal peptide.

[0054] FIG. 5B depicts an alignment of the N-terminal 24 amino acids in murine Prg4-L and Prg4-S splice variants. Specifically, the sequence “MGWKILPVCLSLLLPVVLIQQVSS” (SEQ ID NO: 5) is conserved. The same sequence may be preserved in the recombinant Prg4 protein described herein.

[0055] FIG. 5C depicts exemplary Western Blots (WB) of Prg4 and P-actin performed on primary cultures of PSC isolated from d5 callus. WB was performed on the cells as well as the culture medium. The blot is representative of N = 3. The left panel depicts WB of cultured primary PSCs (cells and medium), showing that PRG4-S is a secreted protein. The right panel depicts WB of PSCs (medium) after treatment using Ctrl or Prg4-S siRNA. N = 4.

[0056] FIG. 5D depicts exemplary data based on RT-qPCR of Prg4 mRNA using RNA isolated from d5 callus of mice treated with control (Ctrl) or Prg4-S siRNA. The level of Prg4 mRNA was normalized to that of P-actin mRNA, and the normalized level in Ctrl-treated mice is defined as 100%. N = 4. Bar graphs represent average, and error bars represent ± SEM. (****) p < 0.001 using unpaired Student’s t-test.Attorney Docket No. 0073605-001029

[0057] FIG. 5E depicts an exemplary fluorescence image of a mouse recovering from a fracture. A hydrogel-nanoparticles-siRNA complex was wrapped around the fracture site to locally deliver siRNA to the callus. SiRNA was labelled using an Alexa Fluor 647 Dye (AF-647).

[0058] FIG. 5F depicts an exemplary immunofluorescence staining (IF) image of dlO callus in Prg4-S KD mice. Soft callus (green), newly formed woven bone (red), and DAPI (4’,6-diamidino-2- phenylindole; blue) are shown respectively.

[0059] FIG. 5G depicts exemplary images based on IF staining of Prg4 (magenta) in dlO callus of mice treated with Ctrl or Prg4-S siRNA. DAPI (blue) stains nuclei. The images are taken in the soft callus area.

[0060] FIG. 5H depicts a quantitation of Prg4 staining shown in FIG. 5G. The area of Prg4 was normalized to the callus area, and the normalized area in Ctrl siRNA-treated mice is defined as 1. N = 7. Bar graphs represent average, and error bars represent ± SEM. (***) P < 0.001 using unpaired Student’s t-test.

[0061] FIG. 51 depicts a representative dot plot of the FC analysis of PSC (i .e., PDGFRoc+SCAl+CD45’CD31' cells) in d5 callus of Ctrl or Prg4-S siRNA-treated mice.

[0062] FIG. 5J depicts an exemplary analysis of flow cytometry (FC) data shown in FIG. 51. The number of PSC was normalized to the total number of singlets in the callus, and the normalized ratio in Ctrl siRNA-treated mice is defined as 1. These results demonstrate the autocrine effects of Prg4-S on PSC proliferation or mobility. N = 3 (5 mice combined in each replicate). Bar graphs represent average, and error bars represent ± SEM. (****) p < 0.001 using unpaired Student’s t-test.

[0063] FIG. 6A depicts an exemplary FC dot plot of total immune (CD45+) cells in d5 callus of Ctrl or Prg4-S siRNA treated mice.

[0064] FIG. 6B depicts an analysis of the FC dot plot in FIG. 6A. The number of CD45+immune cells was normalized to the total number of singlets and presented as %. The scatter plots represent mean ± SEM. N = 3 (5 mice combined in each replicate). (****) P < 0.0001 using unpaired Student’s t-test.

[0065] FIG. 6C depicts an exemplary FC dot plot of myeloid (CD45+CD1 lb+) cells in d5 callus of Ctrl or Prg4-S siRNA treated mice.

[0066] FIG. 6D depicts an analysis of the FC dot plot in FIG. 6C. The number ofCD45+CD1 lb+immune cells was normalized to the total number of singlets and presented as %. The scatter plots represent mean ± SEM. N = 3 (5 mice combined in each replicate). (****) P < 0.0001 using unpaired Student’s t-test.Attorney Docket No. 0073605-001029

[0067] FIG. 6E depicts an exemplary FC dot plot of CD45+CD1 lb+CD11c’ cells in d5 callus of Ctrl or Prg4-S siRNA treated mice.

[0068] FIG. 6F depicts an analysis of the FC dot plot in FIG. 6C. The number of CD45+CD1 lb+CD1 lc immune cells was normalized to the total number of singlets and presented as %. The scatter plots represent mean ± SEM. N = 3 (5 mice combined in each replicate). (****) p < 0.0001 using unpaired Student’s t-test.

[0069] FIG. 7A depicts an exemplary FC dot plot of total macrophages, classical Ly6CH1, and non-classical Ly6CLomonocytes in d5 callus of Ctrl or Prg4-S siRNA treated mice.

[0070] FIG. 7B depicts an analysis of the FC dot plot in FIG. 7A. The number of cells within each population was normalized to the total number of singlets and presented as %. The scatter plots represent mean ± SEM. N = 3 (5 mice combined in each replicate). (**) P < 0.01, (***) P < 0.001, and (****) p < 0.0001 using unpaired Student’s t-test.

[0071] FIG. 7C depicts an exemplary FC dot plot of eosinophils in d5 callus of Ctrl or Prg4-S siRNA treated mice.

[0072] FIG. 7D depicts an analysis of the FC dot plot in FIG. 7C. The number of cells within each population was normalized to the total number of singlets and presented as %. The scatter plots represent mean ± SEM. N = 3 (5 mice combined in each replicate). (**) P < 0.01 using unpaired Student’s t-test.

[0073] FIG. 7E depicts an exemplary FC dot plot of cDC2 and Mo-DC in d5 callus of Ctrl or Prg4-S siRNA treated mice.

[0074] FIG. 7F depicts an analysis of the FC dot plot in FIG. 7E. The number of cells within each population was normalized to the total number of singlets and presented as %. The scatter plots represent mean ± SEM. N = 3 (5 mice combined in each replicate). (*) P < 0.05 and (**) P < 0.01 using unpaired Student’s t-test.

[0075] FIG. 8A depicts an exemplary FC dot plot of CD8+and CD4+T cells in d5 callus of Ctrl or Prg4-S siRNA treated mice.

[0076] FIG. 8B depicts an analysis of the FC dot plot in FIG. 8A. The number of cells within each population was normalized to the total number of singlets and presented as %. The scatter plots represent mean ± SEM. N = 3 (5 mice combined in each replicate). (****) P < 0.0001 using unpaired Student’s t-test.

[0077] FIG. 8C depicts an exemplary FC dot plot of total B cells in d5 callus of Ctrl or Prg4-S siRNA treated mice.Attorney Docket No. 0073605-001029

[0078] FIG. 8D depicts an analysis of the FC dot plot in FIG. 8C. The number of cells within each population was normalized to the total number of singlets and presented as %. The scatter plots represent mean ± SEM. N = 3 (5 mice combined in each replicate). (**) P < 0.01 using unpaired Student’s t-test.

[0079] FIG. 8E depicts an exemplary FC dot plot of mature (B22H1) and immature (B2201°) B cells in d5 callus of Ctrl or Prg4-S siRNA treated mice.

[0080] FIG. 8F depicts an analysis of the FC dot plot in FIG. 8E. The number of cells within each population was normalized to the total number of singlets and presented as %. The scatter plots represent mean ± SEM. N = 3 (5 mice combined in each replicate). (**) P < 0.01 using unpaired Student’s t-test.

[0081] FIG. 9A (left) depicts exemplary images of IF co-staining of Col II (green) and Col I (red) in the soft callus (SC) of Ctrl or Prg4-S siRNA treated mice on dlO post-fracture. Col II marks soft-callus chondrocytes, and Col I is secreted in the soft callus by mineralizing, hypertrophic chondrocytes. DAPI (blue) stains nuclei. The scale bar = 50 pm. FIG. 9A (right) depicts a quantitation of the area of the soft callus. The area of the soft callus in the Ctrl siRNA-treated mice is defined as 1. IF images are representative of 7 mice. Scatter plots are presented as mean ± SEM.(***) P < 0.001 using unpaired Student’s t-test.

[0082] FIG. 9B (left) depicts exemplary images of IF staining of IHH (magenta) in the SC of Ctrl or Prg4-S siRNA treated mice on dlO post-fracture. DAPI (blue) stains nuclei. The scale bar = 20 pm. FIG. 9B (right) depicts a quantitation of the IHH staining. The number of IHH+chondrocytes was normalized to the total number of chondrocytes (defined by DAPI) in the SC shown in FIG. 9A, and the ratio is presented as %. IF images are representative of 7 mice. Scatter plots are presented as mean ± SEM. (*) P < 0.05 using unpaired Student’s t-test.

[0083] FIG. 9C (left) depicts exemplary images of IF staining of MMP13 (magenta) in the SC of Ctrl or Prg4-S siRNA treated mice on dlO post-fracture. DAPI (blue) stains nuclei. The scale bar = 50 pm. FIG. 9C (right) depicts a quantitation of the MMP13 staining. The area of MMP13 staining was normalized to that of the soft callus shown in FIG. 9A, and the normalized area in Ctrl siRNA- treated mice is defined as 1. IF images are representative of 7 mice. Scatter plots are presented as mean ± SEM. (****) P < 0.0001, using unpaired Student’s t-test.

[0084] FIG. 9D (left) depicts exemplary images of IF staining of Col X in the SC of Ctrl or Prg4-S siRNA treated mice on dlO post-fracture. DAPI (blue) stains nuclei. The scale bar = 50 pm. FIG. 9D (right) depicts a quantitation of the Col X staining. The area of Col X staining wasAttorney Docket No. 0073605-001029 normalized to that of the soft callus shown in FIG. 9 A, and the normalized area in Ctrl siRNA- treated mice is defined as 1. IF images are representative of 7 mice. Scatter plots are presented as mean ± SEM. (**) P < 0.01 using unpaired Student’s t-test.

[0085] FIG. 9E (left) depicts exemplary images of IF staining of BrdU (magenta) in the SC of Ctrl or Prg4-S siRNA treated mice on dlO post-fracture. DAPI (blue) stains nuclei. The scale bar = 20 pm. FIG. 9E (right) depicts a quantitation of the BrdU staining. The number of BrdU chondrocytes was normalized to the total number of chondrocytes (defined by DAPI) in the SC shown in FIG. 9A, and the ratio is presented as %. BrdU was injected 24 h prior to harvest, and BrdU uptake was measured as a marker for proliferation. IF images are representative of 7 mice. Scatter plots are presented as mean ± SEM. (*) P < 0.05 using unpaired Student’s t-test.

[0086] FIG. 9F (left) depicts exemplary images of IF staining of apoptotic cells (magenta) in the SC of Ctrl or Prg4-S siRNA treated mice on dlO post-fracture, using a TUNEL staining assay. The white arrows point to TUNEL+cells. DAPI (blue) stains nuclei. The scale bar = 20 pm. FIG. 9F (right) depicts a quantitation of the TUNEL+cells. The number of TUNEL+cells was normalized to the total number of chondrocytes (defined by DAPI) in the SC, as shown in FIG. 9A, and the ratio is presented as %. IF images are representative of 7 mice. Scatter plots are presented as mean ± SEM. (**) P < 0.01 using unpaired Student’ s t-test.

[0087] FIG. 10A depicts exemplary pCT images of dlO callus harvested from Ctrl or Prg4-S siRNA-treated mice.

[0088] FIG. 10B depicts an analysis the images shown in FIG. 10A.

[0089] FIG. 10C (left) depicts exemplary images of IF staining of Brdu (magenta) as an indicator of proliferation, in the dlO callus of Ctrl or Prg4-S siRNA-treated mice. BrdU was injected 24 h prior to harvest. The staining was performed in the woven bone area. The scale bar = 20 pm. FIG. 10C (right) depicts a quantitation of BrdU staining. The number of BrdU+cells was normalized to the total number of cells in the woven bone area (defined by DAPI), and the ratio is presented as %. IF images are representative of 7 mice. Scatter plots are presented as mean ± SEM. Statistical analysis was performed using unpaired Student’s t-test.

[0090] FIG. 10D (left) depicts exemplary images of IF staining of apoptotic cells (magenta) in the SC of Ctrl or Prg4-S siRNA treated mice on dlO post-fracture, using a TUNEL staining assay. The staining was performed in the woven bone area. The scale bar = 20 pm. FIG. 10D (right) depicts a quantification of TUNEL+cells. IF images are representative of 7 mice. Scatter plots are presented as mean ± SEM. Statistical analysis was performed using unpaired Student’s t-test.Attorney Docket No. 0073605-001029

[0091] FIG. 11A depicts exemplary results based on PSC (isolated from d5 callus) subjected to osteogenic differentiation for 2 weeks in the absence (Ctrl) or presence of recombinant murine PRG4-S protein. Alizarin red staining (top) and quantitation (bottom) were performed to measure the efficiency of osteogenic differentiation and mineralization. N = 3. Bar graphs represent mean ± SEM. P < 0.0001 using unpaired Student’s t-test.

[0092] FIG. 1 IB depicts exemplary results based on PSC (isolated from d5 callus) subjected to chondrogenic differentiation for 2 weeks in the absence (Ctrl) or presence of recombinant murine PRG4-S protein. Alcian blue staining (top) and quantitation (bottom) were performed to measure the efficiency of osteogenic differentiation and mineralization. N = 3. Bar graphs represent mean ± SEM. (***) P < 0.001 using unpaired Student’s t-test.

[0093] FIG. 11C depicts a schematic for exon composition (black boxes) for full-length hPrg4 and the engineered hPrg4-S mRNA.

[0094] FIG. 1 ID depicts an alignment of murine PRG4-S vs. the engineered hPRG4-S.

[0095] FIG. 1 IE and hit matrix dot plot based on the alignment of PRG4-S vs. the engineered hPRG4-S shown in FIG. 1 ID.

[0096] FIG. 1 IF depicts exemplary results based on hBMSC (isolated from d5 callus) subjected to differentiation for 3 weeks in the absence (Ctrl) or presence of recombinant hPRG4-S protein. Alizarin red staining (top) and quantitation (bottom) were performed to measure the efficiency of osteogenic differentiation and mineralization. N = 3. Bar graphs represent mean ± SEM. (****) P < 0.0001 using unpaired Student’s t-test.

[0097] FIG. 11G depicts exemplary results based on hBMSC (isolated from d5 callus) subjected to chondrogenic differentiation for 3 weeks in the absence (Ctrl) or presence of recombinant hPRG4- S protein. Alcian blue staining (top) and quantitation (bottom) were performed to measure the efficiency of chondrogenic differentiation and mineralization. N = 3. Bar graphs represent mean ± SEM. (*) < 0.05 using unpaired Student’s t-test.

[0098] FIGS. 12A-B depict cell clusters identified using scRNA-seq on d5 and dlO, respectively, in accordance with FIG. 3C. Three mice were combined each day, and 22,854 cells were sequenced (2207 median gene # per cell).

[0099] FIG. 12C depicts cell clusters identified using scRNA-seq, in accordance with FIG. 3C.

[0100] FIG. 12D depicts a portion of FIG. 12C, showing that Prg4 is exclusively expressed inPostn+stem cells (PSC).Attorney Docket No. 0073605-001029

[0101] FIGS. 12E-G depict additional scRNA-seq violin plots showing the expression of the specified genes in the indicated clusters.

[0102] FIG. 12H depicts an exemplary image of IF staining of chondrocytes in the SC of mice on dlO post -fracture.

[0103] FIG. 13 depicts an exemplary structure of a full-length Prg4.

[0104] FIG. 14 depicts an exemplary sequence alignment that confirms the identity of murinePrg4-S (SEQ ID NOs: 3-4).

[0105] FIG. 15 depicts exemplary RT-PCR data based on total-callus RNA and primer pair 1, showing Prg4-S bands on d7 and dlO post fracture.

[0106] FIG. 16A depicts an exemplary FC dot plot of cDCl and pDC cells in callus of Ctrl or Prg4-S siRNA treated mice.

[0107] FIG. 16B depicts an analysis of the FC dot plot in FIG. 16A. The number of cells within each population was normalized to the total number of singlets and presented as %. (*) P < 0.05 using unpaired Student’s t-test.

[0108] FIGS. 17A-B depict exemplary microscopy images for mice treated with Ctrl and Prg4-S siRNA, respectively.

[0109] FIG. 18A depicts a schematic for exon composition (black boxes) for murine mRNAs that encode Prg4-S and Prg4-L, a human mRNA that encodes full-length human hPRG4, and an engineered human mRNA that encodes hPRG4-S, respectively.

[0110] FIG. 18B depicts an exemplary sequence alignment that confirms the identity of murine Prg4 isoform 2.[OHl] FIG. 19A depicts a schematic illustrating alternative splicing of Prg4, showing a variety of splicing isoforms (and protein motifs) of Prg4 in articular cartilage. The 13 exons “boxes,” connecting introns “lines,” and the 12 coding exons “CDS” are shown. Reported splice variants differ in the exons upstream to exon 7. Exon 7 (-63% of total mRNA length) encodes the mucin-like domain.

[0112] FIG. 19B depicts two splicing isoforms of murine mRNA that encodes two types of murine Prg4, i.e., the pre-mRNA of Prg4-L (NM_021400) and Prg4-S (NM_001110146),

[0113] FIG. 20 depicts exemplary scRNA-seq violin plots showing the expression of various genes in cluster 2 (c2) vs other clusters.

[0114] FIG. 21A depicts an exemplary image showing IF staining of tdTomato (red) in intact bone. DAPI in blue. N = 7. Scale bar = 20 pm. The pool of Pdgfra+cells contain osteochondralAttorney Docket No. 0073605-001029 progenitors that produce the majority of chondrocytes and newly formed bone during fracture healing.

[0115] FIG. 2 IB depicts an exemplary image showing IF staining of tdTomato (red) in d21 callus. DAPI in blue. N=7. Scale bar = 100 pm. The pool of Pdgfra+cells contain osteochondral progenitors that produce the majority of chondrocytes and newly formed bone during fracture healing.

[0116] FIG. 21 C depicts an exemplary image showing IF staining of PRG4 (red) in the soft callus. DAPI in blue. N = 7.

[0117] FIG. 21D depicts an exemplary image showing IF staining of PRG4 (red) in the hard callus. DAPI in blue. N = 7.

[0118] FIG. 22A depicts exemplary results from an analysis of RNA-seq and scRNA-seq data for alternative splicing.

[0119] FIG. 22B depicts a detailed comparison between Prg4-L and Prg4-S. Exons 5, 6, and 7 are spliced out. The prevalent splicing events are consistent with Prg4-S. Prg4-S retains the N- and C-termini domains but lacks the mucin-like domain (with an almost complete loss of glycosylation sites). Therefore, Prg4-S cannot function as a structural lubricant. As a result, Prg4-S is expected to have a reduced interaction with the extracellular matrix and have a larger diffusion coefficient and higher availability in the extracellular space. These features contribute to the role of Prg4-S in cell signaling and its autocrine and paracrine effects.

[0120] FIG. 22C depicts an experimental workflow and exemplary RT-PCR results based on the RNA of isolated callus PSCs.

[0121] FIG. 23 depicts exemplary qPCR results of osteogenic markers following 2 weeks of osteogenic differentiation of hBMSCs in the presence of the indicated recombinant proteins. Control samples (Ctrl) were treated with vehicle. N = 5.

[0122] FIG. 24 depicts exemplary results from qPCR of Prg4 mRNA in day-5 callus. N = 5.

[0123] FIG. 25A depicts exemplary results from RT-qPCR of total Prg4 mRNA in PSCs transduced with Ctrl or PRG4-S lentiviral shRNA.

[0124] FIG. 25B depicts exemplary results from RT-qPCR of the indicated chondrocyte markers in PSCs transduced with Ctrl or PRG4-S short hairpin RNA (shRNA) and subjected to chondrogenic differentiation for 7 days. N = 4. Bar graphs represent mean ± SEM. (****) < 0.0001 using unpaired Student’s t-test.Attorney Docket No. 0073605-001029

[0125] FIG. 26 depicts exemplary images showing tdTomato in the uncal cified articular cartilage of Acan-CreERT:tdTomato mice. Signal was detected directly in OCT samples (middle; red) or by IF staining of FFPE samples using anti-RFP antibody (right; pink). No basal, non-specific Cre activity was detected (left).

[0126] FIG. 27A depicts an exemplary scRNA-seq violin plot showing the expression of the Tgfbr2 gene in the C2 cluster.

[0127] FIG. 27B depicts exemplary results of qPCR of total Prg4 mRNA in PSCs in the presence or absence (Ctrl) of TGFB1.

[0128] FIG. 27C depicts exemplary results of qPCR of total Prg4 mRNA in PSCs in the presence or absence (Ctrl) of TGFB1. TGFB1 can function as a potential regulator of Prg4 expression and splicing in callus PSCs and can induce the expression of Prg4 and reprogram its splicing in PSCs.

[0129] FIG. 28 A depicts examples of transcription factors (TFs) with binding sites in Prg4 promoter. The arrow points to SMAD3.

[0130] FIG. 28B depicts exemplary data based on qPCR of total Prg4 mRNA in PSCs treated with TGFB1 and either Ctrl or SMAD3 shRNA. N = 4. Bar graphs represent mean ± SEM. (**) P < 0.01 using unpaired Student’s t-test.

[0131] FIG. 28C depicts an exemplary scRNA-seq violin plot of Egfir expression in cluster 2 (C2) vs other clusters.

[0132] FIG. 29A depicts a map of predicted Exonic Splicing Silencer (ESS) and Exonic Splicing Enhancer (ESE) in Prg4 exon 7.

[0133] FIG. 29B depicts an exemplary scRNA-seq violin plot of Pcbpl expression in cluster 2 (C2).

[0134] FIG. 29C depicts a chemical structure of the bifunctional crosslinker DTSSP.

[0135] FIG. 29D depicts a WB of Prg4, CD44, and 0-actin in PRG4, and control IgG IP. Only the PRG4-S band was detected in the WB of Prg4.

[0136] The drawings are not necessarily to scale and may be illustrated by phantom lines, diagrammatic representations, and fragmentary views. In certain instances, details that are not necessary for an understanding of the embodiments or that render other details difficult to perceive may have been omitted.Attorney Docket No. 0073605-001029DETAILED DESCRIPTION

[0137] To facilitate the understanding of this invention, a number of terms are defined below and throughout the disclosure. Unless otherwise defined, 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. In case of conflict, the present specification, including definitions, will control. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0138] It is to be understood that any aspect and / or element of any embodiment of the method(s) described herein or otherwise may be combined in any way to form additional embodiments of the method(s), all of which are within the scope of the method(s).

[0139] Where a process is described herein, those of ordinary skill in the art will appreciate that the process may operate without any user intervention. In another embodiment, the process includes some human intervention (e.g., a step is performed by or with the assistance of a human).

[0140] As used herein, including the claims, the phrase “at least some” means “one or more” and includes the case of only one. Thus, e.g., the phrase “at least some ABCs” means “one or more ABCs” and includes the case of only one ABC.

[0141] As used herein, including the claims, the term “at least one” should be understood as meaning “one or more” and therefore includes both embodiments that include one or multiple components. Furthermore, dependent claims that refer to independent claims that describe features with “at least one” have the same meaning, both when the feature is referred to as “the” and “the at least one”.

[0142] As used herein, the term “portion” means some or all. Therefore, for example, “a portion of X” may include some of “X” or all of “X”. In the context of a conversation, the term “portion” means some or all of the conversation.

[0143] As used herein, including the claims, the phrase “using” means “using at least” and is not exclusive. Thus, e.g., the phrase “using X” means “using at least X”. Unless specifically stated by use of the word “only,” the phrase “using X” does not mean “using only X”.

[0144] As used herein, including the claims, the phrase “based on” means “based in part on” or “based, at least in part, on” and is not exclusive. Thus, e g., the phrase “based on factor X” meansAttorney Docket No. 0073605-001029“based in part on factor X” or “based, at least in part, on factor X”. Unless specifically stated by use of the word “only,” the phrase “based on X” does not mean “based only on X”.

[0145] As used herein, including the claims, the phrase “distinct” means “at least partially distinct”. Unless specifically stated, distinct does not mean fully distinct. Thus, e.g., the phrase “X is distinct from Y” means that “X is at least partially distinct from Y” and does not mean that “X is fully distinct from Y”. Thus, as used herein, including the claims, the phrase “X is distinct from Y” means that X differs from Y in at least some way.

[0146] It should be appreciated that the words “first,” “second,” and so on, in the description and claims, are used to distinguish or identify, and not to show a serial or numerical limitation.

[0147] Similarly, letter labels (e.g., “(A),” “(B),” “(C),” and so on, or “(a),” “(b),” and so on) and / or numbers (e.g., “(i),” “(ii),” and so on) are used to assist in readability and to help distinguish or identify, and are not intended to be otherwise limiting or to impose or imply, any serial or numerical limitations or orderings. Similarly, words such as “particular,” “specific,” “certain,” and “given,” in the description and claims, if used, are to distinguish or identify, and are not intended to be otherwise limiting.

[0148] As used herein, including the claims, the terms “multiple” and “plurality” mean “two or more,” and include the case of “two”. Thus, e.g., the phrase “multiple ABCs” means “two or more ABCs” and includes “two ABCs”. Similarly, e.g., the phrase “multiple PQRs” means “two or more PQRs” and includes “two PQRs”.

[0149] The present invention also covers the exact terms, features, values, and ranges, etc., in case these terms, features, values, and ranges, etc., are used in conjunction with terms such as “about,” “around,” “generally,” “substantially,” “essentially,” “at least,” etc. Thus, e.g., “about 3” or “approximately 3” shall also cover exactly 3, and “substantially constant” shall also cover exactly constant.

[0150] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.Attorney Docket No. 0073605-001029

[0151] As used herein, unless stated otherwise, the terms “about” or “approximately” refer to a value that is within 5% above or below the value being described.

[0152] As used herein, including the claims, singular forms of terms are to be construed as also including the plural form and vice versa, unless the context indicates otherwise. Thus, it should be noted that as used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. In other words, terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration.

[0153] Throughout the description and claims, the terms “comprise,” “including,” “having,” “contain,” and their variations should be understood as meaning “including but not limited to” and are not intended to exclude other components unless specifically so stated.

[0154] As used herein, a “subject” includes, but is not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals. The terms “subject” and “patient” may be used interchangeably throughout this disclosure. As a nonlimiting example, a subject or patient may include a human of any age (infant / toddler, child, teenager, young adult, adult, middle age, senior, etc.), sex, gender, race, ethnicity, health record (obesity, diabetes, etc.) , among others, as deemed relevant and / or suitable by a person of ordinary skill in the art, upon reviewing the entirety of this disclosure. The term subject may refer to any individual in need of treatment. While the present invention describes compositions and methods for treatment of human subjects in need thereof, the present invention is not limited to human subjects and the term subject generally includes mammals and birds, such as, but not limited to, non-human primates, cats, dogs, cows, horses, rodents, pigs, sheep, goats, and poultry.

[0155] As used herein, the terms “administration” or “administering” refer to a method of giving a dosage of a compound or pharmaceutical composition to a subject. A composition described herein may be administered to a subject by a single manner of a variety of manners or by a combination of a variety of manners. As a nonlimiting example, a composition may be administered by injection, by surgical implantation, and / or the like.

[0156] As used herein, an “effective amount” or “therapeutically effective amount” is the amount of a composition of this disclosure which, when administered to a subject, is sufficient to effect treatment of a disease or condition in the subject. The amount of a composition of this disclosure which constitutes a “therapeutically effective amount” may vary depending on theAttorney Docket No. 0073605-001029 composition, the condition and its severity, the manner of administration, and the age of the subject to be treated.

[0157] As used herein, the terms “treat,” “treating,” or “treatment” refer to administration of a compound or pharmaceutical composition for a therapeutic purpose. To “treat a disorder” or use for “therapeutic treatment” refers to administering treatment to a patient already suffering from a disease to ameliorate the disease or one or more symptoms thereof to improve the patient’s condition (e.g., by reducing one or more symptoms of a bone fracture). The term “therapeutic” includes the effect of mitigating deleterious clinical effects of certain processes (i.e., consequences of the process, rather than the symptoms of processes). As nonlimiting examples, a treatment may include (i) preventing a disease or condition (e.g., nonunion) from occurring in a subject, in particular, when such subject is predisposed to the condition but has not yet been diagnosed as having it; (ii) inhibiting a disease or condition, i.e., arresting its development; (iii) relieving a disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from a disease or condition, i.e., relieving pain without addressing the underlying disease or condition.

[0158] It will be appreciated that variations to the embodiments of the invention can be made while still falling within the scope of the invention. Alternative features serving the same, equivalent, or similar purpose can replace features disclosed in the specification, unless stated otherwise. Thus, unless stated otherwise, each feature disclosed represents one example of a generic series of equivalent or similar features.

[0159] Use of exemplary language, such as “for instance,” “such as,” “for example,” (“e.g.,”) and the like, is merely intended to better illustrate the invention and does not indicate a limitation on the scope of the invention unless specifically so claimed.

[0160] While the invention has been described in connection with what is presently considered to be the most practical and embodiments thereof are further described in the examples below, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0161] The following description sets forth various examples along with specific details to provide a thorough understanding of claimed subject matter. It will be understood by those skilled in the art, however, that claimed subject matter may be practiced without one or more of the specific details disclosed herein. Further, in some circumstances, well-known methods, procedures, systems, and / or components have not been described in detail in order to avoid unnecessarily obscuringAttorney Docket No. 0073605-001029 claimed subject matter. The illustrative embodiments described in the detailed description and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects of the present disclosure, as generally described herein, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this disclosure.

[0162] The repair of most of the cortical bone fractures proceeds through three main phases, as illustrated in FIG. 1 : i) inflammation, ii) repair that is divided into early cartilaginous and late bony repair, and finally iii) remodeling. During the inflammatory phase immune cells invade the fracture gap and secret cytokines and growth factors that recruit other immune cells as well as stem cells and bone progenitors to set the stage for the repair phase. During the early cartilaginous repair phase, the fracture gap is filled with a fibrocartilaginous (soft) callus composed mainly of proliferating chondrocytes. These chondrocytes undergo hypertrophy and mineralization at later stages, which hardens the fracture gap and allow for formation of new blood vessels across the fracture line. The mineralized soft callus is then replaced by woven bone to form a bony callus at the end of the repair phase. The healing process is then concluded by remodeling the newly formed woven bone to reestablish the characteristic laminar structure of the cortical bone. (Claes, L. et al., “Fracture healing under healthy and inflammatory conditions,” Nat. Rev. Rheumatol. , 2012; 8: 133-143; Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137: 115436)

[0163] The extracellular matrix (ECM) is an integral component of bony tissues. The integrity of extracellular matrix (ECM) is crucial for bone structure and mechanical properties (Alcorta- Sevillano, et al., “Deciphering the Relevance of Bone ECM Signaling,” Cells, 2020; 9(12):2630). The functions of ECM proteins extend beyond their structural roles to regulate variable cellular processes (Alcorta-Sevillano, et al., “Deciphering the Relevance of Bone ECM Signaling,” Cells, 2020; 9(12):2630; Lin, X. et al., “The Bone Extracellular Matrix in Bone Formation and Regeneration,” Front. Pharmacol., 2020; 11 :757).

[0164] Proteoglycans, as shown in FIGS. 2A-B, are a major component of bone ECM and play central roles in keeping tissue integrity (Alcorta-Sevillano, N. et al., “Deciphering the Relevance of Bone ECM Signaling,” Cells, 2020; 9(12) : 2630). Proteoglycans include of a core protein covalently attached to multiple chains of linear, negatively charged polysaccharides glycosaminoglycansAttorney Docket No. 0073605-001029(GAGs) (Farach-Carson, M. C. et al., “Proteoglycans in Mechanobiology of Tissues and Organs: Normal Functions and Mechanopathology,” Proteoglycan Res. 2024;2(2):e21). Proteoglycans are categorized into families based on the core protein, their molecular structures, their interaction with other proteins, and their expression patterns in different tissues, and members of the proteoglycan family often differ in the core protein as well as type and size of attached GAGs. GAGs impart complexity to the proteoglycan structure. Specifically, negatively charged GAGs interact with other ECM components, and the hydrophilic nature of GAGs allows proteoglycans to interact with and attract large amounts of water; these interactions collectively help establish and / or maintain structural integrity of tissues and tissue hydration (Farach-Carson, M. C. et al., “Proteoglycans in Mechanobiology of Tissues and Organs: Normal Functions and Mechanopathology,” Proteoglycan Res. 2024;2(2):e21). In addition, proteoglycans can fit between ECM components and act as cushions and lubricants and can bind to growth factors and regulate their availability throughout various stages of development. Further, proteoglycans can regulate cell signaling via ECM-receptor interactions.

[0165] Proteoglycan 4 protein (Prg4), as shown in FIG. 2C, is an ECM structural protein that was first identified in the synovial fluid (lubricin) of the articular joint and is a proteoglycan that is conserved throughout the animal kingdom (Ikegawa, S., et al., “Isolation, characterization and mapping of the mouse and human PRG4 (proteoglycan 4) genes,” Cytogenet. Cell. Genet. , 2000; 90(3-4):291-297). Prg4 is highly expressed in vertebrate synovial fluid and the superficial zone of the articular cartilage where it acts as a structural protein and a lubricant to reduce shear at the cartilage surface. (Ikegawa, S. et al., “Isolation, characterization and mapping of the mouse and human PRG4 (proteoglycan 4) genes,” Cytogenet. Cell. Genet., 2000; 90(3-4):291-297; Alquraini, A. et al., “The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes,” Arthritis Res. Ther., 2017; 19:89; Swann, D. A. et al., “The molecular structure of lubricating glycoprotein-1, the boundary lubricant for articular cartilage,” J. Biol. Chem., 1981; 256:5921-5925; Schmidt, T. A. et al., “Transcription, translation, and function of lubricin, a boundary lubricant, at the ocular surface,” JAMA Ophthalmol., 2013; 131 :766-776).

[0166] Prg4 has a complex protein structure with a central, heavily glycosylated mucin domain flanked by N-terminal somatomedin B-like domains and a C-terminal hemopexin domain (FIG. 2C). The large and heavily glycosylated mucin domain is key to the lubricating properties of Prg4 within the synovial fluid and its molecular function as a boundary lubricant. The N- and C- termini of Prg4Attorney Docket No. 0073605-001029 are involved in protein-protein interaction, the interaction of Prg4 with receptors, and the adhesion of Prg4 to cell surface. Prg4 is primarily known for its lubricating functions in articular cartilage. Specifically, Prg4 can function as a lubricant to help synovial fluid dissipate strain energy under load, has anti-inflammatory properties that regulate synovial inflammation, and participates in cell signaling by binding to toll-like receptors TLRs and inhibiting proliferation of synovial fibroblasts.

[0167] Prg4 has chondroprotective effects, and deficient or reduced Prg4 expression in the joint is associated with onset of osteoarthritis (OA) (Jay, G. D. et al., “Association between friction and wear in diarthrodial joints lacking lubricin,” Arthritis Rheum., 2007; 56:3662-3669; Waller, K. A. et al., “Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis,” Proc. Natl. Acad. Sci. USA, 2013; 110:5852-5857; Hill, A. et al., “Lubricin restoration in a mouse model of congenital deficiency,” Arthritis Rheumatol., 2015; 67:3070-3081). Re-establishing Prg4 expression is a potential therapeutic approach for OA, and restoration of Prg4 levels using recombinant full-length Prg4 protein has been proposed as an OA disease-modifying therapy (Waller, K. A. et al., “Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis,” Proc. Natl. Acad. Sci. USA, 2013; 110:5852-5857; Cui, Z. et al., “Treatment with recombinant lubricin attenuates osteoarthritis by positive feedback loop between articular cartilage and subchondral bone in ovariectomized rats,” Bone, 2015; 74:37-47; Rhee, D. K. et al., “The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth,” J. Clin. Invest., 2005; 2115:622-631; Alquraini, A. et al., “The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes,” Arthritis Res. Ther., 2017;19:89; Jay, G. D. et al., “Prevention of cartilage degeneration and restoration of chondroprotection by lubricin tribosupplementation in the rat following anterior cruciate ligament transection,” Arthritis Rheum., 2010; 62:2382-2391; Teeple, E. et al., “Effects of supplemental intra-articular lubricin and hyaluronic acid on the progression of posttraumatic arthritis in the anterior cruciate ligament-deficient rat knee,” Am. J. Sports. Med., 2011; 39: 164-172; Jay, G. D. et al., “Prevention of cartilage degeneration and gait asymmetry by lubricin tribosupplementation in the rat following anterior cruciate ligament transection,” Arthritis Rheum., 2012; 64: 1162-1171; Elsaid, K. A. et al., “The impact of forced joint exercise on lubricin biosynthesis from articular cartilage following ACL transection and intra-articular lubricin's effect in exercised joints following ACL transection,” Osteoarthritis Cartilage, 2012; 20:940-948; Elsaid, K. A. et al., “The impact of early intra-articular administration of interleukin- 1 receptor antagonist on lubricin metabolism andAttorney Docket No. 0073605-001029 cartilage degeneration in an anterior cruciate ligament transection model,” Osteoarthritis Cartilage, 2015; 23:114-121).

[0168] Although most of Prg4 studies focused on the cartilage, Prg4 is expressed in other tissues, such as the liver, heart, and bone (Ikegawa, S. et al., “Isolation, characterization and mapping of the mouse and human PRG4 (proteoglycan 4) genes). Consistent with these findings, loss-of-function mutation in Prg4 gene causes the autosomal camptodactyly arthropathy-coxa vara pericarditis (CACP) syndrome (Rhee, D. K. et al., “The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth,” J. Clin. Invest., 2005; 2115:622-631; Rhee, D. K. et al., “Consequences of disease-causing mutations on lubricin protein synthesis, secretion, and post-translational processing,” J. Biol. Chem. 2005; 280:31325-31332; Ciullini Mannurita, S. et al., “CACP syndrome: identification of five novel mutations and of the first case of UPD in the largest European cohort,” Eur. J. Hum. Genet., 2014; 22, 197-201), which is characterized by chronic inflammation and pericarditis in addition to joint degeneration (Ciullini Mannurita, S. et al., “CACP syndrome: identification of five novel mutations and of the first case of UPD in the largest European cohort,” Eur. J. Hum. Genet., 2014; 22, 197-201). These findings indicate that Prg4 plays roles in organs other than the articular joint.

[0169] Recent work started to unravel functions of Prg4 that are independent of its role as a lubricant. These roles include modulating the inflammatory response, regulating the proliferation of progenitors, promoting angiogenesis, and maintaining tissue homeostasis (Das, N. et al., “Proteoglycan 4: From Mere Lubricant to Regulator of Tissue Homeostasis and Inflammation: Does proteoglycan 4 have the ability to buffer the inflammatory response?,” Bioessays, 2019;41(1): 1800166; Krawetz, R. J. et al., “Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration,” NP J Re gen. Med., 2022; 7:32).

[0170] For example, Prg4 appears to be involved in skin wound closure as well. Recent studies started to unravel functions of Prg4 in skin and ear wound closure that are independent of its lubricant effects (Das, N. et al., “Proteoglycan 4: From Mere Lubricant to Regulator of Tissue Homeostasis and Inflammation: Does proteoglycan 4 have the ability to buffer the inflammatory response?” Bioessays, 2019; 41 :el800166; Krawetz, R. J. et al., “Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration,” NPJ Regen. Med., 2022;7:32; Ninkovic, N. et al., “Proteoglycan 4 (PRG4) treatment improves skin wound healing in a porcine model,” FASEB J. 2024; 38:e23547). Importantly, the expression pattern and biological functions of Prg4 during bone healing remain completely uncharacterized prior to the study described herein.Attorney Docket No. 0073605-001029

[0171] Alternative splicing (AS) is a fundamental regulatory process that removes introns from precursor (pre)-mRNAs and connects the exons in different combinations, generating diversified mature mRNAs from the same gene (Faustino, N. A. & Cooper, T. A., “Pre-mRNA splicing and human disease,” Genes Dev., 2003; 17:419-437). AS is a tightly regulated process and plays central roles in maintaining normal tissue homeostasis (Scotti, M. M. & Swanson, M. S., “RNA missplicing in disease,” Nat. Rev. Genet., 2016; 17: 19-32). AS is tissue- and context-specific, and some alternative splicing events occur only in response to particulars signals such as developmental or pathological cues (Nikom, D. & Zheng, S., “Alternative splicing in neurodegenerative disease and the promise of RNA therapies” Nat. Rev. Neurosci., 2023; 24:457-473). AS is under-investigated in the context of bone repair and our knowledge regarding AS makeup within the healing callus is extremely limited. Notably, the cytokines and growth factors secreted by inflammatory and immune cells within the healing hematoma are known to affect and mutually crosstalk with AS (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020;137: 115436; Khajuria, D. K. et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023;14: 1250309; Zhou, J. et al., “The regulatory role of alternative splicing in inflammatory bowel disease,” Front. Immunol., 2023;14: 1095267; Robinson, E. K. et al., “Inflammation drives alternative first exon usage to regulate immune genes including a novel iron-regulated isoform of Aim2,” Elife, 2021; 10:e69431; Wu, W. et al., “The Impact of Pro-Inflammatory Cytokines on Alternative Splicing Patterns in Human Islets,” Diabetes, 2021; 71(1): 116-127; Janssen, W. J. et al., “Inflammation-Induced Alternative Pre-mRNA Splicing in Mouse Alveolar Macrophages,” G3 (Bethesda), 2020;10:555-567), which further emphasizes the importance of studies addressing AS during fracture healing. Different splice variants of Prg4 have been characterized in the articular cartilage and found to be differentially expressed in the anterior load-bearing locations of the femoral medial condyle as compared to the posterior non-load-bearing locations (DuRaine, G. D., et al., “Effects of TGF-pi on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110). However, the functional justification of this anatomical distribution is unclear. Notably, all studies reporting the biological roles or assessing the therapeutic potential of Prg4 in different tissues and disease conditions have focused on the full- length (FL) splice variant, leaving the other variants completely unstudied (Das, N. et al., “Proteoglycan 4: From Mere Lubricant to Regulator of Tissue Homeostasis and Inflammation: DoesAttorney Docket No. 0073605-001029 proteoglycan 4 have the ability to buffer the inflammatory response?,” Bioessays, 2019;41(1): 1800166; Krawetz, R. J. et al., “Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration,” NP J Regen. Med., 2022; 7:32; Ninkovic, N. et al., “Proteoglycan 4 (PRG4) treatment improves skin wound healing in a porcine model,” FASEB J. 2024; 38:e23547).

[0172] However, the expression pattern and roles of Prg4 in tissues other than the articular cartilage as well as the mechanisms that regulate Prg4 expression in these tissues remain largely unknown. Specifically, Prg4 roles in fracture healing have never been characterized prior to the present disclosure, in spite of the fact that all the above-mentioned lubrication-independent roles identified for Prg4 are central to the fracture healing process.

[0173] The present disclosure reveals the expression pattern of Prg4 during fracture healing on a single-cell level and characterizes the splice variants of Prg4 that are specifically expressed in the healing-callus microenvironment as compared to those expressed under the homeostatic conditions in the intact bone. Functional studies were also performed to further define the roles of the callusspecific Prg4 variant in diverse biological processes that play central roles in bone regeneration. Method for Treating Bone Fracture

[0174] An objective of the present disclosure is to provide a method for treating bone fracture or promoting bone growth, bone regeneration, or bone fracture healing in a subject. It should be noted that the method described herein can be used for treatment of any type or pattern of bone fracture, which can include without limitation displaced fractures, nondisplaced fractures, hairline fractures, transverse fractions, oblique fractures, linear fractures, comminuted fractures, spiral fractures, segmental fractures, greenstick fractures, and / or the like. Further, the method described herein can be agnostic to the location of the fracture as well and can accordingly be used for treatment of diaphyseal fractures, metaphyseal fractures, epiphyseal fractures, among others.

[0175] In some embodiments, the method can include administering to the subject a composition including a therapeutically effective amount of a recombinant proteoglycan 4 protein, the recombinant proteoglycan 4 protein having an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1. In some embodiments, the method can include administering to the subject a composition including a therapeutically effective amount of a recombinant proteoglycan 4 protein, the recombinant proteoglycan 4 protein having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. As nonlimiting examples, the recombinant proteoglycan 4 protein can have an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 1.Attorney Docket No. 0073605-001029

[0176] As used herein, a “recombinant” proteoglycan 4 protein is a proteoglycan 4 protein prepared using recombinant DNA technology. As used herein, “recombinant DNA technology,” often referred to as genetic engineering, is a type of technology that involves using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest. Recombinant DNA technology can be used to combine (or splice) DNA from different species or to create genes with new functions. The resulting DNA copies are often referred to as recombinant DNA. Such recombinant DNA can subsequently be propagated in a host cell, such as without limitation a bacterial or yeast cell, whose cellular machinery copies and expresses the recombinant DNA along with its own. Recombinant DNA technology can be used to prepare chemicals or biomolecules that are otherwise challenging to isolate, synthesize, or obtain at a larger scale and / or with a reduced cost.

[0177] In some embodiments, a recombinant DNA encoding the recombinant proteoglycan 4 protein can include or be included in a plasmid. As used herein, a “plasmid” is a circular, doublestranded DNA molecule. Plasmids are distinct from a cell’s chromosomal DNA and are capable of autonomous replication. Plasmids may be used as vectors for insertion, expression, and propagation of foreign genes within a host organism. Such vectors may include specific sequences for an origin of replication, selectable markers, and cloning sites, enabling manipulation and study of genetic material for applications in research, biotechnology, and therapeutic development. As a nonlimiting example, a plasmid can include a nucleic acid sequence that encodes the recombinant proteoglycan 4 protein, e.g., with an amino acid sequence of SEQ ID NO: 1. As another nonlimiting example, a plasmid can include a pET30a(+) plasmid.

[0178] In some embodiments, the recombinant proteoglycan 4 protein may be expressed using host cells such as genetically modified BL21 cells, which is a strain of E. coll. As used herein, a “genetically modified” or “genetically engineered” cell is a cell whose genetic material has been manipulated to alter one or more of its hereditary traits. Such manipulation may include without limitation inserting, deleting, or otherwise modifying one or more specific DNA sequences; as a result of such manipulation, a genetically modified cell may exhibit one or more different traits in its structure, function, or the like, compared to its naturally occurring counterparts. As used herein, “BL21 cell” or “Escherichia coli BL21” is a commonly used protein production strain of Escherichia coli. This strain is derived from the B lineage of Escherichia coli and combines several features that allow for excessive expression of heterologous proteins. As used herein, a “heterologous” protein is a protein that is absent from a naturally occurring species. A heterologousAttorney Docket No. 0073605-001029 protein may originate from a different species or be artificially designed. A heterologous protein may be expressed in a host cell using genetic engineering tools.

[0179] As used herein, a “functional variant” of a base protein or peptide is a protein or peptide that differs from the base protein or peptide by one or more amino acids while maintaining at least 80% of the sequence identity and substantially of the functional characteristics of the base peptide. As a nonlimiting example, a functional variant of a base protein or peptide, when administered to a subject or a sample collected therefrom, can trigger a response similar to the response triggered by the base protein or peptide itself. A functional variant can be created via amino acid substitutions, additions, or deletions, but none of such changes should significantly alter the tertiary structure within the base protein or peptide from which the functional variant is derived.

[0180] In some embodiments, a functional variant can be created via one or more conservative or non-conservative amino acid substitutions. As used herein, a “conservative amino acid substitution” is a change in amino acid sequence where an amino acid is replaced with a different amino acid with broadly similar properties. As used herein, a “non-conservative amino acid substitution” is a change in amino acid sequence where one amino acid is replaced with another amino acid of a different type. As nonlimiting examples, common amino acids can be categorized as follows: nonpolar amino acids include Ala, Vai, Leu, He, Pro, Met, Phe, and Trp; uncharged polar amino acids include Gly, Ser, Thr, Cys, Tyr, Asn, and Gin; acidic amino acids include Asp and Glu; and basic amino acids include Lys, Arg, and His. Since side chains of amino acids in the same category have similar polarities and are capable of establishing or participating in similar electrostatic interaction(s), hydrogen bond(s), and / or van der Waals contact(s), altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide. Non-conservative substitutions are also possible provided that these substitutions do not disrupt the tertiary structure of an epitope within the peptide. Broadly speaking, fewer non- conservative substitutions will be possible without altering the biological activity of the polypeptide. Suitably, functional variants can be at least 80% identical, at least 90% identical, etc., with respect to the base sequence while retaining the immunogenic function of the base sequence. A person of ordinary skill in the art, upon reviewing the entirety of this disclosure, will be able to identify suitable strategies for creating functional variants of a protein or peptide without compromising its tertiary structure.

[0181] Sequence identity between two amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupiedAttorney Docket No. 0073605-001029 by the same amino acid or base, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties. Similarly, sequence identity between two nucleic acid sequences can be determined by comparing an alignment of the sequences as well, as described in further detail below in this disclosure.

[0182] In some embodiments, the method can include administering to the subject a composition including a therapeutically effective amount of a nucleic acid construct, the nucleic acid construct having a nucleic acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 2. In some embodiments, the method can include administering to the subject a composition including a therapeutically effective amount of a nucleic acid construct, the nucleic acid construct having a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 2. As nonlimiting examples, the nucleic acid construct can have a nucleic acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 2.

[0183] As used herein, a “nucleic acid construct” is a chemical species having a nucleic acid sequence containing genetic information that encodes a peptide. As nonlimiting examples, a nucleic acid construct can include or be synthetically derived from a DNA, an RNA, or a peptide nucleic acid (PNA), among others. Accordingly, genetic information can be stored within the nucleic acid construct through unique sequences of nucleobases (e.g., ATGC for DNA and AUGC for RNA). A nucleic acid construct includes a coding sequence that encodes a peptide and one or more regulatory elements that regulate the expression of the coding sequence. Exemplary embodiments of a regulatory element may include a promoter, an enhancer, a silencer, an insulator, an operator, and a response element, among others. As used herein, a “promoter” is a DNA sequence where an RNA polymerase binds to initiate transcription. As used herein, an “enhancer” is a distant element of DNA sequence that increases transcription rates by interacting with a promoter via DNA looping. As used herein, a “silencer” is a DNA sequence that represses transcription when bound by specific proteins.Attorney Docket No. 0073605-001029As used herein, an “insulator” is a DNA sequence that prevents the interaction between one or more enhancers and one or more promoters of neighboring genes. As used herein, an “operator” is a DNA segment that regulates the transcription of adjacent genes. As used herein, a response element is a DNA sequence that responds to external signals, allowing genes to be turned on or off in response to environmental changes. These regulatory elements may work together to ensure a precise gene expression necessary for proper cellular function.

[0184] In some embodiments, a nucleic acid construct can include one or more operons. As used herein, an “operon” is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. It is commonly found in prokaryotes such as bacteria. These genes are transcribed together into a single messenger RNA strand and typically encode proteins that work together in a specific biological pathway. An operon can include a regulatory element, such as an operator as described above, where an activator or repressor protein may bind to increase or inhibit transcription. An operon can include one or more regulatory genes that encode one or more such activator or repressor proteins.

[0185] In some cases, promoter engineering can be used to improve the transcriptional level of a nucleic acid sequence / gene. Promoter engineering modifies the promoter region to increase gene expression levels, thereby improving enzyme production and activity. In one or more embodiments, codon optimization can be used to improve the translational efficiency of a gene. As used herein, “codon optimization” is a technique used in genetic engineering to improve the expression of a gene in a particular host organism. Codon optimization involves altering the DNA sequence of a gene to use codons that are more frequently preferred by the host organism’s translational machinery. A codon optimization process may take into account a codon bias of the host, ensuring that a synthetic gene sequence is translated more efficiently into the desired protein. Codon optimization may enhance the yield and function of a protein, which may be crucial for various applications in biotechnology and synthetic biology. It is worth noting that promoter engineering and codon optimization are distinct yet complementary techniques in genetic engineering. Promoter engineering involves modifying the promoter region of a gene to enhance its expression by improving the binding efficiency of transcriptional machinery. Codon optimization, on the other hand, focuses on altering the coding sequence of a gene to use preferred codons of the host organism, thereby improving translation efficiency. While both aim to increase protein production, promoter engineering targets transcriptional levels, and codon optimization targets translationalAttorney Docket No. 0073605-001029 efficiency. Combining both techniques may in some cases synergistically enhance an overall gene expression.

[0186] As used herein, a “functional variant” of a base nucleic acid is a nucleic acid that differs from the base nucleic acid by one or more nucleotides while maintaining at least 80% of the sequence identity and or at least 80% of the sequence identity in the protein or peptide it encodes.

[0187] In some embodiments, the nucleic acid construct can include a ribonucleic acid (RNA). In some embodiments, the nucleic acid construct can encode a recombinant proteoglycan 4 protein, as described herein. In some embodiments, the recombinant proteoglycan 4 protein encoded by the nucleic acid construct can have an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1, as described herein. In some embodiments, the recombinant proteoglycan 4 protein encoded by the nucleic acid construct can have an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.

[0188] In some embodiments, the recombinant proteoglycan 4 protein can be produced or expressed in the subject as an extracellular protein or secreted protein. In some embodiments, the recombinant proteoglycan 4 protein can be produced or expressed inside a callus of the subject. As used herein, a “callus” is a temporary bridge of tissue that stabilizes fractured bones and promotes their regeneration.

[0189] In some embodiments, the method can include administering the composition locally, e.g., to an articular joint or a callus of the subject. In some embodiments, such administration can be performed a single time in order to reduce pain or discomfort. In some embodiments, such administration can be performed multiple times, if needed. In some embodiments, such administration can be performed via injection (e.g., using a syringe), surgical implantations, and / or the like.

[0190] In some embodiments, the subject can be diagnosed with, or expresses symptoms of, diabetes or obesity. In some embodiments, the subject can be associated with a higher risk of nonunion. As used herein, “nonunion” is a medical condition or situation where a broken bone fails to heal properly and the fractured bone ends cannot form a stable union. Nonunion prevents a broken or fractured bone from regaining its original strength and function.

[0191] In some embodiments, the method can further include inducing or promoting osteogenic or chondrogenic differentiation of mesenchymal stem cells (MSCs) in the subject, e.g., using the composition, recombinant protein, or nucleic acid construct described herein. As used herein, “osteogenic differentiation” is a process by which mesenchymal stem cells (MSCs) and / orAttorney Docket No. 0073605-001029 osteoprogenitor cells transform into mature, bone-forming osteoblasts and osteocytes. Osteogenic differentiation is essential for bone development, fracture healing, and tissue maintenance. As used herein, “chondrogenic differentiation” is a biological process where mesenchymal stem cells (MSCs) transform into chondrocytes, the cells that produce and maintain cartilage tissue.

[0192] In some embodiments, the method can further include maintaining or restoring chondrocyte homeostasis or osteoblast homeostasis in the subject. As used herein, “chondrocyte homeostasis” is a process where cells within articular cartilage, called chondrocytes, maintain a balance between the production and degradation of the cartilage’s extracellular matrix (ECM) to preserve joint health. As used herein, “osteoblast homeostasis” is a process by which osteoblast activity is regulated to maintain bone mass and mineral balance through coordinated communication with osteoclasts, hormonal signals, and mechanical cues, ensuring bone remodeling and overall skeletal health. Chondrocyte homeostasis is disrupted in diseases such as osteoarthritis, and maintaining or restoring normal chondrocyte homeostasis can inhibit the progression of osteoarthritis. The same principle may apply to osteoblast homeostasis, which is disrupted in diseases such as osteoporosis. Therefore, any diagnostic marker related to such diseases can be used to evaluate the effectiveness of Prg4 treatment. A person of ordinary skill in the art, upon reviewing the entirety of this disclosure, will be able to identify suitable or relevant biomarkers for evaluating the effectiveness of Prg4 treatment and / or its ability in maintaining / restoring chondrocyte homeostasis or osteoblast homeostasis.Recombinant Proteoglycan 4 Protein

[0193] Another objective of the present disclosure is to provide a recombinant proteoglycan 4 protein as described herein. In some embodiments, the recombinant proteoglycan 4 protein can have an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1. In some embodiments, the recombinant proteoglycan 4 protein can have an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, etc., to SEQ ID NO: 1, as described herein.

[0194] In some embodiments, the recombinant proteoglycan 4 protein can be administered at a therapeutically effective amount of least 0.1 mg / kg and no greater than 1,000 mg / kg with respect to the body weight of the subject. As nonlimiting examples, the therapeutically effective amount of the recombinant proteoglycan 4 protein can be at least 0.1 mg / kg and no greater than 0.5 mg / kg, at least 0.5 mg / kg and no greater than 1.0 mg / kg, at least 1.0 mg / kg and no greater than 10 mg / kg, at least 10 mg / kg and no greater than 20 mg / kg, at least 20 mg / kg and no greater than 30 mg / kg, at least 30Attorney Docket No. 0073605-001029 mg / kg and no greater than 40 mg / kg, at least 40 mg / kg and no greater than 50 mg / kg, at least 50 mg / kg and no greater than 60 mg / kg, at least 60 mg / kg and no greater than 70 mg / kg, at least 70 mg / kg and no greater than 80 mg / kg, at least 80 mg / kg and no greater than 90 mg / kg, at least 90 mg / kg and no greater than 100 mg / kg, at least 100 mg / kg and no greater than 200 mg / kg, at least 200 mg / kg and no greater than 300 mg / kg, at least 300 mg / kg and no greater than 400 mg / kg, at least 400 mg / kg and no greater than 500 mg / kg, at least 500 mg / kg and no greater than 600 mg / kg, at least 600 mg / kg and no greater than 700 mg / kg, at least 700 mg / kg and no greater than 800 mg / kg, at least 800 mg / kg and no greater than 900 mg / kg, at least 900 mg / kg and no greater than 1,000 mg / kg, and / or the like.

[0195] In some embodiments, the recombinant proteoglycan 4 protein described herein can be used for treating bone fracture. Accordingly, in some embodiments, the recombinant proteoglycan 4 protein described herein can be used in the manufacture of a medicament for the treatment of bone fracture.

[0196] In some embodiments, the recombinant proteoglycan 4 protein described herein can be used for promoting bone growth, bone regeneration, or bone fracture healing. Accordingly, in some embodiments, the recombinant proteoglycan 4 protein described herein can be used in the manufacture of a medicament for promoting bone growth, bone regeneration, or bone fracture healing.

[0197] In some embodiments, the recombinant proteoglycan 4 protein described herein can be used in combination with a full-length proteoglycan 4 protein or a proteoglycan 4 protein having an amino acid sequence longer than that of the recombinant proteoglycan 4 protein for treating osteoarthritis or one or more similar conditions where bone growth or regeneration is desired. Accordingly, in some embodiments, the recombinant proteoglycan 4 protein described herein can be used in the manufacture of a medicament for the treatment of osteoarthritis.

[0198] In some embodiments, the recombinant proteoglycan 4 protein can be used in combination with a stem cell therapy (e.g., as an adjuvant) for treating bone fracture. Accordingly, in some embodiments, the recombinant proteoglycan 4 protein described herein can be used in combination with a stem cell therapy in the manufacture of a medicament for the treatment of bone fracture. Further, in come embodiments, the recombinant proteoglycan 4 protein described herein can be used in combination with a stem cell therapy in the manufacture of a medicament for promoting bone growth, bone regeneration, or bone fracture healing. It should be noted that the recombinant proteoglycan 4 protein described herein can be used in combination with any type ofAttorney Docket No. 0073605-001029 stem cell therapy or therapies deemed suitable or relevant by a person of ordinary skill in the art, upon reviewing the entirety of this disclosure. In some embodiments, the recombinant proteoglycan 4 protein described herein can be used as an adjuvant in combination with stem cell therapy for treating osteoarthritis and / or other cartilage degeneration diseases. Preliminary data suggest that the short isoform of Prg4 stimulates both osteogenic and chondrogenic differentiation of progenitor cells, thereby enhancing their regenerative capacity against bone loss and cartilage degeneration diseases, respectively.Composition for Treatment of Bone Fracture

[0199] Another objective of the present disclosure is to provide a composition for treatment of bone fracture or promoting bone growth, bone regeneration, or bone fracture healing. In some embodiments, the composition can include a therapeutically effective amount of a recombinant proteoglycan 4 protein having an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1 and a delivery vehicle, wherein the therapeutically effective amount of the recombinant proteoglycan 4 protein is stored within a matrix or cavity of the delivery vehicle. In some embodiments, the composition can include a therapeutically effective amount of a recombinant proteoglycan 4 protein having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 and a delivery vehicle, wherein the therapeutically effective amount of the recombinant proteoglycan 4 is stored within a matrix or cavity of the delivery vehicle.

[0200] As used herein, a “delivery vehicle” is a chemical, composition, or formulation that facilitates the transport of a pharmaceutically active substance to its target location in a patient’s body, primarily through increasing the stability and / or controlling the release of the pharmaceutically active substance. It should be noted that the delivery vehicle described herein can include any type of delivery vehicle or delivery system deemed suitable or applicable by a person of ordinary skill in the art, upon reviewing the entirety of this disclosure. Nonlimiting examples of such delivery vehicles can include without limitation micelles, liposomes, nanoparticles, hydrogels (e.g., atelocollagen), or a combination thereof (e.g., a complex of atelocollagen and nanoparticles), among others.

[0201] In some embodiments, the therapeutically effective amount of the recombinant proteoglycan 4 protein can be released from the delivery vehicle over a period of at least seven days and no greater than six months. As nonlimiting examples, the therapeutically effective amount of the recombinant proteoglycan 4 protein can be released from the delivery vehicle over a period of at least ten days, at least fourteen days, at least one month, at least two months, at least three months,Attorney Docket No. 0073605-001029 and / or the like. As further nonlimiting examples, the therapeutically effective amount of the recombinant proteoglycan 4 protein can be released from the delivery vehicle over a period of no greater than three months, no greater than four months, no greater than five months, etc.

[0202] In some embodiments, the composition can include a therapeutically effective amount of a nucleic acid construct having a nucleic acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 2 and a delivery vehicle, wherein the therapeutically effective amount of the nucleic acid construct is stored within a matrix or cavity of the delivery vehicle.

[0203] In some embodiments, the composition can include a therapeutically effective amount of a nucleic acid construct having a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 2 and a delivery vehicle, wherein the therapeutically effective amount of the nucleic acid construct is stored within a matrix or cavity of the delivery vehicle.

[0204] In some embodiments, the composition described herein can be used in the treatment of bone fracture. Accordingly, in some embodiments, the composition described herein can be used in the manufacture of a medicament for the treatment of bone fracture.

[0205] In some embodiments, the composition can be used in promoting bone growth, bone regeneration, or bone fracture healing. Accordingly, in some embodiments, the composition described herein can be used in the manufacture of a medicament for promoting bone growth, bone regeneration, or bone fracture healing.

[0206] In some embodiments, the composition described herein can further include a therapeutically effective amount of a full-length proteoglycan 4 protein or a therapeutically effective amount of a nucleic acid encoding a proteoglycan 4 protein having an amino acid sequence longer than that of the recombinant proteoglycan 4 protein. In some embodiments, the composition described herein can further include a therapeutically effective amount of a nucleic acid encoding the full-length proteoglycan 4 protein or a therapeutically effective amount of a nucleic acid encoding a proteoglycan 4 protein having an amino acid sequence longer than that of the recombinant proteoglycan 4 protein. Accordingly, in some embodiments, such composition can be used in the treatment of osteoarthritis or in the manufacture of a medicament for the treatment of osteoarthritis.

[0207] In some embodiments, the composition described herein can be used in combination with a stem cell therapy for treating bone fracture or promoting bone growth, bone regeneration, or bone fracture healing. In some embodiments, the composition described herein can be used in combination with a stem cell therapy in the manufacture of a medicament for the treatment of bone fracture or for promoting bone growth, bone regeneration, or bone fracture healing. It should beAttorney Docket No. 0073605-001029 noted that the composition described herein can be used in combination with any type of stem cell therapy or therapies deemed suitable or relevant by a person of ordinary skill in the art, upon reviewing the entirety of this disclosure. In some embodiments, the composition described herein can be used as an adjuvant in combination with stem cell therapy for treating osteoarthritis and / or other cartilage degeneration diseases. Preliminary data suggest that the short isoform of Prg4 stimulates both osteogenic and chondrogenic differentiation of progenitor cells, thereby enhancing their regenerative capacity against bone loss and cartilage degeneration diseases, respectively. EXAMPLES EXAMPLE 1

[0208] Proteoglycan 4 (Prg4) is an extracellular matrix protein known for its lubricant effects in articular cartilage. To study the functions of Prg4 during fracture healing, a murine model of tibial fracture was developed, and bulk RNA-seq was performed on multiple post-fracture timepoints. High expression of Prg4 was detected during the first week of healing, which overlaps with the initial inflammatory phase. Single-cell RNA-seq of the healing callus localized Prg4 expression to a population of stem cells that play central roles in fracture healing. Analysis of alternative splicing of callus RNA identified a unique splice variant of Prg4 that lacks 3 coding exons (Prg4-S). Knocking down Prg4-S in vivo using a locally delivered siRNA resulted in multiple defects that culminated in reduced bone formation. These data unravel novel pro-healing roles of a unique splice variant of Prg4, opening multiple translational venues.Results

[0209] The callus progenitors express high levels ofPrg4 during the inflammatory phase.

[0210] Referring now to FIG. 3A, FIG. 3A shows the timepoints and healing phases based on bulk RNA-seq from multiple timepoints that cover the entire healing course of a standard murine model of tibial mid-diaphyseal osteotomy (stabilized by an intramedullary nail) (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14:1250309; Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023; 11 :5). An analysis was performed based on RNA-seq data to identify the expression pattern of Prg4 during fracture healing, and results of the analysis indicated high expression of Prg4 during the initial inflammatory phase (especially on days 3 and 5), which decreased steadily during the cartilaginous phase (days 7-14) and became barely detectable at the later phases (FIG. 3B).Attorney Docket No. 0073605-001029

[0211] The healing hematoma on days 3 and 5 is populated mainly by stem cells / progenitors and immune cells; none of these cell types are known to express Prg4 at the levels detected in the RNA-seq data (FIG. 3B). To localize Prg4 expression to specific cell types, single-cell (sc)RNA-seq was performed on day 5 post-fracture (d5) and on day 10 post-fracture dlO. 32 clusters (i.e., cell types) were identified on the 2 days combined (FIG. 3C; FIGS. 12A-D), among which cluster 2 (C2) was the main source of Prg4 (FIG. 3D). Cluster 2 was identified as skeletal stem cells / osteochondral progenitors based on the expression of the canonical markers, including Prrxl, Pdgfra, and Ly6a (Seal) (Xu, J. et al., “PDGFRalpha reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair,” Bone Res. 2022; 10:7) (FIG. 3E). C2 also expressed high levels of Ctsk and Postn (FIG. 3E), and based on the current knowledge, these markers identify periosteal stem cells (Zou, N. et al., “Cathepsin K+Non-Osteoclast Cells in the Skeletal System: Function, Models, Identity, and Therapeutic Implications,” Front. Cell. Dev. Biol., 2022;10:818462; Yin, B. et al., “Identification of Postn+periosteal progenitor cells with bone regenerative potential,” JCI Insight, 2024; 9(19):el82524; Duchamp de Lageneste, O. et al., “Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin,” Nat. Comimin., 2018; 9:773) These cells may be referred to as Postn+stem cells (PSC) to differentiate them from other clusters detected on d5. In fact, in addition to C2, the 2 clusters C6 and C20 also contained progenitors or mesenchymal stem cell (MSC)-like cells (FIGS. 3C, F). C20 was identified as pericytes (Matthews, B. G. et al., “Heterogeneity of murine periosteum progenitors involved in fracture healing,” Elife, 2021; 10:e58534) (FIG. 3G). C6 was a less identified cluster that exhibited low to undetectable expression levels of Pdgfra, Seal, Cstk, and Postn (FIG. 3E), but expressed a number of other genes reported to mark mesenchymal progenitors, MSC-like cells, and / or activated fibroblasts (FIG. 12E). These findings indicate that C6 is a heterogenous pool that contains mesenchymal-like progenitors distinct from the PSC in C2. Consistently, among the three clusters of stem cells, Prg4 expression was almost restricted to C2 (i.e., PSC) (FIG. 3H). It is noteworthy that purifying Pdgfra+Scal+cells is an effective strategy to isolate mesenchymal stem cells and osteochondral progenitors from the callus, periosteum, or bone marrow (BM) (Duchamp de Lageneste, O. et al., “Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin,” Nat. Commun., 2018; 9:773; Houlihan, D. D., et al., “Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-alpha,” Nat. Protoc., 2012; 7:2103-2111). Given the high expression levels of Pdgfra and Seal in C2 as compared to C6 (FIG. 3E), and the fact that C2Attorney Docket No. 0073605-001029 contained ~10 times more cells than C6 (FIG. 3F), these data indicate that the vast majority of Pdgfra+Scal+cells in d5 callus are PSC.

[0212] An analysis of the dlO results revealed the same 3 clusters of stem cells (FIG. 3F). However, there was a noticeable increase in the relative size of C6 and C20 as compared to d5 (FIG. 3F). Importantly, the expression of Prg4 in C2 was substantially lower than that on d5 (FIG. 3H). This difference explains the substantial reduction in Prg4 expression on dlO vs. d5 observed in the bulk RNA-seq data (FIG. 3B). In addition to C2, Prg4 was also expressed in the soft-callus chondrocytes of dlO callus (C25 and C30; FIGS. 12F-G) but at a significantly lower level than in the PSC in C2 (FIG. 3H). Prg4 expression in the soft-callus chondrocytes was confirmed using immunofluorescence (IF) staining (FIG. 12H). Taken together, these data indicate that PSCs express high levels of Prg4 during the early stages of healing.

[0213] The callus microenvironment induces Prg4 expression.

[0214] To corroborate the scRNA-seq data (FIG. 3D, H) and confirm that PSC is the main source of Prg4 in d5 callus, PSC (CD45“CD31“Pdgfra+Scal+cells) was isolated using fluorescence- activated cell sorting (FACS), and quantitative polymerase chain reaction (qPCR) was employed to quantitate Prg4 expression in PSC versus other cells. Results from these experiments confirmed that the expression of Prg4 in the healing hematoma was almost restricted to PSC (FIG. 4A). To investigate whether the observed high expression of Prg4 is unique to callus PSC or a common feature for these cells regardless of the microenvironment they reside in, FACS was used to isolate CD45“CD31_Pdgfra+Scal+cells from d5 callus or from the contralateral unfractured bone and bone marrow (BM). Interestingly, quantitative polymerase chain reaction (qPCR) indicated that the level of Prg4 was ~ 120-fold and ~ 1300-fold higher in the callus than in intact bone and BM, respectively (FIG. 4B). Taken together, these data suggest that PSC are induced to express high levels of Prg4 when exposed to the callus inflammatory microenvironment during the early phases of healing.

[0215] The callus PSC expresses a unique variant of Prg4 that lacks the mucin domain.

[0216] Human and murine mRNAs encoding the full-length (FL) Prg4 are similar and include 13 exons (FIGS. 4C, D). Exon 7 encodes the central mucin-like domain that contains the glycosylation sites (FIGS. 4C, D; FIG. 13). the N-terminal region of Prg4 upstream to exon 7 encodes two somatomedin-B-like domains and heparin binding sites, while the C-terminal region downstream to exon 7 encodes hemopexin-like domains (FIGS. 4C, D; FIG. 13). The heavily glycosylated central mucin domain allows Prg4 to form a nanofilm and attract water, which are essential properties for the functions of Prg4 as a lubricant at the surface of the articular cartilage. OnAttorney Docket No. 0073605-001029 the other hand, the N- and C-termini are involved in protein-protein interactions and cell signaling. (Ikegawa, S. et al., “Isolation, characterization and mapping of the mouse and human PRG4 (proteoglycan 4) genes,” Cytogenet. Cell. Genet., 2000; 90:291-297; Alquraini, A. et al., “The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes,” Arthritis Res. Ther., 2017;19:89; Swann, D. A., et al., “The molecular structure of lubricating glycoprotein-1, the boundary lubricant for articular cartilage,” J. Biol. Chem., 1981; 256:5921-5925; Schmidt, T. A. et al., “Transcription, translation, and function of lubricin, a boundary lubricant, at the ocular surface,” JAMA Ophthalmol., 2013; 131 :766-776)

[0217] A very limited number of studies have investigated the splice variants of Prg4 in musculoskeletal tissues and were limited to the articular cartilage (DuRaine, G. D. et al., “Effects of TGF-pi on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19:103-110). The reported splice variants of Prg4 lack one or more of exons 3-6 (FIG. 2C, isoforms A-E) (DuRaine, G. D. et al., “Effects of TGF-p i on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110), but the distinctive functions of these variants remain unknown. Importantly, all Prg4 A-E variants contain the central mucin domain. Notably, there are two splice variants of murine Prg4 listed on the Reference Sequence (RefSeq) database of the NCBI and the UCSC genome browser: the FL variant that contains all the exons (NM_021400), which may be referred to as Prg4-Long or Prg4-L (FIG. 4D), and a short variant (NM_001110146), which may be referred to Prg4-short or Prg4-S (FIG. 4D). Prg4-S is distinct from all the A-E variants expressed in the articular cartilage as it lacks exons 5, 6 and the mucin-like repeats in exon 7 (FIG. 4D). Prg4-S has never been detected or studied in any musculoskeletal tissue, and its functions remain unknow.

[0218] Available data on AS during fracture healing are very limited. Thus, the RNA-seq data were further analyzed using rMATS ((Shen, S. et al., “rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data,” Proc. Natl. Acad. Sci. USA, 2014; 111 :E5593-5601) to globally identify splice variants expressed in the callus. Interestingly, our results predicted Prg4-S to be the predominant variant in the callus (FIG. 4E). The bioinformatics results were corroborated by performing RT-PCR on RNA purified from d5 callus using two different primer pairs that span the skipped exons (depicted in FIG. 4F). A major band was detected at the expected size of Prg4-S, while Prg4-L was undetectable (FIG. 4G). The PCR product was sequenced, and the band identity was confirmed as Prg4-S (SEQ ID NOs: 3-4; FIG. 14). The sameAttorney Docket No. 0073605-001029 experiments were performed on RNA isolated from d7 and dlO callus, and results from these experiments demonstrated that Prg4-S remained the major variant (FIG. 15). As expected, Prg4-S was also detected as the major splice variant in the callus PSC (FIG. 4H). In a striking contrast, Prg4-L was the major splice variant expressed in PSC isolated from unfractured bone (FIG. 4H). Therefore, once PSC are mobilized from bone surface to the callus, Prg4 splicing is reprogrammed to favor the expression of Prg4-S, a unique splice variant whose biological functions are uninvestigated thus far.

[0219] Prg4-S mRNA encodes a secreted protein.

[0220] The FL protein encoded in the Prg4-L mRNA is a secreted protein (Jay, G. D. et al., “Association between friction and wear in diarthrodial j oints lacking lubricin,” Arthritis Rheum. , 2007; 56:3662-3669; Waller, K. A. et al., “Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis,” Proc. Natl. Acad. Sci. USA., 2013; 110:5852-5857; Hill, A. et al., “Lubricin restoration in a mouse model of congenital deficiency,” Arthritis Rheumatol. , 2015; 67:3070-3081). In order to determine whether the short splice variant Prg4-S encodes a secreted protein as well, the motifs of PRG4-L protein were analyzed, and the 24 amino acids at the N- terminal were revealed as the signal peptide that targets the protein to the secretory pathway (see SignalP 6.0 results in FIG. 5A) (Teufel, F. et al., “SignalP 6.0 predicts all five types of signal peptides using protein language models,” Nat. BiotechnoL, 2022; 40: 1023-1025; Gutierrez Guarnizo, S. A. et al., “Pathogenic signal peptide variants in the human genome,” NAR Genom. Bioinform., 2- 23; 5(4):lqad093). An alignment between the sequences of Prg4-L and Prg4-S proteins indicates that the signal peptide is retained in PRG4-S (FIG. 5B), which suggests that PRG4-S is a secreted protein. To confirm this hypothesis, primary PSCs (sorted from the callus) were cultured for 2 days. The cells and the medium were harvested from the cell culture, and PRG4 Western blotting (WB) was performed on both the cells and the medium. PRG4-S was mainly detected in the medium (FIG. 5C), which confirms that PRG4-S is indeed a secreted protein.

[0221] PRG4-S increases that abundance of PSC in the healing callus.

[0222] The fact that the callus microenvironment stimulates the expression of PRG4-S in a tissue-specific way (FIG. 4H) inspired the inventors of the present application to investigate the biological functions of PRG4-S in fracture healing. To that end, a complex of atelocollagen and nanoparticles was used to achieve sustained release of Prg4 siRNA locally in the callus (Kawakami, Y. et al., “A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization,” Lab. Invest., 2013; 93: 1036-1053; Yoshizuka, M. et al., “Inhibition ofAttorney Docket No. 0073605-001029 microRNA-222 expression accelerates bone healing with enhancement of osteogenesis, chondrogenesis, and angiogenesis in a rat refractory fracture model,” J. Orthop. Sci., 2016; 21 :852- 858; Ito, Y. et al., “Both microRNA-455-5p and -3p repress hypoxia-inducible factor-2a expression and coordinately regulate cartilage homeostasis,” Nat. Commun., 2021; 12:4148) (see Materials and Methods). The siRNA was designed to be Prg4-S specific. Both qPCR and immunofluorescence (IF) staining indicated efficient siRNA-mediated knockdown (KD) of Prg4 over the first 10 days of healing (FIGS. 5D-H). Analyses were focused on this period, as Prg4 expression was barely detectable in the callus beyond dlO (FIG. 3B).

[0223] The effect of Prg4-S KD on PSC, from which it is secreted, was first analyzed. FC analysis of d5 callus indicated that Prg4-S KD resulted in a ~5-fold reduction in the relative abundance of PSC (FIGS. 51, J). This reduction can be, at least in part, due to the autocrine effects of Prg4-S. Thus, Prg4-S maintains the number of PSC in the callus, which is crucial for normal healing.

[0224] PRG4-S has paracrine immunomodulatory effects.

[0225] Being a secreted protein, PRG4-S has the potential to have paracrine effects that regulate the homeostasis of different cellular populations within the callus. To investigate this potential effect, the impact of Prg4-S KD on the immune response was assessed. An analysis of immune cells during different phases of healing revealed a sharp immune-cell infiltration into the callus on d5 (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14:1250309). Analysis of immune response on d5 indicated that Prg4-S KD increased the total number of immune cells by ~30% (FIGS. 6A-B). The different types of immune cell that populate d5 callus were further analyzed. The myeloid cells were first analyzed because they are the largest constituent of the callus immune populations and play well-established roles in bone healing (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet- induced obesity,” Front. Immunol., 2023; 14: 1250309; Kovtun, A. et al., “The crucial role of neutrophil granulocytes in bone fracture healing,” Eur. Cell. Mater., 2016; 32: 152-162; Kovtun, A. et al., “Neutrophils in Tissue Trauma of the Skin, Bone, and Lung: Two Sides of the Same Coin,” J. Immunol. Res., 2018; 2018:8173983; Bastian, O. W. et al., “Neutrophils contribute to fracture healing by synthesizing fibronectin+extracellular matrix rapidly after injury,” Clin. Immunol., 2016; 164:78-84; Soltan, M. et al., “Monocytes: super cells for bone regeneration,” Implant Dent., 2012; 21(1): 13-20; Yahara, Y. et al., “Monocyte / Macrophage Lineage Cells From Fetal Erythromyeloid Progenitors Orchestrate Bone Remodeling and Repair,” Front. Cell. Dev. BioL, 2021; 9:622035;Attorney Docket No. 0073605-001029Baht, G. S., et al., “The Role of the Immune Cells in Fracture Healing,” Curr. Osteoporos. Rep., 2018; 16:138-145; Starlinger, J. et al., “The influence of M-CSF on fracture healing in a mouse model.,” Set. Rep., 2021; 11 :22326; Frade, B. et al., “The role of macrophages in fracture healing: a narrative review of the recent updates and therapeutic perspectives,” Stem Cell Investig., 2023; 10:4; Schlundt, C. et al., “Macrophages in bone fracture healing: Their essential role in endochondral ossification,” Bone, 2018; 106:78-89). Prg4-S KD increased the relative abundance of CD1 lb+myeloid cells by -25% (FIGS. 6C-D). Within the CD1 lb+myeloid cells, Prg4-S KD increased the relative abundance of the polymorphonuclear granulocytes (PMNs) and myeloid cell progenitors, the largest population within CD1 lb+myeloid cells (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309) by ~35% (FIGS. 6E-F). Therefore, the observed increase in the abundance of total immune cells (FIGS. 6A-B) is largely attributed to expansion in the large CD1 lb+and PMN myeloid populations (FIGS. 6C-F). Analysis of other myeloid cells indicated significant elevation in the relative abundance of macrophages (FIGS. 7A-B), non-classical (Ly6CLo) monocytes (FIGS. 7A-B), and eosinophils (FIGS. 7C-D) in the callus of Prg4-S KD mice, while the relative abundance of classical monocytes was significantly reduced (FIGS. 7A-B).

[0226] Dendritic cells (DC) are closely related populations to the monocyte / macrophage axis, and they act as a crucial linkage between the innate and adaptive immune response (Cabeza- Cabrerizo, M. et al., “Dendritic Cells Revisited,” Ann . Rev. Immunol., 2021; 39: 131-166). Analysis of the two conventional DC (eDC) subpopulations, cDCl and cDC2, and the two effector subpopulations, the plasmacytoid DC (pDC) and monocyte-derived DC (Mo-DC), indicated that Prg4-S KD had diverse effects on DC subpopulations. While a depletion of Prg4 increased the abundance of cDC2 and cDCl (FIG. 7E-F; FIGS. 16A-B), it reduced the abundance of Mo-DC (FIGS. 7E-F) and had no effect on pDC (FIGS. 16A-B).

[0227] Lymphocytes also populate d5 callus (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309). In the T-cell compartment, Prg4-S KD increased the abundance of CD8 T cells but had no effects on CD4 T cells (FIGS. 10A-B). Prg4-S KD also increased the abundance of total B cells (FIGS. 8C-D), an increase that was observed on both immature (B220Lo) and mature (B220H1) B-cell populations (FIGS. 8E-F). Taken together, inhibition of the expression of Prg4-S in the callus resulted in an exacerbated response in numerous the immune-cell compartments.Attorney Docket No. 0073605-001029

[0228] Prg4-S regulates the homeostasis and hypertrophy of soft-callus chondrocytes.

[0229] To further analyze the paracrine effects of PRG4-S, dlO callus were harvested from mice that were treated with either Control or Prg4-S siRNA. Day-10 callus is formed of a bulky cartilaginous (soft) callus that contains a mixture of proliferating and hypertrophic chondrocytes.The soft callus is surrounded by newly formed woven bone (hard callus) at sites distal to the fracture line (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309; Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38:1560-1576). The soft callus was analyzed first, and the analysis revealed that Prg4-S KD resulted in substantial defects in soft callus formation and size (FIG. 9A; FIGS. 17A-B). Prg4-S KD also accelerated chondrocyte hypertrophy as evidenced by increased expression of the pre-hypertrophy marker Indian hedgehog (IHH; FIG. 9B) and the hypertrophy markers MMP13 and Col X (FIGS. 9C, D). Prg4-S KD also inhibited chondrocyte proliferation (FIG. 9E) and induced chondrocyte apoptosis (FIG. 9F). Collectively, these data show that PRG4-S plays critical roles in soft-callus formation and in regulating chondrocyte homeostasis by promoting chondrocyte proliferation and inhibiting premature chondrocyte hypertrophy and apoptosis.

[0230] PRG4-S promotes new hone formation.

[0231] Micro-CT (pCT) analysis of dlO callus indicated significantly reduced bone volume density (BV / TV) and bone mineral density (BMD) in mice treated with Prg4-S siRNA relative to mice treated with Ctrl siRNA (FIGS. 10A-B), demonstrating roles for Prg4-S in new bone formation. Notably, Prg4-S KD had no significant effects on proliferation or apoptosis of cells within the woven bone area of dlO callus (FIGS. 9B, C), suggesting that Prg4-S promotes bone formation via mechanisms other than regulating proliferation and / or viability of bone cells.

[0232] PRG4-S promotes the chondrogenic and osteogenic differentiation ofPSC.

[0233] Experimental data indicated that Prg4-S KD caused defective bone and soft-callus formation (FIGS. 9A, 10A-B). This result might be the outcome of low PSC abundance (FIGS. 51, J), dysregulated immune response (FIGS. 6A-F, 7A-F, 8A-F), or reduced chondrocyte viability and proliferation (FIGS. 9E, F) in Prg4-S siRNA-treated mice. Another possibility to be investigated is whether PRG4-S promotes the differentiation ofPSC. To assess this possibility, primary PSCsAttorney Docket No. 0073605-001029 sorted from d5 callus were cultured and subjected to chondrogenic or osteogenic differentiation conditions. One group of PSCs were differentiated under normal conditions, while the differentiation medium of the other group was conditioned with Prg4-S protein. Results of this experiment demonstrated that Prg4-S significantly boosted both the chondrogenic and osteogenic differentiation of primary PSC (FIGS. 11A, B). Accordingly, PRG4-S increases the abundance of PSC in the callus (FIGS. 51, J) and promotes the differentiation of these PSC as a means to promote fracture healing.

[0234] An engineered human analog of murine Prg4-S promotes the chondrogenic and osteogenic differentiation of mesenchymal stem cells.

[0235] As mentioned above, murine Prg4-S is listed in the murine mRNA databases (FIG. 4D, based on SEQ ID NO: 4). On the other hand, no human analog of this splice variant has ever been listed (FIG. 4D). This discrepancy raised the question of whether the osteogenic and chondrogenic function of this short variant of Prg4 is conserved in humans. To answer this question, the FL human (h)Prg4 was used to clone a variant that lacks exons 5, 6, and 7 and resembles murine Prg4-S (FIG. 11C; FIG. 18A). This engineered human short variant may be referred to as hPrg4-S. The regions cloned in hPrg4-S are highly conserved between mouse and human, which results in high similarity between proteins encoded in murine Prg4-S and the engineered hPrg4-S (FIGS. 11D-E, FIG. 18B). The effects of hPRG4-S on the differentiation of human bone marrow mesenchymal stem cells (hBMSC) were tested, in which Prg4 expression is almost undetectable. Conditioning the differentiation medium with recombinant hPRG4-S promoted both osteogenic and chondrogenic differentiation of hBMSC (FIGS. 11F-G). These findings indicate that the functions of Prg4-S are conserved from mouse to human.Discussion

[0236] The integrity of the ECM is crucial for bone structure and mechanical properties (Alcorta-Sevillano, N., et al., “Deciphering the Relevance of Bone ECM Signaling,” Cells, 2020;9(12):2630). The functions of ECM proteins extend beyond being structural scaffolds to regulate variable cellular processes (Alcorta-Sevillano, N., et al., “Deciphering the Relevance of Bone ECM Signaling,” Cells, 2020;9(12):2630; Lin, X. et al., “The Bone Extracellular Matrix in Bone Formation and Regeneration,” Front. Pharmacol . , 2020; 11 :757); however, many of these functions are not well defined. The present study revealed the novel functions of the ECM component PRG4 and showed that it regulates biological processes that play central roles in bone regeneration. Further, the microenvironment of the fracture callus was found to induce the expression of an uninvestigated splice variant of Prg4.Attorney Docket No. 0073605-001029

[0237] Being a proteoglycan, the FL PRG4 protein is highly glycosylated, and the attached GAG molecules are central to the roles of PRG4 in the articular cartilage. The GAG molecules allow PRG4 to maintain cartilage hydration and to lubricate load bearing surfaces (Swann, D. A. et al., “The molecular structure of lubricating glycoprotein-I, the boundary lubricant for articular cartilage,” J. Biol. Chem., 1981; 256:5921-5925; Schmidt, T. A. et al., “Transcription, translation, and function of lubricin, a boundary lubricant, at the ocular surface,” JAMA Ophthalmol., 2013; 131 :766-776). Glycosylation sites are located in the mucin domain, which is encoded in exon 7 of the Prg4-L mRNA (FIGS. 4C, D; FIG. 13). Exon 7 constitutes ~62% of Prg4-L coding region. The fact that the murine Prg4-S splice isofonn lacks almost all the mucin domain is likely the reason why it remained uninvestigated prior to the present study. The absence of the mucin domain nullifies most of what is known about Prg4 as a structural protein and a lubricant. Prg4-S maintains the bland C-termini of Prg4-L that contain the protein-protein and protein-receptor interaction motifs, which might explain the ability of Prg4-S to act as a signaling molecule and induce several cellular processes in the healing callus (FIGS. 5 A- J, 6A-F, 7A-F, 8A-F, 9A-F, 10A-D, 11-G). The lack of the big, glycosylated mucin domain may have enabled Prg4-S to diffuse easily in the callus area and induce multiple signaling effects in various cellular populations.

[0238] Prg4 is mainly expressed in the callus by a group of cells that express canonical markers for chondro-osteo progenitors. This cluster includes Postn+cells which, according to the current knowledge, represent periosteal progenitors (FIG. 3E). The large relative abundance of this progenitor population, especially during early timepoints (FIG. 3F), is consistent with the significant role of PDGFRa Scal+and of periosteal Postn+progenitors in fracture healing. Populations of Prg4 stem cells have been identified that reside in different tissue during homeostatic conditions, and upon injury, these cells have the ability to migrate to the injury site, expand, and contribute to tissue repair (Massengale, M. et al., “Adult Prg4+progenitors repair long-term articular cartilage wounds in vivo," JOI Jnsight, 2023; 8(17):el67858; He, Q. et al., “Prg4+fibro-adipogenic progenitors in muscle are crucial for bone fracture repair,” Proc. Natl. Acad. Sci. USA, 2025; 122(3 l):e2417806122). These findings identified Prg4 as a marker for stem cells with high regenerative potential. The data of the present study adds an important dimension by showing that the expression of Prg4 in stem cells is strongly induced at the injury site, which causes expansion of stem cells (FIGS. 51- J) and induces their chondrogenic and osteogenic differentiation (FIGS. 11 A, B). These findings can potentially explain why Prg4+stem cells have high regenerative potential. Notably, caution should be practiced in concluding the origin of Prg4-expression stem cells that populate the callus based onAttorney Docket No. 0073605-001029 the profiles of Prg4 expression in intact tissues, because cells are induced in the callus microenvironment to express genes that are normally silenced under homeostatic, normal physiological conditions.

[0239] The clinical situations where a callus is collected during the early healing phases (when Prg4 is expressed) are extremely rare; thus, it may not be feasible to confirm Prg4 expression in the human callus. However, the amino acids sequence and protein domains in murine PRG4-S are highly conserved in hPRG4, which made it possible to engineer a human analogue of murine PRG4- S that lacks exons 5-7 (FIG. 11C; FIGS. 18A, B). hPRG4-S promoted the chondrogenic and osteogenic differentiation of BMSC, which indicates that these functions are conserved from mouse to human. These results have translational potential and open the door for clinical application of hPRG4-S in treating defective healing or as an adjuvant in stem-cell therapy.

[0240] The high expression of Prg4 in the callus was accompanied by splicing reprogramming to favor the expression of Prg4-S over Prg4-L (FIG. 4H). This spatiotemporally regulated and tissuespecific pattern of Prg4-S expression defined the functions of Prg4-S in fracture callus. The full- length Prg4-L isoform can reduce the recruitment of macrophages to different tissues (Krawetz, R. J. et al., “Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration,” NPJ Regen. Med., 2022;7:32; Ninkovic, N. et al., “Proteoglycan 4 (PRG4) treatment improves skin wound healing in a porcine model,” FASEB J. 2024; 38:e23547; Qadri, M. et al., “Proteoglycan-4 is an essential regulator of synovial macrophage polarization and inflammatory macrophage joint infiltration,” Arthritis Res. Ther., 2021; 23:241) and this function of Prg4-L is one of the underpinnings of its use in treating OA. The results provided herein demonstrate that, in the context of fracture healing, Prg4-S moderates macrophage infiltration in the fracture callus (FIGS. 7A-B). The present data also extended beyond monocytes / macrophages to identify regulatory effects of Prg4-S on other immune-cell populations and found that absence of Prg4-S boosts the number of immune cells in the callus, which was mainly attributed to the increased myeloid-cell infiltration (FIGS. 6A-F; FIGS. 7A-D). This increased number of innate-immune cells in the callus is comparable to what is observed in inflammatory diseases typified by delayed healing, such as diet- induced obesity and diabetes (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309). Inhibiting Prg4-S also increased CD8+ / CD4+T-cell ratio (FIGS. 8A-B), a parameter that has been reported to be associated with impaired fracture healing (Schlundt, C. et al., “Individual Effector / Regulator T Cell Ratios Impact Bone Regeneration,” Front. Immunol., 2019;Attorney Docket No. 0073605-00102910: 1954). The data presented herein support important immunomodulatory roles of Prg4-S during fracture healing and suggest that the observed immune dysregulation in the callus of Prg4-S KD mice must have contributed to the overall defective healing in these mice. Notably, some of these immunomodulatory effects can be direct effects of Prg4-S on the corresponding immune populations, while others might be secondary given that disturbing one type of immune cells can cross talk with and impact other immune populations. Future studies will help deconvolute these scenarios. As mentioned above, the small and less complex structure of Prg4-S as compared to Prg4- L might have made Prg4-S a more potent signaling molecule, but proving these points await more comprehensive characterization of the immunomodulatory effects of Prg4-L. This situation also applies to other functions of Prg4-S. For example, Prg4-L has chondrogenic and chondroprotective effects and inhibits chondrocyte hypertrophy; functions that we also observed for Prg4-S (FIGS. 9A- F). However, to what extent each variant inhibits chondrocyte hypertrophy remains unknown. Notably, Prg4-L is expressed in the articular cartilage where chondrocyte hypertrophy must be completely inhibited to avoid OA development, while Prg4-S is expressed during fracture healing where chondrocyte hypertrophy must be temporally controlled but not inhibited, as it constitutes an essential step in the endochondral ossification process. Fracture healing may not be the appropriate physiological context to study the differential functions of Prg4-S and Prg4-L, because Prg4-L is not expressed in the callus (FIG. 4G). Studies identifying the unique functions of each variant in physiological conditions where both variants are expressed are warranted and expected to provide further insights suitable variant(s) to be used in each clinical application (which can be a mixture of more than one variant). These studies are expected to provide more effective, application-tailored therapeutic approaches.

[0241] Prg4 receptors have never been globally characterized in any tissue. Toll-like receptors (TLRs) have been reported to interact with Prg4-L in macrophages. The lack of the mucin-like domain in Prg4-S, and the consequential difference in the 3D structure between Prg4-S and Prg4-L, makes it possible that Prg4-S binds different receptors than those of Prg4-L. Prg4-S receptors remain completely unknown. The heterogeneity of the callus and the wide-spread effects of Prg4-S on different cellular populations raises the possibility that Prg4-S might bind different receptors in different cells. Studies that identify Prg4-S receptors on different cell types will open translational fronts regarding the use of receptor agonists / antagonists to modulate specific cells so as to promote fractur healing.Attorney Docket No. 0073605-001029

[0242] The study presented herein establishes regenerative and pro-healing roles of PRG4-S. The study informs future translational applications of PRG4 in the field of fracture healing and shift the focus from all the reported splice variants, whose expression are completely repressed during normal fracture healing, to the novel PRG4-S variant. The results described herein can inspire future studies assessing the expression and splicing patterns of PRG4 in models of delayed healing and propose PRG4-S as an attractive / novel therapeutic approach for nonunion and defective bone formation. The shorter sequence of PRG4-S mRNA and the smaller size and less complex structure of PRG4-S protein as compared to other variants that contain the mucin domain can facilitate its production and application as RNA or protein therapy.EXAMPLE 2Significance

[0243] Preliminary data described in the present study show high expression of Prg4 in the periosteal bone progenitors that populate the callus during the early inflammatory phase of fracture healing, and that Prg4 plays central roles during different phases of the healing process. Further, the present study can reveal novel roles of Prg4 during fracture healing and signaling pathways that underpin these roles. Collectively, this exemplary study elucidates roles of ECM in cell signaling and tissue homeostasis.

[0244] Characterizing novel roles of alternative splicing (AS) during fracture healing.

[0245] Co- and post-transcriptional regulatory pathways play essential roles in regulating gene expression. Understanding how gene expression is regulated under normal physiological conditions sets the stage for future design of RNA-based therapeutics. Alternative splicing (AS), the process of removing introns from pre-mRNAs and connecting the exons in different combinations, is a fundamental regulatory process that generates diversified mature mRNAs from the same gene (Faustino, N. A. & Cooper, T. A., “Pre-mRNA splicing and human disease,” Genes Dev., 2003;17: 419-437). The roles of AS in tissue homeostasis are well-established, and RNA mis-splicing underlies several diseases (Scotti, M. M. & Swanson, M. S. “RNA mis-splicing in disease,” Nat. Rev. Genet., 2016; 17: 19-32). Although AS has been examined in several biological contexts, it is notably under-investigated in the context of bone repair. The healing process starts with an influx of immune and inflammatory cells in the callus, conditions that are considered to mutually crosstalk with AS (Zhou, J. et al., “The regulatory role of alternative splicing in inflammatory bowel disease,” Front. Immunol., 2023; 14: 1095267; Robinson, E. K. et al., “Inflammation drives alternative firstAttorney Docket No. 0073605-001029 exon usage to regulate immune genes including a novel iron-regulated isoform of Aim2,” Elife, 2021; 10:e69431; Wu, W. et al., “The Impact of Pro-Inflammatory Cytokines on Alternative Splicing Patterns in Human Islets,” Diabetes, 2021; 71(1): 116-127; Janssen, W. J. et al., “Inflammation-Induced Alternative Pre-mRNA Splicing in Mouse Alveolar Macrophages,” G3 (Bethesda), 2020; 10 555-567). In addition, TGFB1 and other cytokines that are expressed and play critical roles during fracture healing are reported to regulate splicing of multiple genes (Tripathi, V et al., “TGF-beta-induced alternative splicing of TAK1 promotes EMT and drug resistance,” Oncogene, 2019; 38:3185-3200; Madne, T. H. & Dockrell, M. E. C., “TGF[31 -mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture,” Cell. Mol. Biol. (Noisy-le-grand) , 2018; 64: 17-24; Tripathi, V. et al., “Direct Regulation of Alternative Splicing by SMAD3 through PCBP1 Is Essential to the Tumor-Promoting Role of TGF- P,” Mol. Cell, 2016; 64(5):549-564). These findings underline the importance of studies that address AS during fracture healing.

[0246] The present study identifies a novel AS isoform of Prg4 secreted by the periosteal progenitors in the callus. Importantly, the periosteal progenitors that reside on the surface of unfractured bone secret different Prg4 AS isoforms and at a substantially lower expression level as compared to the periosteal progenitors that infiltrate the callus. This finding indicates that the callus micro-environment strongly induces the expression of Prg4 and reprograms its splicing. These effects are largely mediated by TGFB1. The present study also indicates that this unique Prg4 AS isoform plays important autocrine and paracrine roles in the callus via which it regulates both the inflammatory and repair phases of healing. Results of the present study i) reveal AS as a crucial gene regulatory mechanism during fracture healing, ii) provide global insights into splicing programming in the healing callus, and iii) specifically reveal novel signaling mechanisms of Prg4 that regulate its expression and splicing.

[0247] Opening new translational avenues for an engineered splice variant of human PRG4.

[0248] The preliminary of the present study indicate that the splice variant of Prg4 identified in the mouse callus has unique pro-healing effects. This finding justifies, at least in part, why splicing generates this isoform in the callus. A human splicing variant analogous to the one identified in mouse callus has never been reported. RefSeq and UCSC human transcriptome libraries do not list this splice variant either, which raised the question of whether this variant is conserved in human. The fact that this variant is more likely to be expressed specifically during the early inflammatory phase of the healing makes it extremely difficult to secure a human sample to validate whether theAttorney Docket No. 0073605-001029 variant is expressed. To overcome this problem, the conservation of PRG4 protein domains in human vs. mouse was studied first. Interestingly, the protein domains that constitute the short callusspecific splicing variant are highly conserved from mouse to human, while other domains that the callus-specific variant lacks are not conserved or show very low conservation level. This finding leads to the discovery of an engineered human splice variant of PRG4 that mimics the one identified in the mouse callus. Interestingly, the engineered human protein strongly enhances the osteogenic differentiation of human bone-marrow mesenchymal stem cells (hBMSCs), while the other isoforms of human PRG4 did not have any osteogenic effects. Thus, the engineered human splice variant of PRG4 has potent osteogenic effects, which has important translational applications and can be harnessed to induce healing in pathological conditions where osteogenesis is impaired.

[0249] Innovative aspects of the present study include without limitation i) discovery of a novel splicing variant of Prg4 that possess osteogenic and pro-healing effects, ii) investigation of the impact of an engineered recombinant human PRG4 variant on rescuing the healing in a mouse model of defective healing, and iii) investigation of the crosstalk between the callus micro-environment and AS; iv) use of complementary, state-of-the-art techniques to study gene expression regulation, which include analyzing AS in a specific cell type, and crosslinking-immunoprecipitation from the callus tissue followed by mass spectrometry, and v) use of techniques to knockdown specific AS isoform locally in the callus.Approach

[0250] Biological variance and scientific rigor.

[0251] All experiments are performed on C57BL / 6J background. Both male and female mice will be used. Adult, 4-mo-old mice are used for both in-vivo studies and isolation of primary cells. Mice that show misalignment of the fracture will be excluded from the study. Analyses are performed by researchers blinded to treatment. Sample size is calculated using 'pwr2' R package, and power and significance levels were provided as 0.8 and 0.05, respectively. The calculated sample size to detect 30-35% difference is 4-5 for in-vitro experiments, 10 for biomechanical testing, and 6- 7 for the rest of experiments. Unpaired Student’s t test is used to compare 2 groups, whereas ANOVA (followed by Tukey’s post hoc test) is used to compare 3 or more groups. Statistical analyses are performed using the GraphPad Prism software.

[0252] Rigor of the Research.

[0253] PRG4 / lubricin is conserved throughout the animal kingdom (Ikegawa, S., et al., “Isolation, characterization and mapping of the mouse and human PRG4 (proteoglycan 4) genes,”Attorney Docket No. 0073605-001029Cytogenet. Cell. Genet., 2000; 90(3-4):291 -297). PRG4 is expressed at high concentration in vertebrate synovial fluid and the superficial zone of the articular cartilage (hence its alias superficial zone protein “STZ”) and acts as a lubricant to reduce friction between load bearing surfaces (hence its alias lubricin) (Swann, D. A. et al., “The molecular structure of lubricating glycoprotein-I, the boundary lubricant for articular cartilage,” J. Biol. Chem., 1981; 256:5921-5925; Schmidt, T. A. et al., “Transcription, translation, and function of lubricin, a boundary lubricant, at the ocular surface,” JAMA Ophthalmol., 2013; 131 :766-776). Mice that lack Prg4 exhibit chondrocyte apoptosis and early onset of osteoarthritis (OA) (Waller, K. A. et al., “Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis,” Proc. Natl. Acad. Sci. USA, 2013; 110:5852-5857; Jay, G. D. et al., “Association between friction and wear in diarthrodial joints lacking lubricin,” Arthritis Rheum., 2007; 56:3662-3669; Hill, A. et al., “Lubricin restoration in a mouse model of congenital deficiency,” Arthritis Rheumatol., 2015; 67:3070-3081). The potential of recombinant PRG4 as an OA disease-modifying therapy has been shown in pre-clinical models (Alquraini, A. et al., “The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes,” Arthritis Res. Ther., 2017;19:89; Cui, Z. et al., “Treatment with recombinant lubricin attenuates osteoarthritis by positive feedback loop between articular cartilage and subchondral bone in ovariectomized rats,” Bone, 2015; 74:37-47; Jay, G. D. et al., “Prevention of cartilage degeneration and restoration of chondroprotection by lubricin tribosupplementation in the rat following anterior cruciate ligament transection,” Arthritis Rheum., 2010; 62:2382-2391; Teeple, E. et al., “Effects of supplemental intra-articular lubricin and hyaluronic acid on the progression of posttraumatic arthritis in the anterior cruciate ligamentdeficient rat knee,” Am. J. Sports. Med., 2011; 39: 164-172; Jay, G. D. et al., “Prevention of cartilage degeneration and gait asymmetry by lubricin tribosupplementation in the rat following anterior cruciate ligament transection,” Arthritis Rheum., 2012; 64: 1162-1171; Elsaid, K. A. et al., “The impact of forced joint exercise on lubricin biosynthesis from articular cartilage following ACL transection and intra-articular lubricin's effect in exercised joints following ACL transection,” Osteoarthritis Cartilage, 2012; 20:940-948; Elsaid, K. A. et al., “The impact of early intra-articular administration of interleukin- 1 receptor antagonist on lubricin metabolism and cartilage degeneration in an anterior cruciate ligament transection model,” Osteoarthritis Cartilage, 2015; 23: 114-121). PRG4 is expressed in tissues other than articular cartilage (Ikegawa, S., et al., “Isolation, characterization and mapping of the mouse and human PRG4 (proteoglycan 4) genes,” Cytogenet. Cell. Genet., 2000; 90(3-4):291-297); however, its biological functions in these tissues remainAttorney Docket No. 0073605-001029 largely unknown. A recent study has shown that Prg4 plays important roles in wound closure and tissue regeneration using an ear wound model (Krawetz, R. J. et al., “Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration,” NPJ Regen. Med., 2022; 7:32), and these roles are independent of Prg4 roles as a lubricant (Krawetz, R. J. et al., “Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration,” NPJ Regen. Med., 2022; 7:32). However, the functions of Prg4 during bone repair remain largely unknown prior to the present study.

[0254] Alternative splicing of Prg4.

[0255] The 4278-nt (full length) mature mRNA of Prg4 (NM_021400) consists of 13 exons, 12 of them (exons 2-13) contain coding sequence (FIGS. 19A, B). The N-terminus of PRG4 contains somatomedin B domain and heparin binding sites, the C-terminus contains a hemopexin (HX) domain (FIG. 19A), while exon 7 in the middle (the longest exon that constitutes -63% of the coding region “CDS”) encodes mucin-like repeats (FIG. 19A). A very limited number of studies have investigated Prg4 splicing variants in musculoskeletal tissues, and these studies were limited to articular cartilage (DuRaine, G. D., et al., “Effects of TGF-pi on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110). Prg4 splicing variants reported in articular cartilage lack one or more of exons 3-6 (FIG. 19A, isoforms A- E) (DuRaine, G. D., et al., “Effects of TGF-pi on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110) and are differentially expressed in the anterior load-bearing locations of the femoral medial condyle as compared to the posterior non-load-bearing locations (DuRaine, G. D., et al., “Effects of TGF-pi on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110). These isoforms are expected to have different pattern and kinetics of interaction with ECM components; however, the distinctive functions of these isoforms have not been investigated until the present study. More importantly, the Prg4 variants that are prevalent in musculoskeletal tissues other than the cartilage have never been investigated until the present study. On the Reference Sequence (RefSeq) data base of the NCBI and the UCSC genome browser, there are 2 splice variants of murine Prg4: i) the full-length variant that contains all the exons (NM_021400 in FIGS. 19A, B), which is termed Prg4-Long or Prg4-L in the present disclosure, and ii) a short AS isoform (NM 001110146 in FIG. 19B, SEQ ID NOs: 3-4), which is termed Prg4-S in the present disclosure. Prg4-S is different from all the A-E variants defined in the articular cartilage (FIGS. 19A, B) as it lacks exons 5, 6, and all of the mucin-like repeats in exon 7 (a comparison between Prg4-LAttorney Docket No. 0073605-001029 and Prg4-S is provided in FIG. 19B). The expression pattern and biological functions of the Prg4-S isoform have never been investigated in any musculoskeletal tissue until the present study.Preliminary data

[0256] Prg4 is exclusively expressed in the periosteal stem cells during the inflammatory phase.Tibial mid-diaphyseal osteotomy supported by an intramedullary nail was performed according to the standard protocol, using 4-mo-old mice (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023; 11 :5; Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576; Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309; Wee, H., et al., “Assessment of Bone Fracture Healing Using Micro-Computed Tomography,” J. Vis. Exp., 2022). First, fracture callus was harvested at time points that cover the entire healing process (days 3-to-35) (FIG. 3B). Then, RNA was extracted to generate high-quality RNA (Le Bleu, et al., “Extraction of high-quality RNA from human articular cartilage,” Anal. Biochem., 2017; 518: 134-138), and total RNA-seq was performed (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309). Analysis of Prg4 expression pattern indicated exceptionally high expression during the inflammatory phase (days 3 and 5 postfracture), which decreased steadily during the cartilaginous callus phase (days 7-14) and became very low at the later phases (FIG. 3B). This observation raised a question about the cell type(s) that exhibits such high expression during the inflammatory phase. Single-cell RNA-seq (scRNA-seq) was then performed on the callus of day 5 post-fracture (d5): the callus / hematoma was digested (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309), and 22,854 cells were sequenced (2207 median gene # per cell; 3 mice combined). The data indicated that Prg4 expression was restricted to one cell cluster (cluster 2, FIG. 20). This cluster was defined as periosteal progenitors / stem cells (PSCs) based on the expression of the established PSC markers, including Pdgfra, Ly6a (Seal), Prrxl, and periostin (Postn) (FIG. 20). All other scRNA-seq clusters were negative for Prg4 expression (FIG. 20). Thus, Pg4 expression observed during theAttorney Docket No. 0073605-001029 inflammatory phase (FIG. 3B) is attributed to PSCs. This pool of PSCs serves as a reservoir of progenitors that differentiate to chondrocytes and osteoblasts during fracture healing (Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576; Xu, J. et al., “PDGFRalpha reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair,” Bone Res., 2022; 10:7). When CD45‘ (to exclude immune cells), CD3 F (to exclude endothelial cells) PDGFRa+SCAl+cells were isolated from the callus or intact bone, these cells showed high chondrogenic and osteogenic potential in vitro (Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576; Xu, J. et al., “PDGFRalpha reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair,” Bone Res., 2022; 10:7). Further, when these PDGFRa+cells were labelled by tdTomato reporter prior to fracture, tdTomato was localized to the periosteum of the unfractured bone (FIG. 21 A) (Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38:1560-1576; Xu, J. et al., “PDGFRalpha reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair,” Bone Res., 2022; 10:7). Following fracture, the majority of callus chondrocytes and bone cells were tdTomato+(FIG. 21B) (Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38:1560-1576; Xu, J. et al., “PDGFRalpha reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair,” Bone Res., 2022; 10:7), indicating that this pool of cells, which is the sole source of Prg4 in the healing hematoma, is the major source of chondrocytes and osteoblasts during fracture healing. This pool is accordingly referred to as PSCs. For FACS isolation, this pool can be more specifically defined as CD45 CD3 F PDGFRa+SCAl+(Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576; Xu, J. et al., “PDGFRalpha reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair,” Bone Res., 2022; 10:7), as PDGFRa and SCA1 are the 2 surface markers expressed on these cells. For rigor, the expression of other markers in this pool is confirmed using RT-qPCR. To corroborate the scRNA-seq data, Prg4 expression was measured using qPCR in PSCs isolated from the callus byAttorney Docket No. 0073605-001029FACS. Results confirmed that Prg4 mRNA was only expressed in PSCs and almost undetectable in other cells on d5 (not shown) as well as d3 post-fracture (FIG. 4A). Cells that express Prg4 during the repair phase were then identified using immunofluorescence (IF) staining. Prg4 was expressed mainly by soft-callus chondrocytes (FIG. 21C) and was detectable at lower levels around bone and some bone marrow (BM) cells (FIGS. 21C, D). These data explain the expression pattern of Prg4 identified by the RNA-seq (FIG. 19A). Taken together, these data indicate that PSCs are the major source of Prg4 during the inflammatory phase, and that Prg4 is expressed at lower levels, mainly by chondrocytes, during the later phases of healing.

[0257] PSCs express only the Prg4-S isoform.

[0258] rMATS (Shen, S. et al., “rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data,” Proc. Natl. Acad. Set. USA, 2014; 111 :E5593-5601) was employed to analyze the bulk RNA-seq data of d5 for AS. The analysis identified Prg4-S as the major AS isoform of Prg4 at d5 callus (FIGS. 22A, B). These results were corroborated using RT- PCR (performed on d5 total-callus RNA) and two different primer pairs that span the skipped exons to differentiate Prg4-L from Prg4-S (depicted in FIG. 4F). A major band was detected at the expected size of Prg4-S, while Prg4-L was undetectable (FIG. 4G; FIG. 22C). The identity of the detected band as Prg4-S (NM_001110146, FIG. 19B) was confirmed by Sanger sequencing (Genewiz). Since PSCs are the sole source of Prg4 at d5 callus (FIGS. 20; FIG. 4A), PSCs from d5 callus were sorted, and the same RT-PCR was performed to identify Prg4 isoforms in PSCs. The same major band of Prg4-S was detected (FIG. 4A), and same results were obtained upon analyzing Prg4 isoforms in PSCs sorted from d3 callus (not shown). Taken together, during the inflammatory phase, Prg4-S is the main AS isoform of Prg4 in PSCs.

[0259] Prg4-S isoform encodes a secreted protein.

[0260] Prg4-L encodes a secreted protein (Alquraini, A. et al., “The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes,” Arthritis Res. Ther., 2017; 19:89). Does Prg4-S mRNA encode a secreted protein as well? To answer this question, different algorithms, including SignalP 6.0 (Teufel, F. et al., “SignalP 6.0 predicts all five types of signal peptides using protein language models,” Nat. Biotechno , 2022; 40: 1023-1025), were used to analyze PRG4-L motifs for a signal peptide that targets a protein to the secretory pathway. All algorithms defined the N-terminus 24 amino acids (FIG. 5A), which consists of the consensus N-H-C structure (FIG. 5A) (Teufel, F. etal., “SignalP 6.0 predicts all five types of signal peptides using protein language models,” Nat.Attorney Docket No. 0073605-001029Biotechnol., 2022; 40: 1023-1025; Gutierrez Guarnizo, S. A. et al., “Pathogenic signal peptide variants in the human genome,” NAR Genom. Bioinform. , 2023; 5(4):lqad093), as the signal peptide. This N-terminus signal peptide is retained in the PRG4-S isoform (FIG. 5B), which suggests that PRG4-S is a secreted protein. To confirm this finding, primary PSCs (sorted from d5 callus) were cultured for 2 days, the cells and the medium were harvested, and PRG4 Western blotting (WB) was performed on both using an antibody that can specifically detect both PRG4-S and PRG4-L (Abeam; ab28484). PRG4-S was mainly detected in the medium (FIG. 5C), which confirms that it is a secreted protein. Consistent with the RNA-seq and RT-PCR data (FIG. 4G; FIG. 22C), PRG4-L band was undetectable. For rigor , the identity of the WB band was confirmed by using a Prg4 siRNA. Prg4 siRNA significantly reduced the WB band intensity (FIG. 5C), which confirms antibody specificity. Importantly, to maintain consistency among in-vitro and in-vivo experiments (see below), the siRNA was designed to specifically target Prg4-S as it spans exon 4-exon 7 junction, which exists in Prg4-S but not Prg4-L (FIG. 19B).

[0261] Prg4-S plays central roles in fracture healing.

[0262] Data collected from the present study and the existing gap of knowledge inspired the investigation of the distinctive roles of Prg4-S in fracture healing. There are no available transgenic mice that allow specific depletion of AS isoforms. In addition, the existing transgenic mice allow constitutive (but not conditional) deletion of Prg4, which causes joint failure, hind paws deformity, and abnormal hopping gait (Rhee, D. K. et al., “The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth,” J. Clin. Invest., 2005; 2115:622-631). Thus, using any of these transgenic mice may complicate interpretation of the healing trajectory. Due to these limitations, a different technique was adopted that allowed specific knockdown (KD) of the Prg4-S isoform locally in the callus, by using a complex of atelocollagen (AteloGene Local Use kit) and 10 pM Prg4-S siRNA. Atelocollagen is a bioabsorbable, non-immunogenic scaffold that allows prolonged release of oligos locally at the transplanted site (Kawakami, Y. et al., “A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization,” Lab. Invest., 2013; 93: 1036-1053; Yoshizuka, M. et al., “Inhibition of microRNA-222 expression accelerates bone healing with enhancement of osteogenesis, chondrogenesis, and angiogenesis in a rat refractory fracture model,” J. Orthop. Sci., 2016; 21 :852-858; Ito, Y. et al., “Both microRNA-455-5p and -3p repress hypoxia-inducible factor-2a expression and coordinately regulate cartilage homeostasis,” Nat. Commun., 2021; 12:4148). A in-vivo ready, Silencer Select (Thermo) siRNA modified by locked nucleic acids (LNA) was used, which confers enhanced specificity and stability and dampensAttorney Docket No. 0073605-001029 the immunogenicity (Hu, B. et al., “Therapeutic siRNA: state of the art,” Signal Transduct Target Ther., 2020; 5: 101). The complex was applied at the fracture site following fracture induction and before suturing the mouse. Ctrl mice received Ctrl siRNA. For rigor, it was confirmed that atelocollagen alone or in complex with Ctrl siRNA did not affect immune response or overall healing (not shown). Although all data define Prg4-S as the main Prg4 AS isoform in the callus, for rigor, a Prg4-S-specific siRNA was used, which had the same sequence as the siRNA used in the in- vitro experiments described in FIG. 5C. Samples were harvested on days 5 and 10 post-fracture. Both RT-qPCR and IF staining indicated successful KD of Prg4 in the callus of Prg4-S siRNA- treated mice at both timepoints (FIGS. 5D, G, H). Importantly, the probe and antibody used in the qPCR and IF staining, respectively, were not specific to Prg4-S and quantitated total Prg4. Thus, the significant KD observed in the Prg4-S siRNA-treated mice (FIGS. 5D, G, H) provides further evidence that Prg4-S is the major splicing isoform of Prg4 in the callus.

[0263] Analysis of d5.

[0264] Since Prg4 is expressed exclusively in the PSCs in d5 callus, the impact of Prg4-S KD on the abundance of PSCs was first analyzed using flow cytometry (FC). Prg4-S KD resulted in ~5- fold reduction in the relative abundance of PSCs (FIGS. 6I-J). To study the paracrine effects of Prg4- S, the total immune (CD45+) cells were analyzed, and a ~50% increase in their relative abundance was observed (FIGS. 6A-B).

[0265] Analysis of dlO.

[0266] Day-10 callus is formed of a bulky cartilaginous (soft) callus that contains a mixture of proliferating and hypertrophic chondrocytes. The soft callus is surrounded by newly formed woven bone (hard callus) at sites distal to the fracture line (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38:1560- 1576; Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309).

[0267] Soft-callus analysis.

[0268] Prg4-S KD resulted in defective soft callus formation (FIG. 9A) and accelerated / pre- mature chondrocyte hypertrophy as evidenced by increased expression of the pre-hypertrophy marker Indian hedgehog (IHH, FIG. 9B) and the hypertrophy markers MMP13 and Col X (FIGS.Attorney Docket No. 0073605-0010299C, D). Prg4-S KD also inhibited chondrocyte proliferation (FIG. 9E) and induced chondrocyte apoptosis (FIG. 9F), and both parameters were analyzed in proliferating chondrocytes (which are negative for hypertrophy markers). These results are consistent with the roles of Prg4 in inhibiting chondrocyte apoptosis and hypertrophy in the articular cartilage (Waller, K. A. et al., “Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis,” Proc. Natl. Acad. Sci. USA, 2013; 110:5852-5857; Jay, G. D. et al., “Association between friction and wear in diarthrodial joints lacking lubricin,” Arthritis Rheum., 2007; 56:3662-3669; Hill, A. et al., “Lubricin restoration in a mouse model of congenital deficiency,” Arthritis Rheumatol., 2015; 67:3070-3081).

[0269] Hard callus analysis.

[0270] pCT exhibited defective bone formation in Prg4-S KD mice (FIGS. 10A-B). Cellular apoptosis and proliferation in the hard callus area were comparable in both groups (FIGS. 10C, 10D). Thus, Prg4-S KD inhibited major aspects of healing. Taken all the preliminary data together, it is speculated that 1) during the inflammatory phase, Prg4 expression is restricted to the PSCs, 2) Prg4-S is the major or sole AS isoform of Prg4 in the callus, 3) Prg4-S encodes a secreted protein, and 4) Prg4-S plays crucial roles in fracture healing. The specific roles of Prg4-S are further investigated in the following Aims.Aim 1 A: Roles of murine Prg4-S in fracture healing.

[0271] Preliminary data.

[0272] IF staining of dlO callus showed expression of Prg4 mainly by chondrocytes, and, at lower levels, by bone and BM cells (FIGS. 21C, D). But what are the Prg4 AS isoforms expressed in the callus at this timepoint? To answer this question, RT-PCR was performed, as shown in FIG. 4G, FIG. 22C, (primer pair 1) using RNA extracted from calli of days 7 and 10. Interestingly, Prg4-S was still the main isoform on both days, and no Prg4-L was detected (FIG. 15), consistent with the above-discussed data showing that Prg4-S siRNA downregulated total Prg4 on dlO to almost undetectable level (FIGS. 5G, H). Collectively, these data show that Prg4-S is the major (or sole) isoform in the callus over the first 10 days of healing (Prg4 expression beyond this time window is very low, FIG. 3B). Thus, Prg4-S is the focus of the studies described herein, and the same Prg4-S- specific siRNA / shRNA discussed above is used throughout the study. In Aim 1, the roles of Prg4-S in fracture healing are comprehensively defined as follows.

[0273] Tibial mid-diaphyseal fractures is performed using 4-mo-old male and female mice, and the mice are treated with either Ctrl or Prg4-S siRNA as detailed above (Khajuria, D. K. et al.,Attorney Docket No. 0073605-001029“Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137:115436; Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023; 11 :5; Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576; Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309). Calli are harvested on days 3, 5, 7, 14, 21, and 28 (in addition to dlO shown in FIGS. 7-8). These timepoints are chosen based on the healing time course in this model (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet- induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023; 11 :5; Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576; Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309; Brown, M. L. et al., “Delayed fracture healing and increased callus adiposity in a C57BL / 6J murine model of obesity-associated type 2 diabetes mellitus,” PLoS One, 2014; 9(6):e99656). Each timepoint is populated based on the Power analysis provided above. Importantly, these studies focus mainly on the inflammatory and cartilaginous phases because the Prg4 expression was barely detectable beyond these phases (FIG. 3B). However, calli from days 21 and 28 are also collected to assess the long-acting effects of inhibiting Prg4-S expression during the early healing phases. Notably, pronounced Prg4 KD was achieved for at least 10 days following implantation of the siRNA-atelocollagen at the surgery time (FIGS. 5D, G, H), which shows that a single application of the complex is sufficient for these studies that address Prg4-S roles during the first 2 weeks of healing. The harvested timepoints are analyzed as follows.

[0274] Analysis of immune response (Days 3, 5, and 21).

[0275] Preliminary data indicate that Prg4-S KD results in increased abundance of immune cells in the callus (FIGS. 6A-B). This result proposes an interesting mechanism whereby PSCs contribute to establishing the inflammatory micro-environment in the healing hematoma.Attorney Docket No. 0073605-001029

[0276] Thus, the analysis is further extended to characterize the impact of Prg4-S on the different populations of callus immune cells (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309). During the inflammatory phase (days 3, 5), activated monocytes and macrophages, as well as callus-specific enrichment of distinct subsets of dendritic cells (DCs) (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309) are defined, which act as a link between the innate and adaptive immune response. Lymphocytes (T and B cells) also infiltrate the callus during the inflammatory phase (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309). Importantly, the innate-immune cells and DCs that populate the inflammatory hematoma induce a wave of activated lymphocytes that infiltrate the callus during the late repair / early remodeling phase (d21 post-fracture) (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309). In all these analyses, the callus immune-cell populations were compared to those of the contralateral unfractured bone and bone marrow to confirm the callus-specific enrichment of analyzed cells (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet- induced obesity,” Front. Immunol., 2023; 14: 1250309). The same multiplexed FC techniques are employed to study the impact of Prg4-S KD on the abundance and activation status of innate and adaptive immune cells during both the first (d3 and d5) and the second (d21) waves of infiltration (Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309). These assays characterize immune-cell populations that underpin the exacerbated immune response on d5 (FIGS. 6A-B) and how changes in the early immune response in Prg4-S KD mice affect the stoichiometry / activation of immune populations in d21 callus (with special focus on lymphocytes on d21).

[0277] Analysis ofPSC viability and proliferation (days 3 and 5).

[0278] Preliminary data indicate that Prg4-S KD decreased the abundance of PSCs in d5 callus.To understand the underpinning reasons, apoptosis and proliferation of PSCs are assessed in Ctrl and Prg4-S siRNA-treated calli using FC. Annexin V and Click-iT Plus kits are used to analyzeAttorney Docket No. 0073605-001029 apoptosis and proliferation, respectively. Based on the results, more mechanistic studies are performed (see Aim 2).

[0279] Analysis of the soft callus formation and mineralization.

[0280] The soft callus in Prg4-S KD mice has a significantly smaller size (FIG. 9A), which can the outcome of several factors, including reduced PSC abundance (FIGS. 5I-J), inhibited chondrogenic differentiation of PSCs (see below), compromised chondrocyte viability and proliferation (FIGS. 9E, F), and / or pre-mature chondrocyte hypertrophy (FIGS. 9B-D). These scenarios are deconvoluted through complementary in-vivo and in-vitro experiments described here and in Aims 2 and 3. In this sub-aim, the soft callus on days 7 and 14 are comprehensively analyzed to corroborate the results of dlO (FIGS. 9A-F). Day-7 callus is mainly formed of proliferating chondrocytes (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone 2020; 137: 115436; Brown, M. L. et al., “Delayed fracture healing and increased callus adiposity in a C57BL / 6J murine model of obesity-associated type 2 diabetes mellitus,” PLoS One, 2014; 9(6):e99656). An analysis of day-7 callus helps elucidate the impact of Prg4-S KD on chondrocyte proliferation and viability, which are achieved by assessing BrdU uptake and TUNEL assay, respectively, as shown in FIGS. 9E, F. Day 7 chondrocytes are also analyzed for early hypertrophy as in FIG. 7B-D. Day-14 soft callus is formed mainly of hypertrophic / mineralizing chondrocytes (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023; 11 :5;Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576) and thus are analyzed for hypertrophy markers and soft-callus mineralization as in FIGS. 9A-F (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet- induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023; 11 :5; Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38:1560-1576).Attorney Docket No. 0073605-001029

[0281] Analysis of bone formation.

[0282] Bone formation is analyzed using p.CT on days 14, 21, and 28 as in FIGS. 10A-D (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38:1560-1576; Wee, H., et al., “Assessment of Bone Fracture Healing Using Micro-Computed Tomography,” J. Vis. Exp., 2022). Masson’s trichome staining (combined with histomorphometry) and IF staining of osteoblast markers are performed (Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576). These analyses can indicate the long-acting effects of inhibiting Prg4-S expression on bone formation during the late healing phases.

[0283] Analysis of Angiogenesis (d7-d21).

[0284] Analysis of Angiogenesis (d7-d21 ) is performed using IF staining of CD31, CD34, andEmcn (Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38:1560-1576).

[0285] The data on dlO indicate inhibited angiogenesis in Prg4-S KD mice (not shown). Such inhibition can be partially due to reduced callus mineralization in the absence of Prg4-S (FIGS. 10A- B). However, Prg4 can promote VEGF expression in endothelial cells during wound healing (Krawetz, R. J. et al., “Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration,” NPJ Regen. Med., 2022; 7:32), which can contribute to the inhibited angiogenesis observed in Prg4-S KD callus. Thus, staining experiments are performed to quantify the expression of VEGF in callus cells, including hypertrophic chondrocytes that are capable of expressing VEGF (Kodama, J. et al., “The role of hypertrophic chondrocytes in regulation of the cartilage-to-bone transition in fracture healing,” Bone Rep., 2022; 17: 101616).

[0286] Osteoclastogenesis (d!0-d21).

[0287] Osteoclastogenesis (d 10-d21) is assessed by TRAP and staining of osteoclast markers(e g., OSCAR).

[0288] Analysis of biomechanical properties of newly formed bone.Attorney Docket No. 0073605-001029

[0289] Biomechanical testing is on day 28 to assess the maximum torque at failure and stiffness of the newly formed bone.

[0290] For rigor, all IF results will be corroborated by RNAScope, which is established in our lab32

[0291] Analysis of biomechanical properties of newly formed bone.

[0292] Biomechanical testing will be performed on day 28 (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576) to assess the maximum torque at failure and stiffness of the newly formed bone.

[0293] For rigor, all IF results are corroborated by RNAScope (Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023).Aim IB. Investigating the pro-healing potential of the engineered human PRG4-S. Preliminary data. Does a human PRG4 analogue have osteogenic effects?

[0294] A human analogue of the murine short splicing variant of Prg4 has never been reported in literature or listed on any database (including RefSeq and UCMC), which might be due to the fact that it is callus-specific and expressed at high levels only during the early phases of healing. Transcriptomic data driven from human callus are not available and do not contribute to the databases of human transcriptome. It is very challenging to confirm that the murine Prg4-S is conserved in human given that the clinical situations where a callus is collected during these early phases are extremely rare. To overcome this problem, a recombinant human (h)PRG4 protein was engineered that mimics murine Prg4-S. This recombinant human (h)PRG4 protein can be referred to as hPRG4-S. A human full-length PRG4 variant (Variant A; FIG. 19A) was also produced, which contains all the exons and is reported to be expressed in cartilage and other tissues. The osteogenic effects of the 2 variants were tested using primary PSCs (isolated from the callus by FACS). Strong osteogenic effects were detected from the engineered recombinant hPRG4-S, while variant A did not show any osteogenic effects (FIG. 23). Same results were obtained when the corresponding mouse variants were tested (not shown). Accordingly, it is speculated that the osteogenic effects of the engineered hPrg4-S can be harnessed to induce healing in patients at high risk of developingAttorney Docket No. 0073605-001029 nonunion. Importantly, most of the healing pathways that are known to be inhibited in pathologies that lead to nonunion are significantly stimulated by PRG4-S in our mouse studies. Such osteogenic effects of the engineered hPrg4-S have high translational and clinical significance, as the use of hPRG4-S as a regenerative therapy can be generalized to various disease conditions. To further support this notion, the expression level of Prg4 were measured in diet-induced obese (DIO) mice; a model of obesity / T2D that suffers from defective healing and recapitulates compromised healing in diabetic patients. DIO mice exhibited healing defects that are similar to those observed in Prg4 KD mice. Interestingly, the expression of Prg4-S in the callus of diet-induced obese (DIO) mice was measured to be only ~13% of its expression in the callus of lean mice (FIG. 24).

[0295] Taken together, it is speculated that Prg4-S is unique splicing variant expressed in the callus and promotes different aspects of fracture healing, and that treating the callus with recombinant murine Prg4-S or the engineered hPrg4-S can rescue healing in the DIO mice. These effects are further investigated through the followings procedures.

[0296] Adult (male and female), 4-mo-old DIO mice (purchased from Jax) are used, which is an established murine model for obesity and early stages of T2D (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309; Brown, M. L. et al., “Delayed fracture healing and increased callus adiposity in a C57BL / 6J murine model of obesity-associated type 2 diabetes mellitus,” PLoS One, 2014; 9(6):e99656). Similar to diabetic patients, DIO mice exhibit defective healing (Khajuria, D. K. et al., “Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model,” Bone, 2020; 137: 115436; Khajuria, D. K et al., “Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity,” Front. Immunol., 2023; 14: 1250309; Brown, M. L. et al., “Delayed fracture healing and increased callus adiposity in a C57BL / 6J murine model of obesity-associated type 2 diabetes mellitus,” PLoS One, 2014; 9(6):e99656). Recombinant hPRG4-S protein is delivered to the healing callus using a delivery system wrapped around the fracture site during the fracture-induction surgery. The recombinant murine PRG4-S is used for comparison. Control lean and DIO mice receive the empty delivery system. Dose is adjusted to re-establish the normal expression of Prg4 in the DIO callus. The effect of each treatment on the overall healing process is determined by monitoring boneAttorney Docket No. 0073605-001029 formation and biomechanical properties of the newly formed bone as described in Aim 1A. Based on the results of knocking down Prg4-S in wild type mice, it is expected that the recombinant murine Prg4-S can largely, if not completely, rescue healing in DIO mice. The pro-healing effects of hPRG4-S are compared to those of murine PRG4-S. Based on the strong osteogenic effects of hPRG4-S observed in vitro, and the >90% homology between murine PRG4-S and hPRG4-S, it is expected that hPRG4-S can rescue healing in DIO mice. Earlier healing phases are also analyzed, as in Aim 1A, to assess the impact of hPRG4-S on healing parameters dysregulated in DIO mice.

[0297] Aim 1 provides comprehensive insights into the roles of Prg4-S in different phases of healing and sets the stage for the mechanistic studies proposed in the following Aims. In addition, results of pre-clinical studies in Aim IB can energize future clinical studies to develop and evaluate a system that locally delivers recombinant hPRG4-S as a novel therapy for delayed healing and nonunion.

[0298] IF and RNAScope are semi-quantitative but provide valuable spatial information, and the state-of-the-art of each is used for the study described herein. A separate cohort of mice can be assigned for RNA extraction (Le Bleu, et al., “Extraction of high-quality RNA from human articular cartilage,” Anal. Biochem., 2017; 518: 134-138) and qPCR, which can provide quantitative data but may lack spatial information. Thus, qPCR analysis is limited to analyzing generalized changes in expression (e.g., Prg4-S in Prg4-S KD callus) or a gene specifically expressed in one cell type (e.g., Col X). It is speculated that analysis of samples from the first two weeks of healing (days 3 to 14) provides sufficient understanding of the roles of Prg4-S in a wide scope of process involved in fracture healing, especially that Prg4 expression beyond dl4 and in bone / BM cells is the lowest in the callus. Notably, repetitive injection of the siRNA-atelocollagen complex to achieve sustained gene silencing in fracture callus or articular cartilage (Yoshizuka, M. et al., “Inhibition of microRNA-222 expression accelerates bone healing with enhancement of osteogenesis, chondrogenesis, and angiogenesis in a rat refractory fracture model,” J. Orthop. Sci., 2016; 21 :852- 858; Ito, Y. et al., “Both microRNA-455-5p and -3p repress hypoxia-inducible factor-2a expression and coordinately regulate cartilage homeostasis,” Nat. Commnn., 2021; 12:4148) can be used if results raise the necessity of sustained Prg4 KD beyond dl4. As mentioned above, no commercially available transgenic mice allow conditional KO of Prg4; however, there is a genetically modified mouse (B6, 129- / 7X7''''2', / "1I‘7J) that carries a floxed gene trap allele of Prg4 gene designed to produce membrane-anchored P-galactosidase instead of Prg4. These mice recapitulate the phenotype of constitutive Prg4 KO mice (described above). Based on preliminary data, these mice are expected toAttorney Docket No. 0073605-001029 develop defective healing. Breeding these mice with, e.g., PDGFRotCreor AcanCrecan allow conditional restoration of Prg4 expression in PSCs or chondrocytes, respectively, upon tamoxifen injection (Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576; Carlson, E. L. et al., “Paroxetine-mediated GRK2 inhibition is a disease-modifying treatment for osteoarthritis” Sei. Transl. Med., 2021; 13(580):eaau8491). This approach can be used to investigate the impact of restoring Prg4 expression on ameliorating the defective healing phenotype. However, the strong phenotype and the abnormal gait of Prg4 KO mice can complicate data interpretation, which makes the siRNA-mediated KD a better approach. In addition, the siRNA approach allowed knocking down a specific AS isoform and has future translational potential.Aim 2: Defining the roles of prg4-S in PSC homeostasis and differentiation.

[0299] Preliminary data.

[0300] As discussed above, Prg4-S KD resulted in reduced abundance of PSCs (FIG. 6C). Was this due to the direct impact of Prg4-S KD on PSCs, or secondary to the dysregulated immune response (FIGS. 6A-B)? Prg4-S KD also caused defective bone and soft callus formation, both might be the outcome of reduced PSC abundance during the inflammatory phase (FIGS. 5I-J). Other factors that contributed to the defective structure of soft callus in Prg4-S KD mice include increased apoptosis, reduced proliferation, and accelerated / pre-mature hypertrophy of chondrocytes (FIGS. 9A-F). But does Prg4-S also regulate PSC differentiation as a means to promote soft callus and bone formation? These questions are addressed first by performing in-vitro experiments on primary PSCs. Prg4-S was knocked down using a lentiviral shRNA designed to target the same Prg4-S exon-exon junction described above (FIG. 5C), which significantly reduced Prg4 expression (FIG. 25A). The PSCs were then subject to chondrogenic differentiation (Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023; 11 :5), and Prg4-S KD substantially inhibited chondrogenesis (FIG. 25B). Thus, regulating PSC differentiation is among the mechanisms whereby Prg4-S regulates fracture healing. In this Aim, the questions posed above are addressed by characterizing further roles of Prg4-S in cellular homeostasis.

[0301] a) Roles of Prg4-S in PSCs.Attorney Docket No. 0073605-001029

[0302] 1) Roles of Prg4-S in osteogenic differentiation of PSCs. First, an assessment is performed to determine whether Prg4-S promotes the osteogenic differentiation of PSCs, which might have contributed to the defective bone formation observed in Prg4-S KD mice (FIGS. 10A-B). primary PSCs (sorted from d5 callus) will be differentiated under osteogenic conditions (Khajuria, D. K. et al., “Transcript shortening via alternative poly adenylation promotes gene expression during fracture healing,” Bone Res., 2023; 11 :5; Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576) in the presence of Prg4-S or Ctrl shRNA. Based on PSC differentiation (Khajuria, D. K. et al., “Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice,” J. Bone. Miner. Res., 2023; 38: 1560-1576), an evaluation is performed to gauge the impact of Prg4-S KD on i) the early phase of osteogenesis by using RT-qPCR by measuring Osterix and Runx2, whose expression increases significantly during the first 3 days of differentiation, ii) formation of mature osteoblasts by measuring Col lai and Alp, whose expression increases after 2 weeks of differentiation, and iii) mineralization by measuring osteocalcin and by Alizarin Red staining, which is performed after 3 weeks of differentiation.

[0303] 2) Roles of Prg4-S in regulating PSC proliferation and viability. MTT (Thermo) andBrdU uptake (Cell Signaling) assays are used to compare the viability and proliferation, respectively, of PSCs in the presence of Prg4-S or Ctrl shRNA. Results of these experiments can corroborate the in-vivo results generated in Aim 1 and deconvolute the pro-healing effects of Prg4-S that stem from its regulatory functions in the PSCs.

[0304] b) Roles of Prg4-S in chondrocyte or osteoblast homeostasis.

[0305] Prg4-S is expressed in the soft callus (FIGS. 21C, 5G, 5H), and Prg4-S KD increased chondrocyte apoptosis and hypertrophy and reduced chondrocyte proliferation (FIGS. 9A-F), which suggests roles of Prg4-S in regulating chondrocyte homeostasis. Prg4-S is lowly expressed in the woven bone area (FIG. 21C) and does not impact the proliferation or apoptosis of bone / BM cells (FIGS. 10C, D). However, it is unclear whether Prg4-S impacts other parameters as bone mineralization. In this sub-aim, these points are further investigated. To study the roles of Prg4-S in regulating chondrocyte homeostasis, PSCs are differentiated produce to mature chondrocytes, which takes 7-10 days (FIG. 25B). Mature chondrocytes are then transduced with lentiviral Ctrl or Prg4-S shRNA, and the impact of Prg4-S KD on chondrocyte proliferation, apoptosis, and hypertrophy are assessed accordingly. The same approach is used to study the impact of Prg4-S KD on osteoblastAttorney Docket No. 0073605-001029 homeostasis and mineralization, and results are corroborated by IF staining of tissue sections generated in Aim 1. Results of these studies corroborate the in-vivo results and unravel new roles of Prg4-S in fracture healing.

[0306] c) Global analysis of the roles of Prg4-S in PSCs and chondrocytes.

[0307] To gain transcriptomic insights into how Prg4-S regulates PSC’s homeostasis, PSCs are sorted from d5 callus treated with either Ctrl or Prg4-S siRNA (both genders). Bulk RNA-seq is performed on isolated PSCs. Global RNA-seq is also performed on chondrocytes sorted from the calli of mice treated with Ctrl or Prg4-S siRNA. To facilitate chondrocyte sorting, a mouse model generated by breeding Ai9 (Yi6Cg-C;i RC)SA)26S()rt,G>d :dddG'nial , / AdJ) with AcanCre / ERTwill be used. Injecting this model with tamoxifen labels the chondrocytes with tdTomato (FIG. 26), which allows chondrocyte sorting from the callus by FACS. Based on the results of Aim 1, the harvest timepoint(s) (d7-to-d!4) can be determined. Gene Ontology and pathway analyses of the RNA-seq provide further insights on how Prg4-S regulates PSCs and chondrocytes and inform the design of studies described in Aim 3. RNA-seq data are confirmed (for genes selected for follow-up studies) using qPCR, IF staining, and RNAScope.

[0308] Aim 2 identifies different mechanisms whereby Prg4-S regulates cellular homeostasis during healing, which lend further insight into the in-vivo data generated in Aim 1.

[0309] In point c, based on our preliminary studies, it is expected that pooling PSCs or chondrocytes from 5 calli (for each replicate) should generate enough RNA for bulk RNA-seq and follow-up qPCR. However, if RNA is not enough, more mice can be pooled. In addition, the purity of the sorted PSCs (CD45 CD3 FPDGFRa+SCAl+) can be crucial for the quality of RNA-seq data. Thus, the FACS panel contains IgG and FMO controls. The main contaminant in sorted callus cells can be immune (CD45+) cells. Therefore, the digested callus can be purified on a CD45+AutoMACS antibody column prior to FACS, which results in highly pure sorted cells with undetectable immunecell content. For rigor, the level of CD45 is measured by qPCR in the RNA prior to RNA-seq. If purity is not satisfactory, another AutoMACS run can be considered. For chondrocyte labeling with tdTomato, 3 consecutive 75 mg / kg doses of tamoxifen are injected to label articular chondrocytes (FIG. 26). The same dose is injected between days 4 and 6 to label callus chondrocytes, which is expected to result in efficient labeling. Otherwise, tamoxifen dosage can be further optimized. If experiments in point b (and Aim 1) indicate roles of Prg4-S in osteoblast homeostasis, bulk RNA- seq can be performed on sorted bone cells, and the Ai9:CollalCre / ERTmouse model can be used to enable bone-cell sorting. In point a.1, the study of impact of Prg4-S KD on the late stages ofAttorney Docket No. 0073605-001029 osteogenesis can be performed in 2 different ways. The first is to transduce cells with a Prg4-S shRNA one time on day 0 (the day we start differentiation). In this case, the level of Prg4-S is expected to be restored to its endogenous level after ~7-10 days. Nevertheless, the impact of Prg4-S KD is expected to be still detectable during the late osteogenic phases (i.e., after 3 weeks of differentiation) due to the long-acting effects of Prg4-S KD. The second way is to keep Prg4-S knocked down during the whole course of differentiation, can be achieved by re-introducing the lentiviral shRNA every 7 days. The level of Prg4-S is monitored by RT-qPCR and WB to confirm KD efficiency.Aim 3. Investigating the mechanisms that regulate Prg4 expression, splicing, and signaling.

[0310] In this Aim, a number of yet unanswered questions are addressed. Is Prg4-S isoform specific to the callus PSCs or also expressed in intact-bone PSCs? What regulates Prg4 expression and splicing? What are the Prg4 receptors that mediate its functions in the callus?

[0311] Preliminary data.

[0312] Preliminary results indicate high expression of Prg4 in the callus PSCs (FIG. 3B, 4A, 20). Is this high expression of Prg4 unique to the callus PSCs, or it also occurs in PSCs that reside on unfractured bone? To answer this question, PSCs were isolated from d5 callus and from the contralateral intact bone. Also, BM-derived mesenchymal stem cells (BMSCs) were isolated according to the standard protocol (Soleimani, M. & Nadri, S., “A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow,” Nat. Protoc., 2009; 4: 102-106; Huang, S. et al., “An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. J. Orthop. Translat., 2015; 3:26-33; Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023;11 :5). RNA was extracted from each pool, and RT-qPCR is performed to measure total Prg4 mRNA. The expression of Prg4 is found to be~12-fold higher in intact-bone PSCs than in BMSCs (FIG. 4B). Interestingly, the expression of Prg4 was ~120-fold higher in callus PSCs than in intact-bone PSCs (FIG. 4B). PCR was then performed to identify Prg4 AS isoforms, and the results indicate that intact-bone PSCs express Prg4-L as the major AS isoform, in a striking contrast to callus PSCs that express Prg4-S (FIG. 4H). These results indicate that once PSCs infiltrate the callus, the expression of Prg4 is highly induced and the splicing of Prg4 is reprogrammed to favor the production of Prg4- S over Prg4-L. It is speculated that the micro-environment of the callus (as compared to that of unfractured bone) induces these effects. The callus at this early stage of healing is characterized byAttorney Docket No. 0073605-001029 hypoxic and inflammatory environment. To investigate how hypoxia impacts Prg4 expression / splicing, PSCs isolated from unfractured bone were incubated in hypoxic (1% oxygen) vs. normoxic (20% oxygen) conditions, and RNA was collected after 1, 2, 4, 8, and 24 h. Hypoxia did not change Prg4 expression or splicing (not shown). Cytokines and growth factors were also investigated, and Tgfbl was evaluated first for the following reasons: i) Tgfbl has roles in the homeostasis and differentiation of skeletal progenitors as well as in bone regeneration (Wu, M. et al., “TGF-P and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease,” Bone Res., 2016; 4: 16009), ii) scRNA-seq data show that Tgfbl receptors are expressed on the PSCs (Tgfbr2 is shown in FIG. 27 A), and iii) Tgfbl can regulate Prg4 in articular chondrocytes (DuRaine, G. D., et al., “Effects of TGF-P 1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110; Cuellar, A. & Reddi, A. H., “Stimulation of Superficial Zone Protein / Lubricin / PRG4 by Transforming Growth Factor-beta in Superficial Zone Articular Chondrocytes and Modulation by Glycosaminoglycans,” Tissue Eng. Part A, 2015; 21 : 1973- 1981 ). Primary PSCs (sorted from unfractured bone) were treated with 10 ng / ml Tgfbl (Coricor, G. & Serra, R., TGF-P regulates phosphorylation and stabilization of Sox9 protein in chondrocytes through p38 and Smad dependent mechanisms,” Sci. Rep., 2016; 6:38616; Han, F. et al., “Transforming growth factor-betal (TGF-betal) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression,” J. Cell. Biochem., 2005; 95:750- 762), which resulted in substantial increase in Prg4 expression (FIG. 27B) as well as splicing reprogramming that shifted the major AS isoform from Prg4-L to Prg4-S (Fig. 27C). Based on these results, it is speculated that the micro-environment of the callus induces Prg4 expression and reprograms Prg4 splicing and that Tgfbl is a major player in this process. In this Aim, as described in detail below, further experiments are performed to answer the questions posted above.Aim 3.1. Identifying how Tgfbl induces Prg4 expression in PSCs.

[0313] Rigor of the research. TGFB1 receptors signal through SMADs (SMAD2 / SMAD3- SMAD4) or MAP kinases (MAPKs) (Wu, M. et al., “TGF-P and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease,” Bone Res., 2016; 4: 16009). Prg4 promoter was analyzed for binding sites of transcription factors (TFs), and SMAD3, among others, was identified as a potential TF (FIG. 28A). Consistent with this finding, TGFB1 has been reported to induce Prg4 expression in articular chondrocytes via SMAD3 (DuRaine, G. D., et al., “Effects of TGF-P 1 on alternative splicing of Superficial Zone Protein in articular cartilageAttorney Docket No. 0073605-001029 cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110). Thus, SMAD3 was knocked down in TGFB1 -treated primary PSCs, which resulted in ~60% downregulation in Prg4 expression (FIG. 28B). Therefore, it is speculated that SMAD3 is a major, but might not be the only, regulator of Prg4 expression downstream to TGFB1 receptor.

[0314] TGFB1 signaling is further investigated as follows.

[0315] a) SMAD3 results are corroborate using SIS3 (Sigma), which specifically inhibits Smad3 phosphorylation and Smad3 -mediated cellular signaling without affecting Smad2 or MAPKs (DuRaine, G. D., et al., “Effects of TGF-pi on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110; Jinnin, M. et al., “Characterization of SIS3, a novel specific inhibitor of Smad3, and its effect on transforming growth factor-beta 1 -induced extracellular matrix expression,” Mol. Pharmacol., 2006; 69:597-607; Ji, X. et al., “Specific Inhibitor of Smad3 (SIS3) Attenuates Fibrosis, Apoptosis, and Inflammation in Unilateral Ureteral Obstruction Kidneys by Inhibition of Transforming Growth Factor beta (TGF- beta) / Smad3 Signaling,” Med. Sci. Monit., 2018; 24: 1633-1641). Inhibition of SMAD3 is confirmed by phospho-SMAD3 WB.

[0316] b) The roles of SMAD2 and MAPKs are tested. MAPKs signaling downstream to TGFBR is mediated mainly through p38oc / |3 or ERK1 / 2. shRNAs are used to knockdown, separately, each of these signaling molecules to determine whether the impact of TGFB1 on Prg4 expression will be blunted (as in FIG. 28B). If the results implicate p38oc / p in regulating Prg4 expression, this finding can be corroborated using VX745 (R&D Systems), the potent and specific inhibitor of p38oc and P (IC50 values are 10 nM and 220 nM, respectively) (Crozier, L. et al., “CDK4 / 6 inhibitor- mediated cell overgrowth triggers osmotic and replication stress to promote senescence,” Mol. Cell, 2023; 83:4062-4077; Singh, R. K. et al., “Differential effect of p38 and MK2 kinase inhibitors on the inflammatory and toxicity biomarkers in vitro,” Hum. Exp. Toxicol., 2018; 37:521-531). VX745 is used at concentrations that inhibit either p38a alone or both. If any of these signaling molecules are involved in regulating Prg4, it can be knocked down concomitantly with SMAD3 to test for synergistic effects.

[0317] Aim 3.1 provides details on TGFB 1 signaling that regulates Prg4 in PSCs.Aim 3.2. Identifying potential roles of EGF in regulating Prg4 expression.

[0318] Tgfbl may not be the only cytokine / growth factor that induces Prg4 expression. EGF regulates the proliferation of skeletal progenitors and induces osteogenesis and bone healing (Basal,Attorney Docket No. 0073605-001029O. et al., “Epidermal growth factor (EGF) promotes bone healing in surgically induced osteonecrosis of the femoral head (ONFH),” Bosn. J. Basic. Med. Sci., 2018;18:352-360; Mangiavini, L. et al., “Epidermal growth factor signalling pathway in endochondral ossification: an evidence-based narrative review,” Arm. Med., 2022; 54:37-50). scRNA-seq data (d5) indicate that EGF receptors are highly and specifically expressed on callus PSCs (FIG. 28C). Primary PSCs are treated with recombinant EGF (R&D), and Prg4 expression and splicing are analyzed, as shown in FIGS. 27B, C. If EGF stimulates Prg4 expression or reprograms its splicing (see Aim 3.4), experiments can be designed as in Aim 3.1 to determine the signaling pathway(s) downstream to EGFR.Aim 3.3. Identifying gene expression signature and splicing map that typify the callus PSCs.

[0319] In Aim 2c, global RNA-seq was performed on Ctrl and Prg4-S KD PSCs isolated from d5 callus to define the impact of Prg4-S KD on the PSC transcriptome and on the stoichiometry of splicing isoforms. In this sub-aim, one group is added to the bulk RNA-seq experiments: PSCs isolated from intact bone. This group is prepared by sorting PSCs from the contralateral unfractured bones of the same mice used to isolate Ctrl callus PSCs in Aim 2C. All groups are sequenced in parallel. Intact-bone PSCs are compared to the control callus PSCs with regard to gene expression. GO analysis of differentially expressed genes helps understand how the homeostasis and fate of PSCs change when they are mobilized from intact periosteum to the healing callus. Importantly, RNA-seq data also indicate the activated signaling pathways and TFs in callus vs intact-bone PSCs. This activation can be evidenced by increased expression of the signaling molecules or TFs.However, it should be noted that activation of signaling molecules or TFs is not necessarily reflected on their expression levels, but rather on their post-translational modification (e.g., phosphorylation). Although RNA-seq may not detect these modifications, activation of signaling pathway s / TFs can be efficiently analyzed via assessing the expression levels of the downstream target genes. These analyses are performed using, e.g., 1PA upstream regulator analysis, which scores activation of a signaling pathway / TF based on the collective changes in expression levels of its responsive genes (Khajuria, D. K. et al., “Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing,” Bone Res., 2023; 11 :5) Outcomes of this study provide global insights into how the callus micro-environment changes the transcriptome of PSCs. Intact-bone PSCs are further compared to the control callus PSCs with regard to global analysis of alternative splicing. rMATS (Shen, S. et al., “rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data,” Proc. Natl. Acad. Sci. USA, 2014; 111 :E5593-5601) isAttorney Docket No. 0073605-001029 employed to globally analyze AS isoforms in both intact-bone and callus PSCs. Outcomes of this study generate a valuable library of callus-specific splicing events in PSCs. These findings extend the knowledge beyond Prg4 and form the basis of future studies regarding how the callus microenvironment reprograms AS and its implications on gene expression and function.Aim 3.4. Identifying potential factors that regulate Prg4 splicing.

[0320] Rigor of the research.

[0321] Preliminary data show that TGFB1-SMAD3 axis induces Prg4 expression (FIG. 26) and that TGFB1 reprograms Prg4 splicing to induce exon skipping, thereby generating Prg4-S instead of Prg4-L (FIGS. 4H, 27C). But what are the splicing regulators that control Prg4 splicing downstream to TGFB1? It has been shown that TGFB1 regulates Prg4 splicing in articular chondrocytes via activating SMAD3 (DuRaine, G. D., et al., “Effects of TGF- 1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110).Furthermore, in cancer cells, the same TGFB1-SMAD3 axis regulates several splicing events via recruiting poly(rC) binding protein 1 (PCBP1) to the target pre-mRNA (Tripathi, V. et al., “Direct Regulation of Alternative Splicing by SMAD3 through PCBP1 Is Essential to the Tumor-Promoting Role of TGF- Mol. Cell, 2016; 64(5):549-564). Does the TGFB1-SMAD3-PCBP1 axis regulatePrg4 splicing?

[0322] PCBP1 belongs to the heteronuclear RNA binding proteins (hnRNPs) and is also known as hnRNP-El (Tripathi, V. et al. “Direct Regulation of Alternative Splicing by SMAD3 through PCBP1 Is Essential to the Tumor-Promoting Role of TGF- 0,” Mol. Cell, 2016; 64(5):549-564;Makeyev, A. V. & Liebhaber, S. A., “The poly(C)-binding proteins: a multiplicity of functions and a search for mechanisms” RNA, 2002; 8:265-278). PCBP1 binds to poly(C) stretches (Tripathi, V. et al. “Direct Regulation of Alternative Splicing by SMAD3 through PCBP1 Is Essential to the Tumor- Promoting Role of TGF- 0,” Mol. Cell, 2016; 64(5):549-564; Makeyev, A. V. & Liebhaber, S. A., “The poly(C)-binding proteins: a multiplicity of functions and a search for mechanisms” RNA, 2002; 8:265-278). Generally, members of the hnRNP group act as splicing regulators that bind exonic splicing silencer (ESS) to repress exon inclusion (i.e., promote exon skipping) (Zhang, Y. et al., “Alternative splicing and cancer: a systematic review,” Signal Transduct Target Ther., 2021;6( 1): 78). Bioinformatic analysis was performed to define potential splicing regulators that bind to the Prg4 pre-mRNA, specifically at the 3 exons skipped in the Prg4-S isoform or the introns around these 3 exons. Results of such bioinformatic analysis indicate that numerous ESS sites areAttorney Docket No. 0073605-001029 distributed throughout the 3 skipped exons, which outnumbered the Exonic Splicing Enhancer (ESE) sites (exon 7 is shown in FIG. 29A). These findings suggest skipping of the 3 exons should the proteins that bind the ESSs be recruited to the pre-mRNA. The potential binding sites of PCBP1 (c- rich stretches) were the most enriched ESS in the skipped exons vs other exons (P = 3.2 * 1 O'24).Also, scRNA-seq data show that Pcbpl is highly expressed in the callus PSCs (FIG. 29B). Thus, it is speculated that TGFB1 acts through a TGFBR-SMAD3-PCBP1 axis to regulate Prg4 splicing in PSCs. This hypothesis is tested as follows to involve more splicing regulators:

[0323] a) Investigation of roles of the TGFBR-SMAD3-PCBP1 axis in Prg4 splicing.

[0324] PSCs isolated from intact bone are treated with TGFB1 (as in FIG. 27B) in the presence or absence of Pcbpl shRNA, and analysis is performed to determine how Pcbpl KD affects Prg4 splicing. It is expected that Pcbpl KD inhibits TGFB1 -mediated splicing reprogramming. If Pcbpl KD results in more than one major AS isoform other than Prg4-S and Prg4-L, the PCR products can be sequenced to investigate which exons are included in the final splicing products. This step characterizes the PCBP1 -bound / regulated exons. The AS isoforms of Prg4 in Smad3 KD cells (FIG. 28B) are also identified. A comparison between the isoforms in Smad3 KD and Pcbpl KD cells can help determine whether SMAD3 is the link between TGFBR and PCBP1.

[0325] b) If results in a) suggest that Pcbpl and Smad3 play a role in regulating Prg4 splicing, the results can be further corroborated by performing immunoprecipitation (IP) of PC BP 1 from primary PSCs as follows.

[0326] PSCs are treated with TGFB1 as in FIG. 29D (untreated cells are used as a control) and formaldehyde crosslinking (CL) is performed prior to IP (Elbarbary, R. A. et al., “Tudor-SN- mediated endonucleolytic decay of human cell microRNAs promotes G(l) / S phase transition,” Science, 2017; 356:859-862; Elbarbary, R. A. et al., “UPF1 helicase promotes TSN-mediated miRNA decay,” Genes Dev. 2017; 31 : 1483-1493; Elbarbary, R. A. et al., “STAU1 binding 3' UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown,” Genes Dev., 2013; 27:1495-1510). Formaldehyde was chosen because it performs protein-to-protein and protein-to-RNA crosslinking, both of which are required for the experiments described herein. Following IP, the following steps are performed:

[0327] i) Extraction of RNA from PCBP1 IP and use of RT-qPCR to measure Prg4 pre-mRNA. The primers are designed to span at least one intron to ensure detection of the pre-mRNA. The following controls are employed: 1) control IgG IP and qPCR of Prg4 pre-mRNA (the pre-mRNA is highly enriched in PCBP1 IP vs. IgG IP), and 2) qPCR to measure GAPDH and actin in PCBP1 IPAttorney Docket No. 0073605-001029(both are highly expressed mRNAs that are not expected to be enriched in PCBP1 IP). These controls can confirm enrichment of Prg4 pre-mRNA in PCBP1 IP above background (Elbarbary, R. A. et al., “Tudor-SN-mediated endonucleolytic decay of human cell microRNAs promotes G(l) / S phase transition,” Science, 2017; 356:859-862; Elbarbary, R. A. et al., “UPF1 helicase promotes TSN-mediated miRNA decay,” Genes Dev. 2017; 31 : 1483-1493; Elbarbary, R. A. et al., “STAU1 binding 3' UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown,” Genes Dev., 2013; 27: 1495-1510). Results of these experiments can corroborate data generated in Aim 3.4. a.

[0328] ii) perform Western Blotting (WB) to detect SMAD3 and p-SMAD3 in PCBP1 IP, which will confirm the formation of p-SMAD3-PCBPl complex. Results are further corroborated by the reciprocal IP of SMAD3 and test for co-IP of PCBP1. In all IP experiments, WB of GAPDH and actin is performed in the target protein IP, and WB of the immuno-precipitated proteins (PCBP1 or SMAD3) is performed in IgG controls to confirm specific IP.

[0329] IF co-staining of p-SMAD3 and PCBP1, combined with confocal microscopy imaging, is performed to assess co-localization in the nucleus in TGFBl-treated cells. Importantly, all the experiments are performed in the presence or absence of TGFB1 to understand howTGFB1 signaling induces the formation of the SMAD3-PCBP1 complex and the consequent deposition of PCBP1 on Prg4 pre-mRNA. All antibodies required for the proposed IP and WB are commercially available.

[0330] PCBP1 can act as a potential regulator of Prg4 splicing, which is assessed in the experiments proposed in Aim 3.4. a. If PCBP1 KD does not affect Prg4 splicing, other splicing regulators identified by bioinformatic analysis can be assessed instead. It is not possible to analyze all the identified regulators, and the identified regulators are prioritized based on the following criteria: 1) the splicing regulator has well established roles in promoting exon skipping (hnRNPs is an important class), 2) the splicing regulator binding sites are enriched in or around the skipped exons, and 3) the splicing regulator is expressed in callus PSCs (based on out scRNA-seq data). Based in all these criteria, the second top candidate is hnRNP Al, another member of the hnRNP group that plays a role in regulating splicing (Bruun, G. H. et al., “Global identification of hnRNP Al binding sites for SSO-based splicing modulation,” BMC Biol., 2016; 14:54). The contribution of a splicing regulator to Prg4 splicing is studied using the canonical way of knocking down the splicing regulator, and analysis is performed to determine how such knockdown impacts Prg4 AS isoforms, which provides a quick assessment. The detailed mechanistic studies described in Aim 3.4.b areAttorney Docket No. 0073605-001029 pursued if the KD results implicate the splicing regulator in Prg4 splicing. If more than one splicing regulator are involved, or if the regulators are differentially involved in the skipping of particular exons, experiments aiming at simultaneous KD of multiple splicing regulators can be designed.Aim 3.5. Identifying Prg4 receptors in the callus, with specific focus on PSCs.

[0331] Rigor of the research.

[0332] Prg4 binds CD44, TLR2, and TLR4 in other tissues (Iqbal, S. M. et al., “Lubricin / Proteoglycan 4 binds to and regulates the activity of Toll-Like Receptors In Vitro," Sci. Rep., 2016; 6: 18910; Al-Sharif, A. et al., “Lubricin / Proteoglycan 4 Binding to CD44 Receptor: A Mechanism of the Suppression of Proinflammatory Cytokine-Induced Synoviocyte Proliferation by Lubricin,” Arthritis Rheumatol., 2015; 67: 1503-1513; Alquraini, A. et al., “The interaction of lubricin / proteoglycan 4 (PRG4) with toll-like receptors 2 and 4: an anti-inflammatory role of PRG4 in synovial fluid,” Arthritis Res. Ther., 2015; 17:353), but the full interactome of this secreted protein remained unidentified prior to the present study. In addition, prior to the present study, it was unclear which AS isoforms of Prg4 have been investigated in most of the published studies as they utilized WB antibodies or PCR primers that quantitated / detected total Prg4. The interactome of PRG4-S is expected to be different than that of PRG4-L due to the skipped coding exons (FIGS. 19A-B). Prior to the present study, the receptors to which PRG4-S binds in the callus remain completely uncharacterized. In the preliminary experiments, PRG4 from d5 calli pooled from 5 mice were immunoprecipitated. The calli with a crosslinker were incubated prior to lysis and IP to detect PRG4-receptor interactions, which are transient in nature. DTSSP (Thermo) in PBS, pH 7.4, was used as a crosslinker at 10x molar excess of DTSSP to protein concentration (Lambert, W. et al., “Thiol-exchange in DTSSP crosslinked peptides is proportional to cysteine content and precisely controlled in crosslink detection by two-step LC-MALDI MSMS,” Protein Sci., 2011; 2: 1682-1691). DTSSP (FIG. 29C) is a homobifunctional, water-soluble, membrane impermeable, and cleavable crosslinker and is used in studying receptor-ligand interaction. A membrane impermeable crosslinker was selected to reduce the background / noise, given that the aim was to detect the interaction between a secreted protein (PRG4) and receptors. Following crosslinking, the calli were pulverized, lysed, and immunoprecipitated (Le Bleu, et al., “Extraction of high-quality RNA from human articular cartilage,” Anal. Biochem., 2017; 518: 134-138; Elbarbary, R. A. et al., “Tudor-SN- mediated endonucleolytic decay of human cell microRNAs promotes G(l) / S phase transition,” Science, 2017; 356:859-862; Elbarbary, R. A. et al., “UPF1 helicase promotes TSN-mediatedAttorney Docket No. 0073605-001029 miRNA decay,” Genes Dev. 2017; 31 : 1483-1493; Elbarbary, R. A. et al., “STAU1 binding 3' UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown,” Genes Dev., 2013; 27: 1495-1510). For IP, a biotin-labelled anti-PRG4 antibody (Bioss) and streptavidin beads (Thermo) were used. Biotin labelled IgG (Abeam) was used as a negative control. Notably, all commercially available anti-PRG4 antibodies bind to both PRG4-S and PRG4-L; however, this did not impact the experiment described herein, as PRG4-L is undetectable in d5 callus. Following IP, the beads were boiled with the WB loading buffer to elute (see below). WB was performed, and PRG4-S was detected in the PRG4 but not the IgG IP, as expected, (FIG. 29D). Notably, multiple WB bands of PRG4 were consistently detected (under reducing WB conditions), which is most likely due to post-translational modifications that occur at multiple sites / domains of PRG4 protein (FIG. 19A) (DuRaine, G. D., et al., “Effects of TGF- 1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures,” Osteoarthritis Cartilage, 2011; 19: 103-110). Consistent with this explanation, the intensity of all these bands decreases substantially upon Prg4 KD (FIG. 5C), which excludes the possibility of non-specific antibody binding. Importantly, CD44 was detected as a co-IP protein and was highly enriched in PRG4 IP as compared to IgG IP (FIG. 29D). P-actin was undetectable in either IP (FIG. 16D). These results indicate specific and efficient CL-IP. CD44 can function as a PRG4 receptor (Al-Sharif, A. et al., “Lubricin / Proteoglycan 4 Binding to CD44 Receptor: A Mechanism of the Suppression of Proinflammatory Cytokine-Induced Synoviocyte Proliferation by Lubricin,” Arthritis Rheumatol., 2015; 67: 1503-1513), and the data described herein further show that the PRG4-S isoform, which lacks 3 coding exons, binds CD44, and that such binding occurs in the context of the healing hematoma. In this sub-Aim, the PRG4-S interactome is further studied and globally defined as follows:

[0333] a) Identification of the full repertoire of PRG4-S receptors and interactome. CL and IP are repeated, as described above, and the sample is prepared for mass spectrometry. To that end, protocol for protein elution from the beads is modified. Specifically, in experiments shown in FIG. 29D, beads were boiled with a reducing loading buffer. This protocol is compatible with WB but releases high levels of streptavidin from the beads into the eluate, which can cause noise in mass spectrometry. Thus, the beads are divided into two portions during the elution step. The first portion is prepared for mass spectrometry by treating the beads with 20 mM DTT (pH 8.5) (as per the manufacturer’s instructions) to cleave DTSSP spacer arms and elute the co-immunoprecipitated proteins. The second portion is boiled with the loading buffer as described above and kept for WB to confirm the mass spectrometry data.Attorney Docket No. 0073605-001029

[0334] b) Selection of receptors for follow-up studies. In (a), the PRG4 interactome is characterized in the whole callus, which opens many questions that can be addressed in future studies. Thew primary focus of the present disclosure are PRG4 receptors in PSCs and the most enriched in the PRG4 IP are prioritized accordingly. scRNA-seq is used to localize receptors to the PSCs (which is confirmed using qPCR and IF).

[0335] c) For proteins / receptors chosen in b) for follow-up, the mass spectrometry data are corroborated by WB of the receptor in the PRG4 IP (as prepared in a) as well as the reciprocal IP of the receptor and WB for PRG4.

[0336] d) Prg4-receptor interaction is further confirmed in primary PSCs using IF co-staining.

[0337] e) The biological significance of Prg4-receptor interaction is assessed by knocking down the receptor in primary PSCs, or using a receptor inhibitor if available, and the results are compared to those generated using the Prg4-S KD approach, as described in Aims 2a, b.

[0338] Outcomes of Aim 3.5 can not only identify, for the first time, the full repertoire of PRG4 receptors and interactome in d5 callus but also characterize PRG4 signaling in PSCs.

[0339] The primary focus of the present studies are receptors of PSC. However, if the studies raise the necessity for further analysis, the same experiments can be performed at later timepoints to define receptors in other cell types, especially chondrocytes. CD44 appears to be a good candidate for follow-up due to the roles of PRG4-CD44 interaction in regulating inflammatory cytokines in the synovium (Al-Sharif, A. et al., “Lubricin / Proteoglycan 4 Binding to CD44 Receptor: A Mechanism of the Suppression of Proinflammatory Cytokine-Induced Synoviocyte Proliferation by Lubricin,” Arthritis Rheumatol., 2015; 67:1503-1513). In addition, CD44 has roles in regulating articular chondrocytes (Ishida, O. et al., “Chondrocytes are regulated by cellular adhesion through CD44 and hyaluronic acid pathway,” J. Bone Miner. Res., 1997; 12: 1657-1663) and MSCs (Zhu, H. et al., “The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix, Stem Cells, 2006; 24:928-935), and scRNA-seq data indicate its expression on the PSCs and on different types of immune cells (not shown). The most interesting receptor(s) to follow-up on can be determined after generating the mass spectrometry data as described in Aim 3.5.b. Pooling 5 calli was sufficient for IP followed by WB (FIG. 29D) and is expected to provide a sufficient amount of protein for mass spectrometry. Otherwise, more mice can be pooled.

[0340] Completion of the present study unravels a novel splicing variant of Prg4 and elucidates its roles in fracture healing. The present study also establishes the potential use of an engineeredAttorney Docket No. 0073605-001029 human Prg4-S variant in inducing fracture healing in pathological conditions and comorbidities that are accompanied by high rates of nonunion.

[0341] It should be understood that modifications to the embodiments disclosed herein can be made to meet a particular set of design criteria. For instance, the number of or configuration of components or parameters may be used to meet a particular objective.

[0342] It will be apparent to those skilled in the art that numerous modifications and variations of the described examples and embodiments are possible in light of the above teachings of the disclosure. The disclosed examples and embodiments are presented for purposes of illustration only. Other alternative embodiments may include some or all the features of the various embodiments disclosed herein. For instance, it is contemplated that a particular feature described, either individually or as part of an embodiment, can be combined with other individually described features, or parts of other embodiments. The elements and acts of the various embodiments described herein can therefore be combined to provide further embodiments.

[0343] It is the intent to cover all such modifications and alternative embodiments as may come within the true scope of this invention, which is to be given the full breadth thereof. Additionally, the disclosure of a range of values is a disclosure of every numerical value within that range, including the end points. Thus, while certain exemplary embodiments of the device and methods of making and using the same have been discussed and illustrated herein, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Claims

1. Attorney Docket No. 0073605-001029CLAIMS1. A method for treating bone fracture or promoting bone growth, bone regeneration, or bone fracture healing in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a recombinant proteoglycan 4 protein, the recombinant proteoglycan 4 protein having an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1.

2. A method for treating bone fracture or promoting bone growth, bone regeneration, or bone fracture healing in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a recombinant proteoglycan 4 protein, the recombinant proteoglycan 4 protein having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.

3. A method for treating bone fracture or promoting bone growth, bone regeneration, or bone fracture healing in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a nucleic acid construct, the nucleic acid construct having a nucleic acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 2.

4. A method for treating bone fracture or promoting bone growth, bone regeneration, or bone fracture healing in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a nucleic acid construct, the nucleic acid construct having a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 2.

5. The method according to claim 3 or 4, wherein the nucleic acid construct comprises a ribonucleic acid.

6. The method according to claim 3 or 4, wherein the nucleic acid construct encodes a recombinant proteoglycan 4 protein.

7. The method according to claim 6, wherein the recombinant proteoglycan 4 protein has an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1.

8. The method according to claim 6, wherein the recombinant proteoglycan 4 protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.

9. The method according to claim 6, wherein the recombinant proteoglycan 4 protein is produced or expressed in the subject as an extracellular protein.

10. The method according to claim 6, wherein the recombinant proteoglycan 4 protein is produced or expressed inside a callus of the subject.Attorney Docket No. 0073605-00102911. The method according to any one of claims 1-4, wherein the composition is administered to an articular joint of the subject.

12. The method according to any one of claims 1-4, wherein the subject is diagnosed with, or expresses symptoms of, diabetes or obesity.

13. The method according to any one of claims 1-4, further comprising inducing or promoting osteogenic or chondrogenic differentiation of mesenchymal stem cells in the subject.

14. The method according to any one of claims 1-4, further comprising maintaining or restoring chondrocyte homeostasis or osteoblast homeostasis in the subject.

15. The method according to any one of claims 1 -4, wherein the subject is a human.

16. A recombinant proteoglycan 4 protein having an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1.

17. A recombinant proteoglycan 4 protein having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.

18. The recombinant proteoglycan 4 protein of claim 16 or 17 for use in the treatment of bone fracture.

19. The recombinant proteoglycan 4 protein of claim 16 or 17 for use in promoting bone growth, bone regeneration, or bone fracture healing.

20. Use of the recombinant proteoglycan 4 protein according to claim 16 or 17 in the manufacture of a medicament for the treatment of bone fracture.

21. Use of the recombinant proteoglycan 4 protein according to claim 16 or 17 in the manufacture of a medicament for promoting bone growth, bone regeneration, or bone fracture healing.

22. Use of the recombinant proteoglycan 4 protein according to claim 16 or 17 for treating bone fracture.

23. Use of the recombinant proteoglycan 4 protein according to claim 16 or 17 for promoting bone growth, bone regeneration, or bone fracture healing.

24. Use of the recombinant proteoglycan 4 protein according to claim 16 or 17 in combination with a full-length proteoglycan 4 protein or a proteoglycan 4 protein having an amino acid sequence longer than that of the recombinant proteoglycan 4 protein in the manufacture of a medicament for the treatment of osteoarthritis.

25. Use of the recombinant proteoglycan 4 protein according to claim 16 or 17 in combination with a full-length proteoglycan 4 protein or a proteoglycan 4 protein having an amino acidAttorney Docket No. 0073605-001029 sequence longer than that of the recombinant proteoglycan 4 protein for treating osteoarthritis.

26. Use of the recombinant proteoglycan 4 protein according to claim 16 or 17 in combination with a stem cell therapy for treating bone fracture.

27. Use of the recombinant proteoglycan 4 protein according to claim 16 or 17 in combination with a stem cell therapy in the manufacture of a medicament for the treatment of bone fracture.

28. Use of the recombinant proteoglycan 4 protein according to claim 16 or 17 in combination with a stem cell therapy in the manufacture of a medicament for promoting bone growth, bone regeneration, or bone fracture healing.

29. A composition comprising: a therapeutically effective amount of a recombinant proteoglycan 4 protein having an amino acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 1; and a delivery vehicle, wherein the therapeutically effective amount of the recombinant proteoglycan 4 protein is stored within a matrix or cavity of the delivery vehicle.

30. A composition comprising: a therapeutically effective amount of a recombinant proteoglycan 4 protein having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1; and a delivery vehicle, wherein the therapeutically effective amount of the recombinant proteoglycan 4 protein is stored within a matrix or cavity of the delivery vehicle.

31. The composition according to claim 29 or 30, wherein the therapeutically effective amount of the recombinant proteoglycan 4 protein is released from the delivery vehicle over a period of at least seven days and no greater than six months.

32. A composition comprising: a therapeutically effective amount of a nucleic acid construct having a nucleic acid sequence that is identical to, or is a functional variant of, SEQ ID NO: 2; and a delivery vehicle, wherein the therapeutically effective amount of the nucleic acid construct is stored within a matrix or cavity of the delivery vehicle.

33. A composition comprising: a therapeutically effective amount of a nucleic acid construct having a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 2; andAttorney Docket No. 0073605-001029 a delivery vehicle, wherein the therapeutically effective amount of the nucleic acid construct is stored within a matrix or cavity of the delivery vehicle.

34. The composition of any one of claims 29, 30, 32, and 33 for use in the treatment of bone fracture.

35. The composition of any one of claims 29, 30, 32, and 33 for use in promoting bone growth, bone regeneration, or bone fracture healing.

36. Use of the composition according to any one of claims 29, 30, 32, and 33 in the manufacture of a medicament for the treatment of bone fracture.

37. Use of the composition according to any one of claims 29, 30, 32, and 33 in the manufacture of a medicament for promoting bone growth, bone regeneration, or bone fracture healing.

38. The composition according to any one of claims 29, 30, 32, and 33, further comprising i) a therapeutically effective amount of a full-length proteoglycan 4 protein or a therapeutically effective amount of a proteoglycan 4 protein having an amino acid sequence longer than that of the recombinant proteoglycan 4 protein or ii) a therapeutically effective amount of a nucleic acid encoding the full-length proteoglycan 4 protein or a therapeutically effective amount of a nucleic acid encoding the proteoglycan 4 protein having an amino acid sequence longer than that of the recombinant proteoglycan 4 protein.

39. The composition of claim 38 for use in the treatment of osteoarthritis.

40. Use of the composition according to claim 38 in the manufacture of a medicament for the treatment of osteoarthritis.

41. Use of the composition according to any one of claims 29, 30, 32, and 33 in combination with a stem cell therapy in the manufacture of a medicament for the treatment of bone fracture or for promoting bone growth, bone regeneration, or bone fracture healing.

42. Use of the composition according to any one of claims 29, 30, 32, and 33 in combination with a stem cell therapy for treating bone fracture or promoting bone growth, bone regeneration, or bone fracture healing.

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