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WO2025039000A1 - Prévention et traitement de la fibrose par inhibition ou régulation positive de protéines spécifiques - Google Patents

Prévention et traitement de la fibrose par inhibition ou régulation positive de protéines spécifiques Download PDF

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WO2025039000A1
WO2025039000A1 PCT/US2024/042952 US2024042952W WO2025039000A1 WO 2025039000 A1 WO2025039000 A1 WO 2025039000A1 US 2024042952 W US2024042952 W US 2024042952W WO 2025039000 A1 WO2025039000 A1 WO 2025039000A1
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lepr
subject
cells
bone
implant
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Xu Yang
Vincentius Jeremy SUHARDI
Anastasia OKTARINA
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Bostrom Mathias PG
New York Society For Relief Of Ruptured And Crippled Maintaining Hospital For Special
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Bostrom Mathias PG
New York Society For Relief Of Ruptured And Crippled Maintaining Hospital For Special
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Orthopedic-related fibrosis is a major cause of orthopedic failure that resulted in significant morbidity and mortality to patients, which included but not limited to implant failure, fracture nonunion, pseudoarthrosis, soft tissue-to-bone failure, soft tissue-to-soft tissue failure, arthrofibrosis, which impacts multiple subspecialties of medicine, often requiring surgical revision 1-4 .
  • Peri-implant fibrosis around joint prostheses is characterized by the formation of fibrotic tissue around the implant instead of bone 3,12,13 .
  • fibrous tissue forms between bone fragments[1], soft-tissue to bone[2], and soft tissue-to- soft tissue [3], respectively.
  • LEPR+ cells Based on their location in the marrow cavity near the ultimate sites of the aforementioned fibrotic processes, we considered LEPR+ cells to be strong candidates for the primary cell type populating orthopedic-related fibrotic tissue. LEPR+ cells largely overlap with CXCL12+ cells and are often also termed CXCL12-abundant reticular (CAR) cells 20 . LEPR+/CAR cells also express high levels of the transcription factors FOXC121 and EBF3 22 .
  • LEPR+ cells have been shown to contribute to bone, adipose, and cartilage tissue formation in adulthood 31,32 , their role in the formation of fibrotic tissue, remains to be elucidated.
  • orthopedic-related fibrosis which includes but is not limited to, peri-implant fibrotic tissue, bone-to-bone fibrosis, bone-to-soft tissue fibrosis, soft tissue-to-soft tissue fibrosis, joint related fibrosis, in a subject in a subject in need thereof.
  • the method includes administering to the subject an inhibitor of ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PER1, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1, or TNNT3.
  • the method includes administering to the subject an activator of BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA.
  • the inhibitor is an antibody to ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PER1, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1, or TNNT3.
  • a method of treating or preventing the formation of fibrotic tissue in bone or bone marrow in a subject in need thereof is provided.
  • the method includes administering a population of isolated cells, optionally wherein the cells have been enriched for one or more of the following markers: CD200+, CD90+, 6C3+, THY1+, PDGFRA+, PDGFRB+, SCA1+, GREM1+, OSX+, CD105+, OCN+, CTSK+, CXCL12+, EBF3+, and MX1+.
  • the method includes administering osteogenic LEPR+ donor cells to the subject.
  • the cells have been enriched for cells showing a phenotype selected from i. LEPR+ BGLAP+, ii. LEPR+ ALPL+, iii. LEPR+ OSX+, iv.
  • FIG.1A – FIG.1P show leptin receptor (LEPR)-expressing cells are abundant in both human and murine peri-implant fibrous tissue.
  • FIG.1A Schematic (FIG.1A) and plain radiograph (FIG.1B) of a patient with peri-implant fibrosis around a total joint replacement.
  • FIG.1C Hematoxylin and eosin staining of mouse peri-implant fibrotic tissue at 14 days post-surgery. Scale bar, 500 ⁇ m. Right, enlarged view of the outlined region (green box).
  • FIG.1D LEPR (red) signal in human peri- implant fibrotic tissue obtained during revision joint replacement surgery.
  • Scale bar 250 ⁇ m.
  • FIG.1E Enlarged view of the outlined region (yellow box) in panel d. Scale bar, 50 ⁇ m.
  • FIG.1F Flow cytometry of cells from digested human peri-implant fibrotic tissue obtained during revision joint replacement surgery to identify Lin-LEPR + cells.
  • FIG.1G Flow cytometry of cells from digested human bone obtained during primary joint replacement surgery to identify Lin-LEPR + cells.
  • FIG.1H There were significantly more Lin-LEPR + cells in human peri-implant fibrotic tissue than in trabecular bone.
  • FIG.1I Plain radiograph of the murine model with peri-implant fibrosis.
  • FIG.1J Hematoxylin and eosin staining of mouse peri- implant fibrotic tissue at 14 days post-surgery. Scale bar, 500 ⁇ m. Right, enlarged view of the outlined region (yellow box). Scale bar, 100 ⁇ m.
  • FIG.1K Tdtomato (red) signal in the proximal tibia of LepR cre ;Rosa26 tdtomato mice on postoperative day 14.
  • White dotted lines correspond to the interface between the implant and fibrous tissue.
  • Scale bar 500 ⁇ m.
  • FIG.1L, FIG.1M Enlarged view of the outlined region (yellow box) in panel k, showing LepR-Tdtomato cells (top) and LepR antibody (LEPR-Ab, bottom) immunofluorescence. Scale bar, 50 ⁇ m.
  • FIG.1N Flow cytometry of cells from digested proximal tibias from mice receiving fibrous-integration surgery (FIG.1N) or osseointegration surgery (FIG.1O) on postoperative day 14 to identify Lin-LepR + cells.
  • FIG.1P There were significantly more Lin-LEPR + cells in the proximal tibia in mice with peri-implant fibrous tissue than in those without fibrous tissue.
  • FIG.2A – FIG.2Q show ablation of LEPR lineage cells resulted in a significant reduction in peri-implant fibrosis.
  • FIG.2A Schematic representation of experiments involving presurgical ablation of Lepr-tdTomato + cells in LepR cre ;Rosa26 tdtomato;iDTR mice with the administration of diphtheria toxin or saline daily from 7 days preoperatively until postoperative day 14. All mice underwent fibrous integration surgery.
  • FIG.2B Microcomputed tomography ( ⁇ CT) of the proximal tibias of mice that received either diphtheria toxin or saline 14 days after fibrous integration surgery. The yellow outline delineates the implant, and the red outline delineates the peri-implant fibrotic area. Scale bars, 500 ⁇ m.
  • FIG.2C Hematoxylin-eosin (left) and immunofluorescence imaging (middle and right) of mice 14 days after undergoing fibrous integration surgery and receiving diphtheria toxin or saline. Scale bar, 500 ⁇ m. Right, enlarged view of the outlined region, Scale bar, 50 ⁇ m.
  • mice receiving diphtheria toxin had significantly fewer Lin-LepR-Tdtomato + cells than mice receiving saline on postoperative day 14.
  • FIG.2I Schematic representation of experiments involving postsurgical ablation of Lepr- tdTomato + cells in LepR cre ;Rosa26 tdtomato;iDTR mice with the administration of diphtheria toxin or saline daily from postoperative day 14 until postoperative day 28. All mice underwent fibrous integration surgery.
  • FIG.2J Microcomputed tomography ( ⁇ CT) of the proximal tibias of mice that received either diphtheria toxin or saline 14 days after fibrous integration surgery. The yellow outline delineates the implant, and the red outline delineates the peri-implant fibrotic area. Scale bars, 500 ⁇ m.
  • FIG.2K Hematoxylin- eosin (left) staining of mice 28 days after undergoing fibrous integration surgery and receiving DT or saline. Scale bar, 500 ⁇ m.
  • FIG.2L Immunofluorescence imaging of mice 28 days after undergoing fibrous integration surgery and receiving diphtheria toxin or saline. Scale bar, 500 ⁇ m. Right, enlarged view of the outlined region. Scale bar, 50 ⁇ m.
  • FIG.2M – FIG.2P Mice in the diphtheria toxin group had less peri-implant fibrotic tissue and more peri-implant bone than those in the saline group, as measured by bone volume/total volume (BV/TV) (FIG.2M), trabecular number (FIG.2N), histological percentage bone area (FIG.2O), and histological percentage fibrotic area (FIG.2P).
  • FIG.3A – FIG.3J show engrafted Lin-LepR-tdTomato + cells from a donor that underwent fibrous integration surgery are more inclined to form fibrous tissue than Lin- LepR-tdTomato + cells from a donor that underwent osseointegration surgery.
  • FIG.3A Schematic representation of kidney and orthotopic engraftment of Lin-LepR-tdTomato + cells from LepR cre ;Rosa26 tdtomato mice that underwent either fibrous integration or osseointegration surgery.
  • FIG.3B Whole-kidney immunofluorescence imaging (far left) and the corresponding enlarged view (second from the left; scale bar, 500 ⁇ m), immunofluorescence imaging of a cryo-sectioned kidney (second from the right; scale bar, 50 ⁇ m) and Movat’s pentachrome-stained slides of kidneys from mice receiving Lin- LepR-tdTomato + cells from a donor that underwent fibrous integration or osseointegration surgery at 2 months post-surgery. Scale bar, 50 ⁇ m.
  • FIG.3C Microcomputed tomography ( ⁇ CT) of the proximal tibias of mice 2 months after fibrous integration surgery with intraoperative peri-implant engraftment of Lin-LepR-tdTomato + cells from a donor that underwent fibrous integration or osseointegration surgery. Scale bar, 500 ⁇ m.
  • FIG.3G Hematoxylin and eosin staining of a recipient mouse’s proximal tibia at 2 months post- surgery. Scale bar, 500 ⁇ m.
  • FIG.3H Immunofluorescence imaging of recipient mice receiving Lin-LepR-tdTomato + cells from donors that underwent either fibrous integration or osseointegration surgery. Scale bar, 500 ⁇ m. Middle, enlarged view of the outlined red box showing the presence of Lepr-tdTomato + aSMA + in the fibrous tissue. Scale bar, 50 ⁇ m. Right, enlarged view of the outlined red box showing the presence of Tdtomato + CD200 + cells in the fibrous tissue. Scale bar, 50 ⁇ m.
  • FIG.4A – FIG.4R show ADGRF5 is abundantly expressed by LEPR + cells in both human and murine peri-implant fibrotic tissue.
  • LEPR red
  • ADGRF5 green
  • Scale bar 250 ⁇ m.
  • Middle row enlarged view corresponding to the area marked by the red box, as depicted in the top row.
  • Scale bar 50 ⁇ m.
  • Bottom row enlarged view corresponding to the area marked by the green box, as depicted in the top row.
  • Scale bar 50 ⁇ m.
  • FIG.4B UMAP plot showing integrated analysis of LEPR-expressing cells. Cells are colored by cluster.
  • FIG.4C – FIG.4E Relative expression of LEPR (FIG.4C) and ADGRF5 (FIG.4D) and coexpression of LEPR and ADGRF5 (FIG.4E).
  • FIG.4G ADGRF5 is more highly expressed by Lin-LEPR + cells in the fibrous membrane than in bone. *p ⁇ 0.05. Unpaired, two-tailed Student’s t test. Data are the mean ⁇ s.e.m.
  • FIG.4H LepR-Tdtomato (red)- and ADGRF5 (green)-expressing cells in LepR cre ;Rosa26 tdtomato mice that underwent fibrous integration surgery at postoperative day 14.
  • Top row scale bar, 500 ⁇ m.
  • Middle row enlarged view corresponding to the area marked by the red box, as depicted in the top row.
  • Scale bar 50 ⁇ m.
  • FIG.4I UMAP plot showing integrated analysis of FACS-sorted Tdtomato + cells from LepR cre ;Rosa26 tdtomato mice. Cells are colored by cluster. The top 20 principal components were chosen for clustering.
  • FIG.4J – FIG.4L Relative expression of Lepr (FIG.4J) and Adgrf5 (FIG.4K) and coexpression of Lepr and Adgrf5 (FIG.4L).
  • FIG.4P Violin plot showing the expression of Lepr, Cxcl12, Ebf3, and Adgrf5 by FACS-sorted Tdtomato + cells from LepR cre ;Rosa26 tdtomato mice.
  • FIG.4R Adgrf5 is more highly expressed in Lin-LepR-Tdtomato + cells from mice that underwent fibrous integration than in those from mice that underwent osseointegration. *p ⁇ 0.05.
  • FIG. 5A – FIG.5M show ablation of Adgrf5 in LEPR lineage cells inhibits peri- implant fibrosis and enhances peri-implant bone formation in vivo.
  • FIG.5A Microcomputed tomography ( ⁇ CT) of the proximal tibias of LepR cre ;Rosa26 tdtomato ; Adgrf5 +/+ (Lepr cre/+ ) and LepR cre ;Rosa26 tdtomato ; Adgrf5 +/+ (Lepr cre/+ ; Adgrd5 f/f ) mice on postoperative day 14. All mice underwent fibrous integration surgery. Scale bar, 500 ⁇ m.
  • FIG.5B Hematoxylin and eosin staining of the proximal tibias of Lepr cre/+ and Lepr cre/+ ; Adgrd5 f/f mice on postoperative day 14. All mice underwent fibrous integration surgery. Scale bar, 500 ⁇ m.
  • FIG.5C – FIG.5E Ablation of Adgrf5 in LepR lineage cells resulted in more peri-implant bone than observed in control mice, as measured by bone volume/total volume (BV/TV) (FIG.5C), trabecular number (FIG.5D), and trabecular thickness (FIG.5E).
  • FIG.5K – FIG.5M Immunofluorescence quantification of peri-implant tissue from control and flox mice on postoperative day 14 demonstrated that ablation of ADGRF5 in LEPR lineage cells resulted in a reduction in tdTomato+ cell numbers (FIG.5K) and ACTA2- expressing LepR lineage cell numbers (FIG.5M) without significant changes in colocalization between tdTomato or CD200 (FIG.5L).
  • FIG.6A – FIG.6P show administration of a neutralizing antibody against ADGRF5 can both prevent and reverse peri-implant fibrosis.
  • FIG.6A Schematic representation of the intraarticular administration of anti-ADGRF5 to prevent peri- implant fibrosis. All LepR cre ;Rosa26 tdtomato mice underwent fibrous integration surgery and were subsequently randomized to receive daily intra-articular injection of anti- ADGRF5 or isotype control.
  • FIG.6B Microcomputed tomography ( ⁇ CT) of the proximal tibias of LepR cre ;Rosa26 tdtomato mice receiving either anti-ADGRF5 or isotype control on postoperative day 14. Scale bar, 500 ⁇ m.
  • FIG.6C Hematoxylin and eosin staining of the proximal tibias of LepR cre ;Rosa26 tdtomato mice receiving either anti- ADGRF5 or isotype control on postoperative day 14. Scale bar, 500 ⁇ m.
  • FIG.6D Immunofluorescence imaging of the proximal tibias of LepR cre ;Rosa26 tdtomato mice receiving either anti-ADGRF5 or isotype control on postoperative day 14.
  • Scale bar 500 ⁇ m.
  • FIG.6E, FIG.6F Mice receiving anti-ADGRF5 have more peri-implant bone than isotype control mice, as measured by bone volume/total volume (BV/TV) (FIG.6E) and trabecular thickness (FIG.6F).
  • FIG.6I Schematic representation of the intraarticular administration of anti-ADGRF5 to reverse peri-implant fibrosis. All LepR cre ;Rosa26 tdtomato mice underwent fibrous integration surgery. On postoperative day 14, the mice were randomized to receive daily intra-articular injection of anti-ADGRF5 or isotype control for 14 days.
  • FIG.6J Microcomputed tomography ( ⁇ CT) of the proximal tibia of LepR cre ;Rosa26 tdtomato mice receiving either anti-ADGRF5 or isotype control on postoperative day 28. Scale bar, 500 ⁇ m.
  • FIG.6K Hematoxylin and eosin staining of the proximal tibia of LepR cre ;Rosa26 tdtomato mice receiving either anti-ADGRF5 or isotype control on postoperative day 28.
  • Scale bar 500 ⁇ m.
  • FIG.6L Immunofluorescence imaging of the proximal tibia of LepR cre ;Rosa26 tdtomato mice receiving either anti- ADGRF5 or isotype control on postoperative day 28.
  • Scale bar 500 ⁇ m.
  • Scale bar 50 ⁇ m.
  • FIG.6M Mice receiving anti- ADGRF5 have more peri-implant bone than isotype control mice, as measured by bone volume/total volume (BV/TV) (FIG.6M) and trabecular thickness (FIG.6N).
  • FIG.6O Histological quantification of the peri- implant tissue of mice receiving anti-ADGRF5 or isotype control on postoperative day 28 demonstrated a significant reduction in peri-implant fibrosis (FIG.6O) and enhanced formation of peri-implant bone (FIG.6P).
  • Images in FIG.6B – FIG.6D and FIG.6J – FIG.6L are representative of at least 3 independent biological replicates.
  • FIG.7A – FIG.7D show LEPR, ACTA2, and ADGRF5 are expressed by peri- implant fibrotic tissue from multiple patients who underwent revision surgery for aseptic loosening.
  • FIG.7A, FIG.7C Expression of LEPR (red), ACTA2 (green), and ADGRF5 (cyan) in peri-implant fibrotic tissue from two different patients suffering from peri- implant fibrosis around prior total hip arthroplasty.
  • Far left overlap between DAPI and LEPR.
  • Second from left overlap between ACTA2 and DAPI.
  • Third from right overlap between LEPR and ACTA2.
  • Second from right overlap between LEPR and ADGRF5.
  • FIG.7B, FIG.7D Expression of LEPR (red), ACTA2 (green), and ADGRF5 (cyan) in peri-implant fibrotic tissue from two different patients suffering from peri-implant fibrosis around prior total knee arthroplasty.
  • Far left overlap between DAPI and LEPR.
  • Second from left overlap between ACTA2 and DAPI.
  • Third from left overlap between ADGRF5 and DAPI.
  • Third from right overlap between LEPR and ACTA2.
  • Second from right overlap between LEPR and ADGRF5.
  • Scale bar 250 ⁇ m.
  • FIG.8A – FIG.8N show a subset of LepR lineage cells in peri-implant fibrotic tissue expresses Cxcl12-GFP.
  • FIG.8A Expression of LepR-tdTomato and Cxcl12-GFP in the peri-implant fibrotic tissue of LepR cre ;Rosa26 tdTomato ; Cxcl12 GFP mice that underwent fibrous integration surgery at postoperative day 14.
  • a subset of LepR-lineage cells (red) colocalized with cells expressing Cxcl12 (green). Scale bar, 500 ⁇ m.
  • FIG.8B – FIG.8E Enlarged view of the outlined blue box of the figure in panel FIG.8A.
  • FIG.8A – FIG.8E are representative of at least 3 independent experiments.
  • FIG.8F Expression of LepR-tdTomato and Cxcl12-GFP in the peri- implant osseous tissue of LepR cre ;Rosa26 tdTomato ; Cxcl12 GFP mice that underwent osseointegration surgery at postoperative day 14.
  • a subset of LepR-lineage cells (red) in the perivascular area colocalizes with cells expressing Cxcl12 (green).
  • Scale bar 500 ⁇ m.
  • Images in FIG.8A are representative of at least 3 independent experiments.
  • FIG.8G – FIG.8J Enlarged view of the outlined blue box of the figure in panel FIG.8F. Scale bar, 50 ⁇ m. Images in FIG.8F – FIG.8J are representative of at least 3 independent experiments.
  • FIG.8K, FIG.8L Expression of CXCL12-GFP by Lin-LepR-tdTomato + cells (FIG.8K) or cells from mice that underwent osseointegration surgery (FIG.8L).
  • FIG.8N Immunofluorescence quantification of colocalization between LepR-tdTomato and Cxcl12-GFP in LepR cre ;Rosa26 tdTomato ; Cxcl12 GFP mice that underwent fibrous integration or osseointegration surgery at postoperative day 14.
  • FIG.9A – FIG.9J show small percentages of Lin-6C3-CD90-CD200 + CD105- (mSSC) and Lin-6C3-CD90-CD200 var CD105 + (BCSP) cells are Lin-LepR-tdTomato + .
  • FIG.9A Schematic of the flow cytometry comparison experiment between LepR cre ;Rosa26 tdtomato mice that underwent fibrous integration and osseointegration implantation surgery at postoperative day 14.
  • FIG.9B – FIG.9D Schematic representation of the strategy used for FACS analysis of Lin-6C3-CD90-CD200 + CD105- (mSSC) and Lin-6C3-CD90-CD200 var CD105 + (BCSP) mice that underwent fibrous integration surgery (FIG.9C) or osseointegration surgery (FIG.9D).
  • FIG.9E, FIG.9F There was no difference in the abundance of mSSC or BCSP in the peri-implant area between mice that underwent fibrous integration and osseointegration surgery. Data are the mean ⁇ s.e.m. Unpaired, two-tailed Student’s t test.
  • FIG.9I LepR-tdTomato + cells at the fibrous integration interface were immunostained for CD200, demonstrating the presence of LepR-Tdtomato + CD200 + cells at the bone-fibrous tissue interface. Scale bar, 500 ⁇ m.
  • FIG.9J LepR-tdTomato + cells at the osseointegration interface were immunostained for CD200, demonstrating the presence of LepR- Tdtomato + CD200 + cells in the perivascular area. Scale bar, 500 ⁇ m.
  • Middle column and right column enlarged views of the outlined red box. Scale bar, 50 ⁇ m. Images in FIG. 9B – FIG.9D and FIG.9I – FIG.9J are representative of at least 3 independent experiments.
  • FIG.10A – FIG.10P show a subset of LepR lineage cells in peri-implant fibrotic tissue expresses Acta2-RFP.
  • FIG.10A Expression of LepR-zsGreen and Acta2-RFP in the peri-implant fibrotic tissue of LepR cre ;Rosa26 Zsgreen ; Acta2 mRFP mice that underwent fibrous integration surgery at postoperative day 14.
  • Scale bar 500 ⁇ m.
  • FIG.10B – FIG.10E Enlarged view of the outlined blue box from the figure in panel FIG.10A. Scale bar, 50 ⁇ m. Images in FIG.10A – FIG.10E are representative of at least 3 independent experiments.
  • FIG.10F Expression of LepR-zsGreen and Acta2-RFP in the peri-implant osseous tissue of LepR cre ;Rosa26 Zsgreen ; Acta2 mRFP mice that underwent osseointegration surgery at postoperative day 14.
  • Scale bar 500 ⁇ m. Images in a are representative of at least 3 independent experiments.
  • FIG.10G – FIG. 10J Enlarged view of the outlined blue box from the figure in panel FIG.10F. Scale bar, 50 ⁇ m. Images in FIG.10F – FIG.10J are representative of at least 3 independent experiments.
  • FIG.10K, FIG.10L Representation of the strategy used for FACS analysis of LepR-zsGreen and Acta2-RFP expression in Lin-6C3-CD90-CD200 + CD105- cells that underwent fibrous integration surgery (FIG.10K) or osseointegration surgery (FIG.10L).
  • FIG.10M, FIG.10N Immunofluorescence quantification of the expression of LepR- zsGreen and Acta2-RFP in LepR cre ;Rosa26 Zsgreen ; Acta2 mRFP mice that underwent fibrous integration or osseointegration surgery at postoperative day 14. Data are the mean ⁇ s.e.m.
  • n 5 for both the osseointegration and fibrous integration models. Unpaired, two- tailed Student’s t test.
  • FIG.11A – FIG.11F show peri-implant fibrosis is persistent for at least 16 weeks in a murine model of peri-implant fibrosis.
  • FIG.11A Microcomputed tomography ( ⁇ CT) of the proximal tibias of LepR cre ;Rosa26 tdtomato mice that underwent fibrous integration surgery at postoperative days 3 and 7 and postoperative weeks 2, 4, 8, and 16. Scale bar, 500 ⁇ m.
  • FIG.11B Hematoxylin and eosin staining of the proximal tibia of LepR cre ;Rosa26 tdtomato mice that underwent fibrous integration surgery at postoperative day 3, day 7, 2 weeks, 4 weeks, 8 weeks, and 16 weeks. Scale bar, 500 ⁇ m.
  • FIG.11C Immunofluorescence imaging of the proximal tibia of LepR cre ;Rosa26 tdtomato mice that underwent fibrous integration surgery at postoperative days 3 and 7 and postoperative weeks 2, 4, 8, and 16. Scale bar, 500 ⁇ m. Bottom row, enlarged view of the outlined red box at each time point. Scale bar, 50 ⁇ m.
  • FIG.11F Immunofluorescence quantification of peri-implant fibrosis (% fibrosis) and peri-implant bone (% bone) for mice at postoperative days 3 and day 7 and postoperative weeks 2, 4, 8, and 16.
  • FIG.11A – FIG.11C are representative of at least 3 independent experiments.
  • FIG.12A – FIG.12F show analysis of peri-implant bone of mice that underwent osseointegration surgery at postoperative days 3 and 7 and postoperative weeks 2, 4, 8, and 16.
  • FIG.12A Microcomputed tomography ( ⁇ CT) of the proximal tibias of LepR cre ; Rosa26 tdtomato mice that underwent osseointegration surgery at postoperative days 3 and 7 and postoperative weeks 2, 4, 8, and 16.
  • Scale bar 500 ⁇ m.
  • FIG.12B Hematoxylin and eosin staining of the proximal tibias of LepR cre ;Rosa26 tdtomato mice that underwent osseointegration surgery at postoperative days 3 and 7 and postoperative weeks 2, 4, 8, and 16.
  • Scale bar 500 ⁇ m.
  • FIG.12C Immunofluorescence imaging of the proximal tibias of LepR cre ;Rosa26 tdtomato mice that underwent osseointegration surgery at postoperative days 3 and 7 and postoperative weeks 2, 4, 8, and 16. Scale bar, 500 ⁇ m. Bottom row, enlarged view of the outlined red box at each time point. Scale bar, 50 ⁇ m.
  • FIG.13A Schematic representation of daily diphtheria toxin administration in LepR cre ;Rosa26 tdtomato;iDTR mice from preoperative day 7 until postoperative day 14. All mice underwent fibrous-integration surgery.
  • FIG.13B Microcomputed tomography ( ⁇ CT) of the proximal tibias of mice 14 days after fibrous-integration surgery and receiving diphtheria toxin. The yellow outline delineates the implant, and the red outline delineates the peri-implant fibrotic area. Scale bars, 500 ⁇ m.
  • FIG.13C, FIG.13D Hematoxylin-eosin (FIG.13C) and immunofluorescence imaging (FIG.13D) of mice 14 days after undergoing fibrous integration surgery and receiving diphtheria toxin. Scale bar, 500 ⁇ m.
  • FIG.13E LepR cre ;Rosa26 tdtomato mice receiving diphtheria toxin had BV/TV values similar to those of LepR cre ;Rosa26 tdtomato;iDTR mice receiving saline.
  • FIG.13B – FIG.13D are representative of at least 3 independent experiments.
  • FIG.14A – FIG.14F show human mSSCs (Lin-PDPN + CD146-CD164 + CD73 + ) are more abundant in bone than in fibrous membranes, but Lin-LEPR + cells are more abundant in fibrous membranes than in bone.
  • FIG.14A Schematic representation of the strategy used for FACS analysis to obtain the Lin- population.
  • FIG.14B, FIG.14C Schematic representation of the strategy used for FACS analysis to obtain a subset of Lin- PDPN + CD146-CD164 + CD73 + LEPR-expressing cells from bone (FIG.14B) or fibrous tissue (FIG.14C).
  • FIG.14F Flow cytometry quantification of human fibrous tissue and bone demonstrates significantly higher numbers of Lin- LEPR + PDPN + CD146- cells in the fibrous membrane than in bone. Data are the mean ⁇ s.e.m.
  • FIG.15A – FIG.15G show single-cell RNA sequencing analysis of Lin-LEPR + cells from human bone.
  • FIG.15A UMAP visualization of different subpopulations of human Lin-LEPR + cells, color-coded based on the subpopulation labels. We identified five clusters based on differential gene expression.
  • DEGs differentially expressed genes
  • FIG.15B Violin plots showing the expression of CXCL12, ADIPOQ, SAT1, HOXC6, SPP1, COL1A1, TIMP3, ACTA2, EGR1, and ANGPTL4 in each cluster.
  • FIG.15C Heatmap showing the relative expression levels (row-wide Z score) of the 20 most significant markers for each cluster (rows) across cells in the 5 clusters (columns). Bars on the top are colored as in FIG.15A.
  • FIG.15D Violin plots showing the expression density levels of salient feature genes of CAR cells in each cluster: Lepr, Cxcl12, Ebf3, and Foxc1.
  • FIG.15E Expression density levels of CXCL12, ACTA2, VIM, FN1, COL1A2, FBLN1, ALPL, BGLAP, WIF1, CLEC3B, LPL, and DUSP1 projected to the UMAP representation, as depicted in FIG. 15A.
  • FIG.15F Coexpression of LEPR and CXCL12, LEPR and ACTA2, LEPR and VIM, LEPR and FN1, LEPR and COL1A2, and LEPR and FBLN1 projected to the UMAP representation.
  • FIG.15G Coexpression among LEPR, CXCL12, and ADGRF5; LEPR, ACTA2 and ADGRF5; LEPR, VIM and ADGRF5; LEPR, FN1, and ADGRF; LEPR, COL1A2, and ADGRF5; and LEPR, FBLN1 and ADGRF5 projected to the UMAP representation.
  • FIG.16A – FIG.16H show single-cell RNA sequencing analysis of FACS-sorted Lin-LepR-Tdtomato + cells from LepR cre ;Rosa26 tdtomato mice.
  • FIG.16A UMAP visualization of all 34,273 cells from the integrated analysis separated based on the dataset source and color-coded based on the subpopulation labels.
  • FIG.16B Violin plots showing the expression density levels of salient feature genes of CAR cells in each cluster: Lepr, Cxcl12, Ebf3, Foxc1, and Kitl.
  • FIG.16C Violin plots showing the expression density levels of osteogenic lineage genes in each cluster: Runx2, Sp7, Alpl, Bglap, Bglap2, and Kitl. Cells.
  • FIG. 16D Violin plots showing the expression of adipogenic genes for each cluster: Adipoq, Lpl, Kng2, Hp, Cebpa, Plin.
  • FIG.16E Violin plots showing the expression of Acta2, Vim, Fn1, Cfl1, Col1a2, and Col3a1 in each cluster.
  • FIG.16F Violin plots showing the expression of chondrogenic genes for each cluster: 16an and Sox9.
  • FIG.16G Violin plots showing the expression of periosteal-associated genes for each cluster: Postn, Ctsk. Cell differentiation trajectory of CAR cells, colored by subpopulation identity.
  • FIG. 16H Slingshot trajectory plots of CAR cells. Top, second from left: periosteal, chondrogenic, and myofibroblastic pathways, as predicted by Slingshot trajectory analysis.
  • FIG.16C Top, far right: Osteogenic pathway, as predicted by Slingshot trajectory analysis. Bottom row: Three adipogenic pathways, as predicted by Slingshot trajectory analysis.
  • FIG.17A – FIG.17I show ablation of Adgrf5 in the LEPR lineage improves implant-host interface strength but does not cause any change in presurgical baseline bone characteristics.
  • FIG.17A, FIG.17B Host bone-implant failure load (FIG.17A) and work to failure (Nm) of LepR cre ;Rosa26 tdtomato ; Adgrf5 +/+ (Lepr cre/+ ) and LepR cre ;Rosa26 tdtomato ; Adgrf5 +/+ (Lepr cre/+ ; Adgrd5 f/f ) mice at postoperative day 28.
  • FIG.17D Hematoxylin and eosin staining of the proximal tibias of unoperated Lepr cre/+ and Lepr cre/+ ; Adgrf5 f/f mice. Scale bar, 500 ⁇ m.
  • FIG.17E Immunofluorescence imaging of unoperated Lepr cre/+ and Lepr cre/+ . Scale bar, 500 ⁇ m.
  • FIG.17F – FIG.17H Ablation of Adgrf5 in LepR lineage cells resulted in peri-implant bone similar to that of control mice, as measured by bone volume/total volume (BV/TV) (FIG.17F), trabecular number (FIG.17G), and trabecular thickness (FIG.17H).
  • BV/TV bone volume/total volume
  • FIG.17G trabecular number
  • FIG.17H trabecular thickness
  • FIG.18A – FIG.18F show administration of a neutralizing antibody reduces LepR-Tdtomato + cell numbers but does not affect the Lin-6C3-CD90-CD200 + CD105- (mSSC) subpopulation.
  • FIG.18A Immunofluorescence imaging of CD200 antibody staining of the proximal tibias of LepR cre ;Rosa26 tdtomato mice receiving either anti- ADGRF5 or isotype control as prophylaxis. Scale bar, 500 ⁇ m. Right column, enlarged view of the outlined red box. Scale bar, 50 ⁇ m.
  • FIG.18B Immunofluorescence imaging of CD200 antibody-stained proximal tibias of LepR cre ;Rosa26 tdtomato mice receiving either anti-ADGRF5 or isotype control as a treatment starting on postoperative day 14.
  • Scale bar 500 ⁇ m.
  • Right column enlarged view of the outlined red box.
  • Scale bar 50 ⁇ m.
  • FIG.18C, FIG.18E The Tdtomato + subset of Lin-6C3-CD90-CD200 + CD105- cells was present at lower abundance in the group of mice receiving anti-ADGRF5, either as prophylaxis (FIG.18C) or as treatment (FIG.18E), than in the isotype control group.
  • FIG.18A – FIG.18B are representative of at least 3 independent biological replicates.
  • FIG.20A-20L demonstrates that ablation of Adgrf5 in LEPR+ resulted in decreased LEPR+ cell proliferation and increased osteogenic differentiation capability.
  • a,b Proliferation of LEPR+ cells in the peri-implant region by in vivo BrdU incorporation assay.
  • c BrdU proliferation assay quantification of LEPR-tdTomato+BrdU+ cells in the peri-implant region.
  • f,i Representative Von Kossa staining for mineralized bone in organoids derived from Lin ⁇ LEPR-tdTomato+ cells from LepRcre/+;Adgrd5f/f (f) and LepRcre/+ (i) mice.
  • Right enlarged view of the outlined region of the yellow box in the left image. Scale bars, 500 ⁇ m (left), 50 ⁇ m (right).
  • g,j Representative immunofluorescence imaging of organoids derived from Lin ⁇ LEPR- tdTomato+ cells from LepRcre/+;Adgrd5f/f (g) and LepRcre/+ (j) mice. Scale bars, 50 ⁇ m.
  • LEPR leptin receptor-expressing
  • Targets to be Enhanced ADGRF5 belongs to Family VI Adhesion G Protein-Coupled Receptors together with ADGRF1-4 and 635 and has been linked to multiple diseases. More than one-third of currently approved drugs target G protein-coupled receptors (GPCRs) due to their properties of highly amenable to modulation by pharmaceuticals. Furthermore, modulation of adhesion GPCR has been shown to reduce number of stem cells associated with multiple diseases, including glioblastoma stem cells, leukemia stem cells, and breast cancer stem cells.
  • GPCRs G protein-coupled receptors
  • compositions and methods for treating or preventing the formation of orthopedic-related fibrosis in a subject in need thereof are provided herein.
  • orthopedic-related fibrosis includes but is not limited to peri-implant fibrotic tissue, bone- to-bone fibrosis, bone-to-soft tissue fibrosis, soft tissue-to-soft tissue fibrosis, joint related fibrosis.
  • the method includes decreasing the amount, expression, or activity of ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PER1, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1, or TNNT3in the subject.
  • the method includes increasing the amount, expression, or activity of BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA in the subject.
  • ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PER1, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1, or TNNT3 is decreased in the subject by administering an antibody.
  • an anti-ADGRR5 antibody is administered.
  • an anti-GREM1 antibody is administered.
  • an anti-GREM1 antibody is administered.
  • an anti-PTPRF antibody is administered.
  • an anti-LAMB1 antibody is administered.
  • an anti-EFEMP1 antibody is administered.
  • an anti-DNMT3b antibody is administered.
  • an anti-RSPO3 antibody is administered.
  • an anti-ACTA2 antibody is administered.
  • an anti-PDGFRA antibody is administered.
  • an anti-PDGFB antibody is administered.
  • an anti-PDGFA antibody is administered.
  • an anti-CX3CR1antibody is administered.
  • anti-TBX18 antibody is administered.
  • the anti- ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, or TBX18 antibody and/or anti- ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PER1, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1, or TNNT3 binding fragment binds with an affinity of at least about 100 nM, or even higher, for example, at least about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM
  • antibody or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen.
  • antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule, and fusions of immunologically active portions of an immunoglobulin molecule.
  • the antibody may be a naturally occurring antibody or may be a synthetic or modified antibody (e.g., a recombinantly generated antibody; a chimeric antibody; a bispecific antibody; a humanized antibody; a camelid antibody; and the like).
  • the antibody may comprise at least one purification tag.
  • the framework antibody is an antibody fragment.
  • antibody fragment includes a portion of an antibody that is an antigen binding fragment or single chains thereof.
  • An antibody fragment can be a synthetically or genetically engineered polypeptide.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • a F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • a Fd fragment consisting
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody.
  • Antibody fragments include, without limitation, immunoglobulin fragments including, without limitation: single domain (Dab; e.g., single variable light or heavy chain domain), Fab, Fab', F(ab')2, and F(v); and fusions (e.g., via a linker) of these immunoglobulin fragments including, without limitation: scFv, scFv2, scFv-Fc, minibody, diabody, triabody, and tetrabody.
  • the antibody may also be a protein (e.g., a fusion protein) comprising at least one antibody or antibody fragment.
  • the inhibitor is a non-antibody.
  • Non-antibody antagonists include antibody mimetics (e.g., Affibody® molecules, affilins, affitins, anticalins, avimers, Kunitz domain peptides, and monobodies) with protein or gene antagonist activity.
  • Affibody® molecules e.g., Affibody® molecules, affilins, affitins, anticalins, avimers, Kunitz domain peptides, and monobodies
  • non-antibody antagonists may be modified to further improve their pharmacokinetic properties or bioavailability.
  • a non-antibody ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PER1, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1, or TNNT3 (protein or gene) antagonist may be chemically modified (e.g., pegylated) to extend its in vivo half-life.
  • aptamer refers to a peptide or nucleic acid that has an inhibitory effect on a target.
  • Inhibition of the target by the aptamer can occur by binding of the target, by catalytically altering the target, by reacting with the target in a way which modifies the target or the functional activity of the target, by ionically or covalently attaching to the target as in a suicide inhibitor or by facilitating the reaction between the target and another molecule.
  • Aptamers can be peptides, ribonucleotides, deoxyribonucleotides, other nucleic acids or a mixture of the different types of nucleic acids.
  • Aptamers can comprise one or more modified amino acid, bases, sugars, polyethylene glycol spacers or phosphate backbone units as described in further detail herein. Other inhibitors include those that use RNA interference.
  • RNA interference refers to any method by which expression of a gene or gene product is decreased by introducing into a target cell one or more double-stranded RNAs, which are homologous to the gene of interest (particularly to the messenger RNA of the gene of interest).
  • Gene therapy i.e., the manipulation of RNA or DNA using recombinant technology and/or treating disease by introducing modified RNA or modified DNA into cells via a number of widely known and experimental vectors, recombinant viruses and CRISPR technologies, may also be employed in delivering, via modified RNA or modified DNA, effective inhibition of ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PER1, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1, or TNNT3 to accomplish the outcomes described herein with the therapies described.
  • the inhibitor is a small molecule.
  • small molecule when applied to a pharmaceutical generally refers to a non-biologic, organic compound that affects a biologic process which has a relatively low molecular weight, below approximately 900 daltons.
  • Small molecule drugs have an easily identifiable structure, that can be replicated synthetically with high confidence.
  • a small molecule has a molecular weight below 550 daltons to increase the probability that the molecule is compatible with the human digestive system’s intracellular absorption ability.
  • Small molecule drugs are normally administered orally, as tablets.
  • the term small molecule drug is used to contrast them with biologic drugs, which are relatively large molecules, such as peptides, proteins and recombinant protein fusions, frequently produced using a living organism.
  • a ADGRF5 small molecule antagonist is administered.
  • a GREM1 small molecule antagonist is administered.
  • a GREM1 small molecule antagonist is administered.
  • a PTPRF small molecule antagonist is administered.
  • a LAMB1 small molecule antagonist is administered.
  • a EFEMP1 small molecule antagonist is administered.
  • a DNMT3b small molecule antagonist is administered.
  • a RSPO3 small molecule antagonist is administered.
  • a ACTA2 small molecule antagonist is administered.
  • a PDGFRA small molecule antagonist is administered.
  • a PDGFB small molecule antagonist is administered.
  • a PDGFA small molecule antagonist is administered.
  • a small molecule antagonist is administered.
  • a TBX18 small molecule antagonist is administered.
  • a DUSP1 small molecule antagonist is administered.
  • a KLF4 small molecule antagonist is administered.
  • a NR1D1 small molecule antagonist is administered.
  • a PER1 small molecule antagonist is administered.
  • a DES small molecule antagonist is administered.
  • a KLHL41 small molecule antagonist is administered.
  • a RYR1 small molecule antagonist is administered.
  • a TNNT3 small molecule antagonist is administered.
  • the antagonist is administered with a pharmaceutically acceptable excipient or carrier.
  • the inhibitor is nintedanib. In another embodiment, the inhibitor is pirfenidone.
  • the inhibitor is Decitabine (5-aza-2'- deoxycytidine) or Azacitidine, which are nucleoside analogs that get incorporated into DNA and trap DNMTs, leading to hypomethylation.
  • the inhibitor is imatinib.
  • the inhibitor is sunitinib.
  • the inhibitor is sorafenib.
  • the inhibitor is AZD8797.
  • the inhibitor is GSK1605786.
  • the inhibitor is dantrolene.
  • the inhibitor is NSC95397.
  • a gene of Table 2 is upregulated.
  • BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA is increased in the subject by administering a nucleic acid which comprises a sequence encoding BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA.
  • a nucleic acid which comprises a sequence encoding BMP2, or functional fragment thereof is provided, as well as expression cassettes and vectors containing same.
  • the nucleic acid encodes the polypeptide sequence of SEQ ID NO: 14, or a sequence sharing at least 90% identity with SEQ ID NO: 14.
  • the sequence encodes a sequence sharing at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14.
  • a nucleic acid which comprises a sequence encoding BMP4, or functional fragment thereof, is provided, as well as expression cassettes and vectors containing same.
  • the nucleic acid encodes the polypeptide sequence of SEQ ID NO: 15, or a sequence sharing at least 90% identity with SEQ ID NO: 15.
  • the sequence encodes a sequence sharing at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15.
  • nucleic acid which comprises a sequence encoding BMP7, or functional fragment thereof, is provided, as well as expression cassettes and vectors containing same.
  • the nucleic acid encodes the polypeptide sequence of SEQ ID NO: 16, or a sequence sharing at least 90% identity with SEQ ID NO: 16.
  • the sequence encodes a sequence sharing at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16.
  • a nucleic acid which comprises a sequence encoding PTHR1, or functional fragment thereof, is provided, as well as expression cassettes and vectors containing same.
  • the nucleic acid encodes the polypeptide sequence of SEQ ID NO: 17, or a sequence sharing at least 90% identity with SEQ ID NO: 17. In another embodiment, the sequence encodes a sequence sharing at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17. In another aspect, a nucleic acid which comprises a sequence encoding OSX, or functional fragment thereof, is provided, as well as expression cassettes and vectors containing same. In one embodiment, the nucleic acid encodes the polypeptide sequence of SEQ ID NO: 18, or a sequence sharing at least 90% identity with SEQ ID NO: 18.
  • the sequence encodes a sequence sharing at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18.
  • a nucleic acid which comprises a sequence encoding RUNX2, or functional fragment thereof, is provided, as well as expression cassettes and vectors containing same.
  • the nucleic acid encodes the polypeptide sequence of SEQ ID NO: 19, or a sequence sharing at least 90% identity with SEQ ID NO: 19.
  • the sequence encodes a sequence sharing at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19.
  • a nucleic acid which comprises a sequence encoding SNCA, or functional fragment thereof, is provided, as well as expression cassettes and vectors containing same.
  • the nucleic acid encodes the polypeptide sequence of SEQ ID NO: 28, or a sequence sharing at least 90% identity with SEQ ID NO: 28.
  • the sequence encodes a sequence sharing at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 28.
  • the nucleic acid which comprises the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA coding sequence is contained within an expression cassette, which further includes additional sequences, such as regulatory sequences which permit expression of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA. These control sequences or the regulatory sequences are operably linked to the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA coding sequence.
  • an “expression cassette” refers to a nucleic acid molecule which comprises coding sequences, promoter, and may include other regulatory sequences therefor, which cassette may be engineered into a genetic element and/or packaged into the capsid of a viral vector (e.g., a viral particle).
  • a viral vector e.g., a viral particle.
  • an expression cassette for generating a viral vector contains the sequences described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.
  • the expression cassette typically contains a promoter sequence as part of the expression control sequences or the regulatory sequences.
  • Promoters such as tissue-specific promoters, viral promoters, constitutive promoters, regulatable promoters [see, e.g., WO 2011/126808 and WO 2013/049493], or a promoter responsive to physiologic cues may be utilized in the vectors described herein.
  • an expression cassette and/or a vector may contain other appropriate “regulatory elements” or “regulatory sequences”, which comprise but are not limited to enhancers; transcription factors; transcription terminators; efficient RNA processing signals such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • suitable polyA sequences include, e.g., SV40, bovine growth hormone (bGH), and TK polyA.
  • the viral vector is an adenoviral vector.
  • Adenoviruses are medium-sized (90–100 nm), nonenveloped (naked) icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome.
  • serotypes There are over 51 different serotypes in humans, which are responsible for 5–10% of upper respiratory infections in children, and many infections in adults as well.
  • the vector is a replication defective adenovirus, in which the E1A and E1B genes are deleted and replaced with an expression cassette comprising the Foxo3 isoform 2 coding sequence.
  • adenoviral vectors are known in the art and include, without limitation, Ad5 based vectors. See, e.g., Wold and Toth, Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy, Curr Gene Ther.2013 Dec; 13(6): 421–433, which is incorporated herein by reference.
  • the viral vector is an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • AAV is composed of an icosahedral protein capsid of ⁇ 26 nm in diameter and a single-stranded DNA genome of ⁇ 4.7 kb that can either be the plus (sense) or minus (anti- sense) strand.
  • the capsid comprises three types of subunit, VP1, VP2 and VP3, totaling 60 copies in a ratio of about 1:1:10 (VP1:VP2:VP3).
  • the genome is flanked by two T- shaped inverted terminal repeats (ITRs) at the ends that largely serve as the viral origins of replication and the packaging signal.
  • AAV vectors include, without limitation, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10 and AAVrh.43 based vectors. See, e.g., Wang et al, Adeno-associated virus vector as a platform for gene therapy delivery, Nature Reviews Drug Discovery, 18: 358–378 (February 2019), which is incorporated herein by reference.
  • BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA is increased in the subject by administering an effective amount of BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA.
  • the polypeptide has the sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, or 28, or a sequence sharing at least 90% identity with SEQ ID NO: 14, 15, 16, 17, 18, 19, or 28.
  • the polypeptide is a functional fragment of BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA.
  • a polynucleotide that encodes a sequence sharing at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14, 15, 16, 17, 18, 19, or 28.
  • the polynucleotide is provided as mRNA.
  • the “effective amount” for a protein or peptide antagonist, e.g., antibody, antibody fragment or recombinant protein or peptide the effective amount can be about 0.01 to 25 mg antibody/injection. In one embodiment, the effective amount is 0.01 to 10 mg antibody/injection. In another embodiment, the effective amount is 0.01 to 1 mg antibody/injection.
  • the effective amount is 0.01 to 0.10 mg antibody/injection. In another embodiment, the effective amount is 0.2, 0.5, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0 up to more than mg antibody/injection. Still other doses falling within these ranges are expected to be useful.
  • an effective amount for the nucleic acid and/or protein inhibitor of composition (a) includes without limitation about 0.001 to about 25 mg/kg subject body weight. In one embodiment, the range of effective amount is 0.001 to 0.01 mg/kg body weight. In another embodiment, the range of effective amount is 0.001 to 0.1 mg/kg body weight.
  • the range of effective amount is 0.001 to 1 mg/kg body weight. In another embodiment, the range of effective amount is 0.001 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 0.001 to 20 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 25 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 0.1 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 1 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 20 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 25 mg/kg body weight.
  • the range of effective amount is 0.1 to 1 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 20 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 25 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 5 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 20 mg/kg body weight. Still other doses falling within these ranges are expected to be useful. As used herein, the term “a therapeutically effective amount” refers an amount sufficient to achieve the intended purpose.
  • an effective amount of a therapy for treating or preventing formation of fibrotic tissue in bone or bone marrow is sufficient to decrease, prevent, or ameliorate the formation of fibrotic tissue associated with a bone disease, bone disorder, procedure, or surgery.
  • An effective amount for treating or ameliorating a disorder, disease, or medical condition is an amount sufficient to result in a reduction or complete removal of the symptoms of the disorder, disease, or medical condition.
  • the effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration. The effective amount in each individual case may be determined by a skilled artisan according to established methods in the art.
  • the “effective amount” for a small molecule can be about 0.01 to 25 mg /dose. In one embodiment, the effective amount is 0.01 to 10 mg /dose. In another embodiment, the effective amount is 0.01 to 1 mg /dose. In another embodiment, the effective amount is 0.01 to 0.10 mg /dose. In another embodiment, the effective amount is 0.2, 0.5, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0 up to more than mg /dose. Still other doses falling within these ranges are expected to be useful. In one embodiment an effective amount for the small molecule inhibitor of composition (a) includes without limitation about 0.001 to about 25 mg/kg subject body weight.
  • the range of effective amount is 0.001 to 0.01 mg/kg body weight. In another embodiment, the range of effective amount is 0.001 to 0.1 mg/kg body weight. In another embodiment, the range of effective amount is 0.001 to 1 mg/kg body weight. In another embodiment, the range of effective amount is 0.001 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 0.001 to 20 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 25 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 0.1 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 1 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 10 mg/kg body weight.
  • the range of effective amount is 0.01 to 20 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 25 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 1 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 20 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 25 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 5 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 20 mg/kg body weight. Still other doses falling within these ranges are expected to be useful.
  • BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA is increased in the subject by administering an effective amount of a BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is an amount ranging from about 0.01 mg/ml to about 10 mg/ml, including all amounts therebetween and end points.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 0.1 mg/ml to about 5 mg/ml, including all amounts therebetween and end points. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 0.3 mg/ml to about 1.0 mg/ml, including all amounts therebetween and end points. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 0.3 mg/ml.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 0.4 mg/ml. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 0.5 mg/ml. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 0.6 mg/ml. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 0.7 mg/ml.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 0.8 mg/ml. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 0.9 mg/ml. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 1.0 mg/ml.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is an amount ranging from about 1 ⁇ M to about 2mM, including all amounts therebetween and end points. In one embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 10 ⁇ M to about 100 ⁇ M, including all amounts therebetween and end points. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 5 ⁇ M.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 10 ⁇ M. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 20 ⁇ M. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 50 ⁇ M. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 100 ⁇ M.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 200 ⁇ M. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 300 ⁇ M. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 400 ⁇ M. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 500 ⁇ M.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 600 ⁇ M. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 700 ⁇ M. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 800 ⁇ M. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 900 ⁇ M.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 1mM. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 1.25 mM. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist about 1.5 mM. In another embodiment, the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 1.75 mM.
  • the effective amount of the BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA agonist is about 2 mM.
  • the methods include administering an agonist, antagonist, etc., with a pharmaceutically acceptable carrier.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., 1990). The formulation should suit the mode of administration. Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the agent may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • the composition is administered to the subject prior to surgery.
  • the composition is administered to the subject after surgery.
  • the composition is administered to the subject during surgery.
  • the methods of treatment include combination with other therapies. As described herein, it has been shown that expression of BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA treats or prevents the formation of fibrotic tissue in bone or bone marrow.
  • a method of treating or preventing the formation of fibrotic tissue in bone or bone marrow in a subject in need thereof includes increasing the expression, amount or activity of BMP2.
  • the method includes increasing the expression, amount or activity of BMP4.
  • the method includes increasing the expression, amount or activity of BMP7.
  • the method includes increasing the expression, amount or activity of PTHR1.
  • the method includes increasing the expression, amount or activity of OSX.
  • the method includes increasing the expression, amount or activity of RUNX2.
  • the method includes increasing the expression, amount or activity of SNCA.
  • the subject may have, or be suspected of having or developing, fibrotic tissue in bone or bone marrow, as described hereinabove.
  • a method of treating or preventing the formation of fibrotic tissue in bone or bone marrow in a subject in need thereof includes administering a population of isolated cells, wherein the cells express one or more of the following markers: CD200 + , CD90 + , 6C3 + , THY1 + , PDGFRA + , PDGFRB + .
  • the cells prior to administration, the cells are expanded.
  • the cells are differentiated. It is to be noted that the term “a” or “an” refers to one or more.
  • Upregulate and “upregulation”, as used herein, refer to an elevation in the level of expression of a product of one or more genes in a cell or the cells of a tissue or organ.
  • the term “agonist” refers to a compound that in combination with a receptor can produce a cellular response.
  • An agonist may be a ligand that directly binds to the receptor.
  • an agonist may combine with a receptor indirectly by for example (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise resulting in the modification of another compound so that the other compound directly binds to the receptor.
  • blocking agent agents, compounds, constructs, small molecules, or compositions that inhibit, either partially or fully, the activity, expression, transcription or production of a target molecule.
  • such antagonists are capable of interrupting the expression, transcription, or activity of the gene in vivo or the activity and function of the protein in vivo.
  • these terms refer to a composition or compound or agent capable of decreasing levels of gene expression, mRNA levels, protein levels or protein activity of the target molecule.
  • antagonists include, for example, proteins, polypeptides, peptides (such as cyclic peptides), antibodies or antibody fragments, peptide mimetics, nucleic acid molecules, antisense molecules, ribozymes, aptamers, RNAi molecules, and small organic molecules.
  • Illustrative non-limiting mechanisms of antagonist inhibition include repression of ligand synthesis and/or stability (e.g., using, antisense, ribozymes or RNAi compositions targeting the ligand gene/nucleic acid), blocking of binding of the ligand to its cognate receptor (e.g., using anti-ligand aptamers, antibodies or a soluble, decoy cognate receptor), repression of receptor synthesis and/or stability (e.g., using, antisense, ribozymes or RNAi compositions targeting the ligand receptor gene/nucleic acid), blocking of the binding of the receptor to its cognate receptor (e.g., using receptor antibodies) and blocking of the activation of the receptor by its cognate ligand (e.g., using receptor tyrosine kinase inhibitors).
  • repression of ligand synthesis and/or stability e.g., using, antisense, ribozymes or RNAi compositions targeting the ligand
  • the blocker or inhibitor may directly or indirectly inhibit the target molecule.
  • a “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or gorilla.
  • the term “patient” may be used interchangeably with the term subject.
  • the subject is a human.
  • the subject may be of any age, as determined by the health care provider.
  • the patient is a subject who has or is at risk of developing fibrotic tissue in bone or bone marrow.
  • the subject may have been treated for a skeletal disease previously, or is currently being treated for the skeletal disease.
  • the subject is an older adult, e.g., over the age of 40. In another embodiment, the subject is at least 45, 50, 55, or 60 years of age. In yet another embodiment, the subject is a senior adult, i.e., over 60 years of age. In certain embodiments, the subject has a bone fracture. In certain embodiments, the subject has received, or will receive, an implant. In certain embodiments, the subject has received, or will receive, a bone fusion procedure. In certain embodiments, the subject has a bone marrow fibrosis. In certain embodiments, the subject has a myelofibrosis.
  • skeletal disease or “skeletal disorder” refers to any condition associated with the bone or joints, including those associated with bone loss, bone fragility, or softening, or aberrant skeletal growth.
  • the term “skeletal disease” refers to a condition associated with osteoclastic activity and/or bone loss.
  • Skeletal diseases include, without limitation, osteoporosis and osteopenia, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, periodontitis, periprosthetic loosening, osteomalacia, hyperparathyroidism, Paget disease of bone, spondyloarthritis, and lupus.
  • the skeletal disease is osteoporosis.
  • the skeletal disease is osteopenia. In another embodiment, the skeletal disease is rheumatoid arthritis. In certain embodiments, the skeletal disease is bone marrow fibrosis. In certain embodiments, the skeletal disease is myelofibrosis. In certain embodiments, the skeletal disease is a bone fracture.
  • “disease”, “disorder” and “condition” are used interchangeably, to indicate an abnormal state in a subject. Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.
  • a reference to “one embodiment” or “another embodiment” in describing an embodiment does not imply that the referenced embodiment is mutually exclusive with another embodiment (e.g., an embodiment described before the referenced embodiment), unless expressly specified otherwise.
  • Specific Embodiments 1.
  • a method of treating or preventing the formation of orthopedic-related fibrosis in a subject in a subject in need thereof comprising administering to the subject an inhibitor of ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PER1, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1, or TNNT3 to the subject.
  • a method of treating or preventing the formation of orthopedic-related fibrosis in a subject in need thereof comprising administering to the subject an activator of BMP2, BMP4, BMP7, PTHR1, OSX, RUNX2, or SNCA, to the subject.
  • the method of embodiment 1 or 2 wherein the subject has a bone fracture.
  • the method of any one of embodiments 1 to 3 wherein the subject has received, or will receive, an implant.
  • the method of any one of embodiments 1 to 4 wherein the subject has received, or will receive, a bone fusion procedure.
  • the method of any one of embodiments 1 to 5 wherein the subject has sustained bone-to-bone fibrosis. 7.
  • the inhibitor is an antibody to ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PER1, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1, or TNNT3.
  • the inhibitor is a small molecule.
  • the inhibitor is nintedanib or pirfenidone.
  • the activator is small molecule. 15.
  • a method of treating or preventing the formation of fibrotic tissue in bone or bone marrow in a subject in need thereof comprising administering a population of isolated cells, optionally wherein the cells have been enriched for one or more of the following markers: CD200+, CD90+, 6C3+, THY1+, PDGFRA+, PDGFRB+, SCA1+, GREM1+, OSX+, CD105+, OCN+, CTSK+, CXCL12+, EBF3+, and MX1+. 18.
  • the method of embodiment 17, wherein the cells are autologous cells. 19. The method of embodiment 17, wherein the cells are allogeneic cells. 20. The method of any one of embodiments 17 to 19, wherein the subject has a bone fracture. 21. The method of any one of embodiments 17 to 20, wherein the subject has received, or will receive, an implant. 22. The method of any one of embodiments 17 to 21, wherein the subject has received, or will receive, a bone fusion procedure. 23. The method of any one of embodiments 17 to 22, wherein the subject has sustained bone-to-bone fibrosis. 24. The method of any one of embodiments 17 to 23, wherein the subject has sustained bone-to-soft tissue fibrosis. 25.
  • a method of reducing orthopedic-related fibrosis which included but not limited to peri-implant fibrotic tissue, bone-to-bone fibrosis, bone-to-soft tissue fibrosis, soft tissue-to-soft tissue fibrosis, joint related fibrosis, in a subject in a subject in need thereof in a subject in need thereof, the method comprising administering osteogenic LEPR+ donor cells to the subject.
  • the cells have been enriched for one or more of the following markers i. LEPR+ BGLAP+, ii. LEPR+ ALPL+, iii. LEPR+ OSX+, iv. LEPR+ CD200+, or v. LEPR+ CD106-.
  • Example 1 Materials and Methods Animals Lepr-cre (Stock 008320), Rosa26-CAG-loxp-stop-loxp-tdTomato (Stock 007909), Rosa26-CAG-loxp-stop-loxp-iDTR (Stock 007900), Acta2-DsRed (Stock 031160), Pdgfr ⁇ -EGFP (Stock 007669), Adgrf5 fl/fl (Stock 022505), and Triple KO (Stock 025730) mice were purchased from Jackson Laboratories.
  • mice were a gift from Takashi Nagasawa (Kyoto University, Japan). All mice were maintained on a C57BL/6 background. All animals were maintained in accordance with the NIH guidelines for the Care and Use of Laboratory Animals and were handled according to protocols approved by the Weill Cornell Medical College subcommittee on animal care (IACUC). Randomization of mice was performed to evaluate the efficacy of anti-ADGRF5 in preventing and reversing peri-implant fibrosis. Blinding to experimental conditions was performed for microcomputer tomography ( ⁇ CT) analyses. The sample sizes were at least 3 animals per experiment. All experiments were performed on 10- to 18-week-old mice.
  • ⁇ CT microcomputer tomography
  • mice were intraperitoneally injected daily with 100 ng of DT (Sigma, cat. D0564) from 7 days before surgery until postoperative day 14 (for prevention of peri-implant fibrosis) or from postoperative day 14 until postoperative day 28 (for reversal of peri-implant fibrosis).
  • PBS was used as a vehicle.
  • Human specimens This study was carried out under the IRB protocol (no.2019-0571). HIPAA authorization was included in the IRB protocol, and all ethical guidelines conformed to the 2008 Helsinki declaration. Leftover peri-implant fibrous tissue was obtained from 15 patients who underwent revision arthroplasty due to aseptic loosening.
  • Peri-implant fibrotic tissue was obtained by bluntly scraping the fibrotic tissue from the proximal femur or acetabulum (total hip arthroplasty) and distal femur or proximal tibia (total knee arthroplasty) adjacent to the implant.
  • the obtained peri-implant tissues consisted of fibrous tissue with a small amount of underlying bone, thus allowing us to evaluate both the bone-to-fibrous tissue and implant- to-fibrous tissue interfaces.
  • Normal bone was obtained from 15 patients who underwent primary total hip (greater trochanter area) or total knee (proximal tibia) replacement. All identifying patient information was scrubbed at the time of sample procurement.
  • Fibrous integration surgery Surgery was carried out with the mice under general anesthesia with isoflurane administered with a nose cone (2% isoflurane, 2 L/min). Under sterile conditions, an 8 mm medial parapatellar incision was made in the knee undergoing surgery.
  • the longitudinal fibers of the quadriceps muscle were released medially to allow lateral subluxation of the patella and exposure of the tibial plateau.
  • the anterior cruciate ligament was released from its tibial attachment, and both menisci were removed.
  • Articular cartilage and proximal epiphysis were removed to the level of the insertion of the posterior cruciate ligament using a fine-tip burr.
  • a 1.4 mm diameter x 2.0 mm length hole was created in the medullary canal.
  • An implant made from medical grade poly(methyl methacrylate) (Simplex P, Stryker, New Jersey, USA) was placed into the hole until the top was flush with the proximal part of the tibia.
  • the dimensions of the implant articulating surface were 2.0 mm for the major axis and 1.5 mm for the minor axis, with a 0.2 mm thickness.
  • the stem was 2.0 mm in length and 1.0 mm in diameter.
  • Full range of motion of the knee was confirmed before closure of the wound.
  • the extensor mechanism and skin were closed in layers with resorbable sutures.
  • the mice were given analgesia (buprenorphine, 0.05 mg/kg subcutaneously) for the first forty-eight hours postoperatively. All mice were allowed to bear full weight on the limb without any activity restriction from the day of surgery until the day of sacrifice. Osseointegration surgery
  • the approach and exposure for the osseointegration surgery were similar to those for the fibrous integration surgery, with the following modifications.
  • a 10.9 mm diameter x 2.0 mm length hole was created in the medullary canal.
  • the implant was made by direct metal laser sintering, and medical grade Ti6Al4V alloy was press-fitted into the hole until the top was flush with the proximal part of the tibia.
  • the dimensions of the implant articulating surface were 2.0 mm for the major axis and 1.5 mm for the minor axis, with a 0.2 mm thickness.
  • the stem was 2.0 mm in length and 1.0 mm in diameter. Closure was then performed as described for mice that underwent fibrous integration surgery.
  • mice were given analgesia (buprenorphine, 0.05 mg/kg subcutaneously) for the first forty-eight hours postoperatively. All mice were allowed to bear full weight on the limb without any activity restriction from the day of surgery until the day of sacrifice. Isolation of mesenchymal cells
  • the proximal tibia (two millimeters from the most proximal surface) was subjected to both mechanical and enzymatic digestion.
  • the harvested tissue was minced using sterile scissors and digested for up to 45 minutes with collagenase P (1 mg/ml; Roche, cat 11213857001) + dispase II (2 mg/ml; Roche; cat 04942078001) in digestion buffer at 37°C with constant agitation.
  • ⁇ MEM containing 2% serum was added to the digestion reaction, and the tubes were centrifuged to obtain cell pellets. After removal of the supernatant, the cell pellet was resuspended in DNAse I (2 units/ml) solution and incubated for 10 minutes at 37°C. ⁇ MEM containing 2% serum was added to the tube, and the digested tissue was resuspended thoroughly and then filtered through 70 ⁇ m nylon mesh. The tubes were then centrifuged, and the resulting cell pellet was subjected to FACS.
  • peri-implant fibrotic tissue or bone was minced using sterile scissors and digested for up to 80 minutes with collagenase P (1 mg/ml; Roche, cat 11213857001) + dispase II (2 mg/ml; Roche; cat 04942078001) in digestion buffer per 100 grams of tissue. Cells were then isolated following the same procedure detailed above. Fluorescence-assisted cell sorting (FACS) Single-cell suspensions were subsequently washed twice with ice-cold FACS buffer (2% FBS + 1 mM EDTA in PBS) and incubated with blocking buffer (1:100 dilution; BD Bioscience 553142 for mouse and 564765 for human) for 30 minutes at 4°C.
  • FACS Fluorescence-assisted cell sorting
  • Fluorescence minus one (FMO) controls were used for further compensation and to assess the background level for each fluorophore. Gates were drawn based on internal FMO controls to separate positive and negative populations for each cell fluorophore. Typically, 1-2.5 million events were recorded for each FACS analysis, and the data were analyzed using FlowJo (version 10.8). Calculations were made in Excel, and bar graphs were generated in GraphPad Prism. Extended Data Figures have been provided to show the dull details of the gating strategies used (FIG.9, FIG.13).
  • FACS antibodies The antibodies for FACS of mouse samples included antibodies specific for CD45 (clone 30-F11, BD Bioscience), CD31 (MEC13.3, BD Bioscience), Ter119 (clone Ter119, BD Biosciences), CD90.2 (clone 53-2.1, BD Biosciences), BP-1 (clone 6C3, eBioscience), Thy-1.2 (clone 53-2.1, BD Biosciences), CD200 (clone OX-90, BD Biosciences), CD105 (clone MJ7/18, BioLegend), CD140 ⁇ (clone APA5, BioLegend), CD51 (clone RMV-7, BD Biosciences), and LeptinR (R&D system, BAF497).
  • CD45 clone 30-F11, BD Bioscience
  • CD31 MEC13.3, BD Bioscience
  • Ter119 clone Ter119, BD Biosciences
  • CD90.2 clone 53-2.1, BD Biosciences
  • the antibodies for FACS of human samples included antibodies specific for CD45 (BioLegend, 304029-BL), CD235a (BioLegend, 306612-BL), CD31 (Thermo Fisher Scientific, 13-0319-82), CD146 (BioLegend, 342010), PDPN (Thermo Fisher Scientific, 17-9381-42), CD90 (THY1; BioLegend, 328110), CD164 (BioLegend, 324808), CD73 (BioLegend, 344016), and LeptinR (R&D system, MAB867).
  • Kidney capsule transplantation model Eight- to ten-week-old male mice were anesthetized and shaved on the left flank and abdomen prior to sterilization of the surgical site. The kidney was exposed through a 1 cm incision and subsequently externalized. A 2-mm pocket in the renal capsule was made, and a 5 ⁇ l Matrigel plug (Corning, cat no 356231) containing 5,000-10,000 cells was implanted underneath the capsule. The hole was sealed via electrocautery before the kidney was returned to the body cavity. Triple KO mice (JAX, Stock 025730) were used as recipients to avoid any potential immunogenicity. Animals were euthanized by CO 2 inhalation after 8 weeks.
  • the cells transplanted in this study originated from FACS-sorted LEPR- tdTomato + cells from the proximal tibia of mice that underwent either osseointegration or fibrous integration surgery. Five to ten proximal tibias from the operated mice were harvested on postoperative day 14 to obtain 5,000-10,000 cells for transplantation into each recipient mouse.
  • Orthotopic engraftment in the proximal tibia transplantation model Surgery was carried out with the mice under general anesthesia with isoflurane administered with a nose cone (2% isoflurane, 2 L/min). Under sterile conditions, an 8 mm medial parapatellar incision was made in the knee that underwent surgery. The longitudinal fibers of the quadriceps muscle were released medially to allow lateral subluxation of the patella and exposure of the tibial plateau. The anterior cruciate ligament was released from its tibial attachment, and both menisci were removed. Articular cartilage and the proximal epiphysis were removed to the level of the insertion of the posterior cruciate ligament using a fine-tip burr.
  • a 1.4 mm diameter x 2.0 mm length hole or 0.9 mm diameter x 2.0 mm length hole was created in the medullary canal of recipient mice subjected to fibrous integration or osseointegration surgery.
  • Two microliters of ⁇ MEM solution containing 5,000-10,000 cells was injected into the distal portion of the hole.
  • Implants made from medical grade poly(methyl methacrylate) (Simplex P, Stryker, New Jersey, USA) were placed into the hole until the top was flush with the proximal part of the tibia.
  • the dimensions of the implant articulating surface were 2.0 mm for the major axis and 1.5 mm for the minor axis, with a 0.2 mm thickness.
  • the stem was 2.0 mm in length and 1.0 mm in diameter.
  • mice Full range of motion of the knee was confirmed before closure of the wound.
  • the extensor mechanism and skin were closed in layers with resorbable sutures.
  • the mice were given analgesia (buprenorphine, 0.05 mg/kg subcutaneously) for the first forty-eight hours postoperatively. All mice were allowed to bear full weight on the limb without any activity restriction from the day of surgery until the day of sacrifice.
  • the cells transplanted in this study originated from FACS-sorted LEPR- tdTomato + cells from the proximal tibia of mice that underwent either osseointegration or fibrous integration surgery. Five to ten proximal tibias of the operated mice were harvested on postoperative day 14 to obtain 5,000-10,000 cells for transplantation into each recipient mouse.
  • the strength of the bone-implant interface was measured via pullout testing as previously described[8]. Briefly, after isolation of the whole tibia, the distal end was placed in polymethylmethacrylate. The bone at the proximal end was dissected to allow the clamp of a custom fixture[8] to hold the implant under its plateau. The long axis of the implant was aligned with the axis of pullout loading. The implant was subsequently pulled out of the tibia with an EnduraTEC ELF3200 system (Bose, EdenPrairie, Minnesota) at a rate of 0.03 mm/sec until the implant was completely removed from the tibia. The maximum pullout load (N) was calculated from the load ⁇ displacement curves.
  • Intra-articular administration of an ADGRF5-blocking antibody We performed daily intra-articular administration of an ADGRF5-blocking antibody to LepR-Cre;tdTomato mice that underwent fibrous integration surgery from postoperative day 0 until postoperative day 14 for the prevention study (FIG.6A) and from postoperative day 14 to postoperative day 28 for the reversal of peri-implant fibrosis study (FIG.6J). For each administration, we injected 8 ⁇ L of 20 ⁇ g/mL anti-ADGRF5 (Abcam, ab111169) or isotype (Abcam, ab171870) solution diluted in PBS.
  • anti-ADGRF5 Abcam, ab111169
  • isotype Abcam, ab171870
  • Sample preparation for cryosectioning Freshly extracted tissue was fixed with 4% paraformaldehyde (PFA) for 6 hours at 4°C. Samples were washed with PBS and decalcified with 0.5 M EDTA for 8 days. Samples were then incubated with infiltration solution (20% sucrose + 2% PVP) with rocking until the samples sank to the bottom of the tube. Embedding was subsequently performed with OCT + 15% sucrose, and samples were stored at -80°C. Fifteen-micron- thick sections were cut using a Leica cryostat.
  • PFA paraformaldehyde
  • Immunohistochemistry Frozen samples were thawed at room temperature (RT), rehydrated with PBS, permeabilized with PBS + 0.3% Triton X-100 for 15 minutes, and blocked for 1 hour with 5% donkey serum in PBS (blocking buffer). Dilutions of primary antibodies were prepared in blocking buffer. Samples were incubated overnight with primary antibodies at 4°C and then washed three times with PBS. Secondary antibodies (1:400 dilutions) were added to the sample for 2 hours, followed by washing three times with PBS. DAPI (300 nM) was then added for 10 minutes, and the samples were mounted with antifade mounting solution (Life Technologies, P36970).
  • the primary antibodies used were specific for murine LEPR (R&D System, AF497, 1:100 dilution), human LEPR (R&D system, MAB867), CXCL12 (R&D System, MAB350, 1:200 dilution), murine CD200 (Abcam, ab33734, 1:100 dilution), murine CD105 (Abcam, ab107595, 1:100 dilution), human PDPN (Invitrogen, MA5-16267, 1:100 dilution), ACTA2 (Abcam, ab5694, 1:200 dilution), and ADGRF5 (Abcam, ab111169, 1:100 dilution).
  • ⁇ CT scanning and analysis ⁇ CT analysis was conducted on a Scanco Medical ⁇ CT 35 system (SCANCO Medical, Bassersdorf, Switzerland) with previously described parameters[8]. Briefly, a Scanco Medical ⁇ CT 35 system with an isotropic voxel size of 6 ⁇ m and a 0.36o rotation step (180o angular range) per view was used to image the tibia. Scans were conducted in PBS and used an X-ray tube potential of 55 kVp, an X-ray intensity of 0.145 mA, and an integration time of 600 ms. The volume of interest was defined as the volume of tissue 200 ⁇ m from the implant’s stem surface. CT analysis was performed by an investigator blinded to the genotypes of the animals.
  • RNA sequencing was performed on sorted LEPR + cell populations isolated from human bone, human peri-implant fibrous tissue, and the peri-implant area of mice on postoperative day 14. RNA isolation from the sorted cells was performed using an RNAeasy Micro Kit (Qiagen, Germantown, MD, USA) in accordance with the protocol provided by the manufacturer. cDNA libraries were generated using the Illumina TruSeq RNA Sample Preparation kit and sequenced on a HiSeq4000 sequencer. The resulting libraries were sequenced using HiSeq2500 with 50-bp single-end reads to a depth of 15- 25 million reads per sample. Read quality assessment and adapter trimming were performed using fastp[9].
  • RNAseq DRaMA Shiny-driven visualization platform
  • Clustering was performed using the first 15 principal components, and clusters were visualized using UMAP projection. Human bone marrow stromal cell single-cell data were obtained from the publicly available dataset GSE147287[14]. After exclusion of hematopoietic and low-quality cells, gene expression for each cell was calculated as transcripts per 10k transcripts. Only cells with LEPR expression > 0 (2198 cells) were used for downstream analysis. The remainder of the data analysis was performed as detailed above. Statistical analyses All data are shown as the mean ⁇ standard deviation (S.D.) or standard error of the mean (S.E.M.), as indicated. Where applicable, we first performed the Shapiro ⁇ Wilk normality test to check normality.
  • S.D. standard deviation
  • S.E.M. standard error of the mean
  • Example 2 LEPR + cells comprise the majority of the cells in peri-implant fibrotic tissue.
  • LEPR + cells significantly contribute to the formation of peri-implant fibrotic tissue.
  • FIG.1A – FIG.1B The diagnosis of aseptic loosening was confirmed by the presence of radiographic peri-implant lucency (FIG.1B) and mechanically loose implants, with the presence of peri-implant fibrotic tissue upon intraoperative assessment.
  • the obtained peri-implant tissues consisted of fibrous tissue with a small amount of underlying bone (FIG.1C), thus allowing us to evaluate both the bone-to-fibrous tissue and implant-to-fibrous tissue interfaces.
  • the peri-implant fibrotic tissue contained spindle-shaped fibroblasts that expressed LEPR (FIG.1D – FIG.1E, FIG.7).
  • the majority of LEPR + cells present in these specimens were in the fibrotic tissue but not the underlying bone, as confirmed by flow cytometry analysis (FIG. 1F – FIG.1H).
  • Example 3 Expression of signature markers of LEPR+ skeletal progenitors in peri- implant fibrotic tissue
  • LEPR + cells similarly comprise peri-implant fibrotic tissue in a murine model and whether signature markers in addition to LEPR (such as CXCL12) are conserved.
  • signature markers in addition to LEPR such as CXCL12
  • This model allows both robust osseointegration upon press-fit insertion of the tibial implant[8] (hereafter osteointegration surgery) or peri-implant fibrosis upon insertion of the tibial implant loosely into an overdrilled site[15] (hereafter fibrous integration surgery), allowing for implant micromotion (FIG.1I – FIG.1J).
  • LEPR lineage cells in peri-implant fibrotic tissue coexpressed other markers previously associated with LEPR + /CAR cells[11, 13], including LEPR (FIG.1M – FIG.1P) and CXCL12 (FIG.8).
  • LEPR + cells in fibrotic tissue can be distinguished from other populations of LEPR + cells by their expression of CD200 (FIG.9I) and ACTA2 (also known as ⁇ SMA) (FIG.10).
  • the peri-implant fibrous tissue expressed CXCL12-GFP (FIG.8) and ACTA2-RFP (FIG.10) when the respective reporter mouse lines were subjected to the fibrous integration surgical model.
  • the peri-implant osseous tissue contained LEPR- tdTomato + CXCL12-GFP + and LEPR-zsGreen + ACTA2-RFP + cells with the expected perivascular distribution (FIG.8E – FIG.8J, FIG.10F – FIG.10J).
  • LEPR-tdTomato + CD200 + cells were osteogenic due to the expression of osteogenesis- associated genes (Alpl, Wif1, Bglap, Sp7)[11], in the fibrous integration context, LEPR- tdTomato + CD200 + cells assume a fibrogenic fate and are accordingly abundant in the peri-implant fibrotic tissue, especially in the transition area from the bone to the fibrotic tissue (FIG.9I).
  • LEPR-lineage cells in the peri-implant fibrotic tissue retain some signature elements of their lineage identity, including expression of LEPR and CXCL12, while also displaying clear phenotypic changes versus the physiologic LEPR cells present at baseline, including increased expression of CD200 and ACTA2 and the acquisition of a fibrogenic fate.
  • peri-implant fibrotic tissue consisted mainly of LEPR-tdTomato- cells with interspersed LEPR-tdTomato + cells, while mature peri-implant fibrotic tissue mostly comprised spindle-shaped LEPR-tdTomato + cells (FIG.1 – FIG.1M, FIG.11C).
  • the thickness of peri-implant fibrotic tissue and the relative amount of Tdtomato + cells plateaued on postoperative day 14 (FIG.11D – FIG.11F).
  • peri-implant bone formation plateaued by postoperative week 2 (FIG.12A – FIG.12B, FIG.12D – FIG.12E).
  • LEPR-tdTomato + cells were present in the immediate peri-implant area, including LEPR-tdTomato+ osteocytes in the peri-implant bone mediating osseointegration, indicating that LEPR lineage cells were one of but not the only source of peri-implant osteocytes (FIG.8C – FIG.8F).
  • Example 3 Ablation of LEPR lineage cells ameliorates peri-implant fibrosis and promotes osseointegration By using a targeted cell ablation approach, we next tested whether peri-implant fibrous tissue is formed predominantly by LEPR + cells.
  • LEPR + cells are required to form fibrotic tissue in the peri- implant area, and ablation of LEPR + cells allowed an alternative set of non-LEPR-lineage skeletal progenitors to form peri-implant bone and mediate osseointegration.
  • LEPR lineage cells are needed to maintain peri-implant fibrosis and whether therapeutic ablation of LEPR lineage cells can attenuate established peri-implant fibrosis.
  • peri-implant fibrosis was allowed to develop for two weeks after surgery.
  • LEPR lineage cells were then ablated by administering diphtheria toxin to LepR-Cre;tdTomato; Rosa26-loxP-stop-loxP-iDTR (iDTR) mice from postoperative week 2 until postoperative week 4.
  • Daily saline injections into LepR-Cre;tdTomato; Rosa26-loxP-stop-loxP-iDTR mice were used as a control (FIG.2I). Both microcomputed tomography and histomorphometric analysis demonstrated that there was more peri-implant bone in the mice that received diphtheria toxin than in the mice in the saline group (FIG.2J, FIG.2M – FIG.2P).
  • Peri-implant fibrosis was significantly reduced in mice that received diphtheria toxin compared to the mice that received the saline control (FIG.2K, FIG.2P). While abundant spindle-shaped LEPR + cells were found in the peri-implant fibrotic tissue of the saline control group, significantly fewer LEPR + cells were present in the iDTR group that received diphtheria toxin (FIG.2K, FIG.2Q). Flow cytometry analysis showed that the majority of cells expressing mouse skeletal stem cell (mSSC) and progenitor markers were LEPR- tdTomato- under both conditions (FIG.9A, FIG.9B – FIG.9H).
  • mSSC mouse skeletal stem cell
  • LEPR- human skeletal stem cell
  • hSSC human skeletal stem cell
  • progenitor markers were LEPR- (FIG.14).
  • LEPR + cells are required to form and maintain peri- implant fibrosis tissue and that ablation of LEPR + cells ameliorates peri-implant fibrotic tissue while allowing an alternative lineage of skeletal progenitors to form peri-implant osseous tissue and mediate osseointegration.
  • Example 4 The fibrotic integration microenvironment reprograms LEPR lineage cells to acquire a fibrogenic fate Subcapsular kidney and orthotopic engraftment were next used to functionally evaluate the differentiation capacity of LEPR lineage cells obtained from profibrotic versus osteogenic microenvironments. Lin-Lepr-tdTomato + cells were isolated from the peri-implant area in the fibrotic integration or osseointegration model and implanted into the kidney capsule of secondary recipients (FIG.3A).
  • LEPR-tdTomato + cells from the osseointegration donors formed bone organoids containing graft-derived osseous and adipose tissue after transplantation (FIG.3B)
  • LEPR-tdTomato + cells from fibrous integration donors exclusively formed graft-derived fibrotic tissue after transplantation, demonstrating a phenotypic switch in differentiation capacity due to cell intrinsic reprogramming that was durable even after removing LEPR cells from the peri-implant microenvironment (FIG.3B, FIG.3D).
  • LEPR cells were sufficient to cause the formation of peri- implant fibrosis when transferred into hosts that would otherwise experience osseointegration.
  • LEPR-tdTomato + cells from the profibrotic microenvironment were reprogrammed toward a fibrogenic fate, and these reprogrammed LEPR cells were sufficient to mediate the peri-implant fibrosis outcome.
  • This increase in fibrogenic capacity was accompanied by a corresponding loss of osteogenic capacity.
  • Example 5 LEPR lineage cells in the fibrous tissue of humans and mice express ADGRF5 Based on our finding that LEPR lineage cells are crucial for the formation of peri- implant fibrous tissue, we asked whether molecular mediators of the fibrogenic reprogramming of LEPR cells could be targeted to therapeutically prevent or reverse peri- implant fibrosis.
  • Lin-LEPR + cells from peri-implant fibrous tissue from both human patient specimens and the mouse fibrotic integration model and compared their RNA expression profile to that of Lin-LEPR + cells sorted from trabecular bone as a baseline comparator.
  • Adgrf5 displayed the highest log fold-change (3.2 and 5.4 in humans and mice, respectively); hence, we prioritized Adgrf5 for further evaluation (FIG.4).
  • ADGRF5 belongs to the Family VI Adhesion G Protein-Coupled Receptors[18] and has been linked to multiple diseases, such as breast cancer[19], gastric cancer[20], and diabetes[21]. From a pharmaceutical perspective, targeting ADGRF5 is advantageous since it belongs to the G protein-coupled receptor family, which has previously been successfully targeted in the treatment of various disorders[22, 23].
  • ADGRF5 immunostaining of human fibrous tissue obtained from patients who underwent revision total hip or total knee replacement surgery for aseptic loosening (FIG.4A, FIG.7). ADGRF5 was expressed by the majority of LEPR + cells in fibrous tissue.
  • LEPR + ADGRF5 + cells were abundant in the fibrous tissue of multiple patients who underwent either revision total hip or total knee replacement surgery, further indicating the strong association of LEPR + ADGRF5 + with peri-implant fibrosis in the set of patient specimens examined.
  • ADGRF5 was expressed abundantly in LEPR lineage cells from peri-implant fibrous tissue (FIG.4H, FIG.4M – FIG.4O).
  • LEPR + ADGRF5 + cells expressed other transcripts commonly expressed by fibroblastic cells, such as VIM, FN1, COL1A2, FBLN1, and adipogenic markers (LPL), but minimally expressed transcripts associated with osteogenesis, such as ALPL, BGLAP, WIF1, and CLEC3B (FIG.15E – FIG.15G).
  • scRNA-seq studies confirmed our gene expression and immunohistochemistry analyses, indicating that there is a subpopulation of LEPR + cells in the human bone compartment that coexpresses markers of fibroblastic cells and ADGRF5.
  • FIG.4I FIG.16
  • BMSC1-3 Lepr + Cxcl12 + bone marrow stromal progenitors
  • OLC 1-2 two populations of osteoprogenitors that express Runx2, Sp7, Alpl, Bglap, Bglap2, and Col1a1
  • Adipoq three populations of adipose progenitors that express Adipoq, Lpl, Kng2, Cebpa, and Plin
  • Adipo Adipo
  • CLC chondroprogenitors that express Acan and Sox9
  • Periosteum Periosteum
  • CXCL12-abundant reticular cell (CAR) markers such as Lepr, Cxcl12, Ebf3, and Foxc1
  • BMSCs bone marrow stromal cell progenitors
  • BMSC1-3 Lepr + Cxcl12 + mouse cells
  • Adgrf5 Adgrf5-expressing LEPR+ cells are distinct from many other recently identified subsets of LEPR/CAR cells, including osteo-CAR and adipo- CAR/MALP cells[26, 27].
  • Example 6 Selective inhibition of ADGRF5 expression attenuates LEPR + cell proliferation and the formation of peri-implant fibrotic tissue Having established that ADGRF5 is expressed by LEPR + cells in both human and murine peri-implant fibrotic tissue, we next aimed to study whether ADGRF5 is necessary for the development of peri-implant fibrosis. To this end, we conditionally knocked out Adgrf5 in LepR-lineage cells (LepR-Cre;tdTomato;Adgfr5 fl/fl , hereafter called LepR-cre +; Adgrf5 f/f mice).
  • LepR-cre + Adgrf5 f/f mice
  • FIG.5B Histomorphometric analysis of the peri-implant tissue demonstrated a significant reduction in peri-implant fibrosis in Lepr-cre +; Adgrf5 f/f mice (FIG.5B, FIG.5F – FIG. 5H).
  • LepR-tdTomato+ cells from the LepRcre/+ donors formed exclusively graft-derived fibrotic tissue after transplantation
  • LEPR-tdTomato+ cells from LepRcre/+;Adgrf5f/f mice formed bone organoids containing graft-derived osseous and adipose tissue after transplantation (Fig.20d–l), showing a phenotypic switch in differentiation capacity due to cell-intrinsic reprogramming that was durable even after removing LEPR+ cells from the peri-implant microenvironment.
  • conditional knockout of Adgrf5 in LEPR+ cells resulted in upregulation of pro-osteogenic genes (Bglap49, Bglap2 (ref.49), Satb2 (ref.50), Enpp6 (ref.51), Serpina9 (ref.52), Pon1 (ref.53), Foxq1 (ref.54), Dusp5 (ref.55), Phex (ref.56), Ifitm5 (ref.57), Cldn1 (ref.58) and Car3 (ref.59)), downregulation of anti-osteogenic-related genes (Pax3 (ref.
  • conditional knockout of Adgrf5 in LEPR+ cells also resulted in downregulation of pro-proliferative genes (Wnt1 (ref.81), Pax3 (ref.82), Cntn2 (ref.83), Tm4sf4 (ref.84), Nat1 (ref.85), Tubb1 (ref.86), Grik3 (ref.87), Piwil4 (ref.88), Nudt10 (ref.89), Epha1 (ref.90), Parm1 (ref.91), Ifit1 (ref.92), Cxcl1 (ref.93), Car9 (ref.94) and Cpxm2 (ref.95)) and upregulation of anti-proliferative genes (Pax5 (ref.96), Spry1 (ref.97), Map3k1 (ref.98), Aldob99, Pon1 (ref.100), Vsig8 (ref.101), Car4 (ref.102), Rassf
  • Conditional knockout of Adgrf5 in LEPR+ cells also resulted in differential expression of several genes involved in mechanotransduction (Pcdh15 (ref.139), Myom2 (ref.140), Cdh5 (ref.141), Notch1 (ref.142), Adgrg1 (ref.143), Ripor2 (ref.144), Rfx3 (ref.145), Septin1 (ref.146), Grk2 (ref.147) and Ackr4 (ref.148)) (Data not shown), showing a potential connection between the local mechanical environment in the peri- implant area to proliferation and differentiation of LEPR+ cells.
  • Example 7 ADGRF5-blocking antibodies attenuate peri-implant fibrosis and improve peri-implant osseointegration
  • conditional genetic deletion of Adgrf5 in Lepr-lineage cells resulted in attenuation of peri-implant fibrosis
  • administration of an anti- ADGRF5-blocking antibody would also prevent peri-implant fibrous tissue formation and promote peri-implant bone formation, therefore mediating a prophylactic effect against peri-implant fibrosis.
  • Anti-ADGRF5 antibodies (previously shown to block ADGRF5 signaling[21]) were administered via intra-articular injection from postoperative day 0 until postoperative week 2 in LepR-Cre;tdTomato mice that underwent fibrous integration surgery (FIG.6A). After a total of 14 days of treatment, ⁇ CT analysis showed an enhancement of peri-implant bone formation in anti-ADGRF5-treated mice compared to mice treated with the isotype control (FIG.6B, FIG.6E – FIG.6F).
  • the ratio of bone volume to total volume in the peri-implant area was more than threefold higher relative to that of controls (FIG.6E), and the trabecular thickness in the peri-implant area was significantly higher than that of controls (FIG.6F). Histomorphometric analysis further showed a reduction in peri-implant fibrous tissue and an increase in peri-implant bone formation in the anti-ADGRF5-treated mice compared to isotype controls (FIG.6C, FIG. 6G – FIG.6H).
  • the ratio of bone volume to total volume and the trabecular thickness in the peri-implant area were significantly higher in the anti-ADGRF5-treated mice than in the isotype controls.
  • Immunofluorescence imaging demonstrated significant depletion of LEPR-tdTomato + and profibrotic LEPR-tdTomato + CD200 + cells in the peri-implant area of anti-ADGRF5-treated mice (FIG.6L, FIG.16B, FIG.16E).
  • the amount of mSSC in the anti- ADGRF5-treated mice was unchanged.
  • Cells of the profibrotic LEPR+ population originate from an osteogenic lineage and retain key features of their identity by maintaining expression of LEPR, CXCL12, BGLAP and ALPL, but they also gain expression of CD200, ACT ⁇ 2, COL3 ⁇ 1, S100A4, SM22 ⁇ , FBLN2 and SDC4 as they acquire a fibrogenic phenotype. This is consistent with the previous finding that Act ⁇ 2-expressing LEPR+ cells are a source of myofibroblasts in primary myelofibrosis40,152,153.
  • peri-implant tissue Most of the cells in peri-implant tissue are LEPR+, and the formation and maintenance of peri-implant fibrotic tissue also requires the continuous presence of LEPR+ cells because ablation of LEPR+ cells attenuates established peri-implant fibrosis. This is in line with studies of fibrosis in other organs, such as studies of cardiac154, kidney155 and lung fibrosis156, in which the presence of the corresponding tissue-resident progenitor cells154–156 was required to initiate and maintain fibrosis. Although tissue-resident stem cells still express markers of their tissue-resident progenitor cells (LEPR in this study), these cells likely undergo genetic reprogramming to become myofibroblasts, lose their multipotency and mediate fibrosis153.
  • fibrotic reprogramming affects a certain subset of progenitor cells while preserving the multipotency of other subsets of progenitor cells153,155,157.
  • the profibrotic microenvironment does not impact all skeletal progenitors equally: it causes LEPR+ cells to bias their differentiation towards a fibrogenic fate while other bone marrow stromal progenitors retain the ability to form bone in the peri-implant region, as observed after inhibition or ablation of LEPR+ cells. This notion is supported by multiple findings from our study.
  • ADGRF5 as a potential mediator of the profibrotic reprogramming of LEPR+ cells in humans and mice.
  • Adgrf5 expression is upregulated at both the RNA- transcript and protein levels in the setting of peri-implant fibrosis in both human specimens and mice.
  • Conditional deletion of Adgrf5 in LEPR+ cells attenuated periimplant fibrosis and augmented peri-implant bone formation through decreased proliferation and increased osteogenic differentiation of the LEPR+ cells.
  • Adgrf5 in LEPR+ cells resulted in differential expression of several genes involved in mechanotransduction (Pcdh15 (ref.139), Myom2 (ref.140), Cdh5 (ref.141), Notch1 (ref. 142), Adgrg1 (ref.143), Ripor2 (ref.144), Rfx3 (ref.145), Septin1 (ref.146), Grk2 (ref. 147) and Ackr4 (ref.148)) (Supplementary Fig.8j) that are associated with a decrease in LEPR+ cell proliferation and increased osteogenic differentiation, resulting in overall decreased peri-implant fibrosis and increased peri-implant osteogenesis.
  • ADGRF5 is primarily activated via its tethered agonistic peptide158 and cleavage at its GPCR proteolytic site is necessary to activate ADGRF5159.
  • cleavage of the ADGRF5 GPCR proteolytic site in LEPR+ cells is a promising clinical strategy to prevent and reverse peri-implant fibrosis.
  • Kanazawa, T., et al. Histomorphometric and ultrastructural analysis of the tendon- bone interface after rotator cuff repair in a rat model. Sci Rep, 2016.6: p.33800. 3. Nichols, A.E.C., K.T. Best, and A.E. Loiselle, The cellular basis of fibrotic tendon healing: challenges and opportunities. Transl Res, 2019.209: p.156-168. 4. Usher, K.M., et al., Pathological mechanisms and therapeutic outlooks for arthrofibrosis. Bone Res, 2019.7: p.9. 5. Stewart, S.K., Fracture Non-Union: A Review of Clinical Challenges and Future Research Needs.

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Abstract

L'invention concerne des procédés de traitement ou de prévention de la formation de fibrose orthopédique, qui comprend, mais sans s'y limiter, un tissu fibrogène péri-implantaire, une fibrose os sur os, une fibrose os sur tissu mou, une fibrose tissu mou sur tissu mou, une fibrose liée à une articulation, chez un sujet en ayant besoin. Dans certains modes de réalisation, le procédé consiste à administrer au sujet un inhibiteur de ADGRF5, GREM1, PTPRF, LAMB1, EFEMP1, DNMT3b, RSPO3, ACTA2, PDGFRA, PDGFB, PDGFA, CX3CR1, TBX18, PERI, NR1D1, RYR1, DES, KLF4, KLHL41, DUSP1 ou TNNT3 au sujet.
PCT/US2024/042952 2023-08-17 2024-08-19 Prévention et traitement de la fibrose par inhibition ou régulation positive de protéines spécifiques Pending WO2025039000A1 (fr)

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SUHARDI VINCENTIUS JEREMY, OKTARINA ANASTASIA, HAMMAD MOHAMMED, NIU YINGZHEN, LI QINGDIAN, THOMSON ANDREW, LOPEZ JUAN, MCCORMICK J: "Prevention and treatment of peri-implant fibrosis by functionally inhibiting skeletal cells expressing the leptin receptor", NATURE BIOMEDICAL ENGINEERING, NATURE PUB. GROUP, vol. 8, no. 10, pages 1285 - 1307, XP093283176, ISSN: 2157-846X, DOI: 10.1038/s41551-024-01238-y *
ZHOU BO O., YUE RUI, MURPHY MALEA M., PEYER JAMES G., MORRISON SEAN J.: "Leptin-Receptor-Expressing Mesenchymal Stromal Cells Represent the Main Source of Bone Formed by Adult Bone Marrow", CELL STEM CELL, ELSEVIER, CELL PRESS, AMSTERDAM, NL, vol. 15, no. 2, 1 August 2014 (2014-08-01), AMSTERDAM, NL , pages 154 - 168, XP093283172, ISSN: 1934-5909, DOI: 10.1016/j.stem.2014.06.008 *

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CN120796387A (zh) * 2025-09-11 2025-10-17 浙江大学 一种特异性示踪心脏瓣膜细胞的小鼠模型构建方法及应用

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