[go: up one dir, main page]

WO2016019225A1 - Modèles de cartilage de génie tissulaire in vitro pour des essais de médicament pour l'ostéoarthrite - Google Patents

Modèles de cartilage de génie tissulaire in vitro pour des essais de médicament pour l'ostéoarthrite Download PDF

Info

Publication number
WO2016019225A1
WO2016019225A1 PCT/US2015/043081 US2015043081W WO2016019225A1 WO 2016019225 A1 WO2016019225 A1 WO 2016019225A1 US 2015043081 W US2015043081 W US 2015043081W WO 2016019225 A1 WO2016019225 A1 WO 2016019225A1
Authority
WO
WIPO (PCT)
Prior art keywords
tgf
modified
sox9
chondrocytes
osteoarthritic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2015/043081
Other languages
English (en)
Inventor
Rosa SERRA
Robert Dalton CHAVEZ
Timothy WICK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UAB Research Foundation
Original Assignee
UAB Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by UAB Research Foundation filed Critical UAB Research Foundation
Publication of WO2016019225A1 publication Critical patent/WO2016019225A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone

Definitions

  • Osteoarthritis is a leading cause of disability in the industrialized world but little is known about mechanisms of cartilage destruction associated with osteoarthritis.
  • TGF- ⁇ Transforming Growth Factor-beta
  • TGF- ⁇ Transforming Growth Factor-beta
  • TGF- ⁇ has many biological effects on multiple tissues making it difficult to use TGF- ⁇ ligand directly as a therapy for cartilage repair or regeneration.
  • changes in TGF- ⁇ signaling as cartilage ages complicate the use of TGF- ⁇ ligand for OA.
  • finding therapeutics that alter the TGF- ⁇ pathway can provide a useful therapy for OA.
  • a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; growing said modified chondrocyte in the absence of the compound; determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and comparing the osteoarthritic characteristic of the modified chondrocytes, wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic.
  • the modified chondrocyte is genetically modified.
  • the modified chondrocyte is a mammalian chondrocyte.
  • the mammalian chondrocyte is a human chondrocyte
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; growing said modified chondrocyte in the absence of the compound; determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and comparing the osteoarthritic characteristic of the modified chondrocytes, wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic, wherein the altered TGF signaling pathway gene is an altered TGFpR gene.
  • the TGFpR gene is TGFpR type II.
  • the altered TGFpR gene comprises a dominant negative mutation in the TGF R gene.
  • the dominant negative mutation is a truncation of the TGF R gene.
  • the truncation of the TGF R gene is the deletion of the kinase domain.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; growing said modified chondrocyte in the absence of the compound; determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and comparing the osteoarthritic characteristic of the modified chondrocytes, wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic, wherein growing the modified chondrocytes comprises culturing the modified chondrocytes on a cell culture dish, multiwell plate, or flask.
  • the growing of the modified chondrocytes comprises culturing the modified chondrocytes on a scaffold.
  • the scaffold is a polylactic acid composition.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; growing said modified chondrocyte in the absence of the compound; determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and comparing the osteoarthritic characteristic of the modified chondrocytes, wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic, wherein the growing of the modified chondrocytes comprises culturing the modified chondrocytes in a bioreactor.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; growing said modified chondrocyte in the absence of the compound; determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and comparing the osteoarthritic characteristic of the modified chondrocytes, wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic, wherein growing the modified chondrocytes results in formation of engineered cartilage tissue.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; growing said modified chondrocyte in the absence of the compound; determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and comparing the osteoarthritic characteristic of the modified chondrocytes, wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic, wherein the osteoarthritic characteristic is a loss of proteoglycans, hypertrophic differentiation of the modified chondrocytes, a decrease in stiffness of the engineered cartilage tissue, or fibrillation of the matrix, clustering of the chondrocytes, or size of the chondrocytes.
  • hypertrophic modified chondrocytes are determined by detecting the presence of a hypertrophy biomarker.
  • the loss of proteoglycans is determined using alcian blue staining or aggrecan staining.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; growing said modified chondrocyte in the absence of the compound; determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and comparing the osteoarthritic characteristic of the modified chondrocytes, wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of an osteoarthritis therapeutic, wherein the reversal of the osteoarthritic characteristic comprises an increase of proteoglycans, a decrease in the hypertrophy biomarker, a return of the stiffness of the modified chondrocytes or a change in histology of the modified chondrocytes or engineered cartilage tissue.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; growing said modified chondrocyte in the absence of the compound; determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and comparing the osteoarthritic characteristic of the modified chondrocytes, wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic, wherein the compound is suspected of being an osteoarthritis therapeutic that alters the TGF- ⁇ pathway.
  • altering the TGF- ⁇ pathway comprises increasing sumoylation of SOX9 or increasing the activity of PAPS synthethase 2 (PAPSS2).
  • FIG. 1 is a schematic of Papss2 gene and regulatory elements.
  • Smad3 binding site (SEQ ID NO: 1) is the sequence shown on the far left and paired Sox9 (SEQ ID NO:2) is the sequence shown on the far right. Arrows indicate direction of binding sequence.
  • FIG. 2 shows that TGF-B regulates Sox9 protein.
  • Sox9 and ⁇ -tubulin proteins were detected in lysates from rib costal chondrocytes treated with TGF- ⁇ (5ng/ml) or vehicle (4mM-HCl with 0.1% BSA) for 2h and 5h. Treatment with TGF-B resulted in increased levels of Sox9 protein.
  • Sox9 and 18S mRNA were examined by RT-PCR of RNA isolated from rib chondrocytes left untreated or treated with TGF- ⁇ (5ng/ml) for 2h and 4h. mRNA levels were not affected by TGF-B.
  • Bovine cells were treated with cyclohexamide with or without TGF- ⁇ treatment. Loss of Sox9 protein was measured over time by western blot.
  • Bovine chondrocytes were infected with Adeno FLAG WT Sox9 for 48h then treated with TGF- ⁇ for 24h followed by IP for FLAG and western for SUMO 1 and FLAG. Western for tubulin (no IP) was used as a control. Treatment with TGF- ⁇ resulted in increased SUMO-Sox9.
  • Figure 3 shows an overall model as related to specific aims in the proposal.
  • Figure 5 shows a modified form of Sox9 is detectable in the nucleus in chondrocytes.
  • A Primary bovine articular chondrocytes were plated for 24h and cellular fractionation was performed. A high MW form of Sox9 was detected in the nucleus by western blot
  • B Bovine chondrocytes were treated with (+) or without (-) 5ng TGF- ⁇ ⁇ / ⁇ . Endogenous Sox9 was IPed from the nuclear lysates. Western blot using anti SUMO antibody confirmed the high MW weight Sox9 band was the sumoylated form. The arrow designates the specific band.
  • FIG. 6 shows an association of PIAS 1 and Sox9 in response to TGF- ⁇ .
  • A Primary bovine chondrocytes were infected with Ad FLAG Sox9. Cells were untreated (-) or treated (+; duplicate samples shown) with TGF- ⁇ . IP was done using anti-FLAG to bring down Sox9 and associated proteins. Western blot was done with anti-PIAS l . There was increased association of FLAG-Sox9 and endogenous PIAS 1 in response to TGF- ⁇ . (Sorry about the bubbles lane 3 is better.)
  • B ATDC5 cells were transfected with FLAG-PIAS 1 and then left untreated (-) or treated (+) with TGF- ⁇ . IP was done using anti-FLAG to bring down PIAS 1. Western blot was done with anti- Sox9 to detect associated endogenous Sox9. There was an increase in association of PIAS 1 and SUMO-Sox9 with TGFR treatment.
  • FIG. 7 Bovine chondrocytes were infected with Ad-GFP or Ad-FLAGSox9. Western blot confirms expression of FLAG Sox9.
  • B Ad-GFP cells were treated with TGF- ⁇ . RNA was used in real time RT-PCR to confirm up-regulation of PRG4 and PAPSS2 in virus infected cells.
  • C Real time RT-PCR shows up regulation of PAPSS2 by FLAG Sox9.
  • D There was no additional increase in Papss2 expression in Ad-Sox9 infected cells treated with TGF-B.
  • Figure 8 shows a concentric cylinder bioreactor schematic diagram depicting bioreactor configuration and construct placement on inner bob.
  • Figures 9 is a graph showing mechanical properties in engineered cartilage (35 days in bioreactor). Shear modulus was measured using a Bohlin CVO rehometer. Low oxygen content lead to constructs with native material properties. * p ⁇ 0.01.
  • Figures 10A and 10B show knee joint histology in wild-type mice showed cartilage with smooth articular surfaces (A), whereas knee joint histology in DNIIR mice showed fibrillated articular surfaces (B) and other OA symptoms.
  • Figures 1 lA-1 ID show that TGF- ⁇ upregulates Papss2 and Prg4 (A and B), maintains type II collagen (C), and blocks type X collagen (D).
  • Figures 12A, 12B, and 12C show chondrocytes were successfully transduced with eGFP and DNIIR adenoviruses (A and B), and the DNIIR adenovirus reduced Papss2 and Prg4 expression as was observed in DNIIR mice (C and D).
  • FIGS 13A-13H show PLLA scaffolds (A) were seeded with chondrocytes and cultivated in a bioreactor (B) for 4, 12, and 20 days (C-E respectively), resulting in tissue formation after 20 days. Histology showed the outermost layer of cells aligned with the tissue surface (F), toluidine blue staining identified proteoglycans in the extracellular matrix (G), and live-dead staining showed the majority of cells were living.
  • Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically
  • polylactic acid composition can be used for a composition made by dissolving polylactic acid in methylene chloride and adding NaCl to form a paste that is dried.
  • the polylactic acid can be poly-D-lactic acid (PDLA), poly-L-lactic acid (PLLA), or a racemic mixture of the two.
  • modified chondrocyte refers to any chondrocyte that has been altered from its native state. In some instances, a modified chondrocyte has an altered TGF signaling pathway gene.
  • osteoarthritic characteristic refers to any characteristic that is used to define or diagnose osteoarthritis.
  • an osteoarthritic characteristic can be a description of the state of osteoarthritic tissue or cells.
  • osteoarthritic characteristics are a loss of proteoglycans, hypertrophic differentiation of the modified chondrocytes, a decrease in stiffness of the engineered cartilage tissue, and fibrillation of the matrix, clustering of the chondrocytes, or size of the chondrocytes
  • Altering the TGF- ⁇ pathway refers to changing the TGF- ⁇ pathway from what is considered normal or what is present in healthy cells or tissue. Altering can comprise an increase or decrease in the amount of a TGF- ⁇ pathway gene or an increase or decrease in the activity of a protein encoded from a TGF- ⁇ pathway gene.
  • each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A-E, B-F, and C- E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
  • steps in methods of making and using the disclosed compositions are if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic.
  • the modified chondrocyte can be a mammalian cell.
  • the mammalian cell can be a human cell.
  • the modified chondrocytes can be chondrocytes that are harvested from a mammal and then modified.
  • the modified chondrocytes can be human chondrocytes.
  • the modified chondrocytes can be multipotent stem cells (MSCs), induced pluripotent stem cells (iPSCs) or any pluripotent cell source.
  • MSCs multipotent stem cells
  • iPSCs induced pluripotent stem cells
  • the modified chondrocyte can be genetically modified.
  • the genetic modification can be a mutation in a TGF signaling pathway gene.
  • the genetic modification can be in the TGF R gene or Smad3 gene.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • the altered TGF signaling pathway gene can be an altered TGFpR gene.
  • the TGFpR gene can be TGFpR type II.
  • the altered TGFpR gene comprises a dominant negative mutation in the TGFpR gene.
  • the dominant negative mutation can be a truncation of the TGFpR gene.
  • TGFpR contains a ligand binding domain, transmembrane domain and juxtamembrane domain and a kinase domain.
  • the truncation of the TGFpR gene can be the deletion of the kinase domain.
  • the deletion can be a deletion of the entire domain or a deletion of a part of the kinase domain, which results in the kinase domain being non-functional.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • the modified chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic; wherein growing the modified chondrocytes comprises culturing the modified chondrocytes on a cell culture dish, multiwell plate, or flask.
  • Any known cell culture apparatus can be used for culturing the modified chondrocytes.
  • the cell culture apparatus can be a bioreactor.
  • the disclosed methods can comprise the growing of the modified chondrocytes comprising culturing the modified chondrocytes in a bioreactor.
  • the growing of the modified chondrocytes comprises culturing the modified chondrocytes on a scaffold.
  • the scaffold can be a poly lactic acid composition.
  • polylactic acid can be poly-D- lactic acid (PDLA), poly-L-lactic acid (PLLA) or a racemic mixture of the two.
  • PDLA poly-D- lactic acid
  • PLLA poly-L-lactic acid
  • any known cell culturing scaffold can be used.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • Engineered cartilage tissue comprises cells and a matrix.
  • the engineered cartilage tissue can have the same properties of natural cartilage tissue.
  • engineered cartilage tissue comprises the natural architecture and extracellular matrix present in native cartilage tissue.
  • Engineered cartilate tissue can comprise type II collagen and aggrecan.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic; wherein the osteoarthritic characteristic is a loss of proteoglycans.
  • the loss of proteoglycans can be determined using alcian blue staining or aggrecan staining. Assays used to determine loss of proteoglycans are well known in the art.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic; wherein the reversal of the osteoarthritic characteristic comprises an increase of proteoglycans.
  • An increase of proteoglycans can be determined using alcian blue staining or aggrecan staining. Assays used to determine the presence or increase of proteoglycans are well known in the art.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • hypertrophic modified chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic; wherein the osteoarthritic characteristic is hypertrophic differentiation of the modified chondrocytes.
  • hypertrophic modified chondrocytes can be determined by detecting the presence of a hypertrophy biomarker. Hypertrophy biomarkers can be type X collagen or MMP 13. In some instances, hypertrophic modified chondrocytes can be determined
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic; wherein the reversal of the osteoarthritic characteristic comprises a decrease in the hypertrophy biomarker.
  • Hypertrophy biomarkers can be type X collagen or MMP 13.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • osteoarthritic characteristic is a decrease in the stiffness of the engineered cartilage tissue.
  • the decrease in stiffness of the engineered cartilage tissue is compared to the stiffness of healthy cartilage tissue whether engineered or native.
  • an osteoarthritic characteristic would be a decrease in the stiffness compared to normal or healthy cartilage tissue.
  • stiffness refers to the Young's elastic modulus which measures the force per unit area that is required to compress a sample.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic; wherein the reversal of the osteoarthritic characteristic comprises a return of the stiffness of the modified chondrocytes.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic; wherein the osteoarthritic characteristic is alterations in normal histology of the cartilage tissue including loss of zonal structure of the tissue, fibrillation of the matrix, clustering of the chondrocytes, or an increase in size of the chondrocytes. Fibrillation of the matrix can be determined by the edges of the tissue not being smooth. Clustering of the chondrocytes can refer to the chondrocytes not having matrix in between them.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic; wherein the reversal of the osteoarthritic characteristic comprises a change in histology of the modified chondrocytes.
  • a change in histology of the modified chondrocytes can be a restoration of the zonal structure of the cartilage tissue, reduced fibrillation of the matrix, reduced clustering of the chondrocytes, or reduced size (hypertrophy) of chondrocytes. Fibrillation of the matrix can be determined by the edges of the tissue not being smooth. Clustering of the chondrocytes can refer to the chondrocytes not having matrix in between them.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • altering the TGF- ⁇ pathway comprises altering sumoylation of SOX9.
  • altering sumoylation of SOX9 can include increasing sumoylation of SOX9.
  • altering the TGF- ⁇ pathway comprises altering protein levels of SOX9.
  • altering protein levels of SOX9 can include increasing protein levels of SOX9.
  • increasing sumoylation of SOX9 can include increasing protein levels of SOX9.
  • sumoylation of SOX9 comprises increase mRNA levels of SOX9.
  • Disclosed are methods for screening potential osteoarthritis therapeutics comprising a) growing a modified chondrocyte comprising an altered TGF signaling pathway gene in the presence of a compound suspected of being an osteoarthritis therapeutic; b) growing said modified chondrocyte in the absence of the compound; c) determining an osteoarthritic characteristic in the modified chondrocyte in the presence of the compound and in the absence of the compound; and d) comparing the osteoarthritic characteristic of the modified
  • chondrocytes wherein a reversal of the osteoarthritic characteristic in the presence of the compound is indicative of a osteoarthritis therapeutic; wherein the compound is suspected of being an osteoarthritis therapeutic that alters the TGF- ⁇ pathway, wherein altering the TGF- ⁇ pathway comprises altering the activity of PAPS synthethase 2 (PAPSS2).
  • PAPSS2 PAPS synthethase 2
  • altering the activity of PAPSS2 can be increasing or decreasing the activity of PAPSS2.
  • the activity of PAPSS2 comprises generating PAPS.
  • PAPSS2 activity can be measured with alcian blue staining.
  • mRNA or protein levels of PAPSS2 can also be measured.
  • Paps can be measured. C. Kits
  • kits for screening potential osteoarthritis therapeutics the kit comprising a compound suspected of being an osteoarthritis therapeutic.
  • kits for screening potential osteoarthritis therapeutics the kit comprising modified chondrocytes.
  • the kits also can contain cell culture media or a cell culture apparatus such as a flask, cell culture dish, multiwall plate, or bioreactor.
  • TGF- ⁇ is a multifunctional peptide that has been shown to regulate cellular differentiation and tissue-specific gene expression.
  • transgenic mice were generated that express a dominant- negative mutation of the TGF- ⁇ type II receptor (Tgfbr2) in articular cartilage.
  • TGF- ⁇ Altered responsiveness to TGF- ⁇ resulted in a progressive skeletal disease that resembled osteoarthritis in humans.
  • Several down-stream targets of TGF- ⁇ have been identified that regulate post- translational processing of the major extracellular matrix proteins in articular cartilage.
  • One in particular, 3-Prime-Phoshoadenosine 5-Prime-Phosphosulfate Synthase 2 (Papss2) has been associated with Spondyloepimetaphyseal Dysplasias in humans and is required for proper sulfation of proteoglycans in cartilage.
  • the transcription factor Sox9 is also associated with the maintenance of mature articular cartilage.
  • TGF- ⁇ enhances the level of Sox9 protein in chondrocytes independently of changes in mR A.
  • the data indicate that treatment with TGF- ⁇ results in sumoylation of Sox9. Sumoylation has been shown to regulate protein stability, activity and cellular localization of proteins.
  • This study is directed to: la) determining which sites on Sox9 are sumoylated in response to TGF- ⁇ and determining the role of sumoylation in TGF- -mediated Sox9 levels, localization, and activity; lb) determining the mechanism of TGF- -mediated sumoylation of Sox9; 2) determining the mechanism of TGF- ⁇ mediated expression of Papss2 and 3) determining if Papss2 activity can alleviate cartilage degeneration when TGF-B signaling is disrupted and determining if activation of TGF-B' s chondroprotective signals can restore biochemical and biomechanical properties to OA cartilage.
  • These studies can identify mechanisms of chondroprotection that can be used as targets for therapies in osteoarthritis.
  • TGF-B Transforming growth factor-beta
  • DNIIR TGF- B type II receptor
  • TGF-B normally prevents joint degeneration, however, the downstream effectors of this chondroprotective effect are not known. Alterations in chondrocyte function and diminished extracellular matrix deposition, leading to cartilage degeneration, have also been associated with a decrease in the expression of the SRY (sex-determining region Y) box 9 (Sox9) transcription factor. Nevertheless, up-stream regulators of Sox9 activity have not been very well characterized. Several down-stream targets of TGF-B have been identified that regulate post-translational processing of the major extracellular matrix proteins in articular cartilage.
  • Papss2 3-Prime-Phoshoadenosine 5-Prime- Phosphosulfate Synthase 2
  • TGF- ⁇ enhances sumoylation of Sox9 protein in chondrocytes, which may alter the stability or activity of the protein.
  • TGF- ⁇ can maintain the differentiated chondrocyte phenotype in permanent cartilages, like articular cartilage, by regulating Sox9 levels and activity via protein sumoylation. Sox9 in turn regulates the expression of specific down-stream targets of TGF-B that have global effects on the extracellular matrix including Papss2. The chondroprotective effects of TGF-B also have reparative functions. Since TGF-B has broad biological activity in many tissues, these studies can elucidate the cartilage specific targets of TGF-B for investigation into new therapies to repair damage to articular cartilage.
  • Sumoylation can affect Sox9 levels, localization, and activity in response to TGF-B. It has been shown that there are three lysine residues in the Sox9 protein that can be sumoylated. The results indicate that TGF-B increases sumoylation of Sox9. Sumoylation has been shown to regulate protein stability, activity and cellular localization. Therefore, FLAG-tagged Sox9- encoding plasmids were generated in which combinations of the three sumoylatable lysine residues were converted to arginine. These mutants as well as wild-type Sox9 can be used to determine the role of sumoylation on TGF-B mediated Sox9 levels, localization, and
  • PIAS 1 can regulate sumoylation of Sox9 by TGF-B. It has been shown that TGF-B can mediate sumoylation of various target proteins through PIAS1. It can be determined if treatment with TGF-B results in activation of PIAS 1 and increased association of PIAS 1 with Sox9. RNA interference as well as a dominant-negative PIASl expression vectors will then be used to examine the requirement of PIASl for Sox9 sumoylation in response to TGF- ⁇ .
  • Sox9 can be required for TGF-B mediated expression of Papss2.
  • TGF-B regulates the expression of Papss2 mRNA in bovine articular chondrocytes and Papss2 mRNA and staining for sulfated proteoglycans is down-regulated in DNIIR mice. This study can determine if Sox9 is sufficient to stimulate expression of Papss2 and is necessary for TGF-B mediated regulation. It can also be determined if sumoylation is also required for TGF-B mediated expression of Papss2.
  • Papss2 activity can alleviate cartilage degeneration when TGF-B signaling is disrupted.
  • Both engineered bovine articular chondrocyte constructs and in vivo transgenic mouse models can be used to test this hypothesis.
  • Bovine cartilage infected with control and DNIIR adenovirus with or without a Papss2 expressing virus can be grown in a bioreactor.
  • Cartilage can be tested to quantify the effect of TGF-B and Papss2 activity in regulating cartilage biochemical biomechanical properties.
  • mice expressing Papss2 under the control of the Col2a promoter can be generated and crossed to DNIIR mice. Biomechanical and biochemical properties can be compared in control and double transgenic mice.
  • human OA cartilage grown in bioreactors can be used to show that stimulation of TGF-B's
  • chondroprotective functions has some reparative effects on OA cartilage.
  • OA is a leading cause of disability in the industrialized world but little is known about mechanisms of cartilage destruction associated with osteoarthritis. Chondrocytes and the extracellular matrix they produce are essential to maintain the normal structure and
  • Articular cartilage has a limited intrinsic capacity for repair placing major obstacles in treatment strategies for OA. Nevertheless, surprisingly little is known about the factors or downstream targets that regulate maintenance of articular cartilage, information that would be the foundation of any prevention or treatment strategies.
  • TGF- ⁇ signaling is critical for maintenance of articular cartilage.
  • TGF- ⁇ Transforming Growth Factor-beta
  • TGF- ⁇ Transforming Growth Factor-beta
  • TGF-Bs signal through heteromeric serine/threonine kinase receptors.
  • the current model is that TGF-B ligand binds to the TGF-B type II receptor (Tgfbr2) on the cell surface. Tgfbr2 is then able to recruit the Type I receptor (Tgfbrl (ALK5)) to form a heterotetrameric complex of two type I and two type II receptors.
  • Tgfbr2 which is a constitutively active kinase, phosphorylates the GS domain of Tgfbrl, activating the type I serine/threonine kinase. Downstream targets of the Tgfbrl kinase then transduce the signal to the nucleus.
  • the Smad family of proteins has been identified as important transducers of TGF-B signaling. Smads are directly phosphorylated by the type I receptor, translocate to the nucleus and act as transcription factors. Receptor associated Smads can be separted into two broad classes: those generally activated by TGF-B/ Activin signaling (Smad 2/3) and those generally activated by BMP signaling (Smadl/5/8).
  • Smads have been shown to bind DNA directly or in some cases cooperate with transcription co-factors or co-repressors. Smad independent, non-canonical signaling pathways have also been identified. These pathways include signaling through Erk, JNK and p38 MAPK pathways, as well as the PI3K pathway, and Rho-like GTPases. Previously, it was shown that dominant-negative interference of Tgf r2 (DNIIR) in mice results in OA-like symptoms in the peripheral joints indicating TGF-B has chondroprotective functions. Similarly, mice deleted for Smad3 or Latent TGF-B Binding Protein 3 (LTBP3), an extracellular mediator of TGF-B signaling demonstrated OA symptoms.
  • DNIIR dominant-negative interference of Tgf r2
  • LTBP3 Latent TGF-B Binding Protein 3
  • TGF-B signaling has a critical role in cartilage homeostasis yet the mechanisms of TGF-B action are not known.
  • This proposal focuses on TGF-B' s chondroprotective effects.
  • TGF- ⁇ has many biological effects on multiple tissues making it difficult to use TGF- ⁇ ligand directly as a therapy for cartilage repair or regeneration.
  • changes in TGF- ⁇ signaling as cartilage ages complicate the use of TGF- ⁇ ligand for OA.
  • Cartilage specific downstream effectors of TGF-B's chondroprotective functions would be good targets for therapy but the molecular targets of TGF- ⁇ signaling in normal articular cartilage are largely unknown.
  • an affymetrix based microarray was performed to find genes in bovine articular chondrocytes grown in micromass culture that were regulated by treatment with TGF- ⁇ (GEO accession # GSE29233). RNA was isolated from three independently derived cultures. RNA was labeled and hybridized to Affymetrix Bovine GeneChips containing 23,000 transcripts according to manufactures instructions.
  • TGF- ⁇ regulated genes were identified on the array including: Transforming growth factor-beta induced protein IG-H3 precursor (BIG-H3), Proteoglycan 4/ Lubricin, and Parathyroid hormone-like hormone.
  • BIG-H3 Transforming growth factor-beta induced protein IG-H3 precursor
  • PAPSS2 PAPS synthethase 2
  • Papss2 was also identified as a TGF- ⁇ responsive gene in a microarray study focused on gene expression in the developing axial skeleton. Regulation of selected genes, including PAPSS2, were verified in bovine cells and in articular cartilage from DNIIR mice using real time RT-PCR.
  • PAPS is created from ATP and inorganic sulfate in a two-step process that involves either Papss l or Papss2 enzymes.
  • Generation of PAPS is the rate-limiting step for most sulfation reactions.
  • Sulfation is a common modification of proteins and carbohydrates but it is especially important in articular cartilage where the large amount of sulfation on the glycosamino glycans linked to the core proteins of proteoglycans provide the necessary biomechanical properties for cartilage to function.
  • Papss2 is highly expressed in cartilage and the importance of this enzyme in maintaining the cartilage phenotype is clear from the largely cartilage specific phenotypes associated with mutations in this gene, nevertheless, almost nothing is known about how enzyme levels are regulated.
  • Sox9 Smad3 binding site was identified 1Kb upstream of the mouse Papss2 start site ( Figure 1). Several individual putative Sox9 and Sox5 binding sites were also observed.
  • a deficiency in Sox9 in humans is associated with Campomelic Dysplasia, a skeletal malformation characterized by generalized hypoplasia in endochondral bones. Sox9 is also expressed in mature articular chondrocytes and high expression levels of Sox9 are associated with enhanced levels of aggrecan and type II collagen and maintenance of articular cartilage.
  • Sox9 usually binds as dimer to paired Sox9 sites to mediate expression of chondrocyte specific genes.
  • Papss2 and Col2al have a similar genomic structure in that they both have a small first exon and very large first intron with many regulatory elements.
  • a strong chondrocyte specific binding site for Sox9 is found in the first intron of the Col2al gene.
  • a putative paired Sox9 site was observed in the large first intron of Papss2 approximately 400bp from the start of exon 2 ( Figure 1). It was previously shown that Papss2 and Sox9 are co-expressed during skeletal development also supporting a potential role for Sox9 in mediating Papss2 expression. The observations indicate a role for Smad3 and Sox9 in regulation of Papss2 expression.
  • Sox9 is post-translationally modified by phosphorylation, ubiquitinization, and sumoylation.
  • Sumoylation is a post-translational modification somewhat similar to
  • ubiquitinization involves the attachment of SUMO, a small peptide, to lysine residues on proteins. Unlike ubiquitinization, sumoylation does not necessarily target a protein for degradation. Sumoylation has varying effects on protein function including altering stability, subcellular localization, or protein-protein interactions. Sumoylation has recently been implicated in altering protein functions during arthritis. Similar to ubiquitinization, sumoylation occurs through the activities of El, E2, and E3 sumoyl ligases. Protein Inhibitor of Activated STAT1 (PIASl) is an E3 sumoyl ligase. PIAS l interacts directly with Sox9 leading to its stabilization.
  • PIASl Protein Inhibitor of Activated STAT1
  • TGF- ⁇ treatment results in up regulation of Sox9 protein without alterations in Sox9 mRNA levels in mouse limb micromass, rib chondrocytes, and bovine articular chondrocytes in culture as well as in cells of the ATDC5 chondrogenic cell line ( Figure 2A, B).
  • the lack of regulation at the mRNA level was confirmed with microarray data.
  • Sox9 mRNA levels are repressed in mesenchymal cells by transcriptional repressors of TGF- ⁇ signaling, including TGIF and SnoN.
  • TGF- ⁇ acting through PIASl mediated sumoylation of Sox9, can act as a chondroprotective factor by regulating the expression of enzymes that globally regulate the biochemical properties of the extracellular matrix, including Papss2 ( Figure 3).
  • OA is one of the most common forms of musculoskeletal disability in the world, treatment options are limited.
  • One of the main functions of articular cartilage in diarthrodial joints is load bearing and the cartilage matrix has to be finely tuned to have the proper biomechanical properties to deal with various types of mechanical load. Surprisingly little is known about how cartilage is maintained even though this information would form the basis of any new prevention or treatment strategies.
  • TGF- ⁇ has been shown to have
  • TGF-B chondroprotective functions in cartilage but the mechanisms are not known. Cartilage specific downstream effectors of TGF-B's chondroprotective signals would make good targets for prevention or treatment of osteoarthritis.
  • Two targets for TGF-Bs action in cartilage have been identified: regulation of Papss2 expression and regulation of Sox9 activity through sumoylation.
  • TGF- ⁇ acting through sumoylation and Sox9 activity can regulate the expression of an important enzyme, Papss2, required for maintaining structural integrity in cartilage.
  • sumoylation through TGF- ⁇ in general is part of the mechanism through which TGF- ⁇ regulates cartilage homeostasis, methods to promote sumoylation could be investigated as treatment strategies, even if the exact targets remain elusive.
  • Another important aspect of the proposed experiments is the innovative use of bioreactors to generate cartilage tissue for biochemical and biomechanical analysis.
  • bioreactors to generate cartilage tissue for biochemical and biomechanical analysis.
  • genetically engineered cartilage tissue can be generated in bioreactors. This tissue can then be used in biomechanical and biochemical assays.
  • the method can be scaled up for treatment screening and is more efficient than generating transgenic mice for experimentation.
  • TGF- ⁇ regulates Sox9 function though PIAS1 mediated sumoylation.
  • TGF- ⁇ results in stabilization of Sox9 protein without alterations in mRNA levels and that treatment with TGF- ⁇ results in increased sumoylation of FLAG-tagged wild type Sox9 protein (Figure 2).
  • Sumoylation was previously shown to regulate stability, localization, and activity of a wide variety of proteins. This study determines the role of sumoylation on the stability, localization, and activity of Sox9 in response to TGF- ⁇ . Since it is known that TGF- ⁇ can activate PIAS 1 in other cell types and PIAS 1 interacts directly with Sox9 this study can determine the part of TGF-B's chondroprotective activity is mediated through the regulation of Sox9 levels, localization, and activity via sumoylation by PIAS1. This would represent a novel mechanism of TGF- ⁇ action in cartilage.
  • bovine articular chondrocytes grown in micromass culture as the model system although mouse costal chondrocytes, limb mesenchyme, C3H10T1/2, or ATDC5 cells can be used as needed.
  • Costal chondrocytes represent a transient cartilage and limb mesenchyme represents an early stage of differentiation.
  • C3H10T1/2 and ATDC5 cells are pluripotent mesenchymal cell lines that can be induced to form cartilage.
  • bovine articular chondrocytes for these studies is that they represent a mature permanent type of cartilage.
  • TGF-B Treatment with TGF-B over seven days resulted in increased alcian blue staining (0.25 vs. 0.55 A620 ⁇ gDNA, p ⁇ 0.0001) and decreased alkaline phosphatase activity (0.29 vs. 0.16 mMol pNp/hr ⁇ g/DNA, p ⁇ 0.002) confirming that TGF- ⁇ promotes the accumulation of cartilage matrix and prevents hypertrophic differentiation.
  • Ad-DNIIR adenovirus containing the dominant-negative TGF-B receptor
  • adenoviruses have been used and generated in the past to study various aspect of skeletal development. Cells were infected with approximately 10 9 pfu of virus for two hours while they were in suspension. Cells were then placed into micromass culture. Viral infection was detected using X-gal staining 48 hours after infection. More recently Ad-GFP has been used as a control. Ad-B-gal and Ad-DNIIR infected cells were stained for Alcian blue and Alkaline Phosphatase as described above Alcian blue staining was reduced in Ad-DNIIR infected cultures when compared to Ad-B-gal infected cultures whereas Alkaline phosphatase staining was increased in Ad-DNIIR infected cultures. The results are in agreement with the phenotype observed in the DNIIR mice. Together the results validate the use of bovine cells and adenovirus vectors to study the molecular mechanisms mediating TGF-B action in articular cartilage.
  • Mouse Sox9 has three sumoylation-targeted lysine residues (K61, K253 and K396).
  • lysine to arginine mutations have been generated in all three sumoylation sites alone (3xKR) or in combination (seven mutants total, see Figure 4), through site-directed mutagenesis, and placed them into expression vectors.
  • Each mutant as well as the WT Sox9 control is FLAG-tagged on the N- terminal for easy identification.
  • Expression plasmids will initially be transfected into
  • C3H10T1/2 or ATDC5cells where transfection efficiencies can be between 30% and 70%, and sumoylation can be measured in response to TGF- ⁇ treatment by immunoprecipitation using FLAG antibody followed by western blot with SUMOl antibody.
  • which or if all of the potential sites are sumoylated in response to TGF-B can be determined by comparing sumoylation in all of the mutants. Since primary chondrocytes generally have lower transfection efficiency (1 to 10%) than the cell lines, all of the mutants as well as WT FLAG-Sox9 are placed into adenovirus vectors and adenoviral transduction can be used for subsequent experiments.
  • Subcellular localization of FLAG-Sox9 proteins can be determined using immunoflourescence and confocal microscopy with specific antibodies against FLAG, and also by cellular fractionation. Data indicates that TGF-B can mediate the localization of Sox9. When limb mesenchyme is treated with TGF-B there is an increase in nuclear localization of Sox9 as seen by immunoflourescent staining. Sox9 is located primarily in the cytoplasm of
  • TGF-B mediated localization of Sox9, mutant, and WT FLAG-Sox9 can be transduced into cells that are either untreated or treated with TGF-B. Localization of FLAG-Sox9 can be determined by confocal microscopy and immunostaining to the FLAG tag. If sumoylation is involved in TGF-B mediated nuclear localization, localization of the mutant Sox9 proteins would not be altered by treatment with TGF-B. It is difficult to quantify changes in nuclear localization using immunoflourescent localization. Therefore nuclear fractionation studies can be used.
  • Sox9 was detected in both nuclear and cytosolic fractions using Sox9-specific antibodies.
  • Sox9-specific antibodies were used to determine the purity of the nuclear and cytosolic fractions respectively.
  • Sox9 bands were detected: one at the same molecular weight as the cytosolic Sox9 and one with higher molecular weight.
  • IP of endogenous nuclear Sox9 and western blot with anti- SUMO Abs was used to confirm that the lOOkD MW weight band in the nucleus was SUMO- Sox9 (Figure 5B). This higher MW band was increased in the nucleus after treatment with TGF- ⁇ ( Figure 5C).
  • virus transduced cells can be treated with or without TGF-B, fractionated, and WT and mutant FLAG-Sox9 can be immunoprecipitated using antibodies to FLAG.
  • Sumoylated protein can be identified by western blot with SUMOl antibodies.
  • Total Sox9 protein levels can be determined by western blot with Sox9 antibodies.
  • the ratio of sumoylated WT and mutant Sox9 protein in nuclear and cytosolic fractions can be compared in samples from untreated and TGF-B treated cells. Treatment with TGF-B can result in an increase in sumoylated WT FLAG-Sox9 in the nuclear fraction; sumoylation mutants of Sox9 can have altered nuclear levels relative to WT Sox9; and treatment with TGF-B would not affect nuclear levels of the sumoylation mutants.
  • transcriptional activity can be determined using co-transfection or co-viral transduction with a luciferase reporter plasmid that contains the Sox9 elements from the type II collagen promoter (Col2Al).
  • a luciferase reporter plasmid that contains the Sox9 elements from the type II collagen promoter (Col2Al).
  • cells can be left untreated or treated with TGF-B and cell lysates can be analyzed for activity.
  • the Dual Luciferase assay from Promega in a 98-well format using a luminometer with the dual injection format can be used.
  • the experiment can be repeated using bovine articular chondrocytes transduced with the appropriate adenoviruses.
  • Cotransfection with WT FLAG-Sox9 can result in increased activity from the Sox9 reporter.
  • Treatment with TGF-B can also result in an increase in activity from the Sox9 reporter and perhaps further increases in the presence of exogenous WT FLAG-Sox9.
  • PIAS1 is an E3 sumoyl ligase.
  • PIAS 1 interacts directly with Sox9 leading to its stabilization and it has been shown that TGF-B acts through PIASl to mediate sumoylation of a variety of transcription factors.
  • IP-Western will be used to determine if treatment with TGF- ⁇ enhances the interaction of tagged PIASl and Sox9 in chondrocytes. Results ( Figure 6) indicated that treatment with TGF-B results in increased association of PIASl and Sox9 in bovine articular chondrocytes and ATDC5 cells.
  • PIAS l activity can be blocked using two methods: shRNA can be used to block PIASl protein expression, and a dominant-negative form of PIASl that lacks the ring finger domain can be used to block activity.
  • shRNA sets with homology to human, mouse and rat genes for PIASl are available in expression vectors and lentiviral particle from Open Blosystems and other vendors. Several shRNAs come in each set. The efficiency of
  • PIASlknock down can be determined by transfecting or transducing cells with shRNA or a scrambled vector control.
  • PIAS 1 levels can be determined using western blot. If none of the shRNAs work in bovine cells either new shRNA sequences can be generated using the published sequence for bovine PIASl or experiments in mouse costal chondrocytes or ATDC5 or C3H10T1/2 cells can be performed. Likewise, the dominant-negative activity of the Ring Finger mutant can be tested using a functional assay for PIASl sumoylation activity.
  • IP- Western can be used to detect native or WT FLAG- Sox9 sumoylation with and without PIAS inhibition in the presence and absence of TGF-B.
  • PIASl is the enzyme responsible for TGF- -mediated sumoylation of Sox9
  • blocking PIAS l activity can prevent sumoylation of Sox9 in response to TGF- ⁇ treatment and potentially affect stability, localization, and activity of Sox9 depending on the degree of PIAS inhibition achieved with the selected tools.
  • TGF-B regulates sumoylation of Sox9 it is likely that TGF-B mediated sumolyation also regulates the chondrocyte phenoytpe. It has been shown that primary articular chondrocytes in culture can become hypertrophic after extended time in culture, similar to phenotypic changes seen in OA. TGF-B can delay hypertrophic differentiation in articular chondrocytes and promote deposition of proteoglycans as measured by Alcian Blue staining.
  • PIASl or sumoylation in general can lead to accelerated hypertrophy in bovine articular chondrocytes and prevent TGF-B mediated protection of the chondrocyte phenotype.
  • cells can be infected with the PIAS 1 ShRNA or the ring finger mutants and untreated or treated with TGF-B described above.
  • cells can be treated with a general antagonist of sumoylation, ginkgolic acid.
  • Cells can be stained with Alcian Blue and for Alkaline Phosphatase activity after seven days in culture.
  • RNA can be isolated from cells at various times after infection and treatment to detect the expression of collagen II, X, and I by RT-PCR.
  • Staining with BrdU can be used to assess cellular proliferation and TUNEL staining can be used to assess apoptosis.
  • Altered sumoylation and specifically PIAS1 activity can mimic the effects of DNIIR on the cartilage phenotype and prevent TGF-B mediated stimulation of Alcian Blue and decrease of Alkaline Phosphatase staining.
  • Bovine articular cartilage is
  • the disadvantages include that primary cells in general are difficult to transfect; however, adenoviruses can be used to transduce genes of interest into cells. More than one Adenovirus vector can infect a cell, but it is important to make sure that the titers of the viruses are the same in any co-transduction experiments so that you get equal amounts of transduction. It is also possible to make adenoviruses that expresse two different products using a double expression cassette.
  • Another disadvantage of the bovine system is that there are not as many reagents available as there are for human or mouse. For example, most commercially tested ShRNAs are directed to human or mouse transcripts.
  • bovine genome has been sequenced and is fairly well annotated at this time so it is possible to generate our own molecular reagents for bovine tissue. Furthermore, most of the Antibodies used that recognize mouse proteins also recognize bovine proteins (for example see Figure 2C with Sox9 antibody). Additional model systems can be used: ATDC5 or C2H10T1/2 cells, mouse costal chondrocytes, and mouse limb
  • PIAS 1 is known to be activated by TGF-B and has been documented to mediate sumoylation of Sox9, other sumoylating enzymes exist. If PIAS 1 is not the sumoylating enzyme responsible for Sox9 post-translational SUMO-modification in response to TGF- ⁇ , other members of the PIAS family of SUMO ligases can be used. For example, PIASy has been shown to mediate sumoylation by TGF- ⁇ in some cases. In addition, the E2 sumoylation ligase
  • Ubc9 is also involved in TGF- ⁇ mediated sumoylation.
  • Papss2 was identified in a microarray screen as a potential mediator of TGF-B's chondroprotective effects.
  • TGF- ⁇ stimulated Papss2 mRNA expression in bovine cells and reduced Papss2 mRNA expression was observed in cartilage from DNIIR mice.
  • Papss2 is a bifunctional enzyme whose main function is to generate PAPS, the sulfate donor for most sulfotransferase reactions.
  • Humans and mice with deficiency in Papss2 exhibit osteoarthritis as well as other skeletal abnormalities. Very little is known about how the levels of this enzyme are regulated even though matrix sulfation is essential for the maintenance of articular cartilage.
  • the Papss2 gene contains a paired Sox9 binding site in the first intron and a Smad3 binding site upstream of the start site. Since TGF- ⁇ regulates Papss2 mRNA expression as well as levels and sumoylation of Sox9 protein, TGF- ⁇ can act through sumoylation and Sox9 to regulate expression of Papss2 mRNA. Smad3 can also be involved.
  • the time course of Papss2 expression can be determined and whether or not regulation by TGF- ⁇ is direct or requires new protein synthesis.
  • Cells in the presence or absence of cyclohexamide can be untreated or treated with TGF- ⁇ for varying times between 0.5 and 8 hours.
  • RNA can be isolated from cells and Papss2 mRNA can be measured by real time RT- PCR.
  • TGF- ⁇ stimulates Papss2 mRNA levels in bovine chondrocytes and in ATDC5 cells. If Papss2 mRNA is still up regulated by TGF- ⁇ in the presence of cyclohexamide, this would indicate that regulation is direct and does not require new protein synthesis. If cyclohexamide blocks up-regulation, then new protein synthesis is required for this response.
  • PAPSS2 expression was measured by real time RT-PCR in bovine chondrocytes infected with Ad-FLAGSox9 or Ad-GFP virus with or without TGF- ⁇ ( Figure 7). Results were analyzed using REST software. In control cells infected with Ad-GFP, treatment with TGF- ⁇ resulted in up regulation of PRG4 and PAPSS2. When gene expression was compared in untreated cells infected with Ad-GFP or Ad-FLAGSox9, PAPSS2 was up regulated in Ad-FLAGSox9 expressing cells. In contrast, PRG4 was not up regulated with expression of Sox9.
  • SUMO-Sox9 should be able to enhance Papss2 mRNA expression over the level of WT FLAG-Sox9 alone.
  • the dominant-negative mutant can result in reduced Papss2 expression and/or attenuate TGF-B mediated Papss2 expression.
  • the sumoylation mutants of Sox9 would not be able to cause up-regulation of Papss2 mRNA. It can also be determine whether sumoylation in general or PIAS 1 specifically is required for TGF-B mediated regulation of Papss2.
  • cells can be treated with an inhibitor of sumoylation, ginkgolic acid, or infected with ShRNA to PIAS 1 or the ring finger mutants described above (Aim #1B).
  • Cells can be left untreated or treated with TGF-B and the levels of Papss2 mRNA can be determined by real time RT-PCR. If sumoylation by PIAS1 is required then the loss of PIAS 1 function can prevent regulation of Papss2 by TGF-B.
  • Smad3 is necessary and/or sufficient to regulate Papss2 expression.
  • a Smad3 binding site was identified in a conserved region 1Kb upstream of the Papss2 start site.
  • Smad3 is part of the canonical TGF-B signaling cascade. It is directly phosphorylated by the TGF-B type I receptor and translocates to the nucleus to regulate gene transcription. WT and dominant-negative forms of Smad2 and Smad3 can be infected into cells and the levels of Papss2 mRNA can be measured in the presence or absence of TGF-B.
  • WT Smad3 should increase Papss2 expression and the dominant- negative forms should reduce expression and block up-regulation in the presence of TGF-B.
  • Smad and Sox9 can also cooperate in regulation of Papss2.
  • WT Sox9 and Smad3 as well as the mutants described above can be ectopically expressed in cells together with and without TGF-B and Papss2 mRNA levels can be measured.
  • Papss2 promoter sequence indicates that there is a strong Smad3 site and a strong Sox9 site in the Papss2 gene ( Figure 1).
  • These DNA elements will be PCR cloned into the promoterless luciferase vector, pGL2 Basic (Promega, Madison, WS).
  • the reporter construct can be introduced into ATDC5 cells using Fugene 6 (Roche), then cells can be left untreated or treated with 5ng/ml of pTGF-Bl for 8 hours. Luciferase assays can be performed using the dual- luciferase reporter system from Promega.
  • the promoterless pGL2 Basic can serve as a control for TGF-B effects on the plasmid backbone, if any. If transcriptional regulation is mediated through the Smad3 or Sox9 DNA regulatory elements in the promoter, more luciferase activity can be seen in TGF-B 1 treated cells relative to the untreated cells.
  • Confirmation can include mutation of the specific binding site and demonstration of loss of activity. It was previously shown that Smad3 can interact with Sox9 and p300 on the Sox9 DNA binding element to regulate chromatin structure and Col2al gene expression. In this case, it does not appear that Smad3 binds to the DNA directly. If TGF-B mediates Papss2 expression through the Sox9 site, experiments can include gel shift and super shift, and chromatin IP (Chip) assays to determine if Sox9 and Smad3 interact on the Papss2 regulatory elements.
  • TGF-B mediates Papss2 expression through the Sox9 site
  • experiments can include gel shift and super shift, and chromatin IP (Chip) assays to determine if Sox9 and Smad3 interact on the Papss2 regulatory elements.
  • mice that express a dominant negative mutation in the TGF-B type II receptor have been generated.
  • the mice developed a condition similar to human osteoarthritis characterized by degeneration of the cartilage.
  • Significant reduction in mechanical properties of DNIIR cartilage has been observed as demonstrated by micro-indentation.
  • Indentation tests were performed on a Bose electro force system. Using an impervious plane-ended cylindrical indenter 178 ⁇ in diameter, tests were comprised of a four-step stress-relaxation protocol, with each step of 5 ⁇ displacement separated by 200s relaxation. A 1 ⁇ tip needle was then pushed through the cartilage surface and thickness was measured from the load-displacement curve. Slopes of stress-strain plots during loading and relaxation steps were calculated as instantaneous and equilibrium stiffness respectively. In the DNIIR mutants, both instantaneous and equilibrium stiffness were reduced at 20 weeks as compared to control littermates.
  • a microarray screen was performed comparing untreated and TGF-B treated bovine cartilage.
  • PAPSS2 was identified in this screen and verified that Papss2 mRNA and protein levels were down regulated in articular cartilage from DNIIR mice.
  • Papss2 is required for sulfation of glycosoaminoglycans on cartilage matrix proteoglycans. Staining with Alcian blue and antibodies specific to unsulfated and sulfated chondroitin indicated the matrix in DNIIR cartilage was hyposulfated. Sulfation provides for many of the biomechanical properties found in cartilage because the sulfate groups attract water to the matrix. Alterations in water content or movement would affect stiffness of the cartilage matrix.
  • chondroprotective effects of TGF-B are mediated through its regulation of Papss2 and sulfation of the matrix.
  • a concentric cylinder bioreactor has been developed and validated for the production of tissue-engineered cartilage.
  • PAPSS2 can restore the biomechanical properties of cartilage with disrupted TGF- ⁇ signaling
  • bovine cartilage expressing genes of interest generated in the bioreactor can be used.
  • cartilage engineered to express DNIIR recapitulates the phenotype seen in mice and in bovine chondrocyte micromass cultures.
  • Bovine articular chondrocytes can be isolated and infected with adenovirus containing DNIIR (Ad-DNIIR) or a control GFP containing adenovirus (Ad-GFP).
  • Each bioreactor can be seeded with 80 million chondrocytes and 16 poly-L-lactic acid (PLLA) cylindrical scaffolds (Figure 8; 1cm in diameter, 2 mm thick, 85-95% porous with pore sizes ranging from 150-300 ⁇ ). Typically, 260 million cells can be obtained per isolation using four metacarpal phalangeal joints. This allows two types of samples (ie DNIIR and control) to be generated (each in a separate bioreactor) with 16 constructs generated for each condition.
  • PLLA poly-L-lactic acid
  • Cartilage constructs are generated in media with 10% FBS under low oxygen conditions (5% 02) as described in (Saini and Wick, 2004). Constructs can be grown for 28 to 35 days at which time they will be removed for analysis. RNA can be isolated from four separate constructs from each condition using the Trizol procedure. DNIIR expression can be confirmed using real time RT-PCR. Regulation of Papss2 in control and DNIIR cartilage can also be compared.
  • protein can be isolated from the cartilage constructs and western blot can be used to confirm DNIIR (via FLAG tag) and Papss2 (Sigma WH0009060M7) expression.
  • four constructs from each condition can be frozen, dried and protease digested to yield four separate lysates from each condition that can be used to determine DNA, GAG, and hydroxyproline content by standard methods.
  • the lysates can also be used for FACE analysis (flourophore assisted charbohydrate electrophoresis) to determine the relative levels of chondroitin sulfate versus non-sulfated chondroitins in DNIIR and control samples.
  • FACE analysis is to measure sulfate incorporation by labeling constructs for the last 24 hours with radioactive [S35]H2S04 following standard protocols.
  • Another set of 4 constructs can be used for histological analysis. Samples can be fixed and embedded in paraffin and cut in cross section. Sections can be stained with hematoxylin and eosin to observe general structure.
  • Sections can also be stained with Alcian blue as described above to observed relative levels of sulfated proteoglycans.
  • Immunostaining can be used to localize chondrotin 4-sulfate and unsulfated chondroitin using specific antibodies obtained from Seikagaku (clone 2-B-6 and 1-B- 5 respectively). The last set of four control and DNIIR constucts can be used for
  • the engineered constructs are larger and thicker than mouse cartilage.
  • Flow dependent behavior of DNIIR and control tissue engineered cartilage can also be measured under confined compression using a Bose ELectroForce ELF (ELF 3300, EnduraTEC, Minnetonka, MN) testing apparatus. Briefly, a 4-mm diameter sample can be cut from the tissue-engineered cartilage, placed in the ELF apparatus, covered with a porous platen. The sample is kept in a bath of PBE with protease inhibitor during the testing. After preloading at 0.01N, stress relaxation testing is initiated at strains of 5, 10, 15, 20% with the stress allowed to reach equilibrium for 15 minutes between strains.
  • the cartilage aggregate modulus is obtained from the slope of the by equilibrium stress versus strain curve.
  • the construct shear modulus can also be measured using a Bohlin CVO® rheometer.
  • Four mm diameter biopsy punches from the tissue-engineered cartilage are placed on the rheometer base plate with a small amount of PBE with protease inhibitors to maintain tissue hydration.
  • the sample is preloaded using an impermeable platen with the rheometer gap set to 90% of the sample thickness.
  • the oscillation test is performed with a frequency sweep in the range of 0.01-1.0 Hz with logarithmic increase in the frequency. A typical test requires 15-20 minutes.
  • This testing protocol causes very little change in the fluid volume of the construct to allow measurement of the flow independent viscoelastic behavior of cartilage.
  • This method has been used to show that low oxygen tension results in engineered cartilage with native material properties (Figure 9). If the engineered cartilage mimics what is seen in mouse articular cartilage alterations in Papss2 expression, hyposulfation in the matrix, and reduced stiffness as well as decreased shear modulus and lower aggregate modulus in the DNIIR samples relative to controls can be seen.
  • the next step can be to see if ectopic expression of Papss2 can prevent alterations observed in DNIIR cartilage.
  • the number of chondrocytes isolated can be scaled up and three bioreactors used for three conditions: control Ad-GFP infected, Ad-DNIIR- Ad-GFP double infected, and Ad-GFP-Ad-Papss2 double infected.
  • Analysis can proceed as described above: verification of DNIIR and Papss2 expression, DNA, GAG, FACE, and hydroxproline assays, histology and immunostaining, as well as microindentation assays.
  • Ectopic expression of Papss2 under the control of the constituative CMV promoter can prevent alterations in matrix sulfation and stiffness observed in DNIIR cartilage.
  • results obtained from the engineered tissue can be compared to that of mouse cartilage in vivo.
  • Transgenic mice that overexpress Papss2 under the control of the Col2a promoter can be generated. After the mice are made and characterized, they can be crossed to DNIIR mice to determine if expression of Papss2 can alleviate joint degeneration observed in these mice. Characterization can include gross, histological, biochemical, and biomechanical
  • the next step can be to determine the effects of Tgfb's chondroprotective signaling and down-stream targets (i.e. PAPSS2) on samples of human OA cartilage.
  • Restoring chondroprotective signals can have some reparative effects on OA cartilage.
  • Cells from normal and OA cartilage grown and maintained in the bioreactors described above can be used.
  • Normal and OA cartilage can be obtained from the National Disease Research Interchange.
  • Tissue can be obtained from a combination of cadaver and surgical discards (for example from joint replacement or amputation surgery). Chondrocytes can be isolated and seeded on PLLA scaffolds and analyzed as described above.
  • normal and OA cartilage can be compared for: 1- DNA, GAG, S04, and hydroxyproline content; 2- histology; 3- mechanical properties; and 4- gene and protein expression, specifically, the expression of Tgfb signaling molecules and known down-stream targets (including Papss2).
  • OA cartilage can have reduced GAG and S04 content and the mechanical properties of the OA cartilage can be altered relative to the control cartilage.
  • human OA chondrocytes can be isolated and infected with adenovirus containing an activated TGF-B Type I receptor (Ad-Tgf rlT202D) or a control GFP containing adenovirus (Ad-GFP).
  • Tgfbrl signaling through Smad2/3 is dominant in healthy cartilage and is thought to promote Tgfb's chondroprotective effects.
  • Cells can be seeded in the bioreactor and analyzed as described above.
  • expression of the activated receptor which contains a FLAG tag can be confirmed by western blot and activity of the transduced receptor can be determined by phospho Smad 3 expression. If Tgfbrl activity results in increased GAG and S04 content then in addition to being chondroprotective, signaling through Tgfbrl/ Smad3 can be reparative. Expression of the activated receptor can result in up- regulation of known targets of Tgfb including Papss2.
  • Adenovirus vectors can be used to transduce genes of interest into bovine articular chondrocytes.
  • the advantages of the adenovirus as a vector for this application include its high level of infectivity and high level of gene expression in chondrocytes.
  • Osteoarthritis and TGF- ⁇ are characterized by the erosion of articular cartilage.
  • Transforming growth factor- ⁇ (TGF- ⁇ ) is a peptide that regulates chondrocyte differentiation and gene expression, and previous work has shown that mice with a dominant- negative mutation of the TGF- ⁇ type II receptor (DNIIR) exhibit OA-like symptoms and a reduction in the expression of genes that may help maintain healthy cartilage ( Figure 10A and 10B).
  • Phosphoadenosine phosphosynthetase 2 (Papss2) is a downstream target of TGF- ⁇ and is required for proper sulfation of chondroitin glycosaminoglycans (GAGs) on cartilage matrix proteoglycans. Sulfation provides many of the biomechanical properties found in cartilage: sulfate groups attract water to cartilage matrix, and alterations in water content and movement affect the stiffness of cartilage matrix. In articular cartilage of DNIIR mice, Papss2 mRNA and protein levels are down-regulated, matrix is hyposulfated, and stiffness is reduced.
  • Proteoglycan 4 is a downstream target of TGF- ⁇ and encodes for lubricin, a protein secreted in joints that helps lubricate the articulating surfaces of cartilage.
  • Prg4 has been shown to have a protective effect on the cartilage of mice with age-related OA.
  • Prg4 mRNA levels are down- regulated.
  • a treatment involving increased expression of Papss2 can result in increased cartilage matrix sulfation and increased cartilage stiffness, possibly rescuing the OA phenotype.
  • Other downstream targets of TGF- ⁇ can also play roles in modulating biochemical and biomechanical properties of OA cartilage. The goal of this research is to determine whether Papss2 and other targets could be used to rescue the OA phenotype.
  • In vitro engineered OA cartilage can be used as a model to identify potential targets because the model is faster and less expensive than using mice and can still be biochemically and mechanically tested.
  • mRNA was harvested after 5 hours, 24 hours, 3 days, and 7 days of TGF- ⁇ treatment, and quantitative real-time RT-PCR (qPCR) was used to determine relative mRNA levels of Papss2, Prg4, type II collagen, and type X collagen ( Figure 1 1).
  • qPCR quantitative real-time RT-PCR
  • Bovine chondrocytes were suspended in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum, L-ascorbic acid, nonessential amino acids, L-proline, penicillin/streptomycin, HEPES buffer, and fungizone. The cells were then dynamically seeded onto 16 poly-L-lactic acid (PLLA) scaffolds in a bioreactor ( Figure 13 A and B) and cultivated in the bioreactor for up to 20 days. Constructs were observed at 4-, 12-, and 20-day time points ( Figure 13 C-H).
  • PLLA poly-L-lactic acid
  • TGF- ⁇ maintains cartilage-specific gene expression over time, and DNIIR adenovirus can reduce Papss2 and Prg4 gene expression in chondrocytes in vitro as in the DNIIR mice. Also, a bioreactor can be used to engineer in vitro 3D cartilaginous tissue.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Toxicology (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pathology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés de criblage d'agents thérapeutiques potentiels de l'ostéoarthrite. Les procédés de criblage peuvent comprendre la croissance d'un chondrocyte modifié comprenant un gène d'une voie de signalisation TGFβ modifié en présence d'un composé supposé être un agent thérapeutique de l'ostéoarthrite ; la croissance dudit chondrocyte modifié en l'absence du composé ; la détermination d'une caractéristique ostéoarthritique dans le chondrocyte modifié en présence du composé et en l'absence du composé ; et la comparaison de la caractéristique ostéoarthritique des chondrocytes modifiés, la sauvegarde de la caractéristique ostéoarthritique en présence du composé étant indicatrice d'un agent thérapeutique de l'ostéoarthrite.
PCT/US2015/043081 2014-08-01 2015-07-31 Modèles de cartilage de génie tissulaire in vitro pour des essais de médicament pour l'ostéoarthrite Ceased WO2016019225A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462032064P 2014-08-01 2014-08-01
US62/032,064 2014-08-01

Publications (1)

Publication Number Publication Date
WO2016019225A1 true WO2016019225A1 (fr) 2016-02-04

Family

ID=55218343

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/043081 Ceased WO2016019225A1 (fr) 2014-08-01 2015-07-31 Modèles de cartilage de génie tissulaire in vitro pour des essais de médicament pour l'ostéoarthrite

Country Status (1)

Country Link
WO (1) WO2016019225A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4230652A4 (fr) * 2019-06-04 2025-05-14 Ewha University-Industry Collaboration Foundation Anticorps anti-oscar pour la prévention ou le traitement de l'arthrose

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011123394A1 (fr) * 2010-03-31 2011-10-06 Isis Pharmaceuticals, Inc. Modulation de l'expression de la métalloprotéinase de matrice 13 (mmp-13)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011123394A1 (fr) * 2010-03-31 2011-10-06 Isis Pharmaceuticals, Inc. Modulation de l'expression de la métalloprotéinase de matrice 13 (mmp-13)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL.: "Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-beta activities.", SCIENCE, vol. 260, no. 5112, 28 May 1993 (1993-05-28), pages 1335 - 1338 *
PIECHA ET AL.: "Novel selective MMP-13 inhibitors reduce collagen degradation in bovine articular and human osteoarthritis cartilage explants.", INFLAMM RES, vol. 59, no. 5, May 2010 (2010-05-01), pages 379 - 389, XP019792877 *
SHEN ET AL.: "Deletion of the transforming growth factor beta receptor type II gene in articular chondrocytes leads to a progressive osteoarthritis-like phenotype in mice.", ARTHRITIS RHEUM, vol. 65, no. 12, December 2013 (2013-12-01), pages 3107 - 3119 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4230652A4 (fr) * 2019-06-04 2025-05-14 Ewha University-Industry Collaboration Foundation Anticorps anti-oscar pour la prévention ou le traitement de l'arthrose

Similar Documents

Publication Publication Date Title
Healy et al. Regulation and role of Sox9 in cartilage formation
Zou et al. In vitro study of enhanced osteogenesis induced by HIF‐1α‐transduced bone marrow stem cells
Stricker et al. Odd-skipped related genes regulate differentiation of embryonic limb mesenchyme and bone marrow mesenchymal stromal cells
Lu et al. Long noncoding RNA H19 accelerates tenogenic differentiation and promotes tendon healing through targeting miR‐29b‐3p and activating TGF‐β1 signaling
Makki et al. A short receptor downregulates JAK/STAT signalling to control the Drosophila cellular immune response
Keyes et al. ΔNp63α is an oncogene that targets chromatin remodeler Lsh to drive skin stem cell proliferation and tumorigenesis
Yu et al. Wnt7b-induced Sox11 functions enhance self-renewal and osteogenic commitment of bone marrow mesenchymal stem cells
Haupt et al. ACVR1R206H FOP mutation alters mechanosensing and tissue stiffness during heterotopic ossification
Filomena et al. Myopalladin promotes muscle growth through modulation of the serum response factor pathway
Zhuang et al. Barx2 and Pax7 have antagonistic functions in regulation of wnt signaling and satellite cell differentiation
Roberto et al. MiR-29a is an enhancer of mineral deposition in bone-derived systems
Baruffaldi et al. Dynamic phosphorylation of the myocyte enhancer factor 2Cα1 splice variant promotes skeletal muscle regeneration and hypertrophy
Meyer et al. Epimuscular fat in the human rotator cuff is a novel beige depot
Scibetta et al. Characterization of the chondrogenic and osteogenic potential of male and female human muscle‐derived stem cells: Implication for stem cell therapy
Chai et al. Complete functional segregation of planarian β-catenin-1 and-2 in mediating Wnt signaling and cell adhesion
Ratnayake et al. Human chondrocytes respond discordantly to the protein encoded by the osteoarthritis susceptibility gene GDF5
Li et al. Fibronectin type III domain containing four promotes differentiation of C2C12 through the Wnt/β‐catenin signaling pathway
Li et al. Joining actions: crosstalk between intermediate filaments and actin orchestrates cellular physical dynamics and signaling
Huang et al. RhoGDIβ inhibits bone morphogenetic protein 4 (BMP4)-induced adipocyte lineage commitment and favors smooth muscle-like cell differentiation
Zhang et al. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle
WO2016019225A1 (fr) Modèles de cartilage de génie tissulaire in vitro pour des essais de médicament pour l'ostéoarthrite
Mulvaney et al. Molecular cloning and functional characterisation of chicken Atonal homologue 1: a comparison with human Atoh1
Abe et al. Hand2 regulates chondrogenesis in vitro and in vivo
US20170016003A1 (en) Composition for promoting chondrocyte differentiation or treating cartilage diseases, containing klf10 expression inhibitor, and method for promoting cartilage differentiation by using same
Wang et al. Whitening of brown adipose tissue inhibits osteogenic differentiation via secretion of S100A8/A9

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15828090

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15828090

Country of ref document: EP

Kind code of ref document: A1