WO2015102351A1 - Composition favorisant une différenciation de chondrocytes ou un traitement de maladies du cartilage, contenant un inhibiteur d'expression du gène klf10, et méthode favorisant une différenciation de cartilage au moyen de cette dernière - Google Patents

Composition favorisant une différenciation de chondrocytes ou un traitement de maladies du cartilage, contenant un inhibiteur d'expression du gène klf10, et méthode favorisant une différenciation de cartilage au moyen de cette dernière Download PDF

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WO2015102351A1
WO2015102351A1 PCT/KR2014/013006 KR2014013006W WO2015102351A1 WO 2015102351 A1 WO2015102351 A1 WO 2015102351A1 KR 2014013006 W KR2014013006 W KR 2014013006W WO 2015102351 A1 WO2015102351 A1 WO 2015102351A1
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klf10
composition
cartilage
differentiation
expression
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PCT/KR2014/013006
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Korean (ko)
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임군일
이종민
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동국대학교 산학협력단
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Priority claimed from KR1020140191592A external-priority patent/KR101642202B1/ko
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Priority to US15/108,525 priority Critical patent/US9963744B2/en
Publication of WO2015102351A1 publication Critical patent/WO2015102351A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • 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
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

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  • the present invention relates to a KLF10 gene expression inhibitor that promotes chondrocyte differentiation, and more particularly, to promote chondrocyte differentiation, and to promote chondrocyte differentiation including a KLF10 gene expression inhibitor that inhibits the thickening and dedifferentiation of chondrocytes. Or it relates to a composition for treating cartilage disease.
  • Existing treatments for injured joints include drug treatment, autologous cartilage transplantation, bone marrow perforation, and arthroplasty.
  • conservative treatment such as drug treatment is limited to restoring limited function by relieving symptoms and used for traumatic defect of articular cartilage.
  • Bone cartilage transplantation causes damage to the donor site due to bone-cartilage harvesting, and the amount of harvesting is limited.
  • bone marrow perforation for moderately advanced osteoarthritis has the disadvantage of poor clinical results by regenerating fibrocartilage instead of the supercartilage, which is the original cartilage tissue.
  • artificial joint replacement is a standard treatment for young patients. In the case of the procedure, the life of the artificial joint is also a problem.
  • stem cells which have self-replicating ability, can be differentiated into various tissues and can be easily collected without a large amount of donor dysfunction, are recognized as an ideal cell source for cell therapy.
  • research is being actively conducted, there is still a lack of clear knowledge about factors, environment, etc. for cartilage formation.
  • Stem cells for articular cartilage regeneration are characterized by their self-proliferative capacity and differentiation ability to differentiate into cells constituting specific tissues, and have recently been proposed as a new cell source for application to articular cartilage treatment. Therefore, theoretically, it is possible to solve the limitations of the conventional cell therapy using chondrocytes and to apply the general degeneration and damage of articular cartilage.
  • adult mesenchymal stem cells and mesenchymal progenitor cells have the advantages of ethical problems and no rejection in vivo upon allogeneic transplantation.
  • chondrocytes The thickening of chondrocytes can be seen at the stage of cartilage cell death and pre-osteolysis in the process of cartilage and cartilage in the growth plate. It is a necessary process in the maturation of other chondrocytes except articular chondrocytes.
  • cartilage tissue engineering aims to regenerate articular cartilage, regenerated cartilage should have the appearance of faux bones seen in normal joints, and cell thickening should not occur.
  • Parathyroid hormone-related protein is a peptide that is involved in the proliferation and maturation of chondrocytes in the growth plate. It is secreted from the periarticular cartilage and spreads to act on the prehypertrophic chondrocytes of the growth plate to inhibit the maturation of chondrocytes. Inhibiting the production of stimulating indian hedgehog protein maintains the chondrocyte phenotype and suppresses thickening.
  • the present inventors first identified that the expression or activity of Indian hedgehog protein (IHH) can be regulated by regulating KLF10 expression or activity, thereby greatly improving the cartilage differentiation ability of bone marrow stem cells and completing the present invention.
  • IHH Indian hedgehog protein
  • miR-892b a microRNA
  • PTHrP a microRNA
  • miR-892b sequentially regulates the expression of Indian hedgehog protein by inhibiting the expression of KLF10 gene, a higher regulatory protein of Indian hedgehog protein. It was found possible.
  • the present invention provides a composition for promoting chondrocyte differentiation comprising a KLF10 (Krueppel-like factor 10) gene expression inhibitor.
  • the present invention provides a composition for treating cartilage disease, comprising KLF10 (Krueppel-like factor 10) gene expression inhibitor.
  • the KLF10 gene may be composed of a base sequence encoding the amino acid sequence described in SEQ ID NO: 1.
  • the expression inhibitor may be selected from the group consisting of micro RNA (miRNA), small interfering RNA (siRNA) and short hairpin RNA (shRNA) that complementarily bind to mRNA of KLF10 gene. .
  • miRNA micro RNA
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • the microRNA may be miR-892b gene consisting of the nucleotide sequence of SEQ ID NO: 4.
  • the short hairpin RNA may be composed of the nucleotide sequence of SEQ ID NO: 2 or 3.
  • the RNA may be inserted into an expression vector.
  • the composition may be to inhibit the thickening and dedifferentiation of chondrocytes.
  • the cartilage disease is from a group consisting of degenerative arthritis, rheumatoid arthritis, fractures, muscle tissue damage, plantar fasciitis, humerus periarthritis, calcification myositis, nonunion of the fracture and joint damage by trauma. May be the disease of choice.
  • the present invention also provides a chondrocyte therapeutic agent comprising the composition.
  • the present invention also provides a method for promoting chondrocyte differentiation in bone marrow stem cells, comprising expressing the composition in bone marrow stem cells.
  • the present invention also provides a method for screening a chondrocyte differentiation promoter or a chondrocyte therapy comprising the following steps:
  • the expression level of step (b) is immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemical analysis (immunohistochemical analysis), RealTime- It may be measured by any one method selected from the group consisting of PCR, qRT-PCR, Western blotting, and flow cytometry (FACS).
  • RIA radioimmunoassay
  • ELISA enzyme immunoassay
  • immunohistochemical analysis immunohistochemical analysis
  • RealTime- It may be measured by any one method selected from the group consisting of PCR, qRT-PCR, Western blotting, and flow cytometry (FACS).
  • the present invention also provides a method of treating cartilage disease comprising administering the composition to a subject.
  • the present invention provides a method for promoting chondrocyte differentiation comprising administering the composition to a subject.
  • the administration may be oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, arterial injection or subcutaneous injection.
  • the subject may be a mammal, including a human.
  • the present invention provides a use for promoting chondrocyte differentiation or a treatment for cartilage disease.
  • KLF10 expression inhibitor of the present invention is expressed in bone marrow stem cells to induce chondrocyte differentiation has an excellent effect to promote chondrocyte differentiation and inhibit cartilage thickening.
  • Figure 1 shows the results of measuring the expression changes of miRNA according to the presence or absence of PTHrP addition in the cartilage differentiation of human bone marrow stem cells by miRNA microarray method.
  • Figure 2 shows a schematic diagram of the production of recombinant lentiviral to express miR-892b in human bone marrow stem cells.
  • Figure 3 shows the results of analyzing the effects of cartilage differentiation promotion and thickening inhibition by miR-892b expression when inducing cartilage differentiation of miR-892b lentiviral infected human bone marrow stem cells.
  • Figure 4 shows the results of analyzing the improvement of cartilage differentiation efficiency and thickening inhibitory mechanism (mechanism) when miR-892b overexpression.
  • Figure 5 shows the results of the vector cloning to identify the target genes (KLF10 and WNT6) of miR892b and the expression inhibition of the target gene expression by miR892b.
  • Figure 6 shows the results of analysis of IHH expression changes by induction of overexpression of KLF10 in bone marrow stem cells.
  • Figure 7 shows the results of analyzing the effect of cartilage differentiation during KLF10 knock-down using shRNA.
  • Figure 8 shows the results of analyzing the effects of cartilage differentiation in KLF10 knock-out mice.
  • Figure 9 shows the results of the cartilage regeneration effect by transplanting miR892b-expressing bone marrow stem cells into a rat cartilage defect model.
  • the present invention provides a composition for promoting chondrocyte differentiation, comprising a KLF10 (Krueppel-like factor 10) gene expression inhibitor, and a composition for treating cartilage diseases comprising a KLF10 (Krueppel-like factor 10) gene expression inhibitor.
  • a composition for promoting chondrocyte differentiation comprising a KLF10 (Krueppel-like factor 10) gene expression inhibitor
  • a composition for treating cartilage diseases comprising a KLF10 (Krueppel-like factor 10) gene expression inhibitor.
  • the present inventors have identified for the first time that the KLF10 gene expression inhibitor exhibits the activity of inhibiting the thickening and dedifferentiation of chondrocytes by reducing the expression of indian hedgehog, a hedgehog signal initiation material of the Indian hedgehog (IHH) signaling system.
  • IHH Indian hedgehog
  • the KLF10 gene is a human (Homo sapiens) KLF10 gene, preferably consisting of a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 1, and most preferably composed of the nucleotide sequence described in SEQ ID NO: 39 But it is not limited thereto.
  • the expression inhibitor may be any material that can inhibit KLF10 expression or activity, but is not limited thereto, and may include micro RNA (miRNA), small interfering RNA (siRNA), or short complementary to the mRNA of the KLF10 gene.
  • miRNA micro RNA
  • siRNA small interfering RNA
  • variants of the nucleotide sequences are also included within the scope of the present invention, specifically, 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95 It may include a base sequence having at least% sequence homology.
  • the "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
  • the RNA as the expression inhibitor may be inserted into a recombinant expression vector.
  • recombinant expression vector means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any vector can be used if it can replicate and stabilize in the host.
  • An important feature of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.
  • the expression vector may be a gene construct comprising essential regulatory elements operably linked to express the gene insert, capable of expressing the protein of interest in a suitable host cell.
  • the term "recombinant” also refers to a cell in which the cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid.
  • Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form.
  • Recombinant cells can also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.
  • Expression vectors comprising such RNA sequences and appropriate transcriptional / translational control signals can be constructed by methods well known to those of skill in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to a suitable promoter in the expression vector. Expression vectors may also include ribosomal binding sites and transcription terminators as translation initiation sites.
  • Expression vectors of the present invention may include a plasmid vector, a cosmid vector, an episomal vector, a viral vector, and the like, and preferably, may be a viral vector.
  • the viral vector may be a vector derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, etc., but is not limited thereto, preferably a lentiviral vector may be used, Most preferably, plasmid vectors such as pCDH, pECFP, or pLKO can be used.
  • cartilage disease refers to a disease caused by cartilage, cartilage tissue and / or joint tissues (stem, articular, subchondral bone, etc.) are injured by mechanical stimulation or an inflammatory response, and includes cartilage damage diseases.
  • cartilage diseases include, but are not limited to, degenerative arthritis, rheumatoid arthritis, fractures, muscle tissue damage, plantar fasciitis, humerus external surgery, calcifying myositis, fracture nonunion or trauma.
  • the present invention can provide a cell therapeutic agent comprising the composition.
  • Cell therapies are cells and tissues prepared by isolation, culture and special chewing from humans and are used for the purpose of treatment, diagnosis and prevention, and are intended to restore living autologous, allogeneic, or heterologous cells to restore the function of cells or tissues. It refers to a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferation selection in vitro or altering the biological characteristics of the cell in other ways.
  • the cell therapeutic agent may be directly injected into the joint of the patient according to a known method, or may be transplanted with a scaffold after three-dimensional culture, and the disease to be treated, the severity of the disease, the route of administration, the weight and age of the patient. And the number of cells to be administered in consideration of various related factors such as sex.
  • composition or cell therapy of the present invention may be inoculated on a support for cartilage formation and applied to cartilage damage.
  • a support for cartilage formation and applied to cartilage damage should be biocompatible, bioabsorbable or remodeled, and provide a framework that facilitates new tissue growth. And exhibit material and mechanical properties compatible with articular cartilage function. Scaffolds that provide a three-dimensional (3D) culture environment affect the ultimate quality of tissue engineered cartilage tissue as well as the proliferation and differentiation of inoculated cells.
  • 3D three-dimensional
  • various materials derived from synthetic or natural materials are used as appropriate supports. These supports are used in various forms, such as sponges, gels, fibers and microbeads, the most commonly used of which can improve cell adhesion rate and maintain a high rate of surface tension with respect to volume. Structure.
  • composition or cell therapeutic agent of the present invention is a cartilage damage part of a non-human mammal such as a human or a non-human organism, such as a cow, monkey, bird, cat, mouse, rat, hamster, pig, dog, rabbit, sheep, horse, etc. It can be used to promote the regeneration (differentiation) of cartilage, or to administer cartilage disease by infusion administration in the joint.
  • the present invention can provide a method for promoting cartilage differentiation or treating cartilage diseases in bone marrow stem cells, comprising the step of expressing the composition in bone marrow stem cells.
  • the present invention can provide a method for screening a chondrocyte differentiation promoter or a chondrocyte therapeutic agent comprising the following steps.
  • (c) preferably, but not limited to, selecting a candidate substance whose expression level of KLF10 is reduced compared to a control group not treated with the candidate substance.
  • the expression level of step (b) is preferably immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemical analysis, RealTime-PCR, qRT-PCR. , Western Blotting, or flow cytometry (FACS), etc., but can be measured using any method of measuring the amount of transcript or protein encoded therefrom, without being limited thereto. have.
  • RIA radioimmunoassay
  • ELISA enzyme immunoassay
  • FACS flow cytometry
  • TGF- ⁇ 3 treatment was performed to induce cartilage differentiation of human bone marrow stem cells for a total of 4 weeks, and further divided into groups treated with PTHrP and untreated PTHrP for the last 2 or 1 weeks, and RNA from cartilage differentiation induced cell pellets.
  • RNA from cartilage differentiation induced cell pellets was isolated from the synthesized cDNA with Cy3 fluorescent dye for the entire miRNA.
  • hybridization was performed on Agilent human miRNA microarray chips coated with DNA sequences complementary to approximately 15,000 human miRNAs, and miRNAs having 1.5-fold or more expression increase only by PTHrP treatment were isolated by Cy3 fluorescence image analysis.
  • RNA sequences of the four miRNAs with increased expression are shown in Table 1 above. Same as In addition, as a result of analyzing the time-dependent expression patterns of the four miRNAs, as shown in FIG. 1B, unlike other miRNAs with a gradually increased pattern, miR-892b showed a temporary increase pattern only for one week after treatment. appear.
  • a 340 bp DNA fragment containing a pre-mature miR-892b and a pCDH lentiviral vector obtained by genomic PCR from human bone marrow stem cell DNA were respectively identified.
  • the pCDH-CMV-miR-892b-EF1-copGFP recombinant vector was prepared as shown in FIG. 2.
  • the lentiviral packaging system containing 3 recombinant vectors (pLP1, pLP2, pVSVG) for the production of the recombinant vector and the lentiviral in a ratio of 1: 1.5: 1 was simultaneously transformed in a ratio of 1: 3 in 293FT cells. After about 48 hours, the culture solution was recovered, and the concentration of virus produced (Titer) was measured and used for infection experiments of human bone marrow stem cells.
  • cartilage differentiation was induced in human bone marrow stem cells infected with miR-892b virus for 4 weeks.
  • miR-892b virus was infected with TGF- ⁇ 3.
  • the ratio of GAG / DNA was similar to that of the control group (Lenti-control: CM-T-Pt) infected with lentiviral and co-treated with PTHrP and TGF- ⁇ 3. Increased a lot.
  • type II collagen (COLII) was increased and the expression levels of cartilage thickening markers type X collagen (COLX) and alkaline phosphatase (ALP) were decreased, indicating that the superior cartilage differentiation efficiency was due to the expression level change. Can be.
  • miR-892b expressing bone marrow stem cells when only TGF- ⁇ 3 is treated without PTHrP treatment when inducing chondrogenic differentiation of mesenchymal stem cells infected with recombinant lentivirus (miR-892b virus) according to the present invention
  • the residual amount of miR-892b was confirmed.
  • cDNA for miRNA was synthesized from RNA isolated from each cartilage pellet tissue, and this was carried out using a forward primer (SEQ ID NO: 9) for miR-892b of Table 2 and qPCR Unversal Reverse primer provided by GenoExplorer. Rq-PCR was performed by repeated amplification for 5 seconds at 60 ° C.
  • 5s rRNA was also used as an internal control to convert the amount of miR-892b per group, and Rq-PCR was simultaneously performed using a forward primer (SEQ ID NO: 10) and qPCR Unversal Reverse primer for 5s rRNA of Table 2 below. .
  • the PTHrP-IHH negative feedback inhibition system according to miR-892b expression as can be seen from above is shown in FIG. 4D.
  • FIG. 5A The binding site of miR-892b in 3'UTR of KLF10 or WNT6 gene predicted as the target gene of miR-892b from the result of Example 2 is shown in FIG. 5A, and in 3'UTR of KLF10 or WNT6 gene.
  • pMIR-luciferase reporter vectors were cloned by synthesizing gene sequences with modified base sequences within the binding sites of miR-892b and their major sites (see FIG. 5B; WT, wild type; MUT, mutated).
  • FIG. 5B The binding site of miR-892b in 3'UTR of KLF10 or WNT6 gene predicted as the target gene of miR-892b from the result of Example 2 is shown in FIG. 5A, and in 3'UTR of KLF10 or WNT6 gene.
  • pMIR-luciferase reporter vectors were cloned by synthesizing gene sequences with modified base sequences within the binding sites of miR-892b and their major sites
  • KLF10 and WNT6 genes are major target genes whose expression can be regulated by miR-892b.
  • IHH Indian hedgehog
  • Human KLF10 cDNA synthesized by reverse transcription from RNA of human bone marrow stem cells was cloned into pECFP (Clontech), an overexpression vector in animal cells, to prepare a KLF10 overexpression vector (pECFP-C1-hKLF10), and a schematic diagram is shown in FIG. 6A.
  • pECFP-C1-hKLF10 an overexpression vector in animal cells
  • FIG. 6A a KLF10 overexpression vector
  • the intracellular distribution of the expressed KLF10 was measured by a fluorescence microscope. It was confirmed that the GFP fused hKLF10 was observed only in the nucleus of the cell after the expression of the transcription factor to be shifted to.
  • hMSC-pECFP transformed bone marrow stem cells
  • hMSC-pECFP-hKLF10 bone marrow stem cells transformed with KLF10 overexpression vector
  • FIG. 6C the expression of Ptch1, Runx2, and Alpl genes remained constant without change during the induction of KLF10, whereas the expression of Indian hedgehog (IHH) was expressed by Rq-PCR. Expression was confirmed to increase about 12-fold.
  • the protein was extracted from the cells according to a predetermined time, and the change in IHH expression according to KLF10 overexpression was confirmed by Western blot. As shown in FIG. 6D, the amount of KLF10 protein with time It was confirmed that the increase can directly induce an increase in the expression of the IHH protein.
  • KLF10 may induce the expression of IHH regardless of PTHrP treatment.
  • KLF10 shRNA capable of inhibiting expression in stem cells was compared and the miR-892b was overexpressed.
  • two KLF10 shRNA overexpression vectors were inserted by inserting KLF10 shRNA sequences (shKLF10-C1 and shKLF10-C2) shown in FIG. 7F into shRNA expressing lentiviral vectors purchased from Addgene, which is schematically shown in FIG. 7A. Prepared.
  • the cartilage differentiation efficiency of shKLF10-C2 lentiviral infected stem cells was compared with that of the negative control group after inducing cartilage differentiation in the presence of TGF- ⁇ 3 for 4 weeks using stem cells infected with shKLF10-C2 lentiviral. .
  • the group overexpressing shKLF10 compared to the negative control group (Lenti-control).
  • cartilage differentiation was induced in the presence of TGF- ⁇ 3.
  • MMSCs wt -CMT or KLF10 K / O bone marrow stem cell group
  • mMSCs KLF10- /-- CMT was confirmed that the highest Safranin-O staining.
  • KLF10 and KLF10 using wild-type or KLF10 K / O cartilage differentiation-inducing pellets induced with cartilage differentiation in the presence of TGF- ⁇ 3
  • cartilage differentiation in the positive control group KLF10 K / O bone marrow stem cell group in which cartilage differentiation was induced in the presence of TGF- ⁇ 3 was negative. It was confirmed that the expression level of IHH is reduced compared to the wild-type bone marrow stem cell group.
  • KLF10 a transcription factor previously unknown when inducing chondrocyte differentiation of adult stem cells, directly regulates IHH expression upon chondrogenic differentiation, thus acting as an initiating protein for inducing chondrocyte thickening.
  • IHH is known as a protein that promotes bone differentiation of mesenchymal stem cells. Therefore, in order to examine the effects of KLF10 gene knock-out on bone differentiation of mesenchymal stem cells, the differentiation medium was replaced every two to three days. Induction of bone differentiation in daily Osteogenic medium (OM) was performed, followed by Alkaline phosphatase (ALP) and Alizarin Red S staining. As a result, as shown in Figure 8g, knock-out of the KLF10 gene was confirmed that about two-fold ALP activity is reduced and about 4.5-fold mineralization is reduced. Therefore, it was confirmed that the expression of KLF10 plays an important role in bone differentiation while affecting the expression of IHH.
  • OM Osteogenic medium
  • ALP Alkaline phosphatase
  • the present inventors examined the cartilage regeneration effect of human miR-892b expressing bone marrow stem cells in a rat cartilage defect model.
  • the bone marrow stem cells are infected with lentivirus (Lenti-control) and miR-892b lentivirus (Lenti-miR-892b), respectively, before transplantation into the rat cartilage defect model, and confirm the in vivo distribution of the transplanted cells.
  • CellVue (Sigma), a far-red fluorescent dye emitting Cy5.5 fluorescence, was secondary to cell staining (CellVue labeling) and analyzed with an IVIS Luminar II fluorescence spectrometer (see FIG. 9A).
  • a 2 ⁇ 2 mm defect in the femur trochlear groove of athymic nude rat (NIH Nude; NTac: NIH-Whn; Taconic) 5 x 10 5 lentiviral or miR-892b lentiviral cells suspended in heparin-conjugated fibrin (HCF) were transplanted thereto, the gels were hardened and then sutured to observe regeneration of each cartilage after 6 weeks. .
  • HCF heparin-conjugated fibrin
  • the experimental group was divided into a total of 5 groups using 5 animals in each group for the experiment: group 1, a negative control without any treatment; Group 2, controls treated with TGF- ⁇ 3 only; Group 3, control lentiviral infected cell treatment; Group 4, control lentiviral infected cells + TGF- ⁇ 3 treatment; Group 5, miR-892b lentiviral infected cells + TGF- ⁇ 3 treatment.
  • group 1 a negative control without any treatment
  • Group 2 controls treated with TGF- ⁇ 3 only
  • Group 3 control lentiviral infected cell treatment
  • Group 4 control lentiviral infected cells + TGF- ⁇ 3 treatment
  • Group 5 miR-892b lentiviral infected cells + TGF- ⁇ 3 treatment were sacrificed and the thigh bones of each experimental group were separated and analyzed by Cy5.5 fluorescence image with an IVIS Luminar II fluorescence spectrometer. As shown in FIG. 9B, their relative quantitative values were compared.
  • the cartilage tissue analysis was performed by demineralizing each cartilage tissue in 14% EDTA solution and then lyophilizing it to a thickness of 10 ⁇ m to perform Safranin-O staining and quantifying it by Wakitani histological scoring analysis.
  • Wakitani histological scoring analysis unlike the ICRS Macroscopic Scoring method, the low score indicates a high degree of cartilage regeneration, as shown in Figures 9d and 9e, it was confirmed that very good cartilage regeneration is achieved in Group 4 and Group 5.
  • the present invention has the advantage that can be used as a chondrocyte therapy for bone marrow stem cells expressing a composition comprising a KLF10 gene expression inhibitor.

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Abstract

La présente invention concerne un inhibiteur d'expression du gène de facteur 10 de type Krueppel (KLF10) favorisant une différenciation de cartilages et concerne plus particulièrement : une composition favorisant une différenciation de chondrocytes ou un traitement de maladies du cartilage, contenant un inhibiteur d'expression du gène KLF10 favorisant une différenciation de cartilages et inhibant l'hypertrophie et la dédifférenciation de chondrocytes ; un agent thérapeutique de cellules contenant la composition ; une méthode favorisant une différenciation de cartilages dans les cellules souches de la moelle osseuse, comprenant une étape consistant à exprimer la composition dans des cellules souches de moelle osseuse ; et une méthode de criblage d'un activateur de différenciation de chondrocytes ou d'un agent thérapeutique de chondrocytes. Selon la présente invention, on a d'abord examiné la génération du mécanisme d'inhibition d'une protéine de hérisson indien (IHH) dont le mécanisme biologique moléculaire n'a pas encore été clairement examiné, et évalué qu'une différenciation de chondrocytes est favorisée et qu'une hypertrophie de chondrocytes est inhibée lorsque la différenciation de chondrocytes est induite par l'expression de l'inhibiteur d'expression du gène KLF10 dans des cellules souches de moelle osseuse. La présente invention a par conséquent un avantage qui est de permettre l'utilisation de cellules souches de moelle osseuse, qui expriment un inhibiteur d'expression du gène KLF10, en tant qu'agent thérapeutique de chondrocytes.
PCT/KR2014/013006 2013-12-31 2014-12-30 Composition favorisant une différenciation de chondrocytes ou un traitement de maladies du cartilage, contenant un inhibiteur d'expression du gène klf10, et méthode favorisant une différenciation de cartilage au moyen de cette dernière WO2015102351A1 (fr)

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US15/108,525 US9963744B2 (en) 2013-12-31 2014-12-30 Composition for promoting chondrocyte differentiation or treating cartilage diseases, containing KLF10 expression inhibitor, and method for promoting cartilage differentiation by using same

Applications Claiming Priority (4)

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KR10-2013-0168736 2013-12-31
KR20130168736 2013-12-31
KR10-2014-0191592 2014-12-29
KR1020140191592A KR101642202B1 (ko) 2013-12-31 2014-12-29 Klf10 발현 억제제를 포함하는 연골세포 분화 촉진 또는 연골질환 치료용 조성물, 및 이를 이용한 연골분화 촉진 방법

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090069013A (ko) * 2007-12-24 2009-06-29 동국대학교 산학협력단 골수기원 중간엽 줄기세포를 연골세포로 분화시키는 방법

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090069013A (ko) * 2007-12-24 2009-06-29 동국대학교 산학협력단 골수기원 중간엽 줄기세포를 연골세포로 분화시키는 방법

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B. HOPWOOD ET AL.: "Gene expression profile of the bone microenvironment in human fragility fracture bone", BONE, vol. 44, 10 September 2009 (2009-09-10), pages 87 - 101 *
GUNIL IM ET AL.: "Overexpression of PTHrP-related miRNA in Human Bone Marrow Derived Stem Cells Emhances Chondrogenesis and Inhibits Hypertrophy", ASBMR ( AMERICAN SOCIETY FOR BONE AND MINERAL RESEARCH) ANNUAL MEETING, 2012 *
YING-JIE GUAN ET AL.: "MiR-365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4", THE FASEB JOURNAL, vol. 25, December 2011 (2011-12-01), pages 4457 - 4466 *

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