WO2020009320A1 - Composition comprenant oct4 pour induire une transdifférenciation directe dans une cellule osseuse - Google Patents

Composition comprenant oct4 pour induire une transdifférenciation directe dans une cellule osseuse Download PDF

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WO2020009320A1
WO2020009320A1 PCT/KR2019/005103 KR2019005103W WO2020009320A1 WO 2020009320 A1 WO2020009320 A1 WO 2020009320A1 KR 2019005103 W KR2019005103 W KR 2019005103W WO 2020009320 A1 WO2020009320 A1 WO 2020009320A1
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bone
oct4
protein
cells
composition
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황석연
김승현
이화진
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SNU R&DB Foundation
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/105—Insulin-like growth factors [IGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/28—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from vascular endothelial cells
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a composition for inducing direct cross-differentiation of bone-related cells including OCT4, or a method for direct cross-differentiation using the same.
  • Induced pluripotent stem cells are artificially produced by artificially expressing and inducing specific genes from adult somatic cells which are non-pluripotent cells.
  • Induced pluripotent stem cells have been cited mainly in stem cell research because somatic cells can be used to obtain pluripotent stem cells without the need for embryos.
  • media such as viruses to modify adult somatic cell traits increases the likelihood of teratoma production, making it difficult to use in vivo tests.
  • To generate induced pluripotent stem cells reprogramming and redifferentiation steps are required. Must go through.
  • One of the aims of the present invention is to provide a means for promoting OCT4 expression in somatic cells.
  • One of the objects of the present invention is to provide a direct cross-differentiation method from somatic cells to bone-related cells.
  • One of the aims of the present invention is to provide bone related cells obtained through direct cross-differentiation and the use thereof.
  • One of the objects of the present invention is to provide a use for the prevention or treatment of bone-related diseases of OCT4 protein through direct cross-differentiation.
  • the present invention provides a means for promoting OCT4 expression in somatic cells, a method for direct cross-differentiation from somatic cells to bone-related cells, bone-related cells obtained through direct cross-differentiation and their use; And the prevention or treatment of bone related diseases of OCT4 protein through direct cross differentiation.
  • One embodiment of the invention is an OCTamer-binding transcription factor 4 (OCT4) expression promoting factor in somatic cells.
  • OCT4 OCTamer-binding transcription factor 4
  • the OCT4 expression promoting factor of the present invention means any physical, chemical, or biological means capable of elevating the expression of OCT4 protein in somatic cells.
  • the OCT4 expression promoting factor includes, but is not limited to, an OCT4 protein, a fusion or carrier thereof, a nucleic acid molecule encoding the protein, and a vector into which the nucleic acid molecule is introduced.
  • OCT4 octamer-binding transcription factor 4 protein
  • POU5F1 protein is also known as POU5F1 protein and is encoded by the POU5F1 gene.
  • OCT4 is one of the homeodomain transcription factors of the POU family.
  • OCT4 protein is known to be involved in autologous division of undifferentiated embryonic stem cells, but there is no information related to direct cross-differentiation from somatic cells to bone-related cells.
  • the genetic sequence of human OCT4 consists of SEQ ID NO: 1 (NM_002701.5), the human OCT4 protein consists of SEQ ID NO: 2 and NP_002692.2), and the gene sequence of mouse OCT4 consists of SEQ ID NO: 3 (NM_013633.3), the amino acid sequence of the mouse OCT4 protein consists of SEQ ID NO: 4 (NP_038661.2), and the OCT4 protein of the present invention has sequence homology or functional similarity with the OCT4 protein known in the art to which the present invention belongs. It may include a protein having.
  • the OCT4 protein may comprise OCT4 protein analogs in which one or more amino acids of the OCT4 protein are genetically and / or chemically modified and retain the biological activity of the parent protein.
  • OCT4 protein fusion means a complex in which a peptide or the like is chemically bonded or fused to one or more amino acids of the OCT4 protein while retaining the biological activity of the OCT4 protein.
  • the OCT4 protein fusion is an OCT4 (OCTamer-binding transcription factor 4) protein fused with a cell permeation transporter.
  • the cell permeation delivery material is a cell-penetrating peptide (CPP).
  • the cell permeable peptide is a peptide that is fused to the OCT4 protein and functions to enable the cellular passage of the OCT4 protein into the desired cell, the meaning of which is known in the art.
  • CPP may be a peptide having a specific sequence called a protein transduction domain (PTD) including a large amount of basic sequences such as arginine or lysine and having a high positively charged 8 to 16 amino acids, but is not limited thereto.
  • PTD protein transduction domain
  • CPP is 30K protein, Antp (Penetratin), HSV-1, VP22, pep-1, PTD4, TAT PTD, Hph-1, Vectocell, Lactoferrin, Sim-2, LPIN3, 2IL-1a, dNP2, nona- arginine (R9), including but not limited to.
  • the cell permeable peptide is a 30K protein.
  • 30K protein refers to a group of 30KDa protein in the silkworm body fluid, and includes the technical field to which the present invention belongs (eg, Korean Patent Application Publication Nos. 10-2011-0003889, 10-2014-0111178 or Korean Patent Registration No. 10-1626343). Is known.
  • the 30K protein may be a recombinant protein obtained from silkworm body fluids or produced from bacteria, plants or plant-derived cells, yeast, fungi, insect cells or vertebrate cells, including E. coli, by genetic recombination method, N- of OCT4 It may be attached at the terminal or C-terminus.
  • the 30K protein may include all analogs, variants, fragments, or subtypes of 30K protein having cell permeable properties in addition to the 30K protein known in the art, for example, may be 30KC19, 30KC ⁇ , PeP-C, etc. have.
  • the 30K protein is 30Kc19.
  • 30K protein has been shown as an example of the cell-penetrating peptide in the present invention, any peptide capable of cell passage of the OCT4 protein by conjugation or fusion to the OCT4 protein and into the desired cell can be used as a CPP for direct cross-differentiation of the present invention. have.
  • the fusion of the OCT4 protein is an OCT4 protein fusion to which bone tissue or bone marrow tissue specific delivery substances are bound.
  • the fusion of the OCT4 protein is an OCT4 protein fusion conjugated or fused to a cell permeable transporter, to which bone tissue or bone marrow tissue specific transporters are bound.
  • OCT4 protein carrier means a delivery material containing an OCT4 protein or a fusion thereof, which can efficiently deliver the OCT4 protein into a desired cell.
  • the OCT4 protein carrier includes, but is not limited to, nanoparticles, liposomes, micelles, or nanoemulsions comprising the OCT4 protein.
  • the nucleic acid molecule encoding the OCT4 protein is delivered intracellularly by methods known in the art, for example, naked DNA in the form of a vector, liposomes, cationic polymers and the like. It can be delivered into the cell using a combination of biological materials such as chemicals or peptides that enable or enable other cell specific targeting.
  • Liposomes are phospholipid membranes prepared by mixing cationic phospholipids, such as DOTMA or DOTAP, for gene delivery. Nucleic acid-liposomal complexes can be formed when cationic liposomes and anionic nucleic acids are mixed in a proportion.
  • vector refers to a gene delivery vehicle that is an expression vector capable of expressing a protein of interest in a suitable host cell, and which includes essential regulatory elements operably linked to express a gene insert.
  • Vectors of the present invention include signal or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers, and can be prepared in various ways depending on the purpose.
  • the promoter of the vector may be constitutive or inducible.
  • the expression vector includes a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin. Vectors can self replicate or integrate into host DNA.
  • Vectors in the present invention include plasmid vectors, cosmid vectors, viral vectors and the like.
  • the vector may be a non-viral vector, specifically, a plasmid.
  • the vector may be, but is not limited to, an plasmid vector comprising an OCT4 gene that induces direct cross-differentiation.
  • One embodiment of the present invention provides an OCT4 expression promoter (OCTamer-binding transcription factor 4) protein, a fusion or carrier thereof, a nucleic acid molecule encoding the protein, and a vector into which the nucleic acid molecule is introduced; And a method for direct cross-differentiation from somatic cells to bone-related cells, comprising the step of treating musculoskeletal growth factors with somatic cells.
  • OCT4 expression promoter OCTamer-binding transcription factor 4
  • direct reprogramming, direct conversion, transdifferentiation is a process of inducing conversion between mature (differentiated) cells having completely different cell types.
  • Direct cross-differentiation process unlike the process of reprogramming into induced Pluripotent Stem Cells (iPSCs) and re-differentiating them into the cells of interest, the conversion to the desired cells without going through the induced pluripotent stem cell stage It is clearly distinguished in that it is derived.
  • iPSCs induced Pluripotent Stem Cells
  • the term "somatic cell” may mean all cells except germ cells, for example, human, horse, sheep, pig, goat, camel, antelope, dog-derived or isolated from mammals Can be. In one embodiment the somatic cells may be isolated in vivo.
  • the somatic cells are fibroblasts, epithelial cells, muscle cells, nerve cells, hair cells, hair follicle cells, hair follicle cells, oral epithelial cells, somatic cells extracted from urine, gastric mucosa cells, goblet cells, G cells, B cells, May be, but is not limited to, endothelial cells, vascular endothelial cells, blood cells, astrocyte blood cells, neural stem cells, hematopoietic stem cells, adipocytes, or mesenchymal stem cells.
  • the somatic cells include, but are not limited to, one or more of the following: endothelial cells, mesenchymal stem cells, adipocytes, and fibroblasts.
  • the present inventors confirmed that bone-related cells induce direct cross-differentiation of OCT4 protein expression in human umbilical vein endothelial (HUVEC) cells, which are human vascular endothelial cells. Analyzes were performed to confirm that they had normal bone related cellular properties (Examples 1 to 3).
  • HUVEC umbilical vein endothelial
  • the bone-related cells are osteoblasts, osteogenic cells, osteoblasts (pre osteoblast), osteocytes (Osteocyte), osteochondroprogenitor cells (osteochondroprogenitor cells), Chondrocytes, osteoclasts (osteoclast), including, but not limited to.
  • the step of treating the somatic cells OCT4 protein expression promoter may further comprise the step of culturing somatic cells.
  • the step of culturing the somatic cells can be performed according to methods known in the art.
  • the medium used for somatic cell culture is any basic medium suitable for animal cell growth, EGM-2 medium, Minimal Essential Medium (MEM), Dulbecco modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), K-SFM (Keratinocyte Serum Free Medium), F12 (Ham's F-12 medium) medium, or DMEM / F12 mixed with DMEM and F12, and the like, but is not limited thereto.
  • EGM-2 medium Minimal Essential Medium (MEM), Dulbecco modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), K-SFM (Keratinocyte Serum Free Medium), F12 (Ham's F-12 medium) medium, or DMEM / F12 mixed with DMEM and F12, and the like, but is not limited thereto.
  • anabolic sources of carbon, nitrogen and micronutrients can be added, including but not limited to serum sources, growth factors
  • the method may further comprise the step of stabilizing the somatic cells in serum-free medium after the step of treating the somatic cells with the OCT4 expression promoting factor.
  • said musculoskeletal growth factor is at least one factor of IGF-1, TGF, BMP, osteogenin, Osx (Osterix), and Run-related transcription factor 2 (RUNX2).
  • said musculoskeletal growth factor is BMP, more specifically BMP4.
  • the musculoskeletal growth factor in the direct cross-differentiation method according to the present invention is a concentration of 0.1 to 500 ng / mL, specifically 0.5 to 100 ng / ml, more specifically 2 to 20 ng / may be added in mL.
  • the method may further comprise the step of culturing the somatic cells in bone differentiation medium.
  • Bone differentiation media that can be used in the present invention are known in the art.
  • One embodiment of the invention provides an OCT4 protein, a fusion or carrier thereof, a nucleic acid molecule encoding the protein, and at least one OCT4 expression promoting factor of the vector into which the nucleic acid molecule is introduced; And musculoskeletal growth factors, and composition for inducing direct cross-differentiation from somatic cells into bone-related cells.
  • the OCT4 expression promoting factor is as defined above.
  • the present invention is bone-related cells produced by performing a direct cross-differentiation method of bone-related cells produced by the direct cross-differentiation method of the present invention.
  • the bone-related cells are osteoblasts, osteogenic cells, osteoblasts (pre osteoblast), osteocytes (Osteocyte), osteochondroprogenitor cells (osteochondroprogenitor cells), It may be chondrocytes or osteoclasts, for example osteoblasts.
  • the expression level of osteoblast specific markers such as cbfa1, ALP and cal1 was confirmed to confirm the differentiation into osteoblasts according to the direct cross-differentiation method (Examples 1-4, Example 2). -6 etc.).
  • One embodiment of the present invention is a cell therapeutic agent for the prevention or treatment of bone-related diseases comprising the bone-related cells produced by the direct cross-differentiation method of the present invention as an active ingredient.
  • the "cell therapeutic agent” is a cell and tissue prepared by separating, culturing and special manipulation from human, and is a medicine used for the purpose of treatment, diagnosis and prevention, and the living autologous to restore the function of the cell or tissue, Drugs used for the purpose of treatment, diagnosis, and prevention are collectively referred to as a series of actions such as proliferating, selecting, or otherwise changing the biological properties of allogeneic or heterologous cells in vitro.
  • Bone-related cells prepared according to the cross-differentiation method of the present invention can be used for the treatment, diagnosis or prevention of bone-related diseases.
  • the bone-related disease is osteoporosis, osteomalacia, osteoopenia, bone atrophy, fibrous dysplasia, Paget's disease , Hypercalcemia, neoplastic destruction of bone, cancer-related bone resorption diseases, fractures, osteolysis, osteoarthritis and rheumatoid arthritis, osteoarthritis (Osteitis), one or more selected from the group consisting of Osteogenesis Imperfecta, but is not limited thereto.
  • the cell therapy agent is added at least one of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, buffers and the like commonly used in the manufacture of pharmaceutical compositions It can be included as.
  • One embodiment of the present invention is a method for screening a bone-related disease therapeutic substance using bone-related cells, including bone-related cells produced by the direct cross-differentiation method of the present invention.
  • it is useful for screening a therapeutic agent for bone-related diseases by a method for confirming the reactivity of bone-related cells prepared by direct cross-differentiation in the somatic cells of the present invention in the presence and absence of a candidate substance for the treatment of bone-related diseases.
  • a method for confirming the reactivity of bone-related cells prepared by direct cross-differentiation in the somatic cells of the present invention in the presence and absence of a candidate substance for the treatment of bone-related diseases. can be used.
  • a method for screening a bone-related disease therapeutic substance using bone-related cells is
  • Preparing a first bone related cell and a second bone related cell by performing a direct cross-differentiation method
  • the candidate substance is a candidate for treating bone-related disease. Determining the drug.
  • the candidate substance may include various small molecule chemical drugs, peptides, proteins, nucleic acid molecules, natural products, extracts of natural products, but is not limited thereto.
  • One embodiment of the invention is a composition for diagnosing bone-related diseases comprising bone-related cells produced by the direct cross-differentiation method of the present invention.
  • a bone-related disease diagnostic composition comprising bone-related cells prepared by a direct cross-differentiation method can be used in the diagnosis of bone-related diseases by contacting a sample collected from an individual and comparing it with a normal control group. have.
  • OCT4 protein for bone prevention or treatment through direct cross differentiation
  • One embodiment of the present invention provides a method for preventing bone-related diseases comprising OCT4 protein, a fusion or carrier thereof, a nucleic acid molecule encoding the protein, and at least one OCT4 expression promoting factor in a vector into which the nucleic acid molecule is introduced.
  • Therapeutic composition comprising OCT4 protein, a fusion or carrier thereof, a nucleic acid molecule encoding the protein, and at least one OCT4 expression promoting factor in a vector into which the nucleic acid molecule is introduced.
  • the composition for preventing or treating bone-related diseases may further include a substance capable of delivering OCT4 expression promoting factor to somatic cells.
  • the OCT4 protein fusion is an OCT4 protein fused to a cell permeable peptide.
  • Cell-permeable peptides are as defined above.
  • the cell permeable peptide is 30K protein, Antp (Penetratin), HSV-1, VP22, pep-1, PTD4, TAT PTD, Hph-1, Vectocell, Lactoferrin, Sim-2, LPIN3 , 2IL-1a, dNP2, nona-arginine (R9).
  • the 30K protein is 30Kc19.
  • the OCT4 protein fusion may be delivered into somatic cells, such as vascular endothelial cells.
  • the composition for preventing or treating bone-related diseases may further include a substance capable of delivering the OCT4 expression promoting factor to a bone site or bone marrow site.
  • said OCT4 protein fusion is an OCT4 protein in which a bone tissue specific delivery agent is fused to a conjugated or fused cell permeable peptide.
  • a bone tissue or bone marrow tissue specific delivery material means a material capable of targeting a substance to be delivered to bone tissue or bone marrow tissue after intravenous injection into an individual.
  • Bone or bone marrow tissue specific delivery materials include, for example, chemicals including bisphosphonate-based compounds, bone marrow homing peptide 1 (BMHP1), Bone or bone marrow homing peptides including PFS (PFSSTKT), and the like. It may be, but is not limited thereto, and may be bone tissue or bone marrow tissue specific delivery materials known in the art.
  • the bone tissue specific delivery material is a bisphosphonate family compound bound to the OCT4 protein fusion.
  • the bisphosphonate-based compound may be bound to, for example, the carboxy group of the OCT4 protein fusion, but is not limited thereto.
  • the bone-related disease is osteoporosis, osteomalacia, osteoopenia, bone atrophy, fibrous dysplasia, Paget's disease , Hypercalcemia, neoplastic destruction of bone, cancer-related bone resorption diseases, fractures, osteolysis, osteoarthritis and rheumatoid arthritis It is 1 or more types chosen from group.
  • the composition for preventing or treating bone related diseases according to the present invention does not increase the expression of Sox2, Klf4, C-Myc or Lin28 protein in somatic cells to be administered.
  • the composition does not include, for example, Sox2, Klf4, C-Myc or Lin28 protein or a fusion thereof, a nucleic acid encoding the protein, or a vector into which the nucleic acid has been introduced.
  • One embodiment of the invention comprises administering to a subject an effective amount of an OCT4 protein or a fusion thereof, a nucleic acid molecule encoding said protein, and at least one OCT4 expression promoting factor in a vector into which said nucleic acid molecule is introduced.
  • a method for preventing or treating a related disease comprises administering to a subject an effective amount of an OCT4 protein or a fusion thereof, a nucleic acid molecule encoding said protein, and at least one OCT4 expression promoting factor in a vector into which said nucleic acid molecule is introduced.
  • in the method of preventing or treating OCT4 expression promoting factor may be administered orally or parenterally.
  • parenteral administration it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration, intrasplenically, or rectal administration.
  • said method of administration is intravenous injection or topical administration to a bone related disease site.
  • Bone-related cells prepared using a composition for inducing direct cross-differentiation from somatic cells comprising OCT4 expression promoting factors according to the present invention to bone-related cells are useful for cell therapy, screening for bone-related diseases, and for diagnosing bone-related diseases.
  • the composition may be administered to a subject to be useful for the prevention or treatment of a bone related disease.
  • FIG. 1 is a diagram showing the process of direct cross-differentiation through the expression of Oct4 gene in vascular endothelial cells.
  • Figure 2 (A) shows the results of confirming the expression of Oct4 gene introduced into vascular endothelial cells by PCR
  • Figure 2 (B) shows the result of confirming the presence or absence of OCT4 protein expressed in the cell by immunocytochemistry. .
  • FIG. 3 shows the results of confirming the expression levels of MBPRIA and BMPRII genes related to BMP4 receptor and SLUG and SNAIL genes related to endothelial-mesenchmal transition.
  • FIG. 7 is a graph of micro-CT photographs and BV / TV% for confirming bone regeneration in a cranial defect mouse model.
  • Figure 8 is a photograph confirming Masson's trichrome staining for confirming bone regeneration in a skull defect mouse model.
  • FIG. 9 shows a vector for preparing a CPP-OCT4 complex protein.
  • 10 to 12 are the results of sieve chromatography (Fast Protein Liquid Chromatography, FPLC) and SDS-PAGE confirming the concentration of the CPP-OCT4 complex protein.
  • Figure 13 is a diagram showing the results of LIVE / DEAD analysis of vascular endothelial cells.
  • vascular endothelial cell-specific genes CD31, VECAD, VEGFR-2
  • mesenchymal stem cell-specific genes VIPIN, TWIST, SLUG
  • CD31 vascular endothelial cell specific protein
  • ⁇ SMA mesenchymal endothelial cell specific protein
  • Figure 17 shows the results of fluorescence staining of cells with paloidine observed the formation of blood vessels.
  • 19 is a photograph confirming the degree of calcification of the cells by allinzarin staining.
  • FIG. 20 is a graph of micro-CT photographs and BV / TV% for confirming bone regeneration in a cranial defect mouse model.
  • 21 is a diagram for confirming the osteoporosis improvement effect according to administration of the CPP-OCT4 complex protein or BP-CPP-OCT4 complex protein.
  • Example 1-1 Osteoblast direct cross-differentiation method
  • HUVEC cells (Lonza, USA) were placed in a 100 mm cell culture dish coated with gelatin 0.1% solution for 20 minutes at 4 ⁇ 10 3 cells / cm. Inoculated at 2 density. Cultivate the cells in EGM-2 medium (Lonza, USA) containing 1% FBS and various growth factors (IGF, VEGF, FGF) until the culture dish is filled to about 90% at 37 ° C., 5% CO 2 culture conditions. It was. The cells in the culture dish were then separated using trypsin at 0.25% concentration.
  • a recombinant plasmid containing the OCT4 (Addgene # 13366) gene was introduced into a DH5alpha bacterial stock (Invitro) given a heat shock at 42 ° C. for 45 seconds.
  • the OCT4 transgenic bacteria were then inoculated in 4 mL LB medium containing 100 ⁇ g / ml ampicillin antibiotic (Sigma, USA) and seeded at 160 rpm in a 37 ° C. shaking incubator for 8 hours. It was.
  • plasmids were obtained from the bacteria using Midi-Prep (Qiagen, USA).
  • the recombinant plasmid was transformed by electroporation in the presence of an R buffer solution of Neon transfection system (Thermo Fisher) and vascular endothelial cells. Electroporation was performed with an electric field strength of 13.5 kV / cm and an impact time of 30 ms so that 15 ⁇ g of recombinant plasmid could be delivered to 2 ⁇ 10 6 cells. Thereafter, immunocytochemistry was performed using OCT4 antibodies (Abcam, USA) targeting OCT4 transcription factors to select transformed cells.
  • OCT4 antibodies Abcam, USA
  • the cells were incubated for 24 hours in serum-free EGM-2 medium at 37 ° C. and 5% CO 2 for stabilization of the cells. Thereafter, 10 ng / mL concentration of BMP4 (RnD systems, USA) was added to the serum-free medium and incubated for 48 hours.
  • BMP4 RnD systems, USA
  • Example 1-2 Expression of OCT4 gene in vascular endothelial cells
  • vascular endothelial cells expressing the OCT4 gene were established according to Example 1-1. RT-PCR and immunocytochemistry were performed on vascular endothelial cells subjected to electroporation.
  • vascular endothelial cells were washed with PBS and treated with Trizol (Thermo, USA) to elute the cells on ice for 20 minutes to separate RNA. Then, chloroform (chloroform, Sigma, USA) was added and shaken for 15 seconds, left at room temperature for 10 minutes, and then centrifuged at 15,000 rpm and 4 ° C. for 20 minutes. The transparent layer was then extracted separately in two separate layers (transparent layer and pink layer) and transferred to a new tube.
  • Trizol Thermo, USA
  • Isopropanol (isopropanol, Sigma, USA) was added and waited for 5 minutes to generate RNA precipitate at room temperature, and the resulting precipitate was separated by centrifugation at 15,000 rpm, 4 ° C. for 20 minutes. In the washing step, 75% ethanol was put into centrifuged RNA pellet, and once again centrifuged at 10,000 rpm and 4 ° C. for 20 minutes. The washed precipitate was denatured in molecular graded water (Sigma, USA) for 10 minutes at 60 °C.
  • RNA of each cell thus obtained was prepared as cDNA using a reverse-transcriptional PCR kit (Enzynomics) according to the manufacturer's protocol.
  • the prepared cDNA was subjected to quantitative real-time PCR using primers of each gene to be analyzed and SYBR green PCR Mastermix (Enzynomics). Primers used are shown in Table 1 below.
  • OCT4 0.1% triton-X100, 5% NGS, and 1% BSA / PBS were mixed at a ratio of 1: 500 and stored overnight at 4 ° C. The next day, wash three times for 10 minutes each day. The remaining first antibody was removed and the second antibody was treated at room temperature for 1.5 hours at a ratio of 1: 200 to the same lysis.
  • DAPI D9541, Sigma-aldrich
  • Example 1-3 Expression of specific genes following BMP4 treatment in transformed cells
  • Example 1-1 the expression levels of the MBPRIA and BMPRII genes associated with the BMP4 receptor and the SLUG and SNAIL genes associated with the endothelial-mesenchmal transition were determined using the RT described in Examples 1-2. Measured via PCR. Primer sequences used for RT-PCR are as follows.
  • Example 1 In order to confirm whether vascular endothelial cells were differentiated into osteoblasts according to the direct cross-differentiation method of Example 1-1, Examples and Comparative Examples (Comparative Example 1: Using vascular endothelial cells as it is, Comparative Example 2: to vascular endothelial cells Only BMP4 treatment, Comparative Example 3: Transformation of vascular endothelial cells with the gene with OCT4 only) was confirmed the expression level of the osteoblast specific markers cbfa1, ALP and cal1 genes.
  • cbfa1 core-binding factor subunit alpha-1
  • RUNX2 Rivt-related transcription factor 2
  • Alkaline phosphatase is present in almost all tissues, especially ALP, which is present in bone tissue, increases its activity when bone growth occurs actively.
  • ALP is an enzyme that plays an important role in osteoid formation and mineralization, and is well known as a marker of osteoblast activity.
  • col1 Type 1 Collagen
  • the cbfa1, ALP and cal1 genes were highly expressed in osteoblasts, and it was determined that vascular endothelial cells were differentiated into osteoblasts.
  • the expression levels of the cbfa1, ALP and cal1 genes were RT-PCR as described in Example 1-2 when the cells of Examples and Comparative Examples were cultured for 1 week or 2 weeks. Primers used are shown in Table 3 below.
  • Example 1 Using vascular endothelial cells as it is, 7 days or 14 days of culture of bone differentiation medium, Comparative Example 2: BMP4 treatment on vascular endothelial cells, Comparative Example 3: OCT4 on vascular endothelial cells Expression of cbfa1, ALP and cal1 genes of only transformation with genes was confirmed (FIG. 4).
  • Example 1 In order to confirm whether vascular endothelial cells were differentiated into osteoblasts according to the direct cross-differentiation method of Example 1-1, the Examples and Comparative Examples (Comparative Example 1) using immunochemical staining (immunocytochemistry) , Comparative Example 2: BMP4 treatment to vascular endothelial cells, Comparative Example 3: OCT4 to the vascular endothelial cells only transgenic) cells were confirmed the presence of osteocalcin (Osteocalcin) markers.
  • Comparative Example 1 Comparative Example 1 using immunochemical staining (immunocytochemistry)
  • Comparative Example 2 BMP4 treatment to vascular endothelial cells
  • Comparative Example 3 OCT4 to the vascular endothelial cells only transgenic cells were confirmed the presence of osteocalcin (Osteocalcin) markers.
  • Example and comparative cells were applied to the skull deficient mouse model to confirm that direct cross-differentiated osteoblasts regenerate bone tissue according to Example 1-1.
  • a skull defect model was made in the form of a 4 mm-sized hole surgically in a skull in an 8-week-old mouse model.
  • the experimental group used the sham-filled scaffold material without the cells in the sham and Example 1 and Comparative Example 1 (Comparative Example 1: using vascular endothelial cells as it is, Comparative Example 2: BMP4 treatment only for vascular endothelial cells , Comparative Example 3: Transformation of the vascular endothelial cells with the gene with OCT4 only. Mice were used as scaffolds containing the cells.
  • Example 2-1 Method for preparing CPP-OCT4 fusion protein complex
  • a CPP-OCT4 complex protein was prepared in which OCT4 protein was fused to cell penetrating protein (Cell Penetrating Peptide, CPP).
  • CPP Cell Penetrating Peptide
  • the cell permeable protein that binds to OCT4 was used 30kc19, which has been reported to have cell permeability.
  • the amino acid sequence of 30Kc19 used in this experiment is as follows (SEQ ID NO: 23).
  • the CPP-OCT4 fusion complex protein was purified by Fast Protein Liquid Chromatography (FPLC) and confirmed by staining with Coomassie Blue on SDS-PAGE gel.
  • the concentration of CPP-OCT4 complex protein was measured by comparing and contrasting the standard BSA concentrations (FIGS. 10-12).
  • Example 1-1 To differentiate vascular endothelial cells cultured according to the method described in Example 1-1 into osteoblasts, 40 ⁇ g of CPP-OCT4 complex protein and 10 ng / mL BMP4 (RnD systems, USA) were used in serum-free EGM-2 medium. ) For 24 hours. BMP4 was treated for 24 hours under the same conditions, and cultured in StemPro Osteogenesis medium (Thermo, USA), a bone differentiation medium, for 14 days to differentiate into osteoblasts.
  • StemPro Osteogenesis medium Thermo, USA
  • the obtained CPP-OCT4 complex protein was treated with vascular endothelial cells at 0, 20, 40, 60, 80 ⁇ g, and then cytotoxicity was confirmed by LIVE / DEAD analysis through staining. Green is live cells stained with Calcein components, and red is dead / dying cells stained with EtBr components (FIG. 13).
  • CPP-OCT4 complex protein is expressed in vascular endothelial cells by using a fluorescent antibody that binds to T7, which is part of the CPP-OCT4 complex protein. Treatment was performed for 24 hours to confirm cell permeation (FIG. 14).
  • vascular endothelial cell-specific genes CD31, VECAD, VEGFR-2
  • mesenchymal stem cell specificity in the cells of Examples and Comparative Examples
  • the expression level of the enemy genes was confirmed by the same RT-PCR as in Experiment 3-1 (Fig. 15).
  • vascular endothelial cell-specific protein CD31
  • ⁇ SMA mesenchymal endothelial cell-specific protein
  • vascular endothelial cell specific protein CD31
  • mesenchymal endothelial cell specific protein ⁇ SMA
  • osteoblast-specific markers Col1 collagen type-1
  • OPN osteopontin
  • Example 2-8 Confirm the regenerative effect of bone tissue
  • Example and Comparative cells were applied to a cranial defect mouse model.
  • Example 3-1 Method for preparing BP-CPP-OCT4 fusion protein complex
  • BP-CPP-OCT4 complex protein was prepared by binding bisphosphonate (BP) to CPP-OCT4 complex protein.
  • Bisphosphonate-based alendronate was used to bind to CPP.
  • BP-CPP-OCT4 complex protein was prepared by combining carboxyl group of CPP protein and amine group of alendronate using 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and Sulfo-NHS (N-hydroxysulfosuccinimide).
  • EDC 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • Sulfo-NHS N-hydroxysulfosuccinimide
  • the prepared BP-CPP-OCT4 complex protein was dissolved in deuterium oxide (D2O) at a concentration of 5 mg / ml to determine whether or not bound by nuclear magnetic resonance.
  • D2O deuterium oxide
  • an animal model of osteoporosis was prepared by resecting the ovary in C3H female mice. After death, the femur was photographed with micor-CT to determine whether osteoporosis developed.
  • BP-CPP-OCT4 complex protein was administered to the osteoporosis-induced animal model.
  • Osteoporosis-induced animal model is an ovarian-resected mouse, divided into two groups of 2.5 mg of BP-CPP-OCT4 complex protein and 0.125 mg of alendronate after two weeks of ovarian ablation for 6 weeks of protein / kg / wk. After intravenous administration, it was confirmed whether osteoporosis was improved (FIG. 21).

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Abstract

La présente invention concerne : un procédé de transdifférenciation directe de cellules somatiques en cellules osseuses, le procédé comprenant une étape consistant à traiter les cellules somatiques avec au moins un facteur favorisant l'expression d'OCT4 (Facteur de transcription 4 de liaison aux octamères) de la protéine OCT4 ou d'une protéine de fusion de celle-ci, une molécule d'acide nucléique codant pour la protéine, et un vecteur transportant la molécule d'acide nucléique, et un facteur de croissance musculo-squelettique ; les cellules osseuses ainsi obtenues ; un produit de thérapie cellulaire utilisant celles-ci ; un procédé de criblage de matériel thérapeutique utilisant celles-ci ; et une composition de diagnostic utilisant celles-ci. La présente invention concerne également : une composition comprenant au moins un facteur favorisant l'expression d'OCT4 de la protéine OCT4 ou une protéine de fusion de celle-ci, une molécule d'acide nucléique codant pour la protéine, et un vecteur transportant la molécule d'acide nucléique en tant que principe actif pour la prévention ou le traitement d'une maladie osseuse ; et une méthode préventive ou thérapeutique faisant appel à celle-ci. En outre, la présente invention concerne une fusion de protéine OCT4 de pénétration cellulaire fusionnée à un transporteur ou une fusion de protéine OCT4 de pénétration cellulaire fusionnée à un transporteur comprenant un transporteur spécifique aux tissus osseux conjugué à celle-ci.
PCT/KR2019/005103 2018-07-04 2019-04-26 Composition comprenant oct4 pour induire une transdifférenciation directe dans une cellule osseuse Ceased WO2020009320A1 (fr)

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