WO2022202930A1 - 組成物および医薬組成物 - Google Patents

組成物および医薬組成物 Download PDF

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WO2022202930A1
WO2022202930A1 PCT/JP2022/013676 JP2022013676W WO2022202930A1 WO 2022202930 A1 WO2022202930 A1 WO 2022202930A1 JP 2022013676 W JP2022013676 W JP 2022013676W WO 2022202930 A1 WO2022202930 A1 WO 2022202930A1
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mir
pka
escs
differentiation
dox
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French (fr)
Japanese (ja)
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潤 山下
朋皓 皆川
哲哉 的場
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Kyoto University NUC
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Kyoto University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to compositions and pharmaceutical compositions for promoting myocardial differentiation for use in the prevention and/or treatment of heart disease.
  • Cardiac regenerative medicine is currently focused on the development of new treatments through the transplantation of cardiomyocytes and heart-like tissues (cell transplantation and tissue transplantation) derived from pluripotent stem cells.
  • cardiomyocytes and heart-like tissues cell transplantation and tissue transplantation
  • a component that promotes proliferation of cardiomyocytes or a mixture such as a culture containing the component is found, administration of the component will be more invasive than transplantation of cultured cardiomyocytes or heart-like tissue. It can be expected to be used for low-cost treatment.
  • Non-Patent Document 1 discloses the research content of transplanting a sphere-like mass of cells called a cardiosphere, which is cultured from heart tissue, to pediatric patients with dilated cardiomyopathy. This article lists microRNA-146 (miR-146) as a possible component functionally involved in this disease.
  • Non-Patent Document 1 mentions miR-146 as a component that may be involved in the function of pediatric patients with dilated cardiomyopathy. The mechanisms involved in proliferation have not been fully elucidated.
  • PSCs pluripotent stem cells
  • EVs extracellular vesicles derived from PSCs in the process of differentiation promote cardiomyocyte differentiation in PSCs and fetuses. Therefore, in order to use components that promote cardiomyocyte differentiation in the treatment of heart disease, we can search for molecules that contribute to concentric muscle differentiation in the components of the EV, mainly in the contents of the EV, and apply them to transportation means. I continued my research.
  • the present invention has been made in view of the above circumstances, and provides a composition for promoting myocardial differentiation that exhibits a high degree of function in promoting cardiomyocyte differentiation and is effective in improving cardiac function after heart disease. and to provide a pharmaceutical composition for promoting myocardial differentiation.
  • Aspect 1 of the present invention is a composition for promoting myocardial differentiation, containing miR-132 as an active ingredient.
  • the composition of aspect 1 is particulate.
  • Aspect 3 of the present invention is the composition of aspect 2, wherein the composition is lactic acid/glycolic acid copolymer (PLGA) nanoparticles or liposomes.
  • PLGA lactic acid/glycolic acid copolymer
  • Aspect 4 of the present invention is a pharmaceutical composition for treating or preventing heart disease, containing miR-132 as an active ingredient.
  • the composition of aspect 4 is particulate.
  • aspects 6 of the present invention is the composition of aspect 5, wherein the composition is lactic acid/glycolic acid copolymer (PLGA) nanoparticles or liposomes.
  • PLGA lactic acid/glycolic acid copolymer
  • Aspect 7 of the present invention is the pharmaceutical composition according to any one of aspects 4 to 6, wherein the heart disease is myocardial infarction.
  • a composition and a pharmaceutical composition that exhibit a high function in promoting cardiomyocyte differentiation and are effective in improving cardiac function after heart disease can be obtained.
  • FIG. 2 is a photographic diagram showing immunostaining of Flk1 during differentiation of PKA-ESCs.
  • FIG. 4 is a graphical representation showing FACS analysis for the appearance of Flk1 in PKA-ESCs.
  • FIG. 4 is a graphical representation showing the percentage of PKA-ESCs expressing Flk1 by FACS analysis.
  • FIG. 11 is a photographic diagram showing immunostaining of Flk1 during differentiation of PKA-ESC and Control-ESC chimeric aggregates.
  • FIG. 4 is a graphical representation showing FACS analysis of Flk1 appearance on chimeric aggregates.
  • FIG. 4 is a graphical representation showing the percentage of chimeric aggregates expressing Flk1 during differentiation. Schematic representation of chimeric aggregate co-culture differentiation system with inhibited EV secretion.
  • FIG. 10 is a graph showing the number of Flk1 cells by treatment with manumycin A, GW4869.
  • FIG. 4 is a graphical representation showing the percentage of PKA-ESCs and Control-ESCs in chimeric aggregates expressing Flk1 under Dox-conditions. The graph on the left shows the results of PKA-ESC and the graph on the right shows the results of Control-ESC in control, manumycin A treatment, and GW4869 treatment, respectively.
  • FIG. 10 is a graph showing the number of Flk1 cells by treatment with manumycin A, GW4869.
  • FIG. 4 is a graphical representation showing the percentage of PKA-ESCs and Control-ESCs in chimeric aggregates expressing Flk1 under Dox-conditions. The graph on the left shows the results of PKA-ESC and the
  • FIG. 11 is a photographic diagram showing immunostaining of Flk1+control-ESCs in chimeric aggregates using manumycin A, GW4869, or DMSO.
  • 1 is a schematic diagram of EV collection and processing;
  • FIG. 11 is a photographic diagram showing immunoblots of CD9, CD63, and CD81 in exosomal protein lysates from 10 mL of conditioned medium.
  • FIG. 2 is a photographic diagram showing transmission electron microscopy (TEM) images of EVs in conditioned medium.
  • FIG. 4 is a graphical representation showing the results of analysis of EV size distribution in Dox+ and Dox ⁇ media.
  • FIG. 10 is a graphical representation showing FACS analysis of Flk1+ cells in EV-treated Control-ESCs on D3.5.
  • FIG. 4 is a graphical representation showing the percentage of control ESCs expressing Flk1.
  • FIG. 11 is a photographic diagram showing immunostaining of mesoderm markers (Flk1, PDGFR ⁇ ) in EV-treated control-ESCs at D3.5.
  • FIG. 4 is a graphical representation showing MA plots summarizing miRNAs differentially expressed in EVs from activated PKA-ESCs (Dox ⁇ ) and inactivated PKA-ESCs (Dox+). Schematic representation of cell chimera experiments and FACS plots showing recipient cells of chimeric aggregates containing miRNA-expressing cell lines on D3.5.
  • FIG. 4 is a graphical representation showing data analyzed for the percentage of Flk1+ recipient cells in aggregates at a 1:3 ratio.
  • FIG. 3 is a graph showing the results of detection of Spry1, Rasa1 and pCREB in capillary Western blot.
  • FIG. 3 is a graph showing the results of pCREB and CREB levels measured by ELISA.
  • FIG. 10 is a graph showing qPCR analysis of ETV2 in Control-ESCs treated with or without EVs from PKA-ESCs (Dox+ or Dox-). Schematic representation of PSyC mechanisms involving EV secretion and miR-132 delivery.
  • 1 is a schematic diagram of the mouse embryo culture of this example.
  • FIG. FIG. 4 is a graphical representation showing FACS analysis of PDGFR ⁇ expression in E6.5 embryos.
  • FIG. 4 is a graphical representation showing the percentage of control ESCs expressing Flk1.
  • FIG. 11 Schematic representation of mouse embryo culture to investigate the effects of EVs (Dox-).
  • FIG. 11 is a photographic diagram showing immunostaining of cTnT in E3.5 mouse embryos.
  • FIG. 4 is a graphical representation showing the percentage of cTnT+ embryos when E3.5 mouse embryos were cultured with undifferentiated ESC-derived EVs (EV(UD)) or early differentiated PKA-ESC-derived EVs (EV(Dox-)) for 10 days.
  • Figure 33 is a graphical representation showing the percentage of beating embryos in cultures similar to Figure 32;
  • FIG. 10 is a photographic representation of a beating mouse embryo cultured from E3.5 for 10 days with miR-132-PLGA.
  • FIG. 2 is a graphical representation showing the percentage of beating of day 8 and day 10 embryos.
  • FIG. 4 is a graphical representation showing cTnT immunostaining in E3.5 mouse embryos cultured for 10 days with or without miR-132-PLGA.
  • FIG. 4 is a graphical representation showing the percentage of cTnT+ embryos at day 10.
  • FIG. 1 is a schematic diagram of aggregate culture used for examining differentiation stages of embryonic stem cells.
  • FIG. 4 is a graphical representation showing FACS analysis of pure PKA-ESC aggregates of PDGFR ⁇ .
  • FIG. 4 is a graphical representation showing data analyzed using the Mann-Whitney U test for the percentage of PDGFR ⁇ +PKA-ESCs.
  • FIG. 11 is a photographic diagram showing immunostaining of PDGFR ⁇ during differentiation of pure PKA-ESC aggregates.
  • FIG. 3 is a graphical representation showing FACS analysis of ESC aggregates.
  • FIG. 3 is a graphical representation showing FACS analysis of ESC aggregates.
  • FIG. 3 is a graphical representation showing FACS analysis of ESC aggregates.
  • FIG. 11 is a photographic diagram showing immunostaining of Flk1 during differentiation of pure Control-ESC aggregates.
  • FIG. 11 is a photographic diagram showing immunostaining of PDGFR ⁇ during differentiation of pure Control-ESC aggregates.
  • FIG. 11 is a photographic diagram showing immunostaining of PDGFR ⁇ during differentiation of chimeric aggregates.
  • FIG. 4 is a graphical representation showing FACS analysis of PDGFR ⁇ expression in chimeric aggregates.
  • FIG. 4 is a graphical representation showing the percentage of differentiating chimeric aggregates expressing PDGFR ⁇ .
  • FIG. 4 is a graphical representation showing the concentration of EV particles in conditioned medium.
  • FIG. 4 is a graph showing concentrations of proteins contained in EVs.
  • FIG. 10 is a graphical representation showing FACS analysis of PDGFR ⁇ expression in Control-ESCs treated with EVs from day 3.5 of differentiation and culture of PKA-ESCs (Dox+ or Dox ⁇ ).
  • FIG. 4 is a graphical representation showing the percentage of PDGFR ⁇ + cells.
  • FIG. 4 is a graphical representation showing FACS analysis of PDGFR ⁇ expression in control-ESCs at day 2.5 of differentiation.
  • FIG. 3 is a graphical representation showing RNA expression levels of Flk1 and PDGFR ⁇ in control-ESCs at day 3.5.
  • FIG. 4 is a graphical representation showing Flk1 mRNA expression levels in day 3.5 control-ESCs treated with or without miR-132-PLGA. Schematic representation of the procedure for the generation of miRNA-PLGA-NP.
  • FIG. 4 is a graphical representation showing a comparison of miR-132-3p amounts for each sample.
  • FIG. 2 is a photomicrograph of miR-132-3p-LNP administration to myocardial ischemia-reperfusion mouse model.
  • FIG. 4 is a graph diagram showing the results of quantification of the stained area;
  • FIG. 3 is a graph showing comparison of miR-132 expression levels by RT-PCR.
  • FIG. 10 is a graph showing a comparison of miR-132 expression levels by RT-PCR when liposomes were intravenously administered.
  • compositions and pharmaceutical composition according to the present invention will be described with reference to embodiments.
  • present invention is not limited to the following embodiments.
  • composition for promoting myocardial differentiation is a composition for promoting myocardial differentiation and contains miR-132 as an active ingredient.
  • miR-132 is a microRNA (miRNA), a class of short non-protein-coding RNA molecules.
  • the miR-132 family is found on the genomes of rodents such as mice and multiple mammals including humans.
  • various miR-132 family molecules can be selected.
  • commercially available miR-132 molecules can be used.
  • Various mutants of miR-132 molecules can also be used as long as they have a function as a microRNA.
  • RNA sequence is SEQ ID NO: 1 (genomic sequence is NCBI RefSeq: NC_000017.11), if mouse is mouse miR-132 (RNA sequence can be selected from SEQ ID NO: 2) (genome sequence is NCBI RefSeq: NC_000077.7), and the like.
  • PSCs pluripotent stem cells
  • EVs extracellular vesicles
  • PKA-ESCs protein kinase A-activated ESCs
  • miR-132 was identified when searching for a functional molecule that can reconstitute PKA activation and promote differentiation of the mesodermal lineage. Furthermore, we found that similar cardiomyocyte induction was reproduced by adding engineered nanoparticles containing miR-132 to ex vivo mouse embryo cultures.
  • miR-132 is a molecule that can have the effect of directly promoting cardiomyocyte induction. Since miR-132 effectively promotes cardiomyocyte induction, compositions containing miR-132 should be widely applied in fields such as cardiomyocyte proliferation, treatment of heart disease, and prevention of heart disease. can be done.
  • miR-132 is a type of microRNA, that is, a small molecule consisting of ten to several dozen nucleic acid residues. Therefore, it is easy to transport via various delivery systems, such as by encapsulating or mixing with other molecules. It can be used for less invasive treatment to patients by delivery via a delivery system mimicking EV, for example, nanoparticles. Also, since miR-132 is a small molecule, it is easy to modulate.
  • the composition of the present embodiment is also preferably particulate.
  • the term "particulate” generally refers to an object with a sufficiently small diameter that is less affected by shape and size. It refers to a massive object in which the difference between the diameter and the longest diameter is relatively small.
  • the largest diameter is preferably nano-sized, preferably 1 to 1000 nm. It is also preferable that the particles have a diameter of 50 to 500 nm. This size makes it easy to contain the microRNA in the particle, and it is not too large to make administration easy.
  • the components constituting the particles contain other components of miR-132 (hereinafter referred to as particle components).
  • This particle component defines to some extent the shape and/or size of the particle.
  • This particle component is preferably a highly biocompatible compound.
  • the particle component conventionally known compounds used for drug delivery and the like can be used.
  • the particle component of the particles is also preferably lactic acid/glycolic acid copolymer (PLGA) nanoparticles or liposomes.
  • PLGA nanoparticles and liposomes are easy to adjust and design in terms of size, etc., have high biocompatibility, and can be suitably applied to compositions used as therapeutic materials.
  • the PLGA compound is not particularly limited in terms of copolymerization ratio, production method, and molecular size used. You can choose.
  • LNPs lipid nanoparticles
  • fatty acids such as phospholipids and cholesterol.
  • the composition of this embodiment when the composition of this embodiment is a particle, it contains miR-132 in said particle.
  • miR-132 may be mixed or complexed with the particle component.
  • miR-132 may be arranged between the molecules of the particle components and fixed by intermolecular force.
  • miR-132 may be immobilized on the particle component. If the particle component and miR-132 are strongly adhered or immobilized, selecting a particle component that decomposes under certain conditions can provide a high degree of selectivity in molecular transport.
  • miR-132 is introduced into the particles.
  • a conventionally known means may be used for the introduction.
  • microRNA is added to the aqueous phase (W phase) and biotin-PLGA is added to the organic phase (O phase).
  • W phase aqueous phase
  • O phase organic phase
  • W/O phase aqueous phase
  • this W/O phase is added dropwise to another aqueous W phase, and the solvent is distilled off to obtain PLGA nanoparticles into which microRNAs have been introduced.
  • miR-132 As a means of introducing miR-132 into LNP, it may be performed along with preparation of liposomes. For example, while stirring the lipid solution containing the lipid described above, an aqueous nucleic acid solution containing miR-132 is added dropwise to prepare a lipid/nucleic acid solution, and this lipid/nucleic acid solution is added dropwise into a buffer solution. can get.
  • composition of the present embodiment can be used for cells in vitro to promote myocardial differentiation, and can also be used for myocardial tissue in vivo. As described above, the composition of the present embodiment can be easily transported and administered, so it is suitable for use in the body of living organisms.
  • the pharmaceutical composition of this embodiment is a pharmaceutical composition for treating or preventing heart disease, and contains miR-132 as an active ingredient.
  • the pharmaceutical composition may be particulate.
  • Particulate pharmaceutical compositions may be polylactic-glycolic acid (PLGA) nanoparticles or liposomes.
  • the composition can be selected from the same composition as described for the composition for promoting myocardial differentiation described above.
  • the pharmaceutical composition of the present embodiment can be used for treating human heart disease (cardiac disease).
  • the heart disease broadly includes diseases in the heart or diseases requiring treatment of the heart.
  • the obtained cardiomyocytes may be administered to a patient, or the obtained cardiomyocytes may be used as a therapeutic agent for heart disease.
  • a method for administering the myocardial cells a means of suspending the cells in a liquid and administering them to the myocardium, or attaching the cells via a sheet, bandage, or the like can be used.
  • the heart disease includes heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic phase hypertrophic cardiomyopathy, dilated cardiomyopathy, etc., or defects due to disorders. It is not limited as long as it is a disease affecting the heart.
  • the heart disease is myocardial infarction.
  • myocardial infarction Many cases of myocardial infarction are currently recognized, and it is a serious problem.
  • the pharmaceutical composition of the present embodiment contains a component for promoting myocardial differentiation, so it can supplement damaged cardiac tissue.
  • treatment can be performed by administering the pharmaceutical composition without resorting to surgery or the like, which reduces the burden on the patient and is particularly effective for treating patients with serious symptoms.
  • a guideline for the effective amount of the pharmaceutical composition of the present embodiment is preferably 0.4-40 ⁇ g/kg as the amount of miRNA-132 for heart disease patients.
  • a pharmaceutical composition containing particles when liposomes are used as the particle component 0.04-4.0 mg/kg is preferable. 0.004-0.4 mg/kg is preferred for pharmaceutical compositions containing particles when PLGA-NP is used as the particle component.
  • the cardiomyocyte proliferation agent may contain other ingredients as appropriate.
  • it may contain ingredients conventionally known as those used in the treatment of diseases of the heart and other circulatory systems.
  • Another aspect of this embodiment is a method of treating or preventing heart disease, comprising administering to a subject the effective amount of the pharmaceutical composition.
  • composition and effective amount of the pharmaceutical composition can be selected from those described above.
  • the heart disease to be treated or prevented according to the present embodiment can be selected from those described above, and the heart disease may be, for example, myocardial infarction.
  • Cardiac regeneration treatment method Another aspect of this embodiment is the cardiac regeneration treatment method of this embodiment, comprising the step of administering the effective amount of the pharmaceutical composition to a subject.
  • the composition and effective amount of the pharmaceutical composition can be selected from those described above.
  • Yet another aspect of this embodiment is a composition comprising miR-132 for use in promoting myocardial differentiation. Yet another aspect of this embodiment is a composition comprising miR-132 for use in treating or preventing heart disease. Yet another aspect of this embodiment is a composition comprising miR-132 for use in cardiac regenerative therapy. Yet another aspect of this embodiment is the use of miR-132 to manufacture a composition that promotes myocardial differentiation. Yet another aspect of this embodiment is the use of miR-132 to manufacture a pharmaceutical composition for treating or preventing heart disease. Yet another aspect of this embodiment is the use of miR-132 to manufacture a composition for use in cardiac regenerative therapy.
  • composition and pharmaceutical composition of the present embodiment contain miR-132 as an active ingredient, promote myocardial differentiation, and have the effect of treating or preventing heart disease.
  • the present inventors in the process of differentiation by cell-to-cell communication mediated by EV, in the process of advancing research on a new biological phenomenon "cell phenotype synchronization (PSyC)" in which cell phenotypes are synchronized, EV identified miR-132, one potential candidate molecule for We have found that this molecule can reconstitute the molecular link of PSyC in recipient cells and reproduce the in vivo effects of EVs to induce mesoderm derivatives.
  • PSyC cell phenotype synchronization
  • EVs are attracting attention as a new intercellular communication modality that acts through hormone-like distant effects. EVs are a heterogeneous group of cell-derived membrane structures, thought to include exosomes and microvesicles, each derived from the endosomal system or shed from the plasma membrane. For example, silencing of TSG101 has been reported to reduce exosome secretion, and consistently, TSG101 knockout inhibits cell proliferation and differentiation, and TSG101KO mice exhibit embryonic lethality before and after the implantation stage.
  • mice knocking out nSMase2 another factor involved in exosome production, show hypoplasia and growth retardation in all tissues, but no fetal lethality.
  • PSyC was significantly, but not completely blocked by nSMase2 inhibitors, suggesting the involvement of other EV generation mechanisms, exosomes and/or microvesicles. .
  • PSyC regulates tissue development and homeostasis, as it establishes cell subsets for building normal tissues through synchronized mechanisms that tightly coordinate cell fate decisions and stages of differentiation. was speculated to be an important mechanism for It was thought that tissues and organs might be established and maintained by more active and bulky molecular trafficking through EV exchange between adjacent cells.
  • the present inventors found that the administration of miR-132-containing nanoparticles tends to suppress the deterioration of cardiac function after myocardial infarction.
  • the myocardial infarction model is an ischemia-reperfusion injury (I/R) model, which is relevant to the clinical situation of recanalization by catheterization after myocardial infarction.
  • I/R ischemia-reperfusion injury
  • administration of nanoparticles is intravenous, which corresponds to normal clinical intravenous injection and drip infusion treatment, and is considered to be less invasive and highly clinically useful. Sufficient therapeutic application can also be expected in the form of transcoronary administration using a catheter.
  • composition and pharmaceutical composition of the present embodiment demonstrate that miR-132 is effective in promoting cardiomyocyte differentiation and improving cardiac function after myocardial infarction, and that nanoparticles are effective as a delivery system for miR-132 to the living body. It is expected that miR-132-containing nanoparticles can be applied as therapeutic agents for cardiac regeneration. In addition, if clinical application is possible in the form of intravenous injection or infusion, cardiac regeneration treatment will be possible even in small-scale medical facilities where cardiac catheterization cannot be performed, and the range of applications will be extremely wide.
  • mice are described by Yamamizu, K.; et al. , (2009), Blood 114, 3707-16.
  • EStTA5-4 cells were prepared by introducing a plasmid carrying the EGFP gene under the control of the CAG promoter by the ES cell Nucleofector kit (VPH-1001, Lonza).
  • a piggyBac vector carrying the primary miRNA gene under the tetracycline response element (TRE) promoter and a transposon vector were introduced using Lipofectamine LTX reagent (15338500, Thermo Fisher Science) to obtain seven mouse ESC lines.
  • TRE tetracycline response element
  • ssODN single-stranded oligodeoxynuclease
  • pX330 plasmids expressing Cas9 and sgRNAs targeting the miR-132 locus were introduced into PKA-ESCs using Lipofectamine LTX reagent.
  • Cells were selected by detecting HiBit protein using the Nano Glo HiBiT Lytic Detection System (N3030, Promega).
  • pX330 was provided by Dr. Feng Zhang (Massachusetts Institute of Technology).
  • ESC lines were cultured in Glasgow minimal essential medium (GMEM; 11710-035, Thermo Fisher) with 10% knockout serum replacement (KSR; 10828-028, Thermo Fisher Scientific), 1% fetal bovine serum (FBS; SAFC Biosciences, USA).
  • GMEM Glasgow minimal essential medium
  • KSR knockout serum replacement
  • FBS fetal bovine serum
  • ESC lines were seeded in low attachment dishes, PrimeSurface Dish 35 mm (MS-9035X, Sumitomo Bakelite), or low cell attachment 96-well plates with U-bottomed conical wells (9,000 cells per well, 100 mL; MS). . Culture used 9096U, Sumilon PrimeSurface plate, Sumitomo Bakelite) differentiation medium. After 24 hours, floating aggregates were collected and plated in multiwell plates (Falcon). To generate chimeric cell aggregates, ESC lines were mixed at the indicated ratios.
  • Tissue staining was performed as follows. Cultured cells were fixed with ice-cold 4% paraformaldehyde solution for 1 hour, blocked with 2% skimmed milk (232100, Becton Dickinson) for 24 hours at 4°C, and incubated with primary antibody for 24 hours at 4°C.
  • primary antibodies include Flk1 (AVAS12, manufactured in-house, 1:500,000), PDGFR ⁇ (3174S, Cell Signaling Technology, 1:500), Oct4 (sc-5279, Santa Cruz Biotechnology, 1:200) and Nanog (RCAB002P, ReproCELL, 1:300) was used.
  • RNA sequencing of miRNA For bulk RNA sequencing, miRCURY RNA Isolation Kit (300110, EXIQON) was used to extract small RNA. Sequencing libraries were prepared using the TruSeq Small RNA Library Prep Kit (Illumina). Libraries were sequenced on the HiSeq2500 in 50-cycle single-read mode. All sequence reads were extracted in FASTQ format using BCL2 FASTQ conversion software 1.8.4 in the CASAVA 1.8.2 pipeline. Sequence reads were mapped to the mm10 reference gene using MirDeep and normalized and quantified using RPKMforGenes (downloaded Dec. 10, 2012). Gene expression levels were expressed as log2(RPKM+1).
  • Isolated EVs were lysed with 2 ⁇ sample buffer solution (Nacalai Tesque, Japan) without 2-ME and incubated at 70° C. for 5 minutes. Samples were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using gradient gels (E-T1020L, Atto) and subsequently electrophoretically transferred to nitrocellulose membranes. After blotting, the membrane was incubated with blocking agent Blocking One (03953-95, Nacalai Tesque) for 1 hour and then incubated with primary antibody for 24 hours at 4°C.
  • blocking agent Blocking One (03953-95, Nacalai Tesque
  • an anti-mouse or rat IgG antibody conjugated with horseradish peroxidase (HRP) was used (1:50,000).
  • HRP horseradish peroxidase
  • Can Get Signal Immunoreaction Enhancer Solution Kit (NKB-101, Toyobo) was used. Immunoreactivity was detected using an enhanced chemiluminescence kit, Immobilon Western (WBKLS0500, Merck Millipore). Signal intensities were calculated using ImageQuant TL software (GE Healthcare Life Sciences).
  • ELISA ELISA
  • RNA isolation, RT-PCR (reverse transcription PCR) Total RNA was isolated from cultured cells using RNeasy (QIAGEN). cDNA was synthesized using SuperScript III (11752-050, Thermo Fisher Scientific) according to the manufacturer's manual. Quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (4367659, Thermo Fisher Scientific) and a StepOnePlus system (Thermo Fisher Scientific). Values for each gene were normalized to the relative amount of GAPDH mRNA in each sample.
  • EVs were suspended in PBS (30 ⁇ g/mL), dropped (20 ⁇ L/drop) onto the membrane side of carbon-stabilized collodion-coated grids (400 mesh; Nisshin-EM) and left at room temperature for 10 minutes. The solution was removed with filter paper and rinsed with distilled water. A 1% solution of uranyl acetate in distilled water was applied to the grid and then left at room temperature for 1 minute. The reagent was then removed with filter paper and dried. EVs were imaged with a transmission electron microscope (TEM; H-7650, Hitachi High-Technologies, Japan).
  • TEM transmission electron microscope
  • E6.5 embryos were dissected for uterus and decidua in Dulbecco's Modified Eagle Medium (DMEM)/F12 (1:1) containing 5% FBS (SAFC Biosciences, USA) (11039-012, Thermo Fisher Scientific). separated by Isolated embryos were 0.4 ⁇ L using 500 ⁇ L DMEM (21063-029, Thermo Fisher Scientific) containing 50% rat serum, 0.1 mM NEAA, 1 mM sodium pyruvate and 0.5 mM 2-ME. Cultures were cultured in 12 mm transwells with ⁇ m pores (3460, Corning) (for low attachment).
  • E3.5 embryos were harvested by washing the uterus with KSOM medium (prepared in-house). Embryos were collected in drops of M2 medium (M7167, Sigma-Aldrich). The zona pellucida was removed by brief exposure to drops of acid Tyrode solution (T1788, Sigma-Aldrich). M2 medium drops and Tyrode's solution drops were overlaid with mineral oil (HiGROW OIL, Fuso Pharmaceutical Co., Ltd.) and pre-equilibrated at 37° C., 5% CO 2 .
  • IVC1 in vitro medium
  • DMEM/F12 Advanced DMEM/F12 (12634-010, Thermo Fisher Penicillin (25 units/mL)/streptomycin (25 ⁇ g/mL), 1 ⁇ insulin-transferrin-selenium-ethanolamine (ITS-X) (51500-056, Thermo Fisher Scientific), 8 nM-estradiol ( E8875, Sigma-Aldrich), 200 ng/mL progesterone (160-24511, Wako, Japan) and 25 ⁇ M N-acetyl-L-cysteine (A7250, Sigma-Aldrich)) was used to remove mineral oil and M2 medium.
  • ITS-X insulin-transferrin-selenium-ethanolamine
  • 8 nM-estradiol E8875, Sigma-Aldrich
  • 200 ng/mL progesterone 160-24511, Wako, Japan
  • 25 ⁇ M N-acetyl-L-cysteine
  • IVC2 medium Advanced DMEM/F12 containing 30% KSR and supplemented with 2 mM L-glutamine and penicillin (25 units/mL)). exchanged for Streptomycin (25 ⁇ g/mL), 1 ⁇ ITS-X, 8 nM-estradiol, 200 ng/mL progesterone and 25 ⁇ M N-acetyl-L-cysteine), embryo culture was performed at 37° C. in 5% CO 2 .
  • Embryos were treated with EV or polymer poly(DL-lactide-co-glycolide) (PLGA) nanoparticles every 2 days from day 0 to day 8. All animal experiments were conducted in accordance with the Japanese Guide for the Care and Use of Laboratory Animals and in accordance with Kyoto University guidelines for animal experiments.
  • PLGA polymer poly(DL-lactide-co-glycolide)
  • Biotinylated PLGA (Nanosoft Polymers, NC) with an average molecular weight of 20,000 Da and a copolymer ratio of lactide to glycolide of 75:25 was used as the nanoparticle matrix.
  • Polyvinyl alcohol (PVA-403, Kuraray) was used as a dispersant.
  • PLGA nanoparticles incorporating mirVana miRNA mimic hsa-mir-132-3p (4464066, Thermo Fisher Science) were prepared in RNAase-free water using the emulsion solvent diffusion method as previously described (Kawashima et al. , (1998), Eur. J. Pharm. Biopharm.45, 41-48).
  • miR-132-3p The sequences of human miR-132-3p and mouse miR-132-3p were identical.
  • miR-132-PLGA contained 11.6% (w/w) miR-132.
  • Particle size was determined using a sample of nanoparticle suspension in distilled water.
  • the median diameter of miR-132-PLGA based on dynamic light scattering was 262 nm.
  • ESCs are derived from the inner cell mass (ICM) of early blastocysts and can be used as a tool to study cell differentiation processes as they can differentiate in vitro from the pluripotent state into all three germ layers.
  • ICM inner cell mass
  • FIG. 1 shows the experimental system for PKA activation.
  • PKA-ESC express the constitutively active form of PKA (CA-PKA) through the doxycycline-regulated expression system (Dox-Off).
  • Dox-Off doxycycline-regulated expression system
  • PKA-ESCs were cultured in the presence of LIF and Dox to maintain an undifferentiated state. LIF is excluded for differentiation induction.
  • PKA-ESCs differentiate faster in Dox ⁇ than Dox+.
  • FIG. 38 shows a schematic diagram of aggregate culture.
  • FIG. 2 shows immunostaining of Flk1 during differentiation of PKA-ESCs.
  • FIG. 3 shows FACS analysis for the appearance of Flk1 in PKA-ESCs.
  • Figure 4 shows the percentage of PKA-ESCs expressing Flk1 by FACS analysis and the data analyzed using the Mann-Whitney U test. Under Dox- conditions, PKA-ESCs show earlier and significantly more Flk1+ cells (*P ⁇ 0.05 versus PKA-ESCs (Dox+)). This was less than 1% activation in non-activated PKA-ESCs and more than 40% in activated PKA-ESCs. Also, FIG. 39 shows FACS analysis of pure PKA-ESC aggregates of PDGFR ⁇ , and FIG.
  • FIG. 40 shows the percentage of PDGFR ⁇ +PKA-ESCs analyzed using the Mann-Whitney U test (*versus PKA-ESC (Dox+) P ⁇ 0.05),
  • FIG. 41 shows immunostaining of PDGFR ⁇ during differentiation of pure PKA-ESC aggregates.
  • PDGFR ⁇ another mesoderm marker, was also enhanced by PKA activation.
  • Figures 42-45 show the percentage of Flkl + Control-ESC and PDGFR ⁇ + Control-ESC in FACS analysis of pure Control-ESC aggregates of Flkl and PDGFR ⁇ .
  • Figures 46 and 47 are immunostainings for Flkl ( Figure 46) and PDGFR ⁇ ( Figure 47) during differentiation of pure Control-ESC aggregates. From these results, the rate of differentiation was not altered by Dox exposure. The amount of Flk1+ cells in both Dox+ and Dox- control ESCs was less than 10% (Fig. 42).
  • FIG. 5 shows a schematic of the chimeric aggregate co-culture differentiation system.
  • PKA-ESCs and Control-ESCs were seeded in low-adhesion dishes at a 3:1 ratio to generate chimeric aggregates, induction of differentiation was initiated by depletion of LIF, and 24 h later, aggregates were re-plated onto regular plates. Plated and allowed cell attachment after about 12 hours.
  • activation of PKA resulted in different differentiation kinetics between the two cell lines.
  • differentiation was expected to be accelerated only in PKA-ESCs after PKA activation with Dox depletion.
  • FIG. 6 shows immunostaining of Flk1 during differentiation of PKA-ESC and Control-ESC chimeric aggregates.
  • FIG. 7 shows FACS analysis of Flk1 appearance on chimeric aggregates. Under Dox- conditions, Flk1+ cells in the PKA-ESC population (blue) appeared earlier, but by D3.5 the proportion of Flk1+ cells in the Control-ESC population (green) was the same as the PKA-ESC population. .
  • FIG. 8 is the percentage of chimeric aggregates expressing Flk1 during differentiation, data are means ⁇ SE. D, data were analyzed using the Mann-Whitney U test (*P ⁇ 0.05 compared to Control-ESC). From these results, in chimeric aggregates, PKA activation in PKA-ESC induces the early appearance of mesoderm cells at the same level not only in PKA-ESC but also in D3.5 Control-ESC. The results were obtained.
  • FIG. 48 shows immunostaining of PDGFR ⁇ during differentiation of chimeric aggregates.
  • the number of PDGFR ⁇ + Control-ESCs (light colors) in chimeric aggregates increased in Dox-conditions.
  • Figure 49 shows FACS analysis of PDGFR ⁇ expression on chimeric aggregates. In each graph, the left side is PKA-ESC and the right side is control-ESC.
  • Figure 50 shows the percentage of differentiating chimeric aggregates expressing PDGFR ⁇ ; represents D. From these results, a similar early and synchronized appearance of PDGFR ⁇ + cells in the Control-ESC population was seen in chimeric cell aggregates after PKA activation. These results indicate that there is a new cellular mechanism that synchronizes the cellular phenotypes of different cell populations (ie PSyC).
  • EV includes exosomes and microvesicles (microvesicles, also called ectosomes).
  • Exosomes are 50-150 nm sized EVs produced by the inward budding of endosomes.
  • Microvesicles are 100-1000 nm sized EVs produced by direct budding of the plasma membrane.
  • Cells transmit several types of biomolecules via EVs, including mRNAs, miRs, and proteins.
  • FIG. 9 shows a schematic of a chimeric aggregate co-culture differentiation system with inhibited EV secretion.
  • PKA-ESCs and Control-ESCs were seeded in low-adhesion dishes at a ratio of 3:1 to generate chimeric aggregates, and induction of differentiation was initiated by depletion of LIF. After 24 hours, the aggregates were replated on regular plates and incubated with EV secretion inhibitors.
  • FIG. 10 shows the number of Flk1 cells upon treatment with manumycin A, GW4869, PKA-ESC and Control-ESC chimeric aggregates on D3.5 under Dox conditions analyzed by FACS.
  • manumycin A and GW4869 are two inhibitors of the enzyme neutral sphingomyelinase 2 (nSMase2) that regulates exosome secretion.
  • the number of Flk1+ cells was reduced by treatment with EV secretion inhibitors manumycin A (10 ⁇ M) or GW4869 (5 ⁇ M).
  • FIG. 11 shows the percentage of PKA-ESCs and Control-ESCs in chimeric aggregates expressing Flk1 under Dox-conditions. Data were analyzed using the Kruskal-Wallis test followed by the Steel-Dwass test. These results indicated that either manumycin A or GW4869 treatment had no effect on PKA-ESCs in chimeric aggregates under PKA activation at D3.5, expressing Flk1+ cells only in the Control-ESC population. was specifically and significantly reduced.
  • FIG. 12 shows immunostaining of Flk1+control-ESC (light) in chimeric aggregates using manumycin A (10 ⁇ M), GW4869 (5 ⁇ M), or DMSO (control). It was found that Flk1 expression by Control-ESCs was decreased by inhibitor treatment. These results suggest that EVs are involved in PSyC.
  • FIG. 13 shows a schematic diagram of EV collection and processing. Control-ESC aggregates were plated without LIF and EVs were collected as pellets from PKA-ESC conditioned media and added to Control-ESC aggregates. To analyze the effects of EVs secreted from PKA-ESCs, EVs were isolated in conditioned media of PKA-ESCs under inactive (EV(Dox+)) and active (EV(Dox-)) PKA conditions.
  • FIG. 14 shows an immunoblot of CD9, CD63, and CD81 in exosomal protein lysates from 10 mL of conditioned medium. These EV markers are expressed mainly in exosomes and occasionally in microvesicles. Separation of EVs was confirmed from the results in the figure.
  • FIG. 15 shows transmission electron microscopy (TEM) images of EVs in conditioned medium.
  • Figure 16 shows the analysis of EV size distribution in Dox+ (left) and Dox- (right) media. EVs were observed as 100-150 nm diameter vesicles by these electron microscopy and EV tracing analyses.
  • FIG. 51 shows EV particles in the conditioned medium
  • FIG. 52 shows the concentration of proteins contained in EVs. These results showed no significant difference in particle number and protein concentration between EV(Dox+) and EV(Dox-).
  • FIG. 17 shows FACS analysis of Flk1 + cells in EV-treated Control-ESCs on D3.5.
  • Figure 18 shows the percentage of control ESCs expressing Flk1 and the data were analyzed using the Kruskal-Wallis test followed by the Steel-Dwass test. Also shown in FIG. 53 is FACS analysis of PDGFR ⁇ expression in EV-treated Control-ESCs from day 3.5 of differentiation and PKA-ESC cultures (Dox+ or Dox ⁇ ).
  • Figure 54 shows the percentage of PDGFR ⁇ + cells and the data were analyzed using the Kruskal-Wallis test followed by the Steel-Dwass test. Data are mean ⁇ S.E.M. represents D. Addition of EVs (Dox-) to pure Control-ESC aggregates was shown to induce a much higher proportion of Flk1+ and PDGFR ⁇ + cells than without EVs or EVs (Dox+).
  • FIG. 19 shows immunostaining of mesoderm markers (Flk1, PDGFR ⁇ ) in EV-treated control-ESCs at D3.5. Data are mean ⁇ S.E.M. represents D. Consistently, immunostaining showed a dramatic increase in Flk1 and PDGFR ⁇ expression after EV(Dox-) treatment. These results indicate that EVs from activated PKA-ESCs promote mesodermal differentiation from Control-ESCs to synchronize differentiation levels.
  • mesoderm markers Flk1, PDGFR ⁇
  • FIG. 20 shows MA plots summarizing miRNAs differentially expressed in EVs from activated PKA-ESCs (Dox ⁇ ) and inactivated PKA-ESCs (Dox+). These results revealed that activated Dox- is enriched for miR-126, miR-132, miR-184, miR-193a, miR-212, and miR-483.
  • FIG. 21 shows a schematic of the experiment and a FACS plot showing recipient cells of chimeric aggregates containing miRNA-expressing cell lines on D3.5.
  • GFP+ miR-expressing cells
  • GFP ⁇ recipient cells
  • a parental mouse ESC line of miR-overexpressing cells lacked miR or GFP genes and was used as recipient cells.
  • Chimeric cell aggregates were generated containing recipient cells and each miR-expressing cell line at a 1:3 ratio.
  • FIG. 22 shows the percentage of Flk1+ recipient cells in aggregates at a 1:3 ratio and the data analyzed using the Kruskal-Wallis test followed by the Steel-Dwass test. According to these results, Flk1+ mesodermal differentiation in recipient cells (GFP ⁇ ) was significantly enhanced only when mixed with miR-132 or multimiR cell lines.
  • FIG. 56 Flk1 and PDGFR ⁇ RNA in day 3.5 control-ESCs treated with EVs from medium (Dox-) of PKA-ESCs, miR-132-KO PKA-ESCs or controls (no EVs). Expression levels are indicated. Values are normalized to controls.
  • FIG. 57 showed Flk1 mRNA expression levels in day 3.5 control-ESCs treated with or without miR-132-PLGA. Values are normalized to controls. Data were analyzed using the Mann-Whitney U test. EVs were prepared from supernatants of PKA-ESCs or miR-132 KO PKA-ESCs after PKA activation (Dox-) and added to differentiating recipient cells.
  • EVs from PKA-ESCs containing Dox- promoted mesoderm differentiation of recipient cells (similar to the results shown in Figure 17), but miR EVs derived from -132 KO PKA-ESCs (Dox-) did not promote mesoderm differentiation.
  • EV(Dox-) miR-132 is a key messenger molecule in enhancing mesodermal differentiation of recipient cells and contributes to PSyC.
  • FIG. 23 shows the results of detection of Spry1, Rasa1 and pCREB in capillary Western blot. Proteins in nuclear fractions were analyzed to determine Spry1 and pCREB expression levels and normalized with Lamin A/C. Cytoplasmic fraction proteins were analyzed to measure Rasa1 expression levels, normalized to ⁇ -actin, and data were analyzed using the Kruskal-Wallis test followed by the Steel-Dwass test. Spry1, an antagonist of the fibroblast growth factor pathway, and Rasa1, a RasGAP activator, are known targets of miR-132. The results in the figure show that treatment of Control-ESCs with EV (Dox-) significantly reduced Spry1 and Rasa1 protein levels, indicating that miR-132 acts on Control-ESCs through EV-mediated transduction.
  • Dox- significantly reduced Spry1 and Rasa1 protein levels
  • Figure 24 shows pCREB and CREB levels as measured by ELISA. Data were analyzed using the Kruskal-Wallis test followed by the Steel-Dwass test.
  • Figure 25 shows qPCR analysis of ETV2 in Control-ESCs treated with or without EVs from PKA-ESCs (Dox+ or Dox-). Data were analyzed using the Kruskal-Wallis test followed by the Steel-Dwass test. miR-132 has been reported to activate Ras/Raf1 signaling by inhibiting Spry1 and Rasa1. Raf1 has also been reported to phosphorylate CREB via the adenylate cyclase/cyclic AMP/PKA and ERK/RSK pathways.
  • pCREB enhances the expression of the ETS mutant transcription factor 2 (ETV2) and Flk1 genes, both of which are mesodermal inducers and miR-132. Therefore, it is hypothesized that decreased Spry1 and Rasa1 protein expression activates PKA signaling through pCREB, leading to mesoderm differentiation. According to the results in Figures 23-25, indeed, CREB phosphorylation and ETV2 mRNA levels in Control-ESCs are increased by the addition of EV (Dox-).
  • ETV2 ETS mutant transcription factor 2
  • Flk1 mesodermal inducers and miR-132
  • PKA-ESC-initiated PKA activation promotes Flk1+ mesodermal cell differentiation and miR-132 expression through CREB phosphorylation.
  • Activated PKA-ESCs then secrete EVs with high miR-132 content.
  • miR-132 is delivered to recipient cells via EV-mediated transfer and represses Spry1 and Rasa1. This activates the PKA pathway through CREB phosphorylation of Control-ESCs.
  • PKA activation initiated in the original PKA-ESC can be reconstituted in Control-ESC via EVs derived from PKA-ESC to evoke PSyC.
  • PSyC is a novel biological mechanism that reconstitutes similar intracellular environments of donor and recipient cells in close proximity via donor-derived EVs.
  • Figure 26 shows a schematic of the PSyC mechanism involving EV secretion and miR-132 delivery that we found.
  • FIG. 27 shows a schematic of mouse embryo culture. E6.5 mouse embryos were collected, subjected to EV (Dox-) treatment and cultured ex vivo for 2 days. Embryos were isolated and examined for PDGFR ⁇ + cells after 2 days of ex vivo culture by FACS.
  • Figure 28 shows FACS analysis of PDGFR ⁇ expression in E6.5 embryos cultured for 2 days in EVs isolated from undifferentiated (UD) control-ESCs or differentiated PKA-ESCs under Dox- conditions.
  • UD undifferentiated
  • Figure 29 shows the percentage of control ESCs expressing Flk1. Data were analyzed using the Mann-Whitney U test. The results in the figure show that significantly more PDGFR ⁇ + cells were observed in embryos when treated with EV(Dox-) compared to the untreated group, indicating that EV(Dox-) stimulated mesoderm differentiation during embryonic development. showed that it can be promoted.
  • FIG. 30 shows a schematic of mouse embryo culture. E3.5 mouse embryos were harvested and cultured ex vivo using EVs. E3.5 mouse embryos isolated from the uterus were cultured on microslides in IVC1 medium. After attaching the mouse embryos to the slides on day 2-3, the medium was changed to IVC2 medium and the embryos were cultured to day 8 or 10. EV was added once every two days.
  • FIG. 30 shows a schematic of mouse embryo culture. E3.5 mouse embryos were harvested and cultured ex vivo using EVs. E3.5 mouse embryos isolated from the uterus were cultured on microslides in IVC1 medium. After attaching the mouse embryos to the slides on day 2-3, the medium was changed to IVC2 medium and the embryos were cultured to day 8 or 10. EV was added once every two days.
  • FIG. 31 shows immunostaining of cTnT in E3.5 mouse embryos cultured for 8 days with EVs isolated from UDControl-ESCs or differentiated PKA-ESCs under Dox-conditions.
  • Figure 32 shows the percentage of cTnT+ embryos when E3.5 mouse embryos were cultured with undifferentiated ESC-derived EVs (EV(UD)) or early differentiated PKA-ESC-derived EVs (EV(Dox-)) for 10 days. rice field.
  • Figure 33 shows the percentage of beating embryos in similar cultures.
  • culturing early embryos with EV(Dox-) dramatically altered embryonic development. Appearance of cardiomyocytes with cardiac Troponin-T positive cells was enhanced in EV (Dox-) treated embryos (5 out of 17 embryos) but collected from undifferentiated Control-ESCs Embryos treated with EV (EV(UD)) were not enhanced (0/19 embryos) and untreated embryos were only slightly enhanced (1/21 embryos).
  • EV(Dox-) may convert cells into mesodermal derivatives, including cardiomyocytes, in the developing embryo.
  • miR-132 similarly affects cell fate decisions. To this end, it is formulated from the biodegradable polymer poly(DL-lactide-co-glycolide) (PLGA), which traps a variety of molecules, including nucleic acids, penetrates cell membranes, and transports the encapsulated contents to cells. In particular, we adopted artificial polymer nanoparticles that can be delivered to the cytoplasm. PLGA nanoparticles containing miR-132 (miR-132-PLGA) were formulated. From the results of FIG.
  • PLGA biodegradable polymer poly(DL-lactide-co-glycolide)
  • miR-132-PLGA added to differentiating mouse ESCs succeeded in upregulating the expression of the mesoderm marker Flk1 mRNA, and miR-132-PLGA demonstrated the function of EV (Dox-). It shows that it is possible to imitate
  • FIG. 34 shows beating mouse embryos cultured with miR-132-PLGA from E3.5 for 10 days and FIG. 35 shows the percentage of embryos beating on days 8 and 10.
  • FIG. 36 showed cTnT immunostaining in E3.5 mouse embryos cultured for 10 days with or without miR-132-PLGA.
  • Table 1 shows the results of quality confirmation when 10 ⁇ g of has-miR-132-3p (mirVana (R) miRNA mimic) was used as the input nucleic acid.
  • the recovery rate is the recovery rate by nucleic acid quantification (Ribogreen), and converted to M (mol/L) assuming that the molecular weight of miRNA is 14000. The same applies to other samples.
  • Table 2 shows the results of quality confirmation when 10 ⁇ g of has-miR-132-3p (mirVana (R) miRNA inhibitor) was used as the input nucleic acid.
  • Table 3 shows the results of quality confirmation when 10 ⁇ g of mirVana (TM) miRNA Inhibitor Negative Control #1 was used as the input nucleic acid.
  • Lyophilizer FDU-1200, using EYELA, using eggplant type $ 29: 300 mL, 500 mL, 1000 mL EYELA as a sample flask, vacuum: ⁇ 10 Pa, temperature: -48 ° C., time: freeze-drying overnight did As a specific operation, a 50 ml tube was immersed in liquid nitrogen, and while rotating, it was confirmed that the pre-frozen sample had been frozen.
  • the miRNA molecular weight is 14,000, Input: miR-132 1800 ⁇ g/PLGA 180 mg Yield: 11.19 mg as miR-132-PLGA-NP Theoretical value: 160.9 ⁇ g miR132-3p/1 mg PLGA-NP Nucleic acid quantification (NanoDrop): 116 ⁇ g RNA/1 mg PLGA-NP Nucleic acid recovery rate 72.1%, encapsulation rate 11.6% Met.
  • miRNA-encapsulating lipid nanoparticles LNPs
  • PLGA nanoparticles as described above were prepared. Also, cultured cells were prepared. Cells were cultured in RAW264.7 (P3), 1.35 ⁇ 10 6 cells/well (24-well plate), medium: DMEM (10% FBS, 1% P/S). The nanoparticles prepared above were adjusted to match the concentration of miRNA. PLGA nanoparticles: 1 mg (powder) was diluted in 60 ml of PBS, and the lipid nanoparticles were used as they were. The miRNA-encapsulated nanoparticles or miRNA (naked) were applied to the medium according to the following concentrations.
  • Fig. 59 shows a comparison of the amount of miR-132-3p for each sample.
  • Administration of miRNA alone (naked) did not result in transfection, but nano-DDS enabled miRNA transfection in a dose-dependent manner.
  • Myocardial ischemia was induced by ligating the left anterior descending artery (LAD) using an 8-0 nylon suture with a silicone tube placed laterally to the LAD. Thirty minutes after occlusion, the silicone tubing was removed to restore blood flow (reperfusion). At the time of reperfusion, miRNA-LNP containing 100 ng of miRNA (mir132-LNP or miRNA-negative control-LNP) diluted with PBS (100 microL in total) was injected through the tail vein. After treatment, mice were placed on a heating board until fully recovered.
  • mice were anesthetized with 2% isoflurane in 2 L/min oxygen and intubated 24 hours after reperfusion.
  • the left anterior descending artery was ligated and 2% Evans blue dye (Sigma-Aldrich, St. Louis, MO, USA) was injected via the inferior vena cava.
  • the heart was then excised, perfused with saline and cut into serial cross sections with a thickness of 1 mm. Sections were incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, Mo., USA) for 10 min at 37° C., then placed in formaldehyde and exposed to a stereomicroscope (Nikon, HC-2500). ) was observed.
  • TTC 2,3,5-triphenyltetrazolium chloride
  • the unstained LV area by TTC was considered the infarct area. as MI regions (TTC negative, white), non-MI regions within the AAR (TTC positive/Evans blue negative, red), non-ischemic regions (TTC positive/Evans blue positive, purple), and AAR (Evans blue negative), These were quantified using ImageJ software (version 1.51h).
  • Fig. 60 shows a micrograph of miR-132-3p-LNP administration to a mouse model of myocardial ischemia-reperfusion.
  • left (a) is control LNP (ie, liposome only)
  • right (b) is miR132-LNP, that is, miR-132 at a ratio of 100 ng/100 ⁇ L was administered.
  • the left (a) shows that the unstained (white and light colors in the figure) area is wide and the infarct area is large, while the right (b) shows that the unstained area is small. That is, in a mouse model of myocardial infarction, administration of liposome particles containing miR-132 reduced the infarcted/ischemic area of the heart.
  • Fig. 61 shows the results of quantification of the stained area.
  • FIG. 63 shows a comparison of miR-132 expression levels by RT-PCR.
  • Endogenous miR-132 expression is enhanced in the ischemic region 24 hours after coronary artery ligation in mice not administered miR-132 (Control LNP administration). It was suggested that endogenous miR-132 may play a protective role in the pathophysiology of acute myocardial infarction.
  • FIG. 64 shows a comparison of miR-132 expression levels by RT-PCR when liposomes were intravenously administered.
  • a total of 4 samples were tested in the non-ischemic area and the ischemic area, with administration of miR-132-Liposome and control (LNP only).
  • miR-132-Liposome was intravenously administered after myocardial infarction, and miR-132 delivery was confirmed in non-ischemic and ischemic areas of cardiac tissue 24 hours later. It was shown that miR-132 encapsulated in Lipid Nanoparticles was delivered to cardiac tissue and contributed to the reduction of myocardial infarction.
  • the present invention provides a composition for promoting myocardial differentiation and a pharmaceutical composition for promoting myocardial differentiation, which exhibits a high function in promoting cardiomyocyte differentiation and is effective in improving cardiac function after heart disease. can provide.

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015524844A (ja) * 2012-08-13 2015-08-27 シーダーズ−サイナイ・メディカル・センターＣｅｄａｒｓ−Ｓｉｎａｉ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ 組織再生のためのエキソソームおよびマイクロリボ核酸
US20170096671A1 (en) * 2014-05-16 2017-04-06 The Trustees Of The University Of Pennsylvania Microrna induction of cardiac regeneration

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015524844A (ja) * 2012-08-13 2015-08-27 シーダーズ−サイナイ・メディカル・センターＣｅｄａｒｓ−Ｓｉｎａｉ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ 組織再生のためのエキソソームおよびマイクロリボ核酸
US20170096671A1 (en) * 2014-05-16 2017-04-06 The Trustees Of The University Of Pennsylvania Microrna induction of cardiac regeneration

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DEVALLIERE JULIE, CHANG WILLIAM G., ANDREJECSK JILLIAN W., ABRAHIMI PARWIZ, CHENG CHRISTOPHER J., JANE‐WIT DAN, SALTZMAN W. MARK, : "Sustained delivery of proangiogenic microRNA‐132 by nanoparticle transfection improves endothelial cell transplantation", THE FASEB JOURNAL, FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, US, vol. 28, no. 2, 1 February 2014 (2014-02-01), US, pages 908 - 922, XP055970832, ISSN: 0892-6638, DOI: 10.1096/fj.13-238527 *
HIRAI KENTA, OUSAKA DAIKI, FUKUSHIMA YOSUKE, KONDO MAIKO, EITOKU TAKAHIRO, SHIGEMITSU YUSUKE, HARA MAYUKO, BABA KENJI, IWASAKI TAT: "Cardiosphere-derived exosomal microRNAs for myocardial repair in pediatric dilated cardiomyopathy", SCIENCE TRANSLATIONAL MEDICINE, vol. 12, no. 573, 1 January 2020 (2020-01-01), pages eabb3336, XP055970831 *
LEE SHARON WEI LING; PAOLETTI CAMILLA; CAMPISI MARCO; OSAKI TATSUYA; ADRIANI GIULIA; KAMM ROGER D.; MATTU CLARA; CHIONO VALERIA: "MicroRNA delivery through nanoparticles", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 313, 14 October 2019 (2019-10-14), AMSTERDAM, NL , pages 80 - 95, XP085925537, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2019.10.007 *
LIU XUELEI, TONG ZHOU, CHEN KEYAN, HU XIAOFANG, JIN HONGXU, HOU MINGXIAO: "The Role of miRNA-132 against Apoptosis and Oxidative Stress in Heart Failure", BIOMED RESEARCH INTERNATIONAL, HINDAWI PUBLISHING CORPORATION, vol. 2018, 1 January 2018 (2018-01-01), pages 1 - 8, XP055970835, ISSN: 2314-6133, DOI: 10.1155/2018/3452748 *
MA TENG, CHEN YUEQIU, CHEN YIHUAN, MENG QINGYOU, SUN JIACHENG, SHAO LIANBO, YU YUNSHENG, HUANG HAOYUE, HU YANQIU, YANG ZIYING, YAN: "MicroRNA-132, Delivered by Mesenchymal Stem Cell-Derived Exosomes, Promote Angiogenesis in Myocardial Infarction", STEM CELLS INTERNATIONAL, HINDAWI PUBLISHING CORPORATION, US, vol. 2018, 9 September 2018 (2018-09-09), US , pages 1 - 11, XP055842550, ISSN: 1687-966X, DOI: 10.1155/2018/3290372 *
MINAKAWA TOMOHIRO, TETSUYA MATOBA,FUMIYOSHI ISHIDATE,TAKAHIRO K. FUJIWARA,SHO TAKEHANA,YASUHIKO TABATA,JUN K. YAMASHITA: "Extracellular vesicles synchronize cellular phenotypes of differentiating cells", JOURNAL OF EXTRACELLULAR VESICLES, vol. 10, no. 11, 17 September 2021 (2021-09-17), pages 1 - 13, XP055970829, DOI: 10.1002/jev2.12147 *
ZHAO ZONGLEI, SONG DU, SHUXIN SHEN, LIXIA WANG: "microRNA-132 inhibits cardiomyocyte apoptosis and myocardial remodeling in myocardial infarction by targeting IL-1β", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 235, no. 3, 17 October 2019 (2019-10-17), pages 2710 - 2721, XP055970839, DOI: 10.1002/jcp.29175 *

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