WO2022113887A1 - Procédé d'évaluation de la maladie rétinienne inflammatoire, thérapie pour la maladie rétinienne inflammatoire, et procédé de criblage pour la thérapeutique de la maladie rétinienne inflammatoire - Google Patents

Procédé d'évaluation de la maladie rétinienne inflammatoire, thérapie pour la maladie rétinienne inflammatoire, et procédé de criblage pour la thérapeutique de la maladie rétinienne inflammatoire Download PDF

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WO2022113887A1
WO2022113887A1 PCT/JP2021/042479 JP2021042479W WO2022113887A1 WO 2022113887 A1 WO2022113887 A1 WO 2022113887A1 JP 2021042479 W JP2021042479 W JP 2021042479W WO 2022113887 A1 WO2022113887 A1 WO 2022113887A1
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cells
fine particles
inflammatory
extracellular
retinal disease
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淳爾 羽室
千恵 外園
茂 木下
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京都府公立大学法人
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility

Definitions

  • the present invention relates to a method for determining an inflammatory retinal disease, a method for screening an inflammatory retinal disease therapeutic agent, and a method for screening an inflammatory retinal disease therapeutic agent.
  • Extracellular fine particles such as exosomes secreted from cells contain lipids and proteins derived from cell membranes on the surface, and nucleic acids derived from intracellular substances (microRNA, messenger RNA, DNA, etc.), proteins, etc. inside. Includes.
  • the microRNA (miRNA) contained in the extracellular fine particles is a non-coding RNA consisting of about 17 to 24 nucleotides that is not translated into protein.
  • miRNA Currently, more than 2500 types of human miRNA have been discovered. This miRNA has the function of suppressing the translation of various genes having target sites complementary to itself, and controls basic biological functions such as cell development, differentiation, proliferation, and cell death. .. In this way, since microRNA regulates the expression of many genes in cells, changes in its expression are thought to be related to the development and progression of diseases. Research is also being conducted to use it for discovery.
  • Patent Document 1 describes a method for detecting bladder cancer using 27 types of miRNA whose expression fluctuates in bladder cancer as an index.
  • miRNA is closely related to diseases such as cancer, and it is considered that miRNA may be used for their diagnosis, prognosis prediction, and the like.
  • age-related macular degeneration is one of the chronic inflammatory retinal diseases and is a major cause of blindness in the elderly in developed countries (see Non-Patent Document 3).
  • various abnormalities occur in the macula in the center of the retina, causing a decrease in visual acuity.
  • Dyschromia and drusen (deposits) in the macula are seen in the early stage of the disease, but the detection of the disease is often delayed because there are few subjective symptoms.
  • abnormal angiogenesis occurs in the macula, and the disorder progresses, causing serious deterioration of visual acuity. Therefore, diagnosis and prognosis of age-related macular degeneration should be made easily and with high accuracy in the early stage of the disease. There is a need for a way to do this.
  • the present invention provides a novel determination method capable of early detection of chronic inflammatory retinal disease such as age-related macular degeneration, determination of severity, determination of therapeutic effect, etc. under the above-mentioned circumstances.
  • the purpose is. It is also an object of the present invention to provide a novel therapeutic agent for inflammatory retinal diseases such as age-related macular degeneration. Furthermore, it is also an object to provide a new screening method for a therapeutic agent for inflammatory retinal disease.
  • the gist of the present invention is as follows.
  • [1] A method for determining an inflammatory retinal disease accompanied by tissue inflammation, using extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE) as an index.
  • RPE retinal pigment epithelial cells
  • [2] The determination method according to [1], wherein the profile of miRNA contained in the extracellular fine particles (EV) is used as an index.
  • [3] The determination method according to [2], wherein the miRNA contains miR494-3p and / or miR1246.
  • the determination method according to any one of [1] to [4], which is an inflammatory disease.
  • a therapeutic agent for an inflammatory retinal disease accompanied by tissue inflammation which contains a function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE) as an active ingredient.
  • RPE retinal pigment epithelial cells
  • the inflammation according to [7] or [8], wherein the function-regulating molecule of the extracellular fine particles (EV) is a function-regulating molecule of miR494-3p and / or miR1246 contained in the extracellular fine particles (EV).
  • the function-regulating molecule of the extracellular fine particle (EV) is any one of [7] to [9], which is at least one selected from the group consisting of nucleic acid oligos, small molecule compounds, peptides and antibodies.
  • the described inflammatory retinal disease therapeutic agent is at least one selected from the group consisting of age-related yellow spot degeneration, central serous chorioretinopathy, proliferative vitreous retinopathy, and diabetic retinopathy.
  • the agent for treating an inflammatory retinal disease according to any one of [7] to [10], which is a disease.
  • Inflammation associated with tissue inflammation characterized by variability in the profile of extracellular fine particles (EV) secreted in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages.
  • RPE retinal pigment epithelial cells
  • a screening method for therapeutic agents for sexual retinal diseases [13] The screening method according to [12], wherein the extracellular fine particles (EV) are secreted from retinal pigment epithelial cells (RPE).
  • RPE retinal pigment epithelial cells
  • the screening method according to [12] or [13], wherein the variation in the profile of the extracellular fine particles (EV) is a variation in the profile of miRNA contained in the extracellular fine particles (EV).
  • the possibility, severity, therapeutic effect, etc. of an inflammatory retinal disease such as age-related macular degeneration can be easily determined with high accuracy. be able to.
  • Age-related macular degeneration has a problem that early detection is difficult because there are few subjective symptoms in the early stage of the disease in patients, but early detection is possible by the determination method of the present invention.
  • the function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE), particularly the function-regulating molecule of specific microRNA is a novel therapeutic agent for chronic inflammatory retinal diseases such as age-related macular degeneration. It is effective as.
  • the screening method of the present invention it is possible to easily search for a substance having a therapeutic effect on inflammatory retinal diseases.
  • the method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention uses extracellular fine particles (EV) produced by the retinal pigment epithelial cells (RPE) of a subject as an index.
  • EV extracellular fine particles
  • extracellular fine particles are fine particles released by cells and are vesicles that can be confirmed with an electron microscope.
  • the extracellular fine particles (EV) contain nucleic acids (microRNA, messenger RNA, DNA, etc.) derived from intracellular substances, proteins, etc., and are covered with a lipid double layer composed of cell-derived lipids and proteins. ..
  • Extracellular microparticles (EVs) are their diameter, intracellular origin, density of microparticles in sucrose, shape, sedimentation rate, lipid composition, protein markers and mode of secretion (ie, after signaling (inducible) or spontaneously (ie). It is classified into multiple types based on structural)) and so on.
  • extracellular fine particles examples include membrane particles, membrane vesicles, microvesicles, exosome-like vesicles, exosomes, ectome-like vesicles, ectosomes, exovesicles and the like.
  • exosomes are vesicles contained in blood, lymph, saliva, urine, breast milk, semen, etc., and released by substantially spherical cells distributed around a diameter of about 100 nm.
  • Molecules such as proteins, mRNA, and miRNA are contained inside the vesicle.
  • Exosomes are sometimes referred to as microvesicles or extracellular vesicles, and there are a plurality of names.
  • the particles are fractionated into 1.13 to 1.19 g / mL, and the particle diameter (diameter) by the dynamic light scattering method or the like is contained in the range of about 40 nm to about 150 nm.
  • CD9, CD63, CD81 and the like are expressed on the membrane of exosomes.
  • the amount of extracellular fine particles (EV) such as exosomes released increases, and in particular, the amount of CD63-positive exosomes released increases and specific microRNAs derived from these. Content increases.
  • the amount of extracellular fine particles (EV) released such as exosomes is significantly increased, and in particular, the amount of CD63-positive exosomes released is further increased and specific micros contained therein are further increased.
  • the RNA content is also increased. Therefore, by using the extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE) as an index, the possibility, severity, therapeutic effect, etc. of inflammatory retinal diseases such as age-related macular degeneration can be determined. It becomes possible to do.
  • the above-mentioned "using extracellular fine particles (EV) as an index” means the amount of extracellular fine particles (EV) released, the amount of CD63-positive exosomes in the extracellular fine particles (EV), and the extracellular fine particles (EV). It means that the profile of a substance contained in EV), particularly a nucleic acid derived from an intracellular substance, particularly miRNA (microRNA), is used as an index.
  • a substance contained in EV particularly a nucleic acid derived from an intracellular substance, particularly miRNA (microRNA)
  • miRNA miRNA
  • the content, expression level, release amount and the like of miR494-3p, miR1246, miR4741 and / or miR7115-p derived from mitochondria are preferable, and among them, miR494-3p and /
  • the content, expression amount, release amount and the like of miR1246 are mentioned as more preferable, and the content, expression amount, release amount and the like of miR494-3p are further mentioned as more preferable.
  • the amount of the specific miRNA (microRNA) contained in the secreted extracellular fine particles (EV) is increased. Therefore, these can be used as indicators.
  • the biological sample of the subject in the determination method of the present invention is not particularly limited as long as it is a sample containing extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE), but for example, the subject's anterior chamber water.
  • EV extracellular fine particles
  • RPE retinal pigment epithelial cells
  • Examples include blood (plasma, serum), tissue fluid and the like.
  • examples of inflammatory retinal diseases associated with tissue inflammation include age-related macular degeneration, central serous chorioretinal disease, proliferative vitreous retinopathy, and diabetic retinopathy.
  • the disease to which the determination method of the present invention is particularly suitable is age-related macular degeneration.
  • the possibility of inflammatory retinal disease, the severity of symptoms, the risk of onset, the risk of aggravation, the therapeutic effect, etc. can be determined.
  • the higher the RNA content than the reference value the higher the possibility, severity, risk of developing, and risk of aggravation of inflammatory retinal disease associated with tissue inflammation in the subject.
  • a reference value for determining the possibility of morbidity an average value, a numerical range, or the like in a healthy person can be used.
  • the value before treatment and the value after treatment are compared, and if the value is low, it is determined that the therapeutic effect was achieved. If the value does not change, it is determined that the therapeutic effect was not seen, and if the value becomes high, it is determined that the treatment has deteriorated.
  • the method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention can also be described as a method including the following steps.
  • Step of preparing a sample from a subject (2) The amount of extracellular fine particles (EV) in the sample of the subject prepared in (1), the amount of CD63-positive exosomes in the extracellular fine particles (EV), substances contained in the extracellular fine particles (EV), particularly.
  • Step of preparing a sample from a subject In this step, a biological sample is collected and prepared from a subject who is judged to have an inflammatory retinal disease accompanied by tissue inflammation.
  • the sample from the subject is not particularly limited as long as it is a sample containing extracellular fine particles (EV) produced by the subject's retinal pigment epithelial cells (RPE), and examples thereof include the subject's anterior chamber water and blood.
  • EV extracellular fine particles
  • RPE retinal pigment epithelial cells
  • miRNA miRNA
  • microRNA miRNA
  • various exosome separation kits pellet down by centrifugation, immunoprecipitation, magnetic
  • Purification with beads, fractionation by particle size, column adsorption, etc. is used to separate extracellular fine particles (EV), and the amount of obtained extracellular fine particles (EV) is measured.
  • the method for separating and recovering extracellular fine particles includes, for example, ultracentrifuging a sample of a subject at about 50,000 to 150,000 G for 0.5 to 2 hours. Prior to ultracentrifugation, the sample can be centrifuged for 0.1-2 hours at about 100-20,000 G. Extracellular fine particles (EV) are freeze-dried at 4 ° C for about 1 week, -20 ° C for about 1 month, and -80 ° C for about 6 months if they are dissolved in a solution such as PBS. If there is, it can be stored at 4 ° C for about 3 years.
  • the method for measuring the amount of extracellular fine particles (EV) is not particularly limited, but for example, a method for measuring the number of particles of extracellular fine particles (EV) in a liquid with a NanoSign LM10V-HS nanoparticle tracking system, Examples include a method for measuring the amount of protein.
  • a method for measuring the amount of CD63-positive exosomes in the obtained extracellular fine particles (EV) for example, protein quantification is performed according to a conventional method, and the amount of protein in each sample is adjusted to be equal to Western blotting. , CD63 is detected, and the amount of CD63 in each sample is analyzed.
  • the profile of the contained microRNA (expression intensity) can be analyzed using 3D-gene human microRNA chips (manufactured by Toray Industries, Inc.) or the like.
  • miRNA As the profile of the microRNA (miRNA), the content, expression level, release amount and the like of miR494-3p, miR1246, miR4741 and / or miR7115-p derived from mitochondria are preferable, and among them, miR494-3p and / Alternatively, the content, expression amount, release amount and the like of miR1246 are mentioned as more preferable, and the content, expression amount, release amount and the like of miR494-3p are further mentioned as more preferable.
  • the numerical value obtained in the step (2) above is compared with the reference value.
  • a reference value for determining the possibility of morbidity an average value, a numerical range, or the like in a healthy person can be used.
  • the value before treatment and the value after treatment are compared, and if the value is low, it is determined that the therapeutic effect was achieved. If the value does not change, it is determined that the therapeutic effect was not seen, and if the value becomes high, it is determined that the treatment has deteriorated.
  • the determination method of the present invention it is possible to easily diagnose and predict the prognosis of age-related macular degeneration in the early stage of the disease with high accuracy.
  • the diagnosis and therapeutic effect of the possibility / risk of developing inflammatory retinal disease and the risk of severe / severe symptoms are determined.
  • the judgment item is not limited to this.
  • Other determination items include determination of pre-illness, initial lesion, or pre-stage (state in which symptoms are not clear) in which a definitive diagnosis can be made for each disease.
  • the condition in which the visual field is lacking on the Amsler examination and new blood vessels are observed on the fluorescence fundus angiography is already severe age-related macular degeneration.
  • the therapeutic agent for inflammatory retinal disease of the present invention contains a function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE) as an active ingredient.
  • RPE retinal pigment epithelial cells
  • the amount of extracellular fine particles (EV) released and the above-mentioned specific miRNA (microRNA) contained therein Due to the increased amount, molecules that regulate (suppress or promote) these functions are expected to have a therapeutic effect on inflammatory retinal diseases associated with tissue inflammation.
  • the retinal pigment epithelial cells When the amount of the specific miRNA (microRNA) released in the retinal pigment epithelial cells (RPE) of a subject who develops inflammatory retinal disease accompanied by tissue inflammation is increased, the retinal pigment epithelial cells (RPE) are used. While it is possible that the specific amount of miRNA in the tissue is excessive, the amount of the specific miRNA in the retinal pigment epithelial cells (RPE) (including those containing mitochondria) decreases due to the increase in the release amount. It is possible that it is. Therefore, it is considered that regulating (suppressing or promoting) the expression of these miRNAs according to the state of the disease of the subject leads to the treatment of the disease.
  • microRNA specific miRNA released in the retinal pigment epithelial cells
  • the function-regulating molecule of the extracellular fine particles (EV) is not particularly limited as long as it is a molecule that regulates the function of the extracellular fine particles (EV), but the production (release) of the extracellular fine particles (EV) is limited. ) Is a broad concept including molecules that regulate. Further, as a function-regulating molecule of extracellular fine particles (EV), a function-regulating molecule of microRNA (miRNA) contained in extracellular fine particles (EV) is given as a preferable example, and among them, extracellular fine particles (EV) are contained.
  • miRNA microRNA
  • a function-regulating molecule of miR494-3p, miR1246, miR4741 and / or miR7110-5p is more preferably a function-regulating molecule of miR494-3p, miR1246, miR4741 and / or miR7110-5p, further preferably a function-regulating molecule of miR494-3p and / or miR1246, and a function-regulating molecule of miR494-3p.
  • these function-regulating molecules include nucleic acid oligos, small molecule compounds, peptides, antibodies, and the like, and specific examples thereof include nucleic acid oligos that regulate the function of the specific microRNA (miRNA). ..
  • nucleic acid oligo introduction of mimic
  • nucleic acid oligo introduction of inhibitor
  • the nucleic acid oligo is preferably modified for the purpose of suppressing decomposition when administered to a living body and / or for the purpose of specifically binding to a diseased site.
  • siRNA means a double-stranded RNA consisting of short strands in a range that does not show toxicity in cells, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs.
  • shRNA is RNA in which a single-stranded RNA constitutes a double strand via a hairpin structure.
  • nucleic acid oligo in the present invention is an oligo composed of natural and unnatural RNA or DNA, and means a nucleic acid oligomer that controls the functions of miR494-3p, miR1246, miR4741 and / or miR7110-5p.
  • Nucleic acid oligos in the present invention include miRNA, siRNA, shRNA, antisense nucleic acid, decoy nucleic acid, and nucleic acid aptamer.
  • SiRNA and shRNA do not have to be exactly the same as the target gene, but have at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence homology.
  • the portion of double-stranded RNA in which RNAs are paired with each other in siRNA and shRNA is not limited to a completely paired one, but is not limited to a completely paired one, but is a mismatch (corresponding bases are not complementary) and bulge (base corresponding to one strand). It may include a non-matching portion due to (there is no) or the like. In the present invention, both the bulge and the mismatch may be contained in the double-stranded RNA region in which the RNAs in the dsRNA are paired with each other.
  • the therapeutic agent for inflammatory retinal disease in the present invention may contain a pharmaceutically acceptable material such as a preservative or a stabilizer.
  • a pharmaceutically acceptable material such as a preservative or a stabilizer.
  • Acceptable means that the material itself may have the above-mentioned therapeutic effect or may not have the above-mentioned therapeutic effect, and may be the material that can be administered together with the above-mentioned therapeutic agent. Means material. Further, a material having no therapeutic effect and having a synergistic effect or an additive stabilizing effect when used in combination with a therapeutic agent for inflammatory retinal disease may be used.
  • Examples of the material permitted in the formulation include sterile water, physiological saline, preservatives, stabilizers, excipients, buffers, preservatives, surfactants, chelating agents (EDTA, etc.), binders, and the like. Can be done.
  • an isotonic solution containing, for example, physiological saline, glucose and other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol) , Sodium chloride), suitable solubilizers such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbitol 80, HCO-50), etc. May be good.
  • a diluent, a solubilizing agent, a pH adjuster, a pain-relieving agent, a sulfur-containing reducing agent, an antioxidant and the like may be further contained.
  • the therapeutic agent for inflammatory retinal disease of the present invention can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylate]), or a colloidal drug delivery system (liposomes, albumin microspheres). , Microemulsions, nanoparticles and nanocapsules, etc.). Further, a method of making a drug a sustained release drug is also known and can be applied to the present invention.
  • the pharmaceutically acceptable carrier to be used is appropriately selected from the above or in combination depending on the dosage form, but is not limited thereto.
  • the therapeutic agent for inflammatory retinal disease of the present invention is used as a human medicine, it is possible to formulate and administer these substances by a known pharmaceutical method, in addition to directly administering these substances to patients. be.
  • the above-mentioned materials that are acceptable for the formulation may be added.
  • the therapeutic agent for inflammatory retinal disease in the present invention can be administered in the form of a pharmaceutical product, and can be administered orally or parenterally systemically or topically.
  • intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral intestinal solvent, etc. can be selected, and the administration method should be appropriately selected according to the age and symptoms of the patient.
  • the effective dose is selected in the range of 0.000001 mg to 1 g per 1 kg of body weight at a time.
  • a dose of 0.00001 to 100 mg, preferably 0.0001 to 50 mg per patient can be selected.
  • Examples of the inflammatory retinal disease in the therapeutic agent for inflammatory retinal disease of the present invention include age-related yellow spot degeneration, central serous chorioretinopathy, proliferative vitreous retinopathy, diabetic retinopathy and the like.
  • the disease for which the therapeutic agent for inflammatory retinal disease of the present invention is particularly effective is age-related macular degeneration.
  • the method for screening a therapeutic agent for an inflammatory retinal disease associated with tissue inflammation of the present invention is a variation in the profile of extracellular fine particles (EV) secreted in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages. Is a feature.
  • RPE retinal pigment epithelial cells
  • monocytes / macrophages constructed by the present inventors as a system for mimicking the state of inflammatory retinal disease accompanied by tissue inflammation, extracellular fine particles secreted.
  • the amount of (EV) increases, and the amount of the specific miRNA (microRNA) contained therein increases.
  • the screening method of the present invention the amount of extracellular fine particles (EV) secreted in the co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages, and the above-mentioned specific miRNA (micro) contained therein.
  • EV extracellular fine particles
  • RPE retinal pigment epithelial cells
  • micro macrophages
  • Substances that are effective in reducing the amount of RNA) can be screened.
  • the substance selected by the screening method of the present invention can be expected to have an effect of improving and treating the symptoms of inflammatory retinal disease accompanied by tissue inflammation.
  • the specific miRNA is preferably mitochondrial-derived miR494-3p, miR1246, miR4741 and / or miR7115-5p, more preferably miR494-3p and / or miR1246p, and miR494-3p. It is even more preferable to have.
  • the screening method of the present invention can also be described as a method including the following steps.
  • Step of preparing a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages (2) Step of adding a test substance to the co-culture system prepared in (1) and culturing for a certain period of time.
  • RPE retinal pigment epithelial cells
  • a substance contained in EV particularly a nucleic acid derived from an intracellular substance, particularly a profile of miRNA (microRNA), specifically, the content of the above-mentioned specific miRNA (microRNA) derived from mitochondria
  • microRNA miRNA
  • Step of preparing a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages As the retinal pigment epithelial cell (RPE), a primary cultured cell of retinal pigment epithelial cell (RPE) collected from human retinal tissue, a cell established from these, or the like may be used, or from iPS cells. Retinal pigment epithelial cells (RPE) (iPS-hRPE cells) produced by induction may be used.
  • RPE retinal pigment epithelial cells
  • iPS-hRPE cells Retinal pigment epithelial cells
  • monocytes / macrophages in this step monocytes / macrophages isolated from human blood, tissues, etc. may be used, or THP-1 cells, which are established cells, may be treated with PMA or the like by a conventional method.
  • THP-1 cells which are established cells, may be treated with PMA or the like by a conventional method.
  • Cells differentiated into macrophages may be used.
  • the retinal pigment epithelial cells for example, iPS-hRPE cells
  • monocytes / macrophages for example, PMA-THP-1 cells
  • co-culture both cells may be mixed and cultured in one incubator, or one may be adherently cultured and the other cell may be seeded after a certain period of time has passed.
  • iPS-hRPE cells and iPS-hRPP cells using Transwell (registered trademark) cell culture inserts (manufactured by Corning Inc.), Nico-1 (registered trademark) culture system (Ginreilab Inc., Uchinada, Ishikawa, Japan) and the like. Co-culture may be carried out under the condition that the cells do not come into direct contact with each other.
  • the medium used in the co-culture is not particularly limited as long as it is usually used for general cell culture or culture of the above-mentioned retinal pigment epithelial cells (RPE) and monocytes / macrophages.
  • Serum such as fetal bovine serum may be added to the medium, but a serum-free medium is preferable in order to eliminate the influence of extracellular fine particles brought in from the serum.
  • the conditions for inducing inflammation by adding the above LPS or the like include, for example, LPS of 1 ng to 500 ng / mL, preferably 10 ng to 200 ng / mL, more preferably 50 ng to 150 ng / mL, still more preferably 80 ng to 120 ng / mL.
  • LPS LPS of 1 ng to 500 ng / mL, preferably 10 ng to 200 ng / mL, more preferably 50 ng to 150 ng / mL, still more preferably 80 ng to 120 ng / mL.
  • the conditions added so as to have a concentration can be mentioned.
  • the number of each cell in the co-culture can be appropriately adjusted according to the size, morphology and the like of the incubator.
  • specific co-culture for example, when a 24-well plate is used, 1.0 ⁇ 10 4 cells to 1.0 ⁇ 10 6 cells / well, preferably 5.0 ⁇ 10 4 cells to 5.0 ⁇ .
  • PMA-THP-1 cells of about 10 5 cells, more preferably about 2.5 ⁇ 10 5 cells are seeded, cultured for about 30 minutes to 12 hours, and then 5.0 ⁇ 10 3 cells to 5.0 ⁇ 10 5 IPS-hRPE cells of cells / well, preferably 2.0 ⁇ 10 4 cells to 2.0 ⁇ 10 5 cells, more preferably 1.0 ⁇ 10 5 cells, can be seeded and co-cultured.
  • test substance A step of adding the test substance to the co-culture system prepared in (1) and culturing for a certain period of time: In this step, the test substance is added to the co-culture system prepared in (1), and the culture is carried out for a certain period of time.
  • the test substance include small molecule compounds, natural products, nucleic acid oligos, peptides, antibodies and the like, and the amount of addition can be appropriately adjusted according to the substance.
  • the culture supernatant after adding the test substance and culturing for a certain period in the above step (2) is collected, and the amount of extracellular fine particles (EV) in the culture supernatant and the extracellular fine particles (EV) are collected.
  • the amount of CD63-positive exosomes, substances contained in extracellular microparticles (EV), especially nucleic acids derived from intracellular substances, especially the profile of miRNA (microRNA), specifically specific miRNA (microRNA) derived from mitochondria. ), Etc. are measured.
  • the amount of extracellular fine particles (EV) in the sample of the subject prepared in step (2) "(1) of the determination method of the present invention described above, in the extracellular fine particles (EV)".
  • the “sample of the subject” can be replaced with the "culture supernatant” and applied.
  • Step of determining whether or not the test substance is suitable as a therapeutic agent for inflammatory retinal diseases accompanied by tissue inflammation In this step, the numerical values obtained in the above step (3) and the numerical values of the negative target are compared. At this time, as the negative target, the numerical value obtained in the test to which the test substance is not added can be adopted. Amount of extracellular fine particles (EV) in the culture supernatant and amount of CD63-positive exosomes in the extracellular fine particles (EV) due to the addition of the test substance, as compared with the values obtained in the test without the addition of the test substance.
  • EV extracellular fine particles
  • EV extracellular fine particles
  • nucleic acids derived from intracellular substances especially the profile of miRNA (microRNA), specifically the content of specific miRNA (microRNA) derived from mitochondria is reduced. If so, it is judged to be a substance that can be expected to be effective as a therapeutic agent for inflammatory retinal diseases.
  • the screening method of the present invention it is possible to easily screen substances that are effective in treating inflammatory retinal diseases associated with tissue inflammation such as age-related macular degeneration.
  • RPE retinal pigment epithelial cells
  • hRPE cells the paper by Sugita et al. (Sugita S. et. Al., Lack of T cell response to iPSC-derivated retinal pigment epithelium cells from epithelium cell6 cell16sor6sol6s. The cells prepared by inducing from iPS cells according to the method described in the above were used.
  • HRPE cells produced by inducing from iPS cells (hereinafter, also referred to as "iPS-hRPE cells”) and PMA-treated THP-1 cells (hereinafter, also referred to as “PMA-THP-1 cells”) are serum-free medium.
  • Co-culture was performed in DMEM / F12 in the presence or absence of 100 ng / mL LPS for 24 hours. Specifically, 2.5 ⁇ 10 5 cells / well PMA-THP-1 cells were seeded on a 24-well plate (manufactured by Corning Inc.) and cultured for 4 hours. Then, 1.0 ⁇ 10 5 cells / well iPS-hRPE cells were seeded and co-cultured for 24 hours (T / R).
  • the comparison targets were PMA-THP-1 cells only (T) or iPS-hRPE cells only (R).
  • the culture supernatant of each well was collected, centrifuged at 2000 ⁇ g for 10 minutes, and the obtained supernatant was passed through a 0.22 ⁇ m filter (manufactured by Millipore) to prepare a sample for quantification of various inflammatory cytokines.
  • the concentrations of IL-6, MCP-1, IL-8, VEGF, TNF- ⁇ , and PEDF in each sample were measured using an ELISA kit (manufactured by BD Bioscience). Four points were measured for each sample, and the average value was taken as the concentration of each sample and is shown in FIG.
  • results of this test 1 show that when retinal pigment epithelial cells and macrophages coexist and inflammation is induced by LPS, the inflammatory cytokines IL-6, MCP-1, IL-8, and VEGF are produced. The amount is significantly increased, indicating that it moves in a direction that further exacerbates inflammation.
  • Test 2 Production of CD63 + exosomes in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages iPS-hRPE cells and PMA-THP-1 cells were placed in a 24-well plate in a serum-free medium (test 2). The cells were co-cultured in the presence or absence of LPS), and the amount of CD63 + exosomes produced was measured. Specifically, 2.5 ⁇ 10 5 cells / well PMA-THP-1 cells were seeded on a 24-well plate (manufactured by Corning Inc.) and cultured for 4 hours.
  • RPE retinal pigment epithelial cells
  • monocytes / macrophages iPS-hRPE cells and PMA-THP-1 cells were placed in a 24-well plate in a serum-free medium (test 2). The cells were co-cultured in the presence or absence of LPS), and the amount of CD63 + exosomes produced was measured. Specifically, 2.5 ⁇ 10 5 cells
  • 1.0 ⁇ 10 5 cells / well iPS-hRPE cells were seeded and co-cultured for 24 hours (T / R).
  • the comparison targets were PMA-THP-1 cells only (T) or iPS-hRPE cells only (R).
  • the culture supernatant of each well was collected, centrifuged at 2,000 ⁇ g for 10 minutes, and the obtained supernatant was passed through a 0.22 ⁇ m filter (manufactured by Millipore). Then, the pellets were collected by ultracentrifugation (100,000 ⁇ g, 90 minutes) using an MLS50 swing rotor (manufactured by Beckman Coulter).
  • the pellet was suspended in PBS and ultracentrifuged (100,000 ⁇ g, 90 minutes) was performed again.
  • the obtained pellet was suspended in PBS and protein quantification was performed according to a conventional method.
  • Western blotting was performed by adjusting the amount of protein in each sample to be equal, CD63 was detected, and the amount of CD63 in each sample was compared. The results are shown in FIG. 2 (a).
  • co-culture was performed using Transwell (registered trademark) cell culture inserts (manufactured by Corning Inc.) under the condition that iPS-hRPE cells and PMA-THP-1 cells do not come into direct contact with each other. Specifically, 700 ⁇ L of 5.0 ⁇ 10 5 cells / mL PMA-THP-1 cells was seeded in the lower chamber, and 300 ⁇ L of 2.0 ⁇ 10 5 cells / mL iPS-hRPE cells was seeded in the upper chamber. , Was co-cultured for 24 hours. A test was also conducted in which the cells seeded in the upper and lower chambers were replaced.
  • Transwell registered trademark
  • the number of fine particles contained in) was measured using a NanoSigt LM10V-HS nanoparticular tracing system (manufactured by Quantum Design).
  • Western blotting was performed on the sample (CS) of each culture supernatant and the sample (UC) obtained by ultracentrifuging the sample (CS) of each culture supernatant by the same method as in Test 2 above, and CD63 was detected. ..
  • the results are shown in FIG.
  • the number of fine particles secreted by co-culturing iPS-hRPE cells and PMA-THP-1 cells as compared with the case of culturing with only one of the cells. It turned out that it increased significantly.
  • FIG. 3 (b) it was found that the proportion of CD63 + exosomes in the fine particles was significantly increased by co-culture. That is, the co-culture increased the amount of CD63 + exosomes secreted among the fine particles.
  • Test 4 Analysis of microRNA in extracellular fine particles whose production is enhanced in a co-culture system of retinal pigment epithelial cells (RPE) and monocytes / macrophages.
  • the profile of the microRNA contained in the cells was analyzed using 3D-gene human microRNA chips (manufactured by Toray Co., Ltd.).
  • Table 1 below, for each microRNA, the expression intensity (THP-1 + RPE) in the co-cultured sample of iPS-hRPE cells and PMA-THP-1 cells is shown by the expression intensity (RPE) in the iPS-hRPE cell single culture sample. The divided value (ratio) is shown. # 0228, # 0259, and # 0619 are independent test numbers.
  • FIG. 4 a Venn diagram shows a set of microRNAs whose expression was increased by co-culture in each test.
  • miR-494-3p showed the largest increase in the first, second, and fourth place among the three measured values (Table 1). See).
  • the expression of the specific microRNA in the secreted microparticles such as exosomes increases, especially miR-494-3p.
  • miR-494-3p was considered to be usable as a marker for inflammatory diseases of the retina in determining the susceptibility, severity, and therapeutic effect of the disease.
  • miR1246 can also be used as a marker for inflammatory diseases of the retina in determining the susceptibility, severity, and therapeutic effect of the disease.
  • the Transwell (registered trademark) system has some limitations. Since the content of the upper chamber is smaller than that of the lower chamber, fine particles may preferentially settle at the bottom of the lower chamber. Also, a 0.40 ⁇ m filter is not sufficient to block cell penetration into other chambers. Therefore, a Nico-1 (registered trademark) system, which is a horizontally connected double chamber, was used, and a comparison was made between the case where a 0.03 ⁇ m filter was attached and the case where a 0.03 ⁇ m filter was attached between the two chambers. Both chambers each hold a final volume of 1.5 mL.
  • iPS-hRPE using Transwell (registered trademark) cell culture cells (manufactured by Corning) and Nico-1 (registered trademark) culture system (Ginreilab Inc., Uchinada, Ishikawa, Japan) with iPS-hRP. Co-culture was performed under the condition that one cell did not come into direct contact with the cell. Specifically, in Transwell®, 700 ⁇ L of 5.0 ⁇ 10 5 cells / mL PMA-THP-1 cells in the lower chamber and 300 ⁇ L of 2.0 ⁇ 10 5 cells / mL iPS-hRPE cells. was seeded in the upper chamber and co-cultured for 24 hours.
  • Transwell® 700 ⁇ L of 5.0 ⁇ 10 5 cells / mL PMA-THP-1 cells in the lower chamber and 300 ⁇ L of 2.0 ⁇ 10 5 cells / mL iPS-hRPE cells.
  • Nico-1® 1.0 ⁇ 10 5 cells PMA-THP-1 cells were seeded in one chamber and 2.0 ⁇ 10 5 cells iPS-hRPE cells were seeded in the other chamber. Co-culture was performed for 24 hours.
  • Nico-1® as described above, comparison was made with and without a 0.03 ⁇ m filter between the two chambers. Western blotting was performed on the sample obtained by ultracentrifuging each culture supernatant by the same method as in Test 2 above, and CD63 was detected. The results are shown in FIGS. 5 (a) and 5 (b).
  • IPS-hRPE cells and PM cells directly with iPS-hRPE cells using Transwell® cell culture plants (Corning) and Nico-1® culture system (Ginreilab Inc., Uchinada, Ishikawa, Japan). Co-culture was performed under the condition of no contact. Specifically, in Transwell®, 700 ⁇ L of 5.0 ⁇ 10 5 cells / mL PMA-THP-1 cells in the lower chamber and 300 ⁇ L of 2.0 ⁇ 10 5 cells / mL iPS-hRPE cells. was seeded in the upper chamber and co-cultured for 24 hours.
  • the concentrations of IL-6, MCP-1, IL-8, VEGF, TNF- ⁇ , and PEDF in each sample were measured using an ELISA kit (manufactured by BD Bioscience). The ELISA measurement was performed in 3 divided doses and repeated as 3 independent experiments. The average value was calculated for each sample and the concentration of each sample is shown in FIGS. 6 (a) and 6 (b).
  • TNF ⁇ Production of TNF ⁇ from macrophages by exosome stimulation derived from retinal pigment epithelial cells (RPE) and production of MCP-1 and IL-6 from RPE cells Cell-cell interaction considered to be involved in pathogenesis
  • RPE retinal pigment epithelial cells
  • MCP-1 and IL-6 production of MCP-1 and IL-6 from RPE cells
  • the concentrations of TNF- ⁇ , MCP-1, and IL-6 in each sample were measured using an ELISA kit (manufactured by BD Bioscience). Particle concentration was measured with the NanoSigt LM10V-HS nanoparticle tracking system. The results are shown in FIGS. 7 (a) and 7 (b).
  • Exosomes derived from iPS-hRPE cells increased the production of TNF- ⁇ from PMA-THP-1 cells in a concentration-dependent manner.
  • the exosomes derived from the co-culture of iPS-hRPE cells and PMA-THP-1 had a greater effect of promoting the production of TNF- ⁇ from PMA-THP-1 cells than the exosomes derived from iPS-hRPE cells alone.
  • the number of particles in the culture supernatant was 3.5 times, and it was supercentrifuged. The concentrated one was 8.5 times, and the co-culture increased remarkably.
  • TNF- ⁇ showed increased production of MCP-1 and IL-6 in parallel with increased production of CD63 + protein in microparticles from iPS-hRPE cells, but increased production of microparticles was confirmed. It was not done (Fig. 7 (b)).
  • the inflammation exacerbation circuit shown in FIG. 7 (c) is involved in the pathogenesis. That is, when an inflammatory stimulus as symbolized by LPS is applied to the RPE, a signal stimulus is applied to the RPE via a receptor related to natural inflammation such as TLR4 existing on the cell. This is thought to lead to EV (fine particle) production in the presence of RPE alone. When this EV is produced, it acts on macrophages that have infiltrated the inflamed area in the vicinity and induces the production of TNF- ⁇ . TNF- ⁇ production is sufficiently induced even with fine particles concentrated twice by ultracentrifugation from the supernatant of the single culture, but the induction activity of the co-culture supernatant is higher.
  • the amount of fine particles produced is considerably enhanced in the co-culture as described above. It is presumed that some Exosomes in the fine particles are responsible for the TNF- ⁇ production-inducing activity. There is no activity in the culture supernatant or the remaining supernatant after ultracentrifugation of the microparticles. This TNF- ⁇ then acts on the nearby RPE, inflammatory cytocan from the RPE, MCP-1 (has the effect of migrating macrophages to the inflammatory site), IL-6 (involved in choroidal tissue fibrosis). , VEGF (involved in angiogenesis) production is thought to be enhanced and EV production induced.
  • EV production from RPE is not significantly enhanced, but CD63-positive exosomes contained in EV are increased. Similarly, it is considered that this circuit is rotated to enhance EV production, inflammatory cytokine production, and local macrophages migration by MCP-1. That is, it is considered that the coexistence of RPE and macrophages and the cell-cell interaction mediated by microparticles (exosomes) establish an inflammatory exacerbation circuit and are involved in the formation of pathological conditions.
  • Exosome miRNAs in the interaction of retinal pigment epithelial cells (RPE) and macrophages The role of microparticles (exosomes) produced in co-culture of iPS-hRPE cells and PMA-THP-1 cells was analyzed in terms of low molecular weight RNA profile in secreted microparticles (exosomes). Fine particle concentration was measured by nanoparticle follow-up analysis. Table 2 summarizes the concentrations of microparticles released per 106 cells in the three cultures (iPS-hRPE cells, PMA-THP-1 cells, co-culture).
  • the base sequence "UGAAACAUACACGGGAAACCUC” in the above miR494-3p mimic shows the sequence of miR-494-3p (SEQ ID NO: 1)
  • the base sequence "AAUGGAUUUUUGGAGCAGG” in the miR1246 mimic shows the sequence of miR-1246 (SEQ ID NO: 2).
  • FCCP Carbonyl cyanide-p-trifluoromethoxyphrydrazone
  • ROT / AA Rotenone / Antimycin A
  • OCR Oxygen Consumption Rate (pmole / min) (oxygen consumption rate), which is a value indicating the oxygen consumption of cells.
  • ECAR Extracellular Acidification Rate (mpH / min) (extracellular acidification rate), which indicates the rate of extracellular pH production by metabolites excreted extracellularly. Both are widely used as indicators of energy metabolism at the cellular level. The results are shown in FIGS. 8 (a) to 8 (c).
  • Mitochondrial function repair by mimic introduction of miR494-3p and miR1246 (primary cultured HRPE cells vs ARPE19 cell line) Human primary retinal pigment epithelial (HRPE) cells (P3) and ARPE19 cell line (P25) were seeded at 16 ⁇ 10 4 cells / well on a 6-well plate, respectively. The next day, the whole medium was replaced with DMEM (+ 10% FBS), and 1 hour later, miR494-3p mimic or miR1246 mimic was transfected. Twenty-four hours after Transfection, cells were seeded on XFe24 plates at 6 ⁇ 10 4 cells / 100 ⁇ L / well.
  • FIG. 9 (a) and Table 3 below primary cultured human RPE cells
  • FIG. 9 (b) and Table 4 below ARPE 19 cell line
  • miR494-3 pinhibitor Anti-miR TM miRNA hsa-miR-494-3p, MH12409, Thermo Fisher Scientific, Waltham, MA, USA
  • miR1246inhibitor Anti-miR TM miRNA hsa-miR-1246, MH13182, Thermo Fisher Scientific, Waltham, MA, USA
  • FIG. 10 (a) and Table 5 below Nic-ARPE cell line
  • FIG. 10 (b) and Table 6 below ARPE 19 cell line).
  • Plasma 6 control samples 100 ⁇ L each, AMD patient 5 samples 100 ⁇ L each
  • Aqueous humor control 5 samples 50 ⁇ L each, AMD patient 5 samples 50 ⁇ L each
  • miRNA was extracted using miRNeasy Serum / Plasma kit (QIAGEN, Hilden, Germany) (extraction amount 10 ⁇ L). Reverse transcription reaction was performed using primers specific for each miRNA, and quantitative PCR was performed. The relative amount of miRNA was quantified by the ⁇ Ct method from each CT value and the CT value of the control using cel-miR-39 as a control (spire-in control) (the number of cycles of quantitative PCR was 40). The results are shown in FIG. 11 (miR-494-3P) and FIG. 12 (miR-1246).
  • the method for determining an inflammatory retinal disease associated with tissue inflammation of the present invention it is possible to easily determine the susceptibility, severity, therapeutic effect, etc. of a chronic inflammatory retinal disease such as age-related macular degeneration.
  • Age-related macular degeneration has a problem that early detection is difficult because there are few subjective symptoms in the early stage of the disease in patients, but early detection is possible by the determination method of the present invention.
  • the function-regulating molecule of extracellular fine particles (EV) produced by retinal pigment epithelial cells (RPE), particularly the function-regulating molecule of specific microRNA is a novel therapeutic agent for chronic inflammatory retinal diseases such as age-related macular degeneration. It is effective as.
  • the screening method of the present invention it is possible to easily search for a substance having a therapeutic effect on inflammatory retinal diseases.

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Abstract

La présente invention a pour objet de fournir un nouveau procédé d'évaluation permettant la détection précoce, l'évaluation de la gravité et l'évaluation de l'effet thérapeutique, etc., des maladies rétiniennes inflammatoires chroniques, y compris la dégénérescence maculaire liée à l'âge. La présente invention vise également à fournir une nouvelle thérapie pour les maladies rétiniennes inflammatoires, y compris la dégénérescence maculaire liée à l'âge, et à fournir un nouveau procédé de criblage pour les thérapies des maladies rétiniennes inflammatoires.　La présente invention concerne un procédé d'évaluation des maladies rétiniennes inflammatoires associées à une inflammation tissulaire, les vésicules extracellulaires (VE) produites par les cellules épithéliales pigmentaires de la rétine (EPR) d'un sujet étant utilisées comme indicateur. Ce procédé d'évaluation se caractérise par le fait que le profil des miARN contenus dans les vésicules extracellulaires (VE) (notamment miR494-3p et/ou miR1246) est utilisé comme indicateur.
PCT/JP2021/042479 2020-11-27 2021-11-18 Procédé d'évaluation de la maladie rétinienne inflammatoire, thérapie pour la maladie rétinienne inflammatoire, et procédé de criblage pour la thérapeutique de la maladie rétinienne inflammatoire WO2022113887A1 (fr)

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