Kit for inducing differentiation into retinal ganglion cell, pharmaceutical composition, and glaucoma model

Abstract

Provided are: a kit for inducing differentiation, which is for inducing the differentiation of a Muller glial cell into a retinal ganglion cell in vivo in a mammal; and a pharmaceutical composition for treating glaucoma or optic neuropathy. The kit and the pharmaceutical composition each include: a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1.

Description

Kit for inducing retinal ganglion cell differentiation, pharmaceutical composition, and glaucoma model

[0001] The present invention relates to a kit for inducing differentiation of retinal ganglion cells, a pharmaceutical composition, and a glaucoma model. More specifically, the present invention relates to a kit for inducing differentiation of mammalian Müller glial cells into retinal ganglion cells in vivo, a pharmaceutical composition, and a glaucoma model. This application claims priority based on Japanese Patent Application No. 2024-020374, filed February 14, 2024, the contents of which are incorporated herein by reference.

[0002] Glaucoma is a disease that damages the optic nerve connecting the eyeball to the brain, resulting in gradual loss of visual field. Approximately 76 million people worldwide suffer from glaucoma. Once vision is lost, it cannot be restored, and it can eventually lead to blindness. Similarly, optic neuropathy (traumatic optic neuropathy, compressive optic neuropathy, ischemic optic neuropathy, toxic optic neuropathy, optic nerve atrophy due to optic neuritis, etc.) damages the optic nerve connecting the eyeball to the brain, causing symptoms such as a sudden loss of vision in one eye, or sometimes both eyes, and a central scotoma, which blocks the center of the visual field, and can even lead to blindness.

[0003] Retinal ganglion cells, which are damaged by glaucoma and optic neuropathy, are located at the innermost part of the retina. They have axons (optic nerves) 35–50 mm long and play an important role in transmitting light information to the brain. Currently, intraocular pressure (IOP) reduction therapy is the only proven effective treatment for glaucoma. However, IOP reduction therapy only slows the progression of glaucoma and does not restore lost nerves, resulting in loss of visual field. Furthermore, there are many glaucoma patients who do not respond to IOP reduction therapy. Optic neuropathy can be treated with conservative treatments, primarily steroids, or surgical procedures, such as optic canal decompression. However, these treatments often fail to restore vision, especially in cases of traumatic optic neuropathy, where the optic nerve remains atrophied. Therefore, the emergence of regenerative medicine to regenerate retinal ganglion cells is anticipated.

[0004] One possible regenerative medicine approach to treating glaucoma and optic neuropathy is to generate retinal ganglion cells from iPS cells and transplant them into the eye. For example, Non-Patent Document 1 reports that iPS cells were differentiated into a three-dimensional retina over approximately 120 days, and that highly purified retinal ganglion cells were isolated from the three-dimensional retina using an immunopanning method. However, Non-Patent Document 2, for example, describes that retinal ganglion cells derived from immature mice rarely survive when transplanted into adult mice.

[0005] Thus, although it is currently possible to generate retinal ganglion cells from iPS cells, transplantation of retinal ganglion cells into the eye and their survival has not been successful, even in mice. Furthermore, from the standpoints of efficiency and cost, it is thought that a therapy in which retinal ganglion cells are generated from iPS cells and transplanted into the eye will be difficult to popularize as a general treatment for patients with glaucoma or optic neuropathy. Furthermore, even if retinal ganglion cells transplanted into the eye could be successfully engrafted, they would still need to propagate their long axons to the brain, making it a long road to clinical application.

[0006] Recently, direct reprogramming, which induces differentiation of somatic cells into target cells without going through the stage of pluripotent stem cells such as iPS cells, has attracted attention. In vertebrates such as fish, Müller glia cells, one of the cells that make up the retina, can be differentiated into retinal ganglion cells even after the retina has matured. However, this pathway was thought to be blocked in mammals.

[0007] In contrast, it has been reported recently that direct reprogramming of Müller glial cells in young mice has been successful in inducing their differentiation into retinal ganglion cells (see, for example, Non-Patent Document 3). Furthermore, Non-Patent Document 4 reports that direct reprogramming of Müller glial cells in young and adult mice (33 to 257 days old) has been successful in inducing their differentiation into amacrine cells, one of the cells that make up the retina, using trichostatin A, a histone deacetylase (HDAC) inhibitor, which is an epigenetic drug.

[0008] Since retinal ganglion cells are damaged in glaucoma, an ideal pathological model of glaucoma would be one in which only retinal ganglion cells are damaged. However, there is currently no ideal glaucoma model in monkeys.

[0009] Models that increase intraocular pressure in monkeys may fail to sufficiently increase intraocular pressure, or may induce excessive intraocular pressure, leading to ciliary shock and hypotony. Ciliary shock is one of the body's defenses against excessive intraocular pressure, and is a phenomenon in which aqueous humor production from the ciliary body is suppressed.

[0010] For example, in a model in which the aqueous humor outflow tract is burned with a laser to induce high intraocular pressure, once high intraocular pressure is reached, the regenerated nerves are also exposed to high intraocular pressure, and there is a high probability of ciliary shock.

[0011] Furthermore, in the silicone oil-induced high intraocular pressure model, it is said that the intraocular pressure can be adjusted by removing the silicone oil, but this model also has a very high probability of causing ciliary shock, which can suppress aqueous humor production and result in low intraocular pressure (see Non-Patent Document 5).

[0012] Furthermore, in models in which retinal ganglion cells are damaged by intravitreal injection of a toxin, there is concern that the toxin may affect tissues other than retinal ganglion cells, and the damage caused by the toxin itself may affect reprogramming. For example, the toxin N-methyl-D-aspartic acid (NMDA) may induce reprogramming itself (see Non-Patent Document 6).

[0013] Kobayashi W., et al., Culture Systems of Dissociated Mouse and Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells Purified by Two-Step Immunopanning, Invest Ophthalmol Vis Sci. 59, 776-787, 2018.Hertz J., et al., Survival and Integration of Developing and Progenitor-Derived Retinal Ganglion Cells Following Transplantation, Cell Transplantation, 23, 855-872, 2014.Ueki Y., et al., Transgenic expression of the proneural transcription factor Ascl1 in Muller glia stimulates retinal regeneration in young mice, PNAS, 112, 13717-13722, 2015.Jorstad N. L., et al., Stimulation of functional neuronal regeneration from Muller glia in adult mice, Nature, 548 (7665), 103-107, 2017.Moshiri A., et al., Silicone Oil-Induced Glaucomatous Neurodegeneration in Rhesus Macaques, Int. J. Mol. Sci. 23, 15896, 2022.Karl M. O., et al., Stimulation of neural regeneration in the mouse retina, PNAS, 105, 19508-19513, 2008.

[0014] The reprogramming efficiency of adult mice is significantly lower than that of immature mice. Non-Patent Document 3 describes that the ability of mouse Müller glial cells to differentiate into retinal ganglion cells by direct reprogramming is lost by 12 days after birth. Furthermore, Non-Patent Document 4 describes that differentiation into amacrine cells was only possible, and differentiation into retinal ganglion cells was not successful.

[0015] Glaucoma is a disease that is common among the elderly, with an estimated incidence of 5% of people over 40 and over 10% of people over 60. Since there have been no reports of successful direct reprogramming in the retina of elderly mice, it is thought that it will be difficult to treat glaucoma in humans using direct reprogramming in the future.

[0016] Furthermore, for future clinical application in humans, it will be necessary to successfully achieve direct reprogramming of retinal ganglion cells not only in rodents such as mice, but also in primates, where direct reprogramming is thought to be far more difficult than in rodents.

[0017] An object of the present invention is to provide a technique for inducing differentiation of mammalian Müller glial cells into retinal ganglion cells in vivo, and a pharmaceutical composition for treating glaucoma or optic neuropathy in humans.

[0018] The present invention includes the following aspects: [1] A differentiation induction kit for inducing differentiation of mammalian Müller glial cells into retinal ganglion cells in vivo, comprising a nucleic acid encoding Neurogenin 2 (NEUROG2); a combination of a nucleic acid encoding Achaete-Scute Family BHLH Transcription Factor 1 (ASCL1), a nucleic acid encoding Brain-Specific Homeobox / POU Domain Protein 3B (BRN3B), and a nucleic acid encoding Atonal BHLH Transcription Factor 7 (ATOH7); or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL LIM Homeobox 1 (ISL1). [2] The differentiation induction kit according to [1], wherein the nucleic acid is carried by an adeno-associated virus vector. [3] The differentiation induction kit according to [1] or [2], which is used to administer the nucleic acid after internal limiting membrane peeling. [4] The differentiation induction kit according to any of [1] to [3], further comprising an epigenetic-related drug. [5] The differentiation induction kit according to [4], which is used to administer the epigenetic-related drug 1 to 45 days after administration of the nucleic acid. [6] The differentiation induction kit according to [4] or [5], wherein the epigenetic-related drug is selected from the group consisting of a histone deacetylase (HDAC) inhibitor, a DOT1 Like Histone Lysine Methyltransferase (DOT1L) inhibitor, a DNA methyltransferase (DNMT) inhibitor, a lysine specific demethylase (LSD) inhibitor, an ALK inhibitor, and a histone acetyltransferase inhibitor.[7] A pharmaceutical composition for inducing differentiation of mammalian Müller glial cells into retinal ganglion cells in vivo, comprising a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1; and a pharmaceutically acceptable carrier. [8] The pharmaceutical composition according to [7], wherein the nucleic acid is carried by an adeno-associated virus vector. [9] The pharmaceutical composition according to [7] or [8], for use in administering the nucleic acid after internal limiting membrane peeling.

[10] The pharmaceutical composition according to [7] or [8], for use in concomitant administration of an epigenetic-related drug.

[11] The pharmaceutical composition according to

[10] , for use in concomitant administration of the epigenetic-related drug 1 to 45 days after administration of the nucleic acid.

[12] The pharmaceutical composition according to

[10] or

[11] , wherein the epigenetic-related drug is selected from the group consisting of an HDAC inhibitor, a DOT1L inhibitor, a DNMT inhibitor, an LSD inhibitor, an ALK inhibitor, and a histone acetyltransferase inhibitor.

[13] A glaucoma model consisting of an animal that has undergone internal limiting membrane peeling.

[0019] According to the present invention, a technique for inducing differentiation of mammalian Müller glial cells into retinal ganglion cells in vivo can be provided.

[0020] FIG. 1 is a schematic diagram illustrating a system for detecting differentiation into retinal ganglion cells prepared in Experimental Example 1. FIG. 2 is a graph showing the results of measuring the number of EGFP-positive cells and the number of EGFP-positive axons in Experimental Example 2. FIG. 3 is a photograph showing the results of fluorescence microscopy of a flat-mount preparation of a mouse eyeball when a combination of Ascl1, Atoh7, and Brn3b was introduced in Experimental Example 2. FIG. 4 is a photograph showing the results of fluorescence microscopy of a flat-mount preparation of a mouse eyeball when Neurog2 was introduced in Experimental Example 2. FIG. 5 is a photograph showing the results of fluorescence microscopy of a flat-mount preparation of a mouse eyeball when a combination of Ascl1, Atoh7, and Brn3b was introduced in Experimental Example 3. FIG. 6 is a photograph showing the results of fluorescence microscopy of a flat-mount preparation of a mouse eyeball when Neurog2 was introduced in Experimental Example 3. FIG. 7 is a graph showing the results of measuring the number of EGFP-positive cells and the number of EGFP-positive axons in Experimental Example 3. FIG. 8 is a graph showing the results of screening epigenetic-related drugs in Experimental Example 4. FIG. 9 is a fluorescent fundus photograph of the retina of a cynomolgus monkey in Experimental Example 6. FIG. 10 is a photograph showing the results of observing EGFP fluorescence in the optic nerve and brain of a cynomolgus monkey that underwent internal limiting membrane peeling and NEUROG2 transfection in Experimental Example 6. FIG. 11 is an image showing the results of three-dimensional evaluation of the retinal morphology of a cynomolgus monkey in Experimental Example 6. FIG. 12 is a fluorescent microscope photograph showing the results of immunostaining of the retina of a cynomolgus monkey that underwent internal limiting membrane peeling and NEUROG2 transfection in Experimental Example 6. FIG. 13 is a schematic diagram explaining the experimental schedule in Experimental Example 7 and a fluorescent fundus photograph of the retina of a young cynomolgus monkey aged 4 to 5 years (equivalent to 20 years in humans). Figure 14 is a schematic diagram illustrating the experimental schedule and fluorescent fundus photographs of the retina of an elderly cynomolgus monkey aged 14 to 15 years (equivalent to 60 years in humans) in Experimental Example 7. Figure 15 is a schematic diagram illustrating the experimental schedule and fundus photographs and fluorescent fundus photographs of the retina of a young cynomolgus monkey aged 4 to 5 years (equivalent to 20 years in humans) in Experimental Example 8.Fig. 16 is a schematic diagram illustrating the experimental schedule in Experimental Example 9, and fundus photographs and fluorescent fundus photographs of the retina of a young cynomolgus monkey aged 4 to 5 years (equivalent to 20 years in humans). Fig. 17 is a schematic diagram illustrating the experimental schedule in Experimental Example 9, and fundus photographs and fluorescent fundus photographs of the retina of an elderly cynomolgus monkey aged 14 to 15 years (equivalent to 60 years in humans). Fig. 18 is a schematic diagram illustrating measurement of the electroretinogram of a cynomolgus monkey in Experimental Example 9. Fig. 19 is a graph showing the results of measurement of the function of retinal nerve cells based on the electroretinogram in Experimental Example 9.

[0021] [Notation of Gene Names and Protein Names] In this specification, human genes and human proteins are represented by capital letters. Mouse genes are represented by an initial capital letter followed by lowercase letters. Mouse proteins are represented by capital letters. However, in some cases, human genes, mouse genes, genes of other species, human proteins, mouse proteins, and proteins of other species may be represented without strict distinction.

[0022] [Differentiation Induction Kit] In one embodiment, the present invention provides a differentiation induction kit for inducing differentiation of mammalian Müller glial cells into retinal ganglion cells in vivo. The differentiation induction kit of this embodiment includes a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1.

[0023] NEUROG2, ASCL1, BRN3B, ATOH7, and ISL1 are transcription factors involved in direct neural reprogramming. The NCBI accession number for the cDNA of the human NEUROG2 gene is NM_024019.4. The NCBI accession number for the cDNA of the mouse Neurog2 gene is NM_009718.2. The NCBI accession number for the cDNA of the human ASCL1 gene is NM_004316.4. The NCBI accession number for the cDNA of the mouse Ascl1 gene is NM_008553.4. The NCBI accession number for the cDNA of the human BRN3B gene is NM_004575.3. The NCBI accession number for the cDNA of the mouse Brn3b gene is NM_138944.2. The NCBI accession number for the cDNA of the human ATOH7 gene is NM_145178.4. The NCBI accession number for the cDNA of the mouse Atoh7 gene is NM_016864.1. The NCBI accession number for the cDNA of the human ISL1 gene is NM_002202.3. The NCBI accession number for the cDNA of the mouse Isl1 gene is NM_021459.4.

[0024] As will be described later in the Examples, administration of the differentiation induction kit of this embodiment to the eye of a mammal can induce differentiation of Müller glia cells present in the retina into retinal ganglion cells in vivo. Furthermore, the axons of the differentiated retinal ganglion cells can be propagated to the brain. Direct reprogramming has not been successful in primates, including the cranial nerve region. In contrast, direct reprogramming was successfully performed in 4- to 5-year-old cynomolgus monkeys (equivalent to 20 years old in humans). Furthermore, direct reprogramming was also successfully performed in 14- to 15-year-old cynomolgus monkeys (equivalent to 60 years old in humans).

[0025] The differentiation induction kit of this embodiment can also be described as an agent for inducing differentiation from Müller glial cells to retinal ganglion cells, a therapeutic agent for glaucoma or optic neuropathy, or the like.

[0026] The differentiation induction kit of this embodiment may contain, as the nucleic acid encoding a transcription factor, a nucleic acid encoding NEUROG2 alone, or a combination of a nucleic acid encoding NEUROG2 and a nucleic acid encoding BRN3B, or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7, or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1.

[0027] Mammals include rodents such as mice and rats, and primates such as monkeys and humans.

[0028] The nucleic acid encoding the transcription factor may be carried in an expression vector such as an adeno-associated virus (AAV) vector. AAVs that target retinal cells, such as AAV1, AAV2, AAV5, AAV6, and AAV6 mutants AAV(ShH10), AAV8, and AAV9, are preferred. AAV(ShH10) is particularly preferred because it is known to transfer genes specifically into Müller glia cells.

[0029] The nucleic acid encoding the transcription factor may be in the form of mRNA. In this case, the nucleic acid encoding the transcription factor is preferably formulated as a pharmaceutical composition together with a carrier commonly used in the formulation of nucleic acid drugs. Specific examples of the carrier include cationic liposomes, non-cationic liposomes, polyethylene glycol (PEG)-modified liposomes, lipid nanoparticles, and block copolymers.

[0030] Examples of materials for liposomes or lipid nanoparticles include, but are not limited to, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (PEG2000-DMG), and the like.

[0031] The pharmaceutical composition may contain an additive, which may be any additive typically used in pharmaceutical preparations, including, but not limited to, buffers such as trometamol and trometamol hydrochloride, pH adjusters such as glacial acetic acid and sodium acetate hydrate, and isotonic agents such as refined sucrose.

[0032] The differentiation-inducing kit of this embodiment is preferably used to administer the nucleic acid encoding the transcription factor after internal limiting membrane peeling. Conventionally, it has been believed that AAV is rarely introduced into the retina of primates, including cynomolgus monkeys. In contrast, as described later in the Examples, the inventors have succeeded in highly efficient AAV introduction into the retina by peeling off the internal limiting membrane of the retina through vitreous surgery.

[0033] The differentiation-inducing kit of this embodiment preferably further comprises an epigenetic drug. As will be described later in the Examples, administering not only a transcription factor but also an epigenetic drug makes it possible to induce direct reprogramming of Müller glial cells into retinal ganglion cells in older mammals.

[0034] The differentiation-inducing kit of this embodiment is preferably used so that an epigenetic-related drug is co-administered 1 to 45 days, for example 5 to 30 days, after administration of a nucleic acid encoding a transcription factor. The epigenetic-related drug may be administered once or twice or more times. The epigenetic-related drug is preferably administered locally intraocularly by intravitreal administration.

[0035] As described later in the Examples, the efficiency of inducing direct reprogramming from Müller glial cells to retinal ganglion cells can be significantly increased by administering not only a nucleic acid encoding a transcription factor but also an epigenetic drug. As described later in the Examples, when the mammal is a mouse, it is preferable to administer the epigenetic drug 7 days after administering the nucleic acid encoding the transcription factor.

[0036] Examples of the epigenetic-related drug include histone deacetylase (HDAC) inhibitors, DOT1-like histone lysine methyltransferase (DOT1L) inhibitors, DNA methyltransferase (DNMT) inhibitors, lysine specific demethylase (LSD) inhibitors, ALK inhibitors, and histone acetyltransferase inhibitors. These may be administered alone or in combination of two or more. The dose of the epigenetic-related drug is preferably about 60 μg / eye to 0.05 ng / eye (3000 μg / kg body weight to 2.5 ng / kg body weight).

[0037] More specific examples of HDAC inhibitors include vorinostat (CAS number: 149647-78-9), Scriptaid (CAS number: 287383-59-9), CHDI00390576 (CAS number: 1629729-98-1), Quisinostat (CAS number: 875320-29-9), valproic acid (CAS number: 99-66-1), sodium butyrate (CAS number: 1 56-54-7), romidepsin (CAS No.: 128517-07-7), tacedinalin (CAS No.: 112522-64-2), entinostat (CAS No.: 209783-80-2), mocetinostat (CAS No.: 726169-73-9), nicotinamide (CAS No.: 98-92-0), trichostatin A (CAS No.: 58880-19-6), etc. Among these, vorinostat, quisinostat, romidepsin, and entinostat are preferred.

[0038] The dosage of vorinostat is preferably 60 μg / eye to 6 ng / eye (3000 μg / kg body weight to 0.3 μg / kg body weight). The dosage of quisinostat is preferably 30 μg / eye to 3 ng / eye (1500 μg / kg body weight to 0.15 μg / kg body weight). The dosage of romidepsin is preferably 8 μg / eye to 0.3 μg / eye (400 μg / kg body weight to 0.15 μg / kg body weight). The dosage of entinostat is preferably 30 μg / eye to 3 ng / eye (1500 μg / kg body weight to 0.15 μg / kg body weight).

[0039] Examples of DOT1L inhibitors include SGC0946 (CAS number: 1561178-17-3), EPZ5676 (CAS number: 1380288-87-8), and EPZ004777 (CAS number: 1338466-77-5). Of these, SGC0946 is preferred. The dosage of SGC0946 is preferably 6 μg / eye to 0.6 ng / eye (300 μg / kg body weight to 0.03 μg / kg body weight).

[0040] DNMT inhibitors include RG108 (CAS number: 48208-26-0), 5-azacytidine (CAS number: 320-67-2), and the like.

[0041] Examples of LSD inhibitors include RN-1 (CAS number: 1781835-13-9), WDR5-0103 (CAS number: 890190-22-4), GSK-LSD1 (CAS number: 1431368-48-7), and UCN1999 (CAS number: 1431612-23-5).

[0042] Examples of ALK inhibitors include SB431542 (CAS number: 301836-41-9) and the like.

[0043] Examples of histone acetyltransferase inhibitors include butyrolactone 3 (CAS number: 778649-18-6).

[0044] [Pharmaceutical Composition] In one embodiment, the present invention provides a pharmaceutical composition for inducing differentiation of mammalian Müller glial cells into retinal ganglion cells in vivo, the pharmaceutical composition comprising: a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1; and a pharmaceutically acceptable carrier. The pharmaceutical composition of this embodiment can be said to be a pharmaceutical composition for treating glaucoma or optic neuropathy.

[0045] In the pharmaceutical composition of this embodiment, the nucleic acid encoding NEUROG2, the nucleic acid encoding ASCL1, the nucleic acid encoding BRN3B, the nucleic acid encoding ATOH7, the nucleic acid encoding ISL1, etc. are the same as those described above.

[0046] The pharmaceutical composition of this embodiment may contain, as the nucleic acid encoding a transcription factor, a nucleic acid encoding NEUROG2 alone, or a combination of a nucleic acid encoding NEUROG2 and a nucleic acid encoding BRN3B, or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7, or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1.

[0047] The nucleic acid encoding the transcription factor may be carried in an expression vector or may be in the form of mRNA.Preferably, the expression vector is an adeno-associated virus (AAV) vector.The AAV vector is the same as described above.When the nucleic acid encoding the transcription factor is in the form of mRNA, it is preferably formulated with a carrier that is commonly used in the formulation of nucleic acid medicines, as described above.

[0048] The pharmaceutical composition of this embodiment can be prepared by a commonly used method using excipients commonly used in the art, i.e., pharmaceutical excipients, pharmaceutical carriers, etc. In one aspect, the pharmaceutical composition of this embodiment contains the nucleic acid encoding the above-mentioned transcription factor and one or more excipients. Examples of dosage forms of these pharmaceutical compositions include injectable pharmaceutical compositions and other parenteral pharmaceutical compositions, and they can be administered by intraocular administration (intravitreal administration or subretinal administration). In one aspect, the pharmaceutical composition of this embodiment is administered intravitreally. The pharmaceutical composition of this embodiment is preferably intended for use in administering the nucleic acid encoding the transcription factor after internal limiting membrane peeling.

[0049] The therapeutically effective amount of the nucleic acid encoding a transcription factor can be optimized as appropriate, taking into consideration the severity of the disease, previous treatments, the administration method, other diseases, etc. When the nucleic acid encoding the transcription factor is carried by an AAV vector, in one aspect, the therapeutically effective amount of the AAV vector is about 0.001 mg / kg body weight to 30 mg / kg body weight. The therapeutically effective amount of the AAV vector can also be expressed as the genome dose of the vector administered per eye (vg / eye). vg can also be expressed as genome copies (GC). In one aspect, the therapeutically effective amount of the AAV vector is about 1 x 10 ６ vg / eye~8×10 １３ vg / eye, approximately 1×10 ６ vg / eye~4×10 １２ vg / eye, and 1 to 3 × 10 １２ When the nucleic acid encoding the transcription factor is in the form of mRNA, the therapeutically effective amount of mRNA to be administered per eye is about 10 ng / eye to 500 μg / eye.

[0050] The pharmaceutical composition of this embodiment can be used in combination with an epigenetic-related drug. The epigenetic-related drug and its dosage are the same as those described above. That is, examples of epigenetic-related drugs include HDAC inhibitors, DOT1L inhibitors, DNMT inhibitors, LSD inhibitors, ALK inhibitors, and histone acetyltransferase inhibitors. These may be used alone or in combination of two or more.

[0051] The epigenetic-related drug may be administered simultaneously with the pharmaceutical composition, or may be administered separately and consecutively, or may be administered at an interval. For example, the epigenetic-related drug may be administered in combination 1 to 45 days after the administration of the pharmaceutical composition of this embodiment. In one aspect, the epigenetic-related drug may be administered in combination 5 to 30 days after the administration of the pharmaceutical composition of this embodiment. The epigenetic-related drug is preferably administered locally intraocularly by intravitreal administration.

[0052] When the pharmaceutical composition of this embodiment is for simultaneous administration of an epigenetic-related drug, the pharmaceutical composition of this embodiment may be a pharmaceutical composition comprising a combination of a nucleic acid encoding the ASCL1 gene, a nucleic acid encoding the ATOH7 gene, and a nucleic acid encoding the BRN3B gene, or a nucleic acid encoding the NEUROG2 gene, an epigenetic-related drug, and a pharmaceutically acceptable carrier.

[0053] [Glaucoma Model] In one embodiment, the present invention provides a glaucoma model comprising an animal that has undergone internal limiting membrane peeling. The internal limiting membrane peeling may be performed by any method commonly performed in the art.

[0054] In the glaucoma model of this embodiment, examples of the animal include monkeys, rabbits, rats, and mice. Since animals other than primates do not have a structure called the macula, it is preferable that the animal be a monkey in order to more closely reflect the pathological condition in humans. Until now, there has been no ideal glaucoma model in monkeys.

[0055] In contrast, as will be described later in the Examples, the inventors have demonstrated that internal limiting membrane peeling can selectively cause mild damage to retinal ganglion cells, and that monkeys that have undergone internal limiting membrane peeling can be used as a glaucoma model.

[0056] An ideal glaucoma model would reflect the pathological condition of human glaucoma, such as elevated intraocular pressure, and have selective damage only to retinal ganglion cells. However, as mentioned above, existing models of elevated intraocular pressure and toxin injection have problems.

[0057] Although the ILM peeling model does not show an increase in intraocular pressure, it is a new, highly reliable, stable, and practical glaucoma model that can selectively damage only retinal ganglion cells without causing ciliary shock.In addition, in recent years, cases have been reported in which retinal ganglion cell damage persists despite sufficient reduction of intraocular pressure, including in normal-tension glaucoma.This ILM peeling model is particularly considered to be a pathological model that can be used to address such cases.

[0058] [Other Embodiments] In one embodiment, the present invention provides a method for treating glaucoma or optic neuropathy, comprising the step of administering to the eye of a patient an effective amount of a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1.

[0059] The treatment method of this embodiment may include a step of administering a nucleic acid encoding NEUROG2 alone, or may include a step of administering a combination of a nucleic acid encoding NEUROG2 and a nucleic acid encoding BRN3B, or may include a step of administering a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7, or may include a step of administering a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1.

[0060] In the treatment method of this embodiment, the nucleic acid encoding NEUROG2, the nucleic acid encoding ASCL1, the nucleic acid encoding BRN3B, the nucleic acid encoding ATOH7, the nucleic acid encoding ISL1, etc. are the same as those described above.

[0061] The treatment method of this embodiment further comprises the step of performing internal limiting membrane peeling, and it is preferable to administer the nucleic acid after performing internal limiting membrane peeling.

[0062] The treatment method of this embodiment preferably further comprises the step of administering an epigenetic-related drug to the patient's eye 1 to 45 days after administering the nucleic acid. The epigenetic-related drug is the same as that described above.

[0063] In one embodiment, the present invention provides a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1 for use in the treatment of glaucoma or optic neuropathy.

[0064] In one embodiment, the present invention provides an epigenetic-related drug for use in the treatment of glaucoma or optic neuropathy. The epigenetic-related drug is the same as described above. As described above, the epigenetic-related drug is preferably used in combination with a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1.

[0065] In one embodiment, the present invention provides the use of a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1; in the manufacture of a therapeutic agent for glaucoma or optic neuropathy.

[0066] In one embodiment, the present invention provides an application of an epigenetic-related drug in the manufacture of a therapeutic drug for glaucoma or optic neuropathy. The epigenetic-related drug is the same as that described above. As described above, the epigenetic-related drug is preferably used in combination with a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1.

[0067] In one embodiment, the present invention provides a method for producing a glaucoma model, the method comprising the step of performing internal limiting membrane peeling on an animal, wherein the animal that has undergone internal limiting membrane peeling is a glaucoma model. The internal limiting membrane peeling may be performed by a method commonly performed in the art.

[0068] In the production method of this embodiment, the animals are the same as those described above, and examples thereof include monkeys, rabbits, rats, mice, etc. Since animals other than primates do not have a structure called the macula, it is preferable that the animal be a monkey in order to more closely reflect the pathological condition of humans.

[0069] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0070] Experimental Example 1 (Preparation of a system for detecting differentiation into retinal ganglion cells) A system was prepared using adeno-associated virus (AAV) to express enhanced green fluorescent protein (EGFP) when Müller glial cells were directly reprogrammed into neurons. The AAV used was AAV (ShH10), which is known to specifically transfer genes into Müller glial cells.

[0071] Figure 1 is a schematic diagram illustrating this system. This system is composed of AAV vector 1 and AAV vector 2. In AAV vector 1, a gene encoding Cre recombinase was placed downstream of the promoter of the glial fibrillary acidic protein (GFAP) gene, which is expressed in Müller glial cells. In addition, in AAV vector 1, a gene encoding EGFP, which was sandwiched between flippase recognition target (FRT) sequences recognized by FLP recombinase and linked in reverse orientation, was placed downstream of the human synapsin 1 (hSYN1) promoter, which is expressed in neurons.

[0072] AAV vector 2 contained a gene encoding a transcription factor, a reporter gene, and a gene encoding FLP recombinase, which were sandwiched between LoxP sequences recognized by Cre recombinase and linked in reverse orientation downstream of the transcription elongation factor 1α (EFS) promoter. These genes were linked via a gene encoding the self-cleaving peptide P2A.

[0073] AAV vectors 1 and 2 also contained a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) that enhances transgene expression.

[0074] When AAV vector 1 and AAV vector 2 are introduced into Müller glia cells, the GFAP promoter is activated and Cre recombinase is expressed. This causes recombination at the LoxP sequence of AAV vector 2, resulting in the inversion and expression of a gene encoding a transcription factor, a reporter gene, and a gene encoding FLP recombinase. This causes recombination at the FRT sequence of AAV vector 1 by the FLP recombinase, resulting in the inversion of the gene encoding EGFP.

[0075] Here, when Müller glia cells are directly reprogrammed into neurons by the transcription factor introduced into AAV vector 2, the hSYN1 promoter is activated and EGFP is expressed. Neurons may be retinal ganglion cells, horizontal cells, bipolar cells, amacrine cells, etc., but since only retinal ganglion cells have long axons, neurons with long axons can be determined to be retinal ganglion cells.

[0076] [Experimental Example 2] (Screening of transcription factors that induce reprogramming from Müller glia cells to retinal ganglion cells) In an in vivo experimental system using mice, transcription factors that induce direct reprogramming from Müller glia cells to retinal ganglion cells were screened using the system prepared in Experimental Example 1. As transcription factors, Ascl1, Atoh7, Brn3b, Neurog2, Isl1, NeuroD1, and several dozen combinations thereof were examined.

[0077] Because AAV vectors are limited in size, only one type of transcription factor can be carried in each AAV vector 2 in Experimental Example 1. Therefore, when examining combinations of transcription factors, multiple AAV vectors containing the desired transcription factors were mixed and administered.

[0078] Two-month-old C57BL / 6J mice were administered 75-100 mg / kg body weight of ketamine hydrochloride and 10 mg / kg body weight of xylazine hydrochloride for general anesthesia. Subsequently, the pupils were dilated with commercially available mydriatic eye drops. Subsequently, intravitreal administration was performed using a 37G microsyringe under a stereomicroscope. First, vitreous aspiration was performed to peel off the internal limiting membrane and reduce intraocular pressure. Subsequently, 1-3.6 μL (2 × 10 １０ vg / eye) was injected.

[0079] Seven and 14 days after intravitreal AAV administration, epigenetic drugs were administered intravitreally. Trichostatin A was administered at 604.7 ng (1 mmol / 2 μL, 30.235 μg / kg body weight). Fludarabine (CAS No.: 75607-67-9), which is known to enhance the reprogramming effect of trichostatin A, was also administered at 1.14 μg / eye (57 μg / kg body weight).

[0080] Forty-five days after intravitreal AAV administration, mouse eyes were enucleated and radial incisions were made to prepare flat-mount specimens. The flat-mount specimens were then observed under a fluorescence microscope to detect EGFP fluorescence. Furthermore, the EGFP fluorescence was separated into neuronal and axonal regions using image processing, and the number of directly reprogrammed ganglion cells and axons was evaluated separately.

[0081] The left graph in Figure 2 shows the results of measuring the number of EGFP-positive cells. The right graph in Figure 2 shows the results of measuring the number of EGFP-positive axons. In both the left and right graphs in Figure 2, the horizontal axis indicates the introduced transcription factor.

[0082] As a result, it was revealed that the combination of Ascl1, Atoh7 and Brn3b, or Neurog2 alone, was particularly effective in inducing direct reprogramming of Müller glial cells into retinal ganglion cells.

[0083] Figure 3 shows a photograph of a flat-mounted specimen transfected with a combination of Ascl1, Atoh7, and Brn3b, and Figure 4 shows a photograph of a flat-mounted specimen transfected with Neurog2, both observed under a fluorescence microscope.

[0084] Experimental Example 3: (Study of reprogramming of Müller glia cells to retinal ganglion cells in mice of various ages) Direct reprogramming of Müller glia cells to retinal ganglion cells was investigated using mice of various ages. As in Experimental Example 2, a combination of Ascl1, Atoh7, and Brn3b or Neurog2 was introduced into the eyes of 2-, 4-, 6-, 10-, and 14-month-old C57BL / 6J mice by intravitreal injection of AAV. Subsequently, 7 and 14 days after intravitreal AAV injection, epigenetic drugs were administered intravitreally. The epigenetic drug used was trichostatin A (604.7 ng, 1 mmol / 2 μL, 30.235 μg / kg body weight). Additionally, fludarabine, which is known to enhance the reprogramming effect of trichostatin A, was administered together with trichostatin A at 1.14 μg / eye (57 μg / kg body weight).

[0085] Forty-five days after intravitreal AAV administration, mouse eyes were enucleated and flat-mounted. The flat-mounted specimens were then observed under a fluorescence microscope to detect EGFP fluorescence. Furthermore, the EGFP fluorescence was separated into neurons and axons using image processing, and the number of directly reprogrammed ganglion cells and axons was evaluated separately.

[0086] Figure 5 shows a photograph of a flat-mounted specimen transfected with a combination of Ascl1, Atoh7, and Brn3b, and Figure 6 shows a photograph of a flat-mounted specimen transfected with Neurog2, both observed under a fluorescence microscope.

[0087] In Figures 5 and 6, the top images are fluorescence microscope images of EGFP fluorescence. The middle images are images of EGFP fluorescence separated into neuronal and axonal regions, showing the neuronal regions. The bottom images are images of EGFP fluorescence separated into neuronal and axonal regions, showing the axonal regions.

[0088] The left side of Fig. 7 is a graph showing the results of measuring the number of EGFP-positive cells based on Fig. 5 and Fig. 6. The right side of Fig. 7 is a graph showing the results of measuring the number of EGFP-positive axons based on Fig. 5 and Fig. 6.

[0089] The results revealed that the efficiency of direct reprogramming from Müller glia to retinal ganglion cells decreased with age. However, previous reports have only shown direct reprogramming from Müller glia to retinal ganglion cells up to approximately one month after birth, whereas in this experiment, direct reprogramming from Müller glia to retinal ganglion cells was efficiently induced up to approximately 10 months after birth. This result is thought to be due to the administration of not only transcription factors but also epigenetic drugs. Furthermore, the inventors have found that without the administration of epigenetic drugs, direct reprogramming from Müller glia to retinal ganglion cells could only be induced in mice up to approximately six days after birth.

[0090] [Experimental Example 4] (Examination of various epigenetic-related drugs) Epigenetic-related drugs that can promote direct reprogramming of Müller glial cells to retinal ganglion cells were screened in aged mice.

[0091] In the same manner as in Experimental Example 2, AAV (2 × 10 １０ Neurog2 was introduced by intravitreal administration of AAV (vg / eye). Subsequently, epigenetic drugs were administered intravitreally 7 and 14 days after intravitreal AAV administration. Epigenetic drugs investigated included histone deacetylase (HDAC) inhibitors, DOT1-like histone lysine methyltransferase (DOT1L) inhibitors, DNA methyltransferase (DNMT) inhibitors, lysine specific demethylase (LSD) inhibitors, ALK inhibitors, and histone acetyltransferase inhibitors.

[0092] HDAC inhibitors include vorinostat (CAS number: 149647-78-9), Scriptaid (CAS number: 287383-59-9), CHDI00390576 (CAS number: 1629729-98-1), Quisinostat (CAS number: 875320-29-9), valproic acid (CAS number: 99-66-1), and sodium butyrate. Thorium (CAS number: 156-54-7), romidepsin (CAS number: 128517-07-7), tacedinalin (CAS number: 112522-64-2), entinostat (CAS number: 209783-80-2), mocetinostat (CAS number: 726169-73-9), and nicotinamide (CAS number: 98-92-0) were investigated.

[0093] As DOT1L inhibitors, SGC0946 (CAS number: 1561178-17-3), EPZ5676 (CAS number: 1380288-87-8), and EPZ004777 (CAS number: 1338466-77-5) were investigated.

[0094] As DNMT inhibitors, RG108 (CAS number: 48208-26-0) and 5-azacytidine (CAS number: 320-67-2) were investigated.

[0095] As LSD inhibitors, RN-1 (CAS number: 1781835-13-9), WDR5-0103 (CAS number: 890190-22-4), GSK-LSD1 (CAS number: 1431368-48-7), and UCN1999 (CAS number: 1431612-23-5) were investigated.

[0096] As an ALK inhibitor, SB431542 (CAS number: 301836-41-9) was investigated.

[0097] Butyrolactone 3 (CAS number: 778649-18-6) was investigated as a histone acetyltransferase inhibitor.

[0098] For comparison, we also prepared a group of 2-month-old mice in which Neurog2 was introduced and trichostatin A was used as an epigenetic drug, and a group of 14-month-old mice in which Neurog2 was introduced and trichostatin A was used as an epigenetic drug.

[0099] Figure 8 is a graph showing the results of the screening. The vertical axis of Figure 8 shows the number of EGFP-positive axons. The results revealed that multiple epigenetic drugs, including vorinostat, quisinostat, and entinostat, can promote the direct reprogramming of Müller glia cells to retinal ganglion cells in aged mice.

[0100] [Experimental Example 5] (Live imaging of retinal ganglion cell regeneration by direct reprogramming) Live imaging of retinal ganglion cell regeneration by direct reprogramming was investigated in a mouse experimental system. As a result, it was revealed that the cornea and lens of the eye are transparent, and live imaging through the cornea is possible using a fundus camera. To date, there have been no reports of successful live imaging of direct reprogramming in all regions, including the cranial nerves and heart.

[0101] Live imaging revealed that not only the expression of transcription factors but also the administration of epigenetic drugs is important for direct reprogramming. While the expression of transcription factors alone is not efficient for the regeneration of retinal ganglion cells, direct reprogramming occurred rapidly immediately after the administration of epigenetic drugs.

[0102] We also investigated the timing of epigenetic drug administration, using trichostatin A. The results showed that the most efficient direct reprogramming was achieved when trichostatin A was administered one week after the administration of AAV for transcription factor expression.

[0103] When trichostatin A administration was delayed by two weeks after AAV administration, the efficiency of direct reprogramming was low until trichostatin A administration, but direct reprogramming occurred rapidly immediately after trichostatin A administration. This demonstrates the importance of administering epigenetics-related drugs, and furthermore, the timing of their administration.

[0104] [Experimental Example 6] (Direct Reprogramming in Monkeys 1) In an in vivo experimental system using cynomolgus monkeys, direct reprogramming from Müller glia cells to retinal ganglion cells was induced using the system prepared in Experimental Example 1. NEUROG2 was used as the transcription factor.

[0105] Young cynomolgus monkeys aged 4-5 years (equivalent to 20 years in humans) were used. Cynomolgus monkeys were administered 10 mg / kg body weight of ketamine hydrochloride for general anesthesia. Subsequently, vitreous surgery (internal limiting membrane peeling) was performed. Seven days after internal limiting membrane peeling, the monkeys were again administered general anesthesia, and AAV (2.2 × 10 １２ vg / eye) was administered intravitreally.

[0106] Epigenetic drugs were administered intravitreally twice, 21 and 35 days after internal limiting membrane peeling. Trichostatin A was administered at 45.4 μg / eye (15 μg / kg body weight) per administration. Fludarabine, which is known to enhance the reprogramming effect of trichostatin A, was also administered at 85.5 μg / eye (28 μg / kg body weight) per administration. EGFP fluorescence was detected by live imaging using a fundus camera 95 days after internal limiting membrane peeling.

[0107] Fig. 9 is a fluorescent fundus photograph of the retina of a cynomolgus monkey, showing EGFP fluorescence. Fig. 10 (left) shows the results for a cynomolgus monkey that underwent internal limiting membrane peeling and NEUROG2 transfection, Fig. 10 (center) shows the results for a cynomolgus monkey that underwent internal limiting membrane peeling but no transcription factor transfection, and Fig. 10 (right) shows the results for a cynomolgus monkey that underwent internal limiting membrane peeling but no NEUROG2 transfection.

[0108] As a result, in cynomolgus monkeys that underwent internal limiting membrane peeling and NEUROG2 transfection, it was observed that regenerated axons with a course similar to that of the original retinal ganglion cells reached the optic nerve head.

[0109] Figure 10 is a photograph showing the results of observing EGFP fluorescence in the optic nerve and brain of a cynomolgus monkey that underwent internal limiting membrane peeling and NEUROG2 transfection. In Figure 10, "control" indicates the results for a cynomolgus monkey that underwent internal limiting membrane peeling but did not undergo transcription factor transfection. As a result, reprogrammed axons were observed to pass through the optic nerve and reach the lateral geniculate body of the head.

[0110] Figure 11 shows images of the results of a three-dimensional evaluation of retinal morphology. Cynomolgus monkeys that underwent internal limiting membrane peeling and NEUROG2 transfection showed increased retinal nerve fiber layer thickness. This result provides evidence that direct reprogramming regenerates retinal ganglion cells and increases the number of axons.

[0111] Figure 12 is a fluorescence micrograph showing the results of immunostaining for Brn3a in the retina of a cynomolgus monkey that underwent internal limiting membrane peeling and NEUROG2 transfection. Brn3a is a marker for retinal ganglion cells. EGFP fluorescence was also observed. Nuclei were also observed by DAPI staining. As a result, immunostaining also showed an increase in the number of retinal ganglion cells and the thickness of the retinal nerve fiber layer, indicating that direct reprogramming regenerated retinal ganglion cells and increased axon numbers.

[0112] [Experimental Example 7] (Direct reprogramming in monkeys 2) In an in vivo experimental system using cynomolgus monkeys, direct reprogramming from Müller glia cells to retinal ganglion cells was induced using the system prepared in Experimental Example 1. NEUROG2 was used as the transcription factor.

[0113] Young cynomolgus monkeys aged 4-5 years (equivalent to 20 years in humans) and elderly cynomolgus monkeys aged 14-15 years (equivalent to 60 years in humans) were used. The cynomolgus monkeys were administered 10 mg / kg body weight of ketamine hydrochloride underwent general anesthesia. Subsequently, vitreous surgery (internal limiting membrane peeling) was performed. Seven days after internal limiting membrane peeling, the monkeys were again administered general anesthesia and injected with AAV (2.2 × 10 １２ vg / eye) was administered intravitreally.

[0114] Twenty-one days after internal limiting membrane peeling, patients were intravitreally administered epigenetic drugs. These drugs included trichostatin A, fludarabine (which is known to enhance the reprogramming effect of trichostatin A), and vorinostat. For comparison, a group not receiving any epigenetic drugs was also prepared.

[0115] Trichostatin A was administered at 45.4 μg / eye (15 μg / kg body weight). Fludarabine was administered at 85.5 μg / eye (28 μg / kg body weight). Vorinostat was administered at 45 μg / eye (15 μg / kg body weight). While epigenetic-related drugs were administered twice in Experimental Example 6, they were administered only once in this experiment.

[0116] EGFP fluorescence was detected by live imaging using a fundus camera 91 days after internal limiting membrane peeling. The upper panel of Figure 13 is a schematic diagram illustrating the experimental schedule. The lower panel of Figure 13 is a fluorescent fundus photograph of the retina of a young cynomolgus monkey aged 4 to 5 years (equivalent to 20 years in humans), showing EGFP fluorescence. In Figure 13, "TSA" represents a combination of trichostatin A and fludarabine, and "SAHA" represents suberoylanilide hydroxamic acid (another name for vorinostat), i.e., vorinostat. The bottom left of Figure 13 shows the results for cynomolgus monkeys that underwent internal limiting membrane peeling, introduced NEUROG2, and were administered trichostatin A and fludarabine; the bottom center of Figure 13 shows the results for cynomolgus monkeys that underwent internal limiting membrane peeling, introduced NEUROG2, and were administered vorinostat; and the bottom right of Figure 13 shows the results for cynomolgus monkeys that underwent internal limiting membrane peeling, introduced NEUROG2, and were not administered an epigenetic-related drug.

[0117] As a result, young cynomolgus monkeys administered vorinostat showed a reprogramming effect comparable to that of cynomolgus monkeys administered trichostatin A and fludarabine.

[0118] The upper panel of Figure 14 is a schematic diagram explaining the experimental schedule. The lower panel of Figure 14 is a fluorescent fundus photograph of the retina of an elderly cynomolgus monkey aged 14 to 15 years (equivalent to 60 years old in humans), showing EGFP fluorescence. In Figure 14, "TSA" represents the combination of trichostatin A and fludarabine, and "SAHA" represents vorinostat. The leftmost result in the bottom row of Figure 14 is a result from a cynomolgus monkey that underwent internal limiting membrane peeling, no transcription factor was introduced, and was administered trichostatin A and fludarabine; the second from the left in the bottom row of Figure 14 is a result from a cynomolgus monkey that underwent internal limiting membrane peeling, NEUROG2 was introduced, and was administered trichostatin A and fludarabine; the third from the left in the bottom row of Figure 14 is a result from a cynomolgus monkey that underwent internal limiting membrane peeling, no transcription factor was introduced, and was administered vorinostat; and the rightmost result in the bottom row of Figure 14 is a result from a cynomolgus monkey that underwent internal limiting membrane peeling, NEUROG2 was introduced, and was administered vorinostat.

[0119] The results showed that in aged cynomolgus monkeys treated with a combination of trichostatin A and fludarabine, direct reprogramming of Müller glia to retinal ganglion cells was barely observed, whereas in cynomolgus monkeys treated with vorinostat, direct reprogramming of Müller glia to retinal ganglion cells was observed.

[0120] [Experimental Example 8] (Direct Reprogramming in Monkeys 3) The timing of internal limiting membrane peeling was investigated. Young cynomolgus monkeys aged 4 to 5 years (equivalent to 20 years in humans) were given general anesthesia and underwent vitreous surgery (internal limiting membrane peeling). Subsequently, 133 days after the internal limiting membrane peeling, they were given general anesthesia again and injected with AAV (2.2 × 10 １２The transcription factor NEUROG2 was introduced by intravitreal administration of 45.4 μg / eye (15 μg / kg body weight). Trichostatin A and fludarabine were administered 147 and 161 days after internal limiting membrane peeling. Trichostatin A was administered at 45.4 μg / eye (15 μg / kg body weight). Fludarabine was administered at 85.5 μg / eye (28 μg / kg body weight). EGFP fluorescence was then detected by live imaging using a fundus camera.

[0121] The upper panel of Figure 15 is a schematic diagram illustrating the experimental schedule. The middle panel of Figure 15 is a fundus photograph of the retina of a cynomolgus monkey. The lower panel of Figure 15 is a fluorescent fundus photograph of the retina of a cynomolgus monkey, showing EGFP fluorescence. In Figure 15, "TSA" represents a combination of trichostatin A and fludarabine. As a result, even when AAV was administered more than four months after internal limiting membrane peeling, EGFP fluorescence was observed that was as strong as when AAV was administered seven days after internal limiting membrane peeling. This result indicates that direct reprogramming is possible even more than four months after internal limiting membrane peeling. In other words, in clinical practice, it is not necessary to perform internal limiting membrane peeling immediately before AAV administration, suggesting that direct reprogramming is possible even in patients who have previously undergone internal limiting membrane peeling.

[0122] [Experimental Example 9] (Direct Reprogramming in Monkeys 4) In an in vivo experimental system using cynomolgus monkeys, direct reprogramming from Müller glia cells to retinal ganglion cells was induced using the system prepared in Experimental Example 1. NEUROG2 was used as the transcription factor.

[0123] Young cynomolgus monkeys aged 4-5 years (equivalent to 20 years in humans) and elderly cynomolgus monkeys aged 14-15 years (equivalent to 60 years in humans) were used. The cynomolgus monkeys were administered 10 mg / kg body weight of ketamine hydrochloride underwent general anesthesia. Subsequently, vitreous surgery (internal limiting membrane peeling) was performed. Seven days after internal limiting membrane peeling, the monkeys were again administered general anesthesia and injected with AAV (2.2 × 10 １２ vg / eye) was administered intravitreally.

[0124] After internal limiting membrane peeling, epigenetic drugs were administered intravitreally. Vorinostat was administered twice or three times as an epigenetic drug. Vorinostat was administered at 45 μg / eye (15 μg / kg body weight) per administration. When vorinostat was administered twice, it was administered 21 and 35 days after internal limiting membrane peeling. When vorinostat was administered three times, it was administered 21, 35, and 49 days after internal limiting membrane peeling. For comparison, a group that did not receive an epigenetic drug was also prepared.

[0125] EGFP fluorescence was detected by live imaging using a fundus camera 63 days after internal limiting membrane peeling. The upper panel of Figure 16 is a schematic diagram illustrating the experimental schedule. The middle panel of Figure 16 is a fundus photograph of the retina of a young cynomolgus monkey aged 4 to 5 years (equivalent to 20 years in humans). The lower panel of Figure 16 is a fluorescent fundus photograph of the retina of a young cynomolgus monkey aged 4 to 5 years (equivalent to 20 years in humans), showing EGFP fluorescence. In Figure 16, "SAHA" represents vorinostat.

[0126] The bottom left of Figure 16 shows the results for cynomolgus monkeys that underwent internal limiting membrane peeling, introduced NEUROG2, and were administered vorinostat twice; the bottom center of Figure 16 shows the results for cynomolgus monkeys that underwent internal limiting membrane peeling, introduced NEUROG2, and were administered vorinostat three times; and the bottom right of Figure 16 shows the results for cynomolgus monkeys that underwent internal limiting membrane peeling, did not introduce a transcription factor, and were not administered an epigenetic-related drug.

[0127] As a result, in young cynomolgus monkeys, good reprogramming effects were observed when vorinostat was administered two or three times.

[0128] The upper panel of Figure 17 is a schematic diagram explaining the experimental schedule. The middle panel of Figure 17 is a fundus photograph of the retina of an elderly cynomolgus monkey aged 14 to 15 years (equivalent to 60 years in humans). The lower panel of Figure 17 is a fluorescent fundus photograph of the retina of an elderly cynomolgus monkey aged 14 to 15 years (equivalent to 60 years in humans), showing EGFP fluorescence. In Figure 17, "SAHA" represents vorinostat.

[0129] The bottom left of Figure 17 shows the results for cynomolgus monkeys that underwent internal limiting membrane peeling, introduced NEUROG2, and were administered vorinostat twice; the bottom center of Figure 17 shows the results for cynomolgus monkeys that underwent internal limiting membrane peeling, introduced NEUROG2, and were administered vorinostat three times; and the bottom right of Figure 17 shows the results for cynomolgus monkeys that underwent internal limiting membrane peeling, did not introduce a transcription factor, and were not administered an epigenetic-related drug.

[0130] As a result, even in aged cynomolgus monkeys, good reprogramming effects were observed when vorinostat was administered two or three times.

[0131] Electroretinography (ERG) is a technique that records the electrical signal (retinal potential) generated when light is shone on the eye and the retina senses the light. By measuring the electroretinogram, the presence or absence of retinal dysfunction can be examined. In the electroretinogram, reprogrammed and regenerated retinal neurons are integrated into neural circuits within the retina, forming synapses, and no electrical signal is generated if they are not functioning.

[0132] As shown in the upper panel of Figure 18, internal limiting membrane peeling damages retinal ganglion cells. In response, direct reprogramming by introducing transcription factors and administering epigenetic-related drugs induces differentiation of Müller glia cells into retinal ganglion cells, thereby increasing the number of retinal ganglion cells. The middle panel of Figure 18 is a schematic diagram illustrating electroretinogram measurements in cynomolgus monkeys. The bottom panel of Figure 18 shows an example of a measured electroretinogram. Photopic negative response (PhNR), a waveform component in the electroretinogram, appears as a negative component following the b-wave of the electroretinogram and is known to represent the function of retinal ganglion cells.

[0133] Electroretinograms were measured 1, 3, 5, 7, 9, and 11 weeks after internal limiting membrane peeling, and the ratio of PhNR to b-wave (PhNR / b-wave) was calculated to examine the functional recovery of retinal nerve cells.

[0134] The upper graph in Figure 19 shows the results for young cynomolgus monkeys aged 4 to 5 years (corresponding to 20 years in humans), the middle graph shows the results for elderly cynomolgus monkeys aged 14 to 15 years (corresponding to 60 years in humans), and the lower graph in Figure 19 is a combined graph of the results for the young cynomolgus monkeys aged 4 to 5 years and the elderly cynomolgus monkeys aged 14 to 15 years. The vertical axis of the graph shows the ratio of PhNR to b-wave (PhNR / b-wave, relative value).

[0135] In Figure 19, "ILM peeling / Neurog2 / Vorinostat" indicates the results of cynomolgus monkeys that underwent internal limiting membrane peeling, NEUROG2 introduction, and two or three doses of vorinostat, while "ILM peeling" indicates the results of cynomolgus monkeys that underwent internal limiting membrane peeling, no transcription factor introduction, and no administration of an epigenetic-related drug. In the lower panel of Figure 19, "*" indicates a significant difference at p<0.05.

[0136] As a result, it was revealed that when internal limiting membrane peeling was performed, NEUROG2 was introduced, and vorinostat was administered two or three times, the decline in retinal nerve cell function caused by internal limiting membrane peeling was restored to the level before internal limiting membrane peeling in not only young but also elderly cynomolgus monkeys.

[0137] These results further support the idea that direct reprogramming of Müller glia into retinal ganglion cells can be induced not only in young but also in aged cynomolgus monkeys by administering transcription factors and epigenetic-related drugs.

[0138] These results also demonstrate that monkeys that have undergone internal limiting membrane peeling can be used as a glaucoma model. Because no ciliary shock or effects on tissues other than retinal ganglion cells were observed in monkeys that have undergone internal limiting membrane peeling, it can be said that monkeys that have undergone internal limiting membrane peeling are currently the most reliable, stable, and practical monkey glaucoma model.

[0139] According to the present invention, a technique for inducing differentiation of mammalian Müller glial cells into retinal ganglion cells in vivo can be provided.

Claims

1. A differentiation induction kit for inducing differentiation of mammalian Müller glial cells into retinal ganglion cells in vivo, comprising: a nucleic acid encoding Neurogenin 2 (NEUROG2); a nucleic acid encoding Achaete-Scute Family BHLH Transcription Factor 1 (ASCL1); a combination of a nucleic acid encoding Brain-Specific Homeobox / POU Domain Protein 3B (BRN3B) and a nucleic acid encoding Atonal BHLH Transcription Factor 7 (ATOH7); or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B and a nucleic acid encoding ISL LIM Homeobox 1 (ISL1).

2. The differentiation induction kit according to claim 1, wherein the nucleic acid is carried in an adeno-associated virus vector.

3. A differentiation induction kit according to claim 1 or 2, which is used to administer the nucleic acid after internal limiting membrane peeling.

4. A differentiation induction kit according to claim 1 or 2, further comprising an epigenetic-related drug.

5. The differentiation induction kit according to claim 4, which is used to administer the epigenetic-related drug 1 to 45 days after administering the nucleic acid.

6. The differentiation induction kit according to claim 4, wherein the epigenetic-related drug is selected from the group consisting of a histone deacetylase (HDAC) inhibitor, a DOT1 Like Histone Lysine Methyltransferase (DOT1L) inhibitor, a DNA methyltransferase (DNMT) inhibitor, a lysine specific demethylase (LSD) inhibitor, an ALK inhibitor, and a histone acetyltransferase inhibitor.

7. A pharmaceutical composition for inducing differentiation of mammalian Muller glial cells into retinal ganglion cells in vivo, comprising: a nucleic acid encoding NEUROG2; a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ATOH7; or a combination of a nucleic acid encoding ASCL1, a nucleic acid encoding BRN3B, and a nucleic acid encoding ISL1; and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein the nucleic acid is carried in an adeno-associated virus vector.

9. A pharmaceutical composition according to claim 7 or 8, for use in administering said nucleic acid after internal limiting membrane peeling.

10. A pharmaceutical composition according to claim 7 or 8 for co-administration with an epigenetic-related drug.

11. The pharmaceutical composition according to claim 10, for use in administering the nucleic acid in combination with the epigenetic-related drug 1 to 45 days after administration.

12. The pharmaceutical composition of claim 10, wherein the epigenetic-related drug is selected from the group consisting of an HDAC inhibitor, a DOT1L inhibitor, a DNMT inhibitor, an LSD inhibitor, an ALK inhibitor, and a histone acetyltransferase inhibitor.

13. Glaucoma model consisting of animals that have undergone internal limiting membrane peeling.

Images