Preventive or therapeutic agent for optic nerve diseases, method for preventing or treating optic nerve diseases, artificial synapse connector or nucleic acid encoding artificial synapse connector, and use thereof

Abstract

An preventive or therapeutic agent for optic nerve diseases comprises an effective amount of an artificial synapse connector or a nucleic acid encoding the artificial synapse connector.

Description

Agents for the prevention or treatment of optic nerve diseases, methods for the prevention or treatment of optic nerve diseases, artificial synaptic connectors or nucleic acids encoding artificial synaptic connectors, and their use

[0001] The present invention relates to an agent for preventing or treating optic nerve diseases, a method for preventing or treating optic nerve diseases, an artificial synaptic connector or a nucleic acid encoding an artificial synaptic connector, and the use of the same.

[0002] Vision is the sense that accounts for the majority of information about the outside world. Millions of people worldwide suffer from visual impairment, which not only affects their quality of life but also poses a significant socioeconomic burden.

[0003] Glaucoma is the leading cause of blindness in adults worldwide and is a neurodegenerative disease characterized by cell death of retinal ganglion cells (RGCs) and loss of optic nerve tissue. The current standard treatment is intraocular pressure (IOP) lowering therapy, which aims to eliminate high intraocular pressure, a contributing factor to neurodegeneration. However, 20% to 30% of cases continue to experience progressive vision loss even after sufficient IOP reduction. Furthermore, in Asians, including Japanese, the majority of glaucoma patients have normal-tension glaucoma, where IOP is within the normal range. Moreover, there are no medications that can restore lost vision or slow its progression, resulting in a significant unmet medical need.

[0004] On the other hand, in many neurodegenerative diseases and traumatic neurological disorders, such as Alzheimer's disease, Parkinson's disease, and spinal cord injury, synaptic damage is known to precede neuronal cell death. Previously, our research group created a fusion protein (hereinafter referred to as "CPTX") by linking the Nrx binding site of the Cbln1 protein and the AMPA-type glutamate receptor binding site of the Nptx1 protein via a trimerizing domain. We found that CPTX functions as an artificial synaptic connector that induces excitatory synapse formation by binding presynaptic neurexin to postsynaptic AMPA receptors. Specifically, we found that a single administration of CPTX to the hippocampus of Alzheimer's disease model mice rapidly increased the number of excitatory synapses, thereby restoring maze learning impairment and contextual fear memory impairment (see Non-Patent Literature 1). Furthermore, administration of CPTX to the spinal cord one week after spinal cord injury restored motor function (see Patent Literature 1). These findings suggest the potential of synaptic therapy for synaptic damage.

[0005] Suzuki et al., Science 369, eabb4853 (2020)

[0006] International Publication No. 2021 / 141118

[0007] The present invention aims to solve the aforementioned conventional problems and achieve the following objectives. Specifically, the present invention aims to provide a preventive or therapeutic agent and a method for the prevention or treatment of optic nerve diseases, an artificial synaptic connector or nucleic acid encoding an artificial synaptic connector for use in the prevention and / or treatment of optic nerve diseases, and the use of an artificial synaptic connector or nucleic acid encoding an artificial synaptic connector in the manufacture of a preventive or therapeutic agent for optic nerve diseases.

[0008] The means for solving the aforementioned problems are as follows: <1> A preventive or therapeutic agent for optic nerve disease, characterized by containing an effective amount of an artificial synaptic connector or a nucleic acid encoding an artificial synaptic connector. <2> The preventive or therapeutic agent for optic nerve disease according to <1>, wherein the artificial synaptic connector comprises a domain that binds to neurexin (Nrx), a multimerization domain, and a domain that binds to the extracellular region of an AMPA receptor. <3> The preventive or therapeutic agent for optic nerve disease according to <1>, wherein the artificial synaptic connector comprises, in this order, a domain that binds to neurexin (Nrx), a multimerization domain, and a domain that binds to the extracellular region of an AMPA receptor. <4> The preventive or therapeutic agent for optic nerve disease according to <1>, wherein the artificial synaptic connector is a protein containing the amino acid sequence of Sequence ID No. 1 or a functional variant thereof. <5> The preventive or therapeutic agent for optic nerve disease according to any one of <1> to <4>, wherein the optic nerve disease is glaucoma, retinitis pigmentosa, and age-related optic nerve loss. <6> The preventive or therapeutic agent for an optic nerve disease according to any one of <1> to <4>, wherein the optic nerve disease is glaucoma. <7> A method for preventing and / or treating an optic nerve disease, comprising the step of administering an artificial synaptic connector or a nucleic acid encoding an artificial synaptic connector to a patient with the optic nerve disease. <8> The preventive or therapeutic method according to <7>, wherein the artificial synaptic connector comprises a domain that binds to neurexin (Nrx), a multimerizing domain, and a domain that binds to the extracellular region of an AMPA receptor. <9> The preventive or therapeutic method according to <7>, wherein the artificial synaptic connector comprises, in this order, a domain that binds to neurexin (Nrx), a multimerizing domain, and a domain that binds to the extracellular region of an AMPA receptor. <10> The preventive or therapeutic method according to <7>, wherein the artificial synaptic connector is a protein containing the amino acid sequence of Sequence ID No. 1 or a functional variant thereof. <11> The method of prevention or treatment according to any one of <7> to <10>, wherein the optic nerve disease is glaucoma, retinitis pigmentosa, or age-related optic nerve loss.<12> The method for prevention or treatment according to any one of <7> to <10>, wherein the optic nerve disease is glaucoma. <13> The method for prevention or treatment according to any one of <7> to <12>, wherein the artificial synaptic connector or the nucleic acid encoding the artificial synaptic connector is administered by intravitreous injection or eye drops. <14> An artificial synaptic connector or a nucleic acid encoding an artificial synaptic connector for use in the prevention and / or treatment of optic nerve diseases. <15> Use of an artificial synaptic connector or a nucleic acid encoding an artificial synaptic connector in the manufacture of an agent for prevention or treatment of optic nerve diseases. <16> The agent for prevention or treatment of optic nerve diseases according to <2> or <3>, wherein the domain that binds to neurexin (Nrx) is a region that can bind to Nrx (Nrx(S4+)) having a splice site 4. <17> The agent for prevention or treatment of optic nerve diseases according to <16>, wherein the region that can bind to Nrx(S4+) is a cysteine-rich region derived from the Cbln1 protein. <18> The preventive or therapeutic agent for optic nerve disease according to <2>, <3>, <16>, or <17>, wherein the domain that binds to the extracellular domain of the AMPA receptor is a region derived from the NPTX1 protein. <19> The preventive or therapeutic agent for optic nerve disease according to any one of <2>, <3>, and <16> to <18>, wherein the multimerizing domain is a trimerizing domain. <20> The preventive or therapeutic agent for optic nerve disease according to <19>, wherein the trimerizing domain is a coiled-coil domain. <21> The preventive or therapeutic agent for optic nerve disease according to <20>, wherein the coiled-coil domain is a triple-stranded GCN4 leucine zipper coiled-coil. <22> The method for preventing or treating optic nerve disease according to <8> or <9>, wherein the domain that binds to neurexin (Nrx) is a region that can bind to Nrx (Nrx(S4+)) having a splice site 4. <23> The method for preventing or treating optic nerve disease according to <22>, wherein the region capable of binding to Nrx(S4+) is a cysteine-rich region derived from the Cbln1 protein.<24> The method for preventing or treating an optic nerve disease according to <8>, <9>, <22>, or <23>, wherein the domain that binds to the extracellular domain of the AMPA receptor is a region derived from the NPTX1 protein. <25> The method for preventing or treating an optic nerve disease according to any one of <2>, <3>, and <22> to <24>, wherein the multimerizing domain is a trimerizing domain. <26> The method for preventing or treating an optic nerve disease according to <25>, wherein the trimerizing domain is a coiled-coil domain. <27> The method for preventing or treating an optic nerve disease according to <26>, wherein the coiled-coil domain is a triple-stranded GCN4 leucine zipper coiled-coil.

[0009] According to the present invention, the aforementioned problems of the conventional era can be solved and the aforementioned objectives can be achieved. The present invention can provide a preventive or therapeutic agent for the prevention and / or treatment of optic nerve diseases, a method for prevention or treatment, an artificial synaptic connector or nucleic acid encoding an artificial synaptic connector for use in the prevention and / or treatment of optic nerve diseases, and the use of an artificial synaptic connector or nucleic acid encoding an artificial synaptic connector in the manufacture of a preventive or therapeutic agent for optic nerve diseases.

[0010] Figure 1A shows the experimental schedule for CPTX administration to a normal-tension glaucoma model. Figure 1B shows the localization of CPTX in the retina of a normal-tension glaucoma model. Figure 1C shows the stratified distribution of CPTX in the retina of a normal-tension glaucoma model. Figure 1D shows the synaptic localization of CPTX in the retina of a normal-tension glaucoma model. Figure 1E shows the synaptic localization of CPTX in the inner plexiform layer (IPL) of the retina of a normal-tension glaucoma model. Figure 1F shows the effect of CPTX on the postsynaptic marker PSD95 in the IPL of a normal-tension glaucoma model. Figure 1G shows the effect of CPTX on the presynaptic marker vGlut1 in the IPL of a normal-tension glaucoma model. Figure 1H shows the effect of CPTX on the retinal ganglion cell marker Rbpms in the IPL of a normal-tension glaucoma model. Figure 2A shows GLAST evaluated by multifocal electroretinography (mfERG). ＋ / － This is a representative three-dimensional heatmap of the mouse's secondary kernel (2K) response. FIG. 2B shows GLAST in mfERG ＋ / － This is a diagram showing quantitative data of the mouse's retinal response. FIG. 2C shows GLAST measured by optokinetic nystagmus (OKR) ＋ / － This is a diagram showing the change over time of the mouse's visual acuity. FIG. 2D is a diagram showing the quantitative analysis at the 14th day after administration in FIG. 2C. FIG. 2E shows GLAST administered with CPTX measured by OKR ＋ / － This is a diagram showing the change over time of the mouse's contrast sensitivity. FIG. 2F is a diagram showing the quantitative analysis at the 14th day after administration in FIG. 2E. FIG. 2G shows GLAST administered with CPTX in the visual cliff test ＋ / －This is a heatmap showing the time spent in each region by mice during the experiment. Figure 2H shows the temporal and quantitative analysis of Figure 2G. Figure 2I shows the quantitative analysis of Figure 2H at 14 days after administration. Figure 3A is a representative image of the silicone oil injection (SIOH) model of intraocular pressure (SIOH model). Figure 3B shows the experimental schedule for evaluating the preventive effect of CPTX in the SIOH model. Figure 3C shows a representative image and quantitative analysis of the PSD95 signal in the inner plexiform layer (IPL) of the SIOH model retina. Figure 3D shows a representative image of the vGlut1 signal in the IPL of the SIOH model and its quantitative analysis of intensity. Figure 3E is a representative heatmap of the 2K response of the SIOH model evaluated by mfERG. Figure 3F shows the quantitative analysis of the retinal response of the SIOH model by mfERG. Figure 3G shows the effect of CPTX on visual acuity in the SIOH model. Figure 3H shows the quantitative analysis of the effect of CPTX on contrast sensitivity in the SIOH model. Figure 3I shows the effect of CPTX on the SIOH model in the Visual Cliff Test. Figure 3J shows the experimental schedule for evaluating the therapeutic effect of CPTX in the chronic SIOH model. Figure 3K shows a representative image of the PSD95 signal in IPL in the chronic SIOH model and a quantitative analysis of its intensity. Figure 3L shows a representative image of the vGlut1 signal in IPL in the chronic SIOH model and a quantitative analysis of its intensity. Figure 3M is a representative heatmap of the 2K response in the chronic SIOH model using mfERG. Figure 3N shows a quantitative analysis of the retinal response in the chronic SIOH model using mfERG. Figure 3O shows a quantitative analysis of the effect of CPTX on visual acuity in the chronic SIOH model. Figure 3P shows a quantitative analysis of the effect of CPTX on contrast sensitivity in the chronic SIOH model. Figure 3Q shows the effect of CPTX on the chronic SIOH model in the Visual Cliff Test. Figure 4A shows the GLAST test for the elderly. ＋ / －This figure shows representative images and quantitative analysis of signal intensity illustrating the effect of CPTX on the postsynaptic marker PSD95 in mice. Figure 4B shows GLAST in elderly mice. ＋ / － This figure shows representative images and quantitative analysis of signal intensity demonstrating the effect of CPTX on the presynaptic marker vGlut1 in mice. Figure 4C shows aged GLAST ＋ / － This figure shows the effect of CPTX on the retinal ganglion cell marker Rbpms in mice. Figure 4D shows aged GLAST mice treated with CPTX. ＋ / － This figure shows the quantitative analysis of retinal responses in mice. Figure 4E shows GLAST in elderly mice treated with CPTX. ＋ / － This figure shows a quantitative analysis of the effect of CPTX on visual acuity in mice. Figure 4F shows aged GLAST mice treated with CPTX. ＋ / － This figure shows a quantitative analysis of the effect of CPTX on contrast sensitivity in mice. Figure 4G shows the results of the Visual Cliff Test in aged mice treated with CPTX. ＋ / －This figure shows the effects of CPTX on mice. Figure 5A shows a quantitative analysis of visual acuity with age. Figure 5B shows a quantitative analysis of contrast sensitivity with age. Figure 5C shows a heatmap and quantitative analysis of the visual cliff test with age. Figure 5D shows the effect of CPTX on retinal ganglion cell number in aged mice. Figure 5E shows the effect of CPTX on the postsynaptic marker PSD95 in aged mice. Figure 5F shows the effect of CPTX on the presynaptic marker vGlut1 in aged mice. Figure 5G shows a quantitative analysis of retinal response in aged mice. Figure 5H shows a quantitative analysis of the effect of CPTX on visual acuity in aged mice. Figure 5I shows a quantitative analysis of the effect of CPTX on contrast sensitivity in aged mice. Figure 5J shows a heatmap and quantitative analysis of the visual cliff test in aged mice. Figure 6A shows a quantitative analysis of the effect of CPTX on visual acuity in an MNU-induced retinal degeneration animal model. Figure 6B shows a quantitative analysis of the effect of CPTX on contrast sensitivity in an MNU-induced retinal degeneration animal model. Figure 6C shows a heatmap and quantitative analysis of the visual cliff test in an MNU-induced retinal degeneration animal model. Figure 7A shows GLAST by OKR. － / － This figure shows the temporal changes in the visual acuity of mice. Figure 7B shows the quantitative analysis 14 days after administration of the results in Figure 7A. Figure 7C shows the GLAST analysis using OKRs. － / － This figure shows the temporal changes in contrast sensitivity in mice. Figure 7D shows the quantitative analysis 14 days after CPTX administration in Figure 7C. Figure 7E shows GLAST － / － This is a heatmap of the visual cliff test in mice. Figure 7F shows GLAST － / －Figure 7G shows the temporal changes in the response to the visual cliff test in mice. Figure 7F shows the quantitative analysis at 14 days after CPTX administration. Figure 8A shows the intraocular pressure (IOP) levels in the eyes of 3-month-old WT mice, both in the normal eye and at 2 and 4 weeks after silicone oil injection. Figure 8B shows a representative heatmap and quantitative analysis of the retinal response of the SIOH model measured by multifocal ERG. Figure 8C shows the visual acuity measurement of the SIOH model by OKR. Figure 8D shows a representative heatmap image and quantitative analysis of the visual cliff test in the SIOH model. Figure 8E shows a representative image of Rbpms immunostaining and quantitative analysis of the number of Rbpms-positive RGCs in the SIOH model pre-administered with CPTX. Figure 8F shows a representative image of Rbpms immunostaining and quantitative analysis of the number of Rbpms-positive RGCs in the SIOH model post-administered with CPTX. Figure 9A shows aged GLAST － / － This figure shows a representative heatmap (left) and quantitative analysis (right) of retinal responses in mice. Figure 9B shows GLAST analysis of elderly mice. － / － This figure shows the quantitative analysis of visual acuity in mice. Figure 9C shows the GLAST analysis of elderly mice. － / － This figure shows a quantitative analysis of the effect of CPTX on contrast sensitivity in mice. Figure 9D shows GLAST in elderly mice. － / － This figure shows a heatmap and quantitative analysis of the visual cliff test in mice.

[0011] (Agent for the prevention or treatment of optic nerve disease) The agent for the prevention or treatment of optic nerve disease of this embodiment is an agent for the prevention and / or treatment of optic nerve disease, comprising an effective amount of artificial synaptic connector or nucleic acid encoding an artificial synaptic connector.

[0012] (Method for preventing or treating optic nerve disease) The method for preventing or treating optic nerve disease according to this embodiment is a method for preventing and / or treating optic nerve disease, comprising the step of administering an artificial synaptic connector or a nucleic acid encoding an artificial synaptic connector to a patient with the optic nerve disease.

[0013] (Use) In one embodiment, an artificial synaptic connector or nucleic acid encoding an artificial synaptic connector is provided for use in the prevention and / or treatment of optic nerve diseases. Also in one embodiment, the use of an artificial synaptic connector or nucleic acid encoding an artificial synaptic connector is provided in the manufacture of a preventive or therapeutic agent for optic nerve diseases.

[0014] <Optical Nerve Diseases> Optic nerve diseases are neurodegenerative diseases that involve a decrease and / or loss of optic nerve cells such as retinal ganglion cells (RGCs). Examples include glaucoma, retinitis pigmentosa, and age-related optic nerve loss. Among these, glaucoma, including normal-tension glaucoma and high-tension glaucoma, is preferred, with normal-tension glaucoma being more preferred.

[0015] As a result of diligent research to solve the above-mentioned objectives, the inventors obtained the following findings, as demonstrated in the examples described later, and based on these findings, the present invention was completed.

[0016] As described above, our research group has suggested that CPTX, an artificial synaptic connector that binds presynaptic neurexin to postsynaptic AMPA receptors, may function as a synaptic treatment for synaptic disorders in neurodegenerative diseases such as Alzheimer's disease and spinal cord injury, as well as traumatic neurological diseases.

[0017] In glaucoma, approximately half of the retinoid cells (RGCs) remain at the time of visual impairment, and it has been reported that atrophy of RGC dendrites and loss of synapses occur before RGC degeneration. Based on this, the inventors hypothesized that synaptic therapy could restore visual function and further suppress the progression of neuronal cell death in retinal neurodegenerative diseases. Therefore, in this embodiment, the effects of intravitreal injection of the artificial synapse connector CPTX were investigated in normal-tension glaucoma, high-tension glaucoma, aging model mice, and photoreceptor degeneration model mice. It was found that pre-administration of CPTX in these disease models not only prevented synaptic damage and neuronal cell death in the retina, but also restored visual function even after the disease had progressed.

[0018] According to the present invention, the aforementioned problems of the conventional era can be solved and the aforementioned objectives can be achieved. The present invention can provide a preventive or therapeutic agent for the prevention and / or treatment of optic nerve diseases, a preventive or therapeutic method, an artificial synaptic connector or nucleic acid encoding an artificial synaptic connector for use in the prevention and / or treatment of optic nerve diseases, and the use of an artificial synaptic connector or nucleic acid encoding an artificial synaptic connector in the manufacture of a preventive or therapeutic agent for optic nerve diseases.

[0019] The following describes the preventive or therapeutic agent, preventive or therapeutic method, artificial synaptic connector, or nucleic acid encoding the artificial synaptic connector in use according to this embodiment.

[0020] <Artificial Synapse Connector> The artificial synapse connector is not particularly limited as long as it is a fusion protein capable of binding presynaptic neurexin and postsynaptic AMPA receptors, and can be appropriately selected according to the purpose. However, it is preferable that it includes a domain that binds to neurexin (Nrx), a multimerizing domain, and a domain that binds to the extracellular domain of the AMPA receptor, and it is more preferable that it includes the domain that binds to neurexin (Nrx), the multimerizing domain, and the domain that binds to the extracellular domain of the AMPA receptor in this order, and it is even more preferable that it includes a protein containing the amino acid sequence of SEQ ID NO: 1 or a functional variant thereof. A functional variant of a protein containing the amino acid sequence of SEQ ID NO: 1 means a variant that retains the function of an artificial synapse connector that induces excitatory synapse formation of the protein containing the amino acid sequence of SEQ ID NO: 1, or a variant that further improves the function of an artificial synapse connector that induces excitatory synapse formation than the protein containing the amino acid sequence of SEQ ID NO: 1. The amino acid sequences of the protein containing the amino acid sequence of SEQ ID NO: 1 and its functional variants are preferably 90% or more identical to each of the known amino acid sequences of SEQ ID NO: 1, and more preferably 95% or more identical.

[0021] [Neurexin] Neurexin (Nrx) is a family of proteins that function as cell adhesion molecules and receptors in the nervous system of vertebrates, and is reported to be localized to the presynaptic region. Nrx has two isoforms, α and β, depending on the promoter used for transcription. The α-Nrx isoform has six laminin G domains (LG domains) extracellularly, and is known to form complexes with proteins such as Cbln1 via the LG domains to facilitate synapse formation.

[0022] [Cbln1] Cbln1 is a protein also known as cerebellin-1. Cbln1 is a secretory protein belonging to the C1q family and is mainly produced and secreted in granule cells of the cerebellum. Cbln1 binds to neurexin (Nrx) at the presynaptic terminal. Cbln1 is essential for the formation of synapses between parallel fibers and Purkinje cells (parallel fiber synapses), and Cbln1 knockout mice exhibit a reduced density of parallel fiber synapses and severe cerebellar ataxia symptoms. Human Cbln1 protein may have the amino acid sequence registered in NCBI Reference Sequence: NP_004343.1.

[0023] In human Cbln1 (NCBI Reference Sequence: NP_004343.1), amino acid sequences 1-21 are a signal sequence, amino acids 34-38 are considered essential for binding to Nrxn1, the cysteine ​​at positions 34 and 38 are used to form disulfide bonds between Cbln1 molecules, and positions 57-193 constitute the C1q domain. Furthermore, amino acids 62-193 are considered necessary for binding to Cbln3 and homotrimerization, and amino acids 122-147 are considered essential for interaction with GLUD2. Human Cbln1 has an Nrx binding site (Nrx-binding domain) in the region of amino acids 22-53. The Nrx-binding domain of the Cbln1 protein may have an amino acid sequence corresponding to the amino acid sequence 22-53 of the human Cbln1 protein. The Nrx-binding domain of the Cbln1 protein can be responsible for binding to Nrx (i.e., Nrx(S4+)) that has splice site 4 (the fourth splice site).

[0024] [Nptx1] Neuronal pentraxin-1, also known as Nptx1 or NP1, is a member of the neuronal pentraxin gene family. Nptx1 binds to the AMPA-type glutamate receptor (AMPAR) and activates AMPAR. Human NPTX1 protein may have the amino acid sequence registered in NCBI Reference Sequence: NP_002513.2.

[0025] In the human NPTX1 protein (NCBI Reference Sequence: NP_002513.2), amino acid sequences 1-22 constitute the signal sequence, and amino acid sequences 222-428 constitute the pentraxin domain. The NPTX1 protein has an AMPAR binding site (AMPAR-binding domain) in the region of amino acids 222-428. The AMPAR-binding domain of the NPTX1 protein may have an amino acid sequence corresponding to amino acid sequences 222-428 of the human NPTX1 protein (NCBI Reference Sequence: NP_002513.2).

[0026] [AMPA-type glutamate receptor (AMPAR)] The AMPA-type glutamate receptor (AMPAR) is a tetrameric excitatory glutamate receptor consisting of four subunits, GluA1 to GluA4, and plays an important role in the plasticity of excitatory synapses in the central nervous system. "AMPA" stands for α-amino-3-hydroxy-5-mesoxazole-4-propionic acid. Each subunit of the human AMPA-type glutamate receptor protein, GluA1, GluA2, GluA3, and GluA4, may have amino acid sequences registered under UniProt IDs P42261, P42262, P42263, and P48058, respectively.

[0027] The NPTX1-binding domain of AMPAR is the amino-terminal domain (ATD) within the extracellular region of human AMPA-type glutamate receptor proteins. Specifically, it may contain amino acid sequences of AMPAR proteins corresponding to amino acid sequences 19-412 of GluA1 (UniProt ID P42261; Ala19-Thr412), 22-419 of GluA2 (UniProt ID P42262; Val22-Thr419), 29-428 of GluA3 (UniProt ID P42263; Gly29-Thr428), and 22-420 of GluA4 (UniProt ID P48058; Ala22-Thr420).

[0028] Throughout this specification, gene names and protein names, whether uppercase or lowercase, are used to include orthologs of all mammalian species. In this specification, the animal species from which a gene name or protein name originates is distinguished by prefixing the gene name or protein name with the animal species name.

[0029] <Nerexin (Nrx) Binding Domain> The neurexin (Nrx) binding domain can be the Nrx binding domain of the Cbln1 protein. In particular, it is the region of the Cbln1 protein that can bind to Nrx having splice site 4 (i.e., Nrx(S4+)). The Nrx binding region of the Cbln1 protein can be, for example, the region of Cbln1 corresponding to the cysteine-rich region (CRR) (J. Elegheert et al., Science353, 295-299 (2016)) of human Cbln1 (GenBank ID NM_004352;Gln22-Ile53) (the region of Cbln1 having the corresponding amino acid sequence).

[0030] <Multimerization Domain> Any multimerization domain capable of multimerizing the fusion protein can be used. In one embodiment, the multimerization domain may be a trimerization domain (or trimer-forming domain). The trimerization domain may be, for example, a coiled-coil domain (especially one capable of forming a coiled-coil triple helix), and is not particularly limited, but may, for example, be a trimerization domain of GCN4 in a preferred embodiment. As trimer domains, trimer domains selected from the group consisting of trimer domains of collagen family proteins (e.g., collagen α1, α2), α-keratin, Clq protein, ACRP30 (an overwintering protein), celeberin, multimerin, collectin, conglutinin, pulmonary surfactant protein A (SP-A), and mannose-binding protein (MBP) can also be used. In preferred embodiments, trimer domains of the Clq protein family and collectin family can be used. The trimer domain may have a collagen-like sequence.

[0031] <Domain that binds to the extracellular domain of the AMPA receptor> The domain that binds to the extracellular domain of the AMPA receptor may be the pentraxin domain of the Nptx1 protein. The region of Nptx1 that binds to the AMPA-type glutamate receptor is, for example, the pentraxin domain (NP1) of human neuronal pentraxin-1. PTX This may be a region of Nptx1 corresponding to ; GenBank ID AC50727.1; Pro224-Ile431 (a region of Nptx1 having the corresponding amino acid sequence).

[0032] <Linker> The artificial synaptic connector may have a domain that binds to neurexin (Nrx), a multimerizing domain, and a domain that binds to the extracellular region of the AMPA receptor linked via a linker. The linker may be a peptide. The linker may also be, for example, a flexible linker. Examples of flexible linkers include -(CH ２ ) ６ - Hydrocarbon linker having (GGGGS) ｎ (SEQ ID NO: 3), KESGSVSSECLAQFRSLD (SEQ ID NO: 4) or EGKSGSGSSESKST (SEQ ID NO: 5), GGGGGGGG (SEQ ID NO: 6), GSAGSAAGSGEF (SEQ ID NO: 7), (GGSG) ｎ (Sequence ID 8) or (GS) ｎ Examples include {wherein n is a natural number from 1 to 5}. In one embodiment of the present invention, in the fusion protein of the present invention, the region of the Cbln1 protein that binds to Nrx, the multimerization domain, and the region of the Nptx1 that binds to the AMPA receptor may be directly linked without the use of a linker.

[0033] Furthermore, the artificial synaptic connector may be a fusion protein further fused with peptides such as tags and protein domains, as long as it can bind the neurexin at the presynaptic site to the AMPA receptor at the postsynaptic site. Examples of tags that can be fused include HA tags and His tags.

[0034] In one embodiment, the artificial synaptic connector may have a multimerizing domain that is a trimerizing domain and may be a trimer. The artificial synaptic connector may have a multimerizing domain that is a trimerizing domain and may be a hexamer formed by the linkage of two trimers. Two trimers may be linked to form a hexamer by the 34th and / or 38th cysteine ​​of the human Cbln1 protein (NCBI Reference Sequence: NP_004343.1).

[0035] In one embodiment, the artificial synaptic connector may be derived from any region or domain of a human protein. In one embodiment, the artificial synaptic connector is a multimer of a fusion protein comprising a region that binds to Nrx of the human Cbln1 protein, a multimerizing domain of the human protein, and a region that binds to the AMPA receptor of the human NPTX1, and is preferably a fusion protein CPTX or a functional variant thereof having the amino acid sequence represented by SEQ ID NO: 1. The multimerizing domain is a trimerizing domain, and the fusion protein may be in trimer or hexamer form.

[0036] <Nucleic acids encoding artificial synaptic connectors> There are no particular restrictions on the nucleic acids encoding artificial synaptic connectors, as long as they have a base sequence that encodes the amino acid sequence of the artificial synaptic connector described above. They can be appropriately selected depending on the purpose. For example, they may be in the form of mRNA or DNA, or they may be incorporated into an expression vector. In any case, they can be appropriately selected depending on the purpose.

[0037] Examples of nucleic acid base sequences encoding artificial synaptic connectors include base sequences encoding the amino acid sequence represented by SEQ ID NO: 1 (e.g., the base sequence represented by SEQ ID NO: 2, or the corresponding mRNA sequence), base sequences containing said base sequences, and base sequences encoding functional variants of CPTX. Here, a functional variant of CPTX refers to a variant of CPTX that functions as an artificial synaptic connector that induces excitatory synapse formation by binding presynaptic neurexin to postsynaptic AMPA receptors. The base sequence encoding the functional variant of CPTX is preferably 90% or more identical to the base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, and more preferably 95% or more identical.

[0038] Furthermore, the nucleic acid encoding the artificial synaptic connector may also be a nucleic acid encoding a fusion protein that combines peptides such as tags and protein domains, as long as it binds neurexin in the presynaptic region to AMPA receptors in the postsynaptic region.

[0039] (Pharmaceutical composition) As a preventive or therapeutic agent, it may be an artificial synaptic connector or the nucleic acid encoding the artificial synaptic connector itself, a protein preparation containing an artificial synaptic connector, a nucleic acid preparation containing the nucleic acid encoding the artificial synaptic connector, or it may contain other drugs or a pharmaceutically acceptable carrier.

[0040] "Pharmacologically acceptable" means a non-toxic component or composition that is physiologically acceptable and, when administered to humans, does not typically cause gastrointestinal disorders, dizziness, or other allergic reactions, or similar reactions. Examples of such carriers include solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes, and biodegradable nanoparticles.

[0041] Prophylactic or therapeutic agents may be formulated with appropriate carriers depending on the route of administration. There are no particular restrictions on the route of administration of prophylactic or therapeutic agents, and they can be appropriately selected depending on the purpose; for example, they may be administered parenterally or orally. Examples of parenteral administration routes include local injection into the eye, such as intravitreal injection, and eye drops.

[0042] Prophylactic or therapeutic agents may be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to the target.

[0043] Prophylactic or therapeutic agents formulated in the manner described above may be administered in an effective dose via various routes, including parenteral, oral, transdermal, subcutaneous, intravenous, or intramuscular. In this context, “effective dose” means the amount of substance administered to a patient that allows for the monitoring of diagnostic or therapeutic effects.

[0044] The dosage of prophylactic or therapeutic agents can be appropriately selected according to the route of administration, target population, target disease and its severity, age, sex, weight, individual differences, and disease state. Depending on the severity of the disease, the content of the active ingredient, artificial synaptic connector or nucleic acid encoding the artificial synaptic connector, can be appropriately selected, but generally, when using adults as a reference, a single dose of 1 μg to 10 μg (e.g., 5 μg) of the active ingredient may be administered repeatedly every few days.

[0045] The nucleic acid encoding the artificial synaptic connector preferably contains mRNA encoding the artificial synaptic connector, and more preferably has a 5' cap structure and a 3' tail structure from the viewpoint of mRNA stabilization. Examples of the 5' cap structure include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (wherein m represents a 2'-O-methyl residue). Examples of the 3' tail structure include a 3' poly(A) tail (e.g., 10 to 200 adenosine nucleotides), a 3' poly(C) tail (e.g., 10 to 200 cytosine nucleotides), and combinations thereof.

[0046] The nucleic acid encoding the artificial synaptic connector may include a 5' untranslated region and / or a 3' untranslated region. The 5' untranslated region and / or 3' untranslated region is, for example, 50 to 500 nucleotides and preferably contains elements that affect mRNA stabilization, translation, etc.

[0047] Nucleic acids encoding artificial synaptic connectors are preferably encapsulated in lipid nanoparticles from the viewpoint of mRNA stabilization. Examples of lipid nanoparticles include cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), OF-02, Target 23, Target 24, ICE, HGT5000, HGT5001, HGT4003, DOTAP (1,2-dioleyl-3-trimethylammoniumpropane), DODAP (1,2-dioleyl-3-dimethylammoniumpropane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammoniumpropane), DLinDMA, DODAC, It is preferable to include a cationic lipid selected from the group consisting of DDAB, DMRIE, DOSPA, DOGS, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLINDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, K-K-DMA, DLin-K-XTC2-DMA, DLin-KC2-DMA, dialkylamino-based, imidazole-based, and guanidine-based cationic lipids, and may further contain non-cationic lipids, PEG-modified lipids, etc.

[0048] The nucleic acid encoding the artificial synaptic connector may consist of unmodified nucleotides or may contain one or more modified nucleotides. Examples of modified nucleotides include pseudouridine, N-1-methylpseudridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 4'-thiouridine, 4'-thiocytidine, and 2-thiocytidine.

[0049] A prophylactic or therapeutic agent containing an expression vector encoding an artificial synaptic connector may contain an expression vector encoding an artificial synaptic connector, other drugs, and a pharmaceutically acceptable carrier.

[0050] There are no particular restrictions on the expression vector encoding the artificial synaptic connector, and any known expression vector can be appropriately selected depending on the purpose. Examples include plasmids, adeno-associated viruses, adenoviruses, retroviruses, poxviruses, baculoviruses, vaccinia viruses, herpes simplex viruses, Epstein or Barr viruses, adenoviruses, geminiviruses, and karimoviruses. Among these, adeno-associated viruses are preferred because they are derived from non-pathogenic viruses, are highly safe, and can efficiently introduce genes into non-dividing cells. The expression vector preferably comprises a promoter, a nucleic acid sequence encoding an interaction inhibitor functionally linked to the promoter, and a 5' untranslated region (UTR). The promoter may be a constitutive expression promoter or a conditionally regulated promoter, and any known promoter can be appropriately selected depending on the purpose.

[0051] A prophylactic or therapeutic agent containing nucleic acids encoding artificial synaptic connectors and / or an expression vector encoding artificial synaptic connectors may be in liquid form or lyophilized powder form. The prophylactic or therapeutic agent and syringe may be pre-filled for single-dose administration. The vial may contain the composition in lyophilized powder or liquid form.

[0052] In one embodiment, the dose of nucleic acid encoding an artificial synaptic connector and / or expression vector encoding an artificial synaptic connector is preferably at least 0.5 mg / kg based on the subject's body weight. Examples of such doses include 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, and 50 mg / kg.

[0053] In one embodiment, the expression of artificial synaptic connectors encoded by nucleic acids and / or expression vectors can be detected at least 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or 1 month after administration.

[0054] When a preventive or therapeutic agent is used in combination with other drugs, the preventive or therapeutic agent and the other drugs may be contained in a single formulation, or they may be contained in separate formulations. If they are in separate formulations, each formulation may be administered simultaneously, or they may be administered separately at different times.

[0055] Methods for evaluating the introduction and / or expression of artificial synaptic connectors include, for example, Western blotting, immunohistochemistry (IHC), immunofluorescence (IF), immunochromatography, enzyme immunosorbent assay (ELISA), chemiluminescent enzyme immunosorbent assay (CLEIA), chemiluminescent enzyme immunosorbent assay (CLIA), fluorescence enzyme immunosorbent assay, aptamer assay, immunoturbidimetry, dye colorimetric assay, immunowax assay, latex agglutination assay, Jaffe assay, and gold colloid colorimetric assay. Methods for evaluating the introduction and / or expression of nucleic acids encoding artificial synaptic connectors include, for example, in situ hybridization, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and spatial transcriptome analysis.

[0056] There are no particular restrictions on the antibodies that can detect artificial synaptic connectors, and they can be appropriately selected depending on the purpose. Furthermore, artificial synaptic connectors with tags such as HA tags can be expressed, and they can be detected using antibodies against the tags.

[0057] There are no particular restrictions on the PCR primers or hybridize probes that can detect nucleic acids and / or expression vectors encoding artificial synaptic connectors, and they can be appropriately selected depending on the purpose.

[0058] The subjects mentioned above are generally mammals, including, for example, humans, non-human primates, dogs, cats, mice, rats, cattle, horses, and pigs. Among these, humans are preferred. The subjects may be adults, infants, children, or the elderly, and subjects suspected of having an optic nerve disease, or subjects diagnosed or confirmed to have an optic nerve disease, are preferred.

[0059] There are no particular restrictions on the subjects who can be evaluated for the introduction and / or expression of artificial synaptic connectors, nucleic acids encoding artificial synaptic connectors, and / or expression vectors. They can be appropriately selected according to the purpose, for example, subjects who have been administered prophylactic or therapeutic agents by intravitreal injection, eye drops, etc.

[0060] The present invention will be described more specifically below based on examples, but the present invention is not limited to the following examples.

[0061] <Methods and Materials> <<Animals>> All animal experiments were conducted in strict accordance with the ARVO Statement for the Ethical Use of Animals in Ophthalmic and Vision Research. The animal experiment protocols were reviewed and approved by the Animal Experiment Committee of the Tokyo Metropolitan Institute of Medical Science (Approval Number: 23-068). C57BL / 6J mice were obtained from SLC Japan Co., Ltd. No specific statistical methods were used to determine sample size, and the number of samples used in this study conformed to the standards of the research field. All mice were housed in a pathogen-free environment, with the temperature maintained at 23°C and the humidity at 55%. Lighting was controlled in a 12-hour cycle (8:00 to 20:00), and the mice were given free access to food and water.

[0062] <<Antibodies>> Primary antibody information is as follows: anti-His (mouse, 1:1000, Medical & Biological Laboratories, Inc. (MBL), D291-3), anti-PSD95 (mouse, 1:1000, BioLegend, 810401), anti-pan-AMPAR (guinea pig, 1:500, Frontier Institute, Af580), anti-vGlut1 (guinea pig, 1:1000, Frontier Institute, Af570), anti-Bassoon (rabbit, 1:500, Synaptic Systems, 141-003, or mouse, 1:500, Enzo Life Sciences, SAP7F407). Secondary antibodies were used as labels for the corresponding primary antibodies, with DyLight 405, Alexa 488, 546, and 647 (Invitrogen or Jackson ImmunoResearch Laboratories) used at a dilution ratio of 1:1000. The cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) at a ratio of 1:2000.

[0063] <<Optokinetic Ocular Reflex (OKR)>> OKR measurements were performed using the OptoMotry system (Cerebral Mechanics) according to the manufacturer's procedures. Mice were placed on a platform 10 cm high, and black and white stripes were displayed on four monitors surrounding them. Visual acuity was measured at 100% contrast, spatial frequency 0.0 c / d to 0.6 c / d, temporal frequency 0.0 Hz to 7.2 Hz, and movement speed 12 deg / s. Contrast sensitivity was measured by varying the contrast while keeping the spatial frequency constant. A positive response was defined as the mouse following the stripe at the same speed as the visual stimulus. Visual acuity and contrast thresholds were measured by gradually changing the stimulus conditions until the response disappeared. Left and right visual acuity was correlated with the direction of stripe rotation.

[0064] <<Visual Cliff Test>> To evaluate visual function, the Visual Cliff Test, developed by Gibson and Walk, was used. The mouse was placed on an acrylic platform (size: 60 cm x 30 cm) and divided into a "shallow side" with a black and white checkerboard pattern and a transparent "deep side". The entire apparatus was positioned at a height of 100 cm and illuminated by a 20 lux LED bar light. The mouse's behavior was recorded for 10 minutes using a LogiCool webcam (C270nd HD 720P), and the time spent in each area was quantified using video tracking software ANY-maze (Stoelting).

[0065] <<Western Blotting>> The amount of CPTX remaining in the retina after intraocular administration was evaluated at each time point. The excised retinas were treated with lysis buffer (2% SDS, 62 mM Tris-HCl [pH 6.8], 15% glycerol, 0.00625% Coomassie Blue G-250, 5% 2-mercaptoethanol) and heated at 100°C for 5 minutes. After electrophoresis with a 5% to 20% gradient SDS-PAGE, the samples were transferred to an Immobilon-P membrane. Blocking was performed with TBS-T (0.05% Tween-20, 50 mM Tris-HCl [pH 7.6], 150 mM NaCl) + 2.5% skim milk powder, and the primary antibody was reacted overnight at 4°C, while the HRP-labeled secondary antibody was reacted at 23°C for 1 hour. Luminescence was detected using Chemi-Lumi One Ultra (Nacalai Tesque Co., Ltd.) and FUSION FX (Vilber Bio Imaging).

[0066] <<Immunofluorescence Staining>> The whole retina or sections were blocked for 1 hour with PBS + 5% horse serum + 1% Triton X-100, and then the primary antibody was reacted in the same buffer at 4°C for 24 to 72 hours. After washing with PBS + 0.1% Triton X-100, the secondary antibody was reacted at room temperature for 1 hour or overnight at 4°C. Fluorescence images were acquired using an Olympus FV3000 confocal microscope (Olympus Corporation). For ganglion cell counting, anti-Rbpms antibody (1:1000, homemade) was used, and cells were counted in the midperiphery (0.5 mm to 1.0 mm from the optic disc) with a 100x objective lens. For synapse formation evaluation, anti-vGlut1 and anti-PSD95 antibodies were used, and staining was performed with a fluorescently labeled secondary antibody. Images were acquired with a 100x objective lens + digital zoom ×2.5.

[0067] <<Measurement of Retinal Response>> Retinal response was evaluated using multifocal electroretinography (mfERG) with the VERIS 6.0 system (Electro-Diagnostic Imaging). Visual stimuli consisted of seven hexagonal patterns displayed at 100 Hz. The amplitude of the second-order kernel (2K) was measured, which is considered an indicator of endoretinal damage and RGC function. Medetomidine (0.3 mg / kg), midazolam (4.0 mg / kg), and butorphanol (5.0 mg / kg) were administered intraperitoneally during anesthesia. Measurements were performed in a light-adapted state. After pupil dilation with eye drops (1% tropicamide + 2.5% phenylephrine), 0.4% benoxil was instilled, and contact lenses with electrodes were placed on the cornea. A reference electrode was subcutaneously inserted into the forehead. Average measurements over 7 minutes were analyzed.

[0068] <<Silicon-Induced Intraocular Pressure Elevation Model (SIOH Model) (High-Tension Glaucoma Model)>> The SIOH model was constructed according to a previously reported method (Zhang et al. eLife 2019 May 15; 8: e45881 https: / / pmc.ncbi.nlm.nih.gov / articles / PMC6533060 / ). Midazolam (4 mg / kg), medetomidine (0.3 mg / kg), and butorphanol (5 mg / kg) were administered intraperitoneally for anesthesia. After instilling 0.4% Benoxil eye drops, the cornea was punctured with a 34G needle and silicone oil (1000 mPa.s) was injected into the anterior chamber.

[0069] <<Intraocular Pressure Measurement (IOP)>> IOP was measured using a rebound tonometer (TonoLab). Anesthesia was administered intraperitoneally with midazolam (4.0 mg / kg), medetomidine (0.3 mg / kg), and butorphanol (5.0 mg / kg), and measurement was performed after the reduction reflex disappeared.

[0070] <<Retinitis Pigmentosa Model>> N-methyl-N-nitrosourea (MNU, 60 mg / kg) was administered intraperitoneally to 3-month-old wild-type mice. The day before, CPTX (7.5 μg / eye) was injected subretinally. Visual function was evaluated using OKRs and visual cliff tests 7 days after MNU administration.

[0071] <<Statistical Analysis>> Statistical analysis was performed using OriginPro (OriginLab). n represents the number of biological replicates. Data are expressed as mean ± SEM. The Mann-Whitney U test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) and Fisher's LSD test were used for comparisons between multiple groups. Post-hoc tests using two-way repeated measures ANOVA and LSD were performed for analysis of changes over time. A p-value < 0.05 was considered statistically significant.

[0072] <Results> <<CPTX shows retinal synaptic protective effects when administered after glaucoma onset in a normal-tension glaucoma model>> First, the therapeutic effect of CPTX was evaluated using GLAST heterozygous knockout (GLAST+ / -) mice (NTG model mice), an established genetic model of normal-tension glaucoma (NTG). In this model mouse, the number of retinal ganglion cells (RGCs) is known to decrease between 3 and 6 weeks of age. Therefore, CPTX was administered intravitreously at 4 weeks of age, corresponding to the early stage of glaucoma onset (Figure 1A). The signal of His-tagged CPTX in the inner plexiform layer (IPL) of wild-type (WT) mouse retinal sections 2 days after administration was significantly stronger compared to the untreated group (Figure 1B). This His signal was distributed in the inner half of the retina, namely the layer of nerve fibers (NFL), ganglion cell layer (GCL), IPL, and outer plexiform layer (OPL) (Figure 1C, left), and colocalized well with the excitatory synaptic marker PSD95 (Figure 1C, right). In other cases, the presynaptic marker Bassoon (Bsn) and postsynaptic pan-AMPAR signaling were observed from the NFL to the OPL, and these also colocalized well with the His signaling (Figures 1D, 1E). These results suggest that CPTX binds to retinal synapses.

[0073] Next, CPTX GLAST ＋ / － The effect of protecting synapses in mice (NTG model mice) was evaluated 2 weeks after administration (i.e., at 6 weeks of age). The result was GLAST ＋ / －In mice, the signal intensities of PSD95 and vGlut1 were significantly reduced, but recovered after CPTX administration (Figure 1F, Figure 1G). This suggests that synaptic disconnection occurred in this model, and that this disconnection was prevented by artificial synaptic connections created by CPTX administration. Furthermore, previous studies have reported that RGCs are protected by inhibiting synaptic disconnection mechanisms such as C1q. Our data also show that CPTX protects GLAST ＋ / － The number of RGCs in mice was shown to be restored (Figure 1H). Physiological synaptic connections are known to induce cytoprotection by activating intracellular signals such as cAMP response element-binding proteins (CREBs), and these results suggest that normalization of synaptic connections by CPTX may induce endogenous cytoprotective mechanisms in RGCs.

[0074] Figures 1A-H show that CPTX restores the number of retinal synapses in the early stages of glaucoma (4 weeks of age) in a normal-tension glaucoma model. Figure 1A shows the experimental schedule for CPTX administration to a normal-tension glaucoma model. GLAST at 4 weeks of age (early stage) ＋ / －Mice were injected intravitreously with His-tagged CPTX (7.5 μg / eye). The contralateral eye was injected with phosphate-buffered saline (PBS) as a control. The distribution of CPTX in the retina was analyzed 2 days after administration, and histological analysis was performed 14 days after administration. Visual function was evaluated by behavioral analysis before injection and 1, 3, 7, 14, 21, and 28 days after injection (Figures 2C-I). Figure 1B shows the localization of CPTX in the retina of a normal-tension glaucoma model. Compared to the no-treat group, a clear punctate His signal was observed in the intraretinal plexiform layer (IPL) after His-CPTX administration. Figure 1C shows the stratified distribution of CPTX in the retina of a normal-tension glaucoma model. After His-CPTX administration, the His signal was detected in the nerve fiber layer (NFL), ganglion cell layer (GCL), and intraretinal plexiform layer (IPL). The His signal co-localized with the PSD95 signal in the IPL. Figures 1D and 1E show the synaptic localization of CPTX in the retina of a normal-tension glaucoma model. In IPL, His-CPTX co-localized with the presynaptic component Bassoon (Bsn) and the postsynaptic component AMPA-type glutamate receptor (pan-AMPAR) signaling pathways. Figure 1F shows the effect of CPTX on the postsynaptic marker PSD95 in a normal-tension glaucoma model. Compared to 6-week-old wild-type (WT) mice, GLAST ＋ / － In mice undergoing IPL, PSD95 signaling was reduced, but CPTX administration restored PSD95 signaling. Representative image (left) and quantitative analysis of PSD95 signaling intensity (right, n = 6 eyes per group, **p < 0.01, ***p < 0.001, one-way ANOVA and Fisher's LSD test). Figure 1G shows the effect of CPTX on the presynaptic marker vGlut1 in a normal-tension glaucoma model. GLAST ＋ / －In mice with IPL, VGluT1 signaling was reduced, but CPTX administration restored VGluT1 signaling. Representative image (left) and quantitative analysis of vGluT1 signaling intensity (right, n = 6 eyes per group, ***p < 0.001, one-way ANOVA and Fisher's LSD test). Figure 1H shows the effect of CPTX on the retinal ganglion cell marker Rbpms in a normal-tension glaucoma model. GLAST compared to 6-week-old wild-type (WT) mice. ＋ / － Rbpms signaling was reduced in mice undergoing IPL, but it was restored by CPTX administration. Representative images (left) and quantitative analysis of Rbpms signaling intensity (right, n = 6 mice, ***p < 0.001, one-way ANOVA and Fisher's LSD test) are shown. Quantitative data are shown as mean ± standard error (SEM).

[0075] Next, CPTX was used in GLAST studies at 6 weeks of age when the onset of glaucoma had progressed. ＋ / － We also investigated whether it has a protective effect on retinal and visual function in mice. Using multifocal electroretinogram (mfERG), GLAST was found in 6-week-old mice. ＋ / － Retinal response in PBS-treated eyes of mice was significantly reduced compared to wild-type mice of the same age. However, retinal response recovered to a level comparable to wild-type mice two weeks after CPTX administration (Figures 2A and 2B). In the longitudinal evaluation of visual acuity using optokinetic response (OKR), CPTX significantly improved visual acuity from day 1 after administration, and this effect lasted for three weeks (Figure 2C). Quantitative analysis 14 days after administration showed that CPTX improved GLAST ＋ / － The mice's vision had almost completely recovered (Figure 2D). Similarly, a time-course analysis of contrast sensitivity using OKRs showed a significant improvement one day after administration, and this effect lasted for three weeks (Figure 2E). Quantitative evaluation at 14 days showed that contrast sensitivity had also almost completely recovered with CPTX administration (Figure 2F).

[0076] Furthermore, a visual cliff test was conducted to evaluate visual function. Heatmap analysis showed that GLAST ＋ / － The mice spent roughly equal amounts of time in shallow and deep areas, suggesting impaired depth perception (Figure 2G). However, after CPTX administration, they began spending more time in shallow areas, and an improvement in visual discrimination was observed. This effect was observed from 7 to 21 days after administration (Figure 2H), and quantitative analysis at 14 days showed that CPTX-treated mice had fully recovered in the visual discrimination test (Figure 2I).

[0077] Furthermore, GLAST homozygous deficiency (GLAST) presents with a more severe phenotype. － / － In mice, CPTX also showed a protective effect (Figures 7A-G).

[0078] Figures 2A-I show that CPTX restores visual function in the early stages of a normal-tension glaucoma model. Figure 2A shows GLAST as evaluated by multifocal electroretinography (mfERG). ＋ / － This is a typical three-dimensional heatmap of the second-order kernel (2K) response in mice. GLAST data from 4 weeks of age. ＋ / － Two weeks after injection into the eyes of mice, a higher response was observed in eyes treated with CPTX than in eyes treated with PBS. Figure 2B shows GLAST in mfERG. ＋ / － This figure shows quantitative data on the visual acuity of mice at 6 weeks of age. ＋ / － In mice, visual acuity was significantly reduced compared to wild-type (WT) mice of the same age, but function was restored with CPTX administration (n = 6 to 12 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 2C shows GLAST measured by optokinetic nystagmus (OKR). ＋ / －This is a figure showing the temporal change in the visual acuity of mice. CPTX showed significant improvement 1 day after administration, and its effect lasted for 3 weeks (n = 5 eyes, *P < 0.05, **P < 0.01, ***P < 0.0001, two-way repeated measures ANOVA and Fisher's LSD test). Figure 2D is a figure showing the quantitative analysis at the 14-day time point of administration in Figure 2C. GLAST administered with PBS ＋ / － In mice, visual acuity was significantly lower compared to WT of the same age, but in GLAST administered with CPTX ＋ / － visual acuity almost completely recovered (n = 5 eyes to 12 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 2E is a figure showing the temporal change in the contrast sensitivity of GLAST administered with CPTX measured by OKR ＋ / － This is a figure showing the temporal change in the contrast sensitivity of mice. After administration of CPTX, CPTX significantly improved contrast sensitivity over 3 weeks (n = 5 eyes, *P < 0.05, **P < 0.01, ***P < 0.0001, two-way repeated measures ANOVA and Fisher's LSD test). Figure 2F is a figure showing the quantitative analysis at the 14-day time point of administration in Figure 2E. GLAST administered with PBS ＋ / － In mice, contrast sensitivity was lower compared to WT of the same age, but in GLAST administered with CPTX ＋ / － in the CPTX-administered eyes of mice, contrast sensitivity was restored by CPTX administration (n = 5 eyes to 12 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 2G is a figure showing GLAST administered with CPTX in the visual cliff test ＋ / － This is a heatmap showing the time that mice stayed in each area during the experiment. GLAST without CPTX injection ＋ / －In mice, they were evenly distributed between the shallow side and the deep side, but in the individuals that received CPTX injection, the time spent on the shallow side increased. Figure 2H is a diagram showing the time-course and quantitative analysis of Figure 2G. CPTX-administered mice tended to stay more on the shallow side during 7 - 12 days after administration (n = 5 mice, **P < 0.01, ***P < 0.0001, two-way repeated measures ANOVA and Fisher's LSD test). Figure 2I is a diagram showing the quantitative analysis at the 14th day of administration in Figure 2H. GLAST administered with PBS ＋ / － Mice had lower selectivity for the shallow side compared to WT, but it was restored by CPTX (n = 10 mice, ***P < 0.001, one-way ANOVA and Fisher's LSD test).

[0079] Figures 7A - G are diagrams showing that CPTX improves visual function at the initial stage of onset in GLAST － / － mice. GLAST ＋ / － mice with significantly lower visual acuity than GLAST － / － mice were used as a more severe normal tension glaucoma (NTG) model. At 4 weeks of age, CPTX was injected at 7.5 μg into the vitreous body of the right eye of GLAST － / － mice. Figure 7A is a diagram showing the temporal change in visual acuity of GLAST － / － mice by OKR. Visual acuity was significantly improved on the 3rd day after CPTX administration, and the effect lasted for 3 weeks (n = 5 eyes, ***P < 0.001, two-factor repeated measures ANOVA and Fisher's LSD test). Figure 7B is a diagram showing the quantitative analysis at the 14th day after CPTX administration in Figure 7A. Compared with the PBS-administered eyes, the CPTX-administered eyes significantly improved the visual response, and the visual acuity approached that of WT mice (n = 5 eyes - 20 eyes, ***P < 0.001, one-factor ANOVA and Fisher's LSD test). Figure 7C is by OKR in GLAST － / －This figure shows the temporal changes in contrast sensitivity in mice. Contrast sensitivity significantly improved 1 day after CPTX administration and remained high for the following 3 weeks (n = 5 eyes, *P < 0.05, ***P < 0.001, two-way repeated-measures ANOVA and Fisher's LSD test). Figure 7D shows the quantitative analysis at 14 days after CPTX administration in Figure 7C. GLAST － / － In mice, contrast sensitivity was almost completely lost, but administration of CPTX significantly improved contrast sensitivity (n = 5 to 20 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 7E shows GLAST － / － This is a heatmap of the visual cliff test in mice. Figure 7F shows GLAST － / － This figure shows the temporal changes in the response to the visual cliff test in mice. CPTX significantly extended the time spent in the shallow area one day after administration, and this effect lasted for three weeks (n = 5 mice, **P < 0.01, ***P < 0.001, two-way repeated measures ANOVA and Fisher's LSD test). Figure 7G shows the quantitative analysis at 14 days after CPTX administration in Figure 7F. CPTX is GLAST － / － It significantly improved the response to the visual cliff test in mice (n = 10 mice, ***P < 0.001, one-way ANOVA and Fisher's LSD test).

[0080] <<Pretreatment with CPTX shows protective effect in a high-tension glaucoma model>> To evaluate the therapeutic effect of CPTX on primary open-angle glaucoma (POAG), for which elevated intraocular pressure (IOP) is the most well-known risk factor, a high-tension model was established by silicone oil injection (SIOH) (Figures 3A and 3B). In 3-month-old wild-type (WT) mice, SIOH increased intraocular pressure to 20 mmHg–25 mmHg under the experimental conditions (Figure 8A, described later), and this was accompanied by a decrease in visual acuity and response to the visual cliff test (Figures 8B–D, described later).

[0081] Two weeks after SIOH treatment, the signal intensities of PSD95 (Figure 3C) and vGlut1 (Figure 3D) were significantly lower compared to 3-month-old WT mice (Naive group), suggesting synaptic degeneration. However, in the group treated with CPTX as a pretreatment before SIOH, these decreases were suppressed, and the levels of PSD95 and vGlut1 were significantly higher compared to the PBS-treated group. Similar to synapses, the number of RGCs also decreased after two weeks with SIOH, but this decrease was partially suppressed by CPTX pretreatment (Figure 8E).

[0082] In terms of functional evaluation, two weeks after SIOH treatment, retinal response (Figure 3E, Figure 3F), visual acuity (Figure 3G), and contrast sensitivity (Figure 3H) were all significantly lower in the PBS pretreatment group compared to the untreated WT (untreated control) group. In contrast, all of these functional parameters were significantly improved in the CPTX pretreatment group, recovering to levels close to those of the untreated control group. This indicates that CPTX has a potent improving effect.

[0083] Furthermore, in the visual cliff test, SIOH treatment significantly reduced the proportion of time mice spent on the shallow side, while CPTX pretreatment significantly increased the proportion of time SIOH-treated mice spent on the shallow side (Figure 3I). These results suggest that CPTX pretreatment improves retinal and visual function that has been impaired by SIOH.

[0084] <<CPTX improves visual function in a chronic hypertensive glaucoma model>> Next, we investigated whether CPTX improves visual function in a chronic pathological state. For this investigation, CPTX was administered 4 weeks after induction of hypertension by SIOH (Figure 3J). 4 weeks after SIOH treatment, intraocular pressure (IOP) was chronically elevated (Figure 8A), and retinal and visual function was significantly impaired (Figures 8B-D). CPTX administration to mice 4 weeks after SIOH treatment did not protect the number of RGCs (retinal ganglion cells) 2 weeks after CPTX administration (Figure 8F), but PSD95 signaling recovered (Figure 3K). On the other hand, the signal intensity of vGlut1 did not decrease significantly even 6 weeks after SIOH treatment (Figure 3L), which is thought to be due to the regeneration response of bipolar cells that form immature synaptic terminals. CPTX administration to mice 4 weeks after SIOH treatment increased the signal intensity of vGlut1 2 weeks after CPTX administration (Figure 3L).

[0085] CPTX administration partially restored chronic retinal responses, visual acuity, and contrast sensitivity impaired by SIOH (Figure 3M-P). Furthermore, CPTX administration completely restored the visual cliff test response in a model of chronic glaucoma (Figure 3Q). These results suggest that CPTX repairs synapses even after chronic damage.

[0086] Figures 3A–Q show the preventive and therapeutic effects of CPTX on retinal and visual impairment in a high-tension glaucoma model. Figure 3A is a representative image of a silicone-injected high-tension model (SIOH model). Figure 3B shows the experimental schedule for evaluating the preventive effect of CPTX in the SIOH model. CPTX was pre-treated on Day 0, and silicone was injected on Day 1. Histological and functional analyses were performed after 2 weeks. Figure 3C shows a representative image (left) and quantitative analysis (right) of the PSD95 signal in IPL of the SIOH model. The PSD95 signal in IPL of 3-month-old wild-type (WT) mice and SIOH models administered with PBS or CPTX was compared (n = 6 eyes in each group, ***p < 0.001, one-way ANOVA + Fisher's LSD test). Figure 3D shows a representative image of the vGlut1 signal in IPL in the SIOH model (left) and a quantitative analysis of its intensity (right) (n = 6 eyes in each group, ***p < 0.001, one-way ANOVA + Fisher's LSD test). Figure 3E is a representative heatmap of the 2K response in the SIOH model evaluated by mfERG. In PBS-treated eyes, the retinal response significantly decreased 2 weeks after SIOH treatment, but a protective effect of CPTX administration was observed. Figure 3F shows a quantitative analysis of the retinal response in the SIOH model using mfERG. In PBS-treated eyes, the retinal response significantly decreased, but a protective effect on the retinal response was observed in CPTX-treated eyes (n = 6 to 16 eyes, **P < 0.01, ***P < 0.001, one-way ANOVA + Fisher's LSD test). Figure 3G shows the effect of CPTX on visual acuity in the SIOH model. While decreased visual acuity was observed in eyes treated with PBS, protective effects on visual acuity were observed in eyes treated with CPTX (n = 6 to 16 eyes, **P < 0.01, ***P < 0.001). Figure 3H shows the quantitative analysis of the effect of CPTX on contrast sensitivity in the SIOH model. While decreased contrast sensitivity was observed in the PBS-treated group, protective effects on contrast sensitivity were observed in the CPTX-treated group (n = 6 to 16 eyes, ***P < 0.001).Figure 3I shows the effect of CPTX administration on the SIOH model in the visual cliff test. The reduction in time spent on the shallow side was protected by CPTX administration (n = 6 eyes to 12 animals, ***p < 0.001). Figure 3J shows the experimental schedule for evaluating the therapeutic effect of CPTX in the chronic SIOH model. CPTX was injected intravitreously 4 weeks after SIOH induction. Individuals that underwent the SIOH treatment described above are hereafter referred to as the chronic SIOH model. The analyses in Figures 3K to 3Q were performed 2 weeks after CPTX administration. Figure 3K shows a representative image (left) of the PSD95 signal in IPL of the chronic SIOH model and a quantitative analysis of its intensity (right) (n = 6 eyes in each group, ***p < 0.001). Figure 3L shows a representative image (left) and a quantitative analysis (right) of the vGlut1 signal in IPL of the chronic SIOH model (n = 6 eyes in each group, ***p < 0.001). Figure 3M is a representative heatmap of the 2K response in a chronic SIOH model using mfERG. Figure 3N shows the quantitative analysis of the retinal response in a chronic SIOH model using mfERG. Four weeks after SIOH treatment, the retinal response was significantly reduced in PBS-administered eyes, but recovered in CPTX-administered eyes (n = 6 to 16 eyes, *P < 0.05, ***P < 0.001). Figure 3O shows the quantitative analysis of the effect of CPTX on visual acuity in a chronic SIOH model. Visual acuity decreased in PBS-administered eyes, but recovered in CPTX-administered eyes (n = 6 eyes, **P < 0.01, ***P < 0.001). Figure 3P shows the quantitative analysis of the effect of CPTX on contrast sensitivity in a chronic SIOH model. In eyes treated with PBS, a decrease in contrast sensitivity was observed, but in eyes treated with CPTX, contrast sensitivity was restored by CPTX administration (n = 6 to 16 eyes, ***P < 0.001). Figure 3Q shows the effect of CPTX administration on a chronic SIOH model in the visual cliff test. The decrease in shallow lateral stay time in SIOH model mice was restored by CPTX administration (n = 6 to 12 mice, ***P < 0.001). Quantitative data are shown as mean ± SEM.

[0087] Figures 8A-8F show the effects of CPTX on a silicone oil-induced intraocular pressure (SIOH) model. Figure 8A shows the intraocular pressure (IOP) levels in normal eyes and eyes 2 weeks and 4 weeks after silicone oil injection in 3-month-old WT mice (n = 20-46 eyes, ***P < 0.001, one-factor ANOVA and Fisher's LSD test). Figure 8B shows a representative heatmap (left) and quantitative analysis (right) of the retinal response of the SIOH model measured by multifocal ERG. The retinal response significantly decreased 2 weeks after SIOH and further decreased 4 weeks (n = 20-46 eyes, ***P < 0.001, one-factor ANOVA and Fisher's LSD test). Figure 8C shows the visual acuity measurement of the SIOH model using OKRs. Visual acuity significantly decreased two weeks after SIOH treatment, but no greater decrease in visual acuity was observed four weeks after SIOH treatment than at two weeks (n = 20–46 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 8D shows representative heatmap images (left) and quantitative analysis (right) of the visual cliff test in the SIOH model. The proportion of time spent in the shallow region significantly decreased two weeks after SIOH treatment, but even four weeks after SIOH treatment, the proportion of time spent in the shallow region did not change beyond the proportion at two weeks (n = 12 animals, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 8E shows representative images (left) of Rbpms immunostaining in retinal flat mounts of the SIOH model pre-treated with CPTX, and quantitative analysis of the number of Rbpms-positive retinal ganglion cells (RGCs) (right). Pretreatment with CPTX two weeks prior to SIOH treatment significantly improved the reduction in RGC count in the SIOH model (n = 6 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 8F shows representative images of Rbpms immunostaining (left) and quantitative analysis of Rbpms-positive RGC count (right) in the SIOH model with post-administration of CPTX. Post-treatment with CPTX four weeks after SIOH treatment did not improve the reduction in RGC count.All quantitative data are shown as mean ± SEM.

[0088] <<CPTX restores visual function in chronic NTG models>> Furthermore, we investigated whether CPTX can restore visual function in the chronic phase of NTG (normal-tension glaucoma). For this purpose, we used GLAST in 12-month-old mice. ＋ / － Mouse (aged GLAST ＋ / － The effect of CPTX on mice was evaluated. Compared to WT mice of the same age, aged GLAST ＋ / － In mice, the signal intensity of the excitatory synaptic markers PSD95 and vGlut1 was decreased (Figure 4A, Figure 4B), which is related to aging GLAST ＋ / － This suggested significant synaptic pathology in mice. Notably, CPTX administration was associated with aged GLAST ＋ / － The signal intensities of PSD95 and vGlut1 in mice were significantly increased, approaching WT levels.

[0089] However, CPTX administration is used in elderly GLAST ＋ / － It did not show any effect on reducing the number of Rbpms-positive cells in mice (Figure 4C). On the other hand, in functional evaluation, CPTX administration was effective in aging GLAST ＋ / － In mice, retinal response (Figure 4D), visual acuity (Figure 4E), and contrast sensitivity as measured by OKR (Figure 4F) were partially restored, and the response to the visual cliff test was almost completely restored (Figure 4G).

[0090] GLAST ＋ / － In mice, the disease progresses during early life (3-6 weeks after birth), so 12 months of age is considered the chronic phase of glaucoma. Furthermore, GLAST － / － Mouse (aged GLAST － / － When the effects of CPTX were also examined in mice, aged GLAST ＋ / －Similar to mice, CPTX is aged GLAST － / － While it partially restored retinal response (Figure 9A) and visual acuity (Figure 9B) in mice, it did not have any effect on contrast sensitivity (Figure 9C) or the visual cliff test response (Figure 9D).

[0091] Therefore, CPTX is thought to have the effect of not only suppressing the progression of glaucoma but also restoring visual function. However, the effectiveness of CPTX may decrease with age and the severity of the disease.

[0092] Figures 4A-G show that CPTX improves chronic visual function in a normal-tension glaucoma model. GLAST － / － Mouse (aged GLAST ＋ / － Mice were used as a model for the chronic phase of normal-tension glaucoma. Figures 4A-B show aged GLAST ＋ / － This figure shows representative images and quantitative analysis of signal intensity illustrating the effect of CPTX on synaptic markers in mice. Representative image (left) and quantitative analysis of signal intensity (right, n = 6 eyes, *P < 0.05, **P < 0.01, one-way ANOVA and Fisher's LSD test). Aged GLAST compared to 12-month-old wild-type (WT) mice. － / － In mice undergoing IPL, PSD95 (Figure 4A) and vGlut1 (Figure 4B) signaling was reduced, but these signals were restored by CPTX administration. Figure 4C shows GLAST in elderly mice. ＋ / － This figure shows the effect of CPTX on retinal ganglion cell markers in mice. CPTX administration was performed on aged mice using GLAST. － / － It did not reverse the decrease in the number of Rbpms-positive cells in mice. Figures 4D-G show that CPTX administration was effective in aged GLAST ＋ / － This study demonstrates the restoration of retinal and visual function in mice using GLAST. － / －In mice, retinal response (Figure 4D), visual acuity (Figure 4E), and contrast sensitivity (Figure 4F) were all reduced, but partial recovery was observed with CPTX administration (n = 6 to 14 eyes, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Also, aged GLAST － / － Performance in the visual cliff test in mice also significantly improved with CPTX administration, approaching the WT level for mice of the same age (Figure 4G) (n = 6-7 mice, **P < 0.01, ***P < 0.001, one-way ANOVA and Fisher's LSD test).

[0093] Figures 9A-D show that CPTX improves visual impairment in chronic GLAST- / - mice. Figure 9A shows 12-month-old GLAST mice. － / － Mouse (aged GLAST － / － This figure shows a representative heatmap (left) and quantitative analysis (right) of retinal response in mice. (Aged GLAST) - / - In mice, a decrease in retinal response was observed, but CPTX administration partially restored the retinal response (n = 6 to 10 eyes, ***P < 0.001, one-factor ANOVA and Fisher's LSD test). Figure 9B shows GLAST in elderly mice. － / － This figure shows the quantitative analysis of visual acuity in mice. (GLAST analysis of aged mice) - / - In mice, vision was almost completely lost, but CPTX administration resulted in a slight but significant improvement (n = 6 to 18 mice, *P < 0.05, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 9C shows aged GLAST － / － This figure shows a quantitative analysis of the effect of CPTX administration on contrast sensitivity in mice. CPTX administration was performed on aged mice using GLAST. - / -It did not improve contrast sensitivity in mice (n = 6 to 18 mice, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 9D shows aged GLAST － / － This figure shows a representative heatmap (left) and quantitative analysis (right) of the time spent in the shallow and deep areas of a mouse's visual cliff test. The bars on the right of the heatmap (left) indicate the time spent in each area, ranging from short to long periods. The graph on the right shows the quantitative analysis results of the percentage of time spent in the shallow area. (Aged GLAST) － / － In mice, CPTX administration did not improve the response to the visual cliff test (n = 6–7 mice, ***P < 0.001, one-way ANOVA and Fisher's LSD test). All data are shown mean ± SEM.

[0094] <<CPTX restores age-related visual decline in wild-type mice>> Visual impairment can be caused not only by eye diseases but also by aging. Therefore, we investigated the effects of CPTX on age-related visual decline. First, we examined the characteristics of age-related visual decline in wild-type (WT) mice. In evaluation using OKR (Optomotor Response), visual acuity gradually declined with age, and 24-month-old mice showed a significantly lower response compared to younger mice (Figure 5A). Contrast sensitivity also significantly decreased with age (Figure 5B). The response to the visual cliff test did not change until 12 months of age, but significantly decreased at 24 months of age (Figure 5C). These results support the natural age-related visual decline in WT mice.

[0095] Next, we evaluated the effect of CPTX administration on the number of retinal ganglion cells (RGCs). While the number of RGCs significantly decreased with age, CPTX did not suppress this decrease (Figure 5D). On the other hand, PSD95 and vGlut1 signal intensities were reduced in aged mice, but significantly improved with CPTX administration (Figures 5E and 5F). Retinal responses were also significantly reduced in 24-month-old mice compared to 3-month-old wild-type mice, but significant improvement was observed with CPTX administration compared to the PBS-treated group (Figure 5G). Visual acuity and contrast sensitivity significantly improved in aged mice treated with CPTX (Figures 5H and 5I), but no improvement was observed in the visual cliff test response (Figure 5J). These results suggest that CPTX may mitigate age-related decline in visual function to some extent.

[0096] Figures 5A–J show that CPTX restores age-related visual impairment in wild-type mice. Figures 5A–C show normal age-related decline in visual function. Visual acuity (Figure 5A) and contrast sensitivity (Figure 5B) gradually declined with age (n = 10 eyes, *P < 0.05, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Responses to the visual cliff test were relatively stable until 12 months of age, but significantly decreased at 24 months of age (Figure 5C, n = 10 mice, ***P < 0.001). Figure 5D shows the effect of CPTX administration on retinal ganglion cell count in 24-month-old wild-type mice (aged mice). The number of Rbpms-positive cells was significantly reduced in aged mice compared to 3-month-old wild-type mice, but no recovery effect on retinal ganglion cell count was observed with CPTX administration in aged mice (n = 6 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figures 5E-5F show the effect of CPTX on synapses in aged mice. Signal intensities of PSD95 and vGlut1 were reduced in aged mice, but were significantly restored with CPTX administration (n = 6 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 5G shows the quantitative analysis of retinal response in aged mice. Retinal response, which was reduced in aged mice, was also restored with CPTX (n = 7-12 eyes, **P < 0.01, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figures 5H–I show the quantitative analysis results of the effects of CPTX administration on visual acuity (Figure 5H) and contrast sensitivity (Figure 5I) in aged mice. Visual acuity and contrast sensitivity were decreased in aged mice but recovered with CPTX administration (n = 6–20 eyes, **P < 0.01, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 5J shows the heatmap and quantitative analysis of the visual cliff test in aged mice.Responses to the visual cliff test were reduced in older mice, but no improvement was observed with CPTX administration (n = 6 to 20 mice, **P < 0.01, ***P < 0.001, one-way ANOVA and Fisher's LSD test).

[0097] <<CPTX also shows protective effects against visual impairment due to photoreceptor degeneration>> To verify whether CPTX is effective against optic nerve diseases other than glaucoma, a retinitis pigmentosa model using N-methyl-N-nitrosourea (MNU), which induces apoptosis of photoreceptor cells, was used. MNU (60 mg / kg) was administered intraperitoneally to 3-month-old WT mice, and CPTX was injected subretinally the day before. MNU administration significantly reduced visual acuity and contrast sensitivity, and the mice became almost blind (Figures 6A and 6B). Pre-administration of CPTX partially but significantly restored these functions. In the visual cliff test, MNU treatment reduced the time spent in the shallow area by 50%, but this reduction was partially restored by pre-administration of CPTX (Figure 6C). These results suggest that CPTX has protective and preventive effects not only against glaucoma but also against other optic nerve diseases such as photoreceptor degeneration.

[0098] Figures 6A-6C show that CPTX improves visual function in methylnitrosourea (MNU)-induced retinal degeneration animal models (retinitis pigmentosa models). Figure 6A shows a quantitative analysis of the effect of CPTX on visual acuity in MNU-induced retinal degeneration animal models. Visual acuity, as measured by optokinetic nystagmus (OKR), decreased significantly with MNU administration, but partially recovered with CPTX pre-administration (n = 5-35 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 6B shows a quantitative analysis of the effect of CPTX on contrast sensitivity in MNU-induced retinal degeneration animal models. Contrast sensitivity, as measured by OKR, also decreased significantly with MNU administration, and partially recovered with CPTX pre-administration (n = 5-22 eyes, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Figure 6C shows a heatmap and quantitative analysis of the visual cliff test in MNU-induced retinal degeneration animal models. In the visual cliff test, the time spent in the shallow area was significantly reduced with MNU administration and partially improved with CPTX pre-administration (n = 3–12 animals, *P < 0.05, ***P < 0.001, one-way ANOVA and Fisher's LSD test). Data are shown mean ± SEM.

[0099] Although embodiments of the present invention have been described in detail above, the present invention is not limited to the specific embodiments described above, and various modifications and changes are possible within the scope of the gist of the present invention as described in the claims.

[0100] This international application claims priority under U.S. Patent Application No. 63 / 701,055, filed on 30 September 2024, which is incorporated herein by reference to the entire contents of U.S. Patent Application No. 63 / 701,055.

Claims

1. A preventive or therapeutic agent for optic nerve disease, characterized by containing an effective amount of artificial synaptic connector or nucleic acid encoding an artificial synaptic connector.

2. The prophylactic or therapeutic agent for optic nerve disease according to claim 1, wherein the artificial synaptic connector comprises a domain that binds to neurexin (Nrx), a multimerizing domain, and a domain that binds to the extracellular region of the AMPA receptor.

3. The prophylactic or therapeutic agent for optic nerve disease according to claim 1, wherein the artificial synaptic connector comprises, in this order, a domain that binds to neurexin (Nrx), a multimerizing domain, and a domain that binds to the extracellular region of the AMPA receptor.

4. The agent for preventing or treating optic nerve disease according to claim 1, wherein the artificial synaptic connector is a protein containing the amino acid sequence of SEQ ID NO: 1 or a functional variant thereof.

5. The optic nerve disease prevention or treatment agent according to claim 1, wherein the optic nerve disease is any of glaucoma, retinitis pigmentosa, and age-related optic nerve loss.

6. The preventive or therapeutic agent for an optic nerve disease according to claim 1, wherein the optic nerve disease is glaucoma.

7. A method for preventing and / or treating an optic nerve disease, comprising the step of administering an artificial synaptic connector or a nucleic acid encoding an artificial synaptic connector to a patient with the optic nerve disease.

8. The method for prevention or treatment according to claim 7, wherein the artificial synaptic connector comprises a domain that binds to neurexin (Nrx), a multimerizing domain, and a domain that binds to the extracellular region of the AMPA receptor.

9. The method for prevention or treatment according to claim 7, wherein the artificial synaptic connector comprises, in this order, a domain that binds to neurexin (Nrx), a multimerizing domain, and a domain that binds to the extracellular region of the AMPA receptor.

10. The method for prevention or treatment according to claim 7, wherein the artificial synaptic connector is a protein containing the amino acid sequence of SEQ ID NO: 1 or a functional variant thereof.

11. The method for prevention or treatment according to claim 7, wherein the optic nerve disease is any of glaucoma, retinitis pigmentosa, and age-related optic nerve loss.

12. The method for prevention or treatment according to claim 7, wherein the optic nerve disease is glaucoma.

13. The method of prevention or treatment according to claim 7, wherein the artificial synaptic connector or the nucleic acid encoding the artificial synaptic connector is administered by intravitreous injection or eye drops.

14. Artificial synaptic connectors or nucleic acids encoding artificial synaptic connectors for use in the prevention and / or treatment of optic nerve diseases.

15. Use of artificial synaptic connectors or nucleic acids encoding artificial synaptic connectors in the manufacture of agents for the prevention or treatment of optic nerve diseases.

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