Artificial excitatory synaptic connector and its use in treating spinal cord injuries

Abstract

To provide a pharmaceutical composition for use in forming new synaptic connections between excitatory interneurons, and to provide a method for producing the same.SOLUTION: Provided are multimers of fusion proteins comprising a region that binds to Nrx of Cbln1 proteins, a multimerization domain, and a region that binds to AMPA receptors of Nptx1 proteins, and pharmaceutical compositions comprising the multimers.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to artificial excitatory synaptic connectors and their use in treating spinal cord injuries. [Background technology]

[0002] Cbln1 is a secretory protein belonging to the C1q family, and is mainly produced and secreted in cerebellar granule cells (Non-Patent Document 1). Cbln1 binds to neurexin (Nrx) at the presynaptic site. Cbln1 is essential for the formation of synapses between parallel fibers and Purkinje cells (parallel fiber synapses) (Non-Patent Documents 2 to 4). Cbln1 knockout (cbln1- / -) mice exhibit a decrease in the density of parallel fiber synapses and severe cerebellar ataxia symptoms (Non-Patent Documents 1 and 5).

[0003] A fusion protein linking the Nrx-binding site of the Cbln1 protein and the AMPA-type glutamate receptor-binding site of the Nptx1 protein via a trimerization domain increased synapses between cerebellar parallel fibers and Purkinje cells in mice exhibiting cerebellar ataxia (cbln1- / -, GluD2- / -) and enhanced electrophysiological synaptic responses. It has also been reported that this fusion protein ameliorates the symptoms of cerebellar ataxia (Non-Patent Document 6). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Hirai, H., Pang, Z., Bao, D. et al.: Cbln1 is essential for synaptic integrity and plasticity in the cerebellum. Nat. Neurosci., 8, 1534-1541 (2005) [Non-patent document 2] Matsuda, K., Miura, E., Miyazaki, T. et al.: Cbln1 is a ligand for an orphan glutamate receptor δ2, a bidirectional synapse organizer. Science, 328, 363-368 (2010) [Non-patent document 3] Matsuda, K. & Yuzaki, M.: Cbln family proteins promote synapse formation by regulating distinct neuroxin signaling pathways in various brain regions. Eur. J. Neurosci., 33, 1447-1461 (2011) [Non-patent document 4] Uemura, T., Lee, SJ, Yasumura, M. et al.: Trans-synaptic interaction of GluRδ2 and Neurexin through Cbln1 mediates synapse formation in the cerebellum. Cell, 141, 1068-1079 (2010) [Non-Patent Document 5] Ito-Ishida, A., Miura, E., Emi, K. et al.: Cbln1 regulates rapid formation and maintenance of excitatory synapses in mature cerebellar Purkinje cells in vitro and in vivo. J. Neurosci., 28, 5920-5930 (2008) [Non-patent document 6] Suzuki, K., et al., Fourth Annual Conference of COST Action ECMNET, October 1st, 2014 Summary of the Invention

[0005] The present invention provides a fusion protein multimer comprising the Nrx-binding region of the Cbln1 protein, a multimerization domain, and the AMPA receptor-binding region of Nptx1, as well as a pharmaceutical composition comprising the multimer for use in forming new synaptic connections between excitatory interneurons (particularly, between excitatory interneurons in the spinal cord).

[0006] The present inventors have discovered that a fusion protein (artificial excitatory synaptic connector) capable of linking Nrx expressed in presynaptic cells with AMPA-type glutamate receptors (AMPARs) expressed in postsynaptic cells generates new connections between excitatory interneurons in the spinal cord and, surprisingly, restores spinal cord injury and motor function. The restoration of motor function by administration of this artificial excitatory synaptic connector was also effective in subacute and chronic spinal cord injury. The present invention is based on these findings.

[0007] According to the present invention, for example, the following inventions are provided. [1] A multimer (preferably a hexamer) of a fusion protein, The fusion protein comprises an Nrx-binding region of the Cbln1 protein, a trimerization domain, and an AMPA receptor-binding region of the Nptx1 protein; Two trimers of the fusion protein are linked by a disulfide bond. Multimers (preferably hexamers). [2] A pharmaceutical composition comprising a multimer (preferably a hexamer) of the fusion protein according to [1] above. [3] A fusion protein comprising the Nrx-binding domain of the Cbln1 protein, a multimerization domain, and the AMPA receptor-binding domain of the Nptx1 protein. A pharmaceutical composition for use in forming new synaptic connections between excitatory interneurons. [4] The pharmaceutical composition according to [3] above, wherein the excitatory interneuron is an excitatory interneuron of the spinal cord. [5] The pharmaceutical composition according to [3] or [4] above for use in treating spinal cord injury. [5A] A multimer of a fusion protein comprising the Nrx-binding region of the Cbln1 protein, a multimerization domain, and the AMPA receptor-binding region of Nptx1; A pharmaceutical composition for use in treating spinal cord injury. [6] The pharmaceutical composition according to any one of [3] to [5] and [5A] above, wherein the multimerization domain is a trimerization domain. [7] The pharmaceutical composition according to any one of the above [3] to [5], [5A] and [6], wherein the fusion protein multimer is a hexamer. [8] The pharmaceutical composition according to any one of the above [2] to [7], which is a therapeutic agent for spinal cord injury. [9] A method for producing a hexamer of the fusion protein according to [1] above or a pharmaceutical composition containing the hexamer, comprising: culturing cells harboring a nucleic acid encoding a fusion protein of the invention, the nucleic acid having a signal sequence, under conditions suitable for expression of the protein; Obtaining a culture supernatant; recovering the hexamer of the fusion protein from the obtained culture supernatant; A method comprising:

[0008] [1A] A multimer of a fusion protein, The fusion protein is a multimer that includes the Nrx-binding region of the Cbln1 protein, a multimerization domain, and the AMPA receptor-binding region of the Nptx1 protein. [2A] The multimer according to [1A] above, wherein the multimerization domain is a trimerization domain and the multimer is a hexamer formed by linking two trimers of the fusion protein by disulfide bonds. [3A] A multimer described in [1A] or [2A] above, wherein the region of the Cbln1 protein that binds to Nrx is a region of the Cbln1 protein that can bind to Nrx having splice site 4 (i.e., Nrx(S4+)). [4A] A multimer described in any of [1A] to [3A] above, wherein the region of Nptx1 that binds to AMPA-type glutamate receptors is a region of Nptx1 that corresponds to the pentraxin domain of human neuronal pentraxin-1. [5A] A multimer described in any of [1A] to [4A] above, wherein the multimerization domain is interposed between the region of the Cbln1 protein that binds to Nrx and the region of Nptx1 that binds to the AMPA-type glutamate receptor, or is sandwiched between the two regions. [6A] A multimer described in any of [1A] to [5A] above, in which the Nrx-binding region of the Cbln1 protein and the multimerization domain, and / or the multimerization domain and the AMPA receptor-binding region of Nptx1 are linked via a linker. [7A] The multimer according to [6A] above, wherein the linker is any one of SEQ ID NOs: 3 to 9. [8A] A nucleic acid encoding the fusion protein defined in any one of [1A] to [5A] above, which has the nucleic acid sequence set forth in SEQ ID NO:1. [9A] The multimer according to any one of [1A] to [5A] above, wherein the fusion protein has the amino acid sequence set forth in SEQ ID NO:2. [10A] A pharmaceutical composition comprising the multimer according to any one of [1A] to [9A] above. [11A-1] A fusion protein comprising a neurexin (Nrx)-binding domain of cerebellin-1 (Cbln1 protein), a multimerization domain, and an AMPA receptor-binding domain of neuronal pentraxin-1 (Nptx1) protein. A pharmaceutical composition for use in forming new synaptic connections between excitatory interneurons. [11A-2] A multimer of a fusion protein comprising a neurexin (Nrx)-binding domain of cerebellin-1 (Cbln1 protein), a multimerization domain, and an AMPA receptor-binding domain of neuronal pentraxin-1 (Nptx1) protein. A pharmaceutical composition for use in forming new synaptic connections between excitatory interneurons. [11A-3] A multimer of a fusion protein comprising a neurexin (Nrx)-binding region of cerebellin-1 (Cbln1 protein), a multimerization domain, and an AMPA receptor-binding region of neuronal pentraxin-1 (Nptx1) protein, wherein the multimerization domain is a trimerization domain, and the multimer is a hexamer formed by two fusion protein trimers linked by disulfide bonds. A pharmaceutical composition for use in forming new synaptic connections between excitatory interneurons.

[0009] Hereinafter, in [12A] and subsequent sections, [11A-1], [11A-2], and [11A-3] will be collectively referred to as [11A]. [12A] The pharmaceutical composition according to either [10A] or [11A] above, wherein the excitatory interneuron is a spinal cord excitatory interneuron. [13A] The pharmaceutical composition according to any one of [10A] to [12A] above, for use in treating spinal cord injury. [14A] The pharmaceutical composition according to any one of [10A] to [13A] above, wherein the multimerization domain is a trimerization domain. [15A] The pharmaceutical composition according to any one of [10A] to [14A] above, wherein the multimerization domain is a trimerization domain and the multimer is a hexamer formed by two trimers of the fusion protein linked by a disulfide bond. [16A] The pharmaceutical composition according to any one of the above [10A] to [15A], which is a therapeutic agent for spinal cord injury. [17A] A method for producing the pharmaceutical composition according to any one of [10A] to [15A] above, culturing cells harboring a nucleic acid encoding a fusion protein of the invention, the nucleic acid having a signal sequence, under conditions suitable for expression of the protein; Obtaining a culture supernatant; recovering the fusion protein multimer from the obtained culture supernatant; A method comprising: [18A] The method according to [17A] above, wherein the multimerization domain is a trimerization domain and the multimer is a hexamer formed by linking two trimers of the fusion protein by a disulfide bond.

[0010] [19A] A conjugate comprising a first molecule that binds to neurexin (Nrx) and a second molecule that binds to neuronal pentraxin-1 (Nptx1), wherein the first molecule and the second molecule are each selected from the group consisting of an antibody, an antigen-binding fragment thereof, and an aptamer. [20A] A pharmaceutical composition comprising the conjugate of [19A] above. [21A] The pharmaceutical composition according to [20A] above, for use in forming new synaptic connections between excitatory interneurons. [22A] The pharmaceutical composition according to [21A] above, wherein the excitatory interneuron is an excitatory interneuron of the spinal cord. [23A] The pharmaceutical composition according to [20A] above, for use in treating spinal cord injury. [24A] The pharmaceutical composition according to [20A] above, which is a therapeutic agent for spinal cord injury. [Brief explanation of the drawings]

[0011] [Figure 1A] Figure 1A shows a schematic diagram of a natural synapse containing presynaptic and postsynaptic cells. In a natural synapse, Nrx(S4+) expressed in the presynaptic cell connects with glutamate D2 receptors (GluD2) expressed in the postsynaptic cell via Cbln1 secreted into the synapse. On the other hand, Nptx1 only binds to AMPA-type glutamate receptors (AMPARs) expressed in the postsynaptic cell, not to the presynaptic cell. [Figure 1B]Figure 1B shows a schematic diagram of the structure and predicted function of an example of a fusion protein (chimeric protein) of the present invention. In the illustrated fusion protein of the present invention, a trimerization domain exists between the Nrx-binding domain of the Cbln1 protein and the AMPAR-binding domain of the Nptx1 protein (see the structure in Figure 1B). The fusion protein (chimeric protein) of the present invention then links Nrx (S4+) in the presynaptic cell to AMPAR in the postsynaptic cell in the synaptic cleft, thereby transmitting excitatory signals from the presynaptic cell to the postsynaptic cell (see the function in Figure 1B). Therefore, the fusion protein of the present invention can act as an artificial excitatory synaptic connector. [Figure 2] FIG. 2 shows that an example of the fusion protein of the present invention forms a trimer, and that two such trimers can be linked to form a hexamer. [Figure 3] Figure 3 shows the results of multi-angle light scattering analysis of the fusion protein of the present invention prepared in the Examples. According to the analysis results, the molecular weight of the fusion protein of the present invention was estimated to be 208.9 ± 3.3 kDa. This estimated molecular weight corresponds to the theoretically predicted molecular weight in Figure 2, suggesting that the fusion protein of the present invention prepared in the Examples forms a hexamer. [Figure 4] Figure 4 is a schematic diagram of the method for preparing a crush model (compression) and a hemisection model (SCI) in mice. [Figure 5A]Figure 5A shows the time course of Basso Mouse Scale (BMS) scores after injection of the fusion protein of the present invention into the spinal cord of a hemisection model immediately after hemisection. In the figure, "Mock" refers to a negative control group administered HEPES buffer after SCI. "ChABC" refers to an enzyme (chondroitinase ABC) that degrades chondroitin sulfate glycosaminoglycans and has a therapeutic effect on spinal cord injury. "Cbln1" refers to the full-length human Cbln1 protein. "Sham" refers to a group in which no spinal cord injury was caused after spinal cord resection. In the figure, "w" refers to weeks. Note that Cbln1 does not have a binding site for AMPA receptors, and therefore, unlike CPTX, cannot form a complex between Nrx(S4+) and AMPA receptors. [Figure 5B] Figure 5B shows the results of a stepping test over time after injection of the fusion protein of the present invention into the spinal cord of a hemisection model immediately after hemisection. Mice were placed on a wire mesh grid and videotaped for 5 minutes. The number of times a mouse stepped off the grid after walking on the grid at least 70 times in 3 minutes was counted. In the figure, "Mock" refers to a negative control group administered HEPES buffer after SCI, "ChABC" refers to an enzyme (chondroitinase ABC) that degrades chondroitin sulfate glycosaminoglycans and has a therapeutic effect on spinal cord injury, "Cbln1" refers to the full-length human Cbln1 protein, and "Sham" refers to a group in which no spinal cord injury was caused after spinal cord resection. In the figure, "w" refers to weeks. Note that Cbln1 does not have a binding site for AMPA receptors and therefore, unlike CPTX, cannot form a complex between Nrx(S4+) and AMPA receptors. [Figure 5C]Figure 5C shows the time course of Basso Mouse Scale (BMS) scores after injection of the fusion protein of the present invention into the spinal cord of a hemisection model one week after hemisection. In the figure, "Mock" refers to a negative control group administered HEPES buffer after SCI, "ChABC" refers to an enzyme (chondroitinase ABC) that degrades chondroitin sulfate glycosaminoglycans and has a therapeutic effect on spinal cord injury, "Cbln1" refers to the full-length human Cbln1 protein, and "Sham" refers to a group in which no spinal cord injury was caused after spinal cord resection. In the figure, "w" refers to weeks. Note that Cbln1 does not have a binding site for AMPA receptors, and therefore, unlike CPTX, cannot form a complex between Nrx(S4+) and AMPA receptors. [Figure 5D] Figure 5D shows the time course of Basso Mouse Scale (BMS) scores after injection of the fusion protein of the present invention into the spinal cord of a crush injury model immediately after injury. In the figure, "Mock" refers to the negative control group administered HEPES buffer after SCI. ChABC refers to an enzyme (chondroitinase ABC) that degrades chondroitin sulfate glycosaminoglycans and has a therapeutic effect on spinal cord injury. ChABC+CPTX refers to the group administered both simultaneously. Cbln1 refers to the full-length human Cbln1 protein. Sham refers to the group in which no spinal cord injury was caused after spinal cord resection. In the figure, "w" refers to weeks. Note that Cbln1 does not have a binding site for AMPA receptors, and therefore, unlike CPTX, cannot form a complex between Nrx(S4+) and AMPA receptors. [Figure 5E] Figure 5E shows the change in Basso Mouse Scale (BMS) score (ΔBMS / week) between week 1 and week 2 after injection of the fusion protein of the present invention into the spinal cord of a hemisection model one week after hemisection. In the figure, Mock represents the negative control group administered HEPES buffer after SCI, and ChABC represents the group administered chondroitin sulfate glycosaminoglycan-degrading enzyme (chondroitinase ABC), which has a therapeutic effect on spinal cord injury. [Figure 5F]Figure 5F shows the time course of Basso Mouse Scale (BMS) scores after local injection of the fusion protein of the present invention into a subacute phase (2 weeks after injury) crush injury model. Cont is a negative control administered with HEPES buffer, and ChABC is an enzyme (chondroitinase ABC) that degrades chondroitin sulfate glycosaminoglycans and has a therapeutic effect on spinal cord injury. The crush injury model was prepared at week 0 (0w) and local administration was performed at 2w. C57 / BL6 mice were used as the model animals. [Figure 5G] Figure 5G shows the time course of Basso Mouse Scale (BMS) scores after local injection of the fusion protein of the present invention into a crush injury model in the chronic phase (4 weeks after injury). In the figure, Cont is a negative control administered with HEPES buffer, and ChABC is an enzyme (chondroitinase ABC) that degrades chondroitin sulfate glycosaminoglycans and has a therapeutic effect on spinal cord injury. The crush injury model was prepared at week 0 (0w) and local administration was performed at 4w. C57 / BL6 mice were used as the model animals. [Figure 5H] Figure 5H shows the time course of Basso Mouse Scale (BMS) scores after local injection of the fusion protein of the present invention into a crush injury model in the chronic phase (4 weeks after injury). In the figure, Cont is a negative control administered with HEPES buffer, and ChABC is an enzyme (chondroitinase ABC) that degrades chondroitin sulfate glycosaminoglycans and has a therapeutic effect on spinal cord injury. The crush injury model was prepared at week 0 (0w) and local administration was performed at 4w. ICR mice were used as the model animals. [Figure 6A] Figure 6A shows a fluorescence microscopy image demonstrating the colocalization of CPTX, Vglut2, and GluA4 after administration of the fusion protein of the present invention (CPTX) to the spinal cord in a hemisection model. Figure 6A shows that CPTX is concentrated around the transection site of the spinal cord. [Figure 6B]Figure 6B shows a fluorescence microscopy image demonstrating the colocalization of CPTX, Vglut2, and GluA4 after administration of the fusion protein of the present invention (CPTX) to the spinal cord of a hemisection model. Figure 6B shows that CPTX is localized between Vglut2 and GluA4. [Figure 7A] Figure 7A shows a fluorescence microscopy image of a tissue section 1.6 mm upstream of the hemisection site in a hemisection model. Figure 7A shows the presence of an overlapping region between the Vglut2 and GluA4 fluorescent signals. CPTX is also localized in this overlapping region. [Figure 7B] Figure 7B shows the effect of CPTX administration on the area of ​​the GluA4 (GR4) and Vglut2 (VT2) fluorescent signal regions and their overlapping areas (see Figure 7B, left). Figure 7B also shows the effect of CPTX administration on the percentage of GR4-positive VT2 regions (see Figure 7B, right). [Figure 8] Figure 8 is a schematic diagram of the predicted recovery process of spinal cord injury using the fusion protein of the present invention. In spinal cord injury, the injury site itself may have irreparable damage. Furthermore, the area surrounding the injury site is not directly damaged, but immune responses and other factors are activated, creating an environment that is unlikely to allow neural circuit reorganization. In contrast, the fusion protein of the present invention can connect excitatory interneurons. Rather than directly repairing the injury site, the fusion protein of the present invention is expected to induce neural circuit reorganization by connecting excitatory interneurons upstream or downstream of the injury site, thereby forming a neural transmission pathway that bypasses the spinal cord injury site and leading to recovery from spinal cord injury. Specific Description of the Invention

[0012] In the present invention, a "subject" is a vertebrate, for example, a bird or a mammal, for example, a mammal, such as a mouse, rat, hamster, guinea pig, horse, cow, pig, goat, sheep, donkey, dog, and cat, as well as a primate, such as a monkey, chimpanzee, gorilla, orangutan, bonobo, or human, particularly a human. As used herein, the term "subject" includes humans, as described above, and the term "non-human" is used to exclude humans.

[0013] As used herein, "treatment" means medical intervention in a subject. Treatment is used to include therapeutic treatment. Therapeutic treatment can produce therapeutic effects such as improvement, inhibition of deterioration, slowing or halting the rate of deterioration, and cure of diseases, disorders, and conditions. As used herein, a "therapeutically effective amount" is an amount that produces a therapeutic effect.

[0014] As used herein, "Cbln1 protein" refers to a protein also known as precerebellin, cerebellin 1 precursor, or cerebellin-1. Cbln1 is a secretory protein belonging to the C1q family, and is primarily produced and secreted in cerebellar granule cells. Cbln1 binds to neurexins (Nrx) at the presynaptic site. Cbln1 is essential for the formation of synapses between parallel fibers and Purkinje cells (parallel fiber synapses). Cbln1 knockout mice exhibit severe cerebellar ataxia symptoms due to reduced density of parallel fiber synapses. Human CBLN1 protein may have the amino acid sequence registered in NCBI Reference Sequence: NP_004343.1. In this human CBLN1, the amino acid sequence at positions 1 to 21 is a signal sequence, the amino acids at positions 34 to 38 are essential for binding to NRXN1, the cysteines at positions 34 and 38 are used to form disulfide bonds within CBLN1, and the C1q domain at positions 57 to 193. Furthermore, in this human CBLN1, the amino acids at positions 62 to 193 are essential for binding to CBLN3 and homotrimerization, and the amino acids at positions 122 to 147 are essential for interaction with GLUD2. Human CBLN1 contains an Nrx-binding site (Nrx-binding domain) in the region at positions 22 to 53. The Nrx-binding domain of the CBLN1 protein may have an amino acid sequence of the CBLN1 protein corresponding to the amino acid sequence at positions 22 to 53 of the human CBLN1 protein (NCBI Reference Sequence: NP_004343.1). The Nrx-binding domain can bind to Nrx with splice site 4 (the fourth splice site) (i.e., Nrx(S4+)). Throughout this specification, gene and protein names, regardless of whether they are written in uppercase or lowercase, are used to include orthologs from all mammalian species. In this specification, the animal species from which a gene or protein name is derived is distinguished by adding the animal species before the gene or protein name.

[0015] As used herein, an amino acid sequence corresponding to a certain amino acid sequence means an amino acid sequence that is located at a corresponding position when aligned with the certain amino acid sequence in a homologue, including an ortholog and a natural variant of the certain amino acid sequence.

[0016] As used herein, "neuronal pentraxin-1" (also known as Nptx1 or NP1) is a member of the neuronal pentraxin gene family. Nptx1 binds to and activates AMPA-type glutamate receptors (AMPARs). The human NPTX1 protein may have the amino acid sequence registered in NCBI Reference Sequence: NP_002513.2. In this human NPTX1 protein, the amino acid sequence at positions 1 to 22 is a signal sequence, and the amino acid sequence at positions 222 to 428 is a pentraxin domain. The NPTX1 protein has an AMPAR-binding site (AMPAR-binding domain) in the region of amino acids 222 to 428. The AMPAR-binding domain of the NPTX1 protein may have the amino acid sequence of the NPTX1 protein corresponding to the amino acid sequence at positions 222 to 428 of the human NPTX1 protein (NCBI Reference Sequence: NP_002513.2). Here, "AMPA" means α-amino-3-hydroxy-5-mesoxazole-4-propionic acid.

[0017] As used herein, the term "multimerization domain" refers to a domain that can multimerize a protein by linking it to the protein. A multimerization domain can be, for example, a coiled-coil domain. Examples of multimerization domains include dimerization domains and trimerization domains. While trimerization domains are involved in protein trimerization in natural proteins, they can also trimerize chimeric proteins formed from the trimerization domain and a target protein. Therefore, a target protein can be trimerized by forming a chimeric protein with the trimerization domain. Examples of trimerization domains include those derived from influenza hemagglutinin, SARS spike, HIV gp41, GCN4, modified GCN4, bacteriophage T4 fibritin, and ATCase. Examples of trimerization domains include those derived from TRAF2, thrombospondin 1, matrilin-4, and matrilin-1. As used herein, a multimer can be a homomultimer.

[0018] According to the present invention, a molecule that can bind to both an antigen presented on the presynaptic membrane surface and an antigen presented on the postsynaptic membrane surface and link them via said molecule has the effect of reconnecting synapses before and after the injury site after spinal cord injury. According to the present invention, in particular, a molecule that links Nrx and AMPA-type glutamate receptors has the effect of reconnecting synapses before and after the injury site in spinal cord injury. Thus, according to the present invention, a fusion molecule of a molecule that binds to Nrx and a molecule that binds to AMPA-type glutamate receptors is provided. In one embodiment, the molecule may be a protein. Those skilled in the art can obtain molecules that bind to specific proteins in the form of antibodies and fragments thereof with antigen specificity (e.g., monoclonal antibodies or fragments thereof, such as bispecific antibodies, bispecific single-chain diabodies, bispecific tandem scFvs, bispecific F(ab)2, and other bispecific antibody fragments), aptamers (DNA aptamers or RNA aptamers {RNA aptamers may be RNA aptamers stabilized by modified nucleic acids}), and protein regions as described below. According to a preferred embodiment of the present invention, a multimer (e.g., a trimer, preferably a hexamer, particularly a hexamer in which two trimers are linked by disulfide bonds) of a fusion protein (sometimes referred to as the "fusion protein of the present invention") comprising the Nrx-binding region of the Cbln1 protein, a multimerization domain, and the AMPA-type glutamate receptor-binding region of Nptx1 is provided.

[0019] According to the present invention, the Nrx-binding region of the Cbln1 protein is a 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 may be, for example, a region of Cbln1 corresponding to the cysteine-rich region (CRR) of human Cbln1 (GenBank ID NM_004352; Gln22-Ile53) (J. Elegheert et al., Science 353, 295-299 (2016)).

[0020] According to the present invention, any multimerization domain (or multimerization domain) can be used as long as it is capable of multimerizing a fusion protein. In one embodiment of the present invention, the multimerization domain can be a trimerization domain (or trimerization domain). The trimerization domain can be, for example, a coiled-coil domain (particularly capable of forming a coiled-coil triple helix), and in a preferred embodiment, can be, but is not limited to, the trimerization domain of GCN4. The trimerization domain may also be selected from the group consisting of a trimerization domain of a collagen family protein (e.g., collagen α1, α2), an α-keratin trimerization domain, a Clq protein trimerization domain, an overwintering protein ACRP30 trimerization domain, a cerebellin trimerization domain, a multimerin trimerization domain, a collectin trimerization domain, a conglutinin trimerization domain, a pulmonary surfactant protein A (SP-A) trimerization domain, and a mannose-binding protein (MBP) trimerization domain. In a preferred embodiment, a trimerization domain of the Clq protein family and a trimerization domain of the collectin family may be used. The trimerization domain may have a collagen-like sequence.

[0021] According to the present invention, the region of Nptx1 that binds to AMPA-type glutamate receptors may be the pentraxin domain of the Nptx1 protein. The region of Nptx1 that binds to AMPA-type glutamate receptors may be, for example, the pentraxin domain of human neuronal pentraxin-1 (NP1 PTX ; GenBank ID AC50727.1; Pro224-Ile431) (a region of Nptx1 having a corresponding amino acid sequence).

[0022] In one embodiment, the fusion protein of the present invention may have a multimerization domain interposed between the Nrx-binding region of the Cbln1 protein and the AMPA-type glutamate receptor-binding region of Nptx1. This makes the fusion protein of the present invention advantageous in mediating the binding of Nrx to AMPA-type glutamate receptors. Furthermore, even if the multimerization domain is located between the two regions, it can still promote multimerization.

[0023] In one embodiment of the present invention, in the fusion protein of the present invention, the Nrx-binding region of the Cbln1 protein, the multimerization domain, and the AMPA receptor-binding region of Nptx1 may be 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 a hydrocarbon linker having -(CH2)6- or (GGGGS) n (SEQ ID NO: 3), KESGSVSSEQLAQFRSLD (SEQ ID NO: 4) or EGKSSGSGSESKST (SEQ ID NO: 5), GGGGGGGG (SEQ ID NO: 6), GSAGSAAGSGEF (SEQ ID NO: 7), (GGSG) n (SEQ ID NO: 8) or (GS) n (SEQ ID NO: 9) {wherein n is a natural number of 1 to 5}. In one embodiment of the present invention, in the fusion protein of the present invention, the Nrx-binding region of the Cbln1 protein, the multimerization domain, and the AMPA receptor-binding region of Nptx1 may be directly linked without a linker.

[0024] In one embodiment of the present invention, the fusion protein of the present invention may have a trimerization domain as the multimerization domain and may be a trimer. The fusion protein of the present invention may have a trimerization domain as the multimerization domain and may be a hexamer formed by linking two trimers. Two trimers may be linked to form a hexamer via the 34th and / or 38th cysteines of the human Cbln1 protein (NCBI Reference Sequence: NP_004343.1).

[0025] In one embodiment of the present invention, any region and domain of the fusion protein of the present invention can be derived from human protein.In one embodiment of the present invention, the fusion protein of the present invention is a fusion protein multimer, comprising the Nrx binding region of human Cbln1 protein, the multimerization domain of human protein, and the AMPA receptor binding region of human Nptx1, wherein the multimerization domain is a trimerization domain, and the fusion protein can be in the form of a trimer or a hexamer.

[0026] In some embodiments of the invention, a fusion protein of the invention may have the amino acid sequence of SEQ ID NO: 2. In some embodiments of the invention, a nucleic acid encoding a fusion protein of the invention may have the nucleic acid sequence of SEQ ID NO: 1 (or the sequence of the corresponding mRNA).

[0027] The fusion proteins or multimers of the present invention link Nrx proteins expressed in the presynaptic terminals of presynaptic cells with AMPARs expressed in the dendrites of postsynaptic cells (see FIG. 1B), thereby creating new connections between excitatory interneurons in the spinal cord. Therefore, the fusion proteins of the present invention can be used to form new connections (e.g., synaptic connections) between excitatory interneurons (e.g., between excitatory interneurons in the spinal cord). According to the present invention, the new connections can be formed around or near the spinal cord injury. The term "around" can refer to, for example, within 0.5 cm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 20 cm, 30 cm, 40 cm, or 50 cm of the injury site. Nearby can be, for example, within 0.5 cm, within 1 cm, within 2 cm, within 3 cm, within 4 cm, within 5 cm, within 6 cm, within 7 cm, within 8 cm, within 9 cm, or within 10 cm of the damaged site.

[0028] The fusion protein or multimer of the present invention can link Nrx(S4+) in presynaptic cells to AMPAR in postsynaptic cells via itself. Therefore, the fusion protein or multimer of the present invention can be used to link Nrx(S4+) in presynaptic cells to AMPAR in postsynaptic cells. Promotion of the link between Nrx(S4+) in presynaptic cells and AMPAR in postsynaptic cells can promote the connection between excitatory interneurons. Therefore, the fusion protein or multimer of the present invention can be used to promote the connection between excitatory interneurons.

[0029] According to the present invention, there is provided a pharmaceutical composition (sometimes referred to as the "pharmaceutical composition of the present invention") comprising the fusion protein of the present invention or a multimer thereof. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable excipient. Examples of excipients include an aqueous solvent, a buffer, a surfactant, an antibacterial agent, and a bulking agent. In some embodiments, the aqueous solvent may be water for injection, physiological saline, or artificial cerebrospinal fluid. The pharmaceutical composition of the present invention may be lyophilized or provided as a lyophilized formulation (or as a kit containing the lyophilized formulation and, if necessary, an aqueous solvent for preparation immediately before use).

[0030] The pharmaceutical composition of the present invention can be used to treat spinal cord injury and to promote connections between nerve cells (especially between excitatory interneurons) in the area surrounding the injury site in the spinal cord.

[0031] The pharmaceutical composition of the present invention can be administered intraspinally. For example, the pharmaceutical composition of the present invention can be administered to a site upstream of the spinal cord injury to improve motor and / or sensory function. Here, "upstream" refers to the direction of the central nervous system from the injury site. Whether motor and / or sensory function has improved can be tested by methods well known to those skilled in the art. For example, improvement in function can be evaluated by neurological assessment methods such as the American Spinal Injury Association (ASIA) motor score, the ASIA impairment scale (AIS), and the Frankel classification.

[0032] In some embodiments of the present invention, the subject is a subject with spinal cord injury, such as a subject with acute spinal cord injury, a subject with subacute spinal cord injury, and / or a subject with chronic spinal cord injury, e.g., a human with spinal cord injury, such as a human with acute spinal cord injury, a human with subacute spinal cord injury, and / or a human with chronic spinal cord injury. The acute phase is a period in which primary injury to the spinal cord due to external force and subsequent biological and biochemical responses are prominent. Secondary injury involves hematoma, ischemia, edema, inflammatory cell infiltration, and cytotoxicity due to neurotransmitter leakage, leading to neuronal and glial cell death. The subacute phase is a period in which acute inflammation subsides, and angiogenesis and tissue repair responses actively occur. The chronic phase is a period in which cavities form at the site of spinal cord injury, resulting in tissue defects, and astrocytes form hard glial scars around the injury site, forming a barrier to axonal regeneration. The subject may be within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 21 days, 28 days, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year, 2 years, 3 years, 4 years, or 5 years or more since suffering a spinal cord injury.

[0033] It is known that in normal human spinal cord injuries, even severe injuries result in the survival of some nerves. Therefore, in certain embodiments of the present invention, the subject may be a subject with partial spinal cord injury. Partial injury means an injury in which the connection between the upstream and downstream of the spinal cord injury site is partially maintained. Nerve connection can be confirmed by anatomical examination and / or electrophysiological examination. Furthermore, in certain embodiments of the present invention, the subject may be a subject with complete spinal cord injury (e.g., complete transection). In cases where the spinal cord is completely injured, for example, a procedure to reconnect the injured surfaces may be performed, and the pharmaceutical composition of the present invention may be administered.

[0034] The fusion protein of the present invention can be a fusion protein of a protein of the same animal species as the animal species to which it is to be administered.

[0035] The present invention provides nucleic acids encoding the fusion proteins of the present invention. Nucleic acids include DNA and RNA.

[0036] The pharmaceutical composition of the present invention may contain, in place of the fusion protein of the present invention, mRNA encoding the fusion protein of the present invention and / or a gene expression vector comprising a nucleic acid encoding the fusion protein of the present invention operably linked to a promoter.

[0037] The fusion proteins of the present invention can be produced from nucleic acids encoding the fusion proteins. The fusion proteins of the present invention can be obtained by expressing the nucleic acids encoding the fusion proteins of the present invention as mRNA in cells (e.g., mammalian cells such as human cells, E. coli, insect cells, etc.). According to the present invention, a gene expression vector (gene expression vector of the present invention) is provided, which comprises a nucleic acid encoding the fusion proteins of the present invention operably linked to a promoter. The gene expression vector can be an episomal vector such as a plasmid vector or a Sendai virus vector, or a vector inserted into a nuclear genome such as a retrovirus vector. The gene expression vector of the present invention can comprise elements selected from the group consisting of a replication origin, a promoter, a nucleic acid encoding the fusion protein of the present invention operably linked to the promoter sequence, and a drug selection marker. The promoter can be any promoter capable of transcribing mRNA encoding the fusion protein of the present invention in cells (e.g., mammalian cells such as human cells, E. coli, insect cells, etc.), and those skilled in the art can appropriately select and use from known promoters (e.g., CMV promoter, RSV promoter, MMT promoter, metallothionein promoter, heat shock promoter, albumin promoter, ApoA1 promoter, human globin promoter, retroviral LTR, human growth hormone promoter, β-actin promoter, adenovirus promoter, thymidine kinase promoter, B19 parvovirus promoter, etc.). A drug selection marker may be present or absent, but in some cases, treating cells with a drug allows only cells carrying the gene expression vector to survive.

[0038] According to the present invention, there is provided a recombinant cell (e.g., mammalian cells such as human cells, E. coli, insect cells, etc.) having a nucleic acid encoding the fusion protein of the present invention. Examples of the cell include Chinese hamster ovary cells (CHO cells) and 293T cells. 293T cells may be cultured in the presence of, for example, the class I α-mannosidase inhibitor kifunensine to express the fusion protein of the present invention. Cells expressing the fusion protein of the present invention also include, for example, GnTI - / - Cells (cells with a homozygous knockout of the GnTI gene), for example, HEK293S-GnTI - / - (e.g., ATCC™ CRL-3022 TM The medium and culture conditions can be selected to be suitable for protein expression, and can be appropriately determined by those skilled in the art.

[0039] The nucleic acid encoding the fusion protein of the present invention may have a signal sequence. The fusion protein or multimer thereof of the present invention may or may not have a signal sequence.

[0040] According to the present invention, multimers of the fusion proteins of the present invention are secreted from cells as multimers. In particular, fusion proteins of the present invention in which the multimerization domain is a trimerization domain can be secreted from cells as hexamers. That is, multimers of the fusion proteins can be obtained by culturing cells expressing the fusion proteins under conditions suitable for multimer formation. Under these conditions, hexamers can be spontaneously formed in the culture medium by self-assembly of monomers. Therefore, the fusion proteins of the present invention can be recovered as multimers (e.g., hexamers) from the culture supernatant of recombinant cells. The obtained multimers can be purified by techniques well known to those skilled in the art. The purified multimers can be formulated while maintaining their multimeric structure. According to the present invention, the fusion protein of the present invention may be incubated under conditions suitable for multimerization, or under conditions suitable for generating disulfide bonds between the fusion proteins, as necessary. According to the present invention, the fusion protein of the present invention may be stored under solution conditions suitable for maintaining the multimeric state. According to the present invention, there is provided a method for producing a hexamer of the fusion protein of the present invention, comprising the steps of: culturing cells harboring a nucleic acid encoding a fusion protein of the invention, the nucleic acid having a signal sequence, under conditions suitable for expression of the protein; Obtaining a culture supernatant; recovering the hexamer of the fusion protein from the obtained culture supernatant; A method comprising: The present invention also provides a method for producing the fusion protein of the present invention, which comprises culturing 293T cells having a nucleic acid encoding the fusion protein of the present invention in the presence of the class I α-mannosidase inhibitor kifunensine, and recovering the hexamer of the fusion protein of the present invention expressed from the cell culture supernatant. The present invention also provides a method for producing the fusion protein of the present invention, which comprises culturing 293T cells having a nucleic acid encoding the fusion protein of the present invention in the presence of the class I α-mannosidase inhibitor kifunensine, and recovering the hexamer of the fusion protein of the present invention expressed from the cell culture supernatant. - / - Cells (e.g., human GnTI - / - Cells, e.g., HEK293S-GnTI - / -The present invention also provides a method for detecting hexamers of fusion proteins of the present invention, comprising culturing fusion protein multimers (cells) under conditions suitable for protein expression and recovering the expressed hexamer of the fusion protein of the present invention from the cell culture supernatant. The above-described method of the present invention may further comprise purifying the recovered hexamer. The above-described method of the present invention may further comprise mixing the purified hexamer with a pharmaceutically acceptable excipient to produce a pharmaceutical composition. The hexamer of the fusion protein of the present invention may be stored under conditions suitable for maintaining the disulfide bond linking the two trimers, such as under non-reducing conditions, because reducing conditions may cleave the disulfide bond linking the two trimers. The method of the present invention may further comprise confirming the number of mers of the fusion protein before recovering the fusion protein multimer. The hexamer of the fusion protein multimer can be confirmed using a multi-angle light scattering detector or the like.

[0041] The present invention provides the use of a multimer of the fusion protein of the present invention in the manufacture of a medicament for use in forming new synaptic connections between excitatory interneurons. The present invention provides the use of a fusion protein of the present invention in the manufacture of a medicament for use in forming new synaptic connections between excitatory interneurons. The present invention provides the use of a nucleic acid encoding a fusion protein of the present invention in the manufacture of a medicament for use in forming new synaptic connections between excitatory interneurons. The present invention provides the use of a recombinant cell carrying a nucleic acid encoding a fusion protein of the present invention in the manufacture of a medicament for use in forming new synaptic connections between excitatory interneurons.

[0042] The present invention provides a trimer of the fusion protein of the present invention. The trimer of the fusion protein of the present invention has a trimerization domain in the fusion protein and is trimerized by the trimerization domain. The present invention also provides a hexamer of the fusion protein of the present invention. The hexamer of the fusion protein of the present invention is formed by linking two trimers of the fusion protein of the present invention via a disulfide bond. The hexamer of the fusion protein of the present invention has one or both sulfhydryl groups of the cysteine ​​residues corresponding to the 34th and 38th cysteines in the amino acid sequence registered in NCBI Reference Sequence: NP_004343.1, and the two trimers are linked by forming a disulfide bond between the sulfhydryl groups. The hexamer of the fusion protein of the present invention can be obtained as a hexamer of the fusion protein of the present invention without a signal sequence by expressing the fusion protein of the present invention in a cell containing a nucleic acid encoding the fusion protein of the present invention with a signal sequence, using the method described above. Therefore, the hexamer of the fusion protein of the present invention can be free of a signal sequence. According to the present invention, there is provided a pharmaceutical composition comprising a hexamer of the fusion protein of the present invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The hexamer of the fusion protein of the present invention is maintained under solution conditions in which the disulfide bond linking two trimers is stable.

[0043] According to the present invention, there is provided a method of treating a subject, the method comprising administering to the subject a therapeutically effective amount of a fusion protein multimer of the present invention. The subject may have a spinal cord injury. The present invention provides a method of treating a subject, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding a fusion protein of the present invention. The subject may have a spinal cord injury. According to the present invention, there is provided a method of treating a subject, comprising administering to the subject a therapeutically effective amount of cells (cells allogeneic or xenogeneic to the subject) comprising a nucleic acid encoding a fusion protein of the present invention. The subject may have a spinal cord injury. According to the present invention, the subject can be administered a multimer of the fusion protein of the present invention in combination with other spinal cord injury therapeutic agents. Also, according to the present invention, the subject can undergo rehabilitation in addition to the treatment of the present invention. [Example]

[0044] Materials and Methods mouse All behavioral experiments were performed in the late afternoon under constant temperature (22 ± 1°C) and humidity (50 ± 10%) during the dark phase of the mouse cycle when the mice were active. Spinal cord injury experiments were performed in accordance with the animal experimentation regulations of Aichi Medical University (approval numbers: 1559, 2019-89, 1642, 2020-48).

[0045] Plasmid Surface plasmon resonance (SPR) assays identified the amino-terminal domain ATD of extracellular human glutamate receptor D2 (GenBank ID NM_001510; GluD2 ATD:Asp24-Gly440), human glutamate receptor A4 ATD (GenBank ID U16129.1; GluA4 ATD:Gly21-Thr416), human β-neurexin-1 LNS6 domain (GenBank NM_NM_138735; β-Nrx LNS6:His85-D265), and the pentraxin domain of human neuronal pentraxin-1 (NP1 PTX The cDNA encoding the pentraxin domain (NP1; GenBank ID AC50727.1; Pro224-Ile431) was fused to the C-terminus of a hexahistidine (His6) tag and cloned into the pHLsec expression vector (AR Aricescu et al., Acta Crystallogr D Biol Crystallogr 62, 1243-1250 (2006)). PTX was fused to the C-terminus of the three-stranded GCN4 leucine zipper coiled-coil sequence (PB Harbury et al., Science 262, 1401-1407 (1993)) to generate NP1PTX-3COIL To produce CPTX, NP1 PTX-3COILwas fused to the C-terminus of the cysteine-rich region (CRR) of human Cbln1 (GenBank ID NM_004352; Gln22-Ile53) (J. Elegheert et al., Science 353, 295-299 (2016)) (see Figure 1B). For in vitro binding and synaptogenesis assays, mouse NP1 (GenBank ID NM_008730.2) was cloned into the pCAGGS vector with a C-terminal HA-tag (kindly provided by Dr. J. Miyazaki, Osaka University). The full-length neurexin cDNA was cloned into the pCAGGS vector with a C-terminal FLAG-tag. The neurexin extracellular domain was cloned into the pCAGGGS vector with a C-terminal human Fc tag. The ATDs of either GluA1-4 were cloned into the modified pDisplay vector (Invitrogen) as previously described (K. Matsuda et al., Neuron 90, 752-767 (2016)). The miR sequence was inserted upstream of the IRES-EGFP sequence in the pCAGGS vector as previously described (Y.H. Takeo et al., J Neurosci 35, 12518-12534 (2015)). The 21-bp target sequences of mouse Nptx1 and Nptxr genes were designed using BLOCK-iT (Invitrogen) as follows: AGA CAA GTT TCA GCT GAC ATT (for NP1, SEQ ID NO: 10) and TGC TCA GTC GCT TCT TCT GTA (for NPR, SEQ ID NO: 11). For immunocytochemical analysis to confirm the selectivity of the anti-NPs antibodies, mouse NP1-full length (GenBank ID NM_008730.2), NP2-full length (GenBank ID NM_016789.3), and NPR-full length (GenBank ID NM_030689.4) were cloned into the pCAGGS vector with a C-terminal HA-tag.For immunoblot analysis to confirm the selectivity of the anti-NP antibodies, mouse NP1 (without signal sequence, aa 1–22), NP2 (without signal sequence, aa 1–14), and NPR (without transmembrane domain, aa 1–23) were cloned into pCAGGS with an Igκ signal sequence followed by an N-terminal 2× HA tag.

[0046] Preparation of recombinant proteins For large-scale protein production, the protein was expressed by transient transfection in HEK293T cells. Five days after transfection, the conditioned medium was collected and buffer-exchanged using a QuixStand benchtop diafiltration system (GE Healthcare). The recombinant protein was secreted into the culture supernatant and is thought to exist in the form of a hexamer consisting of two linked trimers (see Figure 2). The recombinant protein was purified by immobilized metal affinity chromatography (IMAC) using a pre-packed nickel-Sepharose column (GE Healthcare). The protein was concentrated and further purified by size-exclusion chromatography (SEC; Supferdex 200 16 / 60 PG HiLoad column, GE Healthcare) in 10 mM HEPES (4-(2-hydroxyethyl) 1-piperazineethanesulfonic acid) pH 7.40, 150 mM sodium chloride, and 3 mM calcium chloride (HBS-C) for structural studies, or in 10 mM Tris pH 7.4, 150 mM sodium chloride, 3 mM calcium chloride, and 0.005% (v / v) Tween-20 (TBS-CT) for interaction studies using SPR. Note that Cbln1 is thought to form trimers in the absence of the disulfide bond involved in Cbln1 multimerization. However, trimers are poorly secreted outside the cells, whereas hexamers are more commonly secreted.

[0047] Multi-angle light scattering (MALS) Protein samples concentrated to approximately 1.0 g / L were injected onto an HPLC-driven SEC column (Superdex 200 10 / 30 column, GE Healthcare) equilibrated with HBS buffer. The SEC column was coupled to an online UV detector (Shimadzu), an 18-angle light scattering detector (DAWN HELEOS), and a refractive index detector (Optilab T-rEX) (Wyatt Technology). Proteins for MALS contained N-linked oligomannose-type sugars, and molecular weight measurements were performed using an adaptive RI increment (dn / dc standard value; 0.185 mL / g) to account for glycosylation status. Data analysis was performed using ASTRA V software (Wyatt Technology). Multi-angle light scattering analysis revealed that the resulting recombinant protein had a molecular weight of 208.9 ± 3.3 kDa (see Figure 3), suggesting the protein formed a hexamer as expected.

[0048] antibody The origin, dilution, company, and catalog number of the antibodies are as follows: anti-calbindin (goat, 1:500, Frontier Science, Af1040), anti-HIS (mouse, 1:1000, MBL, D291-3 or rabbit, 1:1000, CST, #2365), anti-Myc (rabbit, 1:1000, MBL, 562), anti-FLAG (rabbit, 1:1000, SIGMA, F7425), anti-HA (mouse, 1:1000, BAbCO, MMS-101), synaptophysin (mouse, 1:500, SIGMA, S5768, or G, 1:500, Frontier Science, Af300), anti-neurexin (chicken, 1:500, a gift from Peter Scheiffele (C. Dean et al., Nat Neurosci 6, 708-716) (2003)), anti-GFP (rabbit, 1:1000, Frontier Science, Af-2020), anti-GluA1 (rabbit, 1:100, Calbiochem, PC246, or guinea pig, 1:500, Frontier Science, AF380), anti-GluA2 / 3 (rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (rabbit, 1:1000, Pharmingen, 60666N, or guinea pig, 1:500, Frontier Science, Af640), anti-VGluT1 (rabbit, 1:500, Frontier Science, Af570, or goat, 1:500, Frontier Science, Af310), anti-VGAT (goat, 1:500, Frontier Science, Af620), anti-parvalbumin (goat, 1:500, Frontier Antibodies against NP1 and NPR (NP1: aa 185-227, NPR: aa 226-263) were newly generated by peptide-immunization of guinea pigs. For simultaneous detection of various GluAs (i.e., GluA1-3 or GluA1-4), a mixture of the above primary antibodies was used.Secondary antibodies conjugated with DyLight 405, Alexa 488, 546, 647, and Cy3 (Invitrogen or Jackson Lab) for each primary antibody were used at dilutions (1:1000 for immunocytochemistry and 1:200 for immunohistochemistry).

[0049] Cells and conditioned medium were solubilized in Laemmli buffer (2% SDS, 80 mM Tris-HCl pH 6.8, 10% glycerol, 0.00625% Coomassie Blue G250), and proteins were reduced by boiling in 2% 2-mercaptoethanol for 3 minutes. Samples were subjected to SDS-PAGE on a gradient gel (Wako) and blotted onto a membrane (Millipore). The membrane was blocked with TS-Tween (0.1% Tween 20, 50 mM Tris-HCl pH 7.6, 150 mM NaCl) containing 5% skim milk (Meiji) and incubated with primary antibodies for 2 hours and HRP-conjugated secondary antibodies (GE Healthcare) for 30 minutes. Chemiluminescence was generated using an ImmunoStar detection kit (Wako) or Immobilon (Millipore) and detected using an LAS-3000 mini system (FUJI FILM).

[0050] HEK293 cells were cultured at 37°C in high-glucose DMEM (Sigma) containing 10% FBS (HyClone), 50 units / ml penicillin, 50 mg / ml streptomycin (Invitrogen), and 2 mM L-glutamine. Using Lipofectamine 2000 (Invitrogen), cells were transfected with pDisplay encoding myc-tagged GluA1-GluA4-ATD or pCAGGS encoding FLAG-tagged Nrx or GFP. The next day, transfected cells were detached with PBS containing 5 mM EDTA and aliquoted into 2 × 10 4Cells were seeded at 1000p / well onto PLL-coated 12 mm coverslips. One hour after seeding, cells were treated with vehicle, recombinant Cbln1-HIS, or CPTX-HIS (23.6 nM, final concentration as hexamer) for 4 hours to overnight. For in vitro tripartite binding assays, after the first 4 hours of ligand treatment and one wash with fresh culture medium, cells were treated with conditioned medium containing Nrx1β(+4)-hFc for 4 hours. Cells were fixed with 4% PFA / PBS for 15 minutes and washed three times with PBS. After blocking with 3% BSA / PBS for 30 minutes without permeabilization, cells were incubated with primary antibodies against the tag of the bound ligand (HIS, HA, or hFc) in 3% BSA / PBS for 2 hours at room temperature or 24 hours at 4°C. After three washes with PBS, cells were permeabilized with PBS containing 0.1% TX-100 and 3% BSA. Cells were stained with primary antibodies against the receptor tags (FLAG or Myc) for 2 hours at room temperature or 24 hours at 4°C, followed by PBS washing and staining with each secondary antibody for 30 minutes. After PBS washing, coverslips were mounted on glass slides using Fluoromount-G. Samples in which HEK cells expressed GFP were not stained with the primary antibody against GFP, but were treated with the same primary antibody against the receptor tag (FLAG or Myc), and autofluorescence of the GFP protein was detected by microscopy.

[0051] Immunocytochemistry and immunohistochemistry Spinal cord section.Two to five days after injection, mice were transcardially perfused with 3% glyoxal for 10 minutes under deep pentobarbital anesthesia. The dissected spinal cords were then fixed in 3% glyoxal overnight at 4°C, cryoprotected in 30% sucrose / PBS for several days, and sliced. Spinal cords embedded in Tissue-Tek OCT compound (Sakura Finetek, #4583) were cut horizontally or coronally at 4-5 mm thickness around the epicenter of the injury using a cryostat (Leica). Slices were mounted at 200-400 μm intervals on APS-coated glass slides (Matsunami). After washing with PBS containing 0.1% TX-100, the sections were treated with 10% donkey serum for 30 minutes at room temperature, followed by a mixture of primary and respective secondary antibodies overnight. Finally, the sections were mounted on slides and mounted with Fluoromount-G (Southern Biotech). Fluorescence images of the entire section on the glass slide were captured as virtual slides using a conventional microscope (BX63, Olympus) with a 4x objective to determine the exact center of the injury. Subsequently, for comparison, fluorescence images of the section around the anterior root within the gray matter were captured by confocal microscopy (SD-OSR, Olympus; 63x objective) using the same parameters, including objective lens, laser power, exposure time, gain, and offset. Super-resolution images were obtained using an Airyscan2 with an LSM980 (63x / 1.40 oil immersion with 2.5x digital zoom, 35 nm / px, XY < 120 nm, Z < 350 nm). To define particles of GluA4, VGluT2, and their intersections, fluorescent images were subjected to background subtraction (50px), Laplace filter (9x9), box filter (5x5), autothreshold (Otsu method), particle extraction (>5px), binarization, and watershed segmentation. Using defined particles as ROIs, the size and mean intensity of each channel were quantified. The percentage of VGluT2 fluorescent puncta bearing GluA4 was calculated by dividing the number of VGluT2 particles with defined GluA4 particles greater than 1px in the ROI by the number of all VGluT2 particles. All image processing was performed using ImageJ.

[0052] Intraspinal injection of CPTX injection. CPTX, Cbln1, chondroitinase ABC (Sigma Aldrich, C2905), or vehicle (control drug) was injected proximal to the spinal cord injury site. The drug solution was injected at 0.5 μl (CPTX, Cbln1, vehicle; 1 μg / μl) or 0.5 μg (chondroitinase ABC; U / μl) through a glass capillary (3-000203-G / X, Drummond Scientific Company) using an electric microinjector (BJ110, BEX CO., LTD.). After injection, the muscle layer and skin were closed with sutures.

[0053] Spinal cord injury model Mice (9-11 weeks old, ICR) were subjected to compression-induced (see Figure 4, Crush Model) or hemisection-induced spinal cord injury (SCI; see Figure 4, Hemisection). After anesthesia, the spinal cord was surgically exposed, and the dorsal spinal column at the 10th thoracic vertebra was incised with microscissors or a hemisection scalpel. Alternatively, a commercially available SCI impactor device (Infinite Horizon Impactor; Precision Systems and Instrumentation, Lexington, NY) (K. Takeuchi et al., Nat Commun 4, 2740 (2013)) was used to inflict compression-induced injury on mice. A 70 K dyne impact was used. This device reports data on force versus time and displacement versus time. Immediately after SCI, or 1, 2, or 4 weeks later, 2 μL (1 μL x 2 sites) of 1.6 mg / mL CPTX was administered near the injury site (i.e., 15 pmol) or 1 μL (0.25 μL divided into 4 sites) near the injury site (note that the recovery effect was similar depending on the dose). Other groups also received 15 pmol of CPTX. The muscle layer and skin were closed with sutures. Animals were allowed to recover from anesthesia with the antagonist.

[0054] Behavioral locomotor recovery in SCI-induced animals was assessed using video recordings as previously described (K. Takeuchi et al., Nat Commun 4, 2740 (2013)). Open-field scoring of the Basso Mouse Scale (BMS) (D.M. Basso et al., J Neurotrauma 23, 635-659 (2006)) and footstepping tests were performed weekly. Functional recovery was assessed 6–8 weeks after SCI by at least two researchers blinded to the experimental groups. Mice with incomplete injury (BMS score >0 on that day) were excluded on day 3 after SCI induction. The difference in BMS score between 1 and 2 weeks after administration was calculated as ΔBMS / week (Figure 5E). For each footstepping test, mice were placed on a wire mesh grid and videotaped while walking on the grid for 5 min. Mice that walked on the grid at least 70 times within 3 min were analyzed, and the number of times they stepped off the grid was calculated as the footfall number (Figure 5B).

[0055] result The constructed artificial excitatory synaptic connector (CPTX, a chimeric protein consisting of the Nrx-binding and trimerization domains of Cbln1 and the AMPA receptor-binding domain of Nptx1) artificially connects AMPA receptors (AMPARs) with Nrx, thereby stimulating AMPARs and activating their downstream signals (Figure 1B). As expected, this chimeric protein formed a hexamer and could be recovered from the cell culture supernatant (Figs. 2 and 3). A hemisection model of spinal cord injury was created by cutting the spinal cord in half with scissors. The artificial excitatory synaptic connector was administered immediately after injury. As shown in Figures 5A and 5B, CPTX induced a statistically significant and extremely strong recovery (improvement in BMS score) starting 1-2 weeks after administration. Furthermore, as shown in Figures 5C and 5E, administration one week after injury also induced significant recovery. This is a noteworthy effect, given that no previous treatments have demonstrated significant efficacy when administered one week after injury. Even in spinal cord injury models in which the spinal cord was completely cut with a knife, CPTX still produced a certain degree of recovery from injury when the cut surfaces were kept in contact with each other.

[0056] Next, we created a spinal cord injury model using a crush injury and administered the artificial excitatory synaptic connector CPTX immediately after injury. As shown in Figure 5D, CPTX induced a statistically significant and extremely strong recovery (improvement in BMS score) starting one week after administration. An even stronger effect was observed when CPTX was administered in combination with ChABC. This result suggests that the mechanisms of action of CPTX and the conventional therapeutic drug ChABC are different.

[0057] Next, we administered CPTX to subacute (2 weeks after injury) and chronic (4 weeks after injury) crush injury models to confirm its efficacy. A crush injury model (C57 / BL6 mice, n = 4–5) was created using a spinal cord impactor (70 kDyne, 1.5 mm diameter). Two weeks after injury, a total of 1 μL of 1.7 mg / mL CPTX solution was administered locally at 0.25 μL doses over 1 minute at four sites near the injury site to ensure complete coverage. As shown in Figure 5F, CPTX induced a significant recovery from 1 week after local administration. Furthermore, a more severe crush injury model (C57 / BL6 mice, n = 4) was created using a 90 kDyne, 1.5 mm diameter impactor. Four weeks after injury, a total of 1 μL of 1.7 mg / mL CPTX solution was administered locally at 1 μL per minute at the injury site. As shown in Figure 5G, CPTX induced a strong tendency toward recovery starting one week after local administration. The same experiment as in Figure 5G was performed using ICR mice (n = 2-3). As shown in Figure 5H, CPTX induced extremely strong recovery starting one week after local administration. Thus, CPTX exerted a clear effect not only in the acute phase after injury, but also when administered in the subacute and chronic phases.

[0058] Next, we administered His-tagged CPTX to the SCI injury site and subsequently examined the expression and localization of Vglut2, His, and GluA4 (a subunit of AMPAR) by immunohistochemistry. The administered CPTX accumulated at the injury site, and Vglut2 and GluA4 expression was observed around it (see Figure 6A). Analysis of long-resolution images revealed Vglut2 and GluA4-derived signals above and below His (i.e., CPTX), respectively, suggesting that CPTX bridges Vglut2 and GluA4. Vglut2 was used to observe the presynaptic portion of excitatory synapses.

[0059] In addition, tissue sections 1.6 mm upstream of the hemisection model were observed 1 week after transection (see Figure 7A, upper right). Signals derived from Vglut2 and GluA4 were observed above and below His (i.e., CPTX) in the tissue 1.6 mm upstream, suggesting that CPTX bridges Vglut2 and GluA4. We then calculated the proportion of Vglut2 colocalized with GluA4 (GR4) in the tissue. Specifically, we calculated the proportion of Vglut2 that was GR4-positive. As shown in Figure 7B, CPTX administration significantly increased the proportion of Vglut2 that was GR4-positive. This suggests that neural reorganization and the formation of new neural circuits occurred upstream of the injury site.

[0060] Spinal cord injury is an irreparable injury. CPTX may, at least in part, stimulate the reorganization of neural circuits during the spinal cord injury recovery process, thereby forming new neural circuits and connecting upstream and downstream pathways (see, for example, Figure 8). CPTX's suitability for connecting excitatory interneurons in the spinal cord may be related to the sufficient expression of AMPA-type glutamate receptors on excitatory interneurons.

[0061] Sequence Listing Description SEQ ID NO: 1: Nucleic acid and amino acid sequences of the fusion protein of the present invention prepared in the Examples SEQ ID NO: 2: Amino acid sequence of the fusion protein of the present invention prepared in the Examples {In SEQ ID NO: 2, amino acid numbers 1 to 28 are a signal sequence, amino acid numbers 32 to 63 are the Nrx-binding domain of Cbln1, amino acid numbers 66 to 98 are the trimerization domain, amino acid numbers 110 to 317 are the AMPA receptor-binding domain of NPTX-1 protein, and amino acid numbers 321 to 326 are a 6xHis tag.} SEQ ID NO: 3: Example of an amino acid sequence of a flexible linker SEQ ID NO: 4: Example of an amino acid sequence of a flexible linker SEQ ID NO: 5: Example of an amino acid sequence of a flexible linker SEQ ID NO: 6: Example of an amino acid sequence of a flexible linker SEQ ID NO: 7: Example of an amino acid sequence of a flexible linker SEQ ID NO: 8: Example of an amino acid sequence of a flexible linker SEQ ID NO: 9: Example of an amino acid sequence of a flexible linker SEQ ID NO: 10: Sequence of miRNA for suppressing NP1 expression SEQ ID NO: 11: Sequence of miRNA for suppressing NPR expression

Claims

1. A multimer of a fusion protein, The fusion protein is a multimer comprising the neurexin (Nrx)-binding region and multimerization domain of the cerebellin-1 (Cbln1) protein and the AMPA receptor-binding region of the neuronal pentraxin-1 (Nptx1) protein.

2. The multimer of claim 1, wherein the multimerization domain is a trimerization domain and the multimer is a hexamer formed by two trimers of the fusion protein linked by disulfide bonds.

3. A multimer according to claim 1 or 2, wherein the region of the Cbln1 protein that binds to Nrx is a region of the Cbln1 protein that can bind to Nrx having splice site 4 (i.e., Nrx(S4+)).

4. The multimer according to any one of claims 1 to 3, wherein the region of Nptx1 that binds to an AMPA-type glutamate receptor is a region of Nptx1 that corresponds to the pentraxin domain of human neuronal pentraxin-1.

5. The multimer according to any one of claims 1 to 4, wherein the multimerization domain is interposed between the Nrx-binding region of the Cbln1 protein and the AMPA-type glutamate receptor-binding region of Nptx1, or is sandwiched between the two regions.

6. The multimer according to any one of claims 1 to 5, wherein the Nrx-binding region of the Cbln1 protein and the multimerization domain, and / or the multimerization domain and the AMPA receptor-binding region of Nptx1 are linked via a linker.

7. The multimer of claim 6, wherein the linker is any one of SEQ ID NOs: 3 to 9.

8. A nucleic acid encoding the fusion protein defined in any one of claims 1 to 5, which has the nucleic acid sequence set forth in SEQ ID NO:

1.

9. The multimer according to any one of claims 1 to 5, wherein the fusion protein has the amino acid sequence set forth in SEQ ID NO:

2.

10. A pharmaceutical composition comprising the multimer according to any one of claims 1 to 9.

11. fusion proteins comprising the neurexin (Nrx)-binding region of cerebellin-1 (Cbln1 protein), a multimerization domain, and the AMPA receptor-binding region of neuronal pentraxin-1 (Nptx1) protein; A pharmaceutical composition for use in forming new synaptic connections between excitatory interneurons.

12. The pharmaceutical composition according to claim 10 or 11, wherein the excitatory interneuron is a spinal cord excitatory interneuron.

13. The pharmaceutical composition according to any one of claims 10 to 12, for use in treating spinal cord injury.

14. The pharmaceutical composition according to any one of claims 10 to 13, wherein the multimerization domain is a trimerization domain.

15. The pharmaceutical composition according to any one of claims 10 to 14, wherein the multimerization domain is a trimerization domain, and the multimer is a hexamer formed by two trimers of the fusion protein linked by a disulfide bond.

16. The pharmaceutical composition according to any one of claims 10 to 15, which is a therapeutic agent for spinal cord injury.

17. A method for producing the pharmaceutical composition according to any one of claims 10 to 15, comprising: culturing cells harboring a nucleic acid encoding a fusion protein of the invention, the nucleic acid having a signal sequence, under conditions suitable for expression of the protein; Obtaining a culture supernatant; recovering the fusion protein multimer from the obtained culture supernatant; A method comprising:

18. The method of claim 17, wherein the multimerization domain is a trimerization domain and the multimer is a hexamer formed by two trimers of the fusion protein linked by a disulfide bond.

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