Pharmaceutical composition for treating ciliopathy

Abstract

Provided are: a pharmaceutical composition for treating ciliopathy, the pharmaceutical composition containing, as an active ingredient, a male reproductive cell-related kinase and / or an intestinal cell kinase, or a substance that promotes the expression or function of a male reproductive cell-related kinase and / or an intestinal cell kinase; and a composition for ameliorating ciliary dysfunction, the composition containing, as an active ingredient, a male reproductive cell-related kinase and / or an intestinal cell kinase, or a substance that promotes the expression or function of a male reproductive cell-related kinase and / or an intestinal cell kinase.

Description

Pharmaceutical composition for treating ciliary diseases

[0001] The present invention relates to a pharmaceutical composition for treating ciliopathy.

[0002] Cilia are structures with microtubule axes that protrude from the cell surface and are found in a wide range of organisms, from single-celled organisms to humans. Cilia include motile cilia, such as sperm flagella and cilia in airway epithelia, and non-motile cilia called primary cilia. Cilia are found in most cells and are known to function as antennae that capture signals from outside the cell. A wide variety of receptors are localized in primary cilia, which receive signals from outside the cell, such as light, odor molecules, and Hedgehog signaling, and transmit the information into the cell.

[0003] Cilia component factors, such as receptors, are transported via a protein transport mechanism within cilia called intraflagellar transport (IFT). IFT is essential for ciliary formation and function and consists of three major steps: anterograde transport from the base to the tip of the cilium driven by a kinesin motor, switching of transport direction at the cilial tip, and retrograde transport from the tip to the base of the cilium driven by a dynein motor. Disruption of intraflagellar protein transport leads to abnormalities in ciliary formation and function, which in turn leads to abnormalities in the structure and function of ciliated cells and cell death. Previous research by the present inventors has revealed that the phosphorylation enzyme intestinal cell kinase (Ick) is the master regulator of switching of transport direction at the cilial tip (Non-Patent Document 1).

[0004] It is known that defects in the development or function of cilia in humans, i.e., ciliopathies, cause a group of diseases called ciliopathies, which are characterized by abnormalities in development and sensory organs. However, the mechanisms of ciliopathies development remain unclear, and no truly effective treatments for ciliopathies have been established. As a result, some ciliopathies have been designated as intractable diseases in Japan.

[0005] Human ICK is known to be a causative gene for ciliopathies. The present inventors have demonstrated that Ick-deficient mice are a model for ciliopathies (Non-Patent Document 1). Human male germ cell-associated kinase (MAK) is known to be a causative gene for ciliopathies (retinitis pigmentosa) in Japan and around the world, and is known to be the primary causative gene for retinitis pigmentosa in Europe and the United States in particular. Mak-deficient mice are used as a model for retinitis pigmentosa (Non-Patent Document 2). Furthermore, deficiency of cell cycle-related kinase (Ccrk) is known to cause abnormalities in ciliary length regulation and accumulation of IFT component proteins at the cilia tip, similar to deficiency of Mak or Ick (Non-Patent Document 3). DYNC2LI1 (dynein cytoplasmic 2 light intermediate chain 1), which encodes a subunit of the cytoplasmic dynein 2 complex, is known to be a causative gene for ciliopathies.

[0006] Chaya T, Omori Y, Kuwahara R, Furukawa T. EMBO J (2014) 33:1227-1242.Omori Y, Chaya T, Katoh K, Kajimura N, Sato S, Muraoka K, Ueno S, Koyasu T, Kondo M, Furukawa T, PNAS (2010) 107(52):22671-22676. Taro Chaya, Biochemistry Vol. 93, No. 4 (2021), pp. 494-502.

[0007] An objective of the present invention is to provide a pharmaceutical composition for treating ciliopathies and a composition for improving ciliopathies, as well as a method for screening for substances that improve ciliopathies.

[0008] In order to solve the above-mentioned problems, the present invention encompasses the following inventions: [1] A pharmaceutical composition for treating ciliopathy, comprising a substance that promotes the expression or function of male germ cell-associated kinase and / or intestinal cell kinase, or male germ cell-associated kinase and / or intestinal cell kinase as an active ingredient. [2] The pharmaceutical composition according to [1], wherein the substance that promotes the function of male germ cell-associated kinase and / or intestinal cell kinase is a fibroblast growth factor receptor inhibitor. [3] The pharmaceutical composition according to [2], wherein the fibroblast growth factor receptor inhibitor is rogaratinib, apruzumab, futibatinib, delazantinib, ASP5878, zoligratinib, FP-1039, LY-2874455, RG-7444, fexagratinib, infigratinib, erdafitinib, PD173074, pemigatinib, or a combination thereof. [4] The pharmaceutical composition according to [1], wherein the substance that promotes the function of male germ cell-associated kinase and / or intestinal cell kinase is a cell cycle-related kinase. [5] The pharmaceutical composition according to [1], wherein the substance that promotes the expression of male germ cell-associated kinase is a gene therapy drug that expresses male germ cell-associated kinase. [6] The pharmaceutical composition according to [1], wherein the substance that promotes the expression of intestinal cell kinase is a gene therapy drug that expresses intestinal cell kinase. [7] Ciliopathy includes retinal degeneration, choroidal coloboma, coloboma, retinitis pigmentosa, cone-rod dystrophy, cone dystrophy, macular dystrophy, corneal abnormalities, visual impairment, Leber's congenital amaurosis, Leber's amaurosis, Stargardt's disease, hyperopia, keratoconus, photophobia, cataract, ptosis, nystagmus, microphthalmia, hypertelorism, cyclopia, hydrocephalus, macrocephaly, agenesis of the corpus callosum, holoprosencephaly, cerebellar vermis defects, cerebellar malformation and dysfunction, occipital meningocele, hypoplastic medulloblastoma, hypothalamic hamartoma. , lower brainstem malformations, pituitary gland abnormalities, hypopituitarism, tumors and / or cysts in the central nervous system, brain abnormalities, seizures, psychomotor developmental delay, neuropsychiatric abnormalities, intellectual disability, learning disabilities, behavioral disorders, motor developmental delay, ataxia, feeding disorders, craniectodermal dysplasia, craniosynostosis, facial and digital (toe) anomalies, Weyers tetradactyly, polydactyly, brachymelia, syndactyly, missing fingers, bone and ectodermal abnormalities, bone developmental disorders, pelvic dysplasia, spinal column abnormalities, dental abnormalitiesBronchiectasis, airway abnormalities, lung hypoplasia, pulmonary dysplasia, disorders in lung development and expansion, respiratory disorders, Jeune-asphyxiating thoracic dysplasia, esophageal atresia, bifid epiglottis, cleft uvula, oral abnormalities, dysgeusia, long nose, chronic sinusitis, dysosmia, choanal atresia, tumors and / or cysts in the endolymphatic sac, hearing loss, hearing impairment, liver fibrosis, liver cysts, cystic kidney disease, polycystic kidney disease, chronic tubulointerstitial nephritis, tumors and / or cysts in the kidney cysts, nephronophthisis, tumors and / or cysts in the adrenal gland, tumors and / or cysts in the pancreas, endocrine system abnormalities, short stature, situs inversus, smooth muscle hamartoma, skin and intestinal abnormalities, Hirschsprung's disease, apple-peel small intestinal atresia, imperforate anus, absence or dysplasia of the nails, chest stenosis, congenital heart disease, cardiomyopathy, hypertension, obesity, diabetes, dyslipidemia, morbid obesity and spermatogenesis hypogonadism, hypogonadism, genital abnormalities, genitourinary malformations, infertility, The pharmaceutical composition according to any one of [1] to [6], wherein the ciliopathy is male infertility, decreased sperm count, azoospermia, female infertility, neonatal lethality, fetal hydrops, developmental delay, acrocallosal syndrome, pituitary stalk interruption syndrome, orofacial-digital-limb syndrome-related disease, syndromic craniosynostosis, Greig acupuncture polysyndactyly syndrome, endocrine-brain-bone dysplasia syndrome, COACH syndrome, Joubert syndrome-related disease, Senior-Loken syndrome, Alström syndrome, Carpenter syndrome, Curry-Jones syndrome, Ellis-van Creveld syndrome, Hydroretaras syndrome, Kallmann syndrome, McKusick-Kaufman syndrome, MORM syndrome, Lowe syndrome, Pallister-Hall syndrome, RHYNS syndrome, Ström syndrome, Starr syndrome, Usher syndrome, von Hippel-Lindau syndrome, Bardet-Biedl syndrome-related disease, or Meckel syndrome-related disease. [8] The pharmaceutical composition according to [1], wherein the ciliopathy is a retinal degenerative disease. [9] The pharmaceutical composition according to the above [8], wherein the retinal degenerative disease is retinitis pigmentosa.

[10] A pharmaceutical composition for treating a retinal degenerative disease, comprising a fibroblast growth factor receptor inhibitor as an active ingredient.

[11] The pharmaceutical composition according to the above

[10] , wherein the retinal degenerative disease is retinitis pigmentosa.

[12] A substance that promotes the expression or function of male germ cell-associated kinase and / or intestinal cell kinase,Or a composition for improving cilia disorders, comprising male germ cell-associated kinase and / or intestinal cell kinase as active ingredients.

[13] A method for screening for a substance that improves cilia disorders, comprising the steps of contacting a test substance with cells that express male germ cell-associated kinase and a kinesin family 3A protein, measuring the phosphorylation of the kinesin family 3A protein, and identifying a substance that increases the phosphorylation level of the kinesin family 3A protein compared to that in cells that have not been contacted with the test substance.

[14] A method for screening for a substance that improves cilia disorders, comprising the steps of contacting a test substance with cells that express intestinal cell kinase and a kinesin family 3A protein, measuring the phosphorylation of the kinesin family 3A protein, and identifying a substance that increases the phosphorylation level of the kinesin family 3A protein compared to that in cells that have not been contacted with the test substance.

[15] A method for screening for a substance that improves cilia disorders, comprising the steps of contacting a test substance with cells expressing a cell cycle-related kinase and a male germ cell-related kinase and / or an intestinal cell kinase, measuring the phosphorylation of the male germ cell-related kinase and / or the intestinal cell kinase, and identifying a substance that increases the phosphorylation level of the male germ cell-related kinase and / or the intestinal cell kinase compared to the phosphorylation level in cells not contacted with the test substance.

[0009] The present invention can provide a pharmaceutical composition for treating cilia disorders. The present invention can also provide a composition for improving cilia disorders. Furthermore, the screening method of the present invention can obtain a substance useful for improving cilia disorders, and provide a novel composition for improving cilia disorders.

[0010] Figure 1 shows the results of transfection of Ick-deficient mouse embryonic fibroblasts with plasmids encoding FLAG-tagged EGFP, FLAG-tagged Ick, FLAG-tagged Mak isoform 1 (Mak iso1), or FLAG-tagged Mak isoform 2 (Mak iso2). (A) Immunostaining with DAPI, anti-FLAG antibody, and anti-acetylated α-tubulin (AcTub) antibody. (B) and (C) Measurement of the number (B) and length (C) of cilia stained with anti-AcTub antibody in FLAG-positive cells. Figure 2 shows the results of overexpressing Ick in the retina of Mak-deficient mice. (A) Schedule for subretinal injection of adeno-associated virus (AAV) and retinal harvesting. (B) Immunostaining of harvested retinas with anti-FLAG and anti-rhodopsin antibodies after sectioning. (C) Quantification of the immunofluorescent signal of rhodopsin detected inside photoreceptors. Figure 1 shows the results of observing the retinas of Mak-deficient mice and Mak / Ick double-deficient (DKO) mice. (A) Representative toluidine blue-stained images of control, Mak-deficient (Mak- / -), and Mak / Ick DKO mouse retinas. (B) Measurement of the thickness of the outer nuclear layer (ONL). Figure 2 shows the results of administering a fibroblast growth factor receptor (FGFR) inhibitor (BGJ398) to Mak-deficient mice. (A) Drug administration schedule. (B) Representative toluidine blue-stained images of Mak- / - mouse retinas administered with BGJ398 or vehicle. (C) Measurement of the thickness of the outer nuclear layer (ONL). Figure 3 shows the results of electroretinogram (ERG) analysis of Mak-deficient mice administered with an FGFR inhibitor (BGJ398) or vehicle. (A) shows the a-wave of ERG under dark conditions, (B) shows the b-wave of ERG under dark conditions, (C) shows the a-wave of ERG under light conditions, and (D) shows the b-wave of ERG under light conditions. This figure shows the results of analyzing the phosphorylation of kinesin family 3A protein (Kif3a) in Mak-deficient mice treated with an FGFR inhibitor (BGJ398) or vehicle. (A) shows a Western blotting image of Kif3a.(B) shows the results of quantifying the relative phosphorylation level of Kif3a. (A) shows the results of overexpressing Ick in cells treated with a dynein motor protein inhibitor. (A) shows the results of immunostaining with DAPI, anti-FLAG antibody, and anti-AcTub antibody in NIH3T3 cells transfected with a plasmid encoding FLAG-tagged EGFP or FLAG-tagged Ick, treated with DMSO or a dynein motor protein inhibitor (Ciliobrevin D), and then immunostained with DAPI, anti-FLAG antibody, and anti-AcTub antibody. (B) shows the results of counting the number of cilia stained with anti-AcTub antibody in FLAG-positive cells. (B) shows the results of comparing the expression inhibitory effects of shRNA expression constructs against the dynein motor protein Dync2li1. (C) shows the results of overexpressing Ick in Dync2li1 knockdown cells. (A) NIH3T3 cells were co-transfected with a plasmid encoding Control-shRNA or Dync2li1-shRNA3 and a plasmid encoding FLAG-tagged Ick or FLAG-tagged EGFP, and immunostained with DAPI, anti-FLAG antibody, and anti-AcTub antibody. (B) FLAG-positive cells were stained with anti-AcTub antibody for measuring the length of cilia. (A) FLAG-positive cells were treated with DMSO or an FGFR inhibitor (BGJ398) for 24 hours, and immunostained with anti-FLAG antibody and anti-AcTub antibody for measuring the length of cilia. (B) FLAG-positive cells were stained with anti-AcTub antibody for measuring the length of cilia. (B) FLAG-positive cells were stained with anti-AcTub antibody for measuring the length of FGFR activity. (C) FLAG-positive cells were stained with anti-AcTub antibody for measuring the length of cilia ... FGFR activity. (D) FLAG-positive cells were stained with anti-AcTub antibody for measuring the length of FGFR activity. (A) Cells were treated with DMSO or an FGFR inhibitor (AZD4547) for 24 hours, followed by immunostaining with anti-FLAG and anti-AcTub antibodies. (B) The length of cilia stained with anti-AcTub antibodies was measured in FLAG-positive cells. (C) The figure shows the results of overexpressing a dominant-negative FGFR1 (Fgfr1-A645V) in Dync2li1 knockdown cells. (A) The figure shows the results of immunostaining with anti-FLAG and anti-AcTub antibodies.(B) shows the results of measuring the length of cilia stained with anti-AcTub antibody in FLAG-positive cells. (A) shows the results of analyzing the effects of Ick and Mak overexpression on Hedgehog signaling abnormalities. (B) shows a schematic diagram of a construct expressing NanoLuc luciferase under the control of 8xGli1 binding sites and a minimal promoter. (B) shows the results of measuring luciferase activity in NIH3T3 cells with Dync2li1 knockdown and Ick overexpression. (C) shows the results of measuring luciferase activity in NIH3T3 cells with Dync2li1 knockdown and Mak iso1 or Mak iso2 overexpression. (C) shows the results of observing the retina in retina-specific Ccrk-deficient (Ccrk CKO) mice. (A) shows representative toluidine blue-stained images of control and Ccrk CKO mouse retinas at 14 days (P14) and 1 month (1M) of age. (B) shows the results of measuring the thickness of the outer nuclear layer (ONL). Retinal sections prepared from 14-day-old (P14) wild-type and Ccrk CKO mice were immunostained with DAPI and anti-IFT88 and anti-AcTub antibodies (top), anti-Mak and anti-AcTub antibodies (middle), or anti-IFT88 and anti-Cep164 antibodies (bottom). This figure shows the results of Western blot analysis of Mak phosphorylation levels in Ccrk CKO mouse retinas. This figure shows the results of overexpressing Ccrk in Dync2li1 knockdown cells. (A) shows the results of immunostaining with DAPI, anti-FLAG, and anti-AcTub antibodies after cotransfecting NIH3T3 cells with plasmids encoding control-shRNA or Dync2li1-shRNA3 and plasmids expressing FLAG-tagged Ccrk or FLAG-tagged EGFP. (B) shows the results of measuring the length of cilia stained with anti-AcTub antibody in FLAG-positive cells. (C) shows the results of measuring luciferase activity in NIH3T3 cells with Dync2li1 knockdown and Ccrk overexpression.

[0011] [Pharmaceutical Composition] The present invention provides a substance that promotes the expression or function of male germ cell-associated kinase (hereinafter referred to as Mak) and / or intestinal cell kinase (hereinafter referred to as Ick), or a pharmaceutical composition for treating ciliopathy containing Mak and / or Ick as active ingredients.

[0012] Ciliopathy is a group of diseases in humans characterized by abnormalities in development and sensory organs, and is known to be caused by a malfunction of cilia development or function, i.e., ciliopathy. Therefore, in the present invention, ciliopathy is not particularly limited as long as it is a disease or symptom caused by ciliopathy. Examples of ciliary diseases include retinal degeneration, choroidal coloboma, retinitis pigmentosa, cone-rod dystrophy, cone dystrophy, macular dystrophy, corneal abnormalities, visual impairment, Leber congenital amaurosis, Leber amaurosis, Stargardt disease (juvenile macular degeneration), hyperopia, keratoconus, photophobia, cataract, ptosis, nystagmus, microphthalmia, ocular hypertelorism, cyclopia, hydrocephalus, macrocephaly, agenesis of the corpus callosum, holoprosencephaly, cerebellar vermis defects, cerebellar malformations and dysfunction, occipital meningocele, and desmoplastic medulloblastoma. medulloblastoma), hypothalamic hamartoma, lower brainstem dysplasia, pituitary gland abnormalities, hypopituitarism, tumors and / or cysts in the central nervous system, brain abnormalities, seizures, psychomotor developmental delay, neuropsychiatric abnormalities, intellectual disability, learning disabilities, behavioral disorders, motor developmental delay, ataxia, feeding disorders, craniectodermal dysplasia, craniosynostosis, facial and digital (toe) anomalies, Weyers acrofacial dysostosis (WAD), polydactyly, brachymelia, syndactyly, missing fingers, skeletal and ectodermal abnormalities defects), bone development disorders, pelvic dysplasia, spinal column abnormalities, dental abnormalities, bronchiectasis, airway abnormalities, lung hypoplasia, pulmonary dysplasia, disorders in lung development and expansion, breathing disorders, Jeune-asphyxiating thoracic dystrophy, esophageal atresia, bifid epiglottis, cleft uvula, oral abnormalities, taste disorders, proboscis, chronic sinusitis, smell disorders, choanal atresiaatresia), tumors and / or cysts in the endolymphatic sac, hearing loss, hearing impairment, liver fibrosis, liver cysts, polycystic kidney disease, polycystic kidney disease, chronic tubulointerstitial nephritis, tumors and / or cysts in the kidney, nephronophthisis, tumors and / or cysts in the adrenal gland, tumors and / or cysts in the pancreas, endocrine system abnormalities, short stature, situs inversus, smooth muscle hamartomas, skin and intestinal abnormalities, Hirschsprung's disease, apple-peel small intestinal atresia, imperforate anus, absence or dysplasia of the nails, chest stenosis, congenital heart disease, cardiomyopathy, hypertension, obesity, diabetes mellitus, dyslipidemia, morbid obesity and spermatogenic failure (MOSPGF), hypogonadism, genital anomalies, genitourinary malformations, infertility, male infertility, decreased sperm count, azoospermia, female infertility, neonatal lethality, fetal hydrops, developmental delay, acrocallosal syndrome, pituitary stalk interruption syndrome (PSIS), orofacial-digital-limb syndrome-related disorders, syndromic craniosynostosis, Greig cephalopolysyndactyly syndrome, endocrine-cerebro-osteodysplasia, COACH syndrome (Cerebellar-vermis defect, oligophrenia, ataxia, coloboma, hepatic fibrosis) syndrome), Joubert syndrome-related disorders, Senior-Loken syndrome, Alstrom syndrome, Carpenter syndrome, Curry-Jones syndrome, Ellis-Van Creveld (EVC) syndrome, Hydrolethalus syndrome, Kallmann syndrome, McKusick-Kaufman syndrome, MORM syndrome (Mental retardation, truncalThese include obesity, retinal dystrophy, and micropenis syndrome, Oculocerebrorenal syndrome of Lowe (OCRL), Pallister-Hall syndrome, RHYNS syndrome (Retinitis pigmentosa, Hypopituitarism, Nephronophthisis, and Skeletal dysplasia syndrome), Stromme syndrome, STAR syndrome (Syndactyly-telecanthus-anogenital and renal malformations syndrome), Usher syndrome (USH syndrome), Von Hippel-Lindau (VHL) disease, Bardet-Biedl syndrome-related disorders, and Meckel syndrome-related disorders. Some ciliopathies are designated as intractable diseases in Japan.

[0013] The ciliopathy to be treated by the pharmaceutical composition of the present invention may be a retinal degenerative disease. Retinal degenerative diseases include retinitis pigmentosa, cone-rod dystrophy, cone dystrophy, macular dystrophy, Leber's congenital amaurosis, Leber's amaurosis, and Stargardt's disease. Retinitis pigmentosa is preferred.

[0014] In the present invention, the substance that promotes the expression of Mak and / or Ick may be a substance that promotes the expression of the Mak gene and / or Ick gene, or may be a substance that promotes the expression of the Mak protein and / or Ick protein. In the present invention, the substance that promotes the expression of the Mak gene and / or Ick gene includes a nucleic acid or polynucleotide encoding Mak and / or Ick. Specific examples of polynucleotides encoding Mak and / or Ick include, but are not limited to, DNAs containing all or part of the DNA consisting essentially of the nucleotide sequence of the entire coding region of human MAK (GenBank Accession No. AF505623, SEQ ID NO: 1), the nucleotide sequence of the entire coding region of mouse Mak (GenBank Accession No. X66983, SEQ ID NO: 2), the nucleotide sequence of the entire coding region of human ICK (GenBank Accession No. AF225919, SEQ ID NO: 3), and the nucleotide sequence of the entire coding region of mouse Ick (GenBank Accession No. AF225918, SEQ ID NO: 4).

[0015] In the present invention, a substance that promotes the function of Mak and / or Ick may be a substance that promotes the phosphorylation of Mak and / or Ick, or a substance that promotes the phosphorylation of a downstream factor of Mak and / or Ick. In the present invention, a substance that promotes the phosphorylation of a downstream factor of Mak and / or Ick may be a substance that inhibits the dephosphorylation of a downstream factor of Mak and / or Ick. A representative downstream factor of Mak and / or Ick is kinesin family 3A protein (hereinafter referred to as Kif3a). In the present invention, a substance that promotes the phosphorylation of Mak and / or Ick may be an upstream regulatory factor of Mak and / or Ick, an activator of an upstream regulatory factor of Mak and / or Ick, or a substance that inhibits the dephosphorylation of Mak and / or Ick. An activator of an upstream regulatory factor of Mak and / or Ick may be a substance that promotes the expression or function of an upstream regulatory factor of Mak and / or Ick. In the present invention, a substance that promotes the expression or function of an upstream regulatory factor of Mak and / or Ick may be the upstream regulatory factor of Mak and / or Ick itself. An example of an upstream regulatory factor of Mak and / or Ick is cell cycle-related kinase (hereinafter referred to as Ccrk). In the present invention, a substance that promotes the expression of an upstream regulatory factor of Mak and / or Ick includes a nucleic acid or polynucleotide encoding Ccrk. Specific examples of polynucleotides encoding Ccrk include, but are not limited to, DNAs containing all or part of DNA consisting essentially of the nucleotide sequence of the entire coding region of human CCRK (GenBank Accession No.: AF035013, SEQ ID NO: 5) and the nucleotide sequence of the entire coding region of mouse Ccrk (GenBank Accession No.: AY005133, SEQ ID NO: 6).

[0016] We found that phosphorylation of Mak at threonine 157 was reduced in the retina of retina-specific Ccrk conditional knockout (CKO) mice, and identified Ccrk as a major upstream regulator of Mak and Ick.

[0017] In the present invention, "DNA substantially consisting of" refers to DNA consisting of the above-mentioned specific base sequence, as well as DNA consisting of a base sequence that can hybridize with the above-mentioned DNA consisting of the specific base sequence under highly stringent conditions. Stringent conditions can be calculated by applying appropriate formulas used in the art based on the desired homology, the length of the oligonucleotide, etc. Furthermore, techniques for substituting or deleting any base in the above-mentioned base sequence (e.g., in vitro mutagenesis, site-directed mutagenesis, etc.) may also be used.

[0018] An expression vector functionally containing a polynucleotide encoding Mak and / or Ick can be obtained by inserting the polynucleotide encoding Mak and / or Ick into a plasmid vector, phage vector, or the like, capable of replicating or autonomously replicating in various prokaryotic and / or eukaryotic hosts using appropriate restriction enzyme sites. "Functionally" means that the gene (DNA) is positioned so that it can be transcribed in a host cell compatible with the vector and the encoded protein can be produced. Preferably, the vector has an expression cassette in which a promoter region, an initiation codon, a polynucleotide encoding Mak and / or Ick, a stop codon, and a terminator region are consecutively arranged. The vector may further contain a selection marker gene for selecting transformants. The same procedure can be carried out for an expression vector functionally containing a polynucleotide encoding Ccrk.

[0019] In the present invention, the expression vector is not particularly limited and may be either a non-viral vector or a viral vector. When a non-viral vector is used, known techniques can be used, such as methods for introducing nucleic acid molecules using liposomes (e.g., liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method), microinjection, or methods for transferring nucleic acid molecules into cells together with carriers (metal particles) using a gene gun. When a viral vector is used, viral vectors such as recombinant adenoviruses and retroviruses can be used. Genes can be introduced into cells or tissues by introducing a polynucleotide encoding Mak and / or Ick or a polynucleotide encoding Ccrk into a detoxified DNA or RNA virus such as retrovirus, adenovirus, adeno-associated virus (AAV), herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, or SV40, and then infecting the cells or tissues with the recombinant virus.

[0020] In the present invention, the presence or absence and degree of increase in the transcription level of the Mak gene and / or the Ick gene, or the Ccrk gene can be confirmed by known techniques such as quantitative RT-PCR. Those skilled in the art can appropriately design and prepare primer sequences to be used for quantitative RT-PCR or the like based on gene sequence information obtained from known databases such as GenBank.

[0021] In the present invention, Mak and / or Ick may be Mak and / or Ick proteins, or may be polypeptides or functional fragments of Mak and / or Ick proteins. The amino acid sequences of Mak and / or Ick proteins can be obtained from publicly known databases such as GenBank. For example, the amino acid sequence of human MAK (GenBank Accession No.: AAN16405.1, SEQ ID NO:7), mouse Mak (GenBank Accession No.: CAA47392.1, SEQ ID NO:8), human ICK (GenBank Accession No.: AAF37278.1, SEQ ID NO:9), and mouse Ick (GenBank Accession No.: AAF37277.1, SEQ ID NO:10) have been disclosed. The polypeptides or functional fragments of Mak and / or Ick proteins may have activity similar to or improved from that of wild-type Mak and / or Ick proteins, or may mediate the protein activity of wild-type Mak and / or Ick proteins. In the present invention, Mak and / or Ick proteins or polypeptides may be variants in which one residue is replaced by another with similar properties by conservative amino acid substitutions, or may be part of a larger protein, such as a fusion protein, which may contain additional amino acid sequences, including secretory or leader sequences, prosequences, or other sequences that may aid stability.

[0022] In the present invention, the substance that promotes the expression or function of an upstream regulator of Mak and / or Ick may be the Ccrk protein, or a polypeptide or functional fragment of the Ccrk protein. The amino acid sequence of the Ccrk protein can be obtained from publicly known databases such as GenBank. For example, the amino acid sequence of human CCRK (GenBank accession number: AAC98920.1, SEQ ID NO: 11) and the amino acid sequence of mouse Ccrk (GenBank accession number: AAF89089.1, SEQ ID NO: 12) have been disclosed. The Ccrk protein polypeptide or functional fragment can be used in the same manner as the Mak and / or Ick polypeptide or functional fragment.

[0023] In the present invention, the methods for obtaining or preparing Mak and / or Ick or Ccrk proteins are not particularly limited, and may be naturally occurring proteins, chemically synthesized proteins, or recombinant proteins produced by genetic recombination technology. Naturally occurring Mak and / or Ick or Ccrk proteins can be obtained from cells or tissues expressing Mak and / or Ick or Ccrk proteins by appropriately combining known protein isolation and purification methods. Chemical synthesis of proteins and production of recombinant proteins can be performed by techniques known to those skilled in the art.

[0024] In the present invention, the presence or absence and degree of increase in expression of Mak and / or Ick or Ccrk at the protein level can be confirmed by known techniques such as immunohistological staining. The labeled antibody used in immunohistological staining may be a commercially available antibody or an antibody prepared by known techniques.

[0025] In the present invention, a substance that promotes the function of Mak and / or Ick may be a substance that inhibits the activity of fibroblast growth factor receptor (hereinafter referred to as FGFR). To date, four types of human FGFR have been identified: FGFR1, FGFR2, FGFR3, and FGFR4. Therefore, a substance that inhibits FGFR activity may be an FGFR inhibitor that is selective for one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4. In the present invention, a substance that inhibits FGFR activity may be an FGFR inhibitor, an FGFR kinase inhibitor, a dominant-negative form of FGFR, or an antibody specific to FGFR. Examples of known FGFR inhibitors include, but are not limited to, rogaratinib, apultumab, futibatinib, delazantinib, ASP5878 (Astellas), zoligratinib, FP-1039 (GSK / FivePrime), LY-2874455 (Lilly), RG-7444 (Roche), fexagratinib (AZD4547), infigratinib (BGJ398), erdafitinib, PD173074 (Selleck), pemigatinib, compounds described in International Publication WO 2013 / 108809 A1, compounds described in Patent Publication JP 5639261 B2, and any combinations thereof.

[0026] The present inventors have found that administration of an FGFR inhibitor enhances Kif3a phosphorylation in the retina of Mak-deficient mice, and that FGFR inhibition can activate Ick in the Mak-deficient retina (see Example 3). Thus, FGFR inhibitors are useful as substances that promote the function of Mak and / or Ick.

[0027] Since the FGFR inhibitor, which is the active ingredient of the pharmaceutical composition of the present invention, is already in clinical use, the pharmaceutical composition of the present invention can be safely administered to humans and other mammals (e.g., rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.).

[0028] When an FGFR inhibitor is used as the active ingredient, the daily dose of the pharmaceutical composition of the present invention can be set according to the daily dose of an FGFR inhibitor in clinical use.

[0029] The pharmaceutical preparation of the present invention can be formulated by appropriately blending the above-mentioned active ingredient with a pharmaceutically acceptable carrier and further additives. Specifically, it can be formulated as oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions; or parenteral preparations such as injections, infusions, suppositories, ointments, and patches. The blending ratio of the carrier or additive may be appropriately determined based on the range commonly used in the pharmaceutical field. The carrier or additive that can be blended is not particularly limited, and examples thereof include various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0030] Examples of excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose, and light anhydrous silicic acid. Examples of binders include polymeric compounds such as crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Examples of lubricants include magnesium stearate, calcium stearate, talc, and colloidal silica. Examples of disintegrants include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, and carboxymethylstarch sodium. Examples of wetting agents include, but are not limited to, glycerin, butylene glycol, propylene glycol, sorbitol, and triacetin. If necessary, the tablet may be coated with a coating agent (sucrose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose phthalate, etc.) or may be coated with two or more layers.

[0031] In the case of injections, the active ingredient may be dissolved or dispersed in an aqueous base such as physiological saline or an oily base acceptable for injection to prepare an injection for intravenous administration, intramuscular administration, subcutaneous administration, etc. Additives such as buffers, pH adjusters, isotonicity agents, solubilizers, suspending agents, stabilizers, etc. may be added as needed.

[0032] In the case of injections, examples of aqueous bases include infusion solutions such as physiological saline, water for injection, and Ringer's solution. Examples of oily bases include propylene glycol, polyethylene glycol, sesame oil, soybean oil, corn oil, peanut oil, cottonseed oil, olive oil, and propylene glycol fatty acid esters. Examples of buffers include phosphates, acetates, carbonates, citrates, borates, glutamate, epsilon aminocaproate, and buffer solutions such as Tris buffer. Examples of pH adjusters include inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, and carbonic acid; organic acids such as acetic acid, tartaric acid, lactic acid, citric acid, and succinic acid; inorganic bases such as sodium hydroxide; and organic bases such as sodium citrate and sodium tartrate. Examples of isotonic agents include inorganic salts such as sodium chloride, sugar alcohols such as D-mannitol, sorbitol, and xylitol, sugars such as fructose, glucose, galactose, ribose, xylose, mannose, maltotriose, and maltotetraose, and amino acids such as glycine and arginine. Examples of solubilizing agents include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, lecithin, and nonionic surfactants such as polysorbate 80. Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, and glycerin monostearate, as well as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, and hydroxymethylcellulose. Examples of stabilizers include albumin, globulin, gelatin, sorbitol, ethylene glycol, propylene glycol, and ascorbic acid.

[0033] [Composition for Improving Ciliary Disorders] The present invention provides a composition for improving ciliary disorders, which contains a substance that promotes the expression or function of Mak and / or Ick, or Mak and / or Ick as an active ingredient. The present inventors have confirmed that forced expression of Mak or Ick can improve ciliary abnormalities caused by Ick deficiency (see Example 1), and that forced expression of Ick or administration of an FGFR inhibitor can improve retinal degeneration caused by Mak deficiency (see Examples 2 and 3). The present inventors have also confirmed that forced expression of Ick, Mak, or Ccrk can improve ciliary disorders caused by cytoplasmic dynein inhibition (see Examples 4, 5, and 8). Furthermore, the present inventors have confirmed that inhibition of FGFR activity can improve ciliary disorders caused by cytoplasmic dynein inhibition (see Examples 6 and 7). Therefore, the composition for improving ciliary disorders of the present invention is effective in improving or treating ciliary disorders.

[0034] The composition for improving ciliary disorders of the present invention can contain as an active ingredient the substances described in the above-mentioned embodiments of the pharmaceutical composition. Furthermore, as described in the above-mentioned embodiments of the pharmaceutical composition, the composition for improving ciliary disorders of the present invention can be formulated into various dosage forms and can be safely administered to humans and other mammals (e.g., rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.).

[0035] [Screening Method] The present invention provides a method for screening for a substance that improves cilia dysfunction. The screening method of the present invention may be a method for identifying a substance that activates Mak ex vivo or in vitro, a method for identifying a substance that activates Ick ex vivo or in vitro, or a method for identifying a substance that activates Ccrk ex vivo or in vitro. A substance that activates Mak or Ick may be identified by a method for identifying a substance that enhances Kif3a phosphorylation. A substance that activates Ccrk may be identified by a method for identifying a substance that enhances Mak and / or Ick phosphorylation.

[0036] The test substance to be subjected to the screening method of the present invention is not particularly limited, and may be a nucleic acid, a peptide, a protein, a non-peptide compound, a synthetic compound, a fermentation product, a cell extract, a cell culture supernatant, a plant extract, a mammalian tissue extract, plasma, or the like. The test substance may be a novel substance or a known substance. Furthermore, these test substances may form salts. As the salt of the test substance, a salt with a physiologically acceptable acid or base is preferred.

[0037] A first embodiment of the screening method of the present invention may include the following steps (1) to (3): (1) contacting Mak and Kif3a with a test substance, (2) measuring the phosphorylation of Kif3a, and (3) identifying a substance that increases the phosphorylation level of Kif3a compared to the phosphorylation level of Kif3a in the absence of contact with the test substance.

[0038] Step (1) may be a method of preparing a solution containing purified Mak and purified Kif3a and adding a test substance to the solution, or a method of culturing cells expressing Mak and Kif3a and adding a test substance to the culture medium.

[0039] The Mak and Kif3a used in step (1) may be derived from any organism, including mammals. Examples of mammals include humans, chimpanzees, monkeys, dogs, cattle, mice, rats, and guinea pigs, with humans being preferred. Cells expressing Mak and Kif3a can be prepared by introducing a Mak-encoding gene and a Kif3a-encoding gene into suitable host cells. The prepared Mak- and Kif3a-expressing cells are cultured, and purified Mak and Kif3a can be obtained from the culture supernatant or cell extract using known methods (e.g., affinity columns).

[0040] Information on the amino acid sequences of Mak and Kif3a and the nucleotide sequences of the genes encoding them can be obtained from publicly known databases (e.g., DDBJ, GenBank, EMBL, etc.). For example, the nucleotide sequence (GenBank: AF505623, SEQ ID NO: 1) and amino acid sequence (GenBank: AAN16405.1, SEQ ID NO: 7) of the entire coding region of human MAK, the nucleotide sequence (GenBank: X66983, SEQ ID NO: 2) and amino acid sequence (GenBank: CAA47392.1, SEQ ID NO: 8) of the entire coding region of mouse Mak, the nucleotide sequence (GenBank: AF041853, SEQ ID NO: 13) and amino acid sequence (GenBank: AAC72294.1, SEQ ID NO: 14) of the entire coding region of human KIF3A, and the nucleotide sequence (GenBank: D12645, SEQ ID NO: 15) and amino acid sequence (GenBank: BAA02166.1, SEQ ID NO: 16) of the entire coding region of mouse Kif3a have been disclosed.

[0041] The Mak- and Kif3a-expressing cells used in step (1) may be cells expressing endogenous Mak and endogenous Kif3a, cells expressing recombinant Mak and recombinant Kif3a derived from introduced genes, or cells expressing Mak and Kif3a through a combination of endogenous and recombinant proteins. Cells expressing recombinant Mak and recombinant Kif3a are preferred. Examples of cells expressing endogenous Mak and / or endogenous Kif3a include HEK293T cells, NIH3T3 cells, hTERT-RPE1 cells, and U-251 MG cells. Host cells may be any cells capable of expressing the introduced gene products. Examples include mammalian cells and insect cells. Examples of mammalian cells include HEK293T cells, NIH3T3 cells, Expi293 cells, and hTERT-RPE1 cells. Examples of insect cells include Sf9 cells and Sf21 cells.

[0042] In step (1), when a solution containing purified Mak and purified Kif3a is prepared and a test substance is added to the solution, the amounts of purified Mak and purified Kif3a added to the solution are not particularly limited, but may be approximately a 1:1 molar ratio. The amount of test substance added is not particularly limited as long as the phosphorylation level of Kif3a can be confirmed in the next step. The test substance may be added at multiple concentrations within a range of concentrations.

[0043] The solution used is not particularly limited, and known buffers that can be used in protein phosphorylation reactions can be used. For example, a 50 mM Tris-HCl (pH 7.5) buffer containing 1 mM CaCl, 100 mM NaCl, 1 mg / ml BSA, 0.025% NP-40, and 1 mM DTT can be used. The solution temperature and contact time are not particularly limited, and may be, for example, 37°C and 30 minutes.

[0044] In step (1), when cells expressing Mak and Kif3a are cultured and a test substance is added to the culture medium, the amount of test substance added may be any amount that does not significantly inhibit cell growth or recombinant protein expression. The test substance may be added at multiple concentrations over a range of concentrations. The time period for adding the test substance is not particularly limited and may be from about 0.5 hours to about 48 hours.

[0045] In step (2), the amount of Kif3a phosphorylation may be measured by a known method. For example, electrophoresis, which can separate phosphorylated and non-phosphorylated forms, adding phosphate-labeled ATP to a solution, or using an antibody that recognizes phosphorylation may be used. Specifically, Phos-tag SDS-PAGE (Fujifilm Wako Pure Chemical Industries, Ltd.) 32 P-labeled ATP (PerkinElmer, etc.) can be used.

[0046] In step (3), a substance that increases the phosphorylation level of Kif3a is identified. A method for identifying a substance that increases the phosphorylation level of Kif3a may be performed by comparing the amount of Kif3a phosphorylation in a sample not contacted with the test substance (control group) with the amount of Kif3a phosphorylation in a sample contacted with the test substance (test substance group), and selecting a test substance that increases the amount of phosphorylation. The degree to which a test substance increases the amount of Kif3a phosphorylation is not particularly limited. For example, a test substance that increases the amount of Kif3a phosphorylation by 120% or more, 130% or more, 140% or more, 150% or more, 170% or more, 180% or more, 190% or more, or 200% or more compared to a sample not contacted with the test substance may be selected, or a test substance that shows a significant difference detected by statistical analysis may be selected.

[0047] A second embodiment of the screening method of the present invention may include the following steps (1) to (3): (1): contacting Ick and Kif3a with a test substance; (2): measuring the phosphorylation of Kif3a; and (3): identifying a substance that increases the phosphorylation level of Kif3a compared to the phosphorylation level of Kif3a in the absence of contact with the test substance. Step (1) can be performed in the same manner as step (1) of the first embodiment, except that Ick is substituted for Mak. Steps (2) and (3) can be performed in the same manner as steps (2) and (3) of the first embodiment, respectively.

[0048] The Ick and Kif3a used in the screening methods of the present invention may be derived from any organism, including mammals. Examples of mammals include humans, chimpanzees, monkeys, dogs, cattle, mice, rats, and guinea pigs, with humans being preferred. The amino acid sequence information for Ick and Kif3 and the nucleotide sequence information for the genes encoding them can be obtained from publicly available databases (e.g., DDBJ / GenBank / EMBL). For example, the nucleotide sequence (GenBank accession number: AF225919, SEQ ID NO: 3) and amino acid sequence (GenBank accession number: AAF37278.1, SEQ ID NO: 9) of the entire coding region of human Ick, and the nucleotide sequence (GenBank accession number: AF225918, SEQ ID NO: 4) and amino acid sequence (GenBank accession number: AAF37277.1, SEQ ID NO: 10) of the entire coding region of mouse Ick have been disclosed.

[0049] A third embodiment of the screening method of the present invention may include the following steps (1) to (3): (1): contacting Ccrk, Mak, and / or Ick with a test substance; (2): measuring the phosphorylation of Mak and / or Ick; and (3): identifying a substance that increases the phosphorylation level of Mak and / or Ick compared to the phosphorylation level of Mak and / or Ick in the absence of contact with the test substance. Step (1) can be performed in the same manner as step (1) of the first embodiment, except that Mak and Kif3a are replaced with Ccrk and Mak and / or Ick. Steps (2) and (3) can be performed in the same manner as steps (2) and (3) of the first embodiment, except that kif3a is replaced with Mak and / or Ick in steps (2) and (3) of the first embodiment.

[0050] The Ccrk, Mak, and Ick used in the screening method of the present invention may be derived from any organism, including mammals. Examples of mammals include humans, chimpanzees, monkeys, dogs, cattle, mice, rats, and guinea pigs, with humans being preferred. Amino acid sequence information for Ccrk, Mak, and Ick and the nucleotide sequence information for the genes encoding them can be obtained from publicly available databases (e.g., DDBJ / GenBank / EMBL). For example, the nucleotide sequence (GenBank accession number: AF035013, SEQ ID NO: 5) and amino acid sequence (GenBank accession number: AAC98920.1, SEQ ID NO: 11) of the entire coding region of human CCRK, and the nucleotide sequence (GenBank accession number: AY005133, SEQ ID NO: 6) and amino acid sequence (GenBank accession number: AAF89089.1, SEQ ID NO: 12) of the entire coding region of mouse Ccrk have been disclosed.

[0051] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0052] [Experimental Methods] 1. Animal Experiments All procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, approved by the Osaka University Genetic Modification Experiment Safety Committee (Approval ID 04913) and the Protein Research Institute Animal Experimentation Committee (Approval ID R04-02-0), and in accordance with the facility guidelines. Mice were housed in a temperature-controlled room at 22°C with a 12-hour light / dark cycle, with fresh water and rodent chow available at all times. All animal experiments were performed on either male or female mice.

[0053] 2. Laboratory Animals Mak - / - Mice were provided by Dr. Yoichi Shinkai (RIKEN) (Shinkai Y et al., Mol Cell Biol, 2002, 22(10):3276-80). - / -Mice were generated as previously described (Chaya T et al., EMBO J (2014) 33:1227-1242). Mak Ick DKO mice were generated using the same Mak Ick DKO mice. - / - Ccrk mice were generated by crossing Ick floxed mice (generated as described in Chaya T et al., EMBO J, 2014, 33:1227-1242) with Dkk3-Cre mice (generated as described in Sato S et al., Genesis, 2007, 45(8):502-507). Ccrk CKO mice were generated by crossing Ccrk floxed mice with the Dkk3-Cre mice. Ccrk floxed mice were generated as follows: an 11.5 kb Ccrk genomic fragment was subcloned by PCR using C57BL / 6 genomic DNA. One loxP site was inserted into intron 2 and another loxP site was inserted into intron 4. The targeting vector was then cloned into a modified pBluescript II KS(+) vector (Agilent). The linearized targeting vector was transfected into the JM8A3 embryonic stem (ES) cell line (Pettitt, SJ et al. Nat Methods, 2009, 6:493-495). JM8A3 cell culture, electroporation, and selection were performed as previously described (Muranishi, Y. et al. J Neurosci, 2011, 31:16792-16807). Heterozygous ES cells carrying the targeted gene disruption were microinjected into C57BL / 6 blastocysts to generate chimeric mice. These chimeric mice were then mated with C57BL / 6 mice to generate offspring. Subsequently, the mice were mated with B6-Tg(CAG-FLPe)37 mice (#RBRC01835, kindly provided by the RIKEN BioResource Research Center) and the flippase recognition target (FRT)-flanking neo cassette was removed using flippase (Flp) recombinase.

[0054] 3. Plasmid Constructs Plasmids expressing EGFP, FLAG- or HA-tagged mouse Mak and mouse Ick, and FLAG-tagged EGFP were constructed as previously described (Chaya T et al., EMBO J, 2014, 33:1227-1242; Omori Y et al., Proc Natl Acad Sci USA, 2010, 107:22671-22676; Tsutsumi R et al., J Biol Chem, 2022, 298:101686). The full-length cDNA fragment of mouse Dync2li1 was amplified by PCR using mouse retina cDNA as a template and subcloned into the pCAGGSII-3×FLAG vector (Irie S et al., Mol Cell Biol, 2015, 35:2583-2596; Sanuki R et al., Cell Rep, 2015, 10:796-808). The full-length cDNA fragment of mouse Ccrk was amplified by PCR using mouse testis cDNA as a template and subcloned into the pCAGGSII vector (Omori Y et al., Proc Natl Acad Sci USA, 2010, 107:22671-22676). The full-length cDNA fragment of mouse Fgfr1 was amplified by PCR using mouse brain cDNA as a template. The A645V mutation in mouse Fgfr1 was introduced by PCR using standard methods. Mouse Fgfr1 A645V was subcloned into the pCAGGSII-c-2xHA vector (Ueno A et al., Cell Reports, 2018, 22:3548-3561). For shRNA-mediated Dync2li1 knockdown, Dync2li1-shRNA and Control-shRNA cassettes (Itoh Y et al., Nat Neurosci, 2013, 16:416-425) were subcloned into the pBAsi-mU6 vector (Takara Bio). The target sequences of each shRNA are as follows:Dync2li1 shRNA2: 5'-GCTTTGTGGCACATTACTACG-3' (sequence number 17), Dync2li1 shRNA3: 5'-GCAGGACTGGATTCTTTATGT-3' (sequence number 18), Dync2li1 shRNA5: 5'-GGGAATTAATTGACCCATTTC-3' (sequence number 19), Dync2li1 shRNA6: 5'-GCAAGTCAGAAGCTGTTAC-3' (sequence number 20), Control shRNA: 5'-GACGTCTAACGGATTCGAGCT-3' (sequence number 21). AAV rhodopsin kinase promoter-3×FLAG-Ick was generated by PCR amplification of the full-length mouse Ick cDNA fragment using pCAGGSII-2×HA-mouse Ick (Chaya T et al., EMBO J, 2014, 33:1227-1242) as a template. The full-length cDNA fragment was then subcloned into the pBluescript II KS (+)-3×FLAG vector (modified from the pBluescript II KS (+) vector (Agilent)) to generate pBluescript II KS (+)-3×FLAG-Ick. The excised 3×FLAG-Ick fragment was then ligated into the pAAV-RK-IZsGreen vector (Khani SC et al., Invest Ophthalmol Vis Sci, 2007, 48:3954-3961).

[0055] 4. Cell culture and transfection. HEK293T cells and NIH3T3 cells were cultured at 37°C in a 5% CO2 atmosphere using Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 10% fetal bovine serum or bovine serum, respectively, supplemented with penicillin (100 μg / ml) and streptomycin (100 μg / ml). - / - MEF cells were cultured at embryonic day 13.5 in Ick cells. - / -The cells were cultured at 37°C in a 5% CO2 atmosphere in DMEM containing 10% fetal bovine serum, penicillin (100 μg / ml), and streptomycin (100 μg / ml). Transfection was performed using the calcium phosphate method (HEK293T cells) or the lipofection method using Lipofectamine LTX (Invitrogen) or Lipofectamine 3000 (Invitrogen) (NIH3T3 cells and Ick). - / - Transfection was performed using NIH3T3 cells (MEF cells). To induce ciliogenesis in transfected cells, the medium was replaced with serum-free medium 24 hours after transfection and the cells were cultured in serum-free medium for 24 hours. NIH3T3 cells were incubated for 24 hours in serum-free medium supplemented with 100 nM BGJ398 (ChemScene), 100 nM AZD4547 (MedChemExpress), 100 nM SAG (Smoothened agonist, Calbiochem), or 10 μM Ciliobrevin D (Millipore).

[0056] 5. Cell Fluorescence Immunostaining and Analysis Cells cultured as described above in 4 were washed with PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 5 minutes at room temperature. The fixed cells were then incubated in blocking buffer (PBS containing 5% normal donkey serum and 0.1% or 0.5% Triton X-100) for 30 minutes at room temperature, followed by immunostaining overnight at 4°C in a diluted primary antibody in blocking buffer. The cells were washed with PBS and incubated in blocking buffer containing secondary antibody and DAPI (1:1000, Nacalai Tesque) for 2 hours at room temperature. The cells were then washed three times with PBS and mounted in polyvinyl alcohol (Gelbatol). The primary antibodies used were mouse anti-acetylated α-tubulin (AcTub) antibody (1:1000 or 1:2000 dilution, Sigma, clone 6-11B-1), rabbit anti-FLAG antibody (1:1000 dilution, Sigma, product number F7425), mouse anti-FLAG M2 antibody (1:1000 dilution, Sigma, product number F1804), and rabbit anti-rhodopsin antibody (1:2500 dilution, LSL, product number LB-5597). Secondary antibodies were Cy3-conjugated (1:500 dilution, Jackson ImmunoResearch Laboratories) or Alexa Fluor 488-conjugated (1:500 dilution, Sigma). The specimens were observed under a confocal laser scanning microscope (LSM700, LSM710, or LSM900, Carl Zeiss). Cilia length was measured using ZEN imaging software (ZEN blue edition, Carl Zeiss). Immunofluorescence signal intensity was measured and quantified using NIH ImageJ software. The rhodopsin signal inside the photoreceptor cells was normalized to the total rhodopsin signal (outer segment and inner photoreceptor cells).

[0057] 6. Fluorescent Immunostaining and Analysis of Retinal Sections. Mouse eyes or eyecups were fixed in 4% PFA in PBS for 15 seconds to 30 minutes at room temperature. Fixed eyeball samples were washed with PBS, embedded in Tissue-Tek OCT compound (Sakura Finetech Japan), frozen, and sectioned. Fixed eyecup samples were washed with PBS, incubated overnight at 4°C in 30% sucrose in PBS, embedded in Tissue-Tek OCT compound, frozen, and sectioned. Sections were 14 μm or 20 μm thick. The frozen sections were dried on slides overnight at room temperature and then rehydrated in PBS for 5 minutes. After incubation in blocking buffer (PBS containing 5% normal donkey serum and 0.1% or 0.5% Triton X-100) for 30 minutes, the samples were immunostained overnight at 4°C with diluted primary antibodies in blocking buffer. Slides were washed three times with PBS for 5 minutes each and then incubated in blocking buffer containing fluorescent dye-conjugated secondary antibodies and DAPI for 2 hours at room temperature, protected from light. Sections were washed three times with PBS and mounted with Gelvatol. The primary and secondary antibodies used were the antibodies listed in Section 5 above, as well as the following: rabbit anti-IFT88 antibody (1:500 dilution, Proteintech, 13967-1-AP), guinea pig anti-Mak antibody (1:1000 dilution, Omori Y et al., PNAS, 2010, 107(52):22671-22676), and mouse anti-Cep164 antibody (clone E-9, 1:200 dilution, Santa Cruz, sc-515403). Microscopic observation and analysis were performed as described in Section 5 above.

[0058] 7. Analysis of Retinal Thickness. Retinal sections were washed with PBS and stained for 1 minute in PBS containing 0.1% toluidine blue. After washing the sections with PBS, the slides were covered with a coverslip and immediately observed under a microscope. Retinal thickness was measured and quantified using NIH ImageJ software.

[0059] 8. Electroretinogram (ERG) Measurements After overnight dark adaptation, mice were anesthetized by intraperitoneal injection of 100 mg / kg ketamine and 10 mg / kg xylazine diluted in saline (Otsuka Pharmaceuticals). 0.5% tropicamide and 0.5% phenylephrine hydrochloride were administered topically to dilate the pupils. ERG responses were measured using a PuREC system with LED electrodes (Mayo Corporation). Mice were placed on a heating pad and stimulated with an LED flash. ERG responses ranged from -4.0 to 1.0 log cd sm. -2 After 10 min of light adaptation, mice were exposed to a 1.5 log cd sm stimulus. -2 Photopic ERGs were recorded against a rod-suppressed white background. Photopic ERGs were recorded in the range of −0.5 to 1.0 log cd sm. -2 Four levels of stimulation intensity were used. For dark-adapted ERG recording, the intensity was −4.0 log cd sm -2 8 responses and −3.0 log cd sm -2 For light-adapted ERG recordings, 16 responses were averaged.

[0060] 9. Western blot analysis. HEK293T cells were washed twice with Tris-buffered saline (TBS) and lysed in lysis buffer supplemented with protease inhibitors (Buffer A: 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM PMSF, 2 μg / ml leupeptin, 5 μg / ml aprotinin, 3 μg / ml pepstatin A) or SDS sample buffer. Mouse retinas were lysed in Buffer A or Buffer B (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, supplemented with phosphatase inhibitor cocktail (Roche)). Samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore) using a semi-dry transfer cell (Bio-Rad) or the iBlot system (Invitrogen). The membrane was blocked with blocking buffer (3% skim milk in TBS containing 0.05% Tween 20) and incubated with primary antibodies overnight at 4°C. The PVDF membrane was washed three times for 10 minutes with TBS containing 0.05% Tween 20 and then incubated with secondary antibodies for 2 hours at room temperature. Signals were detected using Chemi-Lumi One L (Nacalai Tesque) or Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific). Phosphorylated Kif3a was detected using 6% SDS-PAGE containing 20 μM Phos-tag acrylamide (Fujifilm Wako Pure Chemical Industries) according to the manufacturer's instructions. Band intensity was quantified using NIH ImageJ software. The primary antibodies used were rabbit anti-Kif3a (1:1500 dilution, Abcam, product number ab11259), mouse anti-FLAG M2 (1:5000 or 1:10000 dilution, Sigma, product number F1804), rabbit anti-GFP (1:2500 dilution, MBL, product number 598), guinea pig anti-Mak (1:150 dilution, Omori Y et al., PNAS, 2010, 107(52):22671-22676), and rabbit anti-phosphorylated Mak (pMak) (Thr1579) (1:1000 dilution, Invitrogen, PA5-105526).The secondary antibodies used were: horseradish peroxidase-labeled anti-mouse IgG (1:10,000 dilution, Zymed), and horseradish peroxidase-labeled anti-rabbit IgG (1:10,000 dilution, Jackson Laboratory).

[0061] 10. Adeno-associated virus (AAV) production and subretinal injection. AAV-293 cells were transfected with the AAV vector plasmid, adenovirus helper plasmid, and AAV helper plasmid (pAAV2-8 SEED) using the calcium phosphate method to produce AAV. 72 hours after transfection, cells were harvested and lysed by four freeze-thaw cycles. The supernatant was collected by centrifugation and treated with Benzonase nuclease (Novagen). Virus was purified using an iodixanol gradient. Iodixanol gradients were formed in ultraclear centrifuge tubes (14 x 95 mm, Beckman) by layering 54% iodixanol (Axis-Shield) in PBS-MK buffer (1x PBS, 1 mM MgCl2, 25 mM KCl) with 40% iodixanol, 25% iodixanol in PBS-MK buffer with phenol red, and 15% iodixanol in PBS-MK buffer with 1 M NaCl in PBS-MK buffer. The tubes were centrifuged at 40,000 rpm in an SW40Ti rotor. The virus-containing fraction between 54% and 40% was collected with an 18-gauge needle and concentrated using an Amicon Ultra Centrifugal Filter Ultracel-100K (Millipore). The titer (vector genome (VG) / ml) of each AAV was measured by qPCR using SYBR GreenER Q-PCR Super Mix (Invitrogen) and a Thermal Cycler Dice Real Time System Single MRQ TP700 (Takara Bio) according to the manufacturer's protocol. Quantification was performed using the Thermal Cycler Dice Real Time System software (Takara Bio). The titer of AAV-RK-N-FLAGx3-Ick was approximately 1.8 x 10 12 0.3 μl of the AAV preparation was injected into 1-day-old (P1) Mak - / - Mice were injected subretinal, and the injected retinas were harvested 14 days later (P14).

[0062] 11. Drug Administration: BGJ398 (ChemScene) was dissolved in dimethyl sulfoxide (DMSO) at 2 mg / ml or 4 mg / ml and diluted with 3.5 mM HCl and 5% DMSO. Mice were injected subcutaneously with 2 mg / kg of BGJ398 daily.

[0063] 12. Luciferase Reporter Assay. Reporter gene assays were performed using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. To generate the reporter construct, a minimal promoter (5'-AGACACTAGAGGGTATATAATGGAAGCTCGACTTCCAG-3', SEQ ID NO: 22) and 8xGli1 binding sites were cloned into the pGL3-Basic vector (Promega), replacing firefly luciferase with NanoLuc luciferase. NanoLuc luciferase was obtained by PCR amplification from the pNLF-N[CMV / Hygro] vector (Promega). The pGL3-Control vector (Promega) was used to normalize transfection efficiency. NIH3T3 cells were transfected with plasmids expressing Dync2li1-shRNA2, Dync2li1-shRNA3, or Dync2li1-shRNA6, and plasmids expressing Ick, Mak iso1, Mak iso2, or Ccrk, along with reporter constructs. 24 hours after transfection, cells were incubated in serum-free medium containing 100 nM SAG for 24 hours and then washed with PBS. Luminescence signals were detected using the GloMax Multi+ Detection System (Promega).

[0064] 13. Statistical Analysis Data were expressed as mean ± SD. Statistical analysis was performed using unpaired t-test, one-way analysis of variance, or two-way analysis of variance. A p value of less than 0.05 was considered statistically significant.

[0065] Example 1: Effect of Mak overexpression in Ick-deficient cells Ick - / -We analyzed the effect of Mak overexpression on ciliary abnormalities in mouse embryonic fibroblasts (MEFs). Plasmids encoding FLAG-tagged EGFP (control), FLAG-tagged Ick, FLAG-tagged Mak isoform 1 (Mak iso1), or FLAG-tagged Mak isoform 2 (Mak iso2) were transfected with Ick. - / - MEFs were transfected and the number and length of cilia were compared. The results are shown in Figure 1. (A) shows the results of immunostaining with anti-FLAG and anti-acetylated α-tubulin (AcTub) antibodies. AcTub is a cilia marker. Nuclei were stained with DAPI. (B) and (C) show the number (B) and length (C) of cilia stained with anti-AcTub antibodies in FLAG-positive cells. Data are shown as mean ± SD. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. Overexpression of Ick, Mak iso1, or Mak iso2 increased the number and length of cilia stained with anti-AcTub antibodies compared with controls. These results indicate that not only Ick expression but also Mak expression can ameliorate ciliary abnormalities caused by Ick deficiency.

[0066] Example 2: Effect of Ick overexpression in the retina of Mak-deficient mice - / - We analyzed the intracellular localization of rhodopsin in photoreceptor cells overexpressing Ick in the mouse retina. The results are shown in Figure 2. (A) shows the schedule for subretinal injection of AAV and retinal harvesting. AAV expressing FLAG-tagged Ick driven by the rhodopsin kinase promoter was injected into 1-day-old (P1) Mack mice. - / -Mak-deficient mice were injected into the retina, and retinas were harvested at 14 days of age (P14) for analysis. (B) Sections of harvested retinas were immunostained with anti-FLAG and anti-rhodopsin antibodies. In the figure, OS indicates the outer segment, and ONL indicates the outer nuclear layer. (C) Quantification of rhodopsin immunofluorescence signals detected inside photoreceptors was performed using ImageJ software (n = 5 retinas from 4 mice). Rhodopsin signals inside photoreceptors were normalized to the total rhodopsin signal (outer segment and inner segment) of the photoreceptor. The rhodopsin signal inside the photoreceptor in the FLAG-positive region was calculated relative to the signal in the FLAG-negative region (normalized to the total signal). Data are shown as mean ± SD. Significance was determined using an unpaired t-test. Although abnormal rhodopsin localization in rod photoreceptors was observed in Mak-deficient retinas, the rhodopsin signal inside the photoreceptor in the FLAG-positive region was significantly reduced. In other words, it was shown that the abnormal localization of rhodopsin caused by Mak deficiency can be improved by the expression of Ick.

[0067] Reference Example 1: Analysis of the retina of Mak / Ick double-deficient (DKO) mice The retina was observed in Mak / Ick DKO mice. The results are shown in Figure 3. Figure 3(A) shows the results of toluidine blue staining of retinal sections prepared from mice aged 14 days (P14) and 1 month (1M). In the figure, OS indicates the outer segment, ONL indicates the outer nuclear layer, INL indicates the inner nuclear layer, and GCL indicates the ganglion cell layer. Figure 3(B) shows the results of measuring the thickness of the outer nuclear layer (ONL). Data are shown as mean ± SD. Significance was tested using an unpaired t-test. In the retinas of Mak / Ick DKO mice, the retinal photoreceptor layer was significantly thinner than in controls, indicating Mak deficiency (Mak - / - ) mice showed more severe photoreceptor degeneration than did mice with glaucoma.

[0068] Example 3: Effect of administration of FGFR inhibitor to Mak-deficient mice - / - Mice were administered an FGFR inhibitor and their effects on retinal tissue were analyzed. The results are shown in Figures 4, 5, and 6. Figure 4(A) shows the drug administration schedule. BGJ398, an FGFR inhibitor, was administered daily from 7 days of age (P7) to 1 month of age (1M).- / - Mice were subcutaneously injected with BGJ398 or vehicle as a control. - / - Retinal sections were prepared from 1M mice and stained with toluidine blue (n = 10 per group). In the figure, ONL denotes the outer nuclear layer, INL denotes the inner nuclear layer, and GCL denotes the ganglion cell layer. Figure 4(C) shows the results of measuring the thickness of the outer nuclear layer (ONL). Data are shown as mean ± SD. Significance was tested using an unpaired t-test. The photoreceptor layer of the retina in BGJ398-treated mice was significantly thicker than that in vehicle-treated mice. These results demonstrate that administration of an FGFR inhibitor can ameliorate retinal degeneration caused by Mak deficiency.

[0069] Figure 5 shows the results of Mak administration of BGJ398 or vehicle as a control. - / - (1M) Electroretinogram (ERG) analysis results for mice. The amplitudes of the a-wave and b-wave are shown as a function of stimulus intensity under dark and light conditions (-4.0 to 1.0 log cd s / m). 2 , -0.5 to 1.0 log cd s / m 2 Data are shown as mean ± SD. Significance was determined by unpaired t-test. Analysis was performed using six dark-controlled (Vehicle) and dark-BGJ398-treated mice, five light-controlled (Vehicle) mice, and six light-BGJ398-treated mice. The BGJ398-treated group showed significantly higher dark ERG a-waves (Figure 5(A)) and b-waves (Figure 5(B)). These results demonstrate that FGFR inhibitors can ameliorate retinal degeneration caused by Mak deficiency.

[0070] Figure 6 shows the results of Mak administration of BGJ398 or vehicle as a control. - / -(1M) Analysis of kinesin family 3A protein (Kif3a) phosphorylation in mouse retinas. Retinas from four mice were analyzed. Retinal lysates were subjected to Phos-tag Western blotting using an anti-Kif3a antibody and standard Western blotting. The relative phosphorylation level of Kif3a was quantified by the ratio of the upper band intensity (indicated by the white arrowhead in Figure 6(A)) to the lower band intensity (indicated by the black arrowhead in Figure 6(A)). Data are shown as mean ± SD. Significance was determined by unpaired t-test. Administration of an FGFR inhibitor enhanced Kif3a phosphorylation in Mak-deficient retinas. Kif3a is known to be a downstream molecule phosphorylated by Mak and Ick, respectively. Therefore, FGFR inhibitors can enhance Ick activity in Mak-deficient retinas.

[0071] Example 4: Effect of Ick overexpression in cells with ciliary damage induced by a dynein motor protein inhibitor. The effect of Ick overexpression was analyzed in cells treated with a dynein motor protein inhibitor. The results are shown in Figure 7. (A) NIH3T3 cells transfected with a plasmid expressing FLAG-tagged EGFP or a plasmid expressing FLAG-tagged Ick were treated with DMSO or the dynein motor protein inhibitor Ciliobrevin D (10 μM) for 24 hours, then harvested and immunostained with anti-FLAG and anti-AcTub antibodies. Nuclei were stained with DAPI. (B) The number of cilia stained with anti-AcTub in FLAG-positive cells was counted. Data are shown as mean ± SD. Significance was tested using two-way ANOVA followed by Tukey's multiple comparison test. The addition of Ciliobrevin D significantly reduced the percentage of ciliated cells, but this was significantly restored by Ick expression. In other words, it was shown that ciliary damage caused by cytoplasmic dynein inhibition can be improved by overexpression of Ick.

[0072] Example 5: Effect of Ick Overexpression in Cells with Ciliary Disorders Caused by Dynein Motor Protein Knockdown (1) Generation of Dync2li1 Knockdown Cells. shRNA expression constructs were constructed to knockdown the dynein motor protein Dync2li1 (dynein cytoplasmic 2 light intermediate chain 1), a subunit of the cytoplasmic dynein 2 complex, and their inhibitory effects were compared. HEK293T cells were co-transfected with plasmids encoding control (Control-shRNA) or shRNAs against Dync2li1 (Dync2li1-shRNA2, Dync2li1-shRNA3, Dync2li1-shRNA5, or Dync2li1-shRNA6), FLAG-tagged Dync2li1, and EGFP-expressing plasmids. Western blot analysis was performed using anti-FLAG and anti-GFP antibodies. GFP was used as an internal control for transfection. The results are shown in Figure 8. All of the shRNAs against Dync2li1 (Dync2li1-shRNA2, Dync2li1-shRNA3, Dync2li1-shRNA5, and Dync2li1-shRNA6) suppressed Dync2li1 expression.

[0073] (2) Ick Overexpression in Dync2li1-Knockdown Cells. The effect of Ick overexpression on cilia length was analyzed in Dync2li1-knockdown cells. The results are shown in Figure 9. (A) Plasmids encoding Control-shRNA or Dync2li1-shRNA3 were co-transfected into NIH3T3 cells in combination with a plasmid expressing FLAG-tagged Ick or a plasmid expressing FLAG-tagged EGFP. Immunostaining was performed with anti-FLAG and anti-AcTub antibodies. Nuclei were stained with DAPI. (B) The length of cilia stained with anti-AcTub in FLAG-positive cells was measured. The n numbers of cilia analyzed were as follows: Control-shRNA; Control (n = 66), Control-shRNA; Ick (n = 63), Dync2li1-shRNA3; Control (n = 67), and Dync2li1-shRNA3; Ick (n = 70). Analysis was performed using samples obtained from triplicate experiments. Data are shown as mean ± SD. Significance was determined by two-way analysis of variance followed by Tukey's multiple comparison test. Dync2li1 knockdown significantly increased cilia length, but the cilia length of Dync2li1 knockdown cells expressing Ick was comparable to that of the control (Control-shRNA; Control or Control-shRNA; Ick). These results suggest that the ciliary damage caused by Dync2li1 knockdown can be improved by overexpressing Ick.

[0074] Example 6: Effect of FGFR Inhibition in Cells with Ciliary Disorders Caused by Dynein Motor Protein Knockdown Dync2li1 knockdown cells were generated and the effect of FGFR activity inhibition on ciliary disorders was examined. (1) Effect of the FGFR Inhibitor BGJ398 NIH3T3 cells were transfected with a plasmid expressing FLAG-tagged EGFP and a plasmid expressing Control-shRNA or Dync2li1-shRNA3 and treated with DMSO or BGJ398 (100 nM) for 24 hours. The results are shown in Figure 10. (A) shows the results of immunostaining with anti-FLAG and anti-AcTub antibodies. (B) shows the results of measuring the length of cilia stained with anti-AcTub antibodies in FLAG-positive cells. The n number of cilia analyzed is as follows: Control-shRNA; DMSO (n = 71), Control-shRNA; BGJ398 (n = 70), Dync2li1-shRNA3; DMSO (n = 74), Dync2li1-shRNA3; BGJ398 (n = 73). Analysis was performed using samples from triplicate experiments. Data are shown as mean ± SD. Significance was determined by two-way ANOVA followed by Tukey's multiple comparison test. Dync2li1 knockdown significantly increased cilia length, but cilia length in BGJ398-treated Dync2li1 knockdown cells was similar to that of controls (Control-shRNA; Control or Control-shRNA; Ick).

[0075] (2) Effect of the FGFR inhibitor AZD4547. NIH3T3 cells were transfected with a plasmid expressing FLAG-tagged EGFP and a plasmid expressing shRNA against Dync2li1 (Dync2li1-shRNA3) and treated with DMSO or AZD4547 (100 nM) for 24 hours. The results are shown in Figure 11. (A) Immunostaining with anti-FLAG and anti-AcTub antibodies. (B) Measurement of the length of cilia stained with anti-AcTub in FLAG-positive cells (Dync2li1-shRNA3: DMSO, n = 46; Dync2li1-shRNA3: AZD4547, n = 43). Data are shown as mean ± SD. Significance was determined by unpaired t-test. Dync2li1 knockdown cells showed longer cilia than normal cells, but AZD4547-treated cells showed a significant decrease in cilia length, confirming that the cilia length approached that of normal cells.

[0076] (3) Effect of Dominant-Negative FGFR1 We analyzed the effect of overexpressing a dominant-negative FGFR1 (Fgfr1-A645V) on cilia dysfunction in Dync2li1-knockdown cells. NIH3T3 cells were transfected with a plasmid expressing Fgfr1-A645V, a plasmid expressing FLAG-tagged EGFP, and a plasmid encoding Dync2li1-shRNA3 and cultured. The results are shown in Figure 12. (A) Immunostaining with anti-FLAG and anti-AcTub antibodies. (B) Measurement of cilia length stained with anti-AcTub antibodies in FLAG-positive cells (Dync2li1-shRNA3: Control, n = 24 and Dync2li1-shRNA3: Fgfr1-A645V, n = 24). Data are shown as mean ± SD. Significance was determined by unpaired t-test. Dync2li1 knockdown cells showed longer cilia than normal cells, whereas cells overexpressing a dominant-negative FGFR1 showed a significant decrease in cilia length, approaching that of normal cells.

[0077] These results indicate that ciliary dysfunction caused by Dync2li1 knockdown can be improved by inhibiting FGFR activity.

[0078] Example 7: Effect of Ick or Mak Overexpression on Hedgehog Signaling Abnormalities in Ciliopathy. Ciliopathy is known to cause abnormalities in Hedgehog signaling. To analyze the effect of Ick or Mak expression on Hedgehog signaling abnormalities in ciliopathy, we performed a luciferase reporter gene assay using an 8xGli1-binding site-minimal promoter-NanoLuc luciferase construct. The results are shown in Figure 13. (A) A schematic diagram of the construct expressing NanoLuc luciferase under the control of 8xGli1-binding sites and a minimal promoter. (B) The results of transfection of NIH3T3 cells with a Dync2li1-shRNA2 expression plasmid, an Ick expression plasmid, a NanoLuc luciferase reporter construct driven by the 8xGli1-binding site and minimal promoter, and a firefly luciferase expression construct driven by the SV40 promoter and enhancer. (C) NIH3T3 cells were transfected with a Dync2li1-shRNA2 expression plasmid, a Mak iso1 or Mak iso2 expression plasmid, a Nanoluc luciferase reporter construct driven by an 8x Gli1 binding site and minimal promoter, and a firefly luciferase expression construct driven by an SV40 promoter and enhancer. Luciferase activity in cell lysates was measured after 24 hours of serum starvation following treatment with 100 nM smoothened agonist (SAG). Nanoluc luciferase activity was normalized to firefly luciferase activity. Data are shown as mean ± SD. Significance was determined by two-way ANOVA followed by Tukey's multiple comparison test. Knockdown of the dynein motor protein Dync2li1 significantly increased luciferase activity, indicating that Dync2li1 knockdown disrupted Hedgehog signaling. On the other hand, Dync2li1 knockdown cells expressing Ick (B) or Mak (C) showed activity comparable to that of the control.In other words, it was shown that expression of Ick or Mak normalized the abnormalities in Hedgehog signaling observed in ciliary disorders caused by Dync2li1 knockdown.

[0079] Reference Example 2: Analysis of Retinas from Retina-Specific Ccrk Conditional Knockout (CKO) Mice. Retinas were observed in Ccrk CKO mice, in which Ccrk was knocked out specifically in the retina. The results are shown in Figures 14, 15, and 16. Figure 14(A) shows the results of toluidine blue staining of retinal sections prepared from 14-day-old (P14) and 1-month-old (1M) mice. In the figure, ONL denotes the outer nuclear layer, INL denotes the inner nuclear layer, and GCL denotes the ganglion cell layer. Figure 14(B) shows the results of measuring the thickness of the outer nuclear layer (ONL). Data are shown as mean ± SD. Significance was tested using an unpaired t-test. The retinal photoreceptor layer in Ccrk CKO mice was significantly thinner than that in wild-type mice, and severe photoreceptor degeneration similar to that observed in Mak / Ick DKO mice (Reference Example 1, Figure 3) was observed.

[0080] Figure 15 shows the results of immunostaining of retinal sections prepared from 14-day-old (P14) wild-type and Ccrk CKO mice with anti-IFT88 and anti-acetylated α-tubulin (AcTub) antibodies (top), anti-Mak and anti-AcTub antibodies (middle), and anti-IFT88 and anti-Cep164 (centriolar protein 164) antibodies (bottom). Nuclei were stained with DAPI. In the Ccrk CKO mouse retinas, cilia were significantly shortened, and only the basal portions of the cilia were observed, with no axonemes observed.

[0081] Figure 16 shows the results of analyzing Mak phosphorylation levels in the retinas of Ccrk CKO mice. Western blotting of retinal lysates using anti-phosphorylated Mak (pMak) antibody and Mak antibody demonstrated reduced phosphorylation of Mak at threonine 157 in the retinas of Ccrk CKO mice. These results indicate that Ccrk is a major upstream regulator of Mak and Ick.

[0082] Example 8: Effect of Ccrk Overexpression in Cells with Ciliary Disorders Caused by Dynein Motor Protein Knockdown (1) Ccrk Overexpression in Dync2li1 Knockdown Cells Dync2li1 knockdown cells were generated, Ccrk was overexpressed, and cilia length was measured. The results are shown in Figure 17. (A) shows the results of immunostaining with anti-FLAG and anti-AcTub antibodies in NIH3T3 cells co-transfected with a plasmid encoding Control-shRNA or Dync2li1-shRNA3, in combination with a plasmid expressing FLAG-tagged Ccrk or a construct encoding FLAG-tagged EGFP. Nuclei were stained with DAPI. (B) shows the results of measuring the length of cilia stained with anti-AcTub antibodies in FLAG-positive cells. The n number of cilia analyzed is as follows: Control-shRNA; Control (n = 100), Control-shRNA; Ccrk (n = 104), Dync2li1-shRNA3; Control (n = 108), Dync2li1-shRNA3; Ccrk (n = 101). Analysis was performed using samples from four experiments. Data are shown as mean ± SD. Significance was determined by two-way ANOVA followed by Tukey's multiple comparison test. Dync2li1 knockdown significantly increased cilia length, whereas Ccrk overexpression significantly decreased cilia length. These results suggest that the ciliary dysfunction caused by Dync2li1 knockdown can be improved by Ccrk expression, as can Ick and Mak expression.

[0083] (2) Luciferase reporter gene assay. A luciferase reporter gene assay was performed. The results are shown in Figure 18. NIH3T3 cells were transfected with plasmids expressing Dync2li1-shRNA2 and Ccrk, a Nanoluc luciferase reporter construct driven by an 8x Gli1 binding site and minimal promoter, and a firefly luciferase expression construct driven by an SV40 promoter and enhancer. Luciferase activity in cell lysates was measured after 24 hours of serum starvation following treatment with 100 nM SAG. Nanoluc luciferase activity was normalized to firefly luciferase activity. Analysis was performed using samples from four experiments. Data are shown as mean ± SD. Significance was determined by two-way analysis of variance followed by Tukey's multiple comparison test. Knockdown of the ciliary protein Dync2li1 significantly increased luciferase activity, whereas expression of Ccrk significantly decreased the activity, indicating that the Hedgehog signaling defects observed in ciliary defects caused by Dync2li1 knockdown were ameliorated by expression of Ccrk.

[0084] These results indicate that Ccrk expression can ameliorate ciliary damage caused by cytoplasmic dynein inhibition.

[0085] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A substance that promotes the expression or function of male germ cell-associated kinase and / or intestinal cell kinase, or a pharmaceutical composition for treating ciliopathy, comprising male germ cell-associated kinase and / or intestinal cell kinase as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the substance that promotes the function of male germ cell-associated kinase and / or intestinal cell kinase is a fibroblast growth factor receptor inhibitor.

3. The pharmaceutical composition of claim 2, wherein the fibroblast growth factor receptor inhibitor is rogaratinib, apruzumab, futibatinib, derazantinib, ASP5878, zoligratinib, FP-1039, LY-2874455, RG-7444, fexagratinib, infigratinib, erdafitinib, PD173074, pemigatinib, or a combination thereof.

4. The pharmaceutical composition according to claim 1, wherein the substance that promotes the function of male germ cell-related kinase and / or intestinal cell kinase is a cell cycle-related kinase.

5. The pharmaceutical composition according to claim 1, wherein the substance that promotes the expression of male germ cell-associated kinase is a gene therapy drug that expresses male germ cell-associated kinase.

6. The pharmaceutical composition according to claim 1, wherein the substance that promotes the expression of enterocyte kinase is a gene therapy drug that expresses enterocyte kinase.

7. Ciliopathy is associated with retinal degeneration, choroidal coloboma, coloboma, retinitis pigmentosa, cone-rod dystrophy, cone dystrophy, macular dystrophy, corneal abnormalities, visual impairment, Leber's congenital amaurosis, Leber's amaurosis, Stargardt disease, hyperopia, keratoconus, photophobia, cataract, ptosis, nystagmus, microphthalmia, hypertelorism, cyclopia, hydrocephalus, macrocephaly, agenesis of the corpus callosum, holoprosencephaly, cerebellar vermis defects, cerebellar malformations and dysfunction, occipital meningocele, hypoplastic medulloblastoma, hypothalamic hamartoma, lower brainstem malformations, pituitary abnormalities, hypopituitarism, tumors of the central nervous system and / or cysts, brain abnormalities, epileptic seizures, psychomotor developmental delay, neuropsychiatric dysfunction, intellectual disability, learning disabilities, behavioral disorders, motor developmental delay, ataxia, feeding disorders, craniectodermal dysplasia, craniosynostosis, facial and digital (toe) malformations, Weyers tetrafacial dysplasia, polydactyly, brachymelia, syndactyly, missing fingers, bone and ectodermal abnormalities, bone development disorders, pelvic dysplasia, spinal column abnormalities, dental abnormalities, bronchiectasis, airway abnormalities, lung hypoplasia, pulmonary dysplasia, disorders of lung development and expansion, respiratory disorders, Jeune-asphyxiating thoracic dysplasia, esophageal atresia, bifid epiglottis, cleft uvula, oral abnormalities, Dysgeusia, long nose, chronic sinusitis, dysosmia, choanal atresia, tumors and / or cysts in the endolymphatic sac, hearing loss, hearing impairment, liver fibrosis, liver cysts, cystic kidney disease, polycystic kidney disease, chronic tubulointerstitial nephritis, tumors and / or cysts in the kidney, nephronophthisis, tumors and / or cysts in the adrenal gland, tumors and / or cysts in the pancreas, endocrine system abnormalities, short stature, situs inversus, smooth muscle hamartoma, skin and intestinal abnormalities, Hirschsprung's disease, apple-peel small intestinal atresia, imperforate anus, absence or dysplasia of the nails, chest narrowing, congenital heart disease, cardiomyopathy, high blood pressure, obesity, diabetes Diarrhea, dyslipidemia, morbid obesity and spermatogenesis failure, hypogonadism, genital anomalies, genitourinary malformations, infertility, male infertility, decreased sperm count, azoospermia, female infertility, neonatal lethality, fetal hydrops, developmental delay, acrocallosal syndrome, pituitary stalk interruption syndrome, orofacial-digital-limbic syndrome-related disorders, syndromic craniosynostosis, Greig apicopolysyndactyly syndrome, endocrine-cerebral-osseous dysplasia syndrome, COACH syndrome, Joubert syndrome-related disorders, Senior-Loken syndrome, Alström syndrome, Carpenter syndrome, Curry-Jones syndrome, Ellis-van Creveld syndrome,The pharmaceutical composition according to any one of claims 1 to 6, wherein the disorder is Hydroretaras syndrome, Kallmann syndrome, McKusick-Kaufman syndrome, MORM syndrome, Lowe syndrome, Pallister-Hall syndrome, RHYNS syndrome, Ström syndrome, Starr syndrome, Usher syndrome, von Hippel-Lindau syndrome, Bardet-Biedl syndrome-related disease, or Meckel syndrome-related disease.

8. The pharmaceutical composition according to claim 1, wherein the ciliopathies are retinal degenerative diseases.

9. The pharmaceutical composition according to claim 8, wherein the retinal degenerative disease is retinitis pigmentosa.

10. A pharmaceutical composition for treating retinal degenerative diseases, comprising a fibroblast growth factor receptor inhibitor as an active ingredient.

11. The pharmaceutical composition according to claim 10, wherein the retinal degenerative disease is retinitis pigmentosa.

12. A substance that promotes the expression or function of male germ cell-associated kinase and / or intestinal cell kinase, or a composition for improving cilia disorders, comprising male germ cell-associated kinase and / or intestinal cell kinase as an active ingredient.

13. A method for screening for a substance that improves cilia disorders, comprising the steps of contacting a test substance with cells that express male germ cell-associated kinase and a kinesin family 3A protein, measuring the phosphorylation of the kinesin family 3A protein, and identifying a substance that increases the phosphorylation level of the kinesin family 3A protein compared to the phosphorylation level in cells that have not been contacted with the test substance.

14. A method for screening for a substance that improves ciliary disorders, comprising the steps of contacting a test substance with cells that express an intestinal cell kinase and a kinesin family 3A protein, measuring the phosphorylation of the kinesin family 3A protein, and identifying a substance that increases the phosphorylation level of the kinesin family 3A protein compared to the phosphorylation level in cells that have not been contacted with the test substance.

15. A method for screening for a substance that improves cilia disorders, comprising the steps of contacting a test substance with cells expressing a cell cycle-related kinase and a male germ cell-related kinase and / or an intestinal cell kinase, measuring the phosphorylation of the male germ cell-related kinase and / or the intestinal cell kinase, and identifying a substance that increases the phosphorylation level of the male germ cell-related kinase and / or the intestinal cell kinase compared to the phosphorylation level in cells not contacted with the test substance.

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