Uses of the CRB1 gene in the treatment of systemic diseases such as eye and intestinal disorders.

Abstract

This document describes the use of the CRB1 gene in the preparation of drugs for treating systemic diseases such as eye and intestinal disorders. It also describes the use of CRB1 gene-based therapeutics in the preparation of drugs for treating systemic diseases such as eye and intestinal disorders.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically involving CRB1 The use of genes in the treatment of systemic diseases such as eye and intestinal disorders. Background Technology

[0002] Crb1 The gene was first discovered in fruit flies and is crucial for maintaining normal apical polarity of cells and the integrity of the embryonic epithelium. Crb1 The phenotypes of retinal diseases caused by mutations vary greatly in spatiotemporal distribution, genetic background, and external influencing factors such as light exposure; no clear genotype-phenotype correlation has been recorded. Additional genetic and / or environmental factors may influence... CRB1 The expression of related diseases is still unclear. Crb1 Do genes play a similar role in regulating cell adhesion and polarization in mammals?

[0003] The CRB complex has four core components: Crb, Stardust (Sdt), PATJ (Protein associated with tight junctions or Pals1-associated tight junction protein), and Lin-7. Crb is a transmembrane protein with a large extracellular structure consisting of 29 epidermal growth factor-like (EGF-like) repeats and 4 laminin-AG-like repeats. The intracellular structure is smaller, consisting of 37 amino acids

[27] . Sdt consists of two Lin-2 / Lin-7 (L27) domains, one PDZ domain, one Src-homology 3 (SH3) domain, and one guanylate kinase (GUK) domain. PATJ consists of 4 PDZ domains and one N-terminal L27 domain. Lin-7 is a relatively short protein consisting of 195 amino acids, containing an N-terminal L27 domain and a C-terminal PDZ domain.

[0004] The human body has multiple mucosal epithelial cells that form a barrier between the external environment and the internal host environment. The gastrointestinal mucosa is a semi-permeable barrier with complex tasks, allowing for nutrient absorption and immune sensing while restricting the transport of potentially harmful antigens and microorganisms. The regulation of this seemingly "paradoxical" task is achieved through interactions between the structural components and molecules of the intestinal mucosa, which operate dynamically to maintain intestinal integrity and immune homeostasis. The function of the intestinal barrier can be impaired by structural damage to the mucosa or subtle changes in barrier regulatory components.

[0005] The intestinal mucosa is composed of multiple elements, mainly including: an outer mucus layer (containing symbiotic gut microbiota, antimicrobial proteins, and secretory immunoglobulin molecules); a central single-cell layer with specialized epithelial cells; and an inner lamina propria (containing innate and adaptive immune cells, such as T cells, B cells, macrophages, and dendritic cells).

[0006] The mucus layer is the first physical defense line encountered by external molecules reaching the intestinal lumen, preventing bacteria from directly contacting epithelial cells. The main component of the mucus layer is highly glycosylated mucin, forming a gel-like sieve structure covering the intestinal epithelium. In both the small and large intestines, mucin 2 is the most abundant mucin protein secreted by goblet cells. Mucin 2 knockout mice spontaneously develop colitis, making mucin 2 expression crucial for disease prevention.

[0007] Beneath the mucus layer lies the strongest physical barrier of the intestine—the intestinal epithelial cell layer. The pluripotent stem cell pool located in the crypts produces five distinct cell types: absorptive intestinal cells, goblet cells, enteroendocrine cells, Paneth cells, and microfold cells. These cells together form a continuous polarized monolayer that separates the lumen from the lamina propria. Because the cell membrane cannot permeate hydrophilic solutes in the absence of specific transport proteins, the transport of such molecules through the intestinal epithelial cells is highly restricted. The uptake of lipophilic or macromolecules depends primarily on diffusion and endocytosis. Molecular transport between intestinal epithelial cells is regulated by the presence of junctional complexes. The three most important junctional complexes are: tight junctions (TJs), adhesive junctions (AJs), and desmosomes. Tight junctions are the junctional complexes at the apex of the epithelial cells, primarily functioning to seal the intercellular spaces. Tight junctions are composed of transmembrane proteins (such as claudins and occludin), peripheral membrane proteins (such as ZO-1 and ZO-2), and regulatory proteins. Adhesive junctions, located beneath tight junctions, along with desmosomes, provide strong adhesive forces to maintain the integrity of epithelial structures. Both tight junctions and adhesive junctions are linked to actin and myosin junctional loops, allowing for regulation of junctions via the cytoskeleton. Summary of the Invention

[0008] On the one hand, this application provides CRB1 The use of genes in the preparation of drugs to treat diseases.

[0009] On the one hand, this application provides CRB1 The use of gene therapy drugs in the preparation of drugs for treating diseases.

[0010] Some of the technical solutions in this application can be used to treat intestinal diseases and / or eye diseases in a targeted and localized manner, which is more effective and has less impact on other organs compared to existing technologies. In addition to intestinal diseases, some of the technical solutions in this application can also treat other diseases of the patient simultaneously, such as eye diseases. Attached Figure Description

[0011] Figure 1 This is a diagram of the AAV2 vector used to express mouse Crb1.

[0012] Figure 2 (A): Immunofluorescence staining of GFP (green) in the colon, liver, spleen, and retina of Rd8 mice treated with AAV2-GFP. (B): Immunofluorescence staining of CRB1 (red) in the retina of Rd8-AAV-GFP and Rd8-AAV-Crb1 mice. (C): Electron micrographs of intestinal epithelial cells from Rd8-AAV-GFP and Rd8-AAV-Crb1 mice, showing microvilli (MV), adhesion junctions (AJ), tight junctions (TJ), and mitochondria (MC). (D–G): In (C), integrity of adhesion junctions, microvilli length, microvilli width, and number of microvilli (per µm) in the epithelial binding complex. (H): Transmission electron micrograph of the Bruch membrane from Rd8-AAV-GFP and Rd8-AAV-Crb1 mice. (I): BM thickness in (H). (J): Serum FITC fluorescence intensity in Rd8-AAV-GFP and Rd8-AAV-Crb1 mice after administration of FITC-glucan. (KL): Flow cytometry images and quantitative analysis of RFP⁺ cells in peripheral blood of Rd8-AAV-GFP and Rd8-AAV-Crb1 mice. Detailed Implementation

[0013] To further illustrate the technical means and effects adopted by this application in order to achieve the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of this application is provided in conjunction with the accompanying drawings and preferred embodiments.

[0014] Terms and Definitions

[0015] To enable those skilled in the art to understand the features and effects of this invention, the terms and expressions mentioned in the specification and claims are generally described and defined below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding this invention, and in case of conflict, the definitions in this specification shall prevail.

[0016] In this document, singular terms refer to one or more. For example, "element" or "one element" both refer to one or more elements. As used herein, the term "multiple" refers to at least two.

[0017] In this document, the terms “comprising,” “including,” “having,” “containing,” or any other similar terms are open-ended conjunctions intended to cover non-exclusive inclusions, indicating that a combination (e.g., apparatus, composition, method, etc.) includes the listed elements (e.g., units of an apparatus, components of a composition, substantial steps of a method, etc.) but does not exclude other elements. For example, a composition or article containing a plurality of elements is not limited to those listed herein but may also include other elements not expressly listed but which are generally inherent to the composition or article. Unless expressly stated to the contrary, the term “or” is inclusive, not exclusive. In this document, the term “substantially constitutes…” when used to define compositions and methods means excluding other elements that have any substantial effect on the combination for the stated purpose, but does not exclude other elements that do not substantially affect the basic and novel features of the invention. In this document, closed-ended conjunctions such as “consisting of…” refer to combinations excluding other elements (units, components, substantial steps, etc.), but unless otherwise stated, do not mean the exclusion of trace amounts of unavoidable impurities. Implementations defined by each of these connecting terms are within the scope of this invention. As a particular implementation thereof, technical solutions that disclose terms including "comprising," "including," "having," "containing," or any other similar terms should also be considered as simultaneously disclosing corresponding technical solutions that include terms "substantially composed of" and "composed of."

[0018] In this document, all features or conditions defined in the form of numerical ranges or percentage ranges are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible subranges and individual values ​​within those ranges, particularly integer values. For example, a range description of "1 to 8" should be considered as specifically disclosing all subranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., particularly subranges defined by all integer values, and should be considered as specifically disclosing individual values ​​within those ranges such as 1, 2, 3, 4, 5, 6, 7, 8, etc. Similarly, a range description of "between 1 and 8" should be considered as specifically disclosing all ranges such as 1 to 8, 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., including their endpoint values. Unless otherwise specified, this method of interpretation applies to all content throughout this invention, regardless of its scope.

[0019] As used herein, the terms "expression vector," "expression construct," or "expression cassette" refer to a nucleic acid comprising a nucleotide sequence, such as a coding sequence and a template sequence, as well as sequences necessary for expressing that coding sequence. Expression vectors can be viral or non-viral. For example, an expression vector may include a nucleic acid construct that, when introduced into a host cell, results in transcription and / or translation of RNA or a polypeptide, respectively. This definition explicitly includes untranslated or untranslatable antisense or sense constructs. Those skilled in the art will recognize that the inserted polynucleotide sequence need not be identical to the gene sequence from which it is derived but may only be substantially similar.

[0020] As used herein, the term "plasmid" generally refers to a non-viral expression vector, such as a nucleic acid molecule encoding a gene and / or a regulatory element necessary for gene expression. As used herein, the term "viral vector" generally refers to a virus-derived nucleic acid capable of transporting another nucleic acid into a cell. When a viral vector is present in a suitable environment, it can direct the expression of one or more proteins encoded by one or more genes carried by that vector.

[0021] Implementation

[0022] On the one hand, it provided CRB1 The use of genes in the preparation of drugs for treating diseases, wherein the diseases are intestinal diseases and / or eye diseases, particularly intestinal diseases. In one aspect, a treatment method for the disease is provided, comprising administering... CRB1 Gene therapy, wherein the disease is an intestinal disease and / or an eye disease, particularly an intestinal disease. In one aspect, it provides a treatment for the disease. CRB1 Gene therapy drugs, wherein the diseases are intestinal diseases and / or eye diseases, particularly intestinal diseases. In one aspect, [the following is provided] CRB1 The use of gene therapy drugs in the treatment of diseases, wherein the diseases are intestinal diseases and / or eye diseases, particularly intestinal diseases. In one aspect, it provides... CRB1 The use of gene therapy drugs in the preparation of medicines for treating diseases, wherein the diseases are intestinal diseases and / or eye diseases, particularly intestinal diseases.

[0023] In some implementations, the application is described as follows: CRB1 Gene therapy includes administering CRB1 Gene therapy drugs. In some embodiments, the... CRB1 Gene therapy drugs include CRB1 Gene-related genetic material. In some embodiments, the genetic material is a nucleotide or peptide, particularly DNA, RNA, or a peptide. In some embodiments, the... CRB1 Gene therapy drugs include CRB1 Expression carrier. In some embodiments, the... CRB1Expression carriers include CRB1 Target gene. In some embodiments, the... CRB1 Gene therapy drugs include CRB1 Target gene.

[0024] In some embodiments, the CRB1 Gene therapy drugs can be viral vectors or non-viral vectors.

[0025] In some embodiments, the CRB1Gene therapy drugs include viral vectors. In some embodiments, the viral vector is a DNA virus or an RNA virus. In some embodiments, the viral vector is replication-defective, replication-deficient, replication-attenuated, or replication-capable. In some embodiments, the viral vector includes one or more viral vectors based on the following viruses: adenovirus (e.g., human adenovirus (e.g., adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), or adenovirus serotype 48 (Ad48))), chimpanzee adenovirus (e.g., ChAd3, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd17, ChAd16, ChAd19, ChAd20, ChAd22, ChAd24, ChAdY25, ChAd26, ChAd28, ChAd30, ChAd31, ChAd37, ChAd38, ChAd43, ChAd44, ChAd55, ChAd...). 63, ChAdV63, ChAd68, ChAd73, ChAd82, ChAd83, ChAd143, ChAd144, ChAd145, or ChAd147), chimpanzee adenovirus (e.g., GC44, GC45, or GC46), rhesus monkey adenovirus (e.g., RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, or RhAd66), bovine adenovirus, canine adenovirus, or porcine adenovirus), adeno-associated virus (e.g., AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8).2. AAV9, AAVrh10, AAV10, AAV11, AAV2 / 8, AAV2 / 5, AAV2 / 6, or AAV2 / 9), retroviruses (e.g., alpha retroviruses (e.g., Rous sarcoma virus), beta retroviruses (e.g., mouse mammary tumor virus), gamma retroviruses (e.g., Moloney mouse leukemia virus, Moloney mouse sarcoma virus, Harvey mouse sarcoma virus, gibberish leukemia virus, feline leukemia virus, Freund's mouse leukemia virus, or mouse stem cell virus), delta retroviruses, epta retroviruses, lentiviruses (e.g., human immunodeficiency virus (type 1 or 2), Medivesna virus, caprine arthritis encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, bovine immunodeficiency virus, or simian immunodeficiency virus), or foamy virus genus viruses), orthomyxoviruses (e.g., influenza virus (e.g., influenza A virus, influenza B virus)). Viruses (such as influenza C virus, or influenza D virus), paramyxoviruses (such as Newcastle disease virus, mumps virus, measles virus, Sendai virus, pneumovirus, Hennipa virus), poxviruses (such as vaccinia virus, vaccinia virus, Ankara vaccinia virus, New York vaccinia virus, Tian Tan strain of vaccinia virus, canarypox virus, fowlpox virus, or myxoma virus), papillomaviruses (such as human papillomavirus, or bovine papillomavirus), piconemaviruses (such as enteroviruses (such as rhinovirus, Coxsackievirus, echovirus, or poliovirus) or Seneca virus), alphaviruses (such as Sindbis virus, Ola virus, Babanken virus, Bama forest virus, Bebaru virus, Bogi River virus, Kabasu virus, Chikungunya virus, Eastern equine encephalitis virus, Swamp virus, Morganburg virus, Getta virus, Highland J virus, Ziragach virus, Mayaro virus, Me Trivirus, Magdeburg virus, Moso das Pedras virus, Mukamb virus, Ndumou virus, Orongrong virus, Pichuna virus, Rio Negro virus, Ross River virus, salmon pancreatic disease virus, Heron Mountain virus, Semliki forest virus, southern elephant seal virus, Tonat virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, or Vodaro River virus), herpesviruses (e.g., herpes simplex virus (type 1 or 2), varicella herpesvirus, Epstein-Barr virus, or cytomegalovirus), arenaviruses (e.g., lymphocytic choroid plexus meningitis). This includes viruses such as mammalian arenavirus, californica arenavirus, guanareto virus, Junin virus, Lassa virus, Luyo virus, Machupo virus, Sabya virus, or Whitewater Arroyo virus; coronaviruses (e.g., A coronavirus, B coronavirus, C coronavirus, or D coronavirus); reoviruses (e.g., rotavirus); rhabdoviruses (e.g., vesicular stomatitis virus, Malaba virus, rabies virus); flaviviruses (e.g., yellow fever virus or Zika virus); polyomaviruses (e.g., simian vacuolar virus 40); baculoviruses; tobacco mosaic virus; or ring viruses.

[0026] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the viral vector is AAV1. In some embodiments, the viral vector is AAV2. In some embodiments, the viral vector is AAV3. In some embodiments, the viral vector is AAV3B. In some embodiments, the viral vector is AAV4. In some embodiments, the viral vector is AAV5. In some embodiments, the viral vector is AAV6. In some embodiments, the viral vector is AAV7. In some embodiments, the viral vector is AAV8. In some embodiments, the viral vector is AAV8.2. In some embodiments, the viral vector is AAV9. In some embodiments, the viral vector is AAVrh10. In some embodiments, the viral vector is AAV10. In some embodiments, the viral vector is AAV11. In some embodiments, the viral vector is AAV2 / 8. In some embodiments, the viral vector is AAV2 / 5. In some embodiments, the viral vector is AAV2 / 6. In some embodiments, the viral vector is AAV2 / 9.

[0027] In some embodiments, the viral vector is an adenovirus vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a poxvirus vector. In some embodiments, the viral vector is an alphavirus vector. In some embodiments, the viral vector includes... CRB1 Target gene.

[0028] In some embodiments, the viral vector includes a promoter. In some embodiments, the viral vector includes an enhancer. In some embodiments, the viral vector includes a silencer. In some embodiments, the viral vector includes a boundary element. In some embodiments, the viral vector includes an insulator.

[0029] In some embodiments, the CRB1 Gene therapy drugs include non-viral vectors. In some embodiments, the... CRB1 Gene therapy agents include plasmids, small circular chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, phages, granules, or F granules. In some embodiments, the... CRB1 Gene therapy drugs include naked plasmid DNA.

[0030] In some embodiments, the CRB1Gene therapy drugs include liposome carriers. In some embodiments, the liposomes comprise trimethyl-2,3-diolenopropylammonium chloride (DOTMA), trimethyl-2,3-dioleoylpropylammonium bromide (DOTAP), and dimethyl-2,3-trifluoroacetic acid. Diolenopropyl-2-(2-Sperminecarbamoylamino)ethylammonium (DOSPA), Trimethyldodecylammonium bromide (DTAB), Trimethyltetradecylammonium bromide (TTAB), Trimethylhexadecylammonium bromide (CTAB), Dimethylbis(octadecylammonium bromide) (DDAB), Dimethyl-2-hydroxyethyl-2,3-diolenopropylammonium bromide (DORI), Dimethyl-2-hydroxyethyl-2,3-diolenopropylammonium bromide (DORIE), Dimethyl-3-hydroxypropyl-2,3-diolenopropylammonium bromide (DORIE-HP), Dimethyl-4-hydroxybutyl-2,3-diolenopropylammonium bromide (DORIE-HB), Dimethyl-5-hydroxypentyl-2,3-diolenopropylammonium bromide (DORIE-HPc) The following are at least one of the following: dimethyl-2-hydroxyethyl-2,3-bishexadecyloxypropylammonium bromide (DPRIE), dimethyl-2-hydroxyethyl-2,3-bisoctadecyloxypropylammonium bromide (DSRIE), dimethyl-2-hydroxyethyl-2,3-bistetradecyloxypropylammonium bromide (DMRIE), N-(2-argininoyl)-N′,N′-bisoctadecylglycineamide (DOGS), 1,2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC), 3β-[N-(N′,N′-dimethylaminoethyl)aminoformyl]choline (DC-Chol), lipid poly-ʟ-lysine (LPLL), stearamide, dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), and cholesterol. In some embodiments, the following... CRB1 Gene therapy drugs include vesicle vectors, particularly extracellular vesicle vectors. In some embodiments, the... CRB1 Gene therapy drugs include exosome vectors. In some embodiments, the... CRB1 Gene therapy drugs include engineered exosome vectors.

[0031] In some embodiments, the CRB1 Gene therapy drugs include microsphere carriers. In some embodiments, the microsphere carriers include polyethylene microspheres, latex microspheres, starch microspheres, gelatin microspheres, or polysaccharide microspheres.

[0032] In some embodiments, the administration is to the intestines. In some embodiments, the administration is to the stomach, jejunum, ileum, cecum, colon, or rectum. In some embodiments, the administration is to the stomach. In some embodiments, the administration is to the jejunum. In some embodiments, the administration is to the ileum. In some embodiments, the administration is to the cecum. In some embodiments, the administration is to the colon. In some embodiments, the administration is to the rectum. In some embodiments, the administration is oral or anal.

[0033] In some embodiments, the drug for treating the disease is a drug administered to the intestines. In some embodiments, the drug for treating the disease is a drug administered to the stomach, jejunum, ileum, cecum, colon, or rectum. In some embodiments, the drug for treating the disease is a drug administered to the stomach. In some embodiments, the drug for treating the disease is a drug administered to the jejunum. In some embodiments, the drug for treating the disease is a drug administered to the ileum. In some embodiments, the drug for treating the disease is a drug administered to the cecum. In some embodiments, the drug for treating the disease is a drug administered to the colon. In some embodiments, the drug for treating the disease is a drug administered to the rectum. In some embodiments, the drug for treating the disease is a drug administered orally or anally.

[0034] In some embodiments, the medicament for treating the disease is selected from at least one of tablets (e.g., sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, or oral tablets), pills, powders, granules, capsules (e.g., soft capsules, or microcapsules), lozenges, syrups, solutions, emulsions, suspensions, controlled-release formulations (e.g., instantaneous-release formulations, sustained-release formulations, or sustained-release microcapsules), aerosols, or films (e.g., oral disintegrating films, or oral mucosal adhesive films).

[0035] In some embodiments, the therapeutic agent for treating the disease includes, selected from sweeteners (e.g., sucrose, xylitol, fructooligosaccharides, cyclamate, stevia, or aspartame), flavoring agents (e.g., fragrances or flavorings), gelling agents (e.g., sodium alginate, gum arabic, gelatin, methylcellulose, or sodium carboxymethylcellulose), clarifying agents (e.g., chitosan, or gelatin), preservatives (e.g., benzoic acid and its salts, sorbic acid and its salts, or parabens), disintegrants (e.g., low-substituted hydroxypropyl cellulose, crospovidone, sodium carboxyacetic acid starch, croscarmellose sodium, or starch), and binders (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose). It comprises at least one of the following: povidone, copovidone, or pregelatinized starch; lubricant (e.g., stearic acid, magnesium stearate, or sodium fumaroyl stearate); wetting agent (e.g., polyoxyethylene sorbitan fatty acid ester, poloxamer, or polyoxyethylene castor oil derivative); suspending agent (e.g., hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, copovidone, sodium carboxymethyl cellulose, or methylcellulose); stabilizer (e.g., citric acid, fumaric acid, or succinic acid); filler (e.g., starch, sucrose, lactose, or microcrystalline cellulose); and binder (e.g., cellulose derivative, alginate, gelatin, or polyvinylpyrrolidone).

[0036] In some embodiments, the intestinal disease is inflammatory bowel disease (IBD) (i.e., enteritis) and / or damage to the intestinal epithelial barrier. In some embodiments, the intestinal disease is inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. In some embodiments, the intestinal disease is damage to the intestinal epithelial barrier. In some embodiments, the intestinal disease is a colonic or rectal intestinal disease. In some embodiments, the intestinal disease is a colonic intestinal disease. In some embodiments, the intestinal disease is a rectal intestinal disease. In some embodiments, the intestinal disease is inflammatory bowel disease of the colon or rectal inflammatory bowel disease.

[0037] In some embodiments, the eye disease is an ocular inflammation. In some embodiments, the eye disease is optic neuritis, uveitis, conjunctivitis, vitreitis, choroiditis, retinitis, retinal vasculitis, sympathetic ophthalmia, retinopathy, glaucoma, cataract, macular degeneration (e.g., age-related macular degeneration), Behçet's disease, or Vogt-Koyanagi-Harada syndrome. In some embodiments, the eye disease is an optic nerve disease, particularly glaucoma, optic neuritis (e.g., optic papillitis or retrobulbar optic neuritis), ischemic optic neuropathy (e.g., non-arteritis-ischemic optic neuropathy or arteritis-ischemic optic neuropathy), inflammatory optic neuropathy, optic disc edema, optic atrophy, or optic disc vasculitis. In some embodiments, the eye disease is a retinal disease, such as retinal tear, retinal detachment, retinopathy (e.g., diabetic retinopathy), epiretinal membrane, macular hole, macular degeneration, or retinitis pigmentosa.

[0038] In some embodiments, the intestinal disease and / or eye disease, particularly the intestinal disease, is related to... CRB1 Genes, especially those related to CRB1 Intestinal and / or eye diseases associated with gene mutations, particularly intestinal diseases. In some embodiments, the intestinal and / or eye diseases, particularly intestinal diseases, are related to... CRB1 Rd8 mutations in the gene are associated with intestinal and / or eye diseases, particularly intestinal diseases.

[0039] In some embodiments, the intestinal disease and / or eye disease, particularly the intestinal disease, is also an intestinal disease and / or eye disease associated with mutations in at least one gene from the group consisting of, particularly, the gene from the group consisting of, particularly the intestinal disease: ABCA4, ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHI1, AIPL1, ALMS1, AMY2B, APC, ARFGEF1, ARL13B, ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1, C2CD3 , CA4, CABP4, CACNA1F, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104, CEP250, CEP290, CEP290, CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC 1. CLCN7, CLN3, CLN5, CLN8, CLRN1, CLRN1, CNGA1, CNGA1, CNGA3, CNGB1, CNGB3, CNNM4, COL11A1, COL11A2, COL18A1, COL2A1, COL4A1, COL9A1, COL9A2, CP , CP, CPLANE1, CRB1, ERCC4, CSPP1, CTNNA1, CYP4V2, DHDDS, DYNC2H1, DYNC2I1, DYNC2I2, ENPP1, ERCC4, EVC2, EYS, EYS, F5, FAM161A, FBN1, FKRP, FKTN, F LG, FLVCR1, FOXE3, FUZ, GLB1, GMPPB, GNAT1, GRK1, GRM6, GUCA1A, GUCA1B, GUCY2D, HADHA, HGSNAT, HPS3, HPS5, IDH3B, IFT122, IFT140, IFT140, IFT43, I FT52, IFT74, IFT80, IFT80, IFT81, IFT88, IKBKG, IMPDH1, IMPG2, INPP5E, INTU, IQCB1, IQCE, IREB2, KCNJ13, KCNQ1, KCNV2, KIAA0586, KIAA0753, KIF7, KIZ, KIZ-AS1, KLHL7, KRIT1, LBR, LCA5, LOC101927157, LOC111365204, LRP2, LRP5, MAK, MAPKAPK3, MATK, MCOLN1, MERTK, MKS1, MPDZ, MT-ATP6, MT-CO3,MT-TE, MT-TL1, MTHFR, MUTYH, MYO7A, MYO7A, NMNAT1, NPHP1, NR2E3, OCA2, OTX2, PANK2, PAX6, PCARE, PC DH15, PDE6A, PDE6B, PDE6B, PDE6D, PEX1, PEX1, PEX12, PEX26, PEX6, PHF3, PITPNM3, PKD2, PLA2G5, POC5 , POMT1, PRCD, PRDM13, PROM1, PRPF3, PRPF31, PRPF8, PRPH2, RAD51C, RBP3, RBP4, RD3, RDH12, RDH5, RGR , RGR, RHO, RIMS1, RLBP1, ROM1, RP1, RP1L1, RP2, RPE65, RPE65, RPGR, RPGRIP1, RPGRIP1L, RS1, SACS, SAG ,SCAPER,SDCCAG8,SIX6,SLC19A1,SLC22A5,SLC26A4,SLC2A9,SLTM,SNRNP200,SPAG17,SPATA7,SPG11 , TFAP2A, TGFB2, TGFBR2, TMEM107, TMEM237, TMEM67, TOGARAM1, TOPORS, TPP1, TRAF3IP1, TREX1, TRIM5 9-IFT80, TSPAN12, TTC21B, TTC21B, TTC8, TULP1, USH1C, USH2A, USH2A, USH2A, USH2A, USH2A-AS1, VAC1 4. VCAN, VCAN, VCAN-AS1, VHL, VPS13B, WDR19, WDR19, WDR35, WDR73, YARS1, ZFYVE26, ZFYVE26, and ZNF408. , Example

[0040] Experimental methods

[0041] mice

[0042] C57BL / 6N mice carrying the Rd8 mutation (Crb1) rd8 / Rd8 (named Rd8 mice) and C57BL / 6J mice (Crb1) wt / wt The mice (named wt mice) were purchased from Jicui Pharmaceutical Co., Ltd. and housed in a standard specific pathogen-free (STORG) level animal laboratory at Zhongshan Ophthalmic Center, Sun Yat-sen University. Purification of the Rd8 mice was performed in the animal laboratory of Huazhong Agricultural University. The purified, germ-free mice were maintained in a sterile state, and fecal samples were tested weekly for microorganisms and parasites to ensure their sterility.

[0043] According to Mattapallil et al. Genotyping of Rd8 and Crb1-KO mutations was performed in 2012, or according to the instructions of Jicui Pharmaceutical Co., Ltd. All animal experiments were reviewed and approved by the Laboratory Animal Management and Use Committee (IACUC) of Zhongshan Ophthalmic Center of Sun Yat-sen University and Huazhong Agricultural University, and complied with the relevant sections of the ARVO Statement on the Use of Research Animals.

[0044] Histology

[0045] Mice were euthanized by cervical dislocation, and the required tissue was removed and fixed in 4% paraformaldehyde (PFA) at 4 °C for 24 h. Samples were washed three times with PBS, embedded in paraffin, and sectioned into 10 µm thick sections. Sections were stained with hematoxylin and eosin (H&E).

[0046] Immunofluorescence

[0047] Mouse heart puncture and perfusion with 4% PFA. Eyeballs were removed and then briefly fixed at room temperature for 15 minutes. Colonic tissue was cut into pieces approximately 5 mm in length and fixed with 4% PFA at room temperature for 4 hours. Colonic and cecal tissues were washed three times with PBS for 15 minutes each time, incubated overnight in 30% sucrose / PBS, embedded in OCT, and then frozen sectioned.

[0048] Frozen tissue sections (12 μm) were incubated at room temperature with blocking buffer (10% donkey serum, 0.1% Triton X-100 in PBS solution) for 30 minutes, then incubated overnight at 4°C with primary antibody diluted in blocking buffer, and finally incubated at room temperature for 2 hours with a fluorescently conjugated secondary antibody or phalloidin, washing thoroughly after each incubation. Fluorescence microscopy images were obtained using a Zeiss LSM880 confocal microscope.

[0049] The primary antibodies used included: anti-CRB1 (PA5-66373, ThermoFisher; 1:50), anti-IBA1 (ab178846, Abcam; 1:500), anti-CD4 (60-0042-U100, Tonbo Biosciences; 1:100), anti-ZO-1 (61-7300, ThermoFisher; 1:500), anti-Occludin (33-1500, ThermoFisher; 1:200), AlexaFluor 488-Phallodin (A12379, ThermoFisher; 1:500), anti-β-Catenin (Cell Signaling Technology, 8480; 1:200), and anti-GFP (ab290, Abcam; 1:500). The specificity of these antibodies was validated prior to immunofluorescence staining.

[0050] Quantitative real-time polymerase chain reaction (qPCR)

[0051] Fresh mouse colon tissue (approximately 1 cm) was rapidly frozen in liquid nitrogen, ground, and lysed with RNA extraction lysis buffer. Total RNA was then purified using the Qiagen RNeasy Plus kit and reverse transcribed into cDNA using the Takara PrimeScript RT kit with gDNA removal enzyme. Expression levels of the corresponding genes were analyzed by qPCR. Data were normalized to β-actin.

[0052] AAV vector preparation

[0053] The enzyme digestion-ligation-transformation method was used to... Crb1 Target gene (SEQ ID NO: 1) or GFP The target gene (SEQ ID NO: 2) was cloned into the target AAV expression vector. Positive clones were obtained and sequenced to confirm the correctness of the inserted fragment. The AAV recombinant vector was transfected into HEK293T cells, and the virus was harvested after 72 hours. After purification, concentration, and detection of viral titers, the encoding... GFP or Crb1 The AAV2 viral particles of the gene serve as AAV2 vectors.

[0054] AAV2 vector delivery

[0055] Rd8 newborn mice were administered rectally with a coded [substance / entity] at 10–12 days of age. GFP or Crb1 Five × 10¹¹ AAV2 viruses containing the gene (dissolved in 20 µl of 1× PBS). The AAV2 vector is as follows: Figure 1 As shown.

[0056] During the enema, a small amount of sterile mineral oil is used to lubricate the exterior of the 10 µL pipette tip, which is secured to the pipette handle. The pipette tip is then inserted into the anus, and the pipette is slowly pressed to release the ultrapure AAV virus solution (…). in vivo class).

[0057] Using the AAV2 virus vector system ( Figure 1 This study obtained CRB1 protein expression at the apical lamina of intestinal epithelial cells, but not in other tissues including the liver, spleen, or retina (see [link to study]). Figure 2 Rd8 mice (A–B) were named Rd8-AAV-Crb1 mice.

[0058] like Figure 2 As shown in C–G, CRB1 expression in intestinal epithelial cells restores the integrity of the adhesion junctions of the colonic epithelial barrier (see Figure C–G). Figure 2 (C–D), while the changes in colonic microvilli in Rd8 mice remained unchanged (see C–D). Figure 2 (E–G).

[0059] Although the structural defects of the Bruch membrane in Rd8-AAV-Crb1 mice were not restored (see... Figure 2 H–I), but intestinal permeability (see Figure 2 J), bacterial transfer in peripheral blood (see J), Figure 2 K–L was significantly reduced in Rd8-AAV-Crb1 mice.

[0060] The results showed that CRB1 expression could be restored in the gut, for example, through viral vectors. CRB1 Gene therapy drugs, when administered to mice, can improve morphological defects in the colonic epithelial barrier, thereby treating intestinal diseases.

[0061] The above embodiments are merely preferred embodiments of this application and should not be construed as limiting the scope of protection of this application. Any non-substantial changes and substitutions made by those skilled in the art based on this application shall fall within the scope of protection claimed by this application.

Claims

1. The sequence is SEQ ID NO: 1 CRB1 The use of genes in the preparation of drugs to treat diseases, wherein the disease is CRB1 Damage to the intestinal epithelial barrier caused by gene mutation.

2. Including the sequence with SEQ ID NO: 1 CRB1 Genetic CRB1 Use of gene therapy drugs in the preparation of drugs for treating diseases, wherein the disease is CRB1 Damage to the intestinal epithelial barrier caused by gene mutation.

3. The use as described in claim 2, characterized in that, The CRB1 Gene therapy drugs can be viral vectors or non-viral vectors.

4. The use as described in claim 2, characterized in that, The CRB1 Gene therapy drugs include non-viral vectors.

5. The use as described in claim 2, characterized in that, The CRB1 Gene therapy drugs include liposome carriers.

6. The use as described in claim 2, characterized in that, The CRB1 Gene therapy drugs include microsphere carriers.

7. The use as described in claim 2, characterized in that, The CRB1 Gene therapy drugs include viral vectors.

8. The use as described in claim 2, characterized in that, The CRB1 Gene therapy drugs include adeno-associated virus vectors.

9. The use as described in claim 8, characterized in that, The adeno-associated virus vector includes one or more viral vectors based on the following viruses: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, AAVrh10, AAV10, AAV11, AAV2 / 8, AAV2 / 5, AAV2 / 6, or AAV2 / 9.

10. The use as described in claim 2, characterized in that, The CRB1 Gene therapy drugs include those with the sequence SEQ ID NO:

1. CRB1 Gene expression vectors.

11. The use as described in claim 10, characterized in that, The CRB1 The expression vector includes the sequence SEQ ID NO:1 CRB1 Target gene.

12. The use as described in claim 1 or 2, characterized in that, The medication used to treat the disease is a medication administered to the intestines.

13. The use as described in claim 1 or 2, characterized in that, The medication used to treat the disease is a medication administered to the colon or rectum.

14. The use as described in claim 1 or 2, characterized in that, The medication used to treat the disease is a medication administered to the colon.

15. The use as described in claim 1 or 2, characterized in that, The disease is CRB1 Damage to the intestinal epithelial barrier of the colon or rectum caused by gene mutation.

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