CN120718885A

CN120718885A - A gRNA, AAV expression vector, and CRISPR-Cas9 system for treating glaucoma

Info

Publication number: CN120718885A
Application number: CN202410378956.9A
Authority: CN
Inventors: 曾震海; 黄锦海; 姚鸣宇; 周行涛; 陈中幸
Original assignee: Eye and ENT Hospital of Fudan University
Current assignee: Eye and ENT Hospital of Fudan University
Priority date: 2024-03-29
Filing date: 2024-03-29
Publication date: 2025-09-30

Abstract

本发明提供了一种用于治疗青光眼的gRNA、AAV表达载体、CRISPR‑CasRx系统，具体地，本发明提供了(a)基因编辑蛋白或其表达载体，所述基因编辑蛋白包括VI型Cas蛋白；和(b)gRNA或其表达载体，所述gRNA为引导所述基因编辑蛋白特异性结合眼睛组织中的房水流出相关基因和/或房水生成相关基因的RNA，所述房水流出相关基因包括Rock1和Rock2，所述房水生成相关基因包括Aqp1和Adrb2。针对房水流出相关基因(比如Rock1和Rock2)和房水生成相关基因(比如Aqp1和Adrb2)的外显子区域设计的gRNA具有编辑效率较高且脱靶率较低的特点，并且将VI型Cas蛋白和本发明设计的上述gRNA组合可有效预防和/或治疗青光眼或高眼压症。The present invention provides a gRNA, an AAV expression vector, and a CRISPR-CasRx system for treating glaucoma. Specifically, the present invention provides (a) a gene editing protein or its expression vector, wherein the gene editing protein includes a type VI Cas protein; and (b) a gRNA or its expression vector, wherein the gRNA is an RNA that guides the gene editing protein to specifically bind to aqueous humor outflow-related genes and/or aqueous humor production-related genes in eye tissue, wherein the aqueous humor outflow-related genes include Rock1 and Rock2, and the aqueous humor production-related genes include Aqp1 and Adrb2. The gRNA designed for the exon regions of aqueous humor outflow-related genes (such as Rock1 and Rock2) and aqueous humor production-related genes (such as Aqp1 and Adrb2) has the characteristics of high editing efficiency and low off-target rate, and the combination of the type VI Cas protein and the above-mentioned gRNA designed by the present invention can effectively prevent and/or treat glaucoma or ocular hypertension.

Description

GRNA, AAV expression vector and CRISPR-CasRx system for treating glaucoma

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a gRNA, an AAV expression vector and a CRISPR-CasRx system for treating glaucoma.

Background

Glaucoma is a disease characterized primarily by RGC death, optic papillary atrophy, and visual field injury. In recent years, the increasing number of glaucoma patients has become a major cause of irreversible vision loss worldwide. Glaucoma has been estimated to have a prevalence of 3.5% in the world population between 40 and 80 years old. As the population and proportion of the elderly continue to increase, it is expected that 1.118 million people will suffer from glaucoma in 20403 years, and a large proportion of these people will face blindness. Glaucoma is generally classified into primary, secondary and congenital glaucoma according to the angle morphology of the anterior chamber, the etiology and the age of onset. Among them, primary glaucoma can be further classified into two types, open-angle type and closed-angle type, according to the anterior chamber angle state at the time of elevation of ocular pressure. Secondary glaucoma may be caused by trauma, corticosteroid use, inflammation, tumor or crystalline capsule false exfoliation. Primary glaucoma is a polygenic complex genetic disease that is affected by environmental factors. Ocular hypertension is the major risk factor for the progression of the disease, while ocular hypotension is the only cure currently available. Common healing methods comprise drug management, surgical intervention, laser and other modes for regulating and controlling the aqueous humor generation and drainage process so as to achieve the effect of reducing intraocular pressure. In addition, existing healing means lack diversified strategies such as effective protection of the optic nerve against damage in addition to controlling high risk factors to alleviate sources of delayed crime for retinal damage.

The regular interval short palindromic repeats (CRISPR) are used as the leading edge gene editing technology of the operating genome, and have wide application prospect in the biomedical field. CRISPR/Cas is used as a third generation gene editing tool, derived from bacterial or archaeal acquired immunity, and RNA is used to guide Cas protein to modify a targeting sequence, so that great attention is paid to the field of gene editing at present, and version optimization and development of new editing tools are continuously underway. As technology advances, CRISPR/Cas is being studied in depth and explored for its diverse application scenarios. Many research results suggest that CRISPR/Cas technology has great potential in the field of disease treatment. CRISPR/Cas9 is a Cas9 endonuclease based on CRISPR-related RNA guidance, and Cas9 is guided to specific positions in complex genomes by searching for sequences on the genome paired with it through a short guide RNA (gRNA), enabling simple, rapid, and accurate editing of mammalian genomes. However, cas9 has serious drawbacks such as irreversible and non-negligible off-target probability as a CRISPR early version tool. In contrast, the novel CRISPR tool CasRx (i.e., cas13 d) mediates targeted silencing of transcript mRNA without altering genomic DNA, and its editing process is reversible, significantly improving gene therapy safety. CasRx-mediated gene silencing has higher specificity (shRNA off-target hundreds, casRx no off-target) and knockdown efficiency (shRNA 65%, CRISPR-CasRx up to 96%) compared to other RNA interference techniques.

However, there is no research on CasRx about glaucoma treatment.

There is therefore a pressing need in the art to develop a new method of treating glaucoma using CRISPR/Cas technology.

Disclosure of Invention

It is an object of the present invention to provide a novel method of treating glaucoma using CRISPR/Cas technology.

In a first aspect of the invention, there is provided a composition comprising:

(a) A gene editing protein or an expression vector thereof, the gene editing protein comprising a type VI Cas protein, and

(B) A gRNA or an expression vector thereof, the gRNA being an RNA that directs the gene-editing protein to specifically bind to an aqueous humor outflow-related gene and/or an aqueous humor-related gene in eye tissue, the aqueous humor outflow-related gene comprising Rock1 and Rock2, the aqueous humor-related gene comprising Aqp1 and Adrb.

In another preferred embodiment, the gRNA is an mRNA that directs the gene-editing protein to specifically bind to a gene associated with aqueous humor outflow and/or an aqueous humor outflow.

In another preferred embodiment, the region targeted by the gRNA is an exon region of an aqueous humor outflow-related gene and/or an aqueous humor-related gene sequence (e.g., exons 1,2, 4, 5, 6, 22, 25, 27).

In another preferred embodiment, the region targeted by the gRNA is the Rock1 Gene sequence (Gene ID: 19877) at position 10,150,247-10,150,276, and/or at position 10136123-10,136,152, and/or at position 10136100-10136129, and/or at position 10134430-10134459, and/or at position 10,134,416-10,134,445.

In another preferred embodiment, the region targeted by the gRNA is the Rock2 Gene sequence (Gene ID: 19878) at position 16,990,492-16,990,521, and/or at position 17,018,628-17,018,657, and/or at position 17024848-17,024,877, and/or at position 17022666-17,022,695, and/or at position 16992791-16,992,820, and/or at position 16998523-16,998,552.

In another preferred embodiment, the region targeted by the gRNA is the Aqp1 Gene sequence (Gene ID: 11826) at position 55313772-55,313,801, and/or at position 55313489-55,313,489, and/or at position 55,313,543-55,313,572, and/or at position 55,323,908-55,323,937, and/or at position 55,324,131-55,324,160, and/or at position 55,323,744-55,323,773.

In another preferred embodiment, the region targeted by the gRNA is position 62311188-62311217, and/or 62311268-62311297, and/or 62312578-62312607, and/or 62312576-62312605 of the Adrb2 Gene sequence (Gene ID: 11555).

In another preferred embodiment, the gRNA directs the gene-editing protein to simultaneously specifically bind RNA of an aqueous humor outflow-related gene in eye tissue.

In another preferred embodiment, the gRNA directs the gene-editing protein to simultaneously specifically bind to RNA of an aqueous humor-producing related gene in eye tissue.

In another preferred embodiment, the gRNA directs the gene-editing protein to bind specifically to both RNA of an aqueous humor outflow-related gene and RNA of an aqueous humor-related gene in eye tissue.

In another preferred embodiment, the genes associated with aqueous humor outflow are Rock1 and Rock2.

In another preferred example, the aqueous humor-related genes are Aqp1 and Adrb.

In another preferred embodiment, the nucleotide encoding the Cas protein of type VI contains the R-X ₄ -H motif, wherein X is 20 natural amino acids.

In another preferred embodiment, the type VI Cas protein comprises a Cas13 protein.

In another preferred embodiment, the type VI Cas protein is selected from the group consisting of type VI-A to type VI-D Cas proteins (i.e., cass 13 a-13D), cas13X, cas Y, or a combination thereof.

In another preferred embodiment, the nucleotide sequence of the gRNA is selected from any one of SEQ ID NOS.1-21, or a combination thereof.

In another preferred embodiment, the ocular tissue comprises trabecular meshwork tissue, ciliary body tissue, retinal tissue, iris tissue, cornea tissue, lens tissue, choroidal tissue, scleral tissue.

In another preferred embodiment, the composition comprises a pharmaceutical composition.

In another preferred embodiment, the composition further comprises:

(c) Other drugs for preventing and/or treating glaucoma or ocular hypertension.

In another preferred embodiment, the glaucoma comprises acute angle closure glaucoma, chronic angle closure glaucoma, secondary angle closure glaucoma, primary open-angle glaucoma, secondary open-angle glaucoma, congenital glaucoma, normal tension glaucoma, or hypersecretion glaucoma.

In another preferred embodiment, the other agent for preventing and/or treating glaucoma or ocular hypertension is selected from the group consisting of prostaglandin analog receptor agonists or prostaglandin compounds such as bimatoprost (amide) or bimatoprost acid, travoprost acid, latanoprost, beta blockers or beta adrenergic antagonists (e.g., timolol, betaxolol, levobunolol or metilol), alpha agonists or alpha adrenergic agonists (e.g., brimonidine, alcalodine, etc.), carbonic anhydrase inhibitors (including brinzolamide, acetazolamide, dorzolamide, methazolamide, or combinations thereof), rho kinase inhibitors, cannabinoid receptor agonists, or combinations thereof.

In another preferred embodiment, the expression vector comprises a viral vector.

In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

In another preferred embodiment, the vector is selected from the group consisting of lentiviruses, adenoviruses, adeno-associated viruses (AAV), or combinations thereof, preferably the vector is an adeno-associated virus (AAV).

In another preferred embodiment, the vector comprises AAV2 or shH10.

In another preferred embodiment, the expression vector of the gene-editing protein and the expression vector of the gRNA are the same vector or different vectors.

In another preferred embodiment, the gene encoding the gene-editing protein is located on the same expression vector as the gRNA or on a different expression vector (e.g., AAV vector).

In another preferred embodiment, the composition is in a dosage form selected from the group consisting of a lyophilized formulation, a liquid formulation, or a combination thereof.

In another preferred embodiment, the composition is in the form of a liquid formulation.

In another preferred embodiment, the composition is in the form of an injectable formulation.

In another preferred embodiment, the composition is a cell preparation.

In another preferred embodiment, the weight ratio of component (a) to component (b) is from 100:1 to 0.01:1, preferably from 10:1 to 0.1:1, more preferably from 2:1 to 0.5:1.

In another preferred embodiment, the content of component (a) in the composition is from 0.001% to 99%, preferably from 0.1% to 90%, more preferably from 1% to 70%.

In another preferred embodiment, the content of component (b) in the composition is 0.001% to 99%, preferably 0.1% to 90%, more preferably 1% to 70%.

In another preferred embodiment, the content of component (c) in the composition is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the components (a) and (b) and optionally (c) comprise from 0.01 to 99.99wt%, preferably from 0.1 to 90wt%, more preferably from 1 to 80wt% of the total weight of the composition.

In a second aspect, the invention provides a kit comprising:

(a1) A first container, and a gene-editing protein or expression vector thereof, or a drug containing a gene-editing protein or expression vector thereof, located in the first container, the gene-editing protein comprising a Cas protein of type VI;

(b1) A second container, and a gRNA or an expression vector thereof or a drug containing a gRNA or an expression vector thereof in the second container, wherein the gRNA is an RNA that directs the gene-editing protein to specifically bind to an aqueous humor outflow-related gene and/or an aqueous humor production-related gene in eye tissue, the aqueous humor outflow-related gene comprises Rock1 and Rock2, and the aqueous humor production-related gene comprises Aqp1 and Adrb.

In another preferred embodiment, the region targeted by the gRNA is an exon region of an aqueous humor outflow-related gene and/or an aqueous humor-related gene sequence.

In another preferred embodiment, the gRNA is an RNA that directs the gene-editing protein to bind specifically to both an aqueous humor outflow-related gene and an aqueous humor-related gene in eye tissue.

In another preferred embodiment, the kit further comprises:

(c1) A third container, and other glaucoma preventing and/or treating agents located in the third container.

In another preferred embodiment, the first container, the second container and the third container are the same or different containers.

In another preferred embodiment, the drug in the first container is a single formulation comprising a gene-editing protein or an expression vector thereof.

In another preferred embodiment, the drug in the second container is a single formulation comprising the gRNA or its expression vector.

In another preferred embodiment, the drug in the third container is a single formulation containing other drugs for preventing and/or treating glaucoma.

In another preferred embodiment, the pharmaceutical dosage form is selected from the group consisting of a lyophilized formulation, a liquid formulation, or a combination thereof.

In another preferred embodiment, the pharmaceutical dosage form is an injectable dosage form.

In another preferred embodiment, the kit further comprises instructions.

In a third aspect the present invention provides the use of a composition according to the first aspect of the invention or a kit according to the second aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of glaucoma or ocular hypertension.

In another preferred embodiment, the composition or kit comprises (a) a gene-editing protein or an expression vector thereof, and (b) a gRNA or an expression vector thereof, and (c) optionally other agents for preventing and/or treating glaucoma or ocular hypertension, and (d) a pharmaceutically acceptable carrier.

In another preferred embodiment, the composition or kit, (a) the gene-editing protein or its expression vector, and (b) the gRNA or its expression vector, and (c) optionally other agents for preventing and/or treating glaucoma comprise 0.01 to 99.99wt%, preferably 0.1 to 90wt%, more preferably 1 to 80wt% of the total weight of the composition or kit.

In a fourth aspect, the present invention provides a method for preventing and/or treating glaucoma or ocular hypertension, comprising:

administering to a subject in need thereof a composition according to the first aspect of the invention, or a kit according to the second aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal suffering from glaucoma or ocular hypertension.

In another preferred embodiment, the non-human mammal comprises a rodent and primate, preferably a mouse, rat, rabbit, monkey.

In a fifth aspect, the present invention provides a viral vector comprising:

(a) A coding sequence for a gene-editing protein comprising a type VI Cas protein, and

(B) A gRNA that is an RNA that directs the gene-editing protein to specifically bind to an aqueous humor outflow-related gene and/or an aqueous humor-related gene in eye tissue, the aqueous humor outflow-related gene comprising Rock1 and Rock2, and the aqueous humor-related gene comprising Aqp1 and Adrb.

In another preferred embodiment, the viral vector is selected from the group consisting of lentiviruses, adenoviruses, adeno-associated viruses (AAV), or combinations thereof, preferably the viral vector is an adeno-associated virus (AAV).

In another preferred embodiment, the viral vector comprises AAV2 or shH10.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the in vitro screening of target genes for highly efficient specific gRNA. Target positions of gRNA in Aqp1, adrb, rock1 and Rock 2. b. gRNA and CasRx expression vectors with fluorescent signals. c. Targeting knockdown efficiency of 5 different grnas of Rock1 gene in N2a cells, with knockdown efficiency of gRNA4 being highest. d. Knockdown efficiency against 6 different grnas of Rock2 gene in N2a cells, with gRNA4 showing the highest knockdown efficiency. e. In stably transfected Aqp1 highly expressed N2a monoclonal cell lines, the knockdown efficiency of 6 different gRNAs targeting the Aqp1 gene was verified, with the knockdown efficiency of gRNA3 being highest. f.4 knockdown efficiencies of gRNAs targeting the Adrb2 gene, with gRNA3 knockdown being most efficient. All values are expressed in mean±sd with p <0.05, p <0.01, p & <0.001, unpaired t-test.

Figure 2 shows an assessment of infection efficiency of AAV viruses of different serotypes. C57BL/6J female mice were intravitreally injected with AAV viruses of different serotypes. The injected virus is AAV2-EFS-EGFP or shH-EFS-EGFP, the titer is 1E+13GC/ml, and the injection dose is 1.5 μl. Viral expression in ciliary body and trabecular meshwork tissue was examined at weeks 1,2 and 3, respectively, post-injection. Arrows mark ciliary body, boxes mark trabecular meshwork, n=3. Scale bar, 100 μm. b. AAV2 virus or shH virus infused into the vitreous cavity for 2 weeks, egfp+ cells were present in the total percentage of trabecular meshwork and ciliary body area. Data are expressed as mean±sem, p <0.05, p <0.01, p <0.001, unpaired t-test.

FIG. 3AAV viral vector construction schematic. Y25 viral vector encoding CasRx, rock1-gRNA4 and Rock2-gRNA4, targeted knockdown of Rock1 and Rock2 genes, Y26 viral vector encoding CasRx, aqp1-gRNA3 and Adrb-gRNA 3, targeted Aqp1 and Adrb genes, non-targeting viral vector (AAV-EFS-CasRx/U6-LacZ) encoding CasRx and gcRNA targeted to LacZ genes.

Figure 4 shows that gene interference mediated by CasRx editing systems effectively reduces ocular pressure elevation in both types of glaucoma models. a. Day and night intraocular pressure (IOP) changes after each operation, Y25 (AAV-EFS-CasRx/U6-Rock 1-Rock 2) virus WT group n=20, loads+saline group n=10, loads+lacz group n=8, loads+y25 group n=20, the values are expressed as mean ± SEM, and the differences between groups are analyzed by one-way variance. Dex modeling and circadian changes in ocular pressure following Y25 virus infection, wtn=20, dex+physiological saline n=20, dex+lacz n=18, dex+y25n=40, all values expressed as mean ± SEM, each group tested using one-way analysis of variance. c. The IOP of each group was changed within 6 weeks after molding of the monocular beads, and Y26 (AAV-EFS-CasRx/U6-Aqp 1-Adrb) virus was injected. WT group n=18, bead+saline group n=8, bead+lacz group n=7, bead+y26 group n=19, values expressed as mean ± SEM, differences between groups were analyzed by one-way variance. day and night intraocular pressure conditions of mice in each group after DEX modeling, WT n=20, DEX+physiological saline n=18, DEX+LacZ n=20, DEX+Y25n=34, all values expressed as mean ± SEM, and differences between groups were analyzed by single factor variance analysis.

Figure 5 shows that a glaucoma model mouse intravitreally injected with therapeutic AAV virus has multiple organs throughout the body that are not infected with the virus. Organ immunofluorescence staining of mice with magnetic bead glaucoma after intravitreal injection of LacZ/Y25/Y26 virus. The brain, heart, liver, spleen, lung and kidney were obtained 10 weeks after molding, and the labeled proteins of the virus were stained and observed by immunofluorescent staining, without a red fluorescent signal co-stained with DAPI. Scale bar, 100 μm.

Detailed Description

The inventors have conducted extensive and intensive studies and, for the first time, unexpectedly found that the exon regions (such as the Gene ID:19877:10,150,247-10,150,276, and/or the Gene ID:19877:10136123-10,136,152, and/or the Gene ID:19877:10136100-10136129, and/or the Gene ID:19877:10134430-10134459, and/or the Gene ID:19877:10,134,416-10,134,445) of the aqueous humor-related genes (such as Aqp1 and Adrb); gene ID of pathogenic Gene Rock2 in 19878:16,990,492-16,990,521, and/or in 19878:17,018,628-17,018,657, and/or in 19878:17024848-17,024,877, and/or in 19878:17022666-17,022,695, and/or in 19878:16992791-16,992,820, and/or in 19878:16998523-16,998,552; the gRNA designed by the pathogenic Gene Aqp1 at positions 11826:553313772-55,313,801 and/or Gene ID at positions 11826:553313489-55,313,489 and/or Gene ID at positions 11826:55,313,543-55,313,572 and/or Gene ID at positions 11826:55,323,908-55,323,937 and/or Gene ID at positions 11826:55,324,131-55,324,160 and/or Gene ID at positions 11826:55,323,744-55,323,773 and the pathogenic Gene Adrb at positions 11555:62311188-62311217 and/or Gene ID at positions 11555:62311268-62311297 and/or Gene ID at positions 11555:62312268-62312607) has the characteristics of higher editing efficiency and lower off-targeting rate, and the combination of the gRNA designed by the type VI protein and the invention can effectively prevent and/or treat ocular hypertension or glaucoma. On this basis, the present inventors have completed the present invention.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of, or" consisting of.

Specifically, the gRNA and CRISPR/CasRx system for treating glaucoma and the AAV expression vector system with high affinity to eye tissue provided by the invention can effectively inhibit pathological damage caused by ocular hypertension of glaucoma. The invention adopts a novel RNA interference tool CRISPR/CasRx to respectively construct CRISPR/CasRx systems aiming at mouse ciliary body aqueous humor generation related genes aquaporin 1 (Aqp 1) and beta 2 adrenergic receptor (Adrb 2) and aqueous humor outflow related genes Rock1 and Rock2, and utilizes a fluorescence quantitative technology (qPCR) to detect the change of RNA level, thereby verifying the knockdown effect in cells. Experimental results show that the highest knockdown efficiency of the CRISPR/CasRx system constructed for the mouse Rock1 gene is 81% + -5% and guided by Rock1-gRNA4, while the highest knockdown efficiency of the CRISPR/CasRx system constructed for the mouse Rock2 gene is 81% + -3% and guided by Rock2-gRNA 4. The highest knock-down efficiency of the CRISPR/CasRx system constructed for the mouse Adrb gene is 93% +/-14% and guided by Adrb2-gRNA3, while the highest knock-down efficiency of the CRISPR/CasRx system constructed for the mouse Aqp1 gene is 95% +/-5% and guided by Aqp1-gRNA 2. The method comprises the steps of injecting AAV (AAV) fluorescent viruses of different serotypes into a vitreous cavity of a mouse, taking materials on days 7, 14 and 21, performing frozen section and immunofluorescent staining, observing fluorescent expression conditions by using a confocal microscope LSM880, screening shH viruses which specifically infect ciliary bodies, and packaging to obtain therapeutic viruses. AAV vectors are delivered to the CRISPR/CasRx system into the eye and expressed continuously.

In the serotype screening and validation experiments, we examined the infection of mice with AAV2 and shH fluorescent-labeled vectors, respectively, and confocal image observations after day 7, day 14 and day 21 revealed that a strong fluorescent signal was generated after shH serotype infection, and furthermore, the presence of a ciliary signal infected with shH fluorescent-labeled vector could still be observed in 6 months of injection of the vector into the balloon sample, and no problems were found with respect to multi-organ safety or with respect to leakage of the vector.

The invention establishes two clinical chronic glaucoma simulating animal models, namely a magnetic bead induced ocular hypertension mouse model and a glucocorticoid induced ocular hypertension mouse model. The established glucocorticoid induces the ocular pressure of the ocular hypertension mouse model to gradually rise and then to be stable. The intraocular pressure of the mice at week 1 after molding was 16.214.+ -. 0.556mmHg to 19.4.+ -. 0.909mmHg at week 10. Glucocorticoid-induced ocular hypertension mice were injected with the therapeutic virus via the vitreous cavity on day 3 after molding, and the ocular tension (13.9±0.277 mmHg) was significantly reduced compared to the positive control group (17.2±0.712 mmHg) from week 1, with a P <0.01, and the ocular tension lowering effect continued for at least 7 weeks. The magnetic bead induced ocular hypertension mouse model established by the invention can quickly rise the daily ocular tension level to 22.944 +/-6.034 mmHg at the 1 st week after the model is made. From week 2 to week 6, the day-time intraocular pressure of the mice was steadily increased and maintained at 20mmHg on average. The ocular hypertension mice induced by the magnetic beads are injected with the therapeutic virus through the vitreous cavity on the 3 rd day after molding, the ocular hypertension (16.2+/-0.467 mmHg) is obviously lower than the positive control group (19.2+/-0.735 mmHg) from the 2 nd week, and the ocular hypertension effect is at least lasting to the 6th week.

In general, the CRISPR/CasRx system of the related genes AQP1 and ADRB2 and related genes Rock1 and Rock2 for aqueous humor outflow of the targeted mice, constructed by the invention, can realize continuous ocular pressure reduction by single injection in ocular hypertension model animals through multiple verification of cell experiments and animal experiments, achieves the effect of long-term treatment by single injection, has no systemic toxicity, and has wide prospect and remarkable significance in clinical application of ocular pressure reduction treatment of glaucoma.

Glaucoma or ocular hypertension

Glaucoma or ocular hypertension is a disease characterized primarily by RGC death, optic papilla atrophy and visual field injury. In recent years, the increasing number of glaucoma patients has become a major cause of irreversible vision loss worldwide. Glaucoma has been estimated to have a prevalence of 3.5% in the world population between 40 and 80 years old. As the population and proportion of the elderly continue to increase, it is expected that 1.118 million people will suffer from glaucoma in 20403 years, and a large proportion of these people will face blindness. Glaucoma is generally classified into primary, secondary and congenital glaucoma according to the angle morphology of the anterior chamber, the etiology and the age of onset. Among them, primary glaucoma can be further classified into two types, open-angle type and closed-angle type, according to the anterior chamber angle state at the time of elevation of ocular pressure. Secondary glaucoma may be caused by trauma, corticosteroid use, inflammation, tumor or crystalline capsule false exfoliation. Primary glaucoma is a polygenic complex genetic disease that is affected by environmental factors. Ocular hypertension is the major risk factor for the progression of the disease, while ocular hypotension is the only cure currently available. Common healing methods comprise drug management, surgical intervention, laser and other modes for regulating and controlling the aqueous humor generation and drainage process so as to achieve the effect of reducing intraocular pressure. In addition, existing healing means lack diversified strategies such as effective protection of the optic nerve against damage in addition to controlling high risk factors to alleviate sources of delayed crime for retinal damage.

The present invention is directed to the treatment of glaucoma or ocular hypertension using CRISPR/Cas technology. Compared with traditional clinical therapy, the invention belongs to gene therapy, and has the advantages of accuracy and extremely high effectiveness for single pathogenic gene regulation. Cas13d is currently the smallest class II CRISPR effector in mammalian cells (20% smaller than Cas13a-c, 33% smaller than Cas9, which is a family member) compared to other gene regulation technologies, facilitating packaging into limited capacity application vectors such as AAV vectors. In addition, cas13d does not rely on PFS sequences (i.e., PAM sequences of Cas9 for DNA) for RNA target cleavage, expanding its range of application and becoming a potential platform for further development of targeted RNA tools. Cas13 d-mediated gene silencing has higher specificity (no off-target phenomenon) and knockdown efficiency (96% vs 65% shrna, 53% crispri) compared to RNA interference techniques. However, cas13 d-mediated gene silencing does not alter genomic DNA, making this silencing reversible, as compared to Cas 9-mediated gene knockout techniques, is more advantageous in the treatment of certain acquired diseases.

Compared with clinical treatment means:

Ocular hypertension is a major risk factor for the progression of glaucoma disease, and ocular hypotension is currently the only clinical treatment for glaucoma. Clinical treatment is mainly to intervene in the circulation process of aqueous humor generation and aqueous humor outflow by means of medicines, operations, lasers and the like so as to reduce intraocular pressure. These treatment regimens have unavoidable adverse consequences such as drug side effects due to arrhythmia, etc., and complications such as low intraocular pressure, cataract, etc. caused by surgery. In addition, existing treatments lack diversity in addition to controlling ocular pressure, a risk factor that alleviates ocular fundus damage and delays disease progression, such as the difficulty of providing an effective neuroprotective strategy against damage.

Compared with other gene editing techniques:

As a CRISPR early version tool, cas9 suffers from serious drawbacks such as irreversible and non-negligible off-target probability. In contrast, the novel CRISPR tool CasRx (i.e., cas13 d) mediates targeted silencing of transcript mRNA without altering genomic DNA, and its editing process is reversible, significantly improving gene therapy safety.

CRISPR-Cas13 is a newly discovered RNA editing system in which Cas13 protein belongs to class 2 class VI CRISPR-Cas system effector proteins, possessing RNA-mediated rnase activity. As the only protein in the second most general class of CRISPR-Cas systems found today that is capable of degrading RNA, it is more specific and efficient than traditional RNA interference tools. It has been determined that 4 members constitute a Cas13 protein family, including Cas13a, cas13b, cas13c, and Cas13d. It is reported that CasRx (RfxCas d) exhibits a stronger and more specific activity compared to other types of Cas13 s. As the current minimum size Cas13 enzyme, casRx can be easily encapsulated into AAVs carriers, where delivery is facilitated. CasRx has been successfully applied in treating mouse models of liver and eye diseases and provides a safer and more reliable RNA editing system suitable for acquired disease treatment methods without changing the genome compared to other gene editing systems.

Genes Rock1 and Rock2 expressed in trabecular meshwork and related to aqueous humor outflow

In the present invention, the terms "Rock1 gene", "Rock1 polynucleotide" are used interchangeably and refer to a nucleic acid sequence having a Rock1 nucleotide sequence.

The full length 164908bp,Gene ID:6093 of the genome of the human Rock1 gene is located Chromosome, nc_000018.10 (20946906..21111813, complex).

The genome of the murine Rock1 gene is full-length 117825bp,Gene ID:19877, position Chromosome, nc_000084.7 (10064401..10182225, complex).

Human and murine Rock1 have 97.46% protein sequence similarity.

In the present invention, the terms "Rock2 gene", "Rock2 polynucleotide" are used interchangeably and refer to a nucleic acid sequence having a Rock2 nucleotide sequence.

The genome of the human Rock2 Gene is 164908bp in length (Gene ID: 9475), position Chromosome, NC_000002.12 (11179759.. 11345437, complex).

The genome of the murine Rock2 Gene is 93468bp in length (Gene ID: 19878) at position Chromosome, NC_000078.7 (16944808.. 17038275).

Human and murine Rock2 have a protein sequence similarity of 98.41%.

It is understood that substitution of nucleotides in the codon is acceptable when encoding the same amino acid. It is further understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions are made by the nucleotide substitutions.

In the case where the amino acid fragments of Rock1 and Rock2 are obtained, a nucleic acid sequence encoding the same can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. For PCR amplification, primers can be designed based on the Rock1, rock2 nucleotide sequences disclosed in the present invention, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is entirely possible to obtain the DNA sequences encoding the proteins of the invention (or fragments, derivatives thereof) by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art.

The polynucleotide sequences of the invention can be used to express or produce recombinant Rock1, rock2 polypeptides by conventional recombinant DNA techniques. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human Rock1 or Rock2 polypeptide of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) Host cells cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present invention, rock1, rock2 polynucleotide sequences may be inserted into recombinant expression vectors. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing Rock1, rock2 coding DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are E.coli, bacterial cells of the genus Streptomyces, fungal cells such as yeast, plant cells, insect cells, animal cells, etc.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl ₂ method using procedures well known in the art. Another approach is to use MgCl ₂. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Genes Aqp1 and Adrb expressed in ciliary body tissue related to house water production

In the present invention, the terms "Aqp1 gene", "Aqp1 polynucleotide", "Aqp1" are used interchangeably and refer to a nucleic acid sequence having an Aqp1 nucleotide sequence.

The genome of the human Aqp1 Gene is 13664bp in length (Gene ID: 358) at position Chromosome, NC-000007.14 (30911853.. 30925516).

The genome of the murine Aqp1 Gene is 12257bp in length (Gene ID: 11826), position Chromosome, NC-000072.7 (55313284.. 55325540).

Human and murine Aqp1, protein sequence similarity was 95.44%.

In the present invention, the terms "Adrb gene", "Adrb2 polynucleotide", "ADRB2" are used interchangeably and refer to a nucleic acid sequence having a nucleotide sequence of Adrb.

The genome of the human Adrb Gene is 2013bp in length (Gene ID: 154), at position Chromosome, NC-000005.10 (148826611.. 148828623).

The genome of the murine Adrb Gene is 2269bp in length (Gene ID: 11555) at position Chromosome, NC-000084.7 (62310784..62313052, complex).

Human and murine Adrb2, protein sequence similarity was 91.15%.

In the case where the amino acid fragments of Aqp1, adrb2 are obtained, a nucleic acid sequence encoding it can be constructed therefrom, and specific probes can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed based on the Aqp1, adrb nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and the relevant sequences can be amplified using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

The polynucleotide sequences of the present invention may be used to express or produce recombinant Aqp1, adrb polypeptides by conventional recombinant DNA techniques. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human Aqp1, adrb2 polypeptide of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) Host cells cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present invention, the Aqp1, adrb polynucleotide sequences may be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the Aqp1, adrb coding DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Gene editing proteins

In the present invention, the gene editing proteins include a CRISPR-Cas13 system, which belongs to the Type VI family, comprising multiple families, wherein the functions of Type a, type B, type D, type X and Type Y families are identified. Cas13 proteins are all single proteins composed of multiple domains, with the function of recognizing crrnas, cleaving RNAs, and even cleaving pre-crrnas. Cas13 proteins have 2 tagged HEPN domains, 2R-X ₄ -H conserved motifs are nuclease active sites of HEPN domains, and most of Cas13 families are known to be mined by letter means based on R-X ₄ -H motif features and crRNA related features. In addition to the Cas13 protein, the Type VI family gene locus also contains CRISPR ARRAY. CRISPR ARRAY consists of a Repeat sequence (Repeat) and a spacer sequence (spacer), CRISPR ARRAY is transcribed to form immature pre-crRNA, which is cleaved to form mature crRNA, leading to Cas13 protein function. Cas1 and cas2 genes may also be present at the Type VI family gene locus, and these 2 genes are associated with bacterial or spacer processes.

In a preferred embodiment of the invention, the gene editing proteins include, but are not limited to, cas proteins of type VI, preferably Cas proteins of type VI-D, i.e., cas13D (CasRx).

Adeno-associated virus

Because Adeno-associated viruses (AAV) are smaller than other viral vectors, are nonpathogenic, and can transfect dividing and non-dividing cells, gene therapy approaches to genetic diseases based on AAV vectors have received considerable attention.

Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus dependovirus of the family picoviridae, and is the simplest class of structurally single-stranded DNA-deficient viruses currently found, requiring helper virus (typically adenovirus) to participate in replication. It encodes cap and rep genes in inverted repeats (ITRs) at both ends. ITRs are decisive for viral replication and packaging. The cap gene encodes viral capsid proteins and the rep gene is involved in viral replication and integration. AAV can infect a variety of cells.

Recombinant adeno-associated virus (rAAV) is derived from non-pathogenic wild adeno-associated virus, and is regarded as one of the most promising gene transfer vectors due to the characteristics of good safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, long time for expressing exogenous genes in vivo, etc., and is widely applied to gene therapy and vaccine research worldwide. Through more than 10 years of research, the biological properties of recombinant adeno-associated viruses have been well understood, and in particular, many data have been accumulated on their utility in various cell, tissue and in vivo experiments. In medical research, rAAV is used for researching gene therapy of various diseases (including in-vivo and in-vitro experiments), and is also widely used as a characteristic gene transfer vector in aspects of researching gene functions, constructing disease models, preparing gene knockout mice and the like.

In a preferred embodiment of the invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can integrate into the genome of the cells they infect in a stable and site-specific manner. They are able to infect a large array of cells without any effect on cell growth, morphology or differentiation, and they do not appear to be involved in human pathology. AAV genomes have been cloned, sequenced and characterized. AAV comprises an Inverted Terminal Repeat (ITR) region of about 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two important regions with encapsidation functions, the left part of the genome containing the rep genes involved in viral replication and viral gene expression, and the right part of the genome containing the cap genes encoding viral capsid proteins.

AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying the vectors can be found, for example, in U.S. Pat. nos. 6566118, 6989264 and 6995006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for in vitro and in vivo transport genes has been described (see, e.g., international patent application publication Nos. WO91/18088 and WO93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535 and 5,139,941, and European patent No.0488528, each of which is incorporated herein by reference in its entirety). These patent publications describe various AAV-derived constructs in which rep and/or cap genes are deleted and replaced by genes of interest, and the use of these constructs to transport genes of interest in vitro (into cultured cells) or in vivo (directly into organisms). Replication-defective recombinant AAV can be prepared by co-transfecting into a cell line infected with a human helper virus (e.g., adenovirus) a plasmid containing a nucleic acid sequence of interest flanked by two AAV Inverted Terminal Repeat (ITR) regions, and a plasmid carrying AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.

In some embodiments, the recombinant vector is encapsidated into a virion (e.g., an AAV virion including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV 16). Thus, the present disclosure includes recombinant viral particles (recombinant as they comprise recombinant polynucleotides) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. patent No.6,596,535.

Pharmaceutical composition

By using the protein of the present invention, substances, particularly inhibitors, which interact with genes related to aqueous humor outflow (e.g., rock1 and Rock 2) and genes related to aqueous humor (e.g., aqp1 and Adrb 2) or proteins can be selected by various conventional screening methods.

Inhibitors (or antagonists) of aqueous humor outflow-related genes (e.g., rock1 and Rock 2) and aqueous humor-related genes (e.g., aqp1 and Adrb 2) useful in the present invention include any substance that can inhibit the expression and/or activity of aqueous humor outflow-related genes (e.g., rock1 and Rock 2) and aqueous humor-related genes (e.g., aqp1 and Adrb) genes or their encoded proteins.

For example, the inhibitors of the aqueous humor outflow-related genes (such as Rock1 and Rock 2) and the aqueous humor production-related genes (such as Aqp1 and Adrb 2) include antibodies to the aqueous humor outflow-related genes (such as Rock1 and Rock 2) and the aqueous humor production-related genes (such as Aqp1 and Adrb), antisense RNA, siRNA, shRNA, miRNA to nucleic acids of the aqueous humor outflow-related genes (such as Rock1 and Rock 2) and the aqueous humor production-related genes (such as Aqp1 and Adrb), gene editors, or inhibitors of the activity of the aqueous humor outflow-related genes (such as Rock1 and Rock 2) and the aqueous humor production-related genes (such as Aqp1 and Adrb 2). Preferred inhibitors of aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor-related genes (such as Aqp1 and Adrb 2) refer to gene editors (such as gene editors comprising gene editing proteins and gRNA) capable of inhibiting the expression of aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor-related genes (such as Aqp1 and Adrb).

Preferably, inhibitors of aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor-related genes (such as Aqp1 and Adrb 2) of the present invention include Gene sequences (such as Gene ID:19877:10,150,247-10,150,276, and/or Gene ID:19877:10136123-10,136,152, and/or Gene ID:19877:10136100-10136129, and/or Gene ID:19877:10134430-10134459, and/or Gene ID:19877:10,134,416-10,134,445) that target aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor-related genes (such as Aqp1 and Adrb); gene ID of pathogenic Gene Rock2 in 19878:16,990,492-16,990,521, and/or in 19878:17,018,628-17,018,657, and/or in 19878:17024848-17,024,877, and/or in 19878:17022666-17,022,695, and/or in 19878:16992791-16,992,820, and/or in 19878:16998523-16,998,552; gene ID of pathogenic Gene Aqp1 is 11826:553313772-55,313,801, and/or Gene ID is 11826:553313489-55,313,489, and/or Gene ID is 11826:55,313,543-55,313,572, and/or Gene ID is 11826:55,323,908-55,323,937, and/or Gene ID is 11826:55,324,131-55,324,160, and/or Gene ID is 11826:55,323,744-55,323,773, and/or Gene ID is 11555:62311188-62311217, and/or Gene ID is 11555:62311268-62311297, and/or Gene ID is 578:623126257, and/or Gene ID is 11555:62312576-62312605). In a preferred embodiment, the method and steps of inhibiting aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor-related genes (such as Aqp1 and Adrb 2) comprise neutralizing their proteins with antibodies to aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor-related genes (such as Aqp1 and Adrb), and silencing aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor production-related genes (such as Aqp1 and Adrb) with shRNA or siRNA or a gene editor carried by a virus (such as adeno-related virus).

The inhibition rate of the aqueous humor outflow-related genes (e.g., rock1 and Rock 2) and the aqueous humor production-related genes (e.g., aqp1 and Adrb 2) is generally at least 50% inhibition, preferably 60%, 70%, 80%, 90%, 95% inhibition, and the inhibition rate of the aqueous humor outflow-related genes (e.g., rock1 and Rock 2) and the aqueous humor production-related genes (e.g., aqp1 and Adrb) can be controlled and detected based on conventional techniques, such as flow cytometry, fluorescent quantitative PCR, or Western blot.

Inhibitors (including antibodies, antisense nucleic acids, gene editors, and other inhibitors) of proteins of aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor-related genes (such as Aqp1 and Adrb) of the present invention, when administered (dosed) therapeutically, can inhibit the expression and/or activity of proteins of aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor-related genes (such as Aqp1 and Adrb), thereby preventing and/or treating glaucoma. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to, topical, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, return after autologous cell extraction culture, and the like.

The invention also provides a pharmaceutical composition comprising a safe and effective amount of an inhibitor of the invention (e.g., an antibody, gene editor, antisense sequence (e.g., siRNA), or inhibitor) and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram-10 milligrams per kilogram of body weight per day.

The main advantages of the invention include:

(1) The present invention unexpectedly found for the first time that the exon regions (such as the GeneID:19877:10,150,247-10,150,276 bits, and/or the GeneID: 19877:10136123-10,136,152 bits, and/or the GeneID: 19877:10136100-10136129 bits, and/or the GeneID:19877:10134430-10134459 bits, and/or the GeneID:19877:10, 134,416-10,134,445 bits) of the aqueous humor outflow-related genes (such as Rock1 and Rock 2) and aqueous humor-related genes (such as Aqp1 and Adrb); gene ID of pathogenic Gene Rock2 in 19878:16,990,492-16,990,521, and/or in 19878:17,018,628-17,018,657, and/or in 19878:17024848-17,024,877, and/or in 19878:17022666-17,022,695, and/or in 19878:16992791-16,992,820, and/or in 19878:16998523-16,998,552; the gRNA designed by the pathogenic Gene Aqp1 at positions 11826:553313772-55,313,801, and/or Gene ID at positions 11826:553313489-55,313,489, and/or Gene ID at positions 11826:55,313,543-55,313,572, and/or Gene ID at positions 11826:55,323,908-55,323,937, and/or Gene ID at positions 11826:55,324,131-55,324,160, and/or Gene ID at positions 11826:55,323,744-55,323,773, and the pathogenic Gene Adrb at positions 11555:62311188-62311217, and/or Gene ID at positions 11555:62311268-62311297, and/or Gene ID at positions 11555:6231231231252-62312607, and/or Gene ID at positions 11555:62312576-62312605) has the characteristics of higher editing efficiency and lower off-targeting rate, and the combination of the above-mentioned gRNA designed by the type VI Cas protein and the present invention can effectively prevent or treat ocular tension or glaucoma.

(2) The present invention is directed to the treatment of glaucoma or ocular hypertension using CRISPR/Cas technology. Compared with traditional clinical therapy, the invention belongs to gene therapy, and has the advantages of accuracy and extremely high effectiveness for single pathogenic gene regulation. Cas13d is currently the smallest class II CRISPR effector in mammalian cells (20% smaller than Cas13 a-c, 33% smaller than Cas9, which is a family member) compared to other gene regulation technologies, facilitating packaging into limited capacity application vectors such as AAV vectors. In addition, cas13d does not rely on PFS sequences (i.e., PAM sequences of Cas9 for DNA) for RNA target cleavage, expanding its range of application and becoming a potential platform for further development of targeted RNA tools. Cas13 d-mediated gene silencing has higher specificity (no off-target phenomenon) and knockdown efficiency (96% vs 65% shrna, 53% crispri) compared to RNA interference techniques. However, cas13 d-mediated gene silencing does not alter genomic DNA, making this silencing reversible, as compared to Cas 9-mediated gene knockout techniques, is more advantageous in the treatment of certain acquired diseases.

(3) The invention applies CasRx to treat glaucoma for the first time by targeting 4 sites on two causative genes of glaucoma simultaneously.

(4) The invention has reversibility of gene regulation because the invention only acts on mRNA of pathogenic genes but not on DNA, thereby having the safety which other gene editing tools do not have.

(5) Compared with the defect that the clinical treatment means needs the combined action of a plurality of medicaments and needs the continuous administration of multiple daily administrations, the invention can realize the effect of one-time treatment for more than 6 weeks.

(6) The invention screens out the gRNA sequence used by CRISPR/CasRx system of the related genes AQP1 and ADRB2 of the ciliary body aqueous humor generation and the related genes Rock1 and Rock2 of the aqueous humor outflow of the targeted mice for the first time, and can realize single injection to continuously reduce intraocular pressure in high intraocular pressure model animals through multiple verification of cell experiments and animal experiments, thereby achieving the effect of long-term treatment of single injection.

(7) The CRISPR/CasRx gene therapy viral vector for glaucoma is constructed for the first time, and has reversibility of gene regulation because the vector only acts on mRNA of pathogenic genes but not on DNA, so that the vector has safety which other gene editing tools do not have.

(8) The invention screens AAV serotypes with targeted mouse ciliary body and trabecular tissue for the first time.

The invention is further described below in conjunction with the specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, a laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are by weight unless otherwise indicated.

The reagents and materials used in the examples of the present invention were all commercially available products unless otherwise specified.

EXAMPLE 1 gRNA selection for treatment of glaucoma and construction of the CRISPR/CasRx plasmid

1.1 Construction of gRNA expression plasmid

The vector used to construct the gRNA expression plasmid (gRNA-Rock 1/Rock2; gRNA-Aqp 1/Adrb), purchased from Wohan vast organism; casRx plasmid (CMV-CasRx, purchased from addgene) carries the mCherry fluorescent signal, the U6 promoter is followed by the gRNA expression cassette with a BsaI single cleavage site between the double DR sequences. The vector skeleton can be obtained after enzyme digestion reaction and purification recovery, then the gRNA sequence (shown in figure 1 a) (table 2) is inserted into the skeleton in a T4 connection mode, and the gRNA expression plasmid can be obtained after transformation, plating, amplification and sequencing.

(1) And (5) performing enzyme digestion reaction to obtain a framework. After the reaction system was prepared as shown in the following table, the reaction was carried out in a 37℃thermostat water bath for 3 to 5 hours.

TABLE 1 cleavage reaction System

(2) And (5) recovering and purifying the framework by gel electrophoresis. Preparing macroporous 1% agarose gel, loading, and respectively adding appropriate DNA MARKER, original plasmid without enzyme digestion reaction and 50 μl of original liquid after enzyme digestion. After running at 200V for 30 minutes, the position of the backbone was determined from DNA MARKER and the position of the original plasmid, and the gel was cut. Purification was then performed using Thermo Scientific GeneJET gel recovery kit.

(3) The formation of phosphodiester bonds between the side-by-side 5-phosphate and 3-hydroxyl ends in duplex DNA was catalyzed by Thermo SCIENTIFIC T DNA ligase, ligated into a complete circular plasmid by T4 ligase, and ligated in a PCR apparatus at 20℃for 30 minutes after the reaction system was configured as shown in Table 1. The actual situation is said to be that the connection time can be prolonged to overnight.

(4) Plasmid transformation and plating were performed with DH 5. Alpha. Competent cells (ex Nanjinopran). After overnight incubation at 37℃2-3 single colonies were isolated, inoculated into 5mL of selective LB liquid medium (from Shanghai Biyun) containing 0.1% ampicillin (50. Mu.g/mL), incubated for 12-16 hours at 37℃on a shaker (about 300 rpm) and the bacterial solution was sent to Sanger for sequencing of hU6-F GAGGGCCTATTTCCCATGATT as sequencing primer. And amplifying and extracting plasmids after the gRNA is sequenced correctly, wherein the CasRx plasmids and the gRNA plasmid structures are shown in figure 1 b.

All plasmids of the CRISPR/CasRx system used in this experiment were constructed and amplified to the amount required for subsequent cell experiments.

TABLE 2CRISPR/CasRx related gRNA targets for the treatment of glaucoma

1.2 Verification of knockdown Effect and efficient in vitro screening of gRNA

The in vivo animal research is carried out by detecting whether the CRISPR/CasRx editing system can effectively reduce the expression level of a target gene by degrading RNA, and screening out proper gRNA with high knocking-down efficiency for virus packaging. The method comprises the following specific steps:

(1) In the control group, 1.5. Mu.g of plasmid was transfected per well and 3 replicate wells were set, and in the experimental group, 1.5. Mu. g CasRx plasmid and 0.5. Mu.g of gRNA plasmid were transfected per well and 3 replicate wells were set per well, using cells transfected with the editing vector (i.e., CRISPR/CasRx system plasmid of example 1.1) and gRNA expression vector.

(2) N2a stable transgenic monoclonal cell lines with a growth density of 80-90% in 10cm dishes were plated one day prior to transfection and passaged 1:4 into 12 well plates with approximately 25-30 ten thousand cells per well. Transfection was performed as described for cell transfection with a plasmid to transfection reagent ratio of 1:2.

(3) Samples were collected 48 hours after transfection. In order to enrich successfully transfected cells and reduce background signal interference, since CasRx plasmids have EGFP enhanced green fluorescent protein and gRNA expression plasmids have mCherry red fluorescent protein, the experiment selects a flow sorting technology to sort out red-green double-positive cells and selects 10% of the strongest fluorescent signal expression to collect 1 ten thousand cells for subsequent detection.

(4) The collected cells were rapidly extracted with RNA, subjected to reverse transcription and qPCR experiments, and analyzed for relevant data.

Experimental results show that the highest knockdown efficiency of the CRISPR/CasRx system constructed for the mouse Rock1 gene is 81% + -5% and guided by Rock1-gRNA4 (FIG. 1 c), while the highest knockdown efficiency of the CRISPR/CasRx system constructed for the mouse Rock2 gene is 81% + -3% and guided by Rock2-gRNA4 (FIG. 1 d). The highest knock-down efficiency of the CRISPR/CasRx system aiming at the mouse Aqp1 gene is 95% +/-5% and is guided by the Aqp1-gRNA2 (figure 1 e), and the highest knock-down efficiency of the CRISPR/CasRx system aiming at the mouse Adrb2 gene is 93% +/-14% and is guided by Adrb-gRNA 3 (figure 1 f). According to experimental results, one gRNA with the best knocking-down effect is selected for subsequent AAV virus packaging plasmid construction.

Example 2 screening of AAV serotypes targeting the ciliary body and trabecular meshwork for the treatment of glaucoma

The AAV viruses of different serotypes have different tissue specificities, and in order to ensure that serotypes which can target ciliary epithelium and trabecular meshwork tissues are selected and influence on non-target tissues is reduced, the invention selects and researches the infection condition of AAV2 and shH and the two serotypes on the ciliary epithelium and trabecular meshwork tissues in eyes. The method comprises the following steps:

(1) AAV2 and shH serotype green fluorescent viruses were ordered from Guangzhou Pi Biotechnology Inc., and were 1E+13GC/mL adjusted for viral titers.

(2) A12-week-old C57BL6/J female mouse (available from Shanghai Jieshiki laboratory animal Co., ltd.) was injected into both eyes by means of intravitreal injection at a dose of 2. Mu.L. 8 mice, 16 eyeballs, were injected with each virus.

(3) 2 Mice are randomly selected from the materials on the 7 th day, the 14 th day and the 21 th day after virus injection, eyeballs are taken as frozen sections to shoot confocal electron microscopes, the infection conditions of different serotypes of viruses are illustrated through green fluorescent signals, and meanwhile, the relation between the virus expression and the time is verified.

(4) And comprehensively evaluating the results, and screening out the most suitable virus serotypes, thereby customizing the packaging viruses by companies.

The present invention found that confocal images after days 7, 14 and 21 gave a strong fluorescent signal after shH a serotype infection (fig. 2 a), and that quantitative analysis of the green fluorescent signal gave a significant difference in the infectivity of ciliary epithelium and trabecular meshwork tissue compared to the control group (fig. 2 b).

Example 3 construction of AAV vector and evaluation of therapeutic Effect for delivery of CRISPR/CasRx System carrying therapeutic gRNA to treat glaucoma

3.1AAV viral packaging plasmid construction

The AAV viral genome is terminated Inverted Terminal Repeats (ITRs) at both ends, and the intermediate genome encodes two proteins, cap, which are viral capsid proteins, and Rep, which are involved in viral replication and integration. Three plasmids are required for AAV packaging, wherein the packaging plasmid is responsible for encoding the gene of interest and two ITR sequences, and the Helper plasmid contains the Cap and Rep genes required for AAV packaging, as well as the adenovirus Helper plasmid. The total AAV capacity is 4.7kb, so that the total size of the gene sequences between ITR sequences, including ITR sequences, of the packaging plasmid cannot exceed 4.7kb when the packaging plasmid is designed, and the shorter the target gene sequence is, the greater the packaging success rate of the virus is. Based on the principle, a smaller broad-spectrum promoter EFS is selected to drive CasRx protein expression in the design process, a U6 promoter is still selected to drive gRNA expression, and a flag tag protein is selected to replace fluorescent protein as an indication signal. In addition, because the experiment relates to a plurality of target genes on the same target tissue, in order to avoid multiple injections and strengthen the treatment effect, the gRNAs of Rock1 and Rock2, ADRB2 and AQP1 are respectively connected in series on the same packaging plasmid in design, and a CRISPR/CasRx editing system for targeting 2 genes is delivered by one virus, so that the treatment effect of efficiently reducing intraocular pressure by one injection is achieved.

Stbl3 competence is a strain recommended by a lentiviral vector system, and can effectively inhibit recombination of a long fragment terminal repeat region and reduce the probability of error recombination. After the packaging plasmid construction was completed, competent transformation with Stbl3 of the indigenous organism was performed. The sequence between ITRs was then sequenced by plating, selection of monoclonal, shaking culture, and Sanger sequencing, confirming that the plasmid was constructed correctly and without mutation, and then passed through the company for virus packaging (FIG. 3).

3.2 Establishment and evaluation of magnetic bead-induced ocular hypertension mouse model

3.2.1 Preparation of magnetic beads

Dynabeads M-450Epoxy was purchased from ThermoFisher, as the beads were coated with Epoxy groups. To prevent any adverse effects such as bead clumping and unwanted molecular interactions, these epoxy groups must first be removed from the microbeads and concentrated to a final concentration of 1.6X10 ⁶ beads/. Mu.L prior to injection surgery, and only 1.5. Mu.L is injected into the anterior chamber to achieve a 2.4X10 ⁶ bead injection.

(1) A solution of 0.02M sodium hydroxide (NaOH, MW 39.997 g/mol) was prepared in 10 XTris buffer (MW 121.14 g/mol).

(2) The magnetic microbead solution (4.5 μm diameter, 4×10 ⁸ beads/mL) was gently vortexed until the beads were uniformly suspended in the solution.

(3) 1ML of the magnetic bead solution was quickly pipetted into 50mL of 10 XTris buffer, 0.02M NaOH.

(4) The beads were spun at room temperature for 24 hours to remove the epoxy groups on the beads.

(5) The beads were collected by fixing a magnet to the bottom of the tube. The mixture was spun at room temperature for another 4 hours.

(6) Using a micropipette, the supernatant was carefully removed without disturbing the beads.

(7) Then 50mL of 10 XTris buffer of 0.02M NaOH was added, and steps (4) - (6) were repeated.

(8) The pellet was gently vortexed in 50ml 10x Tris buffer until the beads were fully suspended.

(9) Repeating the steps (4) - (6).

(10) 5ML of ultrapure water (advanced through 0.22 μm filter) was added, and the beads were washed by gentle swirling for 2 minutes.

(11) The beads were collected using a magnetic rack and the water was carefully removed with a micropipette without disturbing the beads.

(12) Repeating the steps (10) - (11) more than three times.

(13) The remaining steps are performed under sterile conditions in a laminar flow cabinet. With 500. Mu.L of sterile balanced salt solution (BBS)

The beads were blown and washed.

(14) Magnetic beads were collected using a magnetic rack and BSS was carefully removed with a micropipette without disturbing the beads.

(15) Repeating the steps (13) - (14) more than three times.

(16) Add 250. Mu.L BSS and blow the beads up and down to resuspend.

(17) Ensure the magnetic bead solution to be fully and evenly mixed. Then, 25. Mu.L of the suspension was rapidly dispensed into sterile 0.5mL

In the ep tube. The final concentration of bead stock was 1.6X10 ⁶ beads/. Mu.L.

(18) Stored at 4 ℃.

3.2.2. 2 Injection of magnetic beads in the anterior chamber to induce an increase in intraocular pressure in mice

(1) Food and water were obtained ad libitum using 12 week old C57BL6/J female mice (purchased from shanghai jetsche laboratory animals limited) raised in a standard environment.

(2) Mice were measured for daytime and nighttime baseline ocular pressure as described before the experiment.

(3) The mice were anesthetized by intraperitoneal injection of tribromoethanol at a dose of 400mg/kg after weighing.

(4) Topiramate eye drops are used for both eye mydriasis.

(5) The right eye is generally taken as an experimental eye, and local ointment is smeared on the contralateral eye (without operation) so as to avoid cornea dryness in the operation process.

(6) Clean microneedles are connected to injection assemblies of the microinjector pumps.

(7) Person a transfers the anesthesia mouse to the operating platform. Under the operation microscope, the pupil is ensured to be fully dilated, and the eye muscle is relaxed, so that the eyeball does not move. The surface anesthetic is dripped. Later, the eye drops on the eyes are gently rubbed off by a water-absorbing cotton swab.

(8) And B, mixing the prepared magnetic bead solution by up-and-down pipetting. The microneedles were immediately loaded with 1.5 μl of homogeneous magnetic bead solution using a microinjection pump. Ensuring that no bubbles are present at the tips of the microtips. After loading the microneedles, subsequent steps are performed as soon as possible, keeping the magnetic bead solution in a uniform suspension.

(9) The loaded microneedles were placed at a 45 ° angle, anterior to the limbus. Person a supports the eyes using forceps. Ensuring that the angle between the microneedle and the forceps is about 90.

(10) Person B gently prick the cornea with the loaded microneedle, with the tip of the microneedle entering the anterior chamber. Ensuring that the microneedles loaded during penetration remain at a 45 angle to the limbus. Avoiding contact with the lens or iris. Ensuring that the microneedles do not enter the posterior chamber. Person a continues to use forceps to support the eye.

(11) Person A places a magnet beside the eye, opposite the tip of the microneedle, to attract the magnetic bead to the anterior chamber and minimize contact of the bead with the inner surface of the cornea without moving the mouse head. Person B1.5. Mu.L of the magnetic bead solution was injected into the anterior chamber using a microinjection pump. The microbead solution was injected over a period of 15 to 30 seconds. Personnel A continue to face the magnet against the microneedle tip throughout the injection.

(12) Personnel B, after injecting the magnetic beads, slowly remove the microneedles from the eyes. Personnel A to avoid micro

The beads were refluxed and the attraction of the beads to the anterior chamber continued by holding the magnet close to the eye for a further 30 to 60 seconds.

(13) Person a uses a magnet to attract the beads to the corner of the room. Ensuring that the beads form a uniformly distributed ring around the anterior chamber. In this step, the beads are prevented from being attracted to the cornea because they have a tendency to stick to the inner surface of the cornea when contacted.

(14) An antibiotic eye ointment is applied to minimize the risk of infection.

(15) The mice were placed with the eyes facing up and the position was kept waiting for resuscitation. The injected beads are prevented from accumulating under gravity to the inner surface of the cornea.

(16) Intraocular pressure was measured one week after the operation. Once a week, daily and night ocular pressure was monitored at the same time of day to minimize circadian rhythm related fluctuations. Intraocular pressure was monitored in the control group with the same wild type mice remaining untreated.

(17) The slit lamp was photographed one week after the operation, and the distribution of the magnetic beads in the anterior chamber was evaluated.

3.2.3 Glucocorticoid-induced ocular hypertension model

A 10mg/mL suspension of dexamethasone acetate was prepared the day before each injection. 0.01g dexamethasone acetate powder was weighed and mixed with 1mL of solvent in a 2mL sterile ep tube, 25 mm stainless steel balls were added, and mixed in a cryomill at a frequency of 50 times per second for 5 minutes, which would break and homogenize the dexamethasone acetate particles to ensure the fine drug particle size formed. After this step, the drug was transferred into a magnetic stir glass bottle, wrapped in tinfoil paper, protected from light, and magnetically stirred overnight at 4 ℃ for further use.

(1) Animals were fed adaptively for 7 days after entering the animal house, and three eye drops were performed daily with 0.3% levofloxacin eye drops. (2) The basal intraocular pressure was measured by dropping 0.4% obucaine hydrochloride into eyes after anesthetizing the mice. In the prone position, tonometer was used to rapidly measure tonometer values, three times per eye and averaged. (3) Glucocorticoid injection, after completion of anesthesia, the surgical area of the operator was cleaned following a conventional sterilization procedure and 10 μl of DEX-Ac (aladin, china) or the same volume of saline was injected under the conjunctiva of the fornix of both eyes through a 29G needle. An infection-preventing 0.3% levofloxacin eye drop was immediately applied at the injection site. (4) Intraocular pressure monitoring

The intraocular pressure of the experimental eye was measured using TONOLAB rebound type mouse tonometer, mice were anesthetized with isoflurane, the anesthetic concentration was induced to 2.5%, the anesthetic concentration was maintained to 2%, and the intraocular pressure was measured at the center of the pupil. The daytime intraocular pressure was measured from 10 to 14 points. The measurement of ocular tension at night was completed from 21 to 24 points after at least 6 hours of dark field treatment.

The invention discovers that the day intraocular pressure level of the established magnetic bead induced ocular hypertension mouse model can be quickly increased to 22.944 +/-6.034 mmHg at the 1 st week after the model is made. From week 2 to week 6, the day-time intraocular pressure of the mice was steadily increased and maintained at 20mmHg on average. Mice with ocular hypertension induced by magnetic beads were treated by intravitreal injection on day 3 post-molding, and from week 2 onwards, the ocular pressure (16.2±0.467 mmHg) was significantly lower than the positive control group (19.2±0.735 mmHg), P <0.05, with ocular hypotensive effect lasting at least up to week 6 (fig. 4a, c). The established glucocorticoid induces the ocular pressure of the ocular hypertension mouse model to gradually rise and then to be stable. The intraocular pressure of the mice at week 1 after molding was 16.214.+ -. 0.556mmHg to 19.4.+ -. 0.909mmHg at week 10. Glucocorticoid-induced ocular hypertension mice were injected with the therapeutic virus via the vitreous cavity on day 3 post-molding, and the ocular tension (13.9±0.277 mmHg) was significantly reduced compared to the positive control group (17.2±0.712 mmHg) from week 1, with a P <0.01, which ocular tension lowering effect continued for at least 7 weeks (fig. 4b, d).

3.2.4 Safety evaluation

The important organs of the mice are taken at the 10 th week after the virus injection and frozen to see whether the systemic leakage of the virus is caused by the intravitreal injection mode or not, so as to evaluate the safety.

(1) Frozen specimens were subjected to frozen sections, and the thickness of the specimens was selected to be 20. Mu.m.

(2) Placing the cut pieces in a wet box, and marking the samples by using the strokes of the group of painting.

(3) OCT is washed off. The PBS was slowly added around the sample with a pipette without exceeding the range of the brush set. After 10 minutes of soaking, the solution was sucked off and more liquid was sucked off with absorbent paper.

(4) And (5) sealing the piece. Each sample was drip coated with 10. Mu.L of DAPI containing anti-fluorescence quencher, covered with a cover slip and sealed with clear nail polish.

(5) Note the whole procedure in the dark.

(6) The samples were stored at 4 ℃ protected from light and were photographed by confocal electron microscopy as soon as possible.

The invention discovers that the fluorescence labeling signals are not expressed in brain tissue, heart tissue, liver tissue, spleen tissue, lung tissue and kidney tissue by injecting the viruses through the vitreous cavity (figure 5), which shows that the risk of ectopic expression of AAV vector for treating glaucoma by injecting the CRISPR/CasRx system carrying gRNA through the vitreous cavity does not exist, and the invention has better safety.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A composition comprising:

(a) a gene editing protein or an expression vector thereof, wherein the gene editing protein comprises a type VI Cas protein; and

(b) gRNA or its expression vector, wherein the gRNA is an RNA that guides the gene editing protein to specifically bind to aqueous humor outflow-related genes and/or aqueous humor production-related genes in eye tissue, the aqueous humor outflow-related genes include Rock1 and Rock2, and the aqueous humor production-related genes include Aqp1 and Adrb2.

2. The composition of claim 1, wherein the region targeted by the gRNA is an exon region (such as exons 1, 2, 4, 5, 6, 22, 25, 27) of an aqueous humor outflow-related gene and/or an aqueous humor production-related gene sequence.

3. The composition of claim 1, wherein the gRNA guides the gene editing protein to simultaneously and specifically bind to RNA of a gene related to aqueous humor outflow in ocular tissue.

4. The composition of claim 1, wherein the gRNA guides the gene editing protein to simultaneously and specifically bind to RNA of a gene related to aqueous humor production in ocular tissue.

The composition according to claim 1 , wherein the aqueous humor outflow-related genes are Rock1 and Rock2.

The composition according to claim 1 , wherein the aqueous humor production-related genes are Aqp1 and Adrb2.

7 . The composition of claim 1 , wherein the nucleotide encoding the type VI Cas protein contains an RX ₄ -H motif, wherein X is 20 natural amino acids.

8. A medicine box, comprising:

(a1) a first container, and a gene-editing protein or its expression vector, or a drug containing the gene-editing protein or its expression vector, located in the first container, wherein the gene-editing protein comprises a type VI Cas protein;

(b1) a second container, and a gRNA or its expression vector, or a drug containing the gRNA or its expression vector, located in the second container, wherein the gRNA is an RNA that guides the gene editing protein to specifically bind to aqueous humor outflow-related genes and/or aqueous humor production-related genes in ocular tissue, wherein the aqueous humor outflow-related genes include Rock1 and Rock2, and the aqueous humor production-related genes include Aqp1 and Adrb2.

9. Use of the composition according to claim 1 or the medicine kit according to claim 2, characterized in that it is used to prepare a medicament for preventing and/or treating glaucoma or ocular hypertension.

10. A viral vector, comprising:

(a) a coding sequence for a gene editing protein, wherein the gene editing protein comprises a type VI Cas protein; and

(b) gRNA, wherein the gRNA is an RNA that guides the gene editing protein to specifically bind to aqueous humor outflow-related genes and/or aqueous humor production-related genes in eye tissue, the aqueous humor outflow-related genes include Rock1 and Rock2, and the aqueous humor production-related genes include Aqp1 and Adrb2.