Lipid nanoparticle (LNP)-based ocular delivery

Abstract

The content provided herein includes compositions, methods, and systems for delivering CRISPR / Cas mediated gene editing systems to trabecular meshwork cells using lipid nanoparticles (LNPs). Also provided herein are methods, compositions, and systems for treating glaucoma comprising reducing the expression of a Fibroin (MYOC) gene in trabecular meshwork cells of a patient's eye.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 353,374 filed on June 17, 2022 and U.S. Provisional Patent Application No. 63 / 417,233 filed on October 18, 2022 under 35 U.S.C. §119(e), the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to a sequence listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file entitled 80EM-341773-WO_SeqList, which was created on June 16, 2023 and is 479 kilobytes in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety.

[0005] background

[0006] field

[0007] The present disclosure relates generally to the fields of molecular biology and biotechnology, including gene editing.

[0008] Description of the Prior Art

[0009] Treatment of ocular diseases (e.g., glaucoma) requires accurate and effective ocular delivery of therapeutic agents, such as delivery to the trabecular meshwork cells of the patient. Mutations in the myocilin (MYOC) gene encoding myocilin are the cause of some forms of primary open-angle glaucoma (POAG) in adolescents and adults. Myocilin is a secreted 55kDa-57kDa glycoprotein that forms dimers and multimers. It has a myosin-like domain, a leucine zipper region, and an olfactomedin domain. Most mutations identified in patients with POAG are located in the olfactomedin domain, which is highly conserved between species. In the eye, myocilin is expressed in high amounts in the trabecular meshwork (TM), sclera, ciliary body, and iris, and is expressed in relatively low amounts in the retina and optic nerve head (Tamm, Prog Retin Eye Res. 2002 Jul; 21 (4): 395-428).

[0010] Glaucoma is a group of progressive optic neuropathy characterized by the degeneration of retinal ganglion cells and the resulting changes in the optic nerve head. There are several types of glaucoma, including POAG, angle-closure glaucoma, congenital glaucoma and normal-tension glaucoma. The loss of ganglion cells is related to the level of intraocular pressure (IOP), but other factors may also play a role. Lowering IOP is the only proven method for treating the disease. Although treatment usually starts with drops to lower intraocular pressure, laser trabeculoplasty and surgery can also be used to slow disease progression (Weinreb et al., JAMA. 2014 May 14; 311 (18): 1901-1911).

[0011] Overview

[0012] Disclosed herein is a method for delivering a CRISPR / Cas-mediated gene editing system to cells of a subject's eye, the method comprising administering to the subject more than one lipid nanoparticle (LNP) compounded with: (a) a guide RNA for a target gene or a nucleic acid encoding the guide RNA; and / or (b) an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, thereby reducing the expression of the target gene in the cells of the subject's eye. For example, the CRISPR / Cas-mediated gene editing system can be delivered to the subject's trabecular meshwork cells. The target gene can be, for example, a myocilin (MYOC) gene (e.g., a wild-type MYOC or mutant MYOC gene). In some embodiments, the expression of the target gene in the subject's eye, the expression of the protein encoded by the target gene, or both is reduced by at least 20%, at least 40%, at least 70%, or at least 90% after administration. In some embodiments, the expression of the target gene is reduced in the trabecular meshwork cells of the subject's eye.

[0013] The content disclosed herein also includes a method for treating a subject with glaucoma, the method comprising administering to the subject more than one lipid nanoparticle (LNP) complexed with the following: (a) a guide RNA targeting myocilin (MYOC) gene or a nucleic acid encoding the guide RNA; and (b) an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, thereby reducing the expression of the MYOC gene in the subject's eye. In some embodiments, glaucoma is myocilin-associated glaucoma. Glaucoma can be, for example, primary open-angle glaucoma (POAG). In some embodiments, the expression of the MYOC gene is reduced in the trabecular meshwork cells of the subject's eye.

[0014] In the methods described herein, the RNA-guided nuclease can be a Cas9 nuclease, such as a Staphylococcus aureus Cas9 (SaCas9) nuclease or a Streptococcus pyogenes Cas9 (SpCas9) nuclease. In some embodiments, the site targeted by the guide RNA is within exon 1, exon 2, or exon 3 of the MYOC gene. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-27 and 55-115. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 64, 73, 74, 75, 76, and 115. In some embodiments, the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-27 and 55-115. In some embodiments, the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115. In some embodiments, the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 10, 64, 73, 74, 75, 76, and 115. The guide RNA can be, for example, a SaCas9 sgRNA or a SpCas9 sgRNA.

[0015] In some embodiments, the guide RNA comprises a nucleotide sequence selected from SEQ ID NOs: 195-371. In some embodiments, the guide RNA comprises a nucleotide sequence selected from SEQ ID NOs: 258, 267-270, 309, 319, 328-331, 370, and 371.

[0016] In some embodiments, the LNP in more than one LNP comprises an ionizable cationic lipid, an auxiliary lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG-lipid). The LNP may comprise about 20%-60% of an ionizable lipid, about 18.5% to 60% of a sterol, about 0.01% to 30% of an auxiliary lipid, and / or about 0%-10% of a PEG-lipid. In some embodiments, the ionizable cationic lipid is selected from C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, DOTAP, DODAP, DC cholesterol, DLin-DMA, DLin-K-DMA, and DLin-KC2-DMA. In some embodiments, the helper lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diundecanoylphosphatidylcholine (DUPC), phosphatidylcholine (POPC), 1-stearoyl-2-oleoylphosphatidylcholine (DPPC), phosphatidylcholine (POPC), 1-stearoyl-2-oleoylphosphatidylcholine (DPPC), 1-diundecanoylphosphatidylcholine (DU ... In some embodiments, the sterol is selected from cholesterol, beta-sitosterol, beta-sitosterol, phytosterol, fucosterol, zoosterol and ergosterol. In some embodiments, the sterol is selected from cholesterol, sitosterol, campesterol, stigmasterol, fuccasterol, and ergosterol. In some embodiments, the PEG-lipid is DMG-PEG, DSG-PEG, PEG-ceramide, or PEG-phospholipid.In some embodiments, the LNP in more than one LNP comprises about 50mol% C12-200, DLIN-MC3, DODMA or DOTAP, about 10mol% DSPC, about 37.0mol%-39.5mol% cholesterol or sitosterol and about 0.5%-3.0% DMG-PEG. In some embodiments, LNP comprises about 50mol% C12-200, about 10mol% DSPC, about 37.0mol%-39.5mol% sitosterol and about 0.5%-1.5% DMG-PEG. In some embodiments, the average particle size of more than one LNP is about 80nm-100nm, and optionally 85nm-95nm.

[0017] More than one LNP can be administered to the subject, for example, by intravitreal injection or intracameral injection. In some embodiments, the method comprises administering more than one LNP to the subject in a single dose. In some embodiments, MYOC expression in the subject's eye is reduced by at least 20%, at least 40%, at least 70%, or at least 90% after administration. In some embodiments, myocilin in the trabecular meshwork cells of the subject's eye is reduced by at least 20%, at least 40%, at least 70%, or at least 90% after administration.

[0018] As described herein, the subject can be a human. In some embodiments, the LNP is respectively compounded with (a) a guide RNA or a nucleic acid encoding the guide RNA and (b) an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease. In some embodiments, the LNP compounded with (a) a guide RNA or a nucleic acid encoding the guide RNA and the LNP compounded with (b) an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease are different LNPs.

[0019] Disclosed herein are guide RNAs targeting a MYOC gene, the guide RNA comprising a nucleotide sequence specific to a fragment in exon 1, exon 2, or exon 3 of the MYOC gene, wherein the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from SEQ ID NOs: 1-27 and 55-115, or a spacer sequence having one, two, or three mismatches relative to an RNA sequence corresponding to any one of the nucleotide sequences selected from SEQ ID NOs: 1-27 and 55-115. In some embodiments, the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115. In some embodiments, the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from SEQ ID NOs: 10, 64, 73, 74, 75, 76, and 115. In some embodiments, the guide RNA comprises a nucleotide sequence selected from SEQ ID NOs: 195-371. In some embodiments, the guide RNA comprises a nucleotide sequence selected from SEQ ID NOs: 258, 267-270, 309, 319, 328-331, 370, and 371.

[0020] The content disclosed herein also includes a system for treating a subject with glaucoma, the system comprising: (i) a gene editing means for targeting the expression of the myocilin (MYOC) gene in the subject's eye; and (ii) a lipid nanoparticle (LNP), wherein the LNP delivers the gene editing means to the subject's eye. In some embodiments, glaucoma is myocilin-related glaucoma. Glaucoma can be primary open-angle glaucoma (POAG). In some embodiments, the gene editing means is CRISPR / Cas-mediated gene editing. In some embodiments, CRISPR / Cas-mediated gene editing comprises an RNA-guided nuclease and a guide RNA targeting a site in the MYOC gene. In some embodiments, the RNA-guided nuclease is a Cas9 nuclease, for example, a Staphylococcus aureus Cas9 (SaCas9) nuclease or a Streptococcus pyogenes Cas9 (SpCas9) nuclease.

[0021] In some embodiments, the site targeted by the guide RNA is within exon 1, exon 2, and exon 3 of the MYOC gene. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-27 and 55-115. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 64, 73, 74, 75, 76, and 115.

[0022] The guide RNA can be a SaCas9 sgRNA or a SpCas9 sgRNA. In some embodiments, the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 195-371. In some embodiments, the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 258, 267-270, 309, 319, 328-331, 370, and 371. In some embodiments, the LNP comprises an ionizable cationic lipid, an auxiliary lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG-lipid). In some embodiments, the LNP comprises about 20%-60% of an ionizable cationic lipid, about 18.5% to 60% of a sterol, about 0.01% to 30% of an auxiliary lipid, and / or about 0%-10% of a PEG-lipid. In some embodiments, the ionizable cationic lipid is selected from C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, DOTAP, DODAP, DC cholesterol, DLin-DMA, DLin-K-DMA and DLin-KC2_DMA. In some embodiments, the helper lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diundecanoylphosphatidylcholine (DUPC), phosphatidylcholine (POPC), 1-stearoyl-2-oleoylphosphatidylcholine (DPPC), phosphatidylcholine (POPC), 1-stearoyl-2-oleoylphosphatidylcholine (DPPC), 1-diundecanoylphosphatidylcholine (DU ... The 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-diphexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE,18:0-18:1PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dioleoylphosphatidylglycerol (DOPG) and dipalmitoyl-sn-glycero-3-PG (DPPG).

[0023] In some embodiments, sterol is selected from cholesterol, sitosterol, phytosterol, fucosterol, zoosterol and ergosterol. In some embodiments, sterol is selected from cholesterol, sitosterol, β-sitosterol, campesterol, stigmasterol, fucosterol and ergosterol. In some embodiments, PEG-lipid is DMG-PEG, DSG-PEG, PEG-ceramide or PEG-phospholipid. In some embodiments, LNP comprises about 50mol% of C12-200, DLIN-MC3, DODMA or DOTAP, about 10mol% of DSPC, about 37.0mol%-39.5mol% of cholesterol or sitosterol and about 0.5%-3.0% of DMG-PEG. In some embodiments, LNP comprises about 50mol% of C12-200, about 10mol% of DSPC, about 37.0mol%-39.5mol% of sitosterol and about 0.5%-1.5% of DMG-PEG. In some embodiments, the system is administered to the subject by intravitreal injection or intracameral injection.

[0024] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of several embodiments, and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A-1B Explains how to use the guidance number ( Figure 1A ) or by the total indel percentage ( Figure 1B ) Quantification of editing efficiency of sorted SaCas9 sgRNAs targeting the MYOC coding sequence.

[0027] Figure 2A-2B Explains how to use the guidance number ( Figure 2A ) or by total indel percentage ( Figure 2B ) Quantification of editing efficiency of sorted SpCas9 sgRNAs targeting the MYOC coding sequence.

[0028] Figure 3A-3B Source 1 LNP screening in primary trabecular meshwork (TM) cells (Source 1) in culture is illustrated. Figure 3A Includes LNP screening pictures. Figure 3B is a graph showing LNP screening GFP scoring.

[0029] Figure 4A-4B Described is a Source 2 LNP screen performed in an immortalized GTM3 cell line. Figure 4A Includes LNP screening pictures. Figure 4B is a graph showing LNP screening GFP scoring.

[0030] Figure 5A-Figure 5B Source 2 LNP screening in primary TM cells (Source 1) is illustrated. Figure 5A Includes LNP screening pictures. Figure 5B is a graph showing LNP screening GFP scoring.

[0031] Figure 6A-6B Source 2 LNP screening in primary TM cells (Source 2) is illustrated. Fig. 6A Includes LNP screening pictures. Figure 6B is a graph showing LNP screening GFP scoring.

[0032] Figure 7 Demonstrated is the quantification of editing efficiency of selected SpCas9 sgRNAs targeting the MYOC coding sequence using selected lipid nanoparticles for delivery in immortalized GTM3 cells.

[0033] Figure 8 Depicted is the Source 1 LNP screening GFP scoring performed in the mouse trabecular meshwork.

[0034] Fig. 9 Depicted is the GFP scoring of Source 2 LNP screening in the mouse trabecular meshwork.

[0035] Figure 10A-10C An exemplary embodiment of GFP protein expression in trabecular meshwork tissue following in vivo delivery of different LNP / eGFP mRNA in the mouse eye is demonstrated. Fig. 10A : shows IHC of GFP protein expression in mouse trabecular meshwork after delivery of source 1CTX-C12-CT. Fig. 10B : shows IHC of GFP protein expression in mouse trabecular tissue after intravitreal injection of LNP CTX-C12-200-CT / eGFP mRNA in mouse eyes. Fig. 10C : shows IHC of GFP protein expression in mouse trabecular tissue after intravitreal injection of LNP A14 / eGFP mRNA in mouse eyes.

[0036] Fig.11 Describes the delivery of LNP CTX-C12-200-CT / Cas9 mRNA / SpMCh10 sgRNA to the human glaucomatous trabecular meshwork cell line GTM3 MYOC Y437H -Exemplary embodiment of MYOC gene editing and myocilin knockdown after dsRED. Panel A: shows GTM3 MYOC from LNP transfection 7 days later Y437H -MYOC by dsRED Y437H- Epifluorescence microscopy of the dose-responsive decrease in dsRED protein expression. Panel B: shows GTM3MYOC from 5 days after LNP transfection Y437H MYOC-dsRED cell lysates Y437H - Immunoblot showing reduced expression of dsRED protein. Panel C: Myocilin from immunoblot data of the analysis shown in Panel B Y437H -Normalized expression of dsRED protein. Figure D: GTM3 MYOC 5 days after LNP transfection Y437H - Percent editing at both the genomic and transgenic MYOC loci in dsRED.

[0037] Fig.12 Illustrated is an exemplary embodiment of MYOC gene editing and actin knockdown following LNP CTX-C12-200-CT / Cas9 mRNA / SpMCh10 sgRNA delivery to human primary trabecular meshwork cells. Panel A: Percent editing at the genomic MYOC locus in LNP transfection in human primary trabecular meshwork cells. Panel B: Immunoblot showing reduced expression of actin in highly edited samples treated with dexamethasone. Panel C: Normalized expression of actin from immunoblot data of the analysis shown in Panel B.

[0038] Fig.13 An exemplary embodiment of IHC showing GFP protein expression in trabecular meshwork tissue following delivery of LNP CTX-C12-200-CT / eGFP mRNA in ex vivo anterior segment organ culture (ASOC) is illustrated.

[0039] Fig.14 Exemplary embodiments of MYOC gene editing and myocilin knockdown in trabecular meshwork tissue after delivery of LNP CTX-C12-200-CT / Cas9mRNA / MYOC sgRNA in ex vivo anterior segment organ culture (ASOC) are illustrated. Figure A: Percent editing at the genomic MYOC locus in DNA isolated from cornea, sclera, and trabecular meshwork tissue 4 days after LNP perfusion ASOC. Figure B: Immunoblot showing reduced myocilin expression from donor 1656 trabecular meshwork lysate 4 days after LNP perfusion ASOC. Figure C: Normalized expression of myocilin from immunoblot data of the analysis shown in Figure B.

[0040] Fig.15Demonstrates knockdown of α-SMA protein expression in the mouse trabecular meshwork after in vivo delivery of LNP CTX-C12-200-CT / Cas9 mRNA / ACTA2 gRNA in the mouse eye. Panel A: IHC scores for α-SMA protein expression from naive (untreated) and treated animals at 1, 2, and 4 weeks after LNP injection. Panel B: Example of an α-SMA protein expression IHC score of 3 in the trabecular meshwork from a naive (untreated) animal. Panel C: Example of an α-SMA protein expression IHC score of 0 in the trabecular meshwork from an animal treated 4 weeks after LNP injection.

[0041] Details

[0042] Reference is made to the accompanying drawings forming a part of this document in the following detailed description. In the accompanying drawings, similar symbols generally identify similar components unless the context otherwise indicates. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure as generally described herein and illustrated in the accompanying drawings can be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein and form a part of the present disclosure.

[0043] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety with respect to the relevant art.

[0044] Disclosed herein are methods, systems, compositions and kits for treating a subject suffering from glaucoma. The method comprises reducing the expression of an actin gene in trabecular meshwork cells of an eye of the subject.

[0045] definition

[0046] As used herein, the term "about" means plus or minus 5% of the value provided.

[0047] As used herein, the term "RNA-guided endonuclease" refers to a polypeptide that can bind to RNA (e.g., gRNA) to form a complex that targets a specific DNA sequence (e.g., in a target DNA). A non-limiting example of an RNA-guided endonuclease is a Cas polypeptide (e.g., a Cas endonuclease, such as a Cas9 endonuclease). In some embodiments, an RNA-guided endonuclease as described herein targets a specific DNA sequence in a target DNA through an RNA molecule to which it binds. The RNA molecule may include a sequence that is complementary to a target sequence within the target DNA and is capable of hybridizing with the target sequence, thereby allowing the bound polypeptide to be targeted to a specific location within the target DNA.

[0048] As used herein, the term "guide RNA" or "gRNA" refers to a site-specific targeting RNA that can bind to an RNA-guided endonuclease to form a complex and direct the activity of the bound RNA-guided endonuclease (such as a Cas endonuclease) to a specific target sequence within a target nucleic acid. A guide RNA can include one or more RNA molecules.

[0049] As used herein, the "secondary structure" of a nucleic acid molecule (e.g., an RNA fragment or a gRNA) refers to the base pairing interactions within the nucleic acid molecule.

[0050] As used herein, the term "Cas endonuclease" or "Cas nuclease" refers to an RNA-guided DNA endonuclease associated with the CRISPR adaptive immune system.

[0051] Unless otherwise indicated, "nuclease" and "endonuclease" are used interchangeably herein to refer to an enzyme with endonucleolytic catalytic activity for polynucleotide cleavage.

[0052] As used herein, the term "constant region" of gRNA refers to the nucleotide sequence of gRNA associated with the endonuclease guided by RNA. In some embodiments, gRNA includes crRNA and trans-activating crRNA (tracrRNA), wherein crRNA and tracrRNA hybridize with each other to form a duplex. In some embodiments, crRNA includes from 5' to 3': a spacer sequence and a minimum CRISPR repeat sequence (also referred to herein as "crRNA repeat sequence"); and tracrRNA includes a minimum tracrRNA sequence complementary to the minimum CRISPR repeat sequence (also referred to herein as "tracrRNA antisense repeat sequence") and a 3'tracrRNA sequence. In some embodiments, the constant region of gRNA refers to a portion of crRNA that is a minimum CRISPR repeat sequence and tracrRNA.

[0053] As used herein, the term "donor template" refers to a nucleic acid chain containing exogenous genetic material that can be introduced into the genome (e.g., by homology-directed repair) to cause targeted integration of the exogenous genetic material. In some embodiments, the donor template may not have a region homologous to the target position in the DNA, but can be integrated by NHEJ-dependent end connection after cleavage at the target site. The donor template can be single-stranded or double-stranded DNA or RNA, and can be introduced into the cell in a linear or circular form.

[0054] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. A polynucleotide can be a single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid / triple helix, or a polymer containing purine and pyrimidine bases (e.g., the five biologically occurring bases adenine, guanine, thymine, cytosine and uracil) or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. In some embodiments, a nucleic acid or polynucleotide can refer to any nucleic acid, whether comprising phosphodiester linkages or modified linkages, such as phosphotriester, phosphoramide, siloxane, carbonate, carboxymethyl ester, acetamide, carbamate, thioether, bridged phosphoramide, bridged methylenephosphonate, bridged phosphoramide, bridged phosphoramide, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate, or sultone linkages, and combinations of such linkages.

[0055] As used herein, the term "binding" refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). When in a non-covalent interaction state, the macromolecules are said to be "associated" or "interacting" or "bound" (e.g., when molecule X is said to interact with molecule Y, this means that molecule X binds to molecule Y in a non-covalent manner). The binding interaction can be characterized by a dissociation constant (Kd), such as a Kd of or less than: 10 -6 M, 10 -7 M, 10 - 8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15M or a number or range between any two of these values. Kd may depend on environmental conditions, such as pH and temperature. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower Kd.

[0056] As used herein, the term "hybridizing" or "hybridize" refers to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence is joined to a substantially complementary or fully complementary sequence by base pairing to form a hybrid complex. "Hybridizing" or "hybridize" can include denaturing a molecule to destroy one or more intramolecular structures (e.g., one or more secondary structures) in the molecule. In some embodiments, denaturing the molecule includes heating a solution containing the molecule to a temperature sufficient to destroy the intramolecular structure of the molecule. In some cases, denaturing the molecule includes adjusting the pH of the solution containing the molecule to a pH sufficient to destroy the intramolecular structure of the molecule. For the purpose of hybridization, two nucleic acid sequences or sequence segments are "substantially complementary" if at least 80% of the individual bases are complementary to each other. In some embodiments, the splint oligonucleotide sequence is no more than about 50% identical to one of the two polynucleotides (e.g., RNA fragments) designed to be complementary thereto. The complementary portions of each sequence may be referred to herein as "segments," and segments are substantially complementary if they have 80% or greater identity.

[0057] The terms "complementary" and "complementary" refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid based on the traditional Watson-Crick base pairing rules, i.e., adenine (A) pairs with thymine (U), and guanine (G) pairs with cytosine (C). Complementarity can be complete (e.g., complete complementarity) or incomplete (e.g., partial complementarity). Complete or complete complementarity indicates that each and all nucleic acid bases of one chain are able to form hydrogen bonds with corresponding bases in another antiparallel nucleic acid sequence according to the Watson-Crick standard base pairing. Partial complementarity indicates that only a certain percentage of consecutive residues in one nucleic acid sequence can form Watson-Crick base pairing with the same number of consecutive residues in another antiparallel nucleic acid sequence. In some embodiments, complementarity can be at least 70%, 80%, 90%, 100%, or a number or range between any two of these values. In some embodiments, complementarity is complete, i.e., 100%. For example, the complementary candidate sequence segment is completely complementary to the candidate sequence segment, and the sequence of the candidate sequence segment can be deduced from the candidate sequence segment using Watson-Crick base pairing rules.

[0058] As used herein, the term "vector" refers to a polynucleotide construct, typically a plasmid or a virus, for delivering genetic material to a host cell. A vector may be, for example, a virus, a plasmid, a cosmid, or a bacteriophage. A vector as used herein may include DNA or RNA. In some embodiments, a vector includes DNA. An "expression vector" is a vector that can direct the expression of a protein encoded by one or more genes carried by the vector when it is present in an appropriate environment. The vector is preferably capable of autonomous replication. Typically, an expression vector comprises a transcriptional promoter, a gene, and a transcriptional terminator. Gene expression is typically placed under the control of a promoter, and a gene is referred to as being "operably connected" to a promoter.

[0059] As used herein, the term "transfection" or "infection" refers to the introduction of nucleic acid into a host cell, such as by contacting the cell with a recombinant MVA virus or gutless picornaviral particles as described herein.

[0060] As used herein, the term "transgenic" refers to any nucleotide or DNA sequence that is integrated into one or more chromosomes of a target cell by human intervention. In some embodiments, the transgenic comprises a polynucleotide encoding a protein of interest. The polynucleotide encoding the protein is usually operably linked to other sequences that can be used to obtain the desired expression of the gene of interest, such as a transcriptional regulatory sequence. In some embodiments, the transgenic may additionally comprise a nucleic acid or one or more other molecules for marking the chromosome into which it has been integrated.

[0061] As used herein, "treatment" refers to clinical intervention for a disease, disorder, or physiological condition that a patient exhibits or to which the patient may be susceptible. The purposes of treatment include, but are not limited to, alleviating or preventing symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition, and / or alleviating a disease, disorder, or condition. "Treatment" refers to one or both of therapeutic treatment and prophylactic or preventive measures. Subjects in need of treatment include those already affected by a disease, disorder, or undesirable physiological condition, as well as those for whom a disease, disorder, or undesirable physiological condition is to be prevented.

[0062] As used herein, the term "effective amount" or "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce beneficial or desired biological and / or clinical results.

[0063] As used herein, the term "pharmaceutically acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable vehicle, additive, or diluent for administering a compound of interest to a subject. Pharmaceutically acceptable excipients may include substances known as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable vehicles.

[0064] As used herein, "subject" refers to an animal that needs diagnosis, treatment or therapy. In some embodiments, the subject is a mammal. "Mammal" as used herein refers to an individual belonging to the class of mammals, and includes but is not limited to humans, livestock and farm animals, zoo animals, sports animals and pets. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cattle; horses; primates, such as monkeys, chimpanzees and apes, and particularly humans. In some embodiments, mammals are primates. In some embodiments, mammals are humans. In some embodiments, mammals are not humans. In some aspects, the subject may suffer from or be suspected of having cardiovascular disease and / or have one or more symptoms of cardiovascular disease. In some aspects, the subject is a human being diagnosed as having a risk of cardiovascular disease at or after diagnosis. In some cases, the diagnosis of cardiovascular disease risk can be determined based on the presence of one or more mutations that may affect apo (a) protein expression in the genome sequence near the endogenous apolipoprotein (a) (LPA) gene or LPA gene in the genome.

[0065] The present disclosure provides effective compositions, methods, systems and means for delivering CRISPR / Cas mediated gene editing systems to trabecular meshwork cells using lipid nanoparticles (LNPs). As described herein, compositions, methods, systems and means can be used to treat eye diseases, for example, for treating myocilin-related glaucoma.

[0066] Trabecular meshwork (TM) cells are the major cell type that occupies and forms the proximal portion of the conventional outflow pathway, the major outflow pathway for aqueous humor. TM cells have pore-like structures through which aqueous humor circulates to Schlemm's canal. In the eye, the myocilin gene (MYOC) is abundantly expressed in the TM. Wild-type myocilin is secreted into the extracellular matrix (ECM) of the TM, whereas mutants form aggregates that lead to endoplasmic reticulum (ER) stress and death of TM cells. Some of the pathogenic potentials of myocilin include myocilin misfolding / unfolding; overexpression of myocilin; coaggregation of Grp94, which limits autophagy; disruption of ECM homeostasis caused by mutant myocilin; oxidative stress (OS), ER stress, and IL-1 / NF-κB inflammatory stress caused by mutant myocilin; and instability caused by conformational disturbances caused by mutant myocilin.

[0067] Therefore, the present invention provides an effective method for delivering a gene editing system to TM cells of a subject's eye (e.g., treating glaucoma patients with MYOC gene mutations), the method comprising knocking down / reducing the expression of a target gene or a protein encoded by a target gene in TM cells (e.g., myocilin expression). In these methods, mutant and wild-type myocilin alleles are targeted, mutant myocilin accumulated by TM cells is cleared to alleviate ER stress, and aqueous humor (AH) outflow is increased, and intraocular pressure (IOP) is reduced. Clinical readings include measurements of IOP.

[0068] In some embodiments, provided herein are methods and systems for treating a subject with glaucoma by targeted knockdown or knockout of the myocilin (MYOC) gene in the subject's eye, and more specifically, in the trabecular meshwork cells of the subject's eye.

[0069] I. Systems for delivering CRISPR / Cas-mediated gene editing systems to the trabecular meshwork (TM)

[0070] In some aspects, provided herein are compositions, methods and systems for delivering CRISPR / Cas-mediated gene editing systems to TM cells in a subject. The compositions, methods and systems can be used, for example, to treat subjects with glaucoma. Such compositions, methods and systems can include (i) gene editing means for targeting and reducing expression of target genes (e.g., myocilin (MYOC) genes, ACTA2 genes) in the eyes of subjects; and (ii) lipid nanoparticles (LNPs) for delivering gene editing means to the eyes of subjects.

[0071] The reduction of target gene (for example, MYOC gene, ACTA2 gene) expression can be achieved via gene editing (including genome editing), which is a genetic engineering, in which one or more nucleotides / one or more nucleic acids are inserted, deleted and / or replaced in a DNA sequence (such as in the genome of a target cell). Targeted gene editing enables insertion, deletion and / or replacement at a preselected site in the genome of a target cell (for example, in a targeted gene or a targeted DNA sequence). When the sequence of an endogenous gene, for example, is edited by the deletion, insertion or replacement of one or more nucleotides / one or more nucleic acids, the endogenous gene containing the affected sequence can be knocked out or knocked down due to sequence changes. Therefore, targeted editing can be used to destroy endogenous gene expression." Targeted integration" refers to the process of inserting one or more exogenous sequences, with or without the deletion of endogenous sequences at the insertion site. When there is a donor template containing an exogenous sequence, targeted integration can be produced by targeted gene editing.

[0072] (a) Genes edited

[0073] Once the system disclosed herein is delivered to the TM cells of the subject's eye, it will cause a target gene to be destroyed (e.g., MYOC gene, ACTA2 gene). As used herein, a "destroyed gene" refers to a gene that contains an insertion, deletion or substitution relative to an endogenous gene, so that the expression of a functional protein from an endogenous gene is reduced or inhibited. As used herein, "disrupting a gene" refers to a method in which at least one nucleotide / nucleic acid is inserted, deleted or substituted in an endogenous gene so that the expression of a functional protein from an endogenous gene is reduced or inhibited. Methods for destroying genes are known to those skilled in the art and are described herein.

[0074] In some embodiments, cells comprising a disrupted gene do not express (e.g., on the cell surface) detectable levels of a protein encoded by the gene (e.g., in an immunoassay using an antibody that binds to the encoded protein or in an immunoassay performed by flow cytometry). Cells that do not express detectable levels of a protein can be referred to as knockout cells. Cells that express reduced levels of a protein can be referred to as knockdown cells.

[0075] (b) MYOC gene editing

[0076] Actin is a secreted 55kDa-57kDa glycoprotein that forms dimers and multimers. It has a myosin-like domain, a leucine zipper region, and an olfactory modulatory protein domain. In the eye, actin is expressed in high amounts in the TM, sclera, ciliary body, and iris, and in considerably lower amounts in the retina and optic nerve head (Tamm, Prog Retin Eye Res. 2002 Jul; 21(4): 395-428).

[0077] In the present disclosure, the destroyed MYOC gene means that the expression of MYOC in TM cells is substantially reduced / knocked down or completely eliminated. The destroyed MYOC gene can include one or more genetic edits at one or more suitable target sites (e.g., in a coding region or a non-coding regulatory region such as a promoter region) that destroy the expression of the MYOC gene. Such target sites can be identified based on gene editing methods. Exemplary target sites for genetic editing can include exon 1, exon 2 or exon 3 of the MYOC gene, or a combination thereof. In some embodiments, one or more genetic edits may occur in exon 1.

[0078] Genetic editing can be induced by gene editing technology (e.g., CRISPR / Cas technology) using RNA-guided nucleases and guide RNAs targeting sites in the MYOC gene. In some embodiments, the RNA-guided nuclease can be a Cas9 nuclease, including but not limited to Staphylococcus aureus Cas9 (SaCas9) nuclease or Streptococcus pyogenes Cas9 (SpCas9) nuclease.

[0079] In some embodiments, the MYOC site targeted by the guide RNA comprises any one of the nucleotide sequences listed in Table 1 or Table 2 (see Sequence Listing below). In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-27 and 55-115. In some specific examples, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 64, 73, 74, 75, 76, and 115.

[0080] Exemplary MYOC targeting guide RNAs can be deduced from the target sequences listed in Table 1 or Table 2, which are also within the scope of the present disclosure. In some embodiments, the guide RNA can be a SaCas9 sgRNA or a SpCas9 sgRNA.

[0081] II. Gene Editing Methods

[0082] Reduction of expression of target genes (eg, MYOC gene, ACTA2 gene) in TM cells can be achieved via conventional gene editing methods or those described herein.

[0083] (a) Gene editing methods

[0084] Targeted editing can be achieved by nuclease-independent methods or by nuclease-dependent methods. In nuclease-independent targeted editing methods, homologous recombination is guided by homologous sequences flanking the exogenous polynucleotide to introduce endogenous sequences through the enzymatic machinery of the host cell. The exogenous polynucleotide can introduce deletions, insertions, or substitutions of nucleotides in the endogenous sequence.

[0085] Alternatively, the nuclease-dependent method can specifically introduce double-strand breaks (DSBs) by specific rare cutting nucleases (e.g., endonucleases) to achieve targeted editing with higher frequency. Such nuclease-dependent targeted editing also utilizes DNA repair mechanisms, such as non-homologous end joining (NHEJ) that occurs in response to DSB. DNA repair by NHEJ generally results in random insertion or deletion (insertion and deletion (indel)) of a small amount of endogenous nucleotides. Compared with the repair mediated by NHEJ, repair can also occur by homology-directed repair (HDR). When there is a donor template containing an exogenous genetic material flanked by a pair of homology arms, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material.

[0086] In some embodiments, gene disruption occurs by deleting a genomic sequence using two guide RNAs. Methods of using CRISPR-Cas gene editing technology to generate genomic deletions in cells (e.g., knocking out a gene in a cell) are known (Bauer DE et al. Vis. Exp. 2015; 95: e52118).

[0087] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR-Cas9 nucleases (CRISPR / Cas9; clustered regularly interspaced short palindromic repeats-related 9). Additionally, the DICE (Dual Integrase Cassette Exchange) system utilizing phiC31 and Bxb1 integrases can also be used for targeted integration. Some exemplary methods are disclosed in detail below.

[0088] (b) CRISPR-Cas9 gene editing system

[0089] The CRISPR-Cas9 system is a naturally occurring defense mechanism in prokaryotes, and has been reused as a DNA targeting platform for RNA guidance for gene editing. The CRISPR-Cas9 system relies on DNA nuclease Cas9 and two non-coding RNAs, crispr RNA (crRNA) and trans-activating RNA (tracrRNA) to target DNA cleavage. CRISPR is the abbreviation of clustered regular interspaced short palindromic repeats, which is a family of DNA sequences found in the genomes of bacteria and archaea, containing fragments (interval DNA) of DNA similar to the exogenous DNA previously exposed to cells, for example, by viruses that have infected or attacked prokaryotes. These DNA fragments are used by prokaryotes to detect and destroy similar exogenous DNA reintroduced, such as from similar viruses during subsequent attacks. The transcription of the CRISPR locus results in the formation of an RNA molecule comprising a spacer sequence, which is associated with and targets the Cas (CRISPR-related) protein that can recognize and cut foreign exogenous DNA. Various types and classes of CRISPR / Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37:67-78).

[0090] crRNA drives the sequence recognition and specificity of CRISPR-Cas9 complex by Watson-Crick base pairing with 20 nucleotide (nt) sequences in target DNA. Changing the sequence of 5' 20nt in crRNA allows the CRISPR-Cas9 complex to be targeted to a specific locus. If the target sequence is followed by a specific short DNA motif (having the sequence NGG) called the protospacer adjacent motif (PAM), the CRISPR-Cas9 complex only binds to the DNA sequence containing the first 20nt matching with crRNA. TracrRNA hybridizes with the 3' end of crRNA to form an RNA-duplex structure, which is bound by the Cas9 endonuclease to form a catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.

[0091] After the CRISPR-Cas9 complex binds to the DNA at the target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in base pairs (blunt ends). After the CRISPR-Cas9 complex binds to the DNA at a specific target site and forms a site-specific DSB, the next critical step is the repair of the DSB. Cells use two major DNA repair pathways to repair DSBs: non-homologous end joining (NHEJ) and homology-directed repair (HDR).

[0092] NHEJ is a robust repair mechanism that is highly active in most cell types, including non-dividing cells. NHEJ is error-prone and can often result in the removal or addition of between one and hundreds of nucleotides at the site of the DSB, although such modifications are typically <20nt. The resulting insertions and deletions (indels) can damage coding or non-coding regions of genes. Alternatively, HDR uses a long segment of homologous donor DNA provided endogenously or exogenously to repair DSBs with high fidelity. HDR is only active in dividing cells and occurs at relatively low frequencies in most cell types. In many embodiments of the present disclosure, NHEJ is used as a repair operator.

[0093] (c) Endonucleases for use in CRISPR

[0094] In some embodiments, a Cas9 (CRISPR-associated protein 9) endonuclease or a nucleotide sequence encoding a Cas9 endonuclease is used in a CRISPR system. The Cas9 enzyme can be one from Staphylococcus aureus (SaCas9), or one from Streptococcus pyogenes (SpCas9), although other Cas9 homologs can also be used. SaCas9 and SpCas9 DNA sequences are listed in Table 10 (see sequence table below). It should be understood that, as provided herein, wild-type Cas9 can be used or a modified form of Cas9 (e.g., an evolved form of Cas9 or a Cas9 ortholog or variant) can be used. In some embodiments, Cas9 can be replaced by another RNA-guided endonuclease such as Cpf1 (class II CRISPR / Cas system).

[0095] In some embodiments, the CRISPR / Cas system comprises components derived from type I, type II, or type III systems. An updated classification scheme for CRISPR / Cas loci defines class 1 and class 2 CRISPR / Cas systems, with types I to V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). Class 2 CRISPR / Cas systems have single protein effectors. Type II, type V, and type VI Cas proteins are single protein, RNA-guided endonucleases, referred to herein as "class 2 Cas nucleases". Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0096] In some embodiments, the Cas nuclease is from a type II CRISPR / Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is from a class 2 CRISPR / Cas system (a single protein Cas nuclease, such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 protein families are enzymes with DNA endonuclease activity, and as further described herein, they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA.

[0097] In some embodiments, the Cas nuclease comprises more than one nuclease domain. For example, the Cas9 nuclease may comprise at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces DSB in the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified so that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain. In some embodiments, the Cas9 nuclease is modified to contain no functional HNH-like nuclease domain. In some embodiments in which only one nuclease domain is functional, the Cas9 nuclease is a nickase capable of introducing single-strand breaks ("nicks") into the target sequence. In some embodiments, the conservative amino acids in the Cas9 nuclease domain are replaced to reduce or change the nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid substitution in a RuvC-like nuclease domain. Exemplary amino acid substitutions in a RuvC-like nuclease domain include D10A (based on Streptococcus pyogenes Cas9 nuclease). In some embodiments, the nickase comprises an amino acid substitution in a HNH-like nuclease domain. Exemplary amino acid substitutions in a HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on Streptococcus pyogenes Cas9 nuclease).

[0098] In some embodiments, the Cas nuclease is from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease is a component of the Cascade complex of a type I CRISPR / Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type IV CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type V CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type VI CRISPR / Cas system.

[0099] (d) Guide RNA (gRNA)

[0100] CRISPR technology involves the use of a genome-targeting nucleic acid that can guide an endonuclease to a specific target sequence in a target gene to perform gene editing at a specific target sequence. The nucleic acid targeting the genome can be RNA. The RNA targeting the genome is referred to herein as "guide RNA" or "gRNA". The guide RNA comprises a spacer sequence and a CRISPR repeat sequence that hybridizes at least to a target nucleic acid sequence in a target gene for editing.

[0101] In the type II system, the gRNA also includes a second RNA, called a tracrRNA sequence. In the type II gRNA, the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a duplex. In the type V gRNA, the crRNA forms a duplex. In both systems, the duplex binds to the site-directed polypeptide so that the guide RNA and the site-directed polypeptide form a complex. In some embodiments, the genome targeting nucleic acid provides target specificity to the complex through its association with the site-directed polypeptide. Therefore, the genome targeting nucleic acid guides the activity of the site-directed polypeptide.

[0102] As will be appreciated by one of ordinary skill in the art, each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

[0103] In some embodiments, the genome targeting nucleic acid (e.g., gRNA) is a bimolecular guide RNA. The bimolecular guide RNA comprises two chains of RNA molecules. The first chain comprises an optional spacer extension sequence, a spacer sequence, and a minimum CRISPR repeat sequence in the 5' to 3' direction. The second chain comprises a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0104] In some embodiments, the genome targeting nucleic acid (e.g., gRNA) is a single-molecule guide RNA. The single-molecule guide RNA (referred to as "sgRNA") in the II-type system includes an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide joint, a minimum tracrRNA sequence, a 3'tracrRNA sequence, and an optional tracrRNA extension sequence in the 5' to 3' direction. The optional tracrRNA extension sequence may include an element that contributes another function (e.g., stability) to the guide RNA. The single-molecule guide joint connects the minimum CRISPR repeat sequence and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension sequence includes one or more hairpins. The single-molecule guide RNA in the V-type system includes a minimum CRISPR repeat sequence and a spacer sequence in the 5' to 3' direction.

[0105] The spacer sequence in the gRNA is a sequence (e.g., a 20-nucleotide sequence) that defines a target sequence (e.g., a DNA target sequence, such as a genomic target sequence) of a target gene of interest. In some embodiments, the spacer sequence ranges from 15 to 30 nucleotides. For example, the spacer sequence may comprise 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence contains 20 nucleotides.

[0106] The "target sequence" is in the target gene adjacent to the PAM sequence, and is a sequence to be modified by a nuclease (e.g., Cas9) guided by RNA. The "target sequence" is on the so-called PAM-chain in the "target nucleic acid", which is a double-stranded molecule containing a PAM-chain and a complementary non-PAM chain. Those skilled in the art recognize that the gRNA spacer sequence hybridizes with the complementary sequence in the non-PAM chain of the target nucleic acid of interest. Therefore, the gRNA spacer sequence is the RNA equivalent of the target sequence. The spacer of the gRNA interacts with the target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the spacer varies according to the target sequence of the target nucleic acid of interest.

[0107] In the CRISPR / Cas system of this article, the spacer sequence is designed to hybridize with a region of the target nucleic acid located 5' of the PAM that can be recognized by the Cas9 enzyme used in the system. The spacer can completely match the target sequence, or there can be mismatches. Each Cas9 enzyme has a specific PAM sequence that it recognizes in the target DNA. For example, Streptococcus pyogenes Cas9 recognizes a PAM in the target nucleic acid that contains the sequence 5'-NRG-3', where R contains A or G, where N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.

[0108] In some embodiments, the length of the target nucleic acid sequence is 20 nucleotides. In some embodiments, the length of the target nucleic acid is less than 20 nucleotides. In some embodiments, the length of the target nucleic acid is more than 20 nucleotides. In some embodiments, the length of the target nucleic acid is at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the length of the target nucleic acid is at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence has 20 bases immediately adjacent to the first nucleotide 5' of PAM. For example, in the sequence comprising 5'-NNNNNNNNNNNNNNNNNNNN NGG -3' sequence, the target nucleic acid can be a sequence corresponding to N, wherein N can be any nucleotide, and the underlined NGG sequence is the Streptococcus pyogenes PAM.

[0109] The guide RNA disclosed herein can target any sequence of interest via the spacer sequence in the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. In some embodiments, the spacer sequence of the guide RNA is 100% complementary to the target sequence in the target gene. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, such as up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 mismatch.

[0110] For any gRNA sequences provided herein, those not explicitly indicating modification are intended to encompass both unmodified sequences and sequences with any suitable modifications.

[0111] The length of the spacer sequence in any gRNA disclosed herein can depend on the CRISPR / Cas9 system and components for editing any target gene also disclosed herein. For example, different Cas9 proteins from different bacterial species have different optimal spacer sequence lengths. Therefore, the length of the spacer sequence can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides. In some embodiments, the length of the spacer sequence can be 18-24 nucleotides. In some embodiments, the length of the targeting sequence can be 19-21 nucleotides. In some embodiments, the length of the spacer sequence can include 20 nucleotides.

[0112] In some embodiments, the gRNA may be a sgRNA, which may include a spacer sequence of 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA includes a variable length spacer sequence of 17-30 nucleotides at the 5' end of the sgRNA sequence.

[0113] In some embodiments, the sgRNA does not include uracil at the 3' end of the sgRNA sequence. In some embodiments, the sgRNA includes one or more uracils at the 3' end of the sgRNA sequence. For example, the sgRNA can include 1-8 uracil residues at the 3' end of the sgRNA sequence, for example, 1, 2, 3, 4, 5, 6, 7, or 8 uracil residues at the 3' end of the sgRNA sequence.

[0114] Any gRNA disclosed herein (including any sgRNA) may be unmodified. Alternatively, it may contain one or more modified nucleotides and / or a modified backbone. For example, a modified gRNA such as an sgRNA may contain one or more 2'-O-methyl phosphorothioate nucleotides, which may be located at the 5' end, the 3' end, or both.

[0115] In some embodiments, more than one guide RNA can be used with the CRISPR / Cas nuclease system. Each guide RNA can contain different targeting sequences so that the CRISPR / Cas system cleaves more than one target nucleic acid. In some embodiments, one or more guide RNAs can have the same or different properties within the Cas9 RNP complex, such as activity or stability. When more than one guide RNA is used, each guide RNA can be encoded on the same or different vectors. The promoters used to drive the expression of more than one guide RNA are the same or different.

[0116] In some embodiments, the gRNA disclosed herein targets the MYOC gene, for example, a site within any one of exons 1-3 of MYOC, for example, exon 1, exon 2, or exon 3 of the MYOC gene. Such a gRNA may comprise a spacer sequence or a fragment thereof that is complementary (completely or partially) to a target sequence in exon 1 of the MYOC gene. Exemplary target sequences for MYOC are provided in Tables 1-2 (see the sequence table below). Exemplary gRNA sequences can be derived from the target sequence. In some embodiments, the gRNA targets the ACTA2 gene.

[0117] In some embodiments, the gRNA comprises a spacer sequence having an RNA sequence corresponding to any one of the target sequences listed in SEQ ID NO: 1-27 and SEQ ID NO: 55-115 or variants thereof. In some embodiments, the gRNA comprises a spacer sequence having an RNA sequence corresponding to any one of the target sequences listed in SEQ ID NO: 1-27 and SEQ ID NO: 55-115. In some embodiments, the gRNA comprises a spacer sequence having one, two or three mismatches with an RNA sequence corresponding to any one of the target sequences listed in SEQ ID NO: 1-27 and SEQ ID NO: 55-115. In some embodiments, the gRNA comprises a spacer sequence having about, at least or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology with an RNA sequence corresponding to any one of the target sequences listed in SEQ ID NO: 1-27 and SEQ ID NO: 55-115.

[0118] The gRNA spacer sequence is the RNA equivalent of the target sequence. Therefore, the gRNA sequence may include a spacer sequence corresponding to any one of SEQ ID NO: 1-27 and SEQ ID NO: 55-115, wherein "T" is replaced by "U". In some embodiments, the spacer subsequence included in the gRNA sequence may be a variant of a spacer sequence having about, at least or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology with any spacer corresponding to any one of SEQ ID NO: 1-27 and SEQ ID NO: 55-115, wherein "T" is replaced by "U". In some embodiments, the spacer subsequence included in the gRNA sequence may be a variant of a spacer sequence having one, two or three mismatches compared to any spacer corresponding to any one of SEQ ID NO: 1-27 and SEQ ID NO: 55-115, wherein "T" is replaced by "U".

[0119] In some embodiments, the gRNA comprises a spacer sequence having an RNA sequence corresponding to any one of the target sequences listed in SEQ ID NOs: 6, 10, 15, 18, and 26, or variants thereof. In some embodiments, the gRNA comprises a spacer sequence having one, two, or three mismatches with an RNA sequence corresponding to any one of the target sequences listed in SEQ ID NOs: 6, 10, 15, 18, and 26.

[0120] In some embodiments, the gRNA comprises a spacer sequence having an RNA sequence corresponding to any one of the target sequences listed in SEQ ID NO: 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115, or variants thereof. In some embodiments, the gRNA comprises a spacer sequence having one, two, or three mismatches with an RNA sequence corresponding to any one of the target sequences listed in SEQ ID NO: 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115. For example, as shown below and described in the art, the guide RNA or other smaller RNA used in the CRISPR / Cas / Cpf1 system can be easily synthesized by chemical means. Although chemical synthesis methods continue to expand, purification of such RNA by methods such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging because polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One method for producing RNAs of greater length is to produce two or more molecules that are linked together. It is easier to produce much longer RNAs enzymatically, such as RNAs encoding Cas9 or Cpf1 endonucleases. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic production of RNA, for example, modifications that enhance stability, reduce the likelihood or extent of an innate immune response, and / or enhance other properties, as described in the art.

[0121] In some embodiments, the gRNA of the present disclosure is produced by in vitro transcription (IVT), synthesis and / or chemical synthesis methods or a combination thereof. Enzymatic (IVT), solid phase, liquid phase, combinatorial synthesis methods, small area synthesis and ligation methods are used. In some embodiments, gRNA is prepared using IVT enzymatic synthesis methods. Methods for preparing polynucleotides by IVT are known in the art and are described in WO2013 / 151666. Therefore, the present disclosure also includes polynucleotides (e.g., DNA), constructs and vectors for in vitro transcription of the gRNA described herein.

[0122] Various types of RNA modifications can be introduced during or after the chemical synthesis and / or enzymatic generation of RNA, for example, as described in the art, to enhance stability, reduce the possibility or degree of innate immune response and / or enhance other properties. In some embodiments, non-natural modified nucleobases can be introduced into any gRNA disclosed herein during or after synthesis. In some embodiments, the modification is on the internucleoside linkage, purine or pyrimidine base or sugar. In some embodiments, the modification is introduced at the end of the gRNA by chemical synthesis or by a polymerase. Modified nucleic acids and examples of their synthesis are disclosed in WO2013 / 052523. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, Vol. 76, 99-134 (1998).

[0123] The chemically modified gRNA can include one or more thiophosphorylated 2'-O-methyl nucleotides at the 3' end and / or 5' end of the gRNA. In some embodiments, the chemically modified gRNA includes thiophosphorylated 2'-O-methyl nucleotides at the 3' end of the gRNA. In some embodiments, the chemically modified gRNA includes thiophosphorylated 2'-O-methyl nucleotides at the 5' end of the gRNA. In some embodiments, the chemically modified gRNA includes three or four thiophosphorylated 2'-O-methyl nucleotides at the 3' end of the gRNA and / or includes three or four thiophosphorylated 2'-O-methyl nucleotides at the 5' end. In some embodiments, any one of SEQ ID NO: 18-25 and 26-31 can be chemically modified to have one, two, three or four thiophosphorylated 2'-O-methyl nucleotides at the 3' end of the gRNA; One, two or three thiophosphorylated 2'-O-methyl nucleotides at the 5' end of the gRNA, or a combination thereof.

[0124] The number and position of phosphorothioate linkages can vary. In some embodiments, the bond can be between the first and second, second and third, third and fourth positions, fourth and fifth, fifth and sixth, sixth and seventh, seventh and eighth, eighth and ninth, ninth and tenth or other positions at the 5' end of the gRNA. In some embodiments, the bond can be between the first and second, second and third, third and fourth positions, fourth and fifth, fifth and sixth, sixth and seventh, seventh and eighth, eighth and ninth, ninth and tenth or other positions at the 3' end of the gRNA.

[0125] In some embodiments, nucleotide analogs / modifications may include 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate , 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodo-uridine-5 '-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate , 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate or xanthosine-5'-triphosphate. The base modified nucleotides may include 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate and pseudouridine-5'-triphosphate, pyridine-4-ketone ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl uridine, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine, 1-taurine methyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5 -methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wyosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio- Guanosine, 5'-O-(1-phosphorothioate)-adenosine, 5'-O-(1-phosphorothioate)-cytidine, 5'-O-(1-phosphorothioate)-guanosine, 5'-O-(1-phosphorothioate)-uridine, 5'-O-(1-phosphorothioate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine,7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine or 7-deaza-adenosine.

[0126] At least one modified nucleotide and / or at least one nucleotide analog may include 1-methyladenosine, 2-methyladenosine, N6-methyladenosine, 2'-O-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, inosine, 3-methylcytidine, 2-O-methylcytidine, 2-thiocytidine, N4-acetylcytidine, lysine, 1-methylguanosine, 7-methylguanosine, 2'-O-methylguanosine, braided glycoside, epoxy braided glycoside, 7-cyano-7-deazaguanosine, 7-aminomethyl-7-deazaguanosine, pseudouridine, dihydrouridine, 5-methyluridine, 2'-O-methyluridine, 2-thiouridine, 4-thiouridine, 5-methyl-2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 5'-hydroxyuridine, 5-methoxyuridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine or 5-(isopentenylaminomethyl)-2'-O-methyluridine.

[0127] In some embodiments, the chemical modification includes pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the modification comprises 2'-O-methyluridine (2'OMe-rU), 2-O-methylcytidine (2'OMe-rC), 2'-O-methyladenosine (2'OMe-rA), or 2'-O-methylguanosine (2'OMe-rG).

[0128] In some embodiments, enzymatic or chemical ligation methods can be used to conjugate polynucleotides or regions thereof to different functional moieties such as targeting or delivery agents, fluorescent markers, liquids, nanoparticles, etc. Conjugation of polynucleotides and modified polynucleotides is reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0129] In some embodiments, the CRISPR / Cas nuclease system for use in any target gene disclosed herein for genetic editing includes at least one guide RNA. In some embodiments, the CRISPR / Cas nuclease system comprises more than one gRNA, such as 2, 3 or 4 gRNAs. Such more than one gRNA can target different sites in the same target gene. Alternatively, more than one gRNA can target different genes. In some embodiments, the guide RNA and the Cas protein can form a ribonucleoprotein (RNP), for example, a CRISPR / Cas complex. The guide RNA can guide the Cas protein to a target sequence on one or more target genes as disclosed herein, wherein the Cas protein cleaves the target gene at the target site. In some embodiments, the CRISPR / Cas complex is a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex is a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex is a Cas9 / guide RNA complex.

[0130] In some embodiments, the insertion and deletion frequency (editing frequency) of a specific CRISPR / Cas nuclease system comprising one or more specific gRNAs can be determined using TIDE analysis, which can be used to identify efficient gRNA molecules for editing target genes. In some embodiments, efficient gRNA produces a gene editing frequency higher than 80%. For example, if the gRNA produces a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%, the gRNA is considered to be efficient.

[0131] (e) Other gene editing methods

[0132] In addition to the CRISPR methods disclosed herein, additional gene editing methods known in the art can also be used. Some examples include gene editing methods involving zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), restriction endonucleases, meganucleases, homing endonucleases, and the like.

[0133] ZFN is a targeted nuclease comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within a zinc finger binding domain whose structure is stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain that does not exist in nature, and its design / composition is primarily derived from rational criteria, e.g., applying substitution rules and computerized algorithms to process information in a database storing information on existing ZFP designs and binding data. See, e.g., U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO02 / 016536 and WO 03 / 016496. The zinc finger domains selected are domains that do not exist in nature and are generated primarily from empirical processes such as phage display, interaction traps, or hybrid selection. ZFNs are described in more detail in U.S. Pat. Nos. 7,888,121 and 7,972,854. The most recognized example of a ZFN is a fusion of a FokI nuclease with a zinc finger DNA binding domain.

[0134] TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. A "transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain" or "TALE DNA binding domain" is a polypeptide domain of a TAL effector protein that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind effector-specific DNA sequences via their DNA binding domains, and activate gene transcription of these sequences via their transactivation domains. TAL effector DNA binding domain specificity depends on the effector-variable number of incomplete 34 amino acid repeats, which contain polymorphisms at selected repeat positions called repeat variable diresidues (RVDs). TALEN is described in more detail in U.S. Patent Application No. 2011 / 0145940. The most recognized example of TALEN in the art is a fusion polypeptide of the FokI nuclease and the TAL effector DNA binding domain.

[0135] Additional examples of targeted nucleases suitable for use as provided herein include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether used alone or in combination.

[0136] Any of the nucleases disclosed herein can be delivered using a vector system including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors; herpesvirus vectors, and adeno-associated virus vectors, and combinations thereof.

[0137] Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor templates into cells. Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acids, and nucleic acids compounded with delivery vehicles such as liposomes or poloxamers. Viral vector delivery systems include DNA viruses and RNA viruses that have additional genomes or integrated genomes after delivery to cells.

[0138] Non-viral delivery methods for nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virosomes, and agent-enhanced DNA uptake. Acoustic perforation using, for example, the Sonitron 2000 system (Rich-Mar) can also be used for the delivery of nucleic acids. Some specific examples are provided below.

[0139] III. Delivery of guide RNA and nuclease to TM cells

[0140] The CRISPR / Cas nuclease system disclosed herein comprises a guide RNA (gRNA) or a nucleic acid sequence encoding the gRNA and an RNA-guided nuclease or a nucleic acid sequence encoding the RNA-guided endonuclease, which can be delivered to TM cells via conventional methods for genetic editing of target genes (e.g., MYOC gene or ACTA2 gene). In some embodiments, the components of the CRISPR / Cas nuclease system as disclosed herein can be delivered to the target cell individually, simultaneously or sequentially. In other embodiments, the components of the CRISPR / Cas nuclease system can be delivered to the target together, for example as a complex. In some cases, the gRNA and RNA-guided nuclease can be pre-compounded together to form a ribonucleoprotein (RNP), and the RNP can be delivered to the target cell.

[0141] RNP can be used for gene editing, at least because they minimize the risk of promiscuous interactions in a cell environment rich in nucleic acids and protect RNA from degradation. Methods for forming RNP are known in the art. In some embodiments, the RNP containing the nuclease guided by RNA (e.g., Cas nuclease, such as Cas9 nuclease) and the guide RNA targeting the MYOC gene can be delivered to TM cells. In some embodiments, RNP can be delivered to TM cells by electroporation.

[0142] In some embodiments, the RNA-guided nuclease can be delivered to the cell in a DNA vector that expresses the RNA-guided nuclease in the cell. In other examples, the RNA-guided nuclease can be delivered to the cell in an RNA that encodes the RNA-guided nuclease and expresses the nuclease in the cell. Alternatively or additionally, the gRNA targeting the gene can be delivered to the cell as RNA or a DNA vector that expresses the gRNA in the cell.

[0143] Delivery of RNA-guided nucleases, gRNAs and / or RNPs can be by direct injection or cell transfection using known methods (e.g., electroporation or chemical transfection). Other cell transfection methods can be used.

[0144] In some embodiments, one or more of the nucleic acid sequences and / or polypeptides can be delivered to cells in vitro or in vivo via a viral or non-viral based delivery system, including adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, lentiviral vectors, herpes virus vectors, nanoparticles, liposomes, lipid nanoparticles, poxviruses, naked DNA administration, plasmids, cosmids, bacteriophages, encapsulated cell technology, and the like.

[0145] In some embodiments, gRNA and RNA-guided endonucleases or nucleic acids encoding the RNA-guided endonucleases or their compositions can be formulated in liposomes or lipid nanoparticles. In some embodiments, RNA-guided nucleases and guide RNA can be delivered to trabecular meshwork cells by lipid nanoparticles (LNPs). The term "lipid nanoparticle" refers to a nanoscale particle composed of lipids with a size measured in nanometers (e.g., 1-5,000nm). The size of the LNP in the LNP formulations described herein (e.g., CTX-C12-CT LNP formulations) can vary. In some embodiments, LNP has about, at least, at least about, at most or at most about 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm or the average diameter of the numerical value or range between any two of these values. In some embodiments, lipid nanoparticle particle size is a diameter of about 50nm to about 200nm or a diameter of about 70nm to about 180nm or a diameter of about 80nm to about 150nm. In some embodiments, the particle size (e.g., mean diameter) of LNP is in the range of 85nm-95nm. In some embodiments, the particle size (e.g., mean diameter) of LNP is about 190nm, 195nm, 200nm, 205nm or any two of these values. Not bound by any particular theory, it is believed that using small size LNP to deliver payload to trabecular meshwork may be advantageous. For example, using a size of 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm or any two of these values ​​The LNP of the quantity or scope between the CRISPR / Cas-mediated gene editing system is delivered to the trabecular cells of the subject may be advantageous.

[0146] In some embodiments, the lipids included in the lipid nanoparticles include cationic lipids and / or ionizable lipids.Any suitable cationic lipids and / or ionizable lipids known in the art can be used to prepare LNPs for delivery of gRNA and Cas endonucleases to cells.Exemplary cationic lipids include one or more positively charged amine groups.Lipid nanoparticles can also include one or more neutral lipids, charged lipids, sterols, tocopherols, hopanes and polymer-conjugated lipids, such as poly (ethylene glycol) (PEG)-lipids.

[0147] LNP described herein may include one or more ionizable cationic lipids described herein. For example, LNP may include one or more ionizable cationic lipids selected from the group consisting of: C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA or DOTAP. Ionizable cationic lipids may be about 30mol% to about 70mol% (e.g., 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, 55mol%, 60mol%, 65mol%, 70mol%) of the total lipid present in LNP. As used herein, "molar percentage" refers to the molar percentage of the total moles of all lipid components in LNP (i.e., the total moles of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids). In some embodiments, LNP includes about 40% to about 60% ionizable cationic lipids accounting for the total lipids in LNP. For example, lipid nanoparticles can include an ionizable cationic lipid that accounts for about 40%, 45%, 50% or 60% of total lipid by mole (based on 100% total moles of lipid in LNP). In some embodiments, LNP comprises an ionizable cationic lipid described herein of about 50 mole percent.

[0148] The LNP described herein may also include one or more non-cationic lipids (helper lipids). In some embodiments, LNP may also include one or more neutral lipids, charged lipids, sterols, and polymer-conjugated lipids. In some embodiments, lipid nanoparticles include one or more neutral or zwitterionic lipids. The term "neutral lipid" refers to any of a variety of lipid substances that exist in the form of uncharged or neutral zwitterions at physiological pH. The selection of neutral lipids and other non-cationic lipids used in the particles described herein is generally guided by considerations such as lipid particle size and the stability of lipid particles in the bloodstream. In some embodiments, non-cationic lipids include having a carbon chain length in the range of C 1 to C 2. 10 -C 20 In some embodiments, saturated fatty acids having a carbon chain length in the range of C 10 -C 20The non-cationic lipids of mono-or di-unsaturated fatty acids within the scope. Additionally, the non-cationic lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Suitable neutral lipids include but are not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleic acid). diimidomethyl)cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dimyristoylphosphatidylcholine (DMPC), distearoyl-phosphatidyl-ethanolamine (DSPE), SM, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol or a mixture thereof. In some embodiments, the helper lipid is or comprises: PC lipids (e.g., DLPC (12:0), DMPC (14:0), DPPC (16:0), DSPC (18:0), DOPC (18:1), DUPC (18:2), POPC (16:0, 18:1), SOPC (18:0, 18:1)); PE lipids (e.g., DOPE (18:1), DSPE (18:0), DPPE (16:0), DMPE (14:0), SOPE (18:0, 18:1), POPE (16:0, 18:1)); PG lipids (e.g., DOPG (18:1), DPPG (16:0)) or mixtures thereof. In some embodiments, the helper lipid is or comprises 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), DMPC, DPPC, DSPC, DOPC, diundecanoylphosphatidylcholine (DUPC), POPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), DOPE, DSPE, DPPE, DMPE, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE, 18:0-18:1 PE), POPE, dioleoylphosphatidylglycerol (DOPG), dipalmitoyl-sn-glycero-3-PG (DPPG), or a mixture thereof.

[0149] In some embodiments, the neutral lipid can account for about 5 mol% to about 20 mol% (e.g., about 5 mol%, 10 mol%, 15 mol%, 20 mol%) of the total lipid present in the LNP. In some embodiments, the LNP includes a neutral lipid that accounts for about 10% of the total lipid in the LNP by mole (based on 100% total moles of lipid in the LNP).

[0150] LNP can also include sterol, such as cholesterol.Sterol can be about 10mol% to about 60mol% of the total lipid present in LNP, optionally about 20mol% to about 50mol%, more optionally about 30mol% to about 40mol%.In some embodiments, sterol is about 20mol%, 25mol%, 30mol%, 35mol%, 40mol%, 45mol% or 50mol% of the total lipid present in LNP.In LNP described herein, sterol can be cholesterol, sitosterol, campesterol, phytosterol (plant sterol) (also referred to as phytosterol (phytosterol), for example, stigmasterol, beta-sitosterol), from the sterol of algae (for example, fucosterol), from the sterol of animal (also referred to as " zoosterol ") and from the sterol (for example ergosterol) of fungi and protozoa One or more.LNP disclosed herein can include tocopherol and hopane (rebeline and rebeline alcohol) class compound. In some embodiments, tocopherols and hopanes (lipene and lipene alcohol) are used to replace sterols in LNPs. In some embodiments, tocopherols and hopanes (lipene and lipene alcohol) are present in LNPs in addition to sterols.

[0151] LNP can also include the lipid of polymer conjugation, such as the lipid modified by polyethylene glycol (PEG).The lipid that exemplary PEG-conjugates includes, for example, PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEG-dimyristoylglycerol (DMG) or its mixture.In some embodiments, the lipid that PEG conjugates can be about 0mol% to about 10mol% of total lipid in LNP.For example, the lipid that PEG conjugates is about 0mol%, 0.5mol%, 1mol%, 1.5mol%, 2mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol%, 8mol%, 9mol% or 10mol% of the total lipid present in LNP.In some embodiments, the lipid that polymer conjugates (for example, the lipid that PEG conjugates) is about 0mol% to about 5mol% (for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%) of the total lipid present in LNP. The PEG-modified lipid can be or can include, for example, DMG-PEG, DSG-PEG, PEG-ceramide, PEG-phospholipid, or a combination thereof.

[0152] In some embodiments of the LNPs described herein, the ionizable cationic lipid can be C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, or DOTAP; the auxiliary lipid can be 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); the sterol can be cholesterol or sitosterol; and the PEG-lipid can be DMG-PEG.

[0153] In some embodiments, LNP comprises about 50mol% C12-200, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA and / or DOTAP, about 10mol% DSPC, about 37.0mol%-39.5mol% cholesterol or sitosterol and about 0.5%-3.0% DMG-PEG. In some specific examples, LNP comprises about 50mol% C12-200, about 10mol% DSPC, about 37.0mol%-39.5mol% sitosterol and about 0.5%-1.5% DMG-PEG.

[0154] The LNPs can be administered by any suitable route resulting in delivery to the patient's eye. For example, an effective amount of the LNPs can be administered to the patient by intravitreal injection and / or intracameral injection.

[0155] IV. Compositions and Therapeutic Applications

[0156] The Cas9 gRNA system disclosed herein can be administered to a subject for therapeutic purposes, e.g., for the treatment of myocilin-related glaucoma. Disruption of the MYOC gene knocks down / reduces myocilin expression in trabecular meshwork (TM) cells, which in turn leads to the clearance of accumulated mutant myocilin, thereby alleviating ER stress, increasing aqueous humor (AH) outflow, and reducing intraocular pressure (IOP).

[0157] Therefore, a method for treating a subject with glaucoma is provided herein, the method comprising reducing the expression of the myocilin (MYOC) gene in the TM cells of the subject's eyes. In some embodiments, the method for treating a subject with glaucoma comprises administering to the subject more than one lipid nanoparticle (LNP), the lipid nanoparticle being compounded with the following: (a) a guide RNA targeting the myocilin (MYOC) gene or a nucleic acid encoding the guide RNA; and (b) an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, thereby reducing the expression of the MYOC gene in the subject's eyes. In some embodiments, glaucoma may be myocilin-related glaucoma. In some embodiments, glaucoma may be POAG. In some embodiments, the expression of the MYOC gene may be reduced in the TM cells of the subject's eyes.

[0158] As described above and herein, the MYOC gene in TM cells can be destroyed by a CRISPR / Cas-mediated gene editing system, which comprises an RNA-guided nuclease (or a nucleotide sequence encoding the RNA-guided nuclease) and a guide RNA targeting a site in the MYOC gene (e.g., exon 1, 2, or 3 of the MYOC gene). In some embodiments, the CRISPR / Cas-mediated gene editing system can be provided in a pharmaceutical composition. Therefore, the composition may comprise one or more gRNAs (MYOC gRNAs) described herein, RNA-guided endonucleases, or nucleotide sequences encoding the RNA-guided endonucleases. The MYOC gRNA may be any gRNA targeting one or more target sequences of SEQ ID NOs: 1-27 and 55-115 or variants thereof described herein. The RNA-guided endonuclease or the nucleotide sequence encoding the RNA-guided endonuclease may be any RNA-guided endonuclease described herein. In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the Cas9 endonuclease is a Streptococcus pyogenes (SpCas9) nuclease. In some embodiments, the Cas9 endonuclease is a Staphylococcus aureus Cas9 (SaCas9) nuclease.

[0159] In some embodiments, RNA-guided nucleases and guide RNA are delivered to trabecular meshwork cells via lipid nanoparticles (LNPs). LNPs may include one or more ionizable cationic lipids selected from C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA or DOTAP; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) auxiliary lipids; sterols of cholesterol and / or sitosterol; and PEG-lipids of DMG-PEG. LNPs may include one or more PEG glycerides, for example, PEG glycerides in the DMG-PEG and DSG-PEG categories, and in some embodiments, LNPs include one or more DSG-PEGs. LNP can comprise one or more PEG-ceramides, such as C16-PEG 2000 ceramide or C8 PEG 2000 ceramide; one or more PEG phospholipids, such as 14:0PEG 2000PE; or any combination thereof. In some embodiments, LNP comprises PEG-ceramide. In some embodiments, LNP comprises PEG-phospholipids.

[0160] In some embodiments, LNP comprises about 50mol% C12-200, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA and / or DOTAP, about 10mol% DSPC, about 37.0mol%-39.5mol% cholesterol or sitosterol and about 0.5%-3.0% DMG-PEG. In some specific examples, LNP comprises about 50mol% C12-200, about 10mol% DSPC, about 37.0mol%-39.5mol% sitosterol and about 0.5%-1.5% DMG-PEG.

[0161] In some embodiments, the compound of the composition described herein is encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space encapsulated by some or all of the lipid portion of the lipid nanoparticle. Encapsulation can be complete encapsulation, partial encapsulation, or both. In some embodiments, the nucleic acid and / or polypeptide is completely encapsulated in the lipid nanoparticle.

[0162] In some embodiments, one or more compounds described herein are associated with liposomes or lipid nanoparticles via covalent or non-covalent bonds. In some embodiments, any compound in the composition may be contained in a liposome or lipid nanoparticle, either alone or together.

[0163] The LNPs can be administered by any suitable route resulting in delivery to the patient's eye. For example, an effective amount of the LNPs can be administered to the patient by intravitreal injection and / or intracameral injection.

[0164] An effective amount refers to the amount of LNP required to achieve an editing level that prevents or alleviates at least one or more signs or symptoms of a medical condition, and relates to an amount of the composition sufficient to provide the desired effect, for example, to treat a subject with a medical condition. An effective amount also includes an amount sufficient to prevent or delay the development of disease symptoms, to alter the course of disease symptoms (such as, but not limited to, to slow the progression of disease symptoms), or to reverse disease symptoms. It should be understood that for any given situation, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0165] The efficacy of treatment using the means disclosed herein can be determined by a skilled clinician. As just one example, if any one or all of the signs or symptoms of a functional target level are altered in a beneficial manner (e.g., increased by at least 10%), or other clinically acceptable symptoms or markers of a disease (e.g., glaucoma) are improved or ameliorated, then the treatment is considered "effective". Efficacy can also be measured by the absence of deterioration in the subject, as assessed by hospitalization or the need for medical intervention (e.g., the progression of the disease is stopped or at least slowed). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of a disease in a subject, and includes: (1) inhibiting the disease, e.g., preventing or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of symptom development.

[0166] In some embodiments, more than one LNP is administered (e.g., topically) to a subject at a dose of about 0.1 mg / kg-5 mg / kg (determined by the sum of total nucleic acid (e.g., target gene gRNA (e.g., MYOC gRNA) and Cas9 mRNA) per administration, including 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg. / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg or 5 mg / kg, or an amount or range between any two of these values. In some embodiments, more than one LNP is administered to a subject at or about a dose (in some embodiments, determined by the sum of the target gene gRNA (e.g., MYOC gRNA) and the Cas9 mRNA) of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, or 3.0 mg / kg. In some embodiments, more than one LNP is administered at a dose of about 40ug / eye, 45ug / eye, 50ug / eye, 55ug / eye, 60ug / eye, 65ug / eye, 70ug / eye, 75ug / eye, 80ug / eye, 85ug / eye, 90ug / eye, 95ug / eye, 100ug / eye, or an amount or range between any two of these values.

[0167] In some embodiments, the composition described above may also have one or more additional reagents, wherein such additional reagents are selected from a buffer, a buffer for introducing a polypeptide or polynucleotide into a cell, a washing buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for producing a polypeptide from DNA in vitro, an adapter for sequencing, etc. The buffer may be a stabilization buffer, a reconstitution buffer, a dilution buffer, etc. In some embodiments, the composition may also include one or more components that can be used to promote or enhance the target binding or cleavage of the endonuclease to the DNA or improve the specificity of the targeting.

[0168] In some embodiments, any component of the composition is formulated with a pharmaceutically acceptable excipient such as a carrier, solvent, stabilizer, adjuvant, diluent, etc., depending on the specific mode of administration and dosage form. In embodiments, the guide RNA composition is generally formulated to achieve a physiologically compatible pH, and ranges from about pH 3 to about pH 11, about pH 3 to about pH 7, depending on the formulation and route of administration. In some embodiments, the pH is adjusted to a range of about pH 5 to about pH 8.

[0169] Suitable excipients may include, for example, carrier molecules including large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, and inactive viral particles. Other exemplary excipients include antioxidants (such as, but not limited to, ascorbic acid), chelating agents (such as, but not limited to, EDTA), carbohydrates (such as, but not limited to, dextrins, hydroxyalkyl cellulose, and hydroxyalkyl methyl cellulose), stearic acid, liquids (such as, but not limited to, oils, water, saline, glycerol, and ethanol), wetting agents or emulsifiers, pH buffer substances, and the like.

[0170] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that do not contain any substance except the active ingredient and water, or contain buffers such as sodium phosphate, saline or both at physiological pH, such as phosphate buffered saline. Aqueous carriers can contain more than one buffer salt, as well as salts (such as sodium chloride and potassium chloride), dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain a liquid phase in addition to water and a liquid phase without water. Examples of such additional liquid phases are glycerol, vegetable oils such as cottonseed oil and water-oil emulsions. The amount of active compound used in a cell composition that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0171] In some embodiments, the compound described herein of the composition (e.g., RNA-guided endonucleases or nucleic acids encoding RNA-guided endonucleases and / or gRNA) can be delivered via transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, electronuclear transport, chemical transduction, electrotransduction, Lipofectamine-mediated transfection, Effectene-mediated transfection, lipid nanoparticle (LNP)-mediated transfection, or any combination thereof. In some embodiments, lipid nanoparticles are used to introduce the composition into cells via lipid-mediated transfection.

[0172] The compositions described herein can be administered to a subject in need thereof to treat an eye disease (e.g., glaucoma). Therefore, the present disclosure also provides a gene therapy method for treating glaucoma in a patient by reducing the expression of a MYOC gene (e.g., a wild-type MYOC gene or a mutant MYOC gene) in TM cells of the subject's eye. In some embodiments, a method for treating a subject with a type of glaucoma is disclosed. The method includes reducing the expression of a myocilin (MYOC) gene in the subject's eye. In some embodiments, the method includes administering to the subject more than one LNP compounded with: (a) a guide RNA (gRNA) or a nucleic acid encoding a gRNA targeting a MYOC gene, and (b) an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, thereby alleviating glaucoma.

[0173] More than one nanoparticle can be administered to a subject once. In some embodiments, more than one nanoparticle can be administered to a subject twice or more for treatment, for example, twice. Two administrations of nanoparticles to a subject can be spaced apart from a suitable time period. In some embodiments, a suitable time period is the following or about the following: one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, three months, four months, five months, six months, one year, two years, three years or longer. In some embodiments, two of the two or more administrations are spaced apart from about two weeks to about two months, for example, about three weeks. In some embodiments, each of the two or more administrations is spaced apart from about two weeks to about two months, for example, about three weeks. In some embodiments, each of the two or more administrations is spaced apart from about one month to about four months, for example, about two months or three months, or longer. In some embodiments, each of the two or more administrations is spaced apart from about one month to about four months, for example, about two months or three months. In some embodiments, each of the two or more administrations is spaced apart from at least two months or three months. In some embodiments, each of the two or more administrations is spaced apart from at least two months or three months. In some embodiments, the expression level of a target gene (e.g., a MYOC gene) in a subject (e.g., in the eye of a subject) that receives a single administration of a composition described herein can be significantly reduced (e.g., reduced by at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) and maintained at a reduced level for at least two months, three months, four months, six months, ten months, one year, eighteen months, two years, three years, four years, five years, ten years, fifteen years, twenty years or more after administration. In some embodiments, the level of a protein encoded by a target gene (e.g., myocilin levels) in a subject (e.g., in the subject's eye) receiving a single administration of a composition described herein can be significantly reduced (e.g., reduced by at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) and maintained at a reduced level for at least two months, three months, four months, six months, ten months, one year, eighteen months, two years, three years, four years, five years, ten years, fifteen years, twenty years or more after administration. The appropriate time period between two administrations can be the same or different from the appropriate time period between another two administrations.

[0174] In some embodiments, the target tissue of the compositions and methods described herein is trabecular meshwork tissue. In some embodiments, the target cell of the compositions and methods described herein is a trabecular meshwork cell.

[0175] In some embodiments, its pharmaceutical composition can be administered by any suitable approach that can deliver the compound to the target tissue / cell. For example, the pharmaceutical composition can be delivered via intravitreal, intracameral, subconjunctival, subfascial, behind the eyeball, surface (topical), suprachoroidal and / or posterior sclera administration. In some embodiments, the pharmaceutical composition is administered to the subject by intravitreal injection or intracameral injection. Administration can be local. In some embodiments, more than one administration can be used to achieve the desired gene expression level in various intervals (e.g., every day, every week, every month or every year) of the time period.

[0176] Subjects in need thereof may suffer from myocilin-related glaucoma. Myocilin-related glaucoma includes types of glaucoma associated with changes in the myocilin gene (MYOC). In some embodiments, myocilin-related glaucoma includes open-angle glaucoma (OAG). In some embodiments, OAG is primary OAG (POAG). In some embodiments, OAG is juvenile-onset OAG (JOAG).

[0177] In some embodiments, after administration, the expression of the target gene (e.g., MYOC) in the subject's eye is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. In some embodiments, after administration, the expression of the protein (e.g., myocilin, α-SMA protein) encoded by the target gene in the trabecular meshwork cells of the subject's eye is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. The reduction is relative to the expression of myocilin or the concentration of myocilin in the trabecular meshwork cells of the subject (e.g., mammals, NHPs, human subjects) before the administration of the more than one nanoparticle. In some embodiments, the reduction is relative to the expression of the target gene or the protein encoded by the target gene in one or more untreated subjects (e.g., MYOC expression or the concentration of myocilin). In some embodiments, the reduction is relative to a reference level of expression of the target gene (eg, MYOC expression) or concentration of a protein encoded by the target gene (eg, actin) in a healthy and / or unmodified subject.

[0178] In some embodiments, the method can also include measuring the intraocular pressure in the subject before, during and / or after administration. In some embodiments, the method includes identifying a subject in need of treatment. In some embodiments, a subject in need can be identified as having elevated intraocular pressure (IOP). The compositions and methods described herein can reduce the IOP in the subject by at least 20%, at least 40%, at least 70%, or at least 90% after administration.

[0179] The present disclosure also encompasses combination therapies. For example, the methods disclosed herein can be used together with other therapeutic agents to treat the same indication, or to enhance the efficacy of MYOC gene editing in TM cells and / or reduce the side effects of MYOC gene editing in TM cells.

[0180] V. Kit

[0181] The present disclosure also provides a kit for therapeutic use. In some embodiments, the kit provided herein may include components for genetic editing of the MYOC gene in TM cells. The components for genetic editing of the MYOC gene may include suitable endonucleases, such as RNA-guided endonucleases and nucleic acid guides, which guide the endonucleases to cleave one or more suitable genomic sites. For example, the kit may include mRNA encoding Cas enzymes such as Cas9 and one or more gRNAs targeting MYOC. Any gRNA specific to the MYOC gene may be included in the kit.

[0182] Any kit disclosed herein may also include instructions for use. In some embodiments, the included instructions include a description of using the gene editing components to genetically edit the MYOC gene.

[0183] In some embodiments, a kit as disclosed herein may include instructions for administering an LNP as disclosed herein to achieve a desired therapeutic effect. Alternatively or additionally, the kit may also include a description of selecting a subject suitable for treatment based on identifying whether the subject needs treatment. The instructions associated with use typically include information about the dose, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kit of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the contents / components of the kit are used to treat a disease or disorder, delay the onset of a disease or disorder, and / or alleviate a disease or disorder in a subject.

[0184] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Also contemplated are packaging used in combination with specific devices, such as infusion devices for administering the contents. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port.

[0185] The kit may optionally provide additional components, such as buffers and interpretative information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the disclosure provides an article comprising the contents of the above-described kit.

[0186] General techniques

[0187] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis ed., 1989) Academic Press; Animal Cell Culture (RI Freshney ed. 1987); Introduction to Cell and Tissue Culture (JP Thayer and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell ed. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell ed.): Gene Transfer Vectors for Mammalian Cells. Cells (JM Miller and MP Calos, 1987); Current Protocols in Molecular Biology (FM Ausubel et al., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practice approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, ed., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, ed., Harwood Academic Publishers, 1995); DNA Cloning: A practical approach, Volumes I and II (D. N. Glover, ed., 1985); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, ed. (1985); Transcription and Translation (B. D. Hames & S. J. Higgins, ed. (1984); Animal Cell Culture (R. I. Freshney, ed. (1986); Immobilized Cells and Enzymes (lRL Press, (1986); and B. Perbal, A practical guide to molecular cloning (1984); FM Ausubel et al. (eds.).

[0188] Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the above description. Therefore, the following specific examples should be interpreted as being merely illustrative and not limiting the remainder of the present disclosure in any way. All publications cited herein are incorporated herein by reference for the purposes or subject matter cited herein. Example

[0189] Example 1: sgRNA in vitro screening

[0190] This example describes the screening of guide RNAs targeting MYOC by CRISPR / Cas-mediated gene editing.

[0191] HEK293T cells were used for in vitro sgRNA screening and nuclear transfection was performed with Cas9 (SaCas9 or SpCas9) and gRNA ribonucleoprotein complexes. Genomic DNA was extracted, and specific DNA sequences were amplified by PCR, and analysis was performed by tracking indels by decomposition (TIDE, tracking of indels by decomposition). Total insertion / deletion (indel)% and frameshift insertion / deletion (indel)% are reported in Table 6 (for SaCas9) and Table 7 (for SpCas9). Frameshift insertion / deletion% refers to the insertion / deletion (i.e., ±1nt, ±2nt, ±4nt) expected to cause frameshift mutations in the MYOC coding sequence. Therefore, frameshift insertion / deletion% refers to the frequency of total insertion / deletion minus the frequency of insertion / deletions of ±3nt, ±6nt, ±9nt, etc.

[0192] Table 6. Quantification of editing efficiency of SaCas9 sgRNA targeting MYOC coding sequence

[0193]

[0194]

[0195] *Frameshift indel % refers to indels that are expected to result in frameshift mutations in the MYOC coding sequence (ie, ±1 nt, ±2 nt, ±4 nt).

[0196] Thus, the % frameshift indels refers to the frequency of total indels minus the frequency of indels that are ±3 nt, ±6 nt, ±9 nt, etc. All data entered are the average of n=3 replicates.

[0197] Quantitative results of editing efficiency of SaCas9 sgRNA targeting MYOC coding sequence Figure 1A-1B Shown in. Figure 1A The quantitative ordering by guide number is described, and Figure 1B Quantification by insertion-deletion percentage ranking is illustrated. As shown therein, a ribonucleoprotein complex of SaCas9 protein and one of 27 SaCas9 sgRNAs was transfected into HEK293T cells and the cells were harvested at a later time point. After DNA extraction, PCR and TIDE were performed to determine the percentage of insertions and deletions at the cleavage site. Under the conditions tested, the total insertion and deletion percentage ranged from 89.65% for SaMCh10 to 1.48% for SaMCh4. The highest frameshift percentage (80.83%) was also observed in the sample transfected with SaMCh10 sgRNA.

[0198] Table 7. Quantification of editing efficiency of SpCas9 sgRNA targeting MYOC coding sequence

[0199]

[0200]

[0201]

[0202] *Frameshift indel % refers to indels that are expected to result in frameshift mutations in the MYOC coding sequence (ie, ±1 nt, ±2 nt, ±4 nt).

[0203] Therefore, the % frameshift indels refers to the frequency of total indels minus the frequency of indels of ±3 nt, ±6 nt, ±9 nt, etc.

[0204] All data entered are the average of n = 3 replicates.

[0205] Quantitative results of editing efficiency of SpCas9 sgRNA targeting MYOC coding sequence Figure 2A-2B Shown in. Figure 2A The quantitative ordering by guide number is described, and Figure 2B Quantification by indel percentage is illustrated. As shown therein, ribonucleoprotein complexes of SpCas9 protein and one of 61 SpCas9 sgRNAs were transfected into HEK293T cells and the cells were harvested at a later time point. After DNA extraction, PCR and TIDE were performed to determine the percentage of indels at the cut site. Under the conditions tested, the total editing percentage ranged from 91.60% for SpMCh47 to 1.98% for SpMCh58. The highest frameshift percentage was observed in the sample transfected with SpMCh10 sgRNA, which was 84.23% (85.24% total indels).

[0206] Example 2: In vitro screening of lipid nanoparticles

[0207] This example describes an in vitro efficacy study of lipid nanoparticles (LNPs) delivered via different LNPs.

[0208] Several human primary cells and one cell line were used for the study, including primary trabecular meshwork (TM) cells and an immortalized glaucoma TM cell line (GTM3) expressing a myocilin mutant (G364V) protein fused to dsRED. All LNPs were formulated with GFP mRNA and delivered to cells. For all tested formulations, pictures were taken at the same time point and GFP scores were determined using no GFP signal as a score of 0 and the highest GFP signal as a score of 3.

[0209] The characteristics and formulations of the LNPs used in these studies are shown in Table 8 (LNPs obtained from Source 1) and Table 9 (LNPs obtained from Source 2).

[0210] Table 8. Characteristics and formulations of lipid nanoparticles tested

[0211]

[0212] Table 9. Properties of lipid nanoparticles from different sources

[0213]

[0214] The results are Figure 2A / Figure 2B-Figure 6A / Figure 6B and Figure 7 shown in . Specifically, Figure 3A-3B The screening of LNPs obtained from source 1 in primary TM cells is illustrated. LNPs carrying GFP mRNA were transfected into primary TM cells in culture. Photos were taken 22 hours after transfection ( Figure 3A ) and determine the GFP score ( Figure 3B ).

[0215] Figure 4A-4B The screening of LNPs obtained from source 2 in the GTM3 cell line is illustrated. LNPs carrying GFP mRNA were transfected into immortalized glaucoma TM cells in culture. GTM3 cells carry a transgenic form of the mutant G364V myocilin fused to dsRED (not shown). Photos were taken 22 hours after transfection ( Figure 4A ) and determine the GFP score ( Figure 4B ).

[0216] Figure 5A-Figure 5B The figure shows the screening of LNPs obtained from source 2 in primary TM cells obtained from source 1. LNPs carrying GFP mRNA were transfected into primary TM cells in culture. Pictures were taken 22 hours after transfection ( Figure 5A ) and determine the GFP score ( Figure 5B ).

[0217] Figure 6A-6B The screen of LNPs obtained from source 2 in primary TM cells obtained from source 2 is illustrated. LNPs carrying GFP mRNA were transfected into primary TM cells in culture. Photos were taken 22 hours after transfection ( Fig. 6A ) and determine the GFP score ( Figure 6B ).

[0218] Several different sources of trabecular meshwork (TM) cells were used for LNP screening, as TM cells are notoriously difficult to isolate and can be contaminated with other cell types. By using multiple sources, the pattern of LNP transfection efficacy in TM cells could be confirmed.

[0219] Figure 7 The results of quantification of the editing efficiency of selected SpCas9 sgRNAs targeting the MYOC coding sequence in GTM3 cells using selected lipid nanoparticles (LNPs) for delivery are described. GTM3 cells were transfected with either of two (2) forms of CTX-C12-200-CT from source 1: (1) containing 1 mol% DMG-PEG; or (2) containing 1.5 mol% DMG-PEG. Both forms were formulated with Cas9 protein and SpMCh21 sgRNA. Two different doses were transfected in GTM3 cells, which were harvested 2 days after transfection, DNA was extracted, and PCR and TIDE were performed to determine the total indels and frameshift indels percentages for both genomic and transgenic sites. At lower doses, 1 mol% DMG-PEG performed slightly better than 1.5 mol% DMG-PEG. However, at higher doses, they performed similarly, indicating that there was a plateau effect at this dose. This experiment was to confirm the efficacy of one of the leading performing LNP formulations tested with Cas9 / sgRNA in achieving editing in an in vitro cell model.

[0220] Example 3: In vivo screening of lipid nanoparticles

[0221] This example describes the in vivo screening of LNP. BALB / c mice were used for in vivo studies. One (1) microliter of LNP containing GFP mRNA was injected intravitreally, and the entire globe was isolated at a later time point (e.g., at the 24 hour time point). The entire globe was fixed, embedded, and processed for immunohistochemical staining of GFP expression. For all test preparations, photos were taken at the same time point, and the GFP score was determined using no GFP signal as a score of 0 and the highest GFP signal as a score of 3.

[0222] Figure 8 The GFP score of LNP obtained from source 1 in the mouse trabecular meshwork after intravitreal injection of 300ng of LNP is illustrated. LNP carrying GFP mRNA was injected intravitreally into the eyes of BALB / c mice. At 5 hours, mice were euthanized and their eyes were processed for IHC and anti-GFP staining. Photos were taken and GFP scores were determined.

[0223] Fig. 9The GFP score of LNP obtained from source 2 in the mouse trabecular meshwork after intravitreal injection of 300ng of LNP is illustrated. LNP carrying GFP mRNA was injected intravitreally into the eyes of BALB / c mice. At 5 hours, mice were euthanized and their eyes were processed for IHC and anti-GFP staining. Photos were taken and GFP scores were determined.

[0224] Fig. 10A IHC GFP staining of CTX-C12-CT obtained from Source 1 (described in Table 8) delivered in the mouse trabecular meshwork following intravitreal injection of 300 ng of LNPs is illustrated. GFP expression was observed in the trabecular meshwork tissue of this cross section of the Balb / c mouse eye 5 hours after injection of CTX-C12-CT formulated with GFP mRNA. Fig. 10B Demonstrated is the expression of GFP protein in mouse trabecular tissue following intravitreal injection of LNP CTX-C12-200-CT / eGFP mRNA in mouse eyes. Fig. 10C Demonstrated expression of GFP protein in mouse trabecular tissue following intravitreal injection of LNP A14 / eGFP mRNA in mouse eyes. These results demonstrate that intravitreal injection of LNPs in the eye results in expression of the cargo in the trabecular meshwork tissue.

[0225] Example 4: In vitro LNP delivery of Cas9 and sgRNA

[0226] This example describes in vitro LNP delivery of Cas9 mRNA and sgRNA. Immortalized human glaucoma TM cell lines expressing myocilin mutant (G364V or Y437H) proteins fused to dsRED were transfected with LNPs formulated with Cas9 mRNA and sgRNA for MYOC targeting. CTX-C12-CT with 1% or 1.5% DMG-PEG LNP formulations was used to deliver Cas9 mRNA and sgRNA.

[0227] Primary human trabecular meshwork cells were transfected with LNPs formulated with Cas9 mRNA and sgRNA for MYOC targeting for 2 days, and then the cells were treated with dexamethasone to induce myocilin expression and harvested 3 days later.

[0228] Genomic DNA was extracted and subjected to TIDE (Tracking Indels by Decomposition) analysis. The results showed that the combination of one of the leading sgRNAs (SpMCh21) and one of the best LNPs (CTX-C12-CT) could achieve high levels of editing in trabecular cells.

[0229] Fig.11The results show that LNP CTX-C12-200-CT / Cas9 mRNA / SpMCh10 sgRNA can be delivered to the human glaucomatous trabecular meshwork cell line GTM3 MYOC Y437H -MYOC gene editing and myocilin knockdown after dsRED. In this study, human glaucomatous GTM3 MYOC cells were transfected with different amounts of LNPs CTX-C12-200-CT formulated with Streptococcus pyogenes Cas9 mRNA and SpMCh10 targeting the human MYOC gene. Y437H -dsRED.GTM3MYOC Y437H The -dsRED cell line is an immortalized human trabecular meshwork cell line stably expressing the transgenic myocilin mutant Y437H fused to the reporter protein dsRED.

[0230] Targeting the actin mutant sequence was shown to affect expression of the fusion protein, as shown by the reduction of dsRED expression in the edited samples observed by epifluorescence microscopy ( Fig.11 Proteins were extracted 5 days after transfection and immunoblotted using anti-GAPDH and anti-actin to detect actin fused to dsRED ( Fig.11 DNA was also extracted and subjected to PCR and TIDE to determine the percentage of indels at the cleavage site for both the genomic and transgenic loci ( Fig.11 Figure D). Actin mutant-dsRED protein expression correlated with transgenic expression of MYOC, and wild-type expression of actin was not detected in this cell line despite genomic MYOC also being edited. Expression at the transgenic locus ranged from 86.7% (2 ng / μl) to 97.8% (10 ng / μl) compared to unedited controls ( Fig.11 D) resulted in a 83% (2 ng / μl) to 87% (10 ng / μl) decrease in myocilin expression ( Fig.11 These results suggest that a high editing percentage of the MYOC gene results in high myocilin protein knockdown.

[0231] Fig.12MYOC gene editing and myocilin knockdown following delivery of LNP CTX-C12-200-CT / Cas9 mRNA / SpMCh10 sgRNA to human primary trabecular meshwork cells were demonstrated in. In this study, human primary trabecular meshwork cells were transfected with varying amounts of LNP CTX-C12-200-CT formulated with Streptococcus pyogenes Cas9 mRNA and SpMCh10 targeting the MYOC gene. The cells were transfected for 2 days, and then dexamethasone was added to the culture medium to induce the expression of myocilin. After three (3) days, the samples were harvested. DNA was extracted and PCR and TIDE were performed to determine the percentage of indels at the cleavage site of the genomic locus ( Fig.12 A). Proteins were extracted and immunoblotted using anti-GAPDH and anti-actin to detect wild-type actin ( Fig.12 Compared to unedited controls, the range at the genomic locus was 47% (2 ng / μl) to 98% (10 ng / μl) ( Fig.12 A) resulted in a 49% (2 ng / μl) to 85% (10 ng / μl) decrease in myocilin expression ( Fig.12 These results suggest a correlation between the editing percentage at the MYOC locus and actin knockdown.

[0232] Example 5: Ex vivo LNP delivery of Cas9 and sgRNA

[0233] This embodiment describes the in vitro LNP delivery of Cas9 mRNA and MYOC sgRNA. Anterior segment organ culture (ASOC) was established from adult deceased human donors without glaucoma or reported ocular conditions. ASOC was perfused with serum-containing medium at a flow rate of 2.5 μl / ml and intraocular pressure was monitored. The preparation was introduced using a syringe and pump system, and 50 μg of LNP CTX-C12-200-CT / eGFP or LNP CTX-C12-200-CT / Cas9 mRNA / MYOC sgRNA was transfected for 24h (GFP) or 4 days (Cas9 / sgRNA). For GFP, ASOC was fixed, embedded and processed for immunohistochemistry (IHC) staining for GFP expression. For Cas9 mRNA / sgRNA infusion, tissues (cornea, sclera and trabecular meshwork) were separated, and DNA or protein was isolated for TIDE or western blot analysis.

[0234] After LNP CTX-C12-200-CT / eGFP mRNA delivery in isolated anterior segment organ cultures (ASOCs), GFP protein expression in trabecular meshwork tissue was Fig.13The results showed that after LNP transfection, GFP was highly and specifically expressed in the trabecular meshwork tissue.

[0235] Fig.14 MYOC gene editing and myocilin knockdown in trabecular meshwork tissue after delivery of LNP CTX-C12-200-CT / Cas9mRNA / MYOC sgRNA in ex vivo anterior segment organ culture (ASOC) are demonstrated. ASOCs were established from whole eyeballs received from deceased human donors. 50 μg of LNP CTX-C12-200-CT formulated with Streptococcus pyogenes Cas9 mRNA and MYOC sgRNA (SpMCh10 or SpMCh21) was injected into the perfusion system at a flow rate of 2.5 μl / ml. Four (4) days later, cornea, sclera, and trabecular meshwork tissue were isolated from ASOC. DNA was extracted and PCR and TIDE were performed to determine the percentage of indels at the genomic locus cleavage site ( Fig.14 Protein from experiments involving donor #1656 was extracted and immunoblotted using anti-GAPDH and anti-actin to detect wild-type actin ( Fig.14 The percentage of editing in the trabecular meshwork of donors 1580, 1578, and 1656 was 27.6%, 29.7%, and 35.7%, respectively; the percentage of editing in the sclera and cornea was lower (1%-9%) ( Fig.14 Figure A). Actin expression monitored in donor 1656 showed a 44% reduction in protein compared to the contralateral unedited tissue control ( Fig.14 These results provide proof of concept that MYOC gene editing in the targeted trabecular meshwork tissue leads to downregulation of myocilin in this tissue.

[0236] Example 6: In vivo LNP delivery of Cas9 and sgRNA

[0237] This example describes in vivo LNP delivery of Cas9 mRNA and sgRNA in mouse eyes. ACTA2 / α-SMA was chosen as a non-limiting exemplary target gene because this protein is well expressed in the trabecular meshwork and can be easily monitored by IHC.

[0238] One (1) microliter of CTX-C12-CT LNPs formulated with Cas9 / sgRNA was injected intravitreally into the eyes of BALB / c mice. The sgRNA targeting the mouse ACTA2 gene (α-smooth muscle actin or SMA) was chosen due to its high editing efficiency in vitro. Initial samples were harvested one (1), two (2), and four (4) weeks after injection. Whole eyeballs of mice and LNP-injected mice. Whole eyeballs were fixed, embedded and processed for immunohistochemical staining of α-SMA expression. The presence of SMA within the trabecular meshwork was assessed at six (6) locations within the eye. Scores of 0 (no TM cells with SMA signal) to 3 (most TM cells with SMA signal) were assigned to each of the six (6) locations and the average score for each eye was determined.

[0239] After a single injection of LNPs containing sgRNA targeting the ACTA2 mouse gene, the expression of the ACTA2 gene protein α-SMA decreased continuously from week 1 to week 4, as shown in Fig.15 As shown in Figure A. IHC was scored for the expression of α-SMA and the number of cells expressing α-SMA. Fig.15 Figure A shows an example of a score of 3 (the highest score) in a naive / untreated animal, and Fig.15 Panel B shows an example of a score of 0 (lowest score) in the trabecular meshwork of an animal treated four weeks after LNP injection.

[0240] This example demonstrates that LNPs can deliver Cas9 mRNA / sgRNA to the trabecular meshwork in vivo and result in efficient editing at the target sequence by the Cas9 / sgRNA complex and downregulation of protein expression.

[0241] Sequence Listing

[0242] The following table provides detailed information on various nucleotide and amino acid sequences disclosed herein. Table 1. Target sequences of SaCas9 sgRNA in MYOC coding sequence

[0243]

[0244] *Chromosome location of the guide cleavage site in chromosome 1 of the human genome Hg

[0245] Table 2. Target sequences of SpCas9 sgRNA in MYOC coding sequence

[0246]

[0247]

[0248] Table 3. DNA sequences of SpCas9 and SaCas9 tcrRNA sequences used in sgRNA (T is replaced by U in the corresponding tcrRNA sequences)

[0249]

[0250] Table 4. PCR and TIDE primer sequences used to analyze indel frequencies at the cleavage sites corresponding to SaCas9 sgRNAs

[0251]

[0252] Table 5. PCR and TIDE primer sequences used to analyze indel frequencies at the cleavage sites corresponding to SpCas9 sgRNAs

[0253]

[0254] Table 10. Cas9 DNA sequences

[0255]

[0256]

[0257]

[0258] Table 11. SaCas9 sgRNA unmodified sequences for SaCas9 sgRNA targeting MYOC coding sequences

[0259]

[0260]

[0261] Table 12. SaCas9 sgRNA modified sequences for SaCas9 sgRNA targeting MYOC coding sequence (screening / small batch)

[0262]

[0263]

[0264] A, G, U, C, N: RNA nucleotides

[0265] a, g, u, c, n: 2'-O-methyl-nucleotides

[0266] s: phosphorothioate linkage

[0267] Table 13. Unmodified sequences of SpCas9 sgRNAs for SpCas9 sgRNAs targeting MYOC coding sequences

[0268]

[0269]

[0270]

[0271] Table 14. SpCas9 sgRNA modified sequences for SpCas9 sgRNA targeting MYOC coding sequence (screening / small batch)

[0272]

[0273]

[0274]

[0275]

[0276] A, G, U, C, N: RNA nucleotides

[0277] a, g, u, c, n: 2'-O-methyl-nucleotides

[0278] s: phosphorothioate linkage

[0279] Table 15. SpCas9 sgRNA modified sequences for SpCas9 sgRNA targeting MYOC coding sequence (larger batch).

[0280] Take SpMCh10 sgRNA as an example.

[0281]

[0282] Other Implementations

[0283] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an example of a series of general equivalent or similar features.

[0284] From the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and without departing from its spirit and scope, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments fall within the claims.

[0285] Equivalent

[0286] Although several inventive embodiments have been described and illustrated herein, it will be readily apparent to those of ordinary skill in the art that various other devices and / or structures for performing functions and / or obtaining the results and / or one or more advantages described herein will be readily conceived, and each of these changes and / or modifications is considered to be within the scope of the inventive embodiments described herein. More generally, it will be readily understood by those of skill in the art that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on a specific application or applications using inventive teachings. Those skilled in the art will recognize or be able to determine many equivalents of the specific inventive embodiments described herein using no more than conventional experiments. Therefore, it should be understood that the aforementioned embodiments are presented only by way of example, and within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced in a manner different from that specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0287] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0288] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which they are cited, and in some cases, these references, patents, and patent applications may be incorporated in their entirety.

[0289] The indefinite articles "a" and "an" as used herein in the specification and claims should be understood to mean "at least one", unless explicitly indicated to the contrary.

[0290] The phrase "and / or" as used herein in the specification and in the claims should be understood to mean "one or both" of the elements so connected (i.e., elements that are present jointly in some cases and separately in other cases). Multiple elements listed using "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so connected. In addition to the elements specifically indicated by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically indicated. Thus, as a non-limiting example, in one embodiment, reference to "A and / or B" when used in conjunction with open language (such as "comprising") may refer to only A (optionally including elements other than B); in another embodiment, only B (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements); and so on.

[0291] As used in the specification and claims herein, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one of a plurality of elements or a list of elements, but also including more than one, and optionally including additional unlisted items. Only terms that clearly indicate the opposite, such as "only one" or "exactly one", or "consisting of..." when used in the claims, will refer to the inclusion of exactly one element of a plurality of elements or a list of elements. In general, when preceded by exclusive terms such as "any", "one of...", "only one of..." or "exactly one of...", the term "or" as used herein shall only be interpreted as indicating an exclusive alternative (i.e., "one or the other, but not both"). "Substantially consisting of..." when used in the claims shall have its ordinary meaning as used in the field of patent law.

[0292] As used herein, the term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" may mean within an acceptable standard deviation. Alternatively, "about" may mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and still more preferably up to ±1% of a given value. Where specific values ​​are described in the application and claims, the term "about" is implicit, unless otherwise stated, and in the context of this document means within an acceptable error range for the specific value.

[0293] As used herein in the specification and in the claims, the phrase "at least one" when referring to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements and not excluding any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically indicated in the list of elements referred to by the phrase "at least one" may optionally be present, whether related or unrelated to those elements specifically indicated. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") may refer to at least one (optionally including more than one) A, but no B (and optionally including elements other than B) in one embodiment; to at least one (optionally including more than one) B, but no A (and optionally including elements other than A) in another embodiment; to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements) in yet another embodiment; and so on.

[0294] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described unless explicitly indicated to the contrary.

Claims

1. A method for delivering a CRISPR / Cas-mediated gene editing system to cells of an eye of a subject, the method comprising administering to the subject more than one lipid nanoparticle (LNP) complexed with: (a) a guide RNA for a target gene or a nucleic acid encoding the guide RNA; and / or (b) an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, Thereby reducing the expression of the target gene in the cells of the eye of the subject.

2. The method of claim 1, wherein the CRISPR / Cas-mediated gene editing system is delivered to the trabecular meshwork cells of the subject.

3. The method according to any one of claims 1-2, wherein the target gene is the myocilin (MYOC) gene.

4. The method of any one of claims 1-3, wherein the expression of the target gene, the expression of the protein encoded by the target gene, or both in the eye of the subject is reduced by at least 20%, at least 40%, at least 70%, or at least 90% after administration.

5. The method of any one of claims 1-4, wherein the expression of the target gene is reduced in the trabecular meshwork cells of the eye of the subject.

6. A method for treating a subject suffering from glaucoma, the method comprising administering to the subject more than one lipid nanoparticle (LNP) complexed with: (a) a guide RNA targeting the myocilin (MYOC) gene or a nucleic acid encoding the guide RNA; and (b) an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, Thereby reducing the expression of the MYOC gene in the eye of the subject.

7. The method of claim 6, wherein the glaucoma is myocilin-related glaucoma.

8. The method of claim 6 or 7, wherein the glaucoma is primary open-angle glaucoma (POAG).

9. The method according to any one of claims 6-8, wherein the expression of the MYOC gene is reduced in the trabecular meshwork cells of the eye of the subject.

10. The method of any one of claims 1-9, wherein the RNA-guided nuclease is a Cas9 nuclease.

11. The method of claim 10, wherein the Cas9 nuclease is Staphylococcus aureus Cas9 (SaCas9) nuclease or Streptococcus pyogenes Cas9 (SpCas9) nuclease.

12. The method of any one of claims 4-11, wherein the site targeted by the guide RNA is within exon 1, exon 2, or exon 3 of the MYOC gene.

13. The method of any one of claims 4-12, wherein the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-27 and 55-115.

14. The method of any one of claims 4-13, wherein the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115.

15. The method of any one of claims 4-14, wherein the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 64, 73, 74, 75, 76 and 115.

16. The method of any one of claims 4-15, wherein the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-27 and 55-115.

17. The method of any one of claims 4-16, wherein the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115.

18. The method of any one of claims 4-17, wherein the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 10, 64, 73, 74, 75, 76, and 115.

19. The method of any one of claims 1-18, wherein the guide RNA is SaCas9 sgRNA or SpCas9 sgRNA.

20. The method of claim 19, wherein the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 195-371.

21. The method of claim 19 or 20, wherein the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 258, 267-270, 309, 319, 328-331, 370, and 371.

22. The method of any one of claims 1-21, wherein the LNPs in the more than one LNPs comprise an ionizable cationic lipid, a helper lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG-lipid).

23. The method of claim 22, wherein the LNP comprises about 20%-60% ionizable lipid, about 18.5% to 60% sterol, about 0.01% to 30% helper lipid and / or about 0%-10% PEG-lipid.

24. according to the method described in any one of claim 22-23, wherein said ionizable cationic lipid is selected from the group consisting of C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, DOTAP, DODAP, DC cholesterol, DLin-DMA, DLin-K-DMA and DLin-KC2-DMA.

25. The method of any one of claims 22-24, wherein the helper lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diundecanoyl-sn-glycero-3-phosphocholine (DUPC), phosphatidylcholine (POPC). ), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-diphexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE,18:0-18:1PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dioleoylphosphatidylglycerol (DOPG) and dipalmitoyl-sn-glycero-3-PG (DPPG).

26. The method according to any one of claims 22-25, wherein the sterol is selected from the group consisting of cholesterol, sitosterol, β-sitosterol, phytosterol, fucoxantrol, zoostanol and ergosterol.

27. The method of any one of claims 22-25, wherein the sterol is selected from the group consisting of cholesterol, sitosterol, β-sitosterol, campesterol, stigmasterol, fuccasterol and ergosterol.

28. The method of any one of claims 22-27, wherein the PEG-lipid is DMG-PEG, DSG-PEG, PEG-ceramide, or PEG-phospholipid.

29. The method of any one of claims 1-27, wherein the LNPs in the more than one LNPs comprise about 50 mol% C12-200, DLIN-MC3, DODMA or DOTAP, about 10 mol% DSPC, about 37.0 mol%-39.5 mol% cholesterol or sitosterol, and about 0.5%-3.0% DMG-PEG.

30. The method of claim 29, wherein the LNP comprises about 50 mol% C12-200, about 10 mol% DSPC, about 37.0 mol%-39.5 mol% sitosterol, and about 0.5%-1.5% DMG-PEG.

31. The method of any one of claims 1-27, wherein the average particle size of the more than one LNP is about 80 nm-100 nm, and optionally 85 nm-95 nm.

32. The method of any one of claims 1-31, wherein the more than one LNP is administered to the subject by intravitreal injection or intracameral injection.

33. The method of any one of claims 1-32, wherein the method comprises a single administration of the more than one LNP to the subject.

34. The method of any one of claims 1-33, wherein MYOC expression in the subject's eye is reduced by at least 20%, at least 40%, at least 70%, or at least 90% following administration.

35. The method of any one of claims 1-34, wherein the actin in the trabecular meshwork cells of the subject's eye is reduced by at least 20%, at least 40%, at least 70%, or at least 90% following administration.

36. The method of any one of claims 1-35, wherein the subject is a human.

37. The method of any one of claims 1-36, wherein the LNP is complexed with: (a) the guide RNA or a nucleic acid encoding the guide RNA and (b) the RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, respectively.

38. The method of claim 37, wherein the LNP complexed with (a) the guide RNA or a nucleic acid encoding the guide RNA and the LNP complexed with (b) the RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease are different LNPs.

39. A guide RNA targeting a MYOC gene, the guide RNA comprising a nucleotide sequence specific to a fragment in exon 1, exon 2 or exon 3 of the MYOC gene, wherein the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-27 and 55-115, or a spacer sequence having one, two or three mismatches relative to the RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-27 and 55-115.

40. The guide RNA of claim 39, wherein the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115.

41. The guide RNA of claim 39 or 40, wherein the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 10, 64, 73, 74, 75, 76, and 115.

42. The guide RNA of any one of claims 39-41, wherein the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 195-371.

43. The guide RNA of any one of claims 39-42, wherein the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 258, 267-270, 309, 319, 328-331, 370, and 371.

44. A system for treating a subject suffering from glaucoma, the system comprising: (i) gene editing means for reducing the expression of the myocilin (MYOC) gene in the eyes of the subject; and (ii) lipid nanoparticles (LNPs), wherein the LNPs deliver the gene editing means to the eye of the subject.

45. The system of claim 44, wherein the glaucoma is myocilin-related glaucoma.

46. ​​The system of claim 44 or 45, wherein the glaucoma is primary open angle glaucoma (POAG).

47. The system of any one of claims 44-46, wherein the gene editing means is CRISPR / Cas-mediated gene editing.

48. The system of claim 47, wherein the CRISPR / Cas-mediated gene editing comprises an RNA-guided nuclease and a guide RNA targeting a site in the MYOC gene.

49. The system of claim 48, wherein the RNA-guided nuclease is a Cas9 nuclease.

50. The system of claim 49, wherein the Cas9 nuclease is a Staphylococcus aureus Cas9 (SaCas9) nuclease or a Streptococcus pyogenes Cas9 (SpCas9) nuclease.

51. The system of any one of claims 48-50, wherein the sites targeted by the guide RNA are within exon 1, exon 2, and exon 3 of the MYOC gene.

52. The system of any one of claims 48-51, wherein the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-27 and 55-115.

53. The system of any one of claims 48-52, wherein the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 64, 73, 74, 75, 76, and 115.

54. The system of any one of claims 48-53, wherein the guide RNA is a SaCas9 sgRNA or a SpCas9 sgRNA.

55. The system of claim 54, wherein the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 195-371.

56. The system of claim 54 or 55, wherein the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 258, 267-270, 309, 319, 328-331, 370, and 371.

57. The system of any one of claims 44-56, wherein the LNP comprises an ionizable cationic lipid, a helper lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG-lipid).

58. The system of claim 57, wherein the LNP comprises about 20%-60% ionizable cationic lipid, about 18.5% to 60% sterol, about 0.01% to 30% helper lipid and / or about 0%-10% PEG-lipid.

59. The system of any one of claims 57-58, wherein the ionizable cationic lipid is selected from the group consisting of C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, DOTAP, DODAP, DC cholesterol, DLin-DMA, DLin-K-DMA, and DLin-KC2-DMA.

60. The system of claim 57 or 58, wherein the helper lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diundecanoylphosphatidylcholine (DUPC), phosphatidylcholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-diphexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE,18:0-18:1PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dioleoylphosphatidylglycerol (DOPG) and dipalmitoyl-sn-glycero-3-PG (DPPG).

61. The system of any one of claims 57-60, wherein the sterol is selected from the group consisting of cholesterol, sitosterol, phytosterol, fucosterol, zoostanol, and ergosterol.

62. The system of any one of claims 57-60, wherein the sterol is selected from the group consisting of cholesterol, sitosterol, beta-sitosterol, campesterol, stigmasterol, fuccasterol, and ergosterol.

63. The system of any one of claims 57-61, wherein the PEG-lipid is DMG-PEG, DSG-PEG, PEG-ceramide, or PEG-phospholipid.

64. The system of any one of claims 44-63, wherein the LNP comprises about 50 mol% C12-200, DLIN-MC3, DODMA or DOTAP, about 10 mol% DSPC, about 37.0 mol%-39.5 mol% cholesterol or sitosterol, and about 0.5%-3.0% DMG-PEG.

65. The system of claim 64, wherein the LNP comprises about 50 mol% C12-200, about 10 mol% DSPC, about 37.0 mol%-39.5 mol% sitosterol, and about 0.5%-1.5% DMG-PEG.

66. The system of any one of claims 44-65, wherein the system is administered to the subject by intravitreal injection or intracameral injection.

Images