Materials and methods for treating Usher syndrome type 2A

Abstract

The present application provides materials and methods for treating patients with Usher syndrome type 2A in vitro and in vivo; materials and methods for editing USH2A genes in human cells; materials and methods for editing USH2A genes containing IVS40 mutations; materials and methods for treating patients with USH2A genes containing IVS40 mutations; and methods for deleting sequences containing IVS40 mutations within the USH2A gene of cells. The present application also provides one or more gRNAs or sgRNAs for editing USH2A genes containing IVS40 mutations. The present application provides therapeutic agents for treating patients with Usher syndrome type 2A. The present application also provides a kit for treating patients with Usher syndrome type 2A.

Description

[0001] field

[0002] The present application provides materials and methods for treating Usher Syndrome Type 2A.

[0003] Related Applications

[0004] This application claims the benefit of U.S. Provisional Application No. 62 / 609,333, filed December 21, 2017; and U.S. Provisional Application No. 62 / 746,226, filed October 16, 2018, all of which are incorporated herein by reference in their entirety.

[0005] Sequence Listing Incorporated by Reference

[0006] This application contains a sequence listing in computer readable form (file name: 170646PCT_ST25: 11,398,978 bytes -- ASCII text file; created on December 19, 2018), which is incorporated by reference in its entirety and constitutes part of the present disclosure.

[0007] background

[0008] Usher syndrome is a condition that affects both hearing and vision. The main symptoms of Usher syndrome are hearing loss and an eye condition called retinitis pigmentosa, which causes night blindness and loss of peripheral vision through progressive degeneration of the retina. Many people with Usher syndrome also have severe balance problems.

[0009] There are currently no adequate treatments for Usher syndrome that can effectively halt or slow the progression of disease-associated vision loss, and there remains a critical need to develop safe and effective treatments for Usher syndrome. SUMMARY OF THE INVENTION

[0011] The present disclosure presents a novel method for improving (if not eliminating) Usher Syndrome Type 2A. The novel method targets mutations in the USH2A gene, such as IVS40 mutations, using a method that results in disruption of a sequence that serves as a splice donor site encoded by the gene containing the mutation. The splice donor site causes incorrect splicing. In addition, in some cases, treatment can be accomplished with a small number of treatments, and in some cases, can be accomplished with a single treatment. The resulting therapy can improve Usher Syndrome Type 2A associated with IVS40 mutations, or in some cases, can eliminate Usher Syndrome Type 2A associated with IVS40 mutations.

[0012] Provided herein are methods for editing the USH2A gene in human cells, the methods comprising: introducing one or more deoxyribonucleic acid (DNA) endonucleases into human cells, thereby achieving one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the USH2A gene or a DNA sequence encoding a regulatory sequence of the USH2A gene, which results in correction, thereby generating edited human cells.

[0013] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: introducing one or more DNA endonucleases into a human cell, thereby achieving one or more SSBs or DSBs within or near intron 40 of the USH2A gene, which results in correction, thereby generating an edited human cell.

[0014] Also provided herein is a method for editing the USH2A gene in a human cell, the method comprising: introducing one or more DNA endonucleases into the human cell, thereby achieving one or more SSBs or DSBs within or near the USH2A gene or a DNA sequence encoding a regulatory sequence of the USH2A gene, which results in regulation of the expression or function of the USH2A gene, thereby producing an edited human cell.

[0015] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: introducing one or more DNA endonucleases into a human cell, thereby achieving one or more SSBs or DSBs within or near intron 40 of the USH2A gene, which results in regulation of the expression or function of the USH2A gene, thereby generating an edited human cell.

[0016] Also provided herein is an in vivo method for treating a patient with Usher syndrome type 2A, comprising editing the USH2A gene containing the IVS40 mutation in the patient's cells.

[0017] Also provided herein is one or more guide RNAs (gRNAs) for editing the USH2A gene containing an IVS40 mutation in cells from a patient with Usher syndrome type 2A. The one or more gRNAs comprise a spacer sequence selected from the nucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443, and 5446-5461 of the sequence listing.

[0018] Also provided herein is a therapeutic agent for treating a patient with Usher syndrome type 2A, the therapeutic agent comprising at least one or more gRNAs for editing a USH2A gene containing an IVS40 mutation. The one or more gRNAs comprise a spacer sequence selected from the nucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443 and 5446-5461 of the sequence list.

[0019] Also provided herein is a therapeutic agent for treating a patient with Usher syndrome type 2A, the therapeutic agent being formed by a method comprising: introducing one or more DNA endonucleases; introducing one or more gRNAs or one or more single-molecule guide RNAs (sgRNAs) for editing a USH2A gene containing an IVS40 mutation; and optionally introducing one or more donor templates. The one or more gRNAs or sgRNAs comprise a spacer sequence selected from the nucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443, and 5446-5461 of the sequence listing.

[0020] Also provided herein is a kit for treating a patient with Usher syndrome type 2A in vivo. The kit comprises one or more gRNAs or sgRNAs for editing a USH2A gene containing an IVS40 mutation, one or more DNA endonucleases; and optionally one or more donor templates. One or more gRNAs or sgRNAs comprise a spacer sequence selected from the nucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443, and 5446-5461 of the sequence listing.

[0021] Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5321.

[0022] Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5323.

[0023] Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5325.

[0024] Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5327.

[0025] Also provided herein is a gRNA or sgRNA comprising SEQ ID NO: 5328.

[0026] Also provided herein is a gRNA or sgRNA comprising any one of SEQ ID NO: 5321 and SEQ ID NO: 5267-5269.

[0027] Also provided herein is a gRNA or sgRNA comprising any one of SEQ ID NO: 5323 and SEQ ID NO: 5267-5269.

[0028] Also provided herein is a gRNA or sgRNA comprising any one of SEQ ID NO: 5325 and SEQ ID NO: 5267-5269.

[0029] Also provided herein is a gRNA or sgRNA comprising any one of SEQ ID NO: 5327 and SEQ ID NO: 5267-5269.

[0030] Also provided herein is a gRNA or sgRNA comprising any one of SEQ ID NO: 5328 and SEQ ID NO: 5267-5269.

[0031] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a gRNA or sgRNA comprising SEQ ID NO: 5321 to edit a USH2A gene containing an IVS40 mutation.

[0032] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a gRNA or sgRNA comprising SEQ ID NO: 5323 to edit a USH2A gene containing an IVS40 mutation.

[0033] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a gRNA or sgRNA comprising SEQ ID NO: 5325 to edit a USH2A gene containing an IVS40 mutation.

[0034] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a gRNA or sgRNA comprising SEQ ID NO: 5327 to edit a USH2A gene containing an IVS40 mutation.

[0035] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a gRNA or sgRNA comprising SEQ ID NO: 5328 to edit a USH2A gene containing an IVS40 mutation.

[0036] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: editing a USH2A gene containing an IVS40 mutation using a gRNA or sgRNA comprising any one of SEQ ID NO: 5321 and SEQ ID NO: 5267-5269.

[0037] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: editing a USH2A gene containing an IVS40 mutation using a gRNA or sgRNA comprising any one of SEQ ID NO: 5323 and SEQ ID NO: 5267-5269.

[0038] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: editing a USH2A gene containing an IVS40 mutation using a gRNA or sgRNA comprising any one of SEQ ID NO: 5325 and SEQ ID NO: 5267-5269.

[0039] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: editing a USH2A gene containing an IVS40 mutation using a gRNA or sgRNA comprising any one of SEQ ID NO: 5327 and SEQ ID NO: 5267-5269.

[0040] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: editing a USH2A gene containing an IVS40 mutation using a gRNA or sgRNA comprising any one of SEQ ID NO: 5328 and SEQ ID NO: 5267-5269.

[0041] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5321.

[0042] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5323.

[0043] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5325.

[0044] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5327.

[0045] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5328.

[0046] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5321 and SEQ ID NO: 5267-5269.

[0047] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5323 and SEQ ID NO: 5267-5269.

[0048] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5325 and SEQ ID NO: 5267-5269.

[0049] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5327 and SEQ ID NO: 5267-5269.

[0050] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5328 and SEQ ID NO: 5267-5269.

[0051] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5295 and a second gRNA or sgRNA comprising SEQ ID NO: 5279 to delete a sequence containing an IVS40 mutation.

[0052] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5294 and a second gRNA or sgRNA comprising SEQ ID NO: 5300 to delete a sequence containing an IVS40 mutation.

[0053] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5295 and a second gRNA or sgRNA comprising SEQ ID NO: 5300 to delete a sequence containing an IVS40 mutation.

[0054] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5290 and a second gRNA or sgRNA comprising SEQ ID NO: 5300 to delete a sequence containing an IVS40 mutation.

[0055] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5277 and a second gRNA or sgRNA comprising SEQ ID NO: 5300 to delete a sequence containing an IVS40 mutation.

[0056] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5295, and the second gRNA or sgRNA comprises SEQ ID NO: 5279.

[0057] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5294, and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

[0058] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5295, and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

[0059] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5290, and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

[0060] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5277, and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

[0061] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5452 and a second gRNA or sgRNA comprising SEQ ID NO: 5449 to delete a sequence containing an IVS40 mutation.

[0062] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5453 and a second gRNA or sgRNA comprising SEQ ID NO: 5449 to delete a sequence containing an IVS40 mutation.

[0063] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5455 and a second gRNA or sgRNA comprising SEQ ID NO: 5457 to delete a sequence containing an IVS40 mutation.

[0064] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5452 and a second gRNA or sgRNA comprising SEQ ID NO: 5451 to delete a sequence containing an IVS40 mutation.

[0065] Also provided herein is a method for editing a USH2A gene containing an IVS40 mutation. The method comprises: using a first gRNA or sgRNA comprising SEQ ID NO: 5448 and a second gRNA or sgRNA comprising SEQ ID NO: 5449 to delete a sequence containing an IVS40 mutation.

[0066] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5452, and the second gRNA or sgRNA comprises SEQ ID NO: 5449.

[0067] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5453, and the second gRNA or sgRNA comprises SEQ ID NO: 5449.

[0068] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5455, and the second gRNA or sgRNA comprises SEQ ID NO: 5457.

[0069] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5452, and the second gRNA or sgRNA comprises SEQ ID NO: 5451.

[0070] Also provided herein is a method for treating a patient having a USH2A gene containing an IVS40 mutation. The method comprises administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5448, and the second gRNA or sgRNA comprises SEQ ID NO: 5449.

[0071] It should be understood that the invention described in this specification is not limited to the examples outlined in this summary. Various other aspects are described and illustrated herein. This summary is not intended to limit the scope of the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Various aspects of the materials and methods for treating Usher syndrome disclosed and described in this specification may be better understood by reference to the accompanying drawings, in which:

[0074] Figure 1A -B depicts a type II CRISPR / Cas system.

[0075] Figure 1A Depicted is a type II CRISPR / Cas system including a gRNA.

[0076] Figure 1B Depicted is a type II CRISPR / Cas system including sgRNA.

[0077] Figure 2A -F shows the single guide RNA (sgRNA) sequence, the target DNA sequence, and the reverse strand of the target DNA sequence to which the sgRNA binds for each of the 57 sgRNA sequences.

[0078] Figure 2A -B shows the single guide RNA (sgRNA) sequence for each of the 57 sgRNA sequences.

[0079] Figure 2C -D shows the target DNA sequence for each of the 57 sgRNA sequences.

[0080] Figure 2E-F shows the reverse strand of the target DNA sequence to which the sgRNA binds for each of the 57 sgRNA sequences.

[0081] Figure 2G -I shows the single guide RNA (sgRNA) sequence with respect to the sgRNA sequence, the target DNA sequence, and the reverse strand of the target DNA sequence to which the sgRNA binds.

[0082] Figure 2G Single guide RNA (sgRNA) sequences are shown with respect to sgRNA sequences.

[0083] Figure 2H The target DNA sequence is shown with respect to the sgRNA sequence.

[0084] Fig.2I Shown is the reverse strand of the target DNA sequence to which the sgRNA binds, with respect to the sgRNA sequence.

[0085] Figure 2J -L shows the single guide RNA (sgRNA) sequence, the target DNA sequence, and the reverse strand of the target DNA sequence to which the sgRNA binds for each of the 16 sgRNA sequences.

[0086] Figure 2J Single guide RNA (sgRNA) sequences are shown for each of the 16 sgRNA sequences.

[0087] Figure 2K The target DNA sequences for each of the 16 sgRNA sequences are shown.

[0088] Figure 2L For each of the 16 sgRNA sequences, the reverse strand of the target DNA sequence to which the sgRNA binds is shown.

[0089] Figure 3 Shown are diagrams depicting an IVS40 mutation located in intron 40 of the USH2A gene, the results of editing the IVS40 mutation in intron 40 of the USH2A gene, and the results of not editing the IVS40 mutation in intron 40 of the USH2A gene.

[0090] Figure 4A -B shows the IVS40 mutation introduced into genomic DNA via homology directed repair (HDR); the IVS40 mutation is a single nucleotide mutation (A to G) in intron 40 of the human USH2A gene.

[0091] Figure 4A IVS40 mutations introduced into genomic DNA via HDR are shown.

[0092] Figure 4B The IVS40 mutation is shown as a single nucleotide mutation (A to G) in intron 40 of the human USH2A gene.

[0093] Figure 5A -B depicts the binding regions of several sgRNAs targeting USH2A IVS40 mutations, as well as the on-target and off-target editing efficiencies for each of the sgRNAs targeting USH2A IVS40 mutations.

[0094] Figure 5A The binding regions of several sgRNA spacer regions around the IVS40 mutation of the USH2A gene are depicted (SEQ ID NOs: 5321, 5323, 5325, 5327, and 5328). The pointed end is the region adjacent to the PAM sequence in the genomic DNA.

[0095] Figure 5B Shows about Figure 5A The on-target and off-target editing efficiencies of each of the sgRNAs targeting the USH2A IVS40 mutation are depicted in FIG.

[0096] Fig. 6A -C shows the position relative to the IVS40 mutation, the position relative to the IVS40 nucleotide, and the on-target editing efficiency for sgRNAs that: (1) are associated with SpCas9 or SaCas9, and (2) overlap with the IVS40 mutation, bind upstream of the IVS40 mutation, or bind downstream of the IVS40 mutation. Regarding the position relative to the IVS40 nucleotide, -8-(+11) indicates that the sgRNA binds between the position 8 nucleotides upstream of the IVS40 mutation to the position 11 nucleotides downstream of IVS40. Regarding the position relative to the IVS40 nucleotide, -571-590 indicates that the sgRNA binds between the position 571 nucleotides upstream of the IVS40 mutation to the position 590 nucleotides upstream of IVS40. Regarding the position relative to the IVS40 nucleotide, +744-763 indicates that the sgRNA binds between the position 744 nucleotides downstream of the IVS40 mutation to the position 763 nucleotides downstream of IVS40.

[0097] Fig. 6A -B shows the position relative to the IVS40 mutation, the position relative to the IVS40 nucleotide, and the on-target editing efficiency for each of the 52 sgRNAs that are associated with SpCas9 and (1) overlap with the IVS40 mutation, (2) bind upstream of the IVS40 mutation, or (3) bind downstream of the IVS40 mutation.

[0098] Figure 6CShown are the position relative to the IVS40 mutation, the position relative to the IVS40 nucleotide, and the on-target editing efficiency for each of the 16 sgRNAs that are associated with SaCas9 and (1) bind upstream of the IVS40 mutation or (2) bind downstream of the IVS40 mutation.

[0099] Fig. 7A -F shows the binding of the first sgRNA upstream of the IVS40 mutation, and the binding of the second sgRNA downstream of the IVS40 mutation, as well as the five possible editing outcomes from using dual sgRNAs.

[0100] Fig. 7A Depicted are the binding of the first sgRNA upstream of the IVS40 mutation and the binding of the second sgRNA downstream of the IVS40 mutation.

[0101] Figure 7B Unedited genomic DNA comprising an IVS40 mutation is described.

[0102] Figure 7C Depicted are editing of genomic DNA by (1) a first sgRNA that binds upstream of the IVS40 mutation; or (2) a second sgRNA that binds downstream of the IVS40 mutation.

[0103] Fig.7D Described are editing of genomic DNA by both (1) a first sgRNA that binds upstream of the IVS40 mutation; and (2) a second sgRNA that binds downstream of the IVS40 mutation, but the editing does not result in a deletion.

[0104] Fig. 7E Depicted is editing of genomic DNA by both (1) a first sgRNA that binds upstream of the IVS40 mutation; and (2) a second sgRNA that binds downstream of the IVS40 mutation, and the editing results in a deletion.

[0105] Figure 7F Editing of genomic DNA by both (1) a first sgRNA that binds upstream of the IVS40 mutation and (2) a second sgRNA that binds downstream of the IVS40 mutation is described, but the editing does not result in a deletion. Instead, the editing results in an inversion.

[0106] Figure 8 A protocol for performing quantitative analysis of deletions using ddPCR.

[0107] Fig. 9A -B shows the deletion frequency and resulting deletion size for the different dual sgRNAs selected. Fig. 9A- The sgRNAs constituting each dual sgRNA included in B are associated with SpCas9.

[0108] Fig. 9A is a table showing the deletion frequency and resulting deletion size for different dual sgRNAs selected.

[0109] Fig. 9B is a graph showing the deletion frequency and resulting deletion size for different dual sgRNAs selected.

[0110] Fig.10 Depicted is the pAAV-U6 plasmid, which can be engineered to encode a sgRNA associated with SaCas9.

[0111] Fig.11A -B shows the deletion frequency and resulting deletion size for the different dual sgRNAs selected. Fig.11A - The sgRNAs constituting each dual sgRNA included in B are associated with SaCas9.

[0112] Fig.11A is a table showing the deletion frequency and resulting deletion size for different dual sgRNAs selected.

[0113] Fig. 11B is a graph showing the deletion frequency and resulting deletion size for different dual sgRNAs selected.

[0114] Fig. 12A -C shows the splicing reporter plasmid pET01; schematic diagrams of two different USH2A DNA inserts; and gel images of RT-PCR products amplified from mRNA of HEK 293 SpCas9-positive cells transfected with one of two different splicing reporter plasmids and one of four different sgRNAs targeting the USH2A IVS40 mutation.

[0115] Fig. 12A The splicing reporter plasmid pET01 is depicted.

[0116] Fig. 12B Depicted are two different USH2A DNA inserts (part of mutant intron 40 or part of wild-type intron 40) and vector elements surrounding the inserts. Also depicted are the corresponding RNA splice products (mutant splice product or wild-type splice product).

[0117] Fig. 12CShown are gel images of RT-PCR products amplified from mRNA of HEK 293 SpCas9-positive cells transfected with one of two different splicing reporter plasmids (a plasmid containing a portion of mutant intron 40 or a plasmid containing a portion of wild-type intron 40) and one of four different sgRNAs targeting USH2A IVS40 mutations (sgRNA containing 5321, 5323, 5325, or 5327).

[0118] Fig.13A -C shows a construct for a blue fluorescent protein (BFP) splicing reporter assay and the possible effects that genome editing may have on this construct and BFP gene expression therefrom.

[0119] Fig.13A Depicted is a construct expressing BFP using the phosphoglycerate kinase promoter. The BFP gene contains part of the wild-type sequence of intron 40 of the USH2A gene upstream of BFPORF. BFP expression is expected.

[0120] Fig. 13B Depicted is a construct expressing BFP using the phosphoglycerate kinase promoter. The BFP gene contains part of the IVS40 mutant sequence of intron 40 of the USH2A gene upstream of BFPORF. BFP expression is not expected.

[0121] Fig. 13C Depicts a first configuration of a cell from a human genome before genome editing and a second configuration of a cell from a human genome after genome editing. Fig. 13B After genome editing, BFP expression is expected.

[0122] Fig.14 The percentage of live cells expressing BFP after being subjected to a BFP splicing reporter assay is shown. Data for editing strategies using single sgRNA and dual sgRNA are shown. sgRNAs are paired with SpCas9.

[0123] Fig.15 The percentage of live cells expressing BFP after being subjected to a BFP splicing reporter assay is shown. Data for an editing strategy using dual sgRNAs are shown. These sgRNA pairs are paired with SaCas9.

[0124] Fig.16 The percentage of GFP-positive cells expressing BFP after being subjected to a BFP splicing reporter assay is shown. Data on editing strategies using single sgRNA and dual sgRNA are shown. These sgRNAs are paired with SpCas9 or SaCas9. Negative controls for editing are also shown.

[0125] Fig.17A -B shows the sites of primer and probe binding in the ddPCR assay for USH2A transcripts.

[0126] Fig.17A The sequence of the mRNA transcribed from the IVS40 mutant USH2A gene is shown. Also shown are the positions where the primers and probe bind to the corresponding cDNA.

[0127] Fig. 17B The sequence of the mRNA transcribed from the wild-type USH2A gene (or edited USH2A gene) is shown. Also shown are the positions where the primers and probe bind to the corresponding cDNA.

[0128] Fig.18 The percentages of corrected and uncorrected transcripts in IVS40 mutant cells that have been genome edited according to the present disclosure are shown. Data for editing strategies using single sgRNA and dual sgRNA are shown. These sgRNAs are paired with SpCas9. Negative controls are also shown.

[0129] Brief description of sequence listing

[0130] SEQ ID NOs: 1-612 are Cas endonuclease ortholog sequences.

[0131] SEQ ID NOs: 613-4696 are microRNA sequences.

[0132] SEQ ID NOs: 4697-5265 are AAV serotype sequences.

[0133] SEQ ID NO: 5266 is the human USH2A nucleotide sequence.

[0134] SEQ ID NOs: 5267-5269 show the sample sgRNA backbone sequences complexed by SpCas9.

[0135] SEQ ID NO: 5270 is related to Streptococcus pyogenes ( Streptococcus pyogenes ) Sample guide RNA (gRNA) for Cas9 endonuclease.

[0136] SEQ ID NO: 5271 shows the known family of homing endonucleases as classified by their structure.

[0137] SEQ ID NOs: 5272-5319 are 20 bp spacer sequences targeting regions upstream and downstream of the IVS40 mutation within or near intron 40 of the USH2A gene using the Streptococcus pyogenes Cas9 endonuclease.

[0138] SEQ ID NO: 5320 is a 20 bp spacer sequence targeted to a USH2A gene or other DNA sequence encoding a regulatory sequence of the USH2A gene using the Streptococcus pyogenes Cas9 endonuclease.

[0139] SEQ ID NO: 5321 is a 20 bp spacer sequence targeting regions upstream and downstream of the IVS40 mutation within or near intron 40 of the USH2A gene using the Streptococcus pyogenes Cas9 endonuclease.

[0140] SEQ ID NO: 5322 is a 20 bp spacer sequence targeting the USH2A gene or other DNA sequences encoding regulatory sequences of the USH2A gene using the Streptococcus pyogenes Cas9 endonuclease.

[0141] SEQ ID NO: 5323 is a 20 bp spacer sequence targeting regions upstream and downstream of the IVS40 mutation within or near intron 40 of the USH2A gene using the Streptococcus pyogenes Cas9 endonuclease.

[0142] SEQ ID NO: 5324 is a 20 bp spacer sequence targeting a USH2A gene or other DNA sequence encoding a regulatory sequence of the USH2A gene using the Streptococcus pyogenes Cas9 endonuclease.

[0143] SEQ ID NO: 5325 is a 20 bp spacer sequence targeting regions upstream and downstream of the IVS40 mutation within or near intron 40 of the USH2A gene using the Streptococcus pyogenes Cas9 endonuclease.

[0144] SEQ ID NO: 5326 is a 20 bp spacer sequence targeting a USH2A gene or other DNA sequence encoding a regulatory sequence of the USH2A gene using the Streptococcus pyogenes Cas9 endonuclease.

[0145] SEQ ID NOs: 5327-5328 are 20 bp spacer sequences targeting regions upstream and downstream of the IVS40 mutation within or near intron 40 of the USH2A gene using the Streptococcus pyogenes Cas9 endonuclease.

[0146] SEQ ID NOs: 5329-5385 are sequences representing target DNA sequences for each of the 57 sgRNA sequences.

[0147] SEQ ID NOs: 5386-5442 are sequences representing the reverse strand of the target DNA sequence to which the sgRNA binds, for each of the 57 sgRNA sequences.

[0148] SEQ ID NO: 5443 is an 18 bp spacer sequence targeting the region downstream of the IVS40 mutation using the Streptococcus pyogenes Cas9 endonuclease.

[0149] SEQ ID NO: 5444 is an 18 bp sgRNA sequence, representing the sequence of the target DNA sequence.

[0150] SEQ ID NO: 5445 is an 18 bp sgRNA sequence, representing the sequence of the reverse strand of the target DNA sequence to which the sgRNA binds.

[0151] SEQ ID NOs: 5446-5461 are derived from Staphylococcus aureus ( Staphylococcus aureus )Cas9 endonuclease, targeting a 20 bp spacer sequence in the region upstream and downstream of the IVS40 mutation within or near intron 40 of the USH2A gene.

[0152] SEQ ID NOs: 5462-5477 are sequences representing target DNA sequences for each of the 16 sgRNA sequences.

[0153] SEQ ID NOs: 5478-5493 are sequences representing the reverse strand of the target DNA sequence to which the sgRNA binds, for each of the 16 sgRNA sequences.

[0154] SEQ ID NO: 5494 is the single-chain HDR donor sequence.

[0155] SEQ ID NOs: 5495-5506 are PCR primer sequences.

[0156] SEQ ID NOs: 5507-5523 ​​are plasmid sequences expressing sgRNA.

[0157] SEQ ID NO: 5524 is a sequence for pET01, which includes a portion of wild-type intron 40 of USH2A.

[0158] SEQ ID NO: 5525 is a sequence for pET01, which includes a portion of mutant intron 40 of USH2A.

[0159] SEQ ID NOs: 5526-5537 show the sample sgRNA backbone sequences complexed by SaCas9.

[0160] SEQ ID NO: 5538-5549 are plasmid sequences expressing sgRNA. sgRNA is associated with SpCas9.

[0161] SEQ ID NO: 5550-5557 are plasmid sequences expressing sgRNA. sgRNA is associated with SaCas9.

[0162] SEQ ID NOs: 5558 and 5559 are oligonucleotide sequences used to amplify cDNA segments generated from USH2A transcripts.

[0163] SEQ ID NOs: 5560 and 5561 are oligonucleotide probes for detecting corrected or uncorrected USH2A cDNA sequences. DETAILED DESCRIPTION OF THE INVENTION

[0165] Applicants have discovered a novel method for treating Usher Syndrome Type 2A, such as Usher Syndrome Type 2A associated with an IVS40 mutation in the USH2A gene. The method can result in slowing or reversing the progression of Usher Syndrome Type 2A or preventing the progression of the disease in an individual.

[0166] Treatment

[0167] The methods provided herein, whether cell, ex vivo or in vivo methods, may involve one or a combination of the following methods. One method involves disrupting a consensus sequence used as a splice donor site within or near the IVS40 mutation in the USH2A gene by insertion and / or deletion due to a non-homologous end joining (NHEJ) pathway. In another method, the IVS40 mutation located in intron 40 of the USH2A gene is excised. In a third method, the mutant allele (e.g., IVS40 mutation) is corrected by HDR.

[0168] The NHEJ strategy can involve inducing a single-strand break or double-strand break within or near the IVS40 mutation of the USH2A gene with one or more CRISPR nucleases and gRNAs (e.g., cRNA + tracrRNA or sgRNA). This method edits sequences within or near the IVS40 mutation and can destroy sequences that cause incorrect splicing. This method utilizes a gRNA or sgRNA specific for the IVS40 mutation in the USH2A gene.

[0169] The excision strategy may include a set of guide RNAs or sgRNAs that bind within intron 40 of the USH2A gene, upstream and downstream of the IVS40 mutation, and excise the genomic region containing the IVS40 mutation. This strategy can be expected to affect both mutant (Mut) and wild-type (WT) alleles, which are permissive for intronic mutations. The excision strategy may result in a shorter form of the nascent precursor messenger RNA (pre-mRNA, lacking the dominant splice donor to generate the mutation), which may be properly spliced, resulting in expression of WT mRNA and WT usherin protein in edited cells, such as Figure 3 Depicted in. In cases where sufficient supportive retinal structures are still available, improved function and survival of edited cells can be expected. This approach utilizes gRNAs or sgRNAs specific for regions upstream and downstream of the IVS40 mutation within intron 40 of the USH2A gene.

[0170] The size of the deletion produced by the excision strategy can be 50 to 5000 base pairs (bp). For example, the deletion range can be 50-100; 50-250; 50-500; 50-1000; 50-1500; 50-2000; 200-500; 200-750; 200-1000; 200-1100; 500-1,000; 1,000-1,500; 1,500-2,000; 1,000-2,000; 2,000-2,500; 2,500-3,000; 3,000-3,500; 3,500-4,000; 4,000-4,500; 4,500-5,000 or 50-2,900 base pairs.

[0171] The HDR strategy may involve inducing one or more single-strand breaks or double-strand breaks within or near intron 40 of the USH2A gene, upstream and downstream of the IVS40 mutation, using one or more CRISPR endonucleases and gRNAs (e.g., crRNA + tracrRNA or sgRNA) in the presence of an exogenously introduced donor DNA template, or inducing one or more single-strand breaks or double-strand breaks within or near intron 40 of the USH2A gene, upstream and downstream of the IVS40 mutation, using one or more CRISPR endonucleases (Cas9, Cpf1, etc.) and two or more gRNAs to direct the cellular DSB response to homology-based repair. The donor DNA template may be a short single-stranded oligonucleotide, a short double-stranded oligonucleotide, a long single-stranded or double-stranded DNA molecule. The method can provide a gRNA pair that prepares a deletion by cleaving the gene twice: one gRNA that cuts at the 5' end of the IVS40 mutation, and another gRNA that cuts at the 3' end of the IVS40 mutation, which promotes the insertion of a new sequence from a polynucleotide donor template to replace the IVS40 mutation in the USH2A gene. The cleavage can be accomplished by a pair of DNA endonucleases that each make a DSB (one DSB on each end of the IVS40 mutation in or near intron 40), or by multiple nicking enzymes that make DSBs together (one DSB on each end of the IVS40 mutation in or near intron 40). This method utilizes gRNAs or sgRNAs specific for regions upstream and downstream of the IVS40 mutation within intron 40 of the USH2A gene. This method also utilizes a donor DNA molecule.

[0172] The advantages with respect to the above strategies (disruption of RNA splicing consensus sequences, excision and HDR strategies) are similar, in principle including both short-term and long-term beneficial clinical and laboratory effects.

[0173] Such methods use an endonuclease, such as a CRISPR-associated (Cas9, Cpf1, etc.) nuclease, to stably correct the IVS40 mutation within the genomic locus of the USH2A gene. Any CRISPR endonuclease can be used in the methods of the present disclosure, each with its own associated PAM, which may or may not be disease-specific. For example, with the CRISPR / Cas9 endonuclease from Streptococcus pyogenes, the gRNA spacer sequences targeting the IVS40 mutation in the USH2A gene have been identified in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, and 5443 of the sequence listing. For example, with the CRISPR / Cas9 endonuclease from Staphylococcus aureus, the gRNA spacer sequences targeting the IVS40 mutation in the USH2A gene have been identified in SEQ ID NOs: 5446-5461 of the sequence listing.

[0174] The examples described in the present disclosure can induce single-strand breaks or double-strand breaks within or near, upstream and downstream of the IVS40 mutation within intron 40 of the USH2A gene to introduce disruption, excision of the RNA splicing consensus sequence, or correct the IVS40 mutation within the USH2A gene with as little as one treatment (rather than delivering potential therapeutic agents for the patient's lifetime).

[0175] Usher syndrome

[0176] Usher syndrome is an autosomal recessive disorder characterized by sensorineural hearing loss, retinitis pigmentosa (RP), and in some cases vestibular dysfunction. The prevalence of Usher syndrome is estimated to be between 1 / 6,000 and 1 / 25,000.

[0177] Usher syndrome is a clinically and genetically heterogeneous disease, accounting for about half of all cases of combined hereditary deafness-blindness. To date, the disease has been associated with 13 genes. Three clinical forms of the disease (USH I, II, and III) have been identified based on the severity of hearing impairment, the presence or absence of vestibular dysfunction, and the age of disease onset.

[0178] Type II Usher syndrome is the most common clinical form, accounting for approximately 50% of all Usher syndrome cases. Type II Usher syndrome is characterized by congenital hearing loss and progressive vision loss starting in adolescence or adulthood. Hearing loss ranges from mild to severe and primarily affects the ability to hear high-frequency sounds. Vision loss occurs as the light-sensing cells of the retina gradually degenerate. Night vision loss begins first, followed by loss of peripheral vision. Over time, these blind areas expand and merge to produce tubular vision. In some cases, vision is further impaired by cataracts. Many patients become legally blind in the fifth decade of life.

[0179] Usher syndrome type 2A is due to mutations in the USH2A gene and accounts for approximately 80% of all Usher syndrome type 2 cases and 40% of all Usher syndrome cases.

[0180] USH2A gene

[0181] The USH2A gene (e.g., SEQ ID NO: 5266) is 800,503 base pairs and is located on chromosome 1:215,622,893:216,423,395 (Genome Reference Consortium - GRCh38 / hg38) (1q41). The USH2A gene contains 72 exons and encodes two alternatively spliced ​​isoforms of a protein called usherin. The full-length 580 kDa usherin protein (isoform b) is a complex transmembrane protein of 5,202 amino acids with a large extracellular domain. The short 170 kDa usherin protein (isoform a) is translated from a splice variant consisting of only the first 21 coding exons and is seen as an extracellular protein of 1546 amino acids.

[0182] Usherin is localized close to the vesicle loading site at the perifibrillary membrane and may play a role in vesicle transport between the inner and outer segments of the photoreceptor.

[0183] IVS40 mutation

[0184] There are various mutations associated with Usher syndrome, which may be insertions, deletions, missense, nonsense, frameshifts, and other mutations, with the common effect of inactivating the USH2A gene.

[0185] C.7595-2144A>G (IVS40 mutation) in the USH2A gene results in the generation of a splice donor site and the insertion of 152 bp within the USH2A mRNA, which in turn results in a frameshift and a truncated and nonfunctional protein.

[0186] Any one or more mutations can be repaired to restore usherin protein function. For example, the pathological variant IVS40 can be excised or corrected to restore usherin protein expression (see Table 1).

[0187] Table 1

[0188]

[0189] In vivo based therapies

[0190] Provided herein are methods for treating patients with Usher syndrome type 2A. In some aspects, the method is an in vivo cell-based therapy. The materials and methods described herein can be used to edit the chromosomal DNA of cells in patients with Usher syndrome type 2A. For example, the in vivo method can include editing the patient's cells, such as the IVS40 mutation in the USH2A gene in photoreceptor cells or retinal progenitor cells.

[0191] Although certain cells present attractive targets for ex vivo treatment and therapy, increased delivery efficacy can allow direct in vivo delivery to such cells. Ideally, targeting and editing will be directed to relevant cells. Cutting in other cells can also be prevented by using promoters that are only active in certain cells and / or developmental stages. Additional promoters are inducible, and therefore if the nuclease is delivered as a plasmid, it can be controlled in time. The amount of time the delivered RNA and protein remain in the cell can also be adjusted using treatments or domains added to change the half-life. In vivo treatment will eliminate many treatment steps, but lower delivery rates may require higher editing rates. In vivo treatment can eliminate problems and losses from ex vivo treatment and transplantation.

[0192] The advantages of in vivo gene therapy can be the ease of therapeutic agent production and administration. The same treatment method and therapy has the potential to be used to treat more than one patient, for example, many patients sharing the same or similar genotype or allele. In contrast, ex vivo cell therapy generally requires the use of the patient's own cells, which are isolated, manipulated and returned to the same patient.

[0193] Ex vivo based therapies

[0194] Provided herein is a method for treating patients with Usher syndrome type 2A. One aspect of such methods is a therapy based on ex vivo cells. For example, patient-specific induced pluripotent stem cells (iPSC) can be produced. Then, the chromosomal DNA of these iPSC cells can be edited using the materials and methods described herein. For example, the method may include editing within or near the IVS40 mutation in the USH2A gene of the iPSC. Next, the genome-edited iPSC can be differentiated into other cells, such as photoreceptor cells or retinal progenitor cells. Finally, differentiated cells (such as photoreceptor cells or retinal progenitor cells) can be implanted into the patient (i.e., implanted into the patient's eye).

[0195] Another aspect of such methods is based on ex vivo cell therapy. For example, photoreceptor cells or retinal progenitor cells can be isolated from patients. Next, the chromosomal DNA of these photoreceptor cells or retinal progenitor cells can be edited using the materials and methods described herein. For example, the method can include editing within or near the IVS40 mutation in the USH2A gene of photoreceptor cells or retinal progenitor cells. Finally, the genome-edited photoreceptor cells or retinal progenitor cells can be implanted into the patient (i.e., implanted into the patient's eye).

[0196] Another aspect of such methods is based on ex vivo cell therapy. For example, mesenchymal stem cells can be isolated from a patient, which can be isolated from the patient's bone marrow, peripheral blood, adipose tissue or umbilical cord. Next, the chromosomal DNA of these mesenchymal stem cells can be edited using the materials and methods described herein. For example, the method may include editing within or near the IVS40 mutation in the USH2A gene of a mesenchymal stem cell. Next, the genome-edited mesenchymal stem cells can be differentiated into any type of cell, such as a photoreceptor cell or a retinal progenitor cell. Finally, the differentiated cells (e.g., photoreceptor cells or retinal progenitor cells) can be implanted into a patient (i.e., implanted into the patient's eye).

[0197] One advantage of an ex vivo cell therapy approach is the ability to perform a comprehensive analysis of therapeutic agents prior to administration. Nuclease-based therapeutic agents may have a certain level of off-target effects. Performing gene correction in vitro allows characterization of the corrected cell population prior to implantation. The present disclosure includes sequencing the entire genome of the corrected cells to ensure that off-target effects, if any, can be at a genomic location associated with a minimal risk to the patient. In addition, colonies of specific cells, including clonal colonies, may be isolated prior to implantation.

[0198] Another advantage of ex vivo cell therapy involves gene correction in iPSCs compared to other primary cell sources. iPSCs are prolific, making it easy to obtain large numbers of cells required for cell-based therapies. In addition, iPSCs are an ideal cell type for performing clonal isolation. This allows screening for correct genomic corrections without the risk of reduced viability. In contrast, other primary cells, such as photoreceptor cells or retinal progenitor cells, survive only a few passages and are difficult to clone and expand. Therefore, it may be much easier to manipulate iPSCs for the treatment of Usher syndrome type 2A, and the time required to prepare the required gene corrections may be shortened.

[0199] Genome editing

[0200] Genome editing refers to a process of modifying the nucleotide sequence of a genome, for example, in an accurate or predetermined manner. Examples of genome editing methods described herein include using a site-directed nuclease to cut DNA at a precise target position in the genome, thereby producing a method for single-stranded or double-stranded DNA breaks at a specific position in the genome. Such breaks can and are regularly repaired by natural endogenous cell processes (such as HDR and NHEJ). These two main DNA repair processes consist of a series of alternative pathways. NHEJ directly connects the DNA ends caused by double-strand breaks, sometimes accompanied by the loss or addition of nucleotide sequences, which may destroy or enhance gene expression. HDR uses homologous sequences or donor sequences as templates for inserting a defined DNA sequence at the breakpoint. Homologous sequences can be in endogenous genomes, such as sister chromatids. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-stranded oligonucleotide, a double-stranded oligonucleotide, a duplex oligonucleotide, or a virus, which has a region with a high homology to the locus cut by the nuclease, but can also contain additional sequences or sequence changes, including deletions that can be incorporated into the target locus of the cut. A third repair mechanism may be microhomology-mediated end joining (MMEJ), also known as "alternative NHEJ (ANHEJ)", whose genetic outcomes are similar to NHEJ in that small deletions and insertions can occur at the cut site. MMEJ can utilize homologous sequences of several base pairs flanking the DNA break site to drive a more favorable DNA end joining repair outcome, and recent reports have further elucidated the molecular mechanisms of this process. In some cases, it may be possible to predict the likely repair outcome based on analysis of potential microhomologies at the DNA break site.

[0201] Each of these genome editing mechanisms can be used to produce the desired genome changes. One step in the genome editing process can be to produce one or two DNA breaks in the target locus near the expected mutation site, the latter being a double-strand break or two single-strand breaks. As described and shown herein, this can be achieved by using a site-directed polypeptide.

[0202] Site-directed polypeptides, such as DNA endonucleases, can introduce double-strand breaks or single-strand breaks in nucleic acids, such as genomic DNA. Double-strand breaks can stimulate the cell's endogenous DNA repair pathways [e.g., homology-dependent repair (HDR) or non-homologous end joining (NHEJ) or (ANHEJ) or (MMEJ)]. NHEJ can repair the cleaved target nucleic acid without the need for a homologous template. This can sometimes result in small deletions or insertions (indels) in the target nucleic acid at the cleavage site, and can result in disruption or alteration of gene expression.

[0203] HDR can occur when a homologous repair template or donor is available. The homologous donor template can comprise at least a portion of a wild-type USH2A gene or cDNA. At least a portion of the wild-type USH2A gene or cDNA may be exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58 exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, intronic regions, fragments or combinations thereof, or the entire USH2A gene or cDNA.

[0204] The donor template may be a single-stranded or double-stranded polynucleotide. The donor template may be up to 11 kb. The donor template may be up to 10 kb. The donor template may be up to 9 kb. The donor template may be up to 8 kb. The donor template may be up to 7 kb. The donor template may be up to 6 kb. The donor template may be up to 5 kb. The donor template may be up to 4 kb. The donor template may be up to 3 kb. The donor template may be up to 2 kb. The donor template may be up to 1 kb. The donor template may be less than 1 kb. The donor template may be 500 bp to 1000 bp. The donor template may be 250 bp to 500 bp. The donor template may be 100 to 250 bp. The donor template may be delivered by AAV. The homologous donor template may comprise a sequence homologous to a sequence flanking the target nucleic acid cleavage site. For example, the donor template may have arms homologous to the 1q41 region. The donor template may also have arms homologous to the pathological variant IVS40. Sister chromatids may be used by cells as repair templates. However, for the purpose of genome editing, the repair template may be supplied as an exogenous nucleic acid, such as a plasmid, a duplex oligonucleotide, a single-stranded oligonucleotide, a double-stranded oligonucleotide or a viral nucleic acid. Using an exogenous donor template, additional nucleic acid sequences (e.g., transgenes) or modifications (e.g., single or multiple base changes or deletions) may be introduced between the flanking regions of homology so that additional or altered nucleic acid sequences also become incorporated into the target locus. MMEJ may result in genetic outcomes similar to NHEJ because small deletions and insertions may occur at the cleavage site. MMEJ may utilize homologous sequences of several base pairs flanking the cleavage site to drive favorable end-joining DNA repair outcomes. In some cases, it may be possible to predict possible repair outcomes based on analysis of potential microhomologies in the nuclease target region.

[0205] Therefore, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. Exogenous polynucleotide sequences are referred to herein as donor polynucleotides (or donors or donor sequences or polynucleotide donor templates). A portion of a donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide or a copy of a donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that is not naturally present at the target nucleic acid cleavage site.

[0206] Modification of target DNA due to NHEJ and / or HDR can result in, for example, gene correction.

[0207] CRISPR endonuclease system

[0208] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic loci can be found in the genomes of many prokaryotes (such as bacteria and archaea). In prokaryotes, CRISPR loci encode products that act as a class of immune systems to help prokaryotes defend against foreign invaders such as viruses and bacteriophages. There are three stages of CRISPR locus function: new sequences are integrated into the CRISPR loci, expression of CRISPR RNA (crRNA), and silencing of foreign invader nucleic acids. Five types of CRISPR systems have been identified (e.g., Type I, Type II, Type III, Type U, and Type V).

[0209] The CRISPR locus includes many short repetitive sequences, referred to as "repeats". When expressed, repetitions can form secondary structures (e.g., hairpins) and / or contain unstructured single-stranded sequences. Repetitions usually occur in clusters and often diverge between species. Repetitions are regularly spaced by unique intervening sequences called "spacers", resulting in a repeat-spacer-repeat locus architecture. The spacers are equivalent to or have a high degree of homology with known foreign invader sequences. The spacer-repeat unit encodes crispRNA (crRNA), which is processed into a mature form of the spacer-repeat unit. CrRNA includes a "seed" or spacer sequence related to a targeted target nucleic acid (in a form naturally occurring in prokaryotes, the spacer sequence targets foreign invader nucleic acids). The spacer sequence is located at the 5' end or 3' end of the crRNA.

[0210] The CRISPR locus also includes polynucleotide sequences encoding CRISPR-related (Cas) genes. Cas genes encode endonucleases involved in the biogenesis and interference phases of crRNA function in prokaryotes. Some Cas genes include homologous secondary and / or tertiary structures.

[0211] Type II CRISPR systems

[0212] In essence, the crRNA biogenesis in the type II CRISPR system requires trans-activation of CRISPR RNA (tracrRNA). TracrRNA can be modified by endogenous RNase III and then repeatedly hybridized with the crRNA in the precursor crRNA array. Endogenous RNase III can be recruited to cut the precursor crRNA. The cut crRNA can be subjected to exoribonuclease trimming to produce a mature crRNA form (e.g., 5' trimming). TracrRNA can remain hybridized with crRNA, and tracrRNA and crRNA are related to site-directed polypeptides (e.g., Cas9). The crRNA of the crRNA-tracrRNA-Cas9 complex can guide the complex to the target nucleic acid with which crRNA can hybridize. The hybridization of crRNA with the target nucleic acid can activate Cas9 for targeted nucleic acid cutting. The target nucleic acid in the type II CRISPR system is called the protoplast adjacent motif (PAM). In nature, PAM is necessary for promoting the binding of site-directed polypeptides (e.g., Cas9) to target nucleic acids. The type II system (also called Nmeni or CASS4) is further subdivided into type II-A (CASS4) and type II-B (CASS4a). Jinek et al., Science, 337 (6096):816-821 (2012) showed that the CRISPR / Cas9 system can be used for RNA-programmable genome editing, and International Patent Application Publication No. WO2013 / 176772 provides numerous examples and applications of the CRISPR / Cas nuclease system for site-specific gene editing.

[0213] V-type CRISPR system

[0214] The V-type CRISPR system has several major differences from the II-type system. For example, Cpf1 is a single RNA-guided endonuclease, which lacks tracrRNA compared to the II-type system. In fact, the CRISPR array associated with Cpf1 can be processed into mature crRNA without the need for additional trans-activating tracrRNA. The V-type CRISPR array can be processed into a short mature crRNA of 42-44 nucleotides in length, wherein each mature crRNA starts with a direct repeat of 19 nucleotides, followed by a spacer sequence of 23-25 ​​nucleotides. In contrast, the mature crRNA in the II-type system can start with a spacer sequence of 20-24 nucleotides, followed by a direct repeat of about 22 nucleotides. In addition, Cpf1 can utilize the T-rich pro-spacer adjacent motif so that the Cpf1-crRNA complex effectively cuts the target DNA of the short T-rich PAM before, which is in contrast to the G-rich PAM after the target DNA of the II-type system. Therefore, the V-type system cuts at a point away from the PAM, while the II-type system cuts at a point adjacent to the PAM. Additionally, Cpf1 cleaves DNA via staggered DNA double-strand breaks (with 4 or 5 nucleotide 5' overhangs) compared to the type II system. The type II system cleaves via blunt-ended double-strand breaks. Similar to the type II system, Cpf1 contains a predicted RuvC-like endonuclease domain, but lacks a second HNH endonuclease domain, which is the opposite of the type II system.

[0215] Cas gene / peptide and protospacer adjacent motif

[0216] Exemplary CRISPR / Cas polypeptides include Fonfara et al., Nucleic Acids Research, 42 :2577-2590 (2014) in Figure 1. Since the discovery of the Cas gene, the CRISPR / Cas gene naming system has undergone extensive rewriting. Figure 5 of Fonfara above provides the PAM sequences of Cas9 polypeptides from various species.

[0217] Site-directed peptides

[0218] Site-directed polypeptides are nucleases used to cleave DNA in genome editing. Site-directed nucleases can be administered to cells or patients as any of the following: one or more polypeptides, or one or more mRNAs encoding the polypeptides. Any enzyme or ortholog listed in SEQ ID NO: 1-612 or disclosed herein can be used in the methods herein.

[0219] In the context of a CRISPR / Cas or CRISPR / Cpf1 system, the site-directed polypeptide can be associated with a guide RNA, which in turn specifies a site in the target DNA to which the polypeptide is directed. In the CRISPR / Cas or CRISPR / Cpf1 system disclosed herein, the site-directed polypeptide can be an endonuclease, such as a DNA endonuclease.

[0220] The site-directed polypeptide may comprise a plurality of nucleic acid cleavage (i.e., nuclease) domains. Two or more nucleic acid cleavage domains may be linked together via a linker. For example, the linker may comprise a flexible linker. The linker may comprise a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more amino acids.

[0221] The naturally occurring wild-type Cas9 enzyme comprises two nuclease domains: an HNH nuclease domain and a RuvC domain. In this article, "Cas9" refers to both naturally occurring Cas9 and recombinant Cas9. The Cas9 enzyme contemplated herein may comprise an HNH or HNH-like nuclease domain, and / or a RuvC or RuvC-like nuclease domain.

[0222] The HNH or HNH-like domain comprises a McrA-like fold. The HNH or HNH-like domain comprises two antiparallel β strands and an α helix. The HNH or HNH-like domain comprises a metal binding site (e.g., a divalent cation binding site). The HNH or HNH-like domain can cut a strand of the target nucleic acid (e.g., the complementary strand of the crRNA targeting strand).

[0223] RuvC or RuvC-like domains contain RNase H or RNase H-like folds. RuvC / RNase H domains are involved in a variety of nucleic acid-based functions, including functions that act on both RNA and DNA. The RNase H domain contains 5 beta strands surrounded by multiple α-helices. RuvC / RNase H or RuvC / RNase H-like domains contain metal binding sites (e.g., divalent cation binding sites). RuvC / RNase H or RuvC / RNase H-like domains can cut one strand of a target nucleic acid (e.g., a non-complementary strand of a double-stranded target DNA).

[0224] Site-directed polypeptides can introduce double-strand breaks or single-strand breaks in nucleic acids such as genomic DNA. Double-strand breaks can stimulate the endogenous DNA repair pathways of cells (such as HDR or NHEJ or ANHEJ or MMEJ). NHEJ can repair the target nucleic acid of cleavage without the need for a homologous template. This can sometimes result in a small deletion or insertion (indel) in the target nucleic acid at the cleavage site, and can result in the destruction or change of gene expression. When a homologous repair template or donor is available, HDR can occur. The homologous donor template can include a sequence homologous to the sequence of the flanking target nucleic acid cleavage site. Sister chromatids can be used as a repair template by cells. However, for the purpose of genome editing, the repair template can be supplied as an exogenous nucleic acid, such as a plasmid, a duplex oligonucleotide, a single-stranded oligonucleotide or a viral nucleic acid. Using an exogenous donor template, additional nucleic acid sequences (such as transgenics) or modifications (such as single or multiple base changes or deletions) can be introduced between the flanking regions of homology, so that additional or changed nucleic acid sequences also become incorporated into the target locus. MMEJ can result in genetic outcomes similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can utilize homologous sequences of a few base pairs flanking the cleavage site to drive favorable end-joining DNA repair outcomes. In some cases, it may be possible to predict the likely repair outcome based on analysis of potential microhomologies in the nuclease target region.

[0225] Therefore, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. Exogenous polynucleotide sequences are referred to herein as donor polynucleotides (or donors or donor sequences). A donor polynucleotide, a part of a donor polynucleotide, a copy of a donor polynucleotide, or a part of a copy of a donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that is not naturally present at the target nucleic acid cleavage site.

[0226] The site-directed polypeptide may comprise an amino acid sequence that is identical to a wild-type exemplary site-directed polypeptide [e.g., Cas9 from Streptococcus pyogenes, US 2014 / 0068797, SEQ ID No. 8 or Sapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011)], and various other site-directed polypeptides having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity. The site-directed polypeptide may comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 contiguous amino acids to a wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, supra). The site-directed polypeptide may comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 contiguous amino acids to a wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, supra). The site-directed polypeptide may comprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 contiguous amino acids in the HNH nuclease domain of the site-directed polypeptide to a wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, supra). The site-directed polypeptide may comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 contiguous amino acids in the HNH nuclease domain of the site-directed polypeptide to a wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, supra). The site-directed polypeptide may comprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 contiguous amino acids in the RuvC nuclease domain of the site-directed polypeptide to a wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, supra). The site-directed polypeptide may comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 contiguous amino acids in the RuvC nuclease domain of the site-directed polypeptide to a wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, supra).

[0227] The site-directed polypeptide may comprise a modified form of a wild-type exemplary site-directed polypeptide. The modified form of the wild-type exemplary site-directed polypeptide may comprise a mutation that reduces the nucleic acid cleavage activity of the site-directed polypeptide. The modified form of the wild-type exemplary site-directed polypeptide may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type exemplary site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, supra). The modified form of the site-directed polypeptide may not have substantial nucleic acid cleavage activity. When the site-directed polypeptide is a modified form that does not have substantial nucleic acid cleavage activity, it is referred to herein as "enzymatically inactive".

[0228] The modified form of the site-directed polypeptide may include mutations so that it can induce SSB on the target nucleic acid (e.g., by cutting only one of the sugar-phosphate backbones of the double-stranded target nucleic acid). The mutation may result in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% nucleic acid cleavage activity in one or more of the multiple nucleic acid cleavage domains of the wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, supra). The mutation may result in one or more of the multiple nucleic acid cleavage domains retaining the ability to cut the complementary strand of the target nucleic acid, but reducing its ability to cut the non-complementary strand of the target nucleic acid. The mutation may result in one or more of the multiple nucleic acid cleavage domains retaining the ability to cut the non-complementary strand of the target nucleic acid, but reducing its ability to cut the complementary strand of the target nucleic acid. For example, residues such as Asp10, His840, Asn854, and Asn856 in the wild-type exemplary Streptococcus pyogenes Cas9 polypeptide are mutated to inactivate one or more of the multiple nucleic acid cleavage domains (e.g., nuclease domains). The residues to be mutated can correspond to residues Asp10, His840, Asn854, and Asn856 in a wild-type exemplary Streptococcus pyogenes Cas9 polypeptide (e.g., as determined by sequence and / or structural alignment). Non-limiting examples of mutations include D10A, H840A, N854A, or N856A. Mutations other than alanine substitutions may be suitable.

[0229] The D10A mutation can be combined with one or more of the H840A, N854A, or N856A mutations to produce a site-directed polypeptide that is substantially lacking in DNA cleavage activity. The H840A mutation can be combined with one or more of the D10A, N854A, or N856A mutations to produce a site-directed polypeptide that is substantially lacking in DNA cleavage activity. The N854A mutation can be combined with one or more of the H840A, D10A, or N856A mutations to produce a site-directed polypeptide that is substantially lacking in DNA cleavage activity. The N856A mutation can be combined with one or more of the H840A, N854A, or D10A mutations to produce a site-directed polypeptide that is substantially lacking in DNA cleavage activity. Site-directed polypeptides that comprise a substantially inactive nuclease domain are referred to as "nickases."

[0230] The RNA-guided endonuclease, such as the nickase variant of Cas9, can be used to increase the specificity of CRISPR-mediated genome editing. Wild-type Cas9 is usually guided by a single guide RNA, which is designed to hybridize with a specified ~20 nucleotide sequence in a target sequence (e.g., an endogenous genomic locus). However, several mismatches can be tolerated between the guide RNA and the target locus, effectively reducing the length of the required homology in the target site to, for example, as little as 13 nt homology, and thereby causing the potential for binding to increase, and other places in the target genome by double-stranded nucleic acid cutting of the CRISPR / Cas9 complex - also referred to as off-target cutting. Because the nickase variant of Cas9 only cuts one chain each, in order to produce double-strand breaks, a pair of nickases must be closely adjacent to and combined on the opposite strand of the target nucleic acid, thereby producing a pair of nicks, which are equivalent to double-strand breaks. This requires that two separate guide RNAs (one for each nickase) must be closely adjacent to and combined on the opposite strand of the target nucleic acid. This requirement essentially doubles the minimum homology length required for double-strand breaks to occur, thereby reducing the likelihood that double-strand cleavage events will occur elsewhere in the genome where the two guide RNA sites (if they exist) are unlikely to be close enough to each other to allow double-strand breaks to form. As described in the art, nickases can also be used to promote HDR relative to NHEJ. HDR can be used to introduce selected changes into target sites in the genome by using specific donor sequences that effectively mediate the desired changes.

[0231] The mutation considered may include substitution, addition and deletion or any combination thereof. The mutation converts the mutated amino acid to alanine. The mutation converts the mutated amino acid to another amino acid (e.g., glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine or arginine). The mutation converts the mutated amino acid to an unnatural amino acid (e.g., selenomethionine). The mutation converts the mutated amino acid to an amino acid mimic (e.g., a phosphate mimic). The mutation may be a conservative mutation. For example, the mutation converts the mutated amino acid to an amino acid whose size, shape, charge, polarity, conformation and / or rotational isomer (e.g., cysteine / serine mutation, lysine / asparagine mutation, histidine / phenylalanine mutation) similar to the mutated amino acid. The mutation may cause movement in the reading frame and / or the generation of premature stop codons. The mutation may cause changes in the regulatory region of a gene or locus, which may affect the expression of one or more genes.

[0232] The site-directed polypeptide (e.g., a variant, mutation, enzymatically inactive and / or conditionally inactive site-directed polypeptide) can target a nucleic acid. The site-directed polypeptide (e.g., a variant, mutation, enzymatically inactive and / or conditionally inactive endoribonuclease) can target a DNA. The site-directed polypeptide (e.g., a variant, mutation, enzymatically inactive and / or conditionally inactive endoribonuclease) can target an RNA.

[0233] The Argonaute protein can comprise one or more non-native sequences (eg, the Argonaute protein is a fusion protein).

[0234] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from a bacterium (e.g., Streptococcus pyogenes), a nucleic acid binding domain, and two nucleic acid cleavage domains (i.e., a HNH domain and a RuvC domain).

[0235] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from bacteria (e.g., Streptococcus pyogenes), and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain).

[0236] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from bacteria (e.g., Streptococcus pyogenes), and two nucleic acid cleavage domains, wherein one or both of the nucleic acid cleavage domains comprise at least 50% amino acid identity to a nuclease domain of Cas9 from bacteria (e.g., Streptococcus pyogenes).

[0237] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from a bacterium (e.g., Streptococcus pyogenes), two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), and a non-native sequence (e.g., a nuclear localization signal) or a linker connecting the site-directed polypeptide to the non-native sequence.

[0238] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from a bacterium (e.g., Streptococcus pyogenes), two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), wherein the site-directed polypeptide comprises a mutation in one or both of the nucleic acid cleavage domains that reduces the cleavage activity of the nuclease domain by at least 50%.

[0239] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from a bacterium (e.g., Streptococcus pyogenes), and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), wherein one of the nucleic acid cleavage domains comprises a mutation to aspartic acid 10, and / or wherein one of the nuclease domains can comprise a mutation to histidine 840, and wherein the mutation reduces the cleavage activity of the nuclease domain by at least 50%.

[0240] The one or more site-directed polypeptides, such as DNA endonucleases, may include two nickases that together effect one double-strand break at a specific locus in the genome, or four nickases that together effect or cause two double-strand breaks at a specific locus in the genome. Alternatively, one site-directed polypeptide, such as a DNA endonuclease, may effect or cause one double-strand break at a specific locus in the genome.

[0241] Non-limiting examples of Cas9 orthologs from other bacterial strains include, but are not limited to, the Cas proteins identified in: Acaryochloris marina MBIC11017; Arabidopsis halophilus Acetohalobium arabaticum )DSM 5501; Thermophilic acidthiobacillus ( Acidithiobacillus caldus ); Thiobacillus thiooxidans ( Acidithiobacillus ferrooxidans )ATCC 23270; Alicyclobacillus acidocaldarius ( Alicyclobacillus acidocaldarius )LAA1; Alicyclobacillus acidocaldarius subsp. acidocaldarius ( Alicyclobacillus acidocaldarius subsp. acidocaldarius )DSM 446; Chromatids vinifera ( Allochromatium vinosum )DSM 180; Ammonifex degensii KC4; Anabaena variabilis ( Anabaena variabilis )ATCC 29413; Arthrospira maxima ( Arthrospira maxima )CS-328; Arthrospira platensis ( Arthrospira platensis ) strain Paraca ; Arthrospira species ( Arthrospira sp. )PCC 8005; Bacillus pseudomycoides ( Bacillus pseudomycoides )DSM12442; Selenate-reducing Bacillus ( Bacillus selenitireducens )MLS10; Burkholderiales bacteria ( Burkholderiales bacterium )1_1_47; Pyrolytic cellulose bacteria ( Caldicelulosiruptor becscii )DSM 6725; Candidatus Desulforudis audaxviator MP104C; Hydrothermal pyrolysis cellulose bacteria ( Caldicellulosiruptor hydrothermalis )_108; Clostridium phage ( Clostridium phage )c-st; Clostridium botulinum ( Clostridium botulinum ) A3 strain Loch Maree ; Clostridium botulinum Ba4 strain 657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanobacteria species ( Cyanothece sp. )ATCC 51142; Cyanobacteria species CCY0110; Cyanobacteria species PCC 7424; Cyanobacteria species PCC 7822; Exiguobacterium sibiricum ( Exiguobacterium sibiricum )255-15; Fingoldiella major ( Finegoldia magna )ATCC 29328; Clustered Bacteria ( Ktedonobacter racemifer )DSM 44963; Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus )PB2003 / 044-T3-4; Lactobacillus salivarius ( Lactobacillus salivarius )ATCC 11741; Listeria innocua; Sphaerotheca species ( Lyngbya sp. )PCC 8106; Marinobacter species ( Marinobacter sp. )ELB17; Investigation of methanogens ( Methanohalobium evestigatum )Z-7303; Microcystis phage ( Microcystis phage )Ma-LMM01; Microcystis aeruginosa ( Microcystis aeruginosa )NIES-843; Marine Microoscillator ( Microscilla marina )ATCC23134; Microcoleus philoxeroides ( Microcoleus chthonoplastes )PCC 7420; Neisseria meningitidis; Nitrosococcus halophilus Nc4; Nocardia daphni subsp. daphni Nocardiopsis dassonvillei subsp. dassonvillei )DSM 43111; Nodularia foamyssa ( Nodularia spumigena )CCY9414; Nostoc species ( Nostoc sp. )PCC 7120; Oscillatoria species ( Oscillatoria sp. )PCC 6506; Thermopropionic acid degrading bacteria ( Pelotomaculum_thermopropionicum )_SI; Mobilthia spores ( Petrotoga mobilis )SJ95; Pseudomonas naphthotrophicus ( Polaromonas naphthalenivorans )CJ2; Polarmonas species ( Polaromonas sp. )JS666; Pseudoalteromonas marinum ( Pseudoalteromonas haloplanktis )TAC125; Streptomyces spp. Streptomyces pristinaespiralis)ATCC 25486; Streptomyces ATCC 25486; Streptococcus thermophilus; Streptomyces viridis Streptomyces viridochromogenes )DSM 40736; Roseola spp.( Streptosporangium roseum )DSM 43021; ​​Synechococcus species ( Synechococcus sp. )PCC 7335; and Thermus africanus ( Thermosipho africanus )TCF52B ((Chylinski et al., RNA Biol ., 2013;10(5): 726-737.

[0242] In addition to Cas9 orthologs, other Cas9 variants, such as inactive dCas9 and fusion proteins with effector domains of different functions, can serve as platforms for genetic regulation.Any of the aforementioned enzymes may be useful in the present disclosure.

[0243] SEQ ID NO: 1-612 provides further examples of endonucleases that can be utilized in the present disclosure. These proteins can be modified before use, or can be encoded in a nucleic acid sequence such as DNA, RNA or mRNA, or in a vector construct such as a plasmid or adeno-associated virus (AAV) vector taught herein. Further, they can be codon optimized.

[0244] Although nomenclature is used herein to indicate the species of origin for a given site-directed polypeptide, it is understood that the site-directed polypeptide and / or nucleic acid encoding the site-directed polypeptide may be modified compared to the sequence occurring in the species of origin. For example, "SpCas9" indicates that the Cas9 gene / protein in question originates from Streptococcus pyogenes and has been modified, for example, by the addition of an NLS and / or the performance of codon optimization. For example, "SaCas9" indicates that the Cas9 gene / protein in question originates from Staphylococcus aureus and has been modified, for example, by the addition of an NLS and / or the performance of codon optimization.

[0245] Genome-targeted nucleic acids

[0246] The present disclosure provides genome targeting nucleic acids that can direct the activity of related polypeptides (e.g., site-directed polypeptides) to specific target sequences within target nucleic acids. Genome targeting nucleic acids can be RNA. Genome targeting RNA is referred to herein as "guide RNA" or "gRNA". The guide RNA may include at least a spacer sequence and a CRISPR repeat sequence that hybridizes with the target nucleic acid sequence of interest. In a type II system, the gRNA also includes a second RNA called a tracrRNA sequence. In type II gRNA, the CRISPR repeat sequence and the tracrRNA sequence hybridize with each other to form a duplex. In type V gRNA, crRNA forms a duplex. In both systems, the duplex can bind to the site-directed polypeptide so that the guide RNA and the site-directed polypeptide form a complex. Due to its binding to the site-directed polypeptide, the genome targeting nucleic acid can provide target specificity for the complex. Therefore, the genome targeting nucleic acid can guide the activity of the site-directed polypeptide.

[0247] Exemplary guide RNAs include the spacer sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443, and 5446-5461 of the sequence listing ( Figure 2A -B, G and J). The target DNA sequence (5'-3') (see SEQ ID NOs: 5329-5385, 5444 and 5462-5477) can be Figure 2C -D, 2H, and 2K. The reverse strand (5'-3') of the target DNA sequence to which the sgRNA binds can be found in Figure 2E -Found in F, I, and L.

[0248] Each guide RNA can be designed to include a spacer sequence complementary to its genomic target sequence. For example, the spacer sequences in SEQ ID NO: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443 and 5446-5461 of the sequence list can each be placed in a single RNA chimera or crRNA (together with the corresponding tracrRNA). See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

[0249] The gRNA or sgRNA disclosed herein can be combined with a DNA endonuclease to form a ribonucleoprotein complex that can cause stable editing within or near the IVS40 mutation in the USH2A gene. The changes (e.g., edits) in the DNA sequence caused by such editing can be non-transient.

[0250] The genome targeting nucleic acid can be a bimolecular guide RNA ( Figure 1A The genome targeting nucleic acid may be a single molecule guide RNA ( Figure 1B ). Either a double-molecule guide RNA or a single-molecule guide RNA can be modified.

[0251] The bimolecular guide RNA may comprise two RNA strands. The first strand may comprise an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second strand may comprise a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0252] The single molecule guide RNA (sgRNA) in the type II system can include an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single molecule guide linker, a minimal tracrRNA sequence, a 3'tracrRNA sequence, and an optional tracrRNA extension sequence in the 5' to 3' direction. The optional tracrRNA extension can include elements that contribute additional functionality (e.g., stability) to the guide RNA. The single molecule guide linker can connect the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can include one or more hairpins.

[0253] The sgRNA may comprise a variable length spacer sequence of 17-30 nucleotides at the 5' end of the sgRNA sequence (Table 2). In other examples, the sgRNA may comprise a variable length spacer sequence of 17-24 nucleotides at the 5' end of the sgRNA sequence.

[0254] The sgRNA may include a spacer sequence of 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 19 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 18 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 17 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 21 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 22 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 23 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 24 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 25 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 26 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 27 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 28 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 29 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of 30 nucleotides at the 5' end of the sgRNA sequence.

[0255] The sgRNA may not include a uracil at the 3' end of the sgRNA sequence, such as in SEQ ID NO: 5268, 5527, 5530, 5533, or 5536 in Table 2. The sgRNA may include one or more uracils at the 3' end of the sgRNA sequence, such as in SEQ ID NO: 5267, 5269, 5526, 5528, 5529, 5531, 5532, 5534, 5535, or 5537 in Table 2. For example, the sgRNA may include 1 uracil (U) at the 3' end of the sgRNA sequence. The sgRNA may include 2 uracils (UU) at the 3' end of the sgRNA sequence. The sgRNA may include 3 uracils (UUU) at the 3' end of the sgRNA sequence. The sgRNA may include 4 uracils (UUUU) at the 3' end of the sgRNA sequence. The sgRNA may contain 5 uracils (UUUUU) at the 3' end of the sgRNA sequence. The sgRNA may contain 6 uracils (UUUUUU) at the 3' end of the sgRNA sequence. The sgRNA may contain 7 uracils (UUUUUUU) at the 3' end of the sgRNA sequence. The sgRNA may contain 8 uracils (UUUUUUUU) at the 3' end of the sgRNA sequence.

[0256] The sgRNA can be unmodified or modified. For example, a modified sgRNA can include one or more 2'-O-methyl phosphorothioate nucleotides.

[0257] Table 2

[0258]

[0259] The single-molecule guide RNA (sgRNA) in the V-type system can contain a minimal CRISPR repeat sequence and a spacer sequence in the 5' to 3' direction.

[0260] As an illustration, guide RNAs used in CRISPR / Cas / Cpf1 systems, or other smaller RNAs, can be easily synthesized by chemical means, as shown below and described in the art. As chemical synthesis procedures continue to expand, as polynucleotide lengths significantly increase beyond a hundred or so nucleotides, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging. One method for generating RNAs of greater length is to produce two or more molecules linked together. Much longer RNAs, such as those encoding Cas9 or Cpf1 endonucleases, are more easily generated enzymatically. As described in the art, various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic generation of RNA, for example, modifications that enhance stability, reduce the likelihood or degree of an innate immune response, and / or enhance other properties.

[0261] Spacer extension sequence

[0262] In some examples of genome targeting nucleic acids, the spacer extension sequence can modify activity, provide stability and / or provide a location for modifying the genome targeting nucleic acid. The spacer extension sequence can modify on-target or off-target activity or specificity. In some examples, a spacer extension sequence can be provided. The spacer extension sequence can have a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000 or 7000 or more nucleotides. The spacer extension sequence may have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or more nucleotides. The length of the spacer extension sequence may be less than 10 nucleotides. The length of the spacer extension sequence may be between 10-30 nucleotides. The length of the spacer extension sequence may be between 30-70 nucleotides.

[0263] The spacer extension sequence may comprise another portion (e.g., a stability control sequence, an endoribonuclease binding sequence, a ribozyme). The portion may reduce or increase the stability of the nucleic acid targeting nucleic acid. The portion may be a transcription terminator segment (i.e., a transcription termination sequence). The portion may function in eukaryotic cells. The portion may function in prokaryotic cells. The portion may function in both eukaryotic cells and prokaryotic cells. Non-limiting examples of suitable moieties include: a 5' cap (e.g., a 7-methylguanylate cap (m7G)), a riboswitch sequence (e.g., to allow regulated stability and / or regulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, etc.), a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.), and / or a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.).

[0264] Spacer sequence

[0265] The spacer sequence hybridizes with the sequence in the target nucleic acid of interest. The spacer of the genome targeting nucleic acid can interact with the target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer can vary depending on the sequence of the target nucleic acid of interest.

[0266] In the CRISPR / Cas system of this article, the spacer sequence can be designed to hybridize with the target nucleic acid 5' of the PAM of the Cas9 enzyme used in the system. The spacer can be completely matched to the target sequence or can have a mismatch. Each Cas9 enzyme has a specific PAM sequence that it recognizes in the target DNA. For example, Streptococcus pyogenes recognizes a PAM comprising the sequence 5'-NRG-3' in the target nucleic acid, wherein R comprises A or G, wherein N is any nucleotide and N is adjacent to the 3' of the target nucleic acid sequence targeted by the spacer sequence. For example, Staphylococcus aureus Cas9 recognizes a PAM comprising the sequence 5'-NNGRRT-3' in the target nucleic acid, wherein R comprises A or G, wherein N is any nucleotide and N is adjacent to the 3' of the target nucleic acid sequence targeted by the spacer sequence. In some instances, Staphylococcus aureus Cas9 recognizes a PAM comprising the sequence 5'-NNGRRN-3' in the target nucleic acid, wherein R comprises A or G, wherein N is any nucleotide and N is adjacent to the 3' of the target nucleic acid sequence targeted by the spacer sequence. For example, Campylobacter jejuni ( C. jejuni) recognizes a PAM comprising the sequence 5'-NNNNACA-3' or 5'-NNNNACAC-3' in a target nucleic acid, wherein N is any nucleotide and N is immediately 3' to the target nucleic acid sequence targeted by the spacer sequence. In certain examples, the Campylobacter jejuni Cas9 recognizes a PAM comprising the sequence 5'-NNNVRYM-3' or 5'-NNVRYAC-3' in a target nucleic acid, wherein V comprises A, G, or C, wherein R comprises A or G, wherein Y comprises C or T, wherein M comprises A or C, wherein N is any nucleotide and N is immediately 3' to the target nucleic acid sequence targeted by the spacer sequence.

[0267] The target nucleic acid sequence may comprise 20 nucleotides. The target nucleic acid may comprise less than 20 nucleotides. The target nucleic acid may comprise more than 20 nucleotides. The target nucleic acid may comprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The target nucleic acid may comprise at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The target nucleic acid sequence may comprise 20 bases immediately 5' of the first nucleotide adjacent to the PAM. For example, in the sequence comprising 5'-NNNNNNNNNNNNNNNNNNNN NRG -3' (SEQ ID NO: 5270), the target nucleic acid may comprise a sequence corresponding to Ns, wherein N is any nucleotide, and the underlined NRG sequence is the Streptococcus pyogenes PAM.

[0268] The spacer sequence that hybridizes to the target nucleic acid can have a length of at least about 6 nucleotides (nt). The spacer sequence can be at least about 6 nt, at least about 10 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or at least about 40 nt, about 6 nt to about 80 nt, about 6 nt to about 50 nt, about 6 nt to about 45 nt, about 6 nt to about 40 nt, about 6 nt to about 35 nt, about 6 nt to about 30 nt, about 6 nt to about 25 nt, about 6 nt to about 20 nt, about 6 nt to about 19 nt, about 10 nt to about 50 nt, about 10 nt to about 45 nt, about 10 nt to about 40 nt, about 10 nt to about 35 nt, about 10 nt to about 30 nt, about 10 nt to about 25 nt, about 10 nt to about 20 nt, about 10 nt to about 19 In some examples, the spacer sequence may include 24 nucleotides. In some examples, the spacer sequence may include 23 nucleotides. In some examples, the spacer sequence may include 22 nucleotides. In some examples, the spacer sequence may include 21 nucleotides. In some examples, the spacer sequence may include 20 nucleotides. In some examples, the spacer sequence may include 19 nucleotides. In some examples, the spacer sequence can comprise 18 nucleotides. In some examples, the spacer sequence can comprise 17 nucleotides.

[0269] In some instances, the complementarity percentage between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some instances, the complementarity percentage between the spacer sequence and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some instances, the complementarity percentage between the spacer sequence and the target nucleic acid is 100% on the six adjacent 5' nucleotides of the target sequence of the complementary strand of the target nucleic acid. The percent complementarity between the spacer sequence and the target nucleic acid may be at least 60% over about 20 contiguous nucleotides. The length of the spacer sequence and the target nucleic acid may differ by 1 to 6 nucleotides, which may be viewed as one or more bulges.

[0270] Spacer sequences can be designed or selected using computer programs. The computer program can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, genomic frequency (e.g., sequences that are identical or similar but vary in one or more spots due to mismatches, insertions, or deletions), methylation status, presence of SNPs, etc.

[0271] Minimal CRISPR repeat sequence

[0272] A minimal CRISPR repeat sequence can be a sequence that has at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference CRISPR repeat sequence (e.g., crRNA from Streptococcus pyogenes).

[0273] The minimal CRISPR repeat sequence may include nucleotides that can hybridize with the minimal tracrRNA sequence in the cell. The minimal CRISPR repeat sequence and the minimal tracrRNA sequence may form a duplex, i.e., a double-stranded structure of base pairing. Together, the minimal CRISPR repeat sequence and the minimal tracrRNA sequence may bind to the site-directed polypeptide. At least a portion of the minimal CRISPR repeat sequence may hybridize with the minimal tracrRNA sequence. At least a portion of the minimal CRISPR repeat sequence may include at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or 100% complementarity with the minimal tracrRNA sequence. At least a portion of the minimal CRISPR repeat sequence may include at most about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or 100% complementarity with the minimal tracrRNA sequence.

[0274] The minimum CRISPR repeat sequence can have a length of about 7 nucleotides to about 100 nucleotides. For example, the length of the minimum CRISPR repeat sequence is about 7 nucleotides (nt) to about 50 nt, about 7 nt to about 40 nt, about 7 nt to about 30nt, about 7 nt to about 25 nt, about 7 nt to about 20 nt, about 7 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. In some instances, the length of the minimum CRISPR repeat sequence can be about 9 nucleotides. The length of the minimum CRISPR repeat sequence can be about 12 nucleotides.

[0275] The minimal CRISPR repeat sequence can be at least about 60% identical to a reference minimal CRISPR repeat sequence (e.g., a wild-type crRNA from Streptococcus pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimal CRISPR repeat sequence can be at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to a reference minimal CRISPR repeat sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0276] Minimal tracrRNA sequence

[0277] The minimal tracrRNA sequence can be a sequence that has at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a wild-type tracrRNA from Streptococcus pyogenes).

[0278] The minimal tracrRNA sequence may comprise nucleotides that hybridize with the minimal CRISPR repeat sequence in the cell. The minimal tracrRNA sequence and the minimal CRISPR repeat sequence form a duplex, i.e., a double-stranded structure of base pairing. Together, the minimal tracrRNA sequence and the minimal CRISPR repeat can bind to the site-directed polypeptide. At least a portion of the minimal tracrRNA sequence may hybridize with the minimal CRISPR repeat sequence. The minimal tracrRNA sequence may be at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or 100% complementary to the minimal CRISPR repeat sequence.

[0279] The minimum tracrRNA sequence can have a length of about 7 nucleotides to about 100 nucleotides. For example, the minimum tracrRNA sequence can be about 7 nucleotides (nt) to about 50 nt, about 7 nt to about 40 nt, about 7 nt to about 30 nt, about 7 nt to about 25 nt, about 7 nt to about 20 nt, about 7 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25nt. The length of the minimum tracrRNA sequence can be about 9 nucleotides. The length of the minimum tracrRNA sequence can be about 12 nucleotides. The minimal tracrRNA may consist of tracrRNA nt 23-48 as described in Jinek et al., supra.

[0280] The minimal tracrRNA sequence can be at least about 60% identical to a reference minimal tracrRNA (e.g., a wild-type tracrRNA from Streptococcus pyogenes) sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimal tracrRNA sequence can be at least about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical to a reference minimal tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0281] The duplex between the minimal CRISPR RNA and the minimal tracrRNA may comprise a double helix. The duplex between the minimal CRISPR RNA and the minimal tracrRNA may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The duplex between the minimal CRISPR RNA and the minimal tracrRNA may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0282] A duplex may contain mismatches (i.e., the two strands of the duplex are not 100% complementary). A duplex may contain at least about 1, 2, 3, 4, or 5 or mismatches. A duplex may contain up to about 1, 2, 3, 4, or 5 or mismatches. A duplex may contain no more than 2 mismatches.

[0283] Raised

[0284] In some cases, there may be a "bulge" in the duplex between the minimal CRISPR RNA and the minimal tracrRNA. The bulge is an unpaired region of nucleotides in the duplex. The bulge can facilitate the binding of the duplex to the site-directed polypeptide. The bulge can include an unpaired 5'-XXXY-3' on one side of the duplex, where X is any purine and Y includes a nucleotide that can form a wobble pair with a nucleotide on the opposite strand, and an unpaired nucleotide region on the other side of the duplex. The number of unpaired nucleotides on both sides of the duplex can be different.

[0285] In one example, the bulge can comprise an unpaired purine (e.g., adenine) on the minimal CRISPR repeat strand of the bulge. In some examples, the bulge can comprise an unpaired 5'-AAGY-3' of the minimal tracrRNA sequence strand of the bulge, wherein Y comprises a nucleotide that can form a wobble pair with a nucleotide on the minimal CRISPR repeat strand.

[0286] The bulge on the minimal CRISPR repeat side of the duplex may contain at least 1, 2, 3, 4, or 5 or more unpaired nucleotides. The bulge on the minimal CRISPR repeat side of the duplex may contain at most 1, 2, 3, 4, or 5 or more unpaired nucleotides. The bulge on the minimal CRISPR repeat side of the duplex may contain 1 unpaired nucleotide.

[0287] The bulge on the minimal tracrRNA sequence side of the duplex may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more unpaired nucleotides. The bulge on the minimal tracrRNA sequence side of the duplex may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more unpaired nucleotides. The bulge on the second side of the duplex (e.g., the minimal tracrRNA sequence side of the duplex) may contain 4 unpaired nucleotides.

[0288] The bulge may comprise at least one wobble pair. In some instances, the bulge may comprise at most one wobble pair. The bulge may comprise at least one purine nucleotide. The bulge may comprise at least 3 purine nucleotides. The bulge sequence may comprise at least 5 purine nucleotides. The bulge sequence may comprise at least one guanine nucleotide. In some instances, the bulge sequence may comprise at least one adenine nucleotide.

[0289] Hairpin

[0290] In various examples, one or more hairpins can be located 3' of the minimal tracrRNA in the 3' tracrRNA sequence.

[0291] The hairpin may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides 3' from the last paired nucleotide of the minimal CRISPR repeat and minimal tracrRNA sequence duplex. The hairpin may be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides 3' from the last paired nucleotide of the minimal CRISPR repeat and minimal tracrRNA sequence duplex.

[0292] The hairpin may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more consecutive nucleotides. The hairpin may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more consecutive nucleotides.

[0293] The hairpin may comprise a CC dinucleotide (ie, two consecutive cytosine nucleotides).

[0294] The hairpin can comprise duplex nucleotides (e.g., nucleotides hybridized together in a hairpin). For example, the hairpin can comprise a CC dinucleotide hybridized to a GG dinucleotide in the hairpin duplex of the 3' tracrRNA sequence.

[0295] The one or more hairpins can interact with the guide RNA interaction region of the site-directed polypeptide.

[0296] In some instances, there are two or more hairpins, and in other instances, there are three or more hairpins.

[0297] 3' tracrRNA sequence

[0298] The 3' tracrRNA sequence can comprise a sequence having at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a tracrRNA from Streptococcus pyogenes).

[0299] 3' tracrRNA sequence can have a length of about 6 nucleotides to about 100 nucleotides. For example, 3' tracrRNA sequence can have a length of about 6 nucleotides (nt) to about 50 nt, about 6 nt to about 40 nt, about 6 nt to about 30 nt, about 6 nt to about 25 nt, about 6 nt to about 20 nt, about 6 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. 3' tracrRNA sequence can have a length of about 14 nucleotides.

[0300] The 3' tracrRNA sequence can be at least about 60% identical to a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence from Streptococcus pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the 3' tracrRNA sequence can be at least about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical to a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence from Streptococcus pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0301] The 3' tracrRNA sequence may comprise more than one duplex region (eg, hairpin, hybridization region). The 3' tracrRNA sequence may comprise two duplex regions.

[0302] The 3' tracrRNA sequence may comprise a stem-loop structure. The stem-loop structure in the 3' tracrRNA may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more nucleotides. The stem-loop structure in the 3' tracrRNA may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides. The stem-loop structure may comprise a functional portion. For example, the stem-loop structure may comprise an aptamer, a ribozyme, a protein interaction hairpin, a CRISPR array, an intron or an exon. The stem-loop structure may comprise at least about 1, 2, 3, 4 or 5 or more functional portions. The stem-loop structure may comprise at most about 1, 2, 3, 4 or 5 or more functional portions.

[0303] The hairpin in the 3' tracrRNA sequence can include a P domain. In some examples, the P domain can include a double-stranded region in the hairpin.

[0304] tracrRNA extension sequence

[0305] Whether tracrRNA is in the context of a single molecule guide or a double molecule guide, a tracrRNA extension sequence can be provided. The tracrRNA extension sequence can have a length of about 1 nucleotide to about 400 nucleotides. The tracrRNA extension sequence can have a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380 or 400 nucleotides. The tracrRNA extension sequence can have a length of about 20 to about 5000 or more nucleotides. The tracrRNA extension sequence can have a length of more than 1000 nucleotides. The tracrRNA extension sequence may have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or more nucleotides. The tracrRNA extension sequence may have a length of less than 1000 nucleotides. The tracrRNA extension sequence may comprise a length of less than 10 nucleotides. The length of the tracrRNA extension sequence may be 10-30 nucleotides. The length of the tracrRNA extension sequence may be 30-70 nucleotides.

[0306] The tracrRNA extension sequence may include a functional portion (e.g., a stability control sequence, a ribozyme, an endoribonuclease binding sequence). The functional portion may include a transcription terminator segment (i.e., a transcription termination sequence). The functional portion may have a total length of about 10 nucleotides (nt) to about 100 nucleotides, about 10 nt to about 20 nt, about 20 nt to about 30 nt, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. The functional portion may work in a eukaryotic cell. The functional portion may work in a prokaryotic cell. The functional portion may function in both eukaryotic and prokaryotic cells.

[0307] Non-limiting examples of suitable tracrRNA extension functional parts include 3' polyadenylation tails, riboswitch sequences (e.g., to allow stability and / or regulated accessibility regulated by proteins and protein complexes), sequences forming dsRNA duplexes (i.e., hairpins), sequences that target RNA to subcellular locations (e.g., nuclei, mitochondria, chloroplasts, etc.), modifications or sequences that provide for tracking (e.g., direct conjugation with fluorescent molecules, conjugation with moieties that promote fluorescence detection, sequences that allow fluorescence detection, etc.), and / or modifications or sequences that provide binding sites for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.). The tracrRNA extension sequence may include a primer binding site or a molecular index (e.g., a barcode sequence). The tracrRNA extension sequence may include one or more affinity tags.

[0308] Single-molecule guide adapter sequence

[0309] The linker sequence of the single-molecule guide nucleic acid can have a length of about 3 nucleotides to about 100 nucleotides. In Jinek et al., supra, for example, a simple 4-nucleotide "tetraloop" (-GAAA-) is used, Science, 337(6096):816-821(2012). Exemplary linkers have a length of about 3 nucleotides (nt) to about 90 nt, about 3 nt to about 80 nt, about 3 nt to about 70 nt, about 3 nt to about 60 nt, about 3 nt to about 50 nt, about 3 nt to about 40 nt, about 3 nt to about 30 nt, about 3 nt to about 20 nt, about 3 nt to about 10 nt. For example, the adapter can have a length of about 3 nt to about 5 nt, about 5 nt to about 10 nt, about 10 nt to about 15 nt, about 15 nt to about 20 nt, about 20 nt to about 25 nt, about 25 nt to about 30 nt, about 30 nt to about 35 nt, about 35 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt. The adapter of a single-molecule guide nucleic acid can be between 4 and 40 nucleotides. The linker can be at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 nucleotides or more. The linker can be at most about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 nucleotides or more.

[0310] The linker can comprise any of a variety of sequences, although in some instances, the linker does not comprise a sequence having regions of extensive homology to other portions of the guide RNA, which may cause intramolecular binding that may interfere with other functional regions of the guide. In Jinek et al., supra, a simple 4 nucleotide sequence -GAAA- was used, Science, 337(6096):816-821 (2012), but numerous other sequences, including longer sequences, may also be used.

[0311] The joint sequence can include a functional portion. For example, the joint sequence can include one or more features, including an aptamer, a ribozyme, a protein interaction hairpin, a protein binding site, a CRISPR array, an intron or an exon. The joint sequence can include at least about 1, 2, 3, 4 or 5 or more functional portions. In some instances, the joint sequence can include up to about 1, 2, 3, 4 or 5 or more functional portions.

[0312] Complex of genome targeting nucleic acid and site-directed polypeptide

[0313] The genome targeting nucleic acid interacts with an Argonaute (e.g., a nucleic acid-guided nuclease, such as Cas9) to form a complex. The genome targeting nucleic acid directs the Argonaute to the target nucleic acid.

[0314] Ribonucleoprotein complex (RNP)

[0315] The site-directed polypeptide and the genome-targeted nucleic acid can be administered separately to a cell or patient. On the other hand, the site-directed polypeptide can be pre-compounded with one or more guide RNAs, or one or more crRNAs together with tracrRNA. The site-directed polypeptide can be pre-compounded with one or more sgRNAs. The pre-composite can then be administered to a cell or patient. Such pre-composite is referred to as a ribonucleoprotein particle (RNP). The site-directed polypeptide in the RNP can be, for example, a Cas9 endonuclease or a Cpf1 endonuclease. The site-directed polypeptide can be flanked at the N-terminus, the C-terminus, or both the N-terminus and the C-terminus by one or more nuclear localization signals (NLS). For example, the Cas9 endonuclease can be flanked by two NLSs, one NLS at the N-terminus, and the second NLS at the C-terminus. The NLS can be any NLS known in the art, such as an SV40 NLS. The weight ratio of the genome-targeted nucleic acid to the site-directed polypeptide in the RNP can be 1:1. For example, the weight ratio of the sgRNA to the Cas9 endonuclease in the RNP can be 1:1.

[0316] Target sequence selection

[0317] Shifting in the position of the 5' boundary and / or the 3' boundary relative to a particular reference locus can be used to facilitate or enhance a particular application of gene editing, depending in part on the endonuclease system chosen for editing, as further described and illustrated herein.

[0318] In a first non-limiting example of such target sequence selection, many endonuclease systems have rules or criteria that can guide the initial selection of potential target sites for cleavage, such as the requirement for a PAM sequence motif in a specific position adjacent to the DNA cleavage site in the case of CRISPR type II or type V endonucleases.

[0319] In another non-limiting example of target sequence selection or optimization, the frequency of off-target activity for a specific combination of target sequence and gene editing endonuclease can be evaluated relative to the frequency of on-target activity. In some cases, relative to other cells, cells correctly edited at the desired locus have a selection advantage. An illustrative but non-limiting example of selection advantage includes the acquisition of attributes, such as replication of enhanced ratios, persistence, resistance to certain conditions, successful implants of enhanced ratios after introduction into the patient, implantation or in vivo persistence, and other attributes related to the maintenance or increase of such cells or viability. In other cases, cells correctly edited at the desired locus can be actively selected by one or more screening methods for identifying, classifying or otherwise selecting cells correctly edited. Both selection advantage and directional selection methods can utilize phenotypes associated with correction. In some cases, cells can be edited twice or more to produce a second modification, which produces a new phenotype for selecting or purifying an expected cell population. Such a second modification can be produced by adding a second gRNA for a selectable or screenable marker. In some cases, a DNA fragment containing the cDNA as well as a selectable marker can be used to correctly edit the cell at the desired locus.

[0320] No matter be to apply any selection advantage or apply any directional selection in a particular case, it is also possible to guide the target sequence selection by considering the frequency of missing the target, so as to enhance the effectiveness of application and / or reduce the possibility of undesirable changes at the site except the desired target.As further described and shown in this article and in the art, the occurrence of missing the target activity can be affected by multiple factors, and the factors include the similarity and dissimilarity between the target site and the various missing the target sites and the specific endonuclease used.It is available to help predict the bioinformatics tool of missing the target activity, and such tools can often be used to identify the most likely site of missing the target activity, which can then be evaluated in the experimental setting, to assess the relative frequency of missing the target activity and the on-target activity, so as to allow the selection of sequences with higher relative on-target activity.Illustrative examples of such techniques are provided herein, and other technologies are known in the art.

[0321] The gRNA of the present disclosure can guide editing at a genetic locus where editing is desired (e.g., a mutant allele of a USH2A gene). As used herein, "on-target editing," "on-target activity," or "on-target cleavage" means editing at a genetic locus where editing is desired. When the gRNA guides editing of a corresponding mutant allele within or near an IVS40 mutation, the gRNA disclosed herein can have on-target activity.

[0322] The gRNA of the present disclosure can also guide editing at a genetic locus where editing is not desired. As used herein, "off-target editing," "off-target activity," or "off-target cleavage" means editing at a genetic locus where editing is not desired.

[0323] Off-target editing can be editing of the wild-type allele of the USH2A gene. In this article, such off-target editing is referred to as "wild-type off-target editing", "wild-type off-target activity" or "wild-type off-target cutting". When the gRNA guides the editing of the wild-type USH2A allele, the gRNA disclosed herein can have wild-type off-target activity.

[0324] Off-target editing can be editing of a second gene or locus (e.g., editing of a genomic sequence that is not a sequence of a USH2A gene or a regulatory sequence of a USH2A gene). In this article, such off-target editing is referred to as "genomic off-target editing", "genomic off-target activity" or "genomic off-target cleavage". When the gRNA guides the editing of a genomic sequence that is not a sequence of a USH2A gene or a regulatory sequence of a USH2A gene, the gRNA disclosed herein has genomic off-target activity.

[0325] In some examples, the wild-type off-target activity of a gRNA can be "minimal." A gRNA with minimal wild-type off-target activity can be determined using methods known in the art, such as those based on in silico analysis ( in silico ), in vitro methods, or in vivo methods for determining the amount of wild-type off-target editing caused by gRNA. A gRNA with minimal wild-type off-target activity can cause off-target editing in 30% or less cells, such as 25% or less cells, 20% or less cells, 15% or less cells, 10% or less cells, 5% or less cells, 4% or less cells, 3% or less cells, 2% or less cells, 1% or less cells, 0.5% or less cells, 0.25% or less cells, or 0.1% or less cells. In some cases, such determinations can be determined using an in vitro system.

[0326] In some instances, the genome off-target activity of gRNA can be "minimum". The gRNA with minimum genome off-target activity can be determined based on an in silico analysis, an in vitro method, or an in vivo method for determining the amount of genome off-target editing caused by gRNA. The gRNA with minimum genome off-target activity can cause at least one case of genome off-target editing in 30% or less cells, such as 25% or less cells, 20% or less cells, 15% or less cells, 10% or less cells, 5% or less cells, 4% or less cells, 3% or less cells, 2% or less cells, 1% or less cells, 0.5% or less cells, 0.25% or less cells, or 0.1% or less cells. In some cases, such determination can be determined using an in vitro system.

[0327] Another aspect of target sequence selection relates to homologous recombination events. The sequence of the shared homology region can serve as the focus of the homologous recombination events that cause the intervening sequence to be missing. Such recombination events occur during the normal replication process of chromosome and other DNA sequences, and other times when synthetic DNA sequences occur, such as when double-strand breaks (DSBs) are repaired, and they occur regularly during the normal cell replication cycle, but they can also be enhanced by the presence of various events (such as other inducing agents of UV light and DNA fracture) or some reagents (such as various chemical inducing agents). Many such inducing agents force DSB to occur indiscriminately in the genome, and DSB can be regularly induced and repaired in normal cells. In the repair process, the original sequence can be reconstructed with complete fidelity, but in some cases, a small insertion or deletion (called "indels") is introduced at the DSB site.

[0328] As in the case of the endonuclease systems described herein, DSBs can also be specifically induced at particular locations, which can be used to cause directed or preferential gene modification events at selected chromosomal locations. In the context of DNA repair (and replication), the tendency of homologous sequences to undergo recombination can be exploited in many cases and is the basis for one application of gene editing systems such as CRISPR, where homology-directed repair is used to insert a sequence of interest provided by using a "donor" polynucleotide into a desired chromosomal location.

[0329] Regions of homology between specific sequences, which may be small regions of "microhomology" that may contain as little as ten base pairs or less, may also be used to cause desired deletions. For example, a single DSB may be introduced at a site that exhibits microhomology to nearby sequences. During the normal repair process of such DSBs, the result that occurs at high frequency is the deletion of the intervening sequence due to recombination promoted by the DSB and the accompanying cellular repair processes.

[0330] However, in some cases, selecting target sequences within regions of homology may also result in much larger deletions, including gene fusions (when the deletion is in a coding region), which may or may not be desirable depending on the particular situation.

[0331] The examples provided herein further illustrate the selection of various target regions for generating DSBs designed to induce substitutions that result in restoration of usherin protein function, as well as the selection of specific target sequences within such regions designed to minimize off-target events relative to on-target events.

[0332] Homology-directed repair (HDR) / donor nucleotides

[0333] Homology directed repair is a cellular mechanism for repairing double-strand breaks (DSBs). The most common form is homologous recombination. There are alternative pathways to HDR, including single-strand annealing and alternative HDR. Genome engineering tools allow researchers to manipulate the cellular homologous recombination pathway to produce site-specific modifications to the genome. It has been found that cells can repair double-strand breaks using synthetic donor molecules provided in trans. Therefore, by introducing double-strand breaks near specific mutations and providing a suitable donor, targeted changes can be made in the genome. Specific cleavage increases HDR rates by more than 10% over receiving a homologous donor alone. 6 The ratio of HDR to 1 in 10 cells is more than 1,000-fold. The HDR ratio at a specific nucleotide is a function of the distance from the cut site, so it is important to choose an overlapping or closest target site. Gene editing offers an advantage over gene addition because in situ correction leaves the rest of the genome undisturbed.

[0334] The supply donors for editing by HDR are significantly different, but can contain the expected sequence with small or large flanking homology arms to allow annealing to genomic DNA. The homology region of the genetic changes introduced by the flanking can be 30 bp or less, or as large as a multi-kilobase box that can contain promoters, cDNAs, etc. Both single-stranded and double-stranded oligonucleotide donors have been used. The size range of these oligonucleotides is less than 100 nt to more than many kb, although longer ssDNA can also be generated and used. Double-stranded donors can be used, including PCR amplicons, plasmids and minicircles. In general, it has been found that, despite the packaging limit of <5kb for individual donors, AAV vectors can be a very effective means of delivering donor templates. The active transcription of the donor increases HDR by three times, indicating that the inclusion of the promoter may increase conversion. On the contrary, CpG methylation of the donor reduces gene expression and HDR.

[0335] Donor nucleotides used to correct mutations are often small (< 300 bp). This is advantageous because HDR efficiency may be inversely related to the size of the donor molecule. In addition, it is expected that donor templates can be adapted to size-restricted AAV molecules, which have been shown to be an effective means of donor template delivery.

[0336] In addition to wild-type endonucleases, such as Cas9, there are nickase variants that have one or the other nuclease domain inactivated, resulting in cleavage of only one DNA strand. HDR can be directed by individual Cas nickases or using pairs of nickases flanking the target region. The donor can be single-stranded, nicked or dsDNA.

[0337] Donor DNA can be provided with a nuclease or independently by a variety of different methods, such as by transfection, nanoparticles, microinjection or viral transduction. A range of tethering options have been proposed to increase the availability of donors for HDR. Examples include attaching the donor to a nuclease, to a nearby bound DNA binding protein, or to a protein involved in DNA end binding or repair.

[0338] Repair pathway selection can be guided by a variety of culture conditions, such as those that affect the cell cycle, or by targeting DNA repair and related proteins. For example, to increase HDR, key NHEJ molecules such as KU70, KU80 or DNA ligase IV can be suppressed.

[0339] If there is no donor, several non-homologous repair pathways can be used to join ends from a DNA break or ends from different breaks, where the DNA ends are joined with little or no base pairing at the junction. In addition to canonical NHEJ, similar repair mechanisms exist, such as ANHEJ. If there are two breaks, the intervening segment can be deleted or inverted. The NHEJ repair pathway can result in insertions, deletions, or mutations at the junction.

[0340] NHEJ was used to insert a 15-kb inducible gene expression cassette into a defined locus in human cell lines after nuclease cleavage. Methods for the insertion of large inducible gene expression cassettes have been described [Maresca, M., Lin, VG, Guo, N. & Yang, Y., Genome Res 23, 539-546 (2013), Suzuki et al. Nature , 540, 144-149(2016)].

[0341] In addition to genome editing by NHEJ or HDR, site-specific gene insertion using both the NHEJ pathway and HDR has been performed. The combined approach may be applicable in certain settings, possibly including intron / exon boundaries. NHEJ may prove effective for joining in introns, while error-free HDR may be more suitable in coding regions.

[0342] Exemplary modifications within the USH2A gene include substitutions within or near the above-mentioned mutations, such as within a region less than 3 kb, less than 2 kb, less than 1 kb, less than 0.5 kb upstream or downstream of a specific mutation. Considering the relatively wide variation of mutations in the USH2A gene, it will be appreciated that many variations of the above-mentioned substitutions (including but not limited to larger and smaller deletions) are expected to result in restoration of usherin protein function.

[0343] Such variants can include larger than the specific mutation discussed in 5' and / or 3' directions, or smaller replacement in either direction. Accordingly, "near" or "close" to specific replacement, it is expected that the SSB or DSB locus related to the required replacement boundary (also referred to as endpoint in this article) can be in a region less than about 3 kb from the reference locus, such as a mutation site. SSB or DSB locus can be closer, and within 2 kb, within 1 kb, within 0.5 kb or within 0.1 kb. In the case of little replacement, the required endpoint can be at the reference locus or "adjacent" reference locus, and this expected endpoint can be within 100 bp, within 50 bp, within 25 bp or less than about 10 bp to 5 bp from the reference locus.

[0344] Larger or smaller substitutions can provide the same benefit, as long as usherin protein function is restored. Therefore, it is expected that many variations of the substitutions described and shown herein can be effective in improving Usherin syndrome type 2A.

[0345] The term "near" or "close to" an SSB or DSB means that the SSB or DSB is within 2 kb, 1 kb, 0.5 kb, 0.25 kb, 0.2 kb, 0.1 kb, 50 bp, 25 bp, 20 bp, 15 bp, 10 bp, or 5 bp of the IVS40 mutation. The SSB or DSB locus can also be within 2 kb, 1 kb, 0.5 kb, 0.25 kb, 0.2 kb, or 0.1 kb, 50 bp, 25 bp, 20 bp, 15 bp, 10 bp, or 5 bp of intron 40.

[0346] Nucleic acid modifications (chemical and structural modifications)

[0347] In some cases, a polynucleotide introduced into a cell may contain one or more modifications, which may be used individually or in combination, for example, to enhance activity, stability or specificity, to alter delivery, to reduce innate immune responses in the host cell, or for other enhancements, as further described herein and known in the art.

[0348] In certain examples, modified polynucleotides can be used in CRISPR / Cas9 / Cpf1 systems, in which case the guide RNA (single molecule guide or double molecule guide), and / or the DNA or RNA encoding the Cas or Cpf1 endonuclease introduced into the cell can be modified, as described and shown below. Such modified polynucleotides can be used in the CRISPR / Cas9 / Cpf1 system to edit any one or more genomic loci.

[0349] For non-limiting illustrative purposes of such uses using the CRISPR / Cas9 / Cpf1 system, modification of guide RNA can be used to enhance the formation or stability of the CRISPR / Cas9 / Cpf1 genome editing complex, the complex comprising a guide RNA that can be a single molecule guide or a bimolecular guide and a Cas or Cpf1 endonuclease. Modification of guide RNA can also or alternatively be used to enhance the initiation, stability or kinetics of the interaction between the genome editing complex and the target sequence in the genome, which can be used, for example, to enhance on-target activity. Modification of guide RNA can also or alternatively be used to enhance specificity, such as the relative ratio of genome editing at the on-target site compared to the effect at other (off-target) sites.

[0350] Modifications may also or alternatively be used to increase the stability of the guide RNA, for example by increasing its resistance to degradation by ribonucleases (RNases) present in the cell, thereby causing an increase in its half-life in the cell. Modifications that enhance the half-life of the guide RNA may be particularly useful in aspects where a Cas or Cpf1 endonuclease is introduced into a cell to be edited via an RNA that needs to be translated in order to generate the endonuclease, because the increased half-life of the guide RNA introduced simultaneously with the RNA encoding the endonuclease can be used to increase the time that the guide RNA and the encoded Cas or Cpf1 endonuclease coexist in the cell.

[0351] Modification can also or alternatively be used to reduce the possibility or degree that the RNA introduced into the cell triggers an innate immune response. As described below and in the art, such responses fully characterized in the context of RNA interference (RNAi) including small interfering RNA (siRNA) tend to be associated with the reduction of the half-life of RNA and / or the initiation of cytokines or other factors associated with the immune response.

[0352] One or more types of modifications may also be made to the RNA encoding the nuclease introduced into the cell, including but not limited to modifications that enhance the stability of the RNA (e.g., increase its degradation by RNases present in the cell), modifications that enhance the translation of the resulting product (i.e., the nuclease), and / or modifications that reduce the likelihood or extent to which the RNA introduced into the cell will elicit an innate immune response.

[0353] Combinations of the foregoing and other modifications can also be used. For example, in the case of CRISPR / Cas9 / Cpf1, one or more types of modifications (including those exemplified above) can be made to the guide RNA, and / or one or more types of modifications (including those exemplified above) can be made to the RNA encoding the Cas endonuclease.

[0354] As an illustration, the guide RNA used in the CRISPR / Cas9 / Cpf1 system, or other smaller RNA, can be easily synthesized by chemical means, so that many modifications can be easily incorporated, as shown below and described in the art. While the chemical synthesis procedure continues to expand, as the polynucleotide length increases significantly by more than one hundred or so nucleotides, it tends to become more challenging to purify such RNA by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE). One method that can be used to generate chemically modified RNAs of greater length is to produce two or more molecules connected together. Much longer RNAs, such as those encoding Cas9 endonucleases, are more easily enzymatically generated. Although fewer types of modifications can be used in enzymatically generated RNAs, there are still modifications that can be used, for example, to enhance stability, reduce the possibility or degree of innate immune response, and / or enhance other properties, as further described below and in the art; and new types of modifications are often under development.

[0355] As various types of modifications, particularly those frequently used with smaller chemically synthesized RNAs, the modification may include one or more nucleotides modified at the 2' position of the sugar, in some aspects, 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro modified nucleotides. In some instances, RNA modifications may include 2'-fluoro, 2'-amino, or 2'-O-methyl modifications on the ribose of pyrimidines, abasic residues, or inverted bases at the 3' end of the RNA. Such modifications may be routinely incorporated into oligonucleotides, and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxy oligonucleotides for a given target.

[0356] Many nucleotide and nucleoside modifications have been shown to render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than natural oligonucleotides; these modified oligonucleotides survive longer intact than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those oligonucleotides comprising a modified backbone such as a phosphorothioate, a phosphotriester, a methylphosphonate, a short chain alkyl or cycloalkyl sugar bond or a short chain heteroatom or heterocyclic sugar bond. Some oligonucleotides are oligonucleotides with phosphorothioate backbones, and oligonucleotides with heteroatom backbones, particularly CH2-NH-O-CH2, CH,~N(CH3)~O~CH2 (referred to as methylene (methylimino) or MMI backbones), CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2, and ON(CH3)-CH2-CH2 backbones, wherein the natural phosphodiester backbone is represented as O-P-O-CH); amide backbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)]; morpholino backbone structure (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotides are directly or indirectly bound to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3' alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reversed polarity, wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat. Nos. 3,687,808; 4,469 ,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0357] Morpholino-based oligomeric compounds are described in: Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Vol. 30, Issue 3, (2001); Heasman, Dev. Biol., 243:209-214 (2002); Nasevicius et al., Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci., 97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued July 23, 1991.

[0358] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

[0359] Modified oligonucleotide backbones that do not include a phosphorus atom have backbones that are formed by short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatoms or heterocyclic nucleoside linkages. These include those with: morpholino linkages (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formyl and thioformacetyl backbones; methyleneformyl and thioformacetyl backbones; backbones containing olefins; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S and CH2 component parts; see U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,14 1; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0360] One or more substituted sugar moieties may also be included, for example, one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3, wherein n is 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S- or N-alkyl; O-, S- or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkylaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents with similar properties. In some aspects, the modification includes 2'-methoxyethoxy ((2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl)) (Martin et al., HeIv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyl instead of pentofuranosyl.

[0361] In some examples, both the sugar and internucleoside linkage (i.e., main chain) of the nucleotide unit can be replaced by a novel group. The base unit can be maintained for hybridization with an appropriate nucleic acid target compound. A type of such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In a PNA compound, the sugar-main chain of an oligonucleotide can be replaced by a main chain containing an amide, such as an aminoethylglycine main chain. The core base can be retained and directly or indirectly bound to the aza nitrogen atom of the amide portion of the main chain. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254: 1497-1500 (1991).

[0362] The guide RNA may also additionally or alternatively include nucleobase (often referred to in the art as simply "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include those found only rarely or transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine, particularly 5-methylcytosine (also known as 5-methyl-2'deoxycytosine, and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and 2,6-diaminopurine. Kornberg, A., DNA Replication, WH Freeman & Co., San Francisco, pp. 75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). "Universal" bases known in the art, such as inosine, may also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. (Sanghvi, YS, in Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are aspects of base substitution.

[0363] Modified nucleobases may include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine (methylquanine) and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0364] Further, the nucleobase may include those disclosed in U.S. Pat. No. 3,687,808, 'The Concise Encyclopedia of Polymer Science and Engineering', pp. 858-859, Kroschwitz, JI, ed., John Wiley & Sons, 1990, by Englisch et al., Angewandle Chemie, International Edition', 1991, 30, p. 613, and by Sanghvi, YS, Chapter 15, Antisense Research and Applications', pp. 289-302, Crooke, ST and Lebleu, B.ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278), and are an aspect of base substitution, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Modified nucleobases are described in U.S. Patent Nos. 3,687,808, and 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent Application Publication 2003 / 0158403.

[0365] Thus, the term "modified" refers to non-natural sugars, phosphates, or bases incorporated into a guide RNA, an endonuclease, or both a guide RNA and an endonuclease. All positions in a given oligonucleotide need not be uniformly modified, and in fact more than one of the above modifications may be incorporated into a single oligonucleotide, or even into a single nucleoside within an oligonucleotide.

[0366] The guide RNA and / or the mRNA (or DNA) encoding the endonuclease can be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as a cholesterol moiety [Letsinger et al., Proc. Natl. Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al., Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; thioethers, such as hexyl-S-triphenylmethanethiol [Manoharan et al., Ann. NY Acad. Sci., 660: 306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3: 2765-2770 (1993)]; thiocholesterol [Oberhauser et al., Nucl. Acids Res., 20: 533-538 (1992)]; an aliphatic chain, such as dodecanediol or an undecyl residue [Kabanov et al., FEBS Lett., 259: 327-330 (1990) and Svinarchuk et al., Biochimie, 75: 49- 54 (1993)]; phospholipids, such as dihexadecyl-rac-glycerol or triethylammonium 1,2-bis-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. Acids Res., 18: 3777-3783 (1990)]; polyamines or polyethylene glycol chains [Mancharan et al., Nucleosides & Nucleotides, 14: 969-973 (1995)]; adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. Acids Res., 18: 3777-3783 (1990)]; 3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys. Acta, 1264: 229-237 (1995)]; or an octadecylamine or hexylamino-carbonyl-t-hydroxycholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 277: 923-937 (1996)].See also U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5 ,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5 ,082,830;5,112,963;5,214,136;5,082,830;5,112,963;5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241;5,391,723; and 5,688,941.

[0367] Sugar and other moieties can be used to target proteins and complexes (e.g., cationic polyribosomes and liposomes) containing nucleotides to specific sites. For example, hepatocyte directed metastasis can be mediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu et al., Protein Pept Lett. 21(10):1025-30(2014). Other systems known in the art and conventionally developed can be used to target biomolecules and / or their complexes used in this context to specific target cells of interest.

[0368] These targeting moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary hydroxyl or secondary hydroxyl. The conjugate groups of the present disclosure include intercalators, reporters, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyes. In the context of the present disclosure, the group that enhances the pharmacodynamic properties includes a group that improves uptake, enhances resistance to degradation, and / or strengthens sequence-specific hybridization with target nucleic acids. In the context of the present invention, the group that enhances the pharmacokinetic properties includes a group that improves uptake, distribution, metabolism or excretion of the compound of the present disclosure. Representative conjugate groups are disclosed in International Patent Application No. PCT / US92 / 09196 filed October 23, 1992 (published as WO1993007883) and U.S. Pat. No. 6,287,860. Conjugate moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-5-trimethylbenzenethiol, mercaptocholesterol, aliphatic chains such as dodecandiol or undecyl residues, phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-bis-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantaneacetic acid, palmityl moieties or octadecylamine or hexylamino-carbonyl-hydroxycholesterol moieties.See, e.g., U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; and 5,688,941.

[0369] Longer polynucleotides that are less amenable to chemical synthesis and are generally produced by enzymatic synthesis can also be modified by various means. Such modifications may include, for example, the introduction of certain nucleotide analogs, the incorporation of specific sequences or other parts at the 5' or 3' ends of the molecule, and other modifications. As an example, the mRNA encoding Cas9 is about 4kb in length and can be synthesized by in vitro transcription. Modifications to mRNA can be applied to, for example, increase its translation or stability (for example, by increasing its resistance to degradation by cells), or reduce the trend of RNA triggering an innate immune response, which is often observed in cells after exogenous RNA, particularly longer RNA, such as RNA encoding Cas9 is introduced.

[0370] Numerous such modifications have been described in the art, such as the use of a poly A tail, a 5' cap analog (e.g., Anti Reverse Cap Analog (ARCA) or m7G(5')ppp(5')G (mCAP)), a modified 5' or 3' untranslated region (UTR), modified bases (e.g., pseudo-UTP, 2-thio-UTP, 5-methylcytidine-5'-triphosphate (5-methyl-CTP) or N6-methyl-ATP), or treatment with a phosphatase to remove the 5' terminal phosphate. These and other modifications are known in the art, and new modifications of RNA are often under development.

[0371] There are numerous commercial suppliers of modified RNA, including, for example, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon and many other suppliers. As described by TriLink, for example, 5-methyl-CTP can be used to confer desired characteristics, such as increased nuclease stability, increased translation or reduced innate immune receptor interaction with in vitro transcribed RNA. 5-methylcytidine-5'-triphosphate (5-methyl-CTP), N6-methyl-ATP, and pseudo-UTP and 2-thio-UTP have also been shown to reduce innate immune stimulation in culture and in vivo while enhancing translation, as shown in publications by Kormann et al. and Warren et al., mentioned below.

[0372] Chemically modified mRNAs delivered in vivo have been shown to be useful for achieving improved therapeutic effects; see, e.g., Kormann et al., Nature Biotechnology 29, 154–157 (2011). Such modifications can be used, for example, to increase the stability of RNA molecules and / or reduce their immunogenicity. Using chemical modifications such as pseudo-U, N6-methyl-A, 2-thio-U, and 5-methyl-C, it was found that replacing only a quarter of the uridine and cytidine residues with 2-thio-U and 5-methyl-C, respectively, resulted in a significant reduction in toll-like receptor (TLR)-mediated mRNA recognition in mice. These modifications can be used to effectively increase the stability and lifespan of mRNAs in vivo by reducing the activation of the innate immune system; see, e.g., Kormann et al., supra.

[0373] It has also been shown that repeated administration of modified synthetic messenger RNAs designed to bypass innate antiviral responses can reprogram differentiated human cells to pluripotency. See, for example, Warren et al., Cell Stem Cell, 7(5):618-30(2010). Such modified mRNAs acting as primary reprogramming proteins can be an effective means of reprogramming multiple human cell types. Such cells are referred to as induced pluripotent stem cells (iPSCs), and it has been found that RNAs enzymatically synthesized by incorporating 5-methyl-CTP, pseudo-UTP, and anti-reverse cap analogs (ARCA) can be used to effectively evade the antiviral response of cells; See, for example, Warren et al., supra.

[0374] Other modifications of polynucleotides described in the art include, for example, the use of a poly A tail, the addition of a 5' cap analog such as (m7G(5')ppp(5')G(mCAP)), modification of the 5' or 3' untranslated region (UTR), or treatment with phosphatases to remove the 5' terminal phosphate - and new methods are often under development.

[0375] Many compositions and techniques applicable to the generation of modified RNA for use herein have been developed in conjunction with RNA interference (RNAi), including modification of small interfering RNA (siRNA). siRNAs present particular challenges in vivo, as their effects on gene silencing via mRNA interference are generally transient, which may require repeated administration. In addition, siRNAs are double-stranded RNAs (dsRNAs), and mammalian cells have immune responses that have evolved to detect and neutralize dsRNAs, which are often byproducts of viral infections. Thus, there are mammalian enzymes such as PKR (dsRNA-responsive kinase) and possibly retinoic acid-inducible gene 1 (RIG-1), which can mediate cellular responses to dsRNA as well as Toll-like receptors (e.g., TLR3, TLR7, and TLR8), which can trigger the induction of cytokines in response to such molecules; see, e.g., reviews by Angart et al., Pharmaceuticals (Basel) 6(4): 440–468 (2013); Kanasty et al., Molecular Therapy 20(3): 513–524 (2012); Burnett et al., Biotechnol J. 6(9): 1130-46 (2011); Judge and MacLachlan, Hum Gene Ther 19(2): 111-24 (2008).

[0376] As described herein, a variety of modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and / or achieve other benefits that may be useful in conjunction with the introduction of polynucleotides into human cells; see, for example, the following reviews: Whitehead KA et al., Annual Review of Chemical and Biomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, Mini Rev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al., Curr Opin Mol Ther., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic Acid Chem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19 (2008); Fucini et al., Nucleic Acid Ther 22(3): 205–210 (2012); Bremsen et al., Front Genet 3:154 (2012).

[0377] As noted above, there are many commercial suppliers of modified RNA, many of which have specialized in modifications designed to improve the effectiveness of siRNA. Based on the various findings reported in the literature, various methods are provided. For example, Dharmacon points out that replacing non-bridging oxygen with sulfur (phosphorothioate, PS) has been widely used to improve the nuclease resistance of siRNA, as reported by Kole, Nature Reviews Drug Discovery 11: 125-140 (2012). The modification of the 2'-position of ribose has been reported to improve the nuclease resistance of the phosphate bond between nucleotides, while increasing duplex stability (Tm), which has also been shown to provide protection from immune activation. Moderate PS backbone modifications in combination with small, well-tolerated 2'-substitutions (2'-O-methyl, 2'-fluoro, 2'-hydrogen) have been associated with highly stable siRNAs for in vivo applications, as reported by Soutschek et al. Nature 432:173-178 (2004); and 2'-O-methyl modifications have been reported to effectively improve stability, as reported by Volkov, Oligonucleotides 19:191-202 (2009). With regard to reducing the induction of innate immune responses, modification of specific sequences with 2'-O-methyl, 2'-fluoro, 2'-hydrogen has been reported to reduce TLR7 / TLR8 interactions while generally preserving silencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505 (2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine, have also been shown to minimize immune effects mediated by TLR3, TLR7, and TLR8. See, e.g., Kariko, K. et al., Immunity 23:165-175 (2005).

[0378] As is also known in the art and commercially available, a number of conjugates can be applied to the polynucleotides used herein, e.g., RNA, which can enhance their delivery and / or uptake by cells, including, e.g., cholesterol, tocopherol and folic acid, lipids, peptides, polymers, linkers, and aptamers; see, e.g., a review by Winkler, Ther. Deliv. 4:791-809 (2013).

[0379] Codon optimization

[0380] The polynucleotide encoding the site-directed polypeptide can be codon-optimized for expression in cells containing the target DNA of interest according to standard methods in the art. For example, if the intended target nucleic acid is in human cells, a human codon-optimized polynucleotide encoding Cas9 is contemplated for use in producing the Cas9 polypeptide.

[0381] Nucleic acid coding system components

[0382] The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a genome-targeting nucleic acid of the present disclosure, an Argonaute of the present disclosure, and / or any nucleic acid or protein molecule necessary to perform method aspects of the present disclosure.

[0383] Nucleic acids encoding the genome-targeting nucleic acids of the disclosure, the site-directed polypeptides of the disclosure, and / or any nucleic acid or protein molecules necessary to perform method aspects of the disclosure may comprise a vector (e.g., a recombinant expression vector).

[0384] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional nucleic acid segments can be connected. Another type of vector is a viral vector; wherein additional nucleic acid segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell and thereby replicated together with the host genome.

[0385] In some examples, a vector may be capable of directing the expression of a nucleic acid to which it is operably linked. Such vectors are referred to herein as "recombinant expression vectors," or more simply "expression vectors," which perform equivalent functions.

[0386] The term "operably linked" means that the target nucleotide sequence is connected to the regulatory sequence in a manner that allows the expression of the nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct the constitutive expression of nucleotide sequences in many types of host cells, and those that direct the expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the selection of target cells, the desired expression level, and the like.

[0387] Contemplated expression vectors include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for use in eukaryotic target cells include, but are not limited to, vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for use in eukaryotic target cells include, but are not limited to, vectors pCTx-1, pCTx-2, and pCTx-3. Other vectors may be used as long as they are compatible with the host cell.

[0388] In some examples, the vector may contain one or more transcription and / or translation control elements. Depending on the host / vector system utilized, a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc., may be used in the expression vector. The vector may be a self-inactivating vector that inactivates viral sequences or components or other elements of the CRISPR machinery.

[0389] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters that are functional in eukaryotic cells) include those from the following: cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, human elongation factor 1 promoter (EF1), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG), the murine stem cell virus promoter (MSCV), the phosphoglycerate kinase 1 site promoter (PGK), and mouse metallothionein-I.

[0390] For expression of small RNAs, including guide RNAs used in conjunction with Cas endonucleases, various promoters such as RNA polymerase III promoters, including, for example, U6 and H1, may be advantageous. Descriptions and parameters for enhancing the use of such promoters are known in the art, and additional information and methods are often described; see, for example, Ma, H. et al., Molecular Therapy - Nucleic Acids 3 , e161(2014)doi:10.1038 / mtna.2014.12.

[0391] The expression vector may also contain a ribosome binding site and a transcription terminator for translation initiation. The expression vector may also contain an appropriate sequence for amplifying expression. The expression vector may also include a nucleotide sequence encoding a non-natural tag (e.g., a histidine tag, a hemagglutinin tag, a green fluorescent protein, etc.), which is fused to the site-directed polypeptide, thereby resulting in a fusion protein.

[0392] The promoter can be an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). The promoter can be a constitutive promoter (e.g., a CMV promoter, a UBC promoter). In some cases, the promoter can be a spatially restricted and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.).

[0393] Nucleic acids encoding genome targeting nucleic acids and / or site-directed polypeptides of the present disclosure can be packaged into or on a delivery vehicle for delivery to cells. Contemplated delivery vehicles include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. As described in the art, various targeting moieties can be used to enhance the preferential interaction of such vehicles with desired cell types or locations.

[0394] The introduction of the complexes, polypeptides and nucleic acids of the present disclosure into cells can occur by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0395] MicroRNA (miRNA)

[0396] Another class of gene regulatory regions are microRNA (miRNA) binding sites. miRNAs are non-coding RNAs that play a key role in post-transcriptional gene regulation. miRNAs are reported to regulate the expression of a large number of mammalian protein-coding genes. Specific and potent gene silencing by double-stranded RNA (RNAi) has been discovered, coupled with additional small non-coding RNAs (Canver, MC et al., Nature (2015)). The largest class of noncoding RNAs important for gene silencing is miRNA. In mammals, miRNAs are first transcribed as long RNA transcripts, which can be separate transcription units, parts of protein introns, or other transcripts. The long transcripts are called primary miRNAs (pri-miRNAs), which include incomplete base-paired hairpin structures. These pri-miRNAs can be cleaved into one or more shorter precursor miRNAs (miRNA precursors) by the Microprocessor, a protein complex in the nucleus involving Drosha.

[0397] The miRNA precursor is a short stem-loop of ~70 nucleotides in length with a 2-nucleotide 3'-overhang that is exported into a mature 19-25 nucleotide miRNA:miRNA* duplex. The miRNA strand with lower base-pairing stability (the guide strand) can be loaded onto the RNA-induced silencing complex (RISC). The passenger guide strand (marked with *) can be functional but is typically degraded. The mature miRNA tethers the RISC to a partially complementary sequence motif in the target mRNA that is predominantly found within the 3' untranslated region (UTR) and induces post-transcriptional gene silencing (Bartel, DP Cell 136, 215-233(2009); Saj, A. & Lai, E.C. Curr Opin Genet Dev 21, 504-510 (2011)).

[0398] miRNAs may be important in development, differentiation, cell cycle and growth control, and virtually all biological pathways in mammals and other multicellular organisms. miRNAs may also be involved in cell cycle control, apoptosis and stem cell differentiation, hematopoiesis, hypoxia, muscle development, neurogenesis, insulin secretion, cholesterol metabolism, aging, viral replication, and immune response.

[0399] Single miRNA can target hundreds of different mRNA transcripts, and individual transcripts can be targeted by many different miRNAs. More than 28645 microRNAs have been annotated in the latest version of miRBase (v.21). Some miRNAs can be encoded by multiple loci, some of which can be expressed by tandem co-transcription clusters. This feature allows complex regulatory networks with multiple pathways and feedback control. MiRNA may be an integrated part of these feedback and regulatory loops, and can help regulate gene expression by keeping protein production within limits (Herranz, H. & Cohen, SM Genes Dev 24, 1339-1344(2010); Posadas, DM & Carthew, RW Curr Opin Genet Dev 27, 1-6 (2014)).

[0400] miRNAs are also important in a large number of human diseases associated with abnormal miRNA expression. Such associations emphasize the importance of miRNA regulatory pathways. Recent miRNA deletion studies have linked miRNAs to the regulation of immune responses (Stern-Ginossar, N. et al., Science 317, 376-381 (2007)).

[0401] miRNAs also have a strong connection with cancer and can play a role in different types of cancer. It has been found that miRNAs are downregulated in many tumors. miRNAs can be important in the regulation of key cancer-related pathways (such as cell cycle control and DNA damage response) and can therefore be used in diagnosis and can be targeted clinically. miRNAs can finely regulate the balance of angiogenesis, so that experiments that deplete all miRNAs suppress tumor angiogenesis (Chen, S. et al., Genes Dev 28, 1054-1067(2014)).

[0402] As has been shown for protein-coding genes, miRNA genes can also be subject to epigenetic changes that accompany cancer. Many miRNA loci can be associated with CpG islands, increasing the chances that they are regulated by DNA methylation (Weber, B., Stresemann, C., Brueckner, B. & Lyko, F. Cell Cycle 6, 1001-1005 (2007)). Most studies have used treatment with chromatin remodeling drugs to reveal epigenetically silenced miRNAs.

[0403] In addition to their role in RNA silencing, miRNAs can also activate translation (Posadas, DM & Carthew, RW Curr Opin Genet Dev 27, 1-6 (2014). Knocking out these sites can lead to reduced expression of the target gene, while introducing these sites can increase expression.

[0404] Individual miRNAs can be most effectively knocked out by mutating the seed sequence (bases 2-8 of the microRNA), which can be important for binding specificity. Cleavage in this region, followed by error repair by NHEJ, can effectively abolish miRNA function by blocking binding to the target site. miRNAs can also be inhibited by specific targeting of a specialized loop region adjacent to a palindromic sequence. Catalytically inactive Cas9 can also be used to inhibit shRNA expression (Zhao, Y. et al., Sci Rep 4, 3943 (2014). In addition to targeting miRNAs, binding sites can also be targeted and mutated to prevent silencing by miRNAs.

[0405] In accordance with the present disclosure, any miRNA or binding site thereof may be incorporated into the compositions of the present invention.

[0406] The composition can have a region such as, but not limited to, a region comprising the sequence of any miRNA listed in SEQ ID NOs: 613-4696, a reverse complement of a miRNA listed in SEQ ID NOs: 613-4696, or a miRNA anti-seed region of any miRNA listed in SEQ ID NOs: 613-4696.

[0407] The compositions of the present invention may include one or more miRNA target sequences, miRNA sequences or miRNA seeds. Such sequences may correspond to any known miRNA, such as those taught in U.S. Publication No. 2005 / 0261218 and U.S. Publication No. 2005 / 0059005. As non-limiting examples, known miRNAs in the human genome, their sequences and binding site sequences are listed in SEQ ID NO: 613-4696.

[0408] The miRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of a mature miRNA, which has complete Watson-Crick complementarity with the miRNA target sequence. The miRNA seed may comprise positions 2-8 or 2-7 of a mature microRNA. In some examples, the miRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of a mature microRNA), wherein the seed complementary site in the corresponding miRNA target is flanked by an adenine (A) relative to position 1 of the miRNA. In some examples, the miRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of a mature microRNA), wherein the seed complementary site in the corresponding miRNA target is flanked by an adenine (A) relative to position 1 of the microRNA. See, e.g., Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. 2007 Jul 6; 27(1): 91-105. The bases of the miRNA seed have complete complementarity with the target sequence.

[0409] The identification of miRNAs, miRNA target regions, and their expression patterns and roles in biology have been reported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20. doi: 10.1038 / leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al., Cell, 2007 129:1401-1414; Gentner and Naldini, Tissue Antigens. 2012 80:393-403.

[0410] For example, if the composition is not intended to be delivered to the liver but to end up there, miR-122 (an abundant miRNA in the liver) can inhibit the expression of the delivered sequence if one or more target sites for miR-122 are engineered into the polynucleotide encoding the target sequence. One or more binding sites for different miRNAs can be engineered to introduce further reductions in lifespan, stability, and protein translation, thereby providing an additional layer of maintainability.

[0411] As used herein, the term "miRNA site" refers to a miRNA target site or miRNA recognition site, or any nucleotide sequence to which a miRNA is bound or associated. It should be understood that "binding" can follow traditional Watson-Crick hybridization rules, or can reflect any stable binding of a miRNA to a target sequence at or adjacent to the miRNA site.

[0412] Conversely, for the purposes of the compositions of the present disclosure, miRNA binding sites can be engineered out of (i.e., removed from) the sequences in which they naturally occur in order to increase protein expression in specific tissues. For example, the miR-122 binding site can be removed to improve protein expression in the liver.

[0413] Specifically, it is known that miRNA is differentially expressed in immune cells (also referred to as hematopoietic cells), such as antigen presenting cells (APC) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc. Immune cell-specific miRNAs are related to immunogenicity, autoimmunity, immune responses to infection, inflammation, and unwanted immune responses after gene therapy and tissue / organ transplantation. Immune cell-specific microRNAs also regulate many aspects of the development, proliferation, differentiation, and apoptosis of hematopoietic cells (immune cells). For example, miR-142 and miR-146 are exclusively expressed in immune cells and are particularly abundant in myeloid dendritic cells. Introducing a miR-142 binding site into the 3'-UTR of the polypeptides of the present disclosure can selectively suppress gene expression in antigen presenting cells through miR-142-mediated mRNA degradation, limiting antigen presentation in professional APCs (e.g., dendritic cells), and thereby preventing antigen-mediated immune responses after gene delivery (see, Annoni A et al., Blood, 2009, 114, 5152-5161).

[0414] In one example, miRNA binding sites known to be expressed in immune cells, particularly antigen presenting cells, can be engineered into a polynucleotide to suppress polynucleotide expression in APCs through miRNA-mediated RNA degradation, thereby suppressing antigen-mediated immune responses, while expression of the polynucleotide is maintained in non-immune cells that do not express immune cell-specific miRNAs.

[0415] Many miRNA expression studies have been conducted and described in the art to summarize the differential expression of miRNAs in various cancer cells / tissues and other diseases. Some miRNAs are abnormally overexpressed in certain cancer cells, while others are underexpressed. For example, miRNAs are differentially expressed in: cancer cells (WO2008 / 154098, US2013 / 0059015, US2013 / 0042333, WO2011 / 157294); cancer stem cells (US2012 / 0053224); pancreatic cancer and diseases (US2009 / 0131348, US2011 / 0171646, US2010 / 0286232, US8389210); Asthma and inflammation (US8415096); prostate cancer (US2013 / 0053264); hepatocellular carcinoma (WO2012 / 151212, US2012 / 0329672, WO2008 / 054828, US8252538); lung cancer cells (WO2011 / 076143, WO2013 / 033640, WO2009 / 070653, US2010 / 0323357); skin T cell lymphocytes Tumor (WO2013 / 011378); colorectal cancer cells (WO2011 / 0281756, WO2011 / 076142); cancer-positive lymph nodes (WO2009 / 100430, US2009 / 0263803); nasopharyngeal carcinoma (EP2112235); chronic obstructive pulmonary disease (US2012 / 0264626, US2013 / 0053263); thyroid cancer (WO2013 / 066678); Ovarian cancer cells (US2012 / 0309645, WO2011 / 095623); breast cancer cells (WO2008 / 154098, WO2007 / 081740, US2012 / 0214699), leukemia and lymphoma (WO2008 / 073915, US2009 / 0092974, US2012 / 0316081, US2012 / 0283310, WO2010 / 018563).

[0416] Human cells

[0417] As described and shown herein, to improve Usher syndrome type 2A or any condition associated with USH2A, the primary target for gene editing is human cells. For example, in an ex vivo approach, the human cell can be a somatic cell, which can generate differentiated cells, such as photoreceptor cells or retinal progenitor cells, after modification using the described techniques. For example, in an in vivo approach, the human cell can be a photoreceptor cell or a retinal progenitor cell.

[0418] By performing gene editing in autologous cells that are derived from, and therefore fully matched to, the patient in need thereof, it is possible to generate cells that can be safely reintroduced into the patient and effectively produce a cell population that can effectively improve one or more clinical conditions associated with the patient's disease.

[0419] Progenitor cells (also referred to herein as stem cells) can both proliferate and produce more progenitor cells, which in turn have the ability to generate a large number of mother cells, which in turn can produce differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce offspring, which then differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term "stem cell" then refers to cells that have the ability or potential to differentiate into a more specialized or differentiated phenotype under certain circumstances, and retain the ability to proliferate and are not substantially differentiated in certain circumstances. In one aspect, the term progenitor cell or stem cell refers to a broad mother cell, whose offspring (offspring) often specialize in different directions by differentiation (e.g., by obtaining completely individual characteristics), as occurs in the progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process that usually occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, and so on. Although these pluripotent cells can each be regarded as stem cells, the range of cell types that can be produced can be quite different. Some differentiated cells also have the ability to produce cells with greater developmental potential. Such capabilities may be natural, or may be artificially induced following treatment with various factors. In many biological contexts, stem cells may also be "multipotent" in that they can give rise to progeny of more than one different cell type, but this is not required for "stemness."

[0420] Self-renewal can be another important aspect of stem cell.In theory, self-renewal can occur by any of two main mechanisms.Stem cell can asymmetric division, and one of daughter cells retains stem cell state, and another daughter cell expresses some unique other specific functions and phenotypes.Alternately, some stem cells in colony can be symmetrically split into two stems, therefore maintain some stem cells in colony as a whole, and other cells in colony only produce the offspring of differentiation.Generally, " progenitor cell " has more original cell phenotype (that is, in the step earlier than the cell of complete differentiation along developmental pathway or progress).Frequently, progenitor cell also has remarkable or very high proliferation potential.Progenitor cell can produce multiple different differentiated cell types or single differentiated cell types, depending on developmental pathway and cell development and differentiation environment therein.

[0421] In the context of cell ontogeny, the adjective "differentiated" or "differentiation" is a relative term. A "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell to which it is compared. Thus, a stem cell can differentiate into a lineage-restricted precursor cell (e.g., a myocyte progenitor cell), which in turn can differentiate into other types of precursor cells further down the pathway (e.g., a myocyte precursor), and then a terminally differentiated cell, such as a myocyte, which plays a characteristic role in a certain tissue type and may or may not retain the ability to proliferate further.

[0422] Edited human cells

[0423] Provided herein are methods for editing IVS40 mutations in the USH2A gene in human cells. Provided herein are gRNAs for editing IVS40 mutations in the USH2A gene in human cells.

[0424] These methods and / or gRNA disclosed herein can be used to edit a population of human cells. The number of human cells sufficient for treating a patient can be edited within a cell population. For example, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% of the human cells within a cell population can be edited, and are sufficient to treat a patient. In other examples, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or 0.5% of the human cells within a cell population can be edited, and may be sufficient to treat a patient. In various examples, before the edited human cells are applied to a patient, the edited human cells can first be selected and cultured to expand the number of edited cells.

[0425] Induced Pluripotent Stem Cells

[0426] The human cell of genetic modification described herein can be induced pluripotent stem cell (iPSC). The advantage of using iPSC is that the cell can be derived from the same subject to which the progenitor cell is to be applied. That is, somatic cells can be derived from the subject, reprogrammed into induced pluripotent stem cells, and then differentiated into progenitor cells (such as autologous cells) to be applied to the subject. Because the progenitor cell is basically derived from autologous sources, the risk of transplant rejection or allergic response can be reduced compared with the use of cells from another subject or subject group. In addition, the need for cells obtained from embryonic sources is eliminated using iPSC. Therefore, in one aspect, the stem cell used in the disclosed method is not an embryonic stem cell.

[0427] Although differentiation is generally irreversible in a physiological context, several methods have recently been developed to reprogram somatic cells into iPSCs. Exemplary methods are known to those skilled in the art and are briefly described herein below.

[0428] The term "reprogramming" refers to the process of changing or reversing the differentiation state of differentiated cells (e.g., somatic cells). In other words, reprogramming refers to the process of driving the differentiation of cells back to a more undifferentiated or more primitive type of cell. It should be noted that placing many primary cells in culture can lead to some loss of complete differentiation characteristics. Therefore, simply culturing such cells included in the term differentiated cells does not cause these cells to become non-differentiated cells (e.g., undifferentiated cells) or pluripotent cells. The transformation of differentiated cells to pluripotency requires that reprogramming stimulation exceeds the stimulation that causes the partial loss of differentiation characteristics in the culture. Relative to the primary cell parent, the reprogrammed cell also has the ability to extend passage without losing the characteristics of growth potential, and the primary cell parent generally only has the ability to divide in a limited number of cultures.

[0429] Cells to be reprogrammed can be partially differentiated or terminally differentiated before reprogramming. Reprogramming can include reversing the differentiation state of differentiated cells (e.g., somatic cells) completely to a pluripotent state or a multipotent state. Reprogramming can include reversing the differentiation state of differentiated cells (e.g., somatic cells) completely or partially to undifferentiated cells (e.g., embryonic cells). Reprogramming can cause specific genes to be expressed by cells, and the expression of the specific genes further facilitates reprogramming. In some examples described herein, the reprogramming of differentiated cells (e.g., somatic cells) can cause differentiated cells to take an undifferentiated state (e.g., undifferentiated cells). The resulting cells are referred to as "reprogrammed cells" or "induced pluripotent stem cells (iPSC or iPS cells)".

[0430] Reprogramming can involve changes in at least some of the heritable patterns of nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc. that occur during cell differentiation, such as reversal. Reprogramming is different from simply maintaining an existing undifferentiated state of a cell that is already pluripotent, or maintaining an existing less than fully differentiated state of a cell that is already a pluripotent cell (e.g., myogenic stem cell). Reprogramming is also different from promoting self-renewal or proliferation of cells that are already pluripotent or pluripotent, although in some instances, the compositions and methods described herein may also be used for such purposes.

[0431] Many methods are known in the art that can be used to generate pluripotent stem cells from somatic cells. Any such method of reprogramming somatic cells to a pluripotent phenotype is suitable for use in the methods described herein.

[0432] Described is the reprogramming method for generating pluripotent cells using the determined combination of transcription factors. Through the direct transduction of Oct4, Sox2, Klf4 and c-Myc, mouse somatic cells can be converted into ES cell-like cells with expanded developmental potential; See, for example, Takahashi and Yamanaka, Cell 126 (4): 663–76 (2006). iPSC is similar to ES cells because they restore the transcriptional circuits and many epigenetic environments associated with pluripotency. In addition, mouse iPSC meets all standard determinations about pluripotency: specifically, differentiate into cell types of three germ layers in vitro, teratoma formation, contribute to chimeras, germline transmission [see, for example, Maherali and Hochedlinger, Cell Stem Cell. 3 (6): 595-605 (2008)] and tetraploid complementation.

[0433] Human iPSCs can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, have been established as a core set of transcription factors that control pluripotency; see, for example, Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57 (2014); Barrett et al., Stem Cells Trans Med 3:1-6 sctm.2014-0121 (2014); Focosi et al., Blood Cancer Journal 4: e211 (2014). Historically, viral vectors have been used to produce iPSCs by introducing nucleic acid sequences encoding stem cell-related genes into adult somatic cells.

[0434] iPSC can be generated or derived from terminally differentiated somatic cells and adult stem cells or somatic stem cells. That is, non-pluripotent progenitor cells can be caused to become pluripotent or multipotent by reprogramming. In such cases, it is not necessary to include as many reprogramming factors as required for reprogramming of terminally differentiated cells. Further, reprogramming can be induced by the non-viral introduction of reprogramming factors, for example, by introducing protein itself, or by introducing nucleic acids encoding reprogramming factors, or by introducing messenger RNA (see, for example, Warren et al., Cell Stem Cell, 7 (5): 618-30 (2010) that produces reprogramming factors during translation. Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-related genes, including, for example, Oct-4 (also referred to as Oct-3 / 4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klfl, Klf2, Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert and LIN28. Reprogramming using the methods and compositions described herein may further include introducing one or more of Oct-3 / 4, members of the Sox family, members of the Klf family and members of the Myc family into somatic cells. The methods and compositions described herein may further include introducing one or more of Oct-4, Sox2, Nanog, c-MYC and Klf4, respectively, for reprogramming. As noted above, the exact method for reprogramming is not necessarily critical for the methods and compositions described herein. However, when cells differentiated from reprogrammed cells are to be used in, for example, human therapy, in one aspect, reprogramming is not affected by methods that change the genome. Therefore, in such examples, for example, reprogramming can be achieved without the use of viruses or plasmid vectors.

[0435] The efficiency of reprogramming derived from the starting cell population (i.e., the number of reprogrammed cells) can be enhanced by adding various agents such as small molecules, as shown by Shi et al., Cell-Stem Cell 2:525-528 (2008); Huangfu et al., Nature Biotechnology 26(7):795-797 (2008) and Marson et al., Cell-Stem Cell 3:132-135 (2008). Therefore, agents or combinations of agents that enhance the efficiency or rate of induced pluripotent stem cell generation can be used to generate patient-specific or disease-specific iPSCs. Some non-limiting examples of agents that enhance reprogramming efficiency include, among others, soluble Wnt, Wnt-conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, isohydroxamic acid (SAHA), vitamin C, and trichostatin (TSA).

[0436] Other non-limiting examples of reprogramming enhancers include: suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC toxin, Nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutyramide), phenylbutyrate (e.g., sodium phenylbutyrate) and valproic acid ((VP A) and other short chain fatty acids, Scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, Apicidin, sodium butyrate, pivaloyloxymethylbutyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, ester peptide (also known as FR901228 or FK228), benzamide (e.g., CI-994 (e.g., N-acetyl benzophenone) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnamic acid dihydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamidine, 3-Cl-UCHA (e.g., 6-(3-chlorophenylureido)hexanoic acid hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP50. Other reprogramming enhancers include, for example, dominant negative forms of HDAC (e.g., catalytically inactive forms), siRNA inhibitors of HDAC, and antibodies that specifically bind to HDAC. Such inhibitors are available from, for example, BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene and Sigma Aldrich.

[0437] In order to confirm the induction of pluripotent stem cells for use with the methods described herein, the expression of isolated cloned stem cell markers can be tested. Such expression in cells derived from somatic cells identifies cells as induced pluripotent stem cells. Stem cell markers can be selected from the following non-limiting groups including: SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl and Natl. In one case, for example, cells expressing Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods for detecting the presence of encoded polypeptides, such as Western blot or flow cytometry analysis. Detection can involve not only RT-PCR, but also detection of protein markers. Intracellular markers can be best identified via RT-PCR or protein detection methods such as immunocytochemistry, while cell surface markers can be easily identified, for example, by immunocytochemistry.

[0438] The pluripotent stem cell characteristics of the isolated cells can be confirmed by a test evaluating the ability of iPSC to differentiate into the cells of the three germ layers. As an example, teratoma formation in nude mice can be used to evaluate the pluripotent characteristics of isolated clones. Cells can be introduced into nude mice, and histology and / or immunohistochemistry can be performed on tumors arising from cells. For example, the growth of a tumor containing cells from all three germ layers further indicates that the cell is a pluripotent stem cell.

[0439] Retinal progenitor cells and photoreceptor cells

[0440] In some instances, the genetically modified human cells described herein are photoreceptors or retinal progenitor cells (RPCs). RPCs are multipotent progenitor cells that can produce all six types of neurons and Muller glial cells of the retina. Muller glial cells are a class of retinal glial cells and are the main glial components of the retina. Their function is to support neurons of the retina and maintain retinal homeostasis and integrity. Muller glial cells isolated from adult retina have been shown to differentiate into rod photoreceptors. The functional characterization of such Muller glial cell-derived photoreceptors by patch clamp recording has revealed that their electrical properties are comparable to those of adult rods (Giannelli et al., 2011, Stem Cells, (2): 344-56). RPCs are gradually specified as lineage-restricted precursor cells during retinal development, which then mature into terminally differentiated neurons or Muller glial cells. Fetal-derived human retinal progenitor cells (hRPCs) show molecular features indicating retinal progenitor cell states until the sixth generation. They confirmed a gradual decrease in the percentage of KI67, SOX2 and vimentin positive cells from the 1st to the 6th generation, while the continued expression of nestin and PAX6 was visible until the 6th generation. Microarray analysis of the 1st generation hRPC confirmed the expression of early retinal development genes: VIM (vimentin), KI67, NES (nestin), PAX6, SOX2, HES5, GNL3, OTX2, DACH1, SIX6 and CHX10 (VSX2). hRPC is functional in nature and responds to excitatory neurotransmitters (Schmitt et al., 2009, Investigative Ophthalmology and Visual Sciences. 2009; 50 (12): 5901-8). The outermost region of the retina contains a supporting retinal pigment epithelium (RPE) layer, which maintains photoreceptor health by transporting nutrients and recycling detached photoreceptor parts. The RPE is attached to Bruch's membrane, an extracellular matrix structure at the interface between the choroid and the retina. On the other side of the RPE, moving inwards towards the interior of the eye, there are three layers of neurons: light sensing rods and cone photoreceptors, an intermediate layer of connecting neurons (amarine cells, bipolar cells and horizontal cells) and the innermost ganglion cell layer, which transmits signals from the photoreceptor layer through the optic nerve and into the brain. In some aspects, the genetically modified human cells described herein are photoreceptor cells, which are specialized types of neurons found in the retina. Photoreceptors convert light into signals that can stimulate biological processes and are responsible for vision. Rods and cones are two typical photoreceptor cells that contribute to information to the visual system.

[0441] Isolation of retinal progenitor cells and photoreceptor cells

[0442] Retinal cells, including progenitor cells, can be separated according to any method known in the art. For example, human retinal cells are isolated from fresh surgical specimens. By digesting tissue with type IV collagenase, retinal pigment epithelium (RPE) is separated from choroid, and retinal pigment epithelial plaques are cultured. After 100-500 cells are grown from explants, primary cultures ((Ishida M. et al., Current Eye Research 1998; 17 (4): 392-402), and the expression of RPE markers is characterized. Rods are separated by using papain to destroy the retina of biopsy. Precautions are taken to avoid severe damage and improve cell yield. The separated cells are sorted to produce a colony of pure rod cells, and further characterized by immunostaining (Feodorova et al., MethodsX 2015; 2: 39-46).

[0443] To isolate cones, the neural retina was identified, cut out and placed on 10% gelatin. The inner retinal layer was isolated using a laser. The isolated cone monolayer was cultured for 18 hours and compared to untreated retinas by light and transmission microscopy to examine any structural damage. The cells were characterized for the expression of cone-specific markers (Salchow et al., Current Eye Research 2001; 22).

[0444] To isolate retinal progenitor cells, the biopsied retina was minced with a double scalpel and enzymatically digested in an incubator at 37° C. The supernatant of the digested cells was centrifuged and the cells were resuspended in cell-free retinal progenitor cell conditioned medium. The cells were transferred to a fibronectin-coated tissue culture flask containing fresh medium and cultured (Klassen et al., Journal of Neuroscience Research 2004; 77: 334-343).

[0445] Generation of patient-specific iPSCs

[0446] One step of the ex vivo method of the present disclosure may involve generating patient-specific iPS cells, patient-specific iPS cells or patient-specific iPS cell lines. As described in Takahashi and Yamanaka 2006; Takahashi, Tanabe et al. 2007, there are many established methods in the art for generating patient-specific iPS cells. For example, the generation step may include: a) isolating somatic cells, such as skin cells or fibroblasts, from a patient; and b) introducing a set of pluripotency-related genes into the somatic cells so as to induce the cells to become pluripotent stem cells. The set of pluripotency-related genes may be one or more genes selected from the following: OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.

[0447] Perform a biopsy or aspiration of the patient's bone marrow

[0448] A biopsy or aspirate is a sample of tissue or fluid taken from the body. There are many different kinds of biopsies or aspirates. Almost all of them involve using a sharp tool to remove a small amount of tissue. If the biopsy will be on the skin or other sensitive area, a numbing medicine may be applied first. A biopsy or aspiration may be performed according to any method known in the art. For example, in a bone marrow aspiration, a large needle is used to enter the pelvic bone to collect bone marrow.

[0449] Isolation of mesenchymal stem cells

[0450] Mesenchymal stem cells can be separated according to any method known in the art, for example, from the patient's bone marrow or peripheral blood. For example, a bone marrow aspirate can be collected in a syringe with heparin. The cells can be washed and centrifuged on a Percoll density gradient. Density gradient centrifugation medium Percoll can be used to separate cells, such as blood cells, hepatocytes, interstitial cells, macrophages, mast cells and thymocytes. Cells can then be cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% fetal bovine serum (FBS) (Pittinger MF, Mackay AM, Beck SC et al., Science 1999; 284: 143–147).

[0451] Differentiation of genome-edited iPSCs into other cell types

[0452] Another step of the in vitro method of the present disclosure may include differentiating the genome-edited iPSC into photoreceptor cells or retinal progenitor cells. The differentiation step may be performed according to any method known in the art. For example, iPSC can be used to generate retinal organoids and photoreceptors, as described in the art (Phillips et al., Stem Cells, June 2014, 32 (6): pp. 1480-1492; Zhong et al. Nat. Commun., 2014, 5: p. 4047; Tucker et al., PLoS One, April 2011, 6 (4): e18992). For example, hiPSCs are differentiated into retinal progenitor cells using various treatments, including Wnt, Nodal and Notch pathway inhibitors (Noggin, Dk1, LeftyA and DAPT) and other growth factors. Retinal progenitor cells further differentiated into photoreceptor cells by exposure to native retinal cells in a co-culture system, RX+ or Mitf+ by subsequent treatment with retinoic acid and taurine, or by exposure to several exogenous factors including Noggin, Dkk1, DAPT, and insulin-like growth factor (Yang et al., Stem Cells International 2016).

[0453] Genome-edited mesenchymal stem cells differentiate into photoreceptor cells or retinal progenitor cells

[0454] Another step of the ex vivo method of the present disclosure may include differentiating the genome-edited mesenchymal stem cells into photoreceptor cells or retinal progenitor cells. The differentiation step may be performed according to any method known in the art.

[0455] Implanting cells into the patient

[0456] Another step of the in vitro method of the present disclosure can include implanting photoreceptor cells or retinal progenitor cells into a patient. Any implantation method known in the art can be used to complete this implantation step. For example, the genetically modified cells can be injected directly into the patient's blood or otherwise administered to the patient.

[0457] Another step of the in vitro method of the present invention involves implanting photoreceptor cells or retinal progenitor cells into a patient. Any implantation method known in the art can be used to complete this implantation step. For example, the genetically modified cells can be injected directly into the patient's eyes or otherwise administered to the patient.

[0458] Genetically modified cells

[0459] The term "genetically modified cell" refers to a cell comprising at least one genetic modification introduced by genome editing (e.g., using a CRISPR / Cas9 / Cpf1 system). In some in vitro examples herein, the genetically modified cell can be a genetically modified progenitor cell. In some in vivo examples herein, the genetically modified cell can be a genetically modified photoreceptor cell or retinal progenitor cell. Genetically modified cells comprising exogenous genome targeting nucleic acids and / or exogenous nucleic acids encoding genome targeting nucleic acids are contemplated herein.

[0460] The term "control treated population" describes a population of cells that has been treated with equivalent culture medium, viral induction, nucleic acid sequence, temperature, confluence, flask size, pH, etc., except for the added genome editing components. Any method known in the art can be used to measure the restoration of USH2A gene or usherin protein expression or activity, for example, Western blot analysis of usherin protein or real-time PCR for quantification of USH2A mRNA.

[0461] The term "isolated cell" refers to a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. Optionally, the cell may be cultured in vitro, for example under defined conditions or in the presence of other cells. Optionally, the cell may then be introduced into a second organism, or reintroduced into the organism from which it (or a cell from which it is descended) was isolated.

[0462] The term "isolated population" with respect to an isolated cell population refers to a cell population that has been removed and separated from a mixed or heterogeneous cell population. In some cases, the isolated population can be a substantially pure cell population compared to a heterogeneous population from which the cells are isolated or enriched. In some cases, the isolated population can be an isolated human progenitor cell population, such as a substantially pure human progenitor cell population, compared to a heterogeneous cell population comprising human progenitor cells and cells from which the human progenitor cells are derived.

[0463] The term "substantially enhanced" with respect to a particular cell population refers to a cell population in which the presence of a particular type of cell is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 400-fold, at least 1000-fold, at least 5000-fold, at least 20000-fold, at least 100000-fold, or more, relative to a pre-existing level or a reference level, depending on, for example, the desired level of such cells for use in ameliorating Usher Syndrome Type 2A.

[0464] The term "substantially enriched" with respect to a particular cell population refers to cells that make up at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or more of the cell population.

[0465] The term "substantially pure" with respect to a particular cell population refers to a cell population that is at least about 75%, at least about 85%, at least about 90%, or at least about 95% pure with respect to the cells that make up the total cell population. That is, the term "substantially pure" or "substantially purified" with respect to a progenitor cell population refers to a cell population that contains less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than 1% of cells that are not progenitor cells as defined by the terms herein.

[0466] deliver

[0467] Guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotides (RNA or DNA) can be delivered by viral or non-viral delivery vehicles known in the art. Alternatively, endonuclease polypeptides can be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. In further alternative aspects, DNA endonucleases can be delivered as one or more polypeptides, alone or in combination with one or more guide RNAs or one or more crRNAs together with tracrRNA in a pre-complexed manner.

[0468] Polynucleotides can be delivered by non-viral delivery vehicles, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamers-RNA chimeras, and RNA fusion protein complexes. Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene Therapy, 18: 1127–1133 (2011), which focuses on non-viral delivery vehicles for siRNA, which can also be used to deliver other polynucleotides.

[0469] Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding endonucleases, can be delivered to cells or patients via lipid nanoparticles (LNPs).

[0470] LNP refers to any particle with a diameter less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, the size range of the nanoparticles can be 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0471] LNPs can be prepared from cationic, anionic or neutral lipids. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as 'helper lipids' to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy due to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses.

[0472] LNPs may also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0473] Any lipid or lipid combination known in the art can be used to produce LNP. Examples of lipids for producing LNP are: DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE–DPyPE and GL67A–DOPE–DMPE–polyethylene glycol (PEG). Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1 and 7C1. Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE and SM. Examples of PEG-modified lipids are: PEG-DMG, PEG-CerC14 and PEG-CerC20.

[0474] Lipids can be combined in any number of molar ratios to produce LNPs. In addition, polynucleotides can be combined with lipids in a wide range of molar ratios to produce LNPs.

[0475] As previously described, the site-directed polypeptide and the genome-targeting nucleic acid can be administered separately to a cell or patient. On the other hand, the site-directed polypeptide can be pre-compounded with one or more guide RNAs, or one or more crRNAs together with tracrRNA. The pre-composite can then be administered to a cell or patient. Such pre-composite is referred to as a ribonucleoprotein particle (RNP).

[0476] RNA is able to form specific interactions with RNA or DNA. Although this property is used in many biological processes, it also comes with the risk of promiscuous interactions in a cell environment rich in nucleic acids. A solution to this problem is to form ribonucleoprotein particles (RNPs), in which RNA is pre-complexed with endonucleases. Another benefit of RNPs is that RNA is protected from degradation.

[0477] The endonuclease in the RNP can be modified or unmodified. Similarly, gRNA, crRNA, tracrRNA or sgRNA can be modified or unmodified. Numerous modifications are known in the art and can be used.

[0478] The endonuclease and sgRNA can generally be combined in a molar ratio of 1: 1. Alternatively, the endonuclease, crRNA, and tracrRNA can generally be combined in a molar ratio of 1: 1: 1. However, a wide range of molar ratios can be used to produce RNPs.

[0479] AAV (adeno-associated virus)

[0480] Recombinant adeno-associated virus (AAV) vectors can be used for delivery. The technology for producing rAAV particles is a standard technology in the art, wherein the AAV genome to be packaged including the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to the cell. The production of rAAV generally requires the following components to be present in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not therein), and the helper virus function. AAV rep and cap genes can be derived from any AAV serotype from which the recombinant virus can be derived, and can be derived from AAV serotypes different from the rAAV genome ITR, including but not limited to the AAV serotypes described herein. The production of pseudotyped rAAV is disclosed in, for example, International Patent Application Publication No. WO 01 / 83692.

[0481] AAV serotypes

[0482] AAV particles that encode the compositions of the present disclosure, such as polynucleotides encoding the endonucleases, donor sequences, or RNA guide molecules of the present disclosure, may comprise or be derived from any natural or recombinant AAV serotype. According to the present disclosure, the AAV particles may utilize or be based on a serotype selected from any of the following serotypes, and variants thereof, including but not limited to AAV1, AAV10, AAV106.1 / hu.37, AAV11, AAV114.3 / hu.40, AAV12, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.1 / hu.43, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV16.12 / hu. .11, AAV16.3, AAV16.8 / hu.10, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2, AAV2.5T, AAV2-15 / rh.62, AAV22 3.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3 / rh.61, AAV24.1, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV27.3, AAV29.3 / bb.1 , AAV29.5 / bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-11 / rh.53, AAV3-3, AAV33.12 / hu.17, AAV33.4 / h u.15, AAV33.8 / hu.16, AAV3-9 / rh.52, AAV3a, AAV3b, AAV4, AAV4-19 / rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15 , AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AA V43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV4-8 / r11.64, AAV4-8 / rh.64、AAV4-9 / rh.54、AAV5、AAV52.1 / hu.20、AAV52 / hu.19、AAV5-22 / rh.58、AAV5-3 / rh.57、AAV54.1 / hu.21、AAV54.2 / hu.22、AAV54.4R / hu.27、AAV54.5 / hu.23、AAV54.7 / hu.24、AAV58.2 / hu.25、AAV6、AAV6.1、AAV6.1.2、AAV6.2、AAV7、AAV7.2、AAV7.3 / hu.7、AAV8、AAV-8b、AAV-8h、AAV9、AAV9.11、AAV9. 13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAV-b、AAVC1、AAVC2、AAVC5 VCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVcy. V-h、AAVH-1 / hu.1、AAVH2、AAVH-5 / hu.3、AAVH6、AAVhE1.1、AAVhER1.14、AAVhEr1.16、AAVhEr1.18、AAVhER1.23、AAVhEr1.35、AAVhEr1.36、AAVhEr1.5 、AAVhEr1.7、AAVhEr1.8、AAVhEr2.16、AAVhEr2.29、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr2.4、AAVhEr3.1、AAVhu.1、AAVhu.10、AAVhu.11 hu.11、AAVhu.12、AAVhu.13、AAVhu.14 / 9、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.19、AAVhu.2、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23. 2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.3、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.4、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.5、AAVhu.51、AAVhu.52、AAVhu.53、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.6、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.7、AAVhu.8、AAVhu.9、AAVhu.t 19、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVLG-9 / hu.39、AAV-LK01、AAV-LK02、AAVLK03、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK17、AAV-LK18、AAV-LK19、AAVN721-8 / rh.43、AAV-PAEC、AAV-PAEC11、AAV-PAEC12、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAVpi.1、AAVpi.2、AAVpi.3、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.2、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.2R、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.43、AAVrh.44、AAVrh.45、AAVrh.46、AAVrh.47、AAVrh.48、AAVrh.48、AAVrh.48.1. AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh .69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, goat AAV, Japanese AAV 10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK16, AAAV, AAV shuttle 100-1, AAV shuttle 100-2, AAV shuttle 100-3, AAV shuttle 100-7, AAV shuttle 10-2, AAV shuttle 10-6, AAV shuttle 10-8, AAV SM100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and / or AAV SM 10-8.

[0483] In some examples, as described by N Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011), the AAV serotype can be or have a mutation in the AAV9 sequence, such as but not limited to AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.

[0484] In some examples, the AAV serotype can be or have a sequence as described in U.S. Pat. No. US 6156303, such as but not limited to AAV3B (SEQ ID NOs: 1 and 10 of US 6156303), AAV6 (SEQ ID NOs: 2, 7 and 11 of US 6156303), AAV2 (SEQ ID NOs: 3 and 8 of US 6156303), AAV3A (SEQ ID NOs: 4 and 9 of US 6156303) or derivatives thereof.

[0485] In some examples, the serotype can be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12): 5887-5911 (2008)). The amino acid sequence of AAVDJ8 can include two or more mutations to remove the heparin binding domain (HBD). As a non-limiting example, the AAV-DJ sequence described as SEQ ID NO: 1 in U.S. Pat. No. 7,588,772 can include two mutations: (1) R587Q, wherein the arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln), and (2) R590T, wherein the arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). As another non-limiting example, three mutations may be included: (1) K406R, in which the lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q, in which the arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln), and (3) R590T, in which the arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).

[0486] In some examples, the AAV serotype can be or have a sequence as described in International Publication No. WO2015121501, such as, but not limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501), "UPenn AAV10" (SEQ ID NO: 8 of WO2015121501), "Japanese AAV10" (SEQ ID NO: 9 of WO2015121501), or variants thereof.

[0487] According to the present disclosure, the AAV capsid serotype selection or use can be from various species. In one example, the AAV can be an avian AAV (AAAV). The AAAV serotype can be or have a sequence as described in U.S. Pat. No. US 9238800, such as but not limited to AAAV (SEQ ID NO: 1, 2, 4, 6, 8, 10, 12 and 14 of U.S. Pat. No. 9,238,800) or a variant thereof.

[0488] In one example, the AAV may be a bovine AAV (BAAV). The BAAV serotype may be or have a sequence as described in U.S. Pat. No. US 9,193,769, such as but not limited to BAAV (SEQ ID NOs: 1 and 6 of US 9193769) or a variant thereof. The BAAV serotype may be or have a sequence as described in U.S. Pat. No. US7427396, such as but not limited to BAAV (SEQ ID NOs: 5 and 6 of US7427396) or a variant thereof.

[0489] In one example, the AAV may be a goat AAV. The goat AAV serotype may be or have a sequence as described in US Pat. No. US7427396, such as but not limited to goat AAV (SEQ ID NO: 3 of US7427396) or a variant thereof.

[0490] In other examples, AAV can be modified into a hybrid AAV from two or more parental serotypes. In one example, AAV can be AAV2G9, which contains sequences from AAV2 and AAV9. The AAV2G9 AAV serotype can be or have a sequence as described in U.S. Patent Publication No. US20160017005.

[0491] In one example, the AAV can be a serotype generated from an AAV9 capsid library having mutations in amino acids 390-627 (VP1 numbering) as described by Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011). The serotypes and corresponding nucleotide and amino acid substitutions may be, but are not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9. 13(A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T1 521G; W503R), AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289 T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R , W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50(A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134 Q, S469R, A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65 (C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510 A; P504T), AAV9.80 (G1441A,; G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87 (T1 464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T1806C; L 439R, K528I), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L). .

[0492] In one example, the AAV can be a serotype comprising at least one AAV capsid CD8+ T cell epitope. As non-limiting examples, the serotype can be AAV1, AAV2, or AAV8.

[0493] In one example, the AAV can be a variant, such as PHP.A or PHP.B, as described in Deverman. 2016. Nature Biotechnology. 34(2): 204-209.

[0494] In one example, the AAV can be selected from SEQ ID NOs: 4697-5265 and any serotype found in Table 3.

[0495] In one example, the AAV can be encoded by a sequence, fragment or variant as set forth in SEQ ID NOs: 4697-5265 and Table 3.

[0496] The method for generating packaging cells involves generating a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) is integrated into the genome of the cell, and the plasmid comprises an rAAV genome lacking AAV rep and cap genes, an AAV rep and cap gene separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene. The AAV genome has been introduced into a bacterial plasmid by a procedure such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79: 2077-2081), the addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23: 65-73) or by direct blunt end connection (Senapathy & Carter, 1984, J. Biol. Chem., 259: 4661-4666). The packaging cell line can then be infected with a helper virus, such as an adenovirus. This approach has the advantage that the cells are selectable and is amenable to large-scale production of rAAV.Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0497] The general principles of rAAV production are reviewed in, e.g., Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various methods are described in: Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595.

[0498] The AAV vector serotype can be matched to the target cell type. For example, the following exemplary cell types can be transduced by, among other things, the indicated AAV serotypes.

[0499] Table 3: Tissue / cell types and serotypes

[0500] Tissue / cell type Serotype liver AAV3, AA5, AAV8, AAV9 Skeletal muscle AAV1, AAV7, AAV6, AAV8, AAV9 Central nervous system AAV1, AAV4, AAV5, AAV8, AAV9 RPE AAV5, AAV4, AAV2, AAV8, AAV9 AAVrh8r Photoreceptor cells AAV5, AAV8, AAV9, AAVrh8R lung AAV9, AAV5 heart AAV8 pancreas AAV8 kidney AAV2, AAV8

[0501] In addition to adeno-associated virus vectors, other viral vectors can also be used. Such viral vectors include, but are not limited to, lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein-Barr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplex virus.

[0502] In some cases, Cas9 mRNA, sgRNA targeting one or both loci in the USH2A gene, and donor DNA can each be formulated separately into lipid nanoparticles, or all co-formulated into one lipid nanoparticle.

[0503] In some cases, Cas9 mRNA can be formulated into lipid nanoparticles, while sgRNA and donor DNA can be delivered in AAV vectors.

[0504] Options are available for delivering the Cas9 nuclease as a DNA plasmid, mRNA, or protein. The guide RNA can be expressed from the same DNA, or it can also be delivered as RNA. The RNA can be chemically modified to change or improve its half-life, or to reduce the likelihood or extent of an immune response. The nuclease protein can be complexed with the gRNA prior to delivery. Viral vectors allow for efficient delivery; split forms of Cas9 and smaller orthologs of Cas9 can be packaged in AAV, as can donors for HDR. There is also a range of non-viral delivery methods that can deliver each of these components, or non-viral and viral methods can be employed in tandem. For example, nanoparticles can be used to deliver proteins and guide RNA, while AAV can be used to deliver donor DNA.

[0505] Lentivirus

[0506] In some aspects, lentiviral vectors or particles can be used as delivery vehicles. Slow virus is a subgroup of the retroviridae family. Slow virus particles can integrate their genetic material into the genome of the target / host cell. Examples of slow viruses include human immunodeficiency virus: HIV-1 and HIV-2, Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi and caprine arthritis encephalitis virus (CAEV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV). Due to its unique ability to pass through the complete nuclear membrane of the target cell, LV can infect both dividing cells and non-dividing cells, Greenberg et al., University of Berkeley, California; 2006). The lentiviral particles that form the gene delivery vehicle are replication-defective and are generated by weakening HIV virulence genes. For example, genes Vpu, Vpr, Nef, Env and Tat are excised so that the vector is biologically safe. For example, lentiviral vectors derived from HIV-1 / HIV-2 can mediate efficient delivery, integration, and long-term expression of transgenes in non-dividing cells.As used herein, the term "recombinant" refers to vectors or other nucleic acids that contain both lentiviral sequences and non-lentiviral retroviral sequences.

[0507] In order to produce a lentivirus capable of infecting a host cell, three types of vectors need to be co-expressed in the cells producing the virus: a backbone vector containing a target transgene and a self-inactivating 3'-LTR region, a construct expressing viral structural proteins, and a vector encoding vesicular stomatitis virus glycoprotein (VSVG) for encapsulation (Naldini, L. et al., Science 1996; 272, 263-267). The separation of the Rev gene from other structural genes further increases biosafety by reducing the possibility of reverse recombination. Cell lines that can be used to produce high-titer lentiviral particles can include, but are not limited to, 293T cells, 293FT cells, and 293SF-3F6 cells (Witting et al., Human Gene Therapy, 2012; 23: 243-249; Ansorge et al., Journal of Genetic Medicine, 2009; 11: 868-876).

[0508] Methods for generating recombinant lentiviral particles are discussed in the art, for example, WO 2013076309 (PCT / EP2012 / 073645); WO 2009153563 (PCT / GB2009 / 001527); U.S. Patent Nos.: 7,629,153; and 6,808,905.

[0509] Cell types such as photoreceptors, retinal pigment epithelium, and ganglion cells have been successfully targeted using lentiviral (LV) vectors. Delivery efficiency to photoreceptors and ganglion cells is significantly higher with AAV than with LV vectors.

[0510] Pharmaceutically acceptable carrier

[0511] Ex vivo methods of administering progenitor cells to a subject contemplated herein involve the use of a therapeutic composition comprising the progenitor cells.

[0512] The therapeutic composition may contain a physiologically tolerable carrier together with the cell composition and, as an active ingredient, dissolved or dispersed therein, optionally at least one additional biologically active agent as described herein. In some cases, when administered to a mammal or human patient for therapeutic purposes, the therapeutic composition is substantially non-immunogenic unless such is desired.

[0513] In general, the progenitor cells described herein can be administered as a suspension with a pharmaceutically acceptable carrier. Those skilled in the art will recognize that the pharmaceutically acceptable carrier to be used in the cell composition does not include a buffer, compound, cryopreservative, preservative or other agent whose amount substantially interferes with the viability of the cells to be delivered to the subject. The preparation containing the cells may include, for example, a permeability buffer that allows the maintenance of cell membrane integrity, and optionally, nutrients to maintain cell viability or enhance implantation when administered. Such preparations and suspensions are known to those skilled in the art, and / or can be suitable for use with progenitor cells using routine experiments, as described herein.

[0514] The cell composition may also be emulsified or presented as a liposomal composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredients may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredients and in an amount suitable for use in the methods of treatment described herein.

[0515] Additional agents included in the cell composition may include pharmaceutically acceptable salts of the components thereof. Pharmaceutically acceptable salts include acid addition salts (formed from the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or such organic acids as acetic acid, tartaric acid, mandelic acid, etc. Salts formed from free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium, ammonium, calcium or iron, and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0516] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that do not contain any material other than the active ingredient and water, or contain buffers such as sodium phosphate, saline or both at physiological pH, such as phosphate buffered saline. Further, 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 other than and excluding 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 for treating a particular disorder or condition can depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0517] Guide RNA formulation

[0518] The guide RNA of the present disclosure can be formulated with a pharmaceutically acceptable excipient, such as a carrier, a solvent, a stabilizer, an adjuvant, a diluent, etc., depending on the specific mode of administration and dosage form. The guide RNA composition can be formulated to achieve a physiologically compatible pH, and depending on the formulation and route of administration, the range is from about pH 3 to about pH 11, from about pH 3 to about pH 7. In some cases, the pH can be adjusted to a range of about pH 5.0 to about pH 8. In some cases, the composition may include a therapeutically effective amount of at least one compound as described herein, together with one or more pharmaceutically acceptable excipients. Optionally, the composition may include a combination of compounds as described herein, or may include a second active ingredient (such as and not limited to an antibacterial or antimicrobial agent) that can be used to treat or prevent bacterial growth, or may include a combination of agents of the present disclosure.

[0519] Suitable excipients include, for example, carrier molecules, including large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactivated viral particles. Other exemplary excipients may 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, oil, water, saline, glycerol, and ethanol), wetting agents or emulsifiers, pH buffer substances, and the like.

[0520] Application and efficacy

[0521] By causing the introduced cells to be at least partially localized at a desired site, such as a site of injury or repair, so that a desired effect is produced, the terms "administering", "introducing" and "transplanting" may be used interchangeably in the context of placing cells, such as progenitor cells, in a subject. Cells, such as progenitor cells or their differentiated progeny, may be administered by any appropriate route that results in delivery to a desired location in a subject, where at least a portion of the implanted cells or cell components remain viable. After administration to a subject, the survival period of the cells may be as short as a few hours, such as twenty-four hours to a few days, to as long as several years or even the life of the patient, i.e., long-term transplantation. For example, in some aspects described herein, an effective amount of photoreceptor cells or retinal progenitor cells are administered via a systemic route of administration, such as intraperitoneal or intravenous routes.

[0522] The terms "administering", "introducing" and "transplanting" may also be used interchangeably in the context of placing at least one of the gRNA, sgRNA and endonuclease in a subject by causing at least partial localization of the introduced gRNA, sgRNA and / or endonuclease at a desired site, such as a site of damage or repair, so that a desired effect is produced. The gRNA, sgRNA and / or endonuclease can be administered by any appropriate route, the conditions resulting in delivery to the desired location in the subject.

[0523] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any subject for which diagnosis, treatment, or therapy is desired. In some aspects, the subject is a mammal. In some aspects, the subject is a human.

[0524] When provided prophylactically, the progenitor cells described herein can be administered to a subject prior to any symptoms of Usher Syndrome Type 2A. Accordingly, prophylactic administration of a progenitor cell population is useful for preventing Usher Syndrome Type 2A.

[0525] The progenitor cell colony to be administered according to the methods described herein may include allogeneic progenitor cells obtained from one or more donors. Such progenitor cells may have any cell or tissue origin, such as liver, muscle, heart, etc. "Allogeneic" refers to the progenitor cells or biological samples comprising progenitor cells obtained from one or more different donors of the same species, wherein the genes at one or more loci are not equivalent. For example, the photoreceptor cells or retinal progenitor cell colonies to be administered to the subject may be derived from one or more unrelated donor subjects or derived from one or more unequal brothers and sisters. In some cases, isogenic progenitor cell colonies may be used, such as those obtained from genetically identical animals or identical twins. Progenitor cells may be autologous cells; that is, progenitor cells are obtained from or separated from a subject, and are administered to the same subject, i.e., the donor and recipient are the same.

[0526] The term "effective amount" refers to the amount of a population of progenitor cells or their progeny required to prevent or alleviate at least one or more signs or symptoms of Usher Syndrome Type 2A, and relates to a sufficient amount of a composition to provide the desired effect, such as treating a subject with Usher Syndrome Type 2A. Thus, the term "therapeutically effective amount" refers to an amount of such progenitor cells or a composition comprising progenitor cells that, when administered to a typical subject, such as a subject with Usher Syndrome Type 2A or at risk for Usher Syndrome Type 2A, is sufficient to promote a specific effect. 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.

[0527] For use in the various aspects described herein, an effective amount of progenitor cells comprises at least 10 2 progenitor cells, at least 5 x 10 2 progenitor cells, at least 10 3 progenitor cells, at least 5 x 10 3 progenitor cells, at least 10 4 progenitor cells, at least 5 X10 4 progenitor cells, at least 10 5 progenitor cells, at least 2 x 10 5 progenitor cells, at least 3 x 10 5 progenitor cells, at least 4 × 10 5 progenitor cells, at least 5 x 10 5 progenitor cells, at least 6 x 10 5 progenitor cells, at least 7 x 10 5 progenitor cells, at least 8 X10 5 progenitor cells, at least 9 x 10 5 progenitor cells, at least 1 X 10 6 progenitor cells, at least 2 x 10 6 progenitor cells, at least 3X 10 6 progenitor cells, at least 4 × 10 6 progenitor cells, at least 5 x 10 6 progenitor cells, at least 6 x 10 6 progenitor cells, at least 7 x 10 6 progenitor cells, at least 8 x 10 6 progenitor cells, at least 9 x 10 6Progenitor cells or multiples thereof. Progenitor cells can be derived from one or more donors, or can be obtained from autologous sources. In some examples described herein, progenitor cells can be expanded in culture prior to administration to a subject in need thereof.

[0528] Moderate and incremental increases in the level of functional usherin protein expressed in cells of patients with type 2A Usherin syndrome can be beneficial for improving one or more symptoms of the disease, increasing long-term survival, and / or reducing side effects associated with other treatments. After such cells are administered to human patients, the presence of progenitor cells that produce an increased level of functional usherin protein is beneficial. In some cases, the effective treatment of the subject produces at least about 3%, 5%, or 7% functional usherin protein relative to the total usherin in the treated subject. In some instances, functional usherin is at least about 10% of the total usherin. In some instances, functional usherin protein is at least about 20% to 30% of the total usherin protein. Similarly, the introduction of even relatively limited cell subpopulations with significantly elevated levels of functional usherin protein can be beneficial in various patients, because in some cases, standardized cells have a selection advantage relative to diseased cells. However, even moderate levels of progenitor cells with elevated levels of functional usherin protein can be beneficial for improving one or more aspects of type 2A Usherin syndrome in patients. In some examples, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the photoreceptor cells or retinal progenitor cells in a patient to which such cells are administered produce increased levels of functional usherin protein.

[0529] "Administered" refers to the delivery of the progenitor cell composition to a subject by a method or route that results in at least partial localization of the cell composition at a desired site. The cell composition can be administered by any suitable route that results in effective treatment in a subject, i.e., administration results in delivery to a desired location in a subject, where at least a portion of the delivered composition, i.e., at least 1 x 10 4 Cells are delivered to the desired site for a period of time.

[0530] In one aspect of this method, the pharmaceutical composition can be administered by routes such as, but not limited to, enteral (into the intestine), gastrointestinal, epidural (into the dura mater), oral (through the mouth), transdermal, epidural, intracerebral (into the brain), intraventricular (into the ventricle of the brain), epidermal (applied to the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous push, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into intraperitoneal), intravesical infusion, intravitreal (through the eye), intracavernous injection (into a pathological cavity), intracavitary (into the base of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), transvaginal, insufflation (nasal inhalation), sublingual, sublabial, enema, eye drops (on the conjunctiva), ear drops, auris (in or through the ear), buccal (towards the cheek), conjunctiva, skin, dental (to one or more teeth), electroosmosis, intracervical, intrasinus, intratracheal, extracorporeal, hemodialysis, osmotic, interstitial, intraperitoneal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intracystic (intrab ursal), intracartilaginous (in the cartilage), intracaudal (in the cauda equina), intracisternal (in the cerebellomedullary cistern), intracorneal (in the cornea), intracoronary (in the coronary artery), corpora cavernosa (in the expandable space of the corpus cavernosum), intradiscal (in the intervertebral disc), intraductal (in the glandular duct), intraduodenal (in the duodenum), intradural (in or below the dura mater), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (in the stomach), intragingival (in the gums), intraileal (in the distal part of the small intestine), intralesional (in a local lesion or introduced directly into a local lesion), intraluminal (in the lumen), lymphatic intraductal (in lymphatic), intramedullary (in bone marrow cavity), intrameningeal (in meninges), intramyocardial (in myocardium), intraocular (in eye), intraovarian (in ovary), intrapericardial (in pericardium), intrapleural (in pleura), intraprostatic (in prostate), intrapulmonary (in lung or its bronchi), intrasinus (in nasal or periorbital sinuses), intraspinal (in spine), intrasynovial (in synovial cavity of joint), intratendinous (in tendon), intratesticular (in testis), intrathecal (in cerebrospinal fluid at any level of cerebrospinal axis), intrathoracic (in thoracic cavity), intratubular (in tubule of an organ), intratumoral (in tumor), intratympanic (in middle ear (aurus media), intravascular (in one or more blood vessels), intraventricular (in ventricle), iontophoresis (with the aid of an electric current,where ions of soluble salts migrate into body tissues), irrigation (to soak or irrigate an open wound or body cavity), laryngeal (directly on the larynx), nasogastric (through the nose and into the stomach), occlusive dressing techniques (topical routes of administration that are then covered by a dressing that occludes the site), ocular (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, transdermal, periarticular, epidural, perineural, periodontal, rectal, respiratory (by inhalation into the respiratory tract through the mouth or nose for local or systemic effect), retrobulbar (behind the pons or behind the eye), intramyocardial (into the heart muscle), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the tracheal wall), transtympanic (across or through the tympanic cavity), ureteral (to the ureters), urethral (to the urethra), vaginal, sacral blocks, diagnostic, nerve blocks, biliary perfusion, cardiac perfusion, photopheresis, and spinal.

[0531] Modes of administration include injection, infusion, instillation and / or intake. "Injection" includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subretinal, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some instances, the approach is intravenous. For the delivery of cells, administration by injection or infusion can be performed. For the delivery of gRNA, Cas9 and donor templates, administration can be by injection into the subretinal space, close to the photoreceptors.

[0532] The cells can be administered systemically. The phrases "systemic administration," "systemic administration," "peripheral administration," and "peripherally administered" refer to administration of a population of progenitor cells other than directly into a target site, tissue, or organ, such that instead, it enters the subject's circulatory system and is thereby subjected to metabolism and other similar processes.

[0533] The efficacy of a treatment comprising a composition for treating Usher syndrome type 2A can be determined by a skilled clinician. However, if any or all signs or symptoms (as just one example) of functional usherin levels are changed in a beneficial manner (e.g., increased by at least 10%), or other clinically accepted disease symptoms or markers are improved or ameliorated, the treatment is considered "effective treatment". Efficacy can also be measured by the failure of individual deterioration, if evaluated by hospitalization or the need for medical intervention (e.g., disease progression stops or at least slows down). 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 an individual or animal (some non-limiting examples include humans or mammals), and includes: (1) inhibiting the disease, e.g., preventing or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing symptom regression; and (3) preventing or reducing the likelihood of symptom development.

[0534] Treatments according to the present disclosure can improve one or more symptoms associated with Usher syndrome type 2A by increasing, decreasing or altering the amount of functional usherin in an individual. Signs commonly associated with Usher syndrome type 2A include, for example, hearing loss and an eye condition called retinitis pigmentosa, which causes night blindness and loss of peripheral vision through progressive degeneration of the retina. Many people with Usher syndrome also have severe balance problems.

[0535] Reagent test kit

[0536] The present disclosure provides kits for performing the methods described herein. The kits may include one or more of the following: a genome targeting nucleic acid, a polynucleotide encoding a genome targeting nucleic acid, an Argonaute, a polynucleotide encoding an Argonaute, and / or any nucleic acid or protein molecule required to perform aspects of the methods described herein, or any combination thereof.

[0537] The kit may comprise: (1) a vector comprising a nucleotide sequence encoding a genome targeting nucleic acid, (2) an Argonaute or a vector comprising a nucleotide sequence encoding an Argonaute, and (3) reagents for reconstituting and / or diluting the vector and / or the polypeptide.

[0538] The kit may comprise: (1) a vector comprising (i) a nucleotide sequence encoding a genome targeting nucleic acid, and (ii) a nucleotide sequence encoding a site-directed polypeptide; and (2) reagents for reconstituting and / or diluting the vector.

[0539] In any of the above kits, the kit may comprise a single-molecule guide nucleic acid targeting a genome. In any of the above kits, the kit may comprise a bimolecular genome targeting nucleic acid. In any of the above kits, the kit may comprise one or more bimolecular guides or single-molecule guides. The kit may comprise a vector encoding a nucleic acid targeting nucleic acid.

[0540] In any of the above kits, the kit may further comprise a polynucleotide to be inserted to achieve a desired genetic modification.

[0541] The components of the kit may be in separate containers, or combined in a single container.

[0542] Any of the above kits may further comprise 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 wash 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. The kit may also comprise one or more components that may be used to promote or enhance target binding or DNA cleavage by an endonuclease, or to improve targeting specificity.

[0543] In addition to the above components, the test kit may further include instructions for practicing the method using the components of the test kit. The instructions for practicing the method may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic, etc. The instructions may be present in the test kit as a package insert, in the label of the container of the test kit or its components (i.e., related to packaging or sub-packaging), etc. The instructions may be present on a suitable computer-readable storage medium as an electronic storage data file, such as a CD-ROM, a floppy disk, a flash drive, etc. In some cases, actual instructions are not present in the test kit, but may be provided for obtaining instructions from a remote source (e.g., via the Internet). The example of such a case is a test kit comprising a website, in which the instructions may be viewed and / or may be downloaded by it. Like the instructions, this means for obtaining instructions may be recorded on a suitable substrate.

[0544] Other treatments

[0545] Gene editing can be performed using nucleases modified to target specific sequences. So far, there are four main types of nucleases: meganucleases and their derivatives, zinc finger nucleases (ZFNs), transcription activators such as effector nucleases (TALENs) and CRISPR-Cas9 nuclease systems. Nuclease platforms differ in design difficulty, targeting density and mode of action, especially the specificity of ZFN and TALEN is through protein-DNA interactions, while RNA-DNA interactions mainly guide Cas9. Cas9 cutting also requires adjacent motifs PAM, which is different between different CRISPR systems. Cas9 from Streptococcus pyogenes uses NGG PAM cutting, and CRISPR from Neisseria meningitidis can be cut at the site with PAMs including NNNNGATT, NNNNNGTTT and NNNNGCTT. Many other Cas9 orthologs target the front spacer adjacent to the alternative PAM.

[0546] CRISPR endonucleases, such as Cas9, can be used in the methods of the present disclosure. However, the teachings described herein, such as treatment of target sites, can be applied to other forms of endonucleases, such as ZFN, TALEN, HE or MegaTAL, or combinations of nucleases. However, in order to apply the teachings of the present disclosure to such endonucleases, it is particularly necessary to modify proteins directed to specific target sites.

[0547] Additional binding domains can be fused to the Cas9 protein to increase specificity. The target sites of these constructs will map to the identified gRNA-specified sites, but will require additional binding motifs, such as zinc finger domains. In the case of Mega-TAL, a meganuclease can be fused to the TALE DNA binding domain. The meganuclease domain can increase specificity and provide cutting. Similarly, an inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain and requires an adjacent binding site for the sgRNA / Cas9 target site and the DNA binding domain for the fusion. In addition to being catalytically inactive, this may also require some protein modification of dCas9 to reduce binding without additional binding sites.

[0548] Zinc finger nuclease

[0549] Zinc finger nucleases (ZFNs) are modular proteins containing engineered zinc finger DNA binding domains linked to the catalytic domain of the type II endonuclease FokI. Because FokI functions only as a dimer, a pair of ZFNs must be engineered to bind to homologous target "half-site" sequences on opposing DNA strands with precise spacing between them to allow the formation of catalytically active FokI dimers. Upon dimerization of the FokI domains, which themselves have no sequence specificity, DNA double-strand breaks are generated between the ZFN half-sites as the initial step in genome editing.

[0550] The DNA binding domain of each ZFN is typically composed of 3-6 zinc fingers of a rich Cys2-His2 architecture, where each finger primarily recognizes a triplet of nucleotides on one strand of the target DNA sequence, although cross-strand interactions with the fourth nucleotide can also be important. Amino acid changes in the positions where the fingers make critical contacts with the DNA alter the sequence specificity of a given finger. Thus, a four-finger zinc finger protein will selectively recognize a 12 bp target sequence, where the target sequence is a complex of triplet preferences contributed by each finger, although triplet preferences can be affected to varying degrees by adjacent fingers. An important aspect of ZFNs is that they can be easily retargeted to almost any genomic address simply by modifying individual fingers, although considerable expertise is required to do this. In most applications of ZFNs, proteins that recognize 12-18 bp, 4-6 fingers, are used. Thus, a pair of ZFNs typically recognizes a combined target sequence of 24-36 bp, excluding the usual 5-7 bp spacer between half sites. The binding sites can be further separated by larger spacers including 15-17 bp. Assuming that repetitive sequences or gene homologs were excluded during the design process, a target sequence of this length is likely to be unique in the human genome. However, ZFN protein-DNA interactions are not absolute in their specificity, so off-target binding and cleavage events do occur, either as heterodimers between two ZFNs or as homodimers of one or the other ZFN. The latter possibility has been effectively eliminated by engineering the dimerization interface of the FokI domain to produce "positive" and "negative" variants (also called obligate heterodimer variants) that can only dimerize with each other but not with themselves. Forcing obligate heterodimers prevents the formation of homodimers. This has greatly enhanced the specificity of ZFNs and any other nucleases that employ these FokI variants.

[0551] Various ZFN-based systems have been described in the art, modifications of which are frequently reported, and numerous references describe the rules and parameters used to guide ZFN design; see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758-63 (1999); Dreier B et al., J Mol Biol 303(4):489-502 (2000); Liu Q et al., J Biol Chem 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97 (2005); and Dreier et al., J Biol Chem 276(31):29466-78 (2001).

[0552] Transcription activator-like effector nuclease (TALEN)

[0553] Transcription activator-like effector nucleases (TALENs) represent another form of modular nucleases whereby, as with ZFNs, an engineered DNA binding domain is linked to a FokI nuclease domain and a pair of TALENs operate in tandem to achieve targeted DNA cleavage. The main difference from ZFNs is the nature of the DNA binding domain and the associated target DNA sequence recognition properties. The TALEN DNA binding domain is derived from TALE proteins, which were originally developed in the plant bacterial pathogen Xanthomonas ( Xanthomonas ) species. TALE consists of a tandem array of 33-35 amino acid repeats, each of which recognizes a single base pair of up to 20 bp in length in the target DNA sequence, giving a total target sequence length of up to 40 bp. The nucleotide specificity of each repeat is determined by a repeat variable di-residue (RVD), which includes only two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine and thymine are predominantly recognized by four RVDs, respectively: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly. This constitutes a much simpler recognition code than zinc fingers, and therefore represents an advantage over zinc fingers for nuclease design. However, like ZFN, the protein-DNA interaction of TALEN is not absolute in its specificity, and TALEN has also benefited from the use of obligate heterodimer variants of the FokI domain to reduce off-target activity.

[0554] Additional variants of the FokI domain that are inactive in their catalytic function have been produced. If half of the TALEN or ZFN pair contains an inactive FokI domain, only single-stranded DNA cleavage (nicking) occurs at the target site, rather than DSB. The results are comparable to the use of CRISPR / Cas9 / Cpf1 "nickase" mutants (one of the Cas9 cleavage domains is inactive). DNA nicking can be used to drive genome editing by HDR, but the efficiency is lower than DSB. Unlike DSBs that tend to be misrepaired by NHEJ mediation, the main benefit is that off-target nicks are repaired quickly and accurately.

[0555] Various TALEN-based systems have been described in the art, and modifications thereof are frequently reported; see, e.g., Boch, Science 326(5959):1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012); and Moscou et al., Science 326(5959):1501 (2009). The use of TALENs based on the "Golden Gate" platform or cloning scheme has been described by multiple groups; see, e.g., Cermak et al., Nucleic Acids Res 39(12):e82 (2011); Li et al., Nucleic Acids Res. 39(14):6315-25 (2011); Weber et al., PLoS One. 6(2): e16765 (2011); Wang et al., J Genet Genomics 41(6):339-47, Epub 2014 May 17 (2014); and Cermak T et al., Methods Mol Biol 1239:133-59 (2015).

[0556] Homing endonuclease

[0557] Homing endonucleases (HEs) are sequence-specific endonucleases with long recognition sequences (14-44 base pairs) and often cut DNA with high specificity at unique sites in the genome. There are at least six known HE families classified by their structure, including LAGLIDADG (SEQ ID NO: 5271), GIY-YIG, His-Cis box, HNH, PD-(D / E) xK and Vsr-like, which are derived from a wide range of hosts, including eukaryotes, protists, bacteria, archaea, cyanobacteria and bacteriophages. As with ZFN and TALEN, as the starting step in genome editing, HEs can be used to produce DSBs at the target locus. In addition, some natural and modified HEs only cut single strands of DNA, thereby acting as site-specific nickases. The large target sequence of HEs and the specificity they provide have made them attractive candidates for producing site-specific DSBs.

[0558] Various HE-based systems have been described in the art, and modifications thereof are frequently reported; see, e.g., reviews by Steentoft et al., Glycobiology 24(8):663-80 (2014); Belfort and Bonocora, Methods Mol Biol. 1123:1-26 (2014); Hafez and Hausner, Genome 55(8):553-69 (2012).

[0559] MegaTAL / Tev-mTALEN / MegaTev

[0560] As further examples of hybrid nucleases, the MegaTAL platform and the Tev-mTALEN platform use fusions of a TALE DNA binding domain and a catalytically active HE, taking advantage of both the tunable DNA binding and specificity of the TALE, and the cleavage sequence specificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601 (2014); Kleinstiver et al., G3 4: 1155-65 (2014); and Boissel and Scharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

[0561] In a further variation, the MegaTev architecture is a fusion of a meganuclease (Mega) with a nuclease domain derived from the GIY-YIG homing endonuclease I-TevI ​​(Tev). The two active sites are placed ∼30 bp apart on the DNA substrate and generate two DSBs with incompatible sticky ends; see, e.g., Wolfs et al., NAR 42, 8816-29 (2014). It is expected that other combinations of existing nuclease-based methods will be developed and can be used to achieve targeted genome modifications as described herein.

[0562] dCas9-FokI or dCpf1-Fok1 and other nucleases

[0563] Combining the structural and functional properties of the above-mentioned nuclease platform provides a further method of genome editing, which can potentially overcome some inherent defects. For example, the CRISPR genome editing system generally uses a single Cas9 endonuclease to generate DSBs. The targeted specificity is driven by a sequence of 20 or 24 nucleotides in the guide RNA, which undergoes Watson-Crick base pairing with the target DNA (in the case of Cas9 from Streptococcus pyogenes, plus another 2 bases in the adjacent NAG or NGGPAM sequence). Such sequences are long enough to be unique in the human genome, however, the specificity of RNA / DNA interactions is not absolute, and obvious promiscuity is sometimes tolerated, especially in the 5' half of the target sequence, effectively reducing the number of bases that drive specificity. One solution to this is to completely inactivate the Cas9 or Cpf1 catalytic function - retaining only the RNA-guided DNA binding function - and instead fuse the FokI domain to the inactivated Cas9; see, e.g., Tsai et al., Nature Biotech 32: 569-76 (2014); and Guilinger et al., Nature Biotech 32: 577-82 (2014). Because FokI must dimerize to become catalytically active, two guide RNAs are required to tether two FokI fusions in close proximity to form a dimer and cut the DNA. This essentially doubles the number of bases in the combined target site, thereby increasing the stringency of targeting by CRISPR-based systems.

[0564] As a further example, fusions of TALE DNA binding domains with catalytically active HEs (e.g., I-TevI) take advantage of both the tunable DNA binding and specificity of TALEs, and the cleavage sequence specificity of I-TevI, with the expectation that off-target cleavage may be further reduced.

[0565] Methods, compositions, therapeutic agents and kits of the invention

[0566] Accordingly, the present disclosure relates in particular to the following non-limiting inventions:

[0567] In the first method, Method 1, the present disclosure provides a method for editing the USH2A gene in a human cell, the method comprising: introducing one or more DNA endonucleases into the human cell, thereby achieving one or more SSBs or DSBs within or near the DNA sequence of the USH2A gene or the regulatory sequence encoding the USH2A gene, which results in correction, thereby producing an edited human cell.

[0568] In another method, Method 2, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: introducing one or more DNA endonucleases into a human cell to achieve one or more SSBs or DSBs within or near intron 40 of the USH2A gene, which results in correction, thereby producing an edited human cell.

[0569] In another method, Method 3, the present disclosure provides a method as provided in Method 2 for editing a USH2A gene containing an IVS40 mutation in a human cell, wherein the IVS40 mutation is located within intron 40 of the USH2A gene.

[0570] In another method, Method 4, the present disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A, the method comprising: editing a USH2A gene containing an IVS40 mutation in a cell of the patient.

[0571] In another method, method 5, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in method 4, wherein the editing comprises: introducing one or more DNA endonucleases into the cell to achieve one or more SSBs or DSBs within or near intron 40 of the USH2A gene, which results in correction and results in restoration of usherin protein function.

[0572] In another method, Method 6, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of Methods 4 or 5, wherein the IVS40 mutation is located within intron 40 of the USH2A gene.

[0573] In another method, method 7, the present disclosure provides a method as provided in any of methods 1-2 or 5, wherein the one or more DNA endonucleases are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 or Cpf1 nuclease; homologs thereof, recombinants of naturally occurring molecules thereof, codon-optimized or modified forms thereof, and combinations thereof.

[0574] In another method, Method 8, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in Method 7, wherein the method comprises introducing into a cell one or more polynucleotides encoding one or more DNA endonucleases.

[0575] In another method, Method 9, the present disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in Method 7, wherein the method comprises introducing into a cell one or more RNA encoding one or more DNA endonucleases.

[0576] In another method, method 10, the present disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 8 or 9, wherein the one or more polynucleotides or one or more RNAs are one or more modified polynucleotides or one or more modified RNAs.

[0577] In another method, Method 11, the present disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in Method 7, wherein the DNA endonuclease is one or more proteins or polypeptides.

[0578] In another method, method 12, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any one of methods 1-11, wherein the method further comprises: introducing one or more gRNAs into a cell.

[0579] In another method, method 13, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in method 12, wherein the one or more gRNAs are sgRNAs.

[0580] In another method, method 14, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 12-13, wherein the one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs.

[0581] In another method, method 15, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 11-13, wherein the one or more DNA endonucleases are pre-complexed with one or more gRNAs or one or more sgRNAs.

[0582] In another method, method 16, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any one of methods 1-15, further comprising: introducing a polynucleotide donor template into a cell, wherein the polynucleotide donor template comprises at least a portion of a wild-type USH2A gene or cDNA.

[0583] In another method, method 17, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in method 16, wherein at least a portion of the wild-type USH2A gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53 3. Exon 34, Exon 35, Exon 36, Exon 37, Exon 38, Exon 39, Exon 40, Exon 41, Exon 42, Exon 43, Exon 44, Exon 45, Exon 46, Exon 47, Exon 48, Exon 49, Exon 50, Exon 51, Exon 52, Exon 53, Exon 54, Exon 55 , exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, intronic regions, fragments or combinations thereof, or the entire USH2A gene or cDNA.

[0584] In another method, method 18, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 16-17, wherein the donor template is a single-stranded or double-stranded polynucleotide.

[0585] In another method, Method 19, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of Methods 16-17, wherein the donor template has arms homologous to the 1q41 region.

[0586] In another method, method 20, the present disclosure provides a method as provided in any of methods 2 or 5, further comprising: introducing a gRNA into a cell; wherein the one or more DNA endonucleases are one or more Cas9 or Cpf1 endonucleases, which occur at a SSB or DSB at a locus located within or near intron 40 of the USH2A gene; and wherein the gRNA comprises a spacer sequence complementary to a locus segment located within intron 40.

[0587] In another method, method 21, the present disclosure provides a method as provided in any of methods 2 or 5, further comprising: introducing one or more gRNAs into a cell; wherein the one or more DNA endonucleases are one or more Cas9 or Cpf1 endonucleases, which appear in a pair of SSBs or DSBs within or near intron 40 of the USH2A gene, the first at the 5' locus and the second at the 3' locus; and wherein the first guide RNA comprises a spacer sequence complementary to a segment of the 5' locus and the second guide RNA comprises a spacer sequence complementary to a segment of the 3' locus.

[0588] In another method, method 22, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 20 or 21, wherein the IVS40 mutation is located within intron 40 of the USH2A gene.

[0589] In another method, method 23, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-22, wherein the one or more gRNAs are one or more sgRNAs.

[0590] In another method, method 24, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-23, wherein the one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs.

[0591] In another method, method 25, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-24, wherein the one or more DNA endonucleases are pre-complexed with one or more gRNAs or one or more sgRNAs.

[0592] In another method, method 26, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in method 20, further comprising: a polynucleotide donor template comprising at least a portion of a wild-type USH2A gene; wherein a new sequence from the polynucleotide donor template is inserted into chromosomal DNA at a locus located within or near intron 40 of the USH2A gene, which results in correction of the IVS40 mutation in the USH2A gene.

[0593] In another method, method 27, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in method 21, further comprising: a polynucleotide donor template comprising at least a portion of a wild-type USH2A gene; wherein a new sequence from the polynucleotide donor template is inserted into the chromosomal DNA between the 5' locus and the 3' locus within or near intron 40 of the USH2A gene, which results in correction of the chromosomal DNA between the 5' locus and the 3' locus within or near intron 40 of the USH2A gene.

[0594] In another method, method 28, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-27, wherein at least a portion of the wild-type USH2A gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, Exon 33, Exon 34, Exon 35, Exon 36, Exon 37, Exon 38, Exon 39, Exon 40, Exon 41, Exon 42, Exon 43, Exon 44, Exon 45, Exon 46, Exon 47, Exon 48, Exon 49, Exon 50, Exon 51, Exon 52, Exon 53, Exon 54, Exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, intronic regions, fragments or combinations thereof, or the entire USH2A gene or cDNA.

[0595] In another method, method 29, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 20-28, wherein the polynucleotide donor template is a single-stranded or double-stranded polynucleotide.

[0596] In another method, method 30, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 20-29, wherein the polynucleotide donor template has arms homologous to the 1q41 region.

[0597] In another method, method 31, the disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-30, wherein the SSB or DSB is located within 0-1800 nucleotides upstream of intron 40, IVS40 mutation.

[0598] In another method, method 32, the disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-30, wherein the SSB or DSB is located within 0-1100 nucleotides downstream of intron 40, IVS40 mutation.

[0599] In another method, method 33, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 12-15 or 23-25, wherein the gRNA or sgRNA is complementary to a segment of intron 40 of the USH2A gene.

[0600] In another method, method 34, the present disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 1-3 or 5-33, wherein the correction is by HDR.

[0601] In another method, method 35, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 26-27, wherein the donor template has arms homologous to the IVS40 mutation.

[0602] In another method, method 36, the present disclosure provides a method as provided in any one of methods 2 or 5, further comprising: introducing two gRNAs into a cell; wherein the one or more DNA endonucleases are one or more Cas9 or Cpf1 endonucleases, which occur at a pair of double-strand breaks (DSBs) within or near intron 40 of the USH2A gene, the first at the 5' DSB locus and the second at the 3' DSB locus, the double-strand breaks causing a chromosomal DNA deletion between the 5' DSB locus and the 3' DSB locus, which results in a chromosomal DNA deletion between the 5' DSB locus and the 3' DSB locus within or near intron 40 of the USH2A gene; and wherein the first guide RNA comprises a spacer sequence complementary to a segment of the 5' DSB locus, and the second guide RNA comprises a spacer sequence complementary to a segment of the 3' DSB locus.

[0603] In another method, method 37, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in method 36, wherein the IVS40 mutation is located within intron 40 of the USH2A gene.

[0604] In another method, method 38, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 36 or 37, wherein the two gRNAs are two sgRNAs.

[0605] In another method, method 39, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any one of methods 36-38, wherein the two gRNAs or two sgRNAs are two modified gRNAs or two modified sgRNAs.

[0606] In another method, method 40, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 36-39, wherein the one or more DNA endonucleases are pre-complexed with two gRNAs or two sgRNAs.

[0607] In another method, method 41, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 36-40, wherein the 5' DSB within intron 40 is located 0-1800 nucleotides upstream of the IVS40 mutation.

[0608] In another method, method 42, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 36-40, wherein the 3' DSB within intron 40 is located 0-1100 nucleotides downstream of the IVS40 mutation.

[0609] In another method, method 43, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 36-42, wherein the deletion is a deletion of 50 bp to 2900 bp.

[0610] In another method, method 44, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 36-42, wherein the deletion is a deletion of 50 bp to 2000 bp.

[0611] In another method, method 45, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 36-42, wherein the deletion is a deletion of 50 bp to 1000 bp.

[0612] In another method, method 46, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 36-42, wherein the deletion is a deletion of 50 bp to 500 bp.

[0613] In another method, method 47, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 36-42, wherein the deletion is a deletion of 50 bp to 250 bp.

[0614] In another method, method 48, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 36-47, further comprising: a polynucleotide donor template comprising at least a portion of a wild-type USH2A gene.

[0615] In another method, method 49, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-22 and 36-37, wherein the Cas9 or Cpf1 mRNA, gRNA, and donor template are each formulated into separate lipid nanoparticles, or are all co-formulated into lipid nanoparticles.

[0616] In another method, method 50, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-22 and 36-37, wherein the Cas9 or Cpf1 mRNA, gRNA and donor template are each formulated into separate AAV vectors, or are all co-formulated into an AAV vector.

[0617] In another method, method 51, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-22 and 36-37, wherein the Cas9 or Cpf1 mRNA is formulated into lipid nanoparticles and both the gRNA and donor template are delivered to the cell via an AAV vector.

[0618] In another method, method 52, the present disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 20-22 and 36-37, wherein the Cas9 or Cpf1 mRNA is formulated into lipid nanoparticles and the gRNA is delivered to the cell by electroporation and the donor template is delivered to the cell by an AAV vector.

[0619] In another method, method 53, the disclosure provides an in vivo method for treating a patient with Usher syndrome type 2A as provided in any of methods 50-52, wherein the AAV vector is a self-inactivating AAV vector.

[0620] In another method, method 54, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in any of methods 1-53, wherein the USH2A gene is located on chromosome 1: 215,622,893-216,423,395 (Genome Reference Consortium - GRCh38 / hg38).

[0621] In another method, method 55, the disclosure provides a method as provided in any of methods 1-3 or 5-54, wherein the restoration of usherin protein function is compared to wild-type or normal usherin protein function.

[0622] In another method, method 56, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in method 16, wherein the polynucleotide donor template is at most 11 kb.

[0623] In another method, method 57, the present disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in method 56, wherein the polynucleotide donor template is delivered by AAV.

[0624] In another method, method 58, the disclosure provides a method as provided in any of methods 1-3, wherein the human cell is a photoreceptor cell or a retinal progenitor cell.

[0625] In another method, method 59, the disclosure provides an in vivo method for treating a patient with Usher Syndrome Type 2A as provided in methods 4-57, wherein the cell is a photoreceptor cell or a retinal progenitor cell.

[0626] In another method, method 60, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising SEQ ID NO: 5321 to edit a USH2A gene containing an IVS40 mutation.

[0627] In another method, method 61, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising SEQ ID NO: 5323 to edit a USH2A gene containing an IVS40 mutation.

[0628] In another method, method 62, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising SEQ ID NO: 5325 to edit a USH2A gene containing an IVS40 mutation.

[0629] In another method, method 63, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising SEQ ID NO: 5327 to edit a USH2A gene containing an IVS40 mutation.

[0630] In another method, method 64, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising SEQ ID NO: 5328 to edit a USH2A gene containing an IVS40 mutation.

[0631] In another method, method 65, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising any one of SEQ ID NO: 5321 and SEQ ID NO: 5267-5269 to edit a USH2A gene containing an IVS40 mutation.

[0632] In another method, method 66, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising any one of SEQ ID NO: 5323 and SEQ ID NO: 5267-5269 to edit a USH2A gene containing an IVS40 mutation.

[0633] In another method, method 67, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising any one of SEQ ID NO: 5325 and SEQ ID NO: 5267-5269 to edit a USH2A gene containing an IVS40 mutation.

[0634] In another method, method 68, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising any one of SEQ ID NO: 5327 and SEQ ID NO: 5267-5269 to edit a USH2A gene containing an IVS40 mutation.

[0635] In another method, method 69, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a gRNA or sgRNA comprising any one of SEQ ID NO: 5328 and SEQ ID NO: 5267-5269 to edit a USH2A gene containing an IVS40 mutation.

[0636] In another method, method 70, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5321.

[0637] In another method, method 71, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5323.

[0638] In another method, method 72, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5325.

[0639] In another method, method 73, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5327.

[0640] In another method, method 74, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5328.

[0641] In another method, method 75, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5321 and SEQ ID NO: 5267-5269.

[0642] In another method, method 76, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5323 and SEQ ID NO: 5267-5269.

[0643] In another method, method 77, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5325 and SEQ ID NO: 5267-5269.

[0644] In another method, method 78, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5327 and SEQ ID NO: 5267-5269.

[0645] In another method, method 79, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering a gRNA or sgRNA to the patient, wherein the gRNA or sgRNA comprises any one of SEQ ID NO: 5328 and SEQ ID NO: 5267-5269.

[0646] In another method, method 80, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a first gRNA or sgRNA comprising SEQ ID NO: 5295 and a second gRNA or sgRNA comprising SEQ ID NO: 5279 to delete a sequence containing the IVS40 mutation.

[0647] In another method, method 81, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a first gRNA or sgRNA comprising SEQ ID NO: 5294 and a second gRNA or sgRNA comprising SEQ ID NO: 5300 to delete a sequence comprising the IVS40 mutation.

[0648] In another method, method 82, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a first gRNA or sgRNA comprising SEQ ID NO: 5295 and a second gRNA or sgRNA comprising SEQ ID NO: 5300 to delete a sequence containing the IVS40 mutation.

[0649] In another method, method 83, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a first gRNA or sgRNA comprising SEQ ID NO: 5290 and a second gRNA or sgRNA comprising SEQ ID NO: 5300 to delete a sequence containing the IVS40 mutation.

[0650] In another method, method 84, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a first gRNA or sgRNA comprising SEQ ID NO: 5277 and a second gRNA or sgRNA comprising SEQ ID NO: 5300 to delete a sequence containing the IVS40 mutation.

[0651] In another method, method 85, the present disclosure provides the method of any one of methods 80-84, wherein the first gRNA or sgRNA and the second gRNA or sgRNA are administered simultaneously; the first gRNA or sgRNA is administered before the second gRNA or sgRNA; or the second gRNA or sgRNA is administered before the first gRNA or sgRNA.

[0652] In another method, method 86, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5295 and the second gRNA or sgRNA comprises SEQ ID NO: 5279.

[0653] In another method, method 87, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5294 and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

[0654] In another method, method 88, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5295 and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

[0655] In another method, method 89, the present disclosure provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5290 and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

[0656] In another method, method 90, the present invention provides a method for treating a patient having a USH2A gene containing an IVS40 mutation, the method comprising: administering to the patient a first gRNA or sgRNA and a second gRNA or sgRNA, wherein the first gRNA or sgRNA comprises SEQ ID NO: 5277 and the second gRNA or sgRNA comprises SEQ ID NO: 5300.

[0657] In another method, method 91, the present disclosure provides the method of any one of methods 86-90, wherein the first gRNA or sgRNA and the second gRNA or sgRNA are administered simultaneously; the first gRNA or sgRNA is administered before the second gRNA or sgRNA; or the second gRNA or sgRNA is administered to the patient before the first gRNA or sgRNA.

[0658] In another method, method 92, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a first gRNA or sgRNA comprising SEQ ID NO: 5452 and a second gRNA or sgRNA comprising SEQ ID NO: 5449 to delete a sequence containing the IVS40 mutation.

[0659] In another method, method 93, the present disclosure provides a method for editing a USH2A gene containing an IVS40 mutation, the method comprising: using a first gRNA or sgRNA comprising SEQ ID NO: 5453 and a second gRNA or sgRNA comprising SEQ ID NO: 5449 to delete a sequence containing the IVS40 mutation.

[0660] In another method, method 94, the present disclosure provides a method for editing ...

Claims

1. A method for ex vivo editing of a USH2A gene containing an IVS40 mutation, the method comprising: Introduction into human cells: Streptococcus pyogenes Cas9 endonuclease and a guide RNA (gRNA), wherein the gRNA comprises a spacer sequence having a nucleic acid sequence of SEQ ID NO: 5328; Thereby achieving one or more single strand breaks (SSBs) or double strand breaks (DSBs) within intron 40 of the USH2A gene, which results in edited human cells.

2. The method of claim 1, wherein the method comprises introducing a polynucleotide encoding the Cas9 endonuclease into the cell.

3. The method according to claim 1, wherein: (i) the gRNA is a chemically modified gRNA; and / or (ii) The Cas9 endonuclease is pre-complexed with the gRNA.

4. The method according to claim 1, further comprising: A polynucleotide donor template comprising at least a portion of a wild-type USH2A gene is introduced into the cell.

5. The method according to claim 4, wherein: (i) formulating the Cas9 endonuclease, the gRNA and the donor template into separate lipid nanoparticles, or formulating all of them into lipid nanoparticles; (ii) formulating the Cas9 endonuclease, the gRNA, and the donor template into separate adeno-associated virus (AAV) vectors, or formulating all of them into an AAV vector; (iii) the Cas9 endonuclease is formulated into lipid nanoparticles, and both the gRNA and the donor template are delivered to cells via an AAV vector; or (iv) the Cas9 endonuclease is formulated into lipid nanoparticles, the gRNA is delivered to cells by electroporation, and the donor template is delivered to cells by an AAV vector.

6. The method of claim 1, wherein the human cell is a photoreceptor cell or a retinal progenitor cell.

7. A gRNA for editing a USH2A gene containing an IVS40 mutation, comprising a spacer sequence having a nucleic acid sequence of SEQ ID NO: 5328.

8. The gRNA according to claim 7, which is a sgRNA.

9. The gRNA according to claim 7 or 8, wherein the gRNA is a chemically modified gRNA.

10. A gRNA composition for editing a USH2A gene containing an IVS40 mutation, comprising a first gRNA and a second gRNA, wherein the first gRNA comprises a spacer sequence having a nucleic acid sequence of SEQ ID NO: 5328; and the second gRNA comprises a spacer sequence having a nucleic acid sequence of any one of SEQ ID NOs: 5267-5269.

11. Use of the gRNA according to any one of claims 7-9 or the gRNA composition according to claim 10 in the preparation of a medicament for treating a patient with Usher syndrome type 2A by the following method, the method comprising administering to the patient: (i) Streptococcus pyogenes Cas9 endonuclease; and (ii) the gRNA or gRNA composition, wherein the patient comprises a USH2A gene containing an IVS40 mutation.

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