Patent Description:
Described herein are compositions for use in treating subjects with USH2A-associated retinal and/or cochlear degeneration that result from mutations in exon <NUM> of the USH2A gene by deletion of exon <NUM> from the USH2A gene or transcripts, and methods of use thereof, as well as genetically modified animals and cells.

The USH2A gene encodes the transmembrane protein Usherin. Usherin localizes mainly at the periciliary region of mammalian photoreceptors and at the stereocilia or hair bundle of the inner ear hair cells (see, e.g.,<NPL>; <NPL>). The Usherin protein has a large extracellular domain that is proposed to interact with basement membrane collagen IV and fibronectin via laminin domains (see, e.g., Maerker et al. , <NUM>; <NPL>). Usherin also interacts with other proteins of USH1 and USH2 complex to form Usher networks (<NPL>).

Mutations in USH2A are the most common cause of both Usher syndrome type II and autosomal recessive retinitis pigmentosa (arRP), accounting for approximately <NUM>% of the recessive RP cases [<NUM>, <NUM>]. The impairment of both vision and hearing in Usher syndrome results in a reduced ability of the individual to perceive, communicate, and extract vital information from the environment [<NUM>]. Longitudinal regression analysis has showed that the disease course for patients with USH2A mutations can be rapidly progressive, particularly with respect to losing visual field and mobility [<NUM>].

2299delG mutation in exon13 of the USH2A gene is a single basepair deletion that results in a frameshift and premature stop codon, truncating the protein at exon <NUM> and truncates protein causing ciliary defects. Exon <NUM> encodes amino acids <NUM>-<NUM>, which span <NUM> of <NUM> Laminin EGF-like domains in the protein. As not all of these domains appear to be necessary for proper protein function, complete removal of exon <NUM> can be used to correct the disease phenotype by restoring the proper reading frame of the gene. Exons <NUM> and <NUM> are in frame with each other so deletion of exon <NUM> by a dual-cut approach, in which one gRNA directs a double-strand break to intron <NUM> and a second gRNA directs a double-strand break to intron <NUM>, is hypothesized to lead to direct splicing of exon <NUM> to exon <NUM>, thus generating an in-frame coding sequence lacking several of the Laminin EGF-like domains. Alternatively, disrupting the exon <NUM> splice acceptor site using a single gRNA, would provide similar results. As the protein lacking exon <NUM> retains functionality, this approach could also be applied to other exon <NUM> mutations, e.g., as known in the art, e.g., as shown in Table A.

Provided herein are nucleic acids comprising sequences encoding a Cas9 protein, and either: (i) a first gRNA, and a second gRNA, wherein the first gRNA and the second gRNA comprises SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; or SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; or (ii) a gRNA targeted to a splice acceptor site for exon <NUM> of an USH2A gene of the subject, wherein the gRNA targeted to the splice acceptor site for exon <NUM> comprises a targeting domain sequence selected from SEQ ID NOs. <NUM>-<NUM> and <NUM>.

In some embodiments, the nucleic acid encodes S. aureus Cas9, optionally wherein the Cas9 comprises a nuclear localization signal, optionally a C-terminal nuclear localization signal and/or an N-terminal nuclear localization signal; and/or wherein the sequences encoding Cas9 comprises a polyadenylation signal.

In some embodiments, the nucleic acid further comprises a viral delivery vector, optionally wherein the viral delivery vector comprises a promoter for Cas9, preferably a CMV, EFS, or hGRKI promoter, and/or wherein the viral delivery vector comprises an adeno-associated virus (AAV) vector.

In some embodiments, the nucleic acid comprises: (i) a sequence encoding a first gRNA and a second gRNA, wherein the first gRNA and the second gRNA comprise a first and a second targeting domain sequence selected from SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; or SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; or a sequence encoding a single gRNA comprising a targeting domain sequence selected from SEQ ID NOs. <NUM>-<NUM> and <NUM>; (ii) a first and a second inverted terminal repeat sequence (ITR); and (iii) a promoter for driving expression of the Cas9 selected from the group consisting of a CMV, an EFS, or an hGRKI promoter.

Also provided are the nucleic acids described herein for use in therapy; and/or for use in a method of treating a subject who has a condition associated with a mutation in exon <NUM> of USH2A gene, optionally wherein the condition is Usher Syndrome type <NUM> or autosomal recessive retinitis pigmentosa (arRP). In some embodiments, the AAV vector is delivered to a retina of a subject by injection, such as by subretinal injection, or is delivered to the inner ear of a subject by injection, preferably through the round window.

Provided herein are in vitro methods of altering the genome of a cell, the in vitro method comprising using CRISPR editing to forma first double strand break within intron <NUM> of the human USH2A gene and a second double strand within intron <NUM> of the human USH2A gene, wherein the first double strand break is formed between nucleotides <NUM>,<NUM>,<NUM> to <NUM>,<NUM>,<NUM> of human chromosome <NUM> and the step of forming the second double strand break is formed between nucleotides <NUM>,<NUM>,<NUM> and <NUM>,<NUM>,<NUM> of human chromosome <NUM>, wherein the first and second double strand breaks are repaired in a manner that results in the removal of exon <NUM> of the USH2A gene on human chromosome <NUM>, and wherein the step of forming the first strand break comprises contacting the cell with a first gRNA and the step of forming the second strand break comprises contacting the cell with a second gRNA, and wherein the first gRNA and the second gRNA comprise SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; or SEQ ID NO: <NUM> and SEQ ID NO: <NUM>.

In some examples, the method comprises contacting the cell with a recombinant viral particle comprising:.

In some embodiments, the cell is a cell of the eye or the inner ear of a mammal.

In some embodiments, the viral particle is an AAV viral particle.

Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict, between references mentioned herein and the present specification, the present specification, including definitions, will control.

Despite the success of clinical and pre-clinical studies of AAV mediated gene augmentation therapy for multiple genetic types of inherited retinal degeneration [<NUM>-<NUM>], developing gene therapy for the USH2A form of arRP has been challenging, because the large size of the USH2A coding sequence (CDS15602bp, 5202aa) far exceeds the packaging capacity of commonly used AAV viral delivery vectors. The present methods overcome these translational barriers by using a Cas9 gene editing approach for USH2A associated arRP [<NUM>, <NUM>]. The CRISPR/Cas system is capable of maintaining the edited gene under its endogenous regulatory elements by directly altering the genomic DNA, thereby avoiding ectopic expression and abnormal gene production that may occur with conventional viral-mediated gene augmentation therapies [<NUM>, <NUM>].

The usherin protein encoded by USH2A (GenBank Acc No. NC_000001. <NUM>, Reference GRCh38. p7 Primary Assembly, Range <NUM>-<NUM>, complement; SEQ ID NO: <NUM>) is a transmembrance protein anchored in the photoreceptor plasma membrane (<NPL>; <NPL>). Its extracellular portion, which accounts for over <NUM>% of the length of the protein and projects into the periciliary matrix, is thought to have an important structural and a possible signaling role for the long-term maintenance of photoreceptors (<NPL>; <NPL>). Two isoforms of USH2A have been described. Isoform b (GenBank Acc. No. NM_206933. <NUM> (transcript) and NP_996816. <NUM> (protein)) is most abundantly expressed in retina and is used as the canonical, standard sequence in the literature and in this application. Usherin is a protein with a high degree of homologous domain structures (<NPL>). Intracellularly, a PDZ domain has been identified to bind whirlin, whereas extracellularly, several domains are present and in most cases in a repetitive fashion, including10 Laminin EGF-like (LE) domains and <NUM> Fibronectin type <NUM> (FN3) domains. These repetitive domains comprise over <NUM>% of the protein structure combined. The most common mutation c. 2299delG, p. Glu767fs in USH2A gene, which causes approximately <NUM>%-<NUM>% of USH2A cases is USA [<NUM>, <NUM>], is located in exon <NUM> that encodes LE domain <NUM> (aa <NUM>-<NUM>) (<NPL>). Given the high degree of repetitive regions in usherin, it was hypothesized that an usherin protein that lacks one or more of the repetitive domains would retain partial or complete structural integrity and function, such that the abbreviated USH2A can serve as a therapeutic strategy for Usher syndrome type II and autosomal recessive retinitis pigmentosa (arRP) by skipping the mutant exon in USH2A gene.

As shown herein, Ush2a lacking exon <NUM> and with exons <NUM> and <NUM> fused in frame is expressed and localized correctly in the mouse retina and cochlea. When the Ush2a-ΔEx12 allele was expressed on an Ush2a null background, the Ush2a-ΔEx12 protein appeared to rescue the impaired hair cell structure and auditory function as shown by ABR, as compared to Ush2a-/- mice and also showed early signs of at least partial rescue of retinal phenotype. Without wishing to be bound by theory, this data supports the use of the present compositions and methods to restore sight and/or hearing, e.g., at least partially restore sight and/or hearing, in a subject who has Usher syndrome, e.g., associated with a mutation in exon <NUM> of USH2A gene. Thus a CRISPR/Cas9-based exon-skipping gene editing strategy to restore the reading frame of USH2A by deleting exon <NUM> holds therapeutic potential for the treatment of USH2A patients.

In one aspect, an Ush2A nucleic acid molecule includes a nucleotide sequence that is at least about <NUM>% or more identical to the entire length of SEQ ID NO: <NUM>. In some embodiments, the nucleotide sequence is at least about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% identical to SEQ ID NO: <NUM>.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least <NUM>% of the length of the reference sequence, and in some embodiments is at least <NUM>% or <NUM>%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. In another example, the percent identity of two amino acid sequences can be assessed as a function of the conservation of amino acid residues within the same family of amino acids (e.g., positive charge, negative charge, polar and uncharged, hydrophobic) at corresponding positions in both amino acid sequences (e.g., the presence of an alanine residue in place of a valine residue at a specific position in both sequences shows a high level of conservation, but the presence of an arginine residue in place of an aspartate residue at a specific position in both sequences shows a low level of conservation).

For purposes of the present invention, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum <NUM> scoring matrix with a gap penalty of <NUM>, a gap extend penalty of <NUM>, and a frameshift gap penalty of <NUM>.

CRISPR/Cas-based exon-skipping has been successfully used for restoring the expression of functional dystrophin and dystrophic muscle function in the Duchene muscular dystrophy mouse model [<NUM>-<NUM>]. The nucleic acids described herein are useful in methods for the treatment of disorders associated with mutations in exon <NUM> of the USH2A gene. Exemplary mutations, including <NUM> nonsense or frameshift mutations and <NUM> missense mutations on exon <NUM> (LOVD database), as shown in Table A, such as the most common missense mutation c.

In some embodiments, the disorder is Usher syndrome, e.g., type <NUM> Usher syndrome. Subjects with type <NUM> Usher syndrome typically have moderate to severe hearing loss at birth, and vision that becomes progressively impaired starting in adolescence. In some embodiments, the disorder is autosomal recessive retinitis pigmentosa (arRP). Generally, the methods include administering a therapeutically effective amount of a genome editing system as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. The term "genome editing system" refers to any system having RNA-guided DNA editing activity. Genome editing systems of the present disclosure include at least two components adapted from naturally occurring CRISPR systems: a gRNA and an RNA-guided nuclease. These two components form a complex that is capable of associating with a specific nucleic acid sequence in a cell and editing the DNA in or around that nucleic acid sequence, for example by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and/or a base substitution. See, e.g., <CIT> for a full description of genome editing systems.

As used in this context, to "treat" means to ameliorate at least one symptom of the disorder associated with mutations in exon <NUM> of the USH2A gene. Often, these mutations result in hearing loss and/or loss of sight; thus, a treatment comprising administration of a therapeutic gene editing system as described herein can result in a reduction in hearing impairment and/or visual impairment; a reduction in the rate of progression of hearing loss and/or vision loss; and/or a return or approach to normal hearing and/or vision. Hearing and vision can be tested using known methods, e.g., electroretinogram, optical coherence tomography, videonystagmography, and audiology testing.

The nucleic acids can be used to treat any subject (e.g., a mammalian subject, preferably a human subject) who has a mutation in exon <NUM> of the USH2A gene, e.g., the c. 2299delG mutation or c. <NUM>>T mutation, e.g., in one or both alleles of USH2A. As used herein, an "allele" is one of a pair or series of genetic variants of a polymorphism (also referred to as a mutation) at a specific genomic location. As used herein, "genotype" refers to the diploid combination of alleles for a given genetic polymorphism. A homozygous subject carries two copies of the same allele and a heterozygous subject carries two different alleles. Methods for identifying subjects with such mutations are known in the art; see, e.g., <NPL>; <NPL>; or <NPL>. For example, gel electrophoresis, capillary electrophoresis, size exclusion chromatography, sequencing, and/or arrays can be used to detect the presence or absence of the allele or genotype. Amplification of nucleic acids, where desirable, can be accomplished using methods known in the art, e.g., PCR. In one example, a sample (e.g., a sample comprising genomic DNA), is obtained from a subject. The DNA in the sample is then examined to identify or detect the presence of an allele or genotype as described herein. The allele or genotype can be identified or determined by any method described herein, e.g., by Sanger sequencing or Next Generation Sequencing (NGS). Since the exon <NUM> is 643bp in size, thus the genotyping of the patients with exon <NUM> mutations is simple and straight forward using Sanger sequencing or NGS. Other methods can include hybridization of the gene in the genomic DNA, RNA, or cDNA to a nucleic acid probe, e.g., a DNA probe (which includes cDNA and oligonucleotide probes) or an RNA probe. The nucleic acid probe can be designed to specifically or preferentially hybridize with a particular mutation (also referred to as a polymorphic variant).

Other methods of nucleic acid analysis can include direct manual sequencing (<NPL>); <NPL>); <CIT>); automated fluorescent sequencing; single-stranded conformation polymorphism assays (SSCP) (<NPL>)); clamped denaturing gel electrophoresis (CDGE); two-dimensional gel electrophoresis (2DGE or TDGE); conformational sensitive gel electrophoresis (CSGE); denaturing gradient gel electrophoresis (DGGE) (<NPL>)); denaturing high performance liquid chromatography (DHPLC, <NPL>)); infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry (<CIT>); mobility shift analysis (<NPL>)); restriction enzyme analysis (<NPL>); <NPL>)); quantitative real-time PCR (<NPL>)); heteroduplex analysis; chemical mismatch cleavage (CMC) (<NPL>)); RNase protection assays (<NPL>)); use of polypeptides that recognize nucleotide mismatches, e.g., E. coli mutS protein; allele-specific PCR, and combinations of such methods. See, e.g., <CIT> which is incorporated herein by reference in its entirety.

In certain aspects, the present disclosure provides AAV vectors encoding CRISPR/Cas9 genome editing systems, and on the use of such vectors to treat USH2A associated disease. Exemplary AAV vector genomes are schematized in <FIG>, which illustrates certain fixed and variable elements of these vectors: inverted terminal repeats (ITRs), one or two gRNA sequences and promoter sequences to drive their expression, a Cas9 coding sequence and another promoter to drive its expression (an exemplary construct for use in the Method Two described herein would be the same as that illustrated in <FIG>, but with only <NUM> gRNA and U6). Each of these elements is discussed in detail herein. Although <FIG> shows a single vector used to deliver a Cas9 and two gRNAs, in some embodiments a plurality of vectors are used, e.g., wherein one vector is used to deliver Cas9, and another vector or vectors is used to deliver one or more gRNAs (e.g., one vector for one gRNA, one vector for two gRNAs, or two vectors for each of two gRNAs).

Various RNA-guided nucleases can be used in the present methods, e.g., as described in <CIT>. In some embodiments, the RNA-guided nuclease used in the present methods and compositions is a S. aureus Cas9 or a S. pyogenes cas9. In some embodiments of this disclosure a Cas9 sequence is modified to include two nuclear localization sequences (NLSs) (e.g., PKKKRKV (SEQ ID NO:<NUM>) at the C- and N-termini of the Cas9 protein, and a mini-polyadenylation signal (or Poly-A sequence). An exemplary NLS is SV40 large T antigen NLS (PKKKRRV (SEQ ID NO:<NUM>)) and nucleoplasmin NLS (KRPAATKKAGQAKKKK (SEQ ID NO:<NUM>)). Other NLSs are known in the art; see, e.g., <NPL>; <NPL>. An exemplary polyadenylation signal is TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTG TTGGTTTTTTGATCAGGCGCG (SEQ ID NO:<NUM>)). Exemplary S. aureus Cas9 sequences (both nucleotide and peptide) are described in Table <NUM> of <CIT>, e.g., SEQ ID NOs <NUM> and <NUM> therein.

In some embodiments, the gRNAs used in the present disclosure can be unimolecular or modular, as described below. An exemplary unimolecular S. aureus gRNA is shown in <FIG>, and exemplary DNA and RNA sequences corresponding to unimolecular S. aureus gRNAs are shown below:.

It should be noted that, while <FIG> depicts a targeting domain of <NUM> nucleotides, the targeting domain can have any suitable length. As indicated by the "N16-<NUM>" notation in the sequences above, gRNAs used in the various embodiments of this disclosure preferably include targeting domains of between <NUM> and <NUM> (inclusive) bases in length at their <NUM>' ends, and optionally include a <NUM>' U6 termination sequence as illustrated.

The gRNA in <FIG> is depicted as unimolecular, but in some instances modular guides can be used. In the exemplary unimolecular gRNA sequences above, a <NUM>' portion corresponding to a crRNA (bold) is connected by a GAAA linker (lower case) to a <NUM>' portion corresponding to a tracrRNA (double underlined). Skilled artisans will appreciate that two- part modular gRNAs can be used that correspond to the bold and double underlined sections.

Either one of the gRNAs presented above can be used with any of targeting sequences in tables <NUM>-<NUM>, and two gRNAs in a pair do not necessarily include the same backbone sequence. Additionally, skilled artisans will appreciate that the exemplary gRNA designs set forth herein can be modified in a variety of ways, which are described below or are known in the art; the incorporation of such modifications is within the scope of this disclosure.

Described herein are two approaches for treating subjects with mutations in exon <NUM> of USH2A. The first makes use of dual-gRNAs for deletion of exon <NUM>. Two gRNAs (one in intron <NUM>, one in intron <NUM>) are used in combination to cut out a segment of DNA including exon <NUM>. In addition to deleting this segment, it may also be inverted and reinserted. In the present studies, inversion of the exon was seen as commonly as deletion, and the inverted version was equally functional; without wishing to be bound by theory, the rearrangement may remove the functional splice sites, so the protein still lacks exon <NUM> and thus corrects the phenotype.

In some embodiments, this approach uses Staphylococcus aureus Cas9 (SaCas9) and corresponding gRNAs. SaCas9 is one of several smaller Cas9 orthologues that are suited for viral delivery (<NPL>); <NPL>); <NPL>)). The wild type recognizes a longer NNGRRT PAM that is expected to occur once in every <NUM> bps of random DNA; or the alternative NNGRRA PAM. Preferably, the <NUM>' base of each gRNA is a G, and the protospacer is length <NUM>, <NUM> or <NUM> nucleotides, and the target sequence falls in the <NUM>' 1500bp of intron <NUM> or <NUM>' 1500bp of intron <NUM>.

The methods can include using two gRNAs, one that targets intron <NUM>, and one that targets intron <NUM>. The genomic coordinates of introns <NUM> and <NUM> are provided in the following table.

Tables <NUM> and <NUM> provide exemplary sequences for the gRNAs in exons <NUM> and <NUM>, respectively. Note that in the sequences provided herein, the actual gRNA would have U in place of T.

In some embodiments of these methods, any of the intron <NUM> gRNAs in Table <NUM> can be used with any of the intron <NUM> gRNAs in Table <NUM>, though certain combinations may be more suitable for certain applications. It should be noted, notwithstanding the use of "first" and "second" as nomenclature for gRNAs, that any guide in a pair, in intron <NUM> or intron <NUM>, can be placed in either one of the gRNA coding sequence positions illustrated in <FIG>.

In some embodiments, one of the combinations of gRNAs in the following table is used; each row shows a preferred combination (e.g., In12_307 with In12_318).

In any of the methods described herein, the engineered CRISPR from Prevotella and Francisella <NUM> (Cpfl, also known as Cas12a) nuclease can also be used, e.g., as described in <NPL>); <NPL>); <NPL>);<NPL>). Unlike SpCas9, Cpf1/Cas12a requires only a single <NUM>-nt crRNA, which has <NUM> nt at its <NUM>' end that are complementary to the protospacer of the target DNA sequence (Zetsche et al. Furthermore, whereas SpCas9 recognizes an NGG PAM sequence that is <NUM>' of the protospacer, AsCpf1 and LbCp1 recognize TTTN PAMs that are found <NUM>' of the protospacer (Id. In some embodiments, the Cas12a is, e.g., Acidaminococcus sp. BV3L6 Cpfl (AsCpfl, UniProt U2UMQ6. <NUM>) or Lachnospiraceae bacterium ND2006 (LbCpfl, UniProt A0A182DWE3. <NUM>), with corresponding gRNAs.

The second approach makes use of a single gRNA for destruction of the exon <NUM> splice acceptor. Non-homologous end joining (NHEJ)-mediated indels destroy the splice acceptor, thus preventing exon <NUM> from being spliced into mRNA.

Preferably, this method uses Staphylococcus aureus wild type or KKH variant SaCas9 (See<NPL>; <CIT>) or Cas12a and corresponding gRNAs. Table <NUM> provides exemplary target sites in the splice acceptor.

The methods include delivery of a CRISPR/Cas9 genome editing system, including a Cas9 nuclease and one or two guide RNAs, to a subject in need thereof. The delivery methods can include, e.g., viral delivery, e.g., preferably using an adeno-associated virus (AAV) vector that comprises sequences encoding the Cas9 and guide RNA(s). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see <NPL>)). AAV vectors efficiently transduce various cell types and can produce long-term expression of transgenes in vivo. AAV vectors have been extensively used for gene augmentation or replacement and have shown therapeutic efficacy in a range of animal models as well as in the clinic; see, e.g.,<NPL>); <NPL>; <NPL>. AAV vectors containing as little as <NUM> base pairs of AAV can be packaged and can produce recombinant protein expression. For example, AAV2, AAV5, AAV2/<NUM>, AAV2/<NUM> and AAV2/<NUM> vectors have been used to introduce DNA into photoreceptor cells (see, e.g., <NPL>; <NPL>; <NPL>). In some embodiments, the AAV vector can include (or include a sequence encoding) an AAV capsid polypeptide described in <CIT>; for example, a virus particle comprising an AAV capsid polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> of <CIT>, and a Cas9 sequence and guide RNA sequence as described herein. In some embodiments, the AAV capsid polypeptide is an Anc80 polypeptide, e.g., Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; or Anc80L44. In some embodiments, the AAV incorporates inverted terminal repeats (ITRs) derived from the AAV2 serotype. Exemplary left and right ITRs are presented in Table <NUM> of <CIT>. It should be noted, however, that numerous modified versions of the AAV2 ITRs are used in the field, and the ITR sequences shown below are exemplary and are not intended to be limiting. Modifications of these sequences are known in the art, or will be evident to skilled artisans, and are thus included in the scope of this disclosure.

Cas9 expression is driven by a promoter known in the art. In some embodiments, expression is driven by one of three promoters: cytomegalovirus (CMV), elongation factor-<NUM> (EFS), or human g-protein receptor coupled kinase-<NUM> (hGRKl), which is specifically expressed in retinal photoreceptor cells. Nucleotide sequences for each of these promoters are provided in Table <NUM> of <CIT>. Modifications of these sequences may be possible or desirable in certain applications, and such modifications are within the scope of this disclosure.

Expression of the gRNAs in the AAV vector is driven by a promoter known in the art. In some embodiments, a polymerase III promoter, such as a human U6 promoter. An exemplary U6 promoter sequence is presented below:
<IMG>.

In some embodiments, the nucleic acid or AAV vector shares at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or greater sequence identity with one of the nucleic acids or AAV vectors recited above.

The AAV genomes described above can be packaged into AAV capsids (for example, AAV5 capsids), which capsids can be included in compositions (such as pharmaceutical compositions) and/or administered to subjects. An exemplary pharmaceutical composition comprising an AAV capsid according to this disclosure can include a pharmaceutically acceptable carrier such as balanced saline solution (BSS) and one or more surfactants (e.g., Tween <NUM>) and/or a thermosensitive or reverse-thermosensitive polymer (e.g., pluronic). Other pharmaceutical formulation elements known in the art may also be suitable for use in the compositions described here.

Compositions comprising AAV vectors according to this disclosure can be administered to subjects by any suitable means, including without limitation injection, for example, subretinal injection or injection through the round window. The concentration of AAV vector within the composition is selected to ensure, among other things, that a sufficient AAV dose is administered to the retina or inner ear of the subject, taking account of dead volume within the injection apparatus and the relatively limited volume that can be safely administered. Suitable doses may include, for example, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, 6x10<NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> vg/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> vg/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, <NUM>×<NUM><NUM> viral genomes (vg)/mL, or <NUM>×<NUM><NUM> viral genomes (vg)/mL. Any suitable volume of the composition may be delivered to the subretinal or cochlear space. In some instances, the volume is selected to form a bleb in the subretinal space, for example <NUM> microliter, <NUM> microliters, <NUM> microliters, <NUM> microliters, <NUM> microliters, <NUM> microliters, <NUM> microliters, <NUM> microliters, etc..

Any region of the retina may be targeted, though the fovea (which extends approximately <NUM> degree out from the center of the eye) may be preferred in certain instances due to its role in central visual acuity and the relatively high concentration of cone photoreceptors there relative to peripheral regions of the retina. Alternatively or additionally, injections may be targeted to parafoveal regions (extending between approximately <NUM> and <NUM> degrees off center), which are characterized by the presence of all three types of retinal photoreceptor cells. In addition, injections into the parafoveal region may be made at comparatively acute angles using needle paths that cross the midline of the retina. For instance, injection paths may extend from the nasal aspect of the sclera near the limbus through the vitreal chamber and into the parafoveal retina on the temporal side, from the temporal aspect of the sclera to the parafoveal retina on the nasal side, from a portion of the sclera located superior to the cornea to an inferior parafoveal position, and/or from an inferior portion of the sclera to a superior parafoveal position. The use of relatively small angles of injection relative to the retinal surface may advantageously reduce or limit the potential for spillover of vector from the bleb into the vitreous body and, consequently, reduce the loss of the vector during delivery. In other cases, the macula (inclusive of the fovea) can be targeted, and in other cases, additional retinal regions can be targeted, or can receive spillover doses.

For delivery to the inner ear, injection to the cochlear duct, which is filled with high potassium endolymph fluid, could provide direct access to hair cells. However, alterations to this delicate fluid environment may disrupt the endocochlear potential, heightening the risk for injection- related toxicity. The perilymph-filled spaces surrounding the cochlear duct, scala tympani and scala vestibuli, can be accessed from the middle ear, either through the oval or round window membrane (RWM). The RWM, which is the only non-bony opening into the inner ear, is relatively easily accessible in many animal models and administration of viral vector using this route is well tolerated. Administration through the oval window or across the tympanic membrane can also be used. See, e.g., <CIT> and <CIT>.

For pre-clinical development purposes, systems, compositions, nucleotides and vectors according to this disclosure can be evaluated ex vivo using a retinal explant system, or in vivo using an animal model such as a mouse, rabbit, pig, nonhuman primate, etc. Retinal explants are optionally maintained on a support matrix, and AAV vectors can be delivered by injection into the space between the photoreceptor layer and the support matrix, to mimic subretinal injection. Tissue for retinal explanation can be obtained from human or animal subjects, for example mouse.

Explants are particularly useful for studying the expression of gRNAs and/or Cas9 following viral transduction, and for studying genome editing over comparatively short intervals. These models also permit higher throughput than may be possible in animal models, and can be predictive of expression and genome editing in animal models and subjects. Small (mouse, rat) and large animal models (such as rabbit, pig, nonhuman primate) can be used for pharmacological and/or toxicological studies and for testing the systems, nucleotides, vectors and compositions of this disclosure under conditions and at volumes that approximate those that will be used in clinic. Because model systems are selected to recapitulate relevant aspects of human anatomy and/or physiology, the data obtained in these systems will generally (though not necessarily) be predictive of the behavior of AAV vectors and compositions according to this disclosure in human and animal subjects.

Also provided herein are non-human genetically modified animals comprising a mutation in exon <NUM> (or the equivalent exon, for example, exon <NUM> in the mouse) in the USH2A gene. Such animals are useful as models of disease, e.g., of Usher syndrome or arRP, for studying the function and/or activity of USH2A protein and for identifying and/or evaluating potential therapeutic compounds for treating conditions associated with mutations in exon <NUM> of the USH2A gene. As used herein, a "genetically modified animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a modified gene. Other examples of genetically modified animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like.

The genetically modified animals can have a complete deletion of exon <NUM>, an inversion of exon <NUM>, or a mutation that disrupts the exon <NUM> splice acceptor site, integrated into or occurring in the genome of the cells of a genetically modified animal (e.g., in one or both alleles of the gene in the genome). In preferred embodiments, the animal has had both endogenous USH2A alleles replaced with a human USH2A gene, or has had part of both endogenous USH2A alleles containing the relevant exon and flanking intronic regions replaced with a human USH2A exon <NUM> and flanking intronic regions, with a complete deletion of exon <NUM>, an inversion of exon <NUM>, or a mutation that disrupts the exon <NUM> splice acceptor site.

Methods for making genetically modified animals are known in the art; see, e.g., <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>. Such techniques include, without limitation, pronuclear microinjection (See, e.g., <CIT>), retrovirus mediated gene transfer into germ lines (<NPL>)), gene targeting into embryonic stem cells (<NPL>)), electroporation of embryos (<NPL>)), and in vitro transformation of somatic cells, such as cumulus or mammary cells, followed by nuclear transplantation (<NPL>); and <NPL>)); these methods can be modified to use CRISPR as described herein. For example, fetal fibroblasts can be genetically modified using CRISPR as described herein, and then fused with enucleated oocytes. After activation of the oocytes, the eggs are cultured to the blastocyst stage. See, for example, <NPL>).

A founder animal can be identified based upon the presence of a mutation in exon <NUM> of USH2A in its genome and/or expression of USH2A mRNA lacking exon <NUM> in tissues or cells of the animals. A genetically modified founder animal can then be used to breed additional animals carrying the modified gene. Moreover, animals carrying a modified encoding an Ush2A protein lacking exon <NUM> can further be bred to Ush2a knockout animals.

Described herein is a population of cells isolated from an animal as described herein, as well as primary or cultured cells, e.g., isolated cells, engineered to include a mutation in exon <NUM> of human USH2A gene or a deletion of exon <NUM> in the mouse Ush2a gene. The cells can have a complete deletion of exon <NUM> , an inversion of exon <NUM>, or a mutation that disrupts the exon <NUM> splice acceptor site, integrated into or occurs in the genome of the cells. The cells can be from any mammal, e.g., a human or non-human mammal, or other animal.

Further provided herein are nucleic acids (e.g., isolated nucleic acids) that comprise or encode an USH2A mRNA that lacks exon <NUM>, e.g., that have a complete deletion of exon <NUM> , an inversion of exon <NUM>, or a mutation that disrupts the exon <NUM> splice acceptor site, as well as expression and delivery vectors (including viral and non-viral vectors) comprising the nucleic acids, and usherin proteins lacking exon <NUM>. Preferably the sequences are generated using a human USH2A sequence, but they can also be generated from other mammals, including mouse (mRNA: NM_021408. <NUM>; syntenic exon: <NUM>) rat (mRNA: NM_001302219. <NUM>; syntenic exon <NUM>); chimpanzee (mRNA: XM_016938662. <NUM>; syntenic exon: <NUM>); Cynomolgus macaque (macaca fasicularis, mRNA: XM_005540847. <NUM> or XM_005540848. <NUM>) and african green monkey (chlorocebus sabeus, mRNA: XM_007988447.

The invention is further described in the following examples, which do not limit the scope of the invention defined by the claims.

This Examiner describes the generation of an USH2A knockout cell line.

OC-kl cells were selected as a model system for this study. OC-kl cells are derived from mouse cochlea (<NPL>; <NPL>). These are tri allelic, expressing Ush2a protein and its interacting proteins such as Whrn, Vlgr1 in the Usher <NUM> complex (<FIG>), making these cells particularly appropriate for these studies. The preliminary results illustrated that the OC-k1 wild type cells stably expressed the Ush2a protein at the base of primary cilia.

CRISPR/Cas9 technology was used to create the Ush2a null cell model in OC-k1 cells. Guide RNA <NUM>'-GGAATGCAGTACTGCTGAACGG-<NUM>' (SEQ ID NO:<NUM>) for wild-type SpCas9 was designed to target the exon <NUM> of mouse Ush2a gene (<FIG>). U6-sgRNA and CAG-SpCas9-P2A-GFP plasmids were CO-transfected into OC-k1 cells using lipofectamine <NUM>. Two days post-transfection, the GFP positive cells were collected by sorting, plated at low density, and grown to mature colonies. Post genomic DNA isolation from these cells, the region spanning the sgRNA target site was PCR amplified and deep sequencing analysis was performed to get insights into the frequency of NHEJ-induced insertion-deletions (Indels) in Ush2a alleles. A cleavage efficiency of ~<NUM>% was observed with a wide variety of indels with <NUM>% of out-of-frame indels (<FIG>). A total of <NUM> clones were screened with T7E1 assay (<FIG>) and found that the clone <NUM> obtained from first attempt of transfections resulted in a mixed pattern of indels including in-frame, out-of-frame and uncut alleles (<FIG>). In order to knockout all three Ush2a alleles in a single clone, clone <NUM> was re-transfected with the same SpCas9-sgRNA pair and serial dilution of the culture was performed. Of many different single clones analyzed with both T7E1 and NGS analysis, clone J was confirmed to be completely null for Ush2a (two alleles with 7bp and remaining one allele with 1bp out of frame deletions, based on ratios of NGS paired reads) as shown in <FIG>. Other clones, for example clone <NUM>, showed in-frame deletion for one allele and out-of-frame deletion for other two alleles.

Ush2a is expressed at the base of cilia. This experiment investigated whether depletion of Ush2a would affect ciliogenesis. Cells were serum starved and stained for Ush2a and acetylated alpha-tubulin, a ciliary marker. As illustrated in <FIG>, clone <NUM> and clone <NUM> produced cilia as shown by Ace-tubulin staining (<FIG>). They also expressed Ush2a protein at base of cilium, detected by anti-C-term Ush2a antibody (<NPL>). The null clone J failed to produce any detectable cilia (<FIG>). However, a stunted basal body look-like structure was observed in clone J. Cilia length was further measured from the basal body to the tip of cilia using an acetylated tubulin antibody. (<FIG>, n=<NUM>). Significant shortening of cilia in clone <NUM> and J, compared to the wild-type cells was observed, indicating that ciliogenesis is hampered in the Ush2a null OC-kl cells.

In order to determine whether the Ush2a protein that lacks portion of the reparative Laminin EGF like domain (encoded by exon <NUM>) will retain partial or complete biologic function of USH2A protein, full-length and USH2A-ΔEx13 cDNAs were transfected into the Ush2a null line and their effect on ciliogenesis was evaluated. It was observed that both the mouse and human wild-type full length USH2A cDNAs were able to rescue ciliogenesis (<FIG>). In addition, the expression of human USH2A-ΔEx13 was able to increase the length of cilia to <NUM>% of the wild-type cilia (<FIG> and <FIG>). These results indicate that the product of the aberrant USH2A-ΔEx13 retains at least partial biological function of USH2A.

To further assess the cell-based findings in Ush2a null cells in vivo, Ush2a-ΔEx12 mouse lines were generated using CRISPR/Cas9 technology. Pairs of sgRNAs that target the flanking intron <NUM>(11A, 11B and 11C) and intron <NUM> (12A, 12C and 12D) (<FIG>, <FIG>) were designed. All guides were synthesized and in vitro transcribed and tested for cleavage efficiency using an in-vitro cleavage assay. <FIG> illustrates the selected sgRNAs are active and cleaved PCR template derived from Ush2a genomic region surrounding exon <NUM> with efficiencies of <NUM>% to <NUM>%. All three pairs of sgRNAs were microinjected together with SpCas9 protein into the pronuclei of mouse zygotes to generate the Ush2a-ΔEx12 mouse lines in the Genome Modification Facility at Harvard University. A total of <NUM> pups were obtained from the pronuclear injections. With initial genotyping and sequencing, it was confirmed that <NUM> mice (<NUM>%) carry deletion of exon <NUM> and flanking introns with different sizes. Sanger sequence verified <NUM> founders of mice (<FIG>). The male founders were further backcrossed with C57BL6/J females and F1 generations were obtained.

The phenotype of the resulting Ush2a-ΔEx12 mouse lines was characterized histologically and functionally. The localization of the Ush2a-ΔEx12 protein and its interaction with other Usher2 complex proteins at two months of age was determined by immunostaining, and showed that both the wild type and exon <NUM>-skippped Ush2a proteins were localized at the transition zone of photoreceptor sensory cilia in ΔEx12/ΔEx12, ΔEx12/ko, and wt mice (<FIG>).

The therapeutic potential of Ush2a-ΔEx12 was evaluated by transferring this Ush2a-ΔEx12 allele onto an Ush2a null background in Ush2a-/- knockout mice (<NPL>. ) The phenotypes observed in these Ush2a-/- mice include progressive disruption of inner hair cells in the cochlea after <NUM> months of age and diminished inner hair cells at <NUM> months of age; a detectable accumulation of GFAP and mis-localization of cone opsin at <NUM> months of age; gradual outer nuclear layer thinning and photoreceptor abnormalities after <NUM> months of age; <NUM>% loss of photoreceptors and <NUM>% or greater reduction of ERG amplitudes for a- and b- waves by the age of <NUM> months (<NPL>; <NPL>.

Cochleas were isolated from P3 ΔEx12/ΔEx12 and wt mice and stained for phalloidin (top panel), and Ush2a, FM1-<NUM>, and phalloidin (<FIG>), showing essentially normal morphology in the ΔEx12/ΔEx12 mice. Inner and outer hair cell structures were grossly normal in ΔEx12/ΔEx12, ΔEx12/ko, and wt mice on staining with Ush2a, as compared with the knockout (<FIG>). Auditory brain stem recordings showed improved hearing as determined by ABR in the Ush2a ko/ko mice by the Ush2a-Ex12 allele (<FIG>). Phalloidin staining showed disrupted bundles in the ko/ko mice, but not in ΔEx12/ΔEx12, ΔEx12/ko, or wt mice (<FIG>).

In the retina, abnormal accumulation of GFAP in Ush2a ko/ko mice was reduced in the ΔEx12/ko mice at <NUM> months of age, and cone opsin localization was normalized in ΔEx12/ko mice at <NUM> months of age, as compared to ko/ko mice (<FIG>).

These results showed that the protein encoded by Ush2a-ΔEx12 allele rescued the cochlear and retinal phenotypes observed in Ush2a-l- knockout mice.

A humanized USH2A mouse models is developed. Exon <NUM> of the mouse Ush2a gene, along with up to 1500bp of the flanking introns, is replaced with the syntenic human exon <NUM> and up to 1500bp of flanking introns. Two models are generated - one in which the wildtype human exon <NUM> is used and one in which the human exon <NUM> contains the c. 2299delG mutation.

The expectation is that the mouse containing the wildtype human exon <NUM> will be phenotypically normal. This is supported by the high level of similarity between the amino acids encoded by mouse exon <NUM> and human exon <NUM> (<NUM>% exact sequence identity match and <NUM>% sequence similarity match based on amino acid properties).

The expectation is that the mouse containing the c. 2299delG mutation will exhibit an Usher Syndrome disease phenotype. As in Usher Syndrome patients, the c. 2299delG mutation will result in a frameshift and premature stop codon, leading to a prematurely truncated, and non-functional protein. This is therefore expected to mimic the phenotype of the Ush2a knockout mouse (Ush2a-/-) as described above.

The humanized Ush2a mouse models enable pharmacology PK/PD studies with human USH2A-targeted therapeutic guides. In addition, they enable demonstration of correction of disease phenotype. Lead gRNAs, along with Cas9 will be packaged in AAV. An example of what the configuration of this vector could look like is given in <FIG>. Several AAV serotypes could be used, for example, AAV5, AAV8, AAV9 and AAV-Anc80 have all been demonstrated to show strong tropism for photoreceptors. 1ul of AAV will be delivered to mice subretinally at doses ranging from 1E+<NUM> to 1E+<NUM> viral genomes/mL. Mice may be treated at different ages to assess ability to reverse pathology at various stages in disease. Mice will be evaluated at several time points to assess functional and structural rescue of retinal and cochlear phenotypes. Molecular analysis will determine expression levels of Cas9 and gRNAs as well as quantify targeted gene editing rates. ERG will measure photoreceptor function in the retina. Optical coherence tomography (OCT) and histology will examine retinal structure.

A comprehensive list of sgRNAs for SaCas9, SpCas9, their variants, and Cpfl were generated to target the flanking intron <NUM> and <NUM> in the humanized USH2A mice. Those guides are individually screened in the human cell lines. Optimal pairs of sgRNAs are further evaluated for skipping the exon <NUM> in the humanized mice.

Guide RNAs were screened within human USH2A intron <NUM> and intron <NUM> to find the best cutting gRNAs. To this end, ability of <NUM> gRNAs within intron <NUM> and <NUM> gRNAs within intron <NUM> to generate indels in HEK293 cells was evaluated. The cells were transfected with RNPs, gDNA was isolated <NUM> hours later and subjected to PCR amplification and high-throughput sequencing and analysis to determine the editing rates for each gRNA (Table <NUM>).

Following this initial screen, a second screen focused on <NUM> intron <NUM> gRNAs and <NUM> intron <NUM> gRNAs which were identified from the initial screen. All the possible gRNA combinations were screened to determine which worked together to give the highest loss of exon <NUM>. U2OS cells were transfected with plasmids that expressed S. aureus Cas9 and the gRNAs of interest and gDNA was isolated <NUM> hours later for analysis. Editing was determined with a ddPCR assay that measures the presence or absence of USH2A exon <NUM>. Results are shown in <FIG> (N=<NUM>) for all gRNA combinations as well as negative controls (GFP).

For the single gRNA approach, where the aim is to disrupt the exon <NUM> splice acceptor, gRNA that cut near the exon <NUM> splice acceptor were identified (Table <NUM>). The ability of these gRNA to cut and form indels in U20S cells was tested through plasmid transfection of CRISPR Cas9 or Cpfl and the associated gRNA. The genomic DNA was extracted and subject to PCR amplification and sequencing to determine the percentage of indels (<FIG>).

The specificity of the top cutting gRNA for the dual gRNA approach was assessed using three different analyses. First, an in silico screen was conducted to identify all sites in the human genome where the particular guide could potentially cut, allowing for up to <NUM> mismatches or gaps in the protospacer sequence (Tables 6a and 6b).

Next, two different unbiased screens to identify off-target cut sites were completed. Guide-Seq was performed to assess the number and location of all editing events that occurred following treatment of cells with RNPs containing the one of the top gRNA. Guide-Seq was performed in primary human T cells after activation and expansion of the cells. The cells were nucleofected with RNPs and a short doublestranded oligo (<NPL>). gDNA was isolated, sheared, and adapters for PCR amplification were added before PCR amplification. DNA sequences adjacent to the Guide-Seq oligo were aligned to the genome to identify the location where the double-strand oligo was inserted.

Finally, Digenome-Seq was used as a second unbiased method to locate off-target cut sites. In this method, purified genomic DNA is mixed with RNP in a cell-free system (<NPL>). The DNA is then isolated, undergoes high-throughput sequencing, and is aligned to the human genome to identify locations where the DNA was cut (Tables 6a and 6b).

An RTddPCR assay to measure the amount of delta exon <NUM> USH2A transcript relative to WT USH2A was established. The WT assay amplifies the RNA junction between exons <NUM> and <NUM> while the delta <NUM> assay amplifies the junction between exon <NUM> and exon <NUM>, which will only occur if exon <NUM> is precisely skipped (<FIG>). Another transcript ubiquitously expressed from Chromosome <NUM> (C1orf43) was used as a reference in this assay. The assay was validated with plasmids to check the linearity against expected inputs.

The ability of the assay to detect delta exon <NUM> USH2A transcripts after CRISPR/Cas9 mediated editing was tested in human CRL-<NUM> cells, which have been shown to express USH2A. CRL-<NUM> cells were transfected with plasmids expressing Cas9 and gRNA for the single guide approach and DNA and RNA were isolated from the cells <NUM> days after transfection. The DNA was assessed for genomic editing by high-throughput sequencing. RNA was used in the RTddPCR assay to measure the level of WT and delta exon <NUM> USH2A transcripts (<FIG>).

Editing and delta <NUM> USH2A expression was compared with two top pairs of gRNAs. CRL-<NUM> cells were nucleofected with RNPs containing the two gRNA and then DNA and RNA were isolated <NUM> days later (<NUM> biological replicates). Loss of genomic USH2A exon <NUM> was measured by a ddPCR assay (<FIG>) and levels of WT and delta exon <NUM> USH2A transcripts were measured by RTddPCR (Fig. 11E).

AAV5 vectors were cloned and produced to express in12_321 and in13_322 as depicted in <FIG>. Retinal explant punches (<NUM> millimeters) were taken from cadaver eyes within <NUM> hours post-mortem and individually cultured on membranes. <NUM> uL of AAV at a titer of 5e13 vg/mL was added between the neural retina and the membrane such that it created a viral bleb under the retinal tissue. Tissue was incubated for <NUM> days at which point the punches were collected. Each punch was split in half, one half for gDNA and the other half for RNA isolation. PCR amplification and sequencing showed that the punches treated with AAV had between <NUM> - <NUM>% total editing, while untreated control punches had background levels of editing. RNA was used for RTddPCR analysis to determine whether editing had caused production of USH2A delta exon <NUM> transcripts. Analysis showed that between <NUM>-<NUM>% of USH2A transcripts were lacking exon <NUM> (<FIG>).

Human USH2A mRNA lacking exon <NUM>: (exon <NUM> in bold, exon <NUM> double underlined)
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<IMG>.

Translation of this mRNA is expected to result in expression of human Usherin protein lacking part of laminin EGF-like domain <NUM>, all of domains <NUM>, <NUM> and <NUM> and part of domain <NUM>:.

Claim 1:
A nucleic acid comprising sequences encoding a Cas9 protein, and either:
(i) a first gRNA and a second gRNA, wherein the first gRNA and the second gRNA comprise SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; or SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; or
(ii) a gRNA targeted to a splice acceptor site for exon <NUM> of an USH2A gene of the subject, wherein the gRNA targeted to the splice acceptor site for exon <NUM> comprises a targeting domain sequence selected from SEQ ID NOs. <NUM>-<NUM> and <NUM>.