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WO2023194359A1 - Compositions et méthodes de traitement du syndrome d'usher de type 2a - Google Patents

Compositions et méthodes de traitement du syndrome d'usher de type 2a Download PDF

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WO2023194359A1
WO2023194359A1 PCT/EP2023/058797 EP2023058797W WO2023194359A1 WO 2023194359 A1 WO2023194359 A1 WO 2023194359A1 EP 2023058797 W EP2023058797 W EP 2023058797W WO 2023194359 A1 WO2023194359 A1 WO 2023194359A1
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grna
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sequence
cell
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PCT/EP2023/058797
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Antonio CASINI
Laura PEZZÈ
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Alia Therapeutics Srl
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Publication of WO2023194359A1 publication Critical patent/WO2023194359A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • compositions and methods useful for altering USH2A genes in particular USH2A genes having pathogenic mutations such as c.7595- 2144A>G.
  • the disclosed compositions and methods are useful, for example, for cellular manipulations and subject treatments (e.g., of human subjects), particularly of Usher syndrome type 2A subjects.
  • the disclosure provides gRNA molecules (e.g., single guide RNAs (sgRNAs)), for example Type II Cas gRNA molecules, for targeting USH2A genes, for example having the c.7595- 2144A>G mutation.
  • gRNAs can be used, for example, in combination with a Type II Cas protein to edit (e.g., introduce a deletion in) a human USH2A gene having a pathogenic mutation, for example c.7595-2144A>G.
  • the Type II Cas protein comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:1 (such proteins referred to herein as “AIK Type II Cas proteins”).
  • AIK Type II Cas protein sequences are set forth in SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3.
  • the disclosure provides pluralities of gRNA molecules of the disclosure, e.g., a plurality of gRNAs comprising a first gRNA and a second, different gRNA.
  • Pluralities of gRNAs can in some embodiments comprise a gRNA targeting USH2A upstream of the PE40 region and a gRNA targeting USH2A downstream of the PE40 region.
  • Targeting the USH2A gene upstream of the PE40 region and downstream of the PE40 region can introduce a deletion in the USH2A gene that encompasses the PE40 sequence. Without the PE40 sequence, expression of functional usherin protein can be restored.
  • Exemplary features of the gRNAs of the disclosure are described in Section 6.2 and numbered embodiments 1 to 124, infra.
  • Exemplary features of the pluralities of gRNAs of the disclosure are described in Section 6.3 and numbered embodiments 125 to 180, infra.
  • Exemplary features of Type II Cas proteins, for example AIK Type II Cas proteins are described in Section 6.4 and numbered embodiments 201 to 217, infra.
  • the disclosure further provides nucleic acids encoding gRNAs and pluralities of gRNAs of the disclosure, pluralities of nucleic acids and host cells comprising the nucleic acids (including pluralities of nucleic acids) of the disclosure.
  • the nucleic acids further encode a Type II Cas protein, for example an AIK Type II Cas protein. Exemplary features of the nucleic acids and host cells are described in Section 6.5 and numbered embodiments 181 to 222, infra.
  • the disclosure further provides systems and pluralities of systems comprising a gRNA or plurality of gRNAs of the disclosure and a Type II Cas protein, for example, an AIK Type II Cas protein.
  • a Type II Cas protein for example, an AIK Type II Cas protein.
  • Exemplary systems are described in Section 6.6 and numbered embodiments 223 to 224, infra.
  • the disclosure further provides particles and pluralities of particles containing gRNAs of the disclosure, pluralities of gRNAs of the disclosure, nucleic acids (including pluralities of nucleic acids) of the disclosure, systems of the disclosure, and combinations thereof.
  • the disclosure further provides pharmaceutical compositions comprising the gRNAs, plurality of gRNAs, nucleic acids (including pluralities of nucleic acids), systems, and particles (including pluralities of particles), of the disclosure.
  • the disclosure further provides cells (e.g., from a subject having a USH2A gene with a pathogenic mutation) and populations of cells comprising the gRNAs (including pluralities of gRNAs), nucleic acids (including pluralities of nucleic acids), systems, particles (including pluralities of particles), and pharmaceutical compositions of the disclosure.
  • gRNAs including pluralities of gRNAs
  • nucleic acids including pluralities of nucleic acids
  • systems including pluralities of particles
  • particles including pluralities of particles
  • Exemplary particles, pluralities of particles, cells, and populations of cells are described in Section 6.7 and numbered embodiments 227 to 238 and 240 to 250, infra.
  • Exemplary pharmaceutical compositions are described in Section 6.8 and numbered embodiment 239, infra.
  • the disclosure further provides methods of using the gRNAs, pluralities of gRNAs, nucleic acids (including pluralities of nucleic acids), systems (including pluralities of systems), particles (including pluralities of particles), and pharmaceutical compositions of the disclosure for altering cells, for example a human cell having an USH2A gene with a pathogenic mutation, for example c.7595-2144A>G.
  • Methods of the disclosure can be used, for example, to treat subjects having a Usher syndrome type 2A.
  • FIG. 1 shows an exemplary AIK Type II Cas sgRNA scaffold.
  • FIG. 1 shows a schematic representation of the hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of an exemplary sgRNA scaffold (not including the spacer sequence) designed from crRNA and tracrRNA for AIK Type II Cas (sgRNA VI ).
  • Figure discloses SEQ ID N0:9.
  • FIG. 2 shows an exemplary AIK Type II Cas 3’ sgRNA scaffold and exemplary modifications that can be made to produce trimmed scaffolds.
  • Figure discloses SEQ ID NO:9.
  • FIGS. 3A-3B illustrate AIK Type II Cas PAM specificities.
  • FIG. 3A PAM sequence logo for AIK Type II Cas obtained from an in vitro PAM discovery assay.
  • FIG. 3B PAM enrichment heatmap for AIK Type II Cas showing the nucleotide preferences at position 5, 6, 7 and 8 of the PAM.
  • FIG. 4 is a schematic representation of an exemplary targeting strategy to correct the USH2A c.7595-2144A>G deep-intronic mutation using an AIK Type II Cas.
  • Two AIK Type II Cas sgRNAs, one upstream and one downstream the PE40 region, are used in combination to promote the deletion from the cellular genome of the PE40 pseudoexon sequence to avoid its incorporation into the mature USH2A mRNA and prevent loss of function frameshift mutations.
  • FIG. 5 is a schematic representation of the USH2A PE40 locus (corresponding to the genomic coordinates: chr1 :215890982-215891506 as reported by the UCSC Genome Browser, PE40 highlighted in light grey) with the guide RNAs evaluated in Example 2. The position of the c.7595-2144A>G mutation is also indicated.
  • Figure discloses SEQ ID NOS 188-208, 223, and 209-222, respectively, in order of appearance.
  • FIGS. 6A-6B show a schematic representation of the USH2A minigene model exploited in Example 2 to mimic USH2A splicing (FIG. 6A) and a representative agarose gel showing the splicing products detected by RT-PCR after transfection of HEK293 cells with the two minigenes used in Example 2 (FIG. 6B).
  • the minigene includes USH2A exon 40 and exon 41 , as well as the portion of intron 40 giving rise to the pseudoexon 40 (PE40) in the presence of the c.7595-2144A>G mutation.
  • the transcript produced by the mutated minigene is bigger due to the inclusion of PE40.
  • FIG. 7 shows editing activity of AIK Type II Cas guide RNAs targeting the USH2A PE40 region. Indel formation was evaluated after transient transfection of HEK293 stably expressing a single copy of a mutated USH2A minigene recapitulating the aberrant splicing induced by the c.7595-2144A>G mutation with AIK Type II Cas and selected sgRNAs targeting USH2A PE40 region, as indicated on the graph. Editing levels were measured using the TIDE webtool 3 days post-transfection. The dashed vertical line indicates which sgRNAs target the USH2A gene upstream of PE40 and which do downstream. Data are presented as mean ⁇ SEM for n>2 biologically independent runs.
  • FIG. 8 shows PE40 deletion using selected AIK sgRNA couples.
  • the formation of large deletions at the PE40 locus was evaluated using Sanger sequencing and the DECODR webtool after transient transfection of HEK293 cells stably expressing a single copy of the USH2A c.7595-2144A>G mutated minigene with AIK Type II Cas and the indicated sgRNA couples. Indels generated by single guide editing were not taken in consideration for the calculations. Data are presented as mean ⁇ SEM for n>2 biologically independent runs. [0024] FIGS.
  • FIG. 9A-9C shows correction of USH2A splicing after PE40 deletion using selected AIK sgRNA couples.
  • USH2A splicing was evaluated by RT-PCR 6 days after transient transfection of HEK293 cells stably expressing a single copy of the mutated c.7595-2144A>G USH2A minigene with AIK Type II Cas and the indicated sgRNA couples to induce PE40 deletion.
  • the splicing pattern of a wild-type (WT) USH2A minigene is also reported for comparison. Untreated minigene-expressing cells are reported as a negative control.
  • NTC No-Template Control.
  • FIG. 10 shows an exemplary AIK Type II Cas sgRNA scaffold (AIK Type II Cas sgRNA_v5) (SEQ ID NOU 3).
  • the scaffold is based on the AIK Type II Cas sgRNA_v4 scaffold and includes an additionally trimmed stem-loop (substitution with a GAAA tetraloop).
  • FIG. 11 shows a side-by-side comparison of indel formation by AIK Type II Cas and guide RNAs having the AIK Type II Cas sgRNA vl , AIK Type II Cas sgRNA_v4, or AIK Type II Cas sgRNA_v5 scaffold.
  • gRNA molecules e.g., sgRNAs
  • Type II Cas gRNA molecules for example AIK Type II Cas proteins
  • USH2A genes for example having the c.7595-2144A>G mutation.
  • the disclosure provides pluralities of gRNA molecules, for example comprising a first gRNA targeting USH2A upstream of the PE40 region and a second gRNA targeting USH2A downstream of the PE40 region.
  • Pluralities of gRNAs having a first gRNA targeting USH2A upstream of the PE40 region and a second gRNA targeting USH2A downstream of the PE40 region can be used, in combination with a Type II Cas, such as an AIK Type II Cas protein, to promote deletion of the PE40 sequence.
  • gRNAs of the disclosure Exemplary features of the gRNAs of the disclosure, pluralities of gRNAs of the disclosure, and Type II Cas proteins are described in Sections 6.2, 6.3, and 6.4.
  • the disclosure provides nucleic acids encoding gRNAs and pluralities of gRNAs of the disclosure, pluralities of nucleic acids and host cells comprising the nucleic acids (including pluralities of nucleic acids) of the disclosure. Exemplary features of the nucleic acids and host cells are described in Section 6.5.
  • the disclosure provides systems comprising a gRNA or plurality of gRNAs of the disclosure and a Type II Cas protein, for example, an AIK Type II Cas protein.
  • a Type II Cas protein for example, an AIK Type II Cas protein.
  • Exemplary systems are described in Section 6.6.
  • the disclosure provides particles and pluralities of particles containing gRNAs of the disclosure, pluralities of gRNAs of the disclosure, nucleic acids (including pluralities of nucleic acids) of the disclosure, systems of the disclosure, and combinations thereof.
  • the disclosure provides pharmaceutical compositions comprising the gRNAs, plurality of gRNAs, nucleic acids (including pluralities of nucleic acids), systems (including pluralities of systems), and particles (including pluralities of particles) of the disclosure.
  • the disclosure provides cells (e.g., from a subject having a USH2A gene with a pathogenic mutation) and populations of cells comprising the gRNAs (including pluralities of gRNAs), nucleic acids (including pluralities of nucleic acids), systems (including pluralities of systems), particles (including pluralities of particles), and pharmaceutical compositions of the disclosure.
  • Exemplary particles, pluralities of particles, cells, and populations of cells are described in Section 6.7.
  • Exemplary pharmaceutical compositions are described in Section 6.8.
  • the disclosure provides methods of using the gRNAs, pluralities of gRNAs, nucleic acids (including pluralities of nucleic acids), systems (including pluralities of sytems), particles (including pluralities of particles) of the disclosure, and pharmaceutical compositions for altering cells, for example a human cell having an USH2A gene with a pathogenic mutation, for example c.7595-2144A>G.
  • Methods of the disclosure can be used, for example, to treat subjects having a Usher syndrome type 2A. Exemplary methods of altering cells are described in Section 6.9.
  • an agent includes a plurality of agents, including mixtures thereof.
  • an “or” conjunction is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in the alternative (A or B, where the selection of A is mutually exclusive from B) and the selection of features in conjunction (A or B, where both A and B are selected).
  • the term “and/or” is used for the same purpose, which shall not be construed to imply that “or” is used with reference to mutually exclusive alternatives.
  • a Type II Cas protein refers to a wild-type or engineered Type II Cas protein. Engineered Type II Cas proteins can also be referred to as Type II Cas variants. For the avoidance of doubt, any disclosure pertaining to a “Type II Cas” or “Type II Cas protein” pertains to wild-type Type II Cas proteins and Type II Cas variants, unless the context dictates otherwise.
  • a Type II Cas protein can have nuclease activity or be catalytically inactive (e.g., as in a dCas).
  • the percentage identity between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between a pair of aligned sequences by 100, and dividing by the length of the aligned region. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another, nor does it consider substitutions or deletions as matches. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, by manual alignment or using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for achieving maximum alignment.
  • Guide RNA molecule refers to an RNA capable of forming a complex with a Type II Cas protein and which can direct the Type II Cas protein to a target DNA.
  • gRNAs typically comprise a spacer of 15 to 30 nucleotides in length.
  • gRNAs of the disclosure are in some embodiments single guide RNAs (sgRNAs), which typically comprise a spacer at the 5’ end of the molecule and a 3’ sgRNA scaffold.
  • sgRNAs single guide RNAs
  • 3’ sgRNA scaffolds are described in Section 6.2.
  • An sgRNA can in some embodiments comprise no uracil base at the 3’ end of the sgRNA sequence.
  • a sgRNA can comprise one or more uracil bases at the 3’ end of the sgRNA sequence.
  • a sgRNA can comprise 1 uracil (U) at the 3’ end of the sgRNA sequence, 2 uracil (UU) at the 3’ end of the sgRNA sequence, 3 uracil (UUU) at the 3’ end of the sgRNA sequence, 4 uracil (UUUU) at the 3’ end of the sgRNA sequence, 5 uracil (UUUUU) at the 3’ end of the sgRNA sequence, 6 uracil (UUUUU) at the 3’ end of the sgRNA sequence, 7 uracil (UUUUUU) at the 3’ end of the sgRNA sequence, or 8 uracil (UUUUUUUU) at the 3’ end of the sgRNA sequence.
  • uracil can be appended at the 3’ end of a sgRNA as terminators.
  • the 3’ sgRNA scaffolds set forth in Section 6.2 can be modified by adding or removing one or more uracils at the end of the sequence.
  • Peptide, protein, and polypeptide are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.
  • the amino acids may be natural or synthetic, and can contain chemical modifications such as disulfide bridges, substitution of radioisotopes, phosphorylation, substrate chelation (e.g., chelation of iron or copper atoms), glycosylation, acetylation, formylation, amidation, biotinylation, and a wide range of other modifications.
  • a polypeptide may be attached to other molecules, for instance molecules required for function.
  • polypeptides examples include, without limitation, cofactors, polynucleotides, lipids, metal ions, phosphate, etc.
  • polypeptides include peptide fragments, denatured/unstructured polypeptides, polypeptides having quaternary or aggregated structures, etc. There is expressly no requirement that a polypeptide must contain an intended function; a polypeptide can be functional, non-functional, function for unexpected/unintended purposes, or have unknown function.
  • a polypeptide is comprised of approximately twenty, standard naturally occurring amino acids, although natural and synthetic amino acids which are not members of the standard twenty amino acids may also be used.
  • the standard twenty amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine, (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Vai, V).
  • polypeptide sequence or “amino acid sequence” are an alphabetical representation of a polypeptide molecule.
  • Polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers and gRNAs.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine (T) when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • T thymine
  • nucleotide sequence is the alphabetical representation of a polynucleotide molecule.
  • the letters used in polynucleotide sequences described herein correspond to IUPAC notation.
  • nucleotide sequence represents a nucleotide which can be A, T, C, or G in a DNA sequence, or A, U, C, or G in a RNA sequence
  • the letter “R” in a nucleotide sequence represents a nucleotide which can be A or G
  • letter “V” in a nucleotide sequence represents a nucleotide which can be “A, C, or G.
  • Protospacer adjacent motif refers to a DNA sequence downstream (e.g., immediately downstream) of a target sequence on the non-target strand recognized by a Type II Cas protein.
  • a PAM sequence is located 3’ of the target sequence on the non-target strand.
  • Spacer refers to a region of a gRNA molecule which is partially or fully complementary to a target sequence found in the + or - strand of genomic DNA.
  • the gRNA directs the Type II Cas to the target sequence in the genomic DNA.
  • a spacer of a Type II Cas gRNA is typically 15 to 30 nucleotides in length (e.g., 20-25 nucleotides).
  • the nucleotide sequence of a spacer can be, but is not necessarily, fully complementary to the target sequence.
  • a spacer can contain one or more mismatches with a target sequence, e.g., the spacer can comprise one, two, or three mismatches with the target sequence.
  • a target sequence is considered upstream of pseudoexon 40 (PE40) when the target sequence is partially or fully upstream of the USH2A genomic sequence corresponding to PE40.
  • a target sequence is considered downstream of PE40 when the target sequence is partially or fully downstream of the USH2A genomic sequence corresponding to PE40. It should be understood that upstream and downstream are with reference to the + strand, irrespective of whether the target sequence is found in the + or - strand of genomic DNA.
  • treat, treating, treatment, and grammatical variations thereof as used herein include partially or completely delaying, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing, mitigating, and/or reducing the intensity or frequency of one or more diseases or conditions, symptoms of a disease or condition, or underlying causes of a disease or condition, for example Usher syndrome type 2A.
  • Treatments according to the disclosure may be applied prophylactically, palliatively or remedially.
  • Prophylactic treatments can be administered to a subject prior to onset of a symptom, during early onset of a symptom (e.g., upon initial signs and symptoms of Usher syndrome 2A), or after an established development of Usher syndrome type 2A.
  • Prophylactic administration can occur for several days to years prior to the manifestation of a symptom (e.g., prior to vision loss).
  • the terms treat, treating, treatment and grammatical variations thereof include reducing expression of non-functional usherin protein and/or increasing expression of functional usherin protein. Measurements of treatment can be compared with prior treatment(s) of the subject, inclusive of no treatment, or compared with the incidence of such symptom(s) in a general or study population.
  • the disclosure provides gRNA molecules for editing a human USH2A gene.
  • Guide RNA molecules of the disclosure are in some embodiments AIK Type II Cas gRNAs.
  • the gRNAs of the disclosure can be used with Type II Cas proteins, e.g., AIK Type II Cas gRNAs can be used with an AIK Type II Cas protein, to edit genomic DNA, for example mammalian DNA, e.g., human DNA.
  • gRNAs of the disclosure typically comprise a spacer of 15 to 30 nucleotides in length. The spacer can be positioned 5’ of a crRNA scaffold to form a full crRNA. The crRNA can be used with a tracrRNA to effect cleavage of a target genomic sequence.
  • An exemplary crRNA scaffold sequence that can be used for AIK Type II Cas gRNAs comprises GUCUUGAGCACGCGCCCUUCCCCAAGGUGAUACGCU (SEQ ID NO:7) and an exemplary tracrRNA sequence that can be used for AIK Type II Cas gRNAs comprises UCACCUUGGGGAAGGGCGCGGCUCCAGACAAGGGGAGCCACUUAAGUGGCUUACCCGUAAAGUA ACCCCCGUUCAAUCUUCGGAUUGGGCGGGGCGAACUUUUUU (SEQ ID NO:8).
  • gRNAs of the disclosure are in some embodiments single guide RNAs (sgRNAs), which typically comprise the spacer at the 5’ end of the molecule and a 3’ sgRNA scaffold.
  • gRNAs can comprise separate crRNA and tracrRNA molecules.
  • the spacer sequence is partially or fully complementary to a target sequence found in a genomic DNA sequence, for example a human USH2A sequence.
  • a spacer sequence can be partially or fully complementary to a nucleotide sequence in an USH2A gene having a disease-causing mutation, for example a c.7595-2144A>G mutation.
  • a spacer that is partially complementary to a target sequence can have, for example, one, two, or three mismatches with the target sequence.
  • gRNAs of the disclosure can comprise a spacer that is 15 to 30 nucleotides in length (e.g., 15 to 25, 16 to 24, 17 to 23, 18 to 22, 19 to 21 , 18 to 30, 20 to 28, 22 to 26, 22 to 25, or 23 to 25 nucleotides in length).
  • a spacer is 15 nucleotides in length.
  • a spacer is 16 nucleotides in length.
  • a spacer is 17 nucleotides in length.
  • a spacer is 18 nucleotides in length.
  • a spacer is 19 nucleotides in length.
  • a spacer is 20 nucleotides in length.
  • a spacer is 21 nucleotides in length. In other embodiments, a spacer is 22 nucleotides in length. In other embodiments, a spacer is 23 nucleotides in length. In other embodiments, a spacer is 24 nucleotides in length. In other embodiments, a spacer is 25 nucleotides in length. In other embodiments, a spacer is 26 nucleotides in length. In other embodiments, a spacer is 27 nucleotides in length. In other embodiments, a spacer is 28 nucleotides in length. In other embodiments, a spacer is 29 nucleotides in length. In other embodiments, a spacer is 30 nucleotides in length.
  • Type II Cas endonucleases require a specific sequence, called a protospacer adjacent motif (PAM) that is downstream (e.g., directly downstream) of the target sequence on the non-target strand.
  • PAM protospacer adjacent motif
  • spacer sequences for targeting a gene of interest can be identified by scanning the gene for PAM sequences recognized by the Type II Cas protein.
  • Exemplary PAM sequences for AIK Type II Cas proteins are shown in Table 1 .
  • a gRNA of the disclosure targeting USH2A has a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides from a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 16 or more consecutive nucleotides from a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 17 or more consecutive nucleotides from a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 18 or more consecutive nucleotides from a sequence shown in Table 2.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 19 or more consecutive nucleotides from a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 20 or more consecutive nucleotides from a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 21 or more consecutive nucleotides from a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 22 or more consecutive nucleotides from a sequence shown in Table 2.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 23 or more consecutive nucleotides from a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 or more consecutive nucleotides from a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 25 consecutive nucleotides from a sequence shown in Table 2.
  • a gRNA of the disclosure targeting USH2A has a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 16 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 17 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 18 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 19 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 20 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 21 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 22 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 23 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 or more consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 25 consecutive nucleotides from a sequence having one mismatch with a sequence shown in Table 2.
  • a gRNA of the disclosure targeting USH2A has a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 16 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 17 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 18 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 19 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 20 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 21 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 22 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 23 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 or more consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 25 consecutive nucleotides from a sequence having two mismatches with a sequence shown in Table 2.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises the full nucleotide sequence of a spacer sequence shown in Table 2. [0065] In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-01 . In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-02. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-03.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-04. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-05. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-06. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-07.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-07_23nt. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-08. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-09. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-10.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-11 . In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-12. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-13. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-14.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-15. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-15_23nt. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-16. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-17.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-18. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-19. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-20. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-21 .
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-22. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-23. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-24. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-25.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-26. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-27. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-27_22nt. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-27_23nt.
  • a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-28. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-29. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises or consists of the nucleotide sequence of USH-29_22nt.
  • gRNAs of the disclosure can be single-guide RNA (sgRNA) molecules.
  • a sgRNA can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence and an optional tracrRNA extension sequence.
  • the optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA.
  • the single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure.
  • the optional tracrRNA extension can comprise one or more hairpins.
  • the sgRNA can comprise a variable length spacer sequence (e.g., 15 to 30 nucleotides) at the 5’ end of the sgRNA sequence and a 3’ sgRNA segment.
  • Type II Cas gRNAs typically comprise a repeat-antirepeat duplex and/or one or more stem-loops generated by the gRNA’s secondary structure.
  • the length of the repeat-antirepeat duplex and/or one or more stem-loops can be modified in order to modulate (e.g., increase) the editing efficacy of a Type II Cas nuclease, and/or to reduce the size of a guide RNA for easier vectorization in situations in which the cargo size of the vector is limiting (e.g., AAV vectors).
  • the repeat-antirepeat duplex (which in a sgRNA is fused through a synthetic linker to become an additional stem loop in the structure) can be trimmed at different lengths without generally having detrimental effects on nuclease function and in some cases even producing increased enzymatic activity. If bulges are present within this duplex they generally should be retained in the final guide RNA sequence.
  • RNA folding can be obtained by introducing targeted base changes into the stems of the gRNA to increase their stability and folding.
  • Such base changes will preferably correspond to the introduction of G:C couples, which are known to generate the strongest Watson-Crick pairing.
  • these substitutions can consist in the introduction of a G or a C in a specific position of a stem together with a complementary substitution in another position of the gRNA sequence which is predicted to base pair with the former, for example according to available bioinformatic tools for RNA folding such as UNAfold or RNAfold.
  • Stem-loop trimming can also be exploited to stabilize desired secondary structures by removing portions of the guide RNA producing unwanted secondary structures through annealing with other regions of the RNA molecule.
  • FIG. 1 The predicted hairpin structure of an exemplary AIK Type II Cas gRNA scaffold is shown in FIG. 1 , while examples of modifications to that can be introduced in gRNA scaffolds to make trimmed scaffolds are illustrated in FIG. 2.
  • bases 15-50 of the gRNA (which includes the GAAA tetraloop) can be substituted with a GAAA tetraloop to make a trimmed scaffold.
  • the sgRNA (e.g., for use with AIK Type II Cas proteins) can comprise no uracil base at the 3’ end of the sgRNA sequence.
  • the sgRNA comprises one or more uracil bases at the 3’ end of the sgRNA sequence, for example to promote correct sgRNA folding.
  • the sgRNA can comprise 1 uracil (U) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 2 uracil (UU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 3 uracil (UUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 4 uracil (UUUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 5 uracil (UUUUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 6 uracil (UUUUUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 7 uracil (UUUUUUU) at the 3’ end of the sgRNA sequence.
  • the sgRNA can comprise 8 uracil (UUUUUUUU) at the 3’ end of the sgRNA sequence.
  • uracil can be appended at the 3’end of a sgRNA as terminators.
  • the 3’ sgRNA sequences set forth in Table 3 can be modified by adding one or more uracils at the end of the sequence.
  • a sgRNA scaffold for use with an AIK Type II Cas protein comprises the sequence GUCUUGAGCACGCGCCCUUCCCCAAGGUGAGAAAUCACCUUGGGGAAGGGCGCGGCUCCAGACA AGGGGAGCCACUUAAGUGGCUUACCCGUAAAGUAACCCCCGUUCAAUCUUCGGAUUGGGCGGGG
  • a sgRNA scaffold for use with an AIK Type II Cas protein comprises the sequence GUCUUGAGCACGCGCCCUUCCGCAAGGUGAGAAAUCACCUUGCGGAAGGGCGCGGCUCCAGACA AGCGGAGCCACUUAAGUGGCUUACGCGUAAAGUAACCGCCGUUCAAUCUUCGGAUUGGGCGGCG CGAACUUUUUU (SEQ ID NQ:10).
  • a sgRNA scaffold for use with an AIK Type II Cas protein comprises the sequence GUCUUGAGCACGCGAAAGCGGCUCCAGACAAGGGGAGCCACUUAAGUGGCUUACCCGUAAAGUA ACCCCCGUUCAAUCUUCGGAUUGGGCGGGGCGAACUUUUUU (SEQ ID NO:11 ).
  • a sgRNA scaffold for use with an AIK Type II Cas protein comprises the sequence GUCUUGAGCACGCGAAAGCGGCUCCAGACAAGCGGAGCCACUUAAGUGGCUUACGCGUAAAGUA ACCGCCGUUCAAUCUUCGGAUUGGGCGGCGCGAACUUUUUU (SEQ ID NO:12).
  • a sgRNA scaffold for use with an AIK Type II Cas protein comprises the sequence GUCUUGAGCACGCGAAAGCGGCUCCAGACAAGCGGAGCCACUUAAGUGGCUUACGCGUAAAGUA ACCGCCGAAAGGCGCGAACUUUUUU (SEQ ID NO:13).
  • Guide RNAs can be readily synthesized by chemical means, enabling a number of modifications to be readily incorporated, as described in the art.
  • the disclosed gRNA (e.g., sgRNA) molecules can be unmodified or can contain any one or more of an array of chemical modifications.
  • RNAs While chemical synthetic procedures are continually expanding, purifications 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 as polynucleotide lengths increase significantly beyond a hundred or so nucleotides.
  • HPLC high-performance liquid chromatography
  • One approach that can be used for generating chemically modified RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Type II Cas endonuclease, are more readily generated enzymatically.
  • RNAs While fewer types of modifications are available for use in enzymatically produced RNAs, there are still modifications that can be used to, for instance, enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described herein and in the art.
  • modifications can comprise one or more nucleotides modified at the 2' position of the sugar, for instance a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro-modified nucleotide.
  • RNA modifications can comprise 2'-fluoro, 2'-amino or 2'-O-methyl modifications on the ribose of pyrimidines, abasic residues, or an inverted base at the 3' end of the RNA.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2-NH-O-CH2, CH, ⁇ N(CH 3 )-O-CH2 (known as a methylene(methylimino) or MMI backbone), CH2-O-N (CH 3 )-CH 2 , CH 2 -N (CH 3 )-N (CH 3 )-CH 2 and O-N (CH 3 )- CH 2 -CH 2 backbones, wherein the native phosphodiester backbone is represented as O- P- O- CH,); amide backbones (see De Mesmaeker et al. 1995, Ace. Chem.
  • morpholino backbone structures see U.S. Patent No. 5,034,506
  • PNA peptide nucleic acid
  • Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2' ; see U.S.
  • Morpholino-based oligomeric compounds are described in Braasch and David Corey, 2002, Biochemistry, 41 (14):4503-4510; Genesis, Volume 30, Issue 3, (2001 ); Heasman, 2002, Dev. Biol., 243: 209-214; Nasevicius et al., 2000, Nat. Genet., 26:216-220; Lacerra et al., 2000, Proc. Natl. Acad. Sci., 97: 9591 -9596; and U.S. Patent No. 5,034,506.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component parts; see U.S. Patent Nos.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 , OCH 3 O(CH 2 )n CH 3 , O(CH 2 )n NH 2 , or O(CH 2 )n CH 3 , where n is from 1 to about 10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-, S-, or bi- alkyl; O-, S-, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group
  • a modification includes 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl)) (Martin et al., 1995, Helv. Chim. Acta, 78, 486).
  • Other modifications include 2'-methoxy (2'-O-CH 3 ), 2'-propoxy (2'- OCH 2 CH 2 CH 3 ) and 2'-fluoro (2'- F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides can also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group.
  • both a sugar and an internucleoside linkage (in the backbone) of the nucleotide units can be replaced with novel groups.
  • the base units can be maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar- backbone of an oligonucleotide can be replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases can be retained and bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • RNAs such as guide RNAs can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5- methylcytosine (also referred to as 5-methyl-2' deoxy cytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino) adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino) adenine or other heterosub stituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexy
  • Modified nucleobases can comprise other synthetic and natural nucleobases, such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8- thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluor
  • nucleobases can comprise those disclosed in U.S. Patent No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science and Engineering', 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition', 1991 , 30, p. 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications', 289-302, Crooke, S.T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases can be useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by about 0.6-1 .2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds, 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, 276-278) and are aspects of base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • a modified gRNA can include, for example, one or more non-natural sugars, internucleotide linkages and/or bases. It is not necessary for all positions in a given gRNA to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single oligonucleotide, or even in a single nucleoside within an oligonucleotide.
  • the guide RNAs and/or mRNA (or DNA) encoding an endonuclease can be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • moieties comprise, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al. 1989, Proc. Natl. Acad. Sci. USA, 86: 6553-6556); cholic acid (Manoharan et al, 1994, Bioorg. Med. Chem.
  • a thioether e.g., hexyl-S- tritylthiol
  • a thiocholesterol Olet al., 1992, Nucl.
  • Acids Res., 20: 533-538 an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al, 1990, FEBS Lett., 259: 327-330; Svinarchuk etal, 1993, Biochimie, 75: 49- 54); a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate (Manoharan etal., 1995, Tetrahedron Lett., 36: 3651 -3654; and Shea et al, 1990, Nucl.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O- hexadecyl-rac-gly
  • Acids Res., 18: 3777-3783 a polyamine or a polyethylene glycol chain (Mancharan et al, 1995, Nucleosides & Nucleotides, 14: 969-973); adamantane acetic acid (Manoharan et al, 1995, Tetrahedron Lett., 36: 3651 -3654); a palm ityl moiety (Mishra et al., 1995, Biochim. Biophys. Acta, 1264: 229- 237); or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al, 1996, J. Pharmacol. Exp.
  • Sugars and other moieties can be used to target proteins and complexes comprising nucleotides, such as cationic polysomes and liposomes, to particular sites.
  • nucleotides such as cationic polysomes and liposomes
  • hepatic cell directed transfer can be mediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, et al., 2014, Protein Pept Lett. 21 (10):1025-30.
  • ASGPRs asialoglycoprotein receptors
  • Other systems known in the art and regularly developed can be used to target biomolecules of use in the present case and/or complexes thereof to particular target cells of interest.
  • Targeting moieties or conjugates can include conjugate groups covalently bound to functional groups, such as primary or secondary hydroxyl groups.
  • Conjugate groups of the present disclosure include intercalators, reporter molecules, 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 cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present disclosure. Representative conjugate groups are disclosed in International Patent Application Publication WO1993007883, and U.S. Patent No. 6,287,860.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5 -trityl thiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1 ,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl- oxy cholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a
  • the disclosure provides pluralities of two or more different gRNAs (e.g., two different gRNAs) of the disclosure.
  • a plurality of gRNAs can comprise a first gRNA (e.g., sgRNA) targeting an USH2A gene upstream of the PE40 sequence and a second gRNA (e.g., sgRNA) targeting an USH2A gene downstream of the PE40 sequence.
  • a Type II Cas protein can promote deletion of the PE40 sequence from the USH2A gene. Editing the USH2A gene in this manner can result in expression of functional usherin protein, thereby promoting establishment of the correct physiological state of edited cells (e.g., photoreceptors) and tissues and organs containing them (e.g., eyes).
  • Pluralities of gRNAs of the disclosure can comprise two or more different gRNAs described in Section 6.2.
  • the nucleotide sequence of the spacer of the first gRNA can comprise 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of a spacer sequence identified in Table 2 as corresponding to a target sequence upstream of the PE40 sequence
  • the nucleotide sequence of the spacer of the second gRNA can comprise 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of a spacer sequence identified in Table 2 as corresponding to a target sequence downstream of the PE40 sequence.
  • the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GCUUUCAACCAUGCAGUUGCAGGCC (SEQ ID NOU 4). In other embodiments, the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17,
  • the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24) consecutive nucleotides of GCCAGUUGAUUUGUAUAUAGAAUU (SEQ ID NOU 5). In other embodiments, the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17,
  • the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GCUGACUCCUGGCACUCUUUCAAUU (SEQ ID NOU 7). In other embodiments, the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17,
  • the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GUUAAAGCACUAAAUUGAAAGAGUG (SEQ ID NOU 9). In other embodiments, the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GACUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NO:20).
  • the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24) consecutive nucleotides of GCUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NO:21 ). In other embodiments, the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17,
  • the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GUUCCCUAGCCAAAGGAGCUAAUUA (SEQ ID NO:23). In other embodiments, the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17,
  • the spacer of the first gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, or 23) consecutive nucleotides of GUGAUUCUGGAGAGGAAGCUGAA (SEQ ID NO:25).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17,
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17,
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GGUAAGAGAUCAUCUUUAAGAAAA (SEQ ID NO:27).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GCUUGCACUUCAAACCCCCACAAUA (SEQ ID NO:28).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GCUUUAAGAAAAGGCUGUGUAUUGU (SEQ ID NO:29).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GUUUAAGAAAAGGCUGUGUAUUGU (SEQ ID NQ:30). In other embodiments, the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24) consecutive nucleotides of GGCUGUGUAUUGUGGGGGUUUGAA (SEQ ID NO:31 ).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24) consecutive nucleotides of GUAUUGUGGGGGUUUGAAGUGCAA (SEQ ID NO:32). In other embodiments, the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, or 23) consecutive nucleotides of GUGCAAGUUCAUCUCAUUAUCAU (SEQ ID NO:33).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, or 23) consecutive nucleotides of GGAUGUUUCACCCAUAAUACUAU (SEQ ID NO:34). In other embodiments, the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24) consecutive nucleotides of GCUUUUAUUUCUCCUGCAUAUGAU (SEQ ID NO:35).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, or 23) consecutive nucleotides of GGGGGAUAAGGCUUUUAUUUCUC (SEQ ID NO:36). In other embodiments, the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GAUUGGGGGAUAAGGCUUUUAUUUC (SEQ ID NO:37).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GACUAUCAUCAUAUGCAGGAGAAAU (SEQ ID NO:38). In other embodiments, the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17,
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17,
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GUUCAGGGAAAAGAAACAACAAUGA (SEQ ID NO:41 ). In other embodiments, the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) consecutive nucleotides of GUUGUUUCUUUUCCCUGAAGAUGCU (SEQ ID NO:42).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24) consecutive nucleotides of GUGUUUCUUUUCCCUGAAGAUGCU (SEQ ID NO:44). In other embodiments, the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, or 23) consecutive nucleotides of GAAGAUGCUCACAGULJUGAUAGU (SEQ ID NO:45).
  • the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24) consecutive nucleotides of GCUCACAGUUUGAUAGUUCACAUA (SEQ ID NO:46). In other embodiments, the spacer of the second gRNA comprises 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, or 23) consecutive nucleotides of GUCACAGUUUGAUAGUUCACAUA (SEQ ID NO:47).
  • the spacer of the first gRNA has a nucleotide sequence comprising GCCAGUUGAUUUGUAUAUAGAAUU (SEQ ID NO:15) or 15 or more consecutive nucleotides thereof
  • the spacer of the second gRNA has a nucleotide sequence comprising GUGCAAGUUCAUCUCAUUAUCAU (SEQ ID NO:33) or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GAUUUGUAUAUAGAAUUAGAUGA (SEQ ID NO:16) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising GUGCAAGUUCAUCUCAUUAUCAU (SEQ ID NO:33) or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GCUUAAUUAGCUCCUUUGGCUAG (SEQ ID NO:22) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising GUGCAAGUUCAUCUCAUUAUCAU (SEQ ID NO:33) or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GUGAUUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising GGUAAGAGAUCAUCUUUAAGAAAA (SEQ ID NO:27) or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GUGAUUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising GUGCAAGUUCAUCUCAUUAUCAU (SEQ ID NO:33) or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GUGAUUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising GUUUGUUCUCUGUGAUUGGGGGA (SEQ ID NO:39) or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GUGALJUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising
  • GCUUUAAGAAAAGGCUGUGUAUUGU SEQ ID NO:29 or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GUGALJUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising
  • GAUGAUCUCUUACCUUGGGAAAGG SEQ ID NO:26 or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GUGAUUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising
  • GCUUUUAUUUCUCCUGCAUAUGAU (SEQ ID NO:35) or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GUGAUUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising
  • GUUGUUUCUUUUCCCUGAAGAUGCU SEQ ID NO:42 or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GUGAUUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising
  • the spacer of the first gRNA has a nucleotide sequence comprising GACUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NQ:20) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising
  • GCUUUAAGAAAAGGCUGUGUAUUGU SEQ ID NO:29 or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GACUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NQ:20) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising
  • GUGCAAGUUCAUCUCAUUAUCAU SEQ ID NO:33 or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GACUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NQ:20) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising
  • GAUGAUCUCUUACCUUGGGAAAGG SEQ ID NO:26 or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GACUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NQ:20) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising
  • GUUGUUUCUUUUCCCUGAAGAUGCU SEQ ID NO:42 or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising GACUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NQ:20) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising GCUCACAGUUUGAUAGUUCACAUA (SEQ ID NO:46) or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising gCUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NO:21 ) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising GUUGUUUCUUUUCCCUGAAGAUGCU (SEQ ID NO:42) or 15 or more consecutive nucleotides thereof.
  • the spacer of the first gRNA has a nucleotide sequence comprising gCUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NO:21 ) or 15 or more consecutive nucleotides thereof and the spacer of the second gRNA has a nucleotide sequence comprising GCUCACAGUUUGAUAGUUCACAUA (SEQ ID NO:46) or 15 or more consecutive nucleotides thereof.
  • a plurality of gRNAs of the disclosure comprises first and second gRNAs whose spacers comprise the spacer sequences of a pair of gRNAs identified in FIG. 8 or FIGS. 9A-9C.
  • a plurality of gRNAs of the disclosure comprises first and second gRNAs whose full nucleotide sequences comprise the full nucleotide sequences of a pair of gRNAs identified in FIG. 8 or FIGS. 9A-9C.
  • Type II Cas proteins known in the art include Streptococcus pyogenes Cas9 (SpCas9) (NP_269215 (NCBI)), and orthologues thereof, for example S. thermophilus, S. aureus, and N. meningitides Cas9 proteins.
  • SpCas9 Streptococcus pyogenes Cas9
  • NP_269215 NCBI
  • orthologues thereof for example S. thermophilus, S. aureus, and N. meningitides Cas9 proteins.
  • the gRNAs described herein can in some embodiments be used with an AIK Type II Cas protein.
  • Type II Cas proteins can be in the form of fusion proteins and, unless required otherwise by context, disclosures relating to Type II Cas proteins encompass Type II Cas proteins which are not fusion proteins and Type II Cas proteins which are in the form of fusion proteins (e.g., Type II Cas protein comprising one or more nuclear localization signals and/or one or more tags).
  • AIK Type II Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NOU .
  • the AIK Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOU .
  • an AIK Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NOU .
  • Further exemplary AIK Type II Cas protein sequences are set forth in SEQ ID NO:2 and SEQ ID NO:3.
  • AIK Type II Cas protein sequences and nucleotide sequences encoding exemplary AIK proteins are set forth in Table 4.
  • AIK Type II Cas protein can be in the form of fusion proteins comprising a Type II Cas protein sequence fused with one or more additional amino acid sequences, such as one or more a nuclear localization signals and/or one or more non-native tags. Fusion proteins can also comprise an amino acid sequence of a transcriptional activator, a transcriptional repressor, a histone-modifying protein, an integrase, or a recombinase.
  • Non-limiting examples of nuclear localization signals include KRTADGSEFESPKKKRKV (SEQ ID NO:48), PKKKRKV (SEQ ID NO:49), PKKKRRV (SEQ ID NQ:50), KRPAATKKAGQAKKKK (SEQ ID NO:51 ), YGRKKRRQRRR (SEQ ID NO:52), RKKRRQRRR (SEQ ID NO:53), PAAKRVKLD (SEQ ID NO:54), RQRRNELKRSP (SEQ ID NO:55), VSRKRPRP (SEQ ID NO:56), PPKKARED (SEQ ID NO:57), PQPKKKPL (SEQ ID NO:58), SALIKKKKKMAP (SEQ ID NO:59), PKQKKRK (SEQ ID NQ:60), RKLKKKIKKL (SEQ ID NO:61 ), REKKKFLKRR (SEQ ID NO:62), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:63),
  • Exemplary fusion partners include protein tags (e.g., V5-tag (e.g., having the sequence GKPIPNPLLGLDST (SEQ ID NO : 67), FLAG-tag, myc-tag, HA-tag, GST-tag, polyHis-tag, MBP-tag), protein domains, transcription modulators, enzymes acting on small molecule substrates, DNA, RNA and protein modification enzymes (e.g., adenosine deaminase, cytidine deaminase, guanosyl transferase, DNA methyltransferase, RNA methyltransferases, DNA demethylases, RNA demethylases, dioxygenases, polyadenylate polymerases, pseudouridine synthases, acetyltransferases, deacetylase, ubiquitin-ligases, deubiquitinases, kinases, phosphatases, NEDD8-ligases, de-NEDD
  • a fusion protein comprises one or more nuclear localization signals positioned N-terminal and/or C-terminal to an AIK Type II Cas protein sequence.
  • a fusion protein of the disclosure comprises an N-terminal and a C-terminal nuclear localization signal, for example each having the sequence KRTADGSEFESPKKKRKV (SEQ ID NO:48).
  • the Type II Cas protein can be a chimeric Type II Cas protein comprising one or more domains of an AIK Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins).
  • the domain structures of wild-type AIK Cas proteins were inferred by multiple alignment with the amino acid sequences of Type II Cas proteins for which the crystal structure is known and for which it is thus possible to define the boundaries of each functional domain.
  • the domains identified in Type II Cas proteins are: the RuvC catalytic domain (discontinuous, represented by RuvC-l, RuvC-ll, and RuvC-ll I domains), bridge helix (BH), recognition (REC) domain, HNH catalytic domain, wedge (WED) domain, and PAM-interacting domain (PID).
  • a chimeric Type II Cas protein can comprise one of more of the following domains (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more) from an AIK Type II Cas protein and one or more domains from one or more other proteins, for example SaCas9, SpCas9 or a Type II Cas protein described in US 2020/0332273, US 2019/0169648, or 2015/0247150 (the contents of each of which are incorporated herein by reference in their entirety): RuvC-l, BH, REC, RuvC-ll, HNH, RuvC-ll I, WED, PID.
  • the PID domain can be swapped between different Type II Cas proteins to change the PAM specificity of the resulting chimeric protein (which is given by the donor PID domain). Swapping of other domains or portions of them is also within the scope of the disclosure (e.g., through protein shuffling).
  • a Type II Cas protein comprises one, two, three, four, five, six, seven, or eight of a RuvC-l domain, a BH domain, a REC domain, a RuvC-ll domain, a HNH domain, a RuvC-l 11 domain, a WED domain, and a PID domain arranged in the N-terminal to C-terminal direction.
  • all domains are from an AIK Type II Cas protein (e.g., an AIK Type II Cas protein whose amino acid sequence comprises SEQ ID NO:1 , 2, or 3).
  • one or more domains e.g., one domain
  • a PID domain is from another Type II Cas protein.
  • the disclosure provides nucleic acids (e.g., DNA or RNA) encoding the gRNAs of the disclosure and pluralities of gRNAs of the disclosure, and pluralities of nucleic acids, for example comprising one or more nucleic acids encoding a gRNA or more than one gRNA (e.g., two different gRNAs encoded by a single nucleic acid or different nucleic acids) and a nucleic acid encoding a Type II Cas protein.
  • nucleic acids e.g., DNA or RNA
  • nucleic acids e.g., DNA or RNA
  • pluralities of nucleic acids for example comprising one or more nucleic acids encoding a gRNA or more than one gRNA (e.g., two different gRNAs encoded by a single nucleic acid or different nucleic acids) and a nucleic acid encoding a Type II Cas protein.
  • a nucleic acid encoding one or more gRNAs and/or a Type II Cas protein can be, for example, a plasmid or a viral genome (e.g., a lentivirus, retrovirus, adenovirus, or adeno-associated virus genome).
  • Plasmids can be, for example, plasmids for producing virus particles, e.g., lentivirus particles, or plasmids for propagating gRNA and/or Type II Cas coding sequences in bacterial (e.g., E. cell) or eukaryotic (e.g., yeast) cells.
  • a nucleic acid encoding a gRNA can, in some embodiments, further encode a Type II Cas protein, e.g., an AIK Type II Cas described in Section 6.4.
  • a Type II Cas can be encoded by a separate nucleic acid (e.g., DNA or mRNA).
  • Nucleic acids encoding a Type II Cas protein can be codon optimized, e.g., where at least one non-common codon or less-common codon has been replaced by a codon that is common in a host cell.
  • a codon optimized nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system.
  • a human codon-optimized polynucleotide encoding Type II Cas can be used for producing a Type II Cas polypeptide. Exemplary codon-optimized AIK Type II Cas sequences are shown in Table 4.
  • Nucleic acids of the disclosure can comprise one or more regulatory elements such as promoters, enhancers, and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • regulatory elements e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences.
  • Such regulatory elements are described, for example, in Goeddel, 1990, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissuespecific regulatory sequences).
  • a tissue-specific promoter may direct expression primarily in a desired tissue of interest or in particular cell types. Regulatory elements may also direct expression in a temporaldependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
  • a nucleic acid of the disclosure comprises one or more pol III promoter (e.g., 1 , 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1 , 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1 , 2, 3, 4, 5, or more pol I promoters), or combinations thereof, e.g., to express a Type II Cas protein and a gRNA separately.
  • pol III promoters include, but are not limited to, U6 and H1 promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous Sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the CAG promoter, the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, 1985, Cell 41 :521 -530), the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and EF1a promoters (for example, full length EF1a promoter and the EFS promoter, which is a short, intron-less form of the full EF1 a promoter).
  • RSV Rous Sarcoma virus
  • CAG CAG promoter
  • CMV cytomegalovirus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • Exemplary enhancer elements include WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I; SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit p-globin. It will be appreciated by those skilled in the art that the design of an expression vector can depend on such factors as the choice of the host cell, the level of expression desired, etc.
  • vector refers to a polynucleotide molecule capable of transporting another nucleic acid to which it has been linked.
  • polynucleotide vector includes a "plasmid”, which refers to a circular double-stranded DNA loop into which additional nucleic acid segments are or can be ligated.
  • plasmid refers to a circular double-stranded DNA loop into which additional nucleic acid segments are or can be ligated.
  • viral vector Another type of polynucleotide vector; wherein additional nucleic acid segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors can be capable of directing the expression of nucleic acids to which they are operably linked. Such vectors can be referred to herein as “recombinant expression vectors", or more simply “expression vectors”, which serve equivalent functions.
  • operably linked means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence.
  • regulatory sequence is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and 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 constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence 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 can depend on such factors as the choice of the target cell, the level of expression desired, and the like.
  • Vectors can include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus (e.g., AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, AAVrhI O), SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors.
  • retrovirus e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sar
  • vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXTI, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pCTx-l, pCTx-2, and pCTx-3. Other vectors can be used so long as they are compatible with the host cell.
  • a vector can comprise one or more transcription and/or translation control elements.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector.
  • the vector can be a selfinactivating vector that either inactivates the viral sequences or the components of the CRISPR machinery or other elements.
  • Non-limiting examples of suitable eukaryotic promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-l promoters (for example, the full EF1a promoter and the EFS promoter), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-l.
  • CMV cytomegalovirus
  • HSV herpes simplex virus
  • LTRs long terminal repeats
  • human elongation factor-l promoters for example, the full EF1a promoter and the EFS promoter
  • CAG chicken beta-actin promoter
  • MSCV murine stem
  • An expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector can also comprise appropriate sequences for amplifying expression.
  • the expression vector can also include nucleotide sequences encoding non-native tags (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed polypeptide, thus resulting in a fusion protein.
  • a promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline- regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.).
  • the promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter).
  • the promoter can be a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, for example a human RHO promoter or human rhodopsin kinase promoter (hGRK), a cell type specific promoter, etc.).
  • the disclosure also provides a host cell comprising a nucleic acid of the disclosure.
  • host cells can be used, for example, to produce virus particles encoding one or more gRNAs of the disclosure and, optionally, a DNA endonuclease such as a Type II Cas protein.
  • host cells are used to produce virus particles encoding one or more gRNAs (but no Type II Cas protein) and to produce virus particles encoding a Type II Cas protein (but no gRNA).
  • the virus particles encoding one or more gRNAs and the virus particles encoding a Type II Cas can then be used together to deliver the one or more gRNAs and Type II Cas protein to a cell (e.g., by combining the virus particles into a single composition which is then contacted with the cell or by separately contacting the cell with the different virus particles).
  • Host cells can also be used to make vesicles containing one or more gRNAs and, optionally, a DNA endonuclease such as a Type II Cas protein (e.g., as described in Montagna et al., 2018, Molecular Therapy: Nucleic Acids, 12:453-462).
  • Exemplary host cells include eukaryotic cells, e.g., mammalian cells.
  • Exemplary mammalian host cells include human cell lines such as BHK-21 , BSRT7/5, VERO, WI38, MRC5, A549, HEK293, HEK293T, Caco-2, B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, and HT 1080 cell lines.
  • Host cells can be engineered host cells, for example, host cells engineered to express a DNA binding protein such a repressor (e.g., TetR), to regulate virus or vesicle production (see Petris et al., 2017, Nature Communications, 8:15334).
  • a repressor e.g., TetR
  • Host cells can also be used to propagate the gRNA coding sequences of the disclosure.
  • the host cell can be a eukaryote or prokaryote and includes, for example, yeast (such as Pichia pastoris or Saccharomyces cerevisiae), bacteria (such as E. coll or Bacillus subtilis), insect Sf9 cells (such as baculovirus-infected SF9 cells) or mammalian cells (such as Human Embryonic Kidney (HEK) cells, Chinese hamster ovary cells, HeLa cells, human 293 cells and monkey COS-7 cells).
  • yeast such as Pichia pastoris or Saccharomyces cerevisiae
  • bacteria such as E. coll or Bacillus subtilis
  • insect Sf9 cells such as baculovirus-infected SF9 cells
  • mammalian cells such as Human Embryonic Kidney (HEK) cells, Chinese hamster ovary cells, HeLa cells, human 293 cells and monkey CO
  • the disclosure provides systems comprising a gRNA of the disclosure (e.g., as described in Section 6.2) or a plurality of gRNAs of the disclosure (e.g., as described in Section 6.3) and a Type II Cas protein molecule(s) (e.g., as described in Section 6.4).
  • the systems can comprise a ribonucleoprotein particle (RNP) in which a Type II Cas protein is complexed with a gRNA, for example a sgRNA or separate crRNA and tracrRNA.
  • RNP ribonucleoprotein particle
  • Systems of the disclosure can in some embodiments further comprise genomic DNA complexed with the Type II Cas protein and a gRNA.
  • the disclosure provides systems comprising a Type II Cas protein, a genomic DNA, and gRNA, all complexed with one another.
  • Pluralities of systems e.g., a plurality comprising a system comprising a first gRNA and a system comprising a second, different gRNA are also within the scope of the disclosure.
  • the systems (including pluralities of systems) of the disclosure can exist within a cell (whether the cell is in vivo, ex vivo, or in vitro) or outside a cell (e.g., in a particle our outside of a particle).
  • the disclosure further provides particles comprising a gRNA or plurality of gRNAs of the disclosure and a Type II Cas protein (e.g., an AIK Type II Cas protein), particles comprising a gRNA or plurality of gRNAs of the disclosure (e.g., without a Type II Cas protein), particles comprising a system or pluralities of systems of the disclosure, and particles comprising a nucleic acid or plurality of nucleic acids of the disclosure.
  • the particles can comprise a RNP or a combination of RNPs (e.g., a first RNP comprising a first gRNA and a second RNP comprising a second, different gRNA).
  • Exemplary particles include lipid nanoparticles, vesicles, viral-like particles (VLPs) and gold nanoparticles. See, e.g., WO 2020/012335, the contents of which are incorporated herein by reference in their entireties, which describes vesicles that can be used to deliver gRNA molecules and Type II Cas proteins to cells (e.g., complexed together as a RNP).
  • VLPs viral-like particles
  • gold nanoparticles See, e.g., WO 2020/012335, the contents of which are incorporated herein by reference in their entireties, which describes vesicles that can be used to deliver gRNA molecules and Type II Cas proteins to cells (e.g., complexed together as a RNP).
  • the disclosure provides particles (e.g., virus particles) comprising a nucleic acid encoding a gRNA or plurality of gRNAs of the disclosure.
  • the particles can further comprise a nucleic acid encoding a Type II Cas protein.
  • the nucleic acid encoding the gRNA or plurality of gRNAs can further encode the Type II Cas protein.
  • the disclosure further provides pluralities of particles (e.g., pluralities of virus particles).
  • Such pluralities can include, for example, a particle encoding one or more gRNAs (e.g., two), and a different particle encoding a Type II Cas.
  • a plurality can include a particle encoding a first gRNA, and a different particle encoding a second, different gRNA.
  • a plurality of particles can comprise, for example, a virus particle (e.g., a AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhI O virus particle) encoding a Type II Cas protein and a second virus particle (e.g., a AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhl 0 virus particle) encoding one or more gRNAs.
  • a plurality of particles can comprise a plurality of virus particles where each particle encodes one or more gRNAs and a Type II Cas protein.
  • the disclosure further provides cells and populations of cells (e.g., ex vivo cells and populations of cells, for example comprising 10 or more, 50 or more 100 or more, 1 ,000 or more, or 100,000 thousand or more cells) that can comprise a gRNA, plurality of gRNAs, nucleic acid, or plurality of nucleic acids encoding the gRNAs.
  • Such cells and populations can further comprise a Type II Cas protein or a nucleic acid encoding the Type II Cas protein (e.g., DNA or mRNA).
  • such cells and populations are isolated, e.g., isolated from cells not containing the gRNA(s).
  • the cells and populations of cells can be, for example, human cells such as a stem cell, e.g., a hematopoietic stem cell (HSC), a pluripotent stem cell, an induced pluripotent stem cell (iPS) , or an embryonic stem cell.
  • a stem cell e.g., a hematopoietic stem cell (HSC), a pluripotent stem cell, an induced pluripotent stem cell (iPS) , or an embryonic stem cell.
  • HSC hematopoietic stem cell
  • iPS induced pluripotent stem cell
  • embryonic stem cell embryonic stem cell.
  • Methods for introducing proteins and nucleic acids to cells are known in the art.
  • a RNP can be produced by mixing a Type II Cas protein and one or more guide RNAs in an appropriate buffer.
  • An RNP can be introduced to a cell, for example, via electroporation and other methods known in the art.
  • the cell populations of the disclosure can be cells in which gene editing by the systems of the disclosure has taken place, or cells in which the components of a system of the disclosure have been introduced or expressed but gene editing has not taken place, or a combination thereof.
  • a cell population can comprise, for example, a population in which at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cells have undergone gene editing by a system of the disclosure.
  • compositions and medicaments comprising a gRNA or plurality of gRNAs, Type II Cas protein, nucleic acid or plurality of nucleic acids, system, plurality of systems, particle, or plurality of particles of the disclosure together with a pharmaceutically acceptable excipient.
  • Suitable excipients include, but are not limited to, salts, diluents, e.g., Tris-HCI, acetate, phosphate), preservatives ⁇ e.g., Thimerosal, benzyl alcohol, parabens), binders, fillers, solubilizers, disintegrants, sorbents, solvents, pH modifying agents, antioxidants, antinfective agents, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and other components and combinations thereof.
  • Suitable pharmaceutically acceptable excipients can be selected from materials which are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.
  • compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
  • PEG polyethylene glycol
  • metal ions or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc.
  • liposomes such as polyacetic acid, polyglycolic acid, hydrogels, etc.
  • Suitable dosage forms for administration include solutions, suspensions, and emulsions.
  • the components of the pharmaceutical formulation can be dissolved or suspended in a suitable solvent such as, for example, water, Ringer's solution, phosphate buffered saline (PBS), or isotonic sodium chloride.
  • a suitable solvent such as, for example, water, Ringer's solution, phosphate buffered saline (PBS), or isotonic sodium chloride.
  • the formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1 ,3-butanediol.
  • formulations can include one or more tonicity agents to adjust the isotonic range of the formulation.
  • Suitable tonicity agents are well known in the art and include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.
  • the formulations can be buffered with an effective amount of buffer necessary to maintain a pH suitable for parenteral administration.
  • Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.
  • the formulation can be distributed or packaged in a liquid form, or alternatively, as a solid, obtained, for example by lyophilization of a suitable liquid formulation, which can be reconstituted with an appropriate carrier or diluent prior to administration.
  • the formulations can comprise one or more guide RNAs and a Type II Cas (or one or more nucleic acids encoding the gRNA(s) and Type II Cas, where such nucleic acids can be in one or more particles such as AAV particles) in a pharmaceutically effective amount sufficient to edit a USH2A gene having a pathogenic mutation in a cell such as the c.7595-2144A>G mutation.
  • the formulations can comprise one or more guide RNAs and a Type II Cas (or one or more nucleic acids encoding the gRNA(s) and Type II Cas, where such nucleic acids can be in one or more particles such as AAV particles) in a pharmaceutically effective amount sufficient to treat Usher syndrome type 2A.
  • the disclosure further provides methods of using the gRNAs, nucleic acids (including pluralities of nucleic acids), systems (including pluralities of systems), particles (including pluralities of particles), and pharmaceutical compositions of the disclosure for altering cells.
  • Methods of the disclosure can be used, for example, to treat subjects having Usher syndrome type 2A, and in particular subjects having an USH2A gene with the c.7595-2144A>G mutation.
  • a method of altering a cell comprises contacting a human cell having an USH2A gene with a pathogenic mutation (e.g., c.7595-2144A>G) with a nucleic acid (or plurality of nucleic acids), particle (or plurality of particles), system (or plurality of systems) or pharmaceutical composition (or plurality of pharmaceutical compositions) described herein.
  • a pathogenic mutation e.g., c.7595-2144A>G
  • a nucleic acid or plurality of nucleic acids
  • particle or plurality of particles
  • system or plurality of systems
  • pharmaceutical composition or plurality of pharmaceutical compositions
  • Contacting a cell with a disclosed nucleic acid, particle, system, or pharmaceutical composition can be achieved by any method known in the art and can be performed in vivo, ex vivo, or in vitro.
  • the methods can include obtaining one or more cells from a subject prior to contacting the cell(s) with a herein disclosed nucleic acid, particle, system or pharmaceutical composition.
  • the methods can further comprise returning or implanting the contacted cell or a progeny thereof to the subject.
  • Guide RNAs and Type II Cas proteins, as well as nucleic acids encoding gRNAs and nucleic acids encoding Type II Cas proteins can be delivered to a cell by any means known in the art, for example, by viral or non-viral delivery vehicles, electroporation or lipid nanoparticles.
  • Type II Cas proteins can be delivered to a cell as one or more polypeptides, either alone or pre-complexed with one or more guide RNAs, or as a nucleic acid (DNA or RNA) encoding the Type II Cas protein.
  • Polynucleotides such as gRNA and/or a polynucleotide encoding a Type II Cas, can be delivered to a cell (ex vivo or in vivo) by a lipid nanoparticle (LNP).
  • LNPs can have, for example, a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.
  • a nanoparticle can range in size from 1 -1000 nm, 1 -500 nm, 1 -250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.
  • LNPs can be made from cationic, anionic, neutral lipids, and combinations thereof.
  • 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.
  • LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Lipids and combinations of lipids that are known in the art can be used to produce a LNP. Examples of lipids used to produce LNPs 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- CerCI4, and PEG-CerC20.
  • Lipids can be combined in any number of molar ratios to produce a LNP.
  • the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce a LNP.
  • Guide RNAs and/or Type II Cas proteins can be delivered to a cell via an adeno-associated viral vector (e.g., of an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhl 0 serotype), or by another viral vector.
  • adeno-associated viral vector e.g., of an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhl 0 serotype
  • Other viral vectors include, but are not limited to lentivirus, adenovirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplex virus.
  • a Type II Cas mRNA is formulated in a lipid nanoparticle, while a sgRNA is delivered to a cell in an AAV or other viral vector.
  • one or more AAV vectors e.g., one or more AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhl 0 serotype
  • a Type II Cas and a sgRNA are delivered using separate vectors.
  • a Type II Cas and a sgRNA are delivered using a single vector.
  • AIK Type II Cas with their relatively small size, can be delivered with a gRNA (e.g., sgRNA) or plurality of gRNAs (e.g., two sgRNAs) using a single AAV vector.
  • compositions and methods for delivering Type II Cas and gRNAs to a cell and/or subject are further described in PCT Patent Application Publications WO 2019/102381 , WO 2020/012335, and WO 2020/053224, each of which is incorporated by reference herein in its entirety.
  • DNA cleavage can result in a single-strand break (SSB) or double-strand break (DSB) at particular locations within the DNA molecule.
  • SSB single-strand break
  • DSB double-strand break
  • Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-dependent repair (HDR) and non-homologous endjoining (NHEJ).
  • HDR homology-dependent repair
  • NHEJ non-homologous endjoining
  • These repair processes can edit the targeted polynucleotide by introducing a mutation, thereby resulting in a polynucleotide having a sequence which differs from the polynucleotide’s sequence prior to cleavage by a Type II Cas.
  • NHEJ and HDR DNA repair processes consist of a family of alternative pathways.
  • Non- homologous end-joining refers to the natural, cellular process in which a double-stranded DNA- break is repaired by the direct joining of two non-homologous DNA segments. See, e.g. Cahill et al., 2006, Front. Biosci. 11 :1958-1976.
  • DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair.
  • NHEJ repair mechanisms can introduce mutations into the coding sequence which can disrupt gene function.
  • NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with a modification of the polynucleotide sequence such as a loss of or addition of nucleotides in the polynucleotide sequence.
  • the modification of the polynucleotide sequence can disrupt (or perhaps enhance) gene expression.
  • Homology-dependent repair utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point.
  • the homologous sequence can be in the endogenous genome, such as a sister chromatid.
  • the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a double- stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus.
  • a third repair mechanism includes microhomology-mediated end joining (MMEJ), also referred to as “Alternative NHEJ (ANHEJ)”, in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site.
  • MMEJ can make use of homologous sequences of a few base pairs flanking the DNA break site to drive a more favored DNA end joining repair outcome. In some instances, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.
  • Modifications of a cleaved polynucleotide by HDR, NHEJ, and/or ANHEJ can result in, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and/or gene mutation.
  • the aforementioned process outcomes are examples of editing a polynucleotide.
  • the contacting step of the methods of the disclosure results in the editing of a USH2A gene comprising a pathogenic mutation.
  • the editing of the USH2A gene comprising a pathogenic mutation can include deletion (e.g., a large deletion when a plurality of gRNAs comprising a first gRNA targeting upstream of PE40 and a second gRNA targeting downstream of PE40 are used), insertion, or substitution of one or more nucleotides in the USH2A gene.
  • editing of the USH2A gene comprising a pathogenic mutation results in a deletion of one or more nucleotides in the USH2A gene.
  • the deletion is 100 bp to 10 kb in size, for example 100 to 500 bp or 100 to 250 bp.
  • the deletion encompasses the PE40 sequence.
  • the methods can provide for advantageous and/or therapeutic results in the cell and/or the subject in which the cell is located.
  • the methods can reduce expression of nonfunctional usherin protein and/or increase expression of functional usherin protein within the contacted cell and/or progeny thereof.
  • the methods can decrease the rate of or amount of cell death.
  • the methods can delay, slow progression, halt, or reverse onset of Usher syndrome type 2A symptom such as retinitis pigmentosa (RP).
  • RP retinitis pigmentosa
  • the disclosure provides methods for treating a subject having a pathogenic mutation in USH2A using the gRNAs, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure.
  • the methods can comprise editing an USH2A gene in one or more cells from the subject or one or more cells derived from a cell of the subject (e.g., an induced pluripotent stem cell (iPSC)).
  • iPSC induced pluripotent stem cell
  • one or more cells from the subject or one or more cells derived from a cell of the subject can be contacted with a gRNA, nucleic acid, system, particle, or pharmaceutical composition of the disclosure ex vivo, and cells having an edited USH2A gene or progeny thereof can subsequently be implanted into the subject.
  • iPSCs can be generated from epithelial cells of a subject by technologies known to the skilled artisan.
  • the chromosomal DNA of such iPSC cells can be edited using the materials and methods described herein. Repair of the cleaved DNA (e.g., by insertion, deletion, substitution, or frameshift mutations) can result in editing of the USH2A gene at the site of the single- or double-strand break.
  • Edited iPSCs can subsequently be differentiated, for instance into photoreceptor cells or retinal progenitor cells. In some embodiments, resultant differentiated cells can be implanted into the subject.
  • differentiated cells of subject can be used.
  • photoreceptor cells or retinal progenitor cells can be used (e.g., following isolation from the subject).
  • implantation of edited cells can proceed without an intervening differentiation step.
  • Advantages of ex vivo cell therapy approaches include the ability to conduct a comprehensive analysis of the therapeutic prior to administration.
  • Nuclease-based therapeutics can have some level of off-target effects.
  • Performing gene correction ex vivo allows a method user to characterize the corrected cell population prior to implantation, including identifying any undesirable off-target effects. Where undesirable effects are observed, a method user may opt not to implant the cells or cell progeny, may further edit the cells, or may select new cells for editing and analysis.
  • Other advantages include ease of genetic correction in iPSCs compared to other primary cell sources. iPSCs are prolific, making it easy to obtain the large number of cells that will be required for a cell-based therapy. Furthermore, iPSCs are an ideal cell type for performing clonal isolations. This allows screening for the correct genomic correction, without risking a decrease in viability.
  • the disclosure provides in vivo methods for treating a subject with a pathogenic USH2A mutation, e.g., c.7595-2144A>G.
  • the method is an in vivo cell-based therapy.
  • Chromosomal DNA of the cells in the subject can be edited using the materials and methods described herein.
  • the in vivo method can comprise editing a USH2A gene having a pathogenic mutation in a cell of a subject, such as photoreceptor cells or retinal progenitor cells.
  • the in vivo methods comprise administering one or more pharmaceutical compositions of the disclosure to or near the eye of a subject, e.g., by sub-retinal injection or intravitreal injection.
  • a single pharmaceutical composition comprising a first AAV encoding one or more gRNAs (e.g., a gRNA targeting the USH2A gene upstream of the PE40 sequence and a gRNA targeting the USH2A gene downstream of the PE40 sequence) and a second AAV encoding a Type II Cas protein can be used; or alternatively, a first pharmaceutical composition comprising the first AAV and a second, separate pharmaceutical composition comprising the second AAV can be used.
  • a first pharmaceutical composition comprising the first AAV and a second, separate pharmaceutical composition comprising the second AAV can be used.
  • they are preferably administered sufficiently close in time so that the first and second AAVs and/or gRNA(s) and Type II Cas protein(s) encoded thereby are present together in vivo.
  • Additional promoters are inducible, and therefore can be temporally controlled if the Type II Cas is delivered as a plasmid.
  • the amount of time that delivered protein and RNA remain in the cell can also be adjusted using treatments or domains added to change the half-life.
  • In vivo treatment would eliminate a number of treatment steps, but a lower rate of delivery can require higher rates of editing.
  • In vivo treatment can eliminate problems and losses from ex vivo treatment and engraftment.
  • An advantage of in vivo gene therapy can be the ease of therapeutic production and administration.
  • the same therapeutic approach and therapy has the potential to be used to treat more than one patient, for example a number of patients who share the same or similar genotype or allele.
  • ex vivo cell therapy typically requires using a subject’s own cells, which are isolated, manipulated and returned to the same patient.
  • Progenitor cells are capable of both proliferation and giving rise to more progenitor cells, which in turn have the ability to generate a large number of cells that can in turn give rise to differentiated or differentiable daughter cells.
  • the daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential.
  • stem cell refers then to a cell with the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating.
  • progenitor or stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues.
  • Cellular differentiation is a complex process typically occurring through many cell divisions.
  • a differentiated cell can derive from a multipotent cell that itself is derived from a multipotent cell, and so on. While each of these multipotent cells can be considered stem cells, the range of cell types that each can give rise to can vary considerably.
  • Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity can be natural or can be induced artificially upon treatment with various factors.
  • stem cells can also be "multipotent" because they can produce progeny of more than one distinct cell type, but this is not required.
  • Human cells described herein can be induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • An advantage of using iPSCs in the methods of the disclosure is that the cells can be derived from the same subject to which the progenitor cells are to be administered. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then differentiated into a progenitor cell to be administered to the subject (e.g., an autologous cell). Because progenitors are essentially derived from an autologous source, the risk of engraftment rejection or allergic response can be reduced compared to the use of cells from another subject or group of subjects. In addition, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one aspect, the stem cells used in the disclosed methods are not embryonic stem cells.
  • Methods are known in the art that can be used to generate pluripotent stem cells from somatic cells.
  • Pluripotent stem cells generated by such methods can be used in the method of the disclosure.
  • Mouse somatic cells can be converted to ES cell-like cells with expanded developmental potential by the direct transduction of Oct4, Sox2, Klf4, and c-Myc; see, e.g., Takahashi and Yamanaka, 2006, Cell 126(4): 663-76.
  • iPSCs resemble ES cells, as they restore the pluripotency-associated transcriptional circuitry and much of the epigenetic landscape.
  • mouse iPSCs satisfy all the standard assays for pluripotency: specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, contribution to chimeras, germline transmission (see, e.g., Maherali and Hochedlinger, 2008, Cell Stem Cell. 3(6) :595-605), and tetrapioid complementation.
  • iPSCs can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, has been established as the core set of transcription factors that govern pluripotency; see, e.g., 2014, Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57; Barrett et al, 2014, Stem Cells Trans Med 3: 1 -6 sctm.2014-0121 ; Focosi et al, 2014, Blood Cancer Journal 4: e211 .
  • the production of iPSCs can be achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into an adult, somatic cell, historically using viral vectors.
  • iPSCs can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming. In such instances, it may not be necessary to include as many reprogramming factors as required to reprogram a terminally differentiated cell.
  • reprogramming can be induced by the non-viral introduction of reprogramming factors, e.g., by introducing the proteins themselves, or by introducing nucleic acids that encode the reprogramming factors, or by introducing messenger RNAs that upon translation produce the reprogramming factors (see e.g., Warren et al., 2010, Cell Stem Cell, 7(5):6I8- 30.
  • Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes, including, for example, Oct-4 (also known as Oct-3/4 or Pouf5l), Soxl, 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 can further comprise introducing one or more of Oct-3/4, a member of the Sox family, a member of the Klf family, and a member of the Myc family to a somatic cell.
  • the methods and compositions described herein can further comprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYC and Klf4 for reprogramming.
  • the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein.
  • the reprogramming is not affected by a method that alters the genome.
  • reprogramming can be achieved, e.g., without the use of viral or plasmid vectors.
  • Efficiency of reprogramming (the number of reprogrammed cells) derived from a population of starting cells can be enhanced by the addition of various agents, e.g., small molecules, as shown by Shi et al., 2008, Cell-Stem Cell 2:525-528; Huangfu et al., 2008, Nature Biotechnology 26(7):795-797; and Marson etal., 2008, Cell-Stem Cell 3: 132-135.
  • an agent or combination of agents that enhance the efficiency or rate of induced pluripotent stem cell production can be used in the production of patientspecific or disease-specific iPSCs.
  • agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HD AC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.
  • reprogramming enhancing agents include: Suberoylanilide Hydroxamic Acid (SAHA (e.g ., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC Toxin, Nullscript (4-(l,3-Dioxo-IH,3H- benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), 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, pi valoyloxy methyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, Depsipeptide (also known as FR901228 or
  • reprogramming enhancing agents include, for example, dominant negative forms of the HDACs (e.g, catalytically inactive forms), siRNA inhibitors of the HDACs, and antibodies that specifically bind to the HDACs.
  • inhibitors are available, e.g., from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.
  • BIOMOL International Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.
  • Stem cell markers can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdfi, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl.
  • a cell that expresses Oct4 or Nanog is identified as pluripotent.
  • Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. 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 are readily identified, e.g., by immunocytochemistry.
  • Pluripotency of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate into cells of each of the three germ layers.
  • teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones.
  • the cells can be introduced into nude mice and histology and/or immunohistochemistry can be performed on a tumor arising from the cells.
  • the growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.
  • Patient-specific iPS cells or cell line can be created. There are many established methods in the art for creating patient specific iPS cells, e.g., as described in Takahashi and Yamanaka 2006;
  • the creating step can comprise: a) isolating a somatic cell, such as a skin cell or fibroblast, from the patient; and b) introducing a set of pluripotency-associated genes into the somatic cell in order to induce the cell to become a pluripotent stem cell.
  • the set of pluripotency-associated genes can be one or more of the genes selected from the group consisting of OCT4, SOX1 , SOX2, SOX3, SOX15, SOX18, NANOG, KLF1 , KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.
  • a biopsy or aspirate of a subject’s bone marrow can be performed.
  • a biopsy or aspirate is a sample of tissue or fluid taken from the body.
  • biopsies or aspirates There are many different kinds of biopsies or aspirates. Nearly 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, numbing medicine can be applied first.
  • a biopsy or aspirate can be performed according to any of the known methods in the art. For example, in a bone marrow aspirate, a large needle is used to enter the pelvis bone to collect bone marrow.
  • a mesenchymal stem cell can be isolated from a subject.
  • Mesenchymal stem cells can be isolated according to any method known in the art, such as from a subject’s bone marrow or peripheral blood.
  • marrow aspirate can be collected into a syringe with heparin.
  • Cells can be washed and centrifuged on a PercollTM density gradient.
  • Cells, such as blood cells, liver cells, interstitial cells, macrophages, mast cells, and thymocytes can be separated using density gradient centrifugation media, PercollTM.
  • the cells can then be cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% fetal bovine serum (FBS) (Pittinger et. al., 1999, Science 284: 143-147).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • the methods of the present disclosure can also comprise differentiating genome-edited iPSCs into photoreceptor cells or retinal progenitor cells.
  • the differentiating step may be performed according to any method known in the art.
  • iPSCs can be used to generate retinal organioids and photoreceptors as described in the art (Phillips et al., 2014, Stem Cells, 32(6): pgs. 1480-1492; Zhong et al., 2014, Nat. Common., 5:4047; Tucker et al., 2011 , PLoS One, 6(4): e18992).
  • hiPSC can be differentiated into retinal progenitor cells using various treatments, including Wnt, Nodal, and Notch pathway inhibitors (Noggin, Dkl, Lefty A, and DAPT) and other growth factors.
  • the retinal progenitor cells can be further differentiated into photoreceptor cells, the treatment including: exposure to native retinal cells in coculture systems, RX+ or Mitf+ by subsequent treatment with retinoic acid and taurine, or exposure to several exogenous factors including Noggin, Dkkl, DAPT, and insulin-like growth factor (Yang et al., 2016, Stem Cells International 2016).
  • the methods of the present disclosure can also comprise differentiating the genome-edited mesenchymal stem cells into photoreceptor cells or retinal progenitor cells.
  • the differentiating step can be performed according to any method known in the art.
  • the methods of the present disclosure can also comprise implanting the photoreceptor cells or retinal progenitor cells into a subject.
  • This implanting step can be accomplished using any method of implantation known in the art.
  • cells can be injected directly in the subject’s blood or otherwise administered to the subject.
  • Another aspect of the methods can include implanting edited photoreceptor cells or retinal progenitor cells into a subject.
  • the implanting step can be accomplished using any method of implantation known in the art.
  • the genetically modified cells can be injected directly in the subject’s eye or otherwise administered to the patient.
  • Usher syndrome type 2A is a rare genetic disorder leading to progressive vision and hearing loss caused by inactivating mutations affecting both alleles of the USH2A gene. Reactivation of the functionality of one copy of USH2A through gene editing approaches is sufficient to restore usherin protein function and re-establish the correct physiological state of target cells (e.g., photoreceptors).
  • the second most common mutation found in the USH2A gene is the c.7595-2144A>G deep-intronic mutation (affecting 4% of Usher Syndrome type 2A patients) which produces aberrant USH2A splicing leading to inactivating frameshift mutations in the mature mRNA.
  • sgRNA molecules comprising targeting sequences directed towards specific regions of the USH2A gene surrounding PE40 were designed and evaluated. More specifically, the AIK Type II Cas sgRNAs in this Example were designed to edit the USH2A gene either near the cryptic 5’ PE40 splice site or the cryptic 3’ PE40 spice site. The sgRNAs can be used in combination to produce a deletion encompassing the PE40 sequence.
  • one sgRNA targeting the region neighboring the 5’ cryptic splice site can be combined with a sgRNA targeting the region close to the 3’ cryptic splice site to obtain the coordinated cleavage and removal of the intervening genomic region including PE40.
  • the strategy does not discriminate between the two USH2A alleles, but the formation of the deletion per se should not be detrimental on USH2A functionality.
  • both alleles in a patient are already non-functional null alleles and cannot thus be inadvertently inactivated by the edits.
  • a schematic representation of the targeting strategy is reported in FIG. 4.
  • a schematic representation of the positions of the selected guide RNAs is reported in FIG. 5.
  • AIK Type II Cas originates from the genus Collinsella and has a preference for a 3’ N4RHNT, N4RYNT or N4GYNT PAM, in particular N 4 GTTT and N 4 GTGT PAMs (FIGS. 2A-2B).
  • Exemplary sequences of AIK Type II Cas crRNA, tracrRNA, and sgRNA scaffolds are shown in Table 6.
  • AIK Type II Cas coding sequence used was a human codon-optimized synthetic fragment (Genewiz) and was cloned into an expression vector controlled by the CAG promoter, which also contained a U6-driven expression cassette for the AIK sgRNA (obtained by gene synthesis from Genewiz), generating the pAIK-empty plasmid.
  • the AIK nuclease was further modified by the insertion of two nuclear localization signals (both at the N- and C-terminus) and an V5 tag at its N-terminus.
  • the sequences of the AIK sgRNA scaffolds used in this Example are reported in Table 8.
  • Minigene models mimicking the splicing pattern of the wild-type USH2A gene and its mutated counterpart were used.
  • the minigene models are described in Example 11 of PCT publication no. WO 2020/165768, the contents of which are incorporated by reference herein in their entireties.
  • a schematic representation of the minigene construct is reported in FIG. 6A.
  • HEK293 cells were obtained from the American Type Culture Collection (ATCC; www.atcc.org). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS; Life Technologies), 10 U/ml antibiotics (PenStrep, Life Technologies) and 2 mM L-glutamine at 37°C in a 5% CO2 humidified atmosphere.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • PenStrep 10 U/ml antibiotics
  • HEK293 cells stably expressing USH2A wild-type and mutated minigenes were generated by stable transfection of linearized minigene plasmids.
  • Cells were selected with 600 pg/ml of G418 (Invivogen) starting from 48h after transfection.
  • Single cell clones were isolated and characterized for the minigenes copy number and the expression of the minigene constructs. Stable clones were maintained in culture as indicated above with the additional supplementation of 500 pg/ml of G418.
  • the pcDNA3-GAPDH- fragment construct was obtained by blunt-end cloning of a GAPDH fragment amplified using the GAPDH_CN_For and GAPDH_CN_Rev primers reported in Table 9, which were the same primers used for GAPDH qPCR amplification.
  • Transfections were performed in HEK293 cells stably expressing the mutant USH2A minigene seeded (100,000 cells/well) in a 24 well plate. 24 hours after seeding, cells were transfected with 750 ng of the pAIK plasmids each encoding for AIK Type II Cas and the different sgRNAs targeting USH2A using TranslT-LT1 (Mirus Bio) according to manufacturer’s instructions. Cells were split at confluence and collected at 3 days and 6 days post-transfection for DNA and RNA extraction, respectively, to compare editing efficiency and splicing correction within the same samples.
  • Target regions were amplified by PCR with the HOT FIREPol Multiplex Mix (Solis Biodyne) using primers V5tag_For and TEVsite_Rev (reported in Table 10). PCR products were run on 1 .5% agarose gel and images were obtained with the UVIdoc HD5 system (Uvitec Cambridge). 7.1.1.1. Evaluation of indel formation
  • Genomic DNA was extracted from cell pellets using the QuickExtract solution (Lucigen) according to manufacturer’s instructions.
  • the HOT FIREPol Multiplex Mix (Solis Biodyne) was used to amplify the integrated USH2A minigene using primers TIDE-USH2A-PE40-F or TIDE-USH2A-PE40-SA- 250-F (reported in Table 10) and TEVsite_Rev (reported in Table 10), specifically detecting the integrated USH2A minigene.
  • the amplicon pools were Sanger sequenced (EasyRun service, Microsynth) and the indel levels were evaluated using the TIDE webtool (tide.deskgen.com/). When two sgRNAs were used in combination to target the USH2A PE40 pseudoexon, deletion formation was evaluated using the DECODR (decodr.org/).
  • AIK Type II Cas guide RNAs targeting regions both upstream and downstream the cryptic splice sites of PE40 were designed and preliminarily evaluated for their editing activity by transient transfection in HEK293 cells stably expressing a single copy of a USH2A minigene bearing the c.7595-2144A>G mutation in order to determine the best candidates to be used in combination for PE40 deletion.
  • the editing levels measured for the different sgRNAs evaluated are reported in FIG. 7.
  • sgUSH-07, sgUSH-08, sgUSH-11 were selected for further characterization, while among the guides positioned downstream of PE40 sgUSH-12, sgUSH- 13, sgUSH-15, sgUSH-18, sgUSH-20, sgUSH-24, sgUSH-27, sgUSH-29 showed particularly good levels of editing and were thus used in further studies.
  • sgUSH-02 and sgUSH-03 were also included among the guides selected for further evaluation.
  • a “super trimmed” scaffold based on the AIK Type II Cas sgRNA_v4 scaffold was designed.
  • the scaffold, AIK Type II Cas sgRNA_v5 includes the features of the v4 scaffold but includes an additionally trimmed stem-loop (FIG. 10).
  • Indel formation at the DNMT1 and B2M loci was evaluated in an in vitro editing assay using wild-type AIK Type II Cas and gRNAs having the AIK Type II Cas sgRNA_v1 , sgRNA_v4, or sgRNA_v5 scaffold with six 3’ uracils. Results are shown in FIG. 11 .
  • An AIK Type II Cas guide RNA molecule (gRNA) for editing a human USH2A gene optionally comprising a means for binding the human USH2A gene and a sgRNA scaffold.
  • a guide RNA molecule (gRNA) for editing a human USH2A gene which is optionally an AIK Type II Cas gRNA, the gRNA comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is: i) GCUUUCAACCAUGCAGUUGCAGGCC (SEQ ID NO:14); ii) GCCAGUUGAUUUGUAUAUAGAAUU (SEQ ID NO:15);
  • gRNA of embodiment 1 or embodiment 2 which comprises a spacer that is 15 to 30 nucleotides in length.
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GCUUUCAACCAUGCAGUUGCAGGCC (SEQ ID NO:14).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GCUGACUCCUGGCACUCUUUCAAUU (SEQ ID NO:17).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GCUUAUCAUUUUAAAGCACUAAAUU (SEQ ID NO:18).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GCUAAAUUGAAAGAGUGCCAGGAG (SEQ ID NO:21 ).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GUUCCCUAGCCAAAGGAGCUAAUUA (SEQ ID NO:23).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GCCAAAGGAGCUAAUUAAGCUGCU (SEQ ID NO:24).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GGUAAGAGAUCAUCUUUAAGAAAA (SEQ ID NO:27).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GCUUGCACUUCAAACCCCCACAAUA (SEQ ID NO:28).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GCUUUAAGAAAAGGCUGUGUAUUGU (SEQ ID NO:29).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GUUUAAGAAAAGGCUGUGUAUUGU (SEQ ID NQ:30).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GGCUGUGUAUUGUGGGGGUUUGAA (SEQ ID NO:31 ).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GUGCAAGUUCAUCUCAUUAUCAU (SEQ ID NO:33).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GGAUGUUUCACCCAUAAUACUAU (SEQ ID NO:34).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GCUUUUAUUUCUCCUGCAUAUGAU (SEQ ID NO:35).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GGGGGAUAAGGCUUUUAUUUCUC (SEQ ID NO:36).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GAUUGGGGGAUAAGGCUUUUAUUUC (SEQ ID NO:37).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GACUAUCAUCAUAUGCAGGAGAAAU (SEQ ID NO:38).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GUUUGUUCUCUGUGAUUGGGGGA (SEQ ID NO:39). 61. The gRNA of any one of embodiments 2 to 34, wherein the reference sequence is
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GUUCAGGGAAAAGAAACAACAAUGA (SEQ ID NO:41 ).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GUUGUUUCUUUUCCCUGAAGAUGCU (SEQ ID NO:42).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GUGUUUCUUUUCCCUGAAGAUGCU (SEQ ID NO:44).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GAAGAUGCUCACAGUUUGAUAGU (SEQ ID NO:45).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GCUCACAGUUUGAUAGUUCACAUA (SEQ ID NO:46).
  • gRNA of any one of embodiments 2 to 34, wherein the reference sequence is GUCACAGUUUGAUAGUUCACAUA (SEQ ID NO:47).
  • gRNA of embodiment 2 wherein the spacer sequence is GCUUAUCAUUUUAAAGCACUAAAUU (SEQ ID NO:18).
  • gRNA of embodiment 2 wherein the spacer sequence is GCUUAAUUAGCUCCUUUGGCUAG (SEQ ID NO:22).
  • gRNA of embodiment 2 wherein the spacer sequence is GCUUGCACUUCAAACCCCCACAAUA (SEQ ID NO:28).
  • gRNA of embodiment 2 wherein the spacer sequence is GGAUGUUUCACCCAUAAUACUAU (SEQ ID NO:34).
  • gRNA of embodiment 2 wherein the spacer sequence is GCUUUUAUUUCUCCUGCAUAUGAU (SEQ ID NO:35).
  • gRNA of embodiment 2, wherein the spacer sequence is GGGGGAUAAGGCUUUUAUUUCUC (SEQ ID NO:36).
  • gRNA of embodiment 2 wherein the spacer sequence is GAACAACAAUGAUGCAGUUUGUUCU (SEQ ID NQ:40).
  • the gRNA of embodiment 2, wherein the spacer sequence is GAAGAUGCUCACAGUUUGAUAGU (SEQ ID NO:45). 101 . The gRNA of embodiment 2, wherein the spacer sequence is GCUCACAGUUUGAUAGUUCACAUA (SEQ ID NO:46).
  • gRNA of embodiment 2 wherein the spacer sequence is GUCACAGUUUGAUAGUUCACAUA (SEQ ID NO:47).
  • the gRNA of any one of embodiments 2 to 102 which is a AIK Type II Cas gRNA.
  • gRNA of any one of embodiments 1 to 103 which is a single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • the gRNA of embodiment 104 which comprises a 3’ sgRNA scaffold.
  • the gRNA of embodiment 105, wherein the 3’ sgRNA scaffold has a nucleotide sequence comprising a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identical to a scaffold sequence set forth in Table 3.
  • the gRNA of embodiment 106, wherein the 3’ sgRNA scaffold comprises the nucleotide sequence GUCUUGAGCACGCGCCCUUCCCCAAGGUGAGAAAUCACCUUGGGGAAGGGCGCGGCUCCAGACA AGGGGAGCCACUUAAGUGGCUUACCCGUAAAGUAACCCCCGUUCAAUCUUCGGAUUGGGCGGGG CGAAC (SEQ ID NO:68).
  • the gRNA of embodiment 106, wherein the 3’ sgRNA scaffold comprises the nucleotide sequence GUCUUGAGCACGCGCCCUUCCGCAAGGUGAGAAAUCACCUUGCGGAAGGGCGCGGCUCCAGACA AGCGGAGCCACUUAAGUGGCUUACGCGUAAAGUAACCGCCGUUCAAUCUUCGGAUUGGGCGGCG CGAAC (SEQ ID NO:69).
  • the gRNA of embodiment 106, wherein the 3’ sgRNA scaffold comprises the nucleotide sequence GUCUUGAGCACGCGAAAGCGGCUCCAGACAAGGGGAGCCACUUAAGUGGCUUACCCGUAAAGUA ACCCCCGUUCAAUCUUCGGAUUGGGCGGGGCGAAC (SEQ ID NQ:70).
  • the gRNA of embodiment 106, wherein the 3’ sgRNA scaffold comprises the nucleotide sequence GUCUUGAGCACGCGAAAGCGGCUCCAGACAAGCGGAGCCACUUAAGUGGCUUACGCGUAAAGUA ACCGCCGUUCAAUCUUCGGAUUGGGCGGCGCGAAC (SEQ ID NO:71 ).
  • the gRNA of embodiment 106, wherein the 3’ sgRNA scaffold comprises the nucleotide sequence GUCUUGAGCACGCGAAAGCGGCUCCAGACAAGCGGAGCCACUUAAGUGGCUUACGCGUAAAGUA ACCGCCGAAAGGCGCGAAC (SEQ ID NO:72).
  • gRNA of any one of embodiments 1 to 121 wherein the gRNA is an unmodified gRNA.
  • gRNA of any one of embodiments 1 to 121 which comprises one or more modifications.
  • a plurality of gRNA molecules comprising two or more AIK Type II Cas gRNA molecules, optionally wherein the two or more AIK Type II Cas gRNA molecules are gRNA molecules for editing a human USH2A gene.
  • a plurality of gRNA molecules comprising two or more gRNA molecules, wherein each gRNA of the two or more gRNA molecules is independently a gRNA molecule according to any one of embodiments 1 to 124.
  • invention 126 The plurality of gRNA molecules of embodiment 126, which comprises two gRNA molecules.
  • the plurality of gRNA molecules of any one of embodiments 125 to embodiment 127 which comprises a first gRNA molecule comprising a spacer that is partially or fully complementary to a target sequence upstream of the pseudoexon 40 sequence in a human USH2A gene having a c.7595- 2144A>G mutation and a second gRNA molecule comprising a spacer that is partially or fully complementary to a target sequence downstream of pseudoexon 40 sequence in a human USH2A gene having a c.7595-2144A>G mutation.
  • the spacer of the first gRNA has a nucleotide sequence comprising GCUUAAUUAGCUCCUUUGGCUAG (SEQ ID NO:22).
  • the spacer of the second gRNA has a nucleotide sequence comprising GUAUUGUGGGGGUUUGAAGUGCAA (SEQ ID NO:32).
  • the plurality of gRNA molecules of embodiment 129 wherein the spacer of the first gRNA has a nucleotide sequence comprising GUGAUUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) and the spacer of the second gRNA has a nucleotide sequence comprising GGUAAGAGAUCAUCUUUAAGAAAA (SEQ ID NO:27).
  • the plurality of gRNA molecules of embodiment 129 wherein the spacer of the first gRNA has a nucleotide sequence comprising GUGAUUCUGGAGAGGAAGCUGAA (SEQ ID NO:25) and the spacer of the second gRNA has a nucleotide sequence comprising GUUGUUUCUUUUCCCUGAAGAUGCU (SEQ ID NO:42).
  • nucleic acid of embodiment 181 which further comprises a Pol III promoter sequence operably linked to the nucleotide sequence encoding the gRNA.
  • nucleic acid of embodiment 182, wherein the promoter is a U6 promoter.
  • nucleic acid of embodiment 182, wherein the promoter is a H1 promoter.
  • nucleic acid of any one of embodiments 181 to 184 which further encodes a second gRNA, optionally wherein the second gRNA is a gRNA of any one of embodiments 1 to 122.
  • nucleic acid of embodiment 187 which further comprises a Pol III promoter sequence operably linked to the nucleotide sequence encoding each gRNA.
  • nucleic acid of embodiment 188, wherein the promoter is a H1 promoter.
  • nucleic acid of embodiment 192, wherein the viral genome is an adeno-associated virus (AAV) genome.
  • AAV adeno-associated virus
  • nucleic acid of embodiment 193, wherein the viral genome is an AAV2 genome.
  • nucleic acid of embodiment 193, wherein the viral genome is an AAV5 genome.
  • viral genome is an AAV7m8 genome.
  • nucleic acid of embodiment 193, wherein the viral genome is an AAV8 genome.
  • nucleic acid of embodiment 193, wherein the viral genome is an AAV9 genome.
  • nucleic acid of embodiment 193, wherein the viral genome is an AAVrh8r genome.
  • nucleic acid of embodiment 193, wherein the viral genome is an AAVrhl 0 genome.
  • nucleic acid of any one of embodiments 181 to 200 further encoding a Type II Cas protein.
  • the nucleic acid of embodiment 201 , wherein the Type II Cas protein is an AIK Type II Cas protein.
  • the nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • the nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • the nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • the nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • the nucleic acid of embodiment 202, wherein the AIK Type II Cas protein comprises an amino acid sequence that is 100% identical to the full length of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.
  • nucleic acid of embodiment 213, wherein the fusion protein comprises two or more nuclear localization signals.
  • nucleic acid of any one of embodiments 213 to 214 which comprises an N-terminal nuclear localization signal.
  • nucleic acid of any one of embodiments 201 to 217, wherein the nucleotide sequence encoding the Type II Cas protein is operably linked to a promoter, which is optionally a tissue specific promoter or a constitutive promoter.
  • nucleic acid of embodiment 218, wherein the nucleotide sequence encoding the Type II Cas protein is operably linked to a CAG promoter.
  • nucleic acid of embodiment 218, wherein the nucleotide sequence encoding the Type II Cas protein is operably linked to a hGRK promoter.
  • nucleic acid of embodiment 218, wherein the nucleotide sequence encoding the Type II Cas protein is operably linked to a PGK promoter.
  • nucleic acid of embodiment 218, wherein the nucleotide sequence encoding the Type II Cas protein is operably linked to an EF1 alpha promoter, e.g., an EF1 alpha short (EFS) promoter.
  • an EF1 alpha promoter e.g., an EF1 alpha short (EFS) promoter.
  • a system comprising a Type II Cas protein and the gRNA of any one of embodiments 1 to 124 or the plurality of gRNAs of any one of embodiments 125 to 180.
  • a plurality of nucleic acids encoding the system of any one of embodiments 223 to 224 comprising (i) a nucleic acid encoding the Type II Cas protein and (ii) one or more separate nucleic acids encoding the gRNA(s).
  • a particle comprising the gRNA of any one of embodiments 1 to 124, the plurality of gRNAs of any one of embodiments 125 to 180, the nucleic acid of any one of embodiments 181 to 222, the system of any one of embodiments 223 to 224, the nucleic acid of embodiment 225, or the plurality of nucleic acids of embodiment 226.
  • the particle of embodiment 227, wherein the particle is a viral particle, a lipid nanoparticle, a vesicle, or a gold nanoparticle.
  • the particle of embodiment 228, wherein the particle is an adeno-associated virus (AAV) particle.
  • AAV adeno-associated virus
  • the particle of embodiment 230 which is an AAV2 particle.
  • invention 230 which is an AAV5 particle.
  • the particle of embodiment 230 which is an AAV7m8 particle.
  • the particle of embodiment 230 which is an AAV8 particle.
  • the particle of embodiment 230 which is an AAV9 particle.
  • the particle of embodiment 230 which is an AAVrh8r particle.
  • the particle of embodiment 230 which is an AAVrhl 0 particle.
  • a plurality of particles comprising a first particle and a second particle, wherein the first and second particles are each independently a particle according to any one of embodiments 227 to 238.
  • a cell comprising the gRNA of any one of embodiments 1 to 124, the plurality of gRNAs of any one of embodiments 125 to 180, the nucleic acid of any one of embodiments 181 to 222, the system of any one of embodiments 223 to 224, the nucleic acid of embodiment 225, the plurality of nucleic acids of embodiment 226, the particle of any one of embodiments 227 to 237, or the plurality of particles of embodiment 238.
  • the cell of embodiment 240 which is a human cell.
  • the cell of embodiment 240 which is a human retinal cell.
  • the cell of embodiment 240 which is a human retinal epithelial cell.
  • the cell of embodiment 240 which is a human photoreceptor cell.
  • the cell of embodiment 240 which is a human retinal progenitor cell.
  • the cell of embodiment 240 which is a stem cell.
  • the cell of embodiment 240 which is an iPS cell.
  • the cell of embodiment 240 which is a HEK293 cell.
  • a method of altering a human cell comprising a USH2A gene having a pathogenic mutation comprising contacting the cell with the gRNA of any one of embodiments 1 to 124, the plurality of gRNAs of any one of embodiments 125 to 180, the nucleic acid of any one of embodiments 181 to 222, the system of any one of embodiments 223 to 224, the nucleic acid of embodiment 225, the plurality of nucleic acids of embodiment 226, the particle of any one of embodiments 227 to 237, the plurality of particles of embodiment 238, or the pharmaceutical composition of embodiment 239.
  • the method of embodiment 253, wherein the contacting comprises delivering the system to the cell via one or more particles and/or one or more vectors.
  • the contacting comprises delivering the system to the cell via one or more particles.
  • the one or more particles comprise a lipid nanoparticle, a vesicle, or a gold nanoparticle.
  • the one or more viral vectors comprise an adeno-associated virus (AAV), a lentivirus, or an adenovirus.
  • AAV adeno-associated virus
  • the one or more viral vectors comprise an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • the one or more viral vectors comprise one or more AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhI O vectors.
  • the one or more viral vectors comprise an adenovirus.
  • the one or more viral vectors comprise nucleic acid(s) encoding the gRNA(s) and the Type II Cas protein each operably linked to a promoter.
  • invention 300 which further comprises returning the contacted cell or a progeny thereof to the subject.
  • contacting comprises delivering the gRNA, plurality of gRNAs, nucleic acid, plurality of nucleic acids, particle, plurality of particles, system, or pharmaceutical composition to the eye by sub-retinal injection.
  • the method of embodiment 304, wherein the contacting comprises delivering the gRNA, plurality of gRNAs, nucleic acid, plurality of nucleic acids, particle, plurality of particles, system, or pharmaceutical composition to the eye by intravitreal injection.

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Abstract

L'invention concerne des compositions et des méthodes utiles pour modifier des gènes USH2A, par exemple des molécules guides (ARNg) et des pluralités de molécules d'ARNg ciblant un gène USH2A ayant une mutation c.7595-2144 A>G, des acides nucléiques codant pour les ARNg et les pluralités d'ARNg, des particules, des systèmes comprenant un ARNg ou une pluralité d'ARNg conjointement avec une protéine Cas de type II telle qu'une protéine Cas de AIK de type II, des compositions pharmaceutiques et des cellules comprenant ceux-ci et des combinaisons de ceux-ci.
PCT/EP2023/058797 2022-04-04 2023-04-04 Compositions et méthodes de traitement du syndrome d'usher de type 2a WO2023194359A1 (fr)

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