# US 2019/0169652 A1 · Compositions and methods for treating cep290 associated disease

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- Number: US 2019/0169652 A1
- Document ID: US20190169652A1
- Status: Published application
- Kind: A1
- Application number: 16/322,922
- Filed: 2017-08-02
- Published: 2019-06-06
- Earliest claimed: 2016-08-02
- Assignees: Editas Medicine, Inc.
- Applicants: Editas Medicine, Inc.
- Inventors: Morgan Lee MAEDER; Rina J. MEPANI; Michael STEFANIDAKIS
- Web page: https://patentgrep.com/patent/US20190169652A1

## CPC

- C12N 15/907: {in mammalian cells}
- A61K 9/0048: {Eye, e.g. artificial tears}
- A61K 35/761: Adenovirus
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/113: 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 (when used in plants C12N15/8218)}
- C12N 15/86: Viral vectors
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2330/50: Biochemical production, i.e. in a transformed host cell
- C12N 2330/51: Specially adapted vectors
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Nucleic acids and viral vectors, particularly adeno-associated virus (AAV) vectors are provided that encode Cas9 and paired guide RNAs. The nucleic acids and vectors, and compositions that comprise them, can be used in methods to treat subjects, to alter cells in subjects who may suffer from an inherited retinal dystrophy such as CEP290 associated disease or who may be in need of alteration of a cell or a cellular nucleic acid sequence associated with an inherited retinal dystrophy such as the CEP290 gene, and/or to treat inherited retinal dystrophies including CEP290 associated disease.

## Claims

### Claim 1

A nucleic acid encoding a Cas9, a first gRNA and a second gRNA, each gRNA targeted to a CEP290 gene of a subject for use in therapy.

### Claim 2

A nucleic acid encoding a Cas9, a first gRNA and a second gRNA, each gRNA targeted to a CEP290 gene for use in treating a CEP290-associated disease.

### Claim 3 (depends on 1, 2)

The nucleic acid for the use of claim 1 or 2, wherein the first gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 1-3.

### Claim 4 (depends on 1, 2)

The nucleic acid for the use of claim 1 or 2, wherein the second gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 4-6.

### Claim 5 (depends on 1, 2, 3, 4)

The nucleic acid for the use according to any one of claims 1 to 4, wherein the nucleic acid encoding the Cas9, the first gRNA and second gRNA is characterized in that it comprises the following targeting domain sequences
a) SEQ ID NO:1 and SEQ ID NO:5; or
b) SEQ ID NO:1 and SEQ ID NO:6; or
c) SEQ ID NO:2 and SEQ ID NO:4; or
d) SEQ ID NO:3 and SEQ ID NO:4; or
e) SEQ ID NO:3 and SEQ ID NO:5.

### Claim 6

The nucleic acid for the use according to any one of the preceding claims wherein the nucleic acid encodes S. aureus Cas9.

### Claim 7 (depends on 6)

The nucleic acid according to claim 6, wherein the nucleic acid comprises a Cas9 coding sequence according to SEQ ID NO: 10 or encodes a Cas9 comprising the sequence of SEQ ID NO:11.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6)

The nucleic acid for the use according to any one of claims 1 to 6, wherein the Cas9 is a modified Cas9.

### Claim 9

The nucleic acid for the use according to any one of the preceding claims, wherein the Cas9 comprises a nuclear localization signal.

### Claim 10 (depends on 9)

The nucleic acid for the use according to claim 9, wherein the Cas9 comprises a C-terminal nuclear localization signal and an N-terminal nuclear localization signal.

### Claim 11

The nucleic acid for the use according to any one of the preceding claims, wherein the Cas9 comprises a polyadenylation signal.

### Claim 12 (depends on 6, 7)

The nucleic acid for the use according to claim 6 or claim 7, wherein the gRNA is a unimolecular S. aureus gRNA comprising SEQ ID NO:7 or SEQ ID NO: 8, or the corresponding two-part modular S. aureus gRNA, wherein the crRNA component comprises the underlined section and the tracrRNA component comprises the double underlined section of SEQ ID NO:7 or SEQ ID NO:8.

### Claim 13 (depends on 12)

The nucleic acid for the use according to claim 12, wherein the gRNA is a unimolecular S. aureus gRNA of SEQ ID NO:8.

### Claim 14

The nucleic acid for the use according to any one of the preceding claims, wherein the nucleic acid comprises a promoter sequence for driving expression of the Cas9.

### Claim 15 (depends on 14)

The nucleic acid for the use according to claim 14, wherein the promoter sequence is selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 16

The nucleic acid for the use according to any one of the preceding claims wherein the nucleic acid comprises
a) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 5, or
b) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 6, or
c) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 4, or
d) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 4, or.
e) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 5, or
f) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 5, or
g) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 6, or
h) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 4, or
i) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 4, or
j) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 5, or
k) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 5, or
g) an hGRKlpromoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 6, or.
h) an hGRKlpromoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 4, or
i) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 4, or
j) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 5.

### Claim 17 (depends on 14)

The nucleic acid for the use according to claim 14, wherein the promoter sequence is a hGRK1 promoter.

### Claim 18

The nucleic acid for the use according to any one of the preceding claims, wherein the nucleic acid is to be administered as an adeno-associated virus (AAV) vector comprising the nucleic acid.

### Claim 19 (depends on 18)

The nucleic acid for the use according to claim 18, wherein the adeno-associated virus (AAV) vector comprising the nucleic acid is to be administered to a retina of the subject.

### Claim 20 (depends on 19)

The nucleic acid for the use according to claim 19, wherein the AAV vector is delivered to a retina of a subject by injection, such as by subretinal injection.

### Claim 21 (depends on 18, 19, 20)

The nucleic acid for the use according to any one of claims 18 to 20, wherein the AAV vector comprises an AAV5 capsid.

### Claim 22 (depends on 18, 19, 20, 21)

The nucleic acid for the use according to any one of claims 18 to 21, wherein the nucleic acid comprises:
a first and a second inverted terminal repeat sequence (ITR);
a first guide RNA comprising a targeting domain sequence selected from the group consisting of SEQ ID NOS: 1-3;
a second guide RNA comprising a targeting domain sequence selected from the group consisting of SEQ ID NOS: 4-6; and
a promoter for driving expression of the Cas9 comprising a sequence selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 23

The nucleic acid for the use according to any one of the preceding claims, wherein the subject is a human.

### Claim 24 (depends on 23)

The nucleic acid for the use according to claim 23, wherein an AAV vector comprising the nucleic acid is administered and said nucleic acid comprises a first gRNA of SEQ ID NO: 8 and a second gRNA, of SEQ ID NO:8.

### Claim 25

A nucleic acid encoding a Cas9, a first gRNA and a second gRNA, wherein the first gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 1-3 and the second gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 4-6.

### Claim 26 (depends on 25)

The nucleic acid of claim 25, further comprising:
a first and a second inverted terminal repeat sequence (ITR);
a first guide RNA selected from the group consisting of SEQ ID NOS: 1-3;
a second guide RNA selected from the group consisting of SEQ ID NOS: 4-6; and
a promoter for driving expression of the Cas9 comprising a sequence selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 27 (depends on 25, 26)

The nucleic acid of claim 25 or 26, wherein the nucleic acid encoding the Cas9, the first gRNA and second gRNA is characterized in that it comprises the following pairs of targeting domains
a) SEQ ID NO:1 and SEQ ID NO:5; or
b) SEQ ID NO:1 and SEQ ID NO:6; or
c) SEQ ID NO:2 and SEQ ID NO:4; or
d) SEQ ID NO:3 and SEQ ID NO:4; or
e) SEQ ID NO:3 and SEQ ID NO:5.

### Claim 28 (depends on 25, 26, 27)

The nucleic acid of any one of claims 25 to 27, wherein the nucleic acid encodes S. aureus Cas9.

### Claim 29 (depends on 28)

The nucleic acid of claim 28, wherein the nucleic acid comprises a Cas9 coding sequence according to SEQ ID NO: 10 or encodes a Cas9 comprising the amino acid sequence of SEQ ID NO:11.

### Claim 30 (depends on 25, 26, 27)

The nucleic acid of any one of claims 25 to 27, wherein the Cas9 is a modified Cas9.

### Claim 31 (depends on 25, 26, 27, 28, 29, 30)

The nucleic acid of any one of claims 25 to 30, wherein the Cas9 comprises at least one nuclear localization signal.

### Claim 32 (depends on 31)

The nucleic acid of claim 31, wherein the Cas9 comprises a C-terminal nuclear localization signal and an N-terminal nuclear localization signal.

### Claim 33 (depends on 25, 26, 27, 28, 29, 30, 31, 32)

The nucleic acid of any one of claims 25 to 32, wherein the Cas9 comprises a polyadenylation sequence.

### Claim 34 (depends on 28, 29)

The nucleic acid of claim 28 or 29, wherein the gRNA is a unimolecular S. aureus gRNA comprising SEQ ID NO:7 or SEQ ID NO: 8, or the corresponding two-part modular S. aureus gRNA, wherein the crRNA component comprises the underlined section and the tracrRNA component comprises the double underlined section of SEQ ID NO:7 or SEQ ID NO:8.

### Claim 35 (depends on 34)

The nucleic acid of claim 34, wherein the gRNA is a unimolecular S. aureus gRNA of SEQ ID NO:8.

### Claim 36 (depends on 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)

The nucleic acid of any one of claims 25 to 35, further comprising a promoter sequence for driving expression of the Cas9.

### Claim 37 (depends on 36)

The nucleic acid of claim 36, wherein the promoter sequence is selected from the group consisting of SEQ ID NOs: 13-15.

### Claim 38 (depends on 36, 37)

The nucleic acid of claim 36 or 37, wherein the nucleic acid comprises
a) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 5, or targeting domains that are greater than 90% similar thereto; or
b) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 6, or targeting domains that are greater than 90% similar thereto; or
c) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 4, or targeting domains that are greater than 90% similar thereto; or
d) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 4, or targeting domains that are greater than 90% similar thereto; or
e) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 5, or targeting domains that are greater than 90% similar thereto; or
f) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 5, or targeting domains that are greater than 90% similar thereto; or
g) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 6, or targeting domains that are greater than 90% similar thereto; or
h) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 4, or targeting domains that are greater than 90% similar thereto; or
i) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 4, or targeting domains that are greater than 90% similar thereto; or
j) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 5, or targeting domains that are greater than 90% similar thereto; or
k) hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 5, or targeting domains that are greater than 90% similar thereto; or
g) hGRKlpromoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 6, or targeting domains that are greater than 90% similar thereto; or.
h) hGRKlpromoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 4, or targeting domains that are greater than 90% similar thereto; or
i) hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 4, or targeting domains that are greater than 90% similar thereto; or
j) hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 5, or targeting domains that are greater than 90% similar thereto.

### Claim 39 (depends on 36)

The nucleic acid of claim 36, wherein the promoter sequence is a hGRK1 promoter.

### Claim 40 (depends on 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

An AAV vector comprising the nucleic acid of any one of claims 25 to 39.

### Claim 41 (depends on 38)

An AAV vector comprising the nucleic acid of claim 38.

### Claim 42 (depends on 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

An AAV vector comprising the nucleic acid of any one of claims 25 to 39 for use in a method of altering a cell, wherein the method comprises administering the AAV vector to a cell of the subject, thereby altering said cell in the subject.

### Claim 43 (depends on 42)

The AAV vector according to claim 42 for the use specified therein, wherein the AAV vector is administered to a retinal cell.

### Claim 44 (depends on 42)

The AAV vector according to claim 42 for the use specified therein, wherein the subject has a retinal disease.

### Claim 45

A nucleic acid encoding a Cas9, a first gRNA and a second gRNA, wherein the first gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 1-3 and the second gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 4-6 for use in a method of altering a retinal cell, wherein the method comprises administering the nucleic acid to a retinal cell in a subject.

### Claim 46 (depends on 45)

The nucleic acid for the use according to claim 45, wherein the nucleic acid further comprises:
a first and a second inverted terminal repeat sequence (ITR);
a first guide RNA comprising a targeting domain sequence selected from the group consisting of SEQ ID NOS: 1-3;
a second guide RNA comprising a targeting domain sequence selected from the group consisting of SEQ ID NOS: 4-6; and
a promoter for driving expression of the Cas9 comprising a sequence selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 47 (depends on 45, 46)

The nucleic acid for the use according to claim 45 or 46, wherein the nucleic acid encoding the Cas9, the first gRNA and second gRNA is characterized in that it comprises the following pairs of targeting domains
a) SEQ ID NO:1 and SEQ ID NO:5; or
b) SEQ ID NO:1 and SEQ ID NO:6; or
c) SEQ ID NO:2 and SEQ ID NO:4; or
d) SEQ ID NO:3 and SEQ ID NO:4; or
e) SEQ ID NO:3 and SEQ ID NO:5.

### Claim 48 (depends on 45, 46, 47)

The nucleic acid for the use of any one of claims 45 to 47, wherein the nucleic acid encodes S. aureus Cas9.

### Claim 49 (depends on 48)

The nucleic acid for the use of claim 48, wherein the nucleic acid comprises a Cas9 coding sequence according to SEQ ID NO: 10 or encodes an amino acid sequence according to SEQ ID NO:11.

### Claim 50 (depends on 45, 46, 47)

The nucleic acid for the use of any one of claims 45 to 47, wherein the Cas9 is a modified Cas9.

### Claim 51 (depends on 45, 46, 47, 48, 49, 50)

The nucleic acid for the use of any one of claims 45 to 50, wherein the Cas9 comprises at least one nuclear localization signal.

### Claim 52 (depends on 51)

The nucleic acid for the use of claim 51, wherein the Cas9 comprises a C-terminal nuclear localization signal and an N-terminal nuclear localization signal.

### Claim 53 (depends on 45, 46, 47, 48, 49, 50, 51, 52)

The nucleic acid for the use of any one of claims 45 to 52, wherein the Cas9 comprises a polyadenylation sequence.

### Claim 54 (depends on 48, 49)

The nucleic acid for the use of claim 48 or 49, wherein the gRNA is a unimolecular S. aureus gRNA comprising SEQ ID NO:7 or SEQ ID NO: 8, or the corresponding two-part modular S. aureus gRNA, wherein the crRNA component comprises the underlined section and the tracrRNA component comprises the double underlined section of SEQ ID NO:7 or SEQ ID NO:8.

### Claim 55 (depends on 54)

The nucleic acid for the use of claim 54, wherein the gRNA is a unimolecular S. aureus gRNA of SEQ ID NO:8.

### Claim 56 (depends on 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55)

The nucleic acid for the use of any one of claims 45 to 55, further comprising a promoter sequence for driving expression of the Cas9.

### Claim 57 (depends on 56)

The nucleic acid for the use of claim 56, wherein the promoter sequence is selected from the group consisting of SEQ ID NOs: 13-15.

### Claim 58 (depends on 56)

The nucleic acid for the use according to claim 56, wherein the promoter is a human g-protein-coupled receptor kinase-1 (hGRK1) promoter sequence.

### Claim 59 (depends on 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The nucleic acid for the use according to any one of claims 45 to 58 wherein the nucleic acid comprises
a) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 5, or
b) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 6, or
c) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 4, or
d) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 4, or.
e) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 5, or
f) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 5, or
g) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 6, or
h) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 4, or
i) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 4, or
j) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 5, or
k) hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 5, or
g) hGRKlpromoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 6, or.
h) hGRKlpromoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 4, or
i) hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 4, or
j) hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 5.

### Claim 60 (depends on 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)

A nucleic acid for the use according to any one of claims 45 to 59, wherein the retinal cell is a photoreceptor cell.

### Claim 61 (depends on 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

A method of altering a photoreceptor cell in a retinal explant in vitro, comprising:
contacting the retinal cell with a nucleic acid according to claims 25 to 39.

### Claim 62 (depends on 40, 41)

The AAV vector of claim 40 or 41, for use in medicine.

### Claim 63 (depends on 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 61)

The nucleic acid according to any one of claims 25 to 39, or the AAV vector for the use according to claim 61, for use in treatment of CEP290 associated disease.

### Claim 64 (depends on 2, 62)

The nucleic acid for the use according to claim 2, the nucleic acid or the AAV vector for the use according to claim 62 wherein the CEP 290 associated disease is Leber Congenital Amaurosis 10.

### Claim 65 (depends on 2, 62)

The nucleic acid for the use according to claim 2, the nucleic acid or the AAV vector for the use according to claim 62 wherein the CEP 290 associated disease is selected from the group consisting of hereditary retinopathy, retinal dystrophy, Senior Loken syndrome, Meckel Gruber syndrome, Bardet-Biedle syndrome, Joubert Syndrome, Leber Congenital Amaurosis 10.

### Claim 66 (depends on 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The nucleic acid according to any one of claims 25-39, for use in the production of a medicament.

### Claim 67

One or more recombinant viral vectors comprising one or more nucleic acids encoding a Cas9, a first gRNA and a second gRNA for use in a method of altering a nucleotide sequence of the CEP 290 target position,
wherein the method comprises contacting one or more recombinant viral vectors to the retina of a subject,
wherein (a) the first and second gRNAs are adapted to form first and second ribonucleoprotein complexes with the Cas9, and (b) the first and second ribonucleoprotein complexes are adapted to cleave first and second cellular nucleic acid sequences on first and second sides of a CEP290 target position, thereby altering a nucleotide sequence of the CEP290 target position.

### Claim 68 (depends on 67)

One or more recombinant viral vectors for the use according to claim 67, wherein the subject has CEP290 associated disease.

### Claim 69 (depends on 67, 68)

One or more recombinant viral vectors for the use according to claim 67 or 68, wherein the step of contacting the retina of the subject with one or more recombinant viral vectors comprises administering to the subject, a composition comprising the one or more recombinant viral vectors.

### Claim 70 (depends on 68)

One or more recombinant viral vectors for the use according to claim 68, wherein the composition is administered by subretinal injection,

### Claim 71 (depends on 38, 67)

One or more recombinant viral vectors for the use according to claim 67, wherein the one or more recombinant viral vectors are AAV vectors as defined in claim 38.

### Claim 72 (depends on 39, 67)

One or more recombinant viral vectors for the use according to claim 67, wherein the one or more recombinant viral vectors are AAV vectors as defined in claim 39.

### Claim 73 (depends on 67)

One or more recombinant viral vectors for the use according to claim 67, wherein the subject is a primate.

### Claim 74 (depends on 67)

One or more recombinant viral vectors for the use according to claim 67, wherein the subject is a human.

### Claim 75 (depends on 67)

One or more recombinant viral vectors for the use according to claim 67, wherein altering a nucleotide sequence of the CEP290 target position comprises forming an indel in the CEP290 target position.

### Claim 76 (depends on 67)

One or more recombinant viral vectors for the use according to claim 67, wherein altering a nucleotide sequence of the CEP290 target position comprises deleting at least part of the CEP290 target position.

### Claim 77 (depends on 67)

One or more recombinant viral vectors for the use according to claim 67, wherein altering a nucleotide sequence of the CEP290 target position comprises inverting a nucleotide sequence within the CEP290 target position.

### Claim 78 (depends on 1, 2, 3, 4)

The nucleic acid for the use according to any one of claims 1 to 4, wherein the first gRNA and second gRNA are characterized in that they comprise targeting domains differing by no more than 3 nucleotides from:
a) SEQ ID NO: 1 and SEQ ID NO 4; or
b) SEQ ID NO: 2 and SEQ ID NO 5; or
c) SEQ ID NO: 2 and SEQ ID NO 6; or
d) SEQ ID NO: 3 and SEQ ID NO 6.

### Claim 79 (depends on 1, 2)

The nucleic acid for the use of claim 1 or 2, wherein the first gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 7-8.

### Claim 80 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The nucleic acid for the use according to claims 1-15, comprising
a) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4, or
b) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 5, or
c) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 6, or
d) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 6, or.
e) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4, or
f) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 5, or
g) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 6, or
h) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 6, or
i) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4, or
j) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 5, or.
k) an hGRKlpromoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 6, or
l) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 6.

### Claim 81 (depends on 80)

The nucleic acid according to claim 80, comprising an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4.

### Claim 82 (depends on 80)

The nucleic acid according to claim 80, comprising a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4

### Claim 83

A nucleic acid encoding a Cas9, a first gRNA and a second gRNA, wherein each of the first and second gRNAs comprise a sequence having at least 90% sequence identity to one of SEQ ID NOS: 7-8.

### Claim 84 (depends on 36, 37)

The nucleic acid of claim 36 or 37, wherein the nucleic acid comprises
a) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4, or targeting domains that are greater than 90% identical thereto; or
b) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 5, or targeting domains that are greater than 90% identical thereto; or
c) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 6, or targeting domains that are greater than 90% identical thereto; or
d) a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 6, or targeting domains that are greater than 90% identical thereto; or
e) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4, or targeting domains that are greater than 90% identical thereto; or
f) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 5, or targeting domains that are greater than 90% identical thereto; or
g) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 6, or targeting domains that are greater than 90% identical thereto; or
h) an EFS promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 6, or targeting domains that are greater than 90% identical thereto; or
i) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4, or targeting domains that are greater than 90% identical thereto; or
j) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 5, or targeting domains that are greater than 90% identical thereto; or
k) an hGRKlpromoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 2 and 6, or targeting domains that are greater than 90% identical thereto; or
l) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 3 and 6, or targeting domains that are greater than 90% identical thereto.

### Claim 85 (depends on 84)

The nucleic acid according to claim 84, comprising an hGRK1 promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4, or targeting domains that are greater than 90% identical thereto.

### Claim 86 (depends on 80)

The nucleic acid according to claim 80, comprising a CMV promoter for Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs: 1 and 4, or targeting domains that are greater than 90% identical thereto.

### Claim 87 (depends on 84, 85, 86)

An AAV vector comprising the nucleic acid of any one of claims 84-86.

### Claim 88 (depends on 45, 46)

The nucleic acid for the use according to claim 45 or 46, wherein the nucleic acid encoding the Cas9, the first gRNA and second gRNA is characterized in that it comprises the following pairs of targeting domains
a) SEQ ID NO:1 and SEQ ID NO:4; or
b) SEQ ID NO:2 and SEQ ID NO:5; or
c) SEQ ID NO:2 and SEQ ID NO:6; or
d) SEQ ID NO:3 and SEQ ID NO:6.

### Claim 89 (depends on 1, 2)

The nucleic acid for the use of claim 1 or 2, wherein the Cas9 is encoded by a sequence having at least 90%, or 95% sequence identity to SEQ ID NO: 10.

### Claim 90 (depends on 1, 2)

The nucleic acid for the use of claim 1 or 2, wherein the Cas9 comprises an amino acid sequence having at least 90% or 95% sequence identity to SEQ ID NO: 11.

### Claim 91

A nucleic acid encoding a Cas9, a first gRNA and a second gRNA, wherein each of the first and second gRNAs comprise a sequence selected from the group consisting of SEQ ID NOS: 7-8, or a sequence having at least 90% sequence identity thereto.

### Claim 92 (depends on 91)

The nucleic acid of claim 91, further comprising a promoter sequence for driving expression of the Cas9, the promoter sequence selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 93 (depends on 91, 92)

The nucleic acid of any one of claims 91-92, wherein the Cas9 is a modified Cas9.

### Claim 94 (depends on 91, 92, 93)

The nucleic acid of any one of claims 91-93, wherein the Cas9 comprises at least one nuclear localization signal.

### Claim 95 (depends on 91, 92, 93, 94)

The nucleic acid of any one of claims 91-94, wherein the Cas9 comprises a polyadenylation signal.

### Claim 96 (depends on 91, 93, 94, 95)

The nucleic acid of any one of claims 91 and 93-95, further comprising:
a first and a second inverted terminal repeat sequence (ITR);
a first guide RNA selected from the group consisting of SEQ ID NOS: 1-3;
a second guide RNA selected from the group consisting of SEQ ID NOS: 4-6; and
a promoter for driving expression of the Cas9 comprising a sequence selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 97 (depends on 91, 92, 93, 94, 95, 96)

An AAV vector comprising the nucleic acid of any one of claims 91-96.

### Claim 98 (depends on 97)

The AAV vector of claim 97, for use in a method of altering a cellular nucleic acid sequence associated with an inherited retinal dystrophy,
wherein (a) the method comprises contacting one or more recombinant viral vectors to the retina of a subject, and
wherein (b) the first gRNA is adapted to form a first ribonucleoprotein complex with the Cas9, and (c) the first ribonucleoprotein complex is adapted to cleave a first cellular nucleic acid sequence associated with the inherited retinal dystrophy, thereby altering the first cellular nucleic acid sequence, and (d) the second ribonucleoprotein complex is adapted to cleave a second cellular nucleic acid sequence associated with the inherited retinal dystrophy, thereby altering the second nucleic acid sequence.

### Claim 99 (depends on 97)

The AAV vector for the use according to claim 97, further wherein (e) each of the first and second cellular nucleic acid sequences is located within or proximate to a gene associated with the inherited retinal dystrophy, and alteration of the first and second cellular nucleic acid sequences causes the deletion or inversion of an intervening sequence between the first and second cellular nucleic acid sequences.

### Claim 100 (depends on 91, 92, 93, 94, 95, 96)

The nucleic acid according to any one of claims 91-96, for use in the production of a medicament.

### Claim 101

A method of treating a subject, comprising:
administering to the subject a nucleic acid encoding a Cas9, a first gRNA and a second gRNA, each gRNA targeted to a CEP290 gene of the subject.

### Claim 102 (depends on 101)

The method of claim 101, wherein the first gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 1-3

### Claim 103 (depends on 102)

The method of claim 102, wherein the second gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 4-6.

### Claim 104 (depends on 101)

The method of claim 101, wherein the Cas9 is a modified Cas9.

### Claim 105 (depends on 101)

The method of claim 101, wherein the Cas9 comprises a nuclear localization signal.

### Claim 106 (depends on 105)

The method of claim 105, wherein the Cas9 comprises a C-terminal nuclear localization signal and an N-terminal nuclear localization signal.

### Claim 107 (depends on 101)

The method of claim 101, wherein the Cas9 comprises a polyadenylation signal.

### Claim 108 (depends on 1)

The method of claim 1, wherein the nucleic acid comprises a Cas9 coding sequence according to SEQ ID NO: 10.

### Claim 109 (depends on 101)

The method of claim 101, wherein the nucleic acid comprises a promoter sequence for driving expression of the Cas9.

### Claim 110 (depends on 109)

The method of claim 109, wherein the promoter sequence is selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 111 (depends on 101)

The method of claim 101, wherein the step of administering the nucleic acid to the subject comprises administering to the subject an adeno-associated virus (AAV) vector comprising the nucleic acid.

### Claim 112 (depends on 111)

The method of claim 111, wherein the step of administering to the subject an AAV vector comprises delivering the AAV vector to a retina of the subject.

### Claim 113 (depends on 111)

The method of claim 111, wherein the AAV vector comprises an AAV5 capsid.

### Claim 114 (depends on 111)

The method of claim 111, wherein the nucleic acid comprises:
a first and a second inverted terminal repeat sequence (ITR);
a first guide RNA selected from the group consisting of SEQ ID NOS: 1-3;
a second guide RNA selected from the group consisting of SEQ ID NOS: 4-6; and
a promoter for driving expression of the Cas9 comprising a sequence selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 115 (depends on 101)

The method of claim 101, wherein the subject is a human.

### Claim 116

A nucleic acid encoding a Cas9, a first gRNA and a second gRNA, wherein the first gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 1-3 and the second gRNA comprises a targeting domain selected from the group consisting of SEQ ID NOS: 4-6.

### Claim 117 (depends on 116)

The nucleic acid of claim 116, further comprising a promoter sequence for driving expression of the Cas9, the promoter sequence selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 118 (depends on 116)

The nucleic acid of claim 116, wherein the Cas9 is a modified Cas9.

### Claim 119 (depends on 116)

The nucleic acid of claim 116, wherein the Cas9 comprises at least one nuclear localization signal.

### Claim 120 (depends on 116)

The nucleic acid of claim 116, wherein the Cas9 comprises a polyadenylation sequence.

### Claim 121 (depends on 116)

The nucleic acid of claim 116, further comprising:
a first and a second inverted terminal repeat sequence (ITR);
a first guide RNA selected from the group consisting of SEQ ID NOS: 1-3;
a second guide RNA selected from the group consisting of SEQ ID NOS: 4-6; and
a promoter for driving expression of the Cas9 comprising a sequence selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 122 (depends on 121)

An AAV vector comprising the nucleic acid of claim 121.

### Claim 123 (depends on 116)

An AAV vector comprising the nucleic acid of claim 116.

### Claim 124 (depends on 123)

A method of altering a cell in a subject, comprising administering to the subject the AAV vector of claim 123.

### Claim 125 (depends on 124)

The method according to claim 124, wherein the step of administering the AAV vector to the subject comprises contacting a retina of the subject with the AAV vector.

### Claim 126 (depends on 124)

The method according to claim 124, wherein the subject has a retinal disease.

### Claim 127 (depends on 116)

A method of altering a retinal cell, comprising:
contacting the retinal cell with a nucleic acid according to claim 116, wherein expression of the Cas9 is driven by a promoter sequence selected from the group consisting of SEQ ID NOS: 13-15.

### Claim 128 (depends on 116)

A method of altering a photoreceptor cell, comprising:
contacting the photoreceptor cell with a nucleic acid according to claim 116, wherein expression of the Cas9 is driven by a human g-protein-coupled receptor kinase-1 (hGRK1) promoter sequence.

### Claim 129

A method of treating a subject, comprising:
contacting a retina of the subject with one or more recombinant viral vectors comprising one or more nucleic acids encoding a Cas9, a first gRNA and a second gRNA,
wherein (a) the first and second gRNAs are adapted to form first and second ribonucleoprotein complexes with the Cas9, and (b) the first and second ribonucleoprotein complexes are adapted to cleave first and second cellular nucleic acid sequences on first and second sides of a CEP290 target position, thereby altering a nucleotide sequence of the CEP290 target position.

### Claim 130 (depends on 129)

The method of claim 129, wherein the step of contacting the retina of the subject with one or more recombinant viral vectors comprising administering to the subject, by subretinal injection, a composition comprising the one or more recombinant viral vectors.

### Claim 131 (depends on 130)

The method of claim 130, wherein the step of administering the one or more recombinant viral vectors to the subject by subretinal injection comprises inserting a subretinal injection cannula into an exterior surface of the eye, advancing the subretinal injection cannula across the midline of the eye, and inserting a tip of the subretinal cannula into a subretinal space in a parafoveal region of the retina.

### Claim 132 (depends on 129)

The method of claim 129, wherein the subject is a human.

### Claim 133 (depends on 129)

The method of claim 129, wherein altering a nucleotide sequence of the CEP290 target position comprises forming an indel in the CEP290 target position.

### Claim 134 (depends on 129)

The method of claim 129, wherein altering a nucleotide sequence of the CEP290 target position comprises deleting at least part of the CEP290 target position.

### Claim 135 (depends on 129)

The method of claim 129, wherein altering a nucleotide sequence of the CEP290 target position comprises inverting a nucleotide sequence within the CEP290 target position.

### Claim 136

A method of treating a subject having an inherited retinal dystrophy, comprising:
contacting a retina of the subject with one or more recombinant viral vectors comprising one or more nucleic acids encoding a Cas9 and a first gRNA comprising a sequence having at least 90% sequence identity to a sequence selected from SEQ ID NOS: 7 and 8;
wherein (a) the first gRNA is adapted to form a first ribonucleoprotein complex with the Cas9, and (b) the first ribonucleoprotein complex is adapted to cleave a first cellular nucleic acid sequence associated with the inherited retinal dystrophy, thereby altering the first cellular nucleic acid sequence.

### Claim 137 (depends on 136)

The method of claim 136, wherein the recombinant viral vector comprises an AAV5 capsid and a viral genome encoding:
left and right ITRs having at least 90% sequence identity to SEQ ID NOS: 16 and 17;
a U6 promoter sequence having at least 90% sequence identity to SEQ ID NO: 9 and being operably coupled to a sequence encoding the first gRNA;
a promoter sequence selected from the group consisting of:
a CMV promoter sequence having at least 90% sequence identity to SEQ ID NO: 13,
an EFS promoter sequence having at least 90% sequence identity to SEQ ID NO: 14, or
an hGRK promoter sequence having at least 90% sequence identity to SEQ ID NO: 15, the promoter sequence being operably coupled to
a sequence encoding an S. aureus Cas9 comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11.

### Claim 138 (depends on 136)

The method of claim 136, wherein the subject has a retinal dystrophy selected from the group consisting of Senior-Loken syndrome, Meckel Gruber syndrome, Bardet-Biedle syndrome, Joubert Syndrome, and Leber Congenital Amaurosis.

### Claim 139 (depends on 138)

The method of claim 138, wherein the retinal dystrophy is not LCA10.

### Claim 140 (depends on 136)

The method of claim 136, wherein (c) the viral genome further comprises a sequence encoding a second gRNA, (d) the second gRNA is adapted to form a second ribonucleoprotein complex with the Cas9, and (e) the second ribonucleoprotein complex is adapted to cleave a second cellular nucleic acid sequence associated with the inherited retinal dystrophy, thereby altering the second nucleic acid sequence.

### Claim 141 (depends on 140)

The method of claim 140, wherein each of the first and second cellular nucleic acid sequences is located within or proximate to a gene associated with the inherited retinal dystrophy, and cleavage of the first and second cellular nucleic acid sequences causes the deletion or inversion of an intervening sequence between the first and second cellular nucleic acid sequences.