COMPOSITIONS AND METHODS FOR TREATING A CONGENITAL EYE DISEASE

1 . A method of editing a nucleobase of a 290-KD centrosomal protein (CEP290) polynucleotide in a cell, the method comprising contacting the cell with:

(a) a base editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or

(b) one or more polynucleotides encoding the base editor, and one or more guide polynucleotides, or one or more polynucleotides encoding the one or more guide polynucleotides, wherein the one or more guide polynucleotides target said base editor to effect an alteration of the nucleobase of the CEP290 polynucleotide in the cell.

2 . The method of claim 1 , wherein the cell is in a subject having Leber's Congenital Amaurosis-10 (LCA10), and the alteration of the nucleobase of the 290-KD centrosomal protein (CEP290) polynucleotide in the cell treats LCA10 in the subject.

3 . The method of claim 1 , wherein alteration of the nucleobase is associated with any one or more of the following:

an alteration in splicing

an increase in proper splicing of mRNA transcripts transcribed from the CEP290 polynucleotide;

an increase in levels of functional CEP290 polypeptides in the cell;

alleviating one or more symptoms of LCA10 in the subject;

slowing or halting progression of vision loss associated with LCA10 in the subject;

reducing loss of functional rod and/or cone cells associated with LCA10 in the subject.

4 . The method of claim 1 , wherein the base editor effects an alteration of a nucleobase selected from the group consisting of CEP290 c.2991+1651, CEP290 c.2991+1652, and CEP290 c.2991+1655.

5 . The method of claim 4 , wherein the base editor effects an alteration of a nucleobase selected from the group consisting of

CEP290 c.2991+1655G>A;

CEP290 c.2991+1652T>C; and

CEP290 c.2991+1652G>A;

6 . The method of claim 1 , wherein the one or more guide polynucleotides comprise a nucleic acid sequence comprising at least 10 contiguous nucleotides of a spacer corresponding to a nucleic acid sequence selected from the group consisting of:

(SEQ ID NO: 459)

AUACUCACAAUUACAAC;

(SEQ ID NO: 460)

GAUACUCACAAUUACAAC;

(SEQ ID NO: 461)

AGAUACUCACAAUUACAAC;

(SEQ ID NO: 462)

GAGAUACUCACAAUUACAAC;

(SEQ ID NO: 463)

UGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 464)

AUGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 465)

UAUCUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAGAUACU

CACAAUUACAAC;

(SEQ ID NO: 466)

AUCUCACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUGAGAU

ACUCACAAUUACAAC;

(SEQ ID NO: 467)

UAUCUCAUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAUGA

GAUACUCACAAUUACAAC;

(SEQ ID NO: 468)

CUCAUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAUGAG

AUACUCACAAUUACAAC;

(SEQ ID NO: 469)

ACUCACAAUUACAACUG;

(SEQ ID NO: 470)

UACUCACAAUUACAACUG;

(SEQ ID NO: 471)

AUACUCACAAUUACAACUG;

(SEQ ID NO: 472)

GAUACUCACAAUUACAACUG;

(SEQ ID NO: 473)

AGAUACUCACAAUUACAACUG;

(SEQ ID NO: 474)

CAAUUACAACUGGGGCC;

(SEQ ID NO: 475)

ACAAUUACAACUGGGGCC;

(SEQ ID NO: 476)

CACAAUUACAACUGGGGCC;

(SEQ ID NO: 477)

UCACAAUUACAACUGGGGCC;

(SEQ ID NO: 478)

CUCACAAUUACAACUGGGGCC;

and

(SEQ ID NO: 479)

ACUCACAAUUACAACUGGGGCC;

wherein the one or more guide polynucleotides comprise a scaffold comprising a nucleotide sequence with at least about 85% sequence identity to the following sequence:

(SEQ ID NO: 484)

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA

CUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

wherein the one or more guide polynucleotides comprise one or more of a 2′-OMe and a phosphorothioate.

7 . The method of claim 1 , further comprising contacting the cell with a vector comprising polynucleotide(s) encoding the base editor and/or the one or more guide polynucleotides.

8 . The method of claim 1 , wherein the vector is a lipid nanoparticle or an adeno-associated virus (AAV) vector.

9 . The method of claim 1 , wherein the polynucleotide(s) encoding the base editor and/or one or more guide polynucleotides comprises a promoter controlling expression of the base editor and/or one or more guide polynucleotides, wherein the promoter is selected from the group consisting of CMV, PR1.7, hG1.7, hGRK, and U6.

10 . The method of claim 1 , wherein the promoter is selected from the group consisting of PR1.7, hG1.7, and hGRK.

11 . The method of claim 1 , wherein the deaminase domain is an adenosine deaminase, a cytidine deaminase domain, or a cytidine adenosine deaminase domain.

12 . A modified cell comprising an alteration in a nucleobase of a CEP290 polynucleotide, wherein the alteration increases expression and/or activity of the encoded CEP290 polypeptide as compared to a control cell without the alteration, and wherein the cell is prepared according to the method of claim 1 .

13 . A base editor system comprising one or more polynucleotides encoding a base editor polypeptide, wherein the base editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, and one or more guide polynucleotides, or one or more polynucleotides encoding the one or more guide polynucleotides, wherein the one or more guide polynucleotides comprise a spacer sequence comprising at least 10 contiguous nucleotides of a spacer corresponding to a nucleotide sequence selected from the group consisting of:

(SEQ ID NO: 459)

AUACUCACAAUUACAAC;

(SEQ ID NO: 460)

GAUACUCACAAUUACAAC;

(SEQ ID NO: 461)

AGAUACUCACAAUUACAAC;

(SEQ ID NO: 462)

GAGAUACUCACAAUUACAAC;

(SEQ ID NO: 463)

UGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 464)

AUGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 465)

UAUCUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAGAUACU

CACAAUUACAAC;

(SEQ ID NO: 466)

AUCUCACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUGAGAU

ACUCACAAUUACAAC;

(SEQ ID NO: 467)

UAUCUCAUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAUGA

GAUACUCACAAUUACAAC;

(SEQ ID NO: 468)

CUCAUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAUGAG

AUACUCACAAUUACAAC;

(SEQ ID NO: 469)

ACUCACAAUUACAACUG;

(SEQ ID NO: 470)

UACUCACAAUUACAACUG;

(SEQ ID NO: 471)

AUACUCACAAUUACAACUG;

(SEQ ID NO: 472)

GAUACUCACAAUUACAACUG;

(SEQ ID NO: 473)

AGAUACUCACAAUUACAACUG;

(SEQ ID NO: 474)

CAAUUACAACUGGGGCC;

(SEQ ID NO: 475)

ACAAUUACAACUGGGGCC;

(SEQ ID NO: 476)

CACAAUUACAACUGGGGCC;

(SEQ ID NO: 477)

UCACAAUUACAACUGGGGCC;

(SEQ ID NO: 478)

CUCACAAUUACAACUGGGGCC;

and

(SEQ ID NO: 479)

ACUCACAAUUACAACUGGGGCC.

14 . A set of one or more polynucleotides encoding the base editor system of claim 13 , or a component thereof.

15 . A vector comprising the set of one or more polynucleotides of claim 14 .

16 . The vector of claim 15 , wherein the vector comprises a lipid nanoparticle or a viral vector.

17 . A kit comprising a base editor system comprising a base editor polypeptide, or one or more polynucleotides encoding the same, wherein the base editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, and one or more guide polynucleotides, or one or more polynucleotides encoding the one or more guide polynucleotides, wherein the one or more guide polynucleotides comprise a nucleotide sequence selected from the group consisting of:

(SEQ ID NO: 459)

AUACUCACAAUUACAAC;

(SEQ ID NO: 460)

GAUACUCACAAUUACAAC;

(SEQ ID NO: 461)

AGAUACUCACAAUUACAAC;

(SEQ ID NO: 462)

GAGAUACUCACAAUUACAAC;

(SEQ ID NO: 463)

UGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 464)

AUGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 465)

UAUCUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAGAUACU

CACAAUUACAAC;

(SEQ ID NO: 466)

AUCUCACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUGAGAU

ACUCACAAUUACAAC;

(SEQ ID NO: 467)

UAUCUCAUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAUGA

GAUACUCACAAUUACAAC;

(SEQ ID NO: 468)

CUCAUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAUGAG

AUACUCACAAUUACAAC;

(SEQ ID NO: 469)

ACUCACAAUUACAACUG;

(SEQ ID NO: 470)

UACUCACAAUUACAACUG;

(SEQ ID NO: 471)

AUACUCACAAUUACAACUG;

(SEQ ID NO: 472)

GAUACUCACAAUUACAACUG;

(SEQ ID NO: 473)

AGAUACUCACAAUUACAACUG;

(SEQ ID NO: 474)

CAAUUACAACUGGGGCC;

(SEQ ID NO: 475)

ACAAUUACAACUGGGGCC;

(SEQ ID NO: 476)

CACAAUUACAACUGGGGCC;

(SEQ ID NO: 477)

UCACAAUUACAACUGGGGCC;

(SEQ ID NO: 478)

CUCACAAUUACAACUGGGGCC;

and

(SEQ ID NO: 479)

ACUCACAAUUACAACUGGGGCC.

18 . The kit of claim 17 , further comprising written instructions for the use of the kit in the treatment of Leber congenital amaurosis 10 (LCA10).

19 . A pharmaceutical composition comprising an effective amount of a base editor system comprising

(a) a base editor polypeptide, wherein the base editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or

(b) one or more polynucleotides encoding the base editor polypeptide, and one or more guide polynucleotides, or one or more polynucleotides encoding the one or more guide polynucleotides, wherein the one or more guide polynucleotides comprise a nucleotide sequence selected from the group consisting of:

(SEQ ID NO: 459)

AUACUCACAAUUACAAC;

(SEQ ID NO: 460)

GAUACUCACAAUUACAAC;

(SEQ ID NO: 461)

AGAUACUCACAAUUACAAC;

(SEQ ID NO: 462)

GAGAUACUCACAAUUACAAC;

(SEQ ID NO: 463)

UGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 464)

AUGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 465)

UAUCUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAGAUACU

CACAAUUACAAC;

(SEQ ID NO: 466)

AUCUCACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUGAGAU

ACUCACAAUUACAAC;

(SEQ ID NO: 467)

UAUCUCAUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAUGA

GAUACUCACAAUUACAAC;

(SEQ ID NO: 468)

CUCAUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAUGAG

AUACUCACAAUUACAAC;

(SEQ ID NO: 469)

ACUCACAAUUACAACUG;

(SEQ ID NO: 470)

UACUCACAAUUACAACUG;

(SEQ ID NO: 471)

AUACUCACAAUUACAACUG;

(SEQ ID NO: 472)

GAUACUCACAAUUACAACUG;

(SEQ ID NO: 473)

AGAUACUCACAAUUACAACUG;

(SEQ ID NO: 474)

CAAUUACAACUGGGGCC;

(SEQ ID NO: 475)

ACAAUUACAACUGGGGCC;

(SEQ ID NO: 476)

CACAAUUACAACUGGGGCC;

(SEQ ID NO: 477)

UCACAAUUACAACUGGGGCC;

(SEQ ID NO: 478)

CUCACAAUUACAACUGGGGCC;

and

(SEQ ID NO: 479)

ACUCACAAUUACAACUGGGGCC.

20 . A guide polynucleotide, or a polynucleotide encoding the guide polynucleotide, wherein the guide polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(SEQ ID NO: 438)

GAUACUCACAAUUACAACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 439)

gGAUACUCACAAUUACAACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGU

UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 440)

GAGAUACUCACAAUUACAACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG

UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 441)

gGAGAUACUCACAAUUACAACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCC

GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 442)

gUGAGAUACUCACAAUUACAACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC

CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 443)

gAUGAGAUACUCACAAUUACAACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGU

CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 444)

GUAUCUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAGAUACUCACAAUUACAAC

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG

CACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 445)

gAUCUCACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUGAGAUACUCACAAUUAC

AACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG

UGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 446)

gUAUCUCAUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAUGAGAUACUCACAAU

UACAACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA

AAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 447)

gCUCAUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAUGAGAUACUCACAAUU

ACAACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA

AGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 448)

gACUCACAAUUACAACUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 449)

gUACUCACAAUUACAACUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGU

UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 450)

GAUACUCACAAUUACAACUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG

UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 451)

gGAUACUCACAAUUACAACUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCC

GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 452)

GAGAUACUCACAAUUACAACUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC

CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 453)

gCAAUUACAACUGGGGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 454)

gACAAUUACAACUGGGGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGU

UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 455)

gCACAAUUACAACUGGGGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG

UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 456)

gUCACAAUUACAACUGGGGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCC

GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 457)

gCUCACAAUUACAACUGGGGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC

CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 458)

gACUCACAAUUACAACUGGGGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGU

CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;

(SEQ ID NO: 459)

AUACUCACAAUUACAAC;

(SEQ ID NO: 460)

GAUACUCACAAUUACAAC;

(SEQ ID NO: 461)

AGAUACUCACAAUUACAAC;

(SEQ ID NO: 462)

GAGAUACUCACAAUUACAAC;

(SEQ ID NO: 463)

UGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 464)

AUGAGAUACUCACAAUUACAAC;

(SEQ ID NO: 465)

UAUCUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAGAUACU

CACAAUUACAAC;

(SEQ ID NO: 466)

AUCUCACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUGAGAU

ACUCACAAUUACAAC;

(SEQ ID NO: 467)

UAUCUCAUCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCAUGA

GAUACUCACAAUUACAAC;

(SEQ ID NO: 468)

CUCAUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAUGAG

AUACUCACAAUUACAAC;

(SEQ ID NO: 469)

ACUCACAAUUACAACUG;

(SEQ ID NO: 470)

UACUCACAAUUACAACUG;

(SEQ ID NO: 471)

AUACUCACAAUUACAACUG;

(SEQ ID NO: 472)

GAUACUCACAAUUACAACUG;

(SEQ ID NO: 473)

AGAUACUCACAAUUACAACUG;

(SEQ ID NO: 474)

CAAUUACAACUGGGGCC;

(SEQ ID NO: 475)

ACAAUUACAACUGGGGCC;

(SEQ ID NO: 476)

CACAAUUACAACUGGGGCC;

(SEQ ID NO: 477)

UCACAAUUACAACUGGGGCC;

(SEQ ID NO: 478)

CUCACAAUUACAACUGGGGCC;

and

(SEQ ID NO: 479)

ACUCACAAUUACAACUGGGGCC.