CRISPR/CAS-related methods and compositions for treating Leber's Congenital Amaurosis 10 (LCA10)
CRISPR/CAS-related compositions and methods for treatment of Leber's Congenital Amaurosis 10 (LCA10) are disclosed.
1. A method of treating LCA10 in a subject in need thereof comprising administering to said subject:
(i) a viral vector comprising a nucleotide sequence encoding a first gRNA molecule comprising a first targeting domain complementary with a first target domain from the CEP290 gene, wherein said first targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:530 (CEP290-323), SEQ ID NO:555 (CEP290-485), SEQ ID NO:468 (CEP290-490), and SEQ ID NO:538 (CEP290-492); and
(ii) a viral vector comprising a nucleotide sequence encoding a Cas9 molecule;
wherein said viral vector comprising a nucleotide sequence encoding a first gRNA molecule and said viral vector comprising a nucleotide sequence encoding a Cas9 molecule are capable of delivery to a non-dividing cell, and wherein said administration results in NHEJ-mediated alteration of the LCA10 target position in one or more cells of said subject.
2. The method of claim 1 , wherein said NHEJ-mediated alteration is introduction of an indel or deletion of a genomic sequence.
3. The method of claim 1 , wherein said viral vector comprising a nucleotide sequence encoding a first gRNA molecule and said viral vector comprising a nucleotide sequence encoding a Cas9 molecule are selected from the group consisting of an AAV vector, an adenovirus vector, a vaccinia virus vector, and a herpes simplex virus vector.
4. The method of claim 1 , wherein the viral vector comprising a nucleotide sequence encoding a first gRNA molecule and the viral vector comprising a nucleotide sequence encoding a Cas9 molecule are the same viral vector.
5. The method of claim 1 , wherein the viral vector comprising a nucleotide sequence encoding a first gRNA molecule and the viral vector comprising a nucleotide sequence encoding a Cas9 molecule are separate viral vectors.
6. The method of claim 1 , further comprising contacting said cell with (c) a viral vector comprising a nucleotide sequence encoding a second gRNA molecule comprising a second targeting domain complementary with a second target domain from the CEP290 gene, wherein said second targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:558 (CEP290-64), SEQ ID NO:2321 (CEP290-11), SEQ ID NO:2312 (CEP290-230), SEQ ID NO:460 (CEP290-496), SEQ ID NO:586 (CEP290-502), and SEQ ID NO:568 (CEP290-504), and wherein said viral vector comprising a nucleotide sequence encoding a second gRNA molecule is capable of delivery to a non-dividing cell.
7. The method of claim 6 , wherein said first targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:530 (CEP290-323) and said second targeting domain of said second gRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:558 (CEP290-64) and SEQ ID NO:2321 (CEP290-11).
8. The method of claim 6 , wherein said first targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:468 (CEP290-490) and said second targeting domain of said second gRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:460 (CEP290-496), SEQ ID NO:586 (CEP290-502), and SEQ ID NO:568 (CEP290-504).
9. The method of claim 6 , wherein said first targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:538 (CEP290-492) and said second targeting domain of said second gRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:586 (CEP290-502) and SEQ ID NO:568 (CEP290-504).
10. The method of claim 6 , wherein said viral vector comprising a nucleotide sequence encoding a second gRNA molecule is selected from the group consisting of an AAV vector, an adenovirus vector, a vaccinia virus vector, and a herpes simplex virus vector.
11. The method of claim 6 , wherein the viral vector comprising a nucleotide sequence encoding a first gRNA molecule and the viral vector comprising a nucleotide sequence encoding a Cas9 molecule are the same viral vector.
12. The method of claim 6 , wherein the viral vector comprising a nucleotide sequence encoding a first gRNA molecule and the viral vector comprising a nucleotide sequence encoding a second gRNA molecule are the same viral vector.
13. The method of claim 6 , wherein the viral vector comprising a nucleotide sequence encoding a second gRNA molecule and the viral vector comprising a nucleotide sequence encoding a Cas9 molecule are the same viral vector.
14. The method of claim 6 , wherein the viral vector comprising a nucleotide sequence encoding a first gRNA molecule, the viral vector comprising a nucleotide sequence encoding a second gRNA molecule, and the viral vector comprising a nucleotide sequence encoding a Cas9 molecule are all the same viral vector.
15. A method of treating LCA10 in a subject in need thereof comprising administering to said subject a recombinant viral particle comprising:
(i) a nucleotide sequence encoding a first gRNA molecule comprising a first targeting domain complementary with a first target domain from the CEP290 gene, wherein said first targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:530 (CEP290-323), SEQ ID NO:555 (CEP290-485), SEQ ID NO:468 (CEP290-490), and SEQ ID NO:538 (CEP290-492); and
(ii) a nucleotide sequence encoding a Cas9 molecule;
wherein said viral particle is capable of delivery to a non-dividing cell, and wherein said administration results in NHEJ-mediated alteration of the LCA10 target position in one or more cells of said subject.
16. The method of claim 15 , wherein said NHEJ-mediated alteration is introduction of an indel or deletion of a genomic sequence.
17. The method of claim 15 , wherein said recombinant viral particle is selected from the group consisting of an AAV viral particle, an adenovirus viral particle, a vaccinia virus viral particle, and a herpes simplex virus viral particle.
18. The method of claim 15 , wherein said viral particle further comprises a nucleotide sequence encoding a second gRNA molecule comprising a second targeting domain complementary with a second target domain from the CEP290 gene, wherein said second targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:558 (CEP290-64), SEQ ID NO:2321 (CEP290-11), SEQ ID NO:2312 (CEP290-230), SEQ ID NO:460 (CEP290-496), SEQ ID NO:586 (CEP290-502), and SEQ ID NO:568 (CEP290-504).
19. The method of claim 18 , wherein said first targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:530 (CEP290-323) and said second targeting domain of said second gRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:558 (CEP290-64) and SEQ ID NO:2321 (CEP290-11).
20. The method of claim 18 , wherein said first targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:468 (CEP290-490) and said second targeting domain of said second gRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:460 (CEP290-496), SEQ ID NO:586 (CEP290-502), and SEQ ID NO:568 (CEP290-504).
21. The method of claim 18 , wherein said first targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:538 (CEP290-492) and said second targeting domain of said second gRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:586 (CEP290-502) and SEQ ID NO:568 (CEP290-504).
22. A method of treating LCA10 in a subject in need thereof comprising administering to said subject:
(i) a viral vector encoding a first gRNA molecule comprising a first targeting domain complementary with a first target domain from the CEP290 gene, wherein said first targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:530 (CEP290-323), SEQ ID NO:555 (CEP290-485), SEQ ID NO:468 (CEP290-490), and SEQ ID NO:538 (CEP290-492), and wherein said viral vector is capable of delivery to a non-dividing cell; and
(ii) a Cas9 molecule;
wherein said administration results in NHEJ-mediated introduction of an indel or deletion comprising the LCA10 target position in one or more cells of said subject.
23. The method of claim 22 , wherein said viral vector encoding a first gRNA molecule is selected from the group consisting of an AAV vector, an adenovirus vector, a vaccinia virus vector, and a herpes simplex virus vector.
24. The method of claim 22 , further comprising contacting said cell with (c) a viral vector encoding a second gRNA molecule comprising a second targeting domain complementary with a second target domain from the CEP290 gene, wherein said second targeting domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:558 (CEP290-64), SEQ ID NO:2321 (CEP290-11), SEQ ID NO:2312 (CEP290-230), SEQ ID NO:460 (CEP290-496), SEQ ID NO:586 (CEP290-502), and SEQ ID NO:568 (CEP290-504).
25. The method of claim 24 , wherein said first targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:530 (CEP290-323) and said second targeting domain of said second gRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:558 (CEP290-64) and SEQ ID NO:2321 (CEP290-11).
26. The method of claim 24 , wherein said first targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:468 (CEP290-490) and said second targeting domain of said second gRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:460 (CEP290-496), SEQ ID NO:586 (CEP290-502), and SEQ ID NO:568 (CEP290-504).
27. The method of claim 24 , wherein said first targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:538 (CEP290-492) and said second targeting domain of said second gRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:586 (CEP290-502) and SEQ ID NO:568 (CEP290-504).
28. The method of claim 24 , wherein said viral vector encoding a second gRNA molecule is selected from the group consisting of an AAV vector, an adenovirus vector, a vaccinia virus vector, and a herpes simplex virus vector.
29. The method of claim 24 , wherein said viral vector encoding a first gRNA molecule and said viral vector encoding a second gRNA molecule are the same viral vector.