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 recombinant viral vector comprising:
(a) a first nucleotide sequence encoding a first guide RNA (gRNA) molecule comprising a first targeting domain complementary with a first target domain from the CEP290 gene;
(b) a second nucleotide sequence encoding a second gRNA molecule comprising a second targeting domain complementary with a second target domain from the CEP290 gene; and
(c) a third nucleotide sequence encoding a Cas9 molecule;
wherein the recombinant viral vector is capable of altering a cell comprising forming a first DNA double strand break between the 5′ end of the Alu repeat at c.2991+1162 to c.2991+1638 of intron 26 of the CEP290 gene and 500 nucleotides upstream of the 5′ end of the Alu repeat, and forming a second DNA double strand break between the LCA10 target position and 500 nucleotides downstream of the LCA10 target position, and
wherein the first and second DNA double strand breaks are repaired by nonhomologous end joining (NHEJ) in a manner that results in alteration of the LCA10 target position.
2. The recombinant viral vector of claim 1 , wherein the 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).
3. The recombinant viral vector of claim 2 , wherein the 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), and SEQ ID NO:568 (CEP290-504).
4. The recombinant viral vector of claim 1 , wherein the first targeting domain comprises the nucleotide sequence of SEQ ID NO:530 (CEP290-323).
5. The recombinant viral vector of claim 4 , wherein the second targeting domain comprises the nucleotide sequence of SEQ ID NO:558 (CEP290-64).
6. The recombinant viral vector of claim 1 , wherein the second targeting domain comprises the nucleotide sequence of SEQ ID NO:558 (CEP290-64).
7. The recombinant viral vector of claim 6 , wherein the recombinant viral vector is capable of delivery to a non-dividing cell.
8. The recombinant viral vector of claim 6 , wherein the third nucleotide sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 39.
9. The recombinant viral vector of claim 8 , further comprising
a fourth nucleotide sequence selected from the group consisting of:
a CMV promoter sequence having at least 90% sequence identity to SEQ ID NO: 401,
an EFS promoter sequence having at least 90% sequence identity to SEQ ID NO: 402, and
an hGRK promoter sequence having at least 90% sequence identity to SEQ ID NO: 403.
10. The recombinant viral vector of claim 9 , further comprising
a fifth nucleotide sequence comprising a U6 promoter sequence having at least 90% sequence identity to SEQ ID NO: 417 and operably coupled to the first nucleotide sequence encoding the first gRNA.
11. The recombinant viral vector of claim 9 , wherein the fourth nucleotide sequence is the hGRK promoter sequence having at least 90% sequence identity to SEQ ID NO: 403.
12. The recombinant viral vector of claim 6 , wherein the first gRNA comprises a sequence having at least 90% sequence identity to SEQ ID NO: 418.
13. The recombinant viral vector of claim 6 , wherein the recombinant viral vector is selected from the group consisting of an AAV vector, an adenovirus vector, a vaccinia virus vector, and a herpes simplex virus vector.
14. The recombinant viral vector of claim 13 , wherein the recombinant viral vector is the AAV vector.
15. A recombinant viral vector comprising
a first nucleotide sequence encoding a first guide RNA (gRNA) molecule comprising a first targeting domain complementary with a first target domain from the CEP290 gene, the 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
a second nucleotide sequence encoding a Cas9 molecule; and
wherein the first gRNA is adapted to form a first ribonucleoprotein complex with the Cas9 molecule, and the first ribonucleoprotein complex is adapted to cleave a sequence in the CEP290 gene.
16. The recombinant viral vector of claim 15 , wherein the first targeting domain comprises the nucleotide sequence of SEQ ID NO:530 (CEP290-323).
17. The recombinant viral vector of claim 16 , further comprising
a third nucleotide sequence encoding a second gRNA molecule comprising a second targeting domain complementary with a second target domain from the CEP290 gene, the second targeting domain comprising a nucleotide sequence of SEQ ID NO:558 (CEP290-64).
18. The recombinant viral vector of claim 17 , wherein the recombinant viral vector is capable of delivery to a non-dividing cell.
19. The recombinant viral vector of claim 17 , wherein the Cas9 molecule comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26.
20. The recombinant viral vector of claim 19 , further comprising
a fourth nucleotide sequence selected from the group consisting of:
a CMV promoter sequence having at least 90% sequence identity to SEQ ID NO: 401,
an EFS promoter sequence having at least 90% sequence identity to SEQ ID NO: 402, and
an hGRK promoter sequence having at least 90% sequence identity to SEQ ID NO: 403.
21. The recombinant viral vector of claim 20 , wherein the fourth nucleotide sequence is the hGRK promoter sequence having at least 90% sequence identity to SEQ ID NO: 403.
22. The recombinant viral vector of claim 21 , wherein the first gRNA comprises a sequence having at least 90% sequence identity to SEQ ID NO: 418.
23. The recombinant viral vector of claim 22 , wherein the recombinant viral vector 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 recombinant viral vector of claim 23 , wherein the recombinant viral vector is the AAV vector.
25. The recombinant viral vector of claim 15 , further comprising
a third nucleotide sequence encoding a second gRNA molecule comprising a second targeting domain complementary with a second target domain from the CEP290 gene, the second targeting domain comprising 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).
26. The recombinant viral vector of claim 25 , wherein the second targeting domain comprises a nucleotide sequence of SEQ ID NO:558 (CEP290-64).
27. The recombinant viral vector of claim 25 , further comprising
a fourth nucleotide sequence selected from the group consisting of:
a CMV promoter sequence having at least 90% sequence identity to SEQ ID NO: 401,
an EFS promoter sequence having at least 90% sequence identity to SEQ ID NO: 402, and
an hGRK promoter sequence having at least 90% sequence identity to SEQ ID NO: 403.
28. The recombinant viral vector of claim 27 , wherein the fourth nucleotide sequence is the hGRK promoter sequence having at least 90% sequence identity to SEQ ID NO: 403.
29. The recombinant viral vector of claim 15 , wherein the recombinant viral vector is capable of delivery to a non-dividing cell.
30. The recombinant viral vector of claim 15 , wherein the Cas9 molecule comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26.
31. The recombinant viral vector of claim 15 , wherein the first gRNA comprises a sequence having at least 90% sequence identity to SEQ ID NO: 418.
32. The recombinant viral vector of claim 15 , wherein the recombinant viral vector is selected from the group consisting of an AAV vector, an adenovirus vector, a vaccinia virus vector, and a herpes simplex virus vector.
33. The recombinant viral vector of claim 32 , wherein the recombinant viral vector is the AAV vector.
34. A recombinant viral vector comprising
a first nucleotide sequence encoding a first guide RNA (gRNA) molecule comprising a first targeting domain complementary with a first target domain from the CEP290 gene, the first targeting domain comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:558 (CEP290-64), 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
a second nucleotide sequence encoding a Cas9 molecule;
wherein the first gRNA is adapted to form a first ribonucleoprotein complex with the Cas9 molecule, and the first ribonucleoprotein complex is adapted to cleave a sequence in the CEP290 gene.
35. The recombinant viral vector of claim 34 , wherein the first targeting domain comprises the nucleotide sequence of SEQ ID NO:558 (CEP290-64).
36. The recombinant viral vector of claim 34 , further comprising
a third nucleotide sequence encoding a second gRNA molecule comprising a second targeting domain complementary with a second target domain from the CEP290 gene, the second targeting domain comprising 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).
37. The recombinant viral vector of claim 36 , wherein the second targeting domain comprises a nucleotide sequence of SEQ ID NO:530 (CEP290-323).
38. The recombinant viral vector of claim 36 , further comprising
a fourth nucleotide sequence selected from the group consisting of:
a CMV promoter sequence having at least 90% sequence identity to SEQ ID NO: 401,
an EFS promoter sequence having at least 90% sequence identity to SEQ ID NO: 402, and
an hGRK promoter sequence having at least 90% sequence identity to SEQ ID NO: 403.
39. The recombinant viral vector of claim 34 , wherein the second nucleotide sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 39.
40. The recombinant viral vector of claim 34 , wherein the first gRNA comprises a sequence having at least 90% sequence identity to SEQ ID NO: 418.
41. The recombinant viral vector of claim 34 , wherein the recombinant viral vector is selected from the group consisting of an AAV vector, an adenovirus vector, a vaccinia virus vector, and a herpes simplex virus vector.