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 altering a cell comprising contacting said cell with:
(a) a viral vector comprising a nucleotide sequence encoding a first gRNA molecule, wherein the first gRNA molecule comprises a targeting domain which is complementary with a first target domain from the CEP290 gene, and wherein said 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
(b) 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 contacting results in NHEJ-mediated alteration of the LCA10 target position.
2. The method of claim 1 , further comprising contacting said cell with (c) a viral vector comprising a nucleotide sequence encoding a second gRNA molecule, wherein the second gRNA molecule comprises a targeting domain which is complementary with a second target domain from the CEP290 gene, wherein said 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.
3. The method of claim 2 , 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.
4. The method of claim 2 , wherein said targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:530 (CEP290-323) and said 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).
5. The method of claim 2 , wherein said targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:468 (CEP290-490) and said 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).
6. The method of claim 2 , wherein said targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:538 (CEP290-492) and said 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).
7. The method of claim 1 , wherein said cell is from a subject suffering from LCA10, or from a subject having a mutation at the LCA10 target position of the CEP290 gene.
8. The method of claim 1 , wherein the contacting is performed in vivo.
9. 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.
10. The method of claim 9 , 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 AAV vectors.
11. The method of claim 1 , wherein said NHEJ-mediated alteration is introduction of an indel or deletion of a genomic sequence.
12. The method of claim 1 , wherein the contacting is performed ex vivo.
13. 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.
14. 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.
15. A method of altering a cell comprising contacting the cell with a recombinant viral particle comprising:
(a) a nucleotide sequence encoding a first gRNA molecule comprising a targeting domain which is complementary with a first target domain from the CEP290 gene, wherein said 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
(b) a nucleotide sequence encoding a Cas9 molecule;
wherein said viral particle is capable of delivery to a non-dividing cell, and wherein said contacting results in NHEJ-mediated alteration of the LCA10 target position.
16. 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.
17. The method of claim 16 , wherein said recombinant viral particle is an AAV viral particle.
18. The method of claim 15 , wherein said viral particle further comprises a nucleotide sequence encoding a second gRNA molecule comprising a targeting domain which is complementary with a second target domain from the CEP290 gene, wherein said 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 targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:530 (CEP290-323) and said 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 targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:468 (CEP290-490) and said 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 targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:538 (CEP290-492) and said 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. The method of claim 15 , wherein said NHEJ-mediated alteration is introduction of an indel or deletion of a genomic sequence.
23. A CRISPR-Cas9 mediated method 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 said 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, wherein the first and second DNA double strand breaks are repaired by NHEJ in a manner that results in alteration of the LCA10 target position.
24. The method of claim 23 , wherein the step of forming the first and second DNA double strand breaks comprises contacting the cell with a first and a second gRNA molecule configured to form a first and a second complex with a Cas9 molecule, respectively, said first complex configured to form the first DNA double strand break and said second complex configured to form the second DNA double strand break.
25. The method of claim 24 , wherein the step of contacting the cell with the first and second gRNA molecules comprises contacting the cell with a first recombinant viral vector comprising a nucleotide sequence encoding the first gRNA molecule and a second recombinant viral vector comprising a nucleotide sequence encoding the second gRNA molecule, wherein said first and second recombinant viral vectors are capable of delivery to a non-dividing cell.
26. The method of claim 24 , wherein said first gRNA molecule comprises a targeting domain comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:530 (CEP290-323) and SEQ ID NO:555 (CEP290-485).
27. The method of claim 24 , wherein said second gRNA molecule comprises a 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:460 (CEP290-496), and SEQ ID NO:586 (CEP290-502).
28. The method of claim 24 , wherein said first gRNA molecule comprises a targeting domain comprising the nucleotide sequence of SEQ ID NO:530 (CEP290-323) and said second gRNA molecule comprises a targeting domain comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:558 (CEP290-64) and SEQ ID NO:2321 (CEP290-11).
29. The method of claim 24 , wherein the Cas9 molecule is an S. aureus Cas9 molecule.
30. The method of claim 24 , wherein the step of contacting the cell with the first and second gRNA molecules comprises contacting the cell with a recombinant viral vector comprising a nucleotide sequence encoding the first gRNA molecule and a nucleotide sequence encoding the second gRNA molecule, wherein said recombinant viral vector is capable of delivery to a non-dividing cell.
31. The method of claim 30 , wherein the recombinant viral vector further comprises a nucleotide sequence encoding the Cas9 molecule.
32. The method of claim 23 , wherein said alteration results in loss or destruction of a cryptic splice site resulting from a mutation at the LCA10 position.
33. A method of altering a cell comprising contacting said cell with:
(a) a viral vector encoding a first gRNA molecule comprising a targeting domain which is complementary with a first target domain from the CEP290 gene, wherein said 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
(b) a Cas9 molecule;
wherein said contacting results in NHEJ-mediated introduction of an indel or deletion comprising the LCA10 target position.
34. The method of claim 33 , further comprising contacting said cell with (c) a viral vector encoding a second gRNA molecule comprising a targeting domain complementary with a second target domain from the CEP290 gene, wherein said 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).
35. The method of claim 34 , wherein said targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:530 (CEP290-323) and said 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).
36. The method of claim 34 , wherein said targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:468 (CEP290-490) and said 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).
37. The method of claim 34 , wherein said targeting domain of said first gRNA molecule comprises the nucleotide sequence of SEQ ID NO:538 (CEP290-492) and said 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).
38. The method of claim 30 or claim 25 , wherein said recombinant viral vector(s) are selected from the group consisting of an AAV vector, an adenovirus vector, a vaccinia virus vector, and a herpes simplex virus vector.
39. The method of claim 38 , wherein said recombinant viral vector(s) are AAV vectors.