NZ782841B2 - Therapeutic targets for the correction of the human dystrophin gene by gene editing and methods of use - Google Patents

Therapeutic targets for the correction of the human dystrophin gene by gene editing and methods of use

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Publication number
NZ782841B2
NZ782841B2 NZ782841A NZ78284116A NZ782841B2 NZ 782841 B2 NZ782841 B2 NZ 782841B2 NZ 782841 A NZ782841 A NZ 782841A NZ 78284116 A NZ78284116 A NZ 78284116A NZ 782841 B2 NZ782841 B2 NZ 782841B2
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New Zealand
Prior art keywords
seq
binds
grna molecule
nucleotide sequence
targeting domain
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NZ782841A
Other versions
NZ782841A (en
Inventor
Charles A Gersbach
Hamm Jacqueline N Robinson
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Duke University
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Application filed by Duke University filed Critical Duke University
Publication of NZ782841A publication Critical patent/NZ782841A/en
Publication of NZ782841B2 publication Critical patent/NZ782841B2/en

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Abstract

Disclosed herein are therapeutic targets for the correction of the human dystrophin gene by gene editing and methods of use.

Claims (58)

1. A DNA targeting composition , the DNA targeting composition comprising a Staphylococcus aureus Clustered Regularly Interspaced Short Palindromic Repeats associated 9 (Cas9) molecule and a first gRNA molecule and a second gRNA molecule, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of: (i) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 1, and a second gRNA le sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 2; (ii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (iii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a ing domain that binds to the tide sequence of SEQ ID NO: 19; (iv) a first gRNA molecule sing a ing domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; (v) a first gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (vi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (vii) a first gRNA le sing a ing domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; (viii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; 21517931_1 (GHMatters) P108506.NZ.1 (ix) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (x) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 15; (xi) a first gRNA le comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 18; and (xii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 41, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 42.
2. The DNA targeting composition of claim 1, wherein the Cas protein comprises a Cas9 molecule that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25).
3. The DNA ing composition of claim 1, wherein the Cas protein comprises a Staphylococcus aureus Cas9 molecule having an amino acid sequence of SEQ ID NO: 45.
4. The DNA targeting composition of claim 1, wherein the DNA targeting composition ses a nucleotide sequence of SEQ ID NO: 83, a nucleotide sequence of SEQ ID NO: 84, a nucleotide sequence of SEQ ID NO: 37, and/or a nucleotide sequence of SEQ ID NO: 38.
5. An isolated cleotide encoding the DNA targeting composition of claim 1.
6. A vector encoding the DNA targeting ition of claim 1.
7. A vector encoding: (a) a first guide RNA (gRNA) molecule, (b) a second gRNA molecule, and (c) at least one Staphylococcus aureus Cas9 molecule that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25), 31_1 (GHMatters) P108506.NZ.1 wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of: (i) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 1, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 2; (ii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (iii) a first gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (iv) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; (v) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (vi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (vii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA le sing a targeting domain that binds to the nucleotide ce of SEQ ID NO: 18; (viii) a first gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (ix) a first gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a ing domain that binds to the tide sequence of SEQ ID NO: 19; (x) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15; 31_1 (GHMatters) P108506.NZ.1 (xi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; and (xii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 41, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 42.
8. The vector of claim 7, wherein the vector is ured to form a first and a second double strand break in a first and a second intron flanking exon 51 of the human DMD gene.
9. The vector of claim 8, wherein the first gRNA molecule and the second gRNA le are selected from the group consisting of: (i) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 1, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 2; (ii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (iii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (iv) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 18; (v) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (vi) a first gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a ing domain that binds to the tide ce of SEQ ID NO: 19; (vii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; 21517931_1 (GHMatters) P108506.NZ.1 (viii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 4; (ix) a first gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (x) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 15; and (xi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 18.
10. The vector of claim 7, wherein the vector is a viral vector.
11. The vector of claim 10, wherein the vector is an Adeno-associated virus (AAV) vector.
12. The vector of claim 11, wherein the AAV vector is an AAV8 vector or an AAV9 vector.
13. The vector of claim 7, wherein the vector ses a tissue-specific promoter operably linked to the nucleotide sequence encoding the first gRNA molecule, the second gRNA molecule, and/or the Cas9 molecule.
14. The vector of claim 13, wherein the tissue-specific er is a muscle specific er.
15. An isolated cell comprising the DNA targeting composition of any one of claims 1-4.
16. A kit comprising the DNA targeting composition of any one of claims 1-4.
17. Use of a Staphylococcus aureus Clustered Regularly Interspaced Short romic Repeats associated 9 (Cas9) molecule and a first gRNA molecule and a second gRNA molecule in the manufacture of a medicament for correcting a mutant dystrophin gene in a human cell, wherein the first gRNA le and the second gRNA molecule are selected from the group consisting 21517931_1 (GHMatters) P108506.NZ.1 (i) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 1, and a second gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 2; (ii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule sing a ing domain that binds to the nucleotide sequence of SEQ ID NO: 4; (iii) a first gRNA molecule sing a targeting domain that binds to the tide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (iv) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; (v) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (vi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (vii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 18; (viii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (ix) a first gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 19; (x) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15; (xi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; and 21517931_1 (GHMatters) P108506.NZ.1 (xii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 41, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 42.
18. Use of a Staphylococcus aureus Clustered Regularly Interspaced Short Palindromic Repeats ated 9 (Cas9) molecule and a first gRNA molecule and a second gRNA molecule in the cture of a medicament for genome editing a mutant dystrophin gene in a human subject, wherein the first gRNA molecule and the second gRNA molecule are ed from the group consisting of: (i) a first gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 1, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 2; (ii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (iii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (iv) a first gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 18; (v) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (vi) a first gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (vii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 18; (viii) a first gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; 21517931_1 (GHMatters) P108506.NZ.1 (ix) a first gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 19; (x) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15; (xi) a first gRNA molecule comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 11, and a second gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; and (xii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 41, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 42.
19. Use of the DNA targeting composition of any one of claims 1-4 in the manufacture of a medicament for correcting a mutant dystrophin gene in a human cell.
20. Use of the DNA targeting composition of any one of claims 1-4 in the manufacture of a medicament for genome editing a mutant dystrophin gene in a human subject.
21. The use of claim 18 or 20, wherein the DNA targeting composition is to be administered to the subject intramuscularly, enously, or a combination thereof.
22. The use of any one of claims 17-20, wherein correcting or genome editing the mutant phin gene comprises nuclease-mediated non-homologous end joining.
23. Use of a DNA targeting composition comprising a lococcus aureus Clustered Regularly Interspaced Short Palindromic Repeats associated 9 (Cas9) molecule and a first gRNA molecule and a second gRNA molecule in the manufacture of a medicament for treating a human subject in need thereof having a mutant dystrophin gene, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of: (i) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 1, and a second gRNA le comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 2; 21517931_1 (GHMatters) P108506.NZ.1 (ii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 4; (iii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (iv) a first gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 15, and a second gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; (v) a first gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (vi) a first gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (vii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA le comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 18; (viii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (ix) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (x) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15; (xi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 18; and (xii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 41, and a second gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 42. 21517931_1 (GHMatters) P108506.NZ.1
24. Use of the DNA targeting composition of any one of claims 1-4 in the manufacture of a medicament for ng a human t in need thereof having a mutant dystrophin gene.
25. Use of a modified adeno-associated viral vector in the manufacture of a medicament for genome editing a mutant dystrophin gene in a human subject, the modified associated viral vector encoding the DNA targeting composition of any one of claims 1-4, wherein the Cas9 molecule izes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25).
26. The use of claim 25, wherein the modified adeno-associated viral vector comprises the nucleotide ce set forth in SEQ ID NO: 39 or SEQ ID NO: 40.
27. Use of: (a) a first vector comprising a polynucleotide sequence ng a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first Staphylococcus aureus Cas9 molecule that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25), and (b) a second vector comprising a polynucleotide sequence encoding a second gRNA molecule and a polynucleotide sequence encoding a second Staphylococcus aureus Cas9 molecule that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25), in the manufacture of a ment for deleting a segment of a dystrophin gene comprising exon 51 in a human subject, wherein the first vector and second vector are configured to form a first and a second double strand break in a first intron and a second intron flanking exon 51 of the human DMD gene, respectively, thereby ng a segment of the dystrophin gene comprising exon 51, and wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting (i) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 1, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 2; (ii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA le comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 4; 21517931_1 (GHMatters) P108506.NZ.1 (iii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (iv) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; (v) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA le comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 4; (vi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 19; (vii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 14, and a second gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; (viii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (ix) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (x) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15; (xi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 18; and (xii) a first gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 41, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 42.
28. The use of claim 27, wherein the segment has a length of about 50 base pairs to about 2,000 base pairs. 21517931_1 (GHMatters) P108506.NZ.1
29. The use of claim 28, wherein the segment has a length of about 118 base pairs, about 233 base pairs, about 326 base pairs, about 766 base pairs, about 805 base pairs, or about 1611 base pairs.
30. The use of claim 27, wherein the first Cas9 molecule and the second Cas9 le are the same.
31. The use of claim 27, wherein the first Cas9 le and the second Cas9 molecule is a mutant Staphylococcus aureus Cas9 molecule.
32. The use of claim 27, wherein the first Cas9 molecule and the second Cas9 molecule are different.
33. The use of claim 27, wherein the first Cas9 molecule and/or the second Cas9 molecule comprises a SaCas9 molecule having an amino acid sequence of SEQ ID NO: 45.
34. The use of claim 27, wherein the first vector and/or the second vector is a viral vector.
35. The use of claim 34, wherein the first vector and/or the second vector is an Adenoassociated virus (AAV) vector.
36. The use of claim 35, wherein the AAV vector is an AAV8 vector or an AAV9 vector.
37. The use of claim 27, wherein the first vector comprises a nucleotide sequence set forth in SEQ ID NO: 39 and the second vector comprises a nucleotide sequence set forth in SEQ ID NO:
38. The use of claim 27, n the medicament is for ng Duchenne Muscular Dystrophy.
39. The use of claim 27, wherein the DNA targeting composition is configured to form a first and a second double strand break in a first and a second intron flanking exon 51 of the human dystrophin gene, respectively, thereby deleting a segment of the dystrophin gene comprising exon 51, wherein the first gRNA molecule and the second gRNA molecule are selected from the group ting of: 21517931_1 (GHMatters) P108506.NZ.1 (i) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 1, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 2; (ii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (iii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (iv) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 18; (v) a first gRNA molecule comprising a ing domain that binds to the nucleotide ce of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (vi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (vii) a first gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; (viii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 4; (ix) a first gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule sing a targeting domain that binds to the nucleotide ce of SEQ ID NO: 19; (x) a first gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15; and (xi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18. 21517931_1 (GHMatters) P108506.NZ.1
40. The use of claim 39, wherein the mutant dystrophin gene comprises a premature stop codon, disrupted reading frame, an aberrant splice acceptor site, or an aberrant splice donor site.
41. The use of claim 39, wherein the mutant dystrophin gene comprises a frameshift mutation which causes a premature stop codon and a truncated gene t.
42. The use of claim 39, wherein the mutant dystrophin gene comprises a deletion of one or more exons which disrupts the reading frame.
43. The use of claim 39, wherein the correction of the mutant dystrophin gene ses a deletion of a premature stop codon, correction of a ted reading frame, or modulation of splicing by disruption of a splice acceptor site or disruption of a splice donor ce.
44. The use of claim 39, wherein the tion of the mutant dystrophin gene comprises on of exon 51.
45. The use of claim 39, wherein the correction of the mutant phin gene comprises homology-directed repair.
46. The use of claim 45, wherein the DNA targeting composition further comprises a donor
47. The use of claim 39, wherein the correction of the mutant dystrophin gene comprises nuclease mediated non-homologous end joining.
48. The use of claim 39, wherein the correction of the mutant dystrophin gene is in a myoblast cell.
49. The use of claim 39, wherein the subject suffers from Duchenne muscular dystrophy.
50. The use of claim 39, wherein the correction of the mutant phin gene is in a myoblast cell in a human subject suffering from Duchenne muscular dystrophy. 21517931_1 (GHMatters) P108506.NZ.1
51. The use of claim 39, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of: (i) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 1, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 2; (ii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; and (iii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule sing a targeting domain that binds to the nucleotide ce of SEQ ID NO: 19.
52. Use of: (a) a first vector comprising a polynucleotide sequence encoding a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first Staphylococcus aureus Cas9 molecule that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25), and (b) a second vector comprising a polynucleotide sequence encoding a second gRNA molecule and a polynucleotide sequence encoding a second Staphylococcus aureus Cas9 le that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25), in the manufacture of a medicament for treating a human subject in need thereof having a mutant dystrophin gene, wherein the vector is configured to form a first and a second double strand break in a first and a second intron ng exon 51 of the human dystrophin gene, respectively, thereby deleting a segment of the dystrophin gene sing exon 51, and n the first gRNA molecule and the second gRNA molecule are selected from the group consisting of: (i) a first gRNA molecule comprising a ing domain that binds to the nucleotide sequence of SEQ ID NO: 1, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 2; (ii) a first gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; 21517931_1 (GHMatters) P108506.NZ.1 (iii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide ce of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (iv) a first gRNA molecule sing a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; (v) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (vi) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (vii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA le comprising a targeting domain that binds to the tide sequence of SEQ ID NO: 18; (viii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 4; (ix) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 19; (x) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 14, and a second gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 15; (xi) a first gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 11, and a second gRNA le comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 18; and (xii) a first gRNA molecule comprising a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 41, and a second gRNA molecule comprising a ing domain that binds to the nucleotide ce of SEQ ID NO: 42.
53. The use of claim 52, wherein the human subject is suffering from Duchenne muscular 21517931_1 ters) P108506.NZ.1
54. The use of claim 52, wherein the first vector and second vector are to be administered to the human subject intramuscularly, intravenously, or a combination thereof.
55. A method of generating a transgenic rodent embryo having a human dystrophin gene (hDMD) with an exon 52 on (?52), the method comprising administering to a rodent embryo the DNA targeting composition of any one of claims 1-4, wherein the first gRNA molecule comprises a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 41 and the second gRNA molecule comprises a targeting domain that binds to the nucleotide sequence of SEQ ID NO: 42, to delete exon 52 of the human dystrophin gene.
56. The method of claim 55, wherein the rodent embryo is a mouse embryo.
57. The method of claim 55, wherein the transgenic rodent embryo is heterozygous hDMD or heterozygous hDMD-?52.
58. The method of claim 55, r comprising administering to the rodent embryo a Cas n sing an amino acid sequence set forth in SEQ ID NO: 27. 21517931_1 (GHMatters) P108506.NZ.1 \\ N ma?a wmim?w VVVVV Aw,MW, “$523 m am 3mg», Em N Ema?n 333.80 85.86:: 832% Iv Iv meOS— . v H .0: 9.30.). www.mo?g ? 33... 33.... \w\\N uwwwmma??. Q?mwmwm? 3.3 iii. 3......“ $3.6... k"w 9» ?x KW p“; 6‘".-$1 XV“ . \\: R‘. x\\\‘\ k\\~ «Yv \ ‘wk ?‘w $1 \\\' \ 5.» 9‘31'\\ \sz §f$ " \‘ kw \\\ ~ ~\‘\ \ $3\ &‘ w. N‘ \\\‘Q KN k '\ :\\\~ . §§§ §i\'\" ~x w §\\ e?» $0 ;\\\V kw Q .\ Q\\\\ \' ~ § m “\V‘ m {N~‘>\ \‘\\ w «K» a“ ‘ , <‘ \‘ $9 me «\w w \v §x\~\ §w\ \\:k‘ §Ixx T\\\\' WV W Ml} N,‘ \ ~\\\‘ 9“ f\\\ x?x 6 xxx" A\V \\'\'\‘ L\'\'¢ \x\‘ \\\\‘ Q 3\\~\ §w \\'~‘ \ :\\«\ \\‘~- \ §M\‘ ' :xé tQIk'\ \\“ FIG. £t§ '§\V \>‘\\\ tx‘x‘k‘ Qi\\ §\.0 \\\‘ 46, m‘ m $\ .‘\\\ ‘W \ $38359 w. §\\\ 7.".a *
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