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

A composition for deleting a segment of a dystrophin gene comprising exon 51, the composition comprising:

• (a) a first vector comprising a polynucleotide sequence encoding a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first 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 Cas9 molecule that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO:
• 24) or NNGRRV (SEQ ID NO:
• 25),
• wherein each of the first and second gRNA molecules have a targeting domain of 19 to 24 nucleotides in length, and 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 deleting 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 of:
• (i) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 1, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 2;
• (ii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 11, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 4;
• (iii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 19;
• (iv) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 18;
• (v) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 4;
• (vi) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 19;
• (vii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 18;
• (viii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 4;
• (ix) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 11, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 19;
• (x) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15;
• (xi) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 11, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 18;
• and
• (xii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 41, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 42.

A method of correcting a mutant dystrophin gene in a cell, comprising administering to the cell:

• (a) a first vector comprising a polynucleotide sequence encoding a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first 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 Cas9 molecule that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO:
• 24) or NNGRRV (SEQ ID NO:
• 25),
• wherein each of the first gRNA and second gRNA molecules have a targeting domain of 19 to 24 nucleotides in length, wherein the vector 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, and 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 is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 1, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 2;
• (ii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 11, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 4;
• (iii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 19;
• (iv) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising s a nucleotide sequence set forth in SEQ ID NO:
• 15, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 18;
• (v) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 4;
• (vi) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 19;
• (vii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 18;
• (viii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 4;
• (ix) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 11, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 19;
• (x) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15;
• (xi) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 11, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 18;
• and
• (xii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 41, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 42.

A method of treating a subject in need thereof having a mutant dystrophin gene, the method comprising administering to the subject:

• (a) a first vector comprising a polynucleotide sequence encoding a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first 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 Cas9 molecule that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO:
• 24) or NNGRRV (SEQ ID NO:
• 25),
• wherein each of the first gRNA and second gRNA molecules have a targeting domain of 19 to 24 nucleotides in length, wherein the vector 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, and 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 is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 1, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 2;
• (ii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 11, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 4;
• (iii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 19;
• (iv) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 18;
• (v) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 4;
• (vi) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 19;
• (vii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 18;
• (viii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 4;
• (ix) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 11, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 19;
• (x) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 14, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 15;
• (xi) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 11, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 18;
• and
• (xii) a first gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 41, and a second gRNA molecule comprising a targeting domain that is encoded by a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:
• 42.