IP Library › Granted Patent US 12,214,054
Granted Patent B2
US 12,214,054 · App. 15/779,633 · Granted Feb 4, 2025

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

Inventors: Charles A. Gersbach (Durham, NC); Jacqueline N. Robinson-Hamm (Durham, NC)
Assignee: Duke University
A61K48/005A61K31/7105A61K47/26A61K47/549A61K48/0016C12N15/102C12N15/113C12N15/80C12N15/8509C12Q1/68C12N2310/20
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Quick Facts
Patent No.
US 12,214,054
App. No.
15/779,633
Filed
May 29, 2018
Granted
Feb 4, 2025
Kind
B2
Examiner
ZARA, JANE J
Art Unit
1637
USPC
514/44R
Abstract

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

Claims (75)

1. A DNA targeting composition comprising 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 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.

2. The DNA targeting composition of claim 1 , further comprising a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein.

3. An isolated polynucleotide encoding the DNA targeting composition of claim 1 .

4. A vector encoding the DNA targeting composition of claim 1 .

5. A vector encoding:

(a) the DNA targeting composition of claim 1 , and

(b) at least one Cas9 molecule that recognizes a Protospacer Adjacent Motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25).

6. The vector of claim 5 , 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 DMD gene.

7. The vector of claim 5 , wherein the vector is a viral vector.

8. The vector of claim 5 , wherein the vector comprises a tissue-specific promoter operably linked to the nucleotide sequence encoding the first gRNA molecule, and/or the second gRNA molecule, and/or the Cas9 molecule.

9. An isolated cell comprising the DNA targeting composition of claim 5 .

10. A kit comprising the vector of claim 5 .

11. A method of correcting a mutant dystrophin gene in a cell, the method comprising administering to a cell the gRNA of claim 3 .

12. A method of genome editing a mutant dystrophin gene in a subject, the method comprising administering to the subject a genome editing composition comprising the gRNA of claim 3 .

13. A method of treating a subject in need thereof having a mutant dystrophin gene, the method comprising administering to the subject the gRNA of claim 3 .

14. 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.

15. 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.

16. 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.

Assignments (3)
CONFIRMATORY LICENSE Recorded May 4, 2022
From: DUKE UNIVERSITY
To: NATIONAL SCIENCE FOUNDATION
Reel/Frame 059852/0728 →
CONFIRMATORY LICENSE Recorded Sep 17, 2020
From: DUKE UNIVERSITY
To: NATIONAL SCIENCE FOUNDATION
Reel/Frame 053802/0600 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 29, 2018
From: GERSBACH, CHARLES A.; ROBINSON-HAMM, JACQUELINE N.
To: DUKE UNIVERSITY
Reel/Frame 045920/0246 →
Continuity (3)
Provisional Application 62330336 · May 2, 2016
Provisional Application 62260712 · Nov 30, 2015
Related Publication 20180353615A1 · Dec 13, 2018
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