# US 12,214,054 B2 · Therapeutic targets for the correction of the human dystrophin gene by gene editing and methods of use > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,214,054 B2 - Document ID: US12214054B2 - Status: Granted - Kind: B2 - Application number: 15/779,633 - Filed: 2016-11-30 - Published: 2018-12-13 - Granted: 2025-02-04 - Earliest claimed: 2015-11-30 - Assignees: Duke University - Applicants: Duke University - Inventors: Charles A. Gersbach; Jacqueline N. Robinson-Hamm - Web page: https://patentgrep.com/patent/US12214054B2 ## CPC - A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered} - A61K 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links - A61K 47/26: Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin - A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids} - A61K 48/0016: {wherein the nucleic acid is delivered as a 'naked' nucleic acid, i.e. not combined with an entity such as a cationic lipid} - A61P 21/00: Drugs for disorders of the muscular or neuromuscular system - A61P 21/04: for myasthenia gravis - C07K 14/4708: {Duchenne dystrophy} - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 15/102: {Mutagenizing nucleic acids} - C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} - C12N 15/80: for fungi - C12N 15/8509: {for producing genetically modified animals, e.g. transgenic} - C12Q 1/68: involving nucleic acids - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2750/14143: viral genome or elements thereof as genetic vector - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract Disclosed herein are therapeutic targets for the correction of the human dystrophin gene by gene editing and methods of use. ## Claims ### Claim 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. ### Claim 2 (depends on 1) The DNA targeting composition of claim 1, further comprising a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein. ### Claim 3 (depends on 1) An isolated polynucleotide encoding the DNA targeting composition of claim 1. ### Claim 4 (depends on 1) A vector encoding the DNA targeting composition of claim 1. ### Claim 5 (depends on 1) 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). ### Claim 6 (depends on 5) 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. ### Claim 7 (depends on 5) The vector of claim 5, wherein the vector is a viral vector. ### Claim 8 (depends on 5) 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. ### Claim 9 (depends on 5) An isolated cell comprising the DNA targeting composition of claim 5. ### Claim 10 (depends on 5) A kit comprising the vector of claim 5. ### Claim 11 (depends on 3) A method of correcting a mutant dystrophin gene in a cell, the method comprising administering to a cell the gRNA of claim 3. ### Claim 12 (depends on 3) 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. ### Claim 13 (depends on 3) 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. ### Claim 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. ### Claim 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. ### Claim 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.