RNA-GUIDED GENE EDITING AND GENE REGULATION

• US 20160201089A1
• Filed: 06/05/2014
• Published: 07/14/2016
• Est. Priority Date: 06/05/2013
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Gersbach, Charles A., Hilton, Isaac B., Perez-Pinera, Pablo, Kabadi, Ami M., Thakore, Pratiksha I., Ousterout, David G., Black, Joshua B.
• Granted Patent: US 10,704,060 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: C07K 14/4708 Duchenne dystrophy
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2740/16043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/40 Systems of functionally co-operating vectors
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

Disclosed herein are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) 9-based system related compositions and methods of using said CRISPR/Cas9-based system related compositions for altering gene expression and genome engineering. Also disclosed herein are compositions and methods of using said compositions for altering gene expression and genome engineering in muscle, such as skeletal muscle and cardiac muscle.

Claims

1-17. -17. (canceled)

18. A DNA targeting system that binds to a dystrophin gene comprising Cas9 and at least one guide RNA (gRNA).

19. The DNA targeting system of claim 18, wherein the at least one gRNA targets an intron of the dystrophin gene or an exon of the dystrophin gene.

21. The DNA targeting system of claim 18, wherein the at least one guide RNA comprises at least one of SEQ ID NOs:

5-40, 65-144, 492-515, 540-563, and 585-625.

23. An isolated polynucleotide encoding the DNA targeting system of claim 18.

24. A vector comprising the isolated polynucleotide of claim 23.

25. A cell comprising the isolated polynucleotide of claim 23.

44. A method of treating a subject in need thereof having a mutant dystrophin gene, the method comprising administering to the subject the DNA targeting system of claim 18.

45. The method of claim 44, wherein the subject is suffering from Duchenne muscular dystrophy.

46. A method of correcting a mutant dystrophin gene in a cell, the method comprising administering to a cell containing a mutant dystrophin gene the DNA targeting system of claim 18.

47. The method of claim 46, wherein the mutant dystrophin gene comprises a premature stop codon, disrupted reading frame via gene deletion, an aberrant splice acceptor site, or an aberrant splice donor site, and wherein the target region is upstream or downstream of the premature stop codon, disrupted reading frame, aberrant splice acceptor site, or the aberrant splice donor site.

48. The method of claim 46, wherein the correction of the mutant dystrophin gene comprises homology-directed repair.

49. The method of claim 48, further comprising administering to the cell a donor DNA.

50. The method of claim 46, wherein the mutant dystrophin gene comprises a frameshift mutation which causes a premature stop codon and a truncated gene product.

51. The method of claim 50, wherein the correction of the mutant dystrophin gene comprises nuclease mediated non-homologous end joining.

52. The method of claim 46, wherein the correction of the mutant dystrophin gene comprises a deletion of a premature stop codon, correction of a disrupted reading frame, or modulation of splicing by disruption of a splice acceptor site or disruption of a splice donor sequence.

53. The method of claim 52, wherein the correction of the mutant dystrophin gene comprises a deletion of exons 45-55 or exon 51.

78. A composition for genome editing in a muscle of a subject comprising a modified adeno-associated virus (AAV) vector and a nucleotide sequence encoding the DNA targeting system of claim 18, wherein the muscle is skeletal muscle or cardiac muscle.

79. The composition of claim 78, wherein the modified AAV vector has enhanced cardiac and skeletal muscle tissue tropism.

84. A method of genome editing in a muscle of a subject, treating a subject, or correcting a mutant gene in a subject, the method comprising administering to the muscle of the subject the composition of claim 78, wherein the muscle is skeletal muscle or cardiac muscle.

89. The method of claim 84, wherein the subject is suffering from a skeletal muscle condition or a genetic disease.

90. The method of claim 89, wherein the subject is suffering from Duchenne muscular dystrophy.

20. (canceled)

22. (canceled)

26-43. -43. (canceled)

54-77. -77. (canceled)

80-83. -83. (canceled)

85-88. -88. (canceled)

91-130. -130. (canceled)