RNA-guided gene editing and gene regulation

• US 10,704,060 B2
• Filed: 06/05/2014
• Issued: 07/07/2020
• 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.
• Primary Examiner(s): Shin, Dana H
• Application Number:
  US14/895,316
• Publication Number:
  US 20160201089A1
• Time in Patent Office: 2,224 Days
• 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. A DNA targeting system for deleting exon 51 of a dystrophin gene comprising Cas9 and at least one guide RNA (gRNA), wherein the at least one gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO:

65, SEQ ID NO;

679, or SEQ ID NO;

69.

2. An isolated polynucleotide encoding the DNA targeting system of claim 1.

3. A vector comprising the isolated polynucleotide of claim 2.

4. A cell comprising the isolated polynucleotide of claim 2.

5. 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 1, wherein the muscle is skeletal muscle or cardiac muscle.

6. The composition of claim 5, wherein the modified AAV vector has enhanced cardiac and skeletal muscle tissue tropism.

7. The DNA targeting system of claim 1, wherein the DNA targeting system comprises:

a gRNA comprising a polynucleotide sequence corresponding to SEQ ID NO;

65 and a gRNA comprising a polynucleotide sequence corresponding to SEQ ID NO;

69;

or

a gRNA comprising a polynucleotide sequence corresponding to SEQ ID NO;

679 and a gRNA comprising a polynucleotide sequence corresponding to SEQ ID NO;

69.

8. 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 7.

9. The method of claim 8, wherein the subject is suffering from Duchenne muscular dystrophy.

10. 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 7.