Genetic correction of mutated genes

• US 10,711,256 B2
• Filed: 06/27/2017
• Issued: 07/14/2020
• Est. Priority Date: 04/27/2012
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Gersbach, Charles, Ousterout, David
• Primary Examiner(s): Rao, Manjunath N
• Assistant Examiner(s): Liu, Samuel W
• Application Number:
  US15/634,425
• Publication Number:
  US 20170298331A1
• Time in Patent Office: 1,113 Days
• Field of Search: None
• 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: C07K 14/4708 Duchenne dystrophy
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are transcription activator-like effector nuclease (TALEN)-related compositions and methods of using said TALENs for correcting mutant genes.

Claims

1. An engineered transcription activator-like effector nuclease (TALEN) protein that binds to a dystrophin gene, wherein the TALEN protein binds upstream from a premature stop codon on the dystrophin gene, downstream from a premature stop codon on the dystrophin gene, a region in exon 51 of the dystrophin gene, or a region in the 5′

UTR of the dystrophin gene, wherein the TALEN protein comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, and SEQ ID NO;

15.

2. The TALEN protein of claim 1, wherein the TALEN protein binds to a nucleotide sequence comprising one of SEQ ID NOs:

16-46, 50, 52, 58, 59, or a full-length complement thereof.

3. The TALEN protein of claim 2, wherein the TALEN protein comprises a nuclease.

4. The TALEN protein of claim 3, wherein the nuclease comprises FokI.

5. The TALEN protein of claim 1, wherein the TALEN protein comprises 15-19 repeat variable diresidue (RVD) modules.

6. The TALEN protein of claim 1, wherein the dystrophin gene is a human dystrophin gene.

7. A composition comprising two or more TALEN proteins according to claim 1, wherein a first TALEN binds to a first binding region and a second TALEN binds to a second binding region, wherein the first binding region and second binding region are located within a target region and the first binding region and second binding region are not the same.

8. A method of treating a subject in need thereof having a mutant dystrophin gene, the method comprising administering to the subject the TALEN protein of claim 1.

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 the cell comprising a mutant dystrophin gene which comprises a frameshift mutation which causes a premature stop codon and a truncated gene product the TALEN protein of claim 1, thereby the correction restores the mutant dystrophin gene in the cell.

11. The method of claim 10, wherein the correction of the mutant dystrophin gene comprises homology-directed repair.

12. The method of claim 11, further comprising administering to the cell a donor DNA.

13. The method of claim 10, wherein the correction of the mutant dystrophin gene comprises nuclease mediated non-homologous end joining.

14. A method of correcting a mutant gene in a cell, the method comprising administering to the cell containing a mutant gene that has a mutation which causes a premature stop codon and a truncated gene product a first TALEN and a second TALEN, wherein the first TALEN binds to a first binding region and a second TALEN binds to a second binding region, wherein said TALENS are selected from the TALENS of claim 1, wherein the first binding region and second binding region are located within a target region and the first binding region and second binding region are not the same, wherein the correction of the mutant gene comprises nuclease mediated non-homologous end joining, and wherein the correction restores the mutant gene.

15. The method of claim 14, wherein the method comprises administering to a cell containing a mutant dystrophin gene a first TALEN and a second TALEN, wherein the first TALEN binds to a first binding region and a second TALEN binds to a second binding region, wherein the first binding region and second binding region are located within a target region and the first binding region and second binding region are not the same.

16. A kit comprising the TALEN protein of claim 1.