Precise deletion of chromosomal sequences in vivo and treatment of nucleotide repeat expansion disorders using engineered nucleases

Patent metadata

Field	Value
Publication number	US-2025082735-A1
Application number	US-202418770277-A
Country	US
Kind code	A1
Filing date	Jul 11, 2024
Priority date	May 1, 2015
Publication date	Mar 13, 2025
Grant date	—

Abstract

The present invention provides a method of treating a nucleotide repeat expansion disorder comprising delivering a pair of engineered nucleases, or genes encoding engineered nucleases, to the cells of a patient such that the two nucleases excise the nucleotide repeat responsible for the disease permanently from the genome. The invention provides a general method for treating nucleotide repeat expansion disorders and engineered nucleases suitable for practicing the method. The invention further provides vectors and techniques for delivering engineered nucleases to patient cells.

First claim

1 . A method for treating a subject having a nucleotide repeat expansion disorder, wherein said nucleotide repeat expansion disorder is characterized by expansion of a nucleotide repeat in a gene of interest, said method comprising delivering to target cells in said subject:
(a) at least a first engineered nuclease protein and a second engineered nuclease protein; or (b) at least a first nucleic acid encoding said first engineered nuclease and a second nucleic acid encoding said second engineered nuclease, wherein said first engineered nuclease and said second engineered nuclease are expressed in said target cells in vivo; wherein said first engineered nuclease recognizes and cleaves a first recognition sequence positioned 5′ upstream of said nucleotide repeat in said gene of interest; and wherein said second engineered nuclease recognizes and cleaves a second recognition sequence positioned 3′ downstream of said nucleotide repeat in said gene of interest; and wherein an intervening DNA fragment between said first recognition sequence and said second recognition sequence is excised and the number of said nucleotide repeat is reduced in said gene of interest.

2 . The method of claim 1 , wherein said engineered nuclease is an engineered meganuclease, a compact TALEN, or a CRISPR.

3 . The method of claim 1 or claim 2 , wherein said first engineered nuclease and said second engineered nuclease generate complementary overhangs which promote direct re-ligation of said gene of interest.

4 . The method of any one of claims 1-3 , wherein said first recognition sequence and said second recognition sequence are positioned within the same exon, the same intron, or the same untranslated region (UTR) as said nucleotide repeat.

5 . The method of any one of claims 1-4 , wherein said nucleotide repeat is a trinucleotide repeat.

6 . The method of claim 5 , wherein said trinucleotide repeat is selected from the group consisting of CAG, CGG, CCG, GAA, and CTG.

7 . The method of claim 5 or claim 6 , wherein said trinucleotide repeat is GAA and said gene of interest is the frataxin (FXN) gene, wherein said trinucleotide repeat is positioned within intron 1 of the FXN gene.

8 . The method of claim 7 , wherein said first recognition sequence is positioned 5′ upstream in said intron 1 (SEQ ID NO: 74) of said trinucleotide repeat.

9 . The method of claim 7 or claim 8 , wherein said second recognition sequence is positioned 3′ downstream in said intron 1 (SEQ ID NO: 96) of said trinucleotide repeat.

10 . The method of any one of claims 7-9 , wherein said first engineered nuclease is a first engineered meganuclease and said second engineered nuclease is a second engineered meganuclease.

11 . The method of claim 10 , wherein said first recognition sequence comprises any one of SEQ ID NOs: 12-73.

12 . The method of claim 10 or claim 11 , wherein said first recognition sequence comprises SEQ ID NO: 34.

13 . The method of claim 12 , wherein said first engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said first recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said first recognition sequence and comprises a second hypervariable (HVR2) region.

14 . The method of claim 13 , wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of any one of SEQ ID NOs: 155-159, and wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of any one of SEQ ID NOs: 155-159.

15 . The method of claim 13 or claim 14 , wherein said HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 155-159.

16 . The method of any one of claims 13-15 , wherein said HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 155-159.

17 . The method of any one of claims 13-16 , wherein said first subunit comprises residues 7-153 of any one of SEQ ID NOs: 155-159.

18 . The method of any one of claims 13-17 , wherein said second subunit comprises residues 198-344 of any one of SEQ ID NOs: 155-159.

19 . The method of any one of claims 13-18 , wherein said first engineered meganuclease comprises the amino acid sequence of any one of SEQ ID NOs: 155-159.

20 . The method of any one of claims 10-19 , wherein said second recognition sequence comprises any one of SEQ ID NOs: 75-95.

21 . The method of any one of claims 10-20 , wherein said second recognition sequence comprises SEQ ID NO: 89.

22 . The method of claim 21 , wherein said second engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said second recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said second recognition sequence and comprises a second hypervariable (HVR2) region.

23 . The method of claim 22 , wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of any one of SEQ ID NOs: 170-172, or residues 7-153 of SEQ ID NO: 173, and wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of any one of SEQ ID NOs: 170-172, or residues 198-344 of SEQ ID NO: 173.

24 . The method of claim 22 or claim 23 , wherein said HVR1 region comprises residues 215-270 of any one of SEQ ID NOs: 170-172, or residues 24-79 of SEQ ID NO: 173.

25 . The method of any one of claims 22-24 , wherein said HVR2 region comprises residues 24-79 of any one of SEQ ID NOs: 170-172, or residues 215-270 of SEQ ID NO: 173.

26 . The method of any one of claims 22-25 , wherein said first subunit comprises residues 198-344 of any one of SEQ ID NOs: 170-172, or residues 7-153 of SEQ ID NO: 173.

27 . The method of any one of claims 22-26 , wherein said second subunit comprises residues 7-153 of any one of SEQ ID NOs: 170-172, or residues 198-344 of SEQ ID NO: 173.

28 . The method of any one of claims 22-27 , wherein said second engineered meganuclease comprises the amino acid sequence of any one of SEQ ID NOs: 170-173.

29 . The method of any one of claims 1-28 , wherein said method comprises administering to said subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and:
(a) said first nucleic acid encoding said first engineered nuclease of any one of claims 1-28 and said second nucleic acid encoding a second engineered nuclease of any one of claims 1-28 , wherein said first engineered nuclease and said second engineered nuclease are expressed in a target cell in vivo; or (b) said first engineered nuclease protein of any one of claims 1-28 and said second engineered nuclease protein of any one of claims 1-28 .

30 . A pharmaceutical composition for treatment of a subject having a nucleotide repeat expansion disorder, wherein said nucleotide repeat expansion disorder is characterized by expansion of a nucleotide repeat in a gene of interest, said pharmaceutical composition comprising a pharmaceutically acceptable carrier and:
(a) a first nucleic acid encoding a first engineered n

Assignees

• Prec Biosciences Inc

Inventors

• Smith James Jefferson
• Bartsevich Victor
• Jantz Derek

Classifications

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N2320/30

  Special therapeutic applications · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title