Crispr/cas-mediated gene conversion

Patent metadata

Field	Value
Publication number	US-2016281111-A1
Application number	US-201615081456-A
Country	US
Kind code	A1
Filing date	Mar 25, 2016
Priority date	Mar 26, 2015
Publication date	Sep 29, 2016
Grant date	—

Abstract

CRISPR/CAS-related compositions and methods for altering a cell or treating a disease, for example, by gene conversion, are disclosed.

First claim

What is claimed is:

1 . A method of modifying a target gene in a cell, the method comprising:
contacting the cell with a first gRNA molecule, a first enzymatically active Cas9 (eaCas9) molecule, a second gRNA molecule, and a second eaCas9 molecule; wherein the first gRNA molecule and the first eaCas9 molecule associate with the target gene and generate a first single strand cleavage event on a first strand of the target gene; wherein the second gRNA molecule and the second eaCas9 molecule associate with the target gene and generate a second single strand cleavage event on a second strand of the target gene, thereby forming a double strand break having a first overhang and a second overhang; and wherein the first overhang and the second overhang in the target gene are repaired by gene conversion using an endogenous homologous region, thereby modifying the target gene in the cell.

2 . The method of claim 1 , wherein, after repair of the first overhang and the second overhang, the target gene comprises the sequence of the endogenous homologous region.

3 . The method of claim 1 , wherein the cell is not contacted with an exogenous nucleic acid homologous to the endogenous target gene.

4 . The method of claim 1 , wherein the first overhang is a 5′ overhang, and the second overhang is a 5′ overhang.

5 . The method of claim 1 , wherein the method is used to correct a mutation in the target gene, and wherein the mutation in the target gene is located
(a) between the first single strand break and the second single strand break, (b) within fewer than 50 nucleotides of the first single strand break, or (c) within fewer than 50 nucleotides of the second single strand break.

6 . The method of claim 1 , wherein the target gene has at least 90% sequence homology with the endogenous homologous region.

7 . The method of claim 1 , wherein the first eaCas9 molecule is a first nickase molecule and the second eaCas9 molecule is a second nickase molecule.

8 . The method of claim 7 , wherein the first eaCas9 molecule and the second eaCas9 molecule are the same eaCas9 nickase molecule.

9 . The method of claim 8 , wherein the eaCas9 nickase molecule is an HNH-like domain nickase.

10 . The method of claim 9 , wherein the eaCas9 molecule comprises a mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

11 . The method of claim 1 , wherein the method is used to correct a mutation in the endogenous HBB target gene, and wherein the mutation in the endogenous HBB target gene causes sickle cell disease or beta-thalassemia.

12 . The method of claim 11 , wherein the first gRNA molecule is a gRNA molecule comprising SEQ ID NO:387, and wherein the second gRNA molecule is a gRNA molecule comprising SEQ ID NO:16318.

13 . The method of claim 1 , wherein the first gRNA molecule is a gRNA molecule comprising any one of SEQ ID NOs: 387-485, 6803-6871, or 16010-16256, and wherein the second gRNA molecule is a gRNA molecule comprising any one of SEQ ID NOs: 387-485, 6803-6871, or 16010-16256.

14 . The method of claim 1 , wherein the cell is a population of cells, and wherein the first overhang and the second overhang in the target gene are repaired by gene conversion in about 12% to about 45% of the cells in the population of cells.

15 . The method of claim 1 , wherein the cell is a population of cells, and wherein the first overhang and the second overhang in the target gene are repaired by non-homologous end joining (NHEJ) in less than 40% of the cells in the population of cells.

16 . The method of claim 1 , wherein the cell is a mammalian cell.

17 . The method of claim 16 , wherein the cell is a human cell.

18 . The method of claim 1 , wherein the cell is a blood cell or a stem cell.

19 . A composition comprising:
a first non-naturally occurring gRNA molecule, a first non-naturally occurring enzymatically active Cas9 (eaCas9) molecule, a second non-naturally occurring gRNA molecule, and a second non-naturally occurring eaCas9 molecule; wherein the first gRNA molecule and the first eaCas9 molecule are designed to associate with a target gene and generate a first single strand cleavage event on a first strand of the target gene; wherein the second gRNA molecule and the second eaCas9 molecule are designed to associate with the target gene and generate a second single strand cleavage event on a second strand of the target gene, thereby forming a first overhang and a second overhang; and wherein the first gRNA molecule, the first eaCas9 molecule, the second gRNA molecule and the second eaCas9 molecule are designed such that the first overhang and the second overhang in the target gene are repaired by gene conversion.

20 . The composition of claim 19 , wherein the first non-naturally occurring eaCas9 molecule is an HNH-like domain nickase, and wherein the second non-naturally occurring eaCas9 molecule is an HNH-like domain nickase.

21 . The composition of claim 19 , wherein the first non-naturally occurring gRNA molecule comprises SEQ ID NO:387, and wherein the second non-naturally occurring gRNA molecule comprises SEQ ID NO:16318.

22 . The composition of claim 19 , wherein the first gRNA molecule is a gRNA molecule comprising any one of SEQ ID NOs: 387-485, 6803-6871, or 16010-16256, and wherein the second gRNA molecule is a gRNA molecule comprising any one of SEQ ID NOs: 387-485, 6803-6871, or 16010-16256.

23 . A method of treating a disease in a subject having a mutation in an endogenous HBB target gene, the method comprising
contacting a cell from the subject with a first gRNA molecule, a first enzymatically active Cas9 (eaCas9) molecule, a second gRNA molecule, and a second eaCas9 molecule; wherein the first gRNA molecule and the first eaCas9 molecule associate with the HBB target gene and generate a first single strand cleavage event on a first strand of the HBB target gene; wherein the second gRNA molecule and the second eaCas9 molecule associate with the HBB target gene and generate a second single strand cleavage event on a second strand of the HBB target gene, thereby forming a double strand break having a first overhang and a second overhang; and wherein the first overhang and the second overhang in the HBB target gene are repaired by gene conversion using an endogenous homologous region of an endogenous HBD gene which does not comprise the mutation, correcting the mutation in the endogenous HBB target gene in the cell, thereby treating the disease in the subject having the mutation in the endogenous HBB target gene.

24 . The method of claim 23 , wherein the cell is not contacted with an exogenous nucleic acid homologous to the HBB target gene.

25 . The method of claim 23 , wherein the first eaCas9 molecule and the second eaCas9 molecule are each Cas9 nickase molecules.

26 . The method of claim 25 , wherein the first eaCas9 molecule and the second eaCas9 molecule are the same species of eaCas9 nickase molecule.

27 . The method of claim 26 , wherein the eaCas9 nickase molecule comprises a mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

28 . The method of claim 23 , wherein the disease is beta thalassemia or sickle cell disease.

29 . The method of claim 23 , wherein the cell is a b

Assignees

• Editas Medicine Inc

Inventors

• Cotta-Ramusino Cecilia
• Gori Jennifer Leah

Classifications

• A61K48/005

  characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered · CPC title

• A61K38/465 Primary

  acting on ester bonds (3.1), e.g. lipases, ribonucleases · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N9/22

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

• C12N15/102

  Mutagenizing nucleic acids · CPC title