IP Library › Granted Patent US 12,653,860
Granted Patent B2
US 12,653,860 · App. 17/619,949 · Granted Jun 16, 2026

Compositions and methods for editing beta-globin for treatment of hemaglobinopathies

Inventors: Sanjay D'Souza (Cambridge, MA); Jason West (Lexington, MA); Brenda K. Eustace (Brookline, MA); Sudipta Mahajan (Boston, MA)
Assignee: Vertex Pharmaceuticals Incorporated
A61K38/1709A61K35/28A61K40/10A61K40/40A61P7/06A61P37/06C12N5/0636C12N5/0647C12N7/00C12N9/22C12N15/11C12N15/86C12N15/907A61K48/00A61K2239/31A61K2239/38C12N2310/20C12N2500/02C12N2501/125C12N2501/145C12N2501/2303C12N2501/26C12N2750/14143C12N2800/80
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Quick Facts
Patent No.
US 12,653,860
App. No.
17/619,949
Granted
Jun 16, 2026
Kind
B2
Abstract

The disclosure features methods of correcting a mutation in the human beta-globin (HBB) gene in a cell or population of cells. The disclosure also features methods of increasing repair of a DNA double stranded break (DSB) in an HBB gene by the homology-directed repair (HDR) pathway. The disclosure also features compositions for use in the methods.

Claims (47)

1 . A method for homology directed repair (HDR) of a double-strand break (DSB) in a target site in a human beta-globin (HBB) gene in a cell or population of cells, the method comprising contacting the cell or population of cells with:

(a) a S. pyogenes Cas9 endonuclease, an mRNA encoding the S. pyogenes Cas9 endonuclease, or a recombinant expression vector comprising a nucleotide sequence encoding the S. pyogenes Cas9 endonuclease, to generate a double-stranded break (DSB) at the target site in the HBB gene, wherein the S. pyogenes Cas9 endonuclease is a high fidelity Cas9 comprising a R691A mutation;

(b) a single guide RNA (sgRNA) targeting the target site in the HBB gene, the sgRNA comprising a spacer sequence corresponding to a target sequence in the HBB gene consisting of SEQ ID NO: 15;

(c) a recombinant vector comprising a nucleic acid, the nucleic acid comprising from 5′ to 3′ (i) a nucleotide sequence homologous with a region located upstream of the target site in the HBB gene, (ii) a nucleotide sequence homologous with a region of the HBB gene comprising the target site, the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 102, and (iii) a nucleotide sequence homologous with a region located downstream of the target site in the HBB gene; and

(d) a 53BP1 inhibitor; an inhibitor of DNA-PK; or both;

wherein the recombinant vector of (c) comprises the nucleotide sequence of SEQ ID NO: 98;

wherein the double-strand break (DSB) occurs at the target site in the HBB gene and the nucleic acid comprising the nucleotide sequence of SEQ ID NO: 98 is exchanged with a homologous nucleotide sequence of the HBB gene, thereby correcting an E6V mutation in the HBB gene in the cell or population of cells.

2 . A method for correcting an E6V mutation in human beta-globin (HBB) in a cell or population of cells, the method comprising contacting the cell or population of cells comprising an HBB gene encoding the E6V mutation with:

(a) a S. pyogenes Cas9 endonuclease, an mRNA encoding the S. pyogenes Cas9 endonuclease, or a recombinant expression vector comprising a nucleotide sequence encoding the S. pyogenes Cas9 endonuclease to generate a double-stranded break (DSB) at a target site in the HBB gene;

(b) a single guide RNA (sgRNA) targeting the target site in the HBB gene, the sgRNA comprising a spacer sequence corresponding to a target sequence in the HBB gene consisting of SEQ ID NO: 15; and

(c) a recombinant vector comprising a nucleic acid, the nucleic acid comprising from 5′ to 3′ (i) a nucleotide sequence homologous with a region located upstream of a site of the HBB gene encoding the E6V mutation, (ii) a nucleotide sequence which corrects the E6V mutation and is homologous with a site of the HBB gene encoding the E6V mutation, the nucleotide sequence set forth in SEQ ID NO: 102, and (iii) a nucleotide sequence homologous with a region located downstream of the site of the HBB gene encoding the E6V mutation in the HBB gene,

(d) a 53BP1 inhibitor; an inhibitor of DNA-PK; or both;

wherein the recombinant vector of (c) comprises the nucleotide sequence of SEQ ID NO: 98; and

wherein the double-strand break (DSB) occurs at the target site in the HBB gene and the nucleic acid comprising the nucleotide sequence of SEQ ID NO: 98 is exchanged with a homologous nucleotide sequence of the HBB gene, thereby correcting the E6V mutation in HBB in the cell or population of cells.

3 . The method of claim 1 , wherein cleavage of one or more predicted off-target sites in the cell or population of cells is reduced relative to a cell or population of cells contacted with a wild-type S. pyogenes Cas9.

4 . The method of claim 1 , wherein the nucleotide sequence of (c)(i) is homologous with a region located upstream of a mutation in the HBB gene that results in an E6V mutation in HBB and the nucleotide sequence of (c)(iii) is homologous with a region located downstream of the mutation in the HBB gene that results in the E6V mutation in HBB.

5 . The method of claim 1 wherein the 53BP1 inhibitor and/or the inhibitor of DNA-PK increases HDR of the DSB, relative to HDR in a cell or population of cells without the 53BP1 inhibitor and/or inhibitor of DNA-PK.

6 . The method of claim 1 , wherein the high fidelity Cas9 endonuclease comprises at least one NLS.

7 . The method of claim 1 wherein

(i) the 53BP1 inhibitor and/or the inhibitor of DNA-PK increases HDR frequency in the cell population by at least 50% relative to a cell population without the 53BP1 inhibitor and/or the inhibitor of DNA-PK;

(ii) the 53BP1 inhibitor and/or the inhibitor of DNA-PK decreases indel frequency by 2-10 fold in the cell population; or

(iii) both (i) and (ii).

8 . The method of claim 1 wherein the 53BP1 inhibitor is a 53BP1 binding polypeptide that inhibits 53BP1 recruitment to the DSB in the cell, wherein the 53BP1 binding polypeptide comprises an amino acid sequence selected from a group consisting of: SEQ ID NOs: 70, 74, 77, 80, 83 and 86.

9 . The method of claim 1 wherein

(i) the 53BP1 inhibitor comprises a nucleic acid or a vector comprising a nucleotide sequence encoding a 53BP1 binding polypeptide that inhibits 53BP1 recruitment to the DSB site in the cell; or

(ii) the 53BP1 inhibitor comprises a vector comprising a nucleotide sequence encoding the 53BP1 binding polypeptide,

wherein the nucleotide sequence is selected from a group consisting of: SEQ ID NOs: 69, 73, 76, 79, 82, 85 and 88.

10 . The method of claim 1 wherein the inhibitor of DNA-PK targets the catalytic subunit of DNA-PK (DNA-PKcs).

11 . The method of claim 1 wherein the inhibitor of DNA-PK is Nu7441, Compound 984, or Compound 296.

12 . The method of claim 1 , wherein

(i) the nucleotide sequence of (c)(i) is about 0.2 kb to about 3 kb in length;

(ii) the nucleotide sequence of (c)(iii) is about 0.2 kb to about 3 kb in length; or

(iii) both (i) and (ii).

13 . The method of claim 1 , wherein the nucleotide sequence of (c)(i) and/or the nucleotide sequence of (c)(iii) is about 2.2 kb each.

14 . The method of claim 1 , wherein the recombinant vector is an AAV vector.

15 . The method of claim 14 , wherein the AAV vector is about 2.5 kb-4.6 kb in length.

16 . The method of claim 14 , wherein the AAV vector comprises AAV6.

17 . The method of claim 14 , wherein the AAV vector comprises 5′ and 3′ inverted terminal repeats (ITRs) derived from AAV2.

18 . The method of claim 14 , wherein the AAV vector comprises SEQ ID NO: 105.

19 . The method of claim 1 , wherein

(i) the cell or population of cells is a hematopoietic stem or progenitor cell (HSPC) or a population of HSPCs;

(ii) the cell or population of cells is a CD34 expressing cell or a population of CD34 expressing cells; or

(iii) both (i) and (ii).

20 . The method of claim 19 , wherein the cell or the population of cells is isolated from a tissue sample obtained from a human donor.

21 . The method of claim 20 , wherein the tissue sample is a peripheral blood sample.

22 . The method of claim 20 , wherein the human donor has a sickle cell disease.

23 . A cell or population of cells generated by the method of claim 1 .

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 4, 2022
From: EUSTACE, BRENDA K.; MAHAJAN, SUDIPTA
To: VERTEX PHARMACEUTICALS INCORPORATED
Reel/Frame 058885/0260 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 4, 2022
From: D'SOUZA, SANJAY; WEST, JASON
To: CRISPR THERAPEUTICS AG
Reel/Frame 058955/0732 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 4, 2022
From: CRISPR THERAPEUTICS AG
To: VERTEX PHARMACEUTICALS INCORPORATED
Reel/Frame 058956/0073 →
Continuity (2)
Provisional Application 62862539 · Jun 17, 2019
Related Publication 20230022146A1 · Jan 26, 2023
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