Compositions and methods for editing beta-globin for treatment of hemaglobinopathies
AssigneeVertex Pharmaceuticals Incorporated
InventorsSanjay D'Souza, Jason West, Brenda K. Eustace, Sudipta Mahajan
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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.
Claims23
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.
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.
Dependencies are inferred from the claim text.