Compositions and methods for editing beta-globin for treatment of hemaglobinopathies
Inventors
D'Souza, Sanjay • West, Jason • EUSTACE, Brenda K. • Mahajan, Sudipta
Assignees
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Publication Number
US-12653860-B2
Publication Date
2026-06-16
Expiration Date
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.
Core Innovation
The disclosure describes methods for homology-directed repair correction of the E6V mutation in the human beta-globin (HBB) gene in a cell or population of cells. The method uses S. pyogenes Cas9 and an HBB-targeting guide RNA to generate a targeted double-strand break at an HBB target site. A recombinant donor vector provides homologous sequences that correct the mutation, alongside a 53BP1 inhibitor, a DNA-PK inhibitor, or both to favor homology-directed repair over non-homologous end joining.
One independent claim specifically requires high-fidelity S. pyogenes Cas9 comprising an R691A mutation, whereas another claim describes Cas9-mediated correction without that limitation. The methods edit patient-derived CD34-expressing hematopoietic stem or progenitor cells to increase homology-directed repair and decrease indels. Reported cellular outcomes include increased hemoglobin A and reduced hemoglobin S, while the background problem is not explicitly described in patent.
Claims Coverage
The claim coverage includes two inventive features across two independent claims directed to correcting the E6V mutation in the human HBB gene.
HDR correction of the HBB E6V mutation using high-fidelity Cas9
The claim covers homology-directed repair of a double-strand break in human HBB to correct the E6V mutation, specifying high-fidelity S. pyogenes Cas9 comprising an R691A mutation, an HBB-targeting single guide RNA, a recombinant donor vector with HBB-homologous regions and a correction sequence, and an inhibitor of 53BP1, an inhibitor of DNA-PK, or both.
Correction of the HBB E6V mutation using a donor vector and repair inhibitor
The claim covers correcting the E6V mutation in human HBB cells by generating a Cas9-mediated double-strand break with an HBB-targeting single guide RNA, utilizing a recombinant donor vector containing upstream and downstream homologous regions to exchange and correct the mutation, combined with a 53BP1 inhibitor, a DNA-PK inhibitor, or both.
Together, the independent claims cover homology-directed repair of the HBB E6V mutation using targeted Cas9 cleavage, a homologous donor sequence, and inhibition of 53BP1, DNA-PK, or both, with one claim specifically requiring high-fidelity Cas9 comprising an R691A mutation.
Stated Advantages
An inhibitor increases HDR frequency by at least 50% relative to a population without the inhibitor, decreases indel frequency by 2-10 fold, or both.
High-fidelity S. pyogenes Cas9 comprising an R691A mutation retains on-target HDR while substantially reducing off-target cleavage and indels.
The reported use of i53 increases HDR, reduces indels, and corrects the E6V mutation in patient-derived sickle-cell cells.
DNA-PK inhibitors increase HDR and reduce indel formation in HBB-edited CD34+ cells.
Reported cellular outcomes include increased hemoglobin A and reduced hemoglobin S.
Documented Applications
Correction of the E6V sickle mutation in human HBB in a cell or population of cells, including hematopoietic stem and progenitor cells, CD34-expressing cells, and induced pluripotent stem cells for potential treatment of hemoglobinopathies such as sickle cell disease and beta-thalassemia.
Transplantation studies using CD34+ hematopoietic stem and progenitor cells with assessment of marrow and blood chimerism and lineage distribution.
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