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-12497614-B2
Publication Date
2025-12-16
Expiration Date
Abstract
The disclosure features systems and methods for 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 systems and methods for correcting the E6V mutation in human beta-globin (HBB) in a cell or population of cells. The system utilizes Cas9 or material encoding Cas9, an sgRNA targeting a site within HBB intron 1 adjacent to a PAM, and a recombinant vector carrying an HBB-homologous correction nucleic acid encoding E6. Correction of the mutation occurs through homology-directed repair.
The disclosure also describes isolated corrected cells and reports gene editing in CD34+ hematopoietic stem and progenitor cells and sickle-cell samples. Edited cells persist after engraftment and differentiate into erythroid cells. Inhibition of 53BP1 or DNA-PK increases homology-directed repair, while high-fidelity Cas9 variants and intron-targeting guides reduce off-target editing.
Claims Coverage
The claims contain three independent claims directed to three inventive features covering a correction system, a correction method, and isolated cells.
System for correcting the HBB E6V mutation
Claim 1 covers a system comprising Cas9 or material encoding Cas9, an sgRNA targeting a site within HBB intron 1 adjacent to a PAM, and a recombinant vector containing an HBB-homologous correction nucleic acid that encodes E6.
Method for correcting the HBB E6V mutation
Claim 22 covers contacting a cell or cell population comprising an HBB gene encoding the E6V mutation with Cas9 or material encoding Cas9, an sgRNA targeting a site within HBB intron 1, and a recombinant vector carrying an HBB-homologous correction nucleic acid, thereby correcting the mutation.
Isolated cells containing specified HBB sequences
Claim 24 covers an isolated cell or population of isolated cells comprising at least one chromosomal copy of an HBB gene containing one of the specified nucleotide sequences.
The independent claims cover the mutation-correction system, the corresponding method of correction, and isolated cells or populations containing the recited HBB sequences.
Stated Advantages
The disclosure reports improved homology-directed repair, altered INDEL formation, and reduced indels with 53BP1 or DNA-PK inhibition, reducing NHEJ-associated repair by approximately threefold.
High-fidelity Cas9 and intron-targeting guides are reported to reduce off-target editing.
Edited cells retain engraftment and edits in mice, persist after engraftment, and differentiate into erythroid cells.
The method is stated to correct the E6V mutation in the HBB gene in a cell or population of cells.
Documented Applications
Correcting the HBB E6V mutation in cells or populations of cells, including CD34+ hematopoietic stem and progenitor cells and sickle-cell samples.
Ex vivo or in vivo treatment of hemoglobinopathies, including sickle cell disease, using edited cells.
Generation of isolated cells or cell populations containing at least one chromosomal copy of an HBB gene containing one of the specified nucleotide sequences.
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