Lipid-encapsulated dual-cleaving endonuclease for DNA and gene editing
Inventors
EDGELL, David R. • MCMURROUGH, Thomas A. • STEAD, Brent E. • ISRAEL, Odisho K.
Assignees
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Publication Number
US-12460192-B2
Publication Date
2025-11-04
Expiration Date
Abstract
Disclosed herein are methods to edit genes, for example by administering a chimeric nuclease to a cell or organism without the use of a viral vector. Some methods herein include editing DNA of a cell by contacting the cell with a nuclease comprising a Cas9 sequence, where the nuclease edits the DNA of the cell.
Core Innovation
US-12460192-B2 discloses a non-viral gene-editing method for editing DNA of a cell by contacting the cell with a nuclease that includes a Cas9 sequence at least 99% identical to SEQ ID NO: 13 and a glutamate at an amino acid position corresponding to position 10 of SEQ ID NO: 13, where the nuclease edits the DNA of the cell. The disclosure also describes a nuclease architecture in which an I-TevI sequence is linked to a Cas9 sequence, including chimeric nucleases.
The disclosure describes dual-cleaving nuclease concepts based on a modified I-TevI domain linked to a modified RNA-guided saCas9, optionally with donor DNA. The gene modification outcome is characterized by whether donor DNA is provided, including correction via HDR and editing/deletion via NHEJ, and by targeting particular mutations such as CFTR delta F508 and EGFR exon 19 mutations.
The nuclease is delivered using lipid nanoparticles, described as non-viral delivery vehicles suitable for nebulization/inhalation, for editing lung epithelial cells. The disclosure further addresses specificity and activity through specified Cas9-related modifications, including variants and a glutamate at a defined position, and describes edit predictability in terms of deletion size windows for targeted outcomes.
Claims Coverage
The partial content contains one independent claim (clm-00001). The independent claim coverage is centered on sequence identity and a defined amino-acid substitution in a Cas9 nuclease, where the nuclease edits the DNA of a cell. Dependent claims refine the inventive concept by specifying chimeric I-TevI/Cas9 nuclease architecture, permitted I-TevI sequence variants, editing mechanism, target mutations and genes, a spatial constraint on cleavage relative to target mutations, and non-viral lipid nanoparticle formulations.
Cas9 sequence identity and position-10 glutamate
A method for editing DNA of a cell wherein contacting the cell with a nuclease comprising a Cas9 sequence at least 99% identical to SEQ ID NO: 13 and comprising a glutamate at an amino acid corresponding to position 10 of SEQ ID NO: 13 results in editing of the DNA of the cell.
Chimeric I-TevI linked to Cas9 nuclease architecture
The nuclease is a chimeric nuclease comprising an I-TevI sequence coupled to a Cas9 sequence for editing the DNA of the cell.
I-TevI sequence variants with limited amino-acid changes
The I-TevI sequence comprises the amino acid sequence of SEQ ID NO: 6 or a variant with 1, 2, or 3 specified mutations comprising amino acid substitutions, deletions, and/or insertions.
Nicking or double-stranded cutting editing mechanism
The editing is performed by nicking or by double-stranded cutting.
Mutation-targeted editing of CFTR delta F508 or EGFR exon 19
The method uses a dual nuclease that targets and cleaves 10 to 20 nucleotides on either side of either a CFTR delta F508 mutation or an EGFR exon 19 mutation.
Defined lipid nanoparticle lipid composition options
The lipid nanoparticle includes one of three specified lipid compositions with defined molar ratios.
Across the independent claim and refinements in dependent claims, the inventive coverage is grounded in a Cas9 nuclease defined by sequence identity to SEQ ID NO: 13 and a glutamate at a defined position, optionally implemented as an I-TevI linked to Cas9 chimeric nuclease. Further coverage includes permitted I-TevI sequence variants, the editing mechanism, targeting CFTR delta F508 or EGFR exon 19 with a specified cleavage window, and defined non-viral lipid nanoparticle compositions.
Stated Advantages
Dual-site targeting via a dual nuclease approach.
Predictable deletion sizing within a stated ~30–36 bp deletion range.
Improved nuclease specificity/activity through saCas9 activity/specificity modifications as described, including defined amino-acid changes.
Simplified manufacture as described in the disclosure.
Controlled dosing as described in the disclosure.
Non-viral delivery using lipid nanoparticles suitable for nebulization/inhalation as described in the disclosure.
Documented Applications
Editing lung epithelial cells for cystic fibrosis, including CFTR delta F508 correction.
Editing for non-small-cell lung cancer by deleting EGFR exon 19 mutations.
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