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-12312615-B2
Publication Date
2025-05-27
Expiration Date
Abstract
Provided herein are modified Staphylococcus aureus Cas9 nucleases and methods of using modified Staphylococcus aureus Cas9 nucleases. Also provided herein are chimeric nucleases comprising an I-Tevl nuclease domain, and a Staphylococcus aureus Cas9 and methods of using the chimeric nuclease. Also provided herein are methods to edit genes by administering a chimeric nuclease to a cell or organism without the use of a viral vector.
Core Innovation
The invention relates to a TevCas9 chimeric dual-cleaving nuclease system for editing genomic DNA in mammalian cells. The system combines a modified Enterobacteria Phage T4 (I-TevI) nuclease domain, a linker, and an RNA-guided Staphylococcus aureus Cas9 with a guide RNA, and the chimeric nuclease thereby edits genomic DNA of the cell.
The TevCas9 system includes an RNA-guided Staphylococcus aureus Cas9 comprising the sequence of SEQ ID NO: 13 with an aspartic acid to glutamic acid substitution at an amino acid corresponding to position 10 of SEQ ID NO: 13. Dependent refinements further specify additional sequence and amino-acid modifications of the Cas9 and/or modifications to the I-TevI nuclease domain.
The invention further describes editing workflows that include administering the chimeric nuclease to a mammalian cell, with optional exogenous donor DNA, and with non-viral delivery using lipid nanoparticles. The disclosed delivery includes lipid nanoparticles formulated to enable administration without viral vectors, including nebulization/inhalation.
Claims Coverage
The independent claim covers a method of editing genomic DNA in a mammalian cell by administering a specific TevCas9 chimeric nuclease and a guide RNA, where the RNA-guided Staphylococcus aureus Cas9 is a SEQ ID NO: 13 variant containing an Asp-to-Glu substitution at the amino-acid position corresponding to position 10 of SEQ ID NO: 13. Dependent claims refine nuclease sequence details, additional Cas9 mutations, targeting/cleavage span around specific variants, mutant-versus-wild-type selectivity for CFTR, and optionally contacting cells with exogenous donor DNA.
Chimeric TevCas9 dual-cleaving nuclease for genomic editing
Administering a chimeric nuclease comprising a modified Enterobacteria Phage T4 (I-TevI) nuclease domain, a linker, an RNA-guided Staphylococcus aureus Cas9, and a guide RNA to edit genomic DNA of a mammalian cell.
SEQ ID NO: 13 Cas9 Asp-to-Glu substitution at position 10
Using an RNA-guided Staphylococcus aureus Cas9 comprising the sequence of SEQ ID NO: 13 with an aspartic acid to glutamic acid substitution at an amino acid corresponding to position 10 of SEQ ID NO: 13.
Cas9 amino-acid mutation at position corresponding to 557 of SEQ ID NO: 13
Using Staphylococcus aureus Cas9 with an amino acid mutation at a position corresponding to position 557 of SEQ ID NO: 13.
Targeting cleavage span around CFTR Delta F508
Configuring the chimeric nuclease to target and cleave 10 to 20 nucleotides on both sides of the CFTR delta F508 mutation.
CFTR Delta F508 selective cleavage versus wild-type CFTR
Providing selective cleavage of a CFTR gene with the CFTR Delta F508 mutation while not cleaving a CFTR gene with the wild-type CFTR sequence.
Optional exogenous donor DNA for editing
Further including contacting a cell with an exogenous donor DNA.
Overall, the claims coverage centers on administering a TevCas9 chimeric nuclease that includes a modified I-TevI domain and an RNA-guided Staphylococcus aureus Cas9 with a SEQ ID NO: 13 Asp-to-Glu substitution at position corresponding to 10, with further refinements for additional nuclease and Cas9 sequence changes. Dependent claims focus on CFTR Delta F508 cleavage span and selectivity relative to wild-type CFTR and optionally add exogenous donor DNA.
Stated Advantages
Two-site targeting / dual-cleaving capability as described for the TevCas9 system.
Predictable deletion size described as ~30–36 bp deletions.
Donor-directed replacement when exogenous donor DNA is used.
Cas9 activity/specificity mutations, including described Cas9 variants such as D10E, are associated with the disclosed performance.
Non-viral delivery using lipid nanoparticles is described as avoiding viral vectors.
Low toxicity and controlled dosing are described for the non-viral lipid nanoparticle delivery.
Documented Applications
CFTR Delta F508 correction using donor DNA (HDR) as described in connection with targeting/editing CFTR Delta F508.
EGFR exon 19 deletion disruption or selective cleavage as described, including cleavage/disruption using the TevCas9 system without donor DNA relying on cleavage outcomes (e.g., NHEJ-based).
Nebulization/inhalation delivery of the non-viral lipid nanoparticle formulation for mammalian administration is described.
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