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-11814658-B2
Publication Date
2023-11-14
Expiration Date
Abstract
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 non-viral gene editing system using a chimeric dual-cleaving TevCas9 nuclease. The system employs a modified Staphylococcus aureus Cas9 fused to a modified I-TevI domain, coupled via a linker, and used with a guide RNA.
The chimeric nuclease is characterized by specified SEQ ID-based Cas9, I-TevI, and linker elements, including a Staphylococcus aureus Cas9 comprising the sequence of SEQ ID NO: 14. The system is configured to target CFTR and EGFR.
For CFTR, the document describes delta F508 correction with donor-directed HDR using exogenous donor DNA. For EGFR, the document describes deletion/indel formation via dual-site cleavage, including embodiments in which Cas9 can be nicking or cleavage-deficient.
Delivery and practical use are described around lipid nanoparticles encapsulating the nuclease and guide RNA, optionally with exogenous donor DNA. The document further describes lung delivery with nebulization/inhalation and includes mechanistic figure descriptions and example evaluation platforms using CFTR/EGFR cell contexts and functional readouts.
Claims Coverage
Two independent claims are identified: one directed to a modified Staphylococcus aureus Cas9 having a specified sequence, and another directed to a chimeric nuclease comprising I-TevI, a linker, Staphylococcus aureus Cas9, and a guide RNA. Across the independent claims, the inventive features are defined primarily by SEQ ID-specified sequence components and by the presence/arrangement of the I-TevI domain, linker, and guide RNA.
Modified Staphylococcus aureus Cas9 sequence
A nuclease comprising a modified Staphylococcus aureus Cas9 comprising the sequence of SEQ ID NO: 14.
Chimeric nuclease with I-TevI, linker, Cas9, and guide RNA
A chimeric nuclease comprising an I-TevI nuclease domain, a linker, a Staphylococcus aureus Cas9, and a guide RNA, wherein the Staphylococcus aureus Cas9 comprises the sequence of SEQ ID NO: 14.
The independent claims cover (i) a modified Staphylococcus aureus Cas9 defined by SEQ ID NO: 14 and (ii) a chimeric nuclease architecture that combines an I-TevI nuclease domain with a linker, the same SEQ ID NO: 14 Cas9, and a guide RNA. Dependent claims further constrain the nucleases by specifying additional SEQ ID-defined domain sequence and particular amino-acid substitutions/variants tied to referenced sequence positions, as well as linker sequence selections.
Stated Advantages
Dual independent target-site cleavage.
Predictable deletion length.
Rational Cas9 activity/specificity mutation.
Improved replacement efficiency with donor DNA.
Single-contiguous protein manufacturing.
Non-viral LNP delivery with low toxicity.
Nebulization for lung delivery.
Documented Applications
CFTR targeting, including delta F508 correction with donor-directed HDR using exogenous donor DNA.
EGFR exon 19 deletion, including deletion/indel formation via dual-site cleavage.
Lung delivery by nebulization/inhalation in the context of the described non-viral LNP system.
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