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-12297467-B2
Publication Date
2025-05-13
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 approach that uses a lipid-encapsulated dual-cleaving TevCas9 system. The system includes a modified RNA-guided Staphylococcus aureus Cas9 fused via a linker to a modified I-TevI nuclease domain, optionally in a form that further incorporates exogenous donor DNA.
The TevCas9 design targets two independent DNA sites to generate precise deletions, including 30–36 nt deletions, or to facilitate sequence replacement. Sequence replacement is described as being mediated by HDR and/or NHEJ, using the TevCas9/guide RNA targeting with optional donor DNA.
Reprogrammability is enabled by changing elements including the I-TevI/linker/Cas9/guide RNA. The approach also provides rational modulation of Cas9 activity and specificity using specified saCas9 variants and other Cas9 nicking/deficient variants, with the constructs using defined guide RNA sequences to target genomic loci.
The document further describes mechanistic and delivery concepts for non-viral lung tissue application using lipid nanoparticles. It describes uptake and intracellular trafficking concepts including cell uptake, endosomal escape, and nuclear localization, and uses lung delivery by nebulization in treatment concepts for CFTR delta F508 and EGFR exon 19 deletion.
Claims Coverage
The document contains two independent claims directed to a modified Staphylococcus aureus Cas9 nuclease with specified sequence identity and a defined aspartic acid-to-glutamic acid substitution, and to a chimeric nuclease that further includes an I-TevI nuclease domain, a linker, and a guide RNA. Across the independent claims, the inventive features include specific Cas9 sequence constraints, fusion to an I-TevI nuclease domain, and defined linker and guide RNA features.
Modified saCas9 with Glu10 substitution and high sequence identity
A nuclease comprising a modified Staphylococcus aureus Cas9 with an aspartic acid to glutamic acid substitution at an amino acid corresponding to position 10 of SEQ ID NO: 13, wherein the amino acid sequence of the Staphylococcus aureus Cas9 has at least 99% sequence identity to SEQ ID NO: 13.
Chimeric nuclease with I-TevI domain fused to saCas9 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 an aspartic acid to glutamic acid substitution at an amino acid corresponding to position 10 of SEQ ID NO: 13 and the amino acid sequence of the Staphylococcus aureus Cas9 has at least 99% sequence identity to SEQ ID NO: 13.
Overall claim coverage centers on engineered nucleases in which a modified saCas9 with the specified Glu10 substitution and at least 99% sequence identity to SEQ ID NO: 13 is used as a modified nuclease core or incorporated into a chimeric construct that further includes an I-TevI nuclease domain, a linker, and a guide RNA.
Stated Advantages
Reprogrammability via changing the I-TevI/linker/Cas9/guide.
Rational Cas9 activity and specificity modulation using specified saCas9 variants and other Cas9 nicking/deficient variants.
Non-viral delivery using lipid nanoparticles suitable for nebulization to lung tissue.
Documented Applications
Treatment concepts for CFTR delta F508 (cystic fibrosis).
Treatment concepts for EGFR exon 19 deletion (NSCLC).
Non-viral delivery by nebulization to lung tissue using lipid nanoparticles in the described constructs.
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