Engineered target specific base editors
Inventors
Fauser, Friedrich • Miller, Jeffrey C. • Rebar, Edward
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11834686-B2
Publication Date
2023-12-05
Expiration Date
Abstract
Described herein are DNA-editing complexes, particularly DNA-editing complexes that specifically alter a single base pair in target DNA sequence as well as methods of making and using these DNA-editing complexes.
Core Innovation
The disclosure relates to expression vectors for a system that edits an adenine or cytosine base in DNA. The Cas9-free system comprises three zinc finger protein (ZFP)-containing fusion proteins that position split inactive deaminase domains and nickase domains. The first and second inactive domains of an adenosine or cytidine deaminase dimerize to form an active deaminase, while dimerization of catalytically active and inactive nickase domains forms an active nickase.
The system edits target nucleobases without generating a double-strand break across the target DNA sequence. The disclosure encompasses encoding polynucleotides as well as viral or nonviral vectors, specifically including adeno-associated viral vectors suitable for delivery of smaller constructs. In addition, an optional uracil DNA glycosylase inhibitor component and DNA-destabilizing factors are incorporated to support targeted base editing.
Claims Coverage
The relevant claims identify one independent claim comprising four inventive features directed to expression vectors for a DNA base-editing system.
ZFP-containing fusion proteins
The expression vectors express a first fusion protein comprising a first inactive domain of an adenosine or cytidine deaminase and a first ZFP DNA-binding domain; a second fusion protein comprising a second inactive domain of the deaminase, a second ZFP DNA-binding domain, and a catalytically active or inactive nickase domain; and a third fusion protein comprising a catalytically inactive or active nickase domain and a third ZFP DNA-binding domain.
Deaminase activation by dimerization
The first and second inactive domains of the deaminase dimerize to form an active deaminase.
Nickase formation by dimerization
The second and third fusion proteins form an active nickase through dimerization of the catalytically active and inactive nickase domains.
Additional claimed system and vector formats
Dependent claims add a uracil DNA glycosylase inhibitor, specify its placement at an N- or C-terminal end of fusion protein(s), identify adeno-associated viral vectors, and claim kits and cells containing the expression vectors.
The independent claim covers expression vectors encoding three ZFP-containing fusion proteins that reconstitute active deaminase and nickase enzymes through dimerization, with dependent claims reciting inhibitors, vectors, kits, and host cells.
Stated Advantages
The system edits bases without generating a double-strand break.
The system provides improved targeting specificity, expanded target-site and PAM flexibility, and reduced off-target and bystander editing.
Adeno-associated viral vector delivery is identified as a beneficial administration format suitable for smaller constructs.
Examples report enhanced editing in tested constructs, although the described results principally concern Cas9-based editors.
Documented Applications
Editing an adenine or cytosine base in DNA.
Mutation correction, gene knockout, and therapeutic editing.
Correction of disease-associated mutations, including mutations associated with A1AT deficiency and JAK2 V617F.
Interested in licensing this patent?