Methods and compositions for modification of a cystic fibrosis transmembrane conductance regulator (CFTR) gene
Inventors
Conway, Anthony • Paschon, David
Assignees
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Publication Number
US-12043650-B2
Publication Date
2024-07-23
Expiration Date
Abstract
Nucleases and methods of using these nucleases for alteration of a CFTR gene and generation of cells and animal models.
Core Innovation
The invention relates to fusion molecules that comprise a functional domain and a DNA-binding domain that binds to a target site. The DNA-binding domain is implemented as a nuclease system based on a pair of zinc finger nucleases (ZFN), each zinc finger nuclease comprising a zinc finger DNA-binding domain and a cleavage domain, so that binding to specified target sites enables nuclease activity at those sites.
The disclosed approach applies the fusion molecule concept to genome editing at the CFTR locus for Cystic Fibrosis (CF). The content specifies target-site categories in the CFTR locus, including introns such as intron 1, 3, 6–8, and mutation-specific targeting such as a ΔF508 context. The modification can involve nuclease-mediated creation of DNA changes and targeted integration using donor or corrective sequences to generate genetically modified CFTR gene outcomes.
The document further describes genetically modified cells and transgenic animals produced using the disclosed genome-editing concepts. The described embodiments include delivery and composition options using polynucleotides, including plasmid and mRNA, and viral and non-viral vectors including AAV, adenovirus, and integrase-defective lentiviral vector (IDLV). The disclosed scope includes targeted integration of donor/transgene sequences and generation of modified lung/airway cells and stem/progenitor-derived cell contexts, as well as transgenic and embryo editing, including heritable germline editing.
Claims Coverage
The independent claims disclosed in the provided claim set include a fusion molecule claim and additional independent claim coverage for CFTR-focused gene modification and delivery/composition aspects. Across the independent claim coverage, the main inventive features include: a functional-domain/ZFP fusion as a zinc finger nuclease pair; CFTR gene modification in isolated cells with expressed nucleases; donor/transgene integration that yields a functional CFTR protein; mutation-specific donor correction and CFTR intron-target integration locations; and viral or non-viral delivery formats.
Zinc finger nuclease fusion molecule with functional domain
A fusion molecule comprising a functional domain and a DNA-binding domain that binds to a target site, wherein the fusion molecule comprises a nuclease comprising a pair of zinc finger nucleases (ZFN), each zinc finger nuclease comprising a cleavage domain and a zinc finger DNA-binding domain (ZFP).
CFTR gene modification in isolated cells via expressed ZFN polynucleotides
A method modifies one or more CFTR genes in an isolated cell by introducing polynucleotides encoding zinc finger nucleases so the nucleases are expressed to cleave and modify the CFTR genes.
Targeted integration of a CFTR gene transgene fragment for functional CFTR expression
The method further introduces a CFTR gene transgene fragment into the cell such that, upon integration into the CFTR gene, a functional CFTR protein is expressed.
Mutation-specific nuclease cleavage and AF508 donor correction
The method further uses a mutation-specific nuclease to cleave the CFTR gene in a cell carrying an AF508 mutation, and introduces an integrated donor nucleotide sequence that corrects AF508 to a wild-type CFTR sequence.
CFTR transgene integration into specified CFTR introns
The method further integrates a transgene into intron 1, intron 3, intron 7, or intron 8 of the CFTR gene.
Viral vector composition comprising an AAV vector
The claim covers a viral vector that includes an AAV vector as part of the composition.
The provided claim set centers on fusion molecules and ZFN-based DNA binding with cleavage domains, applied to CFTR genome editing. The inventive features further include isolated-cell CFTR modification using expressed ZFN-encoding polynucleotides, integration of a CFTR transgene fragment to produce functional CFTR protein, mutation-specific cleavage with AF508 donor correction, integration into specified CFTR introns, and delivery/composition features including viral vectors containing an AAV vector.
Stated Advantages
Documented Applications
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