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Publication Number
US-11827900-B2
Publication Date
2023-11-28
Expiration Date
Abstract
Described herein are engineered nucleases comprising mutations in the cleavage domain (e.g., FokI or homologue thereof) and/or DNA binding domain (zinc finger protein, TALE, single guide RNA) such that on-target specificity is increased.
Core Innovation
The disclosure describes a polynucleotide encoding an engineered Flavobacterium okeanokoites (FokI) cleavage half-domain comprising one or more specified substitution mutations. The engineered half-domain exhibits at least 90% sequence identity to amino acids 384–579 of the referenced wild-type FokI cleavage domain. This engineered cleavage half-domain is incorporated into artificial nucleases designed to enhance on-target cleavage specificity.
The disclosure addresses nonspecific DNA-backbone interactions associated with off-target cleavage by combining FokI mutations with delivery of cleavage partners at non-one-to-one ratios. Engineered variants demonstrate strong on-target activity alongside reduced off-target cleavage and indels across BCL11A, TRAC, and AAVS1 genomic contexts. Selected engineered nuclease combinations achieve on-target to off-target activity ratios exceeding 1,000, and clinical-scale CD34+ cell testing demonstrates on-target indels without significant modification detected at surveyed off-target loci.
Claims Coverage
The provided claims identify one independent claim and cover four inventive features encompassing the independent claim and dependent-claim subject matter.
Polynucleotide encoding an engineered FokI cleavage half-domain
The independent claim covers a polynucleotide encoding an engineered FokI cleavage half-domain comprising one or more specified substitution mutations and at least 90% sequence identity to amino acids 384–579 of the referenced wild-type FokI cleavage domain.
Artificial nuclease with DNA-binding domains
The claims describe artificial nucleases comprising the engineered cleavage half-domain and DNA-binding domains selected from a zinc finger protein, a TALE domain, or an sgRNA, including heterodimeric configurations.
Compositions comprising polynucleotides with non-one-to-one ratios
The claims describe compositions comprising polynucleotides encoding respective cleavage partners or DNA-binding domains of an artificial nuclease, including configurations where the partner ratio is not one to one.
Genome-cleavage methods
The claims describe methods involving cleavage of cellular chromatin, including cleavage at multiple genomic target sites and donor-assisted homologous recombination.
The claims center on a polynucleotide encoding a specified engineered FokI cleavage half-domain and extend to artificial nucleases, ratio-adjusted compositions, and genome-cleavage methods.
Stated Advantages
The engineered variants reduce off-target cleavage and indels at measured off-target sites while retaining strong on-target activity, improving cleavage specificity.
The nucleases reduce nonspecific DNA-backbone interactions, with selected combinations achieving on-target to off-target ratios above 1,000.
Combining FokI mutations with unequal zinc-finger nuclease partner dosing improves on-target to off-target ratios across diverse mutation profiles and nuclease backgrounds.
Documented Applications
Targeted nuclease cleavage in BCL11A, TRAC, and AAVS1 genomic contexts.
Cleavage of genomic cellular chromatin for genome modification, including donor-assisted homologous recombination.
Cleavage of genomic cellular chromatin at multiple genomic target sites.
Artificial nucleases utilizing DNA-binding domains comprising zinc finger proteins, TALE domains, or sgRNAs.
Clinical-scale testing in CD34+ cells.
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