Homing endonuclease variants
Inventors
JARJOUR, Jordan • HAVENS, Kyle • CHRYSOSTOMOU, CONSTANTINE
Assignees
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Publication Number
US-12404500-B2
Publication Date
2025-09-02
Expiration Date
Abstract
The present disclosure provides homing endonuclease variants and megaTALs reprogrammed to bind and cleave a polynucleotide sequence in the genome. The homing endonuclease variants and megaTALs have been engineered to increase the thermostability and/or activity.
Core Innovation
The disclosure relates to engineered I-OnuI homing endonuclease variants, including I-OnuI/LAGLIDADG homing endonuclease variants, and to megaTAL constructs that incorporate stabilized I-OnuI variants. The core engineering concept is to increase thermostability of an I-OnuI HE variant relative to a reference I-OnuI HE while maintaining at least 97% sequence identity to a reference set of I-OnuI sequences.
The document specifies that the engineered I-OnuI HE variants include at least four amino acid substitutions relative to the reference I-OnuI HE, with substitution patterns defined by amino acid positions relative to a reference sequence. The disclosure further describes claims that refine these substitution patterns by enumerating allowed amino acids at particular numbered positions and by requiring quantitative TM50 increases versus the reference.
Beyond single HE variants, the disclosure teaches megaTALs that combine TAL DNA-binding domains with stabilized I-OnuI variants. The document asserts that stabilized HE variants prolong expression and increase catalytic editing efficiency, and it describes transferring stabilizing mutations between different reprogrammed I-OnuI targets and assessing HE stability using yeast surface display melt curves and expression time-course assays.
Claims Coverage
The independent claims define thermostability-increasing I-OnuI homing endonuclease variants with at least 97% sequence identity to reference I-OnuI sequences and with amino-acid substitution patterns at specified positions. Two independent claims are provided, with one focusing on positional substitution sets and one focusing on enumerated allowed residues at specific positions.
Thermostability-increasing I-OnuI variant with at least 97% identity and positional substitution constraints
An I-OnuI homing endonuclease variant comprising at least 97% sequence identity to a reference I-OnuI comprising any one of SEQ ID NOs: 1-8 and 15, further comprising at least four amino acid substitutions relative to the reference I-OnuI HE that increase the thermostability of the I-OnuI HE variant compared to the reference I-OnuI HE, wherein the at least four amino acid substitutions consist of amino acid substitutions at specified positions in reference to SEQ ID NO: 1 and satisfy listed substitution-pattern conditions for positions selected from provided groups.
Thermostability-increasing I-OnuI variant with enumerated allowed residues at specified positions
An I-OnuI homing endonuclease variant comprising at least 97% sequence identity to a reference I-OnuI comprising any one of SEQ ID NOs: 1-8 and 15, further comprising at least four amino acid substitutions relative to the reference I-OnuI HE that increase the thermostability of the I-OnuI HE variant compared to the reference I-OnuI HE, and wherein the at least four amino acid substitutions are selected from amino acid substitutions at listed amino acid positions in reference to SEQ ID NO: 1 and constrained such that for each listed position the amino acid is selected from the enumerated group for that position.
Targeting a site in a gene from a specified gene group
The I-OnuI HE variant targets a site in a gene selected from the listed group of genes.
Overall claim coverage is centered on engineered I-OnuI HE variants with at least 97% sequence identity to reference sequences that include at least four amino acid substitutions increasing thermostability. The scope is tightened in one independent claim by required allowed amino-acid choices at many numbered positions, and in another by substitution patterns across specified position sets; a dependent feature requires targeting of a site in genes from a provided list.
Stated Advantages
Increases the thermostability of the I-OnuI HE variant compared to the reference I-OnuI HE.
Increases catalytic editing efficiency in disclosed megaTAL examples.
Prolongs expression of stabilized HE variants in disclosed megaTAL examples.
The disclosure asserts thermostability enhancements correlate with increased catalytic activity.
Documented Applications
Genome editing using megaTAL constructs that incorporate stabilized I-OnuI variants, including editing in the PDCD-1 (PD-1) context.
Targeting gene sites using engineered I-OnuI HE variants in selected human genes from a provided list, including BCL11A, PCSK9, PDCD-1, and TCRα/CBLB.
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