Evolved Integrases for Large-Cargo Genome Insertion
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Solution Overview
Problem
Current virus-based gene insertion methods are inefficient for larger cargos and often cause uncontrolled large deletions or chromosomal rearrangements, particularly in non-dividing cells, limiting their therapeutic effectiveness for genetic diseases.
Innovation Solution
Evolved Bxb1 and PhiC31 integrases with specific mutations exhibit enhanced efficiency in integrating large DNA cargos into specific genomic sites, reducing off-target insertion and mutagenesis by using prime editing technologies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If virus-based gene insertion methods are used, then gene insertion can be achieved, but efficiency drops significantly for larger cargos and causes uncontrolled large deletions or chromosomal rearrangements
Solution Approach 1:
The patent applies parameter changes by evolving integrase enzymes through directed evolution to optimize their catalytic activity and specificity. Multiple rounds of mutagenesis and selection generated integrase variants with improved integration efficiency and reduced off-target effects, directly addressing the contradiction between productivity and reliability
Solution Approach 2:
The patent uses integrase enzymes as intermediaries to mediate site-specific recombination between attB and attP sites. This enzymatic mediation provides controlled and precise integration, replacing the uncontrolled virus-based insertion mechanism while maintaining high efficiency for large cargo insertion
2Productivity
If HDR-based insertion methods are used, then homology-directed repair can occur, but insertion efficiency drops significantly for larger cargos and is inefficient or nonfunctional in non-dividing cells
Solution Approach 1:
The patent replaces the HDR mechanical system (which requires DNA double-strand breaks and cellular repair machinery) with an integrase-mediated site-specific recombination system. This substitution eliminates the dependency on cell division and HDR pathway functionality, enabling efficient integration in both dividing and non-dividing cells including neurons and muscle cells
3Productivity
If NHEJ-based insertion methods are used, then insertion can occur, but methods are generally inefficient and rely on unprotected double-strand breaks that cause uncontrolled large deletions or chromosomal rearrangements
Solution Approach 1:
The patent introduces integrase enzymes as intermediaries that catalyze site-specific recombination between defined attB and attP sites. This enzymatic mediation eliminates the need for unprotected double-strand breaks required by NHEJ, providing controlled integration while preventing chromosomal rearrangements and large deletions
Solution Approach 2:
The patent employs preliminary action by first establishing attP integration sites in the genome through integrase-mediated recombination before performing cargo insertion. This pre-positioning of recognition sites enables subsequent controlled integration events without requiring double-strand breaks, thereby preventing chromosomal damage
Data Source
AI summary
Disclosed are evolved Bxb1 and PhiC31 integrases that exhibit increased insertion activity and efficiency as compared to their wild-type counterparts and use of the evolved integrases to modify any sequence within the genome of a cell or subject.