Novel Integrases for Site-Specific Large DNA Integration
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Solution Overview
Problem
Existing integrases exhibit low integration efficiency and potential risks such as chromosomal rearrangement during integration of large-fragment DNA into mammalian genomes.
Innovation Solution
Development of novel integrases, including Cyin10, Cyin11, Cyin12, Cyin13, Cyin14, Cyin15, and Cyin16, with improved integration activity, particularly for large-fragment DNA, and their derived polypeptides, which can integrate exogenous genes into mammalian genomes with high specificity and reduced multi-site integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing integrases are used to integrate large-fragment DNA into mammalian genomes, then integration capability is achieved, but integration efficiency is low and chromosomal rearrangement risks occur
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of integrase proteins through site-directed mutagenesis. Specific residues are mutated to alter the integrase's catalytic properties, thereby improving integration efficiency and reducing chromosomal rearrangement risks while maintaining the core integrase function.
Solution Approach 2:
The patent creates multiple copies of integrase variants with different mutations (e.g., S130R, N131R, D132R mutations at different positions). These copied and slightly modified integrase sequences are tested to identify the optimal variant that achieves high integration efficiency with minimal harmful effects.
2Manufacturing precision
If existing integrases mediate large-fragment integration, then unidirectional integration and site-specificity are achieved, but multi-site integration occurs causing potential risks
Solution Approach 1:
The patent modifies the integrase's substrate recognition parameters by mutating specific amino acid residues that interact with the attB and attP sites. These parameter changes enhance the integrase's specificity for the intended target site, reducing off-target multi-site integration events.
3Duration of action of stationary object
If PhiC31 integrase is used for gene integration, then long-term disease symptom improvement is achieved, but low integration efficiency and multi-site integration remain
Solution Approach 1:
The patent systematically mutates PhiC31 integrase at multiple positions (e.g., positions 130, 131, 132, 192, 193, 194, 258, 259, 260) to optimize its catalytic parameters. These parameter changes aim to maintain the long-term therapeutic effect while significantly improving integration efficiency and reducing multi-site integration.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The novel integrases achieve efficient, site-specific integration of large-fragment DNA into mammalian genomes, minimizing chromosomal rearrangements and enhancing gene therapy applications.
Implementation Method 1
Integrase is a recombinase derived from bacteriophages, and can mediate recombination between the attP site on the bacteriophage genome and the attB site on the bacterial genome, thereby site-specifically integrating the bacteriophage genome into the host genome.
Implementation Method 2
mammalian genomes have pseudo attP sites that are homologous to the attP site, and these pseudo attP sites can also recombine with a plasmid carrying the attB site under the action of integrase, thereby integrating an exogenous gene on a plasmid into a genome.
Data Source
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AI summary
The disclosure relates to an enzyme capable of mediating the integration of large DNA fragments into mammalian genomes and uses thereof. Specifically, novel enzymes that can integrate large DNA fragments into genomes are provided, including integrases as shown in SEQ ID NO: 1-7. The disclosure further relates to polynucleotides containing nucleotide sequences encoding the integrases, and host cells containing these polynucleotides. The disclosure also relates to the integrases and methods for recombining target nucleic acids into the human genome by using the integrases.