Isolated Transposase for Stable Large-Fragment Genome Integration
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Solution Overview
Problem
Current gene therapy methods using viral vectors for large fragment gene integration face limitations such as random genome integration risks, limited gene size capacity, immunogenicity, and complex production processes, necessitating the development of more effective non-viral integration tools.
Innovation Solution
An isolated transposase with specific amino acid sequences or variants, capable of high transposition activity, is provided for integrating exogenous nucleic acid fragments into host cell genomes, offering alternatives to viral methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If viral vectors (lentivirus or retrovirus) are used to integrate gene sequences, then gene integration can be achieved, but random integration creates cancer risk and the virus size limits the exogenous gene capacity
Solution Approach 1:
The patent extracts the transposition function from viral vectors and implements it through a non-viral transposase system. The transposase enzyme performs DNA transposition without requiring viral replication machinery, thereby eliminating the harmful random integration characteristic of viral vectors while maintaining controlled gene integration capability.
Solution Approach 2:
The patent introduces a transposase enzyme as an intermediary protein that mediates the integration process. This enzyme recognizes specific target sequences in the genome and facilitates precise insertion of exogenous genes, replacing the uncontrolled viral integration mechanism with a controlled enzymatic process that reduces cancer risk.
2Quantity of substance
If viral vectors are used for gene integration, then gene transfer can be achieved, but the virus size limits the exogenous gene capacity and immunogenicity affects long-term expression
Solution Approach 1:
The patent employs a non-viral transposase system that avoids the complex, long-lived viral structure. The transposase enzyme can be delivered via simple plasmid vectors or RNA, which are easier to produce, less immunogenic, and do not require maintaining viral replication machinery, thereby reducing immunogenicity and enabling larger exogenous gene insertion.
Solution Approach 2:
The patent replaces the viral mechanical replication and integration system with a biochemical enzymatic system. The transposase enzyme catalyzes DNA transposition through chemical mechanisms rather than viral replication, eliminating the need for viral proteins that trigger immunogenic responses and allowing unrestricted gene size capacity.
3Ease of manufacture
If viral vectors are used for gene integration, then gene therapy can be achieved, but production requires living cells making quality control and downstream processing complex and expensive
Solution Approach 1:
The patent segments the gene therapy system into separate functional components: the transposase enzyme (delivered via plasmid or RNA) and the target DNA. This segmentation eliminates the need for complex viral production systems, allowing independent optimization of each component and simplifying quality control and downstream processing while maintaining therapeutic effectiveness.
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 transposase achieves stable integration and expression of large gene fragments with reduced immunogenicity and simplified production processes, providing enhanced gene therapy options.
Implementation Method 1
A transposon is a DNA sequence that can be inserted into or excised from the genome to transfer its own sequence or a complete copy of its own sequence within or between genomes
Implementation Method 2
Transposases are sequence-specific DNA-binding proteins expressed by DNA transposon sequences, comprising catalytic domains that mediate DNA breakage and ligation
Data Source
AI summary
Provided are an isolated transposase and the use thereof. Provided also are a nucleic acid and a nucleic acid construct encoding the transposase, a nucleic acid set and a nucleic acid set construct, and a composition, a recombinant vector, a recombinant host cell and a kit comprising the transposase. Provided also are a method for introducing an exogenous nucleic acid fragment into the genome of a host cell, a method for editing the genome of a host cell, and a method for obtaining a host cell containing an exogenous nucleic acid fragment in the genome. Provided also are the use of the transposase, the nucleic acid and the nucleic acid construct, the nucleic acid set and the nucleic acid set construct, the composition, the recombinant vector, or the recombinant host cell for introducing an exogenous nucleic acid fragment gene into the genome of a host cell or preparing a drug or a preparation for gene therapy, cell therapy, genome research, or stem cell induction and post-induction differentiation.