Heliothis Transposase Systems for Stable Eukaryotic DNA Integration
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Solution Overview
Problem
Existing piggyBac-like transposases are difficult to identify and implement effectively for stable integration and expression of heterologous DNA in eukaryotic genomes due to host inactivation and lack of active transposases, limiting genomic engineering and gene therapy applications.
Innovation Solution
Development of novel piggyBac-like transposon-transposase systems derived from Heliothis virescens, Agrotis ipsilon, and Helicoverpa armigera, with modified transposases exhibiting enhanced excision and transposition activities, allowing stable integration and expression of heterologous DNA in eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If piggyBac-like transposases are used for integration of heterologous DNA, then transformation efficiency increases, but host inactivation occurs and active transposases become difficult to identify
Solution Approach 1:
The patent modifies the transposase enzyme parameters by introducing specific amino acid mutations (e.g., D268N, D346N, D447N in the DDE motif) to create integration-deficient variants. These parameter changes alter the enzymatic function to perform excision without integration, resolving the contradiction between achieving transformation and maintaining host viability.
Solution Approach 2:
The patent extracts the integration function from the transposase enzyme while retaining the excision function. By creating transposase variants that are deficient in integration activity but retain excision activity, the system allows removal of transposons without permanent genomic disruption, thus maintaining host viability while achieving transformation goals.
2Productivity
If transposases with high excision activity are used, then transposon removal efficiency increases, but ability to integrate into second target sequence is reduced
Solution Approach 1:
The patent segments the transposase function into two distinct activities: excision and integration. By creating transposase variants with specific mutations that enhance excision activity while reducing integration activity, the system can perform excision independently, allowing controlled manipulation of transposon presence without unwanted secondary integrations.
3Adaptability or versatility
If multiple different transposons are integrated into the genome, then genomic engineering capability increases, but transposition specificity decreases and expression properties change
Solution Approach 1:
The patent uses transposon terminal repeat sequences as intermediaries to ensure specific recognition and binding by corresponding transposases. Each transposon system has unique terminal repeats that serve as specific binding sites for their cognate transposases, preventing cross-reactivity and maintaining transposition specificity even when multiple different transposon systems are present in the same genome.
Data Source
AI summary
The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery.