Programmable Transposases for Large Fragment Gene Delivery
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Solution Overview
Problem
Current gene editing technologies face challenges in efficiently delivering and integrating large nucleic acid fragments, such as the Dystrophin and Laminin-α2 genes, into mammalian cells, as existing methods like base editors and CRISPR-based systems are inefficient for large edits and have limitations in precision and scalability.
Innovation Solution
A composition comprising a site-specific DNA binding protein and a modified hyperactive PiggyBac transposase, with specific amino acid mutations, is used to facilitate targeted gene delivery and integration of both small and large nucleic acid fragments into mammalian cells, achieving high efficiency and precision through site-directed integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If base editors or prime editors are used to edit DNA bases, then base-level precision is improved, but the ability to deliver large nucleic acid fragments deteriorates
Solution Approach 1:
The system segments the gene delivery process into two independent functions: (1) a site-specific DNA binding protein creates a targeted double-strand break at the desired genomic location, and (2) a modified hyperactive PiggyBac transposase delivers the large nucleic acid fragment to that location. This segmentation allows each component to optimize for its specific function while working together to achieve both precision and large fragment delivery capability
Solution Approach 2:
The invention merges the targeting capability of CRISPR-Cas systems with the large fragment delivery capability of PiggyBac transposons into a unified gene delivery system. The modified hyperactive PiggyBac transposase is engineered to work synergistically with the site-specific DNA binding protein, combining the precision of programmable DNA targeting with the efficiency of transposon-mediated large fragment integration
2Manufacturing precision
If HDR-based editing is used to insert genetic material, then integration precision is improved, but scalability to large edits deteriorates
Solution Approach 1:
The modified hyperactive PiggyBac transposase acts as an intermediary that bridges the gap between the site-specific DNA binding protein and the large nucleic acid fragment. It captures the fragment at the targeted location created by the DNA binding protein and facilitates its efficient integration, serving as a mediator that enables both precision and scalability for large gene deliveries
3Quantity of substance
If HITI methodology is used for insertions, then delivery capability for large fragments is improved, but integration precision deteriorates
Solution Approach 1:
The system applies local quality by creating a highly localized double-strand break at the precise target site using the site-specific DNA binding protein, then concentrating the integration activity of the modified hyperactive PiggyBac transposase at that same localized site. This localized action ensures both high precision and the ability to handle large fragment sizes
4Ease of operation
If previous PiggyBac or sleeping beauty transposase systems are used, then ease of operation in mammalian cells is improved, but integration precision deteriorates
Solution Approach 1:
The invention creates a composite system by fusing the modified hyperactive PiggyBac transposase with a site-specific DNA binding protein in a fusion protein. This composite structure combines the mammalian cell compatibility and transposition efficiency of PiggyBac with the precise targeting capability of the DNA binding protein, achieving both ease of operation and high integration precision
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 solution achieves high efficiency (5-10%) of site-directed integration in mammalian cells and in vivo mouse liver, demonstrating improved precision and scalability for delivering large nucleic acid fragments compared to existing methods.
Implementation Method 1
a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding said second protein; wherein said transposase is a modified hyperactive PiggyBac
Implementation Method 2
a first protein comprising or consisting of a site-specific DNA binding protein capable of binding and cleaving a target nucleic acid sequence
Data Source
AI summary
The present disclosure provides efficient and precise programmable gene delivery technology based on a composition comprising (i) a first protein comprising or consisting of a site-specific DNA binding protein capable of binding and cleaving a target nucleic acid sequence; or a nucleic acid construct encoding said first protein; and (ii) a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding said second protein; wherein said transposase is a modified hyperactive PiggyBac.