Enhanced Sleeping Beauty Transposons With IR/DR Sequences
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current Sleeping Beauty transposon systems have limitations in transposition efficiency due to aberrant single-ended transposition events, necessitating enhanced IR/DR sequences and cofactors to improve integration rates and genomic stability.
Innovation Solution
The development of polynucleotides with specific IR/DR sequences and the use of cofactors such as FACT complex depletors and ATR signaling modulators to enhance transposition efficiency, specifically targeting SSRP1, SUPT16H, and HSP90, while arresting cell cycles to optimize Sleeping Beauty transposase activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional Sleeping Beauty transposon systems are used, then transposition can occur, but transposition efficiency is limited due to aberrant single-ended transposition events
Solution Approach 1:
The patent modifies the IR/DR sequences of the transposon to optimize transposition efficiency. Specifically, it uses enhanced left and right inverted repeat/direct repeat sequences that improve transposase binding and facilitate proper double-ended transposition, thereby increasing productivity while maintaining genomic stability through reduced aberrant events
Solution Approach 2:
The patent introduces cofactors as intermediaries to enhance transposition. These cofactors mediate the transposition process by facilitating proper transposon end joining and preventing aberrant single-ended events, thus resolving the contradiction between efficiency and reliability
2Productivity
If transposition is enhanced using cofactors, then transposition rates increase 50-fold, but system complexity increases
Solution Approach 1:
The patent employs cofactors that are prepared and introduced before transposition occurs. These cofactors pre-condition the cellular environment by depleting FACT complex components or inhibiting cathepsins, thereby creating optimal conditions for high-efficiency transposition without requiring complex real-time control mechanisms
Solution Approach 2:
The patent achieves 50-fold enhancement in transposition rates by modifying specific parameters: introducing cofactors that alter cellular conditions (FACT complex depletion, cathepsin inhibition), using optimized IR/DR sequences, and controlling cell cycle phase. These parameter changes drive high productivity while keeping the system relatively simple
Data Source
AI summary
The present invention relates to enhanced Sleeping Beauty-type transposons and methods of transposition. In particular the invention relates to a polynucleotide comprising a cargo nucleic acid flanked by a left and a right inverted repeat/direct repeat (IR/DR), wherein IR/DRs, having specific sequences, are recognized by a Sleeping Beauty transposase protein and the polynucleotide is capable of integrating into the DNA of a cell. The invention also relates to a kit for transposing a nucleic acid comprising said polynucleotide as well as to further components such as co-factors of transposition capable of depleting a component of the FACT (facilitates chromatin transcription) complex, namely, SSRP1 and/or SUPT16H/SPT16, or an inhibitor of cathepsin selected from the group comprising H, S, V, and L; or a cofactor capable of depleting or inhibiting HSP90; or a factor temporally arresting cells cell cycle in cell cycle phase G0/G1, G1/S, or G2/M; or a factor inhibiting the ubiquitination of PCNA, or cells wherein these components have been knocked down or inhibited, or the cell cyle arrested in any of said stages. Alternatively or additionally, the kit may comprise as a co-factor of transposition an agent capable of increasing concentration and/or signaling of ATR or a cell wherein concentrationand/or signaling of ATR are increased. The invention further provides methods using said transposon polynucleotide as well as host cells and pharmaceutical compositions. It also relates to use of said co-factors of transposition or specific cells for enhancing transposition efficiencies, e.g., for preparing genetically modified nucleic acids or cells.