Soluble SB Transposase Protein for Controlled Transgene Integration
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Solution Overview
Problem
Current Sleeping Beauty (SB) transposase systems face challenges in manufacturing feasibility and safety, particularly in clinical applications, due to uncontrolled long-term transposition, undesired insertion events, and genome instability, which are exacerbated by the difficulty in producing sufficient quantities and qualities of active SB transposase protein.
Innovation Solution
A variant SB transposase with increased solubility and thermostability is developed through rational mutagenesis, allowing for direct protein delivery, which enhances control over transfection efficiency and safety by minimizing long-term transposition and reducing insertions per cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Sleeping Beauty transposase is used for gene delivery in clinical applications, then gene delivery efficiency is improved, but manufacturing feasibility and safety deteriorate due to uncontrolled long-term transposition and difficulty in producing sufficient quantities of active transposase protein
Solution Approach 1:
The patent introduces specific amino acid mutations (C176S and I212S) in the transposase protein sequence to alter its biophysical properties. These parameter changes in the protein structure dramatically improve solubility and stability, enabling large-scale production while maintaining transposase activity and gene delivery efficiency
Solution Approach 2:
The patent employs a transient expression system where the transposase protein is expressed temporarily in host cells (such as HEK293 cells) and then the cells are discarded. This disposable approach eliminates the need for complex purification and long-term maintenance of transposase production systems, significantly improving manufacturing feasibility while maintaining high gene delivery efficiency
2Adaptability or versatility
If Sleeping Beauty transposase is used for gene delivery, then genetic engineering capability is improved, but safety deteriorates due to uncontrolled long-term transposition and insertional mutagenesis
Solution Approach 1:
The patent utilizes the transient expression of transposase protein, which creates a time-limited window of transposase activity. The protein is expressed temporarily, performs its function of mediating transposition, and then is degraded by cellular proteasomes. This periodic action ensures that transposition events are confined to a specific time period, preventing uncontrolled long-term transposition and reducing insertional mutagenesis while maintaining genetic engineering capability
Solution Approach 2:
The patent exploits the natural feedback mechanism of cellular protein degradation systems. The transposase protein, once expressed, is automatically degraded by the cell's proteasome system after a defined period. This built-in feedback control mechanism limits the duration of transposase activity, thereby controlling transposition events and minimizing harmful insertional mutagenesis while preserving the desired genetic engineering outcomes
3Quantity of substance
If Sleeping Beauty transposase protein is produced in large quantities, then gene delivery capacity is improved, but protein solubility and stability deteriorate
Solution Approach 1:
The patent introduces specific amino acid substitutions (C176S and I212S) that fundamentally change the biophysical parameters of the transposase protein. These mutations reduce aggregation propensity and improve solubility, allowing the protein to be produced in large quantities in soluble form, which can then be purified and stored without precipitation or degradation
Solution Approach 2:
The patent creates a modified version (copy) of the transposase protein with improved properties. The mutated transposase variant serves as a superior copy that retains all necessary catalytic functions while possessing enhanced solubility and stability characteristics, enabling large-scale production that would be impossible with the wild-type protein
Data Source
AI summary
This disclosure provides improvised reagents and methods for inserting a transgene into the genome of a living host cell. Sleeping beauty (SB) transposase is used in protein form rather than as a vector. This has been made possible by using rational mutagenesis in a particular region of the SB crystal structure to develop variant SB transposase protein that is more soluble. The changes increase solubility while maintaining transposase activity, thereby adapting the variant transposase for the purpose of promoting recombinant integration of a transgene into a target cell when used in protein form rather than as a polynucleotide vector. The modified transposase is highly soluble in electroporation buffer and thermostable during storage. When introduced into a host cell, it promotes integration of a transgene into the genome of the cell in a dose-dependent manner. It is degraded within 48 hours, thereby rapidly clearing transposase activity from the host cell.