Soluble Sleeping Beauty Transposase Variant for Controlled Gene Delivery
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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 quality of active SB transposase protein.
Innovation Solution
A variant SB transposase with increased solubility and thermostability is developed by rational mutagenesis, allowing for direct protein delivery, which enhances control over transfection efficiency and safety by promoting recombinant integration of a transgene into target cells without the need for vector-based expression, thereby minimizing off-target activities and cytotoxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Sleeping Beauty transposase is used in vector-based expression, then transgene integration efficiency is achieved, but uncontrolled long-term transposition and genome instability occur
Solution Approach 1:
The transposase protein is pre-produced in a heterologous expression system (insect or mammalian cells) before delivery to target cells, enabling controlled transient expression that limits transposition activity to a specific time window and prevents long-term genomic instability
Solution Approach 2:
A heterologous expression system serves as an intermediary platform for producing the transposase protein, separating the production process from the transgene integration process, thereby enabling better control over transposase activity and duration
2Productivity
If Sleeping Beauty transposase protein is produced in large quantities, then transfection efficiency improves, but protein solubility and quality deteriorate
Solution Approach 1:
Insect or mammalian expression systems serve as intermediary platforms that enable high-level production of soluble, active transposase protein, overcoming the limitations of bacterial systems and enabling large-scale manufacturing with maintained protein quality
Solution Approach 2:
The expression system parameters are optimized (cell type, culture conditions, induction parameters) to maximize both protein yield and solubility, transforming the production process to achieve high quantities of quality protein
3Productivity
If vector-based transposase expression is used, then gene delivery is achieved, but manufacturing feasibility and safety control are reduced
Solution Approach 1:
The transposase protein is pre-produced and purified before delivery to target cells, separating the manufacturing process from the gene therapy process, thereby enabling better quality control, batch consistency, and regulatory compliance
Solution Approach 2:
The transposase gene is extracted from the therapeutic vector and its function is separated into a distinct protein production process, allowing independent optimization of manufacturing parameters and quality attributes
Data Source
AI summary
The present invention relates to improved transposase polypeptides having increased solubility. The enzyme of the invention was developed based on the Sleeping Beauty (SB) transposase. The invention provides further nucleic acids, vectors and recombinant cells encoding or containing the improved transposase, as well as a transposase system. Furthermore provided are medical and non-medical uses of the transposase of the invention for gene delivery. The invention is in particular useful as a tool for gene delivery in genetically modified cell based therapeutic approaches for treating various diseases.