Destabilized SB Transposase Fusion Proteins for Copy Number Control

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Solution Overview

Problem

Current methods to control transposon copy number and transposase activity in genetically engineered cells, such as those using the Sleeping Beauty (SB) transposon system, are inadequate, leading to genotoxicity and transposon remobilization due to high transposase stability and copy numbers, which are not effectively addressed by existing DNA or mRNA regulation strategies.

Innovation Solution

Development of novel SB transposase fusion proteins with destabilizing domains, such as ecDHFR and IKZF3 zinc finger degron-tags, that can be regulated by small molecules like trimethoprim and pomalidomide, allowing precise control over transposase stability and activity, reducing transposon copy number and preventing remobilization.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If Sleeping Beauty transposase is used for gene transfer, then integration efficiency is improved, but transposon copy number increases leading to genotoxicity

Engineering Contradiction:

Improveintegration efficiencyVSAvoidgenotoxicity

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The transposase protein is engineered with a destabilizing domain that allows dynamic control of its stability. Without the inducing agent, the transposase is rapidly degraded, limiting the time window for transposition activity and reducing transposon copy number accumulation, thereby decreasing genotoxicity while maintaining integration efficiency during the active window.

Inventive Principle:

Principle #15Dynamics

Solution Approach 2:

The half-life of the transposase protein is changed from stable to unstable by fusing it with a destabilizing domain. This parameter change allows precise temporal control: the transposase is active only when the destabilizing domain is stabilized by the inducing agent, enabling high integration efficiency during treatment while preventing prolonged activity that would increase copy number and genotoxicity.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If transposase stability is increased to maintain activity, then transposition efficiency is improved, but transposon remobilization increases

Engineering Contradiction:

Improvetransposition efficiencyVSAvoidtransposon remobilization

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The transposase activity is applied periodically rather than continuously. The destabilizing domain ensures that transposase is active only during brief periods when the stabilizing agent is present, enabling transposition efficiency during these windows while preventing remobilization between windows when the transposase is degraded and inactive.

Inventive Principle:

Principle #19Periodic action

3Duration of action of moving object

If DNA-based regulation is used to control transposase expression, then transposase activity is maintained, but control precision is insufficient

Engineering Contradiction:

Improvetransposase activity durationVSAvoidcontrol precision

Core Design Contradiction:

Duration of action of moving objectVSMeasurement precision

Solution Approach 1:

The DNA-based transcriptional regulation system is replaced with a post-translational protein stability control system. Instead of using inducible promoters to control transposase expression, the invention uses a destabilizing domain that responds to small molecule inducers at the protein stability level, providing faster and more precise control over transposase activity duration and levels.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

4Duration of action of stationary object

If mRNA is used to encode transposase, then exposure time is shortened, but control over transposase stability is insufficient

Engineering Contradiction:

Improvetransposase exposure timeVSAvoidcontrol reliability

Core Design Contradiction:

Duration of action of stationary objectVSReliability

Solution Approach 1:

The transposase is constructed as a composite protein by fusing it with a destabilizing domain. This composite structure combines the catalytic function of transposase with the regulated stability properties of the destabilizing domain, enabling reliable control over transposase half-life and activity duration that cannot be achieved with mRNA alone.

Inventive Principle:

Principle #40Composite materials

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 fusion proteins enable rapid and precise control over transposase activity, minimizing genotoxicity and enabling safe, scalable production of genetically engineered cells for clinical applications like CAR T-cell therapy.

Implementation Method 1

the destabilizing domain or degron tag is capable of modulating the half-life of the complex

Methodology Applied

Scientific EffectProtein degradation: Decomposition (biological)

Implementation Method 2

the complex is capable of binding to a signaling molecule or ligand

Methodology Applied

Scientific EffectMolecular binding: Absorption (physical)

Data Source

PatentUS20250361284A1Transposase fusion proteins for use in cell and gene therapy

Publication Date: 2025.11.27 JULIUS MAXIMILIANS UNIV WURZBURG

AI summary

Fusion proteins and methods for conditionally fine-tuning transposase protein stability and activity for clinical applications are provided herein