Minigene Splice Modulator Binding Sequences for Tunable Gene Expression
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Solution Overview
Problem
Gene therapy methods using viral vectors like AAV face challenges in modulating the expression levels of therapeutic proteins, leading to side effects such as loss of drug efficacy or toxicity, due to the inability to control the timing or location of protein expression effectively.
Innovation Solution
The development of minigene nucleotide sequences that include specific exons and introns, along with a splice modulator binding sequence, allows for regulated protein expression by including or excluding certain exons in the mRNA product, enabling controlled expression of proteins using small molecules to turn them on or off.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If viral vectors like AAV are used to deliver therapeutic genes, then gene delivery efficiency is improved, but the ability to modulate protein expression levels is lost
Solution Approach 1:
The gene is divided into multiple exons, with a specific exon containing a splice modulator binding site. This segmentation allows the splicing machinery to selectively include or exclude this exon based on the presence of small molecule modulators, thereby enabling tunable protein expression while maintaining efficient viral delivery
Solution Approach 2:
A splice modulator binding site is introduced as an intermediary element within the gene sequence. Small molecule modulators bind to this site to influence splicing decisions, serving as a controllable intermediary that translates chemical signals into changes in protein expression levels without affecting the viral delivery mechanism
2Reliability
If therapeutic proteins are expressed at high levels, then treatment efficacy is improved, but toxicity increases
Solution Approach 1:
The expression system transitions from static to dynamic control, allowing protein expression levels to be adjusted in real-time based on therapeutic need. Small molecule modulators enable the system to switch between different expression states, optimizing the balance between efficacy and toxicity
Solution Approach 2:
The system changes the parameter of protein expression level from fixed to variable by introducing splice modulator binding sites. By varying the concentration of small molecule modulators, the expression level can be precisely tuned to achieve therapeutic effects while minimizing toxic side effects
3Duration of action of stationary object
If constitutive expression of therapeutic proteins is used, then continuous treatment effect is achieved, but loss of drug efficacy and side effects occur
Solution Approach 1:
Instead of continuous constitutive expression, the system enables periodic or conditional expression through splice modulator binding. The therapeutic protein can be expressed in pulses or only under specific conditions, maintaining continuous treatment effect while allowing periods of reduced expression to prevent drug resistance and side effects
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
This approach allows for precise modulation of protein expression levels, increasing efficacy and reducing side effects by enabling conditional expression of therapeutic proteins, thereby enhancing the safety and effectiveness of gene therapy.
Implementation Method 1
splice modulators to control expression of proteins encoded by heterologous nucleic acids. In particular,splice modulators can be used to control splicing of pre-mRNA transcripts
Data Source
AI summary
Provided herein are compositions comprising minigenes comprising splice modulator binding sequences, for regulatable gene expression, and systems and methods of use thereof.