Aptamer Riboswitch Splicing Cassette for Precise Gene Expression Control
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Solution Overview
Problem
Existing gene expression regulation techniques lack the ability to precisely control and modulate the expression of therapeutic transgenes, which is crucial for safe and effective therapeutic applications.
Innovation Solution
A polynucleotide cassette comprising a riboswitch and an alternatively-spliced exon, flanked by 5′ and 3′ introns, which includes an effector region with a 5′ splice site and an aptamer that binds a small molecule ligand, allowing for controlled splicing and expression modulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional gene expression regulation techniques are used, then gene expression can be controlled, but the precision and modulation capability of therapeutic transgene expression is insufficient
Solution Approach 1:
The patent employs a riboswitch structure that dynamically changes its conformation in response to small molecule ligand binding. The aptamer region binds the ligand, inducing a structural change that propagates to the effector region, thereby dynamically modulating alternative splicing of the target gene. This dynamic response enables precise and versatile control of gene expression levels based on ligand concentration.
Solution Approach 2:
The invention utilizes changes in RNA secondary structure parameters (stem formation, loop configuration) in response to ligand binding. The riboswitch transitions between different structural states (e.g., stem-formed vs. stem-disrupted configurations) that correspond to different splicing outcomes, enabling continuous modulation of gene expression across a wide dynamic range (up to 1687-fold induction).
2Measurement precision
If a riboswitch with aptamer and effector region is introduced, then precise control of gene expression is achieved, but the structural complexity of the polynucleotide cassette increases
Solution Approach 1:
The riboswitch is segmented into distinct functional regions: the aptamer region that binds the small molecule ligand, the effector region containing the 5' splice site, and the alternatively-spliced exon with stop codon. This segmentation allows each region to perform its specific function independently while contributing to the overall regulatory mechanism, making the complex structure manageable and engineerable.
Solution Approach 2:
The riboswitch structure serves multiple functions within a single polynucleotide cassette: ligand sensing, conformational switching, and splicing regulation. The alternatively-spliced exon also serves dual purposes by providing both the stop codon for gene silencing and the splicing regulatory element. This multi-functionality reduces the need for separate regulatory components.
3Reliability
If alternatively-spliced exon with stop codon is used, then gene expression can be silenced when exon is included, but the mechanism requires complex splicing regulation
Solution Approach 1:
The invention converts the potentially harmful effect of constitutive alternative splicing (which would lead to unwanted gene silencing) into a beneficial regulated mechanism. By placing the 5' splice site within the riboswitch effector region, the splicing event itself becomes the mechanism for controlled gene expression. The stop codon in the alternatively-spliced exon, which would be harmful if always present, becomes a useful tool for ligand-inducible gene silencing.
Solution Approach 2:
The riboswitch-splicing system is self-regulating through the coupling of ligand binding to splicing outcomes. The RNA structure automatically responds to ligand presence by forming or disrupting the stem containing the 5' splice site, thereby self-determining whether the alternatively-spliced exon with the stop codon is included in the mature mRNA. This self-service mechanism eliminates the need for external regulatory proteins or complex transcriptional control elements.
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 cassette enables up to 1687-fold induction of target gene expression in response to ligand presence, providing precise control over gene expression levels and timing.
Implementation Method 1
an aptamer that binds a small molecule ligand
Data Source
AI summary
The invention provides a platform and methods of using the platform for the regulation of the expression of a target gene using exposure to an aptamer ligand (for example, a small molecule). The platform features a polynucleotide gene regulation cassette that is placed in the target gene and includes a synthetic riboswitch positioned in the context of a 5′ intron-alternative exon-3′ intron. The riboswitch comprises an effector region and a sensor region (e.g., an aptamer that binds a small molecule ligand) such that the alternative exon is spliced into the target gene mRNA when the ligand is not present thereby preventing expression of the target gene. When the ligand is present, the alternative exon is not spliced into the target gene mRNA thereby providing expression of the target gene.