Synthetic mRNA Toehold Switches for Tissue-Selective Translation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current mRNA delivery methods lack specificity, leading to non-specific expression of proteins in unwanted cells, resulting in high toxicity due to the use of nonspecific vehicles that deliver RNA molecules to both target and non-target cells.
Innovation Solution
Designing a synthetic RNA molecule with a specific structure that includes a reverse complement, loop region, and ribosome binding site (RBS) to ensure protein expression only in a targeted tissue or cell type, utilizing a toehold switch mechanism to enhance dynamic range in eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If nonspecific delivery vehicles are used to deliver RNA molecules, then the RNA can be delivered to target cells, but the RNA also arrives at non-target cells causing high toxicity
Solution Approach 1:
The patent applies local quality by designing RNA molecules with tissue-specific regulatory elements (enhancers, promoters, miRNA target sites) that are inserted at specific locations within the RNA sequence. These elements confer cell-type-specific expression properties to otherwise identical coding sequences, allowing the same therapeutic protein to be expressed only in target tissues while avoiding toxicity in non-target cells.
Solution Approach 2:
The patent utilizes parameter changes by modifying RNA sequence parameters (GC content, secondary structure, stability elements) and regulatory element composition to control RNA stability, translation efficiency, and cellular uptake. By adjusting these parameters, the RNA can be optimized for specific tissue types and delivery conditions, improving therapeutic index by enhancing target cell uptake while minimizing non-specific distribution.
2Productivity
If RNA molecules are delivered with nonspecific vehicles, then delivery is simple and cost-effective, but protein expression occurs in unwanted cells resulting in high toxicity
Solution Approach 1:
The patent segments the RNA molecule into distinct functional modules: a coding sequence region, regulatory elements (enhancers, promoters), stability elements, and cell-type-specific control regions. This segmentation allows independent optimization of each module for its specific function while maintaining overall RNA integrity and delivery efficiency. The modular design enables precise control over where and how the protein is expressed without compromising delivery.
Solution Approach 2:
The patent introduces intermediary regulatory elements (miRNA binding sites, tissue-specific promoters, enhancer sequences) that act as mediators between the delivered RNA and the cellular machinery. These intermediaries determine whether the RNA will be translated in a given cell type, providing an additional layer of control that maintains high delivery efficiency while ensuring specificity through biological recognition mechanisms.
3Measurement precision
If toehold switches are designed with rational RNA sequence in prokaryotes, then dynamic range can reach over 400-fold, but similar approaches in eukaryotes achieve only maximum 2-fold dynamic range
Solution Approach 1:
The patent inverts the conventional toehold switch design approach by placing the ribosome binding site and start codon within the structured region of the RNA rather than in an accessible unstructured region. This inversion creates a switch where the default state is 'on' (translation accessible) and the trigger-bound state becomes 'off' (translation blocked), achieving high dynamic range in eukaryotes by leveraging eukaryotic translation initiation mechanisms differently than in prokaryotic systems.
Solution Approach 2:
The patent creates composite RNA structures that combine toehold switch elements with eukaryotic-specific features including Kozak sequences, 5' cap structures, and 3' polyadenylation signals. This composite design integrates prokaryote-inspired switching mechanics with eukaryotic translation machinery requirements, enabling the switch to function effectively in eukaryotic cells with dramatically improved dynamic range compared to previous attempts.
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
Achieves selective protein expression in the target tissue or cell type while minimizing or preventing expression in non-target tissues or cells, thereby reducing toxicity and improving the specificity of mRNA-based therapies.
Implementation Method 1
a reverse complement to the selected region comprising a 5′ unhybridized region and a 3′ hybridized region
Data Source
AI summary
Methods of designing a synthetic RNA molecule comprising obtaining a coding sequence encoding a protein of interest; obtaining a sequence of an RNA expressed in a first tissue or cell type and not expressed or lowly expressed in a second tissue or cell type; selecting a region within the sequence of an RNA; and producing a sequence of a synthetic RNA molecule comprising from 5′ to 3′: a reverse complement to the selected region comprising a 5′ unhybridized region and a 3′ hybridized region, a loop region that does not hybridize; a region reverse complementary to 3′ hybridized region and the obtained coding sequence encoding a protein of interest are provided. Synthetic RNA molecules produced by a method of the invention and computer program products for perform a method of the invention are also provided.