Polycistronic RNA Co-Expression of Nav Alpha and Beta Subunits
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Solution Overview
Problem
Current methods for expressing voltage-gated sodium channels in host cells are limited due to the large size of Navα subunits and challenges in co-expression with Navβ subunits, leading to suboptimal functional expression and surface localization.
Innovation Solution
A polycistronic RNA message encoding a polyprotein with a voltage-gated sodium channel α subunit and one or more β subunits, separated by 2A self-cleaving peptides, allows for simultaneous expression and assembly of these subunits into functional channels, enhancing expression levels and membrane integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Navα subunits are expressed alone in host cells, then the channels can form functional ion conductors, but the total and functional expression levels are suboptimal due to the large size of Navα subunits
Solution Approach 1:
The patent divides the sodium channel into multiple segments (Navα subunit and Navβ subunit) that are expressed separately but assemble together. The Navβ subunit is expressed as a separate entity from the large Navα subunit, allowing each component to be optimized independently for expression while maintaining functional integrity when assembled.
Solution Approach 2:
The patent creates a composite channel structure by combining Navα and Navβ subunits. This composite approach allows the smaller Navβ subunit to assist in the expression and localization of the larger Navα subunit, improving overall functional expression levels while managing the complexity of the large Navα component.
2Productivity
If Navα subunits are co-expressed with Navβ subunits using conventional methods, then assembly into functional channels is possible, but expression efficiency is limited due to challenges in simultaneous expression
Solution Approach 1:
The patent merges the expression of Navα and Navβ subunits into a single polycistronic RNA molecule. This allows both subunits to be transcribed from a single promoter and translated simultaneously, greatly facilitating co-expression while maintaining the ability to assemble functional channels. The internal ribosome entry sites (IRES) enable independent translation of each subunit from the same RNA transcript.
Solution Approach 2:
The polycistronic RNA design provides multi-functionality by serving as a single expression vehicle for multiple subunits. This universal expression system can accommodate different Navα and Navβ combinations while simplifying the transfection and expression process compared to using separate expression constructs for each subunit.
3Adaptability or versatility
If accessory proteins Navβ subunits are assembled with Navα subunits, then altered voltage dependence and cellular localization are achieved, but the assembly efficiency is reduced due to expression challenges
Solution Approach 1:
The patent uses the polycistronic RNA design to ensure that both Navα and Navβ subunits are produced simultaneously and in appropriate stoichiometric ratios. This preliminary co-production ensures that both subunits are available for assembly at the same time and location, greatly improving assembly efficiency compared to sequential or separate expression methods.
Solution Approach 2:
The IRES elements act as intermediaries that enable independent translation of Navα and Navβ subunits from the same polycistronic RNA. This intermediary mechanism ensures that both subunits are produced from a single transcript without requiring separate promoters or regulatory elements, facilitating efficient co-expression and subsequent assembly.
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 increases the total and functional expression of voltage-gated sodium channels, improving their assembly and localization in host cells, making them more suitable for research and therapeutic applications.
Implementation Method 1
a polycistronic RNA message that encodes a polyprotein that comprises a voltage-gated sodium channel alpha protein (Navα) subunit and one or more voltage-gated sodium channel accessory protein (Navβ) subunits, wherein each subunit is separated from adjacent subunits by a 2A self-cleaving peptide
Data Source
AI summary
A voltage-gated sodium channel expression system is described. The system comprises providing a polycistronic RNA message that encodes a polyprotein comprising a voltage-gated sodium channel alpha protein (Navα) subunit and one or more voltage-gated sodium channel beta protein (Navβ) subunits, each of said subunits being separated by a 2A self-cleaving peptide. During translation, the polyprotein is cleaved into individual subunit proteins which can assemble into a voltage-gated sodium channel. Host cells and lipoparticles comprising the sodium channel expression system are also provided.