UPR-Regulated Intron Control for Low-Background Gene Expression
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Solution Overview
Problem
Existing gene expression systems, particularly for toxic proteins, lack effective control mechanisms that can induce expression at desired times and locations, leading to potential cell death or poor production due to unregulated expression levels.
Innovation Solution
Utilizing a regulatable intron that is spliced out via the unfolded protein response (UPR) mechanism, allowing for controlled expression of proteins or RNA products by splicing out an excisable sequence, thereby regulating translation and minimizing background expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If inducible promoters are used to control gene expression, then expression can be regulated at the transcription level, but there is still background expression that cannot be fully eliminated
Solution Approach 1:
The invention divides the control mechanism into two independent parts: a promoter that drives transcription and a regulatable intron that controls translation. The intron is further segmented into conserved splice sites and a variable central region, allowing modular design and optimization of background expression levels independent of transcriptional control.
Solution Approach 2:
The regulatable intron acts as an intermediary element between the promoter and the coding sequence. It receives the transcribed mRNA and conditionally processes it through splicing based on UPR activation, thereby mediating the transition from transcriptional control to translational control and enabling precise regulation of background expression.
2Productivity
If constitutive expression is used for toxic proteins, then production levels can be high, but cell death occurs due to unregulated expression
Solution Approach 1:
The expression system is designed with a pre-formed but inactive mRNA transcript containing the regulatable intron. The coding sequence is prepared and transcribed in advance, but the toxic protein product is prevented from formation by the intron's presence. Upon UPR activation, splicing removes the intron and translation proceeds, allowing high productivity only when the cell is prepared to handle the toxic product.
Solution Approach 2:
The system changes the functional state of the mRNA from non-translatable to translatable through the parameter change of intron splicing. The same mRNA molecule can exist in two states: with the intron preventing translation (low toxicity) and without the intron allowing translation (high productivity), enabling dynamic control of expression levels to avoid cell death.
3Object-generated harmful factors
If regulatable introns are used to minimize background expression, then translation control is achieved, but the system complexity increases
Solution Approach 1:
The regulatable intron design utilizes universally conserved splicing mechanisms (splice sites recognized by the splicing machinery) combined with a variable central region that can be customized. This universal recognition system allows the same basic intron structure to function across different genes and contexts, reducing overall system complexity while maintaining precise control capabilities.
Solution Approach 2:
The intron system provides dynamic control where the same genetic element can exist in two functional states (spliced or unspliced) based on cellular conditions. The splicing efficiency can be modulated by changing the central region sequence, allowing the system to adapt its background expression level dynamically without requiring completely different structural designs for each application.
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
Enables precise control of gene expression, especially for toxic proteins, by ensuring minimal background expression and significant induction upon UPR activation, enhancing production efficiency and safety.
Implementation Method 1
an intron which comprises an excisable sequence which is capable of being spliced out of a transcript produced from the synthetic expression construct via the unfolded protein response (UPR) system in the cell
Data Source
AI summary
The present invention relates to the use of a regulatory nucleic acid sequences that are able to regulate gene expression in eukaryotic cells and which are responsive to the unfolded protein response (UPR). There are disclosed regulatable introns and UPR-inducible promoters, which are able to regulate gene expression. There are also disclosed recombinant expression constructs comprising such regulatory nucleic acid sequences, whereby expression of the encoded expression product can be induced by invoking the unfolded protein response (UPR) in a eukaryotic cell containing the construct, methods of using such constructs and associated vectors, cells and suchlike.