Synthetic Transcriptional AND Gate Using Pre-mRNA Trans-Splicing
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Solution Overview
Problem
Current gene expression systems using mRNA trans-splicing-based Boolean AND logic gates face challenges in ensuring specific expression of transcripts only in the presence of all required inputs, leading to potential basal expression when one input is active, which can be detrimental in clinical applications.
Innovation Solution
A synthetic nucleic acid expression system is developed, comprising two nucleic acid sequences with distinct promoters, where each sequence encodes an incomplete exon of a transcript of interest, and trans-splicing occurs only when both promoters are active, preventing expression in cells with only one active promoter.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If two synthetic promoters are used to control expression of a master transcription factor, then the system can achieve logic AND gate functionality, but basal expression occurs when only one promoter is active leading to unwanted consequences
Solution Approach 1:
The transcript is divided into two separate exons that are encoded by different promoters. Each promoter controls expression of only one exon, and both exons must be present and expressed simultaneously to produce the complete functional transcript. This segmentation eliminates basal expression because neither exon alone can produce the functional protein.
Solution Approach 2:
The patent combines two separate promoter-exon units into a single integrated expression system where the exons are joined through trans-splicing. The merging occurs at the RNA level where the first exon and second exon are spliced together to form the complete transcript, ensuring that both promoters must be active simultaneously.
2Reliability
If transcriptional targeting is used to regulate gene expression in specific cell populations, then selectivity and safety are enhanced, but implementation complexity increases
Solution Approach 1:
The patent creates a universal logic gate system that can be applied to various therapeutic genes and cell types. The same two-promoter-exon architecture can control different transcripts (e.g., immunomodulatory cassettes, fluorescent reporters) in different cell populations, making the system multi-functional and broadly applicable without requiring redesign.
Solution Approach 2:
The patent uses trans-splicing as an intermediary mechanism to bridge the two promoter-exon units. The splicing process acts as a mediator that joins the exons only when both promoters are active, providing a clean and efficient way to integrate multiple regulatory inputs without complex protein-based control mechanisms.
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 system ensures specific and safe expression of transcripts, such as the mKate2 protein, only in cells where both input signals are present, reducing unwanted expression and enhancing the safety and efficacy of gene-based therapies.
Implementation Method 1
the target sequence is complementary to the guide sequence and hybridization of the target sequence to the guide sequence facilitates trans-splicing
Implementation Method 2
a first RNA sequence required for spliceosome-dependent trans-splicing; and a second RNA sequence required for spliceosome-dependent trans-splicing
Data Source
AI summary
A synthetic nucleic acid expression system for production of a transcript of interest in a predefined cell-state is provided, the system comprising (a) a first nucleic acid sequence comprising a first promoter operably linked to a nucleic acid sequence encoding a first trans-spliceable pre-mRNA sequence comprising at least one exon encoding a 5′ fragment of said transcript of interest and a first RNA sequence required for spliceosome-dependent trans-splicing; and (b) a second nucleic acid sequence comprising a second promoter operably linked to a nucleic acid sequence encoding a second trans-spliceable pre-mRNA sequence comprising at least one exon encoding a 3′ fragment of said transcript of interest and a second RNA sequence required for spliceosome-dependent trans-splicing; wherein said first promoter and said second promoter are different and each one is specifically regulated by said predefined cell-state.