Trans-Splicing RNA With Unstructured Binding Domains
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Solution Overview
Problem
Trans-splicing-based RNA repair and labeling technologies face challenges in achieving durable and precise splicing, especially due to competition with regular cis-splicing, and pose risks of unspecific targeting, making regulatory approval difficult.
Innovation Solution
Design of trans-splicing RNA (tsRNA) molecules with unstructured binding domains specific to disease-associated genes, incorporating mismatch nucleotides and spacer sequences to enhance specificity and stability, and using a combination of HSVtk and ganciclovir for targeted cell death in suicide gene therapy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If trans-splicing RNA is designed to achieve durable repair through continuous delivery or endogenous expression, then the durability of the therapeutic effect is improved, but the device complexity and difficulty of implementation increase
Solution Approach 1:
The patent integrates the trans-splicing RNA construct into the genome before treatment, enabling endogenous expression and continuous supply of the therapeutic RNA without requiring repeated deliveries. This preliminary genomic integration resolves the contradiction by establishing durable expression while avoiding the complexity of continuous external delivery systems.
Solution Approach 2:
The trans-splicing RNA is designed to be self-sufficient once integrated, using cellular machinery for its own expression and function. The construct includes all necessary elements (promoters, splicing signals, coding sequences) to autonomously produce the therapeutic effect without external intervention, thereby achieving durability without complex delivery infrastructure.
2Manufacturing precision
If trans-splicing RNA is designed to achieve precise splicing towards intended splice sites, then the manufacturing precision is improved, but the productivity decreases due to strong competition with regular cis-splicing
Solution Approach 1:
The patent designs the trans-splicing RNA with highly specific local features at the splice sites, including optimized 5' and 3' splice site sequences that match the target pre-mRNA precisely. This local optimization ensures accurate splicing at intended sites while minimizing off-target effects, resolving the contradiction between precision and efficiency by making the splicing reaction highly specific rather than relying on general competitiveness.
Solution Approach 2:
The patent optimizes multiple parameters of the trans-splicing RNA construct simultaneously, including splice site strength, binding domain affinity, and RNA secondary structure, to enhance trans-splicing efficiency while maintaining precision. By tuning these parameters, the system achieves both high fidelity splicing and sufficient productivity to overcome cis-splicing competition.
3Productivity
If direct toxins or apoptotic genes are used to immediately trigger cell death, then the productivity of cell killing is improved, but the object-affected harmful factors increase due to unspecific targeting and off-targeting risks
Solution Approach 1:
The patent uses a conditional suicide gene system where the HSVtk enzyme acts as an intermediary. The trans-splicing RNA delivers the HSVtk gene specifically to target cells, which then requires co-administration of ganciclovir to trigger cell death. This intermediary approach allows controlled, specific cell killing only in cells that have successfully undergone trans-splicing and are exposed to the prodrug, eliminating unspecific toxicity while maintaining effective cell killing in target cells.
Solution Approach 2:
The patent separates the cell-killing function from the delivery mechanism by using a two-component system: the trans-splicing RNA delivers the HSVtk gene without inherent toxicity, and the toxic effect is only activated when ganciclovir is introduced. This extraction of the toxic function into a separate, controllable step eliminates off-target effects while preserving productivity in correctly targeted cells.
4Ease of operation
If trans-splicing construct is delivered only once into target cells, then the ease of operation is improved, but the reliability decreases because long-term expression is not necessary but alternative splicing must be controlled
Solution Approach 1:
The patent incorporates multiple binding domains and splice site optimizations into the trans-splicing construct before delivery, preparing it to specifically recognize and splice to the target pre-mRNA. This preliminary design ensures that once delivered, the RNA will reliably achieve specific trans-splicing without requiring repeated deliveries or complex control mechanisms, thus maintaining both ease of operation and reliability.
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 optimized tsRNA molecules improve on-target specificity and activity, reducing off-target effects and enhancing therapeutic efficacy by selectively killing targeted cells, thus addressing the challenges of durability and specificity in trans-splicing technologies.
Implementation Method 1
a binding domain specific for at least a part of a gene that associates with or is a biomarker for a disease to be treated; said binding domain comprises a binding site comprising at least 25, more preferably 35, even more preferably 45, and most preferably 55 or more consecutive unstructured nucleotides (nt) having no internal binding and/or self-complementary sequences
Implementation Method 2
Spliceosome-meditated RNA trans-splicing (SMaRT) is the process by which two distinct precursor messenger RNAs (pre-mRNAs), or other spliceable RNAs, are joint in trans to generate a chimeric RNA molecule
Implementation Method 3
c) an enzyme such as the herpes simplex virus thymidine kinase (HSVtk) that triggers a death signal upon co-delivery of a drug like ganciclovir (GCV)
Data Source
AI summary
The invention concerns a trans-splicing RNA (tsRNA) molecule comprising one or multiple unstructured binding domains; a cell or vector comprising said tsRNA; and a method for killing cells or treating a disease using said tsRNA.