3' UTR sequences for stabilization of RNA
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Solution Overview
Problem
Current RNA-based cancer vaccines face challenges in achieving high stability and translation efficiency due to the short half-life of RNA and difficulties in cloning and transcription processes, particularly with sequences like the human beta-globin 3'-UTR, which can lead to recombination and mispriming issues.
Innovation Solution
Incorporating specific sequences in the 3'-untranslated region (UTR) of RNA molecules, such as those from FCGRT, LSP1, CCL22, AES, PLD3, MT-RNR1, and HLA-DRB4, and modifying the poly(A) sequence to create an open-ended poly(A) tail, which enhances stability and translation efficiency when used in dendritic cells for antigen-specific immune responses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If two consecutive copies of the human beta-globin 3'-UTR sequence are used in the plasmid DNA, then transcript stability and translational efficiency are improved, but recombination during propagation in E. coli occurs
Solution Approach 1:
The patent uses a single copy of the human beta-globin 3'-UTR sequence (positions 573-853) instead of two consecutive copies, thereby eliminating the recombination risk during E. coli propagation while maintaining the stabilizing effect on the transcript. This single copy serves as the template for in vitro transcription to produce the therapeutic RNA.
2Productivity
If two consecutive copies of the human beta-globin 3'-UTR sequence are used in the plasmid DNA, then translational efficiency is improved, but mispriming during PCR-based amplification occurs leading to omission of one copy
Solution Approach 1:
The patent employs a single copy of the 3'-UTR sequence that can be accurately amplified by PCR without mispriming issues. This single copy design eliminates the cloning accuracy problems associated with repetitive sequences while preserving the ability to generate sufficient RNA for therapeutic applications through efficient in vitro transcription.
3Object-affected harmful factors
If RNA is used for therapeutic applications, then safety risks are reduced compared to DNA, but half-life is shortened
Solution Approach 1:
The patent modifies the RNA molecule by incorporating a specific 3'-UTR sequence (positions 573-853 of human beta-globin) that increases transcript stability and half-life. This parameter change in the RNA structure allows the therapeutic RNA to maintain its safety advantages over DNA while extending its duration of action in vivo.
Solution Approach 2:
The patent creates a composite RNA structure by combining the coding sequence of interest with a stabilizing 3'-UTR element from the beta-globin gene. This composite design integrates the therapeutic function with the stability function, resulting in an RNA molecule that is both safe and long-lasting.
4Stability of the object's composition
If the poly(A) sequence is modified to create an open-ended poly(A) tail, then stability and translation efficiency are enhanced, but cloning complexity increases
Solution Approach 1:
The patent modifies the poly(A) sequence by introducing a type IIS restriction cleavage site that generates an open-ended poly(A) tail in the transcribed RNA. This parameter change in the poly(A) structure enhances both stability and translation efficiency while the cleavage site design facilitates rather than complicates the cloning process.
Data Source
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AI summary
The present invention relates to stabilization of RNA, in particular mRNA, and an increase in mRNA translation. The present invention particularly relates to a modification of RNA, in particular in vitro-transcribed RNA, resulting in increased transcript stability and/or translation efficiency. According to the invention, it was demonstrated that certain sequences in the 3'-untranslated region (UTR) of an RNA molecule improve stability and translation efficiency.