3'-UTR Sequence Selection for mRNA Stability Without Recombination
Find Innovative SolutionsGenerate Solutions
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 during PCR amplification.
Innovation Solution
Incorporating specific sequences from the 3'-untranslated regions of FCGRT, LSP1, CCL22, AES, PLD3, MT-RNR1, and HLA-DRB4 into RNA expression vectors, along with a polyadenyl sequence with an open end, to enhance stability and translation efficiency, and using type IIS restriction cleavage sites for linearization to prevent masking of the poly(A) sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If two consecutive copies of the human beta-globin 3'-UTR sequence are used in the plasmid DNA, then RNA stability and translational efficiency are improved, but recombination occurs during propagation in E. coli and mispriming occurs during PCR amplification
Solution Approach 1:
The patent divides the stabilizing function into separate modular elements: a 3'-UTR sequence from one gene and a poly(A) sequence from another gene. These can be independently selected and combined, allowing optimization of each element without the recombination problems of duplicated sequences. The segmentation allows each functional element to be manufactured and verified separately before assembly.
Solution Approach 2:
The patent introduces a linker sequence as an intermediary element between the 3'-UTR and poly(A) sequence. This linker serves as a buffer zone that prevents unwanted interactions between the two functional elements while maintaining their individual stabilizing effects. The linker acts as a mediator that allows the combination of beneficial elements without causing the recombination issues seen with adjacent identical sequences.
2Manufacturing precision
If the polyadenyl cassette is positioned downstream of the poly(A) sequence, then the poly(A) sequence is not masked, but the cleavage site may still interfere with transcript stability
Solution Approach 1:
The patent extracts the poly(A) sequence from its traditional position within the polyadenyl cassette and places it upstream, followed by the cleavage site and then the 3'-UTR sequence. This repositioning takes out the poly(A) sequence from potential masking by downstream elements while maintaining its functional integrity. The cleavage site is positioned downstream to perform its function without interfering with the poly(A) sequence accessibility.
Solution Approach 2:
The patent changes the spatial arrangement of elements along the transcript by positioning the poly(A) sequence upstream rather than downstream of the cleavage site. This dimensional reorganization allows the poly(A) sequence to be accessible while the cleavage site remains functional, resolving the conflict between accessibility and stability by changing the linear order of elements.
3Manufacturing precision
If type IIS restriction cleavage sites are used for linearization, then the poly(A) sequence is not masked, but the cleavage site remains in the transcript which may affect stability
Solution Approach 1:
The patent creates a functional copy of the poly(A) sequence upstream of the cleavage site, while the downstream polyadenyl cassette serves as a template for proper polyadenylation. This copying approach ensures that the poly(A) sequence is accessible and functional even after cleavage, while the original cassette position allows proper processing. The cleavage site can be removed in subsequent steps if desired, but the upstream copy ensures functionality.
Data Source
Figure 1A
Figure 1B
Figure 2A
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.