Circular RNA Molecule with IRES for Persistent Protein Expression
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Solution Overview
Problem
Current mRNA technologies, such as linear mRNA, face challenges in achieving persistent and high-level protein expression, which is necessary for industrial protein production and therapeutic applications, as they have short expression duration and insufficient protein output.
Innovation Solution
A recombinant nucleic acid molecule is developed that forms circular RNA through specific IRES elements, homology arms, spacers, and introns, enhancing protein expression levels and durability in eukaryotic cells by promoting efficient and long-term protein production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If linear mRNA is used for protein expression, then the production process is simpler and more controllable, but the expression duration is short and protein output is insufficient
Solution Approach 1:
The patent applies circularization of the mRNA molecule, transforming the linear structure into a closed circular form. This structural curvature prevents exonucleolytic degradation at the ends, significantly extending the half-life and expression duration of the mRNA in eukaryotic cells while maintaining high protein expression levels
Solution Approach 2:
The patent modifies key parameters of the mRNA molecule including adding specific 5' and 3' UTR sequences, incorporating IRES elements, and optimizing the circular junction sequence to enhance stability and translation efficiency, thereby achieving both prolonged expression duration and high productivity
2Stability of the object's composition
If circular RNA is formed through back splicing, then the half-life is longer, but the molecular mechanism is complex and difficult to control
Solution Approach 1:
The patent extracts and eliminates the complex back-splicing mechanism by directly designing and synthesizing circular mRNA molecules with predetermined sequences. This approach bypasses the need for intronic sequences and splicing machinery, achieving stable circular RNA with controlled half-life through simplified in vitro synthesis
Solution Approach 2:
The circular mRNA structure itself provides inherent stability and resistance to degradation without requiring additional cellular machinery or complex mechanisms. The closed circular configuration autonomously protects against exonucleases and enables sustained translation without dependence on splicing factors
3Productivity
If IRES elements are incorporated into circular RNA, then protein expression efficiency increases, but the structural design becomes more complex
Solution Approach 1:
The patent segments the circular mRNA into functional modules including 5' UTR with IRES element, coding sequence, and 3' UTR, each optimized independently. This modular segmentation allows systematic design of IRES-containing circular RNAs with controlled complexity and predictable expression efficiency
Data Source
AI summary
The present disclosure relates to a recombinant nucleic acid molecule of the transcriptional circular RNA and its application in protein expression. Specifically, the present disclosure relates to a recombinant nucleic acid molecule of the transcriptional circular RNA, recombinant expression vector, pre-circularized RNA, circular RNA, recombinant host cell, pharmaceutical composition and protein preparing method. The transcription product of the recombinant nucleic acid molecule in this present disclosure is a circular RNA which containing specific IRES element. IRES element can increase the protein expression level of circular RNA in eukaryotic cells, achieve efficient and persistent expression of protein. It has important application value in many fields like: Preparation of mRNA infectious disease vaccines, therapeutic mRNA tumor vaccines, mRNA-based dendritic cell tumor vaccines, mRNA-based gene therapy, mRNA-based chimeric antigen receptor T cell therapy, and protein supplement therapy.