Optimizing mRNA translation efficiency through rational 5'UTR and 3'UTR combinatorial design.

Area of Science:

• Molecular Medicine
• Biotechnology
• Genetic Engineering

Background:

• Messenger RNA (mRNA)-based therapies show potential for treating diseases but face challenges like low translation efficiency and short half-life.
• Untranslated regions (UTRs) critically regulate mRNA stability and translation efficiency, making them key targets for optimization.

Purpose of the Study:

• To enhance exogenous mRNA translation efficiency through de novo design of 5'UTRs and combinatorial screening with 3'UTRs.
• To identify novel UTR sequences and combinations that improve protein expression for mRNA therapeutics.

Main Methods:

• A combinatorial screening strategy was employed to design and test novel 5'UTRs in combination with various 3'UTRs.
• A novel 5'UTR, designated 5UTR05, was designed and its performance compared to a reference mRNA-1273 5'UTR.
• Screening identified synergistic effects when combining 5UTR05 with specific 3'UTRs, including those from immunoglobulin heavy constant gamma 2 (IGHG2) and mitochondrially encoded 12S ribosomal RNA (mtRNR1).

Main Results:

• The novel 5UTR05 demonstrated protein expression levels comparable to the high-performing mRNA-1273 5'UTR.
• Combining 5UTR05 with both IGHG2 and mtRNR1 3'UTRs significantly enhanced mRNA translation efficiency compared to using individual 3'UTRs.
• This synergistic combination resulted in superior protein expression, highlighting the importance of specific UTR pairings.

Conclusions:

• Novel UTR designs and combinatorial strategies can effectively overcome limitations in exogenous mRNA translation efficiency.
• The identified synergistic UTR combination (5UTR05 with IGHG2 and mtRNR1 3'UTRs) offers a promising approach for developing advanced mRNA therapeutics.
• Further exploration of UTR combinations can lead to customized mRNA constructs with optimized expression for diverse therapeutic applications.