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Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
Published on: November 9, 2019
Highly efficient cellular expression of circular mRNA enables prolonged protein expression
Mildred J Unti1, Samie R Jaffrey1
1Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY 10065, USA.
Abstract:
A major problem with mRNA therapeutics is that mRNA is usually degraded within a few hours after entering the cytosol. New approaches for in vitro synthesis of circular mRNA have allowed increased levels and duration of protein synthesis from mRNA therapeutics due to the long half-life of circular mRNA. However, it remains difficult to genetically encode circular mRNAs in mammalian cells. Here, we describe the adaptation of the Tornado (Twister-optimized RNA for durable overexpression) system to achieve in-cell synthesis of circular mRNAs. We screen different promoters and internal ribosomal entry sites (IRESs) and identify combinations that result in high levels of circular mRNA and protein expression. We show that these circular mRNAs can be packaged into virus-like particles (VLPs), thus enabling prolonged protein expression. Overall, these data describe a platform for synthesis of circular mRNAs and how these circular mRNAs can improve VLP therapeutics.
Insights
Researchers developed a new method for creating circular messenger RNA (mRNA) inside cells, leading to longer protein production and improved therapeutic potential for mRNA vaccines and treatments.
Area of Science:
- Biotechnology
- Molecular Biology
- RNA Therapeutics
Background:
- Messenger RNA (mRNA) therapeutics face rapid degradation in cells, limiting their effectiveness.
- Circular mRNA offers a longer half-life, enhancing protein synthesis duration.
- Current methods for producing circular mRNA are primarily in vitro, posing challenges for in-cell applications.
Purpose of the Study:
- To adapt the Tornado system for efficient in-cell synthesis of circular mRNA.
- To identify optimal promoter and internal ribosomal entry site (IRES) combinations for high circular mRNA and protein expression.
- To demonstrate the utility of in-cell synthesized circular mRNA in virus-like particle (VLP) therapeutics.
Main Methods:
- Adaptation of the Tornado (Twister-optimized RNA for durable overexpression) system for intracellular circular mRNA production.
- Screening of various promoters and IRES elements to optimize circular mRNA synthesis.
- Packaging of synthesized circular mRNA into virus-like particles (VLPs).
Main Results:
- Successful adaptation of the Tornado system for in-cell circular mRNA synthesis.
- Identification of specific promoter-IRES combinations yielding high levels of circular mRNA and subsequent protein expression.
- Demonstration that circular mRNA can be effectively packaged into VLPs for sustained protein expression.
Conclusions:
- The developed platform enables efficient in-cell synthesis of circular mRNA.
- This approach significantly enhances protein expression duration compared to linear mRNA.
- In-cell synthesized circular mRNA holds promise for improving VLP-based therapeutics.
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