mRNA Synthesis Messenger RNA (mRNA) serves as the template facilitating protein production based on genetic information from DNA. While it occurs naturally in cells to promote growth, differentiation, and survival, it can be carried out synthetically in cell-free environments. The latter form of mRNA synthesis is realized by in vitro transcription (IVT) and has been widely used in biotechnology applications, from vaccine development to gene therapies. More importantly, IVT-based mRNA synthesis allows researchers customization and precise control for tailored research solutions.

What is mRNA Synthesis?

The role of mRNA in protein synthesis cannot be understated. The information in the DNA, which resides in the nucleus, cannot be used directly to make proteins. It is first transcribed into an mRNA, which transfers the information from the nucleus to the ribosome, where protein synthesis occurs.

mRNA synthesis for protein expression begins with RNA polymerase binding to the start of the gene, called the promoter, with the help of transcription factors. As the enzyme moves along the strand, it links complementary RNA nucleotides to form a single-stranded mRNA molecule until reaching a termination sequence that signifies the end of the gene. Post-transcriptional modifications are used to ensure stability and transfer to the ribosome.

mRNA synthesis steps are tightly regulated to determine which genes become activated and can be translated into essential proteins. Dysregulation in this mechanism can lead to several genetic disorders, chronic diseases, and cancer.

The mRNA Synthesis Process

During IVT, custom mRNA synthesis requires a linearized plasmid DNA (pDNA) template or a synthetic DNA sequence containing promoter sequences that the RNA polymerase can recognize. The DNA template is added to a reaction mix consisting of the buffer, RNA polymerase, and ribonucleotide triphosphates (ATP, UTP, GTP, and CTP). Thus, the RNA polymerase can incorporate the four ribonucleotides to build the complementary mRNA sequence based on the template.

mRNA Maturation

Post-transcriptional modifications are required to ensure the integrity of the newly synthesized mRNA and its successful delivery to the ribosome. These include:

1. 5' capping to enhance stability and translational efficiency. mRNA capping can be conducted co-transcriptionally by adding capping enzymes to the mRNA synthesis reaction mixture. The cap can also be added post-transcriptionally by enzymatic reaction steps.
2. 3' Poly(A) Tail Addition or polyadenylation to prevent degradation by exonucleases, enhance mRNA half-life, and improve ribosomal binding
3. Splicing involves maturing the mRNA by removing the non-coding intron regions and joining the coding exons. This step is essential for accurately reading and translating the mRNA into functional proteins.

Role of RNase and Enzymes

Synthesizing stable mRNA constructs is essential; however, mRNA is still vulnerable to ribonuclease (RNases) degradation. Although key RNases, such as RNase H, can be used to assess mRNA capping efficiency¹, their excessive degradation activity can be detrimental to the mRNA. During mRNA isolation and purification, trace amounts of RNases can co-purify with the mRNA, diminishing the overall yield and altering experimental readouts.

To mitigate the risks associated with RNase contamination, IVT workflows must include RNase inhibitors. Special care must be taken when determining the amount of RNase inhibitor to include in the experimental design.² Furthermore, the laboratory conditions must prevent RNase accumulation. Applying diethylpyrocarbonate (DEPC) treated water on laboratory equipment ensures the inactivation of RNase enzymes.³

Applications of mRNA Synthesis Technologies

Biopharmaceuticals

In vitro–transcribed mRNA offers a fast and scalable way of developing vaccines. Perhaps the most well-known examples are the COVID-19 vaccines made of synthetic mRNA that encodes the SARS-CoV-2 spike protein to stimulate an immune response.^4,5

The success of this strategy prompted more research to develop mRNA-based vaccines for influenza, Zika, and HIV.^6-8 mRNA-based vaccines are also explored for cancer immunotherapy. Here, synthetic mRNA encoding tumor-associated antigens (TAA) helps activate innate and adaptive immune responses against cancer cells.^9,10

Gene Therapy

Besides vaccines, synthetic mRNA is also a powerful tool for various gene therapy modalities, owing to advantages such as transient and safe expression profiles. When compensating for functionally deficient proteins in rare genetic disorders, mRNA-based therapies exhibit short-lived expressions without integrating into the genome, preventing immune rejection and long-term genome instability. mRNA therapies are under development for cystic fibrosis and hemophilia, where the synthetic mRNA encodes functional CFTR protein or clotting factor, respectively.^11,12

Challenges in mRNA Synthesis and Solutions

Common Issues in mRNA Synthesis Processes

Synthesis of mRNA faces several challenges that interfere with its translation to preclinical and clinical applications. These include:

1. RNA degradation by RNases
2. Low mRNA yield, caused by insufficient RNA polymerase activity, defective DNA template design, or disproportionate nucleotide concentrations
3. Double-stranded RNA (dsRNA) formation and aggregation due to self-complementary sequences and RNA secondary structure
4. Incomplete capping or polyadenylation
5. Degradation during freezing and thawing

Solutions to Overcome Degradation

The key objective in mRNA synthesis for protein production is to achieve high-quality mRNA that correctly carries the information provided by the custom DNA template; however, spontaneous degradation can significantly lower the quality and yield.

Several strategies are implemented to overcome degradation. Besides thorough cleaning protocols to eliminate RNase in the laboratory environment, researchers can use RNase inhibitors to regulate RNase activity. Another strategy involves locked nucleic acids (LNAs), which are nucleotides chemically modified to acquire a thermally stable conformation. Thus, mRNA constructs made of LNAs become resistant to nucleases and can evade degradation.¹³

Advancements in the IVT mRNA synthesis workflows can maximize yield, stability, purity, and delivery. Some strategies are as follows:

• High-performance T7 RNA polymerase variants for improved capping efficiency and removal of dsRNA byproducts. ¹⁴
• Nucleotide modifications, such as pseudouridine mRNA, to prevent immune activation and improve mRNA stability ¹⁵
• Optimizing untranslated regions (UTR) to increase half-life and rate of ribosome recruitment. For example, 5' and 3' UTRs can be modified to induce stem-loop structures that are protective against exonucleases. ¹⁶
• Advancements in mRNA purification through high-performance liquid chromatography (HPLC), cellulose chromatography, or RNase III treatment can help remove contaminant dsRNAs. ¹⁷

References

1. Chan SH, Whipple JM, Dai N, Kelley TM, Withers K, Tzertzinis G, et al. RNase H-based analysis of synthetic mRNA 5′ cap incorporation. RNA 2022;28(8):1144-1155.
2. Earl CC, Smith MT, Lease RA, Bundy BC. Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation. Bioengineered 2018;9(1):90-97.
3. Van Pottelberge R, Matthessen R, Salem S, Goffin B, Oien N, Bharti P, et al. Importance of RNase monitoring during large-scale manufacturing and analysis of mRNA-LNP based vaccines. J Pharm Sci 2024.
4. Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Eng J Med 2021;384(5):403-416.
5. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Eng J Med 2020;383(27):2603-2615.
6. Arevalo CP, Bolton MJ, Le Sage V, Ye N, Furey C, Muramatsu H, et al. A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes. Science 2022;378(6622):899-904.
7. Medina-Magües LG, Gergen J, Jasny E, Petsch B, Lopera-Madrid J, Medina-Magües ES, et al. mRNA vaccine protects against zika virus. Vaccines 2021;9(12):1464.
8. Mu Z, Haynes BF, Cain DW. HIV mRNA vaccines—progress and future paths. Vaccines 2021;9(2):134.
9. Huang X, Zhang G, Tang T, Liang T. Identification of tumor antigens and immune subtypes of pancreatic adenocarcinoma for mRNA vaccine development. Mol Cancer 2021;20:1-18.
10. Ma S, Ba Y, Ji H, Wang F, Du J, Hu S. Recognition of tumor-associated antigens and immune subtypes in glioma for mRNA vaccine development. Front Immunol 2021;12:738435.
11. Jackson JJ, Mao Y, White Jr TR, Foye C, Oliver KE. Features of CFTR mRNA and implications for therapeutics development. Front Genet 2023;14:1166529.
12. Arjunan P, Mahalingam G, Sankar P, Kathirvelu D, Suresh S, Rani S, et al. Base-modified factor VIII mRNA delivery with galactosylated lipid nanoparticles as a protein replacement therapy for haemophilia A. Biomater Sci 2024;12(19):5052-5062.
13. Senthilvelan A, Vonderfecht T, Shanmugasundaram M, Pal I, Potter J, Kore AR. Trinucleotide cap analogue bearing a locked nucleic acid moiety: synthesis, mRNA modification, and translation for therapeutic applications. Org Lett 2021;23(11):4133-4136.
14. Miller M, Alvizo O, Baskerville S, Chintala A, Chng C, Dassie J, et al. An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis. Faraday Discuss 2024;252:431-449.
15. Monroe J, Eyler DE, Mitchell L, Deb I, Bojanowski A, Srinivas P, et al. N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation. Nat Commun 2024;15(1):8119.
16. Hashizume M, Takashima A, Iwasaki M. A small stem-loop-forming region within the 3′-UTR of a nonpolyadenylated LCMV mRNA promotes translation. J Biol Chem 2022;298(2).
17. Zhang J, Liu Y, Li C, Xiao Q, Zhang D, Chen Y, et al. Recent advances and innovations in the preparation and purification of in vitro-transcribed-mRNA-based molecules. Pharmaceutics 2023;15(9):2182.

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