General Design Methods for mRNA Drugs

 Image: By Thomas Shafee CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=39630174

With the rapid development of biotechnology and molecular medicine, the introduction of mRNA as a vaccine or therapeutic agent enables the production of almost any desired functional protein/peptide within the human body. This represents an increasingly promising field of precision medicine, holding significant prospects for preventing and treating numerous challenging or genetic diseases. Scientists have been striving to optimize mRNA stability, immunogenicity, translation efficiency, and delivery systems to achieve efficient and safe mRNA delivery.

By Carrier Taylor

Messenger RNA (mRNA) is a single-stranded ribonucleic acid transcribed from the DNA chain, carrying the encoding information for protein synthesis. In vitro transcribed (IVT) mRNA has been successfully transcribed and expressed in mouse skeletal muscle cells, establishing the feasibility of mRNA therapy. When mRNA can be successfully transfected and elicit an immune response by direct injection into the mouse body to express therapeutic proteins in a dose-dependent manner, mRNA-based therapeutic approaches have been proposed. In contrast to DNA-based drugs, mRNA transcripts have relatively high transfection efficiency and low toxicity, as they do not need to enter the cell nucleus to function. Importantly, mRNA carries no potential risks of unintended infection or opportunistic insertion mutations. Compared to traditional transient protein/peptide drugs, mRNA has the ability to continuously translate proteins/peptides, thus holding broad potential in the treatment of various diseases.

Key discoveries and advances in mRNA-based therapeutics (Qin, Shugang, et al. 2022)

General Design Methods for mRNA Drugs

The design and synthesis of mRNA are critical steps in the development of mRNA drugs. mRNA consists of five functional regions, including the 5′ cap structure, 5′ untranslated region (UTR), open reading frame (ORF), and 3′ untranslated region and 3′ poly(A) tail structure, which mediate mRNA translation efficiency and decay rate.

 

Structure of in vitro transcribed (IVT) mRNA and commonly used modification strategies (Wadhwa, Abishek, et al. 2020)

5′ Cap: The 5′ cap is located at the 5′ end of mRNA, and its methylation degree varies. In eukaryotes, the 5′ cap (m7G ppp) contains 7-methylguanosine (m7G), linked to the next nucleotide through a 5′-5′ triphosphate bridge (ppp). During the translation initiation stage, the cap binds to eIF4E through hydrophobic cation-π interactions of m7G and the negatively charged triphosphate bridge. Strategies optimizing m7G or the triphosphate bridge have been employed to achieve cap analogs with high affinity for eIF4E and low sensitivity to decapping enzymes.

Poly(A) Tail: The poly(A) tail typically consists of 10-250 adenine ribonucleotides. Dynamically added to mRNA, the poly(A) tail plays a crucial role in regulating mRNA translation efficiency and protein expression. Mechanistically, the 3′ poly(A) tail binds to poly(A)-binding proteins (PABPs), subsequently interacting with the 5′ cap through translation initiation factors eIF4G and eIF4E, promoting a “closed-loop structure” that regulates mRNA translation efficiency.

In vitro transcribed (IVT) mRNA and translation initiation (Qin, Shugang, et al. 2022)

 

5′-UTR and 3′-UTR: The 3′ and 5′ UTRs of mRNA do not directly encode proteins but play essential roles in regulating mRNA translation and protein expression. UTRs are involved in mRNA subcellular localization, regulating translation efficiency, and mRNA stability. While 5′-UTR and 3′-UTR both regulate protein expression levels, the former primarily participates in translation initiation, while the latter mainly influences mRNA stability and half-life.

 

ORF: ORF design focuses on codon optimization, introducing functional peptides, and the replication process. Codon optimization, a widely used but controversial improvement method, involves replacing rare codons with synonymous codons decoded by abundant tRNAs, enhancing mRNA translation efficiency. However, it may alter protein conformation, generating unknown peptides with in vivo biological activity. Increasing the GC content by substituting rare codons in the ORF protects mRNA from ribonuclease degradation and enhances in vivo mRNA protein expression. Additionally, functional peptides are crucial for mRNA drugs, and signal peptides encoded by mRNA are necessary for proteins to function extracellularly. Optimizing mRNA by introducing signal peptides into the ORF region improves the functionality of therapeutic mRNA.

In conclusion, each step in the quality control of mRNA directly impacts its efficacy. Therefore, the production and preparation of mRNA are crucial for mRNA therapy.

 

mRNA Manufacturing

Synthesis and Optimization of mRNA

In vitro transcribed (IVT) mRNA is synthesized using a linearized plasmid DNA template or a PCR template, requiring at least one promoter and the corresponding mRNA coding sequence. IVT mRNA is generated by adding a polymerase (T7, T3, or SP6), but additional capping is necessary. Uncapped mRNA is rapidly degraded by RNase and contains a 5′-ppp group, causing greater immune stimulation. This can be treated with phosphatase to reduce adverse effects. There are two methods for capping IVT mRNA: co-transcriptional capping and post-transcriptional capping. The Poly(A) tail of IVT mRNA is typically encoded in the DNA template or attached post-synthesis through polyadenylation, with the former providing more precise control over Poly(A) tail length. It’s worth noting that II-type restriction endonucleases used for plasmid template linearization can promote 3′ overhangs, hindering IVT mRNA translation efficiency. This can be avoided by substituting II-type restriction endonucleases with IIS-type endonucleases.

mRNA Purification

Since IVT mRNA is mixed with RNA polymerase and DNA template after synthesis, purification is crucial. This involves removing immune-stimulating contaminants, free ribonucleotides, short mRNA, and DNA templates. DNase is commonly used to degrade excess DNA templates. Commercial purification kits are often employed for purification and separation of synthesized mRNA. The purified mRNA is then precipitated with ethanol or isopropanol to eliminate most contaminants and obtain high-purity mRNA. Further purification methods, such as precipitation with high concentrations of LiCl or alcohol, chromatography, or elution from silica membrane columns, are used to remove proteins, free nucleotides, or other components. However, these methods may not effectively eliminate double-stranded RNA impurities.

RNase III is a novel purification method that has been proposed to eliminate double-stranded RNA (dsRNA) contaminants. It has been shown to significantly reduce the immunogenicity of mRNA and enhance the cytotoxic killing effect of CAR-T cells by reducing the immunogenicity of mRNA through electroporation with RNase III. Its potential drawback is that RNase III may cut double-stranded secondary structures formed by single-stranded RNA.

Various purification methods can be chosen based on research or application purposes, addressing different purity requirements and scales of mRNA. Regardless of the purification method used, stringent mRNA quality control standards are essential to ensure the maximization of therapeutic benefits in mRNA therapy.

References

1.      Boczkowski D, Nair SK, Snyder D, Gilboa E. Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med. 1996 Aug 1;184(2):465-72.

2.      Wadhwa A, Aljabbari A, Lokras A, Foged C, Thakur A. Opportunities and Challenges in the Delivery of mRNA-based Vaccines. Pharmaceutics. 2020 Jan 28;12(2):102.

3.      Qin S, Tang X, Chen Y, Chen K, Fan N, Xiao W, Zheng Q, Li G, Teng Y, Wu M, Song X. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022 May 21;7(1):166.

About the author:

Carrier Taylor

R & D Director and Business Devlopment Director of BOCSCI 

2014 - Present, working in BOCSCI

2012-2014 Study in Rice University, MBA

2004-2008 Study in Rice University,Pharmacy 

Linkedin profile: https://www.linkedin.com/in/carrier-taylor/ 

Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)