Diverse IVT RNA in Vaccine Development: mRNA, saRNA, circRNA

IVT RNA in Vaccine Development

mRNA was discovered in 1961, but it wasn't until the COVID-19 pandemic that its prominent contribution to COVID-19 vaccines garnered significant attention, driving continued interest in this therapy from investors. In 2023, the Nobel Prize in Physiology or Medicine was awarded to the two pioneers of mRNA technology, Katalin Kariko and Drew Weissman, further highlighting its significance. To address the limitations of traditional linear mRNA vaccines, technologies such as self-amplifying mRNA (saRNA/SAM) and trans-amplifying mRNA (taRNA) have emerged. In recent years, circRNA vaccines have also been considered a promising vaccine or therapeutic drug platform due to their structural stability. In vaccine development, in vitro transcription (IVT) plays a crucial role, especially in RNA-based vaccines. IVT involves synthesizing RNA molecules in a laboratory environment using DNA templates and RNA polymerase. This process is applicable to various types of RNA molecules, including mRNA, self-amplifying RNA (saRNA), and circular RNA (circRNA).

• mRNA vaccines utilize IVT to produce RNA molecules encoding the desired antigens. The DNA template containing the antigen sequence is transcribed into mRNA using RNA polymerase. When this mRNA is delivered into host cells, it serves as the blueprint for protein synthesis. mRNA vaccines have shown promise in vaccine development due to their ability to induce robust immune responses against infectious agents such as viruses.
• saRNA vaccines represent another IVT-based approach. In this case, the DNA template encodes not only the antigen sequence but also the mechanisms necessary for self-replication. IVT is used to generate saRNA molecules, which, upon delivery into cells, undergo self-replication, leading to amplified antigen production. This amplification enhances the potency and duration of the immune response, making saRNA vaccines an attractive option for vaccine development.
• While circRNA vaccines are still in the early stages of research, they also benefit from IVT. IVT enables the synthesis of circRNA molecules containing antigen sequences. These circRNA molecules can modulate gene expression or serve as gene therapy vectors in vaccine development efforts.

A timeline of some fundamental discoveries in the development of RNA vaccine technology. (Perenkov, A.D.; et al, 2023)

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What is the Process of RNA IVT?

RNA in vitro transcription, commonly known as RNA IVT, is a technique used in molecular biology to synthesize RNA molecules outside of living cells. It's a crucial method for producing RNA for research purposes and various applications in biotechnology. Essentially, it involves creating RNA from a DNA template in a laboratory setting, allowing scientists to generate large quantities of specific RNA sequences for studying gene expression, developing diagnostic tools, or designing RNA-based therapeutics like mRNA vaccines. This process plays a vital role in advancing our understanding of genetics and molecular biology, as well as in developing innovative solutions for healthcare and biotechnology industries.

Features of Different IVT RNA

IVT mRNA

Non-amplified mRNA (NRM) refers to the mRNA synthesized in vitro without the need for restriction endonucleases or PCR for amplification of DNA. Instead, it directly utilizes linear DNA templates to synthesize mRNA. This process is typically facilitated by bacteriophage T3, T7, or SP6 RNA polymerases. NRM molecules consist of five parts: 5' cap, 5' UTR, ORF, 3' UTR, and poly(A) tail. During the synthesis process, the 5' cap is a methylated guanosine structure crucial for splicing, nuclear export, and translation. Depending on the requirements, RNA capping can be achieved post-transcriptionally or co-transcriptionally. The 5' UTR serves as a platform for transcriptional complex assembly, modulating translation efficiency. The ORF in mRNA determines the encoded sequence governing vaccine functional properties. Meanwhile, the 3' UTR plays a crucial role in mRNA localization, stability, and translation efficiency. Finally, the addition of a poly(A) tail protects mRNA from exonucleolytic degradation, extending mRNA lifespan and enhancing protein production.

IVT Self-Amplifying mRNA & Trans-Amplifying mRNA

• Self-amplifying mRNA (SAM/saRNA) and trans-amplifying mRNA (taRNA) differ in their fundamental composition and replication mechanisms. The key distinction lies in the presence of a replicase downstream of the 5' UTR in SAM, encoding for non-structural proteins (nsPs) that facilitate self-replication. Upon entry into the cytoplasm of target cells, nsPs form early replication complexes, utilizing the SAM template to generate complementary RNA strands. Subsequently, nsP peptides are cleaved into individual proteins, forming late replication complexes to synthesize copies of SAM, thereby increasing the initial quantity of IVT RNA copies. A significant difference between SAM and NRM drugs is the presence of a subgenomic promoter (SGP) region in SAM, located upstream of the gene of interest (GOI). SGP promotes GOI transcription by bypassing the reading of the sequence encoding viral nsP proteins, facilitating the formation and translation of mRNA copies containing only the GOI. Compared to NRM drugs, this replication mechanism can reduce the required dose of SAM by 30-1000 times. However, SAM also has its drawbacks, such as the potential for immune overactivation in host cells due to the inclusion of viral-related sequences, limitations imposed by the length of nsPs on the length of GOI, and complexities in packaging and delivery.
• taRNA, proposed in 2011, is a form of SAM that requires at least two different RNAs, one for the replicase and another for the GOI. By separately encoding the replicase, it effectively circumvents the length limitations of the GOI sequence. Additionally, it reduces the likelihood of producing recombinant viral particles, thereby enhancing safety. Moreover, taRNA exhibits stronger immunogenicity, with lower doses sufficient to evoke considerable immune responses compared to saRNA. Although taRNA technology is still in its early stages, it holds vast potential for applications in the future.

IVT Circular RNA (circRNA)

CircRNA is a single-stranded RNA molecule that forms a closed-loop structure without a 5' cap or poly(A) tail. This structure lacks the terminal sequences required for recognition by exonucleases, making it resistant to degradation by exonucleases and more stable compared to linear RNA. Methods for circRNA synthesis include chemical methods or enzyme-catalyzed methods. Chemical methods often utilize condensing agents like BrCN or EDC to activate RNA circularization. The limitation of this method is that the circularization bond is not a phosphodiester bond, so it cannot mimic the reverse splicing sites in vivo chemically.

• The enzyme-catalyzed method often employs T4 bacteriophage enzymes, where the prerequisite for ligation is that the recipient region on the linear RNA precursor has a 3'-OH, and the donor region has a 5' monophosphate. Commonly used T4 enzymes include T4 DNA ligase (T4 Dnl 1), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2). T4 Dnl and T4 Rnl 1 are suitable for RNA circularization without complex secondary structures, while T4 Rnl 2 is more suitable for linear RNA precursors with double-stranded junction regions. Therefore, the choice of different T4 ligases depends on the secondary structure of the linear RNA precursor in practical applications. It should be noted that all these ligase synthesis methods cannot achieve circularization of large RNA molecules and cannot completely avoid side reactions of intermolecular end ligation.
• Utilizing ribozymes to form circRNA during IVT is also an important enzyme-catalyzed method. Ribozymes are RNA molecules with enzymatic activity, which, in the self-splicing process, produce the desired circRNA product through two consecutive ester exchange reactions at specific sites without the need for additional enzymes. Currently, existing ribozyme methods include the I-type intron self-splicing method (also known as group I intron splicing), the II-type intron self-splicing method, and the hammerhead ribozyme method (HPR). Among these ribozyme methods, the group I intron splicing method (PIE) is the most widely used, requiring only GTP and Mg²⁺ as auxiliary factors in the reaction to produce circRNA in vivo or in vitro, exceeding 5 kb in length. The II-type intron self-splicing mechanism is similar to the I-type, utilizing introns without the need for exons, but its self-splicing forms a 2',5'-phosphodiester bond at the splicing site instead of the natural 3',5'-phosphodiester bond. Another method, HPR, can produce linear RNA molecules using T7 RNA polymerase on a single-stranded circular DNA template through rolling-circle mechanism, further self-cleaving and ligating to form circRNA. This method is particularly suitable for producing high-yield small circRNA molecules. However, the stability of the formed circRNA is poor, and it may retain catalytic active segments of HPR, hence less suitable for large-scale development and utilization.

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Immunogenicity of Different IVT RNA

Immunogenicity poses a significant challenge in RNA technology. To reduce immunogenicity, incorporating various modified nucleotides during IVT process is an effective approach. Commonly used modified nucleotides include N1-methyladenosine (m1A), N6-methyladenosine (m6A), 5-methoxyuridine (mo5U), 2-thiouridine (s2U), pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytidine (m5C), 5-methoxycytidine (5moC), and 5-hydroxymethylcytidine (5hmC). However, it is important to note that the impact of modified nucleotides on the translation process varies across different cells. Moreover, modified nucleotides are mostly used in NRM and taRNA, as they can disrupt the activity of replicases and are therefore not used in SAM. Additionally, unlike linear mRNA, m6A-modified nucleotides are commonly used in circRNA to ensure circularization efficiency and translation levels. Furthermore, to reduce the immunogenicity of products and minimize the generation and removal of dsDNA, dsRNA, ssDNA, and DNA/RNA heterodimers are crucial. Existing methods include using DNase-I to eliminate DNA contaminants, reducing Mg²⁺ concentration or incorporating modified nucleotides to reduce dsRNA formation, using RP-HPLC and cellulose to remove dsRNA, and using RNase R or ribonuclease T to remove linear RNA molecules from circRNA.

The following table shows the basic pattern recognition receptors (PRR) that recognize different nucleic acids.

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Prospects for IVT RNA

In vitro transcription RNA technology plays a crucial role in vaccine development. By synthesizing RNA molecules, this technology facilitates vaccine research and production in several aspects:

• mRNA vaccine preparation: IVT RNA technology is used to synthesize mRNA vectors carrying the information encoding the antigenic proteins required for vaccines, to trigger immune responses.
• Vaccine production: Large quantities of vaccine RNA are synthesized ex vivo to meet global vaccine demands.
• Customized vaccine design: IVT RNA technology is employed to design mRNA sequences tailored to target different pathogenic variations.
• Enhancement of vaccine efficacy and stability: By adjusting RNA structures and sequences, vaccine efficacy and stability are improved.

Looking ahead, the continued improvement of IVT RNA technology, especially RNA platform technologies such as NRM, SAM, taRNA, and circRNA, is anticipated. While the NRM platform is the most mature, its short half-life and limited antigen production present challenges. SAM addresses NRM's shortcomings, but the presence of nsPs restricts the length of the Gene of Interest (GOI). CircRNA, with its unique circular structure, offers a longer half-life and enhanced safety due to the absence of viral sequences. Moreover, circRNA lacks a 5' cap and poly(A) tail, allowing for the use of modified nitrogen-containing bases, which significantly compensate for the deficiencies of NRM. Therefore, the prospects for IVT RNA technology are broad, with the potential to further drive advancements in vaccine research and production to address challenges such as infectious diseases.

Reference

1. Perenkov, A.D.; et al. In Vitro Transcribed RNA-Based Platform Vaccines: Past, Present, and Future. Adv Drug Deliv Rev. Vaccines 2023, 11(10), 160.