Application of Nucleotides in the Direction of RNA Vaccines

Introduction to RNA vaccines

RNA vaccines are composed of the nucleic acid RNA, which encode antigen genes of an infectious agent. When administered to host cells, the RNA is translated into protein antigens that elicit protective immunity against the infectious agent. The mouse encodes influenza virus antigens and exhibits the expected response. However, because the lipid carrier used to inject RNA vaccines was too toxic in the human body at that time, it was not until the discovery of lipid nanoparticle technology that RNA vaccines were put into use. According to the CDC of the US Centers for Disease Control and Prevention, mRNA has become a promising next-generation technology in the past decade. Previously, mRNA vaccines have been studied for preventive vaccines against other emerging infectious diseases such as influenza, Zika virus, rabies and cytomegalovirus (CMV). By the beginning of 2020, the clinical development of RNA vaccines is not very extensive. However, with the emergence of new coronaviruses, in the past 10 months alone, at least six RNA-based new coronary pneumonia vaccines have entered human trials.

Figure 1. Mode of action of mRNA to induce adaptive immune responses.

Advantages of RNA vaccines

Compared with traditional vaccines, mRNA has more safety advantages, it will not insert gene mutations, can be degraded by normal cells, and its half-life can be changed by modifying the regulatory sequence and delivery vectors. More importantly, traditional vaccines are powerless against many new viruses, not to mention diseases such as cancer that seriously threaten human health. The mechanism of mRNA makes it like a meal book. As long as you compile the RNA sequence, you can turn the cell into a small drug factory. The mRNA guides the cell to produce a specific protein to exert its systemic effects. However, today’s RNA vaccine technology still has room for improvement, and its immature development makes large-scale production and use face many challenges. For example, problems such as expensive raw materials, side effects, and the need for cold chain storage at -70°C still need to be resolved urgently.

Application of nucleotides in the direction of RNA vaccines

At present, a variety of modified nucleotide technologies have been used to produce more stable mRNA, and the existing mRNA modification technologies can directly or indirectly affect the immune response. details as follows:

1) Synthesize “cap”-like structures and “capping enzymes” to stabilize mRNA, and enhance protein translation by combining with eukaryotic translation initiation factor 4E (EIF4E);

2) Adding controllable sequences in the 5’ and 3’UTR regions to stabilize the mRNA. The improvement of the 5’ and 3’UTR region sequences is essential to improve mRNA stability and protein expression;

3) Modify Poly(A) “tail” to stabilize mRNA and enhance protein translation;

4) Modify nucleotides to reduce innate immune activation;

5) Separation and (or) purification technology: use RNase III processing and fast protein liquid chromatography (FPLC) purification to reduce immune activation and increase translation rate;

6) Optimize the sequence and codons, and choose codons homologous to tRNA can improve the translation ability of mRNA;

7) Regulation of target cells: co-delivery of translation initiation factors and other methods to optimize translation and immunogenicity

Figure 2. Structural features of mRNA.

Main nucleotide modification methods

During the development of mRNA vaccines, the main modification methods are N6-methyladenosine, pseudouridine and 2′-O-methylation (Nm). N6-Methyladenosine (m6A) plays a role in regulating mRNA stability. The human body’s immune response to mRNA vaccines is mainly related to uridine (partially composed of uracil). Using pseudouracil instead of uracil can reduce the immune system’s recognition of mRNA. The 2′-O-methyl group of the RNA 5’cap Chemical modification can make it escape the host’s antiviral response. Both Moderna and BioNTech use pseudouracil modification to maintain mRNA stability. Pseudouracil modification is the most abundant RNA modification. It is generally produced by the isomerization of uridine. Existing studies have shown that pseudouracil modification of mRNA has three main functions: changing codons, enhancing transcript stability, and enhancing stress response.


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