What is Self-replicating RNA?

The important difference between self-replicating RNA (srRNA, also known as self-amplifying RNA) and regular mRNA is that it can use its own RNA sequence as a template for self-replication. Normally mRNAs encode proteins that need to be expressed, and ribosomes in the cell are used for translation and protein production. In addition to expressing target proteins, srRNA carries a sequence capable of expressing RNA polymerase. After this RNA polymerase is produced, it can use the srRNA as a template to produce more copies of the srRNA.

Fig 1. Schematic illustration of self-replicating RNA alphavirus-based expression systems. (Lundstrom et al., 2021)

How to Self-amplify Designed Self-replicating RNA?

There are currently two designs for making RNA self-amplifying:

• One loads the sequence encoding the RNA polymerase and the sequence expressing the target protein in the same linear mRNA, which is commonly known as an srRNA.
• The other approach is to split the sequence encoding the RNA polymerase and the mRNA sequence encoding the target protein into two parts, which are introduced into the cell separately, a strategy called trans-amplifying.

How does RNA Self Replicate?

The steps of RNA self-replication include the following:

• Template pairing: The RNA replication enzyme first binds to the RNA template, forming a complex. The enzyme moves over the template, pairing new nucleotides to complementary bases on the template by base pairing.
• Synthetic strand extension: The RNA replicase uses the sequence of bases on the template to synthesize a new RNA strand. It does this by adding adapted nucleotides to the newly synthesized strand, sequentially pairing them with the bases on the template. In this way, the new RNA strand will be identical to the original template.
• Separation: After completion of replication, the newly synthesized RNA strand is separated from the template strand. This separation can be accomplished by the enzyme's own activity or assisted by other cofactors.
• Continued replication: The newly synthesized RNA strand can continue to serve as a template and participate in the next round of the replication process. In this way, the replication process can be carried out continuously, thus realizing self-replication of RNA.

Advantages of Self-replicating RNA

• The main advantage of self-replicating RNA is that it can achieve the same level of protein expression as conventional mRNA at a very low dose. As a vaccine, srRNA is hundreds or even thousands of times smaller than conventional messenger RNA. In this case, the same immune response can be generated. Since srRNA can prolong the presence of antigenic proteins in the body, which may enhance the immune response, an srRNA-based vaccine can achieve the effect of two doses of conventional mRNA vaccination in just one shot. In terms of production, the lower effective dose could reduce the cost of srRNA production. As a therapeutic approach, this feature can reduce the amount and number of injections used in mRNA therapy, thereby prolonging the therapeutic effect and reducing the possible toxic side effects of the mRNA and the delivery vehicle.
• Another feature of srRNA is that it may have the potential to stimulate an immune response. There are sensors that recognize the invasion of alien viruses in human cells, and they are called pattern recognition receptors. One of the signals they recognize is the presence of double-stranded RNA in the cytoplasm, as this may represent the replication of viral RNA in the cell. srRNAs form double-stranded RNA during replication, which is very similar to viral RNA during replication, and may stimulate the cell's inherent immune response. This may further enhance the effectiveness of the vaccine.

Applications of Self-replicating RNA

• Vaccine Development

By designing srRNA, cells can be made to produce expression of pathogen proteins, which can trigger an immune response and promote the production of antibodies and cellular immune responses by the immune system.

• Cancer Therapy

Specific srRNAs that can regulate gene expression in cancer cells, inhibit the growth and spread of cancer cells, or promote anti-tumor immune responses.

• Genetic Disease Treatment

srRNAs can alter the expression of specific genes in cells and repair or replace defective genes, thereby correcting abnormal phenotypes caused by genetic diseases.

• Tissue Engineering

srRNA can promote cell proliferation and differentiation, and guide the differentiation of stem cells to specific cell types, thus achieving the purpose of repairing and regenerating damaged tissues.

Reference

1. Lundstrom K. Self-replicating RNA viruses for vaccine development against infectious diseases and cancer[J]. Vaccines, 2021, 9(10): 1187.