By Victoria Zhang
The world has witnessed a shift towards using synthetic RNA platforms for vaccine development during the COVID-19 pandemic, where non-replicating mRNA vaccines play a crucial role.1 But non-replicating mRNA vaccines are not the only type of RNA molecules used in the RNA vaccine family, the other two types being base-modified non-amplifying mRNA(bmRNA) and self-amplifying mRNA(saRNA), which also has a prospect in infectious disease and cancer therapy. 2
The best-studied saRNA molecules are derived from the alphavirus genome. The alphavirus is able to directly deposit its RNA into the cytoplasm and its RNA encodes four non-structural proteins essential to RNA replication. Once these RNA-dependent RNA polymerases (RDRP) are transcribed by the ribosome in the cell, they are able to amplify the gene downstream of the subgenomic promoter within the viral RNA. Thus, in the saRNA vaccine, the saRNA contains a large open reading frame (ORF) coding for the RDRP, subgenomic promoter, and the vaccine antigen. The rest of its components are the same as non-amplifying mRNAs (a cap, 5′ UTR, 3′ UTR, and poly(A) tail of variable length).2 Within the host cell cytoplasm, RDRP is able to replicate multiple copies of the subgenomic RNA for the antigen, which can accumulate to approximately 106 copies per host cell, so many more copies of the antigen can be translated in the cell.1 This results in a host cell antiviral response leading to apoptosis and the release of antigens at high levels. The process is similar to viral infection in cells and can enhance antigen-specific T cell and B cell responses. In addition, the injection of saRNA into the myocytes in the first place will trigger the innate immune response and saRNA will be recognised by the pattern-recognition receptors (PRRs) in antigen-presenting cells like dendritic cells. This activates the adaptive immune responses. However, the mechanism of saRNA self-replication is still being studied to further understand the formation of RDRP and the role of elements encoded in the viral genome that aids RNA replication.2
One of the key processes of saRNA vaccine development is the development of vectors. The saRNA viruses can be classified into two groups based on the polarity of their single-stranded non- fragmented RNA (ssRNA) genome. Alphaviruses and flaviviruses have a positive-sense RNA genome and is often used for saRNA vaccine vectors as their viral RNA can be directly translated in the host cell cytoplasm while negative-strand RNA viruses need mRNA transcription and then translate them into proteins. 1
An innovative development on the saRNA vaccine vector is the use of trans-amplifying RNA(taRNA) in a bipartite vector system by Beissert et al. The system has one non-amplifying mRNA encoding the replicase and another molecule of alphavirus RNA containing the antigen but not the replicase delivered in a separate vector. In mice, a 50ng dose of influenza hemagglutinin antigen-encoding RNA was sufficient to induce neutralizing antibodies and protective immune responses. The taRNA system is safer because pusedotyped alphaviruses used as saRNA vectors have unknown pathogenicity and replicative competency. By the loss of replicase, the expression of viral glycoprotein is blocked. Furthermore, the optimisation is eased as both components can be individually modified and they are easier to manufacture because the RNA length is shortened compared to the large saRNA molecule. 3
The other key aspect of vaccine design is the delivery system, and the large size of the saRNA molecules makes it a major challenge to efficiently deliver them to target cells. Naked saRNA requires a much higher dose for in vivo immunisation, which compromises the advantage of using saRNA vaccines instead of traditional mRNA ones.2 Being 9000 to 15,000 nt long and anionic, saRNAs normally use delivery platforms like polymeric nanoparticles, lipid nanoparticles (LNP), or nanoemulsions. The central dogma of these delivery systems is to condense the saRNA in a cationic carrier to an around 100 nt sized nanoparticle that can be taken up into the cell without being degraded.2
The delivery platform found most potent now is the LNP which only needs 10ng of saRNA to induce a sufficient immune response.4 A comparison between the efficiency of LNP and a bioreducible polymeric nanoparticle pABOL found that LNP formulations can induce a more robust humoral and cellular immune response in mice.5 But Cationic nanoemulsions also has a major advantage of being safer to administrate. This is because the emulsions are similar to the MF59, which is an immunologic adjuvant using oil-in-water emulsion and it has a well-defined safety profile in humans.2
Currently, the saRNA vaccine has been used in vaccine development against many infectious diseases like malaria and COVID-19. By encoding the plasmodium macrophage migration inhibitory factor(PMIF) in the saRNA, the vaccines developed can increase anti-Plasmodium antibody titers, and the number of antigen-experienced memory CD4 T cells and liver-resident CD8 T cells in mice. The vaccine also gives complete protection from re-infection of malaria by the adoptive transfer of CD8 or CD4 T cells. 6
For COVID-19 vaccine development, the trial at Imperial College used saRNA encoding pre-fusion stabilized SARS-CoV-2 spike protein delivered by LNP. It was found that the vaccine can induce robust cellular response and two immunizations of saRNA can trigger significantly higher SARS-CoV-2 specific IgG antibodies in mice than natural infections. 4
Another application of saRNA vaccines is for cancer treatment by introducing a tumour antigen into the saRNA viral vector. IL-12 is able to stimulate CD4+, CD8+ and NK cells to be recruited to the tumour. An in vitro evolution approach has also been used to introduce mutations into the saRNA backbone that enhanced the magnitude and duration of IL-12 protein expression in vivo. This generates a 5.5-fold increase in IL-2 levels and a highly inflamed tumour microenvironment. 7A single administration of saRNA encoding IL-12 delivered by LNP in mice can promote immunogenic cell death and elicit potent rejection on established melanomas. Distal untreated tumours are also eradicated and protective immune memory can also be induced. 8 Trials of saRNA vaccines against other types of cancer like non-small cell lung cancer, colorectal cancer, and solid tumours are still ongoing. 2
saRNA is a promising vaccine platform that has seen outbreaks in more efficient vaccine design and the low dose required for immunisation facilitates manufacture, which implies it is well suited for pandemic settings for faster vaccine manufacture.2 We can thus expect saRNA vaccine being more and more common in future vaccine trials.
References:
(1) Lundstrom K. Self-Replicating RNA Viruses for Vaccine Development against Infectious Diseases and Cancer. Vaccines (Basel). 2021; 9 (10): 1187. 10.3390/vaccines9101187.
(2) Blakney AK, Ip S, Geall AJ. An Update on Self-Amplifying mRNA Vaccine Development. Vaccines (Basel). 2021; 9 (2): 97. 10.3390/vaccines9020097.
(3) Beissert T, Perkovic M, Vogel A, Erbar S, Walzer KC, Hempel T, et al. A Trans-amplifying RNA Vaccine Strategy for Induction of Potent Protective Immunity. Molecular therapy. 2020; 28 (1): 119-128. 10.1016/j.ymthe.2019.09.009.
(4) McKay PF, Hu K, Blakney AK, Samnuan K, Brown JC, Penn R, et al. Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice. Nature communications. 2020; 11 (1): 3523. 10.1038/s41467-020-17409-9.
(5) Blakney AK, McKay PF, Hu K, Samnuan K, Jain N, Brown A, et al. Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines. Journal of controlled release. 2021; 338 201-210. 10.1016/j.jconrel.2021.08.029.
(6) Baeza Garcia A, Siu E, Sun T, Exler V, Brito L, Hekele A, et al. Neutralization of the Plasmodium-encoded MIF ortholog confers protective immunity against malaria infection. Nature communications. 2018; 9 (1): 2714-13. 10.1038/s41467-018-05041-7.
(7) In vitro evolution of enhanced RNA replicons for immunotherapy.
(8) Li Y, Su Z, Zhao W, Zhang X, Momin N, Zhang C, et al. Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity. Nature cancer. 2020; 1 (9): 882-893. 10.1038/s43018-020-0095-6.
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