Trans-amplifying RNA: Translational application in gene therapy

The successful development of COVID-19 mRNA vaccines has increased the demand for relatively high doses of degradation-prone mRNA and extensive large-scale production of RNA. In this issue of Molecular Therapy, Sahin, Beissert, and colleagues propose a potential solution: alphavirus-based trans-amplifying RNA (taRNA), a split-vector derivative of self-amplifying RNA (saRNA).1Perkovic M. Gawletta S. Hempel T. Brill S. Nett E. Sahin U. Beissert T. A trans-amplifying RNA simplified to essential elements is highly replicative and robustly immunogenic in mice.Mol. Ther. 2023; https://doi.org/10.1016/j.ymthe.2023.01.019Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar This approach provides robust RNA self-replication and immunogenicity that could help reduce demand for large-scale RNA production. Moreover, it could translate into lower vaccine doses to decrease or prevent serious rare adverse events such as those reported for COVID-19 mRNA vaccines.2Lundstrom K. Barh D. Uhal B.D. Takayama K. Aljabali A.A.A. Abd El-Aziz T.M. Lal A. Redwan E.M. Adadi P. Chauhan G. et al.COVID-19 vaccines and thrombosis—roadblock or dead-end street?.Biomolecules. 2021; 11: 1020https://doi.org/10.3390/biom11071020Crossref PubMed Scopus (21) Google Scholar,3Kracalik I. Oster M.E. Broder K.R. Cortese M.M. Glover M. Shields K. Creech C.B. Romanson B. Novosad S. Soslow J. et al.Outcomes at least 90 days since onset of myocarditis after mRNA COVID-19 vaccination in adolescents and young adults in the USA: a follow-up surveillance study.Lancet Child. Adolesc. Health. 2022; 6: 788-798https://doi.org/10.1016/S2352-4642(22)00244-9Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Although alphavirus saRNA systems have been useful for vaccine development against both infectious diseases and various cancers,4Lundstrom K. Self-replicating RNA viruses for vaccine development against infectious diseases and cancer.Vaccines. 2021; 9: 1187https://doi.org/10.3390/vaccines9101187Crossref Google Scholar the large saRNA vector size (∼10 kb) presents challenges for scale-up of RNA production and applying reduced doses of saRNA.5Liljeström P. Garoff H. A new generation of animal cell expression vectors based on the Semliki forest virus replicon.Biotechnology. 1991; 9: 1356-1361https://doi.org/10.1038/nbt1291-1356Crossref PubMed Scopus (748) Google Scholar For this reason, the researchers initially engineered a first-generation bipartite taRNA system from Semliki Forest virus (SFV) in which the nsP1-4 replicase genes were deleted, resulting in the transreplicon RNA (TR).6Beissert T. Perkovic M. Vogel A. Erbar S. Walzer K.C. Hempel T. Brill S. Haefner E. Becker R. Türeci Ö. Sahin U. A trans-amplifying RNA vaccine strategy for induction of potent protective immunity.Mol. Ther. 2020; 28: 119-128https://doi.org/10.1016/j.ymthe.2019.09.009Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The replicase activity was supplied in trans by an optimized non-replicating mRNA (nrRNA), requiring less antigen-coding TR than conventional RNA. Moreover, the invariant nrRNA replicase can be produced in large quantities and stored for future co-delivery of low amounts of freshly engineered target-specific TR for vaccine production.6Beissert T. Perkovic M. Vogel A. Erbar S. Walzer K.C. Hempel T. Brill S. Haefner E. Becker R. Türeci Ö. Sahin U. A trans-amplifying RNA vaccine strategy for induction of potent protective immunity.Mol. Ther. 2020; 28: 119-128https://doi.org/10.1016/j.ymthe.2019.09.009Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar This approach resulted in 10- to 100-fold higher transreplicon expression levels than those achieved with standard saRNA, most likely due to higher translational efficiency.2Lundstrom K. Barh D. Uhal B.D. Takayama K. Aljabali A.A.A. Abd El-Aziz T.M. Lal A. Redwan E.M. Adadi P. Chauhan G. et al.COVID-19 vaccines and thrombosis—roadblock or dead-end street?.Biomolecules. 2021; 11: 1020https://doi.org/10.3390/biom11071020Crossref PubMed Scopus (21) Google Scholar Evaluation of the taRNA system in mice demonstrated that 50 ng of influenza virus hemagglutinin (HA) RNA was sufficient to elicit neutralizing antibody responses and provide protection against influenza virus challenges. Nonetheless, to improve upon their initial design, the authors introduced a second-generation taRNA system engineered for alphavirus saRNA vectors by removing redundant non-essential sequences.6Beissert T. Perkovic M. Vogel A. Erbar S. Walzer K.C. Hempel T. Brill S. Haefner E. Becker R. Türeci Ö. Sahin U. A trans-amplifying RNA vaccine strategy for induction of potent protective immunity.Mol. Ther. 2020; 28: 119-128https://doi.org/10.1016/j.ymthe.2019.09.009Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar These modifications further improved taRNA expression and enhanced immunogenicity. An interesting feature of alphavirus replicases is the cross-utilization of RNA templates from different alphaviruses.7Lello L.S. Utt A. Bartholomeeusen K. Wang S. Rausalu K. Kendall C. Coppens S. Fragkoudis R. Tuplin A. Alphey L. et al.Cross-utilisation of template RNAs by alphavirus replicases.Plos Pathog. 2020; 16e1008825https://doi.org/10.1371/journal.ppat.1008825Crossref PubMed Scopus (13) Google Scholar Thus, in the present study, the authors generated shortened TRs (STRs) from alphavirus species such as Fort Morgan virus, Aura virus, Highlands J virus, Madariaga virus, and Chikungunya virus (CHIKV) and compared them to STRs from SFV and Sindbis virus (SINV).1Perkovic M. Gawletta S. Hempel T. Brill S. Nett E. Sahin U. Beissert T. A trans-amplifying RNA simplified to essential elements is highly replicative and robustly immunogenic in mice.Mol. Ther. 2023; https://doi.org/10.1016/j.ymthe.2023.01.019Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar The SFV-replicase showed amplification of all evaluated alphavirus STRs, even demonstrating enhanced expression for all heterologous STRs except for CHIKV. Interestingly, SINV-STR expression was enhanced 100-fold compared with SFV-STR when the SFV-saRNA was used, whereas it was only modestly higher when the SFV-replicase nrRNA was utilized. A directed evolution strategy was also applied for the selection of accelerated TR amplification based on a serial transfer of replicating template RNA. This approach greatly extended the time for replication, and extraction and re-transfection kept replication at high rates, leading to the accumulation of faster replicating mutant templates. The 5′ end extended evolved STR (eSTR) not only accelerated replication but also improved both taRNA expression and immunogenicity. The minimal amount of TR required was further reduced by more than 10-fold without losing in vivo taRNA expression capacity. Moreover, compared with TR-immunized mice, eSTR-immunized mice showed superior antibody titers against taRNA-based influenza virus HA. Although the second-generation taRNA system is a significant improvement, the potential of this platform for vaccine development is currently limited by the lack of appropriate formulations of nanoparticle-encapsulated taRNA, which are needed to improve delivery and extend circulation time in vivo.8Blakney A.K. McKay P.F. Hu K. Samnuan K. Jain N. Brown A. Thomas A. Rogers P. Polra K. Sallah H. et al.Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines.J. Control Release. 2021; 338: 201-210https://doi.org/10.1016/j.jconrel.2021.08.029Crossref PubMed Scopus (28) Google Scholar,9Ly H.H. Daniel S. Soriano S.K.V. Kis Z. Blakney A.K. Optimization of lipid nanoparticles for saRNA expression and cellular activation using a design-of-experiment approach.Mol. Pharm. 2022; 19: 1892-1905https://doi.org/10.1021/acs.molpharmaceut.2c00032Crossref PubMed Scopus (16) Google Scholar In the context of saRNA-based COVID-19 vaccines, lipid nanoparticle encapsulation of saRNA has provided robust immune responses and protection against SARS-CoV-2 challenges in animal models.10Blakney A.K. McKay P.F. Bouton C.R. Hu K. Samnuan K. Shattock R.J. Innate inhibiting proteins enhance expression and immunogenicity of self-amplifying RNA.Mol. Ther. 2021; 29: 1174-1185https://doi.org/10.1016/j.ymthe.2020.11.011Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar However, the size differences of the two co-transfected RNA components presents a challenge for nanoparticle formulations, which needs to be overcome. Another concern is the inhibitory action of innate immunity, which has been observed with saRNA.11Beissert T. Koste L. Perkovic M. Walzer K.C. Erbar S. Selmi A. Diken M. Kreiter S. Türeci Ö. Sahin U. Improvement of in vivo expression of genes delivered by self-amplifying RNA using vaccinia virus immune evasion proteins.Hum. Gene Ther. 2017; 28: 1138-1146https://doi.org/10.1089/hum.2017.121Crossref PubMed Scopus (37) Google Scholar Once optimized nanoparticle formulations are available and the issue of innate immunity has been resolved, the second-generation taRNA system will be an important platform for rapid development of novel efficient vaccines and may better prepare us for future epidemics or pandemics. The author declares no conflict of interest.