A trans-amplifying RNA simplified to essential elements is highly replicative and robustly immunogenic in mice

亚基因组mRNA 生物 核糖核酸 RNA依赖性RNA聚合酶 非编码RNA 分子生物学 病毒学 遗传学 基因
作者
Mario Perković,Stefanie Gawletta,Tina Hempel,Silke Brill,Evelin Nett,Uğur Şahin,Tim Beißert
出处
期刊:Molecular Therapy [Elsevier BV]
卷期号:31 (6): 1636-1646 被引量:20
标识
DOI:10.1016/j.ymthe.2023.01.019
摘要

Trans-amplifying RNA (taRNA) is a split-vector derivative of self-amplifying RNA (saRNA) and a promising vaccine platform. taRNA combines a non-replicating mRNA encoding an alphaviral replicase and a transreplicon (TR) RNA coding for the antigen. Upon translation, the replicase amplifies the antigen-coding TR, thereby requiring minimal amounts of TR for immunization. TR amplification by the replicase follows a complex mechanism orchestrated by genomic and subgenomic promoters (SGPs) and generates genomic and subgenomic amplicons whereby only the latter are translated into therapeutic proteins. This complexity merits simplification to improve the platform. Here, we eliminated the SGP and redesigned the 5′ untranslated region to shorten the TR (STR), thereby enabling translation of the remaining genomic amplicon. We then applied a directed evolution approach to select for faster replicating STRs. The resulting evolved STR (eSTR) had acquired A-rich 5′ extensions, which improved taRNA expression thanks to accelerated replication. Consequently, we reduced the minimal required TR amount by more than 10-fold without losing taRNA expression in vitro. Accordingly, eSTR-immunized mice developed greater antibody titers to taRNA-encoded influenza HA than TR-immunized mice. In summary, this work points the way for further optimization of taRNA by combining rational design and directed evolution. Trans-amplifying RNA (taRNA) is a split-vector derivative of self-amplifying RNA (saRNA) and a promising vaccine platform. taRNA combines a non-replicating mRNA encoding an alphaviral replicase and a transreplicon (TR) RNA coding for the antigen. Upon translation, the replicase amplifies the antigen-coding TR, thereby requiring minimal amounts of TR for immunization. TR amplification by the replicase follows a complex mechanism orchestrated by genomic and subgenomic promoters (SGPs) and generates genomic and subgenomic amplicons whereby only the latter are translated into therapeutic proteins. This complexity merits simplification to improve the platform. Here, we eliminated the SGP and redesigned the 5′ untranslated region to shorten the TR (STR), thereby enabling translation of the remaining genomic amplicon. We then applied a directed evolution approach to select for faster replicating STRs. The resulting evolved STR (eSTR) had acquired A-rich 5′ extensions, which improved taRNA expression thanks to accelerated replication. Consequently, we reduced the minimal required TR amount by more than 10-fold without losing taRNA expression in vitro. Accordingly, eSTR-immunized mice developed greater antibody titers to taRNA-encoded influenza HA than TR-immunized mice. In summary, this work points the way for further optimization of taRNA by combining rational design and directed evolution. A trans-amplifying RNA simplified to essential elements is highly replicative and robustly immunogenic in micePerkovic et al.Molecular TherapyJune 11, 2023In Brief(Molecular Therapy 31, 1636–1646; June 2023) Full-Text PDF A trans-amplifying RNA simplified to essential elements is highly replicative and robustly immunogenic in micePerkovic et al.Molecular TherapyDecember 2, 2023In Brief(Molecular Therapy 31, 1636–1646; June 2023) Full-Text PDF
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