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

Area of Science:

• Molecular Biology
• Vaccinology
• RNA Therapeutics

Background:

• Trans-amplifying RNA (taRNA) is an advanced vaccine platform derived from self-amplifying RNA (saRNA).
• Current taRNA systems rely on complex replication mechanisms involving genomic and subgenomic promoters (SGPs).
• Simplifying taRNA complexity is crucial for improving its efficacy and applicability.

Purpose of the Study:

• To simplify the taRNA platform by eliminating the SGP and redesigning the transreplicon (TR) RNA.
• To enhance taRNA expression and reduce the required dose through rational design and directed evolution.
• To evaluate the immunogenicity of the optimized taRNA construct in a preclinical model.

Main Methods:

• Elimination of the SGP and redesign of the 5' untranslated region to create a shortened TR (STR).
• Application of directed evolution to select for STR variants with accelerated replication.
• In vitro expression analysis and in vivo immunization studies in mice using the evolved STR (eSTR).

Main Results:

• The redesigned STR enabled translation of the genomic amplicon, simplifying the expression mechanism.
• Directed evolution yielded eSTR variants with A-rich 5' extensions, significantly enhancing replication.
• eSTR demonstrated over a 10-fold reduction in the minimal required TR amount for effective in vitro expression.
• Mice immunized with eSTR exhibited superior antibody titers against the encoded influenza HA antigen compared to control TR.

Conclusions:

• The simplified and evolved taRNA (eSTR) platform shows enhanced expression and potent immunogenicity.
• Rational design combined with directed evolution offers a powerful strategy for optimizing taRNA vaccine technology.
• This optimized taRNA system holds promise for developing more efficient and dose-sparing vaccines.