Self-amplifying RNA (saRNA) vaccines are highly advantageous, as they result in enhanced protein expression compared to messenger RNA (mRNA), thus minimizing the required dose. The main findings from this fellowship can be grouped into improving molecular design (WP1-3) and delivery systems for saRNA (WP4-5).
The molecular design (WP1-3) was the first part of this project. saRNA is self-adjuvanting as it activates cellular interferon pathways, which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of protein translation. I screened a library of saRNA constructs with interferon inhibiting proteins (IIPs) derived from other viruses and determine the effect on protein expression and immunogenicity. I observed that two proteins (PIV-5 V and MERS-CoV ORF4a) enhance protein expression 100-500-fold of saRNA in cells. I found that the MERS-CoV ORF4a protein partially abates enhances protein expression of saRNA in vivo . Both the PIV-5 V and MERS-CoV ORF4a proteins were found to increase the number of resident cells in human skin explants expressing saRNA. Finally, I observed that the MERS-CoV ORF4a protein increased the antibody titers of a Rabies saRNA vaccine by ~10-fold in rabbits, but not mice or rats. These experiments provide a proof-of-concept that IIPs can be directly encoded into saRNA vectors and effectively enhance immunogenicity. These molecular advances can be exploited to improve the potency of RNA vaccines.
The research on delivery of saRNA (WP4-5) comprised the second part of this project. Previous RNA delivery strategies were optimized for other types of RNA and do not necessarily deliver saRNA efficiently, thus motivating the development of novel saRNA delivery platforms. I worked with the Stevens group to engineer a positively charged polymer called ‘pABOL’ for saRNA delivery and showed that increasing the size of the polymer enhances delivery both in vitro and in vivo. I demonstrated that pABOL enhances protein expression and cellular uptake via both intramuscular and intradermal injection compared to commercially available polymers in vivo, and that intramuscular injection confers complete protection against influenza challenge. Due to the scalability of polymer synthesis and ease of formulation preparation, we anticipate that this polymer is highly clinically translatable as a delivery vehicle for saRNA for both vaccines and therapeutics. Furthermore, lipid nanoparticles (LNPs) have been widely used for RNA formulations where the prevailing paradigm is to encapsulate RNA within the particle, including FDA-approved small interfering RNA (siRNA) therapy and mRNA vaccines. I compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. I showed that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in cell and in mice. Finally, I showed that cationic and ionizable LNP formulations induce equivalent antibodies against am model HIV-1 antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA. These findings have advanced the state-of-the-art for saRNA delivery.
