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CORDIS - EU research results

Maximization of amplification of next-generation RNA replicon vaccines through synergistic molecular and formulation design

Periodic Reporting for period 1 - RNAmpMax (Maximization of amplification of next-generation RNA replicon vaccines through synergistic molecular and formulation design)

Reporting period: 2019-01-02 to 2021-01-01

Ribonucleic acid (RNA) is a cutting-edge vaccine platform, and the speed and scalability of this strategy has been highlighted during the COVID-19 pandemic. It is possible to make a vaccine against any pathogen with a known protein target, and the currently approved RNA vaccines have remarkably efficacy (>94%). Self-amplifying RNA is a next-generation type of RNA, wherein the RNA is able to make copies of itself once it gets delivered into a host cell, which minimizes the required dose of RNA and therefore cost of the vaccine. However, like all RNA, saRNA is sensed by the cell, which can both help and hinder the immunogenicity of a vaccine. The objectives of this project were to develop new saRNA formulations that increase the amount of RNA that gets into a cell, and to use new molecular designs of RNA to limit the inhibitory cellular sensing of RNA. The findings from this project will enable more potent and efficacious RNA vaccines that increase the accessibility of vaccines for the global population.
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.
The work carried out enhances innovation capacity, creates new market opportunities and has established a proof-of-concept technology that has the potential to have a positive impact on human health. The main outcomes of this research are the knowledge of the state-of-the-art formulations and molecular design for self-amplifying RNA vaccines, as well as the first Phase I/II clinical trial with saRNA. Now that there are two approved RNA vaccines, a monumental gain in the field due to the COVID-19 pandemic, this technology is poised to actually be implemented. We found that a novel, bioreducible polymer developed as part of this work is a highly effective delivery vehicle for self-amplifying RNA vaccines.These findings in this project increase the potency of RNA vaccines and therefore lower the required dose and the cost of vaccines. We also identified new polymeric carriers that are able to target specific cells, elucidating the structure-function relationship between the delivery vehicle and expression of RNA. There is an ongoing Phase I/II clinical trial of saRNA vaccine against COVID-19, and further plans to test a second generation COVID-19 vaccine design with the optimized molecular designs identified from this work. Potential users include those who receive annual vaccines and pharmaceutical companies, both of whom have been made aware of saRNA vaccines through extensive coverage in the media.