Production, Characterization, and Assessment of Permanently Cationic and Ionizable Lipid Nanoparticles for Use in the Delivery of Self-Amplifying RNA Vaccines

Abstract

Africa bears the highest burden of infectious diseases, yet the continent is heavily reliant on First World countries for the development and supply of life-saving vaccines. The COVID-19 pandemic was a stark reminder of Africa’s vaccine dependence and since then great interest has been generated in establishing mRNA vaccine manufacturing capabilities on the African continent. Herein, we explore alphavirus-based self-amplifying RNAs (saRNAs) delivered by lipid nanoparticles (LNPs) as an alternative to the conventional mRNA vaccine platform. The approach is intended to produce dose-sparing vaccines which could assist resource-constrained countries to achieve vaccine independence. Protocols to synthesize high-quality saRNAs were optimized and in vitro expression of reporter proteins encoded by saRNAs was achieved at low doses and observed for an extended period. Permanently cationic or ionizable LNPs (cLNPs and iLNPs, respectively) were successfully produced, incorporating saRNAs either exteriorly (saRNA-Ext-LNPs) or interiorly (saRNA-Int-LNPs). DOTAP and DOTMA saRNA-Ext-cLNPs performed best and were generally below 200 nm with good PDIs (90%. These LNPs allow the delivery of saRNA with no significant toxicity. The optimization of saRNA production and identification of potential LNP candidates will facilitate saRNA vaccine and therapeutic development. The dose-sparing properties, versatility, and manufacturing simplicity of the saRNA platform will facilitate a rapid response to future pandemics.

Keywords

• saRNA
• RNA delivery
• cationic lipid nanoparticles
• lipid film hydration
• microfluidics
• RNA vaccines