Browsing by Author "Kairuz, Dylan Matthew"

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    Developing non-viral vector formulations for the delivery of synthetic self-amplifying RNA
    (University of the Witwatersrand, Johannesburg, 2024) Kairuz, Dylan Matthew
    Conventional vaccines have been instrumental in reducing worldwide morbidity and mortality of infectious diseases. However, some diseases require improvements to their vaccines or do not have an effective vaccine. This has led to the development of next generation vaccines including messenger ribonucleic acid (mRNA) vaccines, a versatile platform allowing simple exchange of encoded antigen sequences for different pathogens in the mRNA. Alternatively, self-amplifying mRNA (saRNA) presents a vaccine platform which replicates in situ using Alphavirus-derived non-structural proteins, mimicking viral replication and prolonging antigen expression. This has the potential to improve immune responses and significantly lower mRNA vaccine doses. Although this is a promising platform, a vector is essential to protect saRNA from ribonuclease degradation and deliver saRNA into cells. Lipid nanoparticles (LNPs) are a safe, highly efficient delivery system, with multiple approved mRNA vaccines. However, saRNA is significantly larger than mRNA, and hence LNPs need to be optimised to achieve efficient delivery. Two main categories of LNPs have been denoted, ionisable (iLNPs) and permanently cationic LNPs (cLNPs). cLNP lipids are less expensive to synthesise and have recently been used successfully to deliver saRNA vaccines. Hence, these were predominantly examined in this study. A library of empty cLNPs were successfully produced using lipid film hydration (LFH) with sizes <150 nanometres (nm). When saRNA was complexed exteriorly to cLNPs (saRNA- Ext-cLNPs) varying sizes were observed depending on the N:P used. N:P ratio is the molar ratio of nitrogen and phosphate on the lipids and RNA respectively, hence larger sizes are expected at a lower N:P. saRNA formulated on the interior of cLNPs (saRNA-Int-cLNPs) were produced by microfluidics or a modified solvent injection (SI) method. Microfluidics formulated saRNA- Int-cLNPs had small sizes and lower polydispersity indices (PDIs) compared to the larger sizes and PDIs of SI formulated cLNPs. The library of saRNA-cLNPs were optimised by transfecting mammalian cells with saRNA encoding reporter genes at a variety of N:P ratios. Generally, the size of the LNPs did not influence the delivery efficiency and the main contributor was N:P ratio for both saRNA-Ext- and saRNA-Int-cLNPs. DOTAP (1,2-dioleoyl-3-trimethylammonium- propane) and DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) performed the best, particularly the F1 cLNPs. Although dimethyldioctadecylammonium (DDA) cLNPs showed poorer delivery efficacy at 24 hours post-transfection, interesting effects on saRNA expression kinetics were observed. A remarkable boost in expression was observed from 24 to 48 hours, and saRNA-Int DDA-cLNPs outperformed DOTAP and DOTMA cLNPs at 48 hours. High encapsulation efficiencies of >90% were observed for all cLNP candidates and the top saRNA-Ext-cLNPs candidates were not toxic. Even though unfavourable results were obtained upon intramuscular injection of saRNA-Ext-cLNPs into mice, this could be as a result of lipid film hydration (LFH) production and could be solved by microfluidics formulation. Although setbacks related to purification of saRNA-Int-cLNPs limited in vivo examination, these showed a strong potential for saRNA delivery for delivery of saRNA vaccines in the future. Overall, the work in this dissertation has developed the protocols for production and characterisation of LNPs. A library of potential cLNP candidates for saRNA vaccine delivery have been optimised in cell culture, with many showing highly efficient saRNA delivery. This will facilitate saRNA vaccine development and examination in a pre-clinical setting in South Africa, reducing reliance on the global North for equitable access of RNA vaccines and improving preparedness for potential future pandemics