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Updated: Aug 1, 2025
Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform
Published on: February 25, 2021
Production, Characterization, and Assessment of Permanently Cationic and Ionizable Lipid Nanoparticles for Use in the
Dylan Kairuz1, Nazia Samudh1, Abdullah Ely1
1Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2050, South Africa.
Insights
Self-amplifying RNA (saRNA) vaccines delivered via lipid nanoparticles offer a dose-sparing alternative for Africa, potentially enabling vaccine independence and rapid pandemic response.
Area of Science:
- Biotechnology
- Vaccinology
- RNA Therapeutics
Background:
- Africa faces a high burden of infectious diseases and significant reliance on external vaccine sources.
- The COVID-19 pandemic highlighted Africa's vulnerability due to vaccine dependence.
- Establishing local vaccine manufacturing is crucial for global health equity.
Purpose of the Study:
- To explore alphavirus-based self-amplifying RNAs (saRNAs) delivered by lipid nanoparticles (LNPs) as a novel vaccine platform.
- To develop dose-sparing vaccines for resource-constrained settings to achieve vaccine independence.
- To optimize saRNA production and identify suitable LNP candidates for vaccine development.
Main Methods:
- Optimized protocols for synthesizing high-quality saRNAs.
- In vitro expression studies of reporter proteins encoded by saRNAs.
- Production and characterization of permanently cationic or ionizable LNPs (cLNPs and iLNPs) encapsulating saRNAs (saRNA-Ext-LNPs and saRNA-Int-LNPs).
- Evaluation of LNP performance based on size, polydispersity index (PDI), N:P ratio, lipid molar ratio, and encapsulation efficiency.
- Assessment of LNP toxicity.
Main Results:
- Achieved in vitro expression of reporter proteins from saRNAs at low doses with prolonged expression.
- Successfully produced cLNPs and iLNPs encapsulating saRNAs with high encapsulation efficiency (>90%).
- Identified optimal LNP formulations (DOTAP/DOTMA saRNA-Ext-cLNPs and DOTAP/DDA saRNA-Int-cLNPs) with good characteristics for saRNA delivery and amplification.
- Demonstrated no significant toxicity associated with LNP delivery of saRNA.
- N:P ratio and lipid molar ratio significantly impacted saRNA expression kinetics.
Conclusions:
- Optimized saRNA production and identified promising LNP candidates for saRNA vaccine and therapeutic development.
- The saRNA platform offers dose-sparing, versatility, and manufacturing simplicity, crucial for rapid pandemic response.
- This approach holds potential to enhance vaccine independence in resource-constrained regions.
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 (<0.3). DOTAP and DDA saRNA-Int-cLNPs performed optimally, allowing for saRNA amplification. These were slightly larger, with higher PDIs as a result of the method used, which will require further optimization. In both cases, the N:P ratio and lipid molar ratio had a distinct effect on saRNA expression kinetics, and RNA was encapsulated at high percentages of >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.
