非洲承担着最大的传染病负担,然而,非洲大陆严重依赖第一世界国家开发和供应拯救生命的疫苗。COVID-19大流行提醒人们非洲对疫苗的依赖,从那时起,人们对在非洲大陆建立mRNA疫苗制造能力产生了极大的兴趣。在这里,我们探索了通过脂质纳米颗粒(LNP)递送的基于甲病毒的自扩增RNA(saRNA),作为常规mRNA疫苗平台的替代方案.该方法旨在生产节省剂量的疫苗,这可以帮助资源有限的国家实现疫苗独立。优化了合成高质量saRNA的方案,并在低剂量下实现了saRNA编码的报告蛋白的体外表达,并在较长时间内观察。永久阳离子或可电离LNP(cLNP和iLNP,分别)成功生产,外部(saRNA-Ext-LNP)或内部(saRNA-Int-LNP)掺入saRNA。DOTAP和DOTMAsaRNA-Ext-cLNP表现最好,通常低于200nm,PDIs良好(<0.3)。DOTAP和DDAsaRNA-Int-cLNP表现最佳,允许saRNA扩增。这些稍微大一点,由于使用了更高的PDI,这将需要进一步优化。在这两种情况下,N:P比和脂质摩尔比对saRNA表达动力学有明显影响,RNA以>90%的高百分比被包封。这些LNP允许在没有显著毒性的情况下递送saRNA。saRNA生产的优化和潜在LNP候选物的鉴定将促进saRNA疫苗和治疗的开发。节省剂量的特性,多功能性,和制造简单的saRNA平台将促进对未来大流行的快速反应。
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.