Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine-ran-propylenimine) (L-PEI_x-ran-PPI_y) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher in vitro saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the in vitro gold standard carrier. This is especially true for our in vitro best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an ex vivo hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based in vivo study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI₂₅₀) demonstrating a higher sustained expression for at least 35 days after injection.

中文翻译：

---

以线性聚丙亚胺和聚氮丙啶-丙亚胺无规共聚物作为非病毒载体在体外和体内高效转染自扩增mRNA

基于信使 RNA (mRNA) 的疫苗已在全球范围内推出，以对抗 Covid-19 大流行。这些疫苗由配制在脂质纳米颗粒 (LNP) 中的非扩增 mRNA 组成。因此，LNP 被认为是核酸递送的基准非病毒载体。然而，这些 mRNA-LNP 纳米颗粒的配制和制造既昂贵又耗时。因此，我们使用自扩增 mRNA (saRNA) 并合成了新型聚合物作为 LNP 的替代非病毒载体平台，从而实现了简单、快速、一锅法的 saRNA-多聚复合物制剂。我们的新型聚合物载体平台由随机连接的乙烯亚胺和丙烯亚胺共聚单体组成，形成具有可控聚合度的线性聚（乙烯亚胺-ran-丙烯亚胺）（L-PEI _x - ran -PPI _y ）共聚物。在此，我们在多种细胞系中证明，与采用 Lipofectamine MessengerMAX™ (LFMM) 的制剂相比，我们的 saRNA 复合物表现出更高的体外saRNA 转染效率和更高的细胞活力，Lipofectamine MessengerMAX™ (LFMM) 是一种商业脂质载体，被认为是体外黄金标准载体。对于我们体外性能最好的 N/P 5 saRNA 聚合复合物来说尤其如此，其特点是尺寸低于 100 nm、正 zeta 电位、接近 100% 的封装效率、高保留能力和保护能力此外，猪红细胞的离体溶血试验表明，saRNA-多聚体表现出可忽略不计的溶血活性。最后，进行了为期 35 天的基于生物发光的体内研究，结果表明，与裸露的 saRNA 和基于 L-PEI 的聚合复合物相比，该聚合物可产生更高且更长的生物发光信号。此外，与 LNP 相比，这些聚合物表现出不同的表达谱，我们的一种新型聚合物 (L-PPI ₂₅₀ ) 在注射后至少 35 天表现出更高的持续表达。