Self-amplifying RNA (saRNA) vectors are a next-generation RNA technology that extends the expression of heterologous genes. Clinical trials have shown the dose-sparing capacity of saRNA vectors in a vaccine context compared to conventional messenger RNA. However, saRNA vectors have historically been based on a limited number of alphaviruses, and only the Venezuelan equine encephalitis virus-based saRNA vaccines have been used clinically. Here, we designed genotypically distinct alphaviral saRNA vectors and characterized their performance in mammalian cell lines, human skin explants and mice. Five of the 12 vectors had substantial luciferase expression in mice with variable pharmacokinetics, enabling modulation of both the magnitude and duration of protein expression. Additionally, we demonstrate that the alphaviral genotype of the saRNA significantly impacts the immunogenicity of saRNA vaccines, including the humoral and cellular responses in mice. Given the differences in RNA reactogenicity and expression between mice and humans, we assessed the saRNA vectors in human skin explants obtained from patients and observed high transgene expression. saRNA bioluminescence and immunogenicity in different mice strains were highly correlative, while minimal correlation was observed when compared to human explants and mammalian cell lines. This work demonstrates that efficacious saRNA vaccines and therapies can be produced by adapting genetically diverse alphaviruses into vectors.

中文翻译：

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自扩增 RNA 的 α 病毒遗传背景增强了蛋白质表达和对 SARS-CoV-2 抗原的免疫原性。


自扩增 RNA （saRNA） 载体是新一代 RNA 技术，可延长异源基因的表达。临床试验表明，与传统信使 RNA 相比，saRNA 载体在疫苗环境中的剂量节省能力。然而，saRNA 载体历来基于有限数量的甲病毒，只有基于委内瑞拉马脑炎病毒的 saRNA 疫苗被用于临床。在这里，我们设计了基因型不同的 α 病毒 saRNA 载体，并表征了它们在哺乳动物细胞系、人皮肤外植体和小鼠中的表现。12 种载体中有 5 种在药代动力学可变的小鼠中具有大量荧光素酶表达，能够调节蛋白质表达的大小和持续时间。此外，我们证明 saRNA 的 α 病毒基因型显着影响 saRNA 疫苗的免疫原性，包括小鼠的体液和细胞反应。鉴于小鼠和人类之间 RNA 反应原性和表达的差异，我们评估了从患者获得的人皮肤外植体中的 saRNA 载体，并观察到高转基因表达。不同小鼠品系中的 saRNA 生物发光和免疫原性高度相关，而与人外植体和哺乳动物细胞系相比，观察到的相关性最小。这项工作表明，可以通过将遗传多样化的甲病毒适应载体来生产有效的 saRNA 疫苗和疗法。