理由：环状 RNA (circRNA) 疗法由于其固有的稳定性和持久的蛋白质翻译能力，作为信使 RNA (mRNA) 疗法的迭代策略具有广阔的前景。然而，RNA 环化的效率仍然是一个重大限制，特别是在建立大规模生产工艺来生产高纯度的 circRNA 方面。因此，当考虑将合成circRNA作为具有前瞻性临床应用的治疗剂时，必须开发一种通用且更有效的RNA环化系统。方法：我们首先开发了基于原始排列内含子-外显子（PIE）的嵌合RNA环化系统，随后建立了高效液相色谱（HPLC）方法以获得高纯度的circRNA。然后我们评估了它们的翻译能力和免疫原性。将表达人乳头瘤病毒 (HPV) E7 肽 (43-62aa) 和 SARS-CoV-2 的二聚化受体结合域 (dRBD) 的 circRNA 封装在脂质纳米粒子 (LNP) 中作为疫苗，然后通过以下方法评估体内功效分别测定抗原特异性T细胞和B细胞反应。结果：我们通过对源自鱼腥藻前 tRNALeu 或 T4 噬菌体胸苷酸 (Td) 合酶基因的 I 组自剪接内含子进行改造，成功开发了通用嵌合排列内含子外显子系统 (CPIE)。在CPIE中，我们有效地提高了RNA环化效率。通过利用尺寸排阻层析，有效分离了circRNA，其表现出低免疫原性和持续有效的蛋白质表达特性。体内数据表明，构建的 circRNA 疫苗可以在小鼠模型中引发针对肿瘤或 SARS-CoV-2 及其变体的强大免疫激活（B 细胞和/或 T 细胞反应）。结论：总的来说，我们提供了一种高效、通用的体外合成circRNA的系统，对于circRNA治疗具有广泛的应用前景。©作者。

Rationale: Circular RNA (circRNA) therapeutics hold great promise as an iteration strategy in messenger RNA (mRNA) therapeutics due to their inherent stability and durable protein translation capability. Nevertheless, the efficiency of RNA circularization remains a significant constraint, particularly in establishing large-scale manufacturing processes for producing highly purified circRNAs. Hence, it is imperative to develop a universal and more efficient RNA circularization system when considering synthetic circRNAs as therapeutic agents with prospective clinical applications. Methods: We initially developed a chimeric RNA circularization system based on the original permuted intron-exon (PIE) and subsequently established a high-performance liquid chromatography (HPLC) method to obtain highly purified circRNAs. We then evaluated their translational ability and immunogenicity. The circRNAs expressing human papillomavirus (HPV) E7 peptide (43-62aa) and dimerized receptor binding domain (dRBD) from SARS-CoV-2 were encapsulated within lipid nanoparticles (LNPs) as vaccines, followed by an assessment of the in vivo efficacy through determination of antigen-specific T and B cell responses, respectively. Results: We have successfully developed a universal chimeric permuted intron-exon system (CPIE) through engineering of group I self-splicing introns derived from Anabaena pre-tRNALeu or T4 phage thymidylate (Td) synthase gene. Within CPIE, we have effectively enhanced RNA circularization efficiency. By utilizing size exclusion chromatography, circRNAs were effectively separated, which exhibit low immunogenicity and sustained potent protein expression property. In vivo data demonstrate that the constructed circRNA vaccines can elicit robust immune activation (B cell and/or T cell responses) against tumor or SARS-CoV-2 and its variants in mouse models. Conclusions: Overall, we provide an efficient and universal system to synthesize circRNA in vitro, which has extensive application prospect for circRNA therapeutics.© The author(s).