MOE Key Laboratory of Coal Environmental Pathogenicity and Prevention, Shanxi Medical University, Taiyuan 030001, China.
Shanxi Academy of Advanced Research and Innovation, Taiyuan 030032, China.
Theranostics. 2024 Sep 9;14(15):5869-5882. doi: 10.7150/thno.98214. eCollection 2024.
: 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. 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 efficacy through determination of antigen-specific T and B cell responses, respectively. We have successfully developed a universal chimeric permuted intron-exon system (CPIE) through engineering of group I self-splicing introns derived from Anabaena pre-tRNA 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. 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. Overall, we provide an efficient and universal system to synthesize circRNA , which has extensive application prospect for circRNA therapeutics.
环状 RNA（circRNA）治疗因其固有的稳定性和持久的蛋白质翻译能力，作为信使 RNA（mRNA）治疗的迭代策略具有巨大的潜力。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高纯度 circRNA 的过程中。因此，当考虑将合成的 circRNA 作为具有潜在临床应用的治疗剂时，开发一种通用且更有效的 RNA 环化系统至关重要。
我们最初开发了一种基于原始改组内含子-外显子（PIE）的嵌合 RNA 环化系统，随后建立了高效液相色谱（HPLC）方法来获得高度纯化的 circRNA。然后，我们评估了它们的翻译能力和免疫原性。表达人乳头瘤病毒（HPV）E7 肽（43-62aa）和 SARS-CoV-2 二聚受体结合结构域（dRBD）的 circRNA 被包裹在脂质纳米颗粒（LNP）中作为疫苗，然后通过测定抗原特异性 T 和 B 细胞反应来评估其疗效。
我们通过工程化来自鱼腥藻前 tRNA 或 T4 噬菌体胸苷酸合酶基因的 I 组自我剪接内含子，成功开发了一种通用的嵌合改组内含子-外显子系统（CPIE）。在 CPIE 中，我们有效地提高了 RNA 环化效率。通过使用尺寸排阻色谱法，有效地分离了 circRNA，其表现出低免疫原性和持续的强大蛋白质表达特性。
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 及其变体的强烈免疫激活（B 细胞和/或 T 细胞反应）。
总之，我们提供了一种高效通用的 circRNA 合成系统，为 circRNA 治疗提供了广阔的应用前景。
