开发增强的嵌合置换式内含子外观系统，用于圆形RNA疗法
Developing an enhanced chimeric permuted intron-exon system for circular RNA therapeutics
影响因子:13.30000
分区:医学1区 Top / 医学：研究与实验1区
发表日期:2024
作者:
Lei Wang, Chunbo Dong, Weibing Zhang, Xu Ma, Wei Rou, Kai Yang, Tong Cui, Shaolong Qi, Lijun Yang, Jun Xie, Guocan Yu, Lianqing Wang, Xiaoyuan Chen, Zhida Liu
摘要
基本原理：圆形RNA（CIRCRNA）疗法由于其固有的稳定性和持久的蛋白质翻译能力，因此在Messenger RNA（mRNA）疗法中作为迭代策略具有很大的希望。然而，RNA循环的效率仍然是一个重大限制，尤其是在建立大规模制造过程中生产高度纯化的CircrNA时。因此，当考虑合成circrNA作为具有前瞻性临床应用的治疗剂时，必须开发通用，更有效的RNA循环系统。方法：我们最初基于原始置换的内含子外观（PIE）开发了一种嵌合RNA圆形系统，然后建立了高性能液相色谱法（HPLC）方法，以获得高度纯化的circrnas。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）和来自SARS-COV-2的二聚受体结合结构域（DRBD）的CIRCRNA被作为疫苗中的脂质纳米颗粒（LNP）封装为疫苗中，随后通过确定Antigen of Antigecic of Antigen and and and vivo效率。结果：我们通过工程化的I组自切开的内含子（CPIE）成功开发了通用嵌合置换的内含子外激素系统（CPIE），这些内包含源自Anabaena pre-trnaleu或T4噬菌体胸甲酰胺基（TD）合酶基因。在CPIE中，我们有效提高了RNA循环效率。通过利用大小排除色谱法，有效地分离了CircrNA，其免疫原性和持续有效的蛋白质表达特性。体内数据表明，构造的ciRCRNA疫苗可以针对肿瘤或SARS-COV-2及其在小鼠模型中的变异引起鲁棒的免疫激活（B细胞和/或T细胞反应）。结论：总的来说，我们提供了一个有效且通用的系统，可以在体外合成Circrna，该系统对Circrna疗法具有广泛的应用前景。
Abstract
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.