开发一种增强的嵌合置换内含子-外显子系统用于环状RNA治疗

Developing an enhanced chimeric permuted intron-exon system for circular RNA therapeutics

Theranostics

影响因子:13.3

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

DOI: 10.7150/thno.98214

摘要

依据：环状RNA（circRNA）治疗在信使RNA（mRNA）治疗中具有巨大潜力，因其固有的稳定性和持续的蛋白质翻译能力。然而，RNA环化效率仍是限制因素，尤其是在建立大规模生产高纯度circRNA的工艺方面。因此，开发一种通用且更高效的RNA环化系统，以便作为具有潜在临床应用的治疗剂显得尤为重要。方法：我们最初基于原始的置换内含子-外显子（PIE）系统开发了嵌合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circRNA。接着，我们评估了其翻译能力和免疫原性。将表达人乳头瘤病毒（HPV）E7肽（43-62氨基酸）和SARS-CoV-2的二聚受体结合域（dRBD）的circRNA封装在脂质纳米粒（LNPs）中作为疫苗，随后通过检测抗原特异性T细胞和B细胞反应评估其体内效果。结果：我们成功开发了通用的嵌合置换内含子-外显子系统（CPIE），通过工程化源自Anabaena预tRNALeu或T4噬菌体胸苷酸合成酶基因的Group I自剪切内含子。在CPIE中，我们有效提升了RNA环化效率。利用尺寸排阻色谱分离出具有低免疫原性和持续高效蛋白表达的circRNA。体内数据显示，所构建的circRNA疫苗能在小鼠模型中引发强烈的免疫激活（B细胞和/或T细胞反应），对抗肿瘤或SARS-CoV-2及其变异株。结论：总体而言，我们提供了一个高效且通用的体外合成circRNA的系统，具有广阔的应用前景，用于circRNA治疗。

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.