Mechanistic insights into circularization via Anabaena group I intron-based scarless circular RNA.

IF 6.1 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Molecular Therapy. Nucleic Acids Pub Date : 2025-07-17 eCollection Date: 2025-09-09 DOI:10.1016/j.omtn.2025.102626

Bangda Fan, Sujia Liu, Yinghuan Xu, Xinghuan Ma, Wenbin Qi, Lei Miao, Lin Liu, Shubo Du, Jiaqi Lin

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引用次数: 0

Abstract

Circular RNA (circRNA) offers significant advantages in stability, storage, manufacturing, and pharmacokinetics, making it an attractive option for therapeutic applications over linear RNA. However, the commonly used permuted intron-exon (PIE) method for constructing circRNA introduces an exogenous "scar" sequence during splicing initiation, potentially compromising circRNA potency and inducing immunogenicity. Through exploration of the molecular mechanism of the Anabaena group I intron splicing, we conclude the sequence characterization of splice sites and the recognition rules of IG sequence. Leveraging these principles, we successfully prepared "scarless" circRNA without sacrificing its circularization efficiency using standard in vitro transcription procedures. Immunogenicity analysis of scarless circRNAs revealed that the scar will not induce an immune response, consistent with previous findings, after complete removal of linear byproducts, that circRNAs are naturally low immunogenicity. Finally, we find that the incorporation of modified nucleotides in circRNAs disrupts not only splicing function but also internal ribosome entry site function, with a low percentage of modified nucleotides destroying translation capacity.

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通过Anabaena I组内含子基无疤痕环状RNA对环状化的机制见解。

环状RNA （circRNA）在稳定性、储存、制造和药代动力学方面具有显著优势，使其成为线性RNA治疗应用的一个有吸引力的选择。然而，通常用于构建circRNA的排列内含子-外显子（PIE）方法在剪接起始过程中引入了外源性“疤痕”序列，可能会损害circRNA的效力并诱导免疫原性。通过探索Anabaena I族内含子剪接的分子机制，我们总结了剪接位点的序列表征和IG序列的识别规律。利用这些原理，我们使用标准的体外转录程序成功制备了“无疤痕”circRNA，而不牺牲其循环效率。无疤痕环状rna的免疫原性分析显示，疤痕不会诱导免疫反应，这与先前的研究结果一致，在完全去除线性副产物后，环状rna天然具有低免疫原性。最后，我们发现在circRNAs中掺入修饰的核苷酸不仅会破坏剪接功能，还会破坏内部核糖体进入位点功能，并且低比例的修饰核苷酸会破坏翻译能力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Molecular Therapy. Nucleic Acids MEDICINE, RESEARCH & EXPERIMENTAL-

CiteScore

15.40

自引率

1.10%

发文量

336

审稿时长

20 weeks

期刊介绍： Molecular Therapy Nucleic Acids is an international, open-access journal that publishes high-quality research in nucleic-acid-based therapeutics to treat and correct genetic and acquired diseases. It is the official journal of the American Society of Gene & Cell Therapy and is built upon the success of Molecular Therapy. The journal focuses on gene- and oligonucleotide-based therapies and publishes peer-reviewed research, reviews, and commentaries. Its impact factor for 2022 is 8.8. The subject areas covered include the development of therapeutics based on nucleic acids and their derivatives, vector development for RNA-based therapeutics delivery, utilization of gene-modifying agents like Zn finger nucleases and triplex-forming oligonucleotides, pre-clinical target validation, safety and efficacy studies, and clinical trials.