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
{"title":"开发用于环状 RNA 治疗的增强型嵌合包被内含子-外显子系统。","authors":"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","DOIUrl":null,"url":null,"abstract":"<p><p><b>Rationale</b>: 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. <b>Methods:</b> 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 <i>in vivo</i> efficacy through determination of antigen-specific T and B cell responses, respectively. <b>Results:</b> 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<sup>Leu</sup> 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. <i>In vivo</i> 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. <b>Conclusions:</b> Overall, we provide an efficient and universal system to synthesize circRNA <i>in vitro</i>, which has extensive application prospect for circRNA therapeutics.</p>","PeriodicalId":22932,"journal":{"name":"Theranostics","volume":null,"pages":null},"PeriodicalIF":12.4000,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11426236/pdf/","citationCount":"0","resultStr":"{\"title\":\"Developing an enhanced chimeric permuted intron-exon system for circular RNA therapeutics.\",\"authors\":\"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\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><b>Rationale</b>: 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. <b>Methods:</b> 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 <i>in vivo</i> efficacy through determination of antigen-specific T and B cell responses, respectively. <b>Results:</b> 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<sup>Leu</sup> 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. <i>In vivo</i> 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. <b>Conclusions:</b> Overall, we provide an efficient and universal system to synthesize circRNA <i>in vitro</i>, which has extensive application prospect for circRNA therapeutics.</p>\",\"PeriodicalId\":22932,\"journal\":{\"name\":\"Theranostics\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":12.4000,\"publicationDate\":\"2024-09-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11426236/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Theranostics\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.7150/thno.98214\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q1\",\"JCRName\":\"MEDICINE, RESEARCH & EXPERIMENTAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Theranostics","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.7150/thno.98214","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
Developing an enhanced chimeric permuted intron-exon system for circular RNA therapeutics.
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.
期刊介绍:
Theranostics serves as a pivotal platform for the exchange of clinical and scientific insights within the diagnostic and therapeutic molecular and nanomedicine community, along with allied professions engaged in integrating molecular imaging and therapy. As a multidisciplinary journal, Theranostics showcases innovative research articles spanning fields such as in vitro diagnostics and prognostics, in vivo molecular imaging, molecular therapeutics, image-guided therapy, biosensor technology, nanobiosensors, bioelectronics, system biology, translational medicine, point-of-care applications, and personalized medicine. Encouraging a broad spectrum of biomedical research with potential theranostic applications, the journal rigorously peer-reviews primary research, alongside publishing reviews, news, and commentary that aim to bridge the gap between the laboratory, clinic, and biotechnology industries.