Clean-PIE: a novel strategy for efficiently constructing precise circRNA with thoroughly minimized immunogenicity to direct potent and durable protein expression
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Abstract
Translatable circular RNAs (circRNAs) are emerging as a crucial molecular format for transient protein expression, with high potential to be an alternative for linear mRNA to reshape the landscape of mRNA pharmaceutical industry. Canonical Anabaena permuted intron-exon ( Ana PIE) format that developed by ORNA is an efficient method for RNA circularization, and the engineered circRNAs direct supreme protein expression in eukaryotic cells. However, recent studies revealed that this method may unavoidably result in a remain of immunogenicity in the circRNA products, albeit after thorough RNA purification. In the current study, we develop a novel strategy for efficient generation of circRNA, via the permuted T4Td introns mediated autocatalytically ribozymatic reaction mediated ligation of the flanking segment sequences that concealing in ORF or translation initiation sequence (normally equal to IRES). This strategy universally realizes around 90% circularization effectivity, and the circRNA products can be purified to around 90% purity by our new purification method, and presented thoroughly minimized immunogenicity, thus is termed “Clean-PIE”. The purified circRNAs are found to direct potent and durable expression of various proteins in vitro and in vivo. The partly purified Fluc circRNA by HPLC-SEC was found to direct Fluc expression in muscle for no less than 20 days. The highly purified circRNA exhibits much stronger protein expression in vitro and in vivo, and presumed a longer duration. Additionally, the scale-up of RNA circularization with the RNA precursors from 1 L transcription revealed high circularization effectivity (around 90%) and a high productivity of the final circRNA products. Collectively, Clean-PIE is a novel circRNA platform that possesses high circularization effectivity, enabled high RNA purity and thoroughly minimized immunogenicity, as well as scaling-up accessibility and directing extreme durability of protein expression, thus has the potential to develop advanced RNA vaccines and therapeutics in pharmaceutical industrial scale.Keywords
This publication has 29 references indexed in Scilit:
- RNA circularization strategies in vivo and in vitroNucleic Acids Research, 2015
- Detecting and characterizing circular RNAsNature Biotechnology, 2014
- Landscape of transcription in human cellsNature, 2012
- In vitro and in vivo production and purification of circular RNA aptamerJournal of Biotechnology, 2009
- Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanismsGenes & Development, 2005
- A universal method to produce in vitro transcripts with homogeneous 3' endsNucleic Acids Research, 2002
- Initiation of Protein Synthesis by the Eukaryotic Translational Apparatus on Circular RNAsScience, 1995
- Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4.Proceedings of the National Academy of Sciences, 1994
- Group I permuted intron-exon (PIE) sequences self-splice to produce circular exonsNucleic Acids Research, 1992
- [5] Synthesis of small RNAs using T7 RNA polymerase1989