Circular RNA: Biosynthesis in vitro

Chen, Xinjie; Lu, Yuan

doi:10.3389/fbioe.2021.787881

Cited by 35 publications

(52 citation statements)

References 92 publications

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“…The advanced property of circRNA, such as stable and directing long-duration of protein expression, makes it to be a preferred alternative to linear RNA 20,[24][25][26][27] . Several circularization strategies have been proposed [28][29][30][31] . The most common methods for in vitro circularization of RNA are mainly chemical methods and enzymatic catalysis methods.…”

Section: Discussionmentioning

confidence: 99%

Clean-PIE: a novel strategy for efficiently constructing precise circRNA with thoroughly minimized immunogenicity to direct potent and durable protein expression

Qiu¹,

Zhao²,

Hou³

et al. 2022

Preprint

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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 via HPLC, and presented thorough 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 high-purity final circRNA products. Collectively, Clean PIE is a novel circRNA platform that possesses high circularization effectivity, enabled high RNA purity and thorough 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.

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Section: Discussionmentioning

confidence: 99%

Clean-PIE: a novel strategy for efficiently constructing precise circRNA with thoroughly minimized immunogenicity to direct potent and durable protein expression

Qiu¹,

Zhao²,

Hou³

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Chemical ligation strategies for oligonucleotides have been developed for decades, and examples for circRNA preparation, such as 5′ or 3′ terminal functionalization with amines, thiols, azides, and others for cyclization [ 11 ], and representative reactions such as ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (EDC)- or cyanogen bromide (BrCN)-based reactions have been summarized well in previous reviews ( Figure 2 a,b). Furthermore, simple click chemistry such as azide-alkyne cycloadditions (ACC) is considered a powerful technique for the fast and efficient cyclization of nucleic acids [ 13 ] ( Figure 2 c).…”

Section: Chemical-based Methodsmentioning

confidence: 99%

“…Although in vivo circRNA synthesis is possible for achieving goals for various applications by delivering DNA for transcription and subsequent circRNA formation through back-splicing or other mechanisms [ 5 ], in vitro circRNA preparation methods would be more suitable for biomedical applications [ 4 ] requiring large-scale production [ 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

Pros and Cons of In Vitro Methods for Circular RNA Preparation

Lee¹,

Kim²,

Lee

2022

IJMS

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mRNA is gaining success as a new therapeutic agent and vaccine. However, mRNA has limitations in stability. To overcome the shortcomings of mRNA, circular RNA is emerging as a new modality. In this review, several current methods of manufacturing circular RNA in vitro are introduced and their advantages and disadvantages are reviewed. Furthermore, this study discusses which fields and directions of research and development are needed for the increase in the efficacy and productivity of circular RNA as a therapeutic agent and vaccine formulation.

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“…For example, compared with the liner mRNAs, the circRNAs are not sensitive to the exonuclease-mediated degradation due to the lack of 5′ or 3′ ends. Additionally, circRNAs are more stable and have a relatively longer half-life compared with linear mRNAs [ 82 , 83 , 84 ]. The circRNAs are less likely to induce undesired immunogenicity by escaping the detection from host Toll-like receptors (TLRs).…”

Section: Vaccine Approach To Prevent Infectionmentioning

confidence: 99%

Newly Emerged Antiviral Strategies for SARS-CoV-2: From Deciphering Viral Protein Structural Function to the Development of Vaccines, Antibodies, and Small Molecules

Zhang

Yang

2022

IJMS

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Coronavirus disease 2019 (COVID-19) caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become the most severe health crisis, causing extraordinary economic disruption worldwide. SARS-CoV-2 is a single-stranded RNA-enveloped virus. The process of viral replication and particle packaging is finished in host cells. Viral proteins, including both structural and nonstructural proteins, play important roles in the viral life cycle, which also provides the targets of treatment. Therefore, a better understanding of the structural function of virus proteins is crucial to speed up the development of vaccines and therapeutic strategies. Currently, the structure and function of proteins encoded by the SARS-CoV-2 genome are reviewed by several studies. However, most of them are based on the analysis of SARS-CoV-1 particles, lacking a systematic review update for SARS-CoV-2. Here, we specifically focus on the structure and function of proteins encoded by SARS-CoV-2. Viral proteins that contribute to COVID-19 infection and disease pathogenesis are reviewed according to the most recent research findings. The structure-function correlation of viral proteins provides a fundamental rationale for vaccine development and targeted therapy. Then, current antiviral vaccines are updated, such as inactive viral vaccines and protein-based vaccines and DNA, mRNA, and circular RNA vaccines. A summary of other therapeutic options is also reviewed, including monoclonal antibodies such as a cross-neutralizer antibody, a constructed cobinding antibody, a dual functional monoclonal antibody, an antibody cocktail, and an engineered bispecific antibody, as well as peptide-based inhibitors, chemical compounds, and clustered regularly interspaced short palindromic repeats (CRISPR) exploration. Overall, viral proteins and their functions provide the basis for targeted therapy and vaccine development.

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Circular RNA: Biosynthesis in vitro

Cited by 35 publications

References 92 publications

Clean-PIE: a novel strategy for efficiently constructing precise circRNA with thoroughly minimized immunogenicity to direct potent and durable protein expression

Clean-PIE: a novel strategy for efficiently constructing precise circRNA with thoroughly minimized immunogenicity to direct potent and durable protein expression

Pros and Cons of In Vitro Methods for Circular RNA Preparation

Newly Emerged Antiviral Strategies for SARS-CoV-2: From Deciphering Viral Protein Structural Function to the Development of Vaccines, Antibodies, and Small Molecules

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