Academic Journals ∣ Characterization and Degradation Pattern of Circular RNA Vaccines

General Information

In July 2024, Institute of Biological Products, Division of Hepatitis and Enterovirus Vaccines, China National Institutes for Food?and Drug Control?and the biomedical R&D team from Vazyme Biotech Co., Ltd. Publish a paper entitled “Study on the Characterization and Degradation Pattern of?Circular RNA Vaccines Using an HPLC Method.[1]”>> https://www.mdpi.com/2227-9040/12/7/120

Background

CircRNAs are covalently closed, single-stranded RNA formed by back-splicing of precursor mRNAs. Compared with linear mRNA, circRNA molecules possess a closed-loop structure that protects them from exonuclease-mediated degradation and thus confers high stability. The existence of differences in production and structure between circRNAs and linear mRNAs suggests that circRNAs may possess unique cellular functions and potential for biological applications.

CircRNAs have recently attracted widespread attention due to their key roles in gene delivery, protein production, and vaccine development. In particular, circRNA vaccines have demonstrated immense potential for applications in infectious disease prevention and cancer treatment. Due to the importance of ensuring the safety and effectiveness of circRNA vaccines, quality control of such vaccines has become a popular topic in research on circRNAs. Researchers have developed various physicochemical analytical methods to characterize the in vitro transcription (IVT) products of circRNAs. For instance, capillary electrophoresis (CE) and gel electrophoresis are convenient and widely used methods for analyzing circRNAs. Certain researchers have attempted to develop HPLC based analytical methods, but these methods have yet to achieve successful identification and separation of circRNAs and nicked circRNAs [2].

Overview

The study investigates the degradation patterns of circRNAs under thermal acceleration conditions and performed biological analysis of degradation products and linear precursors. Here, RP-HPLC method effectively identified circRNAs and nicked circRNAs. With thermal acceleration, circRNAs exhibited a “circular→nicked circRNAs→degradation products”degradation pattern. Biological analysis revealed that the immunogenicity of degradation products significantly decreased, whereas linear precursors did not possess immunogenicity. Thus, our established RP-HPLC method can be used for purity analysis of circRNA vaccines, which contributesto the quality control of circRNA vaccines and promoting the development of circRNA technology.

Study Approach and outcomes

Establishment of the RP-HPLC-Based circRNA Vaccine Purity Analytical Method

Establishment of the RP-HPLC-Based circRNA Vaccine Purity Analytical Method

Based on in vitro transcribed principles of circRNAs, we synthesized EGFP-encoding circRNAs, nicked circRNAs, and linear precursors by adopting the group I intron selfsplicing method (Figure 1A) [3]. CE could detect both the circRNAs and linear precursors but was unable to achieve baseline separation or distinguish between circRNAs and nicked circRNAs (Figure 1B). To achieve the identification of circRNAs and nicked circRNAs, chromatographic column screening and mobile phase were optimized to develop an RP-HPLC method capable of effectively identifying circRNAs, nicked circRNAs, and linear precursors. The results indicated that baseline separation was achieved (Figure 1C). In summary, the RP-HPLC method could sensitively distinguish circRNAs, nicked circRNAs, and linear precursors, while SEP-HPLC could detect the aggregate structures of circRNAs. These two HPLC methods enable considerable enhancement of circRNA testing and can be utilized for quality control analysis of circRNA vaccines.

Degradation Patterns of the circRNA Substance

Although the closed-loop structure of circRNAs makes them more stable than linear mRNAs, the degradation patterns of circRNAs remain unclear. The established RP-HPLC method described earlier demonstrated sensitive detection of circRNAs and nicked circRNAs, thereby providing technical support for exploring the degradation patterns of circRNAs under conditions of thermal acceleration. The RP-HPLC results indicated that under the thermal acceleration treatment conditions, the circRNAs exhibited a “circular→nicked→degradation products” degradation pattern (Figure 2A–C). SEC-HPLC results indicated that circRNAs exhibited degradation of circular RNAs and aggregates accompanied by formation of degradation products. Results of CE could only indicate circRNA degradation and the formation of degradation products in the circRNA samples (Figure 2D).

Expression Activities of Degradation Products of circRNAs

As described in the previous section, the circRNAs exhibited a “circular→nicked→degradation products” degradation pattern. However, the biological activities of degradation products of circRNAs remain unclear. To investigate the expression activities of the degradation products, the circRNAs, nicked circRNAs, aggregates, degradation products, and linear precursors were purified and collected. The expression activities of circRNAs and their degradation products were evaluated at the cellular level by flow cytometry and confocal fluorescence microscopy. The in vitro experiment demonstrated that the nicked circRNAs and aggregates exhibited a significant decrease in EGFP expression activity levels compared with circRNAs, while the degradation products and linear precursors did not induce antigen expression.

In Vivo Immunogenicity and Safety of Degradation Products of circRNA Vaccines

LNPs were used for the encapsulation of unpurified circRNA obtained from IVT, purified circRNAs, nicked circRNAs, aggregates, degradation products, and linear precursors. The encapsulation efficiencies of the various groups exceeded 95% and the average particle size was around 80 nm (Figure 4A). Figure 4B shows the detailed procedure of the animal experiment. The results of inflammatory cytokines and anti-EGFP IgG antibody detection collectively suggest that circRNAs, degradation products, and linear precursors induced strong inflammatory responses. However, the induced specific anti-EGFP lgG antibody levels differed significantly among different groups.

Activated Pattern Recognition Receptors of Degradation Products

Compared with the high-intensity fluorescence signals stimulated by wild-type 293 cells, the fluorescence level induced by circRNAs was significantly reduced after retinoic acid-inducible gene I (RIG-I) knockout (KO). The intensities of fluorescence stimulated by the circRNA in Toll-like receptors (TLR) 3/8 KO cells, stimulated by aggregates and degradation products in TLR7/8 KO cells, and stimulated by linear precursors in TLR3 KO cells were also significantly lower (Figure 5A). These data indicate that the circRNAs could activate RIG-I, nicked circRNAs could activate TLR3 and TLR8, the degradation products and aggregates could activate TLR7 and TLR8, and the linear precursors could activate TLR3, thereby inducing an immune response.

Conclusion

This study focused on the identification and characterization of circRNA vaccines and the investigation of their degradation patterns. IVT products of circRNAs not only consist of target circRNA but may also include nicked circRNAs, aggregates, and residual linear precursors. Commonly used analytical methods are incapable of effectively distinguishing between circRNAs and nicked circRNAs. The RP-HPLC method established and validated in this study demonstrated good discrimination and baseline separation of circRNAs and nicked circRNAs. This study has provided data and support for purity testing and quality control of linear precursors and degradation products during the development of circRNA vaccines. However, these experimental findings were obtained from EGFP-encoding circRNAs and warrant further validation in marketed circRNA vaccines. Further research is also required to investigate the degradation patterns of circRNA molecules in circRNA vaccine products.

About Vazyme-mRNA

1.mRNA synthetase Enzyme materials：Lot-to-Lot Homogeneity、GMP-grade

2.T7 enzyme mutant series：4 relevant patents、High fidelity mRNA、Comprehensively enhance mRNA

3.mRNA QC Reagents ：Complete methodological validation、Competitive Antibodies

4.mRNA-LNP:Well-established and accurate detection methods

5.CircRNA:Well-established and accurate detection methods

Reference

[1] Feiran Cheng, Ji Li, Chaoying Hu, Yu Bai, Jianyang Liu, Dong Liu, Qian He, Qiuheng Jin, Qunying Mao, Zhenglun Liang and Miao Xu. Study on the Characterization and Degradation Pattern of Circular RNA Vaccines Using an HPLC Method. Chemosensors 2024, 12, 120.

[2] Lee, K.H.; Kim, S.; Song, J.; Han, S.R.; Kim, J.H.; Lee, S.W. Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme. Mol. Ther. Nucleic Acids 2023, 33, 587–598.

[3] Nilsen, T.W.; Graveley, B.R. Expansion of the eukaryotic proteome by alternative splicing. Nature 2010, 463, 457–463.