Clean-PIE surpasses ORNA: Curemed unveils the underlying technology of circular RNA looping framework

Currently, there are three main strategies for in vitro RNA cyclization: chemical methods, ligase methods (T4 RNA ligases), and ribozyme methods (group I, group II self-splicing introns). The ligase and ribozyme methods are the most commonly used.

一、I型内含子自剪接

The research team led by Daniel G. Anderson2 at MIT achieved the cyclization of long-coding RNA in vitro by engineering the type I intron of Anabaena. In the presence of magnesium ions and GTP, two ester exchange reactions occurred, forming a cyclic RNA. By optimizing the classic Anabaena PIE and adding homology arms and spacers, the challenge of cyclizing long-coding RNA sequences was addressed, and good cyclization efficiency was achieved. On the other hand, the stability and efficiency of circRNA protein expression in cells were enhanced through HPLC purification strategies and IRES sequence screening. Due to the addition of spacers and longer exon sequences, this cyclization strategy requires the introduction of an additional 186nt base sequence into the final cyclic RNA product.

In March 2022, Professor Wei Wensheng of Peking University successfully prepared a COVID-19 RBD vaccine using the ORNA cyclization framework and achieved excellent protective efficacy against COVID-19 in rhesus monkeys3

In early 2022, Professor Chen Lingling published an article in Molecular Cell, pointing out that circRNA generated by reverse self-splicing of Anabaena PIE or Thymidylate synthase (TD) PIE and T4 ligase method exhibits significantly higher immunogenicity, and demonstrated that this immunogenicity is caused by the additional sequences introduced by Ana PIE or td PIE during the cyclization process. As shown in the figure below:

二、II型内含子自剪接

Group II introns are present in the genomes of protists, fungi, and bacteria. In vivo, group II introns can undergo self-splicing from precursor RNA through two consecutive trans-esterification reactions, connecting the two flanking exons. Many splicing reactions of group II introns are assisted by proteins. Meanwhile, it has been demonstrated that group II introns can also undergo splicing reactions through autocatalysis in vitro.

As shown in the figure below, by utilizing the self-catalytic splicing reaction of group II introns from Clostridium tetani, introducing short exogenous sequences IBS1 and IBS3, or altering the RNA target by controlling the EBS sequence that pairs with the exon during intron insertion, this modified type II intron is termed a "targetron". The Clostridium tetani type II intron and yeast type II intron splicing systems designed in this way enable the artificial in vitro preparation of circRNA without residual exogenous sequences5. As depicted in the figure below, the efficiency of cyclization needs to be improved, and the nicked RNA contained in the cyclization band requires further identification.

三、T4 RNA ligase

The method of T4 DNA/RNA ligase-mediated cyclization in vitro can avoid immunogenicity caused by the introduction of exogenous sequences as mentioned above. However, the relatively low ligation efficiency of T4 ligase and the need for DNA splint sequence assistance in the cyclization method make it difficult to achieve industrial preparation; this method has also developed the use of endogenous splint strands to achieve RNA cyclization3,6. As shown in the figure below:

Secondly, T7 RNA polymerase randomly introduces 1-3 additional guanine (G) nucleotides at the 3' end during transcription,1 making the sequence obtained when applying the T4 RNA ligase method to RNA cyclization in IVT (in vitro transcription) inaccurate;

Thirdly, T4 RNA ligase catalyzes the formation of phosphodiester bonds between and within single-stranded RNA molecules at the 5'-phosphate group and 3'-hydroxyl terminus. Prior to in vitro transcription, the 5’ end of the RNA strand needs to be dephosphorylated, which is a cumbersome operation with low cyclization efficiency. As shown in the figure below:

四、Clean-PIE group I intron自剪接系统

The immunogenicity brought by the introduction of 186nt exogenous sequence through the ORNA group I intron Ana PIE cyclization strategy, the relatively low splicing efficiency of group II introns in Clostridium tetani and yeast in vitro, as well as the sequence inaccuracy and cumbersome operation of the T4 RNA ligase cyclization method, are all barriers restricting the clinical application of circular RNA.

On June 21, 2022, an article published by CureMed on the preprint platform bioRvix unveiled a novel strategy for circular RNA formation known as Clean-PIE. This ingenious study identified the optimal site for circularization by screening protein-coding regions or IRES regions, achieving RNA circularization without the introduction of exogenous sequences by connecting these regions.

Meanwhile, the study also established an automated screening and optimization system for splice sites. By conducting efficient calculations for different sequences, the optimal looping sites were selected. Through evaluating all genes larger than 500bp in Escherichia coli or the human genome, it was found that the optimized system could screen out looping sites with scores above 13 (i.e., differing from the optimal looping sequence by only one base) in 99.9% of H. sapiens genes and 100% of E. coli genes. This proves the universality of this looping strategy.

Meanwhile, the study optimized the sequence composition of circular RNA, enhancing its expression by incorporating polyAC sequences.

The advantage of the Clean-PIE system lies in the absence of exogenous sequence introduction and the precision of the cyclization sequence. The research data presented in this article indicate that, compared to the ORNA cyclization strategy, cells transfected with Clean-PIE circular RNA exhibit lower expression of IFN-β, RIG-1, IL-6, and MDA-5, and the protein expression at the cellular level in vitro is also significantly superior to that of ORNA.

After purification via HPLC-SEC, both Ana PIE and Clean-PIE can achieve sustained protein expression in animals for up to 20 days. The duration of expression in vivo is significantly longer than that of linear mRNA.

In addition, the study further conducted a scaling-up experiment in a 1L reaction system. Capillary electrophoresis results showed a high cyclization efficiency of 96.4%, and agarose gel electrophoresis results also verified the high cyclization efficiency. To further verify the production of nicked RNA during the cyclization process, the impurity residues of precursor and nicked RNA in the purified final product were evaluated. The purified circular RNA was detected by Urea PAGE method, and the results showed that the proportion of circular RNA in the final product exceeded 90%. This indicates that the Clean-PIE system is suitable for industrial mass production and can ensure high cyclization efficiency and relatively pure final products. This lays a foundation for further clinical applications.

In addition, to achieve efficient and tissue-specific expression of circRNA-derived proteins, this study established an efficient IRES screening platform. Over 600 IRES sequences have been screened, among which more than 20 IRES elements exhibited significantly higher expression levels compared to CVB3. Preliminary data on tissue-specific expression of IRES have been obtained.

Since November 2021, Createmed has successively published articles on intratumoral injection of cytokines 7, PROTAC technology based on circular mRNA 8, and a new LNP nucleic acid delivery technology platform 9, demonstrating the comprehensive maturity of the LNP delivery system and circular mRNA platform technology, and rapid progress in project applications.

It is reported that the upcoming IIT research on the FIRST IN CLASS intratumoral injection project of circular RNA is expected to be the first to achieve the world's first circular RNA FIRST IN HUMAN.

The COVID-19 pandemic has accelerated the development of mRNA vaccines. As a platform technology, mRNA is not limited to vaccines and will see increasing applications in tumor immunotherapy, protein replacement, gene therapy, and cell therapy. The circular mRNA technology, as the mRNA2.0 version, has garnered widespread attention due to its advantages such as no need for capping and tailing, no nucleotide modification, greater stability both in vitro and in vivo, higher and longer-lasting protein expression, and has sparked a boom in circular mRNA research.

Internationally, ORNA Therapeutics and Laronde stand as representatives, with the latter notably securing a $440 million Series B funding from Flagship, the incubator of Moderna. ORNA also recently unveiled its latest research advancements at the annual meeting of the American Society of Gene and Cell Therapy (ASGCT), continuing to deeply develop underlying patented technologies such as IRES and LNP.

In China, represented by the Clean-PIE group I intron self-splicing system from CureMed, the group II intron self-splicing system from Huanshi Biotech, the Ana PIE from Yuanyin Biotech, and the T4 RNA ligase cyclization method from Jiesai Biotech, a research and development layout with distinct technical directions has been formed.

It is anticipated that in the near future, there will be an increasing number of circular mRNA development pipelines to meet the huge clinical treatment demand, bringing hope of cure to patients.

[References]

1. Zonghao Qiu, Y. Z. et al. Clean-PIE: a novel strategy for efficiently constructing precise circRNA with thoroughly minimized immunogenicity to direct potent and durable protein expression..

2. Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018).

3. Qu, L. et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell. 185, 1728-1744 (2022).

4. Liu, C. X. et al. RNA circles with minimized immunogenicity as potent PKR inhibitors. Mol. Cell. 82, 420-434 (2022).

5. Chuyun Chen, H. W. Y. Y. A flexible, efficient, and scalable platform to produce circular RNAs as new therapeutics..

6. 梁兴国，陈辉，安然，程凯. 一种制备环状RNA的方法..

7. Jiali Yang, J. S., Jiafeng Zhu, Y. D., Yiling Tan, L. W., Qiangbo Hou, Y. Z. Z. S. & Chijian Zuo. Intratumoral Delivered Novel Circular mRNA Encoding Cytokines for Immune Modulation and Cancer Therapy..

8. Jiali Yang, J. S., Jiafeng Zhu, Y. D., Yiling Tan, L. W., Qiangbo Hou, Y. Z. Z. S. & Chijian Zuo. Circular mRNA encoded PROTAC (RiboPROTAC) as a new platform for the degradation of intracellular therapeutic targets..

9. Ke Huang, N. L. Y. L., Yuping Liu, Q. H. S. G., Ke Wei, C. D. C. Z. & Zhenhua Sun. Delivery of Circular mRNA via Degradable Lipid Nanoparticles against SARS-CoV-2 Delta Variant..