Studies have discovered the immunogenic mechanism of in vitro synthesis of circular RNA and its potential applications

On December 23, Molecular Cell published online the latest research progress of circular RNA by Chen Lingling's research group at the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences.
The title of the paper is RNA circles with minimized immunogenicity as potent PKR inhibitors
.

By comparing the characteristics of different methods of in vitro synthesis of circular RNA, this study clarified the immunogenicity of in vitro synthesis of circular RNA and the molecular mechanism, and found that circular RNA with no immunogenicity and double-stranded structure can effectively inhibit natural immune factors The activation of PKR reveals the application potential of in vitro synthesized circular RNA for the treatment of autoimmune diseases related to abnormal activation of PKR
.

This work lays the foundation for further research on the function and mechanism of circular RNA in natural immunity, and provides a new way for the intervention and treatment of inflammatory autoimmune diseases using circular RNA
.

Most circular RNAs come from reverse splicing of exons to form a closed loop structure with covalent bonds
.

Studies have found that this type of molecule has a unique production and processing process, which is different from the folded conformation of linear RNA, and has high stability
.

These features make circular RNA have important functions in gene expression regulation, including participation in signal transduction, cell proliferation, innate immune regulation, etc.
(see the review Chen, Nat Rev Mol Cell Biol 2020 for details), and have unique application potentials, such as circular RNA can inhibit the abnormal phosphorylation of PKR through the double-stranded region (Liu et al.
, Cell 2019), and can be used as a translation vector for the expression of functional proteins (Wesselhoeft et al.
, Nat Commun 2018; Mol Cell 2019) and so on
.

Two important issues need to be paid attention to when studying the potential applications of circular RNA synthesized in vitro, one is how to improve the efficiency of synthesizing circular RNA in vitro, and the other is whether the synthesized circular RNA is immunogenic
.

On the one hand, some studies have pointed out that the circular RNA synthesized in vitro can be recognized by the innate immune receptor RIG-I, and then cause the innate immune response in the cell (Chen et al.
, Mol Cell 2017; Mol Cell 2019); on the other hand, there are also Researchers believe that the circular RNA synthesized in vitro is not immunogenic (Wesselhoeft et al.
, Mol Cell 2019), and pointed out that the intracellular immune response is caused by the contamination of a small amount of linear RNA in the circular RNA
.

In order to clarify the above problems, Chen Lingling's research group first compared the efficiency and immunogenicity of different methods of in vitro synthesis of circular RNA
.

At present, there are two main ways to synthesize circular RNA in vitro: direct intramolecular linking based on the catalysis of T4 RNA ligase, and self-splicing loop based on type I intron ribozyme
.

The experimental results show that the circular RNA synthesized by T4 RNA ligase, including circular RNA that can be used as a nucleic acid aptamer and translatable circular RNA, will not cause innate immune response in the cell, but through two different type I introns The circular RNA formed by self-splicing of T4 bacteriophage and Anabeana can cause the innate immune response in the cell
.

Further comparison and analysis of the sequence and secondary structure of circular RNA synthesized by different circularization methods found that the circular RNA synthesized based on T4 RNA ligase only introduced 1 to 3 exogenous nucleotides, but the two types of I contained The circular RNA formed by self-splicing in vitro will introduce 74 or 186 exogenous nucleotides, respectively
.

Researchers used the improved circSHAPE-MaP technology (Liu et al.
, Cell 2019) to further analyze the secondary structure of circular RNA synthesized by different pathways, and found that the introduction of these foreign sequences would change the folding conformation of the target circular RNA, such as with circular RNA The internal sequence matches, or the foreign sequence itself forms a stable double-stranded structure
.

Studies have shown that the exogenous sequences introduced by self-splicing into loops of type I introns may be the cause of the innate immune response in cells caused by the synthesis of circular RNA in vitro
.

The previous work of Chen Lingling's research group found that overexpression of endogenous circular RNA containing 16-26bp short double-stranded structure in PBMC or T cells derived from patients with systemic lupus erythematosus (SLE) can inhibit the abnormal activation of PKR and disease marker genes ( Liu et al.
, Cell 2019), suggesting that in vitro synthesis of non-immunogenic circular RNA containing a short double-stranded structure is expected to become a new method for the treatment of autoimmune diseases
.

The small molecule compounds that have been reported to inhibit abnormal phosphorylation activation of PKR, such as 2-Aminopurine (2-AP), oxindole/imidazole derivative compound 16 (C16), etc.
, have limitations
.

The study found that circular RNA synthesized by T4 RNA ligase not only has low immunogenicity, but also retains a short double-stranded structure of 16-26 bp similar to that in the body
.

Further experiments have shown that this type of circular RNA synthesized in vitro can still inhibit the activation of PKR in vitro and in cells, and has an inhibitory effect that is about 1,000 times better than that of small molecule inhibitors
.

In summary, this research lays a foundation for the further application of circular RNA synthesized in vitro, and brings promising prospects for the development of nucleic acid aptamers and gene therapy based on circular RNA technology
.

The research work is supported by the Chinese Academy of Sciences, the Ministry of Science and Technology, and the National Natural Science Foundation of China
.

The immunogenic mechanism and potential applications of circular RNA synthesized in vitro.
Source: Center for Excellence in Molecular and Cellular Science, Chinese Academy of Sciences