Yang et al. established a novel type of circular mRNA called cmRNA. cmRNA contains an echovirus 29-derived internal ribosomal entry point element to promote ribosome binding and a newly designed spacer to enhance translation. Their findings suggest that this type of circular mRNA can mediate strong and persistent expression of various types of proteins. Furthermore, in addition to delivery via lipid nanoparticle (LNP), cmRNAs were discovered suitable for direct intratumoral administration. By giving mice cmRNAs encoding mixed cytokines directly intratumorally, they achieved successful modulation of intratumoral and systemic antitumor immune responses and enhanced anti-programmed cell death protein 1 (PD-1) antibody-induced tumor suppression.
Natural circular RNA is an emerging member of the RNA family that has gained importance in research due to its novel functional role in cell physiology and disease progression.1 Since RNA degradation starts at the tail end and there are no tail ends after cyclization, circular RNAs stay longer than linear ones.2 Various techniques for in vitro circularization of RNAs have been established after decades of research, including enzymatic ligation via T4 ligases, chemical strategies for the ligation of 5' and 3' ends, and a ribozyme technique that creates an intramolecular covalent connection. The greatest potential of circular mRNAs is its ability to meet therapeutic needs that mRNA cannot, which enables long-acting protein replacement or protein overexpression RNA therapies.3 In addition, circular mRNAs are more promising because they are less immunogenic and more readily available.4
In this work, the authors designed a novel cmRNA. It carries echovirus-derived internal ribosome entry site (IRES) elements and spacers screened by point mutations, resulting in sustained and efficient protein expression. Additionally, the authors found that high-performance liquid chromatography (HPLC) purification of cmRNA was necessary to eliminate immunogenicity and promote protein expression. Subsequently, with the help of LNP, sustained protein expression was obtained by administration of cmRNA in vivo and in vitro. To directly alter the intratumoral and systemic anticancer immune response, cmRNA encoding mixed cytokines was injected into tumors, increasing the tumor-suppressive effect of anti- PD-1 antibodies in a mouse model. Moreover, CaCl2 and KCl were found to be essential for the intratumoral delivery of cmRNA. By adjusting the dosage of both, the intratumoral delivery exhibited better anticancer effects and led to substantial infiltration and activation of CD4+ and CD8+ T cells.
One of the most exciting aspects of this manuscript is that naked cmRNA can be delivered directly into tumors and express the relevant proteins by intratumoral injection. In mice tumor models, 6 h after intratumoral injection of cmRNA with luciferase coding sequences, significant luminescence was detected in all transplanted tumors, indicating that cmRNA was delivered and expressed in tumor tissue. This finding implies that cmRNA can be taken up by cancer cells without vectors, eliminating the need for delivery vector development and concerns about material safety.
Nevertheless, cmRNA may not perform well in targeted delivery. Effective luciferase activity was found in the liver and the injection site 24 h after intramuscular injection, indicating that LNPs primarily transported cmRNA to the liver. The LNP technology, although proven safe and effective, is limited in its hepatic targeting,5 hindering the application of cmRNA drugs. Therefore, new delivery strategies are urgently demanded for extrahepatic targeting of cmRNA medicines.
Overall, this work demonstrates the future trends of RNA drugs. First, circular mRNA with better stability and immunogenicity may replace linear mRNA; Second, naked circular mRNA may be injected into tumor tissues through image guidance such as computed tomography (CT) to improve the ability of immune cells to attack cancer cells.
Acknowledgments
We are grateful to the National Natural Science Foundation of China (21574136) and the Youth Innovation Promotion Association Program of CAS (2015008).
Author contributions
J.Z. prepared the original draft, and J.C. reviewed and edited the manuscript.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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