Intratumoral Delivered Novel Circular mRNA Encoding Cytokines for Immune Modulation and Cancer Therapy

Abstract

The application of mRNA as a novel kind of vaccine has been proved recently, due to the emergence use authorization (EUA) by FDA for the two COVID-19 mRNA vaccines developed by Moderna and BioNTech. Both of the two vaccines are based on canonical linear mRNA, and encapsulated by lipid nanoparticle (LNP). Circular mRNA, which is found to mediate potent and durable protein expression, is an emerging technology recently. Owing to its simplicity of manufacturing and superior performance of protein expression, circular mRNA is believed to be a disruptor for mRNA area. However, the application of circular mRNA is still at an initiation stage, proof of concept for its usage as future medicine or vaccine is necessary. In the current study, we established a novel kind of circular mRNA, termed C-RNA, based on Echovirus 29 (E29)-derived internal ribosome entry sites (IRES) and newly designed homology arms and RNA spacers. Our results demonstrated that this kind of circular mRNA is able to mediate strong and durable protein expression, compared to typical linear mRNA. Moreover, for the first time, our study demonstrated that direct intratumoral administration of C-RNA encoding a mixture of cytokines achieved successful modulation of intratumoral and systematic anti-tumor immune responses and finally leading to an enhancement of anti-PD-1 antibody-induced tumor repression in syngeneic mouse model. Additionally, after an optimization of the circular mRNA formulation, a significant improvement of C-RNA mediated protein expression was observed. With this optimized formulation, C-RNA induced enhanced anti-tumor effect via intratumoral administration and elicited significant activation of tumor-infiltrated total T cells and CD8+ T cells. Collectively, we established C-RNA, a novel circular mRNA platform, and demonstrated that it can be applied for direct intratumoral administration for cancer therapy.

Circular mRNA encoded PROTAC (RiboPROTAC) as a new platform for the degradation of intracellular therapeutic targets

“…In our previous studies, we proved that cmRNA can mediated strong and durable protein expression both in vitro and in vivo. Moreover, our studies demonstrated that cmRNA is sufficient for the expression of various types of proteins (9). Therefore, we prospect that cmRNA is probably an excellent vector to deliver and express BioPROTAC molecules intracellularly.…”

“…Since mRNA delivery has been studied extensively, and undergone well-developed (18), we consider that efficient in vivo delivery of cmRNA based RiboPROTAC is highly promising. We have previously shown that direct intratumoral injection of naked cmRNA resulted in durable protein expression in tumor tissues (9). Hence, we used this mouse model to verify RiboPROTAC’s application in vivo.…”

“…Therefore, we considered that, compared to protein-based BioPROTAC, RiboPROTAC owns advantages for in vivo therapeutic applications. Our previous study demonstrated that intratumoral administration of naked cmRNA leads to durable protein expression in syngeneic mouse tumor model (9), in the current study, we evaluated the in vivo delivery and function of RiboPROTAC with the same method. Our results revealed that GFP RiboPROTAC decreased intratumoral GFP protein level, suggesting that intratumoral delivery of naked cmRNA of GFP RiboPROTAC is successful, and targeted GFP degradation occurred efficiently.…”

“…We proposed that BioPROTAC proteins could be encoded and delivered intracellularly by mRNA whose intracellular delivery has been extensively studied and applied in clinical market successfully. In our previous studies, we applied our circular mRNA, termed cmRNA, to express various kinds of proteins with high and durable protein expression, and successfully used in vivo as anti-tumor therapeutic medicine in preclinical mouse models (9). Here, we demonstrated that cmRNA-encoded BioPROTAC, which we termed RiboPROTAC, is a new platform for intracellular target protein degradation, and for the first time, we successfully applied it in vivo as potential anti-tumor therapeutic by degrading the tumor target PCNA.…”

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

“…According to the report from Weesselhoeft et al, the translation initiation sequence, or exactly IRES of Coxsackievirus B3 (CVB3) was identified as the most potent IRES for the direction of circRNA mediated protein expression. In our previous reports, we identified the IRES of Echovirus 29 (E29) and E33 as stronger element to induce protein expression 24 . In the current study, we broadened the screening of IRES in varying strains of virus and attempted to identify better IRES for Clean PIE circRNA.…”