key: cord-0911199-h10081le
authors: Yang, Jiali; Zhu, Jiafeng; Chen, Yiyun; Du, Yaran; Tan, Yiling; Wu, Linpeng; Sun, Jiaojiao; Zhai, Mengting; Wei, Lixiang; Li, Na; Huang, Ke; Hou, Qiangbo; Tong, Zhenbo; Bechthold, Andreas; Sun, Zhenhua; Zuo, Chijian
title: Intratumoral Delivered Novel Circular mRNA Encoding Cytokines for Immune Modulation and Cancer Therapy
date: 2021-11-04
journal: bioRxiv
DOI: 10.1101/2021.11.01.466725
sha: 31e07262d258655e14a456d4196c5109d8831762
doc_id: 911199
cord_uid: h10081le

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.

The To the best of our knowledge, most of the mRNA pipelines that being evaluated in clinical trials all over the world are based on typical linear mRNA, which can be generated by RNA polymerase-mediated in vitro transcription (IVT). Linear mRNA consists of a Cap structure, 5' and 3' untranslational regions (UTRs), protein encoding region and a polyA tail 5 . The 5' cap0 or 5' cap1 structure is generated via enzymatic reaction after IVT procedure or co-transcriptional incorporation of cap analog during IVT procedure 6, 7 . This Capping reaction that replaces the pre-existing 5'-PPP structure reduces its immunogenicity and mediates strong promotion of mRNA translational activity by means of the tight interaction of 5' Cap structure and elongation initiation factors 8 . PolyA tail that generated by polyA polymerase after IVT step, with a length about 200 to 250 bp, facilitates mRNA stability and finally boosts protein production 9 . 5' and 3' UTRs can be engineered to promote mRNA stability and protein expression 10 . To minimize the immunogenicity of mRNA, modified nucleosides can be incorporated into mRNA strand in the step of IVT 11 . The complicated structure of linear mRNA determines the complexity of its mRNA manufacturing and maybe a relative low yield after a few steps of synthesis and purifications.

In addition to linear mRNA, there is an emerging research area focusing on circular mRNA, which is proven to mediate potent and durable protein expression in vitro 12 .

Despite the fact that most of the circular RNAs found in eukaryotic cells so far are considered as non-coding RNAs 13 , some circular RNAs are demonstrated to encode and express proteins 14 . Most of the circular RNAs that have been identified in eukaryotes are derived from in vivo back splicing mechanism 15 . After decades of investigations, methods for in vitro circularization of RNAs have been developed. In summary, circular RNAs can be generated in vitro through different methods 16 , including enzymatically ligation via T4 ligases, chemical strategies for the ligation of 5' end and 3' end, and finally a ribozyme strategy, by which an intra-molecular covalent bond is formed and circular RNAs is generated.

In 1992, M.Puttaraju and Michael D.Been reported that based on Anabaena group I intron, they invented a Group I permuted intron-exon (PIE) system to conduct an ribozyme catalyzed in vitro self-splicing and finally generate an exon-exon covalent -ligated circular RNA with high efficiency. In this case, an insertion of foreign nucleic acid sequences into the sites between exons and introns can generate a circular RNA that containing a foreign RNA fragment 17 (CVB3) as a high efficient one for cap-independent translation. Importantly, they found that this kind of CVB3 IRES-driven circular mRNA conducts stronger and more durable protein expression in vitro, compared to typical linear mRNA, with or without nucleoside modifications 12 . Their further studies indicated that circular mRNA after HPLC-purifications exhibited very low mRNA immunogenicity, suggesting that purified circular mRNA is probably suitable for applications in vivo as vaccines or therapeutic medicines 18 . However, more evidence is required to prove that circular mRNA is a type of proper molecule for clinical usage, especially in vivo experiments and clinical trials.

In the current study, we established a novel kind of circular mRNA, termed "C-RNA", based on Echovirus 29 derived IRES and newly designed homology arms as well as RNA spacers. Compared to typical linear mRNA, C-RNA can mediate strong and durable protein expression. Moreover, for the first time, our study demonstrated that circular mRNA encoding a mixture of cytokines can be directly used for intratumoral administration to modulate intratumoral and systematic anti-tumor immune responses, finally lead to an enhancement of anti-PD-1 antibody induced tumor repression in syngeneic mouse models. Additionally, after an optimization of the circular mRNA formulation, a significant improvement of C-RNA-mediated protein expression in vivo was observed. With this optimized formulation, C-RNA induced better anti-tumor effect via intratumoral administration, and elicited significant activation of tumor-infiltrated CD4 + 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.

Gene cloning and vector construction DNA fragments that containing PIE elements, IRES, coding regions and others were chemically synthesized and cloned into a restriction digestion linearized pUC57 plasmid vector. The vector for linear mRNA containing beta-globin 5' UTR and tandem beta-globin 3' UTR, and coding regions was chemically synthesized and cloned into a restriction digestion linearized pUC57 plasmid. DNA synthesis and gene cloning were customized ordered from Suzhou Genwitz Co.Ltd. (Suzhou, China). circRNA preparations circRNA precursors were synthesized by invitro transcription from a linearized plasmid DNA template using a Purescribe TM T7 High Yield RNA Synthesis Kit (CureMed, Suzhou, China). After in vitro transcription, reactions were treated with DNase I (CureMed, Suzhou, China) for 15 min. After DNaseI treatment, unmodified linear mRNA was column purified using a GeneJET RNA Purification Kit (Thermo Fisher). For circRNA: RNA was purified, after which GTP was added to a final concentration of 2 mM along with a buffer including magnesium (50 mM Tris-HCl, (pH 8.0), 10 mM MgCl 2 , 1 mM DTT; Thermo Fisher). RNA was then heated to 55 °C for 15 min, and then column purified. RNA was separated on 1% agarose gels using the agarose gel systems (Bio-Rad); ssRNA Ladder (Thermo Fisher) was used as a standard.

For high-performance liquid chromatography (HPLC), RNA was run through a 30 × 300 mm size exclusion column with particle size of 5 µm and pore size of 1000 (Sepax Technologies, Suzhou, China) on an SCG protein purification system (Sepure instruments, Suzhou, China). RNA was run in RNase-free Phosphate buffer (pH:6) at a flow rate of 15 mL/minute. RNA was detected and collected by UV absorbance at 260 nm. Concentrate the purified circRNA in an ultrafiltration tube, and then replace the phosphate buffer with an RNase-free water.

The plasmid vector was digested by XbaI for linearization, and transcription was carried out using HyperScribe™ All in One mRNA Synthesis Kit (APExBio), with Cap1 analog incorporation and N1-methyl-pseudouridine as modified nucleoside in transcription. RNA was purified by using a GeneJET RNA Purification Kit (Thermo Fisher).

HEK293T and the murine colon adenocarcinoma cell line MC38 as well as the murine melanoma cell line B16F10 were purchased from Cobier Biosciences (Nanjing, China) and cultured in Dulbecco's modified Eagle's medium (DMEM, BI) supplemented with 10% fetal calf serum (BI) and penicillin/streptomycin antibiotics (100 U/ml penicillin, 100μg/ml streptomycin; Gibco). The human non-small cell lung cancer cell line A549 and NCI-H358 were purchased from Cobier Biosciences (Nanjing， China) and cultured in Dulbecco's modified Eagle's medium (DMEM, BI) and RPMI 1640(BI)respectively supplemented with 10% fetal calf serum (BI) and penicillin/streptomycin antibiotics (100 U/ml penicillin, 100μg/ml streptomycin; Gibco). All cells were maintained at 37 °C, 5% CO 2 , and 90% relative humidity.

For the circRNA transfection in HEK293T, 1×10 5 cells per well were seeded in 24-well plates or 6-well plates. For 24-well plates, 500 ng circRNAs, or for 6-well 

Circular mRNA-LNP complex was generated through microfluidic devices (Micro&Nano Technologies, Shanghai, China). Purified circular mRNA was dissolved in citric acid buffer and lipids were dissolved in ethanol. The liquid flow rate was set as 12 mL/min, mRNA/lipids (v/v) was set as 3:1. Circular mRNA-LNP complex was purified by filtration.

C57BL/6 female mice and CD-1 nude female mice were purchased from Charles River (Beijing, China) at the age of 6-8 weeks and housed in specific-pathogen-free facilities and all experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC).

For tumor implantation, mice were injected with A549, NCI-H358, B16F10 or MC38 (1×106 cells per animal) s.c. in the right flank. Tumor growth was monitored by 2 perpendicular diameters with a digital caliper every 2 to 4 days and tumor volume was calculated by the modified ellipsoidal formula: V = ½ (Length × Width 2 ). Intratumoral injections were performed with 1 mL 29G×1/2 insulin syringe (BD).

Mice were anesthetized in a chamber with 2.5% isoflurane and intra-peritoneal injections of D-luciferin (150mg/kg, Promega). Bioluminescence was measured by an IVIS Spectrum imaging system (PerkinElmer) while maintaining 2.5% isoflurane in the imaging chamber via a nose cone. Images were captured 10 minutes after luciferin administration at indicated time points. The photon flux values(photons/second), corresponding to the ROI (region of interest) marked around the bioluminescence signal, were analyzed using Living IMAGE software.

Tumors were isolated and dissected into around 1 mm3 pieces. Then these pieces were incubated in DMEM medium containing 1mg/mL collagenase-IV (Thermo 

Peripheral blood samples (buffy coats) from healthy volunteers were isolated by Ficoll Hypaque gradient separation (Ficoll-Paque-Plus) and washed 3 times with PBS supplemented with 1 mM EDTA. Freshly isolated PBMC seeded at 96-well plate with the density of 500000/well and treated with cytokine mixture which were collected from different mRNA transfected 293T supernatant for 24 hours. And then the IFN-gamma production was determined by Human IFN-gamma ELISA kit (Neobioscience).

Two-tailed Student t test was used to determine statistical significance for data comparisons at a single time point. Two-way ANOVA was used to determine statistical significance for data comparisons with multiple time points. Mantel-Cox log rank test was performed to determine statistical significance for the comparison of survival curves. Prism version 8.0 (GraphPad) was used for generation of all graphs and performance of statistical analyses. Statistical significance shown for survival curves represents a comparison of the 2 survival curves. Statistical significance is denoted as ns, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.

Due to the fact that CVB3 IRES derived circular mRNA presents high activity for protein translation, we sought to investigate alternative virus-origin IRES that can mediate high and durable protein expression in mammalian cells. In our study, the circular mRNA was generated by a vector the consists of a T7 promoter, PIE system, IRES and novel RNA spacers. After a transcription reaction mediated by T7 RNA polymerase, the RNA precursor was produced. Next, circular mRNA was generated after a further circularization procedure. As shown in Fig.1A consists of E29 IRES, the protein coding region, spacers and Exon2-Exon1 junction that derived from PIE system (Fig.1G) . Besides, we evaluated the expression of C-RNA with or without HPLC-grade purification in A549 cell line 24 h after C-RNA transfection. We found that the purified C-RNA mediated remarkable higher protein expression in A549, compared to the unpurified C-RNA ( Fig.1E and 1F ). The promotion of protein expression may cause by the removal of immunogenic 5'-ppp small intron fragments that generated after circularization reaction. These results suggested that purification by HPLC is a critical step with regards to C-RNA, no matter for elimination of immunogenicity or for promotion of protein expression.

Next, we sought to investigate whether C-RNA is able to direct the expression of different types of proteins in vitro or in vivo. To delineate the localization of in vivo protein expression, C-RNA encoding luciferase encapsulated by lipid nanoparticle (LNP) was delivered to mice, and the expression of luciferase bioluminescence was detected by optical in vivo imaging system. As shown in Fig and 5B). According to the study from J Probst 20 

Ringer's solution, such as CaCl 2 , NaCl, KCl and NaHCO 3 may be vital for mRNA delivery. Thus, removal of the four compositions above in C-RNA solution was performed respectively, and again intratumoral injection to B16F10 tumors. We found that the removal of CaCl 2 and KCl greatly reduced the bioluminescence level of C-RNA injection, and the removal of NaCl and NaHCO 3 led to moderate and mild reduction, respectively ( Fig.5A and 5B) . These results suggest that CaCl 2 and KCl are the two essential components for C-RNA intratumoral delivery. transfecting C-RNAs that encoding four cytokines into 293T cells, the expression of secreted cytokines was quantified by ELISA ( Fig.6A ). High expression of these cytokines was found 24 h after transfection (Fig.6B) . Furthermore, the cell supernatant of each transfection was collected and transferred to PBMC to assess their activities. We found that the incubation with each secreted cytokine resulted in remarkable up-regulation of CD69 positive CD4 + and CD8 + T cells in PBMC presented better control of tumor growth, suggesting that a coordination between cytokine mixture and anti-PD-1 antibody exists (Fig.7C) . Importantly, in the 50-day survival tests, a notable improvement of final survival rate was found after C-RNA mixture monotherapy of combination treatment, featuring with 3/8 complete response (CR) rate, compared to negative control or antibody monotherapy, as shown in Fig.7D .

In summary, these results suggest that cytokines-encoding C-RNA mixture exhibits supreme anti-tumor effect, especially combined with checkpoint blockade agents for tumor therapy.

We proposed that cytokine C-RNA mixture modulates tumor microenvironment, especially the activation of T cells for tumor suppression. To delineate the cellular mechanism for cytokine C-RNA mixture mediated tumor growth suppression, tumor-infiltrated T cells were analyzed by flow cytometry after two doses of combination therapy (Fig.8A) . Firstly, the percentage of infiltrated CD45 + cells were analyzed, and results indicated that the treatment with C-RNA mixture elicited promotion of percentage of CD45 + cells; importantly, the combination treatment with C-RNA mixture and anti-PD-1 antibody led to a potent elevated percentage of CD45 + cells (Fig.8B) , suggesting that C-RNA coordinated with anti-PD-1 antibody to facilitate the infiltration of total leukocytes into tumor. We also assessed the percentage of tumor-infiltrated T cells, as shown in Fig.8C respectively. Collectively, not only there were more immune cells especially T cells recruited to the tumor, but also their activation degrees were higher, thus exerting stronger anti-tumor effects.

The method with PIE system of generating circular RNA in vitro has been developed as early as 1990s 17 , however, the application for protein expression by in vitro-generated circular RNA hasn't been realized until recently. This is probably due to the fact that the notion of circular RNA mediated protein expression has just been However, more in vitro and in vivo evidence is required to prove this concept that circular mRNA can be applied to express varies types of proteins, and probably clinical studies are required to prove its potentiation as vaccines and therapeutics. In the current study, we developed a novel format of circular mRNA, termed C-RNA, which can be generated by PIE system and uses E29 IRES to direct protein translation.

Our experimental results demonstrated that C-RNA mediated high activity of protein translation, as revealed by the high expression of EGFP. Importantly, we found that HPLC-grade RNA purifications resulted in notably higher protein expression in immunogenic sensitive A549 cell line. Due to the fact that immunogenic RNA triggers innate immunity, which may suppress protein expression and caused inflammation of human body 21 , therefore, the purifications of C-RNA by HPLC is crucial for the future development of RNA vaccines and therapeutics. Next, we were up-regulated, suggesting that the tumor microenvironment was actually remodeled by the locally expressed cytokines, and facilitated anti-PD-1 mediated immune therapy. The "cold" B16F10 tumor was turned to be "hot" after intratumoral injection of C-RNA that encoding cytokines. These results are consistent with the outcomes from previous studies of intratumoral mRNA for microenvironment modulations 30 , and suggests that C-RNA is an excellent vector to express immune-modulatory factors intratumorally for cancer therapy.

In summary, we established a novel form of circular mRNA, termed C-RNA, in which the high effective protein expression is driven by E29, an IRES element derived from echovirus 29. This kind of circular mRNA is more stable and mediates higher and more durable protein expression than linear mRNA. The HPLC-grade purification of C-RNA resulted in promotion of protein expression in immunogenic cell line, probably due to the removal of immunogenic 5'-ppp intron fragments, suggesting that C-RNA is safe for in vivo usage as potential vaccines or therapeutics. Furthermore, the successful expression of varies kinds of proteins, including intracellular proteins, secreted proteins and transmembrane proteins, we conclude that C-RNA is a universal vector for protein expression. The intratumoral expression of C-RNA is found to direct higher and more durable protein expression than typical linear mRNA. The intratumoral administration of C-RNA mixture encoding four cytokines elicited notable tumor suppression effect by boosting infiltrated T cells to facilitate immune therapy. Collectively, C-RNA is proven to be a potential platform for the development of mRNA vaccines or therapeutics, may be a preferable RNA vector due to its simplicity for manufacturing and excellent performances for protein expression. 

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