CN118956861A

CN118956861A - An HPV-16 E7circRNA vaccine based on recombinant MS2 virus-like particles delivery

Info

Publication number: CN118956861A
Application number: CN202410986505.3A
Authority: CN
Inventors: 马雁冰; 李书琴
Original assignee: Institute of Medical Biology of CAMS and PUMC
Current assignee: Institute of Medical Biology of CAMS and PUMC
Priority date: 2024-05-24
Filing date: 2024-07-23
Publication date: 2024-11-15

Abstract

The present invention discloses a HPV-16E7circRNA vaccine based on recombinant MS2 virus-like particles. A plasmid carrying both MS2 gene and HPV16E7circRNA gene was constructed by transformation into Escherichia coli, and a technical platform for self-assembly and packaging circRNA in Escherichia coli based on MS2VLP was established. Experimental results show that the designed circRNA can be efficiently cyclized in vitro and resist nuclease degradation; the optimization of UTR sequence and MS2 binding neck ring structure can promote the expression of target gene in cells; E7circRNA-MS2VLP is a 25-30nm nanoparticle; circRNA is encapsulated in the particle and has stability against nuclease cleavage; circRNA VLP can mediate the expression of target gene in DCs and in mice; circRNA-MS2VLP stimulates tumor-specific cellular immune response in TC-1 tumor-bearing mice, promotes CTL production and reduces MDSC response, and inhibits tumor growth.

Description

HPV-16E 7circRNA vaccine based on recombinant MS2 virus-like particle delivery

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to an HPV-16E 7circRNA vaccine based on recombinant MS2 virus-like particle delivery.

Background

Currently, cervical cancer is the third most common cancer in women worldwide, and Human Papillomaviruses (HPV) are responsible for almost all cervical cancer cases.

Although current HPV vaccines are very effective in preventing infections and neoplastic diseases, they are unable to clear established infections. Thus, the current direction of research is the development of therapeutic vaccines. Therapeutic human papillomavirus vaccines are designed to induce cell-mediated immune responses in the host, driven primarily by Th1 cells, aimed at clearing existing viral infections and slowing or inhibiting tumor progression. Currently, several strategies for developing therapeutic vaccines have been tried, including live vectors, nucleic acids, peptides, protein-based and cell-based vaccines, and combinatorial approaches.

Live vector-based vaccines can be classified as bacterial or viral vectors. Live vector-based vaccines can induce strong cellular and humoral immune responses. Live vector-based vaccines are highly immunogenic and there are many vectors available for selection for antigen delivery. However, a major disadvantage of live vector vaccines is that they present a safety risk, especially for immunocompromised individuals. One obstacle to the use of live vector vaccines is the generation of antiviral immune responses and neutralizing antibodies upon initial exposure to the vaccine, which limits the effectiveness of subsequent vaccine administration.

Peptide-based and protein-based vaccines are safe, stable and easy to produce. Peptides and proteins derived from human papilloma virus antigens are processed by dendritic cells and presented on class I or class II Major Histocompatibility Complex (MHC) molecules to stimulate CD8 ⁺ or CD4 ⁺ T cell responses. Peptide-based vaccines are MHC-specific, which makes them unlikely to be candidates for large-scale production and treatment. There are many CD4 ⁺ and CD8 ⁺ T epitopes within protein-based vaccines and the disadvantages of short peptide vaccination are circumvented and no longer subject to MHC restriction. However, antigens are preferentially presented by mhc class ii, which results in the production of antibodies rather than effector Cytotoxic T Lymphocytes (CTLs). Therefore, there is a need to boost endogenous processing by some means of assistance, or to target antigens to DC cells for the purpose of eliciting cellular immunity.

DNA vaccination involves the delivery of plasmid DNA encoding a protein of interest into host tissue, followed by transfection of cells, resulting in the expression of the transgene and the production of proteins that are able to enter cellular processing mechanisms. Has the advantages of simple production, low cost and good thermal stability. DNA vaccines are typically administered by intramuscular injection. After injection, DNA may be taken up by muscle cells or dendritic cells, which play a key role in presenting antigen to CD8 ⁺ cytotoxic T cells by direct transfection through exogenous antigen uptake or vaccination. An important limitation of using DNA vaccines is the low immunogenicity of naked DNA, as it cannot be amplified and spread from transfected cells to surrounding cells.

Therefore, how to develop a novel HPV therapeutic vaccine with high safety and effective inhibition of tumor growth is a problem to be solved.

Disclosure of Invention

In order to overcome the problems of the prior art, the invention provides an HPV-16E 7circRNA vaccine based on recombinant MS2 virus-like particle delivery. As a therapeutic vaccine, the MS2 VLP delivered circRNA immunity can be used as an effective cancer immunotherapy to inhibit tumor growth in vivo.

The purpose of the invention is realized in the following way:

in a first aspect the invention provides an HPV-16E 7circRNA comprising five elements, a vector topology, 5 'and 3' untranslated regions, an Internal Ribosome Entry Site (IRES) and an aptamer (TR) recruiting a viral particle assembly initiation mechanism, the sequences of which are shown in SEQ ID NO. 1.

The second aspect of the invention provides a method for preparing an HPV-16E 7circRNA vaccine based on recombinant MS2 virus-like particle delivery, comprising the following steps:

(1) Sequence design and optimization of HPV-16E 7 circRNA:

The circRNA comprises five elements, namely a vector topological structure, 5 'and 3' untranslated regions, an Internal Ribosome Entry Site (IRES) and an aptamer (TR) recruiting a viral particle assembly initiation mechanism, and the sequences of the five elements are shown as SEQ ID NO. 1;

Adding a pac locus stem loop of 19nt at the 3' end of the circRNA to obtain an optimized circRNA sequence;

(2) Construction of prokaryotic Co-expression plasmid

Inserting a phage MS2 coat protein sequence into NcoI/HindIII cloning sites of pRSF-Duet1, simultaneously inserting the cirRNA sequence optimized in the step (1) into NdeI/NgoMIV cloning sites of pRSF-Duet1, and performing codon optimization to construct a prokaryotic coexpression plasmid;

the nucleic acid sequence of the encoding phage MS2 coat protein is shown as SEQ ID NO. 2;

(3) Prokaryotic expression and purification of MS2 circRNA-VLPs

Converting the prokaryotic co-expression plasmid into competent cells of escherichia coli BL21, inducing the expression of MS2 circRNA-VLPs by adding isopropyl-beta-d-thiogalactoside, and collecting bacterial liquid; after the thalli are broken by ultrasound, PEG6000 is used for concentrating virus-like particles, and further purification is carried out through sucrose density gradient centrifugation and gel chromatography, so that the MS2 circRNA-VLPs are finally obtained, namely the HPV-16E 7circRNA vaccine based on recombinant MS2 virus-like particle delivery.

In a third aspect, the invention provides an HPV-16E 7circRNA vaccine based on recombinant MS2 virus-like particle delivery prepared by the method described above.

The fourth aspect of the invention provides an application of the circRNA vaccine in preparing a medicament for treating cervical cancer.

The invention has the advantages and beneficial effects that:

1. In order to develop an mRNA vaccine resistant to nuclease degradation, the present invention employs an in vivo self-assembly RNA encapsulation method based on MS2 VLPs (phage-like particles). According to the method, the target circRNA is packaged in MS2 phage coat protein, and the obtained HPV-16E 7circRNA vaccine not only shows high yield in an escherichia coli expression system, but also has the advantages of resisting nuclease (keeping stable and non-degradable after being incubated with the nuclease for at least 48 hours), being non-replicable and non-infectious, and being phagocytized by DC cells.

2. The HPV-16E 7circRNA vaccine based on recombinant MS2 virus-like particle delivery has no obvious adverse reaction due to safe and muscle administration; in the immune evaluation of a TC-1 cervical cancer tumor model, an HPV-16E 7circRNA vaccine based on MS2 VLP induces a strong cellular immune response, particularly the generation of antigen specific Cytotoxic T Lymphocytes (CTL) and simultaneously reduces the immune suppression effect of myelogenous suppressor cells (MDSC), and effectively inhibits the tumor growth in a C57BL/6 mouse. MS2 VLP delivery works better and has certain advantages compared to delivery of E7circRNA by LNP.

Drawings

The invention is further described below with reference to the drawings and examples.

FIG. 1 shows EGFP CIRCRNA plasmid design and expression validation; wherein, (a) EGFP CIRCRNA plasmid design map; (b) agarose gel electrophoresis of IVT rnase digestion experiments; (c) IVT RNA plus GTP induced cyclization and enzyme digestion experiments agarose gel electrophoresis, +R: digestion with RNase R; +g: GTP induction cyclization; +h: digestion with RNase H; (d) in vitro circularized RNA expressed in 293T cells;

FIG. 2 shows hGluc circRNA plasmid construction and expression optimization; wherein, (a) hGluc circRNA plasmid restriction enzyme digestion identification; (b) Detecting luciferase expression of hGluc circRNA transfected 293T cells;

FIG. 3 shows the preparation and characterization of EGFP CIRCRNA-VLP prokaryotic expression; wherein, (a) SDS-PAGE analysis of the initially purified VLPs; (b) purification of VLPs, peaks of the protein of interest are indicated by arrows; (c) transmission electron microscopy of VLPs: about 25-30nm; (d) nuclease resistance analysis of VLPs;

FIG. 4 shows the identification of RNAs for MS2 capsid packages; wherein, (a) RT-PCR detection of MS2 capsid packaged RNAs; (b) sanger sequencing;

FIG. 5 shows the functional verification of E7circRNA and E7mRNA expression and the preparation of E7circRNA-MS2 VLPs; (a) Verifying plasmid pVAX-E73' loop of E7circRNA by western blot to construct and express; (b) agarose gel electrophoresis of IVT rnase digestion experiments; +d: digestion with DNaseI; +r: digestion with RNase R; (c) western blot to verify expression of E7circRNA and E7 mRNA; (d) SDS-PAGE analysis of the initially purified E7circRNA-MS2 VLPs; (E) transmission electron microscopy of E7circRNA-MS2 VLPs: about 25-30nm; (f) particle size of E7mRNA LNP and E7CIRCRNA LNP; (g) Zeta potential of E7mRNA LNP, E7CIRCRNA LNP, E7circRNA-MS2 VLP; (h) Ribogreen staining method is used for detecting the standard curve of RNA content and the encapsulation rate of E7mRNA LNP, E7CIRCRNA LNP and E7circRNA-MS2 VLP;

FIG. 6 shows expression of MS2 VLPs packaging exogenous circRNA in mammalian cells; wherein (a) a DC2.4 cell phagocytosis assay of EGFP MS2 VLPs; (b) DC2.4 cell phagocytosis assay of hGluc circRNA-MS2 VLP; (c) in vivo imaging experiments, left: hGluc CIRCRNA MS VLP, in: hGluc circRNA, right: MS2 VLP;

FIG. 7 shows the anti-tumor results of E7 circRNA and different adjuvant mix immunization in TC-1 tumor model; wherein, (a) an immunoassay programming map; (b) a tumor growth curve; (c) Tumor anatomical images (n=5) at day 39 post-treatment of the different groups;

FIG. 8 shows the anti-tumor results of E7 circRNA-LNP in a TC-1 tumor model; wherein, (a) an immunoassay programming map; (b) a tumor growth curve; (c) Tumor anatomical images (n=5) at day 39 post-treatment of the different groups; (d) different sets of tumor and spleen weight statistics (n=5); (e) Flow cytometry analysis of CTL and MDSC cell ratios in tumors;

FIG. 9 shows the anti-tumor results of E7 circRNA-MS2 VLP in TC-1 tumor model; wherein, (a) an immunoassay programming map; (b) a tumor growth curve; (c) Tumor anatomical images (n=5) at day 39 post-treatment of the different groups; (d) different sets of tumor and spleen weight statistics (n=5); (e) Flow cytometry analysis of the proportion of CTL and MDSC cells in tumors.

Detailed Description

The practice of the invention is not limited to the following examples, but is intended to be within the scope of the invention in any form and/or modification thereof. In the present invention, all the equipment, raw materials and the like are commercially available or commonly used in the industry unless otherwise specified. The methods employed in the examples are those generally known in the art, unless otherwise indicated.

For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

Example 1: constructing a circRNA-VLP prokaryotic coexpression plasmid, and carrying out in-vitro expression function and physicochemical characteristic verification through SDS-PAGE electrophoresis, transmission electron microscopy and western blot:

(1) circRNA plasmid design and validation

Determining the sequence of the circRNA, the structure of the circRNA sequence comprises five elements: the vector topology, 5 'and 3' untranslated regions, internal Ribosome Entry Site (IRES) and aptamer (TR) recruiting the viral particle assembly initiation mechanism, sequences shown in SEQ ID NO.1, were synthesized at Sangon Biotech, inc. The circRNA sequence was inserted into the NdeI/XbaI cloning site of pVAX and codon optimized, designated pVAX-EGFP 3' loop.

This example uses the ribozyme approach of intronic self-splicing function, also known as the aligned intron and exon splicing strategy (PIE, permuted intron-exon). The type I intron of the T4 phage thymidylate synthase (Td) gene was used to break it apart, sequentially joining exon 2 to exon 1, inserting a 1.5kb sequence between the exons: the coxsackievirus internal ribosome entry site IRES (IRES), 5'3' utr and green fluorescent protein (EGFP) coding sequences (a in FIG. 1). After transforming the synthesized EGFP CIRCRNA plasmid into E.coli DH5a competent cells, preserving the strain at-80 ℃, extracting the plasmid, and verifying whether the precursor RNA of the In Vitro Transcription (IVT) is successfully self-sheared and cyclized by utilizing an RNase digestion method, wherein the RNA of the IVT comprises linear, cyclized and small fragment RNA, and the self-shearing of the intron does not need the catalytic induction of GTP in the in vitro transcription process as shown in a figure I. Ribonuclease R (RNase R) is derived from the E.coli RNR superfamily and is a 3'-5' ribonuclease with magnesium ion dependence. RNA can be cleaved stepwise from the 3'-5' direction into di-and tri-nucleotides. RNase R is able to digest almost all linear RNA to enrich for circular RNA or lasso-structured RNA, but is not easily digestible to circular RNA, lasso-structured RNA and short double stranded RNA molecules with 3' -end overhangs of less than 7 nucleotides. Ribonuclease H (RNaseH) catalyzes the degradation of the RNA portion of the DNA-RNA hybrid, producing oligoribonucleic acids with different chain lengths, 3 'hydroxyl groups and 5' phosphate ends. As shown in FIG. 1c, the precursor RNA of IVT was efficiently circularized in vitro and was successfully resistant to digestion by the exonuclease RNase R. The efficiency of catalytic induction of RNA cyclization with or without GTP during in vitro transcription is about the same, indicating that intronic self-splicing does not require catalytic induction of GTP during in vitro transcription. The transfection of the synthetic EGFP CIRCRNA plasmid, linearized plasmid and precursor RNA of IVT and circularized RNA into 293T cells also successfully verified that their in vitro circularized RNA was efficiently expressed (d in FIG. 1), and these experimental data indicate that EGFP CIRCRNA plasmid design successfully self-splice circularized IVT RNA precursors and successfully expressed proteins.

(2) Plasmid construction

To pack the target circRNA within MS2 phage coat protein, 19nt of pac sites must be added to the sequence, and to determine the specific location of 19nt of pac site addition and optimize the sequence of the circRNA to be packaged and to enhance translation efficiency, the circRNA sequence is inserted into the NdeI/XbaI cloning site of pVAX and codon optimized to construct a series of plasmids of pVAX-gluc5' loop with 3'5' utr, pVAX-gluc3' loop without 3'5' utr, pVAX-gluc with 5'3' utr without 3' loop.

Experimental results show that the 3'5' UTR and the stem-loop structure have the effect of enhancing the expression of the epiprotein respectively, but have the regulatory effect of 'positive and negative' on the expression of the protein when the 3'5' UTR and the stem-loop structure are added. And the addition of the pac site stem loop of 19nt at the 5 'end severely reduces the expression of the circRNA, and the addition position is preferably at the 3' end. Sequence optimisation results showed that pVAX-gluc3' loop had no optimal expression of 3'5' utr (b in figure 2).

(3) Preparation and characterization of EGFP CIRCRNA-MS2 VLP prokaryotic expression

Phage MS2 coat protein sequence (nucleic acid sequence shown as SEQ ID NO. 2) was inserted into NcoI/HindIII cloning site of pRSF-Duet1, optimized cirRNA sequence was inserted into NdeI/NgoMIV cloning site of pRSF-Duet1 and codon optimized, prokaryotic coexpression plasmid was transformed into competent cells of E.coli BL21 (DE 3) and plated on LB solid plate containing kanamycin resistance, inverted and cultured overnight in incubator at 37 ℃. After bacterial monoclonal growth, one monoclonal was randomly selected and inoculated in LB liquid medium containing Canada resistance, incubated at 180rpm on a shaker at 37℃until OD600 was about 0.6, and induced overnight at 180rpm on a shaker at 37℃with the addition of 1mM isopropyl-D-thiogalactoside (IPTG). The bacterial liquid was collected by centrifugation at 5000rpm at 4℃for 10 min. The cells were sonicated, the sonicated supernatant was directly precipitated with 10% PEG6000 and 500mM NaCl overnight at 4℃and 12000Xg, centrifuged for 1h, and the pellet was resuspended with 1 xPBS. The PEG primary purified sample was subjected to sucrose density gradient using a centrifugal ultracentrifuge at 26000rpm at 4℃for 3 hours. After centrifugation, samples were taken layer by layer from top to bottom, 1 ml/tube, and bottom pellet was also resuspended for sampling.

The position of the VLP protein was observed at 30% -40% sucrose concentration by SDS-PAGE running gel. The purified protein was desalted and replaced by SepRose FF gel chromatography with a mobile phase of 0.01M PBS. (the sucrose background was replaced with PBS). To determine the nucleic acid load of the circRNA-VLPs prepared in this example, MS2 VLPs were prepared in BL21 E.coli transformed with the plasmid pRSF-MS2 without exogenous gene as a control. Purified MS2 VLP-based circRNA vaccine (i.e. EGFP CIRCRNA-MS2 VLP) was incubated with 5U/m L deoxyribonuclease I (DNase I) and 50g/L ribonuclease a (RNase a) for 2, 5, 10, 16, 24, 48 hours and isolated by ethidium bromide staining in 1% agarose gel.

SDS-PAGE electrophoresis shows that EGFP CIRCRNA-VLP is a band with a molecular weight of about 14kDa (a in FIG. 3) as seen in the pre-induction bacterial liquid, post-induction bacterial liquid, ultrasonic supernatant and PEG precipitation weight suspension. The result of VLP GELRED stained agarose gel electrophoresis showed that after incubation of the MS2 VLP-based circRNA with dnase I and rnase a, bands of approximately 750bp size remained in each lane (d in fig. 3), indicating that these circRNAs were successfully encapsulated into the MS2VLP and that the ultra-sonic supernatant was isolated as nuclease resistant VLPs. Furthermore, TEM results indicate that these EGFP CIRCRNA-MS2VLPs are VLPs with diameters of about 25-30nm (c in FIG. 3).

Example 2: identification of RNAs based on MS2 VLPs

In the application, only one pac locus is cloned and is immediately behind the stop codon of the target gene, in order to ensure that the circRNA can be successfully wrapped into the MS2 VLP in a prokaryotic expression system, the total RNA of the VLPs after RNAse digestion is extracted by using a viral RNA extraction kit, and reverse transcription is carried out on cDNA by using corresponding primers for RT-PCR experiments. As in fig. 4a, the PCR products were verified by ethidium bromide stained 1% agarose gel electrophoresis followed by further verification by sequencing, which verified that the introns were circularized at the self-cleavage sites (fig. 4 b). These data indicate that MS2 VLPs produced in e.coli can package target circ RNA of about 1500bp in site-specific integrity with high yield.

The pVAX-E73' loop plasmid was constructed successfully before, and it was verified that the plasmid successfully expressed E7 protein (a in FIG. 5). The pVAX E7mRNA plasmid optimized for the self-design of the department and synthesized by Biotechnology Inc. of Jin Weizhi, an Sheng, GENEWIZ was prepared by in vitro transcription to E7circRNA and E7mRNA, respectively. The products were confirmed by gel-stained 1% agarose gel electrophoresis and as a result, it was found that E7mRNA was digested completely by RNaseR, and the digestion tolerance of E7circRNA to RNaseR further demonstrated the stability of the circRNA vaccine (b in FIG. 5). western blot verifies that E7circRNA and E7mRNA successfully expressed E7 protein (c in FIG. 5), a band with a molecular weight of about 10kDa was seen, and GAPDH protein, an important cellular enzyme, was used as an internal control with a molecular weight of about 36kDa. SDS-PAGE analysis of the initially purified E7circRNA-MS2 VLPs, transmission electron microscopy of E7circRNA-MS2 VLPs: about 25-30nm (e in FIG. 5). E7circRNA and E7mRNA are prepared and encapsulated by LNP, and then key indexes such as particle size, potential and encapsulation efficiency are detected. As shown in FIG. 5 f, E7CIRCRNA LNP and E7mRNA LNP were 185.8 and 158.6nm, respectively, and the LNP had an optimal particle size range of 20-200nm, which was satisfactory. In FIG. 5g shows that the zeta potentials of E7CIRCRNA LNP, E7mRNA LNP, and E7circRNA-MS2VLP are-1.97, -0.92, -9.60, respectively. The surface charges are weak, belonging to neutral particles. For accurate quantification of the encapsulated RNA in E7CIRCRNA LNP, E7mRNA LNP, and E7circRNA-MS2 VLPs, standard curves were established using the RiboGreen dye method (Quant-iT ^TMRiboGreen^TM RNA REAGENT ANDKIT) with RNA standards, as shown in FIG. 5 h with encapsulation rates of 69.13%, 80%, 63.85% for E7CIRCRNA LNP, E7mRNA LNP, and E7circRNA-MS2 VLPs, respectively. In a word, each index of the prepared vaccine meets the requirements, and the vaccine can be used for the next experiment.

Example 3: expression of packaging exogenous circRNA in mammalian cells

To investigate whether MS2 capsid-packaged circRNA can be translated into protein in mouse bone marrow dendritic cells DC2.4, the present example was modeled on EGFP MS2 VLPs and hGluc MS2 VLPs. Phagocytosis assays showed that EGFP CIRCRNA expression was monitored by fluorescence microscopy for 18 hours of incubation (a in fig. 6). After 24, 48 hours incubation, expression of hGluc circRNA was detected (b in fig. 6). These results are different from those observed with the naked circRNA vaccine, which showed maximum expression within 18-24 hours and no expression detected at 48 hours. In vivo imaging experiments 50 ug/mouse injected intravenously with coelenterazine at 20h, 50h, 72h, and taken after waiting 10min, only near the injection site expressed and barely expressed hGluc MS2 VLPs over time, with a possibility that hGluc MS2 VLPs were phagocytized to migrate to other internal organs (fig. 6 c). These results demonstrate the stability of the MS2 VLP CIRCRNA vaccine and the high efficiency of VLP delivery of RNA.

Example 4: evaluation of in vitro antitumor Effect

The experimental mice of the embodiment are derived from SPF-grade C57BL/6 female mice of 6-8 weeks of age in national medical primate research center, and the experimental operation accords with the ethical standard of animal experiments. And has been approved by the committee for animal care and welfare ethics of the institute of medical biology, national academy of medical sciences. Ethical number: DWSP202204001.

Adjuvants are molecules that are commonly used to increase the immune response to a vaccine, but have not been used in RNA vaccines. To investigate whether E7circRNA can enhance its anti-tumor effect by the addition of an adjuvant, anti-tumor immunology studies were performed in the HPV infection-associated tumor mouse TC-1 model. We set up PBS control and E7circRNA, E7circrna+mf59, E7circrna+as01, E7circrna+aloh, E7circrna+ifa experimental groups.

When tumors were as long as about 3×3mm in length and width, the whole experiment was immunized 3 times by injecting 20 μg/dose of vaccine into the right leg muscle of the mice, once every 7 days (a in fig. 7). The antitumor effect of the vaccine was evaluated by measuring the length and width of the tumor of the mice and calculating the volume thereof. Mice tumor tissue was collected after day 39 mice were sacrificed.

Experimental results show that the mixed immunization of the E7 circRNA vaccine with MF59, AS01, alOH did not significantly enhance the anti-tumor immune response, whereas IFA this adjuvant slightly enhanced the anti-tumor immune effect of the E7 circRNA vaccine compared to other adjuvants (b, c in fig. 7). In summary, free E7 circRNA enhances the anti-tumor immune response in tumor-bearing mice and inhibits tumor growth.

Naked RNA cannot be efficiently presented into cells and successfully escape from lysosomes, and in vivo humoral and cellular immunity is low, so there is a need for delivery systems that enhance cellular uptake and improve delivery to cytoplasmic translation mechanisms, thereby enhancing the action of RNA vaccines. To investigate whether E7circRNA enhanced the advantage of immunogenicity compared to linear E7 mRNA. This example examined the anti-tumor effect of delivery of E7circRNA and E7mRNA by LNP we set up PBS control and free E7circRNA and E7mRNA experimental groups, E7circRNA-LNP and E7mRNA LNP experimental groups.

When tumors were as long as about 3×3mm in length and width, the whole experiment was immunized 3 times by injecting 20 μg/dose of vaccine into the right leg muscle of the mice, once every 7 days (fig. 8 a). The antitumor effect of the vaccine was evaluated by measuring the length and width of the tumor of the mice and calculating the volume thereof. Mice on day 39 were sacrificed, tumor tissues of the mice were collected and weighed, and tumor cells were taken for flow cytometry detection experiments to detect T cell and MDSC cell immune response. The results showed that E7CIRCRNALNP had better antitumor effect than the other experimental groups, but the effect of inhibiting tumor growth was not as pronounced (b, c in fig. 8). We analyzed the proportion of immune cells in lymphocytes isolated from tumor tissue by flow cytometry for each experimental group to reveal the intrinsic mechanism of the anti-tumor effect of the E7circRNA vaccine. Flow cytometry analysis of CTL cell level results showed no significant differences in the three PBS, E7circRNA, E7mRNA groups compared to the IFNr +cd8+ T cells, gr-1+cd11b+mdsc cells of the isolated lymphocytes (E in fig. 8). E7 The IFNr +cd8+ T cells of the lymphocytes isolated from the CIRCRNA LNP and E7mRNA LNP group tumors were significantly increased and the Gr-1+cd11b+ mdsc cells were significantly decreased (E in fig. 8). This suggests that LNP delivery of E7circRNA and E7mRNA may slightly promote CTL infiltration in TME, which can produce cytotoxic effects on tumor cells. More importantly, we observed a significant decrease in tumor size and weight in the vaccine group compared to the PBS group (c, d in fig. 8). E7circRNA delivered with LNP was more immunogenic than linear E7mRNA delivered with LNP. Free immunogenicity is not significantly different. LNP delivery of E7circRNA and E7mRNA can slightly promote infiltration of CTL in TME, and can generate cytotoxicity on tumor cells and inhibit tumor growth. In summary, the delivery system is critical for RNA vaccines.

To further investigate the anti-tumor effect of E7circRNA/E7mRNA delivery by LNP, an anti-tumor immunological study was performed in a TC-1 tumor-bearing mouse model. PBS control and E7circRNA-MS2 VLP groups were set up, and when tumors were about 3X 3mm long and wide, the whole experiment was immunized 3 times by injecting 20. Mu.g/dose of vaccine into right leg muscle of mice, 7 days apart (FIG. 9, a). The antitumor effect of the vaccine was evaluated by measuring the length and width of the tumor of the mice and calculating the volume thereof. Mice on day 39 were sacrificed, tumor tissues of the mice were collected and weighed, and lymph node cells were taken for flow cytometry detection experiments to detect T cell and MDSC cell immune response.

The results showed circE MS2 VLPs had better antitumor effect than the E7circRNA group, but inhibited tumor growth more significantly (b, c in fig. 9). The reason for this may be that the circRNA reaches the tumor tissue in small amounts after injection, inhibiting the growth of the tumor. We analyzed the proportion of immune cells in lymphocytes isolated from tumor tissue by flow cytometry for each experimental group to reveal the intrinsic mechanism of the anti-tumor effect of the E7circRNA vaccine. Flow cytometry analysis of CTL cell levels showed a significant increase in IFNr +cd8+ T cells of the lymphocytes isolated from the tumors of the E7 circRNA-MS2VLP group compared to PBS, E7circRNA, and a significant decrease in Gr-1+cd11b+mdsc cells (E in fig. 9). There was no significant difference in the IFNr +cd8+ T cells, gr-1+cd11b+ mdsc cells of the lymphocytes isolated from the tumors of the E7circRNA group compared to the PBS group (E in fig. 9). This suggests that MS2VLP delivery circE RNA is critical in promoting CTL infiltration in TME, and can produce cytotoxic effects on tumor cells. More importantly, we observed a significant decrease in tumor size and weight in the E7circRNA group compared to the PBS group (c, d in fig. 9). E7circRNA may play a key role in activating other immune cell downstream signaling pathways and inhibiting tumor cell growth. MDSC cells, as immunosuppressive immune cells in TME, can down-regulate immune surveillance and anti-tumor immunity, thereby promoting tumor growth. The proportion showed a significant decrease after E7 circRNA-MS2VLP treatment (E in FIG. 9). In conclusion, E7 circRNA-MS2VLP significantly enhanced anti-tumor immunity in tumor-bearing mice, inhibiting tumor growth.

Finally, it should be noted that the above only illustrates the technical solution of the present invention and is not limiting, and although the present invention has been described in detail with reference to the preferred arrangement, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A HPV-16E7circRNA, characterized in that the circRNA contains five elements, namely a vector topology structure, 5' and 3' untranslated regions, an internal ribosome entry site (IRES) and an adapter (TR) for recruiting the viral particle assembly initiation mechanism, and the sequence is shown in SEQ ID NO.1.

2. A method for preparing an HPV-16E7circRNA vaccine based on recombinant MS2 virus-like particles delivery, characterized in that it comprises the following steps:

(1) Sequence design and optimization of HPV-16E7circRNA:

The circRNA contains five elements, namely, the vector topology, 5’ and 3’ untranslated regions, the internal ribosome entry site (IRES), and the adapter (TR) that recruits the viral particle assembly initiation machinery, and the sequence is shown in SEQ ID NO.1;

Adding a 19nt pac site stem loop to the 3' end of the circRNA to obtain an optimized circRNA sequence;

(2) Construction of prokaryotic co-expression plasmid

Insert the bacteriophage MS2 coat protein sequence into the NcoI/HindIII cloning site of pRSF-Duet1, and insert the cirRNA sequence optimized in step (1) into the NdeI/NgoMIV cloning site of pRSF-Duet1 and perform codon optimization to construct a prokaryotic co-expression plasmid;

The nucleic acid sequence encoding the bacteriophage MS2 coat protein is shown in SEQ ID NO.2;

(3) Prokaryotic expression and purification of MS2 circRNA-VLPs

The prokaryotic co-expression plasmid was transformed into Escherichia coli BL21 competent cells, the expression of MS2 circRNA-VLPs was induced by adding isopropyl-β-d-thiogalactoside, and the bacterial liquid was collected; after the bacteria were disrupted by ultrasound, the virus-like particles were concentrated by PEG6000 and further purified by sucrose density gradient centrifugation and gel chromatography to finally obtain MS2 circRNA-VLPs, which is the HPV-16E7circRNA vaccine delivered based on recombinant MS2 virus-like particles.

3. A HPV-16E7circRNA vaccine based on the delivery of recombinant MS2 virus-like particles, characterized in that the vaccine is prepared by the method as described in claim 2.

4. Use of the circRNA vaccine as claimed in claim 3 in the preparation of a drug for treating cervical cancer.