Rabies virus circular RNA vaccine and application thereof

Abstract

The present invention relates to a circular RNA comprising a cyclization regulatory element, an antigen coding element, a Kozak sequence and a dual termination codon wherein the antigen coding element encodes an amino acid sequence of a rabies virus glycoprotein. The invention also provides a composition containing the circular RNA, and an application of the circular RNA or the composition in preparation of a vaccine for preventing rabies.

Description

Technical Field

[0001] This application relates to the field of nucleotide technology applications. Specifically, the present invention relates to a circular RNA comprising a circularization regulatory element, an antigen-coding element, a Kozak sequence, and a double-stop codon, wherein the antigen-coding element encodes an amino acid sequence of a rabies virus glycoprotein. The present invention also provides compositions comprising said circular RNA, and the use of said circular RNA or said composition in the preparation of a vaccine for the prevention of rabies. Background Technology

[0002] Rabies is a fatal zoonotic disease of the central nervous system caused by the rabies virus (RABV). RABV is a single-stranded, non-segmental, negative-sense neurotropic virus. According to the World Health Organization, rabies causes approximately 59,000 deaths globally each year, significantly impacting more than 150 countries. Rabies virus is primarily transmitted through bites or scratches from infected animals, entering the host through the wound. After viral replication, it infects peripheral nerves and further invades the brain; once the virus enters the brain, the mortality rate can reach 100%. The RABV genome encodes five proteins: phosphoprotein (P), matrix protein (M), nucleoprotein (N), glycoprotein (G), and RNA-dependent RNA polymerase (RdRp). RABV-G is a glycoprotein located on the surface of RABV that mediates viral attachment to host receptors and facilitates viral particle entry into host cells through fusion with the cell membrane. Therefore, RABV-G is an ideal target for vaccine development.

[0003] The first live attenuated rabies vaccine was successfully developed by French scientist Louis Pasteur in 1885, and it was recognized by the medical community as having therapeutic potential. The subsequently developed classic inactivated vaccine remains the primary human rabies vaccine on the market; however, protective immunity requires 4-5 doses or more. Post-exposure prophylaxis (PEP) includes rabies vaccination and anti-rabies immunoglobulins (RIG). However, effective neutralizing antibodies must block the virus from infecting neurons. In developing countries, where the disease burden is heaviest due to high costs, there are many cases of failed PEP. Early rabies vaccines were developed in mammalian neural tissue, but these were later superseded by vaccines produced in chicken embryos and tissue cells. Recently, novel vaccines, such as protein subunit vaccines, viral vector vaccines, and DNA vaccines, have shown protective efficacy against rabies infection in preclinical trials, but none have yet been approved for marketing.

[0004] Circular RNA (circRNA), as a novel type of engineered RNA, offers advantages over linear mRNA, primarily including high stability, no need for polyA or capping, and no need for nucleotide modification. Vaccines based on the circRNA platform can effectively prevent SARS-CoV-2, influenza virus, and respiratory syncytial virus infection, and stimulate strong humoral and T-cell immunity. Therefore, the applicant has developed a novel circRNA vaccine against rabies virus by multidimensionally designing immunogens through methods such as introducing a T4 foldon sequence to form a G protein trimer, introducing a pre-fusion conformational H270P point mutation, introducing a tPA signal peptide at the N-terminus, and introducing a helix sequence containing the SARS-CoV-2 spike protein at the C-terminus. Furthermore, the applicant has optimized the circRNA molecule encoding the RABV-G antigen through different circulylation methods. Summary of the Invention

[0005] The object of this invention is to provide a novel circular RNA vaccine against rabies virus. In one aspect, the invention provides a circular RNA comprising a circularization regulatory element, an antigen-coding element, a Kozak sequence, and two stop codons, wherein the antigen-coding element encodes an amino acid sequence of a rabies virus glycoprotein. In another aspect, the invention also provides a composition comprising the circular RNA described herein. In yet another aspect, the invention further provides the use of the circular RNA or the composition described herein in the preparation of a vaccine for the prevention of rabies.

[0006] In some embodiments, the present invention relates to a circular RNA comprising: Circulation control element; An antigen encoding element that encodes the amino acid sequence of a rabies virus glycoprotein, wherein the rabies virus glycoprotein is a wild-type or modified rabies virus glycoprotein. Kozak sequences; and Double stop codon, Optionally, the components are connected to each other via connectors.

[0007] In some embodiments, the present invention relates to a circular RNA comprising: Circulation control element, An antigen encoding element that encodes the amino acid sequence of a rabies virus glycoprotein, wherein the rabies virus glycoprotein is a wild-type or modified rabies virus glycoprotein. Kozak sequence, and Double stop codon, The components are connected to each other via connectors.

[0008] In some embodiments, the present invention relates to a circular RNA, wherein the modified rabies virus glycoprotein is modified by means of: introducing a T4 foldon sequence, introducing an H270P point mutation, introducing a tPA signal peptide at the N-terminus, or introducing a helix sequence containing the SARS-COV-2 spike protein at the C-terminus.

[0009] In some embodiments, the present invention relates to a circular RNA, wherein the sequence of the antigen-coding element is selected from SEQ ID NO: 1-13; preferably, the sequence of the antigen-coding element is SEQ ID NO: 3, 4, 7, 8, 9, 10, 11, 12 or 13; more preferably, the sequence of the antigen-coding element is SEQ ID NO: 9 or 13.

[0010] In some embodiments, the present invention relates to a circular RNA wherein the sequence of the antigen-coding element is selected from SEQ ID NO: 1-13. In some embodiments, the present invention relates to a circular RNA wherein the sequence of the antigen-coding element is SEQ ID NO: 3, 4, 7, 8, 9, 10, 11, 12, or 13. In some embodiments, the present invention relates to a circular RNA wherein the sequence of the antigen-coding element is SEQ ID NO: 9 or 13.

[0011] In some embodiments, the present invention relates to a circular RNA, wherein the circularization regulatory element comprises: an internal ribosome entry site (IRES) or a fragment thereof, or an internal ribosome entry site (IRES) and a type I intron; The internal ribosome entry site (IRES) or a fragment thereof is selected from the group consisting of: Coxsackievirus B3 (CVB3) IRES, Enterovirus 71 (EV71) IRES, Encephalomyocarditis Virus (EMCV) IRES, Picornavir Virus (PV) IRES, Hepatitis C Virus (HCV) IRES, Adenovirus (AdV) IRES, Human Papillomavirus 31 (HPV31) IRES, Human Herpesvirus (HHV) IRES, Rous Sarcoma Virus (RSV) IRES, Classical Swine Fever Virus (CSFV) IRES, FGF9 IRES, SLC7A1 IRES, and RUNX1 IRES; preferably, the internal ribosome entry site (IRES) or a fragment thereof is a CVB3 IRES; more preferably, the sequence of the internal ribosome entry site (IRES) or a fragment thereof is SEQ ID NO: 30; The type I introns include: 5'-intron, Exon2, Exon1 and 3'-intron; preferably, the type I introns are selected from the group consisting of: SEQ ID NO: 33-36.

[0012] In some embodiments, the present invention relates to a circular RNA, wherein the circularization regulatory element is an internal ribosome entry site (IRES) or a fragment thereof, wherein the sequence of the internal ribosome entry site (IRES) or the fragment thereof is SEQ ID NO: 30.

[0013] In some embodiments, the present invention relates to a circular RNA, wherein the circularization regulatory element comprises an internal ribosome entry site (IRES) and a type I intron, wherein the internal ribosome entry site (IRES) or a fragment sequence thereof is SEQ ID NO: 30, and wherein the type I intron is selected from the group consisting of SEQ ID NO: 33-36.

[0014] In some embodiments, the present invention relates to a circular RNA, wherein the Kozak sequence is SEQ ID NO:29.

[0015] In some embodiments, the present invention relates to a circular RNA, wherein the double stop codon is TGATGA.

[0016] In some embodiments, the present invention relates to a circular RNA, wherein the amino acid sequence expressed by the linker is selected from: GA, GS, GG, GGA, GGS, GGG, GGGA, GGGS, GGGG, GGGGA, GGGGS, GGGGG, GGGGA, GGGGS, GGGGG, GGAG, GGSG, AGGGG, SGGG, GAGA, GSGSGS, GAGAGAGA, GSGSGSGS, GAGAGAGAGA, G SGSGSGSGS, GAGAGAGAGAGA, GAGSGSGSGSGS, GGAGGA, GGSGGS, GGAGGAGGA, GGSGGSGGS, GGAGGAGGAGGA, GGSGGSGGSGGS, GGAGGGAG, GGSGGGSGGSG, GGAGGGAGGAGGAG, GGSGGGSGGSG, GGGAGGGAGGAGGA or GGGSGGGSGGSGGS; preferably, the amino acid sequence expressed by the linker is GS.

[0017] In some embodiments, the present invention relates to a circular RNA, wherein the amino acid sequence expressed by the linker is GS.

[0018] In some embodiments, the present invention relates to a circular RNA, wherein the amino acid sequence of the rabies virus glycoprotein is selected from SEQ ID NO: 22-28; preferably, the amino acid sequence of the rabies virus glycoprotein is SEQ ID NO: 23, 26, 27 or 28.

[0019] In some embodiments, the present invention relates to a circular RNA, wherein the amino acid sequence of the rabies virus glycoprotein is SEQ ID NO: 23, 26, 27 or 28.

[0020] In some embodiments, the present invention relates to a circular RNA, wherein the circular RNA is obtained by cyclization of a linear RNA precursor as shown in SEQ ID NO:31 or SEQ ID NO:32 using T4 RNA ligase; or, the circular RNA is obtained by autocatalytic catalysis of a linear RNA precursor as shown in SEQ ID NO:14-17. In some embodiments, The circular RNA is the linear RNA described in SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 14-17, with the 5' end connected to the 3' end to form a loop.

[0021] In some embodiments, the present invention relates to a circular RNA, wherein the circular RNA is obtained by cyclization of a linear RNA precursor, such as SEQ ID NO:31 or SEQ ID NO:32, via T4 RNA ligase. In some embodiments, the present invention relates to a circular RNA, wherein the circular RNA is obtained by autocatalytic catalysis of a linear RNA precursor, such as SEQ ID NO:14-17.

[0022] In some embodiments, the present invention relates to a composition comprising the circular RNA described herein and a pharmaceutically acceptable carrier or excipient.

[0023] In some embodiments, the present invention relates to a composition, wherein the composition is lipid nanoparticles.

[0024] In some embodiments, the present invention relates to a composition wherein the lipid nanoparticles comprise one or more of cationic lipids, neutral lipids, polyethylene glycol lipids, and steroidal lipids, wherein: the cationic lipid is selected from DLin-MC3-DMA or SM102; and the neutral lipid is selected from 1,2-distearyl·sn·glycerol·3-phosphocholine (DSPC), 1,2-dipalmitoyl·sn·glycerol·3-phosphocholine (DPPC), 1,2-dioleoyl·sn·glycerol·3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl·sn·glycerol·3-phosphoethanolamine (DPPE), 1,2-dimyristicoyl·sn·glycerol·3-phosphoethanolamine (DMPE), and 2-dioleoyl·sn·glycerol·3-phosphoethanolamine (1'·rac·glycerol). (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) or more thereof; the polyethylene glycol lipid is selected from: 2-[(polyethylene glycol)·2000]·N,N-tetracosylacetamide (ALC·0159), 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG·DMG), 1,2-distearate-sn-glycerol-3-phosphoethanolamine·N-[amino(polyethylene glycol)] (PEG·DSPE), PEG-disterol glycerol (PEG·DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearate, PEG-diacylglycerol amide (PEG The steroidal lipids are selected from one or more of the following: DAG, PEG·dipalmitoylphosphatidylethanolamine (PEG·DPPE), or PEG·1,2·dimyristoyloxypropyl·3·amine (PEG·c·DMA); the steroidal lipids are selected from one or more of the following: alfalfa sterol, β-sitosterol, brassosterol, ergocalciferol, campesterol, cholesterol, coccidosterol, dehydrocholesterol, sterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, serotonin, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; lanosterol, photosterol, physcosterol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.

[0025] In some embodiments, the present invention relates to a composition, wherein the composition is lipid nanoparticles, wherein the lipid nanoparticles are SM-102.

[0026] In some embodiments, the present invention relates to a composition, wherein the composition is lipid nanoparticles, wherein the lipid nanoparticles are a combination of Dlin-MC3-DMA, DSPC, cholesterol and PEG-DMG, wherein the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and PEG-DMG is 50:10:38.5:1.5.

[0027] In some embodiments, the present invention relates to the use of the circular RNA or compositions described herein in the preparation of a vaccine for the prevention of rabies.

[0028] definition The following lists definitions of various terms used to describe the nucleic acid combinations and compositions disclosed herein. These definitions apply to terms as used throughout this specification and claims, except where otherwise limited in certain circumstances, and these terms are used either individually or as part of a larger group.

[0029] As used in this article, the terms "optional" or "optionally" include both selection and non-selection. For example, "optional modification" includes both being modified and not being modified.

[0030] As used herein, the terms “a” and “the” are generally interpreted to encompass both singular and plural forms.

[0031] The term "comprising" as used herein means the phrase "including (but not limited to)" and may be used interchangeably with it. The term "including" as used herein means the phrase "including (but not limited to)" and may be used interchangeably with it. The technical solutions using "comprising" or "including" in this patent may be further defined as "comprising" or "forming".

[0032] Throughout this specification, the terms "one embodiment," "some embodiments," "implementation," "specific embodiment," "related embodiment," "a particular embodiment," "another embodiment," or "further embodiment," or combinations thereof, refer to a specific feature, structure, or characteristic described in conjunction with that embodiment being included in at least one embodiment of the invention. Therefore, the appearance of these phrases in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0033] As used herein, the terms “circRNA,” “circular RNA,” or “circular polynucleotide” or “circular RNA” are used interchangeably and refer to polynucleotides that form a circular structure through covalent bonds.

[0034] As used in this article, “Type I intron”, “Group I intron” and “Group I Intron” can be used interchangeably.

[0035] As used herein, a "vector" refers to a segment of DNA that is synthesized (e.g., using PCR) or extracted from a virus, plasmid, or cell of a higher organism, into which a foreign DNA fragment may be inserted or has already been inserted for cloning and / or expression purposes.

[0036] The prior art referenced in this specification is incorporated herein by reference in its entirety and is used for all purposes. Attached Figure Description

[0037] Figure 1 . Figure 1 a and 1b show schematic diagrams of the preparation of RABV-GcircRNA plasmids using T4 RNA ligase and type I introns, respectively.

[0038] Figure 2 The expression level of RABV-G protein after transfection with circRNA is shown in human 293T cells.

[0039] Figure 3 The results showed that the circRNA vaccine could induce high levels of RABV-G antibody IgG titers.

[0040] Figure 4 circRNA vaccines encoding wild-type (WT) or prefusion-conformed RABV-G proteins produce higher levels of RABV-G neutralizing antibodies compared to the approved inactivated rabies vaccine Speeda.

[0041] Figure 5 CircRNA vaccination can effectively induce the production of neutralizing antibodies in mice.

[0042] Figure 6 Seven days after the second dose, the circRNA vaccine can produce higher levels of neutralizing antibodies.

[0043] Figure 7 Seven days after the second dose, the circRNA vaccine effectively induced the production of neutralizing antibodies in mice. Detailed Implementation

[0044] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0045] The embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope thereof, but all such modifications and substitutions fall within the protection scope of the present invention. Example 1: In vitro production of circRNA using T4 RNA ligase This embodiment demonstrates the production of circular RNA (circRNA) via in vitro ligation.

[0046] (1) Design and synthesis of circRNA encoding RABV-G protein fragment Design a linear RNA capable of circularization to form a circular RNA, the linear RNA containing the sequence CVB3IRES (386-747)-Kozak-RABV-GCDS-CVB3 IRES (1-385) from the 5' to 3' direction, such as... Figure 1 As shown in a. The linear RNA was designed from 5' to 3' as an IRES sequence (SEQ ID No. 30), a Kozak sequence (SEQ ID No. 29), and a RABV-G coding sequence (SEQ ID No. 5-13), followed by the TGATGA double stop codon.

[0047] Linear RNA that can be circularized to form the circular RNA (circRNA) disclosed in this patent can be prepared using standard laboratory methods and materials. The cDNA sequence encoding the linear RNA can be synthesized using de novo DNA synthesis technology. Gene synthesis can be ordered from Ansonda Life Sciences. The nucleic acid sequence encoding the linear RNA sequence can be cloned into a pUC57 plasmid vector containing a T7 promoter, which has restriction enzyme sites flanking the multiple cloning site, such as SpeI and XhoI. The resulting plasmid can be transformed into chemically competent E. coli. In this example, Trans1-T1 phage-resistant chemically competent cells CD501 are used. The transformation is performed according to the instructions, using 100 ng of plasmid. The procedure is as follows: a. Thaw a tube of competent CD501 cells on ice for 10 minutes.

[0048] b. Add 10 μL of the ligation product to the competent cells, and gently tap the centrifuge tube 4-5 times to thoroughly mix the competent cells and DNA. Do not vortex.

[0049] c. Place the mixture on ice for 30 minutes. Do not stir.

[0050] d. Heat shock at 42°C for 45 seconds. Do not stir.

[0051] e. Place on ice for 2 minutes. Do not stir.

[0052] f. Transfer 800 μL of LB medium stored at room temperature to the mixture.

[0053] g. Place at 37 ℃ for 60 minutes. Shake vigorously (180 rpm).

[0054] h. Heat solid culture plates containing screening antibiotics at 37 °C.

[0055] i. Mix the cells by gently tapping the centrifuge tube and inverting it.

[0056] j. Spread 200 μL of each dilution onto a solid culture medium plate containing the screening antibiotic and incubate overnight at 37°C. k. Select single clones and inoculate them into 5 mL of LB growth medium containing kanamycin. Incubate at 37 °C with vigorous shaking (approximately 220 rpm) for approximately 8 hours. Dilute the starting culture 1 / 1000 in 200 mL of LB medium. Incubate at 37 °C with vigorous shaking (approximately 220 rpm) for 16 hours. Perform large-scale plasmid extraction using an endotoxin-free plasmid extraction kit (QIAGEN, 12362) according to the manufacturer's instructions.

[0057] (2) In vitro transcription and RNA circularization To generate a linearized plasmid DNA template for in vitro transcription (ITT), the plasmid was first linearized using restriction endonucleases (e.g., BspQI, Vazyme, DD4302-PC-02). A typical BspQI restriction endonuclease digestion system contained the following components: 1.0 μg plasmid; 1.0 μL 10× buffer; up to 10 μL ddH2O; incubation at 50°C for 3 hours. The linearized plasmid was purified using a DNA purification and concentration kit (ZYMO, D4034). After purification, the linearized plasmid was quantitatively analyzed using Nanodrop, and the linearization effect was confirmed by agarose gel electrophoresis.

[0058] Unmodified linear RNA was synthesized in vitro from linearized plasmids using the T7 High-Yield RNA Transcription Kit (Vazyme, DD4101-PC-03) according to the kit instructions. DNase I (Vazyme, DD4104-PC-02) was then added, and the mixture was incubated at 37°C for 15 minutes to remove DNA from the IVT transcripts. The transcribed RNA was then purified using an RNA purification and concentration kit (ZYMO, R1018).

[0059] The transcribed linear RNA was treated with T4 RNA ligase (Kactus, TRL-BE103-C1) at 25 °C for 2 hours to generate a clip-ligated circular RNA precursor, which was then purified. The circular RNA was further enriched by treatment with RNase R (Novoprotein, GMP-E224-M001) at 37 °C for 30 minutes. The purified circular RNA product was assessed for RNA quality by agarose gel or denaturing urea gel electrophoresis.

[0060] Example 2: In vitro production of circRNA via type I introns This embodiment demonstrates the in vitro generation of circular RNA via type I intron autocatalysis.

[0061] (1) Design and synthesis of circRNA encoding RABV-G protein fragment Design a linear RNA capable of circularization to form a circular RNA, the linear RNA containing the CVB3IRES-Kozak-RABV-G-opt sequence from 5' to 3', such as... Figure 1 As shown in b, the linear RNA was designed from 5' to 3' with the coding sequences IRES (SEQ ID No. 30), Kozak (SEQ ID No. 29), and RABV-G (SEQ ID No. 1-4), which were then linked together to form type I intron RABV-G precursor RNA (SEQ ID No. 14-17), followed by the TGATGA double stop codons. Subsequently, the DNA fragment encoding the RABV-G sequence was cloned into the corresponding vector.

[0062] De novo synthesized cDNA sequences encoding linear RNA can be cloned into plasmid vectors containing a T7 promoter, with restriction endonuclease sites flanking the multiple cloning site, such as SpeI and AgeI. The resulting plasmids can be transformed into chemically competent *E. coli*. For plasmid isolation, large-scale extraction can be performed using the Qiagen endotoxin-free plasmid extraction kit, following the manufacturer's instructions.

[0063] (2) In vitro transcription and RNA circularization To generate a linearized plasmid DNA template for in vitro transcription, the plasmid was first linearized using a restriction endonuclease, such as PmeI (NEB, R0560). A typical PmeI restriction endonuclease digestion system contains the following components: 2.0 μg plasmid; 1.0 μL 10× buffer; 1.0 μL PmeI; up to 10 μL ddH2O; incubation at 37 °C for 3 hours. The linearized plasmid was purified using a DNA purification and concentration kit (ZYMO, D4034). After purification, the linearized plasmid was quantitatively analyzed using Nanodrop, and the linearization effect was confirmed by agarose gel electrophoresis.

[0064] Unmodified linear RNA was synthesized in vitro from linearized plasmids using the T7 High-Yield RNA Transcription Kit (Vazyme, DD4101-PC-03) according to the kit instructions. DNase I (Vazyme, DD4104-PC-02) was then added, and the mixture was incubated at 37°C for 15 minutes to remove DNA from the IVT transcripts.

[0065] RNA was circularized and purified in vitro. To further obtain circular RNA, the RNA product was treated at 55 °C for 15 min. The circular RNA product was purified using an RNA purification and concentration kit (ZYMO, R1018). The circular RNA was further enriched by treatment with RNase R (Novoprotein, GMP-E224-M001) at 37 °C for 30 min. The purified circular RNA product was assessed for RNA quality by agarose gel or denaturing urea gel electrophoresis.

[0066] Example 3: Expression of circRNA-encoded RABV-G in human HEK293T cells This example demonstrates the ability of circRNA to express proteins in eukaryotic cells. The RABV-G protein can be expressed and secreted in cells transfected with circRNA. Purified circRNA (treated with RNase R) was transfected into human HEK293T cells using Lipofectamine MessengerMAX transfection reagent (Thermo Fisher, LMRNA003).

[0067] Twenty-four hours after transfection, cell lysates and cell culture supernatants were collected for Western blot analysis. Detection was performed using RABV-G antibody (CUSABIO, CSB-PA14899A0Rb). Western blot results showed that the circRNA encoding RABV-G could efficiently express the RABV-G antigen in cells. Figure 3Both circRNAs obtained via T4 RNA ligase and those obtained via type I intron autocatalysis can express RABV-G protein in cell cleavage. GAPDH was used as a control to standardize the protein loading amount.

[0068] Example 4: Preparation of LNP-circRNA particles and in vivo immunization regimen in mice Purified circRNA was encapsulated in lipid nanoparticles (LNPs) and administered to animals via intramuscular injection.

[0069] (1) Circular RNA was encapsulated with lipid nanoparticles (LNPs) according to the following description.

[0070] First, the circRNA was diluted to a final concentration of 114 ng / μL using PNI Formulation Buffer (Precision NanoSystems, NWW0043). Then, LNPs were prepared as follows: DLin-MC3-DMA or SM102, DSPC, DSPE-PEG2k, and cholesterol were dissolved in ethanol. The above lipid mixture was further mixed with the circRNA solution at a 1:3 volume ratio using a NanoAssemblr Ignite (Precision NanoSystems) via an Ignite NxGen Cartridge (Precision NanoSystems, NIT0002). The LNP-circRNA preparation was then diluted 5-fold with PBS buffer (pH 7.2–7.4) in dialysis tubing (Henghuibio, HF132576), dialyzed overnight, and then concentrated using an Amicon® Ultra centrifugal filter (Millipore). The concentration and encapsulation efficiency of circRNA were determined using the Quant-it RiboGreen RNA Assay Kit (Invitrogen, R11490). The size of LNP-circRNA particles was determined using a Malvern Zetasizer Nano-ZS300 (Malvern) dynamic light scattering (DLS) instrument. The samples were illuminated with a red laser, and the scattered light was detected. The results were analyzed using Zetasizer V7.13 software to obtain relevant data.

[0071] (2) Immunization of BALB / c mice (Animal immunization regimen 1) A circRNA vaccine encoding the RABV-G protein (LNP-circRNA) was administered intramuscularly to BALB / c mice. Thirty-nine 8-week-old female BALB / c mice were randomly assigned to 10 groups (n=3 / 4 per group): a placebo (PBS) group, a low-dose group (0.3 μg) of circRNA T4-RABV-G-opti 2, T4-RABV-G-opti 5, and T4-RABV-G-opti 6, a medium-dose group (1 μg) of circRNA T4-RABV-G-opti 2, T4-RABV-G-opti 5, and T4-RABV-G-opti 6, and a high-dose group (3 μg) of circRNA T4-RABV-G-opti 2, T4-RABV-G-opti 5, and T4-RABV-G-opti 6. Immunization was administered in two doses, two weeks apart, starting from week 0. Mouse serum was collected at weeks 0, 2, 3, 4, 6, and 8 (Table 1).

[0072] Table 1: Animal Immunization Program 1 (3) Immunization of BALB / c mice (Animal immunization regimen 2) To better evaluate the efficacy of the RABV-G circRNA vaccine, we selected circRNAs T4-RABV-G-opti2, T4-RABV-G-opti5, and T4-RABV-G-opti9 and compared them with the human purified Vero cell rabies vaccine Speeda (Chengda Biotechnology). The RABV-G circRNA vaccine was administered intramuscularly to BALB / c mice. Thirty-five 8-week-old female BALB / c mice were randomly assigned to seven groups (n=5 per group): placebo (PBS), circRNAs T4-RABV-G-opti2, T4-RABV-G-opti5, T4-RABV-G-opti7, T4-RABV-G-opti8, and T4-RABV-G-opti9 (3 μg), and Speeda (0.25 IU). A two-dose immunization regimen was used, two weeks apart, starting from week 0. Mouse serum was collected at weeks 0, 2, 3, 4 and 5 (Table 2).

[0073] Table 2: Animal Immunization Program 2 (4) Immunization of BALB / c mice (Animal immunization regimen 3) To further evaluate the RABV-G circRNA vaccine, we selected circRNAs T4-RABV-G-opti 2 and T4-RABV-G-opti 9 and compared them with the rabies vaccine Speeda. The RABV-G circRNA vaccine was administered intramuscularly to BALB / c mice. Thirty-five 8-week-old female BALB / c mice were randomly assigned to six groups (n=5 per group): placebo (PBS), circRNA T4-RABV-G-opti 2 (MC3, 3 μg), T4-RABV-G-opti 9 (MC3, 1, 3, 10 μg), and Speeda (0.25 IU). A two-dose immunization regimen was used, one week apart, starting from week 0. Serum samples were collected from mice at weeks 0, 2, and 3 (Table 3).

[0074] Table 3: Animal Immunization Program 3 Example 5: The RABV-G specific antibody response was measured using enzyme-linked immunosorbent assay (ELISA), and the viral neutralizing antibody (VNA) titer was determined using rapid fluorescent focus inhibition assay (RFFIT). A 96-well plate (Corning, 9018) was coated overnight at 4 °C with 3 ng / μL rabies virus glycoprotein G (ACRO Biosystems, RAG-V55H5) in ELISA coating buffer (50 μL / well, Coolaber, SL1337-500ML). The plate was then blocked for 1 hour at room temperature with PBS + 5% skim milk + 0.05% Tween 20. 100 μL of serum diluted with PBS + 5% skim milk was added to the corresponding wells and incubated for 2 hours at room temperature. The plate was then washed four times, and horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin IgG was added at a 1:10000 dilution to PBS + 0.05% Tween 20 and incubated for 1 hour at room temperature. After washing, the sample was developed with TMB substrate (Solarbio, PR1200) at 37 °C for 40 minutes, and absorbance was measured at 450 nm and 630 nm using a microplate reader (Tecan Infinite 200 Pro). The endpoint titer was calculated as the dilution factor at which the optical density (OD) value was higher than 4.1 times the background value. IgG titers below 100 were defined as 100. The specificity of the signal was confirmed by measuring pre-immune serum (background signal).

[0075] Virus neutralizing antibody (VNA) titers were determined using the Rapid Fluorescent Focal Inhibition Assay (RFFIT) from Zhongke Shisheng (Beijing) Pharmaceutical Technology Co., Ltd. Standard serum (0.5 IU / mL) and test serum samples were prepared in 96-well plates, each diluted 3-fold serially, and mixed with 100 TCID50 (tissue culture infection dose) of CVS-11 (50 μL / well). The mixture was then incubated at 37 °C in a 5% CO2 incubator for 1 hour. Next, 50 μL of a suspension containing BSR cells was added to the mixture, and incubation continued at 37 °C for 24 hours. Cells were first fixed with 80% acetone at 4 °C for 30 minutes, followed by staining with FITC-labeled RABV-N antibody. Fluorescence was observed under a microscope, and VNA titers were measured and compared with standard serum values ​​for standardization.

[0076] Following vaccination with 0.3 μg, 1 μg, or 3 μg doses of circRNA vaccine, IgG antibody titers in mouse serum were measured by ELISA at different time points in weeks 3 and 4. The results showed that 0.3 μg, 1 μg, and 3 μg doses of circRNA vaccine maintained high levels of IgG antibody titers in both weeks 3 and 4 post-vaccination, indicating that the circRNA vaccine encoding RABV-G can generate high titers of RABV-G-specific IgG antibodies in a dose-dependent manner. Figure 3 The pre-fusion conformation of RABV-G displays known major neutralizing antibody epitopes and can serve as an ideal antigen. The H270P point mutation stabilizes the pre-fusion conformation of RABV-G. The results also indicate that the H270P mutant protein produces higher IgG antibody titers than the wild-type or fusion proteins containing the tPA signal peptide.

[0077] The Pre1-3 circRNAs encoding the pre-fusion conformation protein were designed to stabilize the pre-fusion conformation of RABV-G by replacing the helix and linker sequences of the spike protein (e.g., SARS-CoV-2). Results from IgG antibody titer assays in immunized mouse serum showed that the circRNA vaccine encoding the pre-fusion conformation RABV-G produced high titers of specific IgG antibodies, with the IgG antibody titer five weeks after vaccination being 5.9 times that of the Speeda rabies vaccine. Figure 4 FAVN test results showed that immunization of mice with 3 μg of circRNA encoding wild-type or pre-fusion Pre-3 protein achieved neutralizing antibody levels higher than the WHO-defined antibody threshold concentration (0.5 IU / mL). Figure 5As shown in the figure. This threshold is also considered an acceptable level of protection after human rabies vaccination. CircRNA vaccines encoding the pre-fusion conformation RABV-G protein produced 4.7 times higher neutralizing antibody titers than the Speeda rabies vaccine, demonstrating better protective efficacy.

[0078] When LNP-circRNA was prepared using DLin-MC3-DMA (Table 3), IgG antibody titers in mouse serum were monitored by ELISA at weeks 2 and 3 after vaccination with 1 μg, 3 μg, or 10 μg of circRNA vaccine encoding the Pre3 protein, and 3 μg of circRNA vaccine encoding the wild-type protein. The results showed that the pre-fusion conformational Pre3 design could generate high titers of specific IgG antibodies in a dose-dependent manner. The antibody titer produced by the 3 μg circRNA vaccine encoding the Pre3 protein (prepared using DLin-MC3-DMA) was 3.8 times higher than that of the Speeda rabies vaccine. Figure 6 FAVN test results showed that immunization of mice with 3 μg of circRNA encoding wild-type or 1 μg, 3 μg, or 10 μg of circRNA encoding the pre-fusion conformation Pre-3 protein resulted in neutralizing antibody levels higher than the antibody threshold concentration (0.5 IU / mL), and the resulting neutralizing antibody titers were more than three times those of the Speeda rabies vaccine. Figure 7 Furthermore, circRNA vaccine administration can effectively induce the production of neutralizing antibodies 7 days after the second immunization in mice.

[0079] Table 4. Sequence information of CTN-1 rabies virus glycoprotein (RABV-G) (GenBank: ACR39382.1)

Claims

1. A circular RNA comprising: Circulation control element; An antigen encoding element that encodes the amino acid sequence of a rabies virus glycoprotein, wherein the rabies virus glycoprotein is a wild-type or modified rabies virus glycoprotein. Kozak sequences; and Double stop codon, Optionally, the components are connected to each other via connectors.

2. The circular RNA according to claim 1, wherein the modification of the modified rabies virus glycoprotein is selected from: introducing a T4 foldon sequence, introducing an H270P point mutation, introducing a tPA signal peptide at the N-terminus, or introducing a helix sequence containing the SARS-COV-2 spike protein at the C-terminus.

3. The circular RNA according to claim 1 or 2, wherein the sequence of the antigen-coding element is selected from SEQ ID NO: 1-13; preferably, wherein the sequence of the antigen-coding element is SEQ ID NO: 3, 4, 7, 8, 9, 10, 11, 12 or 13; more preferably, wherein the sequence of the antigen-coding element is SEQ ID NO: 9 or 13.

4. The circular RNA according to any one of claims 1-3, wherein the circularization regulatory element comprises: Internal ribosome entry site (IRES) or a fragment thereof, or internal ribosome entry site (IRES) and type I intron; The internal ribosome entry site (IRES) or a fragment thereof is selected from the group consisting of: Coxsackievirus B3 (CVB3) IRES, Enterovirus 71 (EV71) IRES, Encephalomyocarditis Virus (EMCV) IRES, Picornavir Virus (PV) IRES, Hepatitis C Virus (HCV) IRES, Adenovirus (AdV) IRES, Human Papillomavirus 31 (HPV31) IRES, Human Herpesvirus (HHV) IRES, Rous Sarcoma Virus (RSV) IRES, Classical Swine Fever Virus (CSFV) IRES, FGF9 IRES, SLC7A1 IRES, and RUNX1 IRES; preferably, the internal ribosome entry site (IRES) or a fragment thereof is a CVB3 IRES; more preferably, the sequence of the internal ribosome entry site (IRES) or a fragment thereof is SEQ ID NO: 30; The type I introns include: 5'-intron, Exon2, Exon1 and 3'-intron; preferably, the type I introns are selected from the group consisting of: SEQ ID NO: 33-36.

5. The circular RNA according to any one of claims 1-4, wherein the Kozak sequence is SEQ ID NO:

29.

6. The circular RNA according to any one of claims 1-5, wherein the double stop codon is TGATGA.

7. The circular RNA according to any one of claims 1-6, wherein the amino acid sequence expressed by the linker is selected from: GA, GS, GG, GGA, GGS, GGG, GGGA, GGGS, GGGG, GGGGA, GGGGS, GGGGG, GGGGA, GGGGS, GGGGG, GGAG, GGSG, AGGGG, SGGG, GAGA, GSGSGS, GAGAGAGA, GSGSGSGS, GAGAGAGAGA, GSGSGSGS, GAGAGAGAGA, GSGSGSGSGS, GAGAGAGAGAGA, GAGSGSGSGSGSGS, GGAGGA, GGSGGS, GGAGGAGGA, GGSGGSGGS, GGAGGAGGAGGA, GGSGGSGGSGGS, GGAGGGAG, GGSGGGSGGSG, GGAGGGAGGAGGAG, GGSGGGSGGSG, GGGAGGGAGGAGGA or GGGSGGGSGGSGGS; preferably, the amino acid sequence expressed by the linker is GS.

8. The circular RNA according to any one of claims 1-7, wherein the amino acid sequence of the rabies virus glycoprotein is selected from SEQ ID NO: 22-28; preferably, the amino acid sequence of the rabies virus glycoprotein is SEQ ID NO: 23, 26, 27 or 28.

9. The circular RNA according to any one of claims 1-8, wherein The circular RNA is obtained by circularization of a linear RNA precursor as shown in SEQ ID NO: 31 or SEQ ID NO: 32 using T4 RNA ligase; or The circular RNA is obtained by autocatalysis from a linear RNA precursor as shown in SEQ ID NO:14-17.

10. A composition comprising the circular RNA according to any one of claims 1-9 and a pharmaceutically acceptable carrier or excipient.

11. The composition of claim 10, wherein the composition is lipid nanoparticles; preferably, wherein the lipid nanoparticles comprise: One or more of the following: cationic lipids, neutral lipids, polyethylene glycol lipids, and steroidal lipids.

12. Use of the circular RNA according to any one of claims 1-9 or the composition according to any one of claims 10-11 in the preparation of a vaccine for the prevention of rabies.

Images