Engineered circular RNA CircH19 and application thereof in preparation of anti-glioma drugs

Abstract

The invention belongs to the field of biological medicine, and relates to an engineered circular RNA CircH19 and application thereof in preparation of a drug for resisting brain glioma. The engineered circular RNA CircH19 provided by the invention can be used as an immunogen component of a vaccine after being successfully cyclized, can effectively activate the anti-tumor immune response of a body through intramuscular injection, prolongs the life of the body, reduces the volume of brain mass tumor, provides an effective scheme for precise treatment of brain glioma, and has a wide application prospect. Wide application prospects are realized in the aspect of brain glioma treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to an engineered circular RNA CircH19 and an application thereof in preparing an anti-glioma drug. Background Art

[0002] Since 2012, with the development of high-throughput sequencing technology and bioinformatics algorithms, researchers have discovered a large number of circular RNA molecules (Circular RNA, CircRNA) in organisms. Circular RNA plays an important role in many life processes such as human diseases and pathogenesis. The functions of circular RNA are also diverse. For example, circular RNA can interact with micro-miRNA and protein molecules, acting as a sponge for miRNA and forming complexes with proteins to play a regulatory role. In addition, natural circular RNA in organisms can also be used as a translation template to directly translate functional proteins.

[0003] Circular RNA has no exposed 5' and 3' ends and can resist RNA exonuclease cleavage. Compared with linear mRNA, the closed structure of circular RNA makes it stable in vivo. Through engineered element sequence design, the ability of circular RNA to translate proteins can be greatly improved, making it possible to use circular RNA as an antigen expression vector to develop disease therapeutic vaccines.

[0004] In 1984, Pachnis et al. cloned and discovered the H19 gene in a mouse fetal liver cDNA library (V Pachnis.etc, doi: 10.1073 / pnas.81.17.5523, PMID: 6206499, PNAS); in 1990, Brannan et al. first reported that H19 could not translate proteins and played a biological function in the form of non-coding RNA. Since the length of the H19 product exceeded 200nt, it was defined as LncRNA (CI Brannan.etc, doi: 10.1128 / mcb.10.1.28-36.1990, PMID: 1688465, Mol Cell Biol). LncRNA H19 is one of the earliest discovered long non-coding RNAs, and plays an important role in various biological functions such as growth and development, tumorigenesis, and development. Summary of the invention

[0005] In one aspect, the present invention provides a recombinant nucleic acid molecule comprising:

[0006] Regulatory element sequences, and

[0007] Coding sequence;

[0008] The coding sequence encodes the amino acid sequence shown in SEQ ID NO:18.

[0009] In some embodiments, the regulatory element sequence comprises: an intron self-splicing ribozyme cyclization sequence, an intron sequence, a 3'UTR sequence, an IRES sequence, a spacer sequence, and / or a kozac sequence.

[0010] In some embodiments, the intron self-splicing ribozyme cyclization sequence comprises the nucleotide sequence shown in SEQ ID NO: 8, and / or SEQ ID NO: 17; in some embodiments, the intron sequence comprises the nucleotide sequence shown in SEQ ID NO: 9, and / or SEQ ID NO: 16; in some embodiments, the 3'UTR sequence comprises the nucleotide sequence shown in SEQ ID NO: 15; in some embodiments, the IRES sequence comprises the nucleotide sequence shown in SEQ ID NO: 12; in some embodiments, the spacer sequence comprises the nucleotide sequence shown in SEQ ID NO: 10; in some embodiments, the kozac sequence comprises the nucleotide sequence shown in SEQ ID NO: 13; in some embodiments, the coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 14.

[0011] In one aspect, the present invention provides a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises the following sequences operably linked from the 5' end to the 3' end:

[0012] The first intron self-splicing ribozyme cyclization sequence, 5' intron sequence, spacer sequence, IRES sequence, kozac sequence, coding sequence, 3' UTR sequence, 3' intron sequence, second intron self-splicing ribozyme cyclization sequence; the coding sequence encodes the amino acid sequence shown in SEQ ID NO:18.

[0013] In some embodiments, a restriction site sequence is further included between the spacer sequence and the IRES sequence. By introducing the restriction site sequence, replacement between different elements or different sequences of the same element becomes possible.

[0014] In some embodiments, the restriction site is one or more of XbaI, EcoRI, BamHI, NheI, and Hind III restriction sites; in some embodiments, the sequence of the restriction site comprises the nucleotide sequence shown in SEQ ID NO: 11.

[0015] In some embodiments, the sequence of the recombinant nucleic acid molecule is shown in SEQ ID NO:4.

[0016] In one aspect, the present invention provides a recombinant expression vector comprising the recombinant nucleic acid molecule.

[0017] In one aspect, the present invention provides a circular RNA formed by cyclization of the recombinant nucleic acid molecule or the recombinant expression vector.

[0018] In some embodiments, the circular RNA comprises a spacer sequence, a restriction site sequence, a coding region sequence, an IRES sequence, a kozac sequence, a coding sequence, and a 3'UTR sequence.

[0019] In some embodiments, the sequence of the circular RNA is as shown in SEQ ID NO:7.

[0020] In one aspect, the present invention provides the use of the recombinant nucleic acid molecule, the recombinant expression vector, or the circular RNA in the preparation or as a vaccine.

[0021] In one aspect, the present invention provides a vaccine comprising the recombinant nucleic acid molecule, the recombinant expression vector, or the circular RNA.

[0022] In some embodiments, the vaccine is a lipid nanoparticle complex comprising the circular RNA.

[0023] On the one hand, the present invention provides the use of the circular RNA or the vaccine in the preparation of a drug for treating brain glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the immunoprecipitation mass spectrometry identification map of H19-256aa binding to HLA-I.

[0025] Figure 2 Design schematic for engineering CircH19 circular RNA vaccine preparation.

[0026] Figure 3 The results of agarose gel electrophoresis of CircH19 obtained after in vitro transcription and circularization. M represents the molecular weight marker. Lane 1 is the agarose gel electrophoresis band of the product of transcription circularization reaction at 37°C for 1 hour, and lane 2 is the agarose gel electrophoresis band of the product of transcription circularization reaction at 37°C for 1 hour and then at 42°C for 2 hours.

[0027] Figure 4 The agarose gel electrophoresis results of the purified CircH19 products without and with RNase R exonuclease treatment. M is the molecular weight marker. Lane 1 is without RNase R, and lane 2 is with RNase R.

[0028] Figure 5 This is the sequencing result of the circularization interface after CircH19 circular RNA was reverse transcribed into cDNA. The black dotted line is the circularization interface position.

[0029] Figure 6 The figure shows the results of detecting the integrity of circular RNA using capillary electrophoresis Agilent 2100.

[0030] Figure 7 This is a graph showing the purity of circular RNA detected by size exclusion high performance liquid chromatography (SEC-HPLC).

[0031] Figure 8 This is the purified LNP-CircH19 solution.

[0032] Fig. 9 The results show that the engineered CircH19 vaccine can effectively activate the anti-tumor immunity of mice in vivo. (a) shows the level of IFN-γ secretion of T cells under different treatments detected by IFN-γ ELISpot; (b) shows the detection of MDSCs and TAMs, which inhibit tumor immunity, in the tumor microenvironment of the vaccine-injected and non-vaccine-injected groups; (c) shows the detection of CD8+, an anti-tumor component in the tumor microenvironment of the vaccine-injected and non-vaccine-injected groups. + T, CD4 + T, TNF-α and IFN-γ detection. vector-vac represents empty vaccine, circH19-vec represents circH19 vaccine, and PMA+Iono (phorbol 12-myristate 13-acetate + ionomycin) treatment is used as positive control. p<0.05 indicates statistically significant difference.

[0033] Fig.10 The therapeutic effect of engineered CircH19 vaccine on tumor mice. (a) shows the survival of mice in the vaccine group and the non-vaccine group; (b) shows the tumor volume of mice in the vaccine group and the non-vaccine group, and the scale bar in the figure is 1mm. vector-vac represents empty vaccine, and circH19-vec represents circH19 vaccine. p=0.005 indicates that there is a significant statistical difference between the two groups. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0035] Definitions and terminology

[0036] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of" are encompassed within the meaning of the expression "comprising."

[0037] As used in the present invention, the connection term "and / or" between multiple mentioned elements should be understood to include individual and combined options. In other words, "and / or" includes "and" and "or". For example, A and / or B includes A, B and A+B. A, B and / or C include A, B, C and any combination thereof, such as A+B, A+C, B+C and A+B+C. More elements defined by "and / or" are understood in a similar manner and include any one of them and any combination thereof.

[0038] Unless otherwise indicated, any numerical value or numerical range, such as concentration or concentration range, is understood to be modified by the term "about" in any case. Therefore, numerical values ​​generally include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0039] As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions within that range, unless the context clearly dictates otherwise.

[0040] As used herein, the terms "circRNA" or "circular polyribonucleotide" or "circular RNA" or "circular RNA polynucleotide" are used interchangeably and refer to polyribonucleotides that form a circular structure through covalent bonds.

[0041] As used herein, the term "linear RNA" refers to an RNA precursor that is capable of circularizing to form a circular RNA.

[0042] As used in the present invention, the anabaena intron belongs to type I intron. "Type I intron" is a large class of self-splicing ribozymes, which have enzyme catalysis and can self-splicing after being transcribed into RNA. It can catalyze its own excision from mRNA, tRNA and rRNA precursors of various organisms. The splicing process of type I intron actually occurs twice in the transfer of phosphodiester bonds, which is characterized by the need for the participation of guanosine.

[0043] As used in the present invention, "3' intron" refers to the 3' end sequence of a group I intron; "5' intron" refers to the 5' end sequence of a group I intron.

[0044] As used herein, "spacer" refers to a region of a polynucleotide sequence ranging from 1 nucleotide to hundreds or thousands of nucleotides that separates two other elements along the polynucleotide sequence. The sequence may be defined or may be random. The spacer is typically non-coding.

[0045] As used herein, the term "IRES" (Internal ribosome entry site, IRES) is also known as an internal ribosome entry site. "Internal ribosome entry site" (IRES) is a translation control sequence that is usually located at the 5' end of the gene of interest and enables translation of RNA in a cap-independent manner. The transcribed IRES can directly bind to ribosomal subunits to orient the mRNA start codon appropriately in the ribosome for translation. The IRES sequence is usually located in the 5'UTR of the mRNA (just upstream of the start codon). IRES functionally replaces the need for various protein factors that interact with the eukaryotic translation machinery.

[0046] As used herein, the term "untranslated region (UTR)" generally refers to a region (non-coding region) in RNA (such as mRNA) that is not translated into an amino acid sequence, or a corresponding region in DNA.

[0047] As used herein, "coding sequence" or "coding region sequence" refers to a nucleotide sequence in a polynucleotide that can be used as a template for synthesizing a nucleotide sequence having a determined nucleotide sequence (e.g., tRNA and mRNA) or a determined amino acid sequence in a biological process. The coding sequence can be a DNA sequence or an RNA sequence. If the mRNA corresponding to the DNA sequence (including the coding strand identical to the mRNA sequence and the template strand complementary thereto) is translated into a polypeptide in a biological process, it can be considered that the DNA sequence or mRNA sequence encodes the polypeptide.

[0048] As used herein, the term "recombinant nucleic acid molecule" includes polynucleotides having sequences that are not linked together in nature. The recombinant polynucleotide may be included in a suitable vector, and the vector may be used to transform into a suitable host cell. The polynucleotide is then expressed in a recombinant host cell or in vitro to produce, for example, a "recombinant polypeptide," "recombinant protein," "fusion protein," and the like.

[0049] As used herein, the term "recombinant expression vector" includes structures for expressing, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) a transcriptional subunit that is suitable for transcription and translation initiation and termination sequences. The recombinant expression vector may be constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, bacteriophages, and transposons.

[0050] As used herein, "vaccine" refers to a composition comprising an active ingredient (e.g., a recombinant nucleic acid molecule, a recombinant expression vector, a circular RNA of the present invention), which can induce an immune response in a vaccinated subject by vaccination. In a specific embodiment, the immune response induced by it can provide immune protection and is sufficient to prevent and / or alleviate at least one symptom associated with a pathogen or disease infection.

[0051] As used herein, "lipid nanoparticle" or "LNP" refers to a lipid vesicle having a uniform lipid core, which is a particle formed by lipids, and the lipid components undergo intermolecular interactions to form a nanostructured entity. Nucleic acids are encapsulated in the lipids.

[0052] As used in the present invention, "treatment" means: after suffering from a disease, the subject is exposed to (e.g., administered) the recombinant nucleic acid molecule, recombinant expression vector, linear RNA, circular RNA or vaccine containing the same (hereinafter also referred to as "the pharmaceutical composition of the present invention"), thereby alleviating the symptoms of the disease compared to when not exposed, and does not mean that the symptoms of the disease must be completely suppressed. Suffering from a disease includes: the body has symptoms of the disease.

[0053] As used in the present invention, "prevention" means that before contracting the disease, by exposing the subject to (e.g., administering) the recombinant nucleic acid molecule, recombinant expression vector, linear RNA, circular RNA, vaccine, etc. of the present invention, the symptoms after contracting the disease are alleviated compared to when not in contact, and it does not mean that the disease must be completely suppressed.

[0054] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0055] The cell and reagent information involved in the examples are shown in Table 1 and Table 2.

[0056] Table 1 Cell information involved in the implementation column

[0057] Cell lines Cell line name Part Number and Company MES28 Human primary glioma stem cells Provided by JeremyRich Lab GL261 Mouse brain tumor cell lines iCell-m063

[0058] Table 2 Reagent information

[0059]

[0060]

[0061] Example 1 Engineering CircH19 Precursor Linear RNA Assembly Design

[0062] In the inventor's previous research, it was found that the human H19 gene can translate a new functional protein of 256 amino acids, which was named H19-256aa. The whole protein of the human primary glioma stem cell line MES28 was used for HLA-I immunoprecipitation. Through mass spectrometry analysis, it was found that the two underlined peptides in H19-256aa, SEQ ID NO: 1: ASVGSSTW (amino acids 19-26) and SEQ ID NO: 2: TRRGGRGGVNEL (amino acids 173-184), can bind to HLA-I ( Figure 1 ).

[0063] H19-256aa protein sequence information SEQ ID NO: 3

[0064] MGPFQAERARGQGGSTGV ASVGSSTW GGTPGLGQTGTWQGTQDRGVPSCHLTHRNSFSSR

[0065] HRGSSGTAFSGLCRSLEGWRAGRQAVLGELQQDVTRRAKRPREGGPGTLRSKEAAGARPA

[0066] GAGPTRHCGPGSGAERAHSGLGDAVCPHRSAPGLADSTASRGVKGMGRDQ TRRGGRG

[0067] GVNEL SGGRMVQAGVRSAAGGEREALASRARGQGGPRGRRRRSRDRCLSVRAGDEARSPA

[0068] GVDVPTSCRRPRRQVRWT

[0069] Amino acids 1-188 of H19-256aa SEQ ID NO: 18

[0070] MGPFQAERARGQGGSTGVASVGSSTWGGTPGLGQTGTWQGTQDRGVPSCHLTHRNSFSS

[0071] RHRGSSGTAFSGLCRSLEGWRAGRQAVLGELQQDVTRRAKRPREGGPGTLRSKEAAGARP

[0072] AGAGPTRHCGPGSGAERAHSGLGDAVCPHRSAPGLADSTASRGVKGMGRDQTRRGGR

[0073] GGVNELSGGR

[0074] The nucleic acid coding region sequence of amino acids 1-188 of H19-256aa (SEQ ID NO: 18), hereinafter referred to as CircH19-188aa, was used for engineering assembly and in vitro transcription and circularization preparation of circular RNA.

[0075] Design of the linear RNA sequence of engineered CircH19 precursor:

[0076] The IRES sequence, a driving translation element, was added to the 5' end of the nucleic acid coding region (ORF) of CircH19-188aa, the 3'UTR sequence and the Spacer sequence were added to the rear end, and the Anabaena intron self-splicing ribozyme cyclization sequence was added to both ends of the 5' and 3' introns; the full-length sequence of the precursor linear RNA of the engineered CircH19 was 1852 nt (SEQ ID NO: 4), as shown in FIG. Figure 2 Shown is a schematic diagram of the precursor linear RNA and circular RNA structure of the engineered CircH19 of the present invention.

[0077] In SEQ ID NO: 4, the underlined positions 926-1489 are the nucleic acid sequence encoding CircH19-188aa, the IRES sequence positions are 294-919, the 3'UTR sequence positions are 1490-1620, the Spacer sequence positions are 226-287, the 288-293 sequence positions are XbaI restriction enzyme cleavage site sequences, which provide the possibility for subsequent replacement of the IRES sequence, and the 920-925 sequence positions are kozac sequences, in order to enhance protein translation efficiency. The Anabaena intron self-splicing ribozyme cyclization sequence positions are 1-94 and 1749-1852. The 95-225 positions are the 5' intron, and the 1621-1748 positions are the 3' intron.

[0078] Detailed sequence information of the engineered CircH19 circular RNA vaccine preparation and design SEQ ID NO: 4:

[0079] CCAAGCTTTAATACGACTCACTATAGGGTGATGTAAGGACGTACAGATTGCATACTCAAAGGACGTCAGCGTCTCATAGAGCTTGCATTACACGAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGGAAATCCGTTAAAAAAAAAAAAACAAAAAAAAAAAAACAAAAAAAAAAAAATAATTGACTAATCTAGATTAAAACAGCGGATGGGTACCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTACTTCGTACCTTTGTACGCCTGTTCTTCCCATTGTACCCTTCCTGAACTTCCAACCCAAGTAACGTTAGAAGCTCAACATTTAGTACAACAGGAAGCACCACATCCAGTGGTGTTTAGTACAAGCACTTCTGTTTCCCCGGAGCGAGGTATAGGCTGTACCCACTGCCAAAAACCTTTAACCGTTATCCGCCAACCAACTACGTAAAAGCTAGTAGTATTATGTTTTTAACTAGGCGTTCGATCAGGTGGATTTCCCCTCCACTAGTTTGGTCGATGAGGCTAGGGATTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCTTATGCTGGGACGCCTTTTTATAGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCTTGTGTCACAAACCAGTGATGATAAGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCTTATTTTTCTTATTATTGTCTTATGGTCACAGCATATATATAACATATACTGTGATCGCCACC ATGGGCCCGTTCCAGGCAGAAAGAGCAAGAGGGCAGGGAGGGAGCACAGGGGTGG CCAGCGTAGGGTCCAGCACGTGGGGTGGTACCCCAGGCCTGGGTCAGACAGGGACATGGCAGGGGACACAGGACAG AGGGGTCCCCAGCTGCCACCTCACCCACCGCAATTCATTTAGTAGCAGGCACAGGGGCAGCTCCGGCACGGCTTTC TCAGGCCTATGCCGGAGCCTCGAGGGCTGGAGAGCGGGAAGACAGGCAGTGCTCGGGGAGTTGCAGCAGGACGTCA CCAGGAGGGCGAAGCGGCCACGGGAGGGGGGCCCCGGGACATTGCGCAGCAAGGAGGCTGCAGGGGCTCGGCCTGC GGGCGCCGGTCCCACGAGGCACTGCGGCCCAGGGTCTGGTGCGGAGAGGGCCCACAGTGGACTTGGTGACGCTGTA TGCCCTCACCGCTCAGCCCCTGGGGCTGGCTTGGCAGACAGTACAGCATCCAGGGGAGTCAAGGGCATGGGGCGAG ACCAGACTAGGCGAGGCGGGCGGGGCGGAGTGAATGAGCTCTCAGGAGGGAGG TGATAATAGCGCGCGGCCGCCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCACTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATG CTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTCGTGTAATGTAAGTTCTATGAGATGCTGATGTAGAAAGAGTATGTAATTTGTATGTGGAATGTAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAGAATTCGC

[0080] Example 2 In vitro transcription and circularization of engineered CircH19 precursor linear RNA

[0081] The full-length sequence of the engineered CircH19 precursor linear RNA designed in Example 1 was commissioned to Guangzhou Jisai Biotechnology Co., Ltd. to obtain fragments through whole-gene chemical synthesis, and then the fragments were ligated into the pUC19 plasmid using EcoRI and BamHI endonucleases. The plasmid was named pUC19-H19-188aa; the prepared pUC19-H19-188aa was linearized with EcoRI endonuclease, and the linearized plasmid product was purified by phenol / chloroform method.

[0082] Step 1: Add an equal volume of DNA extraction solution (phenol: chloroform: isoamyl alcohol = 25:24:1) to the linearized plasmid product, invert and mix thoroughly. Centrifuge at 16000×g for 5 minutes at room temperature to fully separate the aqueous phase and the organic phase, carefully aspirate the upper aqueous phase and transfer it to a new test tube.

[0083] Step 2: Add 1 / 10 volume of NaAc (3M, pH=5.2) to the aqueous phase obtained in step 1, vortex to mix, add 2 volumes of pre-cooled anhydrous ethanol, vortex to mix, and place at -20°C for 30 min.

[0084] Step 3: Centrifuge at 13200rpm for 5min. When a precipitate is visible at the bottom of the test tube, discard the supernatant. Add 1ml of 70% ethanol to the test tube, centrifuge at 12000g for 2min, carefully discard the supernatant, and leave it uncapped at room temperature to dry.

[0085] Step 4: Add an appropriate amount of double distilled water to dissolve the precipitate and determine the concentration of the plasmid product.

[0086] The in vitro transcription circularization reaction system was prepared according to Table 3, and the mixture was mixed by gently pipetting, and the mixture was reacted at 37°C for 1 h, and then at 42°C for 2 h.

[0087] Table 3 Reaction system for in vitro transcription circularization

[0088]

[0089] Through the above cyclization reaction, the CircH19 circular RNA was prepared in vitro using the engineered CircH19 precursor linear RNA designed in Example 1 as a raw material. The cyclization effect was shown in Figure 3 Lane M is the molecular weight marker of agarose gel electrophoresis, lane 1 is the product of reaction at 37°C for 1 h, and lane 2 is the product of reaction at 37°C for 1 h and then at 42°C for 2 h (where 42°C for 2 h is to promote cyclization and improve the efficiency of cyclization). The electrophoresis results show that the precursor linear RNA of engineered CircH19 can effectively self-circularize in vitro.

[0090] Through Figure 3 The linear precursor RNA and circular RNA corresponding bands of the electrophoresis results were quantitatively analyzed by grayscale analysis, and the calculation method was: cyclization efficiency = circular RNA grayscale / (circular RNA grayscale + linear precursor RNA grayscale). Grayscale quantification showed that the in vitro cyclization efficiency was greater than 80%.

[0091] Example 3 Circular RNA Purification and Validation of Circularization Interface

[0092] Step 1: Purify the CircH19 circular RNA in Example 2 by lithium chloride precipitation method. Prepare 100 μL of reaction system in equal proportion according to the system in Table 3 to obtain a reaction mixture, react at 37°C for 1 hour, and then react at 42°C for 2 hours to obtain a reaction product, add 30 μL of RNase-free H2O and 25 μL of LiCl (8M, RNase-free) to the reaction product, mix by inversion, place at -20°C for at least 30 minutes, centrifuge for 15 minutes, and aspirate the supernatant to obtain a circular RNA precipitate, add 500 μL of pre-cooled 70% ethanol to wash the circular RNA precipitate, centrifuge for 5 minutes, aspirate the supernatant, and dissolve the circular RNA precipitate with 100 μL of sodium citrate to obtain purified circular RNA.

[0093] Step 2: RNase R digestion to identify the circular RNA finally obtained in step 1. Use RNase R (Geneseed, Cat. No: R0301) to digest the linear RNA in the circularized product. React at 37° C. for 5 minutes according to the system in Table 4 below.

[0094] Table 4 Identification system of circular RNA

[0095]

[0096] Step 3: After the reaction in step 2 is completed, the circular RNA is finally purified using the NEB RNA purification kit, and the prepared circular RNA is finally identified. Figure 4 The results showed that RNase R enzyme could effectively digest linear precursor RNA and enrich circular RNA, and circular RNA prepared in vitro could withstand RNase R enzyme cleavage.

[0097] Example 4 Validation of CircH19 circular RNA circularization interface

[0098] For the CircH19 circular RNA purified in Example 3, it was reverse transcribed into cDNA by N6 random primer, and PCR primers for reverse amplification were designed. The primer sequences are shown in Table 5. The PCR product was subjected to Sanger sequencing to detect whether the circularization interface was correct. The circularization interface sequencing results are shown in Figure 5 The results showed that the prepared circular RNA was accurately circularized at the designated circularization position, and the circularization interface was as shown in the underlined portion of SEQ ID NO: 7.

[0099] Table 5 Primer information

[0100]

[0101] CircH19 circular RNA nucleotide sequence information SEQ ID NO: 7:

[0102] GAAATCCGTT CTACGGACTT

[0103] Example 5 CircH19 circular RNA integrity and purity detection

[0104] The integrity of the CircH19 circular RNA purified in Example 3 was detected by capillary electrophoresis using an Agilent 2100 bioanalyzer. Figure 6 The results showed that the integrity of the CircH19 circular RNA prepared in vitro was over 90%, no degradation occurred, and the fragment size was in line with expectations.

[0105] The purity of CircH19 circular RNA purified in Example 3 was detected by size exclusion high performance liquid chromatography (SEC-HPLC). Figure 7 The results showed that the purity of CircH19 circular RNA prepared in vitro was greater than 95%.

[0106] Example 6 CircH19 circular RNA cationic lipid carrier LNP encapsulation

[0107] The lipids used were ALC-0315, DSPC, cholesterol, and ALC-0159. All lipids were dissolved in anhydrous ethanol, and the CircH19 circular RNA encoding CircH19-188aa was dissolved in a sodium acetate buffer solution. The molar ratios of the four lipids were 46.3:9.4:42.7:1.6, and the nitrogen-phosphorus ratio (N / P) was 6. The LNP-CircH19 was prepared using the Myana microfluidics preparation instrument INano L, with a water-lipid flow rate ratio of 3:1.

[0108] The prepared LNP-CircH19 was purified by replacing the solution using a 100 kDa ultrafiltration tube. A certain volume of PBS solution was first added to the ultrafiltration tube, and then the LNP solution was added. The mixture was purified by centrifugation at 4°C and 1300 x g. RNA concentration and LNP encapsulation efficiency were determined by using Triton X-100 as a lysing agent and Thermo Quant-iT TM The RiboGreen RNA Quantification Kit was used to measure the RNA in LNPs, and the encapsulation efficiency was calculated as follows:

[0109]

[0110] The purified LNP-CircH19 solution appears uniformly translucent milky white when observed with the naked eye. Figure 8 As shown, the appearance test meets the standard. The concentration of LNP-CircH19 detected by RiboGreen RNA quantification kit is 0.24μg / μL; the encapsulation efficiency is about 96.83%.

[0111] Example 7 Anti-tumor application of CircH19 encoding CircH19-188aa in mice

[0112] Step 1: Use the constructed H19-256aa expression lentivirus to infect mouse glioma cells GL261 to construct mouse glioma cells GL261 that can stably express human H19-256aa (2021, Xinya Gao etc., Nature Cell Biology, PMID: 33664496).

[0113] Step 2: The mouse brain tumor cell line GL261 that stably expresses human H19-256aa constructed in step 1 was injected into immune-competent mice C57BL / 6 (female 6-8 weeks old) using a 10 μL Hamilton syringe and a stereotaxic apparatus. The injection depth was 3 mm to form an orthotopic tumor in the brain and construct an orthotopic mouse brain transplant tumor model.

[0114] Step 3: On the 6th and 16th days after the mouse brain orthotopic transplant tumor model was constructed in step 2, the mice were divided into a non-vaccine group and a vaccine group. The mice in the vaccine group were intramuscularly injected with LNP-encapsulated CircH19 prepared in Example 6, with 10 μg injected per mouse. The study was approved by the Institutional Review Board of the First Affiliated Hospital of Sun Yat-sen University, with the ethics approval number of Lunshendong

[2023] No. 127.

[0115] Step 4: On the 45th day after the establishment of the mouse brain orthotopic transplant tumor model, 4 mice in the vaccine group and 4 mice in the non-vaccine group were euthanized, and the anti-tumor immunity in the mice was detected. The mouse brain tissue was minced and supplemented with 2 ml of digestion solution (collagenase IV 1 mg / ml, DNase I 20U / ml, hyaluronidase 0.01% digested at 37°C for 30 minutes. After digestion, the cells were filtered through a 70 μm filter and the debris was removed using a debris removal solution (130-109-398, MiltenyiBiotec). Then 3 ml of red blood cell lysis solution was added to the cell pellet for red blood cell lysis, and resuspended in PBS solution for mouse anti-tumor immunity detection.

[0116] Step 5: Perform flow cytometry on the cell pellet obtained in step 4 to detect MDSCs, TAMs, and CD8 + T, CD4 + Changes in cell components and cytokine levels such as T, TNF-α and IFN-γ.

[0117] The results showed that the circular RNA CircH19 encoding CircH19-188aa can effectively activate the anti-tumor immunity of mice in vivo. Under the condition of LNP-CircH19 complex treatment, T cells can effectively secrete IFN-γ ( Fig. 9 a); The proportion of MDSCs and TAMs that inhibit tumor immunity decreased in the vaccine group ( Fig. 9 b), CD8 + T, CD4 + Anti-tumor components such as T, TNF-α and IFN-γ were enhanced in the vaccine group ( Fig. 9 c).

[0118] Example 8 Anti-tumor effect of CircH19 in mice

[0119] The mice with orthotopic brain tumors in Example 7 were observed and weighed daily. Once the mice showed neurological symptoms, including hemiplegia, lameness, etc., lost 20% of their body weight, or were dying, they were humanely euthanized to ensure that the intracranial tumor burden did not exceed ethical limits.

[0120] The results showed that injection of LNP-CircH19 complex into the tumor-transplanted mice in Example 7 could significantly prolong the survival of the tumor-transplanted mice ( Fig.10 a) The survival period of mice in the vaccine group was significantly longer than that in the non-vaccine group. The earliest death occurred on the 37th day in the non-vaccine group, and the earliest death occurred on the 51st day in the vaccine group. On the 56th day, all the mice in the non-vaccine group had died, while 4 / 7 (57%) mice in the vaccine group were still alive (7 mice in each group), with a significant statistical difference between the two groups (p = 0.005). In addition, the tumor volume of mice in the vaccine group was significantly smaller than that in the non-vaccine group ( Fig.10 b).

Claims

1. A recombinant nucleic acid molecule, characterized in that Include: Regulatory element sequences, and Coding sequence; The coding sequence encodes the amino acid sequence shown in SEQ ID NO:

18.

2. The recombinant nucleic acid molecule according to claim 1, wherein The regulatory element sequence comprises: an intron self-splicing ribozyme cyclization sequence, an intron sequence, a 3'UTR sequence, an IRES sequence, a spacer sequence, and / or a kozac sequence; Preferably, the intron self-splicing ribozyme cyclization sequence comprises the nucleotide sequence shown in SEQ ID NO: 8, and / or SEQ ID NO: 17; Preferably, the intron sequence comprises the nucleotide sequence shown in SEQ ID NO: 9, and / or SEQ ID NO: 16; Preferably, the 3'UTR sequence comprises the nucleotide sequence shown in SEQ ID NO: 15; Preferably, the IRES sequence comprises the nucleotide sequence shown in SEQ ID NO: 12; Preferably, the spacer sequence comprises the nucleotide sequence shown in SEQ ID NO: 10; Preferably, the kozac sequence comprises the nucleotide sequence shown in SEQ ID NO: 13; Preferably, the coding sequence comprises the nucleotide sequence shown in SEQ ID NO:

14.

3. A recombinant nucleic acid molecule, characterized in that The recombinant nucleic acid molecule comprises the following sequences which are operably linked from the 5' end to the 3' end: The first intron self-splicing ribozyme cyclization sequence, 5' intron sequence, spacer sequence, IRES sequence, kozac sequence, coding sequence, 3' UTR sequence, 3' intron sequence, second intron self-splicing ribozyme cyclization sequence; the coding sequence encodes the amino acid sequence shown in SEQ ID NO:

18.

4. The recombinant nucleic acid molecule according to claim 3, characterized in that A restriction site sequence is also included between the spacer sequence and the IRES sequence; Preferably, the restriction enzyme cutting site is one or more of XbaI, EcoRI, BamHI, NheI, Hind III restriction enzyme cutting sites; Preferably, the sequence of the restriction site comprises the nucleotide sequence shown in SEQ ID NO:

11.

5. The recombinant nucleic acid molecule according to any one of claims 1 to 4, characterized in that: The sequence of the recombinant nucleic acid molecule is shown in SEQ ID NO:

4.

6. A recombinant expression vector, characterized in that: Comprising the recombinant nucleic acid molecule according to any one of claims 1 to 5.

7. A circular RNA, characterized in that Formed by cyclization of the recombinant nucleic acid molecule according to any one of claims 1 to 5, or the recombinant expression vector according to claim 6; Preferably, the circular RNA comprises a spacer sequence, a restriction site sequence, a coding region sequence, an IRES sequence, a kozac sequence, a coding sequence, and a 3'UTR sequence; Preferably, the sequence of the circular RNA is as shown in SEQ ID NO:

7.

8. Use of the recombinant nucleic acid molecule according to any one of claims 1 to 5, the recombinant expression vector according to claim 6, or the circular RNA according to claim 7 in the preparation or as a vaccine.

9. A vaccine comprising the recombinant nucleic acid molecule according to any one of claims 1 to 5, the recombinant expression vector according to claim 6, or the circular RNA according to claim 7; Preferably, the vaccine is a lipid nanoparticle complex comprising the circular RNA as described in claim 7.

10. Use of the circular RNA according to claim 7 or the vaccine according to claim 9 in the preparation of a medicament for preventing and / or treating brain glioma.

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