An engineered circular RNA CircH19 and application thereof in preparation of a drug against brain glioma

Abstract

The application belongs to the field of biological medicine, and relates to an engineered circular RNA CircH19 and application of the engineered circular RNA CircH19 in preparation of an anti-glioma drug. The engineered circular RNA CircH19 provided by the application can be used as an immunogen component of a vaccine after successful circularization, can be effectively injected into muscles, can effectively activate an anti-tumor immune response of the body, can prolong the life of the body, can reduce the volume of glioma, provides an effective scheme for precise treatment of glioma, and has a wide application prospect in the treatment of glioma.

Description

TECHNICAL FIELD

[0001] The present application 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. BACKGROUND

[0002] Since 2012, with the development of high-throughput sequencing technology and bioinformatics algorithm, researchers have found a large number of circular RNA molecules (Circular RNA, CircRNA) existing in organisms. Circular RNA plays an important role in human diseases and pathogenesis and other life processes. The functional mode of circular RNA is also diversified. For example, circular RNA can interact with micro-miRNA and protein molecules, act as a miRNA sponge and form a complex with protein to play a regulatory role. In addition, the natural circular RNA in the organism can also be directly translated into functional proteins as a translation template.

[0003] Circular RNA does not have 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 the organism. Through the design of the sequence of the engineering element, the protein translation ability of the circular RNA can be greatly improved, so that the circular RNA can be developed as an antigen expression vector to develop a disease therapeutic vaccine.

[0004] In 1984, Pachnis et al. cloned H19 gene from mouse fetal liver cDNA library (VPachnis.etc, doi:10.1073 / pnas.81.17.5523, PMID: 6206499, PNAS); In 1990, Brannan et al. first reported that H19 cannot translate protein, and plays a biological function in the form of non-coding RNA. Since the length of H19 product is more than 200 nt, it is 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-chain non-coding RNAs, and plays an important role in various biological functions such as body growth and development, tumor occurrence and development. SUMMARY

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

[0006] a regulatory element sequence, and

[0007] a coding sequence;

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

[0009] In some embodiments, the regulatory element sequence comprises: an intron self-splicing ribozyme circularization 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 circularization sequence comprises a nucleotide sequence as shown in SEQ ID NO: 8, and / or SEQ ID NO: 17; in some embodiments, the intron sequence comprises a nucleotide sequence as shown in SEQ ID NO: 9, and / or SEQ ID NO: 16; in some embodiments, the 3' UTR sequence comprises a nucleotide sequence as shown in SEQ ID NO: 15; in some embodiments, the IRES sequence comprises a nucleotide sequence as shown in SEQ ID NO: 12; in some embodiments, the spacer sequence comprises a nucleotide sequence as shown in SEQ ID NO: 10; in some embodiments, the kozac sequence comprises a nucleotide sequence as shown in SEQ ID NO: 13; in some embodiments, the coding sequence comprises a nucleotide sequence as shown in SEQ ID NO: 14.

[0011] In one aspect, the present application provides a recombinant nucleic acid molecule comprising, in 5' to 3' order, the following sequences operably linked:

[0012] a first intron self-splicing ribozyme circularization sequence, a 5' intron sequence, a spacer sequence, an IRES sequence, a kozac sequence, a coding sequence, a 3' UTR sequence, a 3' intron sequence, a second intron self-splicing ribozyme circularization sequence; the coding sequence encodes an amino acid sequence as 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, it is possible to replace different elements or different sequences of the same element.

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

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

[0016] On the one hand, the present invention provides a recombinant expression vector comprising the recombinant nucleic acid molecule described above.

[0017] On the one hand, the present invention provides a circular RNA formed by circularization of the recombinant nucleic acid molecule or the recombinant expression vector.

[0018] In some embodiments, the circular RNA comprises a spacer sequence, an enzyme cleavage 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 shown in SEQ ID NO: 7.

[0020] On the one hand, 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] On one hand, 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 containing 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 medicament for treating glioma. Attached Figure Description

[0024] Figure 1 The immunoprecipitation mass spectrometry pattern is used to identify the binding of H19-256aa to HLA-I.

[0025] Figure 2 A schematic diagram of the design for the preparation of an engineered CircH19 circular RNA vaccine.

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

[0027] Figure 4 The agarose gel electrophoresis results of purified CircH19 under two conditions: without RNase R exonuclease treatment 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 The results of sequencing the circular interface of CircH19 circular RNA after reverse transcription into cDNA. The black dotted line position is the circular interface position.

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

[0030] Figure 7 The results of size exclusion high performance liquid chromatography (SEC-HPLC) detecting the purity of circular RNA.

[0031] Figure 8 The solution of LNP-CircH19 after purification.

[0032] Figure 9 The results of the engineered CircH19 vaccine effectively activating the anti-tumor immunity of mice in vivo. (a) shows the T cell IFN-γ secretion level detected by IFN-γ ELISpot under different treatments; (b) shows the detection of MDSCs and TAMs, which are components of tumor immunity in the tumor microenvironment of the vaccine injection and non-vaccine injection groups; (c) shows the detection of CD8 + T, CD4 + The detection of T, TNF-α and IFN-γ. vector-vac represents the empty vaccine, circH19-vec represents the circH19 vaccine, and PMA+Iono (Phorbol 12-myristate 13-acetate + ionomycin) treatment as a positive control. p<0.05 means statistically significant difference.

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

[0034] The technical solutions of the present application are further illustrated by specific examples below, and the specific examples do not represent a limitation on the protection scope of the present application. Some non-essential modifications and adjustments made by others according to the concept of the present application still fall within the protection scope of the present application.

[0035] Definitions and Terminology

[0036] As used herein, the expressions "comprising", "containing", "including" and "having" are inclusive or open-ended and specify the presence of stated elements, steps, or components but do not preclude the presence or addition of one or more other, unstated elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially of does not exclude the presence or addition of additional optional elements, steps, or components, provided that such additional optional elements, steps, or components do not materially affect the basic and novel characteristics of the claimed subject matter. It is to be understood that the expressions "consisting essentially of and "consisting of" are encompassed within the meaning of the expression "comprising".

[0037] As used herein, the conjunctive term "and / or" between elements of a plurality is to be understood as including the options of individually and in combination. In other words, "and / or" includes "and" as well as "or". For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. More elements defined with "and / or" are understood in a similar manner and include any of them and any combination thereof.

[0038] Unless otherwise stated, any numerical values or value ranges, such as concentrations or concentration ranges, are to be understood to be modified by the term "about" in any context. Thus, a numerical value typically includes ±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 a numerical range expressly includes all possible subranges, all individual values within the range, including the integers and fractions within the range, unless the context clearly indicates otherwise.

[0040] As used herein, the term "CircRNA" or "circular polyribonucleotide" or "circular RNA" or "circular RNA polynucleotide" are used interchangeably and refer to a polyribonucleotide that forms a circular structure through a covalent bond.

[0041] As used herein, the term "linear RNA" refers to a RNA precursor that can be circularized to form a circular RNA.

[0042] As used herein, the term "Group I intron" belongs to the type I intron. "Type I intron" is a large class of self-splicing ribozymes with enzymatic catalytic function that can self-splice after being transcribed into RNA. It can catalyze its own removal from the mRNA, tRNA, and rRNA precursors of various organisms. The splicing process of type I intron actually occurs twice, which is characterized by the need for guanosine to participate.

[0043] As used herein, "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 region" refers to a region of a polynucleotide sequence ranging from 1 nucleotide to several hundred or several thousand nucleotides that separates two other elements along the polynucleotide sequence. The sequence can be defined or can be random. The spacer region is typically non-coding.

[0045] As used herein, the term "IRES" (Internal ribosome entry site) also known as internal ribosome entry site, is a translational control sequence, usually located at the 5' end of the gene of interest, and allows the translation of RNA in a cap-independent manner. The transcribed IRES can directly bind ribosomal subunits, so that the mRNA start codon is properly oriented in the ribosome for translation. IRES sequences are usually located in the 5' UTR of the mRNA (immediately upstream of the start codon). The 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 of RNA (e.g., mRNA) that is not translated into an amino acid sequence (non-coding region), or the 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 to synthesize a nucleotide sequence having a defined nucleotide sequence (e.g., tRNA and mRNA) or a defined amino acid sequence in a biological process. A coding sequence can be a DNA sequence or an RNA sequence. A DNA sequence or an mRNA sequence corresponding to a DNA sequence (including the coding strand identical to the mRNA sequence and the template strand complementary to it) can be said to encode a polypeptide if the mRNA corresponding to the DNA sequence is translated into the polypeptide in a biological process.

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

[0049] As used herein, the term "recombinant expression vector" includes a construct for expressing, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector can include, for example, a transcriptional unit comprising i) a collection of genetic elements having a regulatory role on gene expression, such as a promoter and an enhancer; ii) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and iii) appropriate transcriptional and translational initiation and termination sequences. The recombinant expression vector can be constructed in any suitable manner. The nature of the vector is not critical and any vector, including plasmids, viruses, bacteriophages, and transposons, can be used.

[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 application) that elicits an immune response in a vaccinated subject by inoculation. In particular embodiments, the immune response it induces provides immunological protection and is sufficient to prevent and / or reduce at least one symptom associated with infection by a pathogen or disease.

[0051] As used herein, "lipid nanoparticle" or "LNP" refers to a lipid vesicle having a uniform lipid core, which is a particle formed from lipids, the lipid components interact with each other to form a nanostructured entity. Nucleic acids are encapsulated in the lipids.

[0052] As used herein, "treatment" refers to: after the onset of a disease, contacting (e.g., administering) a recombinant nucleic acid molecule, a recombinant expression vector, a linear RNA, a circular RNA, or a vaccine containing the same (hereinafter also referred to as "a pharmaceutical composition of the present application") to a subject, thereby reducing the symptoms of the disease compared to not contacting, and does not mean that it is necessary to completely suppress the symptoms of the disease. Onset of a disease includes: the body has symptoms of the disease.

[0053] As used herein, "prevention" refers to: before the onset of a disease, by contacting (e.g., administering) a recombinant nucleic acid molecule, a recombinant expression vector, a linear RNA, a circular RNA, a vaccine, etc. to a subject, thereby reducing the symptoms after the onset of the disease compared to not contacting, and does not mean that it is necessary to completely suppress the disease.

[0054] The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

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

[0056] Table 1 Information on cells involved in the examples

[0057] Cell Lines Cell Line Name Catalog Number and Company MES28 Human primary glioma stem cells Provided by Jeremy Rich lab GL261 Mouse brain tumor cell line iCell-m063

[0058] Table 2 Reagent information

[0059]

[0060]

[0061] Example 1 Precursor linear RNA assembly design of engineered CircH19

[0062] In the inventors’ previous study, it was found that human H19 gene can translate a novel functional protein of 256 amino acids, which was named H19-256aa. Using whole protein of human primary glioma stem cell line MES28 for HLA-I immunoprecipitation, by mass spectrometry analysis, it was found that two underlined peptide segments in H19-256aa, SEQ ID NO: 1: ASVGSSTW (19-26 amino acids) and SEQ ID NO: 2: TRRGGRGGVNEL (173-184 amino acids) can bind to HLA-I. Figure 1

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

[0064] MGPFQAERARGQGGSTGV ASVGSSTW GGTPGLGQTGTWQGTQDRGVPSCHLTHRNSFSSR

[0065] HRGSSGTAFSGLCRSLEGWRAGRQAVLGELQQDVTRRAKRPREGGPGTLRSKEAAGARPA

[0066] GAGPTRHCGPGSGAERAHSGLGDAVCPHRSAPGAGLADSTASRGVKGMGRDQ TRRGGRG

[0067] GVNEL SGGRMVQAGVRSAAGGEREALASRARGQGGPRGRRRSRDRCLSVRAGDEARSPA

[0068] GVDVPTSCRRPRRQVRWT

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

[0070] MGPFQAERARGQGGSTGVASVGSSTWGGTPGLGQTGTWQGTQDRGVPSCHLTHRNSFSS

[0071] ​RHRGSSGTAFSGLCRSLEGWRAGRQAVLGELQQDVTRRAKRPREGGPGTLRSKEAAGARP

[0072] AGAGPTRHCGPGSGAERAHSGLGDAVCPHRSAPGAGLADSTASRGVKGMGRDQTRRGGR

[0073] GGVNELSGGR

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

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

[0076] The nucleic acid coding region (ORF) of CircH19-188aa is added with a driving translation element IRES sequence at the 5' end direction, a 3'UTR sequence and a Spacer sequence at the rear end, and a fishy algae intron self-splicing ribozyme circularization sequence at both ends of the 5' and 3' introns. The full-length sequence of the precursor linear RNA of the engineered CircH19 is 1852nt (SEQ ID NO: 4), as shown in Figure 2 , which is a schematic diagram of the precursor linear RNA and circular RNA structure of the engineered CircH19 of the present application.

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

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

[0079] CCAAGCTTTAATACGACTCACTATAGGGTGATGTAAGGACGTACAGATTGCATACTCAAAGGACGTCAGCGTCTCATAGAGCTTGCATTACACGAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGGAAATCCGTTAAAAAAAAAAAAACAAAAAAAAAAAAACAAAAAAAAAAAAATAATTGACTAATCTAGATTAAAACAGCGGATGGGTACCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTACTTCGTACCTTTGTACGCCTGTTCTTCCCATTGTACCCTTCCTGAACTTCCAACCCAAGTAACGTTAGAAGCTCAACATTTAGTACAACAGGAAGCACCACATCCAGTGGTGTTTAGTACAAGCACTTCTGTTTCCCCGGAGCGAGGTATAGGCTGTACCCACTGCCAAAAACCTTTAACCGTTATCCGCCAACCAACTACGTAAAAGCTAGTAGTATTATGTTTTTAACTAGGCGTTCGATCAGGTGGATTTCCCCTCCACTAGTTTGGTCGATGAGGCTAGGGATTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCTTATGCTGGGACGCCTTTTTATAGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCTTGTGTCACAAACCAGTGATGATAAGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCTTATTTTTCTTATTATTGTCTTATGGTCACAGCATATATATAACATATACTGTGATCGCCACC ATGGGCCCGTTCCAGGCAGAAAGAGCAAGAGGGCAGGGAGGGAGCACAGGGGTGG CCAGCGTAGGGTCCAGCACGTGGGGTGGTACCCCAGGCCTGGGTCAGACAGGGACATGGCAGGGGACACAGGACAG AGGGGTCCCCAGCTGCCACCTCACCCACCGCAATTCATTTAGTAGCAGGCACAGGGGCAGCTCCGGCACGGCTTTC TCAGGCCTATGCCGGAGCCTCGAGGGCTGGAGAGCGGGAAGACAGGCAGTGCTCGGGGAGTTGCAGCAGGACGTCA CCAGGAGGGCGAAGCGGCCACGGGAGGGGGGCCCCGGGACATTGCGCAGCAAGGAGGCTGCAGGGGCTCGGCCTGC GGGCGCCGGTCCCACGAGGCACTGCGGCCCAGGGTCTGGTGCGGAGAGGGCCCACAGTGGACTTGGTGACGCTGTATGCCCTCACCGCTCAGCCCCTGGGGCTGGCTTGGCAGACAGTACAGCATCCAGGGGAGTCAAGGGCATGGGGCGAG ACCAGACTAGGCGAGGCGGGCGGGGCGGAGTGAATGAGCTCTCAGGAGGGAGG TGATAATAGCGCGCGGCCGCGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCACTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTCGTGTAATGTAAGTTCTATGAGATGCTGATGTAGAAAGAGTATGTAATTTGTATGTGGAATGTAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAATTCGC

[0080] In vitro transcriptional circularization preparation of the precursor linear RNA of the engineered CircH19 of Example 2

[0081] The full-length sequence of the precursor linear RNA of the engineered CircH19 designed in Example 1 was entrusted to Guangzhou Jisai Biological Company to obtain the fragment by full gene chemical synthesis, and then the fragment was connected to the pUC19 plasmid by EcoRI and BamHI endonucleases, and the plasmid was named pUC19-H19-188aa; the prepared pUC19-H19-188aa was linearized by EcoRI endonuclease, and the linearized plasmid product was purified by phenol / chloroform method.

[0082] Step 1: Add an equal volume of DNA extraction liquid (phenol: chloroform: isopentanol = 25:24:1) to the linearized plasmid product, invert and mix thoroughly. Centrifuge at 16000xg at room temperature for 5 min to separate the water phase and the organic phase, and carefully aspirate the upper water phase and transfer it to a new test tube.

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

[0084] Step 3: Centrifuge at 13200 rpm for 5 min, and a precipitate appears at the bottom of the test tube. Discard the supernatant. Add 1 ml of 70% ethanol to the test tube, centrifuge at 12000g for 2 min, carefully discard the supernatant, and place it in room temperature open 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] Prepare the in vitro transcription cyclization reaction system according to Table 3. After gently mixing with a pipette, react at 37°C for 1 hour, and then at 42°C for 2 hours.

[0087] Table 3. Reaction system for in vitro transcription cyclization

[0088]

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

[0090] Through the Figure 3 The linear precursor RNA and circular RNA bands from the electrophoresis results were quantified using grayscale analysis. The calculation method was: circularization efficiency = circular RNA grayscale / (circular RNA grayscale + linear precursor RNA grayscale). Grayscale quantification showed that the in vitro circularization efficiency was greater than 80%.

[0091] Example 3: Purification of circular RNA and Validation of the circularization interface

[0092] Step 1: Purify the CircH19 circular RNA from Example 2 using lithium chloride precipitation. Prepare a 100 μL reaction mixture according to the proportions in Table 3 to obtain the reaction mixture. React at 37°C for 1 h, then at 42°C for 2 h to obtain the reaction product. Add 30 μL of RNase-free H2O and 25 μL of LiCl (8M, RNase-free) to the reaction product, invert and mix well, place at -20°C for at least 30 min, centrifuge for 15 min, aspirate the supernatant to obtain the circular RNA precipitate, add 500 μL of pre-cooled 70% ethanol to wash the circular RNA precipitate, centrifuge for 5 min, aspirate the supernatant, dissolve the circular RNA precipitate in 100 μL of sodium citrate to obtain the purified circular RNA.

[0093] Step 2: RNase R digestion and identification of the circular RNA obtained in Step 1. The linear RNA in the circularized product was digested using RNase R (Geneseed, Cat. No: R0301). The reaction was carried out at 37°C for 5 min according to the system in Table 4 below.

[0094] Table 4 Identification system of circular RNA

[0095]

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

[0097] Example 4 Verification of CircH19 circular RNA cyclization interface

[0098] For the CircH19 circular RNA purified in Example 3, reverse transcription into cDNA was performed by N6 random primer, and PCR primers for reverse amplification were designed. The primer sequences are shown in Table 5. The PCR products were subjected to sanger sequencing to detect whether the cyclization interface was correct. The cyclization interface sequencing results are shown in Figure 5 The results showed that the prepared circular RNA was accurately cyclized at the specified cyclization position, and the cyclization interface was as shown by the underlined part of SEQ ID NO: 7.

[0099] Table 5 Primer information

[0100]

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

[0102] GAAATCCGTTCTACGGACTT

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

[0104] The integrity of the CircH19 circular RNA obtained by purification in Example 3 was detected by capillary electrophoresis Agilent 2100 Bioanalyzer, and the results are shown in Figure 6 , which showed that the integrity of the in vitro prepared CircH19 circular RNA reached more than 90%, and no degradation occurred, and the fragment size was as expected.

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

[0106] Example 6CircH19 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 ratio of the four lipids was 46.3:9.4:42.7:1.6, and the nitrogen-phosphorus ratio (N / P) was 6. The LNP-CircH19 was prepared using the INano L microfluidic preparation instrument, and the water-lipid flow rate ratio was 3:1.

[0108] The prepared LNP-CircH19 was purified by liquid exchange 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. Purification was performed at 4°C and 1300xg. RNA concentration and LNP encapsulation rate determination: Triton X-100 was used as a lysing agent, and the Thermo Quant-iT TM RiboGreen RNA quantification kit was used to determine the RNA in the LNP, and the encapsulation rate was calculated as follows:

[0109]

[0110] The purified LNP-CircH19 solution was observed with the naked eye to be a uniform translucent milky white, as shown in Figure 8 , and the appearance detection met the standard. The LNP-CircH19 concentration was 0.24 μg / μL as detected by the RiboGreen RNA quantification kit, and the encapsulation rate was about 96.83%.

[0111] Example 7 Anti-tumor application of CircH19 in vivo in mice

[0112] Step 1: The method for constructing mouse glioma cell GL261 stably expressing human H19-256aa was constructed by using the constructed H19-256aa expression lentivirus to infect mouse glioma cell GL261 (2021, Xinya Gao et al., Nature Cell Biology, PMID: 33664496).

[0113] Step 2: The mouse brain tumor cell line GL261 stably expressing human H19-256aa constructed in step 1 was injected into immunocompetent mice C57BL / 6 (female, 6-8 weeks old) using a 10 μL Hamilton syringe and a stereotaxic apparatus at a depth of 3 mm to form brain tumors in situ, and a mouse brain orthotopic tumor model was constructed.

[0114] Step 3: On the 6th and 16th day after constructing the mouse brain orthotopic tumor model in step 2, the mice were divided into non-vaccine injection group and vaccine injection group. The mice in the vaccine injection group were injected with LNP-encapsulated CircH19 prepared in Example 6 by intramuscular injection, and each mouse was injected with 10 micrograms. The study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University, and the ethical approval number was Lunsanding

[2023] 127.

[0115] Step 4: On the 45th day after constructing the mouse brain orthotopic tumor model, 4 mice in the vaccine injection group and non-vaccine injection group were euthanized, and the anti-tumor immunity in the mice was detected. The mouse brain tissue was chopped and supplemented with 2 ml of digestion solution (collagenase IV 1 mg / ml, DNase I 20 U / ml, hyaluronidase 0.01% 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, Miltenyi Biotec). 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 anti-tumor immune detection of mice.

[0116] Step 5: The cell pellet obtained in step 4 was subjected to flow cytometry detection to detect the changes in the levels of MDSCs, TAMs, CD8 + T, CD4 + T, TNF-α and IFN-γ, etc. in the mouse brain orthotopic tumor.

[0117] Results show that the circular RNA CircH19 encoding CircH19-188aa can effectively activate the anti-tumor immunity of mice in vivo, and under the treatment of LNP-CircH19 complex, T cells can effectively secrete IFN-γ Figure 9 a); the proportion of MDSCs and TAMs and other tumor immune inhibitors in the injection vaccine group decreases Figure 9 b), CD8 + T, CD4 + T, TNF-α and IFN-γ and other anti-tumor components are enhanced in the injection vaccine group Figure 9 c).

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

[0119] The brain orthotopic tumor mice in Example 7 were observed and weighed every day. Once the mice showed neurological symptoms, including hemiplegia, lameness, etc., 20% weight loss or near death, they were humanely euthanized to ensure that the intracranial tumor burden did not exceed the ethical limit.

[0120] Results show that injection of LNP-CircH19 complex into the grafted tumor mice in Example 7 can significantly prolong the survival of the grafted tumor mice Figure 10 a), the survival of mice in the injection vaccine group is significantly longer than that in the non-injection group, the earliest death of mice in the non-injection vaccine group occurred on day 37, and the earliest death of mice in the injection vaccine group occurred on day 51. By day 56, all mice in the non-injection vaccine group had died, while 4 / 7 (57%) mice in the injection vaccine group were still alive (7 mice were counted in each group, respectively), and there was a significant statistical difference between the two groups (p=0.005). And the tumor volume of mice in the injection vaccine group was significantly smaller than that in the non-injection vaccine group Figure 10 b).

Claims

1. A recombinant nucleic acid molecule, characterized in that, The sequence of the recombinant nucleic acid molecule is set forth in SEQ ID NO:

4.

2. The recombinant nucleic acid molecule of claim 1, wherein, comprising an intron self-splicing ribozyme circularization sequence, the intron self- splicing ribozyme circularization sequence set forth in SEQ ID NO: 8 and SEQ ID NO:

17.

3. The recombinant nucleic acid molecule of claim 1, wherein, comprising an intron sequence, the intron sequence set forth in SEQ ID NO: 9, and / or SEQ ID NO:

16.

4. The recombinant nucleic acid molecule of claim 1, wherein, comprising a 3' UTR sequence, the 3' UTR sequence set forth in SEQ ID NO:

15.

5. The recombinant nucleic acid molecule of claim 1, wherein, comprising a spacer sequence, the spacer sequence set forth in SEQ ID NO:

10.

6. The recombinant nucleic acid molecule of claim 1, wherein, comprising a kozac sequence, the kozac sequence being GCCACC.

7. The recombinant nucleic acid molecule of claim 1, wherein, comprising a coding sequence, the coding sequence set forth in SEQ ID NO:

14.

8. The recombinant nucleic acid molecule of claim 1, wherein, comprising a restriction site sequence, the restriction site sequence being TCTAGA.

9. The recombinant nucleic acid molecule of claim 7, wherein, the coding sequence encodes an amino acid set forth in SEQ ID NO:

18.

10. A recombinant expression vector, characterized in that, comprising the recombinant nucleic acid molecule of claim 1.

11. A circular RNA, characterized in that, the sequence of the circular RNA is set forth in SEQ ID NO:

7.

12. A lipid nanoparticle complex, characterized in that, comprising the circular RNA of claim 11.

13. A vaccine comprising a polynucleotide of claim 1. comprising the circular RNA of claim 11 or the lipid nanoparticle complex of claim 12.

14. Use of the circular RNA of claim 11, or the lipid nanoparticle complex of claim 12, or the vaccine of claim 13 in the manufacture of a medicament for treating brain glioma.

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