Recombinant nucleic acid molecule of transcriptional circular RNA and its application in protein expression

Abstract

The present disclosure relates to a recombinant nucleic acid molecule of the transcriptional circular RNA and its application in protein expression. Specifically, the present disclosure relates to a recombinant nucleic acid molecule of the transcriptional circular RNA, recombinant expression vector, pre-circularized RNA, circular RNA, recombinant host cell, pharmaceutical composition and protein preparing method. The transcription product of the recombinant nucleic acid molecule in this present disclosure is a circular RNA which containing specific IRES element. IRES element can increase the protein expression level of circular RNA in eukaryotic cells, achieve efficient and persistent expression of protein. It has important application value in many fields like: Preparation of mRNA infectious disease vaccines, therapeutic mRNA tumor vaccines, mRNA-based dendritic cell tumor vaccines, mRNA-based gene therapy, mRNA-based chimeric antigen receptor T cell therapy, and protein supplement therapy.

Claims

1. A vector comprising a recombinant nucleic acid molecule for making circular RNA, the recombinant nucleic acid molecule comprising, from the 5′ end to the 3′ end, a 3′ intron, a second exon, an internal ribosome entry site (IRES) element, a coding region, a first exon, and a 5′ intron, wherein the IRES element is an Enterovirus EV29 (EV29) IRES comprising the nucleic acid sequence of SEQ ID NO: 10, wherein the recombinant nucleic acid molecule further comprises a 5′ homology arm located upstream of the IRES element, and a 3′ homology arm located downstream of the coding region and complementary to the 5′ homology arm, wherein the recombinant nucleic acid molecule further comprises a 5′ spacer located between the 5′ homology arm and the IRES element, and a 3′ spacer located between the coding region and the 3′ homology arm.

2. A recombinant host cell, wherein the recombinant host cell comprises the vector of claim 1.

3. A pharmaceutical composition, comprising the vector of claim 1.

4. The recombinant nucleic acid molecule of claim 1, wherein the coding region encodes a target polypeptide, and the IRES element increases the expression level of the target polypeptide in eukaryotic cells as compared to the recombinant nucleic acid molecule without the IRES element.

5. A circular RNA molecule produced by the vector of claim 1 comprising EV29 IRES, wherein the EV29 IRES comprises a RNA sequence that is transcribed from the DNA sequence of SEQ ID NO: 10, wherein the circular RNA molecule comprises the first exon adjacent to the second exon, and the coding region, wherein the circular RNA molecule further comprises the 3′ spacer located between the coding region and the first exon, and the 5′ spacer located between the second exon and the EV29 IRES.

6. The recombinant nucleic acid molecule of claim 1, wherein the 5′ homology arm comprises a sequence that is identical to SEQ ID NO: 2 or 3, and the 3′ homology arm comprises a sequence that is identical to SEQ ID NO: 17 or 18.

7. The recombinant nucleic acid molecule of claim 1, wherein the 5′ spacer comprises a sequence that is identical to SEQ ID NO: 6 or 7, and the 3′ spacer comprises a sequence that is identical to SEQ ID NO: 52 or 53.

8. The recombinant nucleic acid molecule of claim 1, wherein the 3′ intron comprises a sequence that is identical to SEQ ID NO: 4, the second exon comprises a sequence that is identical to SEQ ID NO: 5, the first exon comprises a sequence that is identical to SEQ ID NO: 15, and the 5′ intron comprises a sequence that is identical to SEQ ID NO: 16.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic diagram of the process of obtaining circular RNA with a recombinant expression vector (DNA vector) containing a recombinant nucleic acid molecule;

(2) FIG. 2 shows the agarose gel electrophoresis diagram for identifying RNA loops. In FIG. 1: 1. RNA ladder; 2. CVB3-EGFP pre-circularized mRNA; 3. CVB3-EGFP circularized mRNA; 4. EV29-EGFP pre-circularized mRNA 5. EV29-EGFP circularized mRNA; 6. EV29+CVB3v EGFP linearized mRNA; 7. EV29+CVB3v EGFP circularized mRNA; 8. EV33-EGFP linearized mRNA; 9. EV33-EGFP circularized mRNA; 10. EV33+CVB3v EGFP pre-circularized mRNA; 11. EV33+CVB3v EGFP circularized mRNA;

(3) FIG. 3 shows the results of sequencing to identify RNA ring formation;

(4) FIG. 4 shows different IRES elements (Circ-RNA-EV24, Circ-RNA-EV24+CVB3v, Circ-RNA-EV29, Circ-RNA-EV29+CVB3v, Circ-RNA-EV33, Circ-RNA-EV33+CVB3v, Circ-RNA-CVB3)-mediated protein expression level;

(5) FIG. 5 shows the duration of protein expression mediated by different IRES elements (Circ-RNA-EV24, Circ-RNA-EV29, Circ-RNA-EV33, Circ-RNA-EV33+CVB3v, pre-circularized mRNA);

(6) FIG. 6 shows the duration of protein expression mediated by different IRES components (Circ-RNA EV24+CVB3v, Circ-RNA-EV29+CVB3v, Circ-RNA EV33+CVB3v, Circ-RNA CVB3 and linear mRNA)

(7) FIG. 7 shows the agarose gel electrophoresis diagram for identifying RNA loops. In FIG. 7: 1. RNA ladder; 2. CVB3-EGFP pre-circularized mRNA; 3. CVB3-EGFP circularized mRNA; 4. EV29-EGFP H1S1 pre-circularized mRNA; 5. EV29-EGFP H1S1 circularized mRNA; 6. EV29-EGFP H2S2 pre-circularized mRNA; 7. EV29-EGFP H2S2 circularized mRNA;

(8) FIG. 8 shows the protein expression levels mediated by different IRES elements (Circ-RNA-EV24, Circ-RNA-EV29-H1S1, Circ-RNA-EV29-H2S2, Circ-RNA-CVB3);

(9) FIG. 9 shows the duration of protein expression mediated by different IRES elements (Circ-RNA-EV29, Circ-RNA EV2-H1S1, Circ-RNA EV2-H2S2, Circ-RNA CVB3, and pre-circularized mRNA).

STATEMENT REGARDING SEQUENCE LISTING

(10) The Sequence Listing associated with this application is provided in text form in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 52322-0002002.txt. The text file is 79.1 KB, and was created Sep. 27, 2021, and submitted electronically via EFS-Web with this application.

(11) Detailed Ways

(12) When used in conjunction with the term “comprising” in the claims and/or specification, the words “a” or “an” can mean “a”, but can also mean “one or more”, “at least One” and “one or more than one”.

(13) As used in the claims and specification, the words “include”, “have”, “include” or “contain” mean inclusive or open-ended, and do not exclude additional, unquoted elements or methods step.

(14) Throughout the application documents, the term “about” means: a value includes the standard deviation of the error of the device or method used to determine the value.

(15) Although the disclosed content supports the definition of the term “or” only as an alternative and “and/or”, the term “or” in the claims means only alternatives or mutual exclusion between alternatives unless it is clearly stated “and/or”.

(16) As used in the present disclosure, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein and are polymers of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeling components).

(17) As used in the present disclosure, the term “circular RNA” is a closed circular RNA molecule, mainly composed of exons, IRES elements, protein coding regions and spacers. In some preferred embodiments, the circular RNA has the following structure: “second exon E2-spacer-IRES element-coding region-spacer-first exon E1”. The circular RNA used in the present disclosure has protein translation activity and can also be referred to as “circular mRNA”.

(18) As used in the present disclosure, the term “pre-circularized RNA” refers to an RNA precursor capable of circularization to form circular RNA, which is generally formed by transcription of a linear DNA molecule.

(19) As used in the present disclosure, the term “linear RNA” refers to a 5′cap structure (5′Cap), a 3′polyadenosine tail (PolyA tail), and a 5′untranslational region (5′untranslational region, 5′ UTR), 3′untranslational region (3′UTR), and open reading frame (ORF) and other structures with translational function.

(20) As used in the present disclosure, the term “IRES” (Internal ribosome entry site, IRES) is also called internal ribosome entry site. “Internal ribosome entry site” (IRES) is a translation control sequence, usually located at the gene of interest. 5′end, and enables translation of RNA in a cap-independent manner. The transcribed IRES can directly bind to the ribosomal subunit so that the mRNA start codon is properly oriented 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 eukaryotic translation mechanisms. In some preferred embodiments, the IRES element of the present disclosure is selected from EV24 IRES, EV29 IRES, EV33 IRES, CVB3 IRES, or a chimera sequence of CVB3v IRES and any one of EV24 IRES, EV29 IRES, and EV33 IRES. “CVB3v” in the present disclosure refers to the v domain of CVB3 IRES. The chimera sequence in this disclosure includes: the EV24+CVB3v chimera obtained by replacing the v domain of EV24 IRES with the v domain of CVB3 IRES, and the v domain of CVB3 IRES. The EV29+CVB3v chimera obtained by replacing the v-domain of EV29 IRES with the v-domain of CVB3 IRES, and the EV33+CVB3v chimera obtained by replacing the v-domain of EV33 IRES with the v-domain of CVB3 IRES.

(21) As used in the present disclosure, the term “coding region” refers to a gene sequence capable of transcribing messenger RNA and finally translating it into a target polypeptide or protein.

(22) As used in this disclosure, the term “upstream” or “downstream” refers to upstream and downstream along the protein translation direction of the coding region.

(23) In some embodiments, the coding region of the present disclosure encodes a target polypeptide selected from one or more of antigens, antibodies, antigen binding receptors, ligands, fusion proteins, and recombinant proteins.

(24) In some embodiments, the antigen of the present disclosure is selected from virus-derived antibodies or tumor-specific antigens.

(25) In some embodiments, the antibodies of the present disclosure are selected from Fab, Fab′, F(ab′)2, Fv, scFv, sdAb, diabody, camelid antibody, or monoclonal antibody.

(26) In some embodiments, the antigen binding receptors of the present disclosure are selected from chimeric antigen receptors or T cell receptors.

(27) In some embodiments, the target polypeptide of the present disclosure is selected from one or more of antigens, antibodies, antigen binding receptors, ligands, fusion proteins, and recombinant proteins.

(28) As used in the present disclosure, the term “substitution, repetition, deletion or addition of one or more amino acids” wherein substitution refers to the replacement of a nucleotide or amino acid occupying a position with a different amino acid. Deletion refers to the removal of amino acids occupying a certain position. Insertion refers to the addition of amino acids adjacent to and immediately after the amino acid occupying the position. Exemplarily, “mutation” in the present disclosure includes “conservative mutation”.

(29) The term “conservative mutation” in the present disclosure refers to a conservative mutation that can normally maintain the function of a protein. Representative examples of conservative mutations are conservative substitutions. Conservative substitution refers to, for example, when the substitution site is an aromatic amino acid, Phe, Trp, and Tyr are mutually substituted mutations; when the substitution site is a hydrophobic amino acid, Leu, Ile, and Val are mutually substituted In the case of polar amino acids, mutations that replace each other between Gln and Asn; in the case of basic amino acids, mutations that replace each other between Lys, Arg, and His; in the case of acidic amino acids, A mutation that replaces each other between Asp and Glu; in the case of an amino acid having a hydroxyl group, a mutation that replaces each other between Ser and Thr. As the substitutions considered as conservative substitutions, specifically, the substitution of Ala to Ser or Thr, the substitution of Arg to Gln, His, or Lys, the substitution of Asn to Glu, Gln, Lys, His or Asp, the substitution of Asp to Asn, Glu or Gln substitution, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp, Gly to Pro Replacement, replacement of His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met, Val or Phe, Leu to Ile, Met, Val or Phe, Lys to Asn, Glu, Gln, His or Arg, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala, Thr to Ser or Ala, Trp to Phe or Tyr, Tyr to His, Phe or Trp, and Val Replacement to Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations caused by individual differences, differences in strains, and species from which genes are derived.

(30) In the present disclosure, “sequence identity” and “percent identity” refer to the percentage of identical (ie identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides and aligning the aligned polynucleotides or polypeptides The number of positions containing the same nucleotide or amino acid residue is scored and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotide or polypeptide. Polynucleotides may differ at one position, for example, by containing different nucleotides (ie, substitutions or mutations) or deleted nucleotides (ie, nucleotide insertions or nucleotide deletions in one or two polynucleotides). Polypeptides may differ at one position, for example, by containing different amino acids (ie, substitutions or mutations) or missing amino acids (ie, amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

(31) Exemplarily, in the present disclosure, when a sequence comparison algorithm is used or visual inspection measurement is used to compare and align with the greatest correspondence, two or more sequences or subsequences have at least 40%, 50%, 60% %, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide or amino acid residues Identity” or “Percent Identity”. The judgment/calculation of “sequence identity” or “percent identity” can be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In some embodiments, the sequence is substantially the same over the entire length of any one or two compared biopolymers (ie, nucleic acids or polypeptides).

(32) As used in the present disclosure, the term “Reverse Complementary Sequence” means a sequence that is opposite to the sequence of the original polynucleotide and is also complementary to the sequence of the original polynucleotide. Exemplarily, if the original polynucleotide sequence is ACTGAAC, its reverse complementary sequence is GTTCAGT.

(33) As used in this disclosure, the term “polynucleotide” refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in number or concentration, and can pass standards Molecular biology methods (for example, using cloning vectors) identify, manipulate, and restore sequences and their component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (ie A, T, G, C), this also includes an RNA sequence (ie A, U, G, C), where “U” replaces “T”. In other words, “polynucleotide” refers to a polymer of nucleotides removed from other nucleotides (individual fragments or entire fragments), or can be a part or component of a larger nucleotide structure, such as expression Vector or polycistronic sequence. Polynucleotides include DNA, RNA and cDNA sequences. “Recombinant polynucleotide” and “recombinant nucleic acid molecule” belong to one type of “polynucleotide”.

(34) As used in the present disclosure, the term “recombinant nucleic acid molecule” refers to 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 a recombinant host cell to produce, for example, “recombinant polypeptide”, “recombinant protein”, “fusion protein” and the like. In the present disclosure, a recombinant nucleic acid molecule includes a coding region encoding a polypeptide of interest, and an IRES element connected upstream of the coding region. In some specific embodiments, the recombinant nucleic acid molecule of the present disclosure comprises the following sequence structure:

(35) 5′homology arm-3′intron-second exon E2-5′spacer-IRES element-coding region-3′spacer-first exon E1-5′ intron-3′ Homology arm. Utilizing the ribozyme characteristics of introns, under the initiation of GTP, the junction between the 5′intron and the first exon is broken; the ribozyme cleavage of the first exon further attacks the 3′intron and the first exon. At the junction of the two exons, the 3′intron is dissociated, and the first exon and the second exon are connected to form a circular RNA.

(36) As used in this disclosure, the term “vector” refers to a DNA construct that contains a DNA sequence operably linked to a suitable control sequence to express a gene of interest in a suitable host.

(37) As used in the present disclosure, the term “recombinant expression vector” refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide. Recombinant expression vectors may include, for example, i) a collection of genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; ii) structures or coding sequences that are transcribed into mRNA and translated into proteins; and iii) appropriate transcription and the transcription subunits of translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector can be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, such as lentivirus, DNA of viruses such as retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies.

(38) As used in this disclosure, the term “antigen” refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. Any macromolecule, including essentially all proteins or peptides, can be used as an antigen. In the present disclosure, antigens include virus-derived antigens, such as novel coronavirus (SARS-CoV-2) antigens, or tumor-specific antigens.

(39) As used in the present disclosure, the term “antibody” refers to an immunoglobulin or a fragment or derivative thereof, and includes any polypeptide that contains an antigen binding site, regardless of whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, Mutant, grafted antibodies. The term “antibody” also includes antibody fragments such as Fab, F(ab′)2, FV, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function. Normally, such fragments will include antigen-binding fragments.

(40) As used in the present disclosure, the term “single-chain antibody” (scFv) is formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through a short peptide (also called a linker) of a limited number of amino acids. Into antibodies.

(41) As used in the present disclosure, the term “T cell receptor” (T cell receptor, TCR) is a type of T cell receptor capable of targeting heterologous antigens. The TCR of most T cells is composed of α and β peptide chains, and the TCR of a few T cells is composed of γ and δ peptide chains.

(42) As used in this disclosure, the term “chimeric antigen receptor” (CAR) is an artificial receptor that is engineered to contain an immunoglobulin antigen binding domain. Currently, chimeric antigen receptors can include domains such as antigen binding region, hinge region, transmembrane region and intracellular structural region.

(43) The term “host cell” in the present disclosure means any cell type that is easily transformed, transfected, transduced, etc., with a recombinant nucleic acid molecule, circular RNA, or recombinant expression vector containing the present disclosure. The term “recombinant host cell” covers a host cell that is different from the parent cell after the introduction of a recombinant nucleic acid molecule, circular RNA or recombinant expression vector, and the recombinant host cell is specifically achieved by transformation. The host cell of the present disclosure may be a prokaryotic cell or a eukaryotic cell, as long as it is a cell capable of introducing the recombinant nucleic acid molecule, circular RNA or recombinant expression vector of the present disclosure. After introducing the recombinant nucleic acid molecule, circular RNA or recombinant expression vector of the present disclosure, a recombinant host cell expressing the target polypeptide can be obtained.

(44) The terms “transformation, transfection, transduction” in this disclosure have the meaning generally understood by those skilled in the art, that is, the process of introducing 40 foreign DNA into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, and microinjection. Polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.

(45) As used in the present disclosure, “treatment” refers to contacting (eg, administering) the strain and/or macrophages of the present invention or a pharmaceutical composition containing them (hereinafter also It is referred to as the “pharmaceutical composition of the present invention”) to reduce the symptoms of the disease compared with the absence of contact, and does not mean that it is necessary to completely suppress the symptoms of the disease. Suffering from a disease refers to the appearance of symptoms of disease in the body.

(46) As used in the present disclosure, “prevention” refers to: before contracting a disease, by contacting (for example, administering) the pharmaceutical composition of the present invention, etc., the subject can reduce the symptoms after contracting the disease compared with the absence of contact. Does not mean that the disease must be completely suppressed.

(47) As used in this disclosure, the terms “individual”, “patient” or “subject” include mammals. Mammals include, but are not limited to, domestic animals (for example, cattle, sheep, cats, dogs, and horses), primates (for example, human and non-human primates such as monkeys), rabbits, and rodents (for example: Mice and rats).

(48) As used in the present disclosure, the term “high stringency conditions” means that for probes with a length of at least 100 nucleotides, following standard Southern blotting procedures, at 42° C. in 5×SSPE (saline sodium phosphate EDTA) 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA and 50% formamide pre-hybridization and hybridization for 12 to 24 hours. Finally, the carrier material was washed three times with 2×SSC, 0.2% SDS at 65° C., each time for 15 minutes.

(49) As used in the present disclosure, the term “very high stringency conditions” means that for probes with a length of at least 100 nucleotides, following standard Southern blotting procedures, at 42° C. in 5×SSPE (saline sodium phosphate EDTA) 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA and 50% formamide pre-hybridization and hybridization for 12 to 24 hours. Finally, the carrier material was washed three times with 2×SSC, 0.2% SDS at 70° C., each for 15 minutes.

(50) Unless otherwise defined or clearly indicated by the background, all technical and scientific terms in this disclosure have the same meanings as commonly understood by those of ordinary skill in the art to which this disclosure belongs.

(51) Technical Solutions

(52) In the technical solution of the present disclosure, the meanings of the numbers in the nucleotide and amino acid sequence lists in the specification are as follows:

(53) The sequence shown in SEQ ID NO:1 is the nucleotide sequence of the T7 promoter;

(54) The sequence shown in SEQ ID NO: 2 is the nucleotide sequence of 5′homology arm sequence 1 (H1);

(55) The sequence shown in SEQ ID NO: 3 is the nucleotide sequence of 5′homology arm sequence 2 (H2);

(56) The sequence shown in SEQ ID NO: 4 is the nucleotide sequence of the 3′intron of the Type I PIE system;

(57) The sequence shown in SEQ ID NO: 5 is the nucleotide sequence of the second exon (E2) of the class I PIE system;

(58) The sequence shown in SEQ ID NO: 6 is the nucleotide sequence of 5′spacer sequence 1;

(59) The sequence shown in SEQ ID NO: 7 is the nucleotide sequence of 5′spacer sequence 2;

(60) The sequence shown in SEQ ID NO: 8 is the nucleotide sequence of CVB3 IRES;

(61) The sequence shown in SEQ ID NO: 9 is the nucleotide sequence of EV24 IRES;

(62) The sequence shown in SEQ ID NO: 10 is the nucleotide sequence of EV29 IRES;

(63) The sequence shown in SEQ ID NO: 11 is the nucleotide sequence of EV33 IRES;

(64) The sequence shown in SEQ ID NO: 12 is the nucleotide sequence of the chimeric IRES of EV24 and CVB3v;

(65) The sequence shown in SEQ ID NO: 13 is the nucleotide sequence of the chimeric IRES of EV29 and CVB3v;

(66) The sequence shown in SEQ ID NO: 14 is the nucleotide sequence of the chimeric IRES of EV33 and CVB3v;

(67) The sequence shown in SEQ ID NO: 15 is the nucleotide sequence of the first exon (E1) of the class I PIE system;

(68) The sequence shown in SEQ ID NO: 16 is the nucleotide sequence of the 5′intron of the Type I PIE system;

(69) The sequence shown in SEQ ID NO: 17 is the nucleotide sequence of 3′homology arm sequence 1,

(70) The sequence shown in SEQ ID NO: 18 is the nucleotide sequence of 3′homology arm sequence 2;

(71) The sequence shown in SEQ ID NO: 19 is the nucleotide sequence of the XbaI restriction site;

(72) The sequence shown in SEQ ID NO: 20 is the nucleotide sequence of EGFP encoding DNA;

(73) The sequence shown in SEQ ID NO: 21 is the EGFP amino acid sequence;

(74) The sequence shown in SEQ ID NO: 22 is the nucleotide sequence of EGFP circular RNA (CVB3 IRES);

(75) The sequence shown in SEQ ID NO: 23 is the nucleotide sequence of EGFP circular RNA (EV24 IRES);

(76) The sequence shown in SEQ ID NO: 24 is the nucleotide sequence of EGFP circular RNA (EV24+CVB3v IRES);

(77) The sequence shown in SEQ ID NO: 25 is the nucleotide sequence of EGFP circular RNA (EV29 IRES)

(78) The sequence shown in SEQ ID NO: 26 is the nucleotide sequence of EGFP circular RNA (EV29+CVB3v IRES);

(79) The sequence shown in SEQ ID NO: 27 is the nucleotide sequence of EGFP circular RNA (EV33 IRES);

(80) The sequence shown in SEQ ID NO: 28 is the nucleotide sequence of EGFP circular RNA (EV33+CVB3v IRES);

(81) The sequence shown in SEQ ID NO: 29 is the nucleotide sequence of EGFP circular RNA (EV29 IRES+H1S1);

(82) The sequence shown in SEQ ID NO: 30 is the nucleotide sequence of EGFP circular RNA (EV29 IRES+H2S2);

(83) The sequence shown in SEQ ID NO: 31 is the nucleotide sequence of RBD encoding DNA;

(84) The sequence shown in SEQ ID NO: 32 is the amino acid sequence of the RBD protein;

(85) The sequence shown in SEQ ID NO: 33 is the nucleotide sequence of RBD circular RNA (EV29 IRES+H1 S1);

(86) The sequence shown in SEQ ID NO: 34 is the nucleotide sequence of EPO encoding DNA;

(87) The sequence shown in SEQ ID NO: 35 is the amino acid sequence of the EPO protein;

(88) The sequence shown in SEQ ID NO: 36 is the nucleotide sequence of EPO circular RNA (EV29 IRES+H1S1);

(89) The sequence shown in SEQ ID NO: 37 is the nucleotide sequence of the DNA encoding the light chain of the PD-1 monoclonal antibody;

(90) The sequence shown in SEQ ID NO: 38 is the amino acid sequence of the light chain of the PD-1 monoclonal antibody;

(91) The sequence shown in SEQ ID NO: 39 is the nucleotide sequence of the light chain circular RNA (EV29 IRES+H1S1) of the PD-1 monoclonal antibody;

(92) The sequence shown in SEQ ID NO: 40 is the nucleotide sequence of the DNA encoding the heavy chain of the PD-1 monoclonal antibody;

(93) The sequence shown in SEQ ID NO: 41 is the amino acid sequence of the heavy chain protein of the PD-1 monoclonal antibody;

(94) The sequence shown in SEQ ID NO: 42 is the nucleotide sequence of the heavy chain circular RNA of the PD-1 monoclonal antibody (EV29 IRES+H1S1);

(95) The sequence shown in SEQ ID NO: 43 is the nucleotide sequence of IL-15 encoding DNA;

(96) The sequence shown in SEQ ID NO: 44 is the amino acid sequence of IL-15 protein;

(97) The sequence shown in SEQ ID NO: 45 is the nucleotide sequence of IL-15 circular RNA (EV29 IRES+H1S1);

(98) The sequence shown in SEQ ID NO: 46 is the nucleotide sequence of PAP encoding DNA;

(99) The sequence shown in SEQ ID NO: 47 is the amino acid sequence of the PAP protein;

(100) The sequence shown in SEQ ID NO: 48 is the nucleotide sequence of PAP circular RNA (EV29 IRES+H1S1);

(101) The sequence shown in SEQ ID NO: 49 is the nucleotide sequence of CD16 CAR encoding DNA;

(102) The sequence shown in SEQ ID NO: 50 is the amino acid sequence of the CD16 CAR protein;

(103) The sequence shown in SEQ ID NO: 51 is the nucleotide sequence of CD16 CAR circular RNA (EV29 IRES+H1S1);

(104) The sequence shown in SEQ ID NO: 52 is the nucleotide sequence of 3′spacer sequence 1;

(105) The sequence shown in SEQ ID NO:53 is the nucleotide sequence of sequence 2 of the 3′spacer.

(106) The present disclosure has discovered in research that although linear mRNA in the prior art has a high protein expression level, it cannot achieve long-term and persistent protein expression. Although the circular RNA disclosed in Reference 15 has increased the protein expression level and expression time of circular RNA to a certain extent, it still cannot meet the requirements of industrialized protein production. At present, it is necessary to have both high protein expression levels and achieve Circular RNA molecule for long-term protein expression.

(107) In some embodiments, the present disclosure provides a recombinant nucleic acid molecule that is then transcribed to form circular RNA. The recombinant nucleic acid molecule includes a coding region encoding the target polypeptide, and an IRES element connected upstream of the coding region. The IRES element can increase the expression level of the target polypeptide, and the circular RNA transcribed with the above-mentioned recombinant nucleic acid molecule can realize efficient and durable protein expression in eukaryotic cells.

(108) The IRES element includes nucleotide sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity of one or more sequences in the group consisting of any one of SEQ ID NO: 8-11. In some embodiments, the IRES element is CVB3 IRES of the nucleotide sequence shown in SEQ ID NO: 8, EV24 IRES of the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence shown in SEQ ID NO: 10 EV29 IRES, EV33 IRES with the nucleotide sequence shown in SEQ ID NO: 11. In some embodiments, the IRES element comprises a chimera sequence of CVB3v IRES and any one of EV24 IRES, EV29 IRES, and EV33 IRES.

(109) In some specific embodiments, the recombinant nucleic acid molecule of the present disclosure further comprises a 5′ homology arm located upstream of the IRES element, and a 3′ homology arm located downstream of the coding region which is complementary to the 5′ homology arm.

(110) In the present disclosure, the 5′homology arm includes 5′ homology arm 1 (H1) and 5′ homology arm 2 (H2 Specifically, the nucleotide sequence of the 5′homology arm has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared with the sequence shown in any of SEQ ID NO: 2-3.the nucleotide sequence of the 3′homology arm has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared with the sequence shown in any of SEQ ID NO: 17-18. In some specific embodiments, the recombinant nucleic acid molecule of the present disclosure further comprises an IRES element located between the 5′homology arm and the IRES element, and between the coding region and the 3′homology arm.

(111) In the present disclosure, the spacer includes a 5′spacer and a 3′spacer. Specifically, the nucleotide sequence of the 5′spacer has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared with which shown in any one of SEQ ID NOs: 6-7. the nucleotide sequence of the 3′spacer has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared with which shown in any one of SEQ ID NOs: 52-53. The sequences of the 5′homology arm, 3′ homology arm, and spacer in the present disclosure can further improve the circularization efficiency of the circular RNA formed by the recombinant nucleic acid molecule, thereby increasing the protein expression level of the circular RNA.

(112) In some specific embodiments, the recombinant nucleic acid molecule of the present disclosure further comprises a 3′ intron and a second exon located between the 5′ homology arm and the IRES element and the first exon and the 5′intron between the 3′homology arm and the coding region.

(113) In the present disclosure, the nucleotide sequence of the 3′intron has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared with the nucleotide sequence shown in SEQ ID NO: 4. The nucleotide sequence of the second exon (E2) has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity sequence. compared with the nucleotide sequence shown in SEQ ID NO: 5. sequence. The nucleotide sequence of the 5′intron has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared with the nucleotide sequence shown in SEQ ID NO: 16. The nucleotide sequence of the first exon (E1) has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity compared with the nucleotide sequence shown in SEQ ID NO: 15.

(114) In some preferred embodiments, the structure of the recombinant nucleic acid molecule is as follows:

(115) 5′homology arm-3′intron-second exon E2-5′spacer-IRES element-coding region-3′spacer-first exon E1-5′ intron-3′ Homology arm.

(116) In order to enable the recombinant nucleic acid molecule to be further transcribed to form an RNA molecule, the recombinant nucleic acid molecule may also contain regulatory sequences. Exemplarily, the control sequence is the T7 promoter connected to the upstream of the 5′homology arm, and the T7 promoter sequence is the nucleotide sequence shown in SEQ ID NO:1.

(117) In some embodiments, the present disclosure provides a recombinant expression vector comprising the aforementioned recombinant nucleic acid molecule. Among them, the vector for connecting the recombinant nucleic acid molecule can be various vectors commonly used in the art, such as pUC57 plasmid. Further, the recombinant nucleic acid molecule contains restriction enzyme cutting sites, so that the recombinant expression vector is digested to obtain a linearized vector which is suitable for transcription.

(118) In some embodiments, the present disclosure provides a pre-circularized RNA formed by post-transcription of a recombinant nucleic acid molecule or a linearized recombinant expression vector. Preferably, the pre-circularized RNA has the following structure:

(119) 5′homology arm-3′intron-second exon E2-5′spacer-IRES element-coding region-3′spacer-first exon E1-5′ intron-3′ Homology arm.

(120) FIG. 1 shows the process of obtaining circular RNA from a recombinant expression vector (DNA vector) containing a recombinant nucleic acid molecule: firstly, the DNA vector is digested to obtain a linearized vector, and the linearized DNA vector is transcribed to obtain a pre-circularized RNA. Finally, the pre-circularized RNA is circularized through the following process: using the ribozyme characteristics of the intron, under the initiation of GTP, the junction between the 5′intron and the first exon is broken; The ribozyme cleavage of the first exon further attacks the junction between the 3′ intron and the second exon, causing a break at this place, dissociating the 3′ intron, and connecting the first exon and the second exon to form a ring RNA.

(121) In some embodiments, the present disclosure provides a circular RNA formed by circularization of the above-mentioned pre-circularized RNA, or circularization of a recombinant nucleic acid molecule or a recombinant expression vector after transcription. Specifically, under the guidance of the regulatory sequence in the recombinant nucleic acid molecule, the recombinant nucleic acid molecule is transcribed to produce a pre-circularized RNA molecule. Specifically, the 5′ homology arm in the pre-circularized RNA molecule is complementary to the 3′homology arm, and the ribozyme characteristic of the intron is used to make a break occurs between the 3′intron and the second exon E2, and the first an exon E1 and 5′intron, then E1 and E2 are connected to obtain a Circular RNA sequence with the structure of: second exon E2-spacer-IRES element-coding region-spacer-first exon E1.

(122) In some embodiments, one or more target polypeptides selected from antigens, antibodies, antigen-binding receptors, ligands, fusion proteins, and recombinant proteins are expressed.

(123) In some embodiments, the circular RNA expresses the EGFP protein of the amino acid sequence shown in SEQ ID NO: 21, or the amino acid sequence shown in SEQ ID NO: 21 has been substituted, repeated, deleted or added with one or more amino acids, and has EGFP protein active polypeptide. The nucleotide sequence encoding the EGFP protein is shown in SEQ ID NO:20. The circular RNA expressing the EGFP protein contains the nucleotide sequence shown in any one of SEQ ID NO: 22-30.

(124) In some embodiments, the circular RNA expresses viral antigens. Exemplarily, the viral antigen is the RBD protein having the amino acid sequence shown in SEQ ID NO: 32, or the amino acid sequence shown in SEQ ID NO: 32 has been substituted, repeated, deleted or added with one or more amino acids, and has the RBD protein Active peptides. The nucleotide sequence encoding the RBD protein is shown in SEQ ID NO:31. The circular RNA expressing the RBD protein includes the nucleotide sequence shown in SEQ ID NO:33.

(125) In some embodiments, the circular RNA expresses a recombinant humanized protein. Exemplarily, the recombinant humanized protein is specifically the EPO protein with the amino acid sequence shown in SEQ ID NO: 35, or the amino acid sequence shown in SEQ ID NO: 35 has been substituted, repeated, deleted or added with one or more amino acids, and A polypeptide with EPO protein activity. The nucleotide sequence encoding the EPO protein is shown in SEQ ID NO:34. The circular RNA expressing the EPO protein includes the nucleotide sequence shown in SEQ ID NO:36.

(126) In some embodiments, the circular RNA expresses cytokines. The cytokine is specifically IL-15 protein with the amino acid sequence shown in SEQ ID NO: 44, or the amino acid sequence shown in SEQ ID NO: 44 has been substituted, repeated, deleted or added with one or more amino acids, and has IL-15 protein Active peptides. The nucleotide sequence encoding IL-15 protein is shown in SEQ ID NO:43. The circular RNA expressing IL-15 protein includes the nucleotide sequence shown in SEQ ID NO:45.

(127) In some embodiments, the circular RNA expresses tumor-specific antigens, which include CEA AFP PSA PSMA MAGE-A3 PAP protein and the like. Exemplarily, the tumor-specific antigen is the PAP protein having the amino acid sequence shown in SEQ ID NO: 47, or the amino acid sequence shown in SEQ ID NO: 47 has been substituted, repeated, deleted or added with one or more amino acids, and has PAP protein active polypeptide. The nucleotide sequence encoding the PAP protein is shown in SEQ ID NO:46. The circular RNA expressing the PAP protein includes the nucleotide sequence shown in SEQ ID NO:48.

(128) In some embodiments, the circular RNA expresses the chimeric antigen receptor associated proteins, and the chimeric antigen receptor associated proteins include CD19, CD20, CD133, CD138, BCMA, CD16 protein, and the like. Exemplarily, the expresses the chimeric antigen receptor associated proteins is the CD16 protein having the amino acid sequence shown in SEQ ID NO: 50, or the amino acid sequence shown in SEQ ID NO: 50 has been substituted, repeated, deleted, or added one or more Amino acid and a polypeptide with CD16 protein activity. The nucleotide sequence encoding the CD16 protein is shown in SEQ ID NO:49. The circular RNA expressing the CD16 protein includes the nucleotide sequence shown in SEQ ID NO:51.

(129) In some embodiments, the circular RNA expresses monoclonal antibodies. Exemplarily, the monoclonal antibody is PD-1 monoclonal antibody. The light chain of the PD-1 monoclonal antibody is a polypeptide with the amino acid sequence shown in SEQ ID NO: 38, or the amino acid sequence shown in SEQ ID NO: 38 has undergone substitution, repetition, deletion, or addition of one or more amino acids, and has light Chain active polypeptide. The nucleotide sequence encoding the light chain is shown in SEQ ID NO:37. The circular RNA expressing the light chain of the PD-1 monoclonal antibody contains the nucleotide sequence shown in SEQ ID NO:39. The heavy chain of the PD-1 monoclonal antibody is a polypeptide having the amino acid sequence shown in SEQ ID NO: 41, or the amino acid sequence shown in SEQ ID NO: 41 has undergone substitution, repetition, deletion or addition of one or more amino acids, and has a heavy Chain active polypeptide. The nucleotide sequence encoding the heavy chain is shown in SEQ ID NO:40. The circular RNA expressing the heavy chain of the PD-1 monoclonal antibody contains the nucleotide sequence shown in SEQ ID NO:42.

(130) Since the circular RNA contains IRES elements of specific sequence, 5′spacer, 3′spacer, 5′homology arm, 3′homology arm, and all the elements work colloidally, a highly efficient and good durability of protein expression can be obtained through expressing above-mentioned protein with circular RNA of the present disclosure, which is superior to the existing pre-circularized mRNA and circular RNA and other protein expression elements.

(131) In some embodiments, the present disclosure provides a recombinant host cell comprising the aforementioned recombinant nucleic acid molecule, recombinant expression vector, pre-circularized RNA or circular RNA. Preferably, the recombinant host cell is a cell derived from eukaryotes, and the IRES element of the present disclosure can achieve efficient and durable expression of the target polypeptide in eukaryotic cells.

(132) In some embodiments, the present disclosure provides a pharmaceutical composition comprising the above-mentioned recombinant nucleic acid molecule, recombinant expression vector, pre-circularized RNA, circular RNA, recombinant host cell, or protein expressed by it. The circular RNA of the present disclosure can be used as expression elements for viral antigens, recombinant humanized proteins, tumor-specific antigens, chimeric antigen receptors, etc., or as nucleic acid vaccines directly introduced into organisms to produce viral antigens, Tumor-specific antigens, chimeric antigen receptors, etc.

EXAMPLES

(133) Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although representing specific embodiments of the present disclosure) are given for explanatory purposes only, because after reading the detailed description, they are made within the spirit and scope of the present disclosure. Various changes and modifications will become apparent to those skilled in the art.

(134) The experimental techniques and experimental methods used in this example are conventional techniques and methods unless otherwise specified. For example, the experimental methods for which specific conditions are not indicated in the following examples, usually follow conventional conditions such as Sambrook et al., Molecular Cloning: Experiment The conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials and reagents used in the examples can be obtained through formal commercial channels unless otherwise specified.

Example 1: Exploring the Expression of Circular mRNA Mediated by Different IRES in 293T Cells

(135) 1.1 Experimental Methods and Procedures

(136) (1) Plasmid Construction

(137) To construct the EGFP target gene containing different elements, this step entrusts Suzhou Genweiz Biotechnology Co., Ltd. to perform gene synthesis and cloning. The DNA vector used here to construct circular RNA includes T7 promoter, 5′homology arm, 3′intron, second exon E2, 5′spacer, IRES element, EGFP coding region, downstream spacer Region, 5′intron, first exon E1, 3′homology arm, and restriction site XbaI for plasmid linearization. The resulting gene fragment was ligated into the pUC57 vector.

(138) The IRES component information is as follows:

(139) TABLE-US-00001 IRES Content SEQ ID NO: EV24 Enterovirus EV24Included IRES components SEQ ID NO: 9 EV29 Enterovirus EV29Included IRES components SEQ ID NO: 10 EV33 Enterovirus EV33Included IRES components SEQ ID NO: 11 EV24 + The IRES element and v domain of the SEQ ID NO: 12 CVB3v Enterovirus EV24 is replaced with the v domain of the IRES element of CVB3 IRES. EV29 + The IRES element and v domain of the SEQ ID NO: 13 CVB3v Enterovirus EV29 is replaced with the v domain of the IRES element of CVB3 IRES. EV33 + The IRES element and v domain of the SEQ ID NO: 14 CVB3v Enterovirus EV33 is replaced with the v domain of the IRES element of CVB3 IRES.
Among them, the definition of the v domain of the IRES element can be found in the reference (Proc Natl Acad Sci US A. 2009 Jun. 9; 106(23): 9197-9202.) The EGFP-expressing circular RNA sequences obtained from the above IRES elements are as follows:

(140) TABLE-US-00002 IRES Coding region gene SEQ ID NO: CVB3 IRES EGFP SEQ ID NO: 22 EV24 IRES EGFP SEQ ID NO: 23 EV24 + CVB3v IRES EGFP SEQ ID NO: 24 EV29 IRES EGFP SEQ ID NO: 25 EV29 + CVB3v IRES EGFP SEQ ID NO: 26 EV33 IRES EGFP SEQ ID NO: 27 EV33 + CVB3v IRES EGFP SEQ ID NO: 28
(2) Preparation of Linear Plasmid Template
1) Plasmid Extraction
{circle around (1)} Activate the externally synthesized puncture bacteria, under 37° C./220 rpm/3˜4 h
{circle around (2)} Expand culturing with the activated bacteria solution, the culture condition: 37° C./220 rpm/overnight
{circle around (3)} Plasmid extraction (Tiangen Endotoxin free small amount medium extraction kit), determine OD value
2) Plasmid Digestion
Use XbaI single enzyme digestion method to digest the above 1) Prepare plasmid
The digestion system is as follows:

(141) TABLE-US-00003 TABLE 1 Reagent Volume Plasmid 10 μg Enzyme (1000 units) 5 μl 10 × cutsmart buffer 50 μl Nuclease free, H.sub.2O Total, 500 μl
Digestion overnight at 37° C. A universal DNA gel recovery kit (Tiangen Biochemical Technology Co., Ltd.) was used to recover the digested product, determine the OD value, and identify the digested product by 1% agarose gel electrophoresis. The purified linear plasmid template is used for in vitro transcription.
(3) Preparation of Pre-Circularized mRNA by In Vitro Transcription
1) In Vitro Transcription
Use T7 in vitro transcription kit (APExBIO T7 High Yield RNA Synthesis Kit) to synthesize mRNA
The transcription system is as follows:

(142) TABLE-US-00004 TABLE 2 Reagent Volume 10 × Reaction Buffer 2 μl ATP (20 mM) 2 μl CTP (20 mM) 2 μl UTP (20 mM) 2 μl GTP (20 mM) 2 μl Linearized DNA template 1 μg T7 RNA Polymerase Mix 2 μl RNA Nuclease free, H.sub.2O Total 20 μl
Incubate at 37° C. for 2 h, then digest the linear DNA template with DNase I. Digestion conditions: Digestion at 37° C. for 15 minutes.
2) Purification of Pre-Circularized mRNA
The transcription product obtained in 1) above was purified using a silicon membrane spin column method (Thermo, GeneJET RNA Purification Kit), and the OD value was measured and the RNA size was identified by 1% denaturing agarose gel electrophoresis.
The formula of 1% denatured agarose gel is as follows:
{circle around (1)}, Weigh 1 g agarose to 72 ml nuclease-free, H.sub.2O, and heat it in a microwave oven to dissolve;
{circle around (2)} When the above agarose is cooled to 55˜60° C., add 0.1% gel red, 10 ml 10×MOPS, 18 ml formaldehyde in a fume hood, and pour glue.
The process of denaturing agarose gel electrophoresis is as follows: take an equal volume of sample RNA and 2× Loading buffer, and denature at 65˜70° C. for 5˜10 min. The sample was loaded and electrophoresis was carried out under the conditions of 100V/30 min, and then photographed with a gel imaging system.
(4) mRNA Circularization
1) Cyclization Reagent:
GTP Buffer: 50 mM Tris-HCl, 10 mM MgCl.sub.2, 1 mM DTT, pH 7.5
2) Cyclization System and Conditions:

(143) TABLE-US-00005 TABLE 3 Solution Volume mRNA 25 μg RNA solution GTP solution (20 mM) 50 μl GTP buffer Add up to 500 μl
The above solution was heated at 55° C. for 15 minutes, and the circularized RNA product was purified using a silicon membrane spin column method (Thermo, GeneJET RNA Purification Kit). The OD value was measured and the RNA size was identified by 1% denaturing agarose gel electrophoresis.
3) Identification of Circular RNA
{circle around (1)} 1% denatured agarose gel identification:
A. Reagent preparation: Add 1 g of agarose powder to 72 ml of nuclease-free water, heat to melt the agar pond, and add 10 ml of 10×MOPS buffer. Then add 18 ml of fresh 37% formaldehyde in a fume hood, mix well, and pour the gel into the tank.
B. mRNA detection: Take about 500 ng mRNA solution, add an equal volume of 2×RNA loading buffer and mix well, heat at 65° C. for 5 minutes, load the sample for agarose gel detection.
{circle around (2)} Circularized mRNA RT-RCR and sequencing identification
A.) mRNA Reverse Transcription System and Conditions

(144) TABLE-US-00006 TABLE 4 Solution Volume mRNA 1 μg RNA solution RT primer Mix 4.0 μl Primerscript RT Enzye Mix I 1.0 μl 5 × primerscript buffer 2 4.0 μl Nuclease-free water Add up to 20 μl
The experimental group: cyclized mRNA and control group: uncyclized mRNA were prepared according to the above system, heated at 37° C. for 15 minutes, heated at 85° C. for 5 s, and stored at 4° C.
B.) PCR Amplification System and Conditions of Reverse Transcription Products

(145) TABLE-US-00007 TABLE 5 Solution Volume Reverse transcription product 1.0 μl 10 × buffer 2.0 μl dNTP 1.6 μl primer-F (10 μM) 1.0 μl primer-R (10 μM) 1.0 μl Taq Enzyme 0.5 μl Nuclease-free water 12.9 μl 
PCR amplification program: 95° C., 1 min; 95° C., 30 s; 60° C., 30 s; 72° C., 30 s; (35 cycles) 72° C., 7 min; 4° C.
C. PCR Product Cutting and Purification
Nucleic acid electrophoresis, select RT-RCR DNA bands that specifically exist in the experimental group but not in the control group, cut gel and recover, and purify with a universal DNA purification and recovery kit. Take purified DNA and primer EV29-EGFP-F: GTGACAGCAGCAGGAATCACA, Primer EV29-EGFP-R: TGGGATCAACCCACAGGCT was sent to Genweiz Company for forward and reverse sequencing.
(5) Transfection of Circular mRNA Encoding EGFP into 293T Cells and Measurement of Fluorescence Intensity
1) Cell Culture:
293T was inoculated in DMEM high glucose medium containing 10% fetal bovine serum and 1% double antibody, and cultured in a 37° C., 5% CO2 incubator. The cells are subcultured every 2-3 days.
2) Cell Transfection:
Before transfection, 293T cells were seeded in a 24-well plate at 1×105 cells/well and cultured in a 37° C., 5% CO2 incubator. After the cells reach 70-90% confluency, use Lipofectamine MessengerMax (Invitrogen) transfection reagent to transfect mRNA into 293T cells at 500 ng/well. The specific operations are as follows: {circle around (1)} Dilute Messenger MAXTM Reagent

(146) TABLE-US-00008 TABLE 6 Reagent Volume/hole MEM serum-free medium   25 μl Messenger MAXTM Reagent 0.75 μl

(147) After dilution and mixing, incubate at room temperature for 10 min. {circle around (2)} Dilute mRNA

(148) TABLE-US-00009 TABLE 7 Reagent Volume/hole mRNA  1 μl MEM serum-free medium Fill up to 25 μl {circle around (3)} Take mixed and diluted Messenger MAXTM Reagent and mRNA (1:1)

(149) TABLE-US-00010 TABLE 8 Reagent Volume/hole Diluted Messenger MAXTM 25 μl Reagent Diluted mRNA 25 μl

(150) After dilution and mixing, incubate at room temperature for 5 min. {circle around (4)} Take 50 ul of the above mixture and slowly add it to the 24-well plate and incubate in a 37° C., 5% CO2 incubator.
3) Protein Expression Detection

(151) {circle around (1)} Cell fluorescence observation: Observe the expression of EGFP under a 200× fluorescence microscope on 293T cells 1-10 days after transfection.

(152) {circle around (2)} Flow cytometry to detect the average fluorescence intensity of cells: 293T cells 1-10 days after transfection were used to detect the average fluorescence intensity of cells with a flow cytometer.

(153) 1.2 Results

(154) 1) DNA Transcription Template Preparation {circle around (1)} Plasmid extraction concentration: pUC57-CVB3-EGFP: 271.2 ng/μl, pUC57-EV24-EGFP: 245.4 ng/ul, pUC57-EV24+CVB3v-EGFP: 263.8 ng/μl, pUC57-EV29-EGFP: 277.9 ng/μl, pUC57-EV29+CVB3v-EGFP; 249.9 ng/μl, PUC57-EV33-EGFP: 273.0 ng/μl, PUC57-EV33+CVB3v-EGFP: 283.3 ng/μl; {circle around (2)} DNA concentration after plasmid digestion and linearization: pUC57-CVB3-EGFP: 120.6 ng/μl, pUC57-EV24-EGFP: 134.5 ng/ul, pUC57-EV24+CVB3v-EGFP: 125.8 ng/μl, pUC57-EV29-EGFP: 146.1 ng/μl, pUC57-EV29+CVB3v-EGFP: 119.2 ng/μl, pUC57-EV33-EGFP: 141.1 ng/μl, pUC57-EV33+CVB3v-EGFP: 137.9 ng/μl;

(155) 2) mRNA Transcription and Circularization

(156) {circle around (1)} RNA concentration after mRNA transcription and purification: CVB3-EGFP: 1149.8 ng/μl, EV24-EGFP: 1168.5 ng/μl, EV24+CVB3v-EGFP:1284.6 ng/μl, EV29-EGFP: 1245.5 μg/μl, EV29+CVB3vEGFP: 1111.8 ng/μl, EV33-EGFP: 1180.4 ng/μl, EV33+CVB3v-EGFP: 1148.5 ng/μl;

(157) {circle around (2)} RNA concentration after mRNA circularization and purification: CVB3-EGFP: 482.8 ng/μl, EV24-EGFP: 462.4 ng/ul, EV24+CVB3v-EGFP: 532.5 ng/ul, EV29-EGFP: 444.5 μg/μl, EV29+CVB3-EGFP: 447.2 ng/μl, EV33-EGFP: 452.0 ng/μl, EV33+CVB3 EGFP: 415.5 ng/μl;

(158) {circle around (3)} RNA denaturing agarose gel electrophoresis.

(159) As shown in FIG. 2: The electrophoresis of denatured agarose gel showed that the circularized mRNA migrated faster on the gel than the pre-circularized mRNA.

(160) 3) RT-PCR and Gene Sequencing to Identify mRNA Loops

(161) In order to identify whether the RNA obtained from the circularization reaction is circular RNA, RT-PCR and DNA sequencing methods are used for detection. According to the basic principle of RNA circularization, through the ribozyme characteristics of introns, under the initiation of GTP, the junction between the 5′intron and the first exon E1 will be broken, and the nucleic acid at the first exon E1 The breach will attack the junction of the 3′intron and the second exon E2, causing the junction to break, the 3′intron will dissociate, and the first exon E1 and the second exon E2 will form a common Valence link, and finally form circular RNA. Therefore, by amplifying the connecting sequence containing the first exon E1 and the second exon E2 and performing sequencing, it can be used as the final basis for determining whether the RNA is looped. CDNA was obtained by RNA reverse transcription reaction mediated by random primers. Using cDNA as a template, Specific primers are used for PCR amplification. The experimental results showed that no specific amplified bands were seen in the pre-circularized mRNA group, and specific amplified bands were seen in the circular mRNA group. The specific bands are recovered by cutting gel, and DNA sequencing is performed after purification. As shown in FIG. 3, the sequencing results showed that the DNA band contains the ligated E1 and E2 sequences. The circular RNA contains the connected E1-E2 RNA sequence, indicating that the RNA has been connected into a loop.

(162) 4) Protein Expression Detection

(163) The quantitative test results of fluorescence intensity 1-3 days after cell transfection are shown in FIG. 4. Compared with the circular EGFP mRNA containing CVB3 IRES (Circ-RNA CVB3, reference 15), it contains EV24 IRES, and EV24 is chimeric with CVB3v. Cyclic mRNA-mediated cellular fluorescence of IRES (EV24+CVB3v), EV29 IRES, EV29 and CVB3v chimeric IRES (EV29+CVB3v), EV33 IRES, EV33 and CVB3v chimeric IRES (EV33+CVB3v) is stronger, Which shows that the circular mRNA containing different IRES combinations provided by this patent can mediate stronger protein expression.

(164) In order to identify the persistence of this series of circular mRNA-mediated protein expression, the fluorescence intensity of cells 1-5 days after transfection was quantified. The test results are shown in FIG. 5 and FIG. 6: Compared with the circular EGFP mRNA containing CVB3 IRES (Circ-RNA CVB3, cited reference 15), it contains EV24 IRES, EV24+CVB3v IRES, EV29 IRES, EV29+CVB3v IRES, EV33 IRES, EV33+CVB3v IRES circular mRNA-mediated EGFP expression is stronger and longer lasting, and it is also significantly better than linear EGFP mRNA (standard product purchased from APExBio, Cap1 cap structure and PolyA tail) mediated The expression of EGFP is stronger and longer lasting.

Example 2: Expression of Circular mRNA Obtained by Combining EV29 IRES with Different Homology Arms and Spacer Sequences in 293T Cells

(165) 2.1 Experimental Methods and Procedures

(166) On the basis of the above-mentioned Example 1, using the same intron and exon elements as the above-mentioned example, with EV29 IRES (nucleotide sequence shown in SEQ ID NO: 10), a novel 5′homology arm 1 (nucleotide sequence shown in SEQ ID NO: 2), 3′homology arm 1 (nucleotide sequence shown in SEQ ID NO: 17) and 5′spacer 1 sequence (SEQ ID NO: 6 The nucleotide sequence shown) and the 3′spacer 1 sequence (the nucleotide sequence shown in SEQ ID NO: 52) are used as the basic elements of circular mRNA to construct a circular mRNA encoding green fluorescent protein (EGFP) (Circ-RNA EV29 H1S1).

(167) Using the same intron and exon elements as the above circular mRNA, EV29 IRES, 5′homology arm 2 (nucleotide sequence shown in SEQ ID NO: 3), 3′homology arm 2 (SEQ ID NO: ID NO: 18), 5′spacer 2 sequence (SEQ ID NO: 7 nucleotide sequence) and 3′spacer 2 sequence (SEQ ID NO: 53 As the basic element of circular mRNA, a circular mRNA (Circ-RNA EV29 H2S2) encoding green fluorescent protein (EGFP) is constructed.

(168) The DNA sequence encoding EGFP is shown in SEQ ID NO:20. The DNA synthesis was commissioned by Suzhou Genweiz Biotechnology Co., Ltd. to complete. Eventually will contain T7 promoter, class I PIE element, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1, EV29 IRES element, EGFP coding region (or T7 promoter, Class I PIE element, 5′homology arm 2, 3′homology arm 2, 5′spacer 2, 3′spacer 2, EV29 IRES element, EGFP coding region) complete DNA fragments were cloned into pUC57 plasmid.

(169) Plasmid DNA linearization, pre-circularized mRNA in vitro transcription, pre-circularized mRNA purification, mRNA loop reaction, circular mRNA purification, cell culture and transfection, etc., are all the same as in Example 1 in 1.1.

(170) 2.2 Results

(171) 2.2.1 Results

(172) 1) DNA Linearization Template Preparation

(173) {circle around (1)} Plasmid extraction concentration: pUC57-CVB3-EGFP: 356.4 ng/μl, pUC57-EV29-EGFP: 481.9 ng/μl, pUC57-EV29-EGFP H1S1: 283.1 ng/μl, pUC57-EV29-EGFP H2S2: 303.1 ng/μl;

(174) {circle around (2)} Plasmid digestion linearization concentration: pUC57-CVB3-EGFP: 249.6 ng/μl pUC57-EV29-EGFP: 289 ng/μl, pUC57-EV29-EGFP H1S1: 293 ng/μl, pUC57-EV29-EGFP H252: 294 ng/μl;

(175) 2) mRNA Transcription and Circularization

(176) {circle around (1)} Concentration after mRNA transcription and purification: CVB3-EGFP: 506.3 ng/μl, EV29-EGFP: 527.5 ng/μl, EV29-EGFP H1S1: 573.2 ng/μl, EV29-EGFP H2S2: 564.9 ng/μl;

(177) {circle around (2)} Concentration after purification of mRNA circularization: CVB3-EGFP: 257.4 ng/μl, EV29-EGFP: 236.2 ng/μl, EV29-EGFP H1S1: 208.0 ng/μl, EV29-EGFP H2S2: 240.3 ng/μl;

(178) Denaturing agarose gel was used to identify RNA loops. The experimental results are shown in FIG. 7: In the denatured agarose gel electrophoresis, the circularized mRNA of each group migrates faster on the gel than the corresponding pre-circularized mRNA before circularization.

(179) {circle around (3)} Protein expression detection

(180) The 1-3d fluorescence quantification after cell transfection is shown in FIG. 8. The fluorescent protein expression mediated by circular mRNA Circ-RNA EV29-EGFP H1S1 was significantly higher than that of the Circ-RNA EV29-EGFP group, and also significantly higher than the amount corresponding to the Circ-RNA CVB3-EGFP group. It shows that the novel combination of 5′homology arm 1, 3′homology arm 1 and 5′spacer 1, 3′spacer 1 provided by the present invention can effectively improve circular mRNA-mediated protein expression. The expression of fluorescent protein mediated by circular mRNA Circ-RNA EV29-EGFP H2S2 was higher than that of the Circ-RNA EV29-EGFP group, and was significantly higher than that of the Circ-RNA CVB3-EGFP group. It shows that the novel combination of 5′homology arm 2, 3′homology arm 2 and 5′spacer 2, 3′spacer 2 provided by the present invention can increase circular mRNA-mediated protein expression.

(181) In order to identify the persistence of different circular mRNA-mediated protein expression, the fluorescence of the cells was quantified 1-5 days after transfection. As shown in FIG. 9, Circ-RNA EV29-EGFP H1S1 protein mediated fluorescence, intensity and persistence of its expression was higher than Circ-RNA EV29-EGFP group and Circ-RNA CVB3-EGFP group. It shows that the novel combination of 5′homology arm 1, 3′homology arm 1 and 5′spacer 1, 3′spacer 1 provided by the present invention can effectively improve the persistence of circular mRNA-mediated protein expression. The persistence of fluorescent protein expression mediated by Circ-RNA EV29-EGFP H2S2 was comparable to that of the Circ-RNA EV29-EGFP group, but significantly higher than that of the Circ-RNA CVB3-EGFP group. It shows that the novel combination of 5′homology arm 2, 3′homology arm 2 and 5′spacer 2, 3′spacer 2 provided by the present invention, its circular mRNA-mediated protein expression is significantly better than that of Patent Citation 15 design and methods provided. Further, all the above cyclic durability mRNA of EGFP expression were significantly higher than the linear mRNA (purchased from standard APExBio comprising Cap1 PolyA tail and cap structure).

Example 3: Circular mRNA Encoding the New Coronavirus Spike Antigen RBD Protein to Achieve Protein Expression in 293T Cells

(182) 3.1 Experimental Methods and Procedures

(183) On the basis of the above-mentioned Example 2, the EV29 IRES, 5′homology arm 1, 3′homology arm 1 and 5′spacer 1, 3′spacer 1 sequence were used as the basic elements of circular mRNA to construct a new coding RBD domain coronavirus Spike antigen (receptor binding domain) cyclic mRNA. The RBD protein sequence is shown in SEQ ID NO:32, and the DNA sequence encoding RBD is shown in SEQ ID NO:31. The DNA synthesis was commissioned by Suzhou Genweiz Biotechnology Co., Ltd. to complete.

(184) Finally, a complete DNA fragment containing T7 promoter, type I PIE elements, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1, EV29 IRES, and RBD coding region will be cloned into pUC57 plasmid. Plasmid DNA is linearized, pre-circularized mRNA is transcribed in vitro, pre-circularized mRNA is purified, mRNA is circularized, and circular mRNA is purified to obtain circular mRNA with the sequence shown in SEQ ID NO: 33. The cell culture and transfection methods are the same as in Example 1 1.1. The His-tag ELISA detection kit was used to quantitatively detect the secreted His-RBD protein (Nanjing GenScript Biotechnology Co., Ltd.).

(185) 3.2 Result

(186) TABLE-US-00011 TABLE 9 Nucleic acid Concentration pUC57-EV29-RBD-H1S1 plasmid (purified) 342.2 ng/μl pUC57-EV29-RBD-H1S1 linearized plasmid 264.3 ng/μl (purified) EV29-RBD-H1S1 pre-circularized mRNA 508.5 ng/μl (purified) EV29-RBD-H1S1 cyclic mRNA (purified) 236.2 ng/μl

(187) Detected by His-tag ELISA, the amount of protein obtained by expressing RBD-His circular mRNA in 293T for 1-5 days are 21.6, 35.4, 40.3, 28.6, 22.7 ng/ml, respectively, indicating that the circular mRNA of the present disclosure can achieve RBD efficient and persistent expression of the protein.

Example 4: Circular mRNA Encoding EPO to Achieve Protein Expression in 293T Cells

(188) 4.1 Experimental Methods and Procedures

(189) Based on Example 2 above, EV33 IRES, 5′homology arm 1, 3′homology arm 1 and 5′spacer 1, 3′spacer 1 were used as the basic elements of circular mRNA to construct a coding erythropoiesis hormone (EPO) cyclic mRNA. The DNA and protein sequences encoding EPO are shown in SEQ ID NO: 34 and SEQ ID NO: 35, respectively. The DNA synthesis was commissioned by Suzhou Genweiz Biotechnology Co., Ltd. to complete. Ultimately it contains the T7 promoter, Class I PIE elements, 5′ homology arm 3′ homology arm 1,5′ spacer region, 3′ spacer region 1, EV33 IRES, the complete EPO coding region DNA fragment was cloned into the pUC57 plasmid. Plasmid DNA linearization, linearized mRNA in vitro transcription, pre-circularized mRNA purification, mRNA circularization, circular mRNA purification, cell culture and transfection methods are the same as in Example 1. 1.1. EPO ELISA detection kit (Thermo Fisher) was used to quantify the EPO protein expressed by 293T.

(190) 4.2 Result

(191) TABLE-US-00012 TABLE 10 Nucleic acid Concentration pUC57-EV33-EPO-H1S1 plasmid (purified) 297.5 ng/μl pUC57-EV33-EPO-H1S1 linearized plasmid 275.4 ng/μl (purified) EV33-EPO-H1S1 pre-circularized mRNA 375.3 ng/μl (purified) EV33-EPO-H1S1 cyclic mRNA (purified) 286.7 ng/μl

(192) Detected by EPO ELISA, the amount of protein obtained from EPO circular mRNA expression in 293T for 1-5 days is 35.6, 42.8, 56.4, 50.3, 25.7 ng/ml, respectively, indicating that the circular mRNA of the present disclosure can achieve the high efficiency and persistent expression of EPO protein.

Example 5: Circular mRNA Encoding PD-1 Monoclonal Antibody to Achieve Protein Expression in 293T Cells

(193) 5.1 Experimental Methods and Procedures

(194) On the basis of the above-mentioned Example 2, the EV29 IRES, 5′homology arm 1, 3′homology arm 1 and 5′spacer 1, 3′spacer 1 were used as the basic elements of circular mRNA to construct the coding resistance cell death receptor 1 (PD-1) monoclonal antibody) circular mRNA. Encoding the light chain of the monoclonal antibody anti-PD1 DNA and protein sequences, respectively as SEQ ID NO: 37 and SEQ ID NO: 38, the monoclonal antibody anti-PD1 encoding the heavy chain DNA and protein sequences, respectively as SEQ ID NO: 40 and SEQ ID NO: 41 shown. The DNA synthesis was commissioned by Suzhou Genweiz Biotechnology Co., Ltd. to complete. Eventually it will contain T7 promoter, type I PIE elements, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1, EV29 IRES, a complete DNA fragment of the PD1 light chain coding region cloned into the plasmid pUC57. Similarly, it will contain T7 promoter, class I PIE elements, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1, EV29 IRES, the complete heavy chain coding region of PD1 DNA fragment was cloned into the plasmid pUC57. Plasmid DNA linearization, pre-circularized mRNA in vitro transcription, pre-circularized mRNA purification, mRNA loop reaction, circular mRNA purification, to obtain two circular RNAs with the sequence shown in SEQ ID NO: 39 or 42, cell culture, etc. The method is the same as 1.1 of Example 1. Both monoclonal antibody PD1 mRNA encoding the light chain and heavy chain in 1:1 ratio, were transfected into 293T cells. Transfection method described in Example 1. The PD1 protein expressed by 293T was quantified using PD1 ELISA detection kit (Thermo Fisher).

(195) 5.2 Results

(196) TABLE-US-00013 TABLE 11 Nucleic acid Concentration pUC57-EV29-PD1L-H1S1 plasmid (purified) 279.3 ng/μl pUC57-EV29-PD1L-H1S1 linearized plasmid 204.2 ng/μl (purified) EV29-PD1L-H1S1 pre-circularized mRNA (purified) 396.5 ng/μl EV29-PD1L-H1S1 cyclic mRNA (purified) 247.2 ng/μl pUC57-EV29-PD1H-H1S1 plasmid (purified) 268.6 ng/μl pUC57-EV29-PD1H-H1S1 linearized plasmid 201.3 ng/μl (purified) EV29-PD1H-H1S1 pre-circularized mRNA (purified) 304.7 ng/μl EV29-PD1H-H1S1 cyclic mRNA (purified) 207.4 ng/μl

(197) Detected by PD1 monoclonal antibody ELISA, the amount of protein obtained from the expression of PD1 monoclonal antibody circular mRNA in 293T for 1-5 days is 120.3, 234.6, 356.4, 221.6, and 104.8 ng/ml, respectively, indicating that the circular mRNA of the present disclosure can achieve PD1 mAb efficient, long-lasting expression.

Example 6: Circular mRNA Encoding Cytokine IL-15 to Achieve Protein Expression in 293T Cells

(198) 6.1 Experimental Methods and Procedures

(199) Based on the above-mentioned Example 2, the EV29 IRES, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1 were used as the basic elements of circular mRNA to construct an encoding interleukin 15 (IL-15) circular mRNA. The DNA and protein sequences encoding IL-15 are shown in SEQ ID NO: 43 and SEQ ID NO: 44, respectively. The DNA synthesis was commissioned by Suzhou Genweiz Biotechnology Co., Ltd. to complete. Eventually it will contain T7 promoter, class I PIE elements, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1, EV29 IRES, a complete DNA fragment of IL-15 coding region cloned into the plasmid pUC57. Plasmid DNA linearization, pre-circularized mRNA in vitro transcription, pre-circularized mRNA purification, mRNA loop reaction, circular mRNA purification to obtain circular RNA with the sequence shown in SEQ ID NO: 45, cell culture and transfection methods All are the same as 1.1 of Example 1. IL-15 ELISA detection kit (Thermo Fisher) was used to quantify the IL-15 protein expressed by 293T.

(200) 6.2 Results

(201) TABLE-US-00014 TABLE 12 Nucleic acid Concentration pUC57-EV29-1L15-H1S1 plasmid (purified) 286.3 ng/μl pUC57-EV29-1L15-H1S1 linearized plasmid 251.5 ng/μl (purified) EV29-IL15-H1S1 pre-circularized mRNA 311.3 ng/μl (purified) EV29-IL15-H1S1 cyclic mRNA (purified) 274.3 ng/μl

(202) Detected by IL-15 ELISA, the amount of protein obtained from IL-15 circular mRNA expression in 293T for 1-5 days are 38.9, 47.3, 68.4, 51.6, 26.4 ng/ml, respectively, indicating that the circular mRNA of the present disclosure can achieve IL-15 efficient and durable expression.

Example 7: Circular mRNA Encoding the Tumor-Specific Antigen Prostate Cancer PAP Protein to Achieve Protein Expression in 293T Cells

(203) 7.1 Experimental Methods and Procedures

(204) Based on Example 2 above, EV29 IRES, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1 were used as the basic elements of circular mRNA to construct prostate cancer tumor-specific antigens acid phosphatase protein PAP (prostate acid phosphatase) cyclic mRNA. The DNA and protein sequences encoding PAP are shown in SEQ ID NO: 46 and SEQ ID NO: 47, respectively. The DNA synthesis was commissioned by Suzhou Genweiz Biotechnology Co., Ltd. to complete. Finally, a complete DNA fragment containing T7 promoter, type I PIE elements, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1, EV29 IRES, and PAP coding region will be cloned into pUC57 plasmid. Plasmid DNA linearization, pre-circularized mRNA in vitro transcription, pre-circularized mRNA purification, mRNA loop reaction, circular mRNA purification to obtain circular RNA with the sequence shown in SEQ ID NO: 48, cell culture and transfection methods All are the same as 1.1 in Example 1. PAP ELISA detection kit (Thermo Fisher) was used to quantify the PAP protein expressed by 293T.

(205) 7.2 Results

(206) TABLE-US-00015 TABLE 13 Nucleic acid Concentration pUC57-EV29-PAP-H1S1 plasmid (purified) 386.7 ng/μl pUC57-EV29-PAP-H1S1 linearized plasmid 294.5 ng/μl (purified) EV29-PAP--H1S1 pre-circularized mRNA 317.2 ng/μl (purified) EV29-PAP-H1S1 cyclic mRNA (purified) 268.9 ng/μl

(207) According to PAP ELISA detection, the amount of protein obtained from EPO circular mRNA expression in 293T for 1-5 days is 69.3, 86.4, 75.5, 52.4, 38.6 ng/ml, respectively, indicating that the circular mRNA of the present disclosure can achieve the high efficiency and durability of EPO expression.

Example 8: Circular mRNA Encoding Chimeric Antigen Receptor CD16 CAR Protein to Achieve Expression in 293T Cells

(208) 8.1 Experimental Methods and Procedures

(209) Based on the above-mentioned Example 2, EV29 IRES, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1 are used as the basic elements of circular mRNA to construct a chimera cyclic antigen receptor CD16 CAR mRNA. The DNA and protein sequences encoding CD16 CAR are shown in SEQ ID NO:49 and SEQ ID NO:50, respectively. The DNA synthesis was commissioned by Suzhou Genweiz Biotechnology Co., Ltd. to complete. Finally, a complete DNA fragment containing T7 promoter, type I PIE elements, 5′homology arm 1, 3′homology arm 1, 5′spacer 1, 3′spacer 1, EV29 IRES, CD16 CAR coding region will be cloned. To the pUC57 plasmid. Plasmid DNA linearization, pre-circularized mRNA in vitro transcription, pre-circularized mRNA purification, mRNA loop reaction, circular mRNA purification to obtain circular RNA with the sequence shown in SEQ ID NO: 51, cell culture and transfection methods. All are the same as 1.1 of Example 1. Anti-CD16 monoclonal antibody-mediated flow cytometry (Thermo Fisher) was used to test the expression efficiency of the CD16 CAR protein expressed by 293T.

(210) 8.2 Results

(211) TABLE-US-00016 TABLE 14 Nucleic acid Concentration pUC57-EV29-CD16CAR-H1S1 plasmid (purified) 268.5 ng/μl pUC57-EV29-CD16CAR-H1S1 linearized plasmid 221.7 ng/μl (purified) EV29-CD16CAR--H1S1 pre-circularized mRNA 375.4 ng/μl (purified) EV29-CD16CAR-H1S1 cyclic mRNA (purified) 284.3 ng/μl

(212) By using anti-CD16 antibody expression rate of mRNA detected annular mediated CD16CAR in the 293T. The results showed that the positive rates of CD16 CAR expression were 90.4%, 85.6%, 80.3%, 78.4%, and 60.5% in the 1-5 days of circular mRNA transfection, indicating that the circular mRNA of the present disclosure can achieve CD16 antibody efficient, long-lasting expression.

(213) The above-mentioned embodiments of the present disclosure are merely examples to clearly illustrate the present disclosure, and are not intended to limit the implementation of the present disclosure. For those of ordinary skill in the art, other changes or changes in different forms can be made based on the above description. It is unnecessary and cannot be exhaustive of all embodiments. Any modifications within the spirit and principle of the present disclosure, equivalent substitutions and improvements should be included within the scope of the present disclosure claims.