US20220177908A1 - Recombinant nucleic acid molecule of transcriptional circular rna and its application in protein expression - Google Patents

Recombinant nucleic acid molecule of transcriptional circular rna and its application in protein expression Download PDF

Info

Publication number
US20220177908A1
US20220177908A1 US17/337,612 US202117337612A US2022177908A1 US 20220177908 A1 US20220177908 A1 US 20220177908A1 US 202117337612 A US202117337612 A US 202117337612A US 2022177908 A1 US2022177908 A1 US 2022177908A1
Authority
US
United States
Prior art keywords
seq
sequence shown
nucleotide sequence
protein
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US17/337,612
Other languages
English (en)
Inventor
Zhenhua Sun
Chijian ZUO
Jiafeng Zhu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Purecell Biomedical Technology Co Ltd
Original Assignee
Jiangsu Purecell Biomedical Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Purecell Biomedical Technology Co Ltd filed Critical Jiangsu Purecell Biomedical Technology Co Ltd
Priority to US17/486,204 priority Critical patent/US11634727B2/en
Assigned to JIANGSU PURECELL BIOMEDICAL TECHNOLOGY CO. LTD. reassignment JIANGSU PURECELL BIOMEDICAL TECHNOLOGY CO. LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SUN, ZHENHUA, ZHU, Jiafeng, ZUO, Chijian
Publication of US20220177908A1 publication Critical patent/US20220177908A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • C12N2840/206—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES having multiple IRES
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure belongs to the technical fields of molecular biology and bioengineering. Specifically, the present disclosure relates to a recombinant nucleic acid molecule of transcriptional circular RNA and its application in protein expression. More specifically, the present disclosure relates to a recombinant nucleic acid molecule of transcriptional circular RNA, a recombinant expression vector, pre-circularized RNA, circular RNA, recombinant host cells, pharmaceutical compositions, and methods for preparing proteins.
  • mRNA Messenger Ribonucleic Acid
  • mRNA is transiently expressed in cells, there is no risk of integration into the genome, and it is not dependent on the cell cycle, so it has higher security [4] ;
  • mRNA does not have the immune resistance brought by the vector itself, so protein expression is easier to achieve [5] ;
  • the mRNA production process is cell-free. The system only involves enzyme-catalyzed reactions in vitro, so the production process is simpler, more controllable and low-cost [6] .
  • mRNA has shown wide application potential as vaccines, production of therapeutic proteins and as a means of gene therapy.
  • linear mRNA used in both clinical or pre-clinical applications is mainly linear mRNA.
  • the structure of linear mRNA includes 5′ cap structure (5′ Cap), 3′ polyadenosine tail (PolyA tail), and 5′ untranslational sequence (5′ untranslational). region, 5′ UTR), 3′untranslational region (3′ UTR), and open reading frame (ORF), etc. [7] .
  • the 5′ cap structure is the basic feature of eukaryotic mRNA, which is obtained by adding N7-methylguanosine to the 5′ end of the mRNA [8] .
  • the 5′ cap structure promotes mRNA translation by binding to the translation initiation complex eif4E, and can prevent mRNA degradation effectively and reduce mRNA immunogenicity.
  • the main function of the 3′polyadenosine tail is to bind to PolyA binding protein (PolyA binding protein, PABP), which interacts with eiF4G and eiF4E to mediate the formation of a ring of mRNA, promote the translation process, and prevent mRNA degradation [9] .
  • 5′ and 3′ untranslated sequences such as beta-globin 5′ and 3′ untranslated sequences, can effectively prevent mRNA degradation and promote mRNA translation into protein.
  • Circular RNA Circular RNA
  • Naturally occurring circRNAs are mainly produced through a molecular mechanism called “back splicing” in cells. It has been found that eukaryotic circRNAs have a variety of molecular cell regulatory functions [10] .
  • circular RNA can regulate the expression of target genes by binding microRNAs (miRNA); circular RNA can regulate gene expression by directly binding to target proteins.
  • miRNA microRNAs
  • the currently confirmed circular RNAs mainly function as non-coding RNAs.
  • there are also circular RNAs that can encode proteins in nature which is circular mRNAs.
  • Circular mRNA tends to have a longer half-life due to its circular nature, so it is speculated that circular mRNA may have better stability.
  • Methods of forming circular RNA in vitro include chemical methods, protease catalysis and ribozyme catalysis, etc. [11] .
  • the natural type I intron system can undergo cleavage and ligation reactions to form circular intron RNA.
  • the conserved sequence of the specific splicing site located at the 5′end of exon E1 is broken by the nucleophilic attack of the free 3′hydroxyl of guanylic acid triphosphate, resulting in a naked 3′hydroxyl, and guanylic acid binds to on the broken 5′exon E1. Thereafter, the naked 3′hydroxyl at the 5′end of the intron attacked the conserved sequence between the 3′end of the intron and exon E2, and exon E2 was removed, and the intron undergoes a loop reaction in order to obtain the circular intron RNA [12-13] .
  • a modified ribozyme-catalyzed method from Anabaena tRNA introns has been reported to be applied to the formation of circular RNA in vitro [14] , called the “inverted type I intron-exon self-cleavage system” (Group I permuted intron-exon self-splicing system, PIE system).
  • This method can excise introns to form circular RNA containing exons. Therefore, this method has the potential to form expressible circular mRNA.
  • the basic design principle of the PIE system is to connect exon E1 and E2 sequences end to end through molecular cloning to form a continuous circular plasmid.
  • the intron is cut and broken by restriction endonuclease to obtain a linear plasmid. Then inverted T7 promoter upstream of 3′intron was used for in vitro transcription to obtain pre-circularized RNA containing 3′intron-E2-E1-5′ intron structure. Similar to the natural type I intron system, the specific splicing site conservative sequence of exon E1 is broken by the nucleophilic attack of the free guanylic acid 3′hydroxyl group, and exon E1 produces a naked 3′ Hydroxyl, and guanylic acid binds to the broken 5′intron. After that, the naked 3′hydroxyl of exon E1 attacked the conserved sequence between 3′intron and exon E2, removing the 3′intron, and exon E2 and E1 formed a loop reaction to get circular E1-E2 RNA.
  • the PIE system can be used to construct circular RNAs for eukaryotic protein expression.
  • EMCV Extraphalomyocarditis Virus
  • CVB3 Coxsackievirus B3
  • IRES Internal ribosome entry site
  • the coding region and spacer sequence (Spacer) was added between the exons E1.
  • the study referred to the PIE system discovered by M. Puttaraju and Michael D. Been, etc., and used the same Anabaena tRNA PIE system to construct circular mRNA.
  • the EMCV or CVB3 IRES sequence and the coding gene Gluc (Gaussia luciferase) between E1 and E2 of the PIE system, set homology arm sequences at the 5′ and 3′ends of the RNA, respectively, between IRES and exon E2,
  • a spacer sequence is added between the coding region and exon E1, which can form circular mRNA to the greatest extent.
  • circular mRNA is obtained through the autocatalytic reaction of the PIE system under the action of heating and guanylate triphosphate.
  • the circular mRNA finally contains exon E1 and E2 sequences, spacer sequence, IRES and coding gene sequence.
  • E1 and E2 sequences have better mRNA looping characteristics and can enhance protein expression.
  • CVB3 IRES has a high ability to mediate mRNA translation by screening different IRES sequences, and therefore can achieve relatively high protein expression.
  • the present invention provides a recombinant nucleic acid molecule whose circular RNA formed by transcription contains specific IRES elements, which can express target polypeptides in eukaryotic cells continuously and efficiently, and is suitable for preparing mRNA infectious disease vaccines and therapeutic mRNA Tumor vaccines, dendritic cell (DC) tumor vaccines based on mRNA, or for mRNA-based gene therapy (Gene therapy), mRNA-based chimeric antigen receptor T-cell therapy (Chimeric antigen receptor T-cell) therapy, Car-T), protein supplement therapy and other fields.
  • IRES elements specific IRES elements
  • the 5′homology arm comprises the sequence shown in any one of (a1)-(a2) below:
  • the 3′homology arm includes the sequence shown in any one of (b1)-(b2) below:
  • the 5′spacer comprises the sequence shown in any one of (c1)-(c2) below:
  • the 3′spacer includes the sequence shown in any of the following (o1)-(o2):
  • the 3′intron is located upstream of the second exon, and the 5′spacer is included between the second exon and the IRES element; the first exon is located upstream of the 5′intron, and the 3′spacer is included between the first exon and the coding region.
  • the second exon includes the sequence shown in any of the following (e1)-(e2):
  • the first exon includes the sequence shown in any one of (f1)-(f2):
  • the 5′intron includes the sequence shown in any one of (g1)-(g2):
  • the pre-circularized RNA includes 5′homology arm, 3′intron, second exon, 5′spacer, IRES element, coding region, 3′spacer, first exon, 5′′Intron and 3′homology arm.
  • the circular RNA comprises a second exon, a 5′spacer, an IRES element, a coding region, a 3′spacer and a first exon that are sequentially connected.
  • the PD-1 monoclonal antibody comprises any one of the following (j1)-(j6):
  • the recombinant nucleic acid molecule of the present disclosure is transcribed to form a circular RNA containing a specific IRES element.
  • the IRES element can increase the protein expression level of the circular RNA in eukaryotic cells and achieve efficient and persistent protein expression, And the expression efficiency is higher than linear mRNA molecules or other circular RNAs, which can meet the needs of industrialized protein expression.
  • the recombinant nucleic acid molecule of the present disclosure further comprises 5′homology arms, 3′homology arms, 5′spacers, and 3′spacer sequences with specific sequences to make the circular RNA molecule loop.
  • the efficiency and the level of expressed protein are further improved.
  • the circular RNA provided by the present disclosure can increase the expression level of the target polypeptide in eukaryotic cells, and achieve high efficiency and durability for antigens, antibodies, antigen binding receptors, ligands, fusion proteins, or recombinant proteins.
  • sexual expression suitable for preparing therapeutic vaccines, antibodies or chimeric antigen receptors, T cell receptors, pharmaceutical recombinant proteins, etc.
  • 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;
  • FIG. 2 shows the agarose gel electrophoresis diagram for identifying RNA loops.
  • 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;
  • FIG. 3 shows the results of sequencing to identify RNA ring formation
  • 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;
  • 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);
  • 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)
  • FIG. 7 shows the agarose gel electrophoresis diagram for identifying RNA loops.
  • RNA ladder 1.
  • CVB3-EGFP pre-circularized mRNA 3.
  • CVB3-EGFP circularized mRNA 4.
  • EV29-EGFP H1S1 pre-circularized mRNA 5.
  • EV29-EGFP H1S1 circularized mRNA 6.
  • 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);
  • 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).
  • the term “about” means: a value includes the standard deviation of the error of the device or method used to determine the value.
  • polypeptide 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).
  • the term “circular RNA” is a closed circular RNA molecule, mainly composed of exons, IRES elements, protein coding regions and spacers.
  • 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”.
  • 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.
  • 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.
  • 5′Cap 5′cap structure
  • PolyA tail 3′polyadenosine tail
  • ORF open reading frame
  • IRES Internal ribosome entry site
  • IRES Internal ribosome entry site
  • IRES Internal ribosome entry site
  • IRES 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.
  • 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.
  • coding region refers to a gene sequence capable of transcribing messenger RNA and finally translating it into a target polypeptide or protein.
  • upstream or downstream refers to upstream and downstream along the protein translation direction of the coding region.
  • 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.
  • the antigen of the present disclosure is selected from virus-derived antibodies or tumor-specific antigens.
  • the antibodies of the present disclosure are selected from Fab, Fab′, F(ab′)2, Fv, scFv, sdAb, diabody, camelid antibody, or monoclonal antibody.
  • the antigen binding receptors of the present disclosure are selected from chimeric antigen receptors or T cell receptors.
  • 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.
  • 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.
  • “mutation” in the present disclosure includes “conservative mutation”.
  • conservative mutation refers to a conservative mutation that can normally maintain the function of a protein.
  • 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
  • polar amino acids mutations that replace each other between Gln and Asn
  • basic amino acids mutations that replace each other between Lys, Arg, and His
  • 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.
  • 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, Trp to Phe or Tyr, Tyr to His, Phe or Tr Trp, Tyr, Tyr to His, P
  • sequence identity and “percent identity” refer to the percentage of identical (ie identical) nucleotides or amino acids between two or more polynucleotides or polypeptides.
  • 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.
  • sequences or subsequences 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”.
  • sequence identity or “percent identity” can be based on any suitable region of the sequence.
  • 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.
  • the sequence is substantially the same over the entire length of any one or two compared biopolymers (ie, nucleic acids or polypeptides).
  • 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.
  • 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.
  • 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”.
  • 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”.
  • 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.
  • a recombinant nucleic acid molecule includes a coding region encoding a polypeptide of interest, and an IRES element connected upstream of the coding region.
  • the recombinant nucleic acid molecule of the present disclosure comprises the following sequence structure:
  • 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.
  • 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.
  • viral plasmids 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.
  • viral plasmids such as viral plasmids, bacterial plasm
  • 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.
  • antigens include virus-derived antigens, such as novel coronavirus (SARS-CoV-2) antigens, or tumor-specific antigens.
  • 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.
  • single-chain antibody 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.
  • a short peptide also called a linker
  • T cell receptor T cell receptor, TCR
  • TCR T cell receptor
  • the TCR of most T cells is composed of ⁇ and ⁇ peptide chains, and the TCR of a few T cells is composed of ⁇ and ⁇ peptide chains.
  • chimeric antigen receptor is an artificial receptor that is engineered to contain an immunoglobulin antigen binding domain.
  • chimeric antigen receptors can include domains such as antigen binding region, hinge region, transmembrane region and intracellular structural region.
  • 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.
  • 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.
  • transformation, transfection, transduction 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.
  • CaPO4 calcium phosphate
  • CaCl2 calcium chloride
  • 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.
  • 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.
  • 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).
  • 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 ⁇ g/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.
  • 5 ⁇ SSPE saline sodium phosphate EDTA
  • 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 ⁇ g/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.
  • 5 ⁇ SSPE saline sodium phosphate EDTA
  • the sequence shown in SEQ ID NO:1 is the nucleotide sequence of the T7 promoter;
  • the sequence shown in SEQ ID NO: 2 is the nucleotide sequence of 5′homology arm sequence 1 (H1);
  • the sequence shown in SEQ ID NO: 3 is the nucleotide sequence of 5′homology arm sequence 2 (H2);
  • the sequence shown in SEQ ID NO: 4 is the nucleotide sequence of the 3′intron of the Type I PIE system;
  • the sequence shown in SEQ ID NO: 5 is the nucleotide sequence of the second exon (E2) of the class I PIE system;
  • the sequence shown in SEQ ID NO: 6 is the nucleotide sequence of 5′spacer sequence 1;
  • the sequence shown in SEQ ID NO: 7 is the nucleotide sequence of 5′spacer sequence 2;
  • the sequence shown in SEQ ID NO: 8 is the nucleotide sequence of CVB3 IRES;
  • the sequence shown in SEQ ID NO: 9
  • 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.
  • 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.
  • 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.
  • 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.
  • the IRES element comprises a chimera sequence of CVB3v IRES and any one of EV24 IRES, EV29 IRES, and EV33 IRES.
  • 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.
  • 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.
  • 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.
  • the spacer includes a 5′spacer and a 3′spacer.
  • 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.
  • 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.
  • 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.
  • 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.
  • the structure of the recombinant nucleic acid molecule is as follows: 5′homology arm-3′intron-second exon E2-5′spacer-IRES element-coding region-3′spacer-first exon E1-5′ intron-3′ Homology arm.
  • the recombinant nucleic acid molecule may also contain regulatory sequences.
  • 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.
  • the present disclosure provides a recombinant expression vector comprising the aforementioned recombinant nucleic acid molecule.
  • the vector for connecting the recombinant nucleic acid molecule can be various vectors commonly used in the art, such as pUC57 plasmid.
  • 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.
  • the present disclosure provides a pre-circularized RNA formed by post-transcription of a recombinant nucleic acid molecule or a linearized recombinant expression vector.
  • the pre-circularized RNA has the following structure:
  • 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.
  • DNA vector recombinant expression vector
  • 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.
  • 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.
  • 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.
  • one or more target polypeptides selected from antigens, antibodies, antigen-binding receptors, ligands, fusion proteins, and recombinant proteins are expressed.
  • 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.
  • the circular RNA expresses viral antigens.
  • 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.
  • the circular RNA expresses a recombinant humanized protein.
  • 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.
  • 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.
  • the circular RNA expresses tumor-specific antigens, which include CEA AFP PSA PSMA MAGE-A3 PAP protein and the like.
  • 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.
  • 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.
  • 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.
  • the circular RNA expresses monoclonal antibodies.
  • 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.
  • 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.
  • the present disclosure provides a recombinant host cell comprising the aforementioned recombinant nucleic acid molecule, recombinant expression vector, pre-circularized RNA or circular RNA.
  • 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.
  • the present disclosure provides a pharmaceutical composition
  • 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.
  • the experimental techniques and experimental methods used in this example are conventional techniques and methods unless otherwise specified.
  • 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.
  • 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.
  • the IRES component information is as follows:
  • 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 + CVB3v
  • the IRES element and v domain of the Enterovirus EV24 is SEQ ID NO: 12 replaced with the v domain of the IRES element of CVB3 IRES.
  • the IRES element and v domain of the Enterovirus EV29 is SEQ ID NO: 13 replaced with the v domain of the IRES element of CVB3 IRES.
  • the IRES element and v domain of the Enterovirus EV33 is SEQ ID NO: 14 replaced with the v domain of the IRES element of CVB3 IRES.
  • the definition of the v domain of the IRES element can be found in the reference (Proc Natl Acad Sci USA. 2009 Jun. 9; 106(23): 9197-9202.)
  • the EGFP-expressing circular RNA sequences obtained from the above IRES elements are as follows:
  • 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
  • the formula of 1% denatured agarose gel is as follows: ⁇ circle around (1) ⁇ Weigh 1 g agarose to 72 ml nuclease-free, H 2 O, 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
  • GTP Buffer 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM DTT, pH 7.5
  • ⁇ 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 50 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
  • 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
  • 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.
  • 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.
  • Lipofectamine MessengerMax Invitrogen
  • RNA circularization 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.
  • 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.
  • 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.
  • FIG. 4 The quantitative test results of fluorescence intensity 1-3 days after cell transfection are shown in FIG. 4 .
  • the circular EGFP mRNA containing CVB3 IRES (Circ-RNA CVB3, reference 15)
  • 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.
  • Example 2 Expression of Circular mRNA Obtained by Combining EV29 IRES with Different Homology Arms and Spacer Sequences in 293T Cells
  • 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
  • 5′spacer 1 sequence SEQ ID NO: 6
  • the nucleotide sequence shown) and the 3′spacer 1 sequence are used as the basic elements of circular mRNA to construct a circular mRNA encoding green fluorescent protein (EGFP) (Circ-RNA EV29 H1S1).
  • 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
  • a circular mRNA (Circ-RNA EV29 H2S2) encoding green fluorescent protein (EGFP) is constructed.
  • 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.
  • 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.
  • 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.
  • the 1-3 d 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.
  • 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.
  • Circ-RNA EV29-EGFP H2S2 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).
  • 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
  • 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.
  • 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.).
  • 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.
  • EPO erythropoiesis hormone
  • EPO ELISA detection kit (Thermo Fisher) was used to quantify the EPO protein expressed by 293T.
  • 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.
  • 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.
  • PD-1 resistance cell death receptor 1
  • the DNA synthesis was commissioned by Suzhou Genweiz Biotechnology Co., Ltd. to complete.
  • 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.
  • 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.
  • the method is the same as 1.1 of Example 1.
  • the PD1 protein expressed by 293T was quantified using PD1 ELISA detection kit (Thermo Fisher).
  • 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.
  • 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.
  • IL-15 ELISA detection kit (Thermo Fisher) was used to quantify the IL-15 protein expressed by 293T.
  • 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.
  • 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.
  • PAP acid phosphatase protein
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
US17/337,612 2020-12-04 2021-06-03 Recombinant nucleic acid molecule of transcriptional circular rna and its application in protein expression Pending US20220177908A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/486,204 US11634727B2 (en) 2020-12-04 2021-09-27 Recombinant nucleic acid molecule of transcriptional circular RNA and its application in protein expression

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011408937.4 2020-12-04
CN202011408937.4A CN112481289B (zh) 2020-12-04 2020-12-04 一种转录环状rna的重组核酸分子及其在蛋白表达中的应用

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/486,204 Continuation US11634727B2 (en) 2020-12-04 2021-09-27 Recombinant nucleic acid molecule of transcriptional circular RNA and its application in protein expression

Publications (1)

Publication Number Publication Date
US20220177908A1 true US20220177908A1 (en) 2022-06-09

Family

ID=74939493

Family Applications (2)

Application Number Title Priority Date Filing Date
US17/337,612 Pending US20220177908A1 (en) 2020-12-04 2021-06-03 Recombinant nucleic acid molecule of transcriptional circular rna and its application in protein expression
US17/486,204 Active US11634727B2 (en) 2020-12-04 2021-09-27 Recombinant nucleic acid molecule of transcriptional circular RNA and its application in protein expression

Family Applications After (1)

Application Number Title Priority Date Filing Date
US17/486,204 Active US11634727B2 (en) 2020-12-04 2021-09-27 Recombinant nucleic acid molecule of transcriptional circular RNA and its application in protein expression

Country Status (3)

Country Link
US (2) US20220177908A1 (zh)
EP (1) EP4008336A1 (zh)
CN (1) CN112481289B (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230060661A1 (en) * 2021-08-13 2023-03-02 Suzhou Curemed Biomedical Technology Co. Ltd. Tissue-specifically expressed circular rna molecule and application thereof

Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112481289B (zh) 2020-12-04 2023-06-27 苏州科锐迈德生物医药科技有限公司 一种转录环状rna的重组核酸分子及其在蛋白表达中的应用
CN114630909B (zh) * 2020-12-04 2024-05-28 苏州科锐迈德生物医药科技有限公司 环状rna、包含环状rna的疫苗及用于检测新型冠状病毒中和抗体的试剂盒
CN113101363B (zh) * 2021-04-06 2022-08-26 中山大学孙逸仙纪念医院 一种环状rna疫苗及其应用
CN115433733A (zh) * 2021-06-04 2022-12-06 生物岛实验室 环状RNA Circ-ACE2翻译的多肽及其应用
CN114591986A (zh) * 2021-07-29 2022-06-07 苏州科锐迈德生物医药科技有限公司 环状rna分子及其在目标蛋白的靶向降解中的应用
TW202321451A (zh) * 2021-08-18 2023-06-01 北京大學 工程化的 adar 招募 rnas 及其使用方法
CN114277039B (zh) * 2021-10-25 2024-06-21 浙江君怡生物科技有限公司 呼吸道合胞病毒mRNA疫苗及其制备方法和应用
CA3239266A1 (en) * 2021-11-24 2023-06-01 Flagship Pioneering Innovations Vi, Llc Coronavirus immunogen compositions and their uses
AU2022397292A1 (en) * 2021-11-24 2024-05-30 Flagship Pioneering Innovations Vi, Llc Varicella-zoster virus immunogen compositions and their uses
WO2023115732A1 (en) * 2021-12-21 2023-06-29 Peking University Single-pot methods for producing circular rnas
TW202334412A (zh) * 2022-01-11 2023-09-01 北京大學 針對 sars-cov-2 變體的環狀 rna 疫苗及其使用方法
WO2023143541A1 (en) * 2022-01-28 2023-08-03 Beijing Changping Laboratory Circular rna vaccines and methods of use thereof
CN117417937A (zh) * 2022-01-31 2024-01-19 奥明(杭州)生物医药有限公司 一种环状rna分子及应用
CN114438127B (zh) * 2022-03-02 2024-03-19 苏州科锐迈德生物医药科技有限公司 一种重组核酸分子及其在制备环状rna中的应用
CN114507691A (zh) * 2022-03-02 2022-05-17 深圳市瑞吉生物科技有限公司 一种用于制备环状rna的载体及其应用
CN114574483B (zh) * 2022-03-02 2024-05-10 苏州科锐迈德生物医药科技有限公司 基于翻译起始元件点突变的重组核酸分子及其在制备环状rna中的应用
CN114875053A (zh) * 2022-03-11 2022-08-09 杭州师范大学 一种高效稳定环状rna的构建方法及其产品
WO2024051842A1 (en) * 2022-09-10 2024-03-14 Exclcirc (Suzhou) Biomedical Co., Ltd. Circular rnas and preparation methods thereof
CN118360300A (zh) * 2023-01-10 2024-07-19 优环(苏州)生物医药科技有限公司 一种表达尿酸氧化酶的环状rna、制备方法及应用
CN117070564B (zh) * 2023-03-30 2024-05-10 安可来(重庆)生物医药科技有限公司 一种用于合成环形rna的质粒及其构建方法与一种环形rna及其体外合成方法
CN117051043B (zh) * 2023-10-11 2024-01-30 圆因(北京)生物科技有限公司 一种基于环状rna编码耐甲氧西林金黄色葡萄球菌内溶素及其应用

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150291975A1 (en) * 2014-04-09 2015-10-15 Dna2.0, Inc. Enhanced nucleic acid constructs for eukaryotic gene expression
US20200085944A1 (en) * 2017-03-17 2020-03-19 Curevac Ag Rna vaccine and immune checkpoint inhibitors for combined anticancer therapy
WO2019236673A1 (en) * 2018-06-06 2019-12-12 Massachusetts Institute Of Technology Circular rna for translation in eukaryotic cells
JP2020046952A (ja) 2018-09-19 2020-03-26 富士ゼロックス株式会社 情報処理装置及び情報処理プログラム
IL285951B1 (en) * 2019-03-01 2024-04-01 Flagship Pioneering Innovations Vi Llc Polyribonucleotides and their cosmetic uses
WO2020198403A2 (en) * 2019-03-25 2020-10-01 Flagship Pioneering Innovations Vi, Llc Compositions comprising modified circular polyribonucleotides and uses thereof
CN114258430A (zh) * 2019-04-22 2022-03-29 T细胞受体治疗公司 使用融合蛋白进行tcr重编程的组合物和方法
KR20220027855A (ko) * 2019-05-22 2022-03-08 매사추세츠 인스티튜트 오브 테크놀로지 원형 rna 조성물 및 방법
CN111778256B (zh) * 2020-04-20 2022-03-25 广东省微生物研究所（广东省微生物分析检测中心） CircRNA PVT1及其肽段PVT1-104aa在抗衰老方面的应用
CN112481289B (zh) * 2020-12-04 2023-06-27 苏州科锐迈德生物医药科技有限公司 一种转录环状rna的重组核酸分子及其在蛋白表达中的应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230060661A1 (en) * 2021-08-13 2023-03-02 Suzhou Curemed Biomedical Technology Co. Ltd. Tissue-specifically expressed circular rna molecule and application thereof

Also Published As

Publication number Publication date
US20220177910A1 (en) 2022-06-09
EP4008336A1 (en) 2022-06-08
CN112481289A (zh) 2021-03-12
US11634727B2 (en) 2023-04-25
CN112481289B (zh) 2023-06-27

Similar Documents

Publication Publication Date Title
US11634727B2 (en) Recombinant nucleic acid molecule of transcriptional circular RNA and its application in protein expression
US11866510B2 (en) Chimeric antigen receptor with single domain antibody
WO2017215619A1 (zh) 在细胞内产生点突变的融合蛋白、其制备及用途
CN101688222A (zh) 启动子
EP3420090A1 (en) Transposon system and methods of use
WO2022261150A2 (en) Particle delivery systems
US20210047643A1 (en) Gene editing using homology-independent universal genome engineering technology
CN114574483A (zh) 基于翻译起始元件点突变的重组核酸分子及其在制备环状rna中的应用
JP2009538144A (ja) 真核細胞株を用いたタンパク質産生
JP3844656B2 (ja) 動物細胞の形質転換のための方法
CA2706050C (en) Novel att recombination sequences
CN111051509A (zh) 用于电介质校准的含有c2cl核酸内切酶的组合物以及使用其进行电介质校准的方法
WO2023046153A1 (en) Circular rna and preparation method thereof
CN114630909B (zh) 环状rna、包含环状rna的疫苗及用于检测新型冠状病毒中和抗体的试剂盒
US20190233843A1 (en) Transposon system and methods of use
CN113564145B (zh) 用于胞嘧啶碱基编辑的融合蛋白及其应用
CN109320597B (zh) 狐亚科激活素a蛋白及其制备与应用
CN106701764B (zh) 15kDa硒蛋白基因的启动子及其核心区与应用
JP2022513319A (ja) 予測可能かつ安定な導入遺伝子発現を有するｓｓｉ細胞および形成の方法
CN114410644B (zh) 一种环状rna分子及应用
WO2024010028A1 (ja) 環状ｒｎａ分子及びこれを用いた翻訳制御方法、翻訳活性化システム、並びに医薬組成物
WO2024153120A1 (zh) 突变的pd1胞外域片段及含有该片段的car和nk细胞
CN114409760B (zh) 一类结直肠癌的环状mRNA肿瘤免疫类药物
US20220362407A1 (en) A promoter specific for non-pigmented ciliary epithelial cells
WO2020139156A1 (en) Gene therapy dna vector and its application

Legal Events

Date Code Title Description
AS Assignment

Owner name: JIANGSU PURECELL BIOMEDICAL TECHNOLOGY CO. LTD., CHINA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SUN, ZHENHUA;ZUO, CHIJIAN;ZHU, JIAFENG;REEL/FRAME:057684/0082

Effective date: 20210528

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION