WO2020186991A1 - 利用环形rna进行蛋白翻译及其应用 - Google Patents

利用环形rna进行蛋白翻译及其应用 Download PDF

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WO2020186991A1
WO2020186991A1 PCT/CN2020/077026 CN2020077026W WO2020186991A1 WO 2020186991 A1 WO2020186991 A1 WO 2020186991A1 CN 2020077026 W CN2020077026 W CN 2020077026W WO 2020186991 A1 WO2020186991 A1 WO 2020186991A1
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translation initiation
construct
sequence
protein
translation
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French (fr)
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王泽峰
杨赟
樊晓娟
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Shanghai Institute of Nutrition and Health of CAS
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Shanghai Institute of Nutrition and Health of CAS
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Priority to US17/440,774 priority Critical patent/US20220177898A1/en
Priority to CN202080036818.2A priority patent/CN113825837A/zh
Priority to EP20773279.3A priority patent/EP3943601A4/en
Priority to JP2021559486A priority patent/JP7297331B2/ja
Priority to CN202410975296.2A priority patent/CN118834867A/zh
Priority to CN202410975341.4A priority patent/CN118834868A/zh
Application filed by Shanghai Institute of Nutrition and Health of CAS filed Critical Shanghai Institute of Nutrition and Health of CAS
Priority to CN202410093532.8A priority patent/CN117965533A/zh
Publication of WO2020186991A1 publication Critical patent/WO2020186991A1/zh
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Priority to JP2023094068A priority patent/JP7673989B2/ja
Priority to JP2024209709A priority patent/JP2025029079A/ja
Priority to JP2024209710A priority patent/JP2025029080A/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to the field of biotechnology, in particular to the use of circular RNA for protein translation and its application.
  • Common protein replacement or expression therapies include ribonucleic acid (DNA) carrier-based delivery systems, deoxyribonucleic acid (RNA) carrier-based delivery systems, and protein delivery systems. These methods all need to produce protein through messenger RNA translation.
  • the common translation initiation method in eukaryotes is cap-dependent translation, which mainly uses the translation initiation factor to recognize the special cap structure at the 5′ end of the messenger RNA to initiate translation. This type of translation method only exists in linear messenger RNA. .
  • cap-independent translation initiation which mainly initiates translation through the interaction between specific protein factors and RNA elements. This type of translation can be initiated in linear or circular RNA.
  • Common cap-independent translation initiation elements are some elements with specific secondary structure in viral RNA. They can borrow the host cell's translation system to express their desired protein.
  • the internal ribosome entry site (IRES) element contained in RNA such as encephalomyocarditis virus or hepatitis C virus.
  • Circular RNA is a type of single-stranded closed-loop RNA form that is different from linear RNA. Due to its structural specificity, it is not easily degraded by exonuclease, which is more stable than linear RNA. Therefore, the expression of protein through circular RNA translation has the characteristics of more continuous and long-term effect and is an important means to replace linear RNA translation.
  • a common method is to use viral IRES to initiate the translation of circular RNA.
  • pathogenic viral RNA may have immune rejection in the host, the RNA elements derived from viruses basically contain complex RNA secondary structures and long sequences , Which limits the construction of viral IRES-based expression systems and the later application of gene therapy.
  • the purpose of the present invention is to provide a cap-independent translation initiation element of non-viral origin.
  • the first aspect of the present invention provides a circular RNA construct, which has the structure shown in formula I from the 5'-3' direction:
  • TI is the translation initiation element
  • Z1 is an expression cassette for expressing foreign protein
  • Z2 is none or other components
  • each "-" is a bond or a nucleotide connection sequence
  • the length of the TI element is 6-30 nt, preferably, 8-24 nt, more preferably, 10-20 nt;
  • the content of A is ⁇ 35%, preferably, ⁇ 45%, more preferably, ⁇ 60%;
  • the content of T is ⁇ 20%, preferably, ⁇ 30%, more preferably, ⁇ 50%;
  • the content of A+T is ⁇ 65%, preferably, ⁇ 80%, more preferably, ⁇ 90%;
  • the content of G is ⁇ 35%, preferably, ⁇ 25%, more preferably, ⁇ 10%.
  • the circular RNA construct is a circular messenger RNA construct.
  • the content of A in the TI element is 35-100%, preferably, 45-100%, more preferably, 60-100%.
  • the content of T in the TI element is 20-100%, preferably, 30-100%, more preferably, 50-100%.
  • the content of A+T in the TI element is 65-100%, preferably, 80-100%, more preferably, 90-100%.
  • the content of G in the TI element is 0-35%, preferably, 0-25%, more preferably, 0-10%.
  • the TI element contains one or more nucleotide sequences selected from the following group shown in Table 1:
  • the TI element is added with 1-24 (preferably 1-15, more preferably 1-10) at the 5'end and/or 3'end of the nucleotide sequence shown in Table 1.
  • 1-24 preferably 1-15, more preferably 1-10
  • One, more preferably, 1-6 nucleotides, and has the function of TI element.
  • the coding sequence of the TI element is selected from the following group;
  • the TI element has a sequence shown in SEQ ID NO.: 1-40.
  • the coding sequence of the TI element is shown in SEQ ID NO.: 1-40.
  • the Z1 element contains a stop codon.
  • the Z1 element does not contain a stop codon.
  • the coding sequence of the foreign protein is from a prokaryote or eukaryote.
  • the coding sequence of the foreign protein is derived from animals, plants, and pathogens.
  • the coding sequence of the foreign protein is derived from mammals, preferably primates, rodents, including humans, mice, and rats.
  • the encoding sequence of the foreign protein is selected from the group consisting of encoding luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthesis Enzymes, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, foreign DNA of variable regions of antibodies, DNA of luciferase mutants, or a combination thereof.
  • the foreign protein is selected from the following group: luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, variable regions of antibodies, luciferase mutations, ⁇ -amylase, enterobacteria A, hepatitis C virus E2 glycoprotein, insulin precursor , Interferon ⁇ A, interleukin-1 ⁇ , lysozyme, serum albumin, single-chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydr
  • the Z2 element is selected from the group consisting of PolyA, multiple cloning site, aptamer, miRNA binding site, translation enhancement element, or a combination thereof.
  • one or more adenines (A) of the TI element are methylated.
  • sequence of the circular RNA construct is shown in SEQ ID NO.: 61.
  • the second aspect of the present invention provides a vector containing the expression cassette of the construct according to the first aspect of the present invention.
  • the expression cassette contains a first intron and a second intron.
  • first intron and the second intron are completely complementary or not completely complementary.
  • the vector has the sequence shown in SEQ ID NO.: 62.
  • sequence of the first intron is shown in SEQ ID NO.: 63.
  • sequence of the second intron is shown in SEQ ID NO.: 64.
  • the third aspect of the present invention provides a genetically engineered cell in which the nucleic acid construct of the first aspect of the present invention is integrated at one or more sites of the genome of the genetically engineered cell, or the genetically engineered cell contains the present invention
  • the carrier described in the second aspect of the invention is not limited to any particular order.
  • the genetically engineered cells include prokaryotic cells and eukaryotic cells.
  • the eukaryotic cells include higher eukaryotic cells.
  • the genetically engineered cells are selected from the group consisting of human-derived cells (such as Hela cells), Chinese hamster ovary cells, insect cells, wheat germ cells, rabbit reticulocytes, yeast cells, or combinations thereof.
  • the genetically engineered cell is a yeast cell.
  • the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces yeast, or a combination thereof.
  • the Kluyveromyces yeast is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces dobriella, or a combination thereof.
  • the fourth aspect of the present invention provides a reaction system, including:
  • reaction system further includes YTHDF3, PABPC1, and/or hnRNPA1 protein.
  • the reaction system is an in vitro reaction system.
  • the fifth aspect of the present invention provides a method for synthesizing protein in vitro, including the steps:
  • step (ii) Under suitable conditions, incubate the synthesis system of step (i) for a period of T1 to synthesize the protein.
  • the method further includes: (iii) optionally separating or detecting the protein from the in vitro reaction system.
  • the reaction temperature is 25-42°C, preferably, 30-40°C, more preferably, 35-37°C.
  • the reaction time T1 is 1 hour to 20 hours, preferably, 2 hours to 12 hours, more preferably, 3 hours to 6 hours.
  • the sixth aspect of the present invention provides a kit for in vitro protein synthesis, including:
  • the second container, and other components required for the reaction in the second container, the other components are selected from the group consisting of spliceosome, ribosome, translation initiation factor EIF4G2, translation initiation factor EIF4A , The translation initiation factor EIF4B, or a combination thereof; and
  • first container and the second container are the same container or different containers.
  • the kit further includes one or more containers optionally selected from the following group:
  • the seventh aspect of the present invention provides a construct according to the first aspect of the present invention, a vector according to the second aspect of the present invention, a genetically engineered cell according to the third aspect of the present invention, and the fourth aspect of the present invention
  • the reaction system or the use of the kit according to the sixth aspect of the present invention is used for high-throughput in vitro protein synthesis.
  • Figure 1 shows the different cell populations screened by flow cytometry.
  • Figure 2 shows the activity of western blot to detect the characteristic sequence of the translation initiation element.
  • Figure 3 shows the western blot detection of the activity of translation initiation elements produced by anti-learning.
  • the translation initiation element has high translation activity.
  • the translation enlightening element of the present invention was inserted into the circular RNA. Expression vectors can significantly enhance translation efficiency in vitro and in vivo. On this basis, the inventor completed the present invention.
  • the 3'end of the first intron contains a splice acceptor site, which contains a cis element (50bp-300bp in length) that is paired with the second intron.
  • the 5'end of the second intron contains a splice donor site, which contains a cis element (50bp-300bp in length) paired with the first intron.
  • the first aspect of the present invention provides a circular RNA construct, which has the structure shown in formula I from the 5'-3' direction:
  • TI is the translation initiation element
  • Z1 is an expression cassette for expressing foreign protein
  • Z2 is none or other components
  • each "-" is a bond or a nucleotide connection sequence
  • the length of the TI element is 6-30 nt, preferably, 8-24 nt, more preferably, 10-20 nt;
  • the content of A is ⁇ 35%, preferably, ⁇ 45%, more preferably, ⁇ 60%;
  • the content of T is ⁇ 20%, preferably, ⁇ 30%, more preferably, ⁇ 50%;
  • the content of A+T is ⁇ 65%, preferably, ⁇ 80%, more preferably, ⁇ 90%;
  • the content of G is ⁇ 35%, preferably, ⁇ 25%, more preferably, ⁇ 10%.
  • the content of A in the TI element is 35-100%, preferably, 45-100%, more preferably, 60-100%.
  • the content of T in the TI element is 20-100%, preferably, 30-100%, more preferably, 50-100%.
  • the content of A+T in the TI element is 65-100%, preferably, 80-100%, more preferably, 90-100%.
  • the content of G in the TI element is 0-35%, preferably, 0-25%, more preferably, 0-10%.
  • the selection of the encoding sequence of the foreign protein is not particularly limited.
  • the encoding sequence of the foreign protein is selected from the following group: encoding luciferin, or luciferase (such as firefly luciferase), green Fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody variable region foreign DNA, luciferase mutant DNA , Or a combination thereof.
  • the coding sequence of the foreign protein can also encode a protein selected from the group consisting of: ⁇ -amylase, enterobacteria A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon ⁇ A, interleukin-1 ⁇ , bacteriolysis Enzyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
  • ⁇ -amylase enterobacteria A
  • hepatitis C virus E2 glycoprotein insulin precursor
  • interferon ⁇ A interleukin-1 ⁇
  • bacteriolysis Enzyme bacteriolysis Enzyme
  • serum albumin serum albumin
  • scFV single chain antibody fragment
  • transthyretin tyrosinase
  • xylanase xylanase
  • nucleic acid construct of the present invention is circular.
  • nucleic acid construct of the present invention is single-stranded.
  • nucleic acid construct of the present invention is RNA.
  • sequence of the circular RNA construct of the present invention is shown in SEQ ID NO.: 61.
  • Circular RNA sequence using GFP as an example is an example.
  • sequence of the circular RNA precursor (containing the first intron and the second intron) taking GFP as an example is as follows:
  • the TI element of the present invention contains the nucleotide sequence shown in Table 1 selected from the following group:
  • the coding sequence of the TI element of the present invention is shown in SEQ ID NO.: 1-40.
  • the circular RNA construct of the present invention has high translation activity and can significantly enhance the translation efficiency in vivo or in vitro.
  • the present invention provides a reaction system, including:
  • reaction system further includes YTHDF3, PABPC1, and/or hnRNPA1 protein.
  • the reaction system may be in vitro or in vivo.
  • the present invention provides a kit for in vitro protein synthesis, including:
  • the second container, and other components required for the reaction in the second container, the other components are selected from the group consisting of spliceosome, ribosome, translation initiation factor EIF4G2, translation initiation factor EIF4A , The translation initiation factor EIF4B, or a combination thereof; and
  • first container and the second container are the same container or different containers.
  • coding sequence of foreign protein and “foreign DNA” are used interchangeably, and both refer to exogenous DNA molecules used to direct protein synthesis.
  • the DNA molecule is linear or circular.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • examples of the sequence encoding the foreign protein include (but not limited to): genomic sequence, cDNA sequence.
  • the sequence encoding the foreign protein also contains a promoter sequence, a 5'untranslated sequence, and a 3'untranslated sequence.
  • the selection of the exogenous DNA is not particularly limited.
  • the exogenous DNA is selected from the following group: encoding luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein , Aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA in the variable region of an antibody, luciferase mutant DNA, or a combination thereof.
  • the exogenous DNA can also be selected from the following group: encoding ⁇ -amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon ⁇ A, interleukin-1 ⁇ , lysozyme, serum white Exogenous DNA of protein, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
  • the foreign DNA encodes a protein selected from the group consisting of green fluorescent protein (enhanced GFP, eGFP), yellow fluorescent protein (YFP), and E. coli ⁇ -galactosidase ( ⁇ -galactosidase, LacZ), human lysine-tRNA synthetase (Lysine-tRNA synthetase), human leucine-tRNA synthetase (Leucine-tRNA synthetase), Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate) dehydrogenase), mouse catalase, or a combination thereof.
  • GFP green fluorescent protein
  • eGFP enhanced green fluorescent protein
  • YFP yellow fluorescent protein
  • E. coli ⁇ -galactosidase ⁇ -galactosidase, LacZ
  • human lysine-tRNA synthetase Lysine-t
  • the present invention provides an in vitro protein synthesis method, including the steps:
  • step (ii) Under suitable conditions, incubate the synthesis system of step (i) for a period of T1 to synthesize the protein.
  • the method further includes: (iii) optionally separating or detecting the protein from the reaction system.
  • the present invention has developed a set of methods for designing and synthesizing new artificially synthesized eukaryotic translation initiation elements of non-viral origin with high translation activity and controllable sequence structure and length. Drive the translation of circular RNA.
  • the present invention screens for the first time a specific translation initiation element, which is very short, only 6-30 nt, but has high translation activity, and inserts the translation initiation element into circular RNA expression
  • the carrier can significantly enhance translation efficiency both in vivo and in vitro.
  • the circular RNA reporter gene can be expressed under the drive of the translation initiation element to produce green fluorescent protein) to construct a library containing millions of different sequences, through Cell transfection and flow cytometry screen different cell populations (negative: no green fluorescence, positive: green fluorescence with different intensities). Perform amplicon sequencing on the collected different cell populations and analyze the sequence information contained in negative and different positive cells in combination with computational biology analysis, and extract sequence features of different lengths from the sequence information;
  • a high-throughput screening system based on circular RNA separates cell populations with different green fluorescence intensities (positive) and cell populations without fluorescence (negative).
  • results are shown in Table 1 and Figure 1.
  • the results show that the circular RNA system can express green fluorescent protein and be used for screening. At the same time, the system can isolate cell populations with different fluorescence intensities, indicating that different inserted sequences in the library have a differential effect on the translation initiation of circular RNA.
  • Table 1 Sequence features of translation initiation elements contained in cell populations with high green fluorescence signals (the first 100 sequence features displayed in the form of 6 bases)
  • Translation initiation elements (take 12 bases as an example) produced by anti-learning based on the characteristic sequences of positive and negative cell populations. List the top 20 sequences with different translational activity strengths.
  • the highly active translation initiation elements are basically AT-enriched sequences, as shown in Table 2.
  • the results show that the anti-learning method of the present invention can effectively infer the activity of translation initiation elements, and the method can be used to generate translation initiation elements of different lengths and strengths, and the translation initiation elements of the present invention have high translation activity and can Significantly enhance translation efficiency.

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PCT/CN2020/077026 2019-03-20 2020-02-27 利用环形rna进行蛋白翻译及其应用 Ceased WO2020186991A1 (zh)

Priority Applications (10)

Application Number Priority Date Filing Date Title
CN202410093532.8A CN117965533A (zh) 2019-03-20 2020-02-27 利用环形rna进行蛋白翻译及其应用
CN202080036818.2A CN113825837A (zh) 2019-03-20 2020-02-27 利用环形rna进行蛋白翻译及其应用
EP20773279.3A EP3943601A4 (en) 2019-03-20 2020-02-27 TRANSLATION OF PROTEINS USING CIRCULAR RNA AND THEIR USE
JP2021559486A JP7297331B2 (ja) 2019-03-20 2020-02-27 環状ｒｎａを使用したタンパク質翻訳およびその応用
CN202410975296.2A CN118834867A (zh) 2019-03-20 2020-02-27 利用环形rna进行蛋白翻译及其应用
US17/440,774 US20220177898A1 (en) 2019-03-20 2020-02-27 Protein translation using circular rna and application thereof
CN202410975341.4A CN118834868A (zh) 2019-03-20 2020-02-27 利用环形rna进行蛋白翻译及其应用
JP2023094068A JP7673989B2 (ja) 2019-03-20 2023-06-07 環状ｒｎａを使用したタンパク質翻訳およびその応用
JP2024209710A JP2025029080A (ja) 2019-03-20 2024-12-02 環状ｒｎａを使用したタンパク質翻訳およびその応用
JP2024209709A JP2025029079A (ja) 2019-03-20 2024-12-02 環状ｒｎａを使用したタンパク質翻訳およびその応用

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2025077734A1 (en) * 2023-10-09 2025-04-17 Shanghai Circode Biomed Co., Ltd. Constructs and methods for preparing circular rnas and uses thereof
US12338474B2 (en) 2021-09-17 2025-06-24 Flagship Pioneering Innovations Vi, Llc Compositions and methods for producing circular polyribonucleotides

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CN112608946B (zh) * 2021-01-17 2023-07-28 楷拓生物科技(苏州)有限公司 一种环状rna载体及其在疫苗中的应用
CN121752588A (zh) 2023-08-29 2026-03-27 上海环码生物医药有限公司 编码vegf多肽的环状rna、制剂和使用方法

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