Polypeptides Translated by Circular RNA Circ-ACE2 and Uses Thereof

Abstract

Provided are circular RNA Circ-ACE2 translated polypeptides and uses thereof, the circular RNA molecule comprising at least one of: 1) an RNA sequence as set forth in SEQ ID NO:1; 2) an RNA sequence having at least 70% identity to 1), preferably an RNA sequence having at least 80% identity, preferably an RNA sequence having at least 85% identity, preferably an RNA sequence having at least 90% identity, preferably an RNA sequence having at least 95% identity, more preferably an RNA sequence having at least 99% identity.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to patent applications filed with the State Intellectual Property Office of China on June 4, 2021, bearing application number 202110624105.4, and filed with the State Intellectual Property Office of China on July 7, 2021, bearing application number 202110765444.4, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of biomedicine. Specifically, the present invention relates to polypeptides translated by circular RNA Circ-ACE2 and uses thereof. More specifically, the present invention relates to isolated circular RNA molecules, polypeptides encoded by isolated circular RNA molecules, fusion proteins, isolated nucleic acids encoding fusion proteins, and uses of polypeptides encoded by circular RNA molecules, fusion proteins, and isolated nucleic acids encoding fusion proteins in the manufacture of drugs or kits, methods for inhibiting infection of cells by novel coronavirus, methods for treating or preventing novel coronavirus pneumonia, and uses in the treatment or prevention of novel coronavirus pneumonia. [Background technology]

[0003] The placenta is not only an important, temporary organ for the exchange of materials between the fetus and the mother during pregnancy, but it can also protect the fetus from infection by pathogenic microorganisms during fetal development, and placental cells may play an important role in suppressing viral transmission from the pregnant mother to the fetus. The COVID-19 pandemic has had a significant impact on global health, and some research groups have reported that there is some limitation to vertical transmission of the COVID-19 virus from mother to child. Data suggests that the rate at which newborns transmit COVID-19 from their mothers is less than 5%, suggesting that placental tissue contains unique antiviral substances that suppress transmission of the virus from infected mothers to the fetus.

[0004] Polypeptide drugs have been widely used in clinical practice, and insulin polypeptide drugs are a typical example.The drug metabolites of polypeptides are amino acids, which are essential elements for the human body, and polypeptide drugs are similar to protein drugs, so polypeptide drugs have low toxicity and high safety.Compared with large-scale protein and antibody drugs, polypeptide drugs have low immunogenicity and are easy to artificially synthesize, and are suitable for injection and nasal atomization administration, and have excellent drug discovery potential.

[0005] Recent research has discovered that circular RNA molecules exist in large quantities in many organisms, and that circular RNA is widely present in eukaryotes and has important biological functions in individual development and the occurrence and development of diseases.The novel coronavirus epidemic has already spread worldwide, and there are currently no drugs that can treat the diseases it causes with specific efficacy and minimal side effects.Therefore, it is necessary to take a new perspective and discover and develop ideal drugs to combat the novel coronavirus. Summary of the Invention

[0006] The present application was prepared based on the inventor's discovery and recognition of the following facts and problems.

[0007] The novel coronavirus infects cells by binding to ACE2 (angiotensin 2). Through in-depth mining and experiments on the circular RNA circAtlas integrated database, the inventors discovered that the ACE2 gene specifically undergoes cyclization shearing in human placental tissue to form circular RNA Circ-ACE2. The polypeptide encoded by this circular RNA and the recombinant polypeptide obtained using this polypeptide have the ability to inhibit COVID-19 from infecting eukaryotic cells.

[0008] Thus, in a first aspect, the present invention provides an isolated circular RNA molecule. According to an embodiment of the present invention, the circular RNA molecule comprises: 1) a sequence encoding SEQ ID NO:1 (SEQ ID NO: 1)1) an RNA sequence having at least 70% identity to 1), preferably an RNA sequence having at least 80% identity, preferably an RNA sequence having at least 85% identity, preferably an RNA sequence having at least 90% identity, preferably an RNA sequence having at least 95% identity, more preferably an RNA sequence having at least 99% identity.

[0009] According to an embodiment of the present invention, the SEQ ID NO: 1 (SEQ ID NO: 1) The specific sequence of CGCCCAACCCAAGUUCAAAGGCUGAUAAGAGAGAAAAUCUCAUGAGGAGGUUUUAGUCUAGGGAAAGUCAUUCAGUGGAUGUGAUCUUGGCUCACAGGGGACGAUGUCAAGCUCUUCCUGGCUCCUUCUCAGCCUUGUUGCUGUAA CUGCUGCUCAGUCCACCAUUGAGGAACAGGCCAAGACAUUUUUGGACAAGUUUAACCACGAAGCCGAAGACCUGUUCUAUCAAAGUUCACUUGCUUCUUGGAAUUAUAACACCAAUAUUACUGAAGAGAAUGUCCAAAACAUG.

[0010] The polypeptides obtained by translation of the circular RNA sequences according to the embodiments of the present invention can significantly suppress the ability of the novel coronavirus to infect cells.

[0011] According to an embodiment of the present invention, the circular RNA molecule may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the circular RNA molecule is formed by circularization of the second exon alone of the mRNA sequence encoding angiotensin converting enzyme 2.

[0013] According to an embodiment of the present invention, said cyclization occurs in placental tissue.

[0014] According to an embodiment of the present invention, the circular RNA molecule is formed by linking the initiating nucleotide and the terminal nucleotide of the nucleotide sequence shown in 1) or 2), and the initiating nucleotide is set as the first nucleotide of the circular RNA molecule.

[0015] According to an embodiment of the present invention, the circular RNA coding region comprises nucleotides 104 to 45 of the circular RNA molecule.

[0016] According to an embodiment of the present invention, the nucleic acid sequence of the circular RNA coding region is: 1) SEQ ID NO:2 (SEQ ID NO: 2) 1) an RNA sequence having at least 70% identity to 1), preferably an RNA sequence having at least 80% identity, preferably an RNA sequence having at least 85% identity, preferably an RNA sequence having at least 90% identity, preferably an RNA sequence having at least 95% identity, more preferably an RNA sequence having at least 99% identity.

[0017] According to an embodiment of the present invention, the SEQ ID NO:2 (SEQ ID NO: 2) The specific sequence of AUGUCAAGCUCUUCCUGGCUCCUUCUCAGCCUUGUUGCUGUAACUGCUGCUCAGUCCACCAUUGAGGAACAGGCCAAGACAUUUUUGGACAAGUUUAACCACGAAGCCGAAGACCUG UUCUAUCAAAGUUCACUUGCUUCUUGGAAUUAUAACACCAAUAUUACUGAAGAGAAUGUCCAAACAUGCGCCCAACCCAAGUUCAAAGGCUGAUAAGAGAGAAAAUCUCAUGA.

[0018] The polypeptides obtained by translating the nucleic acid sequence of the circular RNA coding region according to the embodiments of the present invention can significantly suppress the ability of the novel coronavirus to infect cells.

[0019] In a second aspect, the present invention provides a polypeptide, the amino acid sequence of which is selected from the group consisting of: 1) SEQ ID NO:3 (SEQ ID NO: 3) 1) an amino acid sequence having at least 70% identity to 1), preferably at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, more preferably at least 99% identity. According to an embodiment of the present invention, the polypeptide can significantly suppress the ability of novel coronavirus to infect cells.

[0020] According to an embodiment of the present invention, the SEQ ID NO:3 (SEQ ID NO: 3) The specific sequence of MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMRPTQVQRLIREKIS.

[0021] According to an embodiment of the present invention, the above polypeptide may further comprise at least one of the following additional technical features:

[0022] According to an embodiment of the present invention, the polypeptide comprises a signal peptide sequence.

[0023] According to an embodiment of the present invention, the amino acid sequence of the signal peptide is MSSSSWLLLSLVAVTAA.

[0024] According to an embodiment of the invention, the polypeptide is present in placental tissue.

[0025] In a third aspect of the present invention, there is provided a fusion protein. According to an embodiment of the present invention, the fusion protein comprises the polypeptide according to the second aspect and Fc, and the C-terminus of the polypeptide is connected to the N-terminus of Fc. According to an embodiment of the present invention, the fusion protein can significantly suppress the ability of novel coronavirus to infect cells.

[0026] According to an embodiment of the present invention, the fusion protein may further comprise at least one of the following additional technical features:

[0027] According to an embodiment of the present invention, the fusion protein comprises: 1) a polypeptide having SEQ ID NO:4 (SEQ ID NO: 4) 1) an amino acid sequence having at least 70% identity to 1), preferably at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, more preferably at least 99% identity.

[0028] According to an embodiment of the present invention, the SEQ ID NO:4 (SEQ ID NO: 4) The specific sequence of MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMRPTQVQRLIREKISLVPRGSGGGGDPEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0029] In a fourth aspect, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid encodes the polypeptide described in the second aspect or the fusion protein described in the third aspect. The isolated nucleic acid sequence according to this embodiment of the present invention can be expressed under appropriate conditions to obtain large amounts of the polypeptide or fusion protein, and either the polypeptide or fusion protein translated from the isolated nucleic acid sequence can significantly suppress the ability of novel coronavirus to infect cells.

[0030] According to an embodiment of the present invention, the isolated nucleic acid may further comprise at least one of the following additional technical features:

[0031] According to an embodiment of the present invention, the nucleic acid encoding the fusion protein is selected from the group consisting of: 1) SEQ ID NO:5 (SEQ ID NO: 5) 2) a nucleotide sequence having at least 70% identity to 1), preferably at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, more preferably at least 99% identity.

[0032] According to an embodiment of the present invention, the SEQ ID NO:5 (SEQ ID NO: 5) The specific sequence of ATGTCAAGCTCTTCCTGGCTCCTTCTCAGCCTTGTTGCTGTAACTGCTGCTCAGTCCACCATTGAGGAACAGGCCAAGACATTTTTGGACAAGTTTAACCACGAAGCCGAAGACCTGTTCTATCAAAGTTCACTTGCTTCTTGGAATTATAACACCAATATTACTGAAGAGAATGTCCAAAACATGCGCCCAACCCAAGTTCAAAGGCTGATAAGAGAGAAAATCTCACTGGTGCCCAGAGGCTCCGGCGGCGGCGGCGATCCTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA is as follows.

[0033] In a fifth aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the RNA molecule described in the first aspect or the isolated nucleic acid described in the fourth aspect. The polypeptide translated by the expression vector according to this embodiment of the present invention can significantly suppress the ability of novel coronavirus to infect cells.

[0034] In a sixth aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries an RNA molecule according to the first aspect, an isolated nucleic acid according to the fourth aspect, or an expression vector according to the fifth aspect. The recombinant cell has the ability to strongly resist novel coronavirus infection.

[0035] In a seventh aspect of the present invention, there is provided a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises an RNA molecule according to the first aspect, a polypeptide according to the second aspect, a fusion protein according to the third aspect, an isolated nucleic acid according to the fourth aspect, an expression vector according to the fifth aspect, or a recombinant cell according to the sixth aspect. According to an embodiment of the present invention, the pharmaceutical composition is used to resist infection of cells by a novel coronavirus.

[0036] According to an embodiment of the present invention, the pharmaceutical composition may further include at least one of the following additional technical features:

[0037] According to an embodiment of the present invention, the pharmaceutical composition is in the form of at least one of a solution, a powder, a microsphere, and a microcapsule, and is easy to administer and suitable for maintaining the efficacy of the pharmaceutical composition.

[0038] According to the embodiment of the present invention, the dosage of the drug is not particularly limited and can be flexibly selected in practice depending on the health condition of the subject.

[0039] In an eighth aspect of the present invention, there is provided use of the RNA molecule according to the first aspect, the polypeptide according to the second aspect, the fusion protein according to the third aspect, the isolated nucleic acid according to the fourth aspect, the expression vector according to the fifth aspect, the recombinant cell according to the sixth aspect, or the pharmaceutical composition according to the seventh aspect in the preparation of a medicament or kit for use in inhibiting infection of cells by a novel coronavirus. According to an embodiment of the present invention, the medicament or kit can significantly inhibit infection of cells by a novel coronavirus, and the medicament or kit can be used in clinical diagnosis or scientific research.

[0040] In a ninth aspect of the present invention, there is provided a method for inhibiting infection of cells by a novel coronavirus, the method comprising the step of contacting a novel coronavirus or an infected cell with an RNA molecule described in the first aspect, a polypeptide described in the second aspect, a fusion protein described in the third aspect, an isolated nucleic acid described in the fourth aspect, an expression vector described in the fifth aspect, a recombinant cell described in the sixth aspect, or a pharmaceutical composition described in the seventh aspect. According to an embodiment of the present invention, the method can significantly inhibit infection of cells by a novel coronavirus.

[0041] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0042] According to an embodiment of the present invention, the contacting can be in vivo or ex vivo. For example, the in vivo contacting can be achieved by at least one of direct microinjection, oral, nasal, transdermal, intravenous, respiratory inhalation, and rectal administration, i.e., the RNA molecule described in the first aspect, the polypeptide described in the second aspect, the fusion protein described in the third aspect, the isolated nucleic acid described in the fourth aspect, the expression vector described in the fifth aspect, the recombinant cell described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect is introduced into the body of a subject by the above methods and then contacted with infected target cells, such as lung epithelial cells. For example, the ex vivo contacting can be achieved by directly mixing, incubating, or contacting the RNA molecule described in the first aspect, the polypeptide described in the second aspect, the fusion protein described in the third aspect, the isolated nucleic acid described in the fourth aspect, the expression vector described in the fifth aspect, the recombinant cell described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect with the novel coronavirus or isolated infected target cells in vitro.

[0043] According to a specific embodiment of the present invention, the contact is carried out by: 1) performing a first mixing process on the novel coronavirus and the RNA molecule described in the first aspect, the polypeptide described in the second aspect, the fusion protein described in the third aspect, the isolated nucleic acid described in the fourth aspect, the expression vector described in the fifth aspect, the recombinant cell described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect; and 2) performing a second mixing process on the mixture obtained in step 1) and the target cells to be infected, thereby inhibiting infection of the cells by the novel coronavirus.

[0044] According to an embodiment of the present invention, the final concentration of the polypeptide or fusion protein in the mixture is 10 μg / mL or 160 μg / mL. According to an embodiment of the present invention, when the final concentration of the polypeptide in the mixture is 10 μg / mL, the ability of the SARS-CoV-2 pseudovirus to infect cells is inhibited by 91.61%, and when the final concentration is 160 μg / mL, the ability of the SARS-CoV-2 pseudovirus to infect cells is inhibited by 95.50%. When the final concentration of the fusion protein (recombinant polypeptide) in the mixture is 10 μg / mL, the ability of the SARS-CoV-2 pseudovirus to infect cells is inhibited by 52.11%, and when the final concentration is 160 μg / mL, the ability of the SARS-CoV-2 pseudovirus to infect cells is inhibited by 92.18%. The final concentrations of the polypeptide or fusion protein administered in the mixture according to the embodiments of the present invention are suitable for maintaining the efficacy of the drug.

[0045] In a tenth aspect, the present invention provides a method for treating or preventing novel coronavirus pneumonia. According to an embodiment of the present invention, a patient is administered a pharmaceutically acceptable RNA molecule, polypeptide, fusion protein, or isolated nucleic acid. The method according to an embodiment of the present invention can effectively treat or prevent novel coronavirus pneumonia.

[0046] In an eleventh aspect of the present invention, the present invention provides use of the above-described RNA molecule, polypeptide, fusion protein or above-described isolated nucleic acid in the treatment or prevention of novel coronavirus pneumonia.

[0047] Additional aspects and advantages of the present invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the present invention. [Brief explanation of the drawings]

[0048] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the following description of the embodiments taken in conjunction with the drawings. [Figure 1]FIG. 1 is a distribution diagram of the expression levels of ACE2 mRNA molecules in various tissues of the human body according to an embodiment of the present invention. [Figure 2] This is a distribution map of the expression levels of circular RNA circ-ACE2 in various human tissues according to an embodiment of the present invention. [Figure 3] 1 is a schematic diagram of the formation of circular RNA Circ-ACE2 according to an embodiment of the present invention, in which ACE2 RNA undergoes shearing and circularization at the second exon (Exon 2) position to form circular RNA Circ-ACE2. [Figure 4] This is a diagram showing the verification results of the circular RNA Circ-ACE2 cyclization sequence by sequencing the PCR product of RNA Circ-ACE2 according to an embodiment of the present invention, where the direction from left to right in the PCR result diagram is the direction from the 3' end to the 5' end of the PCR product sequence. [Figure 5] 1 is a diagram illustrating an analysis of the circular RNA Circ-ACE2 interface across the coding reading frame according to an embodiment of the present invention, where the coding sequence of the circular RNA molecule is the nucleotide sequence from nucleotide 104 to nucleotide 45 of the circular RNA molecule sequence across the circularization interface, and the specific sequence is the underlined sequence in the diagram. [Figure 6] This is a comparative analysis diagram of the amino acid sequences of the CircACE2-76aa polypeptide according to an embodiment of the present invention and the protein encoded by ACE2. Compared with the ACE2 protein amino acid sequence, CircACE2-76aa contains 62 amino acids at the N-terminus of ACE2 and a C-terminus carrying a specific 14 amino acids (RPTQVQRLIREKIS). [Figure 7] FIG. 1 shows the results of mass spectrometry identification of polypeptides obtained by circular RNA Circ-ACE2 molecule translation according to an embodiment of the present invention. [Figure 8] FIG. 1 is an online program analysis diagram of the amino acid sequence of CircACE2-76aa according to an embodiment of the present invention. [Figure 9] This is a cellular localization diagram of CircACE2-76aa under a fluorescence microscope according to an embodiment of the present invention. [Figure 10]This is a graph showing the results of detecting the fold difference in expression level of the EGFP gene carried by the pseudovirus in an experiment in which the chemically synthesized polypeptide CircACE2-76aa according to an embodiment of the present invention was used in a cell infected with a novel coronavirus pseudovirus. [Figure 11] This is a graph showing the results of detecting the fold difference in expression level of the EGFP gene carried by the pseudovirus in an experiment in which the recombinant protein CircACE2-76aa-FC according to an embodiment of the present invention was used in a novel coronavirus pseudovirus-infected cell experiment. DETAILED DESCRIPTION OF THE INVENTION

[0049] DETAILED DESCRIPTION OF THE INVENTION The following detailed description of the present invention will be given of embodiments of the present invention, examples of which are shown in the drawings. The embodiments described below with reference to the drawings are illustrative and are for explaining the present invention, but should not be understood as limitations on the present invention.

[0050] Terminology

[0051] In this text, the terms "novel coronavirus," "coronavirus," and "COVID-19" all refer to the pathogen that causes novel coronavirus pneumonia.

[0052] Regarding identity, in the present invention, for comparing two or more nucleotide sequences, the percent of "sequence identity" between a first sequence and a second sequence can be calculated by [dividing the number of identical nucleotides at corresponding positions in the first sequence], subtracting [the total number of nucleotides in the first sequence] from [the nucleotides in the second sequence], and multiplying by [100%], where each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence is considered a single nucleotide (position) difference relative to the first nucleotide sequence.

[0053] Alternatively, the degree of sequence identity between two or more nucleotide sequences is calculated using known computer algorithms used for sequence comparisons, such as NCBI Blast v2.0, using standard settings.

[0054] Several other techniques, computer algorithms and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0055] Regarding identity, in the present invention, for comparing two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence is calculated by dividing the number of amino acid residues in the first amino acid sequence by the same number as the amino acid residue at the corresponding position in the second amino acid sequence, which is the total number of nucleotides in the first amino acid sequence, and then multiplying by 100%, and each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered to be a difference in a single amino acid residue (position), i.e., an "amino acid difference" as defined herein.

[0056] Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as the algorithm for determining the degree of sequence identity of nucleotide sequences described above, again using standard settings.

[0057] Generally, for purposes of determining the percentage of "sequence identity" between two amino acid sequences based on the calculation method outlined above, the amino acid sequence having the largest number of amino acid residues is referred to as the "first" amino acid sequence, and the other amino acid sequence is referred to as the "second" amino acid sequence.

[0058] Similarly, when determining the degree of sequence identity between two amino acid sequences, a skilled artisan may consider so-called "conservative" amino acid substitutions, which can usually be described as replacing an amino acid residue therein with a substituted amino acid. The other amino acid residue, having a similar chemical structure, has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are known in the art, and include, for example, WO 04 / 037999, GB-A-2357768, WO 98 / 49185, WO 00 / 46383, WO 01 / 09300, and WO 01 / 09300. The type and / or combination of such substitutions may be selected and / or (preferably selected) based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0059] The inventors of this embodiment conducted in-depth analysis and mining of the circular RNA circAtlas integrated database and discovered that the ACE2 gene specifically formed a circular RNA molecule in placental tissue. This circular RNA molecule was formed by single circularization in the second exon of ACE2 RNA, which the inventors named Circ-ACE2. Sequence analysis and experimental verification of the circular RNA Circ-ACE2 showed that CircACE2 could translate a polypeptide containing a secretory signal peptide. This polypeptide was a new polypeptide molecule, which the inventors named CircACE2-76aa. The polypeptide was 76 amino acids long. The present invention obtains the chemically synthesized polypeptide CircACE2-76aa and the recombinant polypeptide CircACE2-76aa-FC from the eukaryotic cell-expressed fused human immunoglobulin G FC fragment. The antiviral function of CircACE2-76aa was verified using a protocol for in vitro infection of human hACE2-293T cells with the novel coronavirus pseudovirus. The chemically synthesized CircACE2-76aa polypeptide and the recombinant polypeptide CircACE2-76aa-FC significantly suppressed the cell infectivity of the novel coronavirus pseudovirus. The CircACE2-76aa polypeptide and the recombinant polypeptide CircACE2-76aa-FC of the present invention can be candidate molecules for suppressing COVID-19 virus infection.

[0060] The following examples are specifically described. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0061] Example 1 Identification and identification of circular RNA Circ-ACE2 in isolated placental tissue

[0062] The human placental tissue in this example was derived from discarded placentas of pregnant women in clinical obstetrics and gynecology following spontaneous delivery.

[0063] 1. Identification of circular RNA Circ-ACE2 in isolated placental tissue

[0064] ACE2 mRNA is widely expressed in multiple human tissues, and the expression distribution of this gene in various human tissues is shown in Figure 1.

[0065] By performing in-depth mining of the circular RNA circAtlas integrated database, the inventors discovered that the ACE2 gene was specifically circularized in isolated placental tissue to form circular RNA Circ-ACE2, as shown in Figure 2.

[0066] 2. Identification of circular RNA Circ-ACE2 by sequencing in isolated placental tissue

[0067] PCR detection primers were designed to specifically detect the circular RNA Circ-ACE2. The upstream sequence of the primers was 5' GAAGCCGAAGACCTGTTCTA 3', and the downstream sequence was 5' TCTTATCAGCCTTTGAACTTGG 3'. The amplified fragment size was 114 bp. Total RNA was extracted from placental tissue samples isolated using the Trizol method, and the RNA was reverse-transcribed to cDNA using a random primer reverse transcription kit (Vazyme). The PCR amplification reaction system and conditions were as follows: a 50 μL system, specifically, 25 μL of 2x PCR mix (Vazyme), 2 μL each of 10 mM upstream and downstream primers, 1 μL of cDNA template, and sterile water to make up to 50 μL. The reaction conditions were as follows: cDNA pre-denaturation at 95°C for 3 minutes; first-step amplification: denaturation at 95°C for 30 seconds; annealing at 60°C for 30 seconds; extension at 72°C for 30 seconds; 35 cycles; PCR reaction cycles were followed by extension at 72°C for 3 minutes and then storage at 16°C. The PCR product was separated on a 1.5% agarose gel, purified by cleavage, and then subjected to Sanger DNA sequencing. As can be seen in Figure 3, the circular RNA Circ-ACE2 is a circular RNA molecule formed by circular shearing of the second solitary exon of ACE2 mRNA. As shown in Figure 4, the inventors used Sanger DNA sequencing to identify the exact circularization interface of the circular RNA Circ-ACE2. The sequence length of Circ-ACE2 RNA is 289 nt, and the specific nucleic acid sequence is SEQ ID NO:1 (SEQ ID NO: 1) Shown below.

[0068] 3. Analysis of the coding potential of RNA Circ-ACE2

[0069] Based on the nucleic acid sequence of RNA Circ-ACE2, coding potential analysis was performed using the ORF finder online program and it was found that the circular RNA Circ-ACE2 contains a reading frame that crosses the circular RNA interface. As shown in Figure 5, the coding region of the polypeptide translated by circular RNA Circ-ACE2 consists of 231 bases, and the base sequence is SEQ ID NO:2 (SEQ ID NO: 2) The length of the RNA Circ-ACE2 translated polypeptide is 76 amino acids, which is designated as CircACE2-76aa in the present invention, and the amino acid sequence is SEQ ID NO:3 (SEQ ID NO: 3) The protein amino acid sequences encoded by CircACE2-76aa and RNA ACE2 were compared and analyzed, and the results, as shown in Figure 6, indicate that CircACE2-76aa contains 62 amino acids from the N-terminus of ACE2 and a C-terminus carrying a specific 14 amino acids (RPTQVQRLIREKIS).

[0070] Example 2 Identification of Polypeptide CircACE2-76aa by Circular RNA Circ-ACE2 Translation

[0071] 1. Design and construction of circular RNA Circ-ACE2 expression plasmid

[0072] Based on the circular RNA Circ-ACE2 sequence information, the target sequence was obtained by the method of total gene chemical synthesis, and then the sequence was constructed into the circular RNA expression vector pCD5-ciR (Gisei Bio) by the restriction enzymes EcoRI and BamHI.

[0073] 2. Construction of red fluorescent protein fusion expression plasmid CircACE2-76aa-mcherry

[0074] Based on the circular RNA expression design method of the CircRNA Mini Vector (Addgene ID: #60648), CircACE2-76aa and the red fluorescent protein mcherry were fused to the circular RNA expression vector CircACE2-76aa-mcherry. The initiation codon ATG and termination codon of the mcherry gene fragment itself were removed and inserted at the end of the termination codon of CircACE2-76aa. The circularization mediator sequences of the CircRNA Mini Vector were added to both ends of the sequence. The final expression framework was SEQ ID NO:7 (SEQ ID NO: 7)After designing the CircACE2-76aa-mcherry circular RNA fusion expression framework, we obtained the expression framework by total gene chemical synthesis and then constructed the expression framework into the pcDNA3.1(+) expression vector with EcoRI and BamHI restriction enzyme sites.

[0075] 3. CircACE2-76aa-mcherry transfected hACE2-293 T cells

[0076] After transfection of the circular RNA Circ-ACE2 expression plasmid into 293T cells, the circular RNA Circ-ACE2 expression product was subjected to SDS-PAGE protein electrophoresis and Cauma Brilliant Blue staining, and then the rubber was cut for mass spectrometry identification. As shown in Figure 7, the peptide segment traversing the cyclization interface translated by the circular RNA Circ-ACE2 was identified by mass spectrometry. The sequence of the peptide segment analyzed by mass spectrometry was QNMRPTAVQR, which indicates the specific amino acid sequence of the terminal portion not translated by the circular RNA Circ-ACE2. Amino acid sequence analysis of the CircACE2-76aa polypeptide was performed using the signalP-4.1 online program. As shown in Figure 8, it was found that CircACE2-76aa does not contain a membrane-spanning amino acid sequence but contains a typical secretory signal peptide sequence. The secretory signal peptide sequence of CircACE2-76aa consists of 17 amino acids at the N-terminus and has the sequence MSSSSWLLLSLVAVTAA. Fluorescence microscopy using a fused mcherry red fluorescent protein revealed that the CircACE2-76aa polypeptide is secreted outside the cell. The results are shown in Figure 9.

[0077] Example 3 Chemical synthesis of polypeptide CircACE2-76aa

[0078] In this example, the chemical synthesis of the polypeptide was outsourced to Shanghai Bohai Biotechnology Co., Ltd., and the polypeptide was synthesized using solid-phase synthesis. Peptide chain synthesis: Fmoc / PyBOP method was used. A 30% hexahydropyridine solution in DMF was used to remove the Fmoc protecting group, and a peptide cleavage reagent (trifluoroacetic acid / crystalline phenol / water / ethylenedithiol / methylethyl sulfide / =81.5 / 5 / 5 / 5 / 2.5 / 1) was used to cleave the peptide chain from the resin. Polypeptide purification and detection: C18 reversed-phase column. Conditions: Phase A was 95% water (methanol blend ratio), Phase B was 95% methanol (methanol blend ratio), followed by the addition of 0.1% TFA. Typical conditions: the column was equilibrated in Phase A for 15 minutes before loading the sample, and then the sample was loaded and a 25-minute gradient was applied from Phase A to Phase B. Detection wavelength: 220 nm, flow rate: 1 mL / min. The column is equilibrated with solution A, and the sample is loaded. After that, the sample is eluted with a gradient from solution A to solution B for 25 min. The target peptides are collected, and the synthesized polypeptides are subjected to mass spectrometry identification using a mass spectrometer.

[0079] Example 4 Construction of recombinant expression plasmid for circACE2-76aa-FC polypeptide and eukaryotic cell expression and purification

[0080] 1. Construction of recombinant polypeptide expression plasmid

[0081] Based on the coding nucleic acid sequence of circACE2-76aa and the human immunoglobulin IGg sequence, a recombinant expression framework was designed, and the nucleic acid sequence is SEQ ID NO:5 (SEQ ID NO: 5) As shown in Figure 1, a segmented PCR amplification and overlapping PCR splicing amplification scheme was used to obtain the circACE2-76aa-FC expression framework, and the framework was then ligated into the pcDNA3.1(+) vector at the EcoRI and BamHI endonuclease sites.

[0082] 2. Expression of Plasmids in Eukaryotic Cells

[0083] Grow cells to a density of 2.5-3.0 million / mL and then culture 500mL of cells in a 2L culture flask at 37°C, 120 rpm, and 8% CO2. Transfect 0.5mg of plasmid per 500mL of cells, using 2.7x the amount of PEI. First, prepare 10mL of fresh medium (for example, 500mL), add 50µg of plasmid, mix thoroughly, and slowly add the equivalent amount of PEI. Incubate for 5-10 minutes (until the medium becomes slightly cloudy instead of its original clear state). After incubation, add the PEI to the cells. Express the cells at 33°C, 8% CO2, and 120 rpm. After 6 days of expression, collect the cell supernatant and expression is complete.

[0084] 3. Purification of circACE2-76aa-FC Recombinant Polypeptide

[0085] 5 mL of neutralizing solution (1 M Tris-HCl, pH 8.0) was added to each collection tube. An AKTA prue column (AKTA prue column: protein A (Cytiva, catalog number 17040301)) was equilibrated with binding buffer (20 mM phosphate). 500 mL of cell supernatant was added to the purification column in a volume of 1500 mL. After loading onto the column, 80 mL of impurity protein was washed off with binding buffer (20 mM phosphate). 100 mL of elution buffer (0.1 M citric acid, pH 3.0) was added. 5 mL of collection tubes were used. The collection tubes with the peak positions were taken and run on a gel to determine which collection tubes contained the protein. The protein was concentrated and finally replaced with PBS.

[0086] Example 5: Experimental cell infection with COVID-19 pseudovirus using CircACE2-76aa polypeptide and recombinant CircACE2-76aa-FC

[0087] By using a commercially available recombinant pseudovirus containing the novel coronavirus S protein and stable overexpression of the full-length human ACE2 gene in the 293T cell line hACE2-293T (Guangzhou Pazhen Biotechnology Co., Ltd.), we were able to simulate the process of virus entry into cells, and chemically synthesized CircACE2-76aa polypeptide (SEQ ID NO: 6 (SEQ ID NO: 6) ) and the recombinant polypeptide CircACE2-76aa-FC (SEQ ID NO: 4) obtained by eukaryotic expression in 293T. (SEQ ID NO: 4) ) were mixed with the COVID-19 pseudovirus and incubated, and then infected with hACE2-293 T cells. The specific procedure was to plate hACE2-293 T cells in a 96-well plate at a density of 1 × 10 4 Cells were plated at 100 μL per well. Cell adhesion was allowed for 12 hours, and then a pseudovirus COVID-19 infection experiment was performed. 5 μL of pseudovirus (approximately 10,000 virus particles) and different concentrations of polypeptide were added to the cells in each well. The pseudovirus and polypeptide were mixed in vitro in 100 μL of DMEM complete medium and left at room temperature for 30 minutes. Before the pseudovirus infected the cells, polybrene amine was added to a final concentration of 8 μg / mL.

[0088] Using the EGFP gene sequence carried by the novel coronavirus pseudovirus, genomic DNA extracted from infected cells was used for QPCR fluorescence detection to quantitatively detect the effect of chemically synthesized CircACE2-76aa polypeptide and recombinant CircACE2-76aa-FC on cell infection by the novel coronavirus pseudovirus COVID-19. The specific procedure was as follows: after 15 hours of infection of hACE2-293T cells with the pseudovirus, the cell culture medium was removed, 100μL of cellular genomic DNA was directly added to each well to extract the lysate, the cells were kneaded in a 96-well plate, and then transferred to a 1.5mL centrifuge tube. Next, the genomic DNA was extracted, the extracted DNA was dissolved in sterile water, and 1μL of genomic DNA was used for QPCR fluorescence detection to detect the integration of the EGFP gene carried by the pseudovirus into the cellular genome, and the GAPDH gene was used as an internal reference to evaluate the ability of the novel coronavirus pseudovirus to infect hACE2-293T cells.

[0089] QPCR detection: For QPCR detection, the reaction system was prepared according to the specifications of the fluorescent quantitative reaction kit (Vazyme). The QPCR reaction system consisted of 20 μL and 10 μL of 2x SYBR Green PCR Master Mix, 0.4 μL each of upstream and downstream primers (10 μM), 1 μL of template DNA, and finally topped up to 20 μL with sterile deionized water. The fluorescent quantitative PCR reaction conditions were as follows: cDNA pre-denaturation at 95°C for 5 min; first amplification step: denaturation at 95°C for 10 s; annealing at 60°C for 35 s (fluorescent signal collected during this step); 40 cycles; then, a melting curve analysis was performed, collecting the fluorescent signal from 60°C to 95°C. The sequences of the QPCR detection primers were EGFP-upstream primer: 5′ TTCAAGGAGGACGGCAACAT 3′, EGFP-downstream primer: 5′ TGGCGGATCTTGAAGTTCAC 3′, and the primer amplification size was 119 bp.

[0090] As can be seen from Figure 10, the chemically synthesized polypeptide CircACE2-76aa inhibited the cell infection ability of the SARS-CoV-2 pseudovirus by 91.61% at a concentration of 10 μg / mL, and by 95.50% at a concentration of 160 μg / mL. As can be seen from Figure 11, the recombinant CircACE2-76aa-FC inhibited the cell infection ability of the SARS-CoV-2 pseudovirus by 52.11% at a concentration of 10 μg / mL, and by 92.18% at a concentration of 160 μg / mL. The specific results are shown in Figure 11.

[0091] [Table 1-1]

[0092] [Table 1-2]

[0093] [Table 1-3]

[0094] In the description herein, references such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" mean that the specific features, structures, materials, or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In the description herein, the terms "exemplary" and "specific examples" do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, if not in conflict with each other, those skilled in the art may combine and combine features of different embodiments or examples described herein.

[0095] Although embodiments of the present invention have been presented and described, the above embodiments are illustrative and should not be construed as limiting the present invention, and those skilled in the art will appreciate that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. An isolated circular RNA molecule, wherein the circular RNA molecule has the nucleotide sequence shown in 1) or 2) below: 1) the RNA sequence shown in SEQ ID NO: 1, or 2) an RNA sequence having at least 90% identity to 1); and a terminal nucleotide of the formula: The isolated circular RNA molecule is characterized in that the initiation nucleotide is set as the first nucleotide of the circular RNA molecule, and the polypeptide encoded by the isolated circular RNA molecule competitively binds to the COVID-19 novel coronavirus in cells, thereby inhibiting infection of cells by the COVID-19 novel coronavirus.

2. The circular RNA molecule of claim 1, wherein the circular RNA molecule is formed by single circularization of the second exon of the mRNA sequence encoding angiotensin-converting enzyme 2.

3. The circular RNA molecule of claim 2, wherein the coding region of the circular RNA molecule comprises nucleotides 104 to 45 of the circular RNA molecule.

4. The nucleic acid sequence of the coding region of the circular RNA molecule is 1) the RNA sequence shown in SEQ ID NO: 2, or 2) an RNA sequence having at least 90% identity to 1), the circular RNA molecule of claim 3.

5. A polypeptide, the amino acid sequence of which is 1) the amino acid sequence shown in SEQ ID NO: 3, or 2) an amino acid sequence having at least 90% identity with 1); and The polypeptide is characterized in that it inhibits infection of cells by the COVID-19 novel coronavirus by competitively binding to the COVID-19 novel coronavirus.

6. The polypeptide of claim 5, wherein the polypeptide comprises a signal peptide sequence.

7. The polypeptide of claim 6, wherein the amino acid sequence of the signal peptide is MSSSSWLLLLSLVAVTAA.

8. A fusion protein comprising the polypeptide of claim 5 and Fc, wherein the C-terminus of the polypeptide is linked to the N-terminus of Fc.

9. The fusion protein comprises: 1) the amino acid sequence set forth in SEQ ID NO: 4, or 9. The fusion protein according to claim 8, comprising at least one of the following sequences: 2) an amino acid sequence having at least 90% identity with 1).

10. An isolated nucleic acid, characterized in that it encodes the fusion protein of claim 8.

11. The nucleic acid encoding the fusion protein 1) the nucleotide sequence set forth in SEQ ID NO: 5, or 2) A nucleotide sequence having at least 90% identity to 1), characterized in that the nucleic acid comprises at least one of the following sequences:

12. A drug or kit for inhibiting infection of cells by a novel coronavirus, comprising an RNA molecule described in claim 1 or 2, a polypeptide described in claim 5 or 6, a fusion protein described in claim 8 or 9, or an isolated nucleic acid described in claim 10 or 11.

13. A therapeutic agent for treating or preventing novel coronavirus pneumonia, comprising an RNA molecule described in claim 1 or 2, a polypeptide described in claim 5 or 6, a fusion protein described in claim 8 or 9, or an isolated nucleic acid described in claim 10 or 11.

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