RNA enhancers

Abstract

The present invention provides immunomodulators for enhancing RNA expression, improving RNA stability, and / or prolonging RNA expression time. The invention provides a composition and application thereof. The composition comprises one or more immunomodulatory factors or active variants thereof, wherein the immunomodulatory factors are selected from the group consisting of ORF6, M protein, A46R, SOCS1, SOCS2, SOCS3, NSP5, NS5A, VP35, NS4b, tat, DHX58, B18R, V protein, VP3 and NS1; or a polynucleotide encoding the same; or a vector comprising a polynucleotide encoding the same.

Description

Technical Field

[0001] The present invention belongs to the field of gene therapy, and in particular relates to RNA enhancers. Background Art

[0002] RNA molecules have a wide range of drug targets, including proteins, RNA, and genomes. Therefore, RNA therapy is widely used in the prevention and treatment of many diseases, including infectious diseases, cancer, immune diseases, and genetic diseases. However, RNA therapy also has certain limitations: because RNA has certain immunogenicity, it easily activates the body's immune response after entering the cell, resulting in rapid degradation or inhibition of RNA expression (such as mRNA and circular RNA). Although B18R from vaccine viruses and NS1 from influenza viruses have been used to enhance the expression of mRNA, the field urgently needs reagents with better effects for enhancing mRNA expression, improving mRNA stability, and prolonging mRNA expression time. Summary of the Invention

[0003] The present invention relates to an immunomodulator for enhancing RNA expression, improving RNA stability and / or prolonging RNA expression time.

[0004] In a first aspect, the present invention provides a composition comprising one or more immunomodulatory factors or active variants thereof selected from the group consisting of: ORF6, M protein (membrane protein, M pro), A46R, SOCS1, SOCS2, SOCS3, NSP5, NS5A, VP35, NS4b, tat, DHX58, B18R, V protein (Vpro), VP3, NS1; or

[0005] A polynucleotide encoding the same; or a vector comprising the polynucleotide encoding the same.

[0006] In some embodiments, ORF6 is an immunomodulatory factor from SARS-CoV-2. In some embodiments, the M protein (membrane protein) is an immunomodulatory factor from a coronavirus (such as SARS-CoV, MERS, SARS-CoV-2). In some embodiments, A46R is an immunomodulatory factor from vaccinia virus. In some embodiments, NSP5 is an immunomodulatory factor from SARS-CoV-2. In some embodiments, NS5A is an immunomodulatory factor from hepatitis C virus. In some embodiments, VP35 is an immunomodulatory factor from Ebola virus. In some embodiments, NS4b is an immunomodulatory factor from MERS-CoV. In some embodiments, tat is an immunomodulatory factor from HIV. In some embodiments, the V protein is an immunomodulatory factor from Nipah virus. In some embodiments, VP3 is an immunomodulatory factor from rotavirus. In some embodiments, NS1 is an immunomodulatory factor from influenza virus. In some embodiments, SOCS1, SOCS2, SOCS3, and DHX58 are derived from host immune regulatory factors. In some embodiments, B18R is derived from a Vaccinia virus immune regulatory factor.

[0007] In some embodiments, the polynucleotide is RNA, such as mRNA, circular RNA (circRNA), or siRNA.

[0008] In some embodiments, the vector is a plasmid vector or a viral vector, such as AAV (adeno-associated virus), ADV (adenovirus) or LV (lentiviral vector).

[0009] In some embodiments, the RNA molecule is an mRNA comprising a 5' untranslated region, a 3' untranslated region, and / or a polyA tail.

[0010] In some embodiments, the RNA molecule is an mRNA comprising a chemical modification, preferably a pseudouridine modification and / or a 5-methylcytosine. Also preferably, each U in the mRNA molecule is a pseudouridine, and / or each C is a 5-methylcytosine.

[0011] In some embodiments, the RNA molecule has a nucleotide sequence selected from the following, or a nucleotide sequence having at least 65% sequence identity thereto:

[0012] SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 and / or SEQ ID NO:35.

[0013] In some embodiments, the immunomodulatory factor is a protein.

[0014] In some embodiments, the immunomodulatory factor is one or more immunomodulatory factors or active variants thereof having an amino acid sequence selected from the following or an amino acid sequence having at least 65% sequence identity thereto:

[0015] SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and / or SEQ ID NO:36.

[0016] In some embodiments, the active variant is a truncated form, fragment, mutant, or ortholog thereof, as long as its immunomodulatory activity, such as immunosuppressive activity, is retained.

[0017] In some embodiments, the composition further comprises a polynucleotide encoding a protein of interest, or a vector comprising a polynucleotide encoding the same.

[0018] The vector is a plasmid vector or a viral vector, such as AAV (adeno-associated virus), ADV (adenovirus) or LV (lentiviral vector).

[0019] In some embodiments, the target protein may be a protein used to treat or prevent a disease, and the disease may be an inflammatory disease, an infectious disease, an autoimmune disease, or cancer.

[0020] In some embodiments, the polynucleotide is RNA, such as mRNA, circRNA, or siRNA.

[0021] In some embodiments, the RNA molecule is an mRNA comprising a 5' untranslated region, a 3' untranslated region, and / or a polyA tail.

[0022] In some embodiments, the RNA molecule is an mRNA that comprises chemical modifications, preferably pseudouridine and / or 5-methylcytosine modifications. Also preferably, each U in the mRNA molecule is a pseudouridine, and / or each C is a 5-methylcytosine modification.

[0023] In some embodiments, at least one mRNA in the composition is complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles.

[0024] In some embodiments, the composition further comprises an adjuvant.

[0025] In some embodiments, the composition is formulated for oral administration, transdermal administration, or parenteral administration; preferably, the composition is formulated as a solution, emulsion, suspension, tablet, capsule, powder, pill, or aerosol.

[0026] In a second aspect, the present invention provides a polynucleotide comprising an immune regulatory domain and a target protein encoding domain, wherein the immune regulatory domain encodes one or more immune regulatory factors or active variants thereof selected from the following: ORF6, M protein (membrane protein), A46R, SOCS1, SOCS2, SOCS3, NSP5, NS5A, VP35, NS4b, tat, DHX58, B18R, V protein, VP3, NS1.

[0027] The target protein coding domain encodes a protein for treating or preventing a disease, which may be an inflammatory disease, an infectious disease, an autoimmune disease, a gene defect disease (such as cystic fibrosis, hemophilia, phenylketonuria, sickle cell anemia, thalassemia, albinism, etc.), an ischemic disease, an allergic disease (such as allergic rhinitis, allergic asthma, allergic purpura, anaphylactic shock and allergic dermatitis, etc.), a chronic disease (such as chronic obstructive pulmonary disease, pulmonary fibrosis, asthma, etc.) or a cancer, etc.

[0028] The target protein domain encodes one or more proteins for treating or preventing diseases.

[0029] In some embodiments, the protein for treating or preventing a disease is selected from pathogen proteins, tumor antigens, cytokines, growth factors, or proteins for treating chronic diseases, ischemic diseases, or gene defect diseases.

[0030] In some embodiments, the pathogen protein is selected from bacteria, fungi, viruses, mycoplasmas, chlamydia, rickettsia, etc., such as influenza virus (NP, HA, NA), coronavirus (spike), hepatitis C virus, Nipah virus, hepatitis B virus (HBsAg), Ebola virus (envelope protein GP, ​​ribonucleoprotein NP, matrix protein VP24 and VP40), human papillomavirus (E6, E7 protein), immunodeficiency virus (envelope protein), rabies virus (glycoprotein G and nucleoprotein N), rotavirus (VP7 and VP4), poliovirus (C antigen and D antigen), Coxsackie virus ( PEP32, PEP37, PEP55, PEP71 and PEP91), hand, foot and mouth disease virus (VP1 and VP2), herpes zoster virus (glycoprotein gE), poxvirus (A35R, M1R), measles virus, respiratory syncytial virus (G protein), Mycobacterium tuberculosis (ESAT6 and Ag85B), Escherichia coli, Salmonella, Streptococcus pneumoniae, Klebsiella pneumoniae, Staphylococcus aureus, Vibrio cholerae, Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Chlamydia psittaci, Chlamydia pneumoniae, Chlamydia trachomatis, Candida, Aspergillus, Cryptococcus, Mucor, Histoplasma, etc.

[0031] In some embodiments, the combination of the pathogen protein and the RNA construct of the immune regulatory factor can be selected from: SOCS1 and the new coronavirus (e.g., spike), Mpro and the new coronavirus (e.g., spike), A46R and the new coronavirus (e.g., spike), ORF6 and the new coronavirus (e.g., spike), SOCS1 and influenza virus (e.g., SOCS1 and NP, SOCS1 and HA, SOCS1 and NA, SOCS1 and HA, NP and NA), Mpro and influenza virus (e.g., Mpro and NP, Mpro and HA, Mpro and NA, Mpro and HA, NP and NA), A46R and influenza virus (e.g., A46R and NP, A46R and HA, Mpro and NA, A46R and HA, NP and NA), ORF6 and influenza virus (e.g., ORF6 and NP, ORF6 and HA, ORF6 and NA, ORF6 and HA, NP and NA), SOCS1 and respiratory syncytial virus (e.g., SOCS1 and G protein), Mpro and respiratory syncytial virus (e.g., Mpro and G protein), ORF6 and respiratory syncytial virus (e.g., ORF6 and G protein), A46R and respiratory syncytial virus (e.g., A46R and G protein).

[0032] In some embodiments, the immunomodulatory domain encodes one or more immunomodulatory factors or active variants thereof.

[0033] In some embodiments, the immune regulatory domain encodes one or more of the following immune regulatory factors or active variant combinations thereof: such as SOCS1 and M pro, SOCS1 and ORF6, M pro and ORF6, M pro and ORF6 and SOCS1, SOCS1 and A46R, M pro and A46R, ORF6 and A46R, M pro and ORF6, SOCS1 and A46R, etc., combined with proteins for treating or preventing diseases.

[0034] In some embodiments, the immune regulatory factor is derived from a virus or a primate immune negative regulatory protein.

[0035] In some embodiments, the immune modulator is selected from coronavirus open reading frame 6 (ORF6), and the coronavirus is selected from Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2, HcoV-229E, HcoV-OC43, HcoV-NL63, HcoV-HKU1.

[0036] In a preferred embodiment, the immune regulatory factor is selected from SARS-CoV-2 ORF6.

[0037] In some embodiments, the immune regulatory factor is selected from coronavirus membrane proteins, and the coronavirus is selected from Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2, HcoV-229E, HcoV-OC43, HcoV-NL63, HcoV-HKU1; in a preferred embodiment, the immune regulatory factor is selected from SARS-CoV-2M protein.

[0038] In some embodiments, the immune modulator is selected from a non-structural protein of a virus, and the virus is selected from a coronavirus, a rotavirus, a hepatitis virus, and an influenza virus.

[0039] In some embodiments, the coronavirus is selected from Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2, HcoV-229E, HcoV-OC43, HcoV-NL63, HcoV-HKU1.

[0040] In some embodiments, the influenza virus is selected from type A, type B, type C, and type D; further, in a preferred embodiment, the influenza A virus is selected from H1N1, H3N2, and H7N9.

[0041] In some embodiments, the immune regulatory factor is selected from non-structural proteins nsp2, nsp3 and nsp4; in a preferred embodiment, the immune regulatory factor is selected from SARS-CoV-2 NSP5.

[0042] In one embodiment, the immune regulatory factor is selected from NS1, NS2, NS3, NS4a, NS4b, NS5a and NS5b of hepatitis virus, Ebola virus, coronavirus, and influenza virus; in a preferred embodiment, the immune regulatory factor is selected from NS5A of hepatitis C virus (Hepacivirus C), MERS-CoVNS4b, and influenza virus (influenza) NS1.

[0043] In one embodiment, the immune regulatory factor is selected from poxvirus A46R, and the poxvirus is selected from vaccinia virus, monkeypox virus, smallpox virus, and cowpox virus; in a preferred embodiment, the immune regulatory factor is selected from vaccinia virus A46R.

[0044] In one embodiment, the immune regulatory factor is selected from poxvirus, and the poxvirus is selected from vaccinia virus, monkeypox virus, smallpox virus, and cowpox virus; in a preferred embodiment, the immune regulatory factor is selected from vaccinia virus B18R.

[0045] In one embodiment, the immune modulator is selected from tat of HIV.

[0046] In one embodiment, the immune modulator is selected from the V protein of Nipah virus.

[0047] In one embodiment, the immune modulator is selected from VP3 of rotavirus.

[0048] In one embodiment, the immune modulator is selected from NS1 of influenza virus.

[0049] In some embodiments, the polynucleotide is RNA, such as mRNA, circular RNA, or siRNA.

[0050] In some embodiments, the RNA molecule is an mRNA comprising a 5' untranslated region, a 3' untranslated region, and / or a polyA tail.

[0051] In some embodiments, the RNA molecule is an mRNA that comprises chemical modifications, preferably pseudouridine and / or 5-methylcytosine modifications. Also preferably, each U in the mRNA molecule is a pseudouridine, and / or each C is a 5-methylcytosine modification.

[0052] In some embodiments, the polynucleotide is complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles.

[0053] In some embodiments, the polynucleotide is formulated with an adjuvant.

[0054] In some embodiments, the polynucleotide is formulated for oral administration, transdermal administration, or parenteral administration; preferably, the composition is formulated as a solution, emulsion, suspension, tablet, capsule, powder, pill or aerosol.

[0055] In a third aspect, the present invention provides a method for expressing a target protein in a cell, comprising the following steps:

[0056] (i) introducing RNA encoding the target protein into the cell, and (ii) providing the cell with one or more immunomodulatory factors or active variants thereof selected from the following: ORF6, M protein (membrane protein), A46R, SOCS1, SOCS2, SOCS3, NSP5, NS5A, VP35, NS4b, tat, DHX58, B18R, V protein, VP3, NS1; or polynucleotides encoding the same; or vectors comprising the polynucleotides encoding the same.

[0057] In some embodiments, the RNA encoding the target protein and the RNA encoding the immunomodulatory factor or its active variant are two separate RNAs, or an RNA construct connected directly or via a linker (such as 2A or IRES).

[0058] In some embodiments, the RNA construct is, for example, mRNA, circular RNA, or siRNA.

[0059] In some embodiments, the vector is a plasmid vector or a viral vector, such as AAV (adeno-associated virus), ADV (adenovirus) or LV (lentiviral vector).

[0060] In some embodiments, the RNA comprises a 5' untranslated region, a 3' untranslated region, and / or a polyA tail.

[0061] In some embodiments, the RNA comprises chemical modifications, preferably pseudouridine and / or 5-methylcytosine modifications. Also preferably, each U in the mRNA molecule is a pseudouridine, and / or each C is a 5-methylcytosine modification.

[0062] In some embodiments, the active variant is a truncated form, fragment, mutant, or ortholog thereof, as long as its immunomodulatory activity, such as immunosuppressive activity, is retained.

[0063] In some embodiments, the target protein can be a protein used to treat or prevent a disease, and the disease can be an inflammatory disease, an infectious disease, an autoimmune disease, a genetic defect (such as cystic fibrosis (CFTR), hemophilia, phenylketonuria, sickle cell anemia, thalassemia, albinism, etc.), an ischemic disease, an allergic disease, a chronic disease (such as chronic obstructive pulmonary disease, pulmonary fibrosis, asthma, etc.) or cancer, etc.

[0064] In a fourth aspect, the present invention provides use of the composition of the first aspect or the polynucleotide of the second aspect in the preparation of a medicament for treating or preventing a disease.

[0065] The disease can be an inflammatory disease, an infectious disease, an autoimmune disease, a genetic defect (such as cystic fibrosis, hemophilia, phenylketonuria, sickle cell anemia, thalassemia, albinism, etc.), an ischemic disease, an allergic disease, a chronic disease (such as chronic obstructive pulmonary disease, pulmonary fibrosis, asthma, etc.) or cancer, etc.

[0066] In a fifth aspect, the present invention provides a method for increasing the expression level of a target protein, the method comprising constructing a polynucleotide comprising an immune regulatory domain and a target protein encoding domain, wherein the immune regulatory domain encodes one or more immune regulatory factors or active variants thereof selected from the following: ORF6, M protein (membrane protein), A46R, SOCS1, SOCS2, SOCS3, NSP5, NS5A, VP35, NS4b, tat, DHX58, B18R, V protein, VP3, NS1.

[0067] In a sixth aspect, the present invention provides use of the composition of the first aspect or the polynucleotide of the second aspect in the preparation of a drug for enhancing angiogenesis.

[0068] The present invention discovered that some viral immune regulatory factors can enhance target gene expression. These RNAs can be used as enhancers for RNA (mRNA, self-replicating RNA, circular RNA, and siRNA) therapy. The enhancers can be in the form of mRNA (with varying capping, base modification, and codon optimization), circRNA, plasmids, or proteins (full-length or truncated functional domains).

[0069] These enhancers have negative immune regulatory functions and can serve as enhancers for RNA therapy, as well as anti-inflammatory drugs. Therefore, by inhibiting the immune response induced by RNA transfection, they can enhance the stability and duration of RNA expression, thereby enhancing the therapeutic effect of RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1Shows the effect of different RNAs on enhancing mCherry mRNA expression in HUVECs cells.

[0071] Figure 2 The results show that different RNAs enhance the expression of Guassialuciferase mRNA in HUVECs cells.

[0072] Figure 3 The results show that different RNAs enhance the expression of Gaussia luciferase mRNA in 293T cells.

[0073] Figure 4 The results show that different RNAs enhance the stability of mCherry mRNA in HUVECs cells.

[0074] Figure 5 The results show that different SOCSs enhance the expression of Gaussia luciferase mRNA in HUVECs cells.

[0075] Figure 6 The results show that different coronavirus membrane mRNAs (Mpro) in HUVECs cells enhance the expression of Gaussia luciferase mRNA.

[0076] Figure 7 The anti-inflammatory effects of different RNAs in HUVEC cells were shown. Figure 7 A shows the inhibitory effects of several RNAs on LPS-induced IL-6 mRNA levels. Figure 7 B shows the inhibitory effects of several RNAs on LPS-induced IL-1α mRNA levels.

[0077] Figure 8 Shows the effect of different RNAs on enhancing the expression of firefly luciferase in vivo. Figure 8 A shows representative in vivo imaging results. Figure 8 B shows the quantitative results of luciferase expression from two days of in vivo imaging.

[0078] Figure 9 The results showed that SOCS1 enhanced the expression of ETV2 mRNA.

[0079] Figure 10 The results showed that SOCS1 can promote ETV2 angiogenesis in mice. Figure 10 A is a photo of Matrigelplugs. Figure 10 B is the H&E staining photos of three groups of Matrigel plugs.

[0080] Figure 11 The results showed that SOCS1 can promote LMO2 angiogenesis in mice. Figure 11 A is a photo of Matrigelplugs. Figure 11 B is the H&E staining photos of two groups of Matrigel plugs. DETAILED DESCRIPTION

[0081] In some embodiments, the immune modulators described herein are derived from viruses.

[0082] The immunomodulatory factors described in the present invention are:

[0083] ORF6 is an immune regulatory factor from SARS-CoV-2; M protein (membrane protein) is an immune regulatory factor from three coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2); A46R is an immune regulatory factor from vaccinia virus; NSP5 is an immune regulatory factor from SARS-CoV-2; NS5A is an immune regulatory factor from hepatitis C virus; VP35 is an immune regulatory factor from Ebola virus; NS4b is an immune regulatory factor from MERS-CoV; tat is an immune regulatory factor from HIV; V protein is an immune regulatory factor from Nipah virus; VP3 is an immune regulatory factor from rotavirus; B18R is an immune regulatory factor from vaccinia virus; and NS1 is an immune regulatory factor from influenza virus. SOCS1, SOCS2, SOCS3, and DHX58 are immune regulatory factors from the host.

[0084] The nucleotide sequence and amino acid sequence of the immune regulatory factor disclosed in the present invention are shown in Table 1.

[0085] Table 1. Nucleotide and amino acid sequences of immunomodulatory factors

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] In one embodiment of the present invention, RNA encoding a target protein (which is mRNA encoding a target protein) and RNA encoding a virus-derived factor (which is mRNA encoding a virus-derived factor) are co-transfected.

[0097] In one embodiment of the present invention, RNA encoding a target protein (which is a replicon RNA encoding a target protein) and RNA encoding a virus-derived factor (which is an mRNA encoding a virus-derived factor) are co-transfected.

[0098] The RNA molecules according to the present invention may involve a 5'-cap, a 5'-UTR, a 3'-UTR, a poly(A) sequence and / or codon optimization.

[0099] "Variants" include all splice variants, post-translationally modified variants, conformers, isomers and orthologs, especially those naturally expressed by the cell.

[0100] In the present invention, mRNA refers to messenger RNA, and also refers to transcripts produced using a DNA template and encoding a peptide or protein. Typically, mRNA comprises a 5'-UTR, a protein coding region, a 3'-UTR, and a poly(A) sequence. mRNA can be produced from a DNA template by in vitro transcription. In vitro transcription methods are known to those skilled in the art.

[0101] definition

[0102] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0103] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0104] As used herein, the expression "about" is understood by one of ordinary skill in the art and varies within certain limits depending on the context in which it is used. If the use of the term is not understood by one of ordinary skill in the art based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0105] "Sequence identity" refers to the "percent sequence identity" or "percent identity" between two polypeptides or two polynucleotides, that is, the number of identical matching positions shared by the sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted because gaps are not nucleotides. At least 65% sequence identity includes contiguous segments having at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity over the entire length of the sequence.

[0106] Comparison that is used to determine nucleotide sequence or amino acid sequence identity percentage ratio can realize in the various ways within the scope of this area technology, for example, use publicly available computer software, for example EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine to be used to measure the appropriate parameters of comparison, be included in and realize maximum required any algorithm of comparison on the sequence full length being compared.

[0107] In the present disclosure, the linker refers to a connecting sequence consisting of nucleotides, which is used to connect multiple ORFs driven by a single promoter for simultaneous expression. The length of the linker anywhere can be 1 to 500 nucleotides or 3 to 1000 nucleotides. It can be repeated 1 to 10 times, or about 8 times, or about 6 times, or about 5 times, or 4 times, or 3 times, or 2 times. In some embodiments, examples of the linker include 2A linkers, such as P2A, T2A, E2A, and F2A. In some embodiments, another example of a cleavable linker includes an internal ribosome entry site (IRES). Those skilled in the art will readily understand other linkers that can be used herein.

[0108] In the present disclosure, immunomodulation refers to the process by which an immune response is regulated to a desired level, for example by inducing, enhancing or suppressing an immune response. In the present disclosure, immunosuppression refers to the inhibition or reduction of an immune response.

[0109] In the present disclosure, the disease may be an infectious disease, an allergic disease, an autoimmune disease or cancer.

[0110] In the present disclosure, the cancer includes but is not limited to: solid tumors, lymphomas, blastomas, sarcomas and leukemias. More specifically, the cancer includes but is not limited to bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular malignant melanoma, cervical cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, liver cancer, colon cancer, breast cancer, prostate cancer, Hodgkin's disease (Hodgkin'sDisease), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasms, neuroectodermal cancer, spinal axis tumors, gliomas, meningiomas and pituitary adenomas.

[0111] In the present disclosure, the infectious diseases include, but are not limited to, AIDS (HIV infection), hepatitis A, hepatitis B, hepatitis C or hepatitis D, herpes, herpes zoster, measles (rubella virus), yellow fever, dengue fever and other flavivirus infections, influenza virus infection, hemorrhagic infectious diseases (Marburg or Ebola virus) infection, and severe acute respiratory syndrome (SARS), SARS-CoV-2; bacterial infectious diseases, such as Legionnaire's disease (Legionella spp.), sexually transmitted diseases (e.g., Chlamydia or gonorrhea), gastric ulcer (Helicobacter), cholera (Vibrio), tuberculosis, diphtheria, E. coli, Staphylococci, Salmonella or Streptococci (tetanus); protozoan pathogen infections, such as malaria, sleeping sickness, leishmaniasis; toxoplasmosis, i.e., infections with Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or fungal infections, such as infections caused by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

[0112] In the present disclosure, the autoimmune diseases include but are not limited to Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, etc.

[0113] In the present disclosure, the inflammatory diseases include but are not limited to inflammation of substantial organs such as the liver, kidney, heart and brain, for example, encephalitis, hepatitis, nephritis, myocarditis, etc.; serous catarrhal inflammation, serositis, loose connective tissue inflammation, rheumatoid arthritis, bacterial dysentery, diphtheria, pneumonia; suppuration and empyema, phlegmonous inflammation, abscess, hemorrhagic inflammation; general proliferative inflammation; granulomatous inflammation.

[0114] In the present disclosure, the allergic diseases include but are not limited to allergic rhinitis, allergic asthma, allergic purpura, anaphylactic shock and allergic dermatitis, etc.

[0115] The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that, for those skilled in the art, any modifications and improvements made based on this inventive concept fall within the scope of protection of the present invention. The reagents used, for which the manufacturer is not indicated, are all commercially available conventional products.

[0116] Example

[0117] Example 1: Co-transfection experiment of mCherry mRNA and 15 kinds of RNA

[0118] mRNA preparation: Using a DNA fragment containing a T7 promoter and the CDS sequence of the target protein as a template, In vitro transcription was performed according to the instructions of the T7 High-Efficiency RNA Synthesis Kit (New England Biolabs, E2040S). The product was digested with DNase I (Promega, M6101) to remove the template DNA. The digested product was then incubated with oligo d(T) magnetic beads (New England Biolabs, T2050L) and UltraBio TM The products were purified successively using TBE-Urea PAGE precast gel (Aladdin, T753824-A1-10EA), and the concentration of the final products was quantified using nanodrop.

[0119] 2000 HUVECs (ATCC) cells were plated per well of a 384-well plate. 24 hours later, mCherry mRNA (0.003 μg) prepared using the above method was co-transfected with 15 types of RNA (0.0006 μg) (sequences see Table 1), of which firefly luciferase mRNA served as a control group (CT-Fluc). The fluorescence intensity of mCherry was captured using an Incucyte S3 live cell imager 12, 24, and 48 hours after transfection, and the fluorescence intensity was quantified relative to the CT-Fluc group. The results are shown in Figure 2. Figure 1 The results showed that compared with the control RNA (Luc) group, the 14 RNAs could significantly increase the fluorescence intensity of mCherry at three time points and prolong the expression duration of mCherry mRNA.

[0120] Example 2: Co-transfection experiment of Guassia luciferase mRNA and 15 kinds of RNA

[0121] 5×10 4 HUVECs cells. 24 hours later, guassia luciferase mRNA (0.03 μg) was co-transfected with 15 types of RNA (0.006 μg) (sequences see Table 1), of which firefly luciferase mRNA served as the control group (CT-Fluc). 24 hours after transfection, cell supernatant was collected and Guassia substrate (GeneCopoeia Inc, LF062) was added. The Guassia luciferase content was read using a multifunctional microplate reader, and relative quantification was performed with the CT-Fluc group as 1. The results are shown in Figure 2. Figure 2 The results showed that compared with the control RNA (Luc) group, except for VP3, the other 13 species could significantly increase the expression of Guassia luciferase at 24 hours.

[0122] Example 3: Co-transfection experiment of Guassia luciferase mRNA and 6 kinds of RNA

[0123] 5×10 4 293T cells (ATCC). 24 hours later, Guassia luciferase mRNA (0.03 μg) was co-transfected with six RNAs (0.006 μg) (sequences in Table 1), with firefly luciferase mRNA serving as the control group (Luc). 12 hours after transfection, cell supernatants were collected, Guassia substrate was added, and guassia was quantified using a multifunctional microplate reader. The results are shown in Figure 2. Figure 3The results showed that compared with the control RNA (Luc) group, all RNAs could significantly enhance the expression of Guassialuciferase, and the effects of SOCS1, ORF6, M protein, and A46R on the expression efficiency of guassia were greater than that of NS1.

[0124] Example 4: Co-transfection experiment of mCherry mRNA and 6 kinds of RNA

[0125] 1×10 5 HUVECs cells. 24 hours later, mCherry mRNA (0.03 μg) was co-transfected with 6 types of RNA (0.006 μg) (sequences see Table 1), of which firefly luciferase mRNA was used as the control group (Luc). At the same time, an mCherry single transfection group (0.03 μg) was set up. Cells were collected at 6, 9, and 12 hours after transfection, RNA was extracted, and mCherry mRNA was quantified. The results are shown in Figure 2. Figure 4 The results showed that, except for the control RNA (Luc), the other five RNAs could increase the stability of mCherry mRNA (compared with the Luc group, SOCS1, M Pro, ORF6, A46R and VP35 increased by 3.096, 1.546, 2.956, 1.646 and 0.764 respectively).

[0126] Example 5: Co-transfection experiment of Guassia luciferase mRNA and SOCS1, 2 or 3

[0127] 1×10 5 HUVECs cells were transfected with guassialuciferase mRNA (0.03 μg) and SOCS1, 2, or 3 (0.006 μg) (sequences are shown in Table 1) 24 hours later, and firefly luciferase mRNA was used as a control (Luc). Cell supernatants were collected 12 and 24 hours after transfection, and guassialuciferase substrate was added. Guassialuciferase was quantified using a multifunctional microplate reader. The results are shown in Figure 1. Figure 5 The results showed that compared with the control RNA group, the three SOCSs could increase the expression of Guassia luciferase at two time points.

[0128] Example 6: Co-transfection experiment of Guassia luciferase mRNA and coronavirus membrane mRNA

[0129] 1×10 5HUVECs cells were co-transfected with 0.03 μg of Guassia luciferase mRNA and 0.006 μg of membrane mRNA from three coronaviruses (sequences in Table 1). Firefly luciferase mRNA was used as a control (Luc). Cell supernatants were collected 12 and 24 hours after transfection, and Guassia luciferase substrate was added. The amount of Guassia luciferase was quantified using a multifunctional microplate reader. The results are shown in Figure 2. Figure 6 It shows that compared with the control group, the membrane proteins of the three coronaviruses can increase the expression of Guassialuciferase at two time points.

[0130] Example 7: Experimental study on the effects of 15 mRNAs on LPS-induced inflammatory response

[0131] 1.5×10 5 HUVECs cells were transfected with 15 mRNAs (0.2 μg) 24 hours later (sequences are shown in Table 1), of which the same dose of mCherry mRNA was used as the mRNA control group, and the group treated with the same transfection reagent was the blank control group (CT). Fresh culture medium was replaced 6 hours after transfection. 18 hours after transfection, all groups were stimulated with culture medium containing LPS (1 μg / mL) for 6 hours. Cells were collected, RNA was extracted, and the mRNA levels of cytokines IL-1α and IL-6 were quantified. The results are shown in Figure 2. Figure 7 The results showed that compared with the negative control mCherrymRNA group, all 14 mRNAs could inhibit the LPS-induced inflammatory response and had anti-inflammatory effects.

[0132] Example 8: LNP preparation

[0133] Dlin-MC3-DMA (MedChemExpress, HY-112251), cholesterol (Avanti Polar Lipids, 700000P), DOPE (Avanti Polar Lipids, 850725P), and DMG-PEG 2000 (Avanti Polar Lipids, 880151P) were dissolved in anhydrous ethanol and mixed at a molar ratio of 35:46.5:16:2.5. The different mRNAs were dissolved in 10 mM citrate buffer. The aqueous and oil phases were rapidly mixed at a volume ratio of 3:1 to prepare LNPs@mRNA. The product was dialyzed against PBS buffer at 4°C for 2 h.

[0134] Example 9: In vivo expression enhancement experiment of firefly luciferase by two mRNAs

[0135] Firefly luciferase mRNA was encapsulated by ionizable lipid nanoparticles (LNPs) alone or co-encapsulated with SOCS1 or Gaussia luciferase mRNA. The three LNP drugs were injected intramuscularly into male C57BL / 6 mice (20 μL LNPs containing 4 μg mRNA per mouse) (n=8), and luciferase expression was detected by in vivo imaging on days 1 and 2 after injection. The results are shown in Figure 2. Figure 8 The results showed that compared with the Gaussia luciferase mRNA control group, SOCS1 could significantly enhance the expression of firefly luciferase at both time points.

[0136] Example 10: Co-transfection experiment of ETV2 and LMO2 with firefly Luciferase, SOCS1, M protein or ORF6

[0137] 1. ETV2 and LMO2 were co-transfected with firefly luciferase, SOCS1, M protein or ORF6, respectively. 24 hours after transfection, the cells were collected and the expression levels of ETV2 and LMO2 proteins were detected by western blot to determine the effects of several RNAs on ETV2 or LMO2 expression.

[0138] 2×10 5 HUVECs cells. 24 hours later, ETV2 mRNA (0.5 μg) was co-transfected with firefly luciferase (CT-Fluc) or SOCS1 mRNA (0.1 μg) (sequences see Table 1), and the same dose of ETV2 mRNA alone was transfected as a control. 24 hours after transfection, cells were collected, lysed, and protein precipitates were collected. 6× loading buffer (Biyuntian) was added, and SDS-PAGE electrophoresis was performed. The membrane was transferred and incubated with ETV2 antibody (Abcam), and finally developed. The results are shown in Figure 2. Figure 9 Shows that compared with the Luciferase control group, SOCS1 increased the expression of ETV2 mRNA.

[0139] 2. ETV2 mRNA (10 μg) was co-encapsulated with firefly Luciferase (CT-Fluc) or SOCS1 mRNA (2 μg) into LNPs and named CT-Fluc and SOCS1. LNPs without mRNA were named Vehicle. Vehicle, CT-Fluc and SOCS1 (50 μL) were mixed with Matrigel (300 μL) (Corning, 354234), VEGF (100 ng / mL) (Peprotech, 100-20-1 mg) and heparin (60 IU / mL) (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.) (PBS was added to 400 μL) and injected subcutaneously into the abdomen of 8-week-old male C57BL / 6 mice (n=3). After 5 days, Matrigel plugs were collected, photographed, fixed with paraformaldehyde, and sliced ​​and stained with H&E. Figure 10 The results showed that compared with the vehicle control group, ETV2 mRNA promoted angiogenesis in Matrigel, and compared with the CT-Fluc group, SOCS1 further enhanced the angiogenesis ability of ETV2.

[0140] 3. LMO2 mRNA (10 μg) was co-encapsulated with firefly luciferase (CT-Fluc) or SOCS1 mRNA (2 μg) into LNPs and named CT-Fluc and SOCS1. CT-Fluc and SOCS1 were mixed with Matrigel (300 μL), VEGF (100 ng / mL), and heparin (60 IU / mL), respectively, and injected subcutaneously into the abdomen of 8-week-old male C57BL / 6 mice (n=4). After 5 days, Matrigel plugs were collected, photographed, fixed with paraformaldehyde, sectioned, and stained with H&E. Figure 11 It was shown that compared with the CT-Fluc group, SOCS1 could enhance the angiogenesis ability of LMO2.

[0141] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A composition comprising one or more immunomodulatory factors or active variants thereof selected from the group consisting of ORF6, M protein, A46R, SOCS1, SOCS2, SOCS3, NSP5, NS5A, VP35, NS4b, tat, DHX58, B18R, V protein, VP3, NS1; or A polynucleotide encoding the same; or a vector comprising the polynucleotide encoding the same.

2. The composition according to claim 1, wherein the immunomodulatory factor is selected from the group consisting of the following amino acid sequences, or one or more immunomodulatory factors or active variants thereof having at least 65% sequence identity thereto: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and / or SEQ ID NO:

36.

3. The composition according to claim 1, further comprising a polynucleotide encoding a target protein, or a vector comprising the polynucleotide encoding the same.

4. A polynucleotide comprising an immune regulatory domain and a target protein encoding domain, wherein the immune regulatory domain encodes one or more immune regulatory factors or active variants thereof selected from the following: ORF6, M protein, A46R, SOCS1, SOCS2, SOCS3, NSP5, NS5A, VP35, NS4b, tat, DHX58, B18R, V protein, VP3 and / or NS1.

5. A vector comprising the polynucleotide of claim 4.

6. A method for expressing a target protein in a cell, comprising the following steps: (i) introducing RNA encoding the target protein into the cell, and (ii) providing the cell with one or more immunomodulatory factors or active variants thereof selected from the group consisting of ORF6, M protein, A46R, SOCS1, SOCS2, SOCS3, NSP5, NS5A, VP35, NS4b, tat, DHX58, B18R, V protein, VP3, NS1; or polynucleotides encoding the same; or vectors comprising the polynucleotides encoding the same.

7. The composition according to any one of claims 1 to 3, or the polynucleotide according to claim 4, or the vector according to claim 5, or the method according to claim 6, wherein the polynucleotide is mRNA, circular RNA or siRNA; Preferably, the mRNA, circular RNA or siRNA comprises a 5' untranslated region, a 3' untranslated region, and / or a polyA tail; Preferably, the mRNA, circular RNA or siRNA comprises chemical modifications, more preferably pseudouridine and / or 5-methylcytosine modifications.

8. The composition according to any one of claims 1 to 3, the vector according to claim 5, or the method according to claim 6, wherein The vector is a plasmid vector or a viral vector, Preferably, the viral vector is AAV, ADV or LV.

9. The composition according to any one of claims 1 to 3, the polynucleotide according to claim 4, the vector according to claim 5, or the method according to claim 6, wherein The polynucleotide encoding the immunomodulatory factor or the immune regulatory domain has a nucleotide sequence selected from the following, or a nucleotide sequence having at least 65% sequence identity therewith: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 and / or SEQ ID NO:

35.

10. The composition according to any one of claims 1 to 3, the polynucleotide according to claim 4, the vector according to claim 5, or the method according to claim 6, wherein The target protein is a protein used to treat or prevent a disease, and the disease is an inflammatory disease, a chronic disease, an ischemic disease, a gene defect disease, an infectious disease, an autoimmune disease or a cancer.

11. The composition according to any one of claims 1 to 3, the polynucleotide according to claim 4 or the vector according to claim 5, The polynucleotide is complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles.

12. The composition according to any one of claims 1 to 3, the polynucleotide according to claim 4 or the vector according to claim 5, which is formulated into a pharmaceutical composition for oral administration, transdermal administration, or parenteral administration; preferably, the pharmaceutical composition is formulated into a solution, emulsion, suspension, tablet, capsule, powder, pill or aerosol form. Preferably, the pharmaceutical composition further comprises an adjuvant.

13. Use of the composition of any one of claims 1 to 3, the polynucleotide of claim 4 or the vector of claim 5 in the preparation of a medicament for treating or preventing a disease, wherein the disease is an inflammatory disease, an infectious disease, a chronic disease, an ischemic disease, a gene defect disease, an autoimmune disease or cancer.

14. Use of the composition according to any one of claims 1 to 3, the polynucleotide according to claim 4 or the vector according to claim 5 in the preparation of a drug for enhancing angiogenesis.

Images