RNA construct

Abstract

This invention relates to RNA constructs encoding (i) at least one therapeutic biomolecule; and (ii) at least one innate inhibitor protein (IIP). The constructs are RNA replicons and saRNA molecules, and the invention comprises genetic constructs or vectors encoding such RNA replicons. The invention extends to the use of such RNA constructs and replicons in therapy, such as in treating diseases and / or in vaccine delivery. The invention extends to pharmaceutical compositions comprising such RNA constructs, as well as methods and uses thereof.

Description

Technical Field

[0001] This invention relates to RNA constructs, and particularly, though not exclusively, to RNA replicons and saRNA molecules, as well as genetic constructs or vectors encoding such RNA replicons. The invention extends to the use of such RNA constructs and replicons in therapy, such as in treating diseases and / or in vaccine delivery. The invention also extends to pharmaceutical compositions comprising such RNA constructs, methods thereof, and their uses. Background Technology

[0002] Messenger RNA (mRNA) is a promising tool for biotherapy. However, while mRNA therapy has proven highly effective in small animals, results are not linearly scaled when these formulations are translated in dose-escalation studies in humans. Furthermore, adverse events associated with inducing interferon responses have limited the escalation of potentially effective RNA doses in humans. The reasons for this inconsistency are unclear, but the inventors hypothesize that differences in innate perception in humans constitute an obstacle to the translation of RNA therapy from the laboratory to the clinical. In addition, innate perception of RNA has been associated with protein expression inhibition. To date, the main approach to overcoming innate recognition of exogenous RNA has been the use of modified ribonucleotides that are not easily detected through innate perception mechanisms. However, modified mRNA is not entirely undetectable and still leads to some degree of interferon activation, protein silencing, and reduced tolerability for human use.

[0003] Another approach is to use self-amplifying or saRNA vectors, typically based on an alphavirus backbone that has the ability to self-amplify its own RNA by encoding polymerase activity within its non-structural proteins. Existing methods involve replacing the structural proteins of these vectors with a target gene (GOI), which is a vaccine construct or encodes a therapeutic protein. Other versions of saRNA have been based on picornaviruses, flaviviruses, and coronaviruses. When saRNA is taken up into the cytoplasm of target cells, this results in RNA amplification via the encoded polymerase system and very high GOI expression levels. Therefore, saRNA has been shown to induce immune responses at much lower doses (10-100 times lower) than mRNA and to result in prolonged protein expression in mice for up to 60 days.

[0004] However, a drawback of saRNAs is that they can still be sensed through innate recognition, thereby triggering antiviral responses that restrict protein expression and self-amplification of these existing saRNA technologies. Due to their large size (typically >5000 bases) and deep secondary structure containing double-stranded regions (dsRNA), the innate sensing of saRNAs differs from that of mRNAs. Long double-stranded RNAs trigger innate responses via the MDA5 (melanoma differentiation-associated protein 5) pathway. This is facilitated by the binding of PACT (PKR activator protein), which promotes MDA5 oligomerization, to long dsRNAs, and the subsequent triggering of a downstream signaling cascade that inhibits saRNA replication and expression.

[0005] Therefore, there is a need in the art for new methods that can deliver and express RNA therapy in patients, enabling them to overcome the innate immune system's perception of RNA. Summary of the Invention

[0006] The inventors have developed a novel self-amplifying RNA (saRNA) that, by expressing innate inhibitory proteins that block or reduce innate immune system mechanisms, leads to increased protein expression and self-amplification, thereby advantageously overcoming the innate immune system's perception of RNA.

[0007] Therefore, in a first aspect of the invention, an RNA construct is provided that encodes (i) at least one therapeutic biomolecule; and (ii) at least one innate inhibitor protein (IIP).

[0008] RNA replicons or constructs are considered potential tools for the delivery and expression of target genes in vaccines and therapies. However, double-stranded RNA (dsRNA) is detected intracellularly by innate sensing mechanisms that trigger responses, which inhibit protein translation. Therefore, the expression of target genes encoded in replicons is significantly impaired, thus limiting the therapeutic potential of RNA replicons. Advantageously, the RNA constructs of the present invention overcome this problem because they encode one or more innate inhibitory proteins, i.e., IIPs, which eliminate downstream inhibition of transgene expression. Interferon induction is a downstream consequence of innate recognition, but it should be understood that other molecules and pathways can be induced, any of which are inhibited by one or more IIPs contained in the RNA construct. The only previously published method for eliminating interferon responses with saRNA uses interferon inhibitory proteins derived from vaccinia virus E3, K3, and B18. However, in this study, these interferon inhibitory proteins were delivered and formulated as separate mRNA molecules in combination with saRNA. This requires the manufacture of both saRNA and mRNA, and must use 3-6 times the amount of vaccine mRNA in the replicon RNA construct according to the present invention to ensure co-delivery into the same cell and to provide any observable enhancement of protein expression. Furthermore, the expression kinetics of mRNA and saRNA differ, meaning that any beneficial effects of IIP expressed by mRNA will have a very short duration (up to 72 hours) compared to the RNA construct of the present invention (up to 60 days).

[0009] Advantageously, in the RNA construct of the first aspect, the presence of one or more IIPs enables dual protein expression with the target peptide or protein. Compared to the prior art, which delivers two different RNA strands encoding a target peptide / protein and a single strand encoding an IIP, the construct of the present invention delivers only a single strand to the target cell, thereby ensuring co-localization of the RNA and the innate inhibitor protein. The IIP inhibits innate sensing of the RNA, resulting in higher protein expression, and the IIP itself is amplified due to co-expression with the target gene (i.e., the therapeutic biomolecule) on a subgenomic strand. As described in the examples, it has been surprisingly shown that the RNA constructs encoding luciferase of the present invention (also known as “Stealthicons”) increase luciferase protein expression levels by up to two orders of magnitude in vitro in human cell lines, and in BL / 6 mice, also increase both the amount and duration of luciferase protein expression compared to conventional VEEV RNA replicons. Those skilled in the art will readily understand that the luciferase reporter is a true representative of therapeutic biomolecules, as it demonstrates that the RNA constructs are capable of expressing the genes contained on the RNA molecules of the present invention in vivo. Therefore, luciferase provides strong proof-of-concept evidence that the saRNA construct of the present invention can be used to express any biomolecule with therapeutic activity.

[0010] Those skilled in the art will understand that the RNA construct can also be referred to as a self-replicating RNA viral vector or an RNA replicon. The RNA construct can be double-stranded or single-stranded. Preferably, the RNA construct comprises self-amplifying RNA (saRNA), and more preferably is a saRNA construct.

[0011] Preferably, the RNA construct comprises or is derived from a positive-sense RNA virus selected from the group consisting of the following genera: alphavirus; microvirus; flavivirus; rubellavirus; plaguevirus; hepatitis virus; calicivirus; or coronavirus.

[0012] Suitable wild-type alphavirus genera are well-known. Representative examples of suitable alphavirus genera include Auravirus, Bibaruvirus, Kabasovirus, Chikungunyavirus, Eastern Equine Encephalitis Virus, FortMorganvirus, Gaitavivirus, Ziragachvirus, Mayaro, Mayaro Virus, Middleburg, Mukambvirus, Ndumouvirus, Pixuna virus, Ross River Virus, Semliki Forest Virus, Sindbisvirus, Thunatvirus, Trinitivirus, Una, Venezuelan Equine Encephalitis, Western Equine Encephalitis, Watarooa Virus, and Y-62-33.

[0013] Preferably, the RNA construct comprises or is derived from a virus selected from the group consisting of: Venezuelan equine encephalitis virus (VEEV); enterovirus 71; encephalomyocarditis virus; Cuting virus; and Middle East respiratory syndrome virus. Preferably, the vector is derived from VEEV.

[0014] The RNA construct contains a sequence encoding at least one therapeutic biomolecule. The at least one therapeutic biomolecule may comprise either a vaccine construct or a therapeutic protein. Those skilled in the art will understand that the therapeutic protein refers to any protein that preferably has therapeutic applications in humans. Exemplary therapeutic biomolecules that can be encoded by RNA molecules include proteins and peptides derived from pathogens such as bacteria, viruses, fungi, protozoa, and / or parasites. Preferably, the proteins and peptides are antigens.

[0015] Proteins and peptides derived from viruses can be viral antigens. These viral antigens can be derived from viruses selected from the group consisting of: orthomyxoviruses; paramyxoviridae viruses; metapneumoviruses and measles viruses; pneumoviruses; paramyxoviruses; poxviruses; metapneumoviruses; measles viruses; picornaviruses; enteroviruses; Bunyaviruses; sandfly viruses; Nairoviruses; hepatotropic RNA viruses; cloacal viruses; alphaviruses; arteritis viruses; flaviviruses; plague viruses; hepatotropic DNA viruses; rhabdoviruses; caliciviruses; coronaviruses; retroviruses; respiratory enteroviruses; parvoviruses; hepatitis D virus (HDV); hepatitis E virus (HEV); human herpesviruses and papillomaviruses.

[0016] Orthomyxoviruses can be influenza A, influenza B, and influenza C. Paramyxoviridae viruses can be pneumoviruses (RSV) and paramyxoviruses (PIV). Metapneumoviruses can be measles viruses (such as measles). Pneumoviruses can be respiratory syncytial virus (RSV), bovine respiratory syncytial virus, mouse pneumonia virus, or turkey rhinotracheitis virus. Paramyxoviruses can be parainfluenza viruses (PIV) types 1-4, mumps virus, Sendai virus, simian virus 5, bovine parainfluenza virus, Nipah virus, Henney virus, or Newcastle disease virus. Poxviridae can be smallpox viruses, such as large and smallpox. Metapneumoviruses can be human metapneumovirus (hMPV) or avian metapneumovirus (aMPV). Measles viruses can be measles. Picornaviridae viruses can be enteroviruses, rhinoviruses, hepatotropic RNA viruses, paraenteric orphanviruses, heart viruses, and foot-and-mouth disease viruses. Enteroviruses can be polioviruses type 1, 2, or 3; Coxsackieviruses 1 through 22 and 24; Coxsackieviruses 1 through 6; ECHO viruses 1 through 9, 11 through 27, and 29 through 34; or Enteroviruses 68 through 71. Bunyaviruses can be California encephalitis viruses. Sandfly viruses can be Rift Valley fever viruses. Nairoviruses can be Crimean-Congo hemorrhagic fever viruses. Hepatotropic RNA viruses can be hepatitis A viruses (HAV). Cloaked viruses can be rubella viruses. Flavivirosis viruses can be tick-borne encephalitis (TBE) viruses, dengue fever (types 1, 2, 3, or 4) viruses, yellow fever viruses, Japanese encephalitis viruses, Khosanur forest viruses, West Nile encephalitis viruses, St. Louis encephalitis viruses, Russian spring-summer encephalitis viruses, or Poissan encephalitis viruses. Plague viruses can be bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), or border disease virus (BDV). Hepatotropic DNA viruses can be hepatitis B virus or hepatitis C virus. Rhabdoviruses can be rabies virus (Rabies virus) or vesicular virus (VSV). Caliciviridae can be norovirus, or norovirus-like viruses, such as Hawaiian virus and snow mountain virus. Coronaviruses can be SARS CoV-1, SARS-CoV-2, MERS, human respiratory coronavirus, avian infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), or porcine transmissible gastroenteritis virus (TGEV). Retroviruses can be tumor viruses, lentiviruses, or foam viruses. Respiratory enteroviruses can be orthoreoviruses, rotaviruses, circoviruses, or Koroviruses. Parvoviruses can be parvovirus B19. Human herpesviruses can be herpes simplex virus (HSV), varicella-zoster virus (VZV), Ebola virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), or human herpesvirus 8 (HHV8). Papillomaviruses can also be papillomavirus, polyomavirus, adenovirus, or arenavirus.

[0017] As in the examples andFigure 13 As shown, in a preferred embodiment, the viral antigen may be a rabies virus antigen, preferably a rabies virus glycoprotein. In another preferred embodiment, as illustrated in the examples and... Figure 29 As shown, the viral antigen can be a coronavirus antigen. Preferably, the coronavirus antigen is a surface glycoprotein, more preferably the SARS-CoV-2 surface glycoprotein.

[0018] Proteins and peptides derived from bacteria can be bacterial antigens.

[0019] The bacterial antigen can be derived from bacteria selected from the group consisting of: Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis, Burkholderia spp. (e.g., Burkholderia melioides, Burkholderia cepacia, and Burkholderia cepacia), Staphylococcus aureus, and Haemophilus influenzae. Clostridium tetani (tetanus), Clostridium perfringens, Clostridium botulinum, Corynebacterium diphtheriae (diphtheria), Pseudomonas aeruginosa, Legionella pneumophila, Coxsella burgdorferi, Brucella (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella neotomae, Brucella ovis, Brucella suis, and Brucella pinnipediae), and J. Francisella. (e.g., *F. novicida*, *F. philomiragia*, *F. tularensis*), *Streptococcus agalactiae*, *Neisseria gonorrhoeae*, *Chlamydia trachomatis*, *Treponema pallidum* (syphilis), *Haemophilus ducreyi*, *Enterococcus faecalis*, *Enterococcus faecium*, *Helicobacter pylori*, *Staphylococcus saprophyticus*, *Yersinia enterocolitica*, *Escherichia coli*, *Bacillus anthracis* (anthrax), *Yersinia pestis* (plague), *Mycobacterium tuberculosis*, *Rickettsia*, *Listeria*, *Chlamydia pneumoniae*, *Vibrio cholerae*, *Salmonella typhi* (typhoid fever), *Treponema pallidum*, *Porphyromonas*, and *Klebsiella*.

[0020] Proteins and peptides derived from fungi can be fungal antigens.

[0021] The fungal antigen may be derived from fungal antigens of fungi selected from the group consisting of: dermatophytes, including: *Epidermophytes*, *Microsporum audouinii*, *Microsporum canis*, *Microsporum distortum*, *Microsporum equineii*, *Microsporum gypsum*, *Microsporum suis*, *Trichophyton concentricum*, *Trichophyton equineii*, *Trichophyton foetida*, *Trichophyton gypseum*, *Trichophyton mentagrophytes*, *Trichophyton quinckeanum*, *Trichophyton rubrum*, *Trichophyton schwannosum*, *Trichophyton tonsurans*, *Trichophyton verrucous*, *Trichophyton verrucous* var. *white*, *Trichophyton discoidum*, *Trichophyton ochre*, *Trichophyton violaceum*, and / or *Trichophyton faviforme*; or derived from *Aspergillus fumigatus*, *Aspergillus spp.* Aspergillus kavus, Aspergillus niger, Aspergillus terreus, Aspergillus sydowi, Aspergillus kavatus, Aspergillus glaucus, blastomonas, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parakwsei, Candida lucida, Candida pseudotropica, Candida guillezoniflora, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Cladosporium, Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi), Brachiolaspp., Microsporidia, Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp., Paracoccidioides brasiliensis, Pneumocystis carinii, Pythium cryptanatum, Malassezia ovalis, Saccharomyces boulardii, Saccharomyces cerevisiae, Sedodes scintillans, Sporothrix schenckii, Trichosporium bleutiferum, Toxoplasma gondii, Penicillium marneffei, Malassezia, Chromophycetes, Wangiellaspp.The genera *Sporothrix*, *Sporothrix*, *Auricularia*, *Rhizopus*, *Mucor*, *Pterocytotrichum*, *Morchella*, *Cunninghamella spp.*, *Pyrhodotorula*, *Alternaria*, *Curvularia spp.*, *Helicobacter*, *Fusarium*, *Aspergillus*, *Penicillium*, *Sclerotinia*, *Rhizoctonia*, *Penicillium*, *Pyrrosia*, and *Cladosporium*.

[0022] Proteins and peptides derived from protozoa can be protozoan antigens.

[0023] The protozoan antigen may be derived from protozoa selected from the group consisting of: Shigella, Giardia lamblia, Cryptosporidium, Cyclosporium caietatum, and Toxoplasma gondii.

[0024] The therapeutic biomolecules may be plant-derived proteins and peptides. Preferably, the proteins and peptides are plant antigens. The plant antigens may be derived from castor beans.

[0025] In another embodiment, the therapeutic biomolecule may be an immunogen or antigen. Preferably, the immunogen or antigen is a tumor immunogen or antigen or a cancer immunogen or antigen. The tumor immunogen and antigen may be peptide-containing tumor antigens, such as polypeptide tumor antigens or glycoprotein tumor antigens.

[0026] The tumor antigen may be (a) a full-length molecule associated with cancer cells, (b) its homologues and modified forms, including molecules with deleted, added and / or substituted parts, and (c) its fragments.

[0027] Suitable tumor immunogens include: class I restriction antigens recognized by CD8+ lymphocytes or class II restriction antigens recognized by CD4+ lymphocytes.

[0028] The tumor antigen may be a cancer-related antigen, and the cancer is selected from the group consisting of: testicular cancer, melanoma, lung cancer, head and neck cancer, NSCLC, breast cancer, gastrointestinal cancer, bladder cancer, colorectal cancer, pancreatic cancer, lymphoma, leukemia, kidney cancer, liver cancer, ovarian cancer, stomach cancer, and prostate cancer.

[0029] The tumor antigen can be selected from:

[0030] (a) Cancer-testis antigens such as NY-ESO-I, SSX2, SCP1 and RAGE, BAGE, GAGE ​​and MAGE family peptides, such as GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6 and MAGE-12 (e.g., which can be used to treat melanoma, lung tumors, head and neck tumors, NSCLC, breast tumors, gastrointestinal tumors and bladder tumors);

[0031] (b) Mutated antigens, such as p53 (associated with various solid tumors, such as colorectal cancer, lung cancer, and head and neck cancer), p21 / Ras (associated with, for example, melanoma, pancreatic cancer, and colorectal cancer), CDK4 (associated with, for example, melanoma), MUM1 (associated with, for example, melanoma), cysteine ​​aspartate protease-8 (associated with, for example, head and neck cancer), CIA0205 (associated with, for example, bladder cancer), HLA-A2-R1701, β-catenin (associated with, for example, melanoma), TCR (associated with, for example, T-cell non-Hodgkin's lymphoma), BCR-abl (associated with, for example, chronic myeloid leukemia), triose phosphate isomerase, KIA0205, CDC- 27 and LDLR-FUT;

[0032] (c) Overexpressed antigens, such as galactoglobulin 4 (associated with, for example, colorectal cancer), galactoglobulin 9 (associated with, for example, Hodgkin's disease), proteinase 3 (associated with, for example, chronic myeloid leukemia), WT1 (associated with, for example, various types of leukemia), carbonic anhydrase (associated with, for example, renal cell carcinoma), aldolase A (associated with, for example, lung cancer), PRAME (associated with, for example, melanoma), HER-2 / neu (associated with, for example, breast cancer, colon cancer, lung cancer, and ovarian cancer), alpha-fetoprotein (associated with, for example, liver cancer), KSA (associated with, for example, colorectal cancer), gastrin (associated with, for example, pancreatic cancer and gastric cancer), telomerase catalytic protein, MUC-I (associated with, for example, breast cancer and ovarian cancer), G-250 (associated with, for example, renal cell carcinoma), p53 (associated with, for example, breast cancer and colon cancer), and carcinoembryonic antigen (associated with, for example, breast cancer, lung cancer, and gastrointestinal cancers such as colorectal cancer).

[0033] (d) Shared antigens, such as melanoma-melanocyte differentiation antigens, such as MART-1 / Melan A, gplOO, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-associated protein-1 / TRPl and tyrosinase-associated protein-2 / TRP2 (associated with melanoma, for example).

[0034] (e) Prostate-associated antigens, such as PAP, PSA, PSMA, PSH-P1, PSM-P1, and PSM-P2, are associated with, for example, prostate cancer; and / or

[0035] (f) Individual immunoglobulin genotype (e.g., associated with myeloma and B-cell lymphoma).

[0036] The therapeutic biomolecule may be a eukaryotic polypeptide. In one embodiment, the eukaryotic polypeptide is a mammalian polypeptide. The mammalian polypeptide may be selected from the group consisting of: enzymes; enzyme inhibitors; hormones; immune system proteins; receptors; binding proteins; transcription or translation factors; tumor growth inhibitory proteins; structural proteins; and hemoglobins.

[0037] The enzyme may be selected from the group consisting of: rennet; gastric lipase; tissue plasminogen activator; streptokinase; cholesterol biosynthesis or steroid degradation enzymes; kinases; phosphodiesterases; methylases; demethylases; dehydrogenases; cellulases; proteases; lipases; phospholipases; aromatases; cytochromes; adenosine or guanylate cyclases and ceramides.

[0038] The enzyme inhibitor may be a tissue inhibitor of metalloproteinases (TIMP). The hormone may be growth hormone.

[0039] The immune system proteins can be selected from the group consisting of: cytokines; chemokines; lymphokines; erythropoietin; integrins; addressins; selectins; homing receptors; T cell receptors; and immunoglobulins.

[0040] The cytokines may be interleukins, such as IL-2, IL-4 and / or IL-6; colony-stimulating factor (CSF); granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor (TNF).

[0041] The chemokines may be macrophage inflammatory protein-2 and / or plasminogen activator.

[0042] The lymphokine may be interferon.

[0043] The immunoglobulin may be a natural, modified, or chimeric immunoglobulin or a fragment thereof. Preferably, the immunoglobulin is a chimeric immunoglobulin with dual activity, such as an antibody-enzyme or an antibody-toxin chimera.

[0044] The hormones may be selected from the group consisting of: insulin, thyroid hormones, catecholamines, gonadotropins, tropa hormones, prolactin, oxytocin, dopamine, bovine growth hormone, leptin, growth hormone (e.g., human growth hormone), and growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.).

[0045] The receptor may be a steroid hormone receptor or a peptide receptor. Preferably, the receptor is a growth factor receptor.

[0046] The binding protein may be a growth factor binding protein.

[0047] The tumor growth inhibitory protein may be a protein that inhibits angiogenesis.

[0048] The structural proteins may be selected from the group consisting of: collagen; fibroin; fibrinogen; elastin; tubulin; actin and myosin.

[0049] The blood proteins may be selected from the group consisting of: thrombin; serum albumin; factor VII; factor VIII; insulin; factor IX; factor X; tissue plasminogen activator; protein C; von Willebrand factor; antithrombin III; glucocerebrosidase; erythropoietin-granulocyte colony-stimulating factor (GCSF) or modified factor VIII; and anticoagulants.

[0050] In a preferred embodiment, the therapeutic biomolecule is a cytokine capable of regulating lymphatic homeostasis, preferably a cytokine involved in and more preferably inducing or enhancing the development, initiation, proliferation, differentiation, and / or survival of T cells. Therefore, preferably, the cytokine is an interleukin. Most preferably, IL-2, IL-7, IL-12, IL-15, or IL-21.

[0051] The therapeutic biomolecule may be a protein capable of enhancing the reprogramming of somatic cells into cells with stem cell characteristics.

[0052] Proteins that can enhance the reprogramming of somatic cells into cells with stem cell characteristics can be selected from the following group: OCT4, SOX2, NANOG, LIN28, p53, ART-4, BAGE, ss-catenin / m, Bcr-abL. CAMEL, CAP-1, CASP-8, CDC27 / m, CD4 / m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, GaplOO, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, MAGE-B, MAGE-C, MART-l / Melan-A, MC1R, Myosin / m, MUC1, MUM-1,-2,-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90minor BCR-abL, Plac-1, Pml / RARa, PRAME, protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE and WT, preferably WT-1.

[0053] Preferably, MAGE-A is selected from the group consisting of: MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 or MAGE-A12.

[0054] Preferably, the proteins that can enhance the reprogramming of somatic cells into stem cell-like structures are OCT4, SOX2, LF4, c-MYC, NANOG, and LIN28.

[0055] The therapeutic biomolecule can be a biomolecule used for in vitro modification of cells for cell therapy indications. Therefore, preferred therapeutic biomolecules may be selected from the group consisting of free immunoglobulins, T cell receptors, and NK receptors.

[0056] The therapeutic biomolecule may be an RNA molecule capable of regulating the expression of endogenous host genes, such as interfering RNA, like small RNA, siRNA, or microRNA.

[0057] Preferably, the RNA construct comprises a gene encoding at least one innate inhibitor protein (IIP) capable of reducing or blocking the innate immune response to RNA. Preferably, the reduction or blocking of the innate immune response to RNA is achieved by the IIP through reducing or blocking the recognition of RNA (preferably long RNA, which those skilled in the art will understand to mean RNA of at least 1 kb in length) or dsRNA produced by host cells containing the RNA construct of the present invention. More preferably, the innate inhibitor protein is an innate inhibitory protein that enables the reduction or blocking of the innate response to RNA, preferably the RNA construct of the first aspect. The innate inhibitor protein can reduce or prevent the recognition of cytosolic saRNA by pattern recognition receptors, which leads to the activation of interferon regulators 3 and 7 (IRF3 and IRF7) and NF-κB transcription factors, directly triggering the direct activation of a series of antiviral genes (e.g., IFIT1-3, MX1, MX2, known to inhibit saRNA expression), pro-inflammatory genes whose products coordinate the innate immune response, and any upstream classical IFN-stimulated genes (ISGs) in an interferon-dependent cascade. These pathways can be enhanced by inducing type I and type III interferons, which provide positive feedback loops that further amplify many antiviral responses.

[0058] The RNA can be single-stranded RNA or double-stranded RNA. Preferably, the RNA is saRNA.

[0059] The at least one innate inhibitor protein is capable of: (i) reducing or blocking the action of melanoma differentiation-associated protein 5 (MDA5), for example by preventing the oligomerization of MDA5 and the binding of MDA5 to RNA, and / or (ii) blocking or reducing the binding of PACT to RNA, also known as the binding of PKR activating proteins to RNA. Those skilled in the art will understand that these sensor proteins transmit signals to transduce signals to downstream mitochondrial conjugates, mitochondrial antiviral signaling (MAVS) activation of downstream cascades, including the activation of transcription factors (NF-κB, IRF-3, IRF-7). This, in turn, leads to appropriate antiviral signaling responses and activation of type I interferon-stimulated genes encoding molecules with antiviral activity, including IFIT1, which is known to inhibit saRNA expression.

[0060] At least one innate inhibitor protein that blocks the action of MDA5 may be selected from the group consisting of: paramyxovirus V protein, Coxsackievirus A16, Coxsackievirus A6 and enterovirus D68 3C protein, double RNA virus VP3 protein, porcine delta coronavirus accessory protein NS6; and encephalocarditis virus 2C protein; and their orthologs.

[0061] Preferably, the at least one innate inhibitor protein that blocks the action of MDA5 is a paramyxovirus V protein. Most preferably, the at least one innate inhibitor protein that blocks the action of MDA5 is parainfluenza virus type 5 V protein (PIV5V).

[0062] The at least one innate inhibitor protein that blocks or reduces the binding of PACT to RNA may be selected from the group consisting of: ORF4a (NS4a) of any coronavirus, ORF3b of any coronavirus, or nucleocapsid protein of mouse hepatitis virus and SARS (coronavirus); and their orthologs.

[0063] Preferably, ORF4a (NS4a) is Middle East Respiratory Syndrome Coronavirus (MERS Coronavirus) (ORF4a).

[0064] Preferably, the coronavirus ORF3b is SARS-CoV2ORF3b.

[0065] The following provides the protein, DNA, and RNA sequences for each of PIV5V, ORF4a, and ORF3b.

[0066] In one embodiment, the present invention provides the PIV5V polypeptide of SEQ ID NO: 11, as follows:

[0067]

[0068] Accordingly, preferably, the PIV5V polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 11 or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 11 or a biologically active variant or fragment thereof.

[0069] In one embodiment, the PIV5V polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 12, as follows:

[0070]

[0071] Accordingly, preferably, the PIV5V polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 12 or a variant or fragment thereof.

[0072] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 47, as follows:

[0073]

[0074] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 47 or a variant or fragment thereof.

[0075] In one embodiment, this document provides the MERS-CoV ORF4a peptide as SEQ ID NO: 15, as follows:

[0076]

[0077] Accordingly, preferably, the MERS-COVORF4a polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 15 or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 15 or a variant or fragment thereof.

[0078] In one embodiment, the MERS-COVORF4a polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 16, as follows:

[0079]

[0080] Accordingly, preferably, the MERS-CoVORF4a polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 16 or a variant or fragment thereof.

[0081] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 48, as follows:

[0082]

[0083] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 48 or a variant or fragment thereof.

[0084] In one embodiment, this document provides the SARS-CoV-2ORF3b peptide as SEQ ID NO: 20, as follows:

[0085]

[0086] Accordingly, preferably, the SARS-CoV-2ORF3b polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 20 or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 20 or a variant or fragment thereof.

[0087] In one implementation, the SARS-CoV-2ORF3b polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 55 (accession number NC_045512.2; nucleotides 25814-26050), as follows:

[0088]

[0089] Accordingly, preferably, the SARS-CoV-2ORF3b polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 55 or a variant or fragment thereof.

[0090] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 56, as follows:

[0091]

[0092] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence or a variant or fragment thereof substantially as shown in SEQ ID NO: 56.

[0093] In another embodiment, the at least one innate inhibitor protein may be able to inhibit downstream pathways activated by MDA5 or block downstream pathways recognized by dsRNA via MDA / PACT.

[0094] The at least one innate inhibitor protein capable of inhibiting downstream pathways activated by MDA5 or blocking downstream pathways recognized by dsRNA via MDA / PACT can be selected from the group consisting of: HSV-2Us1; HSV-1Us1; HSV-1Us11; OV20.0L; BVDV Npro; Langat virus NS5; and influenza NS1.

[0095] Those skilled in the art will understand that the at least one innate inhibitor protein capable of inhibiting downstream pathways activated by MDA5 or blocking downstream pathways of MDA / PACT recognition of dsRNA can be used in combination with the at least one innate inhibitor protein that blocks the action of MDA5 and / or the at least one innate inhibitor protein that blocks or reduces the binding of PACT to RNA.

[0096] In one embodiment, this document provides the HSV-2Us1 polypeptide represented by SEQ ID NO: 1, as follows:

[0097]

[0098] Accordingly, preferably, the HSV-2Us1 polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 1, or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 1, or a biologically active variant or fragment thereof.

[0099] In one embodiment, the HSV-2Us1 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 2, as follows:

[0100]

[0101] Accordingly, preferably, the HSV-2Us1 polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 2 or a variant or fragment thereof.

[0102] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 42, as follows:

[0103]

[0104] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 42 or a biologically active variant or fragment thereof.

[0105] In one embodiment, this document provides the HSV-1Us1 polypeptide represented by SEQ ID NO: 3, as follows:

[0106]

[0107] Accordingly, preferably, the HSV-1Us1 polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 3 or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 3 or a biologically active variant or fragment thereof.

[0108] In one embodiment, the HSV-1Us1 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 4, as follows:

[0109]

[0110] Accordingly, preferably, the HSV-1Us1 polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 4 or a variant or fragment thereof.

[0111] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 43, as follows:

[0112]

[0113] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 43 or a variant or fragment thereof.

[0114] In one embodiment, this document provides the HSV-1Us11 polypeptide represented by SEQ ID NO: 5, as follows:

[0115]

[0116] Accordingly, preferably, the HSV-1Us11 polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 5 or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 5 or a variant or fragment thereof.

[0117] In one embodiment, the HSV-1Us11 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 6, as follows:

[0118]

[0119] Accordingly, preferably, the HSV-1Us11 polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 6 or a variant or fragment thereof.

[0120] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 44, as follows:

[0121]

[0122] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence or a variant or fragment thereof that is substantially as shown in SEQ ID NO: 44.

[0123] In one embodiment, the present invention provides the OV20.0L polypeptide as SEQ ID NO: 7, as follows:

[0124]

[0125] Accordingly, preferably, the OV20.0L polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 7 or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 7 or a variant or fragment thereof.

[0126] In one embodiment, the OV20.0L polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 8, as follows:

[0127]

[0128] Accordingly, preferably, the OV20.0L polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 8 or a variant or fragment thereof.

[0129] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 45, as follows:

[0130]

[0131] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 45 or a variant or fragment thereof.

[0132] In one embodiment, this document provides the BVDV Npro polypeptide represented by SEQ ID NO: 9, as follows:

[0133]

[0134] Accordingly, preferably, the BVD VNpro polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 9 or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 9 or a variant or fragment thereof.

[0135] In one embodiment, the BVDV Npro polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 10, as follows:

[0136]

[0137] Accordingly, preferably, the BVDV Npro polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 10 or a variant or fragment thereof.

[0138] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 46, as follows:

[0139]

[0140] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 46 or a variant or fragment thereof.

[0141] In one embodiment, the Langate NS5 peptide of SEQ ID NO: 17 is provided herein as follows:

[0142]

[0143] Accordingly, preferably, the Langat NS5 polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 17 or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 17 or a variant or fragment thereof.

[0144] In one embodiment, the Langat NS5 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 18, as follows:

[0145]

[0146] Accordingly, preferably, the Langat virus NS5 polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 18 or a variant or fragment thereof.

[0147] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 49, as follows:

[0148]

[0149] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 49 or a variant or fragment thereof.

[0150] In one embodiment, the influenza NS1 polypeptide (accession number DQ508893) of SEQ ID NO: 13 is provided herein as follows:

[0151]

[0152] Therefore, preferably, the influenza NS1 polypeptide comprises an amino acid sequence substantially as shown in SEQ ID NO: 13 or a biologically active variant or fragment thereof. Therefore, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 13 or a variant or fragment thereof.

[0153] In one embodiment, the influenza NS1 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 14, as follows:

[0154]

[0155] Accordingly, preferably, the influenza NS1 polypeptide is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 14 or a variant or fragment thereof.

[0156] Therefore, the RNA construct may contain the RNA nucleotide sequence of SEQ ID NO: 19, as follows:

[0157]

[0158] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 19 or a variant or fragment thereof.

[0159] Preferably, the innate inhibitor protein is selected from the group consisting of: HSV-2Us1; HSV-1Us11; OV20.0L; BVDV Npro; PIV5V; MERS-CoV ORF4a; SARS-CoV-2ORF3b; Langat virus NS5 and influenza NS1.

[0160] Preferably, the innate inhibitor protein is selected from the group consisting of: HSV-2Us1; HSV-1Us11; OV20.0L; BVDV Npro; PIV5V; MERS-CoV ORF4a; and Langat virus NS5.

[0161] Preferably, the innate inhibitor protein is selected from the group consisting of: HSV-2Us1; HSV-1Us11; OV20.0L; BVDV Npro; PIV5V; and MERS-CoV ORF4a.

[0162] As described in the examples, through a process of screening a wide variety of IIPs, the inventors identified two highly effective IIPs that surprisingly significantly enhanced saRNA expression both in vitro and in vivo: an inhibitor of parainfluenza virus type 5 V protein (PIV-5)-MDA5 activation and an inhibitor of Middle East respiratory syndrome (MERS) coronavirus ORF4a-PACT / MDA activation. The inventors used these two novel IIPs, PIV-5 and MERS-CoV ORF4a, which had not previously been used to attenuate innate sensing of replicon RNA. The inventors have demonstrated that these constructs can serve as next-generation RNA replicons for enhancing the potency of RNA vaccines and therapies in humans. The inventors believe that they will have significant practical applications regardless of expression in replicons of alphaviruses, picornaviruses, flaviviruses, or coronaviruses.

[0163] Therefore, most preferably, the innate inhibitor protein is PIV-5 and / or MERS-CoV ORF4a, which those skilled in the art will understand can also be referred to as NS4a.

[0164] Such constructs exhibit several advantages over constructs described in the prior art, including:

[0165] i) PIV-5 and ORF4a proteins are directly inserted into the VEEV replicon, thereby achieving dual protein expression of VPII protein and target gene.

[0166] ii) Instead of providing one RNA strand encoding the target gene (GOI) (i.e., the therapeutic biomolecule) and another encoding the IIP, one strand is delivered to ensure co-localization of the RNA and the innate inhibitor protein;

[0167] iii) IIP inhibits innate RNA sensing, thereby achieving higher protein expression;

[0168] iv) IIP expression itself is self-amplified due to co-expression with GOI on the subgenomic strand; and / or

[0169] v) Compared to the traditional VEEV RNA replicon construct, it improves both protein expression levels and duration.

[0170] The sequence encoding the at least one innate inhibitor protein can be placed anywhere within the RNA construct or replicon sequence, for example, the sequence encoding the at least one target peptide or protein can be placed at the 5' or 3' position of the sequence encoding the at least one innate inhibitor protein.

[0171] However, preferably, the sequence encoding the at least one target peptide or protein is positioned at the 5' position of the sequence encoding the at least one innate inhibitor protein.

[0172] Preferably, the RNA construct according to the first aspect contains at least one genomic or subgenomic promoter. Preferably, the promoter is a subgenomic promoter.

[0173] Those skilled in the art will understand that a subgenomic promoter is a promoter operatively linked to a sequence encoding at least one therapeutic biomolecule and at least one innate inhibitor protein, thereby enabling transcription of the nucleotide sequence encoding the therapeutic biomolecule and at least one innate inhibitor protein.

[0174] Preferably, the subgenomic promoter is 26S, which is provided herein as SEQ ID NO: 57, as follows:

[0175]

[0176] Therefore, the preferred promoter (preferably a subgenomic promoter) is essentially as shown in SEQ ID NO: 57 or a variant or fragment thereof.

[0177] In one embodiment, the same promoter is operatively linked to a sequence encoding the at least one target peptide or protein and a sequence encoding the at least one innate inhibitor.

[0178] The inventors’ design (in which both GOI (i.e., therapeutic biomolecule) and IIP are encoded on one strand) advantageously enables the use of smaller doses of RNA because it ensures expression in the same cell that senses the RNA and can also replicate the protein, thus having the additional aspect of amplifying the innate repressive component.

[0179] Therefore, in one embodiment, a promoter is positioned at the 5' of the sequence encoding the target peptide or protein (i.e., the therapeutic biomolecule) and the sequence encoding the at least one innate inhibitor protein, such that the promoter is operatively linked to these two sequences.

[0180] In another embodiment, a first promoter is operatively linked to a sequence encoding at least one target peptide or protein (i.e., a therapeutic biomolecule), and a second promoter is operatively linked to a sequence encoding at least one innate inhibitor protein.

[0181] The RNA construct may encode at least two, three, four, or five IIPs. In embodiments where there is more than one sequence encoding an innate repressor protein, a single promoter may be operatively linked to all sequences encoding the innate repressor protein. Alternatively, the promoter may be linked to each of at least one other sequence encoding an innate repressor protein, such that each innate repressor protein is operatively linked to an independent promoter. In this embodiment, the independent promoters may contain the same promoter sequence or different promoter sequences. In another embodiment, different promoters are operatively linked to each sequence encoding an innate repressor protein.

[0182] The RNA construct may further include a linker positioned between a sequence encoding the at least one target peptide or protein (i.e., a therapeutic biomolecule) and a sequence encoding the at least one innate inhibitor protein.

[0183] In one embodiment, the linker sequence comprises a sequence encoding a peptide spacer configured to be digested to thereby separate the at least one therapeutic biomolecule encoded by the target gene and the at least one innate inhibitor protein. Therefore, preferably, the spacer sequence is positioned between the sequence encoding the at least one target peptide or protein and the sequence encoding the at least one innate inhibitor protein.

[0184] Therefore, the spacer sequence is preferably a cleavable peptide, such as a 2A peptide. Suitable 2A peptides include porcine swine cisvirus-12A (P2A)-ATNFSLLKQAGDVEENPGP (SEQ ID NO: 21), Thosea asigna virus 2A (T2A)-QCTNYALLKLAGDVESNPGP (SEQ ID NO: 22), equine rhinitis virus A 2A (E2A), and foot-and-mouth disease virus 2A (F2A)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). Preferably, the 2A peptide is Thosea asigna virus 2A (T2A).

[0185] In another embodiment, the cleavable peptide is a self-cleaving peptide. Preferably, the self-cleaving peptide is a furin / 2A peptide. The furin sequence can be positioned at the 3' or 5' of the 2A sequence. However, it is preferred that the furin sequence be positioned at the 5' of the 2A sequence, and preferably that a GSG spacer is provided between the furin and the 2A sequences.

[0186] Those skilled in the art will understand that furin is a ubiquitous calcium-dependent protoprotein convertase located in the secretory pathway (primarily in the Golgi apparatus and trans-Golgi network), cleaving the precursor protein at a specific recognition sequence—the standard RXR / K / XR (SEQ ID NO: 24)—and cleaving the protoprotein after the final R. Therefore, in one embodiment, the furin sequence is RXR / K / XR. However, preferably, the furin sequence is the optimized sequence RRRRRR (SEQ ID NO: 25)-GSG sequence. Preferably, the GSG spacer is positioned at the 3' of the furin sequence and the 5' of the 2A sequence.

[0187] Therefore, preferably, the spacer sequence is furin / T2A, as provided in NCBI reference sequence GenBank: AAC97195.1, and is provided herein as the spacer sequence SEQ ID NO: 26, as follows:

[0188]

[0189] Therefore, preferably, the spacer sequence comprises an amino acid sequence substantially as shown in SEQ ID NO: 26, or a variant or fragment thereof.

[0190] In embodiments in which the RNA construct or replicon contains more than one sequence encoding an innate inhibitor protein, the replicon may have a linker sequence positioned between each sequence encoding an innate inhibitor protein or positioned between only some IIPs.

[0191] In one embodiment, the sequence encoding the at least one therapeutic biomolecule and the at least one innate inhibitor protein may be separated by a stop codon following an internal ribosome entry site (IRES) sequence capable of initiating translation of a downstream sequence. Typical IRES sequences include those of encephalomyocarditis virus or vascular endothelial growth factor and type I collagen-inducing protein (VCIP), and are known to those skilled in the art. Therefore, preferably, the IRES sequence is positioned between the sequence encoding the at least one target peptide or protein and the sequence encoding the at least one innate inhibitor protein. When using multiple sequences encoding the at least one innate inhibitor protein, the spacer sequence may include a combination of known cleavage sequences and / or IRES sequences.

[0192] In another embodiment, the sequence encoding the at least one therapeutic biomolecule and the at least one innate inhibitor protein may be separated by a stop codon of a second subgenomic promoter sequence capable of initiating transcription of downstream sequences.

[0193] The RNA construct may encode at least one non-structural protein (NSP), wherein the at least one NSP is located at the 5' or 3' position of the sequence encoding the at least one target peptide or protein and the at least one innate inhibitor protein. Preferably, the sequence encoding the at least one NSP is located at the 5' position of the sequence encoding the target peptide or protein and the at least one innate inhibitor protein. Therefore, preferably, the sequence encoding the at least one NSP is located at the 5' end of the RNA construct.

[0194] At least one non-structural protein encoded by the RNA construct may be the RNA polymerase nsP4. Preferably, the construct encodes nsP1, nsP2, nsP3, and nsP4. Those skilled in the art will understand that nsP1 is a viral capping enzyme and a membrane anchoring site for the replicon complex (RC), nsP2 is an RNA helicase and a protease responsible for processing ns polyproteins, nsP3 interacts with several host proteins and can regulate poly- and mono-ADP-ribosylation of proteins, and nsP4 is a core viral RNA-dependent RNA polymerase.

[0195] In one implementation, nsP1 for SEQ ID NO: 27 is provided below:

[0196]

[0197] Accordingly, nsP1 preferably comprises an amino acid sequence substantially as shown in SEQ ID NO: 27 or a biologically active variant or fragment thereof.

[0198] In one implementation, nsP1 is encoded by the nucleotide sequence defined in SEQ ID NO: 28, as follows:

[0199]

[0200] Accordingly, nsP1 is preferably encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 28 or a variant or fragment thereof.

[0201] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 50 or a variant or fragment thereof.

[0202]

[0203] In one implementation, nsP2, designated as SEQ ID NO: 29, is provided herein as follows:

[0204]

[0205] Accordingly, nsP2 preferably comprises an amino acid sequence substantially as shown in SEQ ID NO: 29 or a biologically active variant or fragment thereof.

[0206] In one implementation, nsP2 is encoded by the nucleotide sequence defined in SEQ ID NO: 30, as follows:

[0207]

[0208] Accordingly, preferably, nsP2 is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 30 or a variant or fragment thereof.

[0209] Therefore, the RNA construct may contain SEQ ID NO: 51, as follows:

[0210]

[0211] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 51 or a variant or fragment thereof.

[0212] In one implementation, nsP3, designated SEQ ID NO: 31, is provided herein as follows:

[0213]

[0214] Accordingly, nsP3 preferably comprises an amino acid sequence substantially as shown in SEQ ID NO: 31 or a biologically active variant or fragment thereof.

[0215] In one implementation, nsP3 is encoded by the nucleotide sequence defined in SEQ ID NO: 32, as follows:

[0216]

[0217] Accordingly, preferably, nsP3 is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 32 or a variant or fragment thereof.

[0218] Therefore, the RNA construct may contain SEQ ID NO: 52, as follows:

[0219]

[0220] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 52, or a variant or fragment thereof. In one embodiment, nsP4 as SEQ ID NO: 33 is provided herein as follows:

[0221]

[0222] Accordingly, nsP4 preferably comprises an amino acid sequence substantially as shown in SEQ ID NO: 33 or a biologically active variant or fragment thereof.

[0223] In one implementation, nsP4 is encoded by the nucleotide sequence defined in SEQ ID NO: 34, as follows:

[0224]

[0225] Accordingly, preferably, nsP4 is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 34 or a variant or fragment thereof.

[0226] Therefore, the RNA construct may contain SEQ ID NO: 53, as follows:

[0227]

[0228] Accordingly, preferably, the RNA construct comprises an RNA nucleotide sequence substantially as shown in SEQ ID NO: 53 or a variant or fragment thereof.

[0229] Preferably, together with proteins present in the host cell, a non-structural protein encoded by the RNA construct of the present invention forms an enzyme complex, said enzyme complex being essential for genome replication and transcription of sequences encoding the at least one target peptide or protein and the at least one innate inhibitor protein. For example, the one or more non-structural proteins may encode a polymerase to enable the construct to amplify the nucleotide sequences encoding the at least one target peptide or protein and the at least one innate inhibitor protein.

[0230] The host cell can be a eukaryotic or prokaryotic host cell. Preferably, the host cell is a eukaryotic host cell. More preferably, the host cell is a mammalian host cell.

[0231] The RNA construct may further include a promoter located at the 5' of at least one non-structural protein, such that the promoter is operatively linked to the sequence encoding the at least one non-structural protein, and that the at least one non-structural protein can be expressed in the host cell.

[0232] Preferably, the promoter comprises a 5'UTR conserved sequence element, which may be referred to herein as SEQ ID NO: 54, as follows:

[0233]

[0234] Accordingly, preferably, the UTR is located at the 5' of the at least one non-structural protein and comprises a nucleotide sequence substantially as shown in SEQ ID NO: 54 or a fragment or variant thereof.

[0235] Preferably, the replicon includes a poly A tail. More preferably, the poly A tail is located at the 3' end of the replicon. The replicon may further include a 5' cap. In the context of this invention, the term "5'-cap" includes 5'-cap analogues that resemble RNA cap structures and are modified to, preferably in vivo and / or in cells, have the ability to stabilize RNA and / or enhance RNA translation if attached to RNA.

[0236] RNA with a 5'-cap can be obtained by in vitro transcription of a DNA template in the presence of the 5'-cap, wherein the 5'-cap is co-transcribed into the resulting RNA strand. Alternatively, the RNA can be generated, for example, by in vitro transcription, and the 5'-cap can be ligated to the transcribed RNA using a capping enzyme (e.g., a capping enzyme from vaccinia virus). In the capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked to the 5' position of the subsequent base ("second base") of the RNA molecule via a phosphodiester bond.

[0237] In one embodiment, the RNA construct preferably comprises, from 5' to 3', a promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, and at least one sequence encoding an innate inhibitor protein.

[0238] In another embodiment, the RNA construct preferably comprises, from 5' to 3', a promoter, a sequence encoding at least one non-structural protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, and at least one sequence encoding an innate inhibitor protein.

[0239] In yet another embodiment, the RNA construct preferably comprises, from 5' to 3', a promoter, a sequence encoding at least one non-structural protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, a sequence encoding at least one innate inhibitor protein, and a polyA tail.

[0240] In yet another embodiment, the RNA construct preferably comprises, from 5' to 3', a promoter, a sequence encoding at least one non-structural protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, a sequence encoding at least one innate inhibitor protein, optionally a spacer sequence between each of the at least one innate inhibitor protein sequences, and a polyA tail.

[0241] In yet another embodiment, the RNA construct preferably comprises, from 5' to 3', a 5' cap, a promoter, a sequence encoding at least one non-structural protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, a sequence encoding at least one innate inhibitor protein, optionally a spacer sequence between each of the at least one innate inhibitor protein sequences, and a polyA tail.

[0242] In yet another embodiment, the RNA construct preferably comprises, from 5' to 3', a 5' cap, a promoter, a sequence encoding at least one non-structural protein, a spacer sequence, a sequence encoding at least one innate inhibitor protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, and a polyA tail.

[0243] In yet another embodiment, the RNA construct preferably comprises, from 5' to 3', a 5' cap, a promoter, a sequence encoding at least one non-structural protein, a spacer sequence, a sequence encoding at least one innate inhibitor protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, a sequence encoding at least one innate inhibitor protein, optionally a spacer sequence between each sequence encoding at least one innate inhibitor protein, and a polyA tail.

[0244] It is believed that the V protein of PIV5 directly binds to MDA5 to prevent oligomerization, while ORF4a is believed to block the binding of PACT to dsRNA. The inventors incorporated these protein-coding genes into saRNA, separated by a T2A cleavage site after the target gene (GOI) (i.e., the therapeutic biomolecule), to generate a construct that advantageously bypasses innate sensing mechanisms. GOI and IIP are paired into a single open reading frame that is cleaved by endogenous protease (T2A) upon expression, maximizing their expression in the same cell at a predetermined ratio and with identical kinetics.

[0245] Therefore, preferably, the RNA construct, from 5' to 3', comprises a 5' cap, a promoter containing 51 conserved nucleotide sequence elements, nsP1, nsP2, nsP3v, nsP4, a subgenomic promoter 26S, a sequence encoding a therapeutic biomolecule, a T2A spacer sequence, a sequence encoding PIV5V and / or MERS-CoV ORF4a, and a polyA tail.

[0246] Therefore, in one embodiment, the RNA construct may comprise or consist of SEQ ID NO: 38, as follows:

[0247]

[0248]

[0249]

[0250]

[0251] Accordingly, preferably, the RNA construct comprises a nucleotide sequence substantially as shown in SEQ ID NO: 38 or a fragment or variant thereof.

[0252] Therefore, in another embodiment, the RNA construct may comprise or consist of SEQ ID NO: 39, as follows:

[0253]

[0254]

[0255]

[0256]

[0257] Therefore, preferably, the RNA construct comprises a nucleotide sequence substantially as shown in SEQ ID NO: 39 or a fragment or variant thereof.

[0258] In a second aspect of the invention, a nucleic acid sequence encoding the RNA construct of the first aspect is provided.

[0259] In one embodiment, the nucleic acid sequence may contain or consist of SEQ ID NO: 40, as follows:

[0260]

[0261]

[0262]

[0263]

[0264] Accordingly, preferably, the nucleic acid sequence comprises a nucleotide sequence substantially as shown in SEQ ID NO: 40 or a fragment or variant thereof.

[0265] In one embodiment, the nucleic acid sequence may contain or consist of SEQ ID NO: 41, as follows:

[0266]

[0267]

[0268]

[0269]

[0270] Accordingly, preferably, the nucleic acid sequence comprises a nucleotide sequence substantially as shown in SEQ ID NO: 41 or a fragment or variant thereof.

[0271] In the third aspect, an expression cassette containing a nucleic acid sequence according to the second aspect is provided.

[0272] The nucleic acid sequence of the present invention is preferably contained in a recombinant vector, for example, a recombinant vector for delivery into a target host cell to produce an RNA construct.

[0273] Therefore, in the fourth aspect, a recombinant vector containing an expression box according to the third aspect is provided.

[0274] In one embodiment, the vector may include the nucleic acid sequence of SEQ ID NO: 35, as follows, wherein “GOI” represents the position of the coding sequence for a therapeutic biomolecule:

[0275]

[0276]

[0277]

[0278] Accordingly, preferably, the vector comprises a nucleotide sequence substantially as shown in SEQ ID NO: 35 or a variant or fragment thereof.

[0279] In one embodiment, the vector comprises a nucleic acid sequence encoding an RNA construct containing MERS-CoV ORF4a, and the vector may comprise the nucleic acid sequence of SEQ ID NO: 36, as follows, where “GOI” represents the position of the therapeutic biomolecule coding sequence:

[0280]

[0281]

[0282]

[0283] Accordingly, preferably, the vector comprises a nucleotide sequence substantially as described in SEQ ID NO: 36 or a variant or fragment thereof.

[0284] In one embodiment, the vector comprises a nucleic acid sequence encoding an RNA construct containing PIV5, and the vector may include the nucleic acid sequence of SEQ ID NO: 37, as follows, wherein “GOI” represents the position of the sequence encoding a therapeutic biomolecule:

[0285]

[0286]

[0287]

[0288]

[0289] Accordingly, preferably, the vector comprises a nucleotide sequence substantially as shown in SEQ ID NO: 37 or a variant or fragment thereof.

[0290] Usable Figure 7 Alternatively, the DNA plasmid shown in Figure 8 can be used as a template to create the saRNA construct of this invention. RNA copies can then be produced by in vitro transcription using a polymerase (such as T7 polymerase), with the T7 promoter shown... Figure 7 Upstream of the saRNA in plasmid diagram 8. Therefore, it can be used as follows: Figure 7The saRNA construct of the first aspect can be prepared using a DNA plasmid having any of the nucleic acid sequences or variants or fragments shown in SEQ ID NO: 35-37 as a template. It is understood, of course, that other RNA polymerases, such as SP6 or T3 polymerases, can be used instead of T7 polymerase, in which case the saRNA construct can replace the SP6 or T3 promoter.

[0291] The vector encoding the fourth aspect of the RNA construct of the first aspect can be, for example, a plasmid, granule, or bacteriophage and / or viral vector. Such recombinant vectors are very useful in the delivery system of the present invention for transforming cells with a nucleotide sequence. The nucleotide sequence is preferably a DNA sequence that encodes the RNA sequence forming the RNA construct of the first aspect.

[0292] Recombinant vectors encoding RNA constructs of the first aspect may also contain other functional elements. For example, they may further contain a variety of other functional elements, including suitable promoters for initiating transgenic expression after introduction of the vector into a host cell. For example, the vector is preferably capable of autonomous replication in the nucleus of a host cell (e.g., a bacterial cell). In this case, elements that induce or regulate DNA replication may be needed in the recombinant vector. Alternatively, the recombinant vector may be designed to integrate into the genome of the host cell. In this case, DNA sequences that facilitate directed integration (e.g., through homologous recombination) are envisioned. Suitable promoters may include the SV40 promoter, CMV, EF1a, PGK, viral long terminal repeats, and inducible promoters, such as tetracycline-inducible systems. Expression cassettes or vectors may also include terminators, such as β-globulin, SV40 polyadenylated sequences, or synthetic polyadenylated sequences. Recombinant vectors may also include promoters, regulators, or enhancers to control nucleic acid expression as needed.

[0293] Vectors can also contain DNA encoding genes that can be used as selectable markers during cloning, i.e., to enable selection of already transfected or transformed cells, and to enable selection of cells containing vectors with integrated heterologous DNA. For example, consider ampicillin, neomycin, puromycin, or chloramphenicol resistance. Figure 7 and Figure 8 The vector shown contains an ampicillin resistance marker, which is useful for selecting plasmids in bacteria. Additionally, optional marker genes can be used in different vectors alongside the transgenic vector. Expression cassettes or vectors may also contain DNA involved in regulating nucleotide sequence expression or for targeting expressed polypeptides to a specific part of the host cell.

[0294] The purified vector can be directly inserted into host cells via appropriate methods, such as direct endocytosis. The vector can also be introduced directly into host cells (e.g., eukaryotic or prokaryotic cells) through transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, or ballistic bombardment. Alternatively, the vector of this invention can be directly introduced into host cells using a particle gun.

[0295] Nucleic acid molecules can (but are not necessarily) be a type of nucleic acid molecule that is integrated into the DNA of a host cell. Undifferentiated cells can be stably transformed, leading to the production of genetically modified daughter cells (in which case, regulation of expression in the subject may be necessary, such as with specific transcription factors or gene activators). Alternatively, delivery systems can be engineered to favor unstable or transient transformation of differentiating cells. In this case, regulating expression may be less important because DNA expression will cease when the transformed cells die or stop expressing proteins.

[0296] Alternatively, delivery systems can deliver nucleic acid molecules to host cells without integrating them into a vector. For example, nucleic acid molecules can be integrated into liposomes or viral particles. Furthermore, "naked" nucleic acid molecules can be inserted into host cells via appropriate methods, such as direct endocytosis.

[0297] In a fifth aspect, a pharmaceutical composition is provided, comprising an RNA construct of the first aspect, a nucleic acid sequence of the second aspect, an expression cassette of the third aspect or a vector of the fourth aspect, and a pharmaceutically acceptable excipient.

[0298] In a sixth aspect, a method for manufacturing a pharmaceutical composition according to the fifth aspect is provided, the method comprising contacting an RNA construct of the first aspect, a nucleic acid sequence of the second aspect, an expression cassette of the third aspect, or a vector of the fourth aspect with a pharmaceutically acceptable excipient.

[0299] In a seventh aspect, a method for preparing the RNA construct of the first aspect is provided, the method comprising:

[0300] a)i) Introducing the fourth aspect's vector into the host cell; and

[0301] ii) Culture host cells under conditions that lead to the production of the RNA construct of the first aspect; or

[0302] b) Transcription originates from RNA constructs based on the vector in aspect four.

[0303] The host cell in step a) can be a eukaryotic or prokaryotic host cell. Preferably, the host cell is a eukaryotic host cell. More preferably, the host cell is a mammalian host cell, such as human embryonic kidney 293 cells or Chinese hamster ovary (CHO) cells. Step (b) can be performed in vitro or in vivo, preferably in vitro.

[0304] Suitable in vitro transcription methods are well known in the art and are also known to those skilled in the art. For example, as described in Molecular Cloning, Laboratory Manual, Second Edition (1989), edited by C. Nolan, Cold Spring Harbor Laboratory Press.

[0305] The first type of RNA replicon is particularly suitable for therapeutic use.

[0306] While the inventors envision generating an RNA construct for in vivo treatment via in vitro transcription, those skilled in the art will recognize that an RNA construct for treatment can also be generated in vivo in a subject by delivering a nucleic acid according to the second aspect, an expression cassette according to the third aspect, and a vector according to the fourth aspect into the subject.

[0307] Therefore, according to the eighth aspect, RNA constructs according to the first aspect, nucleic acids according to the second aspect, expression cassettes according to the third aspect, vectors according to the fourth aspect, or pharmaceutical compositions according to the fifth aspect are provided for use as drugs or for treatment.

[0308] In a ninth aspect of the invention, an RNA construct according to the first aspect, a nucleic acid according to the second aspect, an expression cassette according to the third aspect, a vector according to the fourth aspect, or a pharmaceutical composition according to the fifth aspect are provided for use in the prevention, improvement, or treatment of protozoan, fungal, bacterial, or viral infections.

[0309] An infection of a protozoan, fungus, bacterium, or virus may be an infection of a protozoan, fungus, bacterium, or virus as defined in the first aspect.

[0310] In a tenth aspect of the invention, an RNA construct according to the first aspect, a nucleic acid according to the second aspect, an expression cassette according to the third aspect, a vector according to the fourth aspect, or a pharmaceutical composition according to the fifth aspect are provided for the prevention, improvement, or treatment of cancer.

[0311] Cancer can be defined as in the first aspect.

[0312] In an eleventh aspect of the invention, a method for treating protozoan, fungal, bacterial, or viral infections is provided, the method comprising administering to a subject in need a therapeutically effective amount of an RNA construct according to the first aspect, a nucleic acid according to the second aspect, an expression cassette according to the third aspect, a vector according to the fourth aspect, or a pharmaceutical composition according to the fifth aspect.

[0313] The protozoan, fungal, bacterial, or viral infection to be treated can be an infection of protozoa, fungi, bacteria, or viruses as defined in the first aspect.

[0314] In a twelfth aspect of the invention, a method for treating cancer is provided, the method comprising administering to a subject in need a therapeutically effective amount of an RNA construct according to the first aspect, a nucleic acid according to the second aspect, an expression cassette according to the third aspect, a vector according to the fourth aspect, or a pharmaceutical composition according to the fifth aspect.

[0315] Cancers awaiting treatment can be defined as described in the first aspect.

[0316] The RNA constructs described in this article provide an effective method for vaccinating subjects against viral infections and cancer.

[0317] Therefore, in a thirteenth aspect of the present invention, a vaccine is provided comprising an RNA construct according to the first aspect, a nucleic acid according to the second aspect, an expression cassette according to the third aspect, a vector according to the fourth aspect, or a pharmaceutical composition according to the fifth aspect.

[0318] Preferably, the vaccine contains a suitable adjuvant.

[0319] The adjuvant may be an adjuvant encoded in an RNA construct sequence according to the first aspect, a nucleic acid according to the second aspect, an expression cassette according to the third aspect, a vector according to the fourth aspect, or a drug composition according to the fifth aspect, or an adjuvant incorporated into a delivery formulation as an adjuvant.

[0320] Encoding molecular adjuvants may encode cytokines (e.g., IL-12, GM-CSF, IL-2, IFN-g) or effector proteins (e.g., CD40L, Flt-3) or microbial proteins (e.g., flagellin or cholera toxin B).

[0321] The adjuvant incorporated into the delivery formulation may be selected from the group consisting of: bacterial lipopeptides, lipoproteins and lipoteichoic acid; mycobacterial lipotoxin, yeast polysaccharides, porin, lipopolysaccharide, lipid A, monophospholipid A (MPL), flagellin, CpG DNA, Plasmodium pigment, saponins (Quil-A, QS-21, lycopene, ISCOM, ISCANATRIX™), squalene emulsions, polymers such as PEI, carbomer, lipid nanoparticles and bacterial toxins (CT, LT).

[0322] In a fourteenth aspect of the invention, an RNA construct according to the first aspect, a nucleic acid according to the second aspect, an expression cassette according to the third aspect, a vector according to the fourth aspect, or a pharmaceutical composition according to the fifth aspect is provided for stimulating an immune response in a subject.

[0323] According to the definition in the first aspect, an antigen can stimulate an immune response against protozoa, bacteria, viruses, fungi, or cancer.

[0324] According to the fifteenth aspect, an RNA construct according to the first aspect, a nucleic acid according to the second aspect, an expression cassette according to the third aspect, a vector according to the fourth aspect, or a pharmaceutical composition according to the fifth aspect are provided for use in stem cell therapy.

[0325] Stem cell therapy can involve reprogramming somatic cells into cells with stem cell characteristics.

[0326] Somatic cells are reprogrammed by delivering one or more proteins that enhance the reprogramming of somatic cells to have stem cell characteristics as defined in the first aspect.

[0327] According to the sixteenth aspect, a method for modifying cells in vitro or in vitro is provided, the method comprising delivering an RNA construct according to the first aspect, a nucleic acid according to the second aspect, an expression cassette according to the third aspect, a vector according to the fourth aspect, or a pharmaceutical composition according to the fifth aspect to the cells.

[0328] Preferably, the method is performed in vitro.

[0329] The cell can be a eukaryotic cell or a prokaryotic cell. Preferably, the cell is a eukaryotic cell. More preferably, the cell is a mammalian host cell. Most preferably, the cell is a human cell.

[0330] Preferably, the modified cells are suitable for indications of cell therapy.

[0331] In the seventeenth aspect, modified cells obtained from or obtainable by the method of the sixteenth aspect are provided.

[0332] In the eighteenth aspect, the modified cells of the seventeenth aspect are provided for use in treatment, optionally in cell therapy.

[0333] It is understood that the RNA construct according to the first aspect, the nucleic acid according to the second aspect, the expression cassette according to the third aspect, the vector according to the fourth aspect, or the pharmaceutical composition according to the fifth aspect (referred to herein as an active agent) can be used in a drug that can be used as a monotherapy (i.e., using the active agent) to treat, improve, or prevent disease or to administer a vaccine. Furthermore, the active agent according to the invention can be used as an adjunct to, or in combination with, known therapies for treating, improving, or preventing disease.

[0334] Depending on, in particular, the manner of use of the composition, the RNA constructs, nucleic acid sequences, expression cassettes, vectors, or pharmaceutical compositions of the present invention can be combined in compositions having many different forms. Thus, for example, the composition may be in the form of powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle solution, transdermal patch, liposome suspension, polyplex, emulsion, lipid nanoparticles (with RNA on the surface or with encapsulated RNA), or any other suitable form that can be administered to humans or animals requiring treatment or vaccination. It will be understood that the excipients of the pharmaceuticals according to the present invention should be excipients that are well tolerated by the subject to whom the pharmaceutical excipient is administered.

[0335] The RNA constructs, nucleic acid sequences, expression cassettes, vectors, or pharmaceutical compositions of the present invention can also be incorporated into sustained-release or delayed-release devices. Such devices, for example, can be inserted onto or under the skin to release the drug over several weeks or even months. The device may be located at least near the treatment site. Such devices may be particularly advantageous when long-term treatment with gene constructs or recombinant vectors is required and frequent administration (e.g., at least daily injections) is typically necessary.

[0336] However, in a preferred embodiment, the drug according to the invention can be administered to the subject by injection into the bloodstream, muscle, skin, or directly into the site of treatment. Most preferably, the drug, including the RNA construct, is injected into the muscle. The injection can be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), or intramuscular (bolus or infusion).

[0337] Understandably, the required amount of RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition is determined by its biological activity and bioavailability, which in turn depends on the route of administration, the physicochemical properties of the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition, and whether it is used as a monotherapy or in combination therapy. The frequency of administration will also be affected by the half-life of the active agent in the treated subject. The optimal dose that can be administered is determined by those skilled in the art and will vary depending on the specific RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition used; the strength of the pharmaceutical composition; the route of administration; and the type and progression of the viral infection. Other factors depending on the specific subject being treated will necessitate dose adjustments, including the subject's age, weight, sex, diet, and timing of administration.

[0338] Generally, depending on the active agent used, the RNA constructs, nucleic acid sequences, expression cassettes, vectors or pharmaceutical compositions of the present invention, at daily doses of 0.001 μg / kg body weight to 10 mg / kg body weight or 0.01 μg / kg body weight to 1 mg / kg body weight, can be used to treat, improve or prevent diseases.

[0339] The daily dose can be administered in a single-dose manner (e.g., a single injection or nasal spray per day). Alternatively, the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition may require two or more administrations per day. As an example, the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition can be administered in two (or more, depending on the severity of the disease being treated) daily doses ranging from 0.07 μg to 700 mg (i.e., assuming a body weight of 70 kg). The treated patient may take the first dose upon waking, followed by the second dose at night or 3 or 4 hours later (if a two-dose regimen is used). Alternatively, a sustained-release device can be used to deliver the optimal dose of the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition according to the invention to the patient without the need for repeated doses.

[0340] However, preferably, the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition according to the invention can be administered at a weekly dose, more preferably at a bi-weekly dose.

[0341] Known procedures, such as those commonly used in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to form specific formulations and precise treatment regimens (e.g., daily doses and frequency of administration of drugs) for RNA constructs, nucleic acid sequences, expression cassettes, or vectors according to the present invention.

[0342] The “subject” can be a vertebrate, mammal, or livestock. Therefore, the compositions and medicines according to the invention can be used to treat any mammal, such as livestock (e.g., horses), pets, or for other veterinary applications. However, most preferably, the subject is a human.

[0343] The "therapeuticly effective amount" of an RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition means any amount of the aforementioned amount required to improve, prevent, or treat any given disease when administered to a subject.

[0344] For example, the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition of the present invention can be used in amounts from about 0.0001 mg to about 800 mg, preferably from about 0.001 mg to about 500 mg. Preferably, the amount of the replicon, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition is from about 0.01 mg to about 250 mg, most preferably from about 0.01 mg to about 1 mg. Preferably, the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition according to the present invention is administered at a dose of 1-200 μg.

[0345] The term "pharmaceuticalally acceptable excipient" as used herein refers to any known compound or combination of known compounds that is known to those skilled in the art as useful in the formulation of pharmaceutical compositions.

[0346] In one embodiment, the pharmaceutically acceptable excipient may be a solid, and the composition may be in the form of a powder or tablet. Solid pharmaceutically acceptable excipients may include one or more substances that can also function as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, flow aids, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrating agents. The excipient may also be an encapsulation material. In powders, the excipient is a finely ground solid mixed with a finely ground active agent according to the invention. In tablets, the active agent (e.g., an RNA construct, nucleic acid sequence, expression cassette, carrier, or pharmaceutical composition according to the invention) may be mixed in appropriate proportions with an excipient having the necessary compressibility and compressed into the desired shape and size. Powders and tablets preferably contain up to 99% active agent. Suitable solid excipients include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low-melting-point waxes, and ion exchange resins. In another embodiment, the pharmaceutical excipient may be a gel, and the composition may be in the form of a cream, etc.

[0347] However, pharmaceutical excipients can be liquids, and pharmaceutical compositions are in solution form. Liquid excipients are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and compressed compositions. RNA constructs, nucleic acid sequences, expression cassettes, vectors, or pharmaceutical compositions according to the invention can be dissolved or suspended in pharmaceutically acceptable liquid excipients, such as water, organic solvents, mixtures of both, or pharmaceutically acceptable oils or fats. Liquid excipients may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, coloring agents, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Suitable examples of liquid excipients for oral and parenteral administration include water (partially containing the above-mentioned additives, such as cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric and polyhydric alcohols, such as ethylene glycol) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). For parenteral administration, excipients can also be oleic esters, such as ethyl oleate and isopropyl myristate. Sterile liquid excipients are useful in sterile liquid compositions administered parenterally. The liquid excipient in compressed compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0348] Liquid pharmaceutical compositions (as sterile solutions or suspensions) can be utilized by, for example, subcutaneous injection, intradermal injection, intrathecal injection, epidural injection, intraperitoneal injection, intravenous injection, and especially intramuscular injection. The nucleic acid sequences or expression cassettes of the present invention can be prepared as sterile solid compositions, which can be dissolved or suspended in sterile water, physiological saline, or other suitable sterile injection media upon administration.

[0349] The RNA constructs, nucleic acid sequences, expression cassettes, vectors, or pharmaceutical compositions of the present invention can be administered orally in the form of sterile solutions or suspensions containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, gum arabic, gelatin, sorbitan monooleate, polysorbate 80 (an oleate copolymer of sorbitan and its anhydrides with ethylene oxide), etc. The RNA constructs, nucleic acid sequences, expression cassettes, vectors, or pharmaceutical compositions of the present invention can also be administered orally in the form of liquid or solid compositions. Compositions suitable for oral administration include solid and liquid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms for parenteral administration include sterile solutions, emulsions, and suspensions.

[0350] It is understood that this invention extends to any nucleic acid or polypeptide or its variants, derivatives or analogs thereof that substantially comprise the amino acid or nucleic acid sequence of any sequence mentioned herein, including its variants or fragments. The terms “substantially amino acid / nucleotide / peptide sequence,” “variant,” and “fragment” can mean a sequence having at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any sequence mentioned herein, such as a sequence having at least 40% sequence identity with a sequence defined as SEQ ID NO: 1-55, etc.

[0351] Amino acid / polynucleotide / peptide sequences with sequence identity to any sequence mentioned herein are also envisioned, wherein the sequence identity is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80%. Preferably, the amino acid / polynucleotide / peptide sequence has at least 85% identity to any sequence mentioned herein, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity to any sequence mentioned herein.

[0352] Those skilled in the art will understand how to calculate the percentage identity between two amino acid / polynucleotide / peptide sequences. To calculate the percentage identity between two amino acid / polynucleotide / peptide sequences, the two sequences must first be aligned, and then the sequence identity value must be calculated. The percentage identity of two sequences can have different values ​​depending on: (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used in the alignment method, such as local vs. global alignment, the pair-score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, such as functional forms and constants.

[0353] After alignment, there are many different methods to calculate the percentage identity between two sequences. For example, we can divide the number of consistent sequences by: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-empty positions; or (v) the number of equivalent positions excluding prominent overhangs. Furthermore, it is understood that percentage identity is also highly length-dependent. Therefore, the shorter a pair of sequences, the higher the expected probability of coincidental sequence identity.

[0354] Therefore, it is understandable that accurate alignment of protein or DNA sequences is a complex process. The widely used multiplex alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for generating the protein or DNA multiplex alignments of this invention. Suitable parameters for ClustalW can be as follows: For DNA alignment, vacancy opening penalty = 15.0, vacancy extension penalty = 6.66, and matrix = Identity. For protein alignment: vacancy opening penalty = 10.0, vacancy extension penalty = 0.2, and matrix = Gonnet. For both DNA and protein alignment: ENDGAP = -1 and GAPDIST = 4. Those skilled in the art will recognize that it may be necessary to modify these and other parameters to achieve optimal sequence alignment.

[0355] Preferably, the percentage identity between two amino acid / polynucleotide / peptide sequences can be calculated using an alignment such that (N / T)*100, where N is the number of positions in the sequences that share the same residues, and T is the total number of comparison positions, including vacancies and those including or excluding protruding sticky ends. Preferably, protruding sticky ends are included in the calculation. Therefore, the most preferred method for calculating the percentage identity between two sequences includes: (i) preparing the sequence alignment using the ClustalW program with appropriate parameter settings (e.g., as described above); and (ii) substituting the values ​​of N and T into the following formula: Sequence Identity = (N / T)*100.

[0356] Other methods for identifying similar sequences are known to those skilled in the art. For example, substantially similar nucleotide sequences would be encoded by a sequence that hybridizes with a DNA sequence or its complement under stringent conditions. The stringent conditions referred to by the inventors mean hybridization of the nucleotides with the DNA or RNA bound to the filter membrane in 3× sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing at least once in 0.2×SSC / 0.1% SDS at about 20–65°C. Additionally, substantially similar polypeptides may differ from, for example, sequences shown in SEQ ID NO: 1 to 57 by at least one, but fewer than 5, 10, 20, 50, or 100 amino acids.

[0357] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be altered or modified to provide functional variants without substantially affecting the sequence of the protein it encodes. Suitable nucleotide variants are those sequences that produce silent (synonymous) changes by substituting different codons encoding the same amino acids within the sequence. Other suitable variants are those sequences that have homologous nucleotide sequences but contain all or part of a sequence that produces a conserved change by substituting different codons encoding amino acids in side chains with biophysical properties similar to the amino acids they have replaced. For example, small nonpolar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar, hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is understandable that which amino acids can be replaced with amino acids having similar biophysical properties, and those skilled in the art know the nucleotide sequences encoding these amino acids.

[0358] All features described herein (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any way with any of the foregoing aspects, except for combinations in which at least some of such features and / or steps are mutually exclusive. Attached Figure Description

[0359] To better understand the present invention and to show how to implement embodiments thereof, reference is now made to the accompanying drawings with reference to embodiments, wherein:

[0360] Figure 1 This diagram illustrates one implementation of a self-amplifying RNA replicon or construct based on the Venezuelan equine encephalitis virus (VEEV) backbone. The so-called "stealthicon" vector is a saRNA replicon / construct encoding non-structural proteins (NSP1-4) and innate repressor proteins (IIP) located upstream or downstream of the GOI (target gene).

[0361] Figure 2This shows that genomic replicons produce dsRNAs recognized by the sensor molecules MDA5 and PACT. These sensor molecules transmit signals to activate downstream cascades, including the activation of transcription factors (NF-κB, IRF-3, IRF-7) and restriction factors that directly inhibit RNA amplification and protein expression (dashed lines). Expression of PIV-V or ORF4a blocks the innate recognition of dsRNAs by MDA5 and PACT, thereby preventing the activation of downstream cascades that restrict replicon RNA amplification and protein expression of synthetic RNA.

[0362] Figure 3 shows a) screening of VEEV replicons encoding IIP in vitro. Cells were transfected with two batches of RNA containing luciferase as a reporter protein, and protein expression was assessed after 24 hours. Compared to HEK, HeLa and MRC5 are known to have a more complete IFN expression pathway, and b) shows the fold change in expression relative to wild-type. Figure 3a The data.

[0363] Figure 4 The use of firefly luciferase as a reporter protein in BALB / c and BL / 6 mice was demonstrated to screen for replicons encoding IIP (#6 and #8). BL / 6 mice are known to express more IFN than BALB / c mice, thus IIP achieved higher luciferase expression at 3 days and longer expression duration >14 days.

[0364] Figure 5 The study demonstrates the use of Gaussian luciferase protein as a reporter protein to screen replicons (#6 and #8) encoding IIP in BALB / c and BL / 6 mice. BL / 6 mice are known to express more IFN than BALB / c mice, thus IIP enables longer expression of the soluble reporter protein, lasting >14 days.

[0365] Figure 6 A construction diagram of one embodiment of an expression vector encoding an RNA construct containing GOI is shown.

[0366] Figure 7 A construction diagram of one embodiment of an expression vector encoding an RNA construct containing GOI-MERS-CoV ORF4a is shown.

[0367] Figure 8 A construction diagram of one embodiment of an expression vector encoding an RNA construct containing GOI-PIV5 is shown.

[0368] Figure 9The diagram shows the constructs of the present invention and in vitro protein expression from wild-type and IIP VEEV replicons. a) Schematic diagram of wild-type and cis-encoding IIP VEEV replicons. b) Schematic diagram of innate sensing of self-amplified RNA. c) In vitro transfection of firefly luciferase saRNA in HEK 293T.17, HeLa, and MRC5 cells, measured in relative light units (RLU). Bars represent the mean fold change ± standard deviation normalized to wild-type VEEV control, n = 3.

[0369] Figure 10 Dosage adjustments for WT and MERS COV-2 replicons in C57BL6 / J mice are shown. Protein expression was quantified on days 7 and 10 following intramuscular injection of 0.2, 2, or 20 μg RNA. Each point represents one mouse, bars represent mean ± SEM, n = 10. * indicates significance of P < 0.05 as assessed using the Kruskal-Wallis test with multiple comparisons.

[0370] Figure 11 The co-formulation of WT and MERS CoV_2 replicons with the JAK inhibitor ruxotinib in C57BL6 / J mice is shown. Protein expression was quantified on days 4, 7, 10, and 14 (a, b, c, and d, respectively) following intramuscular injection of 5 μg RNA and 100 μg ruxotinib. Each dot represents one mouse leg, bars represent mean ± SEM, n = 10.

[0371] Figure 12 Protein expression in human skin explants is shown for eGFP ± MERS-CoV_2 RNA (corresponding to MERS-CoV ORF4a) (0.2, 2, or 20 μg) (a, b) or ± ruxotinib (0.1, 1, 10, or 100 μg) (c, d). The number of eGFP-expressing cells (%GFP+ cells) (a, c) and total protein expression per cell (median fluorescence intensity (MFI) of GFP) (b, d) were quantified at 72 hours post-injection. Each point represents mean ± SEM, n = 3. * indicates significance of P < 0.05 as assessed using the Kruskal-Wallis test with multiple comparisons.

[0372] Figure 13 Immunogenicity of RABV±MERS-CoV_2 (corresponding to MERS-CoV ORF4a) in rabbits was demonstrated. a) RABV antigen-specific IgG antibody titers after intramuscular immunization with 20 μg at weeks 0 and 4, and booster immunization, n=5. b) Neutralizing IC50 against pseudotyped RABV virus. 50n=5, and the gray dashed line represents the detection limit. * indicates significance of p<0.05 as assessed using the Kruskal-Wallis test with multiple comparisons.

[0373] Figure 14 The in vitro transfection of firefly luciferase saRNA in HEK 293T.17, HeLa, and MRC5 cells is shown, measured in relative light units (RLU). Bars represent mean ± standard deviation controls, n = 3. 'a' and 'b' indicate two separate batches of RNA prepared.

[0374] Figure 15 The in vitro transfection of WT fLuc, MERS-CoV_2ORF4a, and PIV-5 RNA in a) mouse (MEF), b) rabbit (RK13), c) non-human primate (LLC), and d) human (MRC5) cells is shown in relative optical units (RLU). Bars represent controls with mean ± standard deviation, n = 3.

[0375] Figure 16 In vivo luciferase expression of intracellular proteins (firefly luciferase) and secreted proteins (gaussian luciferase) in BALB / c and C57BL6 / J mice is shown. Protein expression in muscle (a,c,e) or serum (b,d,f) was quantified using an in vivo imaging system (IVIS) on days 3 (a,b), 7 (c,d), and 14 (e,f). Each point represents one mouse, bars represent mean ± SEM, and n = 10 for firefly luciferase (fLuc) and n = 5 for Gaussian luciferase (gLuc).

[0376] Figure 17 The study showed that human skin explants treated with 0.2 μg eGFP RNA contained eGFP superimposed on them. + t-distributed random neighborhood embedding (tSNE) plot of unsupervised live cell clusters (gray) that are gated (green), separated by phenotype (blue), and genotype-separated.

[0377] Figure 18 The study showed that human skin explants treated with 2 μg of eGFP RNA contained eGFP superimposed on them. + t-distributed random neighborhood embedding (tSNE) plot of unsupervised live cell clusters (gray) that are gated (green), separated by phenotype (blue), and genotype-separated.

[0378] Figure 19 The study showed that human skin explants treated with 20 μg eGFP RNA contained eGFP superimposed on them. +t-distributed random neighborhood embedding (tSNE) plot of unsupervised live cell clusters (gray) that are gated (green), separated by phenotype (blue), and genotype-separated.

[0379] Figure 20 The study showed that human skin explants treated with 0.2 μg eGFP-PIV-5 RNA contained eGFP superimposed on them. + t-distributed random neighborhood embedding (tSNE) plot of unsupervised live cell clusters (gray) that are gated (green), separated by phenotype (blue), and genotype-separated.

[0380] Figure 21 The study showed that human skin explants treated with 2 μg eGFP-PIV-5 RNA contained eGFP superimposed on them. + t-distributed random neighborhood embedding (tSNE) plot of unsupervised live cell clusters (grey) gated and phenotype-separated (blue) by cells (green).

[0381] Figure 22 The study showed that human skin explants treated with 20 μg eGFP-PIV-5 RNA contained eGFP superimposed on them. + t-distributed random neighborhood embedding (tSNE) plot of unsupervised live cell clusters (gray) that are gated (green), separated by phenotype (blue), and genotype-separated.

[0382] Figure 23 The image shows that human skin explants treated with 0.2 μg eGFP-MERS-COV_2 RNA (corresponding to MERS-CoV ORF4a) contained eGFP superimposed on them. + t-distributed random neighborhood embedding (tSNE) plot of unsupervised live cell clusters (gray) that are gated (green), separated by phenotype (blue), and genotype-separated.

[0383] Figure 24 The image shows that human skin explants treated with 2 μg of eGFP-MERS-CoV_2 RNA contained eGFP superimposed on them. + t-distributed random neighborhood embedding (tSNE) plot of unsupervised live cell clusters (gray) that are gated (green), separated by phenotype (blue), and genotype-separated.

[0384] Figure 25 The image shows that human skin explants treated with 20 μg eGFP-MERS-COV_2 RNA (corresponding to MERS-CoV ORF4a) contained eGFP superimposed on them. + t-distributed random neighborhood embedding (tSNE) plot of unsupervised live cell clusters (gray) that are gated (green), separated by phenotype (blue), and genotype-separated.

[0385] Figure 26The following diagram shows the phenotypic identification of cells present in human skin explants after intradermal (ID) injection of eGFP±ruxo formulation, as determined by flow cytometry: a) Cell identification of GFP-expressing skin cells from the total cell population extracted from human skin explants and from explants treated with 2 μg eGFP-encoding saRNA±0.1, 1, 10, or 100 μg ruxo, n=3. b) Percentage of cells expressing each GFP phenotype. Cells identified using the following antibodies: epithelial cells (CD45-), fibroblasts (CD90+), NK cells (CD56+), leukocytes (CD45+), Langerhans cells (CD1a+), monocytes (CD14+), dendritic cells (CD11c+), T cells (CD3+), and B cells (CD19+).

[0386] Figure 27 Immunogenicity of RABV±MERS-CoV_2 (corresponding to MERS-CoV ORF4a) in mice and rats was shown. a) RABV antigen-specific IgG antibody titers in mice after intramuscular immunization with 1 μg of primary and booster immunization at weeks 0 and 4, n=5. b) Neutralizing IC50 against pseudotyped RABV virus in mice. 50 n=5, the gray dashed line represents the detection limit. a) RABV antigen-specific IgG antibody titers after intramuscular immunization of rats with 20 μg of primary and booster immunization at weeks 0 and 4, n=5. b) Neutralizing IC50 against pseudo-RABV virus in rats. 50 n=5, and the gray dashed line represents the detection limit.

[0387] Figure 28 A schematic diagram showing the formulation mechanism of PIV-5V and MERS-CoV ORF4a sensing saRNA is shown.

[0388] Figure 29 Median fluorescence intensity (MFI) data are shown, indicating that in HeLa cells, the expression of SARS-CoV-2 glycoprotein from saRNA according to one embodiment of the invention is increased when co-expressed with the innate repressor protein MERS-ORF4a, compared to saRNA that only encodes SARS-CoV-2 glycoprotein (i.e., without IIP). Example

[0389] The inventors hypothesized that viral cis-encoded proteins that inhibit innate recognition of saRNA suppress innate perception and enhance protein expression and immunogenicity of saRNA vaccines. Therefore, the inventors designed and tested a series of RNA replicons containing an innate repressor protein (IIP) and a target gene, and then characterized whether these replicons enhanced intracellular protein expression and the expression of secretory proteins (encoded by the target gene).

[0390] Materials and methods

[0391] Cloning of the innate repressor protein (IIP) replicon

[0392] As previously stated (53) Self-amplifying RNA encoding firefly luciferase, Gaussian luciferase, enhanced green fluorescent protein (eGFP), rabies virus glycoprotein (RABV), and replicase from Venezuelan equine encephalitis was cloned into plasmid vectors. Interferon repressor protein libraries were cloned into these vector backbones as part of target genes (fLuc, GLuc, eGFP, or RABV) with T2A cleavage sites (GenBank accession number #AAC97195.1). Interferon-inhibiting proteins can be found using the following GenBank accession numbers: HSV-2Us1 (Z86099.2), HSV-1Us1 (AWO69381.1), HSV-1Us11 (YP_009137147.1), OV20.0L (AF053969.1), BVDV Npro (AIE38066.1), PIV-5V (YP_138513.1), MERS-CoV M (AHC74104.1), MERS-CoV ORF4a (AHC74090.1), Langat virus NS5 (AF253420), and influenza virus NS1 (DQ508893.1). In mouse studies, the PIV-5V protein with the N100D mutation was used. (45) .

[0393] in vitro transcription of saRNA

[0394] Self-amplified RNA was produced using in vitro transcription. Plasmid DNA (pDNA) was transformed into *E. coli* (New England BioLabs, UK) and cultured in 100 mL of Luria Broth (LB) containing 100 μg / mL carbenicillin (Sigma Aldrich, UK). pDNA was then isolated using the Plasmid Plus MaxiPrep kit (QIAGEN, UK), and the final concentration of pDNA was measured on a NanoDrop One (ThermoFisher, UK). pDNA was linearized at 37°C for 3 hours using MLUL. The pDNA was then processed using an mMachine. TM RNA for in vitro transfection was prepared using 1 μg of linearized pDNA template in a T7 transcriptome (Invitrogen, UK) and MEGAclear was used. TM The transcription cleanup kit (Invitrogen, UK) was used for purification according to the manufacturer's instructions. As previously stated... (2) RNA was prepared for in vitro and in vivo experiments. 1 μg of linearized pDNA template was used, and MEGAScript was employed. TM Uncapped RNA transcripts were produced at 37°C for 2 hours using a T7 transcriptome (Invitrogen, UK) according to the manufacturer's instructions. The transcripts were then purified by overnight LiCl precipitation at -20°C, followed by centrifugation at 14,000 RPM for 20 minutes at 4°C to precipitate the RNA. The RNA was washed once with 70% EtOH, centrifuged again at 14,000 RPM for 5 minutes at 4°C, and then resuspended in UltraPure H2O (Ambion, UK). ScriptCap was used. TM The Cap 1 Capping System Kit (CellScript, WI, USA) was used to cap the purified transcripts at 37°C for 2 hours according to the manufacturer's instructions. The capped transcripts were then purified a final time using LiCl precipitation as described above, and resuspended in RNA storage buffer (10 mM HEPES, 0.1 mM EDTA, and 100 mg / mL trehalose) and stored at -80°C until further use.

[0395] saRNA preparation

[0396] Use the titration method as described previously. (2)fLuc, gLuc, and eGFP saRNAs used for protein expression experiments were compounded with 100 kDa pABOL. RABV saRNAs used for in vivo immunogenicity experiments were compounded with 8 kDa pABOL. In short, RNA and pABOL were diluted in HEPES buffer (20 mM HEPES, 5 wt.% glucose aqueous solution, pH 7.4) and compounded on a NanoAssemblr benchtop formulation device (Precision Nanosystems, Inc., Vancouver, Canada) at a volume ratio of 4:1 (RNA to polymer) and a flow rate of 10 mL / min. The final polymer to saRNA ratio was 45:1 (w / w). Polyplexes were freshly prepared and used within 1 hour of preparation. For co-formulation, ruxotinib (ruxo, Selleck Chemicals, UK) was added directly to the polymers at the specified dose.

[0397] In vitro transfection

[0398] Transfection was performed on HEK293T.17 cells (ATCC, USA), HeL cells (ATCC, USA), MRC5 cells (ATCC, USA), mouse embryonic fibroblast (MEF) cells (SigmaAldrich, UK), RK13 rabbit kidney cells (Public Health England, UK), and LLC-MK2 rhesus monkey kidney cells (ATCC, USA). Cells were cultured in complete Dulbecco modified Eagle medium (cDMEM) (Gibco, Thermo Fisher, UK) containing 10% (v / v) fetal bovine serum (FBS), 5 mg / mL L-glutamine, and 5 mg / mL penicillin / streptomycin (Thermo Fisher, UK) (HEK, HeLa, MEF cells); complete modified Eagle medium (cMEM) (cMEM) containing 10% (v / v) fetal bovine serum (FBS), 5 mg / mL L-glutamine, and 5 mg / mL penicillin / streptomycin (Thermo Fisher, UK) (MRC5, RK13 cells); or complete medium 199 (cM199, Sigma Aldrich, UK) containing 1% horse serum (Gibco, Thermo Fisher, UK) (LLC cells). Twenty-four hours before transfection, cells were seeded at a density of 50,000 cells per well in clear 96-well plates. The culture medium was then completely removed and replaced with 50 μL of preheated transfection medium (DMEM + 5 mg / mL L-glutamine, MEM + 5 mg / mL L-glutamine, or M199). 100 μL of polymerase solution (containing 100 ng saRNA) was then added to each well and incubated for 4 hours. The transfection medium was then completely removed and replaced with cDMEM, cMEM, or CM199. After 24 hours, 50 μL of medium was removed from each well, and 50 μL of ONE-Glo D-fluorescein substrate (Promega, UK) was added and mixed thoroughly by pipetting. The total volume from each well was then transferred to a white 96-well plate (Costar) for analysis and quantified using a FLUOstar OMEGA microplate reader (BMG LABTECH, UK). The background fluorescence from the control wells was subtracted from each well.

[0399] Luciferase expression in mice

[0400] All animal handling complied with the UK Home Office Animal Science Procedures Act 1986 and was conducted with a project license (P63FE629C) and individual license (IC37CBB8F) approved by the local ethics committee and the UK government. Food and water were provided freely. Female BABL / c mice (Charles River, UK) or C57BL / 6 mice (Charles River, UK), 6–8 weeks old, were housed in groups (5 mice per cage) in a fully acclimatized room. Mice were intramuscularly injected with 50 μL of a total volume of 5 μg fLuc saRNA (both hind legs) in combination with pABOL (one hind leg). After 3, 4, 7, 10, or 14 days, the mice were administered the following treatment as previously described. (54，55) For fLuc imaging in mice, or for GLuc analysis using blood samples, the GausSia Luciferase Glow assay kit (Pierce, Thermo Scientific, UK) was performed according to the manufacturer's instructions. Serum protein expression was quantified using a FLUOstar OMEGA microplate reader (BMG LABTECH, UK). Background fluorescence from control wells was subtracted from each well. For fLuc analysis, mice were intraperitoneally injected (IP) with 150 μL of XenoLight RediJect D-fluorescein substrate (PerkinElmer, UK) and allowed to rest for 10 minutes. Mice were then anesthetized with isoflurane and imaged for 2 minutes using an In Vivo Imaging System (IVIS) FX Pro (Kodak Co., Rochester, NY, USA) equipped with Molecular Imaging Software version 5.0 (Carestream Health, USA). Signal at each injection site was quantified using the molecular imaging software and expressed as total flux (p / s).

[0401] Expression of Gaussian luciferase in mice

[0402] Female BALB / c or C57BL / 6 mice (Charles River, UK) aged 6–8 weeks were randomly divided into groups (n = 5) and housed in fully adapted rooms. Mice were intramuscularly injected (IM) with 5 μg of gLuc (from a single hind leg) in a total volume of 50 μL. Blood was exsanguinated via the tail vein at 3, 7, and 14 days. Blood was allowed to clot and centrifuged at 10,000 RPM for 5 minutes, after which the serum was collected. The serum was then processed using Pierce... TMThe GausSia Luciferase Glow assay kit was used to detect serum at all time points using 20 μL of serum and 100 μL of working solution prepared according to the manufacturer's instructions on a single 96-well white plate (Costar). Luciferin was analyzed using a FLUOstar Omega microplate reader (BMG LABTECH, UK), and the background from the naive animal was subtracted from each sample.

[0403] Vaccination of mice, rats and rabbits

[0404] BALB / c mice, Sprague-Dawley rats, and New Zealand white rabbits were immunized intramuscularly (IM) with 1 μg (mice) or 20 μg (rats, rabbits) saRNA encoding RABV, prepared with pABOL, at a total volume of 50 μL (mice) or 100 μL (rats, rabbits) on one hind leg. A booster injection was given 4 weeks after the initial immunization. Blood was collected at 0, 4, and 6 weeks after the start of the study and centrifuged at 10,000 RPM for 5 minutes. The serum was then decanted and stored at -80°C until further analysis.

[0405] RABV-specific ELISA

[0406] According to the previous description (56)A semi-quantitative immunoglobulin ELISA protocol was performed. Briefly, 0.5 μg / mL RABV-coated ELISA plates were blocked with 1% (w / v) bovine serum albumin (BSA) and 0.05% (v / v) Tween-20 in PBS. After washing, diluted serum samples were added to the plates and incubated for 2 hours. The plates were then washed, and for mouse ELISA, 1:4000 dilution of anti-mouse IgG-HRP (Southern Biotech, UK) was added; for rat ELISA, 1:4000 dilution of goat anti-rat IgG-HRP (Southern Biotech, UK) was added; and for rabbit ELISA, 1:10000 dilution of mouse anti-rabbit IgG-HRP (Sigma, UK) was added. Mouse standards were prepared as follows: ELISA plate wells were coated with anti-mouse κ (1:1,000) and λ (1:1,000) light chains (Serotec, UK), blocked with 1% (w / v) BSA / 0.05% (v / v) Tween-20 PBS solution, washed, and purified IgG (Southern Biotech, UK) was added, starting at 1000 ng / mL, and titrated down in a 5-fold serial dilution series. Rat standards were prepared as follows: ELISA plate wells were coated with purified rat IgG (R&D Systems, UK), starting at 1000 ng / mL, and titrated down in a 5-fold serial dilution series. Rabbit standards were prepared as follows: ELISA plate wells were coated with 1:1250 anti-rabbit IgG Fc (Milipore), blocked with 1% (w / v) BSA / 0.05% (v / v) Tween-20 PBS solution, washed, and purified rabbit IgG (AbD Serotech, UK) was added, starting at 1000 ng / mL, and titrated serially down-diluted in 5-fold increments. Samples and standards were developed using 3,3',5,5'-tetramethylbenzidine (TMB). The reaction was stopped with stop solution (InSight Biotechnologies, UK) after 5 minutes. Absorbance was read using a spectrophotometer (VersaMax, Molecular Devices, UK) with SoftMax Pro GxP V5 software.

[0407] RABV microneutralization analysis

[0408] Samples from weeks 0, 4, and 6 were microneutralized with pseudotyped rabies virus. BHK-21 cells were seeded at 10,000 cells / well in cDMEM in 96-well plates. Serum was heat-inactivated at 56°C and then serially diluted 1:5 in cDMEM. Samples were then diluted with an equal volume of 50 μL of 100 TCID50 solution. 50The pseudovirus was diluted, incubated at 37°C for 1 hour, and then added to BHK-21 cells, which were cultured at 37°C for 48 hours. Cells were then lysed, and luciferase activity was quantified using the Bright-Glo luciferase assay (Promega, UK). The total volume from each well was then transferred to a white 96-well plate (Costar) for analysis, and quantification was performed using a FLUOstar OMEGA microplate reader (BMG LABTECH, UK), calculating the IC50 for each sample. 50 .

[0409] Human skin explant culture and injection

[0410] In this ex vivo study, surgically removed human skin tissue specimens were collected at Charing Cross Hospital, Imperial NHS Trust, London, UK. All tissues were collected under a protocol approved by the Local Research Ethics Committee (MED_RS_II_014) of Imperial College London, after receiving informed consent from patients undergoing elective abdominoplasty or mastectomy. The skin tissue was refrigerated and cut into 1cm pieces before use. 2 The explants were cultured in 12-well plates at 37°C and 5% CO2 using 2 mL of cDMEM. Explants were also injected intradermally (ID) at a dose of 2 μg saRNA in a total volume of 50 μL using a Micro-Fine Demi 0.3 mL syringe (Becton Dickinson, UK). The culture medium was changed daily during the culture period.

[0411] Flow cytometry

[0412] 72 hours after injection, the skin explants were trimmed to remove the subcutaneous fat layer. The epidermis and dermis were then minced with scissors and cultured in 2 mL of DMEM supplemented with 1 mg / mL collagenase P (Sigma Aldrich, UK) and 5 mg / mL dispersin II (Sigma Aldrich, UK) at 37°C for 4 hours on a rotating shaker. The digest was then filtered through a 70 μm cell filter and centrifuged at 1,750 RPM for 5 minutes. The cells were then resuspended in 100 μL of FACS buffer (PBS + 2.5% FBS) and stained on ice for 20 minutes with a 1:400 diluted fixable Aqua live / dead cell staining agent (ThermoFisher, UK) in FACS buffer. The samples were then washed with 1 mL of FACS buffer, centrifuged at 1,750 RPM for 5 minutes, and stained with a mixture of the following antibodies: CD3-V450 (BioLegend, UK), CD14-Qdot605 (BioLegend, UK), CD19-BV650 (BioLegend, UK), CD56-BV711 (BioLegend, UK), CD1a-PerCP-eFluor710 (BioLegend, UK), CD11c-PE (BioLegend, UK), CD90-PE-Cy7 (BioLegend, UK), and CD45-AF700 (BioLegend, UK). The samples were then washed with 1 mL of FACS buffer, centrifuged at 1,750 RPM for 5 min, resuspended in 250 μL of PBS, and fixed with 250 μL of 3% paraformaldehyde to a final concentration of 1.5%. The samples were then refrigerated until flow cytometry analysis. The samples were analyzed on an LSR Tortessa (BD Biosciences, UK) flow cytometer equipped with FACSDiva software (BD Biosciences, UK) and 100,000 cell events. As previously described. (58) Implement a gating strategy and use FlowJo version 10 (FlowJo LLC, Oregon, USA) for GFP. + Cell phenotypic identification was quantified. Unsupervised t-distributed random neighborhood embedding (tSNE) analysis of live cell clusters was performed in FlowJo using 1000 iterations, a perplexity of 30, a learning rate of 15196, the Exact (dominance tree) KNN algorithm, and the Barnes-Hut gradient algorithm.

[0413] In vitro working of SARS-CoV-2 glycoprotein

[0414] Based on the genome of the Trinidad donkey Venezuelan equine encephalitis virus (VEEV) alpha virus strain, the inventors synthesized a self-amplifying RNA (saRNA) replicon using a plasmid vector. The viral structural proteins driven by the subgenomic promoter were replaced with the surface 'spike' glycoprotein of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (GenBank accession number QHD43416.1). The inventors synthesized oligonucleotide fragments encoding the SARS-CoV-2 gene using oligonucleotide strings (GeneArt, Germany) and assembled these fragments into the plasmid vector using the Gibson assembly method (NEB Ltd, UK). In further modification of the SARS-CoV-2 replicon vector, an oligonucleotide string encoding MERS-CoV ORF4a (AHC74090.1) (GeneArt, Germany) was synthesized and its 3' end was inserted into the SARS-CoV-2 coding region, which was ligated into a continuous open reading frame using a variant sequence of furin / T2A (SEQ ID NO: 26), generating a novel SARS-CoV-2 ORF4a vector. Cells were transfected with SARS-CoV-2 and SARS-CoV-2 ORF4a saRNA, respectively, and then stained with polyclonal antibodies to examine expression. Briefly, cells were harvested 24 hours after transfection and processed at 1 × 10⁻⁶. 7Resuspend the cells at a concentration of 100 cells / mL in 1 mL of FACS buffer (PBS + 2.5% FBS). Add 100 μL of the resuspended cells to a FACS tube and stain on ice with 50 μL of live / dead fixed Aqua dead cell stain (Thermo Fisher Scientific, UK) at a dilution of 1:400 for 20 minutes. Then wash the cells with 2.5 mL of FACS buffer and centrifuge at 1750 RPM for 7 minutes. After centrifugation, stain the cells on ice with 2.5 μg of SARS-CoV spike protein polyclonal antibody (PA1-41165, Thermo Fisher Scientific, UK) for 30 minutes, then wash with 2.5 mL of FACS buffer and centrifuge at 1750 RPM for 7 minutes. Then stain the cells on ice with 0.4 μg of FITC goat anti-rabbit IgG (BD Pharmigen, UK) for 30 minutes. After incubation, cells were washed with 2.5 mL of FACS buffer, centrifuged at 1750 RPM for 7 min, and resuspended in 250 μL of PBS. Cells were fixed with 250 μL of 3% paraformaldehyde to a final concentration of 1.5%. Samples were analyzed on an LSR Torterssa (BD Biosciences, UK) with FACSDiva software (BD Biosciences, UK). Data were analyzed using FlowJo version 10 (FlowJo LLC, USA), and the median fluorescence intensity (MFI) of positive cell populations was measured, with the negative / positive cutoff value set for cells gated as viable and single, as well as cells that underwent simulated transfection using the same method as described above but did not contain SARS-CoV-2 or SARS-CoV-2 ORF4a replicon saRNA.

[0415] Statistical analysis

[0416] Create graphs and statistics in GraphPad Prism, version 8. Analyze statistical differences using two-way ANOVA or the adjusted Kruskal-Wallis test for multiple comparisons, and consider p < 0.05 as significant.

[0417] Results and discussion

[0418] RNA replicons are considered potential tools for delivering and expressing target genes in vaccines and therapies. However, double-stranded RNA (dsRNA) is detected within cells by an innate sensing mechanism that triggers a signal cascade that inhibits protein translation. As a result, the expression of the target gene encoded in the replicon is significantly impaired, thus limiting the therapeutic potential of RNA replicons.

[0419] The inventors tackled this problem by developing an RNA replicon encoding an innate inhibitor protein to attenuate innate recognition of saRNA. The only previously published method for attenuating interferon responses with saRNA was the use of interferon inhibitor proteins from vaccine viruses, E3, K3, and B18. However, in this study, the interferon inhibitor protein was delivered and formulated as a separate mRNA molecule in combination with the saRNA. This requires the manufacture of both saRNA and mRNA, and necessitates the use of 3–6 times the amount of vaccine mRNA as the replicon RNA to ensure co-delivery to the same cell and to provide any observable enhancement of protein expression. Furthermore, the expression kinetics of mRNA and saRNA differ, therefore any beneficial effects of IIP expressed from the mRNA are transient compared to those from the accompanying replicon.

[0420] Given the current difficulties in using saRNA in treatment, the inventors have designed a novel saRNA that will more effectively limit the immune response to it than existing methods, thereby improving its efficacy in vaccination and treatment.

[0421] Using PIV-5 and ORF4a as novel IIPs

[0422] PIV-5 and ORF4a are known to block MDA-5, a cytoplasmic RNA helicase that signals via a conjugate molecule called MAV, leading to the induction of interferon regulators 3 and 7 (IRF3 and IRF7), which respectively result in the production of restriction factors that reduce the translation of introduced synthetic saRNAs. Figure 1 These two IIPs were identified in a preliminary in vitro screening of 10 IIPs from a range of different viruses.

[0423] l.HSV-2Us1- inhibits the production of IFN-B by inhibiting the binding of IRF-3 to the IFN-β promoter [1].

[0424] 2. HSV-1Us1 (a regulatory factor from HSV-1)

[0425] 3. HSV-1Us11 - Blocks RIG-I signal conduction [2,3].

[0426] 4. OV20.0L- binds to dsRNA and inhibits both PKR and PACT, blocking RIG-I signaling [4,5].

[0427] 5. BVDV Npro: Blocks IRF3 phosphorylation and S100A9 signal transduction [6,7].

[0428] 6. PIV5V: Blocks MDA-5 and IRF3 by binding to MDA-5 [8,9]

[0429] 7. MERS-CoV M: Interacts with TRAF3, disrupts TRAF3-TBK1 binding, leading to reduced IRF3 activation [10-12].

[0430] 8. MERS-CoV ORF4a: binds to dsRNA (with a preference for long RNA) and inhibits PACT triggering and stress response of MDA5, RIG-I, and PKR [13,14].

[0431] 9. Langat virus NS5: downregulates IFNA1R and impairs JAK-STAT signaling [15-16].

[0432] 10. Influenza NS1: binds to dsRNA and blocks RIG-I signaling

[17] .

[0433] These IIPs, along with luciferase which serves as a GOI and is used as an expression marker, were integrated into the inventors' standard VEEV saRNA replicon. Figure 1 These constructs were evaluated in three human cell lines: HEK293T cells with impaired innate sensory pathways, HeLa cells, and primary MRC5 embryonic epithelial cells. Figure 3a (b) In the absence of innate recognition, all IIP candidate saRNAs replicated in a manner similar to wild-type saRNAs in HEK293T cells. A range of IIPs (except MERS-CoV M and influenza NS1) were able to enhance expression in HeLa cells, but the enhancement observed was most significant (3 logs) for PIV-V and ORF4a. The most important evaluation in primary MRC5 cells showed that only PIV-V and ORF4a were able to increase luciferase expression by 2 logs. These data suggest that PIV-V and ORF4a are unique in their ability to enhance GOI expression in human primary cells. The identification of PIV-5 and ORF4a contained in RNA vectors is based on these experimental data, and their activity is unpredictable, considering that other IIPs believed to be evaluated work through similar mechanisms. To further support their application in gene delivery, the inventors conducted in vivo experiments in mice. In this study, the inventors utilized Black 6 (BL6) mice, which are known to possess stronger innate sensing mechanisms and downstream interferon responses than BalBc mice, thus providing an insightful comparison between the two models. The inventors used firefly luciferase (fLuc) as a reporter protein to assess the relative expression of WT, PIV-5, and ORF4a replicons in BL / 6 mice. Figure 4 ).

[0434] Because fLuc is expressed intracellularly, the expression of the target gene can be visualized as luciferase-based expression after intraperitoneal (IP) injection with the substrate D-luciferin, and expressed as total throughput (p / s) (see Methods). These in vivo experiments demonstrated that PIV5 and ORF4a prolonged the duration of luciferase expression in BL / 6 cells to 14 days, while the WT construct was negative at this time point. In further studies, the inventors evaluated the effects of these two RNA replicon constructs on Gaussian luciferase (GLuc as GO I) expression. Figure 5 The effects of PIV5 and ORF4a on luciferase expression were investigated. GLuc is secreted as a soluble protein, and its activity was measured in the blood (see Methods). These in vivo experiments demonstrated that PIV5 and ORF4a prolonged the duration of luciferase expression in BL / 6 cells to 14 days. At day 14, gLuc expression was significantly higher in both PIV5 and ORF4a RNA replicons (p = 0.0244 and 0.00422, respectively). The area under the curve (AUC) calculations for the period exceeding 14 days showed the following AUC values: fLuc BALBc = 1950; PIV5 BALBc = 2742; ORF4a BALBc = 1596; fLuc BL6 = 2012; PIV5 BL6 = 4513; ORF4a BL6 = 6972 (total flux (p / s)). While these in vivo experiments are supportive, it is important to note the significant differences between humans and mice in the specificity of innate limiting factors and interferon-stimulated genes (of which >100). Therefore, based on in vitro observations of human cell lines, these data likely underestimate the potential impact on humans.

[0435] Interferon inhibitory protein enhances the protein expression of saRNA in vitro.

[0436] The inventors sought to determine whether an interferon-inhibiting protein library could enhance the in vitro protein expression of firefly luciferase (fLuc). They prepared libraries containing a wide variety of cytoplasmic interferon targets (Table 1), including IRF-3, MDA5, RIG-I, and JAK / STAT. Figure 9 b) a saRNA VEEV replicon library, in which IIP is separated from fLuc by the T2A cleavage site ( Figure 9 a).

[0437]

[0438] Table 1. Interferon inhibition of VEEV replicons and associated IFN targets.

[0439] Then, the inventors used pABOL ( Figure 9c) saRNA was transfected into HEK293T.17, HeLa, and MRC5 cells. pABOL is a polymer delivery system previously characterized as producing relatively high protein expression, but is relatively immune-silencing due to its bioreducible nature (2). The inventors chose these three cell lines because they differ in the integrity of the IFN pathway; HEK293T.17 cells lack the complete pathway due to the absence of endogenous RIG-I and MDA5 expression (37), and should therefore be less sensitive to proteins affecting this pathway, while HeLa and MRC5 are more discriminative (38, 39). The inventors observed that no IIP replicon enhanced protein expression in HEK293T.17 cells, but interestingly, both langate and influenza IIP significantly reduced protein expression by 0.06-fold, with P = 0.0097 and 0.0061, respectively. In HeLa cells, many IIPs enhanced protein expression; in terms of fLuc expression, HSV-2, HSV-1_1, HSV-1_2, Orf, and BVDV showed increases ranging from 20 to 150-fold. However, PIV-5V and MERS-CoV ORF4a proteins showed the greatest enhancement in protein expression, at 796-fold and 893-fold, respectively, although this was only statistically significant in the PIV-5 group (p = 0.0272), while it was not in the ORF4a group (p = 0.0689). In MRC5 cells, the inventors also observed the greatest enhancement in PIV-5V and MERS-CoV ORF4a proteins, with 72-fold and 109-fold higher fLuc expression, respectively, where P = 0.0485 and 0.025. RNA from two separately prepared batches was used in all cell types and for each construct. Figure 14 There is good consistency between the expression levels of )

[0440] The inventors further investigated how two mutations in the PIV-5V and MERS-CoV ORF4a proteins in mice (MEF), rabbits (RK13), non-human primates (LLC), and human cells (MRC5) affect protein expression. Figure 15 The R172A mutation in PIV-5V eliminates the ability to block MDA5 but not STAT (40), while the K63A / K67A mutation in MERS-CoV ORF4a blocks binding to dsRNA (41). The inventors observed that neither PIV-5V nor MERS-CoV ORF4a proteins enhanced protein expression in MEF or RK13 cells. MERS-CoV ORF4a protein did indeed enhance protein expression in LLC and MRC5 cells (41). Figure 15(c, d) The K63A / K67A mutation significantly reduced protein expression. In MRC5 cells, PIV-5V protein expression was enhanced, but not in LLC cells, while the R172A mutation reduced protein expression in MRC5 cells. Overall, these data indicate that PIV-5V and MERS-CoV ORF4a proteins enhance protein expression in interferon-competent human cells, while K63A / K67A and R172A substitution mutations weaken saRNA expression.

[0441] The MERS-CoV ORF4a protein partially mitigates the nonlinear increase in dose in vivo.

[0442] Given the enhanced protein expression in vitro by PIV-5V and MERS-CoV ORF4a proteins, the inventors subsequently sought to determine whether these constructs could enhance in vivo protein expression and mitigate the nonlinearity of increasing saRNA dosage. The inventors tested saRNAs encoding both firefly luciferase (an intracellular protein) and Gaussian luciferase (an in vivo secreted protein) (Table 2).

[0443]

[0444] Table 2. Area under the curve (AUC) of total luciferase expression in BALB / c and C57BL6 / J over a 14-day period, n = 5.

[0445] Due to differences in interferon production capacity, the inventors chose to test these constructs in BALB / c and C57BL / 6 mice: BALB / c is a poor producer of IFN, while C57BL / 6 mice have previously been found to be high producers of IFN-α / β and IFN-γ (42), similar to the differences in HEK293T.17 and HeLa / MRC-5 cells in vitro. The inventors observed that integration of PIV-5V and MERS-CoV ORF4a proteins in BALB / c mice did not enhance protein expression of fLuc or GLuc (Table 2, ...). Figure 16 The inventors observed that the area under the curve (AUC) of total fLuc protein expression was slightly enhanced in C57BL / 6 mice with MERS-CoV ORF4a protein, while the area under the curve (AUC) of total GLuc protein expression was slightly increased in C57BL / 6 mice with PIV-5V and MERS-CoV ORF4a proteins, although this difference was not statistically significant.

[0446] The inventors had previously observed that increasing the dose of saRNA ultimately led to lower protein expression levels, and therefore attempted to characterize MERS-CoV ORF. 4aWhether the protein can attenuate the in vivo nonlinear dose-dependent expression of saRNA. The inventors tested wild-type fLuc and fLuc+MERS-CoV ORF4a replicons at doses of 0.2, 2, and 20 μg, and quantified protein expression on days 7 and 10 after intramuscular injection (IM). Figure 10 The inventors observed that after 7 days, both constructs exhibited similar protein expression (~5000 p / s) at a dose of 0.2 μg. When the dose was increased to 2 μg, protein expression increased in both constructs (WT ~50000 p / s for the MERS-CoV ORF4a construct and ~200,000 p / s for the MERS-CoV ORF4a construct), but integration of the MERS-CoV ORF4a protein resulted in a 4-fold increase in protein expression (p = 0.0029). Interestingly, after 7 days, both constructs showed lower protein expression at a dose of 20 μg, but the WT was 18-fold lower than that of the MERS-CoV construct (p < 0.0001). After 10 days, protein expression levels at the 2 μg dose were equal, and no expression was observed at the 0.2 and 20 μg doses. Without wishing to be bound by any particular theory, these data suggest that the MERS-CoV ORF4a protein can partially rescue the in vivo nonlinear dose-dependent nature of saRNA.

[0447] Ruxotinib enhanced the protein expression of saRNA in vivo.

[0448] Given the role of the JAK / STAT pathway in downstream interferon responses, the inventors subsequently sought to characterize how the combination of saRNA, MERS-CoV ORF4a interferon inhibitor protein, and ruxotinib (a potent, selective inhibitor of JAK1 and JAK2 protein kinases (36)) affected protein expression in vivo (Figure 3). The inventors injected mice with 100 μg of saRNA encoding fLuc±MERS-CoV ORF4a, either with or without 100 μg of ruxotinib, and quantified protein expression at 4, 7, 10, and 14 days post-injection. Four days later (… Figure 11 a) with WT or MERS-CoV ORF4a constructs (~5x10⁻¹⁰) 5 Compared to p / s, the two formulations containing ruxolitinib showed slightly higher protein expression (~10 p / s). 6The p / s values ​​were not statistically significant. However, after 7 days, both formulations containing ruxolitinib showed higher protein expression levels compared to the parallel group containing only saRNA, with p = 0.0347 and 0.0447, respectively. By day 10, these groups still showed slight increases, but the differences were no longer statistically significant. After 14 days, no protein expression was observed in the saRNA group without ruxolitinib, while only a few positive samples were observed in the ruxolitinib group. Without being bound by any particular theory, these data suggest that ruxolitinib can increase the depth of saRNA protein expression, but there is no synergistic effect between MERS-CoV ORF4a protein and ruxolitinib when combined.

[0449] In human skin explants, PIV-5V and MERS-CoV ORF4a proteins attenuated the nonlinear increase in in vitro dose.

[0450] Because the inventors observed differences in protein expression of IIPs in vitro based on cell type, they sought to test the saRNA IIP construct in a more clinically meaningful human skin explant model. The inventors characterized the amount of protein expression (eGFP) in resident human skin cells incorporating PIV-5V and MERS-CoV ORF4a proteins (referred to as MERS-COV_2 in the figure) and in those co-formulated with ruxolitinib. + (Percentage of cells) and quality (median eGFP fluorescence intensity per cell) Figure 12 The inventors tested eGFP saRNA containing PIV-5V and MERS-CoV ORF4a proteins at doses of 0.2, 2, and 20 μg. Figure 12 a), b), and observed that increasing the dose of the WT construct from 0.2 μg to 2 μg resulted in eGFP + The percentage of cells increased from 10% to 18%, but when the dose was increased to 20 μg, eGFP... + The percentage of cells dropped sharply to ~5%. However, for the PIV-5 and MERS-CoV ORF4a constructs, the dose increased linearly with increasing saRNA dosage. For both constructs, a dose of 0.2 μg also resulted in eGFP. + The concentration was approximately 12%, further increased to 15% at 2 μg, and to 25% at 20 μg. At these concentrations, both the PIV-5 and MERS-CoV ORF4a constructs exhibited statistically significantly higher eGFP levels. +Cell percentage, where for both, P < 0.0001. Interestingly, neither the dose nor the integration of PIV-5 or MERS-CoV ORF4a protein affected the eGFP MFI, which was ~350 for all samples. Figure 12 b).

[0451] The inventors further characterized which cells were expressing saRNA using t-distributed random neighborhood embedding, a principle-based component analysis type for flow cytometry data that allows visualization of unsupervised cell clustering by employing superimposed, defined protein and phenotypic gating. Figure 17-25 (43). The inventors observed that, at the highest dose of saRNA (20 μg), PIV-5V and MERS-CoV ORF4a proteins were expressed in immune cells, including T cells, dendritic cells, monocytes, B cells, Langerhans cells, leukocytes, and NK cells, but not in resident epithelial cells and fibroblasts.

[0452] Next, the inventors tested how the integrated dose of ruxolitinib (0-100 μg) affected saRNA expression in human skin resident cells. They observed that the co-formulation of ruxolitinib and saRNA had an effect on eGFP expression. + The percentage of cells has no effect. Figure 12 c) Although there was a slight trend that increasing the dose of ruxolitinib actually decreased the percentage of eGFP+ cells from ~8% to ~5%, the inventors did observe a profound effect on the quality of eGFP expression in each cell type. Figure 12 d) Increasing the dose of ruxolitinib, at a dose of 10 μg, the eGFP MFI increased from ~100 to ~2000, although at a dose of 100 μg, the MFI decreased to ~1000. Similar to cells expressing saRNA PIV-5 and MERS-CoV ORF4a proteins, the inventors found that ruxolitinib enhanced protein expression in immune cells but not in epithelial cells and fibroblasts, particularly increasing uptake in T cells, Langerhans cells, leukocytes, and NK cells. Figure 26 a,b).

[0453] In summary, without being bound by any particular theory, these data show that IIP replicons enhance expression in immune cells by increasing the proportion of cells expressing saRNA, while ruxolitinib enhances protein expression on a per-cell basis.

[0454] In rabbits, the MERS-CoV ORF4a protein enhances the immunogenicity of the RABV glycoprotein.

[0455] Since protein expression in nucleic acid preparations is not always a direct predictor of immunogenicity (2), the inventors subsequently attempted to characterize the immunogenicity of a model protein (i.e., the rabies virus glycoprotein (RABV) represented by GeneBank ID NP_056796.I) as a therapeutic biomolecule when combined with the MERS-CoV ORF4a protein (as an innate repressor protein or IIP) in the saRNA construction of the present invention. Furthermore, a RABV protein modified with F318V, which has amino acid substitutions and prevents binding to the cell P75NTR surface receptor, was used. The inventors injected rabbits with a primary dose of 20 μg of saRNA and a booster dose after 4 weeks, then sampled their blood for RABV-specific IgG antibodies at 0, 4, and 6 weeks. Figure 13 a). The inventors observed that seroconversion occurred in all rabbits, both wild-type and those using the MERS-CoV ORF4a construct, after a single injection. Four weeks later, the IgG titer in the MERS-CoV group (~10) 4 (ng / mL) compared to wild type (~5×10) 3 The antibody titer (ng / mL) was slightly higher, but not statistically significant. However, after 6 weeks, the antibody titer in the MERS-CoV ORF4a group was ~10 ng / mL. 5 ng / mL) and WT group (~10 4 The levels were significantly higher than those in ng / mL, with p = 0.0061. RABV pseudotype neutralization reflects the trend of antibody ( Figure 13 b). After 4 weeks, the average IC of the WT group 50 ~10 3 The IC of the MERS-CoV group 50 ~10 4 Six weeks later, the IC50 of the MERS-CoV group... 50 Much higher, ~10 5 The WT group has stabilized at ~10 3 The inventors also compared the immunogenicity of WT RABV and RABV-MERS-CoV ORF4a saRNA in mice and rats. Figure 27 However, no differences in antibody titers and neutralizing IC were observed in these species. 50 Any differences between them. Without wishing to be bound by any particular theory, these data suggest that the MERS-CoV ORF4a protein enhances the immunogenicity of the RABV glycoprotein encoded by saRNA in rabbits.

[0456] MERS-CoV ORF4a protein enhances the in vitro expression of SARS-CoV-2 glycoprotein.

[0457] The inventors also characterized the immunogenicity of another model protein (i.e., a therapeutic biomolecule, namely the SARS-CoV-2 glycoprotein represented by Genbank ID No: QHD43416.1) when combined with the MERS-CoV ORF4a protein (i.e., the innate inhibitor protein or IIP) in the saRNA construction of the present invention.

[0458] Twenty-four hours after transfection into the HeLa cell line, the surface expression levels of SARS-CoV-2 (without IIP) and SARS-CoV-2 combined with MERS-CoV ORF4a (IIP) were compared. (See reference...) Figure 29 The median fluorescence intensity of positive cell populations was measured, with the negative / positive cutoff value set at the gate of live, single, and simulated transfected cells. Surprisingly, the MERS-CoV ORF4a protein nearly doubled the expression level of SARS-CoV-2 glycoprotein per cell compared to saRNA that only encodes SARS-CoV-2 glycoprotein (i.e., without IIP).

[0459] discuss

[0460] The inventors screened a self-amplifying RNA library containing cis-encoded interferon-inhibiting proteins for protein expression in vitro, in human skin explants, and in mice in mouse, rabbit, non-human primate, and human cells, as well as for immunogenicity in mice, rats, and rabbits. The inventors observed that PIV-5V and MERS-CoV ORF4a proteins enhanced protein expression 100-500-fold in vitro in IFN-competent HeLa and MRC5 cells. They found that MERS-CoV ORF4a protein partially attenuated dose-nonlinearity in vivo, and that ruxotinib, rather than IIP, enhanced saRNA protein expression in vivo. In human skin explants expressing saRNA, both PIV-5V and MERS-CoV ORF4a proteins increased the percentage of resident cells and completely rescued the dose-nonlinearity of saRNA, while ruxotinib increased protein expression on a per-cell basis. Finally, the inventors observed that MERS-CoV ORF4a reduced RABV-specific IgG titers and neutralized IC50 in rabbits. 50 It increased by 10-fold, but this result was not observed in mice or rats.

[0461] The protein design, cell and mutation characterization in these experiments provided insights into the mechanisms by which PIV-5V and MERS-CoV ORF4a proteins enhance protein expression. PIV-5V blocks MDA-5 and IRF3 by binding to MDA-5 (26, 27), while MERS-CoV ORF4a binds to dsRNA and inhibits PACT triggering of MDA-5 and RIG-I (29–31). Following our in vitro screening, the inventors chose to continue using the MERS-CoV ORF4a replicon because screening all 10 candidates in vivo was not feasible, and PIV-5V is not conserved across species (e.g., the N100D mutation required for mouse adaptation (44)), while ORF4a is more highly conserved across species (29–31). The inventors observed that the R172A mutation in PIV-5V (which eliminates the ability to block binding to MDA-5 rather than STAT (45)) slightly inhibited protein expression in MRC5 cells. Figure 15 d), thus indicating that binding to MDA5 is partly responsible for the enhanced protein expression. Similarly, the K63A / K67A mutation in the MERS-CoV ORF4a protein restricts its ability to bind dsRNA (46, 47), and this mutation was observed to reduce protein expression in both non-human primates and human cells. Figure 15 (c, d). Although various IIPs inhibit interferon through mechanisms similar to those of PIV-5V and MERS-CoV ORF4a, the observed mechanisms of action do not necessarily predict the enhancement of protein expression.

[0462] The inventors had previously observed that protein expression in saRNA formulations does not necessarily predict immunogenicity (2), and therefore also characterized how the MERS-CoV ORF4a protein affects the immunogenicity of rabies virus glycoproteins in mice, rats, and rabbits. In rabbits, the inventors observed that the use of saRNA encoding RABV and MERS-CoV ORF4a resulted in antibody titers and neutralizing IC50 values. 50 All increased ( Figure 13 a, b), but no increase in immunogenicity was observed in mice or rats. While no preclinical animal model perfectly predicts human response, rabbits are considered more immunologically similar to humans than mice or rats (48-50). Furthermore, the inventors did not observe any enhancement in protein expression caused by PIV-5V or MERS-CoV ORF4a proteins in mouse cells ( Figure 15a) Therefore, the lack of enhanced immunogenicity is not surprising. The inventors paired features from preclinical animal models with human transplantation models, where cells were in native tissue structures possessing the inherent human IFN response. To the best of their knowledge, the inventors were the first to observe that IIP increased the percentage of cells expressing saRNA, while ruxolitinib enhanced expression per cell. Given these promising results, the inventors believe that MERS-CoV ORF in humans... 4a Proteins can enhance the immunogenicity of saRNA vaccines and are also useful for the application of saRNA in protein replacement therapy (51, 52).

[0463] These experiments provide proof-of-concept that IIP can be directly encoded into saRNA vectors and effectively reduce nonlinear dose-dependency and enhance immunogenicity. As the mechanistic studies have shown, different aspects of the interferon pathway can be targeted and increase saRNA expression, prompting exploration of combinations of IIP with other IFN inhibitory strategies, such as ruxolitinib.

[0464] Statement of Financial Support

[0465] The project for which this application was submitted was funded by the EU Horizon 2020 research and innovation program under Marie Sklodowska-Curie authorization agreement No. 794059.

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ACS Nano 13, 5920-5930 (2019). sequence list <110> Imperial College Innovation Ltd. <120> RNA construct <130> FP214242GB <150> GB1908729.5 <151> 2019-06-18 <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 196 <212> PRT <213> Human herpesvirus 2 <400> 1 Val Arg Asp Cys Tyr Leu Met Gly Tyr Cys Arg Thr Arg Leu Gly Pro 1 5 10 15 Arg Thr Trp Gly Arg Leu Leu Gln Ile Ser Gly Gly Thr Trp Asp Val 20 25 30 Arg Leu Arg Asn Ala Ile Arg Glu Val Glu Ala His Phe Glu Pro Ala 35 40 45 Ala Glu Pro Val Cys Glu Leu Pro Cys Leu Asn Ala Arg Arg Tyr Gly 50 55 60 Pro Glu Cys Asp Val Gly Asn Leu Glu Thr Asn Gly Gly Ser Thr Ser 65 70 75 80 Asp Asp Glu Ile Ser Asp Ala Thr Asp Ser Asp Asp Thr Leu Ala Ser 85 90 95 His Ser Asp Thr Glu Gly Gly Pro Ser Pro Ala Gly Arg Glu Asn Pro 100 105 110 Glu Ser Ala Ser Gly Gly Ala Ile Ala Ala Arg Leu Glu Cys Glu Phe 115 120 125 Gly Thr Phe Asp Trp Thr Ser Glu Glu Gly Ser Gln Pro Trp Leu Ser 130 135 140 Ala Val Val Ala Asp Thr Ser Ser Ala Glu Arg Ser Gly Leu Pro Ala 145 150 155 160 Pro Gly Ala Cys Arg Ala Thr Glu Ala Pro Glu Arg Glu Asp Gly Cys 165 170 175 Arg Lys Met Arg Phe Pro Ala Ala Cys Pro Tyr Pro Cys Gly His Thr 180 185 190 Phe Leu Arg Pro 195 <210> 2 <211> 588 <212> DNA <213> human herpesvirus 2 <400> 2 gtgagagact gctacctgat gggctactgc cggacaagac tgggacctag aacatggggc 60 agactgctgc agatcagcgg cggaacatgg gatgtgcggc tgagaaacgc catcagagag 120 agactgctgc agatcagcgg cggaacatgg gatgtgcggc tgagaaacgc catcagagag 120 gtggaagccc acttcgagcc tgccgctgaa cctgtgtgtg aactgccctg tctgaacgct 180 gtggaagccc acttcgagcc tgccgctgaa cctgtgtgtg aactgccctg tctgaacgct 180 agaagatacg gccctgagtg cgacgtgggc aacctggaaa caaatggcgg cagcaccagc 240 agaagatacg gccctgagtg cgacgtgggc aacctggaaa caaatggcgg cagcaccagc 240 gacgacgaga tttccgatgc caccgacagc gacgatacac tggccagcca cagcgataca 300 gacgacgaga tttccgatgc caccgacagc gacgatacac tggccagcca cagcgataca 300 gaaggcggac catctcctgc cggaagagag aatcctgagt ctgcctctgg cggagccatt 360 gaaggcggac catctcctgc cggaagagag aatcctgagt ctgcctctgg cggagccatt 360 gccgctagac tggaatgcga gttcggcacc ttcgactgga caagcgagga aggctctcag 420 gccgctagac tggaatgcga gttcggcacc ttcgactgga caagcgagga aggctctcag 420 ccttggctgt ctgctgtggt ggccgataca agctctgccg agagaagtgg acttcctgct 480 ccttggctgt ctgctgtggt ggccgataca agctctgccg agagaagtgg acttcctgct 480 cctggcgcct gtagagctac agaggctcct gaaagagagg acggctgcag aaagatgcgg 540 cctggcgcct gtagagctac agaggctcct gaaagagagg acggctgcag aaagatgcgg 540 ttccctgccg cctgtcctta tccttgcggc cacacatttc tgcggccc 588 ttccctgccg cctgtcctta tccttgcggc cacacatttc tgcggccc 588 <210> 3<210> 3 <211> 395 <211> 395 <212> PRT <212> PRT <213> 人类疱疹病毒1(human herpesvirus 1) <213> Human herpesvirus 1 <400> 3 <400> 3 Val Arg Asp Cys Tyr Leu Met Gly Tyr Cys Arg Thr Arg Leu Gly Pro Val Arg Asp Cys Tyr Leu Met Gly Tyr Cys Arg Thr Arg Leu Gly Pro 1 5 10 15 1 5 10 15 Arg Thr Trp Gly Arg Leu Leu Gln Ile Ser Gly Gly Thr Trp Asp Val 20 25 30 Arg Leu Arg Asn Ala Ile Arg Glu Val Glu Ala His Phe Glu Pro Ala 35 40 45 Ala Glu Pro Val Cys Glu Leu Pro Cys Leu Asn Ala Arg Arg Tyr Gly 50 55 60 Pro Glu Cys Asp Val Gly Asn Leu Glu Thr Asn Gly Gly Ser Thr Ser 65 70 75 80 Asp Asp Glu Ile Ser Asp Ala Thr Asp Ser Asp Asp Thr Leu Ala Ser 85 90 95 His Ser Asp Thr Glu Gly Gly Pro Ser Pro Ala Gly Arg Glu Asn Pro 100 105 110 Glu Ser Ala Ser Gly Gly Ala Ile Ala Ala Arg Leu Glu Cys Glu Phe 115 120 125 Gly Thr Phe Asp Trp Thr Ser Glu Glu Gly Ser Gln Pro Trp Leu Ser 130 135 140 Ala Val Val Ala Asp Ile Arg Asp Cys Tyr Leu Met Gly Tyr Cys Arg 145 150 155 160 Ala Arg Leu Ala Pro Arg Thr Trp Cys Arg Leu Leu Gln Val Ser Gly 165 170 175 Gly Thr Trp Gly Met His Leu Arg Asn Thr Ile Arg Glu Val Glu Ala 180 185 190 Arg Phe Asp Ala Thr Ala Glu Pro Val Cys Lys Leu Pro Cys Leu Glu 195 200 205 Thr Arg Arg Tyr Gly Pro Glu Cys Asp Leu Ser Asn Leu Glu Ile His 210 215 220 Leu Ser Ala Thr Ser Asp Asp Glu Ile Ser Asp Ala Thr Asp Leu Glu 225 230 235 240 Ala Ala Gly Ser Asp His Thr Leu Ala Ser Gln Ser Asp Thr Glu Asp 245 250 255 Ala Pro Ser Pro Val Thr Leu Glu Thr Pro Glu Pro Arg Gly Ser Leu 260 265 270 Ala Val Arg Leu Glu Asp Glu Phe Gly Glu Phe Asp Trp Thr Pro Gln 275 280 285 Glu Gly Ser Gln Pro Trp Leu Ser Ala Val Val Ala Asp Thr Ser Ser 290 295 300 Val Glu Arg Pro Gly Pro Ser Asp Ser Gly Ala Gly Arg Ala Ala Glu 305 310 315 320 Asp Arg Lys Cys Leu Asp Gly Cys Arg Lys Met Arg Phe Ser Thr Ala 325 330 335 Cys Pro Tyr Pro Cys Ser Asp Thr Phe Leu Arg Pro Thr Ser Ser Ala 340 345 350 Glu Arg Ser Gly Leu Pro Ala Pro Gly Ala Cys Arg Ala Thr Glu Ala 355 360 365 Pro Glu Arg Glu Asp Gly Cys Arg Lys Met Arg Phe Pro Ala Ala Cys 370 375 380 Pro Tyr Pro Cys Gly His Thr Phe Leu Arg Pro 385 390 395 <210> 4 <211> 597 <212> DNA <213> Human herpesvirus 1 <400> 4 atcagagact gctacctgat gggctactgc cgggctagac tggcccctag aacatggtgc 60 agactgctgc aagtgtctgg cggcacatgg ggcatgcacc tgagaaacac catcagagag 120 gtggaagcca gattcgacgc cacagccgag cctgtgtgca agctgccttg tctggaaact 180 cggagatacg gccccgagtg cgacctgagc aatctggaaa ttcacctgag cgccaccagc 240 gacgacgaga tttctgatgc caccgacctg gaagccgccg gatctgatca tacactggcc 300 agccagagcg acaccgagga tgctccatct ccagtgactc tggaaacccc tgagcctaga 360 ggatctctgg ccgtgcgact ggaagatgag ttcggcgagt tcgactggac ccctcaagag 420 ggatctcagc cttggctgtc tgccgtggtg gccgatacaa gcagcgtgga aagacccgga 480 cctagcgatt ctggtgctgg cagagccgcc gaggatagaa agtgcctgga tggctgccgg 540 aagatgcggt tctctaccgc ctgtccatat ccttgcagcg acaccttcct gcggcct 597 <210> 5 <211> 161 <212> PRT <213> Human herpesvirus 1 (human herpesvirus 1) <400> 5 Put Ser Gln Thr Gln Pro Pro Here Pro Val Gly Pro Gly Asp Pro Asp 1 5 10 15 Val Tyr Leu Lys Gly Val Pro Ser Ala Gly Met His Pro Arg Gly Val 20 25 30 His Ala Pro Arg Gly His Pro Arg Met Ile Ser Gly Pro Pro Gln Arg 35 40 45 Gly Asp Asn Asp Gln Ala Ala Gly Gln Cys Gly Asp Ser Gly Leu Leu 50 55 60 Arg Val Gly Ala Asp Thr Thr Ile Ser Lys Pro Ser Glu Ala Val Arg 65 70 75 80 Pro Pro Thr Ile Pro Arg Thr Pro Arg Val Pro Arg Glu Pro Arg Val 85 90 95 Pro Arg Pro Pro Arg Glu Pro Arg Glu Pro Arg Val Pro Arg Ala Pro 100 105 110 Arg Asp Pro Arg Val Pro Arg Asp Pro Arg Asp Pro Arg Gln Pro Arg 115 120 125 Ser Pro Arg Glu Pro Arg Ser Pro Arg Glu Pro Arg Ser Pro Arg Glu 130 135 140 Pro Arg Thr Pro Arg Thr Pro Arg Glu Pro Arg Thr Ala Arg Gly Ser 145 150 155 160 Val <210> 6 <211> 483 <212> DNA <213> human herpesvirus 1 <400> 6 atgagccaga cacagcctcc agctccagtt ggacctggcg accctgatgt gtatctgaag 60 ggcgtgccaa gcgccggcat gcatcctaga ggtgttcatg cccctagagg acaccccaga 120 atgatctctg gccctcctca gagaggcgac aacgatcagg ctgctggaca gtgtggcgat 180 agcggactgc tgagagtggg cgccgatacc acaatcagca agccatctga ggctgtgcgg 240 cctcctacaa tccccagaac acctagagtg ccccgcgagc caagagtgcc tagactcct 300 agagagccca gagaacccag agtgccaagg gctcccagag atcctagagt ccccgggac 360 cctagggacc agagccaacc tegtcaccc agagagccctc ggaggcccaag agagccaag 420 agccctaggg aaccccggac accagaca cccagggaac ctagaccgc cagaggcagc 480 gtg 483 <210> 7 <211> 183 <212> PRT <213> Orf virus <400> 7 Met Ala Cys Glu Cys Ala Ser Leu Ileu Glu Leu Leu Arg Lys Ser 1 5 10 15 Asp Asp Lys Pro Ala Lys Gln Ile Ala Lys Glu Leu Gly Ile Ser 20 25 30 Lys His Glu Ala Asn Arg Gln Leu Tyr Arg Leu Asp Ser Asp Glu 35 40 45 Val Cys Cys Glu Asp Gly Asn Pro Pro Arg Trp Phe Val Glu Cys Ala 50 55 60 Pro Ser Ala Pro Thr Glu Glu Asp Glu Asn Ser Asp Thr Glu Pro Met 65 70 75 80 Glu Thr Glu Ala Gly Cys Asp Thr Leu Phe Gly Gly Asp Ile Asp Ile 85 90 95 Met Thr Gln Ser Ala Val Ile Arg Leu Lys Ser Leu Asn Pro Val Ser 100 105 110 Ala Val Asn Glu Phe Cys Met Met Thr His Arg Pro Leu Glu Phe Cys 115 120 125 Glu Thr Arg Ala Gly Gly Glu Asp His Cys Pro Arg Phe Thr Cys Thr 130 135 140 Ile Thr Ile Ser Gly Lys Val Val Ala Val Ala Asp Gly Ala Ser Lys 145 150 155 160 Lys Leu Ala Arg His Thr Ala Cys Ser Ser Ala Leu Thr Ile Leu Ile 165 170 175 Asn Asn Cys Gly Ile Ser Phe 180 <210> 8 <211> 549 <212> DNA <213> Orf virus <400> 8 atggcctgtg aatgcgccag cctgatcctg gaactgctga gaaagagcga cgacaagctg 60 cccgccaagc agatcgccaa agagctgggc atctctaagc acgaggccaa ccggcagctg 120 taccggctgc tggattctga cgaagtgtgc tgcgaggacg gcaatcctcc tcgttggttc 180 gtggaatgtg cccctagcgc tcccaccgaa gaggacgaga atagcgacac cgagcctatg 240 gaaaccgagg ccggctgcga tacactgttt ggcggagaca tcgacatcat gacccagagc 300 gccgtgatcc ggctgaagtc cctgaatcct gtgtccgccg tgaacgagtt ctgcatgatg 360 acccaccggc ctctggaatt ttgcgagaca agagccggcg gagaggatca ctgccccaga 420 ttcacctgta ccatcaccat cagcggcaag gtggtggctg ttgccgatgg cgcctctaag 480 aaactggcca gacacaccgc ctgtagcagc gccctgacaa tcctgatcaa caactgcggc 540 atcagcttc 549 <210> 9 <211> 168 <212> PRT <213> pestivirus type 1 <400> 9 Met Glu Leu Ile Thr Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys 1 5 10 15 Pro Val Gly Val Glu Glu Pro Val Tyr Asp Gln Ala Gly Asp Pro Leu 20 25 30 Phe Gly Glu Arg Gly Ala Val His Pro Gln Ser Thr Leu Lys Leu Pro 35 40 45 His Lys Arg Gly Glu Arg Asp Val Pro Thr Asn Leu Ala Ser Leu Pro 50 55 60 Lys Arg Gly Asp Cys Arg Thr Gly Asn Ser Arg Gly Pro Val Ser Gly 65 70 75 80 Ile Tyr Leu Lys Pro Gly Pro Leu Phe Tyr Gln Asp Tyr Lys Gly Pro 85 90 95 Val Tyr His Arg Ala Pro Leu Glu Leu Phe Glu Glu Gly Ser Met Cys 100 105 110 Glu Thr Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu 115 120 125 Tyr His Ile Tyr Val Cys Ile Asp Gly Cys Ile Ile Ile Lys Ser Ala 130 135 140 Thr Arg Ser Tyr Gln Arg Val Phe Arg Trp Val His Asn Arg Leu Asp 145 150 155 160 Cys Pro Leu Trp Val Thr Ser Cys 165 <210> 10 <211> 504 <212> DNA <213> pestivirus type 1 <400> 10 atggaactga tcaccaacga gctgctgtac aagacctaca agcagaaacc cgtgggcgtc 60 gaggaacccg tgtatgatca agctggcgac cctctgtttg gcgagagagg cgctgttcac 120 cctcagagca cactgaagct gccccacaag cggggcgaaa gagatgtgcc taccaacctg 180 gccagcctgc ctaagagagg cgattgcaga accggcaata gcagaggccc tgtgtccggc 240 atctacctga aacctggacc actgttctac caggactaca agggacccgt gtaccacaga 300 gcccctctgg aactgtttga agagggcagc atgtgcgaaa ccaccaagcg gatcggaaga 360 gtgaccggct ctgacggcaa gctgtaccac atctacgtgt gcatcgacgg ctgcatcatc 420 atcaagagcg ccaccagatc ctaccagcgg gtgttcagat gggtgcacaa cagactggac 480 tgccctctgt gggtcaccag ctgc 504 <210> 11 <211> 222 <212> PRT <213> Simian parainfluenza virus 5 <400> 11 Met Asp Pro Thr Asp Leu Ser Phe Ser Pro Asp Glu Ile Asn Lys Leu 1 5 10 15 Ile Glu Thr Gly Leu Asn Thr Val Glu Tyr Phe Thr Ser Gln Gln Val 20 25 30 Thr Gly Thr Ser Ser Leu Gly Lys Asn Thr Ile Pro Pro Gly Val Thr 35 40 45 Gly Leu Leu Thr Asn Ala Ala Glu Ala Lys Ile Gln Glu Ser Thr Asn 50 55 60 His Gln Lys Gly Ser Val Gly Gly Gly Ala Lys Pro Lys Lys Pro Arg 65 70 75 80 Pro Lys Ile Ala Ile Val Pro Ala Asp Asp Lys Thr Val Pro Gly Lys 85 90 95 Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Pro Ser Thr Gln 100 105 110 Thr Val Leu Asp Leu Ser Gly Lys Thr Leu Pro Ser Gly Ser Tyr Lys 115 120 125 Gly Val Lys Leu Ala Lys Phe Gly Lys Glu Asn Leu Met Thr Arg Phe 130 135 140 Ile Glu Glu Pro Arg Glu Asn Pro Ile Ala Thr Ser Ser Pro Ile Asp 145 150 155 160 Phe Lys Arg Gly Arg Asp Thr Gly Gly Phe His Arg Arg Glu Tyr Ser 165 170 175 Ile Gly Trp Val Gly Asp Glu Val Lys Val Thr Glu Trp Cys Asn Pro 180 185 190 Ser Cys Ser Pro Ile Thr Ala Ala Ala Arg Arg Phe Glu Cys Thr Cys 195 200 205 His Gln Cys Pro Val Thr Cys Ser Glu Cys Glu Arg Asp Thr 210 215 220 <210> 12 <211> 666 <212> DNA <213> Simian parainfluenza virus 5 <400> 12 atggacccta ccgacctgag cttcagcccc gacgagatca acaagctgat cgagacaggc 60 ctgaacaccg tggaatactt caccagccag caagtgaccg gcacaagcag cctgggcaag 120 aacacaattc ctccaggcgt gaccggcctg ctgacaaatg ctgccgaggc caagatccaa 180 gagagcacca accaccagaa gggctctgtt ggaggcggag ccaagcctaa gaagcccaga 240 cctaagatcg ccatcgtgcc cgccgacgat aagacagtgc ctggcaagcc cattcctaat 300 cctctgctgg gcctcgacag cacccctagc acacagacag tgctggatct gagcggcaag 360 acactgccta gcggcagcta taagggcgtg aagctggcca agttcggcaa agaaaacctg 420 atgacccggt tcatcgagga acccagagag aaccctatcg ccaccagctc tcccatcgac 480 ttcaagagag gcagagacac cggcggcttc cacagaagag agtacagcat tggctgggtc 540 ggagatgaag tgaaagtgac cgagtggtgc aaccccagct gcagccctat tacagccgcc 600 gctagaagat tcgagtgcac ctgtcaccag tgtcctgtga cctgtagcga gtgcgagcgg 660 gacaca 666 <210> 13 <211> 230 <212> PRT <213> Influenza virus <400> 13 Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His Val Arg Lys Gln Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe 20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly Ser 35 40 45 Thr Leu Gly Leu Asn Ile Glu Thr Ala Thr Cys Val Gly Lys Gln Ile 50 55 60 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Phe Arg Met Thr 65 70 75 80 Met Ala Ser Ala Leu Ala Ser Arg Tyr Leu Thr Asp Met Thr Ile Glu 85 90 95 Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Ala 100 105 110 Gly Pro Leu Cys Val Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile 115 120 125 Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Asp Arg Leu Glu Thr Leu 130 135 140 Thr Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Ile Val Gly Glu Ile 145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn 165 170 175 Ala Ile Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Asp Asn Thr Val 180 185 190 Arg Val Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser Asn Glu 195 200 205 Asn Gly Gly Pro Pro Leu Thr Pro Thr Gln Lys Arg Lys Met Ala Gly 210 215 220 Lys Ile Arg Ser Glu Val 225 230 <210> 14 <211> 693 <212> DNA <213> Influenza virus <400> 14 atggattcca acactgtgtc aagctttcag gtagattgct tccttttggca tgtccgcaaa 60 caagttgcag accagagct aggtgatgcc ccattccttg atcggctcg ccgagatcag 120 aagtccctaa agggaaggg cagcactctc ggtctgaaca tcgaacagc cacctgtgtt 180 ggaaagcaaa tagtagagag gattctgaag gagaatccg atgaggcatt tgaatgacc 240 atggcctccg cacttgcttc gcgataccta actgacatga ctattgaaga gatgtcagg 300 gactggttca tgctcatgcc caagcagaaa gtggcaggcc ctcttgtgt cagaatggac 360 caggcgataa tggataagaa catcatactg aaagcgaatt tcagtgtgat tttgaccgg 420 ttggagactc tgacattact aagggctttc accgaagagg gagcaatgt tggcgaatt 480 tcaccattgc cttctcttcc aggacatact atgaggatg tcaaaatgc aattggggtc 540 ctcatcgggg gacttgaatg gatgataac acagttcgag tcttgaac tctacagaga 600 ttcgcttgga gaagcagtaa tgagaatgggg ggacctccac tcactccac acagaaacgg 660 aaaatggcgg gaaaattag gtcagaagtt tga 693 <210> 15 <211> 109 <212> PRT <213> Middle East respiratory syndrome-related coronavirus <400> 15 Met Asp Tyr Val Ser Leu Leu Asn Gln Ile Trp Gln Lys Tyr Leu Asn 1 5 10 15 Ser Pro Tyr Thr Thr Cys Leu Tyr Ile Pro Lys Pro Thr Ala Lys Tyr 20 25 30 Thr Pro Leu Val Gly Thr Ser Leu His Pro Val Leu Trp Asn Cys Gln 35 40 45 Leu Ser Phe Ala Gly Tyr Thr Glu Ser Ala Val Asn Ser Thr Lys Ala 50 55 60 Leu Ala Lys Gln Asp Ala Ala Gln Arg Ile Ala Trp Leu Leu His Lys 65 70 75 80 Asp Gly Gly Ile Pro Asp Gly Cys Ser Leu Tyr Leu Arg His Ser Ser 85 90 95 Leu Phe Ala Gln Ser Glu Glu Glu Glu Ser Phe Ser Asn 100 105 <210> 16 <211> 327 <212> DNA <213> Middle East respiratory syndrome-related coronavirus <400> 16 atggactacg tgtccctgct gaaccagatt tggcagaagt acctgaacag cccctacacc acctgtctgt acatccccaa gcctaccgcc aagtacacac ctctcgtggg cacatctctg caccccgtgc tgtggaattg ccagctgagc tttgccggct acaccgagtc tgccgtgaac 180 agcacaaagg ccctggccaa acaggacgcc gctcagaga ttgcctggct gctgcacaag gatggcggca tccctgatgg ctgtagcctg tacctgagac acagcagcct gttcgcccag 300 stirring stirringcctt stirring <210> 17 <211> 381 <212> PRT <213> Anti-inflammatory (Langat virus) <400> 17 Val Phe Lys Asp Lys Val Asp Thr Lys Ala Gln Glu Pro Gln Pro Gly 1 5 10 15 Thr Lys Ile Ile Met Arg Ala Val Asn Asp Trp Leu Leu Glu Arg Leu 20 25 30 Val Lys Light Ser Arg Pro Arg Met Cys Ser Arg Glu Glu Phe Ile Ala 35 40 45 Lys Val Arg Ser Asn Ala Ala Leu Gly Ala Trp Ser Asp Glu Gln Asn 50 55 60 Lys Trp Lys Ser Ala Arg Glu Ala Val Glu Asp Pro Glu Phe Trp Ser 65 70 75 80 Leu Val Glu Ala Glu Arg Glu Arg His Leu Gln Gly Arg Cys Ala His 85 90 95 Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe 100 105 110 Gly Val Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ser 115 120 125 Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp 130 135 140 Ala Ser Arg Ala Ser Ser Gly Ala Gly Val Glu Gly Ile Ser Leu Asn 145 150 155 160 Tyr Leu Gly Trp His Leu Lys Lys Leu Ala Ser Leu Ser Gly Gly Leu 165 170 175 Phe Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Lys Ile Thr Asn Ala 180 185 190 Asp Leu Asp Asp Glu Glu Gln Ile Leu Arg Tyr Met Asp Gly Asp His 195 200 205 Lys Lys Leu Ala Ala Thr Val Leu Arg Lys Ala Tyr His Ala Lys Val 210 215 220 Val Arg Val Ala Arg Pro Ser Arg Glu Gly Gly Cys Val Met Asp Ile 225 230 235 240 Ile Thr Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Val Thr Tyr Ala 245 250 255 Leu Asn Thr Ile Thr Asn Ile Lys Val Gln Leu Val Arg Met Met Glu 260 265 270 Gly Glu Gly Val Ile Glu Val Ala Asp Ser His Asn Pro Arg Leu Leu 275 280 285 Arg Val Glu Lys Trp Leu Glu Glu His Gly Glu Glu Arg Leu Ser Arg 290 295 300 Met Leu Val Ser Gly Asp Asp Cys Val Val Arg Pro Val Asp Asp Arg 305 310 315 320 Phe Ser Lys Ala Leu Tyr Phe Leu Asn Asp Met Ala Lys Thr Arg Lys 325 330 335 Asp Thr Gly Glu Trp Glu Pro Ser Thr Gly Phe Ala Ser Trp Glu Glu 340 345 350 Val Pro Phe Cys Ser His His Phe His Glu Leu Val Met Lys Asp Gly 355 360 365 Arg Ala Leu Val Val Pro Cys Arg Asp Gln Asp Glu Leu 370 375 380 <210> 18 <211> 1143 <212> DNA <213> Langat virus <400> 18 gtgttcaagg acaaggtgga caccaaggct caagagcctc agcctggcac caagatcatc 60 atgagagccg tgaacgactg gctgctggaa cggctggtca agaaaagcag accccggatg 120 tgcagccggg aagagtttat cgccaaagtg cggagcaatg ccgctctcgg agcttggagt 180 gacgagcaga acaagtggaa gtccgccaga gaagccgtgg aagatcccga gttttggagc 240 ctggtggaag ccgagagaga gaggcatctg cagggaagat gtgcccactg cgtgtacaac 300 atgatgggca agagagagaa gaagctgggc gagttcggag tggccaaagg cagcagagcc 360 atctggtata tgtggctggg cagccgcttc ctggaatttg aggccctggg cttcctgaac 420 gaggatcact gggctagcag agcctcttct ggtgctggcg tggaaggcat cagcctgaat 480 tatctcggct ggcacctgaa gaaactggcc tctctgtctg gcggcctgtt ctacgccgat 540 gatacagccg gatgggacac aaagatcacc aacgccgacc tggacgacga ggaacagatc 600 ctgagatata tggacggcga ccacaaaaag ctggccgcca ccgtgctgag aaaggcctat cacgccaagg tcgtcagagt ggccagacct aggravate gcggctgcgt gatggacatc 720 atcaccaga gggaccagcg cggctctggc caggttgtga catacgccct cggctctc accaacatca aggtgcagct cgtgcggatg atggaaggcg agggcgtgat cgaagtggcc gacagccata atcctcggct gctgagagtg gaaaagtggc tggagaca cggcgaga cggctgagca gaatgctggt gtccggcgac gattgtgttg tgcggcccgt ggacgacaga 960 ttcagcaagg ccctgtactt tctgaatgac atggccaaga ccagaaagga caccggcgag tggggagcctt ctacaggctt tgccagctgg gaagaagtgc ctttctgcag ccaccacttc 1080 ccgagctgg tcatgaagga tggcagagcc ctggtggtgc cctgcagaga tcaggacgaa ctg 1143 <210> 19 <211> 693 <212> RNA <213> Influenza virus (Influenza virus) <400> 19 60. auggauucca acacuguguc aagcuuucag guagauugcu uccuuuggca uguccgcaaa 120 aagucccuaa agggaagagg cagcacucuc ggucugaaca ucgaaacagc caccuguguu 180 ggaaagcaaa uaguagagag gauucugaag gaagaauccg augaggcauu uagaaugacc 240 auggccuccg cauugcuuc gcgauaccua agucaauga cuauugaa gauguagg 300 gacugguuca ugcucaugcc caagcagaaa guggcaggcc cucuuuugu cagaauggac 360 caggcgauaa uggauaagaa caucauacug aaagcgaauu ucagugugau uuuugaccgg 420 uuggagacuc ugacauuacu aagggcuuuc accgaagagg gagcaauugu uggcgaaauu 480 ucaccauugc cuucucuucc aggacauacu aaugaggaug ucaaaaaugc aauugggguc 540 cucaucgggg gacuugaaug gaaugauaac acaguucgag ucucugaaac ucuaacagaga 600 uucgcuugga gaagcaguaa ugagaauggg ggaccuccac ucacuccaac acagaaacgg 660 aaaauggcgg gaaaaaauuag your voice is 693 <210> 20 <211> 78 <212> PRT <213> Severe acute respiratory syndrome coronavirus 2 <400> 20 Met Met Pro Thr Ile Phe Phe Ala Gly Ile Leu Ile Val Thr Thr Ile 1 5 10 15 Val Tyr Leu Thr Ile Val Gln Leu Leu Gln Leu Ser Leu Leu Gln Val 20 25 30 Met Ala Gln Gln Val Leu Phe Leu Asn Met Thr Thr Arg Leu Val Val 35 40 45 Ile Leu Lys Asn Gly Asn Leu Glu Gln Lys Thr Val Leu Tyr Tyr Thr 50 55 60 Val Thr Ser Leu Gln Thr Ile Thr Ser Cys Thr Gln Leu Asn 65 70 75 <210> twenty one <211> 19 <212> PRT <213> Porcine teschovirus <400> twenty one Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> twenty two <211> 20 <212> PRT <213> East Asian virus (Thosea asigna virus) <400> twenty two Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> twenty three <211> twenty two <212> PRT <213> Equine Rhinovirus type 1 <400> twenty three Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <210> twenty four <211> 4 <212> PRT <213> Artificial Sequence <220>

Claims

1. A self-amplifying RNA (saRNA) construct encoding (i) at least one therapeutic biomolecule; and (ii) at least one innate inhibitor protein (IIP), wherein the at least one innate inhibitor protein is Middle East Respiratory Syndrome Coronavirus (MERS-CoV) ORF4a with the amino acid sequence represented by SEQ ID No: 15, or parainfluenza virus type 5 V protein (PIV5 V) with the amino acid sequence represented by SEQ ID No:

11.

2. The saRNA construct according to claim 1, wherein, The saRNA construct comprises or is derived from a positive-sense RNA virus selected from the following genera: alphavirus; microvirus; flavivirus; rubellavirus; plaguevirus; hepatitis virus; calicivirus; and coronavirus.

3. The saRNA construct according to claim 1 or 2, wherein, The saRNA construct comprises or is derived from a virus selected from the group consisting of the following species: Venezuelan equine encephalitis virus (VEEV); enterovirus 71; encephalomyocarditis virus; Cuting virus and Middle East respiratory syndrome virus.

4. The saRNA construct according to claim 1, wherein, The construct is derived from VEEV.

5. The saRNA construct according to claim 1, wherein, The at least one therapeutic biomolecule is a vaccine construct or a therapeutic protein.

6. The saRNA construct according to claim 1, wherein, The at least one therapeutic biomolecule: (i) is an RNA molecule that can regulate the expression of endogenous host genes; (ii) Derived from bacteria, viruses, fungi, protozoa, or parasites; (iii) Select from the following groups: enzymes; enzyme inhibitors; hormones; immune system proteins; receptors; binding proteins; transcription or translation factors; tumor growth inhibitory proteins; structural proteins and hemoglobins; (iv) is an antigen.

7. The saRNA construct according to claim 6, wherein, The antigen in question is a tumor antigen.

8. The saRNA construct according to claim 1, wherein, The at least one therapeutic biomolecule encoded by the saRNA molecule is a protein or peptide derived from a pathogen selected from the group consisting of bacteria, viruses, fungi, protozoa, and parasites.

9. The saRNA construct according to claim 8, wherein, The protein or peptide is a viral antigen derived from a virus selected from the group consisting of: orthomyxoviruses; paramyxoviridae viruses; poxviridae viruses; microRNA viruses; Bunyaviruses; sandfly viruses; Nairoviruses; cloacal viruses; arteritis viruses; flaviviruses; plague viruses; hepatotropic DNA viruses; rhabdoviruses; caliciviruses; coronaviruses; retroviruses; respiratory enteroviruses; parvoviruses; hepatitis D virus (HDV); hepatitis E virus (HEV); human herpesviruses; and papillomaviruses.

10. The saRNA construct according to claim 9, wherein, The paramyxoviridae family of viruses includes metapneumovirus, measles virus, pneumonia virus, and paramyxovirus.

11. The saRNA construct according to claim 9, wherein, The small RNA viruses include enteroviruses and hepatotropic RNA viruses.

12. The saRNA construct according to claim 9, wherein, The enveloped viruses include alphaviruses.

13. The saRNA construct according to claim 9, wherein, The coronaviruses mentioned are SARS-CoV-1, SARS-CoV-2, MERS, human respiratory coronavirus, avian infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), or porcine transmissible gastroenteritis virus (TGEV).

14. The saRNA construct according to claim 1, wherein, The at least one innate inhibitor protein is capable of: (i) reducing or blocking the action of melanoma differentiation-associated protein 5 (MDA5) and / or (ii) blocking or reducing the binding of PKR-activating proteins to RNA.

15. The saRNA construct according to claim 1, wherein, The saRNA construct contains RNA nucleotide sequences as shown in SEQ ID NO: 47 and / or SEQ ID NO:

48.

16. The saRNA construct according to claim 1, wherein, The at least one innate inhibitor protein can inhibit one or more downstream pathways activated by MDA5, or block downstream pathways recognized by MDA / PACT of one or more dsRNAs.

17. The saRNA construct according to claim 1, wherein, The saRNA construct contains a promoter or subgenomic promoter operatively linked to a sequence encoding the at least one therapeutic biomolecule and the at least one innate inhibitor protein, enabling it to transcribe nucleotide sequences encoding the therapeutic biomolecule and the at least one innate inhibitor protein.

18. The saRNA construct according to claim 17, wherein, The promoter is 26S.

19. The saRNA construct according to claim 18, wherein, The promoter contains a nucleotide sequence as shown in SEQ ID NO:

57.

20. The saRNA construct according to claim 1, wherein, The saRNA construct includes a linker sequence positioned between the sequence encoding the therapeutic biomolecule and the sequence encoding at least one innate inhibitor protein.

21. The saRNA construct according to claim 20, wherein, The linker sequence encodes a peptide spacer, which is configured to be digested to thereby separate the at least one therapeutic biomolecule from the at least one innate inhibitor protein.

22. The saRNA construct according to claim 21, wherein, The peptide spacer is a 2A peptide.

23. The saRNA construct according to claim 21, wherein, The peptide spacer is furin / 2A peptide.

24. The saRNA construct according to claim 1, wherein, The saRNA construct contains a nucleotide sequence as shown in SEQ ID NO: 38 or 39.

25. A nucleic acid sequence encoding the saRNA construct according to any one of claims 1-24, wherein, The sequence is as shown in SEQ ID NO: 40 or 41.

26. An expression cassette comprising the nucleic acid sequence according to claim 25.

27. A recombinant vector comprising the expression cassette according to claim 26.

28. The recombinant vector according to claim 27, wherein, The vector contains nucleotide sequences as shown in any of SEQ ID NO: 35 to 37.

29. A pharmaceutical composition comprising an saRNA construct according to any one of claims 1 to 24, a nucleic acid sequence according to claim 25, an expression cassette according to claim 26 or a vector according to claim 27 or 28, and a pharmaceutically acceptable excipient.

30. A method for preparing the saRNA construct according to any one of claims 1 to 24, the method comprising: a) i) Introducing the vector according to claim 27 or 28 into a host cell; as well as ii) Culture the host cells under conditions that result in the production of the saRNA construct according to any one of claims 1 to 24; or b) Transcription of saRNA constructs derived from the vector according to claim 27 or 28.

31. Use of the saRNA construct according to any one of claims 1 to 24, the nucleic acid according to claim 25, the expression cassette according to claim 26, the vector according to claim 27 or 28, or the pharmaceutical composition according to claim 29 in the preparation of a medicament for the prevention of rabies virus infection, wherein, The therapeutic biomolecule is a rabies virus glycoprotein.

32. A vaccine comprising an saRNA construct according to any one of claims 1 to 24, a nucleic acid according to claim 25, an expression cassette according to claim 26, a vector according to claim 27 or 28, or a pharmaceutical composition according to claim 29.

33. The vaccine according to claim 32, further comprising an adjuvant.

34. A method for modifying cells in vitro or in vitro, comprising delivering an saRNA construct according to any one of claims 1 to 24, a nucleic acid according to claim 25, an expression cassette according to claim 26, a vector according to claim 27 or 28, or a pharmaceutical composition according to claim 29 to the cells.

35. A modified cell obtained from the method of claim 34.

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