RNA construct

Abstract

To provide new means by which RNA therapeutics may be delivered and expressed in patients, such that they are able to overcome innate immune system sensing of RNA.SOLUTION: The 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 includes genetic constructs or vectors encoding such RNA replicons. The invention extends to the use of such RNA constructs and replicons in therapy, for example in treating diseases and / or in vaccine delivery. The invention extends to pharmaceutical compositions comprising such RNA constructs, and methods and uses thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to RNA constructs, particularly, but not exclusively, 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, for example, in the treatment of disease and / or vaccine delivery. The invention extends to pharmaceutical compositions comprising such RNA constructs, as well as methods and uses thereof. [Background technology]

[0002] Messenger RNA (mRNA) is a promising tool for biological therapy. However, while mRNA therapy has been shown to be highly effective in small animals, the translation of these formulations into dose-escalation studies in humans has not yielded linear results. Furthermore, adverse events associated with the induction of an interferon response have been rate-limiting for increasing RNA doses that are likely to be effective in humans. While the reasons for this discrepancy are unclear, we hypothesize that inherent differences in human innate sensing pose a barrier to the translation of RNA therapy from the laboratory to the clinic. Furthermore, innate sensing of RNA has been associated with the inhibition of protein expression. To date, the primary approach to overcoming innate recognition of exogenous RNA has been the use of modified ribonucleotides that are less detectable by innate sensing mechanisms. However, modified mRNAs are not completely undetectable, yet they still result in some interferon activation, protein silencing, and reduced tolerability for human use.

[0003] Another approach is the use of self-amplifying or saRNA vectors, which are typically based on an alphavirus backbone, which has the ability to self-amplify its own RNA by encoding polymerase activity within its nonstructural proteins. Prior art methods involve replacing the structural proteins of these vectors with a gene of interest (GOI), which is expected to be a vaccine construct or encode a therapeutic protein. Other versions of saRNA are based on picornaviruses, flaviviruses, and coronaviruses. Once saRNA is incorporated into the cytoplasm of target cells, it leads to amplification of the RNA by the encoded polymerase machinery and very high levels of expression of the GOI. As a result, saRNA has been shown to induce immune responses at lower doses (10-100 times lower) than mRNA, resulting in prolonged protein expression of up to 60 days in mice.

[0004] However, a drawback of saRNA is that it is also sensed by the innate recognition system, which triggers an antiviral response that limits the protein expression and self-amplification of these prior art saRNAs. The innate sensing of saRNA differs from that of mRNA due to its large size (typically >5000 bases) and deep secondary structure, including double-stranded regions (dsRNA). Long double-stranded RNAs trigger the innate response via the MDA5 (melanoma differentiation-associated protein 5) pathway. This is facilitated by the binding of PACT (PKR activating protein) to long dsRNA RNAs, which promotes MDA5 oligomerization, followed by the initiation of downstream signaling cascades that inhibit saRNA replication and expression. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Molecular Cloning, A Laboratory Manual, 2nd edition (1989) editor C Nolan, Cold Spring Harbor Laboratory Press [Non-patent document 2] Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680 [Non-patent document 3] Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882 Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need in the art to produce new means by which RNA therapeutics can be delivered and expressed in patients so that they can overcome the detection of the RNA by the innate immune system. [Means for solving the problem]

[0007] The present inventors have developed novel self-amplifying RNAs (saRNAs) that advantageously overcome innate immune system RNA sensing by expressing native inhibitory proteins that block or reduce innate immune system mechanisms, resulting in improved protein expression and self-amplification.

[0008] Thus, in a first aspect of the present invention, there is provided an RNA construct encoding (i) at least one therapeutic biomolecule; and (ii) at least one innate inhibitory protein (IIP). DETAILED DESCRIPTION OF THE INVENTION

[0009] RNA replicons or constructs have been hypothesized to be promising tools for the delivery and expression of genes of interest for vaccines and therapies. However, double-stranded RNA (dsRNA) is detected intracellularly by a sensing mechanism that triggers a response that inhibits protein translation. As a result, expression of the gene of interest encoded by the replicon is significantly impaired, thus limiting the therapeutic potential of RNA replicons. Advantageously, the RNA constructs of the present invention overcome this problem by encoding one or more innate inhibitory proteins (IIPs), which eliminate downstream inhibition of transgene expression. While interferon induction is a downstream consequence of innate recognition, it will be understood that other molecules and pathways may be induced, and any of these may be inhibited by one or more IIPs encapsulated in the RNA construct. The only previously published approach to abrogating the interferon response with saRNA has used interferon inhibitory proteins derived from vaccinia virus, E3, K3, and B18. However, in this study, the interferon inhibitory protein was delivered and formulated as a separate mRNA molecule combined with saRNA. This required the production of both saRNA and mRNA, and the use of 3-6 times more vaccinia mRNA than the replicon RNA construct of the present invention to ensure co-delivery to the same cells and provide any observable enhancement in protein expression. Furthermore, the kinetics of expression differ between mRNA and saRNA, such that any beneficial effects of IIPs expressed from mRNA will only last for a very short period (up to 72 hours) compared to the RNA construct of the present invention (up to 60 days).

[0010] Advantageously, the presence of one or more IIPs in the RNA construct of the first embodiment allows for dual protein expression with a peptide or protein of interest. In contrast to delivering two different strands of RNA, one encoding the peptide / protein of interest and the other encoding the IIP, as described in the prior art, using the constructs of the present invention, only a single strand is delivered to the target cell, thereby ensuring co-localization of the RNA with the native inhibitory protein. The IIP inhibits native sensing of the RNA, thus allowing for greater protein expression, and IIP expression itself is self-amplified by being co-expressed with the gene of interest, i.e., the therapeutic biomolecule, on the subgenomic strand. As described in the Examples, the RNA constructs of the present invention encoding luciferase (also known as "Stealthicons") have surprisingly been shown to increase luciferase protein expression levels in vitro by up to two orders of magnitude in human cell lines in vivo, and also to increase both the magnitude and duration of luciferase protein expression in vivo in BL / 6 mice compared to conventional VEEV RNA replicons. Those skilled in the art will readily recognize that luciferase reporters truly represent therapeutic biomolecules, demonstrating that RNA constructs can express genes linked to the RNA molecules of the present invention in vivo. Thus, luciferase provides strong proof-of-concept evidence that the saRNA constructs of the present invention can be used to express any therapeutically active biomolecule.

[0011] Those skilled in the art will understand that the RNA construct may also be referred to as a self-replicating RNA virus vector or an RNA replicon. The RNA construct may be double-stranded or single-stranded. Preferably, the RNA construct comprises a self-amplifying RNA (saRNA), and is preferably a saRNA construct.

[0012] Preferably, the RNA construct comprises or is derived from a positive strand RNA virus selected from the group of genera consisting of: alphavirus; picornavirus; flavivirus; rubivirus; pestivirus; hepacivirus; calicivirus or coronavirus.

[0013] Suitable wild-type alphavirus sequences are well known. Representative examples of suitable alphaviruses include Aura, Bebaru virus, Cabassou, Chikungunya virus, Eastern equine encephalomyelitis virus, Fort Morgan, Getah virus, Kyzylagach, Mayaro, Mayaro virus, Middleberg, Mukambo virus, Ndumu, Pixuna virus, Ross River virus, Semliki Forest, Sindbis virus, Tonate, Triniti, Una, Venezuelan equine encephalomyelitis, Western equine encephalomyelitis, Whataroa, and Y-62-33.

[0014] Preferably, the RNA construct comprises or is derived from a virus selected from the group of species consisting of Venezuelan equine encephalitis virus (VEEV); Enterovirus 71; Encephalomyocarditis virus; Kunjin virus; and Middle East respiratory syndrome virus. Preferably, the vector is derived from VEEV.

[0015] The RNA construct comprises a sequence encoding at least one therapeutic biomolecule. The at least one therapeutic biomolecule may comprise or be a vaccine construct or a therapeutic protein. Those skilled in the art will understand that a therapeutic protein refers to any protein that has therapeutic use, preferably in humans. Exemplary therapeutic biomolecules that can be encoded by the RNA molecule include proteins and peptides derived from pathogens, such as bacteria, viruses, fungi, protozoa, or parasites. Preferably, the proteins and peptides are antigens.

[0016] The viral proteins and peptides may be viral antigens, which may be derived from a virus selected from the group consisting of orthomyxoviruses, Paramyxoviridae viruses, metapneumoviruses and morbilliviruses, pneumoviruses, paramyxoviruses, poxviridae, metapneumoviruses, morbilliviruses, picornaviruses, enteroviruses, bunyaviruses, phleboviruses, nairoviruses, hepadnaviruses, togaviruses, alphaviruses, arteriviruses, flaviviruses, pestiviruses, hepadnaviruses, rhabdoviruses, caliciviridae, coronaviruses, retroviruses, reoviruses, parvoviruses, hepatitis delta virus (HDV), hepatitis E virus (HEV), human herpesviruses, and papovaviruses.

[0017] The orthomyxovirus may be influenza A, B, or C. The virus of the Paramyxoviridae family may be pneumovirus (RSV) or paramyxovirus (PIV). The metapneumovirus may be morbillivirus (e.g., measles). The pneumovirus may be respiratory syncytial virus (RSV), bovine respiratory syncytial virus, pneumonia virus of mice, or turkey rhinotracheitis virus. The paramyxovirus may be parainfluenza virus types 1-4 (PIV), mumps, Sendai virus, Simian virus 5, bovine parainfluenza virus, Nipah virus, Henipavirus, or Newcastle disease virus. The poxviridae family may be true smallpox, e.g., variola major and variola minor. The metapneumovirus may be human metapneumovirus (hMPV) or avian metapneumovirus (aMPV). The measles virus may be measles. The picornavirus may be an enterovirus, rhinovirus, hepadnavirus, parechovirus, cardiovirus, or aphthovirus. The enterovirus may be poliovirus type 1, 2, or 3, coxsackie A virus types 1-22 and 24, coxsackie B virus types 1-6, echovirus (ECHO virus) types 1-9, 11-27, and 29-34, or enterovirus 68-71. The bunyavirus may be California encephalitis virus. The phlebovirus may be Rift Valley fever virus. The nairovirus may be Crimean-Congo hemorrhagic fever virus. The hepadnavirus may be hepatitis A virus (HAV). The togavirus may be rubivirus. The flavivirus may be tick-borne encephalitis (TBE) virus, dengue fever (type 1, 2, 3, or 4) virus, yellow fever virus, Japanese encephalitis virus, Kyasanur Forest disease virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, or Powassan encephalitis virus. The pestivirus may be bovine viral diarrhea (BVDV), classical swine fever (CSFV), or border disease virus (BDV). The hepadnavirus may be hepatitis B virus or hepatitis C virus.The rhabdovirus may be a lyssavirus (rabies virus) or a vesiculovirus (VSV). The caliciviridae may be a Norwalk virus or a Norwalk-like virus, such as Hawaii virus and Snow Mountain virus. The coronavirus may be SARS-CoV-1, SARS-CoV-2, MERS, human respiratory coronavirus, infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), or transmissible gastroenteritis virus (TGEV). The retrovirus may be an oncovirus, lentivirus, or spumavirus. The reovirus may be an orthoreovirus, rotavirus, orbivirus, or coltivirus. The parvovirus may be parvovirus B19. The human herpesvirus may be herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), or human herpesvirus 8 (HHV8). The papovavirus may be a papillomavirus, polyomavirus, adenovirus, or arenavirus.

[0018] In a preferred embodiment, the viral antigen may be a rabies virus antigen, preferably a rabies virus glycoprotein, as shown in the Examples and Figure 13. In another preferred embodiment, and as shown in the Examples and Figure 29, the viral antigen may be a coronavirus antigen. Preferably, the coronavirus antigen is a surface glycoprotein, more preferably a surface glycoprotein of SARS-CoV-2.

[0019] Proteins and peptides derived from bacteria may be bacterial antigens.

[0020] Bacterial antigens include Neisseria meningitides, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis, Burkholderia species (e.g., Burkholderia mallei, Burkholderia pseudomallei, and Burkholderia cepacia), Staphylococcus aureus, Haemophilus influenzae, Clostridium tetani (tetanus), Clostridium perfringens, and the like. perfringens, Clostridium botulinums, Cornynebacterium diphtheriae (diphtheria), Pseudomonas aeruginosa, Legionella pneumophila, Coxiella burnetii, Brucella species (e.g., B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis, and B. pinnipediae), Francisella species (e.g., F. novicida, F. novicida), F. philomiragia and F. tularensis (F.tularensis), Streptococcus agalactiae, Neiserria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum (syphilis), Haemophilus ducreyi, Enterococcus faecalis, Enterococcus faecium, Helicobacter pylori, Staphylococcus saprophyticus, Yersinia enterocolitica, E. coli, Bacillus anthracis The antiviral agent may be derived from a bacterium selected from the group consisting of: Yersinia anthracis (anthrax), Yersinia pestis (plague), Mycobacterium tuberculosis, Rickettsia spp., Listeria spp., Chlamydia pneumoniae, Vibrio cholerae, Salmonella typhi (typhoid fever), Borrelia burgdorferi, Porphyromonas spp., and Klebsiella spp.

[0021] Proteins and peptides derived from fungi may be fungal antigens.

[0022] Fungal antigens include dermatophytes, such as: Epidermophyton koccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini. megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and / or Trichophyton faviforme faviforme, etc.; or Aspergillus fumigatus, Aspergillus kavus, Aspergillus niger, Aspergillus nidulans, etc.nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus kavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae pneumoniae, order Microsporidia, species of the genus Encephalitozoon, Septata intestinalis and Enterocytozoon bienusibieneusi; Brachiola spp., Microsporidium spp., Nosema spp., Pleistophora spp., Trachypleistophora spp., Vitaforma spp., Paracoccidioides brasiliensis, Pneumocystis carinii, Pythium insidiosum, Pityrosporum ovale, Saccharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiospermum apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia species, Fonsecaea species, Wangiella species , Sporothrix species, Basidiobolus species, Conidiobolus species, Rhizopus species, Mucor species, Absidia species, Mortierella species, Cunninghamella species, Saxenaea species, Alternaria species, Curvularia species, Helminthosporium species, Fusarium species, Aspergillus species, Penicillium species, Monolinia species, Rhizoctonia species, Paecilomyces species, Pithomyces species, and Cladosporium species.

[0023] Proteins and peptides derived from protozoa may be protozoan antigens.

[0024] The protozoan antigen may be derived from a protozoan selected from the group consisting of Entamoeba histolytica, Giardia lambli, Cryptosporidium parvum, Cyclospora cayatanensis, and Toxoplasma.

[0025] The therapeutic biomolecules may be proteins and peptides derived from plants. Preferably, the proteins and peptides are plant antigens. The plant antigens may be derived from the castor plant (Ricinus communis).

[0026] 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. Tumor immunogens and antigens may be peptide-containing tumor antigens, such as polypeptide tumor antigens or glycoprotein tumor antigens.

[0027] Tumor antigens may be (a) full-length molecules associated with cancer cells, (b) homologs and modified forms thereof, such as molecules with deleted, added, and / or substituted portions, and (c) fragments thereof.

[0028] Suitable tumor immunogens include class I restricted antigens recognized by CD8+ lymphocytes or class II restricted antigens recognized by CD4+ lymphocytes.

[0029] The tumor antigen may be an antigen associated with a cancer 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, gastric cancer, and prostate cancer.

[0030] Tumor antigens are (a) cancer-testis antigens, such as NY-ESO-I, SSX2, SCP1, as well as RAGE, BAGE, GAGE, and MAGE family polypeptides, such as GAGE-I, GAGE-2, MAGE-I, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used to treat, for example, melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors); (b) mutated antigens, such as p53 (associated with various solid tumors, e.g., colorectal, lung, and head and neck cancers), p21 / Ras (e.g., associated with melanoma, pancreatic cancer, and colorectal cancer), CDK4 (e.g., associated with melanoma), MUM1 (e.g., associated with melanoma), caspase-8 (e.g., associated with head and neck cancer), CIA0205 (e.g., associated with bladder cancer), HLA-A2-R1701, beta-catenin (e.g., associated with melanoma), TCR (e.g., associated with T-cell non-Hodgkin's lymphoma), BCR-abl (e.g., associated with chronic myeloid leukemia), triosephosphate isomerase, KIA0205, CDC-27, and LDLR-FUT; (c) overexpressed antigens, such as galectin 4 (e.g., associated with colorectal cancer), galectin 9 (e.g., associated with Hodgkin's disease), proteinase 3 (e.g., associated with chronic myeloid leukemia), WT1 (e.g., associated with various leukemias), carbonic anhydrase (e.g., associated with kidney cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., associated with breast, colon, lung, and ovarian cancer), alpha-fetoprotein (e.g., associated with liver cancer), KSA (e.g., associated with colorectal cancer), gastrin (e.g., associated with pancreatic and gastric cancer), telomerase catalytic protein, MUC-I (e.g., associated with breast and ovarian cancer), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast and colon cancer), and carcinoembryonic antigen (e.g., associated with cancers of the gastrointestinal tract, such as breast, lung, and colorectal cancer); (d) common antigens, e.g., melanoma-melanocyte differentiation antigens, e.g., MART-1 / MelanA, gplOO, MClR, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1, and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma); (e) prostate-associated antigens, such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, e.g., those associated with prostate cancer; and / or (f) immunoglobulin idiotypes (e.g., associated with myeloma and B-cell lymphoma) may be selected from:

[0031] 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 suppressor proteins, structural proteins, and blood proteins.

[0032] The enzyme may be selected from the group consisting of chymosin; gastric lipase; tissue plasminogen activator; streptokinase; cholesterol biosynthetic or degrading steroidogenic enzymes; kinases; phosphodiesterases; methylases; demethylases; dehydrogenases; cellulases; proteases; lipases; phospholipases; aromatase; cytochromes; adenylate or guanylate cyclases and neuramidases.

[0033] The enzyme inhibitor may be a tissue inhibitor of metalloproteinases (TIMPs).The hormone may be growth hormone.

[0034] The immune system protein may be selected from the group consisting of cytokines; chemokines; lymphokines; erythropoietin; integrins; addressins; selectins; homing receptors; T cell receptors and immunoglobulins.

[0035] The cytokine may be an interleukin, such as IL-2, IL-4 and / or IL-6, a colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or tumor necrosis factor (TNF).

[0036] The chemokine may be macrophage inflammatory protein-2 and / or plasminogen activator.

[0037] The lymphokine may be an interferon.

[0038] The immunoglobulin may be a natural, modified, or chimeric immunoglobulin or fragment thereof. Preferably, the immunoglobulin is a chimeric immunoglobulin with dual activity, such as an antibody-enzyme or antibody-toxin chimera.

[0039] The hormone may be selected from the group consisting of insulin, thyroid hormone, catecholamines, gonadotropins, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin; growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.).

[0040] The receptor may be a steroid hormone receptor or a peptide receptor. Preferably, the receptor is a growth factor receptor.

[0041] The binding protein may be a growth factor binding protein.

[0042] The tumor growth suppressor protein may be a protein that inhibits angiogenesis.

[0043] The structural protein may be selected from the group consisting of collagen; fibroin; fibrinogen; elastin; tubulin; actin; and myosin.

[0044] The blood protein 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.

[0045] In a preferred embodiment, the therapeutic biomolecule is a cytokine capable of regulating lymphocyte homeostasis, preferably a cytokine involved in, preferably inducing or enhancing, the development, priming, expansion, differentiation and / or survival of T cells. Thus, preferably, the cytokine is an interleukin. Most preferably, it is IL-2, IL-7, IL-12, IL-15 or IL-21.

[0046] The therapeutic biomolecule may be a protein capable of enhancing the reprogramming of somatic cells into cells with stem cell characteristics.

[0047] Proteins that can enhance the reprogramming of somatic cells into cells with stem cell characteristics include 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-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 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.

[0048] 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.

[0049] Preferably, the proteins capable of enhancing the reprogramming of somatic cells into cells with stem cell characteristics are OCT4, SOX2, LF4; c-MYC; NANOG; LIN28.

[0050] The therapeutic biomolecule may be a biomolecule utilized for ex vivo cell modification for the indication of cell therapy. Preferably, therefore, the therapeutic biomolecule may be selected from the group consisting of immunoglobulins, T cell receptors, and NK receptors.

[0051] The therapeutic biomolecule may be an RNA molecule capable of regulating the expression of an endogenous host gene, for example an interfering RNA, such as a small RNA, siRNA or microRNA.

[0052] Preferably, the RNA construct comprises a gene encoding at least one innate inhibitory protein (IIP) capable of reducing or blocking the innate immune response to RNA. Reduction or blocking of the innate immune response to RNA is preferably achieved by the IIP by reducing or blocking recognition of RNA (preferably long RNA (which will be understood by those skilled in the art to mean RNA that is at least 1 kb in length) or dsRNA) by a host cell harboring the RNA construct of the present invention. More preferably, the innate inhibitory protein is an innate inhibitory protein such that it is capable of reducing or blocking the innate response to RNA, preferably to the RNA of the RNA construct of the first aspect. Innate inhibitory proteins may be able to reduce or prevent recognition of cytosolic saRNA by pattern recognition receptors, which lead to activation of interferon regulatory factors 3 and 7 (IRF3 and IRF7) and NF-κB transcription factors, directly drive various antiviral genes (e.g., IFIT1-3, Mx1, Mx2, which are known to suppress saRNA expression), pro-inflammatory genes whose products orchestrate innate immune responses, and directly activate IFN-stimulated genes (ISGs), typically upstream of any interferon-dependent cascade. These pathways may also be reinforced by the induction of type I and III interferons, which provides a positive feedback loop that further amplifies many of the antiviral responses.

[0053] The RNA may be single-stranded or double-stranded RNA. Preferably, the RNA is saRNA.

[0054] At least one native inhibitory protein may be capable of either (i) reducing or blocking the action of melanoma differentiation-associated protein 5 (MDA5), for example, by preventing MDA5 oligomerization and binding to RNA, and / or (ii) blocking or reducing the binding of PACT, sometimes referred to as PKR-activating protein, to RNA. Those skilled in the art will appreciate that these sensors transmit signals that are transduced into downstream mitochondrial adaptors, and mitochondrial antiviral signaling (MAVS) activates downstream cascades, including activation of transcription factors (NF-κB, IRF-3, and -7). This, in turn, results in an appropriate antiviral signaling response and activation of type I interferon-stimulated genes encoding molecules with antiviral activity, including IFIT1, which is known to suppress saRNA expression.

[0055] The at least one native inhibitory 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 virus 3C protein; birnavirus VP3 protein; porcine deltacoronavirus accessory protein NS6; encephalomyocarditis virus 2C protein; and orthologs thereof.

[0056] Preferably, the at least one native inhibitory protein that blocks the action of MDA5 is a paramyxovirus V protein. Most preferably, the at least one native inhibitory protein that blocks the action of MDA5 is a parainfluenza virus type 5 V protein (PIV5V).

[0057] The at least one native inhibitory protein that blocks or reduces 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 orthologs thereof.

[0058] Preferably, the ORF4a (NS4a) is Middle East Respiratory Syndrome coronavirus MERS coronavirus (ORF4a).

[0059] Preferably, the coronavirus ORF3b is SARS-CoV2 ORF3b.

[0060] Provided below are the protein, DNA and RNA sequences for PIV5V, ORF4a and ORF3b, respectively.

[0061] In one embodiment, the PIV5V polypeptide is provided herein as SEQ ID NO: 11, as follows: MDPTDLSFSPDEINKLIETGLNTVEYFTSQQVTGTSSLGKNTIPPGVTGLLTNAAEAKIQESTNHQKGSVGGGAKPKKPRPKIAIVPADDKTVPGKPIPNPLLGLDSTPSTQTVLDLSGKTLPSGSYKGVKLAKFGKENLMTRFIEEPRENPIATSSPIDFKRGRDTGGFHRREYSIGWVGDEVKVTEWCNPSCSPITAAARRFECTCHQCPVTCSECERDT [SEQ ID NO: 11]

[0062] Thus, preferably, the PIV5V polypeptide comprises an amino acid sequence substantially as set forth in SEQ ID NO: 11, or a biologically active variant or fragment thereof. Thus, 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.

[0063] In one embodiment, the PIV5V polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 12, as follows: [SEQ ID NO: 12]

[0064] Thus, preferably, the PIV5V polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 12, or a variant or fragment thereof.

[0065] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 47, as follows: [SEQ ID NO: 47]

[0066] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 47 or a variant or fragment thereof.

[0067] In one embodiment, the MERS-CoV ORF4a polypeptide is provided herein as SEQ ID NO: 15, as follows: MDYVSLLNQIWQKYLNSPYTTCLYIPKPTAKYTPLVGTSLHPVLWNCQLSFAGYTESAVNSTKALAKQDAAQRIAWLLHKDGGIPDGCSLYLRHSSLFAQSEEEESFSN [SEQ ID NO: 15]

[0068] Thus, preferably, the MERS-CoV ORF4a polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 15, or a biologically active variant or fragment thereof. Thus, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 15, or a variant or fragment thereof.

[0069] In one embodiment, the MERS-CoV ORF4a polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 16, as follows: ATGGACTACGTGTCCCTGCTGAACCAGATTTGGCAGAAGTACCTGAACAGCCCCTACACCACCTGTCTGTACATCCCCAAGCCTACCGCCAAGTACACACCTCTCGTGGGCACATCTCTGCACCCCGTGCTGTGGAATTGCCAGCTGAGCTTTGCCGGCTACACCGA GTCTGCCGTGAACAGCACAAAGGCCCTGGCCAAACAGGACGCCGCTCAGAGAATTGCCTGGCTGCTGCACAAGGATGGCGGCATCCCTGATGGCTGTAGCCTGTACCTGAGACACAGCAGCCTGTTCGCCCAGAGCGAGGAAGAGGAATCCTTCAGCAAC [SEQ ID NO: 16]

[0070] Thus, preferably, the MERS-CoV ORF4a polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 16, or a variant or fragment thereof.

[0071] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 48, as follows: AUGGACUACGUGUCCCUGCUGAACCAGAUUUGGCAGAAGUACCUGAACAGCCCCUACACCACCUGUCUGUACAUCCCCAAGCCUACCGCCAAGUACACACCUCUCGUGGGCACAUCUCUGCACCCCGUGCUGUGGAAUUGCCAGCUGAGCUUUGCCGGCUACACCGA GUCUGCCGUGAACAGCACAAAGGCCCUGGCCAAACAGGACGCCGCUCAGAGAAUUGCCUGGCUGCUGCACAAGGAUGGCGGCAUCCCUGAUGGCUGUAGCCUGUACCUGAGACACAGCAGCCUGUUCGCCCAGAGCGAGGAAGAGGAAUCCUUCAGCAAC [SEQ ID NO: 48]

[0072] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 48, or a variant or fragment thereof.

[0073] In one embodiment, the SARS-CoV-2 ORF3b polypeptide is provided herein as SEQ ID NO: 20, as follows: MMPTIFFAGILIVTTIVYLTIVQLLQLSLLQVMAQQVLFLNMTTRLVVILKNGNLEQKTVLYYTVTSLQTITSCTQLN [SEQ ID NO: 20]

[0074] Thus, preferably, the SARS-CoV-2 ORF3b polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 20, or a biologically active variant or fragment thereof. Thus, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 20, or a variant or fragment thereof.

[0075] In one embodiment, the SARS-CoV-2 ORF3b polypeptide is encoded by the nucleotide sequence of SEQ ID NO:55 (Wuhan-Hu-1 Accession No. NC_045512.2; nucleotides 25814-26050), as follows: ATGATGCCAACTATTTTCTTTGCTGGCATACTAATTGTTACGACTATTGTATACCTTACAATAGTGCAACTTCTTCAATTGTCATTACTTCAGGTGATGGCACAACAAGTCCTATTTCTGAACATGACTACCAGATTGGTGGTTATACTGAAAAATGGGAATCTGGAGCAAAAGACTGTGTTGTATTACACAGTTACTTCACTTCAGACTATTACCAGCTGTACTCAACTCAATTGA [SEQ ID NO: 55]

[0076] Thus, preferably, the SARS-CoV-2 ORF3b polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 55, or a variant or fragment thereof.

[0077] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 56, as follows: AUGAUGCCAACUAUUUUCUUUGCUGGCAUACUAAUUGUUACGACUAUUGUAUACCUUACAAUAGUGCAACUUCUUCAAUUGUCAUUACUUCAGGUGAUGGCACAACAAGUCCUAUUUCUGAA CAUGACUACCAGAUUGGUGGUUAUACUGAAAAAUGGGAAUCUGGAGCAAAAGACUGUGUUGUAUUACACAGUUACUUCACUUCAGACUAUUACCAGCUGUACUCAACUCAAUGA [SEQ ID NO: 56]

[0078] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 56, or a variant or fragment thereof.

[0079] In another embodiment, the at least one native inhibitory protein may be capable of inhibiting a downstream pathway of MDA5 activation or of blocking a downstream pathway of MDA / PACT recognition of dsRNA.

[0080] The at least one native inhibitory protein capable of inhibiting the downstream pathway of MDA5 activation or blocking the downstream pathway of MDA / PACT recognition of dsRNA may be selected from the group consisting of HSV-2 Us1; HSV-1 Us11; OV20.0L; BVDV Npro; Langat virus NS5; and influenza NS1.

[0081] Those skilled in the art will understand that at least one native inhibitory protein capable of inhibiting a downstream pathway of MDA5 activation or blocking a downstream pathway of MDA / PACT recognition of dsRNA may be used in combination with at least one native inhibitory protein that blocks the action of MDA5 and / or at least one native inhibitory protein that blocks or reduces binding of PACT to RNA.

[0082] In one embodiment, the HSV-2 Us1 polypeptide is provided herein as SEQ ID NO: 1, as follows: VRDCYLMGYCRTRLGPRTWGRLLQISGGTWDVRLRNAIREVEAHFEPAAEPVCELPCLNARRYGPECDVGNLETNGGSTSDDEISDATDSDDTLASHSDTEGGPSPAGRENPESASGGAIAARLECEFGTFDWTSEEGSQPWLSAVVADTSSAERSGLPAPGACRATEAPEREDGCRKMRFPAACPYPCGHTFLRP [SEQ ID NO: 1]

[0083] Thus, preferably, the HSV-2 Us1 polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 1, or a biologically active variant or fragment thereof. Thus, 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.

[0084] In one embodiment, the HSV-2 Us1 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:2, as follows: [SEQ ID NO: 2]

[0085] Thus, preferably, the HSV-2 Us1 polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:2, or a variant or fragment thereof.

[0086] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 42, as follows: [SEQ ID NO: 42]

[0087] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 42, or a biologically active variant or fragment thereof.

[0088] In one embodiment, the HSV-1 Us1 polypeptide is provided herein as SEQ ID NO: 3, as follows: VRDCYLMGYCRTRLGPRTWGRLLQISGGTWDVRLRNAIREVEAHFEPAAEPVCELPCLNARRYGPECDVGNLETNGGSTSDDEISDATDSDDTLASHSDTEGGPSPAGRENPESASGGAIAARLECEFGTFDWTSEEGSQPWLSAVVADIRDCYLMGYCRARLAPRTWCRLLQVSGGTWGMHLRNTIREVEARFDATAEPVCKLPCLETRRYGPECDLSNLEIHLSATSDDEISDATDLEAAGSDHTLASQSDTEDAPSPVTLETPEPRGSLAVRLEDEFGEFDWTPQEGSQPWLSAVVADTSSVERPGPSDSGAGRAAEDRKCLDGCRKMRFSTACPYPCSDTFLRPTSSAERSGLPAPGACRATEAPEREDGCRKMRFPAACPYPCGHTFLRP [SEQ ID NO: 3]

[0089] Thus, preferably, the HSV-1 Us1 polypeptide comprises an amino acid sequence substantially as set forth in SEQ ID NO: 3, or a biologically active variant or fragment thereof. Thus, 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.

[0090] In one embodiment, the HSV-1 Us1 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:4, as follows: [SEQ ID NO: 4]

[0091] Thus, preferably, the HSV-1 Us1 polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 4, or a variant or fragment thereof.

[0092] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 43, as follows:

[0093] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 43, or a variant or fragment thereof.

[0094] In one embodiment, the HSV-1 Us11 polypeptide is provided herein as SEQ ID NO:5, as follows: MSQTQPPAPVGPGDPDVYLKGVPSAGMHPRGVHAPRGHPRMISGPPQRGDNDQAAGQCGDSGLLRVGADTTISKPSEAVRPPTIPRTPRVPREPRVPRPPREPREPRVPRAPRDPRVPRDPRDPRQPRSPREPRSPREPRSPREPRTPRTPREPRTARGSV [SEQ ID NO: 5]

[0095] Thus, preferably, the HSV-1 Us11 polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 5, or a biologically active variant or fragment thereof. Thus, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 5, or a variant or fragment thereof.

[0096] In one embodiment, the HSV-1 Us11 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:6, as follows: ATGAGCCAGACACAGCCTCCAGCTCCAGTTGGACCTGGCGACCCTGATGTGTATCTGAAGGGCGTGCCAAGCGCCGGCATGCATCCTAGAGGTGTTCATGCCCCTAGAGGACACCCCAGAAT GATCTCTGGCCCTCCTCAGAGAGGCGACAACGATCAGGCTGCTGGACAGTGTGGCGATAGCGGACTGCTGAGAGTGGGCGCCGATACCACAATCAGCAAGCCATCTGAGGCTGTGCGGCCTCC TACAATCCCCAGAACACCTAGAGTGCCCCGCGAGCCAAGAGTGCCTAGACCTCCTAGAGAGCCCAGAGAACCCAGAGTGCCAAGGGCTCCCAGAGATCCTAGAGTCCCTCGGGACCCTAGGG ACCCAAGACAACCTAGATCACCCAGAGAGCCTCGGAGCCCAAGAGAGCCAAGAAGCCCTAGGGAACCCCGGACACCAAGAACACCCAGGGAACCTAGAACCGCCAGAGGCAGCGTG [SEQ ID NO: 6]

[0097] Thus, preferably, the HSV-1 Us11 polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 6, or a variant or fragment thereof.

[0098] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 44, as follows: AUGAGCCAGACACAGCCUCCAGCUCCAGUUGGACCUGGCGACCCUGAUGUGUAUCUGAAGGGCGUGCCAAGCGCCGGCAUGCAUCCUAAGAGGUGUUCAUGCCCCUAGAGGACACCCCAGAAU GAUCUCUGGCCCUCCUCAGAGAGGCGACAACGAUCAGGCUGCUGGACAGUGUGGCGAUAGCGGACUGCUGAGAGUGGGCGCCGAUACCACAAUCAGCAAGCCAUCUGAGGCUGUGCGGCCUCC UACAAUCCCCAGAACACCUAGAGUGCCCCGCGAGCCAAGAGUGCCUAGACCUCCUAGAGAGCCCAGAGAACCCAGAGUGCCAAGGGCUCCCAGAGAUCCUAGAGUCCCUCGGGACCCUAGGGA CCCAAGACAACCUAGAUCACCCAGAGAGCCUCGGAGCCCAAGAGAGCCAAGAAGCCCUAGGGAACCCCGGACACCAAGAACACCCAGGGAACCUAGAACCGCCAGAGGCAGCGUG [SEQ ID NO: 44]

[0099] Therefore, preferably the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 44 or a variant or fragment thereof.

[0100] In one embodiment, the OV20.0L polypeptide is provided herein as SEQ ID NO:7, as follows: MACECASLILELLRKSDDKLPAKQIAKELGISKHEANRQLYRLLDSDEVCCEDGNPPRWFVECAPSAPTEEDENSDTEPMETEAGCDTLFGGDIDIMTQSAVIRLKSLNPVSAVNEFCMMTHRPLEFCETRAGGEDHCPRFTCTITISGKVVAVADGASKKLARHTACSSALTILINNCGISF [SEQ ID NO: 7]

[0101] Thus, preferably, the OV20.0L polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 7, or a biologically active variant or fragment thereof. Thus, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 7, or a variant or fragment thereof.

[0102] In one embodiment, the OV20.0L polypeptide is encoded by the nucleotide sequence of SEQ ID NO:8, as follows: [SEQ ID NO: 8]

[0103] Thus, preferably, the OV20.0L polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:8, or a variant or fragment thereof.

[0104] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 45, as follows: [SEQ ID NO: 45]

[0105] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 45, or a variant or fragment thereof.

[0106] In one embodiment, the BVDV Npro polypeptide is provided herein as SEQ ID NO: 9, as follows: MELITNELLYKTYKQKPVGVEEPVYDQAGDPLFGERGAVHPQSTLKLPHKRGERDVPTNLASLPKRGDCRTGNSRGPVSGIYLKPGPLFYQDYKGPVYHRAPLELFEEGSMCETTKRIGRVTGSDGKLYHIYVCIDGCIIIKSATRSYQRVFRWVHNRLDCPLWVTSC [SEQ ID NO: 9]

[0107] Thus, preferably, the BVDV Npro polypeptide comprises an amino acid sequence substantially as set forth in SEQ ID NO: 9, or a biologically active variant or fragment thereof. Thus, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 9, or a variant or fragment thereof.

[0108] In one embodiment, the BVDV Npro polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 10, as follows: ATGGAACTGATCACCAACGAGCTGCTGTACAAGACCTACAAGCAGAAACCCGTGGGCGTCGAGGAACCCGTGTATGATCAAGCTGCGACCCTCTGTTTGGCGAGAGAGGCGCTGTTCACCCTCAGAG CACACTGAAGCTGCCCCACAAGCGGGGCGAAAGAGATGTGCCTACCAACCTGGCCAGCCTGCCTAAGAGAGGCGATTGCAGAACCGGCAATAGCAGAGGCCCTGTGTCCGGCATCTACCTGAACCTG GACCACTGTTCTACCAGGACTACAAGGGACCCGTGTACCACAGAGCCCCTTGGAACTGTTTGAAGAGGGCAGCATGTGCGAAACCACCAAGCGGATCGGAAGAGTGACCGGCTCTGACGGCAAGCTG TACCACATCTACGTGTGCATCGACGGCTGCATCATCATCAAGAGCGCCACCAGATCCTACCAGCGGGTGTTCAGATGGGTGCACAACAGACTGGACTGCCCTCTGTGGGTCACCAGCTGC [SEQ ID NO: 10]

[0109] Thus, preferably, the BVDV Npro polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 10, or a variant or fragment thereof.

[0110] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 46, as follows: AUGGAACUGAUCACCAACGAGCUGCUGUACAAGACCUACAAGCAGAACCCGUGGGCGUCGAGGAACCCGUGUAUGAUCAAGCUGCGACCCUCUGUUUGGCGAGAGAGGCGCUGUUCACCCUCAGAG CACACUGAAGCUGCCCCACAAGCGGGGCGAAAGAGAUGUGCCUACCAACCUGGCCAGCCUGCCUAAGAGAGGCGAUUGCAGAACCGGCAAUAGCAGAGGCCCUGUGUCCGGCAUCUACCUGAACCUG GACCACUGUUCUACCAGGACUACAAGGGACCCGUGUACCACAGAGCCCCUUGGAACUGUUUGAAGAGGGCAGCAUGUGCGAAACCACCAAGCGGAUCGGAAGAGUGACCGGCUCUGACGGCAAGCUG UACCACAUCUACGUGUGCAUCGACGGCUGCAUCAUCAUCAAGAGCGCCACCAGAUCCUACCACGGGGUUCAGAUGGGUGCACAACAGACUGGACUGCCCUCUGUGGGUCACCAGCUGC [SEQ ID NO: 46]

[0111] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 46, or a variant or fragment thereof.

[0112] In one embodiment, the Langat NS5 polypeptide is provided herein as SEQ ID NO: 17, as follows: VFKDKVDTKAQEPQPGTKIIMRAVNDWLLERLVKKSRPRMCSREEFIAKVRSNAALGAWSDEQNKWKSAREAVEDPEFWSLVEAERERHLQGRCAHCVYNMMGKREKKLGEFGVAKGSRAIWYMWLGSRFLEFEALGFLNEDHWASRASSGAGVEGISLNYLGWHLKKLASLSGGLFYADDTAGWDTKITNADLDDEEQILRYMDGDHKKLAATVLRKAYHAKVVRVARPSREGGCVMDIITRRDQRGSGQVVTYALNTITNIKVQLVRMMEGEGVIEVADSHNPRLLRVEKWLEEHGEERLSRMLVSGDDCVVRPVDDRFSKALYFLNDMAKTRKDTGEWEPSTGFASWEEVPFCSHHFHELVMKDGRALVVPCRDQDEL [SEQ ID NO: 17]

[0113] Thus, preferably, the Langat NS5 polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 17, or a biologically active variant or fragment thereof. Thus, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 17, or a variant or fragment thereof.

[0114] In one embodiment, the Langat NS5 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 18, as follows:

[0115] Thus, preferably, the Langat NS5 polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 18, or a variant or fragment thereof.

[0116] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 49, as follows:

[0117] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 49 or a variant or fragment thereof.

[0118] In one embodiment, the influenza NS1 polypeptide (accession number DQ508893) is provided herein as SEQ ID NO: 13, as follows: MDSNTVSSFQVDCFLWHVRKQVADQELGDAPFLDRLRRDQKSLKGRGSTLGLNIETATCVGKQIVERILKEESDEAFRMTMASALASRYLTDMTIEEMSRDWFMLMPKQKVAGPLCVRMDQAIMDKNIILKANFSVIFDRLETLTLLRAFTEEGAIVGEISPLPSLPGHTNEDVKNAIGVLIGGLEWNDNTVRVSETLQRFAWRSSNENGGPPLTPTQKRKMAGKIRSEV [SEQ ID NO: 13]

[0119] Thus, preferably, the influenza NS1 polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 13, or a biologically active variant or fragment thereof. Thus, the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence encoding SEQ ID NO: 13, or a variant or fragment thereof.

[0120] In one embodiment, the influenza NS1 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 14, as follows: [SEQ ID NO: 14]

[0121] Thus, preferably, the influenza NS1 polypeptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 14, or a variant or fragment thereof.

[0122] Thus, the RNA construct may comprise the RNA nucleotide sequence of SEQ ID NO: 19, as follows: [SEQ ID NO: 19]

[0123] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 19, or a variant or fragment thereof.

[0124] Preferably, the native inhibitory protein is selected from the group consisting of HSV-2 Us1; HSV-1 Us11; OV20.0L; BVDV Npro, PIV5V; MERS-CoV ORF4a; SARS-CoV-2 ORF3b; Langat virus NS5 and influenza NS1.

[0125] Preferably, the native inhibitory protein is selected from the group consisting of HSV-2 Us1; HSV-1 Us11; OV20.0L; BVDV Npro, PIV5V; MERS-CoV ORF4a; and Langat virus NS5.

[0126] Preferably, the native inhibitory protein is selected from the group consisting of HSV-2 Us1; HSV-1 Us11; OV20.0L; BVDV Npro, PIV5V; and MERS-CoV ORF4a.

[0127] As described in the Examples, through a broad screening method for IIPs, the inventors surprisingly identified two highly potent IIPs that could significantly enhance saRNA expression in vitro and in vivo: parainfluenza virus type 5 V protein (PIV-5), an inhibitor of MDA5 activation, and Middle East respiratory syndrome (MERS) coronavirus ORF4a, an inhibitor of PACT / MDA activation. The inventors utilized these two novel IIPs, PIV-5 and MERS-CoV ORF4a, which have not previously been used to mitigate innate sensing of replicon RNA. The inventors demonstrated that these constructs could be next-generation RNA replicons that would enable higher efficacy of RNA vaccines and therapies in humans. The inventors believe that they will have significant utility regardless of whether they are expressed in alphavirus, picornavirus, flavivirus, or coronavirus replicons.

[0128] Thus, most preferably, the native inhibitory protein is PIV-5 and / or MERS-CoV ORF4a, which those skilled in the art will understand may also be referred to as NS4a.

[0129] Such constructs exhibit a number of advantages over constructs described in the prior art, including: i) Insertion of PIV-5 and ORF4a proteins directly into the VEEV replicon, which allows dual protein expression of the VPII protein and the gene of interest; ii) as opposed to delivering two different strands of RNA, a single strand is delivered, one strand encoding the gene of interest (GOI), i.e., the therapeutic biomolecule, and one strand encoding the IIP, thus ensuring co-localization of the RNA with the native inhibitory protein; iii) IIPs inhibit innate sensing of RNA, thus allowing greater protein expression; iv) IIP expression itself is self-amplified by co-expression with the GOI on the subgenomic strand; and / or v) Increased both the magnitude and duration of protein expression compared to conventional VEEV RNA replicon constructs.

[0130] The sequence encoding at least one native inhibitory protein may be positioned anywhere within the RNA construct or replicon sequence, such that the sequence encoding at least one peptide or protein of interest may be positioned 5' or 3' to the sequence encoding at least one native inhibitory protein.

[0131] Preferably, however, the sequence encoding at least one peptide or protein of interest is positioned 5' to the sequence encoding at least one native inhibitory protein.

[0132] Preferably, the RNA construct according to the first aspect comprises at least one promoter, which is either a genomic promoter or a subgenomic promoter. Preferably, the promoter is a subgenomic promoter.

[0133] Those skilled in the art will understand that a subgenomic promoter refers to a promoter that is operably linked to sequences encoding at least one therapeutic biomolecule and at least one native inhibitory protein such that it allows for transcription of the nucleotide sequences encoding the therapeutic biomolecule and at least one native inhibitory protein.

[0134] Preferably, the subgenomic promoter is 26S, which is provided herein as SEQ ID NO: 57, as follows: GGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACAT [SEQ ID NO: 57]

[0135] Thus, preferably the promoter (preferably a subgenomic promoter) is substantially as set forth in SEQ ID NO: 57, or a variant or fragment thereof.

[0136] In one embodiment, the same promoter is operably linked to a sequence encoding at least one peptide or protein of interest and a sequence encoding at least one native inhibitor.

[0137] Our design, in which both the GOI (i.e., therapeutic biomolecule) and the IIP are encoded on a single strand, advantageously allows for the use of much smaller doses of RNA, ensuring that the protein is expressed in the same cells that sense the RNA and are also capable of replicating, thus having the added aspect of amplifying the innate inhibitory component.

[0138] Thus, in one embodiment, a promoter is positioned 5' to a sequence encoding a peptide or protein of interest (i.e., a therapeutic biomolecule) and a sequence encoding at least one native inhibitory protein such that the promoter is operably linked to both sequences.

[0139] In another embodiment, a first promoter is operably linked to a sequence encoding at least one peptide or protein of interest (i.e., a therapeutic biomolecule), and a second promoter is operably linked to a sequence encoding at least one native inhibitory protein.

[0140] The RNA construct may encode at least two, three, four, or five IIPs. In embodiments where there are sequences encoding more than one native inhibitory protein, a single promoter may be operably linked to all of the sequences encoding the native inhibitory proteins. Alternatively, a promoter may be linked to each of at least one other sequence encoding a native inhibitory protein, such that each native inhibitory protein is operably linked to a separate promoter. In this embodiment, the separate promoters may comprise the same promoter sequence or different promoter sequences. In another embodiment, a different promoter is operably linked to each sequence encoding a native inhibitory protein.

[0141] The RNA construct may further comprise a linker sequence disposed between the sequence encoding at least one peptide or protein of interest (i.e., a therapeutic biomolecule) and the sequence encoding at least one native inhibitory protein.

[0142] In one embodiment, the linker sequence comprises a sequence encoding a peptide spacer configured to separate, upon digestion, at least one therapeutic biomolecule encoded by the gene of interest from at least one native inhibitory protein. Therefore, preferably, a spacer sequence is disposed between the sequence encoding at least one peptide or protein of interest and the sequence encoding at least one native inhibitory protein.

[0143] Therefore, the spacer sequence is preferably a cleavable peptide, such as a 2A peptide. Suitable 2A peptides include porcine teschovirus-12A (P2A)-ATNFSLLKQAGDVEENPGP (SEQ ID NO: 21), thosea asigna virus 2A (T2A)-QCTNYALLKLAGDVESNPGP (SEQ ID NO: 22), equine rhinitis A virus 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).

[0144] In another embodiment, the cleavable peptide is a self-cleaving peptide. Preferably, the self-cleaving peptide is a furin / 2A peptide. The furin sequence may be located 3' or 5' of the 2A sequence. However, preferably, the furin sequence is located 5' of the 2A sequence, preferably with a GSG spacer located between the furin and 2A sequences.

[0145] Those skilled in the art will appreciate that furin is a ubiquitous calcium-dependent proprotein convertase located in the secretory pathway (primarily the Golgi and trans-Golgi network) that cleaves precursor proteins at a specific recognition sequence, typically RXR / K / XR (SEQ ID NO: 24), cleaving the precursor protein after the last R. Thus, in one embodiment, the furin sequence is RXR / K / XR. Preferably, however, the furin sequence is the optimized sequence RRRRRR (SEQ ID NO: 25), which is a GSG sequence. Preferably, a GSG spacer is located 3' of the furin sequence and 5' of the 2A sequence.

[0146] Thus, preferably, the spacer sequence is furin / T2A as provided by NCBI Reference Sequence: GenBank: AAC97195.1, provided herein as SEQ ID NO: 26, as follows: RRRRRRGSGEGRGSLLTCGDVEENPGP [SEQ ID NO: 26]

[0147] Thus, preferably, the spacer sequence comprises an amino acid sequence substantially as set out in SEQ ID NO: 26, or a variant or fragment thereof.

[0148] In embodiments in which the RNA construct or replicon contains more than one sequence encoding a native inhibitory protein, the replicon may have a linker sequence located between each sequence encoding a native inhibitory protein, or only between some of the IIPs.

[0149] In one embodiment, the therapeutic biomolecule and at least one sequence encoding at least one native inhibitory protein may be separated by a stop codon followed by an internal ribosome entry site (IRES) sequence capable of initiating translation of downstream sequences. Typical IRES sequences include, for example, the IRES sequences of encephalomyocarditis virus or vascular endothelial growth factor and type 1 collagen-induced protein (VCIP), which will be known to those skilled in the art. Therefore, preferably, the IRES sequence is positioned between the sequence encoding at least one peptide or protein of interest and the sequence encoding at least one native inhibitory protein. When multiple sequences encoding at least one native inhibitory protein are used, the spacer sequence may include a combination of known cleavage sequences and / or IRES sequences.

[0150] In another embodiment, the sequences encoding at least one therapeutic biomolecule and at least one native inhibitory protein may be separated by a stop codon followed by a second subgenomic promoter sequence capable of initiating transcription of the downstream sequence.

[0151] The RNA construct may encode at least one nonstructural protein (NSP) located 5' or 3' of the sequence encoding at least one peptide or protein of interest and at least one native inhibitory protein. Preferably, the sequence encoding at least one NSP is located 5' of the sequence encoding the peptide or protein of interest and at least one native inhibitory protein. Thus, preferably, the sequence encoding at least one NSP is located at the 5' end of the RNA construct.

[0152] At least one nonstructural protein encoded by the RNA construct may be RNA polymerase nsP4. Preferably, the construct encodes nsP1, nsP2, nsP3, and nsP4. Those skilled in the art will understand that nsP1 is the viral capping enzyme and membrane anchor of the replication complex (RC), while nsP2 is an RNA helicase and protease involved in processing the ns polyprotein. nsP3 can interact with several host proteins and modulate protein poly- and mono-ADP-ribosylation, and nsP4 is the core viral RNA-dependent RNA polymerase.

[0153] In one embodiment, nsP1 is provided herein as SEQ ID NO: 27, as follows: [SEQ ID NO: 27]

[0154] Thus, nsP1 preferably comprises an amino acid sequence substantially as set forth in SEQ ID NO: 27, or a biologically active variant or fragment thereof.

[0155] In one embodiment, nsP1 is encoded by the nucleotide sequence defined in SEQ ID NO:28 as follows:

[0156] Thus, nsP1 is preferably encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 28, or a variant or fragment thereof.

[0157] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 50, or a variant or fragment thereof.

[0158]

[0159] In one embodiment, nsP2 is provided herein as SEQ ID NO: 29, as follows: [SEQ ID NO: 29]

[0160] Thus, nsP2 preferably comprises an amino acid sequence substantially as set forth in SEQ ID NO: 29, or a biologically active variant or fragment thereof.

[0161] In one embodiment, nsP2 is encoded by the nucleotide sequence defined in SEQ ID NO: 30 as follows:

[0162] Thus, preferably, nsP2 is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 30, or a variant or fragment thereof.

[0163] Thus, the RNA construct may comprise SEQ ID NO: 51, as follows:

[0164] Therefore, preferably the RNA construct therefore comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 51, or a variant or fragment thereof.

[0165] In one embodiment, nsP3 is provided herein as SEQ ID NO: 31, as follows: [SEQ ID NO: 31]

[0166] Thus, preferably, nsP3 comprises an amino acid sequence substantially as set forth in SEQ ID NO: 31, or a biologically active variant or fragment thereof.

[0167] In one embodiment, nsP3 is encoded by the nucleotide sequence defined in SEQ ID NO: 32 as follows:

[0168] Thus, preferably, nsP3 is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 32, or a variant or fragment thereof.

[0169] Thus, the RNA construct may comprise SEQ ID NO: 52, as follows:

[0170] Thus, preferably, the RNA construct therefore comprises an RNA nucleotide sequence substantially as set forth as SEQ ID NO: 52, or a variant or fragment thereof. In one embodiment, nsP4 is provided herein as SEQ ID NO: 33, as follows: [SEQ ID NO: 33]

[0171] Thus, preferably, nsP4 comprises an amino acid sequence substantially as set forth in SEQ ID NO: 33, or a biologically active variant or fragment thereof.

[0172] In one embodiment, nsP4 is encoded by the nucleotide sequence defined in SEQ ID NO: 34 as follows:

[0173] Thus, preferably, nsP4 is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 34, or a variant or fragment thereof.

[0174] Thus, the RNA construct may comprise SEQ ID NO: 53, as follows:

[0175] Therefore, preferably the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 53, or a variant or fragment thereof.

[0176] Preferably, the nonstructural proteins encoded by the RNA construct of the invention, together with proteins present in the host cell, form an enzyme complex necessary for genome replication and transcription of the sequences encoding at least one peptide or protein of interest and at least one native inhibitor protein. For example, one or more of the nonstructural proteins may encode a polymerase, allowing the construct to amplify nucleotide sequences encoding at least one peptide or protein of interest and at least one native inhibitor protein.

[0177] The host cell may 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.

[0178] The RNA construct may further comprise a promoter operably linked to the sequence encoding the at least one nonstructural protein and positioned 5' of the at least one nonstructural protein so as to permit expression of the at least one nonstructural protein in the host cell.

[0179] Preferably, the promoter comprises a conserved sequence element in the 5'UTR, which may be referred to herein as SEQ ID NO: 54, as follows: AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAA [SEQ ID NO: 54]

[0180] Thus, preferably, a UTR is located 5' of at least one nonstructural protein and comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 54, or a fragment or variant thereof.

[0181] Preferably, the replicon comprises a polyA tail. Preferably, the polyA tail is located at the 3' end of the replicon. The replicon may further comprise a 5' cap. In the context of the present invention, the term "5'-cap" includes 5'-cap analogs that resemble the RNA cap structure and have been modified to have the ability to stabilize RNA and / or enhance translation of RNA when attached thereto, preferably in vivo and / or in cells.

[0182] RNA with a 5'-cap can be achieved by in vitro transcription of a DNA template in the presence of a 5'-cap, in which case the 5'-cap is incorporated into the co-transcribed RNA strand, or RNA can be generated, for example, by in vitro transcription, and a 5'-cap can be attached to the RNA post-transcription using a capping enzyme, e.g., vaccinia virus capping enzyme. In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked to the 5' position of the subsequent base (the "second base") of the RNA molecule via a phosphodiester bond.

[0183] In one embodiment, the RNA construct comprises, preferably from 5' to 3', a promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, and at least one sequence encoding a native inhibitory protein.

[0184] In another embodiment, the RNA construct comprises, preferably from 5' to 3', a promoter, a sequence encoding at least one nonstructural protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, and at least one sequence encoding a native inhibitory protein.

[0185] In yet another embodiment, the RNA construct comprises, preferably from 5' to 3', a promoter, a sequence encoding at least one nonstructural protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, at least one sequence encoding a native inhibitory protein, and a polyA tail.

[0186] In another embodiment, the RNA construct comprises, preferably from 5' to 3', a promoter, a sequence encoding at least one nonstructural protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, at least one sequence encoding a native inhibitory protein, optionally a spacer sequence between each of the at least one sequence encoding a native inhibitory protein, and a polyA tail.

[0187] In one embodiment, the RNA construct preferably comprises, from 5' to 3', a 5' cap, a promoter, a sequence encoding at least one nonstructural protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, at least one sequence encoding a native inhibitory protein, optionally a spacer sequence between each of the at least one sequence encoding a native inhibitory protein, and a polyA tail.

[0188] In one embodiment, the RNA construct comprises, preferably from 5' to 3', a 5' cap, a promoter, a sequence encoding at least one nonstructural protein, a spacer, at least one sequence encoding a native inhibitory protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, and a polyA tail.

[0189] In one embodiment, the RNA construct preferably comprises, from 5' to 3', a 5' cap, a promoter, a sequence encoding at least one nonstructural protein, a spacer, at least one sequence encoding a native inhibitory protein, a subgenomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, a sequence encoding at least one native inhibitory protein, optionally a spacer sequence between each sequence encoding at least one native inhibitory protein, and a poly-A tail.

[0190] The V protein of PIV5 is thought to directly bind to MDA5, preventing oligomerization, while ORF4a is thought to block PACT from binding to dsRNA. The inventors incorporated these protein-encoding genes into saRNA after the gene of interest (GOI) (i.e., therapeutic biomolecule), separated by a T2A cleavage site, to generate constructs that advantageously circumvent the innate sensing mechanism. By pairing the GOI and IIP into a single open reading frame that is cleaved by endogenous protease (T2A) when expressed, expression of both in the same cell at a set ratio with the same kinetics is maximized.

[0191] Thus, preferably, the RNA construct comprises, from 5' to 3', a 5' cap, a promoter containing a 51-nucleotide conserved sequence element, nsP1, nsP2, nsP3v, nsP4, the 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.

[0192] Thus, in one embodiment, the RNA construct may comprise or consist of SEQ ID NO: 38, as follows:

[0193] Thus, preferably the RNA construct comprises a nucleotide sequence substantially as set out in SEQ ID NO: 38, or a fragment or variant thereof.

[0194] In another embodiment, the RNA construct may comprise or consist of SEQ ID NO: 39, as follows:

[0195] Thus, preferably the RNA construct comprises a nucleotide sequence substantially as set out in SEQ ID NO: 39, or a fragment or variant thereof.

[0196] In a second aspect of the invention, there is provided a nucleic acid sequence encoding the RNA construct of the first aspect.

[0197] In one embodiment, the nucleic acid sequence may comprise or consist of SEQ ID NO: 40, as follows:

[0198] Thus, preferably, the nucleic acid sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO: 40, or a fragment or variant thereof.

[0199] In one embodiment, the nucleic acid sequence may comprise or consist of SEQ ID NO: 41, as follows:

[0200] Thus, preferably, the nucleic acid sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO: 41, or a fragment or variant thereof.

[0201] In a third aspect, there is provided an expression cassette comprising a nucleic acid sequence according to the second aspect.

[0202] The nucleic acid sequences of the present invention are preferably comprised in a recombinant vector, eg, for delivery to a host cell of interest to enable production of an RNA construct.

[0203] Thus, in a fourth aspect, there is provided a recombinant vector comprising an expression cassette according to the third aspect.

[0204] In one embodiment, the vector may comprise the nucleic acid sequence of SEQ ID NO: 35, as follows, where "GOI" represents the location of the sequence encoding the therapeutic biomolecule:

[0205] Thus, preferably the vector comprises a nucleotide sequence substantially as set out in SEQ ID NO: 35, or a variant or fragment thereof.

[0206] In embodiments in which the vector comprises a nucleic acid sequence encoding an RNA construct comprising MERS-CoV ORF4a, the vector may comprise the nucleic acid sequence of SEQ ID NO: 36, as follows, where "GOI" represents the location of the sequence encoding the therapeutic biomolecule:

[0207] Thus, preferably the vector comprises a nucleotide sequence substantially as set out in SEQ ID NO: 36, or a variant or fragment thereof.

[0208] In embodiments in which the vector comprises a nucleic acid sequence encoding an RNA construct comprising PIV5, the vector may comprise the nucleic acid sequence of SEQ ID NO: 37, as follows, where "GOI" represents the location of the sequence encoding the therapeutic biomolecule: GAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAA [SEQ ID NO: 37]

[0209] Thus, preferably the vector comprises a nucleotide sequence substantially as set out in SEQ ID NO: 37, or a variant or fragment thereof.

[0210] The saRNA constructs of the present invention may be made using the DNA plasmid shown in Figure 7 or 8 as a template. An RNA copy may then be made by in vitro transcription using a polymerase, such as T7 polymerase, with the T7 promoter shown upstream of the saRNA in the plasmid map of Figure 7 or 8. Thus, the saRNA constructs of the first embodiment may be made using, as a template, a DNA plasmid having a nucleic acid sequence as set forth in any one of SEQ ID NOS: 35-37 shown in Figure 7 or 8, or a variant or fragment thereof. Of course, it will be understood that instead of T7 polymerase, other RNA polymerases may be used, such as SP6 or T3 polymerase, in which case the saRNA construct may instead include an SP6 or T3 promoter.

[0211] The vector of the fourth aspect encoding the RNA construct of the first aspect may be, for example, a plasmid, cosmid or phage, and / or may be a viral vector. Such recombinant vectors are highly useful in the delivery system of the present invention for transforming cells with a nucleotide sequence. The nucleotide sequence may preferably be a DNA sequence, and it is this DNA sequence that encodes the RNA sequence that forms the RNA construct of the first aspect.

[0212] Recombinant vectors encoding the RNA constructs of the first embodiment may also contain other functional elements. For example, they may further contain various other functional elements, such as a suitable promoter for initiating transgene expression upon 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 for inducing or regulating DNA replication may be required 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 favoring targeted integration (e.g., via homologous recombination) are envisioned. Suitable promoters include, for example, the SV40 promoter, CMV, EF1a, PGK, viral long terminal repeats, and inducible promoters, such as the tetracycline-inducible system. The cassette or vector may also contain a terminator, e.g., beta globin, an SV40 polyadenylation sequence, or a synthetic polyadenylation sequence. The recombinant vector may also contain a promoter, regulator, or enhancer for controlling nucleic acid expression, as needed.

[0213] A vector may also contain DNA encoding a gene that can be used as a selectable marker in the cloning process, i.e., a gene that can be used to allow for the selection of transfected or transformed cells and to allow for the selection of cells harboring a vector that has incorporated heterologous DNA. For example, ampicillin, neomycin, puromycin, or chloramphenicol resistance are contemplated. The vectors shown in Figures 7 and 8 contain an ampicillin resistance marker useful for selecting plasmids in bacteria. Alternatively, the selectable marker gene may be in a different vector so that it can be used simultaneously with the vector containing the transgene. The cassette or vector may also contain DNA involved in regulating the expression of a nucleotide sequence or DNA for targeting the expressed polypeptide to a specific portion of the host cell.

[0214] Purified vectors may be directly inserted into host cells by suitable means, such as direct endocytic uptake. Vectors may also be directly introduced into host cells (e.g., eukaryotic or prokaryotic cells) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, or ballistic bombardment. Alternatively, vectors of the invention may be directly introduced into host cells using a particle gun.

[0215] The nucleic acid molecule may, but need not, become incorporated into the DNA of the host cell. Undifferentiated cells can be stably transformed, resulting in the production of genetically modified daughter cells (in which case regulation of expression in the subject may require, for example, specific transcription factors or gene activators). Alternatively, delivery systems can be designed to favor unstable or transient transformation of differentiated cells. In this case, regulation of expression may be less important, since expression of the DNA molecule will cease when the transformed cell dies or ceases to express the protein.

[0216] Alternatively, the delivery system can provide the nucleic acid molecule to the host cell without it being incorporated into a vector. For example, the nucleic acid molecule may be incorporated into a liposome or viral particle. Alternatively, the "naked" nucleic acid molecule may be inserted into the host cell by suitable means, such as by direct endocytic uptake.

[0217] In a fifth aspect, there is provided a pharmaceutical composition comprising the RNA construct of the first aspect, the nucleic acid sequence of the second aspect, the expression cassette of the third aspect or the vector of the fourth aspect, and a pharmaceutically acceptable vehicle.

[0218] In a sixth aspect, there is provided a method for making a pharmaceutical composition according to the fifth aspect, 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 vehicle.

[0219] In a seventh aspect, there is provided a method for preparing an RNA construct of the first aspect, comprising the steps of: a) i) introducing the vector of the fourth aspect into a host cell; and ii) culturing the host cell under conditions that result in the production of the RNA construct of the first aspect; or b) transcribing an RNA construct from a vector according to the fourth aspect A method is provided, comprising:

[0220] The host cell in step a) may 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 a human embryonic kidney 293 cell or a Chinese hamster ovary (CHO) cell. Step (b) may be carried out in vitro or in vivo, preferably in vitro.

[0221] Suitable methods for in vitro transcription are well known in the art and will be known to those skilled in the art, for example as described in Molecular Cloning, A Laboratory Manual, 2nd Edition (1989) editor C Nolan, Cold Spring Harbor Laboratory Press.

[0222] The RNA replicon of the first aspect is particularly suitable for therapy.

[0223] Although the inventors have envisaged that the RNA construct of the first aspect will be produced by in vitro transcription for in vivo use in therapy, those skilled in the art will recognise that the RNA construct may be produced in vivo in a subject involved in therapy by delivering to the subject in vivo a nucleic acid according to the second aspect, an expression cassette according to the third aspect, or a vector according to the fourth aspect.

[0224] Thus, according to an eighth aspect there is provided 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 for use as a medicament or in therapy.

[0225] In a ninth aspect of the present invention there is provided 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 for use in the prevention, amelioration or treatment of a protozoan, fungal, bacterial or viral infection.

[0226] The protozoan, fungal, bacterial or viral infection may be a protozoan, fungal, bacterial or viral infection as defined in the first aspect.

[0227] In a tenth aspect of the present invention there is provided 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 for use in the prevention, amelioration or treatment of cancer.

[0228] The cancer may be as defined in the first aspect.

[0229] In an eleventh aspect of the present invention there is provided a method for treating a protozoan, fungal, bacterial or viral infection, the method comprising administering to a subject in need thereof 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.

[0230] The protozoan, fungal, bacterial or viral infection to be treated may be a protozoan, fungal, bacterial or viral infection as defined in the first aspect.

[0231] In a twelfth aspect of the present invention there is provided a method for treating cancer, the method comprising administering to a subject in need thereof 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.

[0232] The cancer to be treated may be as defined in the first aspect.

[0233] The RNA constructs described herein provide an effective means of vaccinating subjects against viral infections and cancer.

[0234] Accordingly, in a thirteenth aspect of the present invention there is provided a vaccine 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.

[0235] Preferably, the vaccine comprises a suitable adjuvant.

[0236] The adjuvant may be encoded in the RNA construct sequence of 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, or as an adjuvant incorporated into the delivery formulation, or may be an encoded molecular adjuvant.

[0237] The encoded molecular adjuvant may encode a cytokine such as IL-12, GM-CSF, IL-2, IFN-g, or an effector protein such as CD40L, Flt-3, or a microbial protein such as flagellin or cholera toxin B.

[0238] Adjuvants incorporated into the delivery formulation may be selected from the group consisting of bacterial lipopeptides, lipoproteins and lipoteichoic acids; mycobacterial lipoglycans; yeast zymosan, porins, lipopolysaccharides, lipid A, monophosphoryl lipid A (MPL), flagellin, CpG DNA, hemozoin, saponins (Quil-A, QS-21, tomatine, ISCOM, ISCOMATRIX™), squalene-based emulsions, PEI, polymers such as carbopol, lipid nanoparticles and bacterial toxins (CT, LT).

[0239] In a fourteenth aspect of the present invention there is provided 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 for use in stimulating an immune response in a subject.

[0240] As an antigen as defined in the first aspect, an immune response may be stimulated against a protozoan, a bacterium, a virus, a fungus or a cancer.

[0241] According to a fifteenth aspect, there is provided 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 for use in stem cell therapy.

[0242] Stem cell therapy may involve reprogramming somatic cells into cells with stem cell characteristics.

[0243] Somatic cells may be reprogrammed by delivering one or more proteins capable of enhancing the reprogramming of somatic cells into cells with stem cell characteristics as defined in the first aspect.

[0244] According to a sixteenth aspect there is provided a method of modifying a cell ex vivo or in vitro, the method comprising delivering to the cell 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.

[0245] Preferably, the method is carried out ex vivo.

[0246] The cell may be a eukaryotic or 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.

[0247] Preferably, the modified cells are suitable for cell therapy indications.

[0248] In a seventeenth aspect, there is provided a modified cell obtained from or obtainable by the method of the sixteenth aspect.

[0249] In an eighteenth aspect, there is provided a modified cell of the seventeenth aspect for use in therapy, optionally cell therapy.

[0250] It will be appreciated 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 (known herein as active agents) can be used in medicine, and they can be used as a monotherapy (i.e. active agent use) for treating, ameliorating or preventing disease or for vaccination. Alternatively, the active agents according to the invention can be used as an adjunct to or in combination with known therapies for treating, ameliorating or preventing disease.

[0251] The RNA constructs, nucleic acid sequences, expression cassettes, vectors, or pharmaceutical compositions of the present invention may be combined into compositions having a number of different forms, depending on the particular manner in which the composition will be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposomal suspension, polyplex, emulsion, lipid nanoparticles (having RNA on their surface or encapsulated), or any other suitable form that can be administered to humans or animals in need of treatment or vaccination. It will be understood that the pharmaceutical vehicle of the present invention should be well tolerated by the subject to which it is administered.

[0252] The RNA constructs, nucleic acid sequences, expression cassettes, vectors, or pharmaceutical compositions of the present invention may also be incorporated into sustained- or delayed-release devices. Such devices may, for example, be inserted above or below the skin, and the medication may be released over a period of weeks or even months. The device may be placed at least adjacent to the treatment site. Such devices may be particularly advantageous when long-term treatment with a genetic construct or recombinant vector is required, which typically requires frequent administration (e.g., at least daily injections).

[0253] However, in a preferred embodiment, the medicament of the present invention can be administered to a subject by injection into the bloodstream, muscle, skin, or directly into the area requiring treatment. Most preferably, the medicament containing the RNA construct is injected into muscle. The injection can be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), or intramuscular (bolus or infusion).

[0254] It will be understood that the amount of RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition required will be determined by its biological activity and bioavailability, which in turn will depend on the mode of administration, the physiochemical properties of the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition, and whether it is used as a monotherapy or combination therapy. The frequency of administration will also be affected by the half-life of the active agent within the subject being treated. The optimal dosage to be administered can be determined by one skilled in the art and is expected to vary depending on the particular RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition used, the strength of the pharmaceutical composition, the mode of administration, and the type and progression of the viral infection. It is expected that the dosage will need to be adjusted depending on additional factors depending on the specific subject being treated, such as the subject's age, weight, sex, diet, and time of administration.

[0255] Generally, a daily dose of between 0.001 μg / kg and 10 mg / kg of body weight, or between 0.01 μg / kg and 1 mg / kg of body weight of an RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition of the invention can be used to treat, ameliorate or prevent a disease, depending on the active agent used.

[0256] The daily dose may be given as a single administration (e.g., a single daily injection or inhalation of a nasal spray). Alternatively, the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition may require administration two or more times during the day. By way of example, the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition may be administered as two (or more, depending on the severity of the disease being treated) daily doses of between 0.07 μg and 700 mg (i.e., assuming a body weight of 70 kg). The patient receiving treatment may take a first dose while awake and then a second dose in the evening (in the case of a two-dose regimen), or at intervals of three or four hours thereafter. Alternatively, a delayed-release device may be used to provide the patient with an optimal dose of the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition of the present invention, without the need for repeated doses.

[0257] Preferably, however, the RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition according to the invention may be given as a once-weekly dose, more preferably once every two weeks.

[0258] Known procedures, such as those routinely employed by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to formulate specific formulations of the RNA constructs, nucleic acid sequences, expression cassettes or vectors of the invention and precise treatment regimens (e.g., daily doses and frequency of administration of drugs).

[0259] A "subject" may be a vertebrate, a mammal, or a domestic animal. Thus, the compositions and medicaments of the present invention can be used to treat any mammal, such as livestock (e.g., horses), pets, or for other veterinary purposes. Most preferably, however, the subject is a human.

[0260] A "therapeutically effective amount" of an RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition is any amount that, when administered to a subject, is the amount of the foregoing necessary to ameliorate, prevent, or treat any given disease.

[0261] For example, about 0.0001 mg to about 800 mg, preferably about 0.001 mg to about 500 mg, of the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition of the present invention may be used. The amount of the replicon, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition is preferably about 0.01 mg to about 250 mg, and most preferably about 0.01 mg to about 1 mg. Preferably, the RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition of the present invention is administered at a dose of 1 to 200 μg.

[0262] A "pharmaceutically acceptable vehicle," as referred to herein, is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0263] In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances that may act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, pigments, coatings, or tablet disintegrants. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid in admixture with a finely divided active agent of the present invention. In tablets, the active agent (e.g., an RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition of the present invention) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compressed to the desired shape and size. Powders and tablets preferably contain up to 99% of the active agent. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting point waxes, and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream, etc.

[0264] However, the pharmaceutical vehicle may be liquid, and the pharmaceutical composition may be in the form of a liquid formulation. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The RNA construct, nucleic acid sequence, expression cassette, vector, or pharmaceutical composition of the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity regulators, stabilizers, or osmolality regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives such as those described above, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (such as monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid compositions for parenteral administration. The liquid vehicle for pressurized compositions may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0265] Liquid pharmaceutical compositions are sterile solutions or suspensions, which can be utilized by, for example, subcutaneous, intradermal, intrathecal, epidural, intraperitoneal, intravenous, and particularly intramuscular injection. The nucleic acid sequences or expression cassettes of the invention can be prepared as sterile solid compositions that can be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0266] The RNA constructs, nucleic acid sequences, expression cassettes, vectors, or pharmaceutical compositions of the invention may be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, gum arabic, gelatin, sorbitan monoleate, polysorbate 80 (oleic acid esters of sorbitol and its anhydrides copolymerized with ethylene oxide), etc. The RNA constructs, nucleic acid sequences, expression cassettes, vectors, or pharmaceutical compositions of the invention may be orally administered in either liquid or solid composition form. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0267] It will be understood that the present invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, comprising substantially the amino acid or nucleic acid sequence of any of the sequences referred to herein, including variants or fragments thereof. The terms "substantially an amino acid / nucleotide / peptide sequence," "variant," and "fragment" may refer to a sequence having at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any one of the sequences referred to herein, for example, a sequence having 40% identity with the sequences identified as SEQ ID NOs: 1-55.

[0268] Also contemplated are amino acid / polynucleotide / polypeptide sequences that have greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and even more preferably greater than 80% sequence identity with any of the sequences mentioned. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences mentioned, 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 with any of the sequences mentioned herein.

[0269] A skilled artisan will know how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. To calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, the two sequences must first be aligned, and then the sequence identity value is calculated. The percentage identity of two sequences may vary 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 by the alignment method, such as local versus global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and gap penalties, such as functional forms and constants.

[0270] Once an alignment is made, there are many different ways to calculate the percentage identity between two sequences. For example, one can divide the identity number 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-gap positions; or (v) the number of equalized positions excluding overhangs. Furthermore, it will be understood that the percentage identity is also strongly dependent on length. Therefore, the shorter the sequence pair, the higher the chance of sequence identity occurring by chance.

[0271] It will be appreciated, therefore, that accurate alignment of protein or DNA sequences is a complex process. The popular multiple 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 protein or DNA multiple alignments in accordance with the present invention. Suitable parameters for ClustalW may be as follows: for DNA alignments: GAP open penalty = 15.0, GAP extension penalty = 6.66, and matrix = Identity; for protein alignments: GAP open penalty = 10.0, GAP extension penalty = 0.2, and matrix = Gonnet; for DNA and protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will recognize that these and other parameters may need to be modified for optimal sequence alignment.

[0272] Preferably, the percentage identity between two amino acid / polynucleotide / polypeptide sequences can then be calculated from such an alignment as (N / T) x 100, where N is the number of positions where the sequences share identical residues, and T is the total number of positions compared, including gaps, and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percentage identity between two sequences includes (i) preparing a sequence alignment using the ClustalW program with a suitable set of parameters, such as those detailed above; and (ii) inserting the values of N and T into the following formula: sequence identity = (N / T) x 100.

[0273] Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence would be encoded by a sequence that hybridizes to a DNA sequence or its complement under stringent conditions. Stringent conditions, as defined by the inventors, mean that the nucleotides hybridize to filter-bound DNA or RNA in 3× sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by at least one wash in 0.2× SSC / 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ from a sequence set forth, for example, in SEQ ID NOs: 1-57 by at least one, but fewer than 5, 10, 20, 50, or 100, amino acids.

[0274] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be altered or varied to provide functional variants thereof without substantially affecting the sequence of the protein encoded thereby. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons encoding the same amino acid within the sequence, thereby producing a silent (synonymous) change. Other suitable variants include those having a homologous nucleotide sequence but having all or part of the sequence altered by the substitution of different codons encoding amino acids having side chains with similar biophysical properties to the substituted amino acid, resulting in a conservative change. 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. It is expected that it will be understood which amino acids can be replaced with amino acids having similar biophysical properties, and the skilled artisan will know the nucleotide sequences encoding these amino acids.

[0275] All of the features described in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0276] For a better understanding of the present invention and to show how an embodiment thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief explanation of the drawings]

[0277] [Figure 1] Schematic diagram of one embodiment of a self-amplifying RNA replicon or construct based on the Venezuelan Equine Encephalitis Virus (VEEV) backbone. The so-called "stearchicon" vector is a saRNA replicon / construct that encodes nonstructural proteins (NSP1-4) and innate inhibitory proteins (IIPs), upstream or downstream of the GOI (gene of interest). [Figure 2] (Figure 1) Genome replication results in dsRNA that is recognized by the sensor molecules MDA5 and PACT. These sensors signal to activate downstream cascades, including transcription factors (NF-κB, IRF-3, and -7) and activation of restriction factors (dotted lines), which directly inhibit RNA amplification and protein expression. Expression of PIV-V or ORF4a blocks innate recognition of dsRNA by MDA5 and PACT, preventing activation of downstream cascades that limit replicon RNA amplification and protein expression from synthetic RNA. [Figure 3a] (Figure 1) Screening of IIP-encoding VEEV replicons in vitro. Cells were transfected with two batches of RNA containing luciferase as a reporter protein and assessed for protein expression 24 hours later. HeLa and MRC5 are known to have a more intact IFN expression pathway compared to HEK. [Figure 3b] FIG. 3 shows data for FIG. 3 a, presented as fold change in expression compared to wild type. [Figure 4] Screening of IIP-encoding replicons (#6 and #8) in BALB / c and BL / 6 mice using firefly luciferase as a reporter protein. BL / 6 mice are known to express more IFN than BALB / c mice, and therefore IIP allowed for more luciferase expression at 3 days and longer expression lasting longer than 14 days. [Figure 5]Screening of IIP-encoded replicons (#6 and #8) in BALB / c and BL / 6 mice using Gaussia luciferase as a reporter protein. BL / 6 mice are known to express more IFN than BALB / c mice, and therefore IIP allowed for longer expression of the soluble reporter protein, lasting longer than 14 days. [Figure 6] FIG. 1 shows a construct map of one embodiment of an expression vector encoding an RNA construct containing a GOI. [Figure 7] FIG. 1 shows a construct map of one embodiment of an expression vector encoding an RNA construct containing GOI-MERS-CoV ORF4a. [Figure 8] FIG. 1 shows a construct map of one embodiment of an expression vector encoding an RNA construct containing GOI-PIV5. [Figure 9a] Figure 1 shows the constructs of the invention and a schematic representation of protein expression from wild-type and IIP VEEV replicons in vitro. Schematic representation of wild-type and cis-encoded IIP VEEV replicons. [Figure 9b] Figure 1 shows the constructs of the invention and a schematic of protein expression from wild-type and IIP VEEV replicons in vitro. Schematic of innate sensing of self-amplifying RNA. [Figure 9c] Figure 1 shows a schematic representation of the constructs of the invention and protein expression from wild-type and IIP VEEV replicons in vitro. Transfection of firefly luciferase saRNA in HEK293T.17, HeLa, and MRC5 cells measured as relative light units (RLU) in vitro. Bars represent the mean fold change ± standard deviation normalized to the wild-type VEEV control for n=3. [Figure 10]Figure 1 shows dose titration of WT and MERS CoV-2 replicons in C57BL6 / J mice. Protein expression was quantified on days 7 and 10 after intramuscular injection of either 0.2, 2, or 20 μg of RNA. Each dot represents a single mouse, and bars represent mean ± SEM for n=10. * indicates significance of p<0.05 as assessed using the Kruskal-Wallis test with multiple comparisons. [Figure 11] Figure 1 shows co-formulation of WT and MERS CoV_2 replicons with the JAK inhibitor ruxolitinib in C57BL6 / J mice. Protein expression was quantified on days 4, 7, 10, and 14 (a, b, c, and d, respectively) after intramuscular injection of either 5 μg of RNA and 100 μg of ruxolitinib. Each dot represents a single mouse leg, and bars represent the mean ± SEM for n=10. [Figure 12] Figure 1 shows protein expression of eGFP ± MERS CoV_2 RNA (which corresponds to MERS-CoV ORF4a) (0.2, 2, or 20 μg) (a, b) or ± ruxolitinib (0.1, 1, 10, or 100 μg) (c, d) in human skin explants. The number of eGFP-expressing cells (% of GFP+ cells) (a, c) and total protein expression per cell (median GFP fluorescence intensity (MFI)) (b, d) were quantified 72 hours post-injection. Each dot represents the mean ± SEM with n=3. * indicates significance of p<0.05 as assessed using the Kruskal-Wallis test with multiple comparisons. [Figure 13] Figure 1 shows the immunogenicity of RABV ± MERS-CoV_2 (which corresponds to MERS-CoV ORF4a) in rabbits. a) Antibody titers of RABV antigen-specific IgG after intramuscular immunization with 20 μg prime and boost at weeks 0 and 4, n=5. b) Neutralizing IC50 against pseudotyped RABV virus, n=5; gray dotted line represents limit of detection. * indicates significance of p<0.05 as assessed using the Kruskal-Wallis test with multiple comparisons. [Figure 14]Figure 1 shows transfection of firefly luciferase saRNA in HEK293T.17, HeLa, and MRC5 cells in vitro, measured as relative light units (RLU). Bars represent mean ± standard deviation control, n=3. "a" and "b" represent two independently prepared batches of RNA. [Figure 15] Figure 1 shows transfection of WT fLuc, MERS-CoV_2 ORF4a, and PIV-5 RNA in a) mouse (MEF), b) rabbit (RK13), c) non-human primate (LLC), and d) human (MRC5) cells in vitro measured as relative light units (RLU). Bars represent mean ± standard deviation control, n=3. [Figure 16] Figure 1 shows luciferase expression of intracellular (firefly luciferase) and secreted (Gaussia luciferase) proteins in BALB / c and C57BL6 / J mice in vivo. Protein expression was quantified in either muscle (a, c, e) or serum (b, d, f) using an in vivo imaging system (IVIS) on days 3 (a, b), 7 (c, d), and 14 (e, f). Each dot represents a single mouse, and bars represent the mean ± SEM, with n = 10 for fLuc and n = 5 for gLuc. [Figure 17] FIG. 1 shows t-distributed stochastic neighbor embedding (tSNE) plots of unsupervised clusters of live cells (gray) overlaid with gating on eGFP+ cells (green) separated by phenotype (blue) in human skin explants treated with 0.2 μg eGFP RNA. [Figure 18] FIG. 1 shows t-distributed stochastic neighbor embedding (tSNE) plots of unsupervised clusters of live cells (gray) overlaid with gating on eGFP+ cells (green) separated by phenotype (blue) in human skin explants treated with 2 μg of eGFP RNA. [Figure 19]FIG. 1 shows t-distributed stochastic neighbor embedding (tSNE) plots of unsupervised clusters of live cells (gray) overlaid with gating on eGFP+ cells (green) separated by phenotype (blue) in human skin explants treated with 20 μg of eGFP RNA. [Figure 20] Figure 1 shows t-distributed stochastic neighbor embedding (tSNE) plots of unsupervised clusters of live cells (gray) overlaid with gating on eGFP+ cells (green) separated by phenotype (blue) in human skin explants treated with 0.2 μg of eGFP-PIV-5 RNA. [Figure 21] Figure 1 shows t-distributed stochastic neighbor embedding (tSNE) plots of unsupervised clusters of live cells (gray) overlaid with gating on eGFP+ cells (green) separated by phenotype (blue) in human skin explants treated with 2 μg of eGFP-PIV-5 RNA. [Figure 22] Figure 1 shows a t-distributed stochastic neighbor embedding (tSNE) plot of unsupervised clusters of live cells (gray) overlaid with gating on eGFP+ cells (green) separated by phenotype (blue) in human skin explants treated with 20 μg of eGFP-PIV-5 RNA. [Figure 23] Figure 1 shows a t-distributed stochastic neighbor embedding (tSNE) plot of unsupervised clusters of live cells (gray) overlaid with gating on eGFP+ cells (green) separated by phenotype (blue) in human skin explants treated with 0.2 μg of eGFP-MERS-CoV_2 RNA (which corresponds to MERS-CoV ORF4a). [Figure 24] t-SNE plot of unsupervised clusters of live cells (gray) overlaid with gating on eGFP+ cells (green) separated by phenotype (blue) in human skin explants treated with 2 μg of eGFP-MERS-CoV_2 RNA. [Figure 25]Figure 1 shows a t-distributed stochastic neighbor embedding (tSNE) plot of unsupervised clusters of live cells (gray) overlaid with gating on eGFP+ cells (green) separated by phenotype (blue) in human skin explants treated with 20 μg of eGFP-MERS-CoV_2 RNA (which corresponds to MERS-CoV ORF4a). [Figure 26a] Figure 1 shows the phenotypic identity of cells present in human skin explants and GFP+ cells after intradermal (ID) injection of eGFP±ruxo formulations as determined by flow cytometry. Identification of cells in the population of total cells extracted from human skin explants and GFP-expressing skin cells from explants treated with 2 μg of eGFP-encoding saRNA±0.1, 1, 10, or 100 μg of ruxo, n=3. [Figure 26b] Figure 1 shows the phenotypic identity of cells present in human skin explants and GFP+ cells after intradermal (ID) injection of eGFP±ruxo formulations as determined by flow cytometry. Percentage of cells of each phenotype expressing GFP. 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+). [Figure 27] Figure 1 shows the immunogenicity of RABV ± MERS-CoV_2 (which corresponds to MERS-CoV ORF4a) in mice and rats. a) Antibody titers of RABV antigen-specific IgG after intramuscular immunization of mice with 1 μg prime and boost at weeks 0 and 4, n=5. b) Neutralization IC50 of mice against pseudotyped RABV virus, n=5, the grey dotted line represents the limit of detection. a) Antibody titers of RABV antigen-specific IgG after intramuscular immunization of rats with 20 μg prime and boost at weeks 0 and 4, n=5. b) Neutralization IC50 of rats against pseudotyped RABV virus, n=5, the grey dotted line represents the limit of detection. [Figure 28]FIG. 1 shows a schematic diagram of the proposed mechanism of PIV-5 V and MERS-CoV ORF4a in saRNA sensing. [Figure 29] FIG. 1 shows median fluorescence intensity (MFI) data demonstrating increased expression of SARS-CoV-2 glycoprotein from saRNA according to one embodiment of the present invention in HeLa cells compared to saRNA encoding only SARS-CoV-2 glycoprotein (i.e., no IIP) when expressed together with the native inhibitory protein, MERS-ORF4a. [Example]

[0278] We hypothesized that cis-encoded proteins from viruses known to inhibit innate recognition of saRNA would reduce innate sensing and enhance both protein expression and immunogenicity of saRNA vaccines. Therefore, we designed and tested RNA replicons containing various innate inhibitory proteins (IIPs) and genes of interest, and then characterized whether these replicons enhanced the expression of both intracellular and secreted proteins (encoded by the genes of interest).

[0279] Materials and Methods Cloning of innate inhibitory protein (IIP) replicons Self-amplifying RNAs encoding firefly luciferase, Gaussia luciferase, enhanced green fluorescent protein (eGFP), rabies glycoprotein (RABV), and replicase from Venezuelan equine encephalitis were cloned into plasmid vectors as previously described (53). Libraries of interferon inhibitor proteins were cloned into these vector backbones as part of the gene of interest (fLuc, GLuc, eGFP, or RABV) containing a T2A cleavage site (GenBank accession number AAC97195.1). Interferon inhibitory proteins can be found under 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). For studies in mice, the PIV-5V protein with the N100D mutation was used (45).

[0280] In vitro transcription of saRNA Self-amplifying RNA was produced using in vitro transcription. Plasmid DNA (pDNA) was transformed into Escherichia coli (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 a Plasmid Plus MaxiPrep kit (QIAGEN, UK), and the final pDNA concentration was measured using a NanoDrop One (ThermoFisher, UK). pDNA was linearized with MluI for 3 hours at 37°C. RNA for in vitro transfection was prepared using 1 μg of linearized pDNA template in an mMachine™ T7 transcription kit (Invitrogen, UK) and purified using the MEGAclear™ Transcription Cleanup Kit (Invitrogen, UK) according to the manufacturer's protocol. RNA for ex vivo and in vivo experiments was prepared as previously described (2). Uncapped RNA transcripts were produced using 1 μg of linearized pDNA template in a MEGAScript™ T7 transcription reaction (Invitrogen, UK) at 37°C for 2 h using the manufacturer's protocol. Transcripts were then purified by LiCl precipitation overnight at -20°C, centrifuged at 14,000 RPM for 20 min at 4°C to pellet the RNA, rinsed once with 70% EtOH, centrifuged again at 14,000 RPM for 5 min at 4°C, and resuspended in UltraPure HO (Ambion, UK). Purified transcripts were capped for 2 h at 37°C using the ScriptCap™ Cap1 Capping System Kit (CellScript, WI, USA) using the manufacturer's protocol.The capped transcripts were then finally purified by LiCl precipitation as described above, 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.

[0281] saRNA formulation For protein expression experiments, fLuc, gLuc, and eGFP saRNAs were complexed with 100 kDa pABOL using a titration method as previously described (2). For in vivo immunogenicity experiments, RABV saRNA was complexed with 8 kDa pABOL. Briefly, RNA and pABOL were diluted in HEPES buffer (20 mM HEPES, 5 wt.% glucose in HO, pH 7.4) and combined in a NanoAssemblr benchtop compounding device (Precision Nanosystems, Inc., Vancouver, Canada) at a volume ratio of 4:1 (RNA:polymer) at 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 h of preparation. For co-formulation, ruxolitinib (ruxo, Selleck Chemicals, UK) was added directly to the polyplexes at the specified doses.

[0282] In vitro transfection Transfections were performed in 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 macaque kidney cells (ATCC, USA). Cells were cultured in complete Dulbecco's modified Eagle's medium (cDMEM) (Gibco, ThermoFisher, UK) containing 10% (v / v) fetal bovine serum (FBS), 5 mg / mL L-glutamine, and 5 mg / mL penicillin / streptomycin (ThermoFisher, UK) (HEK, HeLa, MEF cells), complete modified Eagle's medium (cMEM) containing 10% (v / v) fetal bovine serum (FBS), 5 mg / mL L-glutamine, and 5 mg / mL penicillin / streptomycin (ThermoFisher, UK) (MRC5, RK13 cells), or complete Medium 199 (cM199, SigmaAldrich, UK) containing 1% horse serum (Gibco, ThermoFisher, UK) (LLC cells). Twenty-four hours prior to transfection, cells were plated 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 prewarmed transfection medium (DMEM + 5 mg / mL L-glutamine, MEM + 5 mg / mL L-glutamine, or M199). 100 μL of polyplex solution (containing 100 ng of 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-luciferin substrate (Promega, UK) was added and mixed well 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 plate reader (BMG LABTECH, UK). Background fluorescence from control wells was subtracted from each well.

[0283] f-luciferase expression in mice in vivo All animals were operated in accordance with the UK Home Office Animals Scientific Procedures Act 1986 and project licenses (P63FE629C) and personal licenses (IC37CBB8F) approved by the local ethical committee and the UK government. Food and water were provided ad libitum. Six- to eight-week-old female BABL / c mice (Charles River, UK) or C57BL / 6 mice (Charles River, UK) were group-housed (n = 5 per cage) in a well-acclimated room. Mice were injected intramuscularly (IM) with either 5 μg of fLuc saRNA complexed with pABOL in both hind limbs or 5 μg of GLuc in one hind limb, in a total volume of 50 μL. After 3, 4, 7, 10, or 14 days, mice were imaged for fLuc as previously described (54, 55), or blood was collected for GLuc analysis using a Gaussia luciferase glow assay kit (Pierce, Thermo Scientific, UK) according to the manufacturer's protocol. Protein expression in serum was quantified using a FLUOstar OMEGA plate reader (BMG LABTECH, UK). Background fluorescence from control wells was subtracted from each well. For fLuc analysis, mice were injected intraperitoneally (IP) with 150 μL of XenoLight RediJect D-luciferin substrate (PerkinElmer, UK) and allowed to rest for 10 min. Mice were then anesthetized using isoflurane and imaged for 2 min using an In Vivo Imaging System (IVIS) FX Pro (Kodak Co., Rochester, NY, USA) with Molecular Imaging software version 5.0 (Carestream Health, USA). The signal from each injection site was quantified using Molecular Imagine software and expressed as total flux (p / s).

[0284] g luciferase expression in mice in vivo Six- to eight-week-old female BALB / c or C57BL / 6 mice (Charles River, UK) were housed in groups (n=5) in a well-conditioned room. Mice were injected intramuscularly (IM) with 5 μg of gLuc in a total volume of 50 μL into one hind limb. After 3, 7, and 14 days, mice were bled via the tail vein. Blood was allowed to clot, centrifuged at 10,000 RPM for 5 minutes, and serum was removed. Serum from all time points was then assayed in a single 96-well white plate (Costar) using the Pierce™ Gaussia Luciferase Glow Assay Kit, using 20 μL of serum and 100 μL of working solution prepared according to the manufacturer's protocol. Luminescence was analyzed using a FLUOstar OMEGA plate reader (BMG LABTECH, UK), and background from naive animals was subtracted from each sample.

[0285] Vaccination of mice, rats and rabbits. BALB / c mice, Sprague-Darley rats, and New Zealand White rabbits were immunized intramuscularly in one hind limb with 1 μg (mice) or 20 μg (rats, rabbits) of RABV-encoding saRNA formulated with pABOL in a total volume of 50 μL (mice) or 100 μL (rats, rabbits). A boost injection was given 4 weeks after the initial prime. Blood was collected 0, 4, and 6 weeks after the start of the study and centrifuged at 10,000 RPM for 5 minutes. Serum was then decanted and stored at -80°C until further analysis.

[0286] RABV-specific ELISA. A semiquantitative immunoglobulin ELISA protocol was performed as previously described (56). Briefly, ELISA plates coated with 0.5 μg / mL RABV 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 h. The plates were then washed, and for mouse ELISA, a 1:4000 dilution of anti-mouse IgG-HRP (Southern Biotech, UK) was added; for rat ELISA, a 1:4000 dilution of goat anti-rat IgG-HRP (Southern Biotech, UK) was added; and for rabbit ELISA, a 1:10000 dilution of mouse anti-rabbit IgG-HRP (Sigma, UK) was added. Mouse standards were prepared by coating ELISA plate wells with anti-mouse kappa (1:1,000) and lambda (1:1,000) light chains (Serotec, UK), blocking with 1% (w / v) BSA / 0.05% (v / v) Tween-20 in PBS, washing, and adding purified IgG (Southern Biotech, UK) starting at 1000 ng / mL and titrating downward in a series of 5-fold dilutions. Rat standards were prepared by coating ELISA plate wells with purified rat IgG (R & D Systems, UK) starting at 1000 ng / mL and titrating downward in a series of 5-fold dilutions. Rabbit standards were prepared by coating ELISA plate wells with 1:1250 anti-rabbit IgG Fc (Milipore), blocking with 1% (w / v) BSA / 0.05% (v / v) Tween-20 in PBS, washing, and adding purified rabbit IgG (AbD Serotech, UK) starting at 1000 ng / mL and titrating downward with a series of 5-fold dilutions. Samples and standards were developed using 3,3',5,5'-tetramethylbenzidine (TMB). The reaction was stopped after 5 minutes with stop solution (Insight Biotechnologies, UK).Absorbance was read on a spectrophotometer (VersaMax, Molecular Devices, UK) using SoftMax Pro GxP v5 software.

[0287] RABV microneutralization assay. Pseudotyped rabies microneutralization was performed on samples from weeks 0, 4, and 6. BHK-21 cells were seeded in 96-well plates at 10,000 cells / well in cDMEM. Serum was heat-inactivated at 56°C and then diluted in 1:5 serial dilutions in cDMEM. Samples were then challenged with an equal volume of pseudovirus at 100 TCID in 50 μL. 50 The mixture was diluted to a concentration of 0.01% and incubated at 37°C for 1 hour, then added to BHK-21 cells and 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 quantified on a FLUOstar OMEGA plate reader (BMG LABTECH, UK). IC values were calculated for each sample. 50 was calculated.

[0288] Human skin explant culture and injection. For ex vivo studies, surgically excised samples of human skin tissue were collected at Charing Cross Hospital, Imperial NHS Trust, London, UK. All tissue was collected after receiving signed informed consent from patients undergoing elective abdominoplasty or mastectomy at Imperial College London under a protocol approved by the local research ethics committee (MED_RS_11_014). Skin tissue was frozen until use and cut into 1 cm sections. 2The explants were cut into 100 mm pieces and cultured in 12-well plates with 2 mL of cDMEM at 37°C with 5% CO2. The explants were injected intradermally (ID) with a dose of 2 μg of saRNA in a total volume of 50 μL using a Micro-Fine Demi 0.3 mL syringe (Becton Dickinson, UK). The medium was changed daily throughout the culture period.

[0289] Flow cytometry Seventy-two hours after injection, skin explants were trimmed to remove the subcutaneous fat layer, and the epidermis and dermis were thoroughly minced with scissors and incubated for 4 hours at 37°C in 2 mL of DMEM supplemented with 1 mg / mL collagenase P (Sigma-Aldrich, UK) and 5 mg / mL dispase II (Sigma-Aldrich, UK) on a rotary shaker. The digest was then filtered through a 70 μm cell strainer and centrifuged at 1,750 RPM for 5 minutes. Cells were then resuspended in 100 μL of FACS buffer (PBS + 2.5% FBS) and stained with fixable aqua live / dead cell stain (ThermoFisher, UK) diluted 1:400 in FACS buffer for 20 minutes on ice. Samples were then washed with 1 mL of FACS buffer, centrifuged at 1,750 RPM, 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). Samples were then washed with 1 mL of FACS buffer, centrifuged at 1,750 RPM for 5 minutes, resuspended in 250 μL of PBS, and then fixed with 250 μL of 3% paraformaldehyde to a final concentration of 1.5% paraformaldehyde and frozen until flow cytometry analysis. Samples were analyzed on an LSRFortessa (BD Biosciences, UK) flow cytometer using FACSDiva software (BD Biosciences, UK) using 100,000 acquired cell events. The gating strategy was performed as previously described (58) and included GFP. +The phenotypic identity of cells was quantified using FlowJo version 10 (FlowJo LLC, Oregon, USA). Unsupervised t-distributed stochastic neighbor embedding (tSNE) analysis of clusters of live cells was performed in FlowJo using 1000 iterations, a perplexity of 30, a learning rate of 15196, the Exact (Vantage Point Tree) KNN algorithm, and the Barnes-Hutt gradient algorithm.

[0290] SARS-CoV-2 glycoproteins in vitro We used a plasmid vector to synthesize a self-amplifying RNA (saRNA) replicon based on the Trinidad donkey Venezuelan equine encephalitis virus (VEEV) alphavirus genome. The viral structural proteins, driven from a subgenomic promoter, were replaced with the surface "spike" glycoprotein of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): GenBank accession number QHD43416.1. We synthesized oligonucleotide fragments encoding SARS-CoV-2 genes using oligonucleotide chains (GeneArt, Germany) and assembled them into a plasmid vector using the Gibson assembly method (NEB Ltd, UK). In a further modification of the SARS-CoV-2 replicon vector, oligonucleotide sequences encoding MERS-CoV ORF4a (AHC74090.1) were synthesized (GeneArt, Germany) and inserted 3' into the linked SARS-CoV-2 coding region in a continuous open reading frame using a variant of the furin / T2A sequence (SEQ ID NO: 26), generating a new SARS-CoV-2 ORF4a vector. Cells were transfected separately with both SARS-CoV-2 and SARS-CoV-2 ORF4a saRNAs and then stained with polyclonal antibodies to examine expression. Briefly, 24 hours after transfection, cells were harvested and 1 x 10 cells were placed in 1 mL of FACS buffer (PBS + 2.5% FBS). 7The cells were resuspended at a concentration of 100 cells / mL. One hundred microliters of the resuspended cells were added to a FACS tube and stained with 50 μL of Live / Dead Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific, UK) at a 1:400 dilution for 20 minutes on ice. The cells were then washed with 2.5 mL of FACS buffer and centrifuged at 1750 RPM for 7 minutes. After centrifugation, the cells were stained with 2.5 μg of SARS-CoV spike protein polyclonal antibody (PA1-41165, Thermo Fisher Scientific, UK) for 30 minutes on ice, then washed with 2.5 mL of FACS buffer and centrifuged at 1750 RPM for 7 minutes. The cells were then stained with 0.4 μg of FITC goat anti-rabbit IgG (BD Pharmigen, UK) for 30 minutes on ice. After incubation, cells were washed with 2.5 mL of FACS buffer, centrifuged at 1750 RPM for 7 minutes, 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 using FACSDiva software (BD Biosciences, UK) on an LSR Forterssa (BD Biosciences, UK). Data were analyzed using FlowJo version 10 (FlowJo LLC, USA) to measure the median fluorescence intensity (MFI) of the positive population of cells, using the same procedure as above, except that SARS-CoV-2 or SARS-CoV-2 ORF4a replicon saRNA was not used, with negative / positive cutoffs set for cells gated as live single cells and mock-transfected cells.

[0291] Statistical analysis. Graphs and statistical tables were generated with GraphPad Prism, version 8. Statistical differences were analyzed using either two-way ANOVA or the Kruskal-Wallis test adjusted for multiple comparisons, with p<0.05 used to indicate significance.

[0292] Results and Discussion RNA replicons have been hypothesized to be promising tools for the delivery and expression of genes of interest for vaccines and therapy. However, double-stranded RNA (dsRNA) is detected intracellularly by a sensing mechanism that initiates a signal transduction cascade that inhibits protein translation. As a result, the expression of the gene of interest encoded in the replicon is significantly impaired, thus limiting the therapeutic potential of RNA replicons.

[0293] We attempted to overcome this problem by developing an RNA replicon encoding a native inhibitory protein to reduce native recognition of saRNA. The only previously published approach to reducing the interferon response using saRNA used interferon inhibitory proteins derived from vaccinia virus, E3, K3, and B18. However, in this study, the interferon inhibitory protein was delivered and formulated as a separate mRNA molecule combined with the saRNA. This required the production of both saRNA and mRNA, and the use of 3-6 times more vaccinia mRNA than replicon RNA to ensure co-delivery to the same cells and provide any observable enhancement in protein expression. Furthermore, the kinetics of expression differ between mRNA and saRNA, such that any beneficial effects of IIPs expressed from mRNA are of shorter duration compared to the associated replicon.

[0294] In light of the current difficulties in using saRNA in therapy, the inventors have designed novel saRNAs that are expected to limit immune responses more effectively than prior art methods, increasing their usefulness in vaccination and therapy.

[0295] Use of PIV-5 and ORF4a as a novel IIP PIV-5 and ORF4a are known to block MDA-5, a cytoplasmic RNA helicase that signals through adaptor molecules called MAVs, leading to the induction of interferon regulatory factors 3 and 7 (IRF3 and IRF7), respectively, which cause the production of restriction factors that reduce translation of the introduced synthetic saRNA (Figure 1). These two IIPs were identified in an initial in vitro screen of 10 IIPs from a variety of different viruses.

[0296] 1. HSV-2 Us1 - This inhibits IFN-B production by suppressing IRF-3 association with the IFN-β promoter [1] 2. HSV-1 Us1 (HSV-1 derived regulatory factor) 3. HSV-1 Us11 - Prevents RIG-I signaling [2, 3] 4. OV20.0L - binds to dsRNA, inhibits both PKR and PACT, and blocks RIG-I signaling [4, 5]. 5. BVDV Npro: Blocks IRF3 phosphorylation and S100A9 signaling [6, 7] 6. PIV5V: Blocks MDA-5 and IRF3 by binding to MDA-5 [8, 9] 7. MERS-CoV M: Interacts with TRAF3 and disrupts TRAF3-TBK1 association, resulting in reduced IRF3 activation [10-12]. 8. MERS-CoV ORF4a: Binds to dsRNA (with preference for long RNAs), inhibits PACT initiation of MDA5 and RIG-I, PKR, and stress responses [13, 14]. 9. Langat virus NS5: Downregulates IFNA1R and impairs JAK-STAT signaling [15-16] 10. Influenza NS1: Binds to dsRNA and blocks RIG-I signaling

[17] .

[0297] These IIPs were incorporated into our standard VEEV saRNA replicon (Figure 1) along with luciferase as the GOI and used as a marker for expression. The constructs were evaluated in three human cell lines: HEK293T cells, HeLa cells, and primary MRC5 fetal epithelial cells (Figures 3a and 3b), which are impaired in the innate sensing pathway. All IIP candidate saRNAs replicated similarly to wild-type saRNAs in HEK293T cells in the absence of innate recognition. While various IIPs (except MERS-CoV M and influenza NS1) were able to enhance expression in HeLa cells, the most significant enhancement (3 logs) was seen with PIV-V and ORF4a. Most importantly, evaluation in primary MRC5 cells showed that only PIV-V and ORF4a were able to enhance luciferase expression by 2 logs. These data suggest that PIV-V and ORF4a are unique in their ability to enhance GOI expression in primary human cells. The identification of PIV-5 and ORF4a for inclusion in RNA vectors was based on these experimental data; their activity was unpredictable given that the other IIPs evaluated appear to function via similar mechanisms. To further support their use in gene delivery, we performed in vivo experiments in mice. Here, we utilized Black 6 (BL6) mice, which are known to have more robust innate sensing mechanisms and downstream interferon responses than BalBc mice; therefore, a comparison between the two models is informative. We assessed the relative expression of WT, PIV-5, and ORF4a replicons in BL6 mice using firefly luciferase (fLuc) as a reporter protein (Figure 4).

[0298] Because fLuc is expressed intracellularly, expression of this gene of interest was visualized based on luciferase expression after intraperitoneal (IP) injection of the substrate D-luciferin and expressed as total flux (p / s) (see Methods). These in vivo experiments demonstrate that PIV5 and ORF4a increased the duration of luciferase expression in BL / 6 cells up to 14 days, while the WT construct was negative up to this time point. In further studies, we evaluated the effect of these two RNA replicon constructs on the expression of gluciferase (Figure 5) or gLuc (as the GOI). gLuc is secreted as a soluble protein, and its activity is indicated in the blood (see Methods). These in vivo experiments demonstrate that PIV5 and ORF4a increased the duration of luciferase expression in BL / 6 cells up to 14 days. Expression of gLuc was significantly higher for both the PIV5 and ORF4a RNA replicons at day 14 (p=0.0244 and 0.00422, respectively). Calculation of the "area under the curve" over 14 days showed the following values. AUCs were as follows: 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 may be supportive, it is important to note that there are significant differences between humans and mice with respect to the specificity of innate restriction factors and interferon-stimulated genes (>100 significant differences); therefore, these data likely underestimate the potential impact in humans based on in vitro observations seen in human cell lines.

[0299] Interferon inhibitory proteins enhance protein expression of saRNA in vitro. We attempted to determine whether a library of interferon-inhibitory proteins enhances firefly luciferase (fLuc) protein expression in vitro. We prepared a library of saRNA VEEV replicons containing IIPs isolated from fLuc with a T2A cleavage site (Figure 9a) using various cytoplasmic interferon targets (Table 1), including IRF-3, MDA5, RIG-I, and JAK / STAT (Figure 9b).

[0300] [Table 1]

[0301] We then transfected the saRNA into HEK293T.17, HeLa, and MRC5 cells using pABOL (Figure 9c, supplemental Figure 1). pABOL is a polymeric delivery system previously characterized as resulting in relatively high protein expression but relatively immunosilent due to its susceptibility to biological degradation (2). We selected these three cell lines because of their variation in the integrity of the IFN pathway; HEK293T.17 cells lack endogenous RIG-I and MDA5 expression and therefore do not possess an intact pathway (37) and should therefore be less sensitive to proteins affecting this pathway, whereas HeLa and MRC5 cells are more sensitive (38, 39). We observed that none of the IIP replicons enhanced protein expression in HEK293T.17 cells (Fig. 1c). Interestingly, both Langat and influenza IIPs significantly reduced protein expression by 0.06-fold, with p = 0.0097 and 0.0061, respectively. In HeLa cells, many of the IIPs enhanced protein expression; HSV-2, HSV-1_1, HSV-1_2, Orf, and BVDV, and increased fLuc expression by 20- to 150-fold. However, PIV-5V and MERS-CoV ORF4a proteins enhanced protein expression by the highest factor of 796- and 893-fold, respectively; however, only the PIV-5 group was statistically significant (p = 0.0272), whereas the ORF4a group did not (p = 0.0689). In MRC5 cells, we also observed the greatest enhancement from PIV-5V and MERS-CoV ORF4a proteins, with 72- and 109-fold greater fLuc expression, respectively, with p=0.0485 and 0.025. In all cell types, and for each construct, there was good concordance between expression levels from two independently generated batches of RNA (Figure 14).

[0302] We further investigated how two mutations in the PIV-5V and MERS-CoV ORF4a proteins affected protein expression in mouse (MEF), rabbit (RK13), non-human primate (LLC), and human cells (MRC5) (Figures 15a-d). The R172A mutation in PIV-5V abolishes its ability to block MDA5 but not STAT (40), and the K63A / K67A mutation in MERS-CoV ORF4a blocks binding to dsRNA (41). We observed that the PIV-5V and MERS-CoV ORF4a proteins did not enhance protein expression in MEF or RK13 cells. The MERS-CoV ORF4a protein did not enhance protein expression in LLC and MRC5 cells (Figures 15c-d), and the K63A / K67A mutation significantly reduced protein expression. The PIV-5V protein enhanced protein expression in MRC5 cells but not in LLC cells, and the R172A mutation reduced protein expression in MRC5 cells. Overall, these data indicate that the PIV-5V and MERS-CoV ORF4a proteins enhance protein expression in interferon-competent human cells, and mutating the proteins with K63A / K67A and R172A substitutions attenuates saRNA expression.

[0303] MERS-CoV ORF4a protein partially reduces the nonlinearity of dose escalation in vivo. Given the enhanced protein expression from PIV-5V and MERS-CoV ORF4a proteins in vitro, we next sought to determine whether these constructs could enhance protein expression in vivo and reduce the nonlinearity of saRNA dose escalation. We tested saRNAs encoding both the intracellular protein firefly luciferase and the secreted protein Gaussia luciferase in vivo (Table 2).

[0304] [Table 2]

[0305] We chose to test these constructs in both BALB / c and C57BL / 6 mice due to differences in their ability to produce interferon: BALB / c mice are poor producers of IFN, whereas C57BL / 6 mice have previously been found to be high producers of IFN-α / β and IFN-γ (42), similar to the imbalance observed in HEK293T.17 and HeLa / MRC5 cells in vitro. We observed that incorporation of PIV-5V and MERS-CoV ORF4a proteins did not enhance protein expression of either fLuc or GLuc in BALB / c mice (Table 2, Figure 16). We observed a slight enhancement in the total area under the curve (AUC) of protein expression of fLuc with MERS-CoV ORF4a protein and of protein expression of GLuc with both PIV-5V and MERS-CoV ORF4a protein in C57BL / 6 mice, but the difference was not statistically significant.

[0306] We previously observed that increasing the dose of saRNA ultimately resulted in lower levels of protein expression, and therefore sought to characterize whether the MERS-CoV ORF4a protein could reduce the nonlinear dose-dependence of saRNA in vivo. We tested doses of 0.2, 2, and 20 μg of wild-type fLuc and fLuc + MERS-CoV ORF4a replicon and quantified protein expression at 7 and 10 days after intramuscular (IM) injection (Figure 10). We observed that both constructs showed similar protein expression (approximately 5000 p / s) at a dose of 0.2 μg after 7 days, and increasing the dose to 2 μg increased protein expression for both constructs (approximately 50,000 p / s for WT and approximately 200,000 p / s for the MERS-CoV ORF4a construct). However, incorporation of the MERS-CoV ORF4a protein enhanced protein expression by 4-fold (p=0.0029). Interestingly, both constructs showed lower protein expression at a dose of 20 μg after 7 days, but WT was 18-fold lower than the MERS-CoV construct (p<0.0001). After 10 days, protein expression levels leveled out at the 2 μg dose, and no expression was observed at the 0.2 and 20 μg doses. Without wishing to be bound to any particular theory, these data indicate that the MERS-CoV ORF4a protein enables a nonlinear, dose-dependent partial recapture of saRNA in vivo.

[0307] Ruxolitinib enhances the protein expression of saRNA in vivo. Given the role of the JAK / STAT pathway in downstream interferon responses, we next sought to characterize how combining saRNA, the MERS-CoV ORF4a interferon inhibitor protein, and ruxolitinib, a potent, selective inhibitor of JAK1 and JAK2 protein kinases (36), affected protein expression in vivo (Figure 3). They injected mice IM with 5 μg of saRNA encoding fLuc±MERS-CoV ORF4a, with or without co-formulation with 100 μg of ruxolitinib, and quantified protein expression 4, 7, 10, and 14 days after injection. After 4 days (Figure 11a), both ruxolitinib-containing formulations significantly increased the expression of WT or MERS-CoV ORF4a constructs (approximately 5 × 10 5 p / s) compared to slightly more protein expression (approximately 10 6 p / s), but it was not statistically significant. However, after 7 days, both ruxolitinib-containing formulations showed greater protein expression compared to the parallel saRNA-only group, with p=0.0347 and 0.0447, respectively. By day 10, these groups still showed a slight increase, but the difference was no longer statistically significant. After 14 days, no protein expression was observed in the saRNA-free group, and very few positive samples were observed in the ruxolitinib group. Without wishing to be bound by any particular theory, these data indicate that ruxolitinib allows for a significant increase in saRNA protein expression, but that in combination, there is no additive effect between MERS-CoV ORF4a protein and ruxolitinib.

[0308] PIV-5V and MERS-CoV ORF4a proteins reduce the nonlinearity of dose escalation in ex vivo human skin explants. Because we observed that IIPs in vitro exhibited differences in protein expression depending on the cell type, we sought to test our saRNA IIP constructs in a more clinically relevant human skin explant model. We characterized both the quantity (% of eGFP+ cells) and quality (median eGFP fluorescence intensity per cell) of protein expression in resident human skin cells using incorporation of PIV-5V and MERS-CoV ORF4a proteins (referred to as MERS-CoV_2 in the figure), as well as co-formulation with ruxolitinib (Figure 12). We tested eGFP saRNA at doses of 0.2, 2, and 20 μg with PIV-5V and MERS-CoV ORF4a proteins (Figures 12a and 12b). We observed that increasing the dose of the WT construct from 0.2 to 2 μg resulted in an increase in the percentage of eGFP+ cells from 10% to 18%, but increasing the dose to 20 μg resulted in a drop in the percentage of eGFP+ cells to approximately 5%. However, for the PIV-5 and MERS-CoV ORF4a constructs, the dose increased linearly with increasing dose of saRNA. The 0.2 μg dose similarly increased eGFP+ cells by approximately 12% for both of these constructs. + This further increased to 15% at 2 μg and 25% at 20 μg, so that at this time point both the PIV-5 and MERS-CoV ORF4a constructs had a statistically significantly higher percentage of eGFP+ cells, p<0.0001 for each. Interestingly, neither the dose nor the incorporation of PIV-5 or MERS-CoV ORF4a protein affected the MFI of eGFP, which was approximately 350 in all samples (Figure 12b).

[0309] We further characterized which cells expressed the saRNA using t-distributed stochastic neighbor embedding, a type of principal component analysis for flow cytometry data that allows visualization by unsupervised clustering of cells, overlaid with defined protein and phenotypic gating (Figures 17-25) (43). We observed that at the highest dose of saRNA (20 μg), PIV-5V and MERS-CoV ORF4a proteins enabled protein expression in immune cells, including T cells, dendritic cells, monocytes, B cells, Langerhans cells, leukocytes, and NK cells, in contrast to resident epithelial cells and fibroblasts.

[0310] Next, we examined how ruxolitinib doses ranging from 0 to 100 μg affected saRNA expression in human skin resident cells. We observed that co-formulation of ruxolitinib with saRNA had no effect on the percentage of eGFP+ cells (Figure 4c), but in fact there was a slight tendency for increasing the ruxolitinib dose to decrease the percentage of eGFP+ cells from approximately 8% to approximately 5%. However, we observed a significant effect on the per-cell quality of eGFP expression (Figure 12d); increasing the ruxolitinib dose increased the eGFP MFI from approximately 100 to approximately 2000 at a 10 μg dose of ruxolitinib, but decreased the MFI to approximately 1000 at a 100 μg dose of ruxolitinib. Similar to cells expressing saRNA PIV-5 and MERS-CoV ORF4a proteins, we found that ruxolitinib enhanced protein expression in immune cells, as opposed to epithelial cells and fibroblasts, and specifically increased uptake in T cells, Langerhans cells, leukocytes, and NK cells (Figures 26a and 26b).

[0311] Taken together, and without wishing to be bound by any particular theory, these data indicate that the IIP replicon enhances expression in immune cells by increasing the percentage of cells expressing the saRNA, while ruxolitinib enhances protein expression depending on the cell.

[0312] MERS-CoV ORF4a protein enhances the immunogenicity of RABV glycoproteins in vivo in rabbits Because protein expression of a nucleic acid formulation is not necessarily a direct predictor of immunogenicity (2), we next attempted to characterize the immunogenicity of a model protein acting as a therapeutic biomolecule, i.e., rabies glycoprotein (RABV), represented by GeneBank identification number NP_056796.1, when combined with the MERS-CoV ORF4a protein (acting as an innate inhibitory protein or IIP) in our saRNA construct. Additionally, we used a RABV protein with an amino acid substitution, the F318V modification, that prevents binding to the cellular p75NTR surface receptor. We injected rabbits with a primary dose of 20 μg of saRNA, followed by a booster injection 4 weeks later, and then sampled their blood for RABV-specific IgG antibodies at 0, 4, and 6 weeks later (Figure 13a). We observed that all rabbits seroconverted after a single injection of both the wild-type and MERS-CoV ORF4a constructs. After 4 weeks, the IgG titers in the MERS-CoV group were significantly higher than those in the wild-type (approximately 5 × 10 3 ng / mL), but was slightly higher (approximately 10 4 ng / mL), which was not statistically significant. However, after 6 weeks, the antibody titers of animals in the MERS-CoV ORF4a group were significantly higher than those of WT (approximately 10 4 ng / mL), the results were significantly higher (approximately 10 5 RABV pseudotyping neutralization mirrored the antibody trends (Fig. 13b). After 4 weeks, the WT group was approximately 10 3 Average IC 50 The MERS-CoV group had approximately 104 IC 50 After 6 weeks, the MERS-CoV group had approximately 10 5 Higher IC 50 The WT group had approximately 10 3 These were also compared to the immunogenicity of WT RABV and RABV-MERS-CoV ORF4a saRNA in mice and rats (Figure 27), which did not result in any significant increase in antibody titers or neutralizing IC50 in either of these species. 50 Without wishing to be bound by any particular theory, these data indicate that the MERS-CoV ORF4a protein enhances the immunogenicity of the saRNA-encoded RABV glycoprotein in rabbits.

[0313] MERS-CoV ORF4a protein enhances expression of SARS-CoV-2 glycoproteins in vitro The inventors also characterized the immunogenicity of another model protein (i.e., therapeutic biomolecule), the SARS-CoV-2 glycoprotein represented by Genbank identification number QHD43416.1, when combined with the MERS-CoV ORF4a protein (i.e., innate inhibitory protein or IIP) in the saRNA constructs of the present invention.

[0314] Twenty-four hours after transfection into the HeLa cell line, we compared the levels of surface expression between SARS-CoV-2 (without IIP) and SARS-CoV-2 combined with MERS-CoV ORF4a (IIP). Referring to Figure 29, the median fluorescence intensity of the positive population of cells was measured, where negative / positive cutoffs were set for cells gated as live single cells and mock-transfected cells. Surprisingly, the MERS-CoV ORF4a protein nearly doubled the per-cell expression level of the SARS-CoV-2 glycoprotein when compared with saRNA encoding only the SARS-CoV-2 glycoprotein (i.e., without IIP).

[0315] Consideration We screened a library of self-amplified RNAs using cis-encoded interferon inhibitory proteins (IPPs) for protein expression in mice, rabbits, non-human primates, and human cells in vitro, ex vivo in human skin explants, and in mice in vivo, as well as for immunogenicity in mice, rats, and rabbits. We observed that PIV-5V and MERS-CoV ORF4a proteins enhanced protein expression by 100- to 500-fold in IFN-competent HeLa and MRC5 cells in vitro. We found that MERS-CoV ORF4a protein partially reduced dose nonlinearity in vivo, and that ruxolitinib, but not IIPS, enhanced protein expression of saRNA in vivo. Both PIV-5V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells expressing saRNA in human skin explants, fully reversing the dose-nonlinearity of saRNA, while ruxolitinib increased protein expression in a cell-dependent manner. Finally, we demonstrated that MERS-CoV ORF4a significantly increased RABV-specific IgG titers and neutralizing IC50 in rabbits. 50 We observed an approximately 10-fold increase in IL-1 in mice but not in rats.

[0316] The protein design, cellular, and mutational analyses characterized in these experiments provide insight into the mechanisms by which the PIV-5V and MERS-CoV ORF4a proteins increase protein expression. The PIV-5V protein blocks MDA-5 and IRF3 by binding to MDA-5 (26, 27), whereas the MERS-CoV ORF4a protein binds to dsRNA and inhibits MDA-5 and RIG-I from driving PACT (29-31). After our in vitro screening, it was not feasible to screen all 10 candidates in vivo. Because the PIV-5V protein is not conserved across species (e.g., the N100D mutation required for mouse adaptation (44)), whereas the ORF4a protein is more highly conserved across species (29-31), we chose to proceed with the MERS-CoV ORF4a replicon. We observed that the R172A mutation in the PIV-5V protein, which eliminates its ability to block binding to MDA-5 but not STATs (45), slightly inhibited protein expression in MRC5 cells (Fig. 15d), thus indicating that binding to MDA5 is partially responsible for enhanced protein expression. Similarly, the K63A / K67A mutation in the MERS-CoV ORF4a protein limits its ability to bind dsRNA (46, 47), which was observed to reduce protein expression in both non-human primate and human cells (Fig. 15c, 15d). Although various IIPs inhibit interferon by mechanisms similar to those of PIV-5V and MERS-CoV ORF4a, the mechanism of action predicting enhanced protein expression is not always observed.

[0317] Because we previously observed that protein expression of saRNA formulations does not necessarily predict immunogenicity (2), we also characterized how the MERS-CoV ORF4a protein affects the immunogenicity of the rabies glycoprotein in mice, rats, and rabbits. We measured antibody titers and neutralization IC40 in rabbits using saRNA encoding RABV and MERS-CoV ORF4a. 50 We observed an increase in both PIV-5V and MERS-CoV ORF4a protein expression in mice (Figures 13a and 13b), but not in immunogenicity in mice or rats. While no preclinical animal model perfectly predicts human responses, rabbits are considered more immunologically similar to humans than mice or rats (48-50). Furthermore, we did not observe any enhancement of protein expression by either PIV-5V or MERS-CoV ORF4a protein in mouse cells (Figure 15a), so the lack of enhanced immunogenicity is not unexpected. We matched our characterization of preclinical animal models to human explant models in which cells have a natural tissue structure and an intrinsic human IFN response. To our knowledge, we are the first to observe that IIPs enhance the percentage of cells expressing saRNA, whereas ruxolitinib enhances expression in a cell-dependent manner. Considering these promising results, we hypothesize that the MERS-CoV ORF4a protein can enhance the immunogenicity of saRNA vaccines in humans and may also be useful for the application of saRNA in protein replacement therapy (51, 52).

[0318] These experiments provide proof of concept that IIPs can be directly encoded into saRNA vectors, effectively reducing nonlinear dose-dependence and enhancing immunogenicity. As shown by mechanistic studies, different aspects of the interferon pathway can be targeted to increase saRNA expression, motivating the exploration of combining IIPs with other IFN-inhibitory strategies, such as ruxolitinib.

[0319] Financial Aid Statement The remaining projects in this application have received funding from the European Union's Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement no. 794059.

[0320] [References] JPEG2025118871000003.jpg232170JPEG2025118871000004.jpg182170

Claims

1. (i) at least one therapeutic biomolecule; and (ii) an RNA construct encoding at least one innate inhibitory protein (IIP).

2. The RNA construct of claim 1, wherein the RNA construct comprises a self-amplifying RNA (saRNA), preferably a saRNA construct.

3. The RNA construct of claim 1 or 2, wherein the RNA construct comprises or is derived from a positive-strand RNA virus selected from the group of genera consisting of alphaviruses; picornaviruses; flaviviruses; rubiviruses; pestiviruses; hepaciviruses; caliciviruses and coronaviruses.

4. 4. The RNA construct of any one of claims 1 to 3, wherein the RNA construct comprises or is derived from a virus selected from the group of species consisting of Venezuelan equine encephalitis virus (VEEV); Enterovirus 71; Encephalomyocarditis virus; Kunjin virus; and Middle East respiratory syndrome virus.

5. 5. The RNA construct of claim 1, wherein the construct is derived from VEEV.

6. 6. The RNA construct of claim 1, wherein the at least one therapeutic biomolecule is a vaccine construct or a therapeutic protein.

7. At least one therapeutic biomolecule is (i) Is it an RNA molecule capable of regulating the expression of an endogenous host gene? (ii) is derived from a bacterium, virus, fungus, protozoan, or parasite; (iii) is selected from the group consisting of enzymes; enzyme inhibitors; hormones; immune system proteins; receptors; binding proteins; transcription or translation factors; tumor growth suppressor proteins; structural proteins; and blood proteins; (iv) An RNA construct according to any one of claims 1 to 6, which is an antigen, optionally a tumor antigen.

8. 8. The RNA construct of any one of claims 1 to 7, wherein at least one therapeutic biomolecule encoded by the RNA molecule is a protein or peptide derived from a pathogen selected from the group consisting of bacteria, viruses, fungi, protozoans, and parasites.

9. The RNA construct of claim 8, wherein the protein or peptide is a viral antigen derived from a virus selected from the group consisting of orthomyxoviruses; viruses of the Paramyxoviridae family; metapneumoviruses and morbilliviruses; pneumoviruses; paramyxoviruses; poxviridae; metapneumoviruses; morbilliviruses; picornaviruses; enteroviruses; bunyaviridae; phleboviruses; nairoviruses; hepadnaviruses; togaviruses; alphaviruses; arteriviruses; flaviviruses; pestiviruses; hepadnaviruses; rhabdoviruses; caliciviridae; coronaviruses; retroviruses; reoviruses; parvoviruses; hepatitis delta virus (HDV); hepatitis E virus (HEV); human herpesviruses and papovaviruses.

10. 10. The RNA construct of claim 9, wherein the coronavirus is SARS-CoV-1, SARS-CoV-2, MERS, human respiratory coronavirus, infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), or transmissible gastroenteritis virus (TGEV).

11. 11. An RNA construct according to any one of claims 1 to 10, wherein at least one native inhibitory protein is capable of either (i) reducing or blocking the action of melanoma differentiation associated protein 5 (MDA5) and / or (ii) blocking or reducing the binding of PKR activating protein to RNA.

12. 12. The RNA construct of any one of claims 1 to 11, wherein at least one native inhibitory protein is Middle East Respiratory Syndrome coronavirus MERS coronavirus (ORF4a).

13. 13. The RNA construct of any one of claims 1 to 12, wherein at least one native inhibitory protein is parainfluenza virus type 5 V protein (PIV5V).

14. 14. The RNA construct of any one of claims 1 to 13, wherein at least one native inhibitory protein is coronavirus ORF3b, preferably SARS-CoV-2 ORF3b.

15. 15. The RNA construct of any one of claims 1 to 14, wherein the RNA construct comprises an RNA nucleotide sequence encoding SEQ ID NO: 11, SEQ ID NO: 15 and / or SEQ ID NO: 20, or a variant or fragment thereof.

16. 16. The RNA construct of any one of claims 1 to 15, wherein the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID NO: 47, SEQ ID NO: 48 and / or SEQ ID NO: 56, or a variant or fragment thereof.

17. 17. The RNA construct of any one of claims 1 to 16, wherein at least one native inhibitory protein is capable of inhibiting one or more downstream pathways of MDA5 activation or blocking one or more downstream pathways of MDA / PACT recognition of dsRNA.

18. The RNA construct of claim 17, wherein at least one native inhibitory protein is selected from the group consisting of HSV-2 Us1; HSV-1 Us1; HSV-1 Us11; OV20.0L; BVDV Npro; Langat virus NS5; and influenza NS1.

19. 19. An RNA construct described in any one of claims 1 to 18, comprising a promoter or subgenomic promoter operably linked to sequences encoding at least one therapeutic biomolecule and at least one native inhibitory protein so as to enable transcription of the nucleotide sequences encoding the therapeutic biomolecule and at least one native inhibitory protein.

20. 20. The RNA construct of claim 19, wherein the promoter is 26S, and optionally the promoter comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 57, or a variant or fragment thereof.

21. 21. The RNA construct of claim 1, wherein the RNA construct comprises a linker sequence positioned between the sequence encoding the therapeutic biomolecule and the sequence encoding at least one native inhibitory protein, and optionally the linker sequence encodes a peptide spacer configured to separate the at least one therapeutic biomolecule and the at least one native inhibitory protein upon digestion, preferably the peptide spacer is a 2A peptide or a furin / 2A peptide.

22. 22. The RNA construct of any one of claims 1 to 21, wherein the RNA construct comprises a nucleotide sequence substantially as set out in SEQ ID NO: 38 or 39, or a fragment or variant thereof.

23. 23. A nucleic acid sequence encoding the RNA construct of any one of claims 1 to 22, optionally wherein the sequence is substantially as set forth in SEQ ID NO: 40 or 41, or a fragment or variant thereof.

24. 24. An expression cassette comprising the nucleic acid sequence of claim 23.

25. 25. A recombinant vector comprising the expression cassette of claim 24, optionally comprising a nucleotide sequence substantially as set out in any one of SEQ ID NOs: 35 to 37, or a variant or fragment thereof.

26. 26. A pharmaceutical composition comprising an RNA construct according to any one of claims 1 to 22, a nucleic acid sequence according to claim 23, an expression cassette according to claim 24 or a vector according to claim 25, and a pharmaceutically acceptable vehicle.

27. 23. A method for preparing an RNA construct according to any one of claims 1 to 22, comprising: a) i) introducing the vector of claim 25 into a host cell; and ii) culturing the host cell under conditions that result in the production of an RNA construct according to any one of claims 1 to 22; or b) transcribing the RNA construct from the vector of claim 25 A method comprising:

28. 27. An RNA construct according to any one of claims 1 to 22, a nucleic acid according to claim 23, an expression cassette according to claim 24, a vector according to claim 25 or a pharmaceutical composition according to claim 26 for use as a medicament or in therapy.

29. 27. An RNA construct according to any one of claims 1 to 22, a nucleic acid according to claim 23, an expression cassette according to claim 24, a vector according to claim 25, or a pharmaceutical composition according to claim 26 for use in the prevention, amelioration or treatment of a protozoan, fungal, bacterial or viral infection.

30. 27. An RNA construct according to any one of claims 1 to 22, a nucleic acid according to claim 23, an expression cassette according to claim 24, a vector according to claim 25, or a pharmaceutical composition according to claim 26 for use in the prevention, amelioration or treatment of cancer.

31. 27. A vaccine comprising an RNA construct according to any one of claims 1 to 22, a nucleic acid according to claim 23, an expression cassette according to claim 24, a vector according to claim 25, or a pharmaceutical composition according to claim 26, and optionally an adjuvant.

32. 27. An RNA construct according to any one of claims 1 to 22, a nucleic acid according to claim 23, an expression cassette according to claim 24, a vector according to claim 25, or a pharmaceutical composition according to claim 26, for use in stimulating an immune response in a subject.

33. 27. An RNA construct according to any one of claims 1 to 22, a nucleic acid according to claim 23, an expression cassette according to claim 24, a vector according to claim 25, or a pharmaceutical composition according to claim 26 for use in stem cell therapy.

34. 27. A method for modifying cells ex vivo or in vitro, comprising delivering to a cell an RNA construct according to any one of claims 1 to 22, a nucleic acid according to claim 23, an expression cassette according to claim 24, a vector according to claim 25, or a pharmaceutical composition according to claim 26.

35. 29. A modified cell obtained from or obtainable by the method of claim 28.

36. 36. The modified cell of claim 35 for use in therapy, optionally cell therapy.

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