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  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
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  • C12N2760/00011—Details
  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2760/00011—Details
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  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20111—Lyssavirus, e.g. rabies virus
  • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2770/00011—Details
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  • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2770/00011—Details
  • C12N2770/36011—Togaviridae
  • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
  • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
  • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to RNA constructs, and particularly, although not exclusively, to RNA replicons and saRNA molecules, and to 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.
• RNA messenger RNA
• mRNA messenger RNA
• mRNA therapeutics have been shown to be highly effective in small animals, the outcomes do not scale linearly when these formulations are translated in dose- escalation studies in humans.
• adverse events associated with the induction of interferon responses have been rate-limiting with respect to the increased doses of RNA likely to be effective in humans.
• the reason for this inconsistency is unclear, but the inventors hypothesize that inherent differences in human innate sensing pose a barrier to the translation of RNA therapeutics from the lab to the clinic.
• innate sensing of RNA has been associated with the inhibition of protein expression.
• modified ribonucleotides that are less detectable by innate sensing mechanisms.
• modified mRNA is not completely undetectable, and still results in some activation of interferon, protein silencing and reduced tolerability for human use.
• saRNA vectors which are typically based on an alphavirus backbone that have the capacity to self-amplify their own RNA by encoding polymerase activity within their non-structural proteins.
• Prior art methods have involved replacing the structural proteins of these vectors by a gene of interest (GOI), be it a vaccine construct, or encoding a therapeutic protein.
• GOI gene of interest
• Other versions of saRNA have been based on picornaviruses,flaviviruses, and coronaviruses. When saRNA is taken up into the cytoplasm of target cells, this leads to amplification of the RNA by the encoded polymerase machinery and very high expression levels of the GOI.
• saRNA has been shown to induce immune responses with lower doses of saRNA than mRNA (10- to 100-fold lower) and results in prolonged protein expression for up to 60 days in mice.
• a drawback with saRNA is that it is also sensed by innate recognition, triggering antiviral responses that limit protein expression and self-amplification of these prior art saRNAs. Innate sensing of saRNA differs to that of mRNA due to its large size (typically >5000 bases) and profound secondary structure, including double stranded regions (dsRNA). Long and double stranded RNA triggers innate responses through the MDA5 ((Melanoma Differentiation-Associated protein 5) pathway.
• MDA5 (Melanoma Differentiation-Associated protein 5) pathway.
• PACT (PKR activating protein)
• RNA construct encoding (i) at least one therapeutic biomolecule; and (ii) at least one innate inhibitor protein (IIP).
• RNA replicons or constructs have been postulated to be potential tools for the delivery and expression of genes of interest for vaccines and therapeutics.
• double stranded RNA dsRNA
• dsRNA double stranded RNA
• innate sensing mechanisms that trigger responses, which inhibit protein translation.
• expression of genes of interest encoded in the replicon is significantly impaired and thus the therapeutic potential of RNA replicons is limited.
• the RNA constructs of the invention overcome this problem because they encode one or more innate inhibiting protein(s), i.e. IIP, which ablates the downstream inhibition of transgene expression.
• interferon inhibiting proteins from the vaccinia virus, E3, K3 and B18.
• the interferon inhibiting proteins were delivered and formulated as separate mRNA molecules that were combined with the saRNA.
• RNA construct of the first aspect of one or more IIP enables dual protein expression with the peptide or protein of interest.
• the construct of the invention As opposed to delivering two different strands of RNA as described in the prior art, one encoding the peptide/protein of interest and one encoding the IIP, using the construct of the invention, only a single strand is delivered to the target cell, thereby ensuring colocalization of the RNA and the innate inhibiting protein.
• the IIP inhibits the innate sensing of RNA, thus enabling higher protein expression, and the IIP expression itself is self-amplified by virtue of being co-expressed on the subgenome strand with the gene of interest, i.e. the therapeutic biomolecule.
• RNA constructs of the invention also known as“Stealthicons” encoding luciferase have surprisingly been shown to increase luciferase protein expression levels up to two orders of magnitude in human cell lines in vitro, and also to increase both the magnitude and duration of protein expression of luciferase compared to a conventional VEEV RNA replicon in vivo in BL/6 mice.
• the skilled person would readily appreciate that the luciferase reporter is truly representative of the therapeutic biomolecule, because it proves that the RNA construct is able to express in vivo the gene harboured on the RNA molecule of the invention.
• RNA construct can 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.
• the RNA construct comprises self- amplifying RNA (saRNA), and is preferably an saRNA construct
• the RNA construct comprises or is derived from a positive stranded RNA virus selected from the group of genus consisting of: alphavirus; picornavirus;
• flavivirus flavivirus
• rubivirus pestivirus
• hepacivirus calicivirus or coronavirus.
• Suitable wild-type alphavirus sequences are well-known.
• suitable alphaviruses include Aura, Bebaru virus, Cabassou, Chikungunya virus, Eastern equine encephalomyelitis virus, Fort Morgan, Getah virus, Kyzylagach, Mayaro, Mayaro virus, Middleburg, Mucambo 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.
• the RNA construct comprises or is derived from a virus selected from the group of species consisting of: Venezuelan Equine Encephalitis Virus (VEEV);
• VEEV Venezuelan Equine Encephalitis Virus
• the vector is derived from VEEV.
• the RNA construct comprises a sequence which encodes the at least one therapeutic biomolecule.
• the at least one therapeutic biomolecule may comprise or be a vaccine construct, or a therapeutic protein.
• therapeutic protein relates to any protein that has therapeutic application preferably in human.
• 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.
• the protein and peptide is an antigen.
• the protein and peptide derived from a virus may be a viral antigen.
• the viral antigen maybe derived from a virus selected from the group consisting of Orthomyxoviruses; Paramyxoviridae viruses; Metapneumovirus and Morbilliviruses; Pneumoviruses; Paramyxoviruses; Poxviridae; Metapneumoviruses; Morbilliviruses; Picomaviruses; Enteroviruseses; Bunyaviruses; Phlebovirus; Nairovirus; Hepamaviruses; Togaviruses; Alphavirus; Arterivirus; Flaviviruses; Pestiviruses; Hepadnaviruses; Rhabdoviruses; Caliciviridae; Coronaviruses; Retroviruses; Reoviruses; Parvoviruses; Delta hepatitis virus (HDV); Hepatitis E virus (HEV); Human Herpesviruses and Papovaviruses.
• Orthomyxoviruses Paramyxoviridae viruses
• the Orthomyxoviruses may be Influenza A, B and C.
• the Paramyxoviridae virus may be Pneumoviruses (RSV), Paramyxoviruses (PIV).
• the Metapneumovirus may be Morbilliviruses (e.g., measles).
• the Pneumovirus maybe Respiratory syncytial virus (RSV), Bovine respiratory syncytial virus, Pneumonia virus of mice, or Turkey
• the Paramyxovirus maybe Parainkuenza virus types 1 - 4 (PIV), Mumps, Sendai viruses, Simian virus 5, Bovine parainkuenza virus, Nipahvirus, Henipavirus or Newcastle disease virus.
• the Poxviridae may be Variola vera, for example Variola major and Variola minor.
• the Metapneumovirus may be human
• the Morbillivirus may be measles.
• the Picornaviruses maybe Enteroviruses, Rhinoviruses, Hepamavirus, Parechovirus, Cardioviruses and Aphthoviruses.
• the Enteroviruses maybe Poliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24, Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus) types 1 to 9, 11 to 27 and 29 to 34 or Enterovirus 68 to 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 Hepamaviruses maybe Hepatitis A virus (HAV).
• the Togaviruses maybe Rubivirus.
• the Flavivirus maybe Tick-borne encephalitis (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest 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 (BDV).
• the Hepadnavirus maybe Hepatitis B virus or Hepatitis C virus.
• the Rhabdovirus may be Lyssavirus (Rabies virus) or Vesiculovirus (VSV).
• the Caliciviridae may be Norwalk virus, or Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus.
• the Coronavirus maybe SARS C0V-1, SARS-COV-2, MERS, Human respiratory coronavirus, Avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV), or Porcine transmissible gastroenteritis virus (TGEV).
• the Retrovirus maybe Oncovirus, a Lentivirus or a Spumavirus.
• the Reovirus may be an Orthoreo virus, a Rotavirus, an Orbivirus, or a Coltivirus.
• the Parvovirus maybe Parvovirus B 19.
• the Human Herpesvirus maybe Herpes Simplex Viruses (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), or Human Herpesvirus 8
• HSV Herpes Simplex Viruses
• VZV Varicella-zoster virus
• EBV Epstein-Barr virus
• CMV Cytomegalovirus
• HHV6 Human Herpesvirus 6
• HHV7 Human Herpesvirus 7
• the Papovavirus maybe Papilloma viruses, Polyomaviruses, Adenoviruess or Arenaviruses.
• the viral antigen may be a Rabies virus antigen, preferably Rabies virus glycoprotein.
• the viral antigen may be a Coronavirus antigen.
• the Coronavirus antigen is a surface glycoprotein, more preferably SARS-C0V-2 surface glycoprotein.
• the protein and peptide derived from bacteria may be a bacterial antigen.
• the bacterial antigen may derived from a bacterium selected from the group consisting of: Neisseria meningitid.es, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Bor detella pertussis, Burkholderia sp. ⁇ e.g., Burkholderia mallei,
• Burkholderia pseudomallei and Burkholderia cepacia Staphylococcus aureus
• Staphylococcus saprophyticus Yersinia enter ocolitica, E. coli, Bacillus anthracis (anthrax), Yersinia pestis (plague), Mycobacterium tuberculosis, Rickettsia, Listeria, Chlamydia pneumoniae, Vibrio cholerae, Salmonella typhi (typhoid fever), Borrelia burgdorfer, Porphyromonas s and Klebsiella sp.
• the protein and peptide derived from a fungus may be a fungal antigen.
• the fungal antigen maybe derived from a fungus selected from the group consisting of Dermatophytres, including: Epidermophyton koccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T verrucosum var.
• Dermatophytres including: Epidermophyton koccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum g
• album var. discoides, var. ochraceum, Trichophyton violaceum, and/ or Trichophyton faviforme; or from Aspergillus fumigatus, Aspergillus kavus, Aspergillus niger,
• Aspergillus nidulans Aspergillus terreus, Aspergillus sydowi, Aspergillus kavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida par apsilosis, 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, Microsporidia, Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi; Brachiola spp, Microsporidium
• the protein and peptide derived from a protozoan maybe a protozoan antigen.
• 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.
• the therapeutic biomolecule maybe a protein and peptide derived from a plant.
• the protein and peptide is a plant antigen.
• the plant antigen may be derived from Ricinus communis.
• the therapeutic biomolecule may be an immunogen or an antigen.
• the immunogen or an antigen is a tumour immunogen or antigen, or cancer immunogen or antigen.
• the tumour immunogens and antigens may be peptide-containing tumour antigens, such as a polypeptide tumour antigen or glycoprotein tumour antigens.
• tumour antigens maybe (a) full length molecules associated with cancer cells, (b) homologs and modified forms of the same, including molecules with deleted, added and/or substituted portions, and (c) fragments of the same.
• Suitable tumour immunogens include: class I-restricted antigens recognized by CD 8+ lymphocytes or class II-restricted antigens recognized by CD4+ lymphocytes.
• the tumour antigen maybe an antigen that is associated with a cancer selected from the group consisting of: a testis cancer, melanoma, lung cancer, head and neck cancer,
• NSCLC nucleophilicity parameter function determining breast cancer, gastrointestinal cancer, bladder cancer, colorectal cancer, pancreatic cancer, lymphoma, leukaemia, renal cancer, hepatoma, ovarian cancer, gastric cancer and prostate cancer.
• tumour antigen maybe selected from:
• cancer-testis antigens such as NY-ESO-I, SSX2, SCPl as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE- 1, GAGE- 2, MAGE-I, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE- 12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumours);
• cancer-testis antigens such as NY-ESO-I, SSX2, SCPl as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE- 1, GAGE- 2, MAGE-I, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE- 12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumours);
• mutated antigens for example, P53 (associated with various solid tumours, e.g., colorectal, lung, head and neck cancer), p2i/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUMl (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T- cell non-Hodgkins lymphoma), BCR- abl (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase,
• P53 associated with various solid tumours, e.g., colore
• (c) over-expressed antigens for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myelogenous leukemia), WT 1 (associated with, e.g., various leukemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2/neu (associated with, e.g., breast, colon, lung and ovarian cancer), alpha- fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein, M
• prostate-associated antigens such as PAP, PSA, PSMA, PSH-Pl, PSM-Pl, PSM-P2, associated with e.g., prostate cancer; and/or
• immunoglobulin idiotypes associated with myeloma and B cell lymphomas, for example.
• the therapeutic biomolecule may be a eukaryotic polypeptide.
• the eukaryotic polypeptide is a mammalian polypeptide.
• the mammalian polypeptide may be selected from the group consisting of: an enzyme; an enzyme inhibitor; a hormone; an immune system protein; a receptor; a binding protein; a transcription or translation factor; tumour growth supressing protein; a structural protein and a blood protein.
• the enzyme maybe selected from the group consisting of: chymosin; gastric lipase; tissue plasminogen activator; streptokinase; a cholesterol biosynthetic or degradative steriodogenic enzyme; kinases; phosphodiesterases; methylases; de-methylases;
• dehydrogenases cellulases; proteases; lipases; phospholipases; aromatases;
• cytochromes adenylate or guanylaste cyclases and neuramidases.
• the enzyme inhibitor maybe tissue inhibitor of metalloproteinase (TIMP).
• TIMP tissue inhibitor of metalloproteinase
• the hormone may be growth hormone.
• the immune system protein may be selected from the group consisting of: a cytokine; a chemokine; a lymphokine; erythropoietin; an integrin; addressin; selectin; homing receptors; T cell receptors and immunoglobulins.
• the cytokine maybe an interleukin, for example IL-2, IL-4 and/or IL-6, colony stimulating factor (CSF), granulocyte colony stimulating factor (G- CSF), granulocyte- macrophage colony stimulating factor (GM-CSF) or tumour necrosis factor (TNF).
• the chemokine may be a macrophage inflammatory protein-2 and/ or a plasminogen activator.
• the lymphokine may be an interferon.
• the immunoglobulin may be a natural, modified or chimeric immunoglobulin or a fragment thereof.
• the immunoglobulin is a chimeric immunoglobulin having dual activity such as antibody enzyme or antibody-toxin chimera.
• the hormone may be selected from the group consisting of: insulin, thyroid hormone, catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins; growth hormones (e.g., human grown hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor and the like).
• the receptor maybe a steroid hormone receptor or a peptide receptor.
• the receptor is a growth factor receptor.
• the binding protein maybe a growth factor binding protein.
• the tumour growth suppressing protein maybe a protein that inhibits angiogenesis.
• the structural protein may be selected from the group consisting of: collagen; fibroin; fibrinogen; elastin; tubulin; actin; and myosin.
• the blood protein maybe selected from the group consisting of thrombin; serum albumin; Factor VII; Factor VIII; insulin; Factor IX; Factor X; tissue plasminogen activator; protein
• the therapeutic biomolecule is a cytokine which is capable of regulating lymphoid homeostasis, preferably a cytokine which is involved in and preferably induces or enhances development, priming, expansion, differentiation and/or survival of T cells.
• the cytokine is an interleukin.
• IL-2, IL-7, IL-12, IL-15, or IL-21 are interleukins.
• the therapeutic biomolecule may be protein that is capable of enhancing
• the protein that is capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics may be selected from the group consisting of: OCT4, SOX2, NANOG, LIN28, p53, ART-4, BAGE, ss- catenin/m, Bcr-abL CAMEL, CAP-i, CASP-8, CDC27/ m, CD 4/m, CEA, CLAUDIN-12, c- MYC, CT, Cyp-B, DAM, ELF2M, ETV6-
• MAGE-A is selected from the group consisting of: MAGE-A 1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE- A7, MAGE-A8, MAGE-A9, MAGE-A 10, MAGE-A 11, or MAGE-A 12
• the protein that is capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics is OCT4, SOX2, LF4; c-MYC; NANOG; LIN28.
• the therapeutic biomolecule may be a biomolecule that is utilised for the modification of cells ex vivo for cell-therapy indications.
• the therapeutic biomolecule may be a biomolecule that is utilised for the modification of cells ex vivo for cell-therapy indications.
• the therapeutic biomolecule may be a biomolecule that is utilised for the modification of cells ex vivo for cell-therapy indications.
• the biomolecule may be selected from the group consisting of an immunoglobulin, a T-cell receptor and NK receptor.
• the therapeutic biomolecule may be an RNA molecule that is capable of regulating expression of endogenous host genes, for example an interfering RNA, such as small RNAs, siRNA or microRNAs.
• the RNA construct comprises a gene, which encodes the at least one innate inhibitor protein (IIP), which is capable of reducing or blocking the innate immune response to RNA.
• IIP innate inhibitor protein
• the 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 would be understood by the skilled person to mean RNA that is at least 1 kb in length) or dsRNA) by a host cell harbouring the RNA construct of the invention.
• the innate inhibitor protein is an innate inhibiting protein such that it is capable of reducing or blocking the innate response to RNA, preferably the RNA of the RNA construct of the first aspect.
• the innate inhibitor protein maybe capable of reducing or preventing the recognition of cytosolic saRNAby pattern recognition receptors leading to activation of interferon regulatory factor 3 and 7 (IRF3 and IRF7) and NF-KB transcription factors, directly triggering a range of antiviral genes (e.g. IFIT1-3, Mxi, MX2 known to suppress saRNA expression), proinflammatory genes whose products orchestrate the innate immune response, and direct activation of canonically IFN-stimulated genes (ISGs) upstream of any interferon dependent cascade.
• IRF3 and IRF7 interferon regulatory factor 3 and 7
• IRF7 interferon regulatory factor 3 and 7
• NF-KB transcription factors directly triggering a range of antiviral genes (e.g. IFIT1-3, Mxi, MX2 known to suppress saRNA expression), proinflammatory genes whose products orchestrate the innate immune response, and direct activation of canonically IFN-stimulated genes (ISGs) upstream of any inter
• the RNA may be single stranded RNA or double stranded RNA.
• the RNA is saRNA.
• the at least one innate inhibitor protein may be capable of either: (i) reducing or blocking the action of Melanoma Differentiation-Associated protein 5 (MDA5), for example by preventing oligomerization of MDA5 and binding of MDA5 to RNA, and/or (ii) blocking or reducing the binding of PACT to RNA, which may also be referred to as PKR activating protein, to RNA.
• MDA5 Melanoma Differentiation-Associated protein 5
• the at least one innate inhibitor protein blocking the action of MDA5 may be selected from the group consisting of: paramyxovirus V protein, 3C Proteins of Coxsackievirus A16, Coxsackievirus A6, and Enterovirus D68 viruses; the VP3 protein of Birnavirus; the Accessory Protein NS6 of Porcine Deltacoronavirus; and the 2C protein of
• Encephalomyocarditis virus and orthologues thereof.
• the at least one innate inhibitor protein blocking the action of MDA5 is a paramyxovirus V protein.
• the at least one innate inhibitor protein blocking the action of MDA5 is Parainfluenza virus type 5 V protein (PIV5 V).
• the at least one innate inhibitor protein blocking or reducing the binding of PACT to RNA maybe selected from the group consisting of: ORF4a (NS4a) of any coronavirus, ORF3b of any coronavirus, or the nucleocapsid proteins of mouse hepatitis virus and SARS ( coronavirus ); and orthologues thereof.
• the ORF4a is Middle East respiratory syndrome coronavirus MERS coronavirus (ORF4a).
• the coronavirus ORF3b is SARS-C0V2 ORF3b.
• the PIV5 V polypeptide is provided herein as SEQ ID No: 11, as follows:
• the PIV5 V polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 11, or a biologically active variant or fragment thereof.
• the RNA construct of the first aspect preferably comprises a RNA nucleotide sequence which encodes SEQ ID No: 11, or a biologically active variant or fragment thereof.
• the PIV5 V polypeptide is encoded by the nucleotide sequence of SEQ ID No: 12, as follows:
• the PIV5 V polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 12, or a variant or fragment thereof.
• RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 47, as follows:
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No 47 or a variant or fragment thereof.
• MERS-CoV ORF4a polypeptide is provided herein as SEQ ID No: 15, as follows:
• 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.
• the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence which encodes SEQ ID No: 15, or a variant or fragment thereof.
• the MERS-CoV ORF4a polypeptide is encoded by the nucleotide sequence of SEQ ID No: 16, as follows:
• the MERS-CoV ORF4a polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 16, or a variant or fragment thereof.
• the RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 48, as follows:
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No: 48, or a variant or fragment thereof.
• SARS-C0V-2 ORF3b polypeptide is provided herein as SEQ ID No: 20, as follows:
• the SARS-C0V-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.
• the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence which encodes SEQ ID No: 20, or a variant or fragment thereof.
• the SARS-C0V-2 ORF3b polypeptide is encoded by the nucleotide sequence (Wuhan-Hu-i Accession no. NC_045512.2; nucleotides 25814-26050) of SEQ ID No: 55, as follows:
• the SARS-C0V-2 ORF3b polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 55, or a variant or fragment thereof.
• RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 56, as follows:
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No: 56, or a variant or fragment thereof.
• the at least one innate inhibitor protein may be capable of inhibiting pathways downstream of MDA5 activation or blocking the downstream pathways of MDA/PACT recognition of dsRNA.
• the at least one innate inhibitor protein that is capable of inhibiting pathways downstream of MDA5 activation or blocking the downstream pathways of MDA/PACT recognition of dsRNA maybe selected from the group consisting of: HSV-2 Usi; HSV-i Usi; HSV-i Usii; OV20.0L; BVDV Npro; Langat virus NS5; and Influenza NSi
• the at least one innate inhibitor protein that is capable of inhibiting pathways downstream of MDA5 activation or blocking the downstream pathways of MDA/PACT recognition of dsRNA maybe used in
• HSV-2 Usi polypeptide is provided herein as SEQ ID No: 1, as follows:
• the HSV-2 Usi polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 1, or a biologically active variant or fragment thereof.
• the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence which encodes SEQ ID No: 1 or a biologically active variant or fragment thereof.
• the HSV-2 Usi polypeptide is encoded by the nucleotide sequence of SEQ ID No: 2, as follows:
• the HSV-2 Usi polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 2, or a variant or fragment thereof.
• RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 42, as follows: - I8 -
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No: 42, or a biologically active variant or fragment thereof.
• the HSV-i Usi polypeptide is provided herein as SEQ ID No: 3, as follows:
• the HSV-i Usi polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO:3, or a biologically active variant or fragment thereof.
• the RNA construct of the first aspect preferably comprises an RNA nucleotide sequence which encodes SEQ ID No: 3, or a biologically active variant or fragment thereof.
• the HSV-i Usi polypeptide is encoded by the nucleotide sequence of SEQ ID No: 4, as follows: ATCAGAGACTGCTACCTGATGGGCTACTGCCGGGCTAGACTGGCCCCTAGAACATGGTGCAGACTGCTGCAAGTGTCT GGCGGCACATGGGGCATGCACCTGAGAAACACCATCAGAGAGGTGGAAGCCAGATTCGACGCCACAGCCGAGCCTGTG TGCAAGCTGCCTTGTCTGGAAACTCGGAGATACGGCCCCGAGTGCGACCTGAGCAATCTGGAAATTCACCTGAGCGCC ACCAGCGACGACGAGATTTCTGATGCCACCGACCTGGAAGCCGCCGGATCTGATCATACACTGGCCAGCCAGAGCGAC
• the HSV-i Usi polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 4, or a variant or fragment thereof.
• RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 43, as follows:
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No: 43, or a variant or fragment thereof.
• the HSV-i Usii polypeptide is provided herein as SEQ ID No: 5, as follows:
• the HSV-i Usii polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 5, or a biologically active variant or fragment thereof.
• the RNA construct of the first aspect preferably comprises a RNA nucleotide sequence which encodes SEQ ID No: 5, or a variant or fragment thereof.
• the HSV-i Usii polypeptide is encoded by the nucleotide sequence of SEQ ID No: 6, as follows: ATGAGCCAGACACAGCCTCCAGCTCCAGTTGGACCTGGCGACCCTGATGTGTATCTGAAGGGCGTGCCAAGCGCCGGC ATGCATCCTAGAGGTGTTCATGCCCCTAGAGGACACCCCAGAATGATCTCTGGCCCTCCTCAGAGAGGCGACAACGAT CAGGCTGCTGGACAGTGTGGCGATAGCGGACTGCTGAGAGTGGGCCGATACCACAATCAGCAAGCCATCTGAGGCT GTGCGGCCTCCTACAATCCCCAGAACACCTAGAGTGCCCCGCGAGCCAAGAGTGCCTAGACCTCCTAGAGCCCAGA GAACCCAGAGTGCCAAGGGCTCCCAGAGATCCTAGTCCCTCGGGACCCTAGGGACCCAAGACAACCTAGATCACCC AGAGCCTCGGAGCCCAAGACAACCTAGATCACCC AGAGCCTCGGAGCCCAAGACAACCTAGATCACCC AGAGCC
• HSV-i Usii polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 6, or a variant or fragment thereof.
• RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 44, as follows:
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No: 44 or a variant or fragment thereof.
• the OV20.0L polypeptide is provided herein as SEQ ID No: 7, as follows:
• 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.
• the RNA construct of the first aspect preferably comprises a RNA nucleotide sequence which encodes SEQ ID No: 7 or a variant or fragment thereof.
• the OV20.0L polypeptide is encoded by the nucleotide sequence of SEQ ID No: 8, as follows:
• the OV20.0L polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 8, or a variant or fragment thereof.
• the RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 45, as follows:
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No: 45, or a variant or fragment thereof.
• the BVDV Npro polypeptide is provided herein as SEQ ID No: 9, as follows:
• the BVDV Npro polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 9, or a biologically active variant or fragment thereof.
• the RNA construct of the first aspect preferably comprises a RNA nucleotide sequence which encodes SEQ ID No: 9 or a variant or fragment thereof.
• the BVDV Npro polypeptide is encoded by the nucleotide sequence of SEQ ID No: 10, as follows:
• the BVDV Npro polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 10, or a variant or fragment thereof.
• RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 46, as follows: AUGGAACUGAUCACCAACGAGCUGCUGUACAAGACCUACAAGCAGAAACCCGUGGGCGUCGAGGAACCCGUGUAUGAU CAAGCUGGCGACCCUCUGUUUGGCGAGAGAGGCGCUGUUCACCCUCAGAGCACACUGAAGCUGCCCCACAAGCGGGGC GAAAGAGAUGUGCCUACCAACCUGGCCAGCCUGCCUAAGAGAGGCGAUUGCAGAACCGGCAAUAGCAGAGGCCCUGUG UCCGGCAUCUACCUGAAACCUGGACCACUGUUCUACCAGGACUACAAGGGACCCGUACCACAGAGCCCCUCUCUGGAA CUGUUUGAAGAGGGCAGCAUGUGCGAAACCACCAAGCGGAUCGGAAGUGACCGGCUCUGACGGCAAGCUGUACCAC AUCUACCAGACGGGUGCAUCGACGGCUGCAUCAUCAUCAAGAGCGCCACCAGAUCCUACCAGCGGGUUCACCACA
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No: 46, or a variant or fragment thereof.
• the Langat NS5 polypeptide is provided herein as SEQ ID No: 17, as follows:
• VFKDKVDTKAQEPQPGTKI IMRAVNDWLLERLVKKSRPRMCSREEFIAKVRSNAALGAWSDEQNKWKSAREAVEDPEF WSLVEAERERHLQGRCAHCVYNMMGKREKKLGEFGVAKGSRAIWYMWLGSRFLEFEALGFLNEDHWASRAS SGAGVEG I SLNYLGWHLKKLASLSGGLFYADDTAGWDTKI TNADLDDEEQILRYMDGDHKKLAATVLRKAYHAKWRVARPSREG GCVMD I I TRRDQRGSGQWTYALNT I TNI KVQLVRMMEGEGVI EVADSHNPRLLRVEKWLEEHGEERLSRMLVSGDDC WRPVDDRFSKALYFLNDMAKTRKDTGEWEP STGFASWEEVPFCSHHFHELVMKDGRALWPCRDQDEL
• 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.
• the RNA construct of the first aspect preferably comprises a RNA nucleotide sequence which encodes SEQ ID No: 17, or a variant or fragment thereof.
• the Langat NS5 polypeptide is encoded by the nucleotide sequence of SEQ ID No: 18, as follows:
• the Langat NS5 polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 18, or a variant or fragment thereof.
• RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 49, as follows:
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No: 49 or a variant or fragment thereof.
• influenzaza NSi polypeptide (Accession number DQ508893) is provided herein as SEQ ID No: 13, as follows:
• the Influenza NSi polypeptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 13, or a biologically active variant or fragment thereof.
• the RNA construct of the first aspect preferably comprises a RNA nucleotide sequence which encodes SEQ ID No: 13, or a variant or fragment thereof.
• the Influenza NSi polypeptide is encoded by the nucleotide sequence of SEQ ID No: 14, as follows: atggattccaacactgtgtcaagctttcaggtagattgcttcctttggcatgtccgcaaacaagttgcagaccaagag ctaggtgatgccccattccttgatcggcttcgccgagatcagaagtccctaagggaagaggcagcactctcggtctg aacatcgaaacagccacctgtgttggaaagcaatagtagagaggattctgaaggaagaatccgatgaggcatttaga atgaccatggcctccgcacttgcttcgcgatacctaactgacatgactattgaagagatgtcaagggactggttcatg cccacca
• the Influenza NSi polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 14, or a variant or fragment thereof.
• the RNA construct may comprise an RNA nucleotide sequence of SEQ ID No: 19, as follows: auggauuccaacacugugugucaagcuuucagguagauugcuuccuuuggcauguccgcaaacaaguugcagaccaagag cuaggugaugccccauuccuugaucggcuucgccgagaucagaagucccuaaagggaagaggcagcacucucggucug aacaucgaaacagccaccuguguuggaaagcaaauaguagagaggauucugaaggaagaauccgaugaggcauuuaga augaccauggccuccgcacuugcuucgcgauaccuaacugacaugacuauugaagagaugucaagggacugguucaug cucaugcccaagcagaaaguggcaggcccucuuugugucagaauggaccaggcgauaagggac
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No: 19, or a variant or fragment thereof.
• the innate inhibitor protein is selected from the group consisting of HSV-2 Usi; HSV-i Usii; OV20.0L; BVDV Npro, PIV5 V; MERS-CoV ORF4a; SARS-C0V-2 ORF3b;Langat virus NS5 and Influenza NSi.
• the innate inhibitor protein is selected from the group consisting of HSV-2 Usi; HSV-i Usii; OV20.0L; BVDV Npro, PIV5 V; MERS-CoV ORF4a; and Langat virus NS5.
• the innate inhibitor protein is selected from the group consisting of HSV-2 Usi; HSV-i Usii; OV20.0L; BVDV Npro, PIV5 V; and MERS-CoV ORF4a.
• the inventors identified two highly potent IIPs that were surprisingly able to 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, and inhibitor of PACT/MDA activation.
• the inventors have 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 have shown that these constructs can be the next-generation RNA replicons that will enable higher efficacy of RNA vaccines and therapeutics in humans. The inventors believe that they will have significant utility, whether expressed in alphavirus, picornaviruses, flaviviruses, or coronaviruses replicons.
• the innate inhibitor protein is PIV-5 and/or MERS-CoV ORF4a, which the skilled person would understand may also be referred to as NS4a.
• PIV-5 and/or MERS-CoV ORF4a which the skilled person would understand may also be referred to as NS4a.
• Such constructs display many advantages over those described in the prior art, including: i) insertion of PIV-5 and ORF4a proteins directly into the VEEV replicon, enabling dual protein expression of the VPII protein and the gene of interest;
• RNA as opposed to delivering two different strands of RNA, one encoding the gene of interest (GOI), i.e. the therapeutic biomolecule, and one encoding the IIP, a single strand is delivered thus ensuring colocalization of the RNA and the innate inhibiting protein; iii) the IIP inhibits innate sensing of RNA, thus enabling higher protein expression; iv) the IIP expression itself is self-amplified by virtue of being co-expressed on the sub genome strand with the GOI; and/or
• the sequence encoding the at least one innate inhibitor protein maybe disposed anywhere within the RNA construct or replicon sequence, such that the sequence encoding the at least one peptide or protein of interest maybe disposed 5’ or 3’ to the sequence encoding the at least one innate inhibitor protein.
• sequence encoding the at least one peptide or protein of interest is disposed 5’ to the sequence encoding the at least one innate inhibitor protein.
• the RNA construct according to the first aspect comprises at least one promotor, either genomic or subgenomic.
• the promoter is a sub genomic promoter.
• the sub genomic pro motor relates to a promoter that is operably linked to the sequences encoding the at least one therapeutic biomolecule and the at least one innate inhibitor protein, such that it enables the transcription the nucleotide sequence encoding the therapeutic bio molecule and the at least one innate inhibitor protein.
• the sub genomic promoter is 26S, which is provided herein as SEQ ID No: 57, as follows:
• the promoter (preferably, a sub genomic promoter) is as substantially as set out in SEQ ID NO: 57, or a variant or fragment thereof.
• the same promotor is operably linked to the sequence encoding the at least one peptide or protein of interest and the sequence encoding the at least one innate inhibitor.
• the promoter is disposed 5’ of the sequence encoding the peptide or protein of interest (i.e. the therapeutic biomolecule) and the sequence encoding the at least one innate inhibitor protein such that the promoter is operably linked to both sequences.
• a first promotor is operably linked to the sequence encoding the at least one peptide or protein of interest (i.e. the therapeutic biomolecule) and a second promotor is operably linked the sequence encoding the at least one innate inhibitor protein.
• the RNA construct may encode at least two, three, four or five IIPs.
• a single promotor maybe operably linked to all sequences encoding an innate inhibitor protein.
• a promotor may be linked to each of the at least one other sequence encoding an innate inhibitor protein such that each innate inhibitor protein is operably linked to a separate promoter.
• the separate promoters may comprise the same promotor sequence or different promoter sequences.
• different pro motors are operably linked to each sequence encoding an innate inhibitor protein.
• the RNA construct may further comprise a linker sequence disposed between the sequence encoding the at least one peptide or protein of interest (i.e. the therapeutic biomolecule) and the sequence encoding the at least one innate inhibitor protein.
• the linker sequence comprises a sequence that encodes a peptide spacer that is configured to be digested to thereby separate the at least one therapeutic biomolecule encoded by the gene of interest and the at least one innate inhibitor protein. Therefore, preferably the spacer sequence is disposed between the sequence encoding the at least one peptide or protein of interest and the sequence encoding at least one innate inhibitor protein. As such, the spacer sequence is preferably a cleavable peptide, for example a 2A peptide.
• Suitable 2A peptides include the porcine teschovirus-i 2A (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).
• the 2A peptide is thosea asigna virus 2A (T2A).
• the cleavable peptide is a self-cleaving peptide.
• the self-cleaving peptide is a furin/ 2A peptide.
• the furin sequence may be disposed 3’ or 5’ of the 2A sequence.
• the furin sequence is disposed 5’ of the 2A sequence, and preferably with a GSG spacer disposed between the furin and 2A sequence.
• furin is a ubiquitous calcium-dependent proprotein convertase located in the secretory pathway (mainly in the golgi and trans- golgi network) that cleaves precursor proteins at a specific recognition sequence - canonically R-X-R/K/X-R (SEQ ID No: 24), and cleaving the proprotein after the final R.
• the furin sequence is R-X-R/K/X-R.
• the furin sequence is the optimised sequence RRRRRR (SEQ ID No: 25) a GSG sequence.
• the GSG spacer is disposed 3’ of the furin sequence and 5’ of the 2A sequence.
• the spacer sequence is the furin/T2A, as provided by NCBI Reference Sequence: GenBank: AAC97195.1, and provided herein as SEQ ID No: 26, as follows:
• the spacer sequence comprises an amino acid sequence substantially as set out in SEQ ID NO: 26, or a variant or fragment thereof.
• the replicon may have linker sequences disposed between each sequence encoding an innate inhibitor protein, or only between some IIPs.
• the at least one sequence encoding the therapeutic biomolecule and the at least one innate inhibitor protein may be separated by a stop codon followed by an internal ribosome entry site (IRES) sequence capable of initiating translation of the downstream sequence.
• IRES sequences include those such as the IRES sequence of encephalomyocarditis virus or vascular endothelial growth factor and type l collagen-inducible protein (VCIP), and would be known to those skilled in the Art.
• the IRES sequence is disposed between the sequence encoding the at least one peptide or protein of interest and the sequence encoding at least one innate inhibitor protein.
• spacer sequences may include combinations of known cleavage sequences and/or IRES sequences.
• sequence encoding the at least one therapeutic biomolecule and the at least one innate inhibitor protein may be separated by a stop codon followed by a second subgenomic promotor sequence capable of initiating transcription of the downstream sequence.
• the RNA construct may encode at least one non-structural protein (NSP), disposed 5’ or 3’ of the sequence encoding the at least one peptide or protein of interest and the at least one innate inhibiting protein.
• NSP non-structural protein
• the sequence encoding the at least one NSP is disposed 5’ of the sequences encoding the peptide or protein of interest and the at least one innate inhibiting protein.
• the sequence encoding the at least one NSP is disposed at the 5’ end of the RNA construct.
• the at least one non-structural protein which is encoded by the RNA construct, may be the RNA polymerase nsP4.
• the construct encodes nsPi, nsP2, nsP3 and nsP4-
• nsPi is the viral capping enzyme and membrane anchor of the replication complex (RC)
• nsP2 is an RNA helicase and the protease responsible for the ns polyprotein processing.
• nsP3 interacts with several host proteins and may modulate protein poly- and mono-ADP-ribosylation
• nsP4 is the core viral RNA-dependent RNA polymerase.
• nsPi is provided herein as SEQ ID No: 27, as follows: MEKVHVD IEEDSPFLRALQRSFPQFEVEAKQVTDNDHANARAFSHLASKLI ETEVDP SDTI LD IGSAPARRMYSKHKY HCI CPMRCAEDPDRLYKYATKLKKNCKEI TDKELDKKMKELAAVMSDPDLETETMCLHDDESCRYEGQVAVYQDVYAV DGP TSLYHQANKGVRVAYWIGFDTTPFMFKNLAGAYP SYSTNWADETVL TARNIGLCSSDVMERSRRGMS I LRKKYLK PSNNVLFSVGS TI YHEKRDLLRSWHLP SVFHLRGKQNYTCRCETIVSCDGYVVKRIAI SPGLYGKPSGYAATMHREGF LCCKVTDTLNGERVSFPVCTYVPATLCDQMTGI LATDVSADDAQKLLVGLNQRIWNGRTQRNTNTMKNYLLPWAQA FARWAKEY
• nsPi preferably comprises an amino acid sequence as substantially as set out in SEQ ID No: 27, or a biologically active variant or fragment thereof.
• nsPi is encoded by a nucleotide sequence a defined in SEQ ID No: 28, as follows:
• nsPi is preferably encoded by a nucleotide sequence as substantially as set out in SEQ ID No: 28, or a variant or fragment thereof. Accordingly, therefore, preferably the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No 50 or a variant or fragment thereof.
• nsP2 is provided herein as SEQ ID No: 29, as follows:
• nsP2 preferably comprises an amino acid sequence as substantially as set out in SEQ ID No: 29, or a biologically active variant or fragment thereof.
• nsP2 is encoded by a nucleotide sequence a defined in SEQ ID No: 30, as follows:
• nsP2 is encoded by a nucleotide sequence as substantially as set out in SEQ ID No: 30, or a variant or fragment thereof.
• RNA construct may comprise SEQ ID No 51, as follows:
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No 51 or a variant or fragment thereof.
• nsP3 is provided herein as SEQ ID No: 31, as follows:
• nsP3 comprises an amino acid sequence as substantially as set out in SEQ ID No: 31, or a biologically active variant or fragment thereof.
• nsP3 is encoded by a nucleotide sequence a defined in SEQ ID No: 32, as follows: GCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAA GGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTA GGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAG GTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTA GCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGAC TAACCCAATCATTGAACCATTTGCTGACA GCTTTAGACACCACTGATGCAGATGTAGCCATATACTGGGACAAAATGGGAAATGACTCTCAAG
• nsP3 is encoded by a nucleotide sequence as substantially as set out in SEQ ID No: 32, or a variant or fragment thereof.
• RNA construct may comprise SEQ ID No 52, as follows:
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No 52 or a variant or fragment thereof.
• nsP4 is provided herein as SEQ ID No: 33, as follows:
• nsP4 comprises an amino acid sequence as substantially as set out in SEQ ID No: 33, or a biologically active variant or fragment thereof.
• nsP4 is encoded by a nucleotide sequence a defined in SEQ ID No: 34, as follows:
• nsP4 is encoded by a nucleotide sequence as substantially as set out in SEQ ID No: 34, or a variant or fragment thereof.
• RNA construct may comprise SEQ ID No 53, as follows: UACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACGGUGCUAUCCGAAGUGGUG UUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUCGACCAAGAAAAAGAAGAAUUACUACGCAAGAAAUUA CAGUUAAAUCCCACACCUGCUAACAGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUAACAGCUAGA CGUAUUCUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUGCAUCCUGUUCCU UUGUAUUCAUCUAGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCAGUGGAAGCCUGUAACGCCAUGUUGAAAGAG AACUUUCCGACUGUGGCUUCUUACUGUAUUAUUAUUCCAAGGUCGCAGUGGAAGCCUGUAACGCCAUGU
• the RNA construct comprises an RNA nucleotide sequence substantially as set out as SEQ ID No 53 or a variant or fragment thereof.
• the non-structural proteins encoded by the RNA construct of the invention form an enzyme complex that is required for genome replication and transcription of the sequences encoding the at least one peptide or protein of interest and the at least one innate inhibitor protein.
• the one or more non-structural protein may encode a polymerase to enable the construct to amplify the nucleotide sequences encoding the at least one peptide or protein of interest and the at least one innate inhibitor protein.
• the host cell may be a eukaryotic or prokaryotic host cell.
• the host cell is a eukaryotic host cell. More preferably, the host cell is a mammalian host cell.
• the RNA construct may further comprise a promoter disposed 5’ of the at least one non- structural protein, such that the promoter is operably linked to sequence encoding the at least one non-structural protein and enables expression of the at least one non- structural protein in a host cell.
• the promoter comprises a 5’ UTR conserved sequence element, which may be referred to herein as SEQ ID No: 54, as follows:
• the UTR is disposed 5’ of the at least one non-structural protein and comprises a nucleotide sequence substantially as set out in SEQ ID No: 54, or a fragment or variant thereof.
• the replicon comprises a polyA tail.
• the polyA tail is disposed at the 3’ end of the replicon.
• the replicon may further comprise a 5’ cap.
• the term "s'-cap” includes a 5'-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA and/or enhance translation of RNA if attached thereto, preferably in vivo and/ or in a cell.
• RNA with a 5’-cap may be achieved by in vitro transcription of a DNA template in presence of said 5'-cap, wherein said 5'-cap is co- transcriptionally incorporated into the generated RNA strand, or the RNA maybe generated, for example, by in vitro
• RNA post-transcriptionally may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
• capping enzymes for example, capping enzymes of vaccinia virus.
• the 3' position of the first base of a (capped) RNA molecule is linked to the 5' position of the subsequent base of the RNA molecule ("second base") via a phosphodiester bond.
• the RNA construct comprises, preferably 5’ to 3’, a promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, and at least one sequence encoding an innate inhibitor protein.
• the RNA construct comprises, preferably 5’ to 3’, a promoter, a sequence encoding at least one non-structural protein, a sub genomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, and at least one sequence encoding an innate inhibitor protein.
• the RNA construct comprises, preferably 5’ to 3’, a promoter, a sequence encoding at least one non-structural protein, a sub genomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, at least one sequence encoding an innate inhibitor protein, and a polyAtail.
• the RNA construct comprises, preferably 5’ to 3’, a promoter, a sequence encoding at least one non-structural protein, a sub genomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, at least one sequence encoding an innate inhibitor protein, optionally a spacer sequence between each at least one sequence encoding an innate inhibitor protein, and a polyA tail.
• the RNA construct comprises, preferably 5’ to 3’, a 5’ cap, a promoter, a sequence encoding at least one non-structural protein, a sub genomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, at least one sequence encoding an innate inhibitor protein, optionally a spacer sequence between each at least one sequence encoding an innate inhibitor protein, and a polyA tail.
• the RNA construct comprises, preferably 5’ to 3’, a 5’ cap, a promoter, a sequence encoding at least one non-structural protein, a spacer, at least one sequence encoding an innate inhibitor protein, a sub genomic promoter, a sequence encoding at least one therapeutic biomolecule and a polyA tail.
• the RNA construct comprises, preferably 5’ to 3’, a 5’ cap, a promoter, a sequence encoding at least one non-structural protein, a spacer, at least one sequence encoding an innate inhibitor protein, a sub genomic promoter, a sequence encoding at least one therapeutic biomolecule, a spacer sequence, a sequence encoding at least one an innate inhibitor protein, optionally a spacer sequence between each sequence encoding at least one innate inhibitor protein, and a polyA tail.
• the V protein of PIV5 is thought to directly bind to MDA5 preventing oligomerization, while ORFqa is thought to block the binding of PACT to dsRNA.
• the inventors incorporated these protein-encoding genes into saRNA following the gene of interest (GOI) (i.e. the therapeutic biomolecule), separated by a T2A cleavage site, to generate constructs that are advantageously able to bypass innate sensing mechanisms. Pairing the GOI and IIPs into a single open reading frame that is cleaved by endogenous proteases (T2A) upon expression, maximize expression of both within the same cells at a set ratio with the same kinetics.
• GOI gene of interest
• T2A endogenous proteases
• the RNA construct comprises, 5’ to 3’, a 5’ cap, a promoter comprising a 51 nucleotide conserved sequence element, nsPi, nsP2, nsP3v, nsP4, the sub genomic promoter 26S, a sequence encoding a therapeutic biomolecule, a T2A spacer sequence, a sequence encoding PIV5 V and/or MERS-CoV ORF4a and a polyAtail.
• a promoter comprising a 51 nucleotide conserved sequence element, nsPi, nsP2, nsP3v, nsP4, the sub genomic promoter 26S, a sequence encoding a therapeutic biomolecule, a T2A spacer sequence, a sequence encoding PIV5 V and/or MERS-CoV ORF4a and a polyAtail.
• the RNA construct may comprise or consist of SEQ ID No: 38 as follows:
• the RNA construct comprises a nucleotide sequence substantially as set out in SEQ ID No: 38, or a fragment or variant thereof.
• the RNA construct may comprise or consist of SEQ ID No: 39, as follows:
• the RNA construct comprises a nucleotide sequence substantially as set out in SEQ ID No: 39, or a fragment or variant thereof.
• nucleic acid sequence may comprise or consist of SEQ ID No: 40, as follows:
• nucleic acid sequence comprises a nucleotide sequence substantially as set out in SEQ ID No: 40, or a fragment or variant thereof.
• nucleic acid sequence may comprise or consist of SEQ ID No: 41, as follows:
• nucleic acid sequence comprises a nucleotide sequence substantially as set out in SEQ ID No: 41, or a fragment or variant thereof.
• an expression cassette comprising a nucleic acid sequence according to the second aspect.
• nucleic acid sequences of the invention are preferably harboured in a recombinant vector, for example a recombinant vector for delivery into a host cell of interest to enable production of the RNA construct. Accordingly, in a fourth aspect, there is provided a recombinant vector comprising the expression cassette according to the third aspect.
• the vector may comprise the nucleic acid sequence of SEQ ID No: 35, as follows, where“GOI” represents the position of the therapeutic biomolecule encoding sequence: CGCCAGCAACGCGAGCTCTAATACGACTCACTATAGATGGGCGGCGCATGAGAAGCCCAGACCAATTACCTACCCAAAA TGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAG AAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGG TGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCT GTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTG ATAAGAAATGAAAATGAAGGAAGGAAGGAAGGAAG
• the vector comprises the nucleotide sequence substantially as set out in SEQ ID NO: 35, or a variant or fragment thereof.
• the vector may comprise the nucleic acid sequence of SEQ ID No: 36, as follows, where“GOI” represents the position of the therapeutic biomolecule encoding sequence: CGCCAGCAACGCGAGCTCTAATACGACTCACTATAGATGGGCGGCGCATGAGAAGCCCAGACCAATTACCTACCCAAAA TGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAG AAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGG TGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCT GTCCGATGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACT
• the vector comprises the nucleotide sequence substantially as set out in SEQ ID NO: 36, or a variant or fragment thereof.
• the vector may comprise the nucleic acid sequence of SEQ ID No: 37, as follows, where“GOI” represents the position of the sequence encoding the therapeutic bio molecule:
• the vector comprises the nucleotide sequence substantially as set out in SEQ ID NO: 37, or a variant or fragment thereof.
• the saRNA constructs of the invention may be made using a DNA plasmid, which is shown in Figures 7 or 8, as a template. RNA copies may then be made by in vitro transcription using a polymerase, such as T7 polymerase, and the T7 promoter is shown upstream of the saRNA in the plasmid map in Figures 7 or 8.
• a polymerase such as T7 polymerase
• the saRNA constructs of the first aspect may be made using the DNA plasmid having a nucleic acid sequence as set out in any one of SEQ ID No: 35-37, or a variant or fragment thereof, which is shown in Figure 7 or 8, as the template.
• RNA polymerases could be used instead of T7 polymerase, for example the SP6 or the T3 polymerase, in which case the saRNA construct may comprise the SP6 or T3 promoter instead.
• the vector of the fourth aspect encoding the RNA construct of the first aspect may for example be a plasmid, cosmid or phage and/or be a viral vector. Such recombinant vectors are highly useful in the delivery systems of the invention for transforming cells with the nucleotide sequences.
• the nucleotide sequences may preferably be a DNA sequence, and it is this DNA sequence which encodes the RNA sequence forming the RNA construct of the first aspect.
• Recombinant vectors encoding the RNA construct of the first aspect may also include other functional elements.
• they may further comprise a variety of other functional elements including a suitable promoter for initiating transgene expression upon introduction of the vector in a host cell.
• the vector is preferably capable of autonomously replicating in the nucleus of the host cell, such as a bacterial cell.
• elements which induce or regulate DNA replication maybe required in the recombinant vector.
• the recombinant vector maybe designed such that it integrates into the genome of a host cell. In this case, DNA sequences which favour targeted integration (e.g. by homologous recombination) are envisaged.
• Suitable promoters may include the SV40 promoter, CMV, EFia, PGK, viral long terminal repeats, as well as inducible promoters, such as the Tetracycline inducible system, as examples.
• the cassette or vector may also comprise a terminator, such as the Beta globin, SV40 polyadenylation sequences or synthetic polyadenylation sequences.
• the recombinant vector may also comprise a promoter or regulator or enhancer to control expression of the nucleic acid as required.
• the vector may also comprise DNA coding for a gene that maybe used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA.
• a selectable marker for example, ampicillin, neomycin, puromycin or chloramphenicol resistance is envisaged.
• the vectors shown in Figure 7 and 8 include an ampicillin resistant marker, which is useful for selecting the plasmid in bacteria.
• the selectable marker gene maybe in a different vector to be used simultaneously with the vector containing the transgene(s).
• the cassette or vector may also comprise DNA involved with regulating expression of the nucleotide sequence, or for targeting the expressed polypeptide to a certain part of the host cell.
• Purified vector may be inserted directly into a host cell by suitable means, e.g. direct endocytotic uptake.
• the vector may be introduced directly into a host cell (e.g. a eukaryotic or prokaryotic cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion or ballistic bombardment.
• vectors of the invention maybe introduced directly into a host cell using a particle gun.
• the nucleic acid molecule may (but not necessarily) be one, which becomes incorporated in the DNA of the host cell.
• Undifferentiated cells may be stably transformed leading to the production of genetically modified daughter cells (in which case regulation of expression in the subject may be required e.g. with specific transcription factors or gene activators).
• the delivery system maybe designed to favour unstable or transient transformation of differentiated cells. When this is the case, regulation of expression may be less important because expression of the DNA molecule will stop when the transformed cells die or stop expressing the protein.
• the delivery system may provide the nucleic acid molecule to the host cell without it being incorporated in a vector.
• the nucleic acid molecule may be incorporated within a liposome or virus particle.
• a“naked” nucleic acid molecule maybe inserted into a host cell by a suitable means e.g. direct endocytotic uptake.
• 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.
• a process for making the pharmaceutical composition according to the fifth aspect comprising contacting 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, with a pharmaceutically acceptable vehicle.
• a method of preparing the RNA construct of the first aspect comprising:
• RNA construct of the first aspect ii) culturing the host cell under conditions to result in the production of the RNA construct of the first aspect
• the host cell of step a) may be a eukaryotic or prokaryotic host cell.
• the host cell is a eukaryotic host cell.
• the host cell is a mammalian host cell such as Human embryonic kidney 293 cells or Chinese hamster ovary (CHO) cells.
• Step (b) may be performed in vitro or in vivo, preferably in vitro.
• the RNA replicon of the first aspect is particularly suitable for therapy.
• RNA construct of the first aspect would be generated by in vitro transcription for in vivo use in therapy
• those experienced in the art will recognise that the RNA construct can be generated in vivo in a subject for therapy, by in vivo delivery of the nucleic acid according to the second aspect, the expression cassette according to the third aspect, the vector according to the fourth aspect to a subject.
• 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
• the protozoan, fungal, bacterial or viral infection maybe an infection of a protozoa, fungus, bacterium or virus as defined in the first aspect.
• 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, for use in the prevention, amelioration or treatment of cancer.
• the cancer may be as defined in the first aspect.
• a method for treating a protozoan, fungal, bacterial or viral infection comprising administering, to a subject in need thereof, a therapeutically effective amount of 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
• the protozoan, fungal, bacterial or viral infection to be treated maybe an infection of a protozoa, fungus, bacterium or virus as defined in the first aspect.
• a method for treating cancer comprising administering, to a subject in need thereof, a therapeutically effective amount of 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.
• the cancer to be treated maybe as defined in the first aspect.
• the RNA construct described herein provides an effective means of vaccinating a subject against a viral infection and cancer.
• a vaccine comprising 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.
• the vaccine comprises a suitable adjuvant.
• the adjuvant may be an encoded molecular adjuvant that is 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 adjuvant incorporated into a delivery formulation.
• the encoded molecular adjuvant may encode a cytokine, for example IL-12, GM-CSF, IL-2, IFN-g, or an effector protein such as CD40L, Flt-3 or microbial protein, e.g. flagellin or cholera toxin B.
• a cytokine for example IL-12, GM-CSF, IL-2, IFN-g
• an effector protein such as CD40L, Flt-3 or microbial protein, e.g. flagellin or cholera toxin B.
• the adjuvant incorporated into a delivery formulation may be selected form the group consisting of a bacterial lipopeptide, lipoprotein and lipoteichoic acid; mycobacterial lipoglycan; yeast zymosan, porin, Lipopolysaccharide, Lipid A, monophosphoryl lipid A (MPL), Flagellin, CpG DNA, hemozoin, Saponins (Quil-A, QS-21, Tomatine, ISCOM, ISCOMATRIXTM), squalene based emulsions, polymers such as PEI, Carbopol, lipid nanoparticles and bacterial toxins (CT, LT).
• 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
• composition according to the fifth aspect for use in stimulating an immune response in a subject.
• RNA construct according to the first aspect may relate to the reprogramming somatic cells to cells having stem cell characteristics.
• Somatic cells may be reprogrammed by delivering one or more proteins that are capable of enhancing reprogramming of somatic cells to cells having stem cell characteristics as defined in the first aspect.
• a method of modifying a cell ex vivo or in vitro comprising delivering, to the cell, 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.
• the method is performed ex vivo.
• the cell may be a eukaryotic or prokaryotic cell.
• the cell is a eukaryotic cell. More preferably, the cell is a mammalian host cell. Most preferably, the cell is a human cell.
• the modified cell is suitable for cell-therapy indications.
• a modified cell obtained from, or obtainable by, the method of the sixteenth aspect.
• the modified cell of the seventeenth aspect for use in therapy, optionally cell therapy.
• 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 may be used in a medicament, which maybe used as a monotherapy (i.e. use of the active agent), for treating, ameliorating, or preventing disease or vaccination.
• the active agents according to the invention maybe used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing disease.
• RNA construct nucleic acid sequence, expression cassette, vector or
• compositions of the invention maybe combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
• the composition maybe in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension, polyplex, emulsion, lipid
• nanoparticles with RNA on the surface or encapsulated
• any other suitable form that may be administered to a person or animal in need of treatment or vaccination.
• vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given.
• RNA construct nucleic acid sequence, expression cassette, vector or
• composition of the invention may also be incorporated within a slow- or delayed-release device.
• Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
• the device may be located at least adjacent the treatment site.
• Such devices maybe particularly advantageous when long-term treatment with the genetic construct or the recombinant vector is required and which would normally require frequent administration (e.g. at least daily injection).
• medicaments according to the invention may be administered to a subject by injection into the blood stream, muscle, skin or directly into a site requiring treatment. Most preferably, the medicaments, including the RNA construct, are injected into muscle. Injections maybe intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion), or intramuscular (bolus or infusion).
• RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition that is required is determined by its biological activity and bioavailability, which in turn depends 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 being used as a monotherapy or in a combined therapy.
• the frequency of administration will also be influenced by the half-life of the active agent within the subject being treated.
• Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular the RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition in use, the strength of the pharmaceutical composition, the mode of administration, and the type and advancement of the viral infection. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
• a daily dose of between o.ooiug/kg of body weight and lomg/kg of body weight, or between o.oipg/kg of body weight and lmg/kg ofbody weight, of the RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition of the invention may be used for treating, ameliorating, or preventing a disease, depending upon the active agent used.
• RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition may require administration twice or more times during a day.
• the RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition may be administered as two (or more depending upon the severity of the disease being treated) daily doses of between 0.07 pg and 700 mg (i.e. assuming a body weight of 70 kg).
• a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
• a slow release device may be used to provide optimal doses of the RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition according to the invention to a patient without the need to administer repeated doses.
• RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition according to the invention may be given as a weekly dose, and more preferably a fortnightly dose.
• RNA constructs such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the RNA construct, nucleic acid sequence, expression cassette or vector according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration).
• compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or maybe used in other veterinary applications. Most preferably, however, the subject is a human being.
• A“therapeutically effective amount” of the RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition is any amount which, when administered to a subject, is the amount of the aforementioned that is needed to ameliorate, prevent or treat any given disease.
• the RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition of the invention maybe used maybe from about 0.0001 mg to about 800 mg, and preferably from about 0.001 mg to about 500 mg. It is preferred that the amount of the replicon, nucleic acid sequence, expression cassette, vector or pharmaceutical composition is an amount from about 0.01 mg to about 250 mg, and most preferably from about 0.01 mg to about 1 mg.
• the RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition according to the invention is administered at a dose of i-200pg.
• A“pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
• 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 which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
• the vehicle may also be an encapsulating material.
• the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
• the active agent e.g.
• RNA construct, nucleic acid sequence, expression cassette, vector or pharmaceutical composition according to the invention may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
• vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
• the powders and tablets preferably contain up to 99% of the active agents.
• Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
• the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
• the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
• 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 according to the invention maybe dissolved or suspended in a
• liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
• the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers,
• liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g.
• the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
• Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
• the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
• Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, subcutaneous, intradermal, intrathecal, epidural,
• the nucleic acid sequence, or expression cassette of the invention maybe prepared as a sterile solid composition that maybe dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
• RNA construct nucleic acid sequence, expression cassette, vector or
• composition of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
• solutes or suspending agents for example, enough saline or glucose to make the solution isotonic
• bile salts for example, enough saline or glucose to make the solution isotonic
• bile salts for example, enough saline or glucose to make the solution isotonic
• bile salts for example, enough saline or glucose to make the solution isotonic
• acacia gelatin
• sorbitan monoleate sorbitan monoleate
• polysorbate 80 oleate esters of sorbitol and its
• 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. It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof.
• amino acid/nucleotide/peptide sequence can be a sequence that has at least 40% sequence identity with the amino acid/ nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID Nos: 1-55 and so on.
• amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
• sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
• amino acids referred to amino acids
• acid/polynucleoti de/polypeptide sequence has at least 85% identity with any of the sequences referred to, 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 referred to herein.
• the skilled technician will appreciate howto calculate the percentage identity between two amino acid/polynucleoti de/polypeptide sequences.
• an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.
• the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g.
• acid/ polynucleotide/ polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs.
• overhangs are included in the calculation.
• a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the
• a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or too amino acids from the sequences shown in, for example, SEQ ID Nos:i to 57 ⁇ Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
• Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change.
• Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a
• small non-polar, hydrophobic amino acids include gfycine, alanine, leucine, isoleucine, valine, proline, and methionine.
• Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
• the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
• the positively charged (basic) amino acids include lysine, arginine and histidine.
• the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
• Figure 1 shows a schematic of one embodiment of a self-amplifying RNA replicon or construct based on the Venezuelan Equine Encephalitis Virus (VEEV) backbone.
• the so- called‘Stealthicon’ vector is an saRNA replicon/ construct encoding Non-structural Proteins (NSP1-4), and an innate inhibitory protein (IIP), which is upstream or downstream of the GOI (Gene of Interest).
• NSP1-4 Non-structural Proteins
• IIP innate inhibitory protein
• Figure 2 shows that genome replication results in dsRNA, which is recognized by the sensor molecules MDA5 and PACT. These sensors transmit signals to activate
• downstream cascades including the activation of transcription factors (NF-KB, IRF-3, -7), restriction factors which directly inhibit RNA amplification and protein expression (dotted line).
• transcription factors NF-KB, IRF-3, -7
• restriction factors which directly inhibit RNA amplification and protein expression (dotted line).
• Expression of PIV-V or ORF4a block innate recognition of dsRNA by MDA5 and PACT preventing the activation of the downstream cascade that limits replicon RNA amplification and protein expression from the synthetic RNA.
• Figure 3 shows a) screening of IIP-encoding VEEV replicons in vitro.
• Cells were transfected with two batches of RNA containing luciferase as a reporter protein and assess for protein expression after 24 hours.
• HeLa and MRC5 are known to have more intact IFN expression pathways compared to HEK and b) Data for figure 3a shown as fold change in expression relative to wild type.
• Figure 4 shows 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, and thus the IIPs enabled higher luciferase expression at 3 days, and longer expression, lasting >14 days.
• Figure 5 shows screening of IIP-encoding 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, and thus the IIPs enabled longer expression of a soluble reporter protein, lasting >14 days.
• Figure 6 shows the construct map of one embodiment of an expression vector encoding the RNA construct comprising an GOI.
• Figure 7 shows the construct map of one embodiment of an expression vector encoding the RNA construct comprising GOI - MERS-CoV ORF4a.
• Figure 8 shows the construct map of one embodiment of an expression vector encoding the RNA construct comprising GOI - PIV5.
• Figure 11 shows co-formulation of WT and MERS CoV_2 replicons with JAK inhibitor ruxolitinib in C57BL6/J mice. Protein expression was quantified at days 4, 7, 10 and 14 (a, b, c, and d, respectively) after intramuscular injection of either 5 pg of RNA with 100 ug of ruxolitinib. Each dot represents a single mouse leg and the bar represents the mean ⁇
• Figure 12 shows protein expression of eGFP ⁇ MERS CoV_2 RNA (which corresponds to MERS-CoV ORF4a) (0.2, 2 or 20 pg) (a,b) or ⁇ ruxolitinib (0.1, 1, 10 or too pg) (c,d) in human skin explants.
• IVIS In Vivo Imaging System
• Figure 17 shows t-Distributed Stochastic Neighbor Embedding (tSNE) plots of unsupervised cluster of live cells (grey), overlaid with gating for eGFP+ cells (green), separated by phenotype (blue), for human skin explants treated with 0.2 pg of eGFP RNA.
• Figure 18 shows t- Distributed Stochastic Neighbor Embedding (tSNE) plots of unsupervised cluster of live cells (grey), overlaid with gating for eGFP + cells (green), separated by phenotype (blue), for human skin explants treated with 2 pg of eGFP RNA.
• Figure 19 shows t-Distributed Stochastic Neighbor Embedding (tSNE) plots of unsupervised cluster of live cells (grey), overlaid with gating for eGFP + cells (green), separated by phenotype (blue), for human skin explants treated with 20 pg of eGFP RNA.
• tSNE t-Distributed Stochastic Neighbor Embedding
• Figure 20 shows t-Distributed Stochastic Neighbor Embedding (tSNE) plots of unsupervised cluster of live cells (grey), overlaid with gating for eGFP+ cells (green), separated by phenotype (blue), for human skin explants treated with 0.2 pg of eGFP-PIV- 5 RNA.
• tSNE t-Distributed Stochastic Neighbor Embedding
• Figure 21 shows t-Distributed Stochastic Neighbor Embedding (tSNE) plots of unsupervised cluster of live cells (grey), overlaid with gating for eGFP+ cells (green), separated by phenotype (blue), for human skin explants treated with 2 pg of eGFP-PIV-5 RNA.
• tSNE t-Distributed Stochastic Neighbor Embedding
• Figure 22 shows t-Distributed Stochastic Neighbor Embedding (tSNE) plots of unsupervised cluster of live cells (grey), overlaid with gating for eGFP + cells (green), separated by phenotype (blue), for human skin explants treated with 20 pg of eGFP-PIV-5 RNA.
• tSNE t-Distributed Stochastic Neighbor Embedding
• Figure 23 shows t-Distributed Stochastic Neighbor Embedding (tSNE) plots of unsupervised cluster of live cells (grey), overlaid with gating for eGFP + cells (green), separated by phenotype (blue), for human skin explants treated with 0.2 pg of eGFP- MERS-COV_2 RNA (which corresponds to MERS-CoV ORF4a).
• tSNE t-Distributed Stochastic Neighbor Embedding
• Figure 24 shows t-Distributed Stochastic Neighbor Embedding (tSNE) plots of unsupervised cluster of live cells (grey), overlaid with gating for eGFP+ cells (green), separated by phenotype (blue), for human skin explants treated with 2 pg of eGFP-MERS- CoV_2 RNA.
• tSNE Stochastic Neighbor Embedding
• Figure 25 shows t-Distributed Stochastic Neighbor Embedding (tSNE) plots of unsupervised cluster of live cells (grey), overlaid with gating for eGFP + cells (green), separated by phenotype (blue), for human skin explants treated with 20 pg of eGFP- MERS-COV_2 RNA (which corresponds to MERS-CoV ORF4a).
• tSNE t-Distributed Stochastic Neighbor Embedding
• CD45- epithelial cells
• fibroblasts CD90+
• NK cells CD56+
• leukocytes CD45+
• Langerhans cells CDia+
• monocytes CD14+
• dendritic cells CDiic+
• T cells CD3+
• B cells CD19+
• Figure 28 show a schematic drawing of proposed mechanism of PIV-5 V and MERS-CoV ORF4a on saRNA sensing.
• Figure 29 shows Median Fluorescent Intensity (MFI) data showing the increased expression of SARS-C0V-2 glycoprotein from an saRNA according to one embodiment of the invention in Hela cells when co-expressed with the innate inhibitor protein, MERS- ORF4a, when compared to an saRNA encoding an SARS-C0V-2 glycoprotein only (i.e. no IIP).
• MFI Median Fluorescent Intensity
• RNA replicons containing innate inhibiting proteins IIPs
• a gene of interest RNA replicons containing innate inhibiting proteins
• IIP innate inhibitory proteins
• RNA encoding firefly luciferase, Gaussia luciferase, enhanced green fluorescent protein (eGFP), rabies glycoprotein (RABV) and the replicase derived from the Venezuelan equine encephalitis were cloned into a plasmid vector, as previously described (53) ⁇
• the library of interferon inhibiting proteins was cloned into these vector backbones as part of the gene of interest (fLuc, GLuc, eGFP or RABV) with a T2A cleavage site
• GenBank accession # AAC97195.1 The interferon inhibiting proteins can be found with the following GenBank accession numbers: HSV-2 Usi (Z86099.2), HSV-i Usi
• RNA for in vitro transfections was prepared using 1 pg of linearized pDNA template in a mMachineTM T7 Transcription
• RNA for ex vivo and in vivo experiments was prepared as previously described (2). Uncapped RNA transcripts were produced using 1 pg of linearized pDNA template using a MEGAScriptTM T7 Transcription reaction (Invitrogen, UK) for 2h at 37 °C using the manufacturer’s protocol. Transcripts were then purified by overnight LiCl precipitation 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 H 2 0 (Ambion, UK).
• RNA storage buffer (10 mM HEPES, o.i mM EDTA and too mg/mL trehalose) and stored at - 80 °C until further use saRNA formulation
• RNA and pABOL were diluted in HEPES buffer (20 mM HEPES, 5 wt.% glucose in H 2 0, pH 7.4) and combined on a NanoAssemblr benchtop formulation unit (Precision Nanosystems, Inc., Vancouver, Canada) at a volume ratio of 4:1 (RNA to polymer) with at flow rate of 10 mL/min. The final ratio of polymer to saRNA was 45:1 (w/w).
• Transfections were performed in HEK293T.17 cells (ATCC, USA), HeL cells (ATCC, USA), MRC5 cells (ATCC, USA), mouse embryonic fibroblasts (MEF) cells (SigmaAldrich, UK), RK13 rabbit kidney cells (Public Health England, UK) and LLC-MK2 rhesus macaque kidney cells (ATCC, USA).
• mice After 24 h, 50 uL of medium was removed from each well and 50 uL 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 on a FLUOstar OMEGA plate reader (BMG LABTECH, UK). Background fluorescence from the control wells was subtracted from each well. In vivo fLuciferase expression in mice
• IM intramuscularly
• 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.
• the protein expression in the sera was quantified on a FLUOstar OMEGA plate reader (BMG LABTECH, UK). Background fluorescence from the control wells was subtracted from each well.
• the mice were injected intraperitoneally (IP) with 150 pL of XenoLight RediJect D-luciferin substrate (PerkinElmer, UK) and allowed to rest for 10 min.
• IP intraperitoneally
• mice were then anesthetized using isoflurane and imaged on an In Vivo Imaging System (RTS) FX Pro (Kodak Co., Rochester, NY, USA) equipped with Molecular Imaging software version 5.0 (Carestream Health, USA) for 2 min. Signal from each injection site was quantified using Molecular Imagine software and expressed as total flux (p/s).
• RTS In Vivo Imaging System
• mice were bled via the tail vein.
• the blood was allowed to clot and centrifuged for 5 min at 10,000 RPM and the sera removed.
• the serum from all timepoints was then assayed on a single 96-well white plate (Costar) using the PierceTM Gaussia Luciferase Glow Assay Kit using 20 pL of sera and 100 uL of Working Solution prepared according to the manufacturer’s protocol.
• the luminescne was analysed a FLUOstar Omega plate reader (BMG LABTECH, UK) and background from naive animals was subtracted from each sample. Vaccination of Mice, Rats and Rabbits.
• mice BALB/c mice, Sprague Dawley rats and New Zealand white rabbits were immunized with 1 ug (mice) or 20 pg (rats, rabbits) of RABV-encoding saRNA formulated with pABOL in a total volume of 50 uL (mice) or too uL (rats, rabbits) IM in one hind leg.
• a boost injection was given 4 weeks after the initial prime. Blood was collected after o, 4 and 6 weeks from study onset and centrifuged at 10,000 RPM for 5 min. Sera was then decanted and stored at -80 °C until further analysis.
• bovine serum albumin BSA
• Tween-20 0.05% (v/v) Tween-20 in PBS.
• diluted serum samples were added to the plates, incubated for 2 h.
• the plates were then washed and a 1:4000 dilution of anti-mouse IgG-HRP (Southern Biotech, UK) was added for the mouse ELISAs, a 1:4000 dilution of goat anti-rat IgG-HRP (Southern Biotech, UK) was added for the rat ELISAs, and a 1:10000 dilution of mouse anti-rabbit IgG-HRP
• 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 down with a 5-fold dilution series.
• Rat standards were prepared by coating ELISA plate wells with purified rat IgG (R & D Sytems, UK) starting at 1000 ng/mL and titrating down with a 5-fold dilution series.
• Rabbit standards were prepared by coating ELISA plate wells with a 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 rabiit IgG (AbD Serotech, UK) starting at 1000 ng/ mL and titrating down with a 5-fold dilution series. Samples and standard were developed using 3,3’,5,5’-tetramethylbenzidine (TMB). The reaction was stopped after 5 min with stop solution (Insight Biotechnologies, UK). Absorbance was read on a spectrophotometer (VersaMax, Molecular Devices, UK) with SoftMax Pro GxP V5 software.
• TMB 3,3’,5,5’-tetramethylbenzidine
• Pseudotyped rabies microneutralization was performed on week o, 4 and 6 samples.
• BHK-21 cells were seeded at 10,000 cells/well in cDMEM in a 96-well plate.
• Sera was heat-inactivated at 56 °C and then diluted in a 1:5 serial dilution in cDMEM.
• Samples were then diluted with an equal volume of pseudo-virus at a concentration of 100 TCID 50 in 50 uL, incubated for 1 h at 37 °C and then added to BHK-21 cells and cultured for 48 h at 37 °C.
• Cells were then lysed and luciferase activity was quantified using a 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) and the IC
• Explants were injected intradermally (ID) using a Micro-Fine Demi 0.3 mL syringe (Becton Dickinson, UK) with a dose of 2 pg of saRNA in a total volume of 50 pL. Media was replaced daily for the duration of culture.
• FACS buffer PBS + 2.5% FBS
• fixable aqua live/dead cell stain ThermoFisher, UK
• CD3-V450 BioLegend, UK
• CDi4-Qdot6o5 BioLegend, UK
• CD19-BV650 BioLegend, UK
• CD56-BV711 BioLegend, UK
• CDia-PerCP-eFluor7io BioLegend, UK
• CDiic-PE BioLegend, UK
• CD90-PE-Cy7 BioLegend, UK
• CD45-AF700 BioLegend, UK
• t-Distributed Stochastic Neighbor Embedding (tSNE) analysis of unsupervised 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-Hut gradient algorithm.
• SARS-COV-2 Glycoprotein In Vitro work 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-Hut gradient algorithm.
• the inventors used a plasmid vector to synthesize a self-amplifying RNA (saRNA) replicon, based on the Trinidad donkey Venezuelan equine encephalitis virus strain (VEEV) alphavirus genome.
• saRNA self-amplifying RNA
• VEEV Venezuelan equine encephalitis virus strain
• an oligonucleotide string was synthesised (GeneArt, Germany) encoding the MERS-CoV ORF4a (AHC74090.1), and inserted 3’ to the SARS-C0V-2 coding region connected using a variant of the furin/T2A sequence (SEQ ID No: 26) in a continuous open reading frame, generating a new SARS- C0V-2 ORF4a vector.
• Cells were transfected separately with both the SARS-C0V-2 and the SARS-COV-2 ORF4a saRNA and then stained with a polyclonal antibody to examine expression.
• FACS buffer PBS + 2.5 % FBS
• concentration of 1 x to 7 cells / mL One hundred microliters of the resuspended cells were added to a FACS tube and stained with 50 uL of Live/Dead Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific, UK) at a 1:400 dilution on ice for 20 min. Cells were then washed with 2.5 mL of FACS buffer and centrifuged at 1750 RPM for 7 min.
• RNA replicons have been postulated to be potential tools for the delivery and expression of genes of interest for vaccines and therapeutics.
• dsRNA dsRNA
• RNA replicons that encode innate inhibiting proteins to abate the innate recognition of saRNA.
• the only previously published approach to abating the interferon response with saRNA used interferon inhibiting proteins from the vaccinia virus, E3, K3 and B18.
• the interferon inhibiting proteins were delivered and formulated as separate mRNA molecules that were combined with the saRNA. This requires the manufacture of both saRNA and mRNA and necessitates the use of 3-6 times as much vaccinia mRNA as replicon RNA to ensure co-delivery into the same cells and provide any observable enhancement in protein expression.
• the kinetics of expression differ for mRNA and saRNA such that any beneficial effects of the IIPs expressed from mRNA would be of short duration in comparison the accompanying replicon.
• PIV-5 and ORF4a are known to block MDA-5, a cytoplasmic RNA helicase that signals through an adaptor molecule called MAVs that results in the induction of interferon regulatory factor 3 and 7 (IRF3 and IRF7), which respectively leads to the production of restriction factors that decrease the translation of the introduced synthetic saRNA ( Figure l).
• IRF3 and IRF7 interferon regulatory factor 3 and 7
• Figure l These two IIPs were identified in an initial in vitro screen of to IIPs from a range of different viruses.
• HSV-2 USI - inhibits IFN-B production by suppressing association of IRF-3 with IFN-b promoter [1]
• HSV-i Usi modulatory factor from HSV-i
• BVDV Npro blocks and IRF3 phosphorylation and S100A9 signalling [6, 7]
• PIV5 V blocks MDA-5 and IRF3 by binding to MDA-5 [8,9]
• MERS-CoV M interacts with TRAF3 and disrupt TRAF3-TBK1 association leading to reduced IRF3 activation [10-12]
• MERS-CoV ORF4a binds to dsRNA (has a preference for long RNA), suppresses
• Influenza NSi binds to dsRNA, blocks RIG-I signalling [17] These IIPs were incorporated within the inventor’s standard VEEV saRNA replicon
• Figure 1 together with fLuciferase as the GOI and used as a marker of expression.
• the constructs were assessed in three human cell lines: HEK293T cells that have impaired innate sensing pathways, HeLa cells and primary MRC5 embryonic epithelial cells (Figure 3a and b). All of the IIP candidate saRNA replicated similarly to wild type saRNA in HEK293T cells in the absence of innate recognition.
• a range of IIPs (with the exception of MERS-CoV M and Influenza NSi) were able to enhance expression in HeLa cells, however the most pronounced enhancement (3 logs) was seen for PIV-V and ORF4a.
• IP intraperitoneally
• the inventors sought to determine whether the library of interferon inhibiting proteins enhanced firefly luciferase (fLuc) protein expression in vitro. They prepared a library of saRNA VEEV replicons with an IIP separated from the fLuc with a T2A cleavage site ( Figure 9a), with a variety of cytoplasmic interferon targets (Table 1), including IRF-3, MDA5, RIG-I, and JAK/STAT ( Figure 9b).
• pABOL Figure 9c, Supplementary Figure 1
• pABOL a polymeric delivery system that has previously been characterized to yield relatively high protein expression but is relatively immune silent due to its bioreducible nature (2).
• the inventors chose these three cell lines for their variation in completeness of the IFN pathway; HEK293T.17 cells do not have a complete pathway as they lack endogenous RIG-I and MDA5 expression (37) and thus should be less sensitive to proteins affecting this pathway, whereas HeLa and MRC5 are more discriminatory (38, 39).
• ORF4a protein affected protein expression in mouse (MEF), rabbit (RK13), nonhuman primate (LLC) and human cells (MRC5) (figure I5a-d).
• the R172A mutation in PIV-5 V abrogates ability to block MDA5 but not STAT (40), and the K63A/K67A mutations in MERS-CoV ORF4a block binding to dsRNA (41).
• the inventors observed that the PIV-5 V and MERS-CoV ORF4a proteins did not enhance protein expression in MEF or RK13 cells.
• the MERS-CoV ORF4a protein did enhance protein expression in LLC and MRC5 cells (figure 15 c,d), and the K63A/K67A mutation greatly decreased the protein expression.
• the PIV-5 V protein enhanced protein expression in MRC5 cells but not LLC cells, and the R172A mutation decreased protein expression in MRC5 cells.
• the PIV-5 V and MERS-CoV ORF4a proteins enhanced protein expression in interferon-competent human cells, and mutating the proteins with the K63A/K67A and R172A substitutions muted saRNA expression.
• the inventors observed slight enhancement of total area under the curve (AUC) protein expression of fLuc in C57BL/6 mice with the MERS-CoV ORF4a protein, and GLuc with both the PIV-5 V and MERS-CoV ORF4a proteins, although the differences were not statistically significant.
• AUC total area under the curve
• the inventors have previously observed that increasing the dose of saRNA eventually results a lower level of protein expression, and thus sought to characterize whether the MERS-CoV ORF4a protein could abate the nonlinear dose dependency of saRNA in vivo.
• the inventors tested doses of 0.2, 2 and 20 pg of the wild-type fLuc and the fLuc + MERS- CoV ORF4a replicon and quantified protein expression at days 7 and 10 after
• Rnxolitinib Enhances Protein Expression of saRNA In Vivo.
• both of the formulations containing ruxolitinib had slightly higher protein expression ( ⁇ io 6 p/s) compared to the WT or MERS-CoV ORF4a constructs ( ⁇ 5xio 5 p/s), although it was not statistically significant.
• 14 days there was no protein expression observed in the saRNA groups without ruxolitinib, and only a few positive samples for the ruxolitinib groups.
• the inventors observed that the IIPs exhibit differences in protein expression depending on the species of the cell type in vitro, they sought to test the saRNA IIP constructs in a more clinically relevant human skin explant model.
• the inventors characterized both the quantity (% of eGFP + cells) and the quality of protein expression (median eGFP fluorescent intensity per cell) in resident human skin cells with
• the inventors further characterized which cells were expressing the saRNA using t- Distributed Stochastic Neighbour Embedding, a type of principle component analysis for flow cytometry data that allows for visualization by unsupervised clustering of cells with overlaid defined protein and phenotype gating ( Figures 17-25) (43).
• the inventors observed that at the highest dose of saRNA (20 ug), the PIV-5 V and MERS-CoV ORF4a proteins enabled protein expression in the immune cells, including T cells, dendritic cells, monocytes, B cells, Langerhans cells, leukocytes and NK cells, as opposed to resident epithelial cells and fibroblasts.
• ruxolitinib enhanced protein expression in the immune cells, as opposed to epithelial cells and hbroblasts, and specifically increased uptake in T cells, Langerhans cells, leukocytes and NK cells ( Figure 26 a,b).
• RABV rabies glycoprotein
• the inventors injected rabbits with a primary dose of 20 pg of saRNA and a boost after 4 weeks, and then sampled the RABV-specific IgG antibodies in their blood after o, 4, and 6 weeks ( Figure 13a).
• the inventors observed that all the rabbits for both the wild type and MERS-CoV ORF4a constructs seroconverted after a single injection.
• the RABV pseudotyped neutralization reflected the antibody trends ( Figure 13b).
• the WT group had an average IC 50 of ⁇ io 3 whereas the MERS-CoV group had an IC 50 of ⁇ io 4 .
• the MERS-CoV group had a much higher IC 50 of ⁇ io 5 , whereas the WT group had stabilized at ⁇ io 3 .
• CoV ORF4a protein enhances the immunogenicity of the RABV glycoprotein encoded by saRNA in rabbits.
• the inventors also characterized the immunogenicity of another model protein (i.e.
• the SARS-C0V-2 glycoprotein represented by Genbank ID No: QHD43416.1 when combined with the MERS-CoV ORF4a protein (i.e. the innate inhibitor protein or IIP) in an saRNA construct of the invention.
• MERS-CoV ORF4a protein i.e. the innate inhibitor protein or IIP
• the levels of surface expression were compared between SARS-C0V-2 (with no IIP) and SARS-C0V-2 combined with MERS-CoV ORF4a (IIP).
• IIP MERS-CoV ORF4a
• the MERS-CoV ORF4a protein almost doubled the per cell expression levels of the SARS-C0V-2 glycoprotein, when compared to an saRNA encoding an SARS-C0V-2 glycoprotein only (i.e. no IIP).
• the inventors screened a library of self-amplifying RNA with cis-encoded interferon inhibiting proteins for protein expression in vitro in mouse, rabbit, nonhuman primate and human cells, ex vivo in human skin explants and in vivo in mice, as well as immunogenicity in mice, rats and rabbits.
• the inventors observed that the PIV-5 V and MERS-CoV ORF4a proteins enhanced protein expression 100-500-fold in vitro in IFN- competent HeLa and MRC5 cells. They found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, but not the UPS, enhances protein expression of saRNA in vivo.
• MERS-CoV ORF4a Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA, while ruxolitinib increases the protein expression on a per cell basis. Finally, the inventors observed that the MERS-CoV ORF4a increased the RABV-specific IgG titer and neutralization IC 50 by -lo-fold in rabbits, but not mice or rats.
• the protein designs, cells and mutations characterized in these experiments offer insights into the mechanism by which the PIV-5 V and MERS-CoV ORF4a proteins increase protein expression.
• the PIV-5 V protein blocks MDA-5 and IRF3 by binding to MDA-5 (26, 27), whereas the MERS-CoV ORF4a protein binds to dsRNA and suppresses PACT triggering of MDA-5 and RIG-I (29-31).
• the inventors chose to move forward with the MERS-CoV ORF4a replicon as it was not feasible to screen all 10 candidates in vivo, and the PIV-5 V protein isn’t conserved between species (e.g.
• the inventors have previously observed that protein expression of saRNA formulations is not necessarily predictive of immunogenicity (2), and thus also characterized how the MERS-CoV ORF4a protein affected the immunogenicity of the rabies glycoprotein in mice, rats and rabbits.
• the inventors observed an increase in both the antibody titer and neutralization IC 50 with the saRNA encoding RABV and MERS-CoV ORF4a ( Figure 13a, b) in rabbits, but did not observe an increase in immunogenicity in mice or rats. While no preclinical animal model is perfectly predictive of human responses, rabbits are regarded as more immunologically similar to humans than mice or rats (48-50).
• the inventors did not observe any enhancement of protein expression by either the PIV-5 V or MERS-CoV ORF4a proteins in murine cells ( Figure 15a), and thus the lack of enhancement of immunogenicity is not unexpected.
• the inventors paired characterization in preclinical animal models with a human explant model, in which the cells are in a native tissue architecture and possess the inherent human IFN response. To their knowledge, the inventors are the first to observe that the IIPs enhance the percentage of cells expressing saRNA, whereas ruxolitinib enhanced the expression per cell.
• MERS-CoV ORF4a protein may enhance immunogenicity of saRNA vaccines in humans, and may also be useful for saRNA application to protein replacement therapies (51, 52).
• IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.
• different aspects of the interferon pathway can be targeted and increase saRNA expression, thus motivating probing of combinations of IIPs and other IFN inhibitions strategies, such as ruxolitinib.
• A. S. Devasthanam Mechanisms underlying the inhibition of interferon signaling by viruses.
• LGP2 disrupts MDA5 signaling enhancement but is not relevant to LGP2-mediated RLR signaling inhibition. J Virol 88, 8180-8188 (2014).
• M The structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists. Protein Cell 4, 951- 961 (2013).
• MERS-CoV Middle East respiratory syndrome coronavirus
• RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response. Journal of virology 88, 4866-4876 (2014).

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