WO2021259883A1 - Molécule d'acide nucléique pour le traitement de maladies provoquées par un nidovirus - Google Patents

Molécule d'acide nucléique pour le traitement de maladies provoquées par un nidovirus Download PDF

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WO2021259883A1
WO2021259883A1 PCT/EP2021/066890 EP2021066890W WO2021259883A1 WO 2021259883 A1 WO2021259883 A1 WO 2021259883A1 EP 2021066890 W EP2021066890 W EP 2021066890W WO 2021259883 A1 WO2021259883 A1 WO 2021259883A1
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nucleic acid
nidovirus
acid molecule
synthetic nucleic
cell
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PCT/EP2021/066890
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English (en)
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Ingo Jordan
Sven KRÜGENER
Volker Sandig
Alexander Karlas
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Probiogen Ag
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae

Definitions

  • the present invention relates to the therapy of diseases caused by a nidovirus by administering a synthetic nucleic acid molecule.
  • This therapy is to assist the immune system in combatting the nidovirus and, thereby, preventing and ameliorating symptoms of diseases caused by a nidovirus.
  • Nidovirales is a large order of positive sense, single-stranded RNA (ssRNA) viruses that consists of many genera and species in the families Coronaviridae , Arteriviridae , Roniviridae and Mesoniviridae . Although the genomes of nidoviruses vary in length, ranging from 13 to 32 kilobases (kb), the organization of the genomes are similar across the entire order. The 5' end of the genome encodes two replicase polyproteins (p la and pplab). The name Nidovirales is derived from the Latin nidus, to nest, and refers to the 3' coterminal nested set of subgenomic (sg) viral mRNAs produced during infection. Sequence comparisons of the replicase genes suggest that the Nidovirales have evolved from a common ancestor despite their substantial differences.
  • ssRNA single-stranded RNA
  • Nidoviruses infect a broad range of hosts including humans and other mammals, birds, fish, insects, and crustaceans.
  • Coronaviruses are nidoviruses. Coronaviruses cause about 30% of common colds in humans and, unlike rhinoviruses, cause both upper and lower respiratory infections, the latter being a more serious affliction. In addition, coronaviruses cause gastroenteritis and diarrhea in humans and many other serious diseases in non-human animals including mice, chickens, pigs and cats.
  • Important pathogens affecting livestock or companion animals are IBV (causes avian infectious bronchitis of chicken), TGEV (transmissible gastroenteritis of pigs), and FCoV (feline infectious peritonitis).
  • human pathogens are HCoV-229E and HCoV- OC43 (both cause common cold), SARS-CoV (severe acute respiratory syndrome), MERS- CoV (Middle East respiratory syndrome), and SARS-CoV-2 (coronavirus disease 2019).
  • SARS-CoV pandemic started in 2002 with an outbreak that initiated in China and infected more than 8000 patients until it disappeared in 2004. The virus was later determined to probably have jumped from the bat reservoir to a civet cat, and from there (in a wet market) into the human population. During the outbreak, SARS-CoV caused nearly 800 fatalities at a mortality rate of almost 10%. SARS-CoV caused an atypical pneumonia characterized by fever, cough and infiltrates with ground-glass opacity on X-ray or computed tomography (CT). Severe disease is typically associated with acute diffuse alveolar damage with oedema together with fibrin and hyaline membranes in the alveolar spaces as a sign of acute lung injury. Late-term disease progression was found to be rather associated with immunopathological damage than with viraemia.
  • CT computed tomography
  • MERS-CoV another zoonotic coronavirus was observed. This virus repeatedly entered the human population via contact with dromedary camels in the Arabian Peninsula. To date, cases of MERS-CoV have been detected in 27 countries and almost 2500 laboratory-confirmed cases of infection and more than 850 deaths were counted, representing a case fatality rate in humans of approximately 35%. MERS-CoV enters the cell via the dipeptidyl peptidase 4 (DPP4) and the virus-induced disease is particularly severe in aged patients and those with pre-existing co morbidities. Most people, confirmed to have MERS-CoV infection, have had severe respiratory illness with symptoms of fever, cough and shortness of breath caused by pneumonia.
  • DPP4 dipeptidyl peptidase 4
  • Both virus variants can lead to an atypical pneumonia characterized by fever, cough and infiltrates in the lung.
  • patients suffer from acute respiratory distress syndrome that necessitate intensive care treatment including mechanical ventilation.
  • COVID-19 is not only restricted to the lung tissue.
  • SARS-CoV-2 could also be detected in the heart, liver, brain and kidneys, which can explain the multi-organ failure which is partly associated with COVID-19.
  • a high viral burden of SARS-CoV-2 was also shown in the upper respiratory tract.
  • even asymptomatic carriers can spread SARS-CoV-2 to others, a property which dramatically complicates efforts to control spread by quarantine.
  • coronaviruses are known to mutate and recombine often, they present an ongoing challenge for disease control.
  • No vaccines for coronaviruses or arteriviruses are presently available and no effective antiviral therapies are available to treat an infection.
  • available treatment involves supportive measures such as anti pyretics to keep fever down, fluids, antibiotics for secondary bacterial infections and respiratory support as necessary.
  • the present inventors generated a new synthetic/artificial nucleic acid molecule containing some nucleotide sequences of a nidovirus and not containing others. Said synthetic nucleic acid molecule surprisingly acts as a replication competitor against a nidovirus. It can be given to a subject infected with a nidovirus (either with symptoms or without, but with active virus replication) or not infected with a nidovirus (preventative). When given to a subject infected with a nidovirus, it ends up together with the nidovirus in a cell of the subject.
  • the new synthetic/artificial nucleic acid molecule costs the nidovirus resources and, thus, interferes with the normal kinetic of the nidovirus infection or disease. Even if nidovirus replication is not reduced below limits of detection, any interference significantly can reduce the infectious burden for the subject so that transmission of nidovirus to others will be interrupted. Furthermore, a lower infectious burden allows the immune system to better cope with virus replication and delays or even suppresses symptoms caused of an over-reactive response such as cytokine storms.
  • the present invention relates to a synthetic/artificial nucleic acid molecule comprising a polynucleotide (i) containing a nucleotide sequence of the 5’ untranslated region (UTR) of a nidovirus, a nucleotide sequence of the 3’ untranslated region (UTR) of a nidovirus, and a nucleotide sequence of a packaging signal localized in ORFlb of a nidovirus, and (ii) not containing a nucleotide sequence encoding a RNA-dependent RNA polymerase of a nidovirus or a functionally active variant or fragment thereof, and a nucleotide sequence encoding at least one structural protein of a nidovirus or a functionally active variant or fragment thereof.
  • a polynucleotide i) containing a nucleotide sequence of the 5’ untranslated region (UTR) of a n
  • the present invention relates to particles comprising the synthetic nucleic acid molecule according to the first aspect.
  • Said particles are preferably free of any structural protein of a nidovirus.
  • the present invention relates to a cell comprising the synthetic nucleic acid molecule according to the first aspect or the particles according to the second aspect.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a synthetic nucleic acid molecule according to the first aspect or particles according to the second aspect and one or more pharmaceutical acceptable excipient(s), diluent(s), and/or carrier(s).
  • the present invention relates to a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect or a pharmaceutical composition according to the fourth aspect for use as a medicament.
  • the present invention relates to a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect or a pharmaceutical composition according to the fourth aspect for use in the treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus.
  • the present invention relates to a combination comprising
  • the present invention relates to a combination comprising
  • the present invention relates to a virus like particle comprising a synthetic nucleic acid molecule according to the first aspect and an envelope of a nidovirus.
  • the present invention relates to a method of producing a virus like particle comprising a synthetic nucleic acid molecule according to the first aspect and an envelope of a nidovirus comprising the steps of:
  • the present invention relates to a combination comprising
  • a virus like particle comprising a synthetic nucleic acid molecule according to the first aspect and an envelope of a nidovirus
  • the present invention relates to a combination comprising
  • a nidovirus or a combination according to the eleventh aspect for use in the treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus and for vaccination against nidovirus infection.
  • nidovirus reters to a positive sense, single- stranded RNA ((+) ssRNA) enveloped virus.
  • (+) ssRNA single- stranded RNA
  • the genomes of nidovi ruses vary in length, ranging from 13 to 32 kilobases (kb), the organization of the genomes are similar across the entire order.
  • the genomes comprise two untranslated sections on each side of the coding sequence. The section upstream of the first start codon is called 5’ untranslated region (5’ UTR) (or leader sequence) and the section downstream of the last stop codon is called 3’ untranslated region (3’ UTR) (or trailer sequence).
  • the 5’ UTR comprises promoter sequences for replication and transcription.
  • the 3’ UTR comprises promoter sequences for antigenomic and subgenomic copies and the circularization signal for the cognate 5' UTR.
  • the 5’ two-thirds of the nidovirus genome encodes non-structural proteins, in particular two polyproteins, ppla and pplab, collectively termed the replicase. These polyproteins are cleaved into 16 non-structural proteins including RNA-dependent RNA polymerase (RdRp) by two essential viral proteases, 3C-like protease (3CLpro) and papain-like protease (PLpro).
  • RdRp RNA-dependent RNA polymerase
  • the 3’ one-third of the nidovirus genome encodes 4 essential structural proteins which can all interfere with the host innate immune response.
  • the 4 structural proteins are spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N).
  • S protein spike protein
  • E envelope protein
  • M membrane protein
  • N nucleocapsid protein
  • the spike protein (S protein) is responsible for receptor-recognition, attachment to the host cell, infection via the endosomal pathway, and the genomic release driven by fusion of viral and endosomal membranes. Though sequences between the different family members vary, there are conserved regions and motifs within the S protein making it possible to divide the S protein into two subdomains: SI and S2. While the S2, with its transmembrane domain, is responsible for membrane fusion, the SI domain recognizes the virus-specific receptor and binds to the target host cell.
  • the envelope protein (E) is only present in small quantities and most likely function as ion channels, not necessarily needed for viral replication but essential for pathogenesis.
  • the membrane (M) protein is the most abundant protein in the virus structure and is responsible for viral membrane curvature and binding to the nucleocapsid.
  • the nucleocapsid (N) protein binds to the viral RNA genome and ensures the maintenance of the RNA in a ‘beads-on-a-string’ conformation.
  • a variable number of virus-specific accessory proteins that augment adaptation to the host can also be encoded by the 3' terminal region of the genome.
  • the accessory genes are usually at intergenic positions and may also partially overlap with other genes.
  • Nidoviruses enter the cell via specific receptors.
  • Certain coronaviruses enter the cell via the angiotensin-converting enzyme 2 (ACE2) receptor.
  • ACE2 angiotensin-converting enzyme 2
  • S viral
  • S2 binds to the viral (S) protein and allows the cellular entry. More specifically, the S protein is cleaved into two subunits, SI and S2, by an extracellular protease.
  • TMPRSS2 host surface-associated transmembrane protease serine 2
  • the translational machinery of the host is hijacked for the translation of the two replicase polyproteins (ppla and pplab) and the essential viral proteases (3CLpro and PLpro).
  • the two replicase polyproteins (ppla and pplab) are then cleaved into 16 non- structural effector proteins by the viral proteases (3CLpro and PLpro) allowing them to form the replication complex together with the RNA-dependent RNA polymerase, which synthesizes a full-length negative RNA strand template. This is used to replicate the complete RNA genome and generate the individual subgenomic mR A templates needed for the translation of the viral structural and accessory proteins.
  • the mRNAs for the genes downstream of ORF la/lb share a 5’ leader and a body consisting of the respective first and all subsequent ORFs.
  • the replicase first transcribes subgenomic RNAs with genomic RNA as template.
  • transcription proceeds towards an element termed transcription regulating sequence (TRS-B) upstream of each gene in the body of the genome (the region downstream of ORF la/lb).
  • TRS-B transcription regulating sequence upstream of each gene in the body of the genome (the region downstream of ORF la/lb).
  • TRS-L transcription regulating sequence
  • the mRNAs are next transcribed as plus-strand copies. Translation occurs at the rough endoplasmic reticulum (ER). The translated structural and accessory proteins are then trafficked from the ER through the Golgi apparatus, after which new virions assemble in budding Golgi vesicles.
  • the packaging signal of nidoviruses is located in the 3’ half of ORF lb. After packaging, the mature virions are exocytosed and released from the host cell into the surrounding environment to repeat the infection cycle.
  • Predominant hosts of the nidoviruses are mammals and birds ( Coronaviridae and Arterivirdae), crustaceans ( Roniviridae ) and mosquitos ( Mesoniviridae ).
  • Important pathogens in the Arteriviridae are viruses that cause porcine reproductive and respiratory syndrome (PRRS) and equine viral arteritis (EVA).
  • the nidovirus is a coronavirus.
  • the nidovirus is a coronavirus.
  • Orthocoronavirinae one subfamily is termed Orthocoronavirinae, and the four genera therein are alpha-coronavirus, beta-coronavirus, delta-coronavirus and gamma- coronavirus.
  • the alpha- and beta-coronaviruses appear to have evolved in bat reservoirs, and the delta- and gamma-coronaviruses in avian reservoirs.
  • the coronavirus is an alpha-coronavirus or a beta-coronavirus. It is even more preferred that the coronavirus is a respiratory coronavirus, i.e. a coronavirus that causes a respiratory disease.
  • alpha-coronaviruses are HCoV-229E and CoV-NL63
  • beta- coronaviruses are SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-HKUl, and HCoV-OC43.
  • the coronavirus is a SARS coronavirus, wherein preferably the SARS coronavirus is a virus of the species “severe acute respiratory syndrome-related coronavirus” as classified by the International Committee on Taxonomy of Viruses (ICTV). In a most preferred embodiment, it is SARS-CoV-2.
  • nidovi ruses infect a broad range of hosts including humans and other mammals, birds, fish, insects and crustaceans.
  • coronavi ruses cause about 30% of common colds in humans and, unlike rhinoviruses, cause both upper and lower respiratory infections, the latter being a more serious affliction.
  • coronavi ruses cause gastroenteritis and diarrhea in humans and many other serious diseases in non-human animals including mice, chickens, pigs and cats.
  • a nidovirus, e.g. coronavirus, infection is defined by the entry of nidovirus, e.g. coronavirus, into at least one cell of a subject and its replication in the at least one cell.
  • the infection can be an infection of the respiratory tract, including the upper respiratory tract (nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords)) and/or the lower respiratory tract (portion of the larynx below the vocal folds, trachea, bronchi, bronchioles and the lungs including the respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli).
  • the infection can also be characterized immunologically by the presence of at least one nidovirus-antigen-specific immune factor, preferably selected from the group consisting of B cells, follicular helper T cells (TFH cells), activated CD4+ T cells and CD8+ T cells (particularly also CD38+HLA-DR+ ), IgM antibodies, and IgG antibodies.
  • at least one nidovirus-antigen-specific immune factor preferably selected from the group consisting of B cells, follicular helper T cells (TFH cells), activated CD4+ T cells and CD8+ T cells (particularly also CD38+HLA-DR+ ), IgM antibodies, and IgG antibodies.
  • a nidovirus-specific cytokine profile preferably selected from the group consisting of B cells, follicular helper T cells (TFH cells), activated CD4+ T cells and CD8+ T cells (particularly also CD38+HLA-DR+ ), IgM antibodies, and IgG antibodies.
  • a nidovirus infection may or may not cause symptoms of a nidovirus disease in a subject.
  • the terms “nidovirus infection” and “nidovirus disease” are distinguished herein by the presence of at least one nidovirus disease symptom. As long as the infection is not accompanied by at least one symptom of nidovirus disease, it (or the subject) is asymptomatic (includes presymptomatic).
  • the term “nidovirus disease” as used herein requires the presence of a nidovirus infection and at least one symptom of nidovirus disease (also referred to herein as “symptomatic infection”).
  • Nidovirus symptoms include dry cough, fever (>J V.8°C), runny and/or blocked nose, fatigue, breathing difficulty, pneumonia, organ (e.g. heart, lung, liver and/or kidney) failure, itchy throat, headache, joint pain, nausea, diarrhoea, shivering, lymphophenia, loss of smell and/or loss of taste.
  • the nidovirus disease is characterized by the presence of two or more, three or more, or four or more symptoms, preferably including one or two or more of dry cough, fever (>37.8°C), breathing difficulty, loss of smell and/or loss of taste.
  • the breathing difficulties may require intensive medical care including artificial ventilation.
  • Nidoviruses are mainly transmitted by infected persons when they cough or sneeze. Protection is possible by washing hands frequently and avoiding touching the face. Also keeping a distance from people who feel unwell helps. Someone can also get infected by touching surfaces or things where the virus is present and then touching eyes, nose or mouth.
  • a nidovirus disease can occur in a mild form (no severe symptoms) or in a severe form.
  • nidovirus disease refers to a nidovirus disease associated with breathing difficulty in particular acute respiratory distress syndrome, pneumonia, organ (e.g. heart, lung, liver and/or kidney) failure and high fever (>40°C).
  • the severe form of a nidovirus disease can be life-threatening.
  • a life-threatening form of a nidovirus disease is usually associated with septic shock and/or multiple organ (e.g. heart, lung, liver and/or kidney) failure.
  • the nidovirus disease is preferably a coronavirus disease, in particular a respiratory disease (e.g. SARS or MERS), more preferably Covid-19.
  • subject refers to any individual which may receive the nucleic acid molecule, particles, or pharmaceutical composition of the present invention.
  • the subject may be infected by a nidovirus or may suffer from a nidovirus disease. Alternatively, the subject may have a risk to be infected by a nidovirus (e.g. clinical personnel).
  • the subject is a vertebrate.
  • the vertebrate is a mammal (e.g. a canine, a feliformia, a rodent such as a mouse, rat or hamster, an ovine, a caprine, a pig, a bat such as a megabat or microbat, or a human/non-human primate such as a monkey or a great ape), a fish (e.g. carp, tench, zander, or pike), an amphibian, a reptile, or an avian (e.g. a chicken, quail, goose, or duck).
  • a mammal e.g. a canine, a feliformia, a rodent such as a mouse, rat or hamster, an ovine, a caprine, a pig, a bat such as a megabat or microbat, or a human/non-human primate such as a monkey or a great
  • the mammal is a mouse, a rat, a cat, a pig, a bat or a human/non-human primate. Humans are most preferred.
  • the term “(host) cell”, as used herein, reters to a cell which is infected by a nidovirus or may be infected (being at risk to be infected) by a nidovirus.
  • the (host) cell is a vertebrate cell. More the vertebrate cell is a mammalian cell (e.g.
  • a canine a feliformia, a rodent such as a mouse, rat or hamster, an ovine, a caprine, a pig, a bat such as a megabat or microbat, or a human/non-human primate such as a monkey or a great ape cell
  • a fish cell e.g. carp, tench, zander, or pike cell
  • an amphibian cell e.g. a reptilian cell
  • an avian cell e.g. a chicken, quail, goose, or duck cell.
  • the mammalian cell is a mouse, a rat, a cat, a pig, a bat or a human/non-human primate cell.
  • Human cells are most preferred.
  • the (host) cell may be part of the subject defined above. It may also be an isolated cell, i.e. isolated from the subject defined above.
  • the cell may be part of a cell culture, e.g. an adherent cell culture or a non-adherent cell culture.
  • the cell may also be part of a cell line, e.g. a suspension cell line.
  • isolated (host) cell refers to a cell that is removed from its native or culturing environment.
  • an isolated cell may be free of some or all native or culture components, i.e. components of the organism in which the cell naturally occurs (e.g. organ particularly tissue) or in which it is cultured (e.g. culture medium or culture-related impurities such as culture remnants).
  • the cell may be infected with the nidovirus as described herein.
  • the cell may further comprise the nucleic acid molecule or the particle of the present invention. Techniques how to infect or transfect a cell are known to the skilled person.
  • infectious refers to the ability of a nidovirus to replicate in a (host) cell and to produce viral particles. Infectivity can be evaluated either by detecting the virus load or by observing disease progression in a subject.
  • treatment refers to any therapy which improves the health status and/or prolongs (increases) the lifespan of a subject suffering from a nidovirus disease.
  • Said therapy may eliminate the nidovirus disease in a subject, arrest or slow the development of the nidovirus disease in a subject, inhibit the development of the nidovirus disease in a subject, decrease the severity of symptoms in a subject suffering the nidovirus disease, and/or decrease the recurrence in a subject who currently has or who previously has had a nidovirus disease.
  • prophylactic treatment refers to the preparation of a subject for a (potential/likely) nidovirus infection.
  • the subject is conditioned for a (potential/likely) nidovirus infection.
  • the subject is not yet infected by a nidovirus.
  • the “prophylactic treatment”, “preventative treatment” or “preventive treatment” is meant to avoid/prohibit the development of a nidovirus disease.
  • a subject being infected with a nidovirus and having at least one symptom of nidovirus disease can also be designated as patient.
  • treatment refers to a substance/combination/composition used in therapy, i.e. in treating, ameliorating or preventing a disease or disorder.
  • transgene refers to a heterologous nucleotide sequence that is not normally found intimately associated with a nidovirus. The transgene rather encodes a molecule of interest.
  • molecule of interest refers to a macromolecule, such as but not limited to DNA, RNA, a peptide and a polypeptide or protein. The molecule of interest is preferably therapeutically active.
  • therapeutically active molecule refers to a compound having a therapeutic/pharmacologic effect when administered appropriately to a subject suffering from a disease or disorder.
  • Such therapeutic/pharmacologic effect is one that is expected to be related to a beneficial effect on the course or a symptom of the disease or disorder.
  • the transgene may encode a molecule having reporter activity, anti-inflammatory activity, anti-coagulation activity, and/or anti-viral activity.
  • peptide generally relates to molecules which include at least 2, at least 3, at least 4, at least 6, at least 8, at least 10, at least 12 or at least 14 and preferably up to 8, 10, 12, 14, 16, 18, 20, 25, 30, or 50 consecutive amino acids which are connected together by peptide bonds.
  • polypeptide and protein as used herein, relate to large peptides, preferably peptides having more than 50 amino acids, but the terms “peptide”, “polypeptide” and “protein” may be used interchangeably herein.
  • polynucleotide refers to DNA or RNA molecules, preferably with more than 10 nucleotide residues.
  • the polynucleotide is a DNA polynucleotide.
  • the polynucleotide is a RNA polynucleotide.
  • DNA relates to a molecule, which comprises deoxyribonucleotide residues and preferably is entirely or substantially composed of deoxyribonucleotide residues.
  • Deoxyribonucleotide relates to a nucleotide, which lacks a hydroxyl group at the T -position of a b-D-ribofuranosyl group.
  • DNA comprises isolated DNA such as partially or completely purified DNA, essentially pure DNA, synthetic DNA, and recombinantly generated DNA and includes modified DNA, which differs from naturally occurring DNA by addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of a DNA or internally, for example at one or more nucleotides of the DNA.
  • Nucleotides in DNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides. These altered DNAs can be referred to as analogs or analogs of naturally occurring DNA.
  • RNA relates to a molecule, which comprises ribonucleotide residues and preferably is entirely or substantially composed of ribonucleotide residues.
  • “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2’-position of a b-D-ribofuranosyl group.
  • fragment refers to a continuous element.
  • a part of a structure such as an amino acid sequence or a nucleotide sequence, refers to a continuous element of said structure.
  • a fragment of an amino acid sequence preferably comprises a sequence of at least 10, in particular at least 15, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, or 500 consecutive amino acids of the amino acid sequence.
  • a fragment of a nucleotide sequence preferably comprises a sequence of at least 6, in particular at least 8, 12, 15, 20, 30, 50, 100, 150, 160, 170, 180, 190, or 200 consecutive nucleotides of the nucleotide sequence.
  • variants of an amino acid sequence or nucleotide sequence comprise amino acid/nucleotide insertion variants, amino acid/nucleotide addition variants, amino acid/nucleotide deletion variants and/or amino acid/nucleotide substitution variants.
  • Amino acid/nucleotide insertion variants comprise insertions of one or two or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids/nucleotides in a particular amino acid sequence or nucleotide sequence.
  • Amino acid/nucleotide addition variants comprise additions of one or two or more (e.g.
  • amino acids/nucleotides to a particular amino acid sequence or nucleotide sequence.
  • Amino acid/nucleotide deletion variants comprise deletions of one or two or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids/nucleotides from a particular amino acid sequence or nucleotide sequence.
  • Amino acid/nucleotide substitution variants are characterized by at least one residue (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) in the sequence being removed and another residue being inserted in its place.
  • structural protein refers to a protein required for production of viral particles, such as those encoded by the spike (S), envelope (E), membrane (M), and nucleocapsid (N) genes of a nidovirus.
  • RNA-dependent RNA polymerase also designated as RNA replicase
  • the term “RNA-dependent RNA polymerase” refers to an enzyme that catalyzes the replication of RNA from an RNA template.
  • the RNA-dependent RNA polymerase is an essential protein encoded in the genome of the RNA-containing viruses with no DNA stage, i.e. of the RNA viruses such as nidoviruses. It catalyzes synthesis of the RNA strand complementary to a given RNA template
  • DNA-dependent RNA polymerase refers to an enzyme that catalyses the transcription of RNA from a DNA template.
  • synthetic nucleic acid molecule refers to a molecule produced in vitro. It is constructed by joining nucleic acid sequences together. It may comprise chemically or by other means synthesized or amplified nucleotide sequences.
  • the synthetic nucleic acid molecule of the present invention may be produced from a DNA template that can be obtained by polymerase chain reaction (PCR) on another DNA template.
  • PCR polymerase chain reaction
  • One primer for the PCR amplification may be designed such, that it also contains the sequence for a bacteriophage promoter, for example the T7 promoter (see examples).
  • a PCR-derived template may be further used for in vitro transcription.
  • In vitro transcription may comprise the enzymatic polymerisation to yield a synthetic RNA molecule using a DNA template, an isolated DNA- dependent RNA polymerase (e.g. the bacteriophage T7 polymerase), and nucleotides in an in vitro reaction.
  • the nucleotides therein include natural and non-natural nucleotides that are modified by methylation (e.g. m6A, N 6 -methyladenosine).
  • the nucleotides may also include di- or tri-nucleotides such as m 7 G(5')ppp(5')G or m 7 G(5')ppp(5')Athat can serve as cap analoga.
  • the nucleotides may also include analoga such as those containing a thiophosphate modification that are capable of forming RNA molecules with increased resistance against RNAses.
  • the resulting synthetic RNA may be further enzymatically modified with, for example, vaccinia virus derived caping enzyme (for example, New England BioLabs (NEB), Cat. M2080S), 2 ⁇ methyltransferase (for example, NEB, M0366), and/or E. coli poly(A) polymerase (for example, NEB, Cat. M0276S).
  • vaccinia virus derived caping enzyme for example, New England BioLabs (NEB), Cat. M2080S
  • 2 ⁇ methyltransferase for example, NEB, M0366
  • E. coli poly(A) polymerase for example, NEB, Cat. M0276S.
  • the synthetic nucleic acid molecule of the present invention is incapable of autonomous replication. Reason for this is that it does not comprise a nucleic acid sequence encoding a (functionally active) RNA-dependent RNA polymerase (also designated as RNA replicase) of a nidovirus or a functionally active variant or fragment thereof. Tests to determine the functional activity of a RNA-dependent RNA polymerase or a variant or fragment thereof are known to the skilled person. For example, one could contact the cognate RNA template and the corresponding RNA-dependent polymerase in the presence of nucleotides labelled by radioactive atoms (e.g. 32 P) or fluorescent dye or functional group suitable for immunodetection.
  • radioactive atoms e.g. 32 P
  • fluorescent dye or functional group suitable for immunodetection e.g. 32 P
  • RNA template and polymerase can be done in vitro in a suitable reaction environment (buffer, temperature, other nucleotides known in the art) or by transfection into cultured cells.
  • the total RNA is purified after 60 minutes and visualized by gel electrophoresis.
  • Activity of the RNA-dependent polymerase is revealed if labeled nucleotides have been incorporated into the purified RNA molecules.
  • the tests aim to determine whether the RNA-dependent RNA polymerase ot a nidovirus or the variant or fragment thereof is able to perform its natural function. For example, the tests aim to determine whether the RNA-dependent RNA polymerase or the variant or fragment thereof is able to catalyze the replication of RNA from an RNA template.
  • the nucleic acid molecule of the present invention does not encode a RNA-dependent RNA polymerase of a nidovirus or a variant or fragment thereof which is able to catalyze the replication of RNA from an RNA template.
  • the synthetic nucleic acid molecule of the present invention is further incapable of providing all necessary structural proteins required for the formation of a nidovirus envelope.
  • Reason for this is that it does not contain a nucleotide sequence encoding at least one (functionally active) structural protein of a nidovirus or a functionally active variant or fragment thereof.
  • Tests to determine the functional activity of the spike (S), envelope (E), membrane (M), and/or nucleocapsid (N) protein of a nidovirus or a variant or fragment thereof are known to the skilled person.
  • a recombinant coronavirus genome could be constructed that contains the particular active or inactive structural protein or variant or fragment thereof that is to be tested.
  • efficacy for rescue of a replication competent virus with that particular recombinant genome is determined in a parallel experiment. If the tested gene for the structural protein or variant thereof is not active, then no more than 10 % of infectious activity compared to the parallel rescue of the wildtype can be obtained. Specifically, such tests aim to determine whether the proteins or protein variants or fragments thereof are able to perform their natural functions.
  • the tests aim to determine whether the S protein or the S protein variant or fragment thereof is able to recognize the cell receptor, the E protein or the E protein variant or fragment thereof is able to form ion channels, the M protein or the M protein variant or fragment thereof is able to bind the nucleocapsid, and/or the N protein or the N protein variant or fragment thereof is able to bind the nidovirus genome and ensure the maintenance of the nidovirus genome in a ‘beads-on-a-string’ conformation.
  • ORFlb refers to the open reading frame lb which is naturally comprised in the nidovirus genome. It encodes, together with ORFla, the non- structural proteins of the nidovirus. In particular, it encodes the main RNA-dependent RNA polymerase (nspl2), the helicase/triphosphatase (nspl3), two unusual ribonucleases (nspl4, nspl5) and RNA-cap methyltransferases (nspl4, nspl6). The nidovirus packaging signal is also localized in ORFlb.
  • packaging signal refers to a conserved cis-regulatory element found in nidoviruses. It has an important role in regulating the packaging of the viral genome into the capsid/envelop. As part of the viral life cycle, within the infected cell, the viral genome becomes associated with viral structural proteins and assembles into new infective progeny viruses. This process is called packaging and is important for viral replication.
  • nidoviruses The ORFla and ORFlb of nidoviruses are translated by “ribosomal frameshifting”.
  • programmed -1 ribosomal frameshifting -1 PRF
  • RDRP RNA-dependent RNA polymerase
  • proteases that are thought to prepare the infected cell for takeover by the virus.
  • the ribosomal frameshifting signal of nidoviruses comprises a “slippery site”.
  • the generally accepted mechanism of -1 PRF is as follows: (1) the mRNA secondary structure forces elongating ribosomes to pause, and the length of the linker is such that the ribosomal A- and P-site bound aminoacyl- (aa-) and peptidyl-tRNAs are positioned over the slippery site, (2) the sequence of the slippery site allows for re-pairing of the tRNAs to the -1 frame codons after they “simultaneously slip” by one base in the 5' direction along the mRNA, and (3) subsequent melting of the downstream mRNA secondary structure allows the ribosome to continue elongation of the nascent polypeptide in the new translational reading frame.
  • hepatitis delta virus (HDV) ribozyme refers to a non-coding nucleic acid molecule found in the hepatitis delta virus that is necessary for viral replication and is the only known human virus that utilizes ribozyme activity to infect its host.
  • the ribozyme acts to process the RNA transcripts to unit lengths in a self-cleavage reaction during replication of the hepatitis delta virus, which is thought to propagate by a double rolling circle mechanism.
  • the nucleic acid molecule comprises a nucleotide sequence of a hepatitis delta virus (HDV) ribozyme in order to obtain the authentic 3’ UTR.
  • nucleic acid molecule particle refers to a structure allowing or improving cell penetration of the nucleic acid molecule of the present invention which is comprised therein.
  • the nucleic acid molecule particle is formulated for cell transfection.
  • the particle comprising the nucleic acid molecule is free of any structural protein of a nidovirus.
  • the skilled person knows techniques to formulate a nucleic acid molecule for cell transfection. One of these techniques is the use of calcium phosphate. HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the nucleic acid molecule to be transfected.
  • HeBS HEPES-buffered saline solution
  • a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the nucleic acid molecule to be transfected on its surface.
  • the suspension of the precipitate is then added to the cells to be transfected.
  • the cells take up some of the precipitate, and with it, the nucleic acid molecule.
  • cationic polymers such as DEAE-dextran or polyethylenimine (PEI) may be used.
  • PEI polyethylenimine
  • the nucleic acid molecule may also be complexed with liposomes which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer.
  • the nucleic acid molecule may further be contacted with dendrimers. The dendrimers bind the nucleic acid molecule to form dendriplexes that then penetrate the cells.
  • Attenuated virus refers to a virus with compromised virulence in the intended recipient, e.g. subject as defined herein.
  • highly attenuated virus refers to a virus with blocked virulence in the intended recipient, e.g. subject as defined herein.
  • virus-like particle refers to a non-replicating viral vehicle.
  • VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface and/or envelope proteins. They contain functional viral proteins responsible for cell penetration by the virus, which ensures efficient cell entry.
  • the virus-like particle of the present invention comprises the nucleic acid molecule of the present invention which is incapable of autonomous replication. It further comprises an envelope of a nidovirus.
  • the envelope of a nidovirus comprises the structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N).
  • the present invention refers to nucleotide sequences according to SEQ ID NO: 1 to SEQ ID NO: 12 or variants thereof.
  • the term “variant of a nucleotide sequence according to SEQ ID NO: 1 to SEQ ID NO: 12”, as used herein, can be characterized by a certain degree of sequence identity to the (parent) nucleotide sequence from which it is derived. More precisely, a nucleotide sequence variant in the context of the present invention may exhibit at least 80% sequence identity to its (parent) nucleotide sequence from which it is derived.
  • the term “at least 80% identical to”, as used herein, refers to a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective (parent/reference) nucleotide sequence from which it is derived.
  • the nucleotide sequence in question and the (parent/reference) nucleotide sequence from which it is derived exhibit the indicated sequence identity over the entire length ot the (parent/reference) nucleotide sequence from which it is derived.
  • the similarity of nucleotide sequences can be determined via sequence alignments.
  • sequence alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson J. D. et al. Nucleic Acids Res. 1994, 22:4673-80) available e.g.
  • sequence identity may be calculated using e.g.
  • BLAST BLAT or BlastZ (or BlastX).
  • BLASTN Altschul et al. J. Mol. Biol. 1990, 215:403-410.
  • Gapped BLAST is utilized as described in Altschul et al. Nucleic Acids Res. 1997, 25:3389-3402.
  • Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154- 162) or Markov random fields.
  • the term “functional variant” as used in connection with the nucleotide sequences according to SEQ ID NO: 1 to SEQ ID NO: 12 means that it is functional in the sense that it has the same or essentially the same activity (in particular properly initiation of replication, transcription, packaging, if applicable) as the respective (parent/reference) nucleotide sequence from which it is derived.
  • the term “functional variant” in connection with the nucleotide sequence of the 5’ UTR of a nidovirus refers to a variant of said nucleotide sequence which is functional in the sense that it allows the replication and/or transcription of the nucleic acid molecule to the same or essentially the same degree as the respective (parent/reference) nucleotide sequence from which it is derived.
  • the variations are outside of the promoter regions comprised in the 5’ UTR allowing replication and/or transcription of the nucleic acid molecule.
  • nucleotide sequence of the 3’ UTR of a nidovirus refers to a variant of said nucleotide sequence which is functional in the sense that it allows the formation of antigenomic and/or subgenomic copies of the nucleic acid molecule and/or the circulation of the 3’ UTR with the 5’ UTR to the same or essentially the same degree as the respective (parent/reference) nucleotide sequences from which it is derived.
  • the variations are outside of the promoter regions comprised in the 3’ UTR for antigenomic and subgenomic copies and/or the circularization signal for the cognate 5' UTR comprised in the 3’ UTR.
  • the term “functional variant” in connection with the nucleotide sequence of a packaging signal localized in ORFlb of a nidovirus refers to a variant of said nucleotide sequence which is functional in the sense that it allows/results in packaging of the nucleic acid molecule to the same or essentially the same degree as the respective (parent/reference) nucleotide sequences from which it is derived.
  • the term “functional variant” in connection with the nucleotide sequence of a ribosomal slippage site at the junction of ORFla and lb of a nidovirus refers to a variant of said nucleotide sequence which is functional in the sense that it allows the expression of the N gene and/or the nucleation of the N protein to the same or essentially the same degree as the respective (parent/reference) nucleotide sequences from which it is derived.
  • the variations are outside of these regions.
  • the present invention further provides a pharmaceutical composition comprising the nucleic acid molecule of the present invention or the particle free of any structural protein of a nidovirus of the present invention.
  • the pharmaceutical composition in accordance with the present invention may further comprise one or more excipient(s), diluent(s), and/or carrier(s), all of which are preferably pharmaceutically acceptable.
  • pharmaceutically acceptable refers to the non-toxicity of a material, which, preferably, does not interact with the action of the active component of the pharmaceutical composition, i.e., the nucleic acid molecule of the present invention or the particle free of any structural protein of a nidovirus of the present invention.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • excipient is intended to indicate all substances in a pharmaceutical composition which are not active ingredients such as binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, or colorants.
  • diluting and/or thinning agent relates to a diluting and/or thinning agent.
  • the term “diluent” includes a solution, suspension (e.g. liquid or solid suspension) and/or media.
  • carrier as used herein, relates to one or more compatible solid or liquid fillers, which are suitable for an administration, e.g. to a human.
  • carrier relates to a natural or synthetic organic or inorganic component which is combined with an active component in order to facilitate the application of the active component.
  • carrier components are sterile liquids such as water or oils, including those which are derived from mineral oil, animals, or plants, such as peanut oil, soy bean oil, sesame oil, sunflower oil, etc. Salt solutions and aqueous dextrose and glycerin solutions may also be used as aqueous carrier compounds.
  • Pharmaceutically acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).
  • suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • suitable diluents include ethanol, glycerol, and water.
  • Pharmaceutical carriers, diluents, and/or excipients can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • compositions of the present invention may comprise as, or in addition to, the carrier(s), excipient(s) or diluent(s) any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilising agent(s).
  • suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, com sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose, and polyethylene glycol.
  • Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Preservatives, stabilizers, dyes, and even flavoring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • terapéuticaally effective amount means that the amount of the nucleic acid molecule or particle of the present invention contained in the pharmaceutical composition administered is of sufficient quantity to achieve the intended purpose, such as, in this case, to cause treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus.
  • the treatment of nidovirus infection or disease may be measured immunologically by determining the presence of at least one nidovirus-antigen-specific immune factor, preferably selected from the group consisting of B cells, follicular helper T cells (TFH cells), activated CD4+ T cells and CD8+ T cells (particularly also CD38+HLA-DR+ ), IgM antibodies, and IgG antibodies.
  • nidovirus-antigen-specific immune factor preferably selected from the group consisting of B cells, follicular helper T cells (TFH cells), activated CD4+ T cells and CD8+ T cells (particularly also CD38+HLA-DR+ ), IgM antibodies, and IgG antibodies.
  • a nidovirus-specific cytokine profile preferably selected from the group consisting of B cells, follicular helper T cells (TFH cells), activated CD4+ T cells and CD8+ T cells (particularly also CD38+HLA-DR+ ), IgM antibodies, and
  • vaccination describes the process of administering a nidovirus (e.g. attenuated nidovirus) to a subject with the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.
  • a nidovirus e.g. attenuated nidovirus
  • the present inventors surprisingly generated a new synthetic/artificial nucleic acid molecule containing some nucleotide sequences of a nidovirus and not containing others.
  • Said synthetic nucleic acid molecule acts as a replication competitor against a nidovirus. It can be given to a subject infected with a nidovirus (either with symptoms or without, but with active virus replication) or not infected with a nidovirus (preventative). When given to a subject infected with a nidovirus, it ends up together with the nidovirus in a cell of the subject.
  • the new synthetic/artificial nucleic acid molecule costs the nidovirus resources and, thus, interferes with the normal kinetic of the nidovirus infection or disease. Even if nidovirus replication is not reduced below limits of detection, any interference significantly can reduce the infectious burden for the subject so that transmission of nidovirus to others will be interrupted. Furthermore, a lower infectious burden allows the immune system to better cope with virus replication and delays or even suppresses symptoms caused of an over-reactive response such as cytokine storms.
  • the first aspect of the present invention relates to a synthetic/artificial nucleic acid molecule comprising, consisting essentially of, or consisting of a polynucleotide
  • the at least one (functionally active) structural protein of a nidovirus or functionally active variant or fragment thereof is selected from the group consisting of a spike (S) protein or a functionally active variant or fragment thereof, an envelope (E) protein or a functionally active variant or fragment thereof, a membrane (M) protein or a functionally active variant or fragment thereof, and a nucleocapsid (N) protein or a functionally active variant or fragment thereof.
  • the nucleotide sequence encoding the S, M, E, or N protein the nucleotide sequences of the S and M proteins, the S and E proteins, the S and N proteins, the S, M, and E proteins, the S, M, and N proteins, the S, E, and N proteins, the M and E proteins, the M and N proteins, the M, E, and N proteins or the S, M, E, and N proteins are not contained.
  • the polynucleotide does not contain a nucleotide sequence encoding a (functionally active) spike (S) protein of a nidovirus or a functionally active variant or fragment thereof, a (functionally active) enveloped (E) protein of a nidovirus or a functionally active variant or fragment thereof, and a (functionally active) membrane (M) protein of a nidovirus or a functionally active variant or fragment thereof.
  • the polynucleotide does not contain a nucleotide sequence encoding a (functionally active) spike (S) protein of a nidovirus or a functionally active variant or fragment thereof, a (functionally active) enveloped (E) protein of a nidovirus or a functionally active variant or fragment thereof, and a (functionally active) membrane (M) protein of a nidovirus or a functionally active variant or fragment thereof, and contains a nucleotide sequence encoding a (functionally active) nucleocapsid (N) protein of a nidovirus or a functionally active variant or fragment thereof.
  • the inventors of the present invention found that the presence of the N protein helps encapsidation of the synthetic RNA (or its amplified derivatives) within a cell. Such encapsidated RNAs are a better substrate for further amplification.
  • Nucleotide sequences encoding (functionally active) accessory proteins of a nidovirus or functionally active variants or fragments thereof are preferably also not present/contained.
  • the synthetic nucleic acid molecule comprises a polynucleotide (i) containing a nucleotide sequence of the 5’ untranslated region (UTR) of a nidovirus, a nucleotide sequence of the 3’ untranslated region (UTR) of a nidovirus, a nucleotide sequence of a packaging signal localized in ORFlb of a nidovirus, and a nucleotide sequence encoding a (functionally active) nucleocapsid (N) protein of a nidovirus or a functionally active variant or fragment thereof, and
  • nucleotide sequence encoding a (functionally active) spike (S) protein of a nidovirus or a functionally active variant or fragment thereof a nucleotide sequence encoding a (functionally active) enveloped (E) protein of a nidovirus or a functionally active variant or fragment thereof, a nucleotide sequence encoding a (functionally active) membrane (M) protein of a nidovirus or a functionally active variant or fragment thereof, and nucleotide sequences encoding (functionally active) accessory proteins of a nidovirus or functionally active variants or fragments thereof.
  • the polynucleotide only contains as protein/polypeptide encoding nucleotide sequence(s): a nucleotide sequence encoding a (functionally active) nucleocapsid (N) protein of a nidovirus or a functionally active variant or fragment thereof and optionally a nucleotide sequence encoding a molecule of interest (i.e. transgene).
  • the polynucleotide does not contain nucleotide sequences encoding (functionally active) non- structural proteins of a nidovirus or functionally active variants or fragments thereof and nucleotide sequences encoding (functionally active) structural proteins of a nidovirus or functionally active variants or fragments thereof.
  • only a transgene may be present as a sequence encoding a protein/polypeptide.
  • Nucleotide sequences encoding (functionally active) accessory proteins of a nidovirus or functionally active variants or fragments thereof are preferably also not present.
  • the synthetic nucleic acid molecule comprises a polynucleotide
  • nucleotide sequences encoding (functionally active) non- structural proteins of a nidovirus or functionally active variants or fragments thereof nucleotide sequences encoding (functionally active) structural proteins of a nidovirus or functionally active variants or fragments thereof, and nucleotide sequences encoding (functionally active) accessory proteins of a nidovirus or functionally active variants or fragments thereof.
  • transgene may be present as a sequence encoding a protein/polypeptide.
  • the synthetic nucleotide acid molecule of the present invention comprises, consists essentially of, or consist of between 1200 bp and 3500 bp, more preferably between 1251 bp and 2898 bp.
  • the synthetic nucleic acid molecule of the present invention comprises specific elements of a nidovirus, i.e. a nucleotide sequence of the 5’ untranslated region (UTR) of a nidovirus, a nucleotide sequence of the 3’ untranslated region (UTR) of a nidovirus, and a nucleotide sequence of a packaging signal localized in ORFlb of a nidovirus.
  • Other elements usually comprised in the nucleic sequence/genome of a nidovirus are missing/not present.
  • the 5’ UTR comprises promoter sequences for replication and transcription.
  • the 5’ UTR comprises a leader and a transcription regulating sequence (TRS) (as close to the leader also designated as TRS-L element).
  • TRS transcription regulating sequence
  • the 5’ UTR comprised in the polynucleotide has a nucleotide sequence according to SEQ ID NO: 6 or a nucleotide sequence having at least 80% sequence identity thereto.
  • the polynucleotide comprises, in addition to the nucleotide sequence of the 5’ UTR, a nucleotide sequence of the ORFla to also include a promoter element for amplification of a full-length RNA.
  • the polynucleotide comprises a nucleotide sequence of the 5’UTR which extends into ORFla.
  • this element has a nucleotide sequence according to SEQ ID NO: 1 or a nucleotide sequence having at least 80% sequence identity thereto.
  • the 3’UTR comprises promoter sequences for antigenomic and subgenomic copies and the circularization signal for the cognate 5' UTR.
  • the 3’ UTR has a nucleotide sequence according to SEQ ID NO: 7 or a nucleotide sequence having at least 80% sequence identify thereto.
  • the polynucleotide comprises a nucleotide sequence including the intergenic region upstream of the N gene, the N protein coding sequence and the 3’ UTR.
  • this element has a nucleotide sequence according to SEQ ID NO: 4 or a nucleotide sequence having at least 80% sequence identity thereto.
  • the polynucleotide comprises a nucleotide sequence including regulating stem loops but no sequences of the open reading frame of N.
  • this element has a nucleotide sequence according to SEQ ID NO: 8 or a nucleotide sequence having at least 80% sequence identity thereto.
  • the polynucleotide preferably further comprises a ribosomal slippage site at the junction of ORFla and ORFlb. More preferably, this element has a nucleotide sequence according to SEQ ID NO: 2 or a nucleotide sequence having at least 80% sequence identity thereto, or a nucleotide sequence according to SEQ ID NO: 10 or a nucleotide sequence having at least 80% sequence identity thereto.
  • the polynucleotide comprises a nucleotide sequence of a packaging signal localized in ORFlb of a nidovirus. It has an important role in regulating the packaging of the viral genome into the capsid/envelop.
  • this element has a nucleotide sequence according to SEQ ID NO: 3 or a nucleotide sequence having at least 80% sequence identity thereto.
  • the polynucleotide contains elements having nucleotide sequences according to SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 6 or nucleotide sequences having at least 80% sequence identity thereto.
  • the polynucleotide contains elements having nucleotide sequences according to SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 7 or nucleotide sequences having at least 80% sequence identity thereto.
  • the polynucleotide contains elements having nucleotide sequences according to SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 8 or nucleotide sequences having at least 80% sequence identity thereto.
  • the polynucleotide contains elements having nucleotide sequences according to SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4 or nucleotide sequences having at least 80% sequence identity thereto.
  • the polynucleotide contains elements having nucleotide sequences according to SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 7 or nucleotide sequences having at least 80% sequence identity thereto.
  • the polynucleotide contains elements having nucleotide sequences according to SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 8 or nucleotide sequences having at least 80% sequence identity thereto.
  • the above mentioned polynucleotides further contain the element having a nucleotide sequence according to SEQ ID NO: 2 or a nucleotide sequence having at least 80% sequence identity thereto, or a nucleotide sequence according to SEQ ID NO: 10 or a nucleotide sequence having at least 80% sequence identity thereto. In one even more preferred embodiment, the above mentioned polynucleotides further contain the element having a nucleotide sequence according to SEQ ID NO: 9 or a nucleotide sequence having at least 80% sequence identity thereto.
  • the nucleic acid molecule comprises a polynucleotide containing a nucleotide sequence according to SEQ ID NO: 5 or a nucleotide sequence having at least 80% sequence identity thereto.
  • the polynucleotide further comprises a transgene. It is particularly located between the 5’ UTR and 3’ UTR. It is preferred that the transgene encodes a therapeutic molecule, e.g. a molecule having reporter activity, anti-inflammatory activity, anti-coagulation activity, and/or anti-viral activity.
  • the therapeutic molecule may be a polypeptide or protein, e.g. a polypeptide or protein that interferes with inflammation or coagulation disorders associated with a nidovirus infection/disease.
  • the polynucleotide contains a transcription regulating sequence (TRS) and the transgene is located downstream of said sequence (as close to the coding sequence the TRS is also designated as TRS-B).
  • TRS transcription regulating sequence
  • the transgene is operably linked to this sequence.
  • the nucleic acid molecule is a DNA molecule.
  • the DNA molecule preferably further comprises a promoter of a DNA-dependent RNA polymerase.
  • the promoter is operably linked to the polynucleotide.
  • the DNA-dependent RNA polymerase is selected from the group consisting of a an eukaryotic polymerase II, a bacteriophage polymerase, and a poxviral polymerase.
  • Suitable promoters of a DNA-dependent RNA polymerase are known to the skilled person.
  • the promoters of human or mouse cytomegalovirus (hMCV or mCMV), or the bacteriophage T7 or Sp6 promoters may be used.
  • the nucleic acid molecule is a RNA molecule.
  • the RNA molecule is a single-stranded RNA molecule in sense (+) orientation (relative to the nidovirus genome).
  • the single-stranded RNA molecule in sense (+) orientation preferably contains a 5ppp5 mG cap structure at the 5’ end of the polynucleotide and/or a poly(A)tail at the 3’ end of the polynucleotide.
  • the polynucleotide preferably further comprises a sequence encoding a hepatitis delta virus (HDV) ribozyme downstream of the poly(A)tail.
  • the RNA molecule is a single-stranded RNA molecule in antisense (-) orientation (relative to the nidovirus genome).
  • a DNA molecule is used and administered to the subject.
  • the synthetic nucleic acid molecule may be a linear nucleic acid molecule, a circular nucleic acid molecule, comprised in/part of a plasmid, comprised in/part of a viral vector, or formulated or condensed into particles allowing or improving cell penetration of the nucleic acid molecule. These particles are preferably free of any structural protein of a nidovirus.
  • the nucleic acid molecule is a DNA molecule which is comprised in/part of a viral vector of the family o f Herpes viridae , Poxviridae, Parvoviridae, Adenoviridae, or Retroviridae .
  • the nucleic acid molecule is a RNA molecule which is comprised in/part of a viral vector of the family of Paramyxoviridae .
  • the synthetic nucleic acid molecule of the present invention comprises specific elements of a nidovirus, i.e. a nucleotide sequence of the 5’ untranslated region (UTR) of a nidovirus, a nucleotide sequence of the 3’ untranslated region (UTR) of a nidovirus, and a nucleotide sequence of a packaging signal localized in ORFlb of a nidovirus.
  • Other elements usually comprised in the nucleic sequence/genome of a nidovirus are missing/not present.
  • the nidovirus is a virus of the family of Coronaviridae, Arteriviridae, Roniviridae , or Mesoniviridae .
  • the virus of the family of Coronaviridae is selected from the group consisting of MERS-CoV, SARS-CoV, SARS-CoV-2, a coronavirus that gains entry by the ACE2 receptor, and an Infectious Bronchitis Virus (IBV).
  • the virus of the family of Arteriviridae is selected from the group consisting of a virus that causes porcine reproductive and respiratory syndrome (PRRS) and a virus that causes equine viral arteritits (EVA).
  • PRRS porcine reproductive and respiratory syndrome
  • EVA equine viral arteritits
  • the nucleic acid molecule of the present invention does not express a reading frame larger than 9 amino acids.
  • a packaging signal of a virus other than a nidovirus may be used.
  • the present invention relates to particles comprising, consisting essentially of, or consisting of a synthetic nucleic acid molecule according to the first aspect.
  • Said particles are preferably free of any structural protein of a nidovirus. Said particles allow or improve cell penetration of the nucleic acid molecule which is comprised therein.
  • the nucleic acid molecule is formulated for cell transfection.
  • the skilled person knows techniques to formulate a nucleic acid molecule for cell transfection. One of these techniques is the use of calcium phosphate. HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the nucleic acid molecule to be transfected.
  • HeBS HEPES-buffered saline solution
  • a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the nucleic acid molecule to be transfected on its surface.
  • the suspension of the precipitate is then added to the cells to be transfected.
  • the cells take up some of the precipitate, and with it, the nucleic acid molecule.
  • cationic polymers such as DEAE-dextran or polyethylenimine (PEI) may be used.
  • PEI polyethylenimine
  • the nucleic acid molecule may also be complexed with liposomes which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer.
  • the nucleic acid molecule may further be contacted with dendrimers.
  • the dendrimers bind the nucleic acid molecule to form dendriplexes that then penetrate the cells.
  • the particles comprise the nucleic acid molecule complexed with calcium phosphate, liposomes, DEAE-dextran, polyethylenimine (PEI), or dendrimers.
  • the nucleic acid molecule according to the first aspect can alternatively be delivered using electroporation, with gene guns or with aerosols.
  • the present invention relates to a (host) cell comprising a synthetic nucleic acid molecule according to the first aspect or particles according to the second aspect.
  • the (host) cell may be infected by a nidovirus.
  • the (host) cell may have a risk to be infected or may potentially be infected by a nidovirus.
  • the (host) cell may be part of a subject, e.g. a patient suffering from a nidovirus infection or disease.
  • the (host) cell may also be an isolated cell, i.e. isolated from a subject, e.g. a patient suffering from a nidovirus infection or disease.
  • the cell may be part of a cell culture, e.g. an adherent cell culture or a non-adherent cell culture.
  • the cell may also be part of a cell line, e.g. a suspension cell line.
  • the (host) cell is a vertebrate cell.
  • the vertebrate cell is a mammalian cell (e.g. a canine, a feliformia, a rodent such as a mouse, rat or hamster, an ovine, a caprine, a pig, a bat such as a megabat or microbat, or a human/non-human primate such as a monkey or a great ape cell), a fish cell (e.g. carp, tench, zander, or pike cell), an amphibian cell, a reptilian cell, or an avian cell (e.g. a chicken, quail, goose, or duck cell).
  • the mammalian cell is a mouse, a rat, a cat, a pig, a bat or a human/non-human primate cell. Human cells are most preferred.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of, or consisting of a synthetic nucleic acid molecule according to the first aspect or particles according to the second aspect and one or more pharmaceutical acceptable excipient(s), diluent(s), and/or carrier(s).
  • Said pharmaceutical composition may be administered locally or systemically. It is preferred that the pharmaceutical composition is formulated for local administration or systemic administration.
  • the local administration is by parenteral administration, e.g. by oral, mucosal, intranasal, intravenous, subcutaneous, intradermal, or intramuscular administration, and the systemic administration is by intraarterial administration.
  • the composition is administered orally, intranasal, intramuscular, or mucosal.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. Aerosols or sprays may also be used.
  • the quantity of active component in a unit dose preparation administered in the use of the present invention may be varied or adjusted from about 1 mg to about 1000 mg per m 2 , preferably about 5 mg to about 150 mg/m 2 according to the particular application and the potency of the active component.
  • the compounds employed in the medical use of the invention are administered at an initial dosage of about 0.05 mg/kg to about 20 mg/kg daily.
  • a daily dose range of about 0.05 mg/kg to about 2 mg/kg is preferred, with a daily dose range of about 0.05 mg/kg to about 1 mg/kg being most preferred.
  • the dosages may be varied depending upon the requirements of the subject, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
  • the present invention relates to a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect for use as a medicament.
  • the synthetic nucleic acid molecule according to the first aspect, the particles according to the second aspect, or the pharmaceutical composition according to the fourth aspect is/are introduced into a subject or administered to a subject.
  • the subject receiving the synthetic nucleic acid molecule according to the first aspect, the particles according to the second aspect, or the pharmaceutical composition according to the fourth aspect may be infected by a nidovirus or may suffer from a nidovirus disease.
  • the subject may have a risk to be infected by a nidovirus (e.g. clinical personnel).
  • the subject is a vertebrate. More preterably, the vertebrate is a mammal (e.g.
  • a canine a feliformia, a rodent such as a mouse, rat or hamster, an ovine, a caprine, a pig, a bat such as a megabat or microbat, or a human/non-human primate such as a monkey or a great ape), a fish (e.g. carp, tench, zander, or pike), an amphibian, a reptile, or an avian (e.g. a chicken, quail, goose, or duck).
  • the mammal is a mouse, a rat, a cat, a pig, a bat or a human/non- human primate. Humans are most preferred. If the subject suffers from a nidovirus disease, it may also be designated as patient.
  • This aspect can alternatively be reworded as follows: Use of a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect for the manufacture of a medicament.
  • the present invention relates to a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect for use in the treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus.
  • the synthetic nucleic acid molecule according to the first aspect, the particles according to the second aspect, or the pharmaceutical composition according to the fourth aspect is introduced into a subject or administered to a subject.
  • the subject receiving the synthetic nucleic acid molecule according to the first aspect, the particles according to the second aspect, or the pharmaceutical composition according to the fourth aspect may be infected by a nidovirus or may suffer from a nidovirus disease.
  • the subject may have a risk to be infected by a nidovirus (e.g. clinical personnel).
  • the subject is a vertebrate. More preferably, the vertebrate is a mammal (e.g.
  • a canine a feliformia, a rodent such as a mouse, rat or hamster, an ovine, a caprine, a pig, a bat such as a megabat or microbat, or a human/non-human primate such as a monkey or a great ape), a fish (e.g. carp, tench, zander, or pike), an amphibian, a reptile, or an avian (e.g. a chicken, quail, goose, or duck).
  • the mammal is a mouse, a rat, a cat, a pig, a bat or a human/non- human primate. Humans are most preferred. If the subject suffers from a nidovirus disease, it may also be designated as patient.
  • a nidovirus infection may or may not cause symptoms of a nidovirus disease in a subject.
  • a nidovirus infection and a nidovirus disease are distinguished herein by the presence of at least one nidovirus disease symptom. As long as the infection is not accompanied by at least one symptom of nidovirus disease, it (or the subject) is asymptomatic (includes presymptomatic).
  • a nidovirus disease requires the presence of a nidovirus infection and at least one symptom of nidovirus disease (also referred to herein as “symptomatic infection”).
  • Nidovirus symptoms include dry cough, fever (>37.8°C), runny and/or blocked nose, fatigue, breathing difficulty, pneumonia, organ (e.g. heart, lung, liver and/or kidney) failure, itchy throat, headache, joint pain, nausea, diarrhoea, shivering, lymphophenia, loss of smell and/or loss of taste.
  • the nidovirus disease is characterized by the presence of two or more, three or more, or four or more symptoms, preferably including one or two or more of dry cough, fever (>37.8°C), breathing difficulty, loss of smell and/or loss of taste.
  • This aspect can alternatively be reworded as follows: Use of a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect for the manufacture of a medicament for the treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus.
  • this aspect can alternatively be reworded as follows: A method for the treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus comprising the steps of: (i) providing a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect and (ii) administering the synthetic nucleic acid molecule according to the first aspect, the particles according to the second aspect, or the pharmaceutical composition according to the fourth aspect (in a therapeutically effective amount) to a subject in need thereof.
  • the novel synthetic/artificial nucleic acid molecule acts as replication competitor against a nidovirus and can be given (preferably oral or intranasal) to an subject infected with a nidovirus (either with symptoms or without, but with active virus replication) or not yet infected with a nidovirus (as a preventive measure).
  • nucleic acid molecule according to the first aspect, the particles according to the second aspect, or the pharmaceutical composition according to the fourth aspect can be given in any way that they end up together with nidovirus in a cell.
  • Nidoviruses infect a wide range of epithelial and endothelial cells. Thus, an active targeting is not necessary.
  • the dose can be very low as the nucleic acid molecule according to the first aspect will be amplified within the subject. There is also no need for re-dosing because the nucleic acid molecule according to the first aspect will spread as long as nidovirus is present in the cell.
  • the presence of the nucleic acid molecule according to the first aspect will cost the nidovirus resources and, thus, will interfere with the normal kinetic of the nidovirus infection.
  • the nidovirus acts here like a helper virus providing any missing non- structural and/or structural proteins and elements required for virus propagation. Even if nidovirus replication is not reduced below limits of detection, any interference will significantly reduce the infectious burden for the subject so that transmission of nidovirus to others (including medical personnel) will be interrupted.
  • a lower infectious burden allows the immune system to better cope with nidovirus replication and will delay or even suppress symptoms caused of an over-reactive response such as cytokine storms.
  • a lower number of actively replicating nidoviruses will also facilitate novel therapeutic options. For example, susceptibility to antiviral drugs increases in a depleted nidovirus population so that medications with lower specific activity may be used in combination therapies.
  • nucleic acid molecule according to the first aspect also expresses a therapeutic protein (from a transgene comprised therein) (e.g. factors that interfere with inflammation or coagulation disorders) then therapeutic efficacy of the artificial nucleic acid molecule according to the first aspect may be even higher.
  • a therapeutic protein from a transgene comprised therein
  • therapeutic efficacy of the artificial nucleic acid molecule according to the first aspect may be even higher.
  • nucleic acid molecule according to the first aspect as medication is that this construct can be given at any time to an infected subject. It is not a vaccine and will not interfere with immunity but will be active at the site of viral replication, including the mucosa that is notoriously difficult to reach with medical interventions. There is no risk of ADE or other immune-mediated enhancement of disease.
  • the nucleic acid molecule of the present invention reduces viral load in subjects independent of the immune status of the subjects and can be expected to be safe also for subjects in chemotherapy or co-morbidities.
  • the nucleic acid molecule according to the first aspect depends on the presence of the wildtype nidovirus in the cell. That means, if the nidovirus infection or disease is cleared then the nucleic acid molecule according to the first aspect cannot be renewed/ repli cated .
  • the nidovirus naturally comprised in the cell may also be designated as wild-type virus.
  • nucleic acid molecule according to the first aspect, the particles according to the second aspect or the pharmaceutical composition according to the fourth aspect can be used/administered alone or in combination with another therapeutic substance/drug (i.e. combination therapy) as a medicament.
  • another therapeutic substance/drug i.e. combination therapy
  • the present invention relates to a combination comprising, consisting essentially of, or consisting of
  • the other therapeutic substance/drug may be an immune stimulatory substance/drug (e.g. an adjuvant, a TLR agonist, a PRR, e.g. a cytosolic PRR such as RIG-I-like receptors including RIG-I, MDA5 and LGP2, or a mitogen, particularly a T cell mitogen or activator such as phytohemagglutinin (PHA), Concanavalin (Con) A, wheat germ agglutinin (WGA), pokeweed mitogen (PWM), a lectin or an anti-CD3 25 antibody or antigen-binding fragment thereof), or a B cell mitogen or activator such as PWM, lipopolysaccharide (LPS), an anti-CD40 antibody or antigen-binding fragment thereof, or an anti-(B cell antigen receptor) antibody, or antigen binding fragment thereof.
  • the immune stimulatory agent preferably promotes an antiviral cell state.
  • the other therapeutic substance/drug may further be an antiviral substance/drug (e.g. IFITM3), an antibiotic, an adjuvant, a glucocorticoid, an antihypertensive drug (e.g. an ACE inhibitor), a hypoglycaemic drug, an anti-shock drug, and a pain reliever, a sedative, an anti-fever drug, an antiinflammatory drug, a cough medication, and an immunosuppressant.
  • an antiviral substance/drug e.g. IFITM3
  • an antibiotic e.g. an antibiotic
  • an adjuvant e.g. an glucocorticoid
  • an antihypertensive drug e.g. an ACE inhibitor
  • a hypoglycaemic drug e.g. an anti-shock drug
  • a pain reliever e.g. an sedative
  • an anti-fever drug e.g. an antiinflammatory drug
  • an antiinflammatory drug e.g
  • the other therapeutic substance/drug is an immune stimulating substance/drug or an antiviral substance/drug.
  • TMPRSS2 inhibitors e.g. ammonium chloride, serine protease inhibitor camostat mesylate
  • CatB/L inhibitor e.g. E-64d
  • Favipiravir Pimodivir
  • Baloxavir 15 optionally together with Marboxil
  • IL-6 blockers e.g. sarilumab, tocilizumab
  • Sarilumab Zitivekumab
  • lopinavir optionally together with ritonavir
  • chloroquine hydroxychloroquine
  • Remdesivir a- Ketoamides
  • IFN-I Type I interferons
  • the components of the combination i.e. the nucleic acid molecule according to the first aspect, particles according to the second aspect or pharmaceutical composition according to the fourth aspect and the other therapeutic substance/drug, may be administered together or independent from each other (e.g. one after the other).
  • the combination is a composition. More preferably, the combination is a composition, e.g. aqueous solution, and the nucleic acid molecule according to the first aspect, the particles according to the second aspect or the pharmaceutical composition according to the fourth aspect and the other therapeutic substance/drug are administered together.
  • This aspect can alternatively be reworded as tollows: Use of a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect and another therapeutic substance/drug for the manufacture of a medicament.
  • nucleic acid molecule according to the first aspect, the particles according to the second aspect or the pharmaceutical composition according to the fourth aspect can be used/administered alone or in combination with another therapeutic substance/drug for the treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus.
  • the present invention relates to a combination comprising, consisting essentially of, or consisting of
  • the other therapeutic substance/drug may be an immune stimulatory substance/drug (e.g. an adjuvant, a TLR agonist, a PRR, e.g. a cytosolic PRR such as RIG-I-like receptors including RIG-I, MDA5 and LGP2, or a mitogen, particularly a T cell mitogen or activator such as phytohemagglutinin (PHA), Concanavalin (Con) A, wheat germ agglutinin (WGA), pokeweed mitogen (PWM), a lectin or an anti-CD3 25 antibody or antigen-binding fragment thereof), or a B cell mitogen or activator such as PWM, lipopolysaccharide (LPS), an anti-CD40 antibody or antigen-binding fragment thereof, or an anti-(B cell antigen receptor) antibody, or antigen-binding fragment thereof.
  • the immune stimulatory agent preferably promotes an antiviral cell state.
  • the other therapeutic substance/drug may be an antiviral substance/drug (e.g. IFITM3), an antibiotic, an adjuvant, a glucocorticoid, an antihypertensive drug (e.g. an ACE inhibitor), a hypoglycaemic drug, an anti-shock drug, and a pain reliever, a sedative, an anti-fever drug, an antiinflammatory drug, a cough medication, and an immunosuppressant.
  • an antiviral substance/drug e.g. IFITM3
  • an antibiotic e.g. IFITM3
  • an adjuvant e.g. an glucocorticoid
  • an antihypertensive drug e.g. an ACE inhibitor
  • a hypoglycaemic drug e.g. an anti-shock drug
  • a pain reliever e.g. an sedative
  • an anti-fever drug e.g. an antiinflammatory drug
  • an antiinflammatory drug e.
  • the other therapeutic substance/drug is an immune stimulating substance/drug or an antiviral substance/drug.
  • TMPRSS2 inhibitors e.g. ammonium chloride, serine protease inhibitor camostat mesylate
  • CatB/L inhibitor e.g. E-64d
  • Favipiravir Fimodivir
  • Baloxavir 15 optionally together with Marboxil
  • IL-6 blockers e.g. sarilumab, tocilizumab
  • Sarilumab Zitivekumab
  • lopinavir optionally together with ritonavir, chloroquine, hydroxychloroquine, Remdesivir, a- Ketoamides (e.g.
  • IFN-I Type I interferons
  • the components of the combination i.e. nucleic acid molecule according to the first aspect, particles according to the second aspect or a pharmaceutical composition according to the fourth aspect and the other therapeutic substance/drug, may be administered together or independent from each other (e.g. one after the other).
  • the combination is a composition. More preferably, the combination is a composition, e.g. aqueous solution, and the nucleic acid molecule according to the first aspect, the particles according to the second aspect or the pharmaceutical composition according to the fourth aspect and the other therapeutic substance/drug are administered together.
  • a composition e.g. aqueous solution
  • the nucleic acid molecule according to the first aspect, the particles according to the second aspect or the pharmaceutical composition according to the fourth aspect and the other therapeutic substance/drug are administered together.
  • This aspect can alternatively be reworded as follows: Use of a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect and another therapeutic substance/drug for the manufacture of a medicament for the treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus.
  • this aspect can alternatively be reworded as follows: A method for the treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus comprising the steps of: (i) providing a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect and another therapeutic substance/drug, and (ii) administering the synthetic nucleic acid molecule according to the first aspect, the particles according to the second aspect, or the pharmaceutical composition according to the fourth aspect and the other therapeutic substance/drug (in a therapeutically effective amount) to a subject in need thereof.
  • the present invention relates to a virus like particle comprising, consisting essentially of, or consisting of a synthetic nucleic acid molecule according to the first aspect and an envelope of a nidovirus.
  • the nucleic acid molecule according to the first aspect is incapable of autonomous replication. It further comprises an envelope of a nidovirus.
  • the envelope of a nidovirus comprises the structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N).
  • the nidovirus may be a naturally occurring nidovirus, an attenuated nidovirus, or a nidovirus pseudotyped.
  • the nidovirus is preferably a replication competent nidovirus. More preferably, the nidovirus is attenuated SARS-CoV-2 with deletions at the S1/S2 junction.
  • the present invention relates to a(n) (in vitro or in vivo ) method of producing a virus like particle comprising a synthetic nucleic acid molecule according to the first aspect and an envelope of a nidovirus comprising, consisting essentially of, or consisting of the steps of:
  • nucleic acid according to the first aspect in case that it is delivered as DNA is operably linked to a promoter allowing transcription.
  • the (host) cell is a vertebrate cell.
  • the vertebrate cell is a mammalian cell (e.g. a canine, a feliformia, a rodent such as a mouse, rat or hamster, an ovine, a caprine, a pig, a bat such as a megabat or microbat, or a human/non-human primate such as a monkey or a great ape cell), a fish cell (e.g. carp, tench, zander, or pike cell), an amphibian cell, a reptilian cell, or an avian cell (e.g. a chicken, quail, goose, or duck cell).
  • a mammalian cell e.g. a canine, a feliformia, a rodent such as a mouse, rat or hamster, an ovine, a caprine, a pig, a bat such as a megabat or microbat, or
  • the mammalian cell is a mouse, a rat, a cat, a pig, a bat or a human/non- human primate cell.
  • Human cells are most preferred.
  • the (host) cell may be part of a subject. It may also be an isolated cell, i.e. isolated from a subject. Alternatively, the cell may be part of a cell culture, e.g. an adherent cell culture or a non-adherent cell culture. The cell may also be part of a cell line, e.g. a suspension cell line.
  • the nidovirus may be a naturally occurring nidovirus, an attenuated nidovirus, or a nidovirus pseudotyped.
  • the nidovirus is preferably a replication competent nidovirus.
  • the nidovirus is attenuated SARS-CoV-2 with deletions at the S1/S2 junction and the cell line is Vero or recombinant Vero (with stabile integration of nucleic acid molecule according to the first aspect).
  • the nucleic acid molecule according to the first aspect may be transiently or continually expressed in the cell.
  • the cell may further be a cell line stably expressing the nucleic acid molecule according to the first aspect.
  • a cell suspension comprising both a virus like particle comprising a synthetic nucleic acid molecule according to the first aspect and an envelope of a nidovirus as well as a nidovirus.
  • the present invention relates to a combination comprising, consisting essentially of, or consisting of
  • a virus like particle comprising a synthetic nucleic acid molecule according to the first aspect and an envelope of a nidovirus
  • the nidovirus may be a naturally occurring nidovirus, an attenuated nidovirus, or a nidovirus pseudotyped.
  • the nidovirus is preferably a replication competent nidovirus. More preferably, the nidovirus is attenuated SARS-CoV-2 with deletions at the S1/S2 junction.
  • the combination may be a composition, e.g. an aqueous solution.
  • the combination according to the eleventh aspect may also be used in the treatment, prophylaxis, and/or prevention of an infection or disease caused by a nidovirus and for vaccination against nidovirus infection.
  • the present invention relates to a combination comprising, consisting essentially of, or consisting of
  • a nidovirus or a combination according to the eleventh aspect for use in the treatment, prophylaxis, and/or prevention of an infection or disease caused by a nidovirus and for vaccination against nidovirus infection.
  • the nidovirus may be a naturally occurring nidovirus, an attenuated nidovirus, or a nidovirus pseudotyped.
  • the nidovirus is preferably a replication competent nidovirus. More preferably, the nidovirus is attenuated SARS-CoV-2 with deletions at the S1/S2 junction.
  • the components of the combination i.e. the nucleic acid molecule according to the first aspect, particles according to the second aspect or pharmaceutical composition according to the fourth aspect and the nidovirus may be administered together or independent from each other (e.g. one after the other).
  • the components of the combination i.e. the virus like particle comprising a synthetic nucleic acid molecule according to the first aspect and an envelope of a nidovirus and the nidovirus (combination according to the eleventh aspect) may be administered together or independent from each other (e.g. one after the other).
  • the combination is a composition, e.g. an aqueous solution. More preferably, the combination is a composition and the nucleic acid molecule according to the first aspect, the particles according to the second aspect or the pharmaceutical composition according to the fourth aspect and the nidovirus are administered together. Alternatively, the combination is a composition and the virus like particle comprising a synthetic nucleic acid molecule according to the first aspect and an envelope of a nidovirus and the nidovirus are administered together.
  • This aspect can alternatively be reworded as follows: Use of a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect and a nidovirus or a combination according to the eleventh aspect for the manufacture of a medicament for the treatment, prophylaxis, and/or prevention of an infection or disease caused by a nidovirus and for vaccination against nidovirus infection.
  • this aspect can alternatively be reworded as follows: A method for the treatment, prophylaxis, and/or prevention of an infection or a disease caused by a nidovirus and for vaccination against nidovirus infection comprising the steps of: (i) providing a synthetic nucleic acid molecule according to the first aspect, particles according to the second aspect, or a pharmaceutical composition according to the fourth aspect and a nidovirus, or a combination according to the eleventh aspect, and (ii) administering the synthetic nucleic acid molecule according to the first aspect, the particles according to the second aspect, or the pharmaceutical composition according to the fourth aspect and the nidovirus, or the combination according to the eleventh aspect (in a therapeutically effective amount) to a subject in need thereof.
  • the polynucleotide of the nucleic acid molecule according to the first aspect does not encompass a nucleotide sequence of a packaging signal localized in ORFlb of a nidovirus.
  • the present invention relates to a synthetic/artificial nucleic acid molecule comprising a polynucleotide
  • nucleotide sequences encoding (functionally active) non- structural proteins of a nidovirus or functionally active variants or fragments thereof nucleotide sequences encoding (functionally active) structural proteins of a nidovirus or functionally active variants or fragments thereof
  • nucleotide sequences encoding (functionally active) accessory proteins of a nidovirus or functionally active variants or fragments thereof nucleotide sequences encoding (functionally active) accessory proteins of a nidovirus or functionally active variants or fragments thereof.
  • this nucleic acid molecule still acts/works as replication competitor against a nidovirus and can be given (preferably oral or intranasal) to an subject infected with a nidovirus (either with symptoms or without, but with active virus replication) or not yet infected with a nidovirus (as a preventive measure).
  • This nucleic acid molecule may alternatively be used in the second to twelfth aspect.
  • SEQ ID NO: 1 5 UTR extending into ORFl to also include a promoter element for amplification of a full-length RNA of SARS-CoV-2,
  • SEQ ID NO: 2 element including the ribosomal slippage site at the junction of ORFl a and lb of SARS-CoV-2,
  • SEQ ID NO: 5 combination of elements according to SEQ ID NO:l to SEQ ID NO: 4 of
  • SEQ ID NO: 6 5 UTR of SARS-CoV-2
  • SEQ ID NO: 7 3 UTR of SARS-CoV-2
  • SEQ ID NO: 8 3 UTR with stem loops but no sequences of the ORF of N of SARS- CoV-2
  • SEQ ID NO: 9 hepatitis delta virus (HDV) ribozyme
  • SEQ ID NO: 10 element including the ribosomal slippage site at the junction of ORF la and lb, short of SARS-CoV-2,
  • SEQ ID NO: 11 CMV-promoter, SEQ ID NO: 12 SV40 polyA signal, SEQ ID NO: 13 screen-Pf primer, SEQ ID NO: 14 screen- IBr primer, SEQ ID NO: 15 screen-Nr primer,
  • the novel and artificial sequence was constructed stepwise into a mosaic of 4 sequences. With this length of the sequences we speculated to include potentially necessary cis-acting viral elements. At the same time, the fusion of the mosaic fragments provides an uninterrupted coding sequence to avoid degradation by cellular mechanisms that have evolved to remove non-coding RNAs that, by chemical appearance such as 5' cap and polyadenylation, should perform as mRNA.
  • the individual mosaic sequences are as follows:
  • Mosaic-1 (SEQ ID NO: 1) includes the 5' UTR with proposed promoter sequences for replication and the TRS-L element. The sequence extends into ORF la to also include a promoter element for amplification of a full-length RNA.
  • Mosaic-2 (SEQ ID NO: 2) includes the ribosomal slippage site at the junction of ORF la and lb. This site was included to obtain a pause sequence that may be helpful to express the N gene further downstream. This site may also be helpful for N protein nucleation.
  • Mosaic-3 (SEQ ID NO: 3) contains a 3' terminal fragment of ORF lb containing the packaging signal. This element contains at least 11 stem loops (Gruber, 2008, PMID 18424795).
  • Mosaic-4 (SEQ ID NO: 4) represents the 3' terminal region of the genomic RNA of SARC-CoV-2 starting with the stop codon of ORF 8. This sequence, thus, includes the intergenic region upstream of the N gene, the N protein coding sequence and the 3' UTR.
  • Mosaic- 1 can be shortened to contain only the 5' UTR and not contain any ORF la sequence.
  • Mosaic-2 can be omitted or mutagemzed in the stem-loop that programs the ribosomal frameshift.
  • Mosaic-2 can be further designed to include an additional packaging signal.
  • Mosaic-3 can be omitted to avoid packaging, or can contain the packaging signal of a virus other than isolate Wuhan-Hu-1.
  • Mosaic-4 can be deleted for the N gene, either including the intergenic sequence or only the coding sequence of N.
  • Mosaic-4 can also be designed such that no antigenomic copy can be generated.
  • Mosaic- 1 through -4 were obtained from a synthetic DNA. Expression of this synthetic DNA can be achieved with a polymerase II expression system.
  • the CMV enhancer and promoter (SEQ ID NO: 11) was attached to mosaic- 1 precisely at the +1 mRNA start position. Polymerase-II-driven transcription is terminated with a polyadenylation signal.
  • the well- known signal of the SV40 virus (SEQ ID NO: 12) was used.
  • the hepatitis delta virus (HDV) ribozyme was inserted precisely after a hard coded short polyadenosyl-tract.
  • T7 polymerase promoter was shortened to allow a more authentic transcription of the coronaviral 5' UTR, and the above described HDV ribozyme was also maintained to obtain the expected polyadenylated 3' UTR.
  • the shortened T7 promoter used has a nucleotide sequence according to SEQ ID NO: 16.
  • the T7 terminator used has a nucleotide sequence according to SEQ ID NO: 17.
  • RNA molecule was generated by a T7 RNA polymerase provided by a virus, preferably by a vaccinia virus, as this would ensure capping while the RNA is being produced in a cell.
  • the schema of the T7 expression construct thus, is: T7
  • In vitro transcripts were obtained in a reaction of 1 pg of expression plasmid with 2 pL of Hi- T7 RNA polymerase (New England Biolabs #M0658, at 50 U/pL) in the presence of 0.5 mM each of ATP, CTP, and UTP, 0.5 mM of a 1:4 mix of GTP and anti-reverse cap analog (ARCA, 3'-0-Me-m7G(5') ppp(5')G, NEB #S1411), 0.5 pL (1 U) RNase inhibitor, and 2 pL 10X Hi-T7 RNA Polymerase Reaction Buffer in a total volume of 20 pL. Transcription was allowed at 50 °C for 1 hour.
  • the transcription reaction was purified with the ReliaPrepTM RNA Clean-Up and Concentration System (Promega Corporation, Cat. Z1071) according to the manufacturer's instructions. The reaction was eluted into 2 x 15 pL of TE buffer. The concentration of the RNA was determined with a spectrophotometer (NanoDrop; ThermoFisher Scientific)
  • the Lipofectamine MessengerMAX kit ThermoFisher Scientific, Cat. LMRNA003
  • Opti-MEM medium ThermoFisher, Cat. 31985062
  • Nucleic acid molecules were further amplified by transfection of RNA coding for the mimic into cells infected with SARS-CoV-2. Presence of mimic RNA in cells productively infected with SARS-CoV-2 releases a mixed population of mimic RNA that is packaged into authentic SARS-CoV-2 virions, and the authentic SARS-CoV-2 infectious units.
  • the primers used for RT-PCR for discrimination of the two entities were screen-Pf (aggctatgccttcgaaca, SEQ ID NO: 13) with screen-lBr (ccgtaggcaaccactgtc, SEQ ID NO: 14) for detection of wildtype virus, and screen-Pf with screen-Nr (tcaaggctccctcagttg, SEQ ID NO: 15) for detection of the nucleic acid molecule.
  • the expected amplicons are 629 bp for screen- Pf + screen- IBr on the viral genome and 499 bp for screen-Pf + screen-Nr on the mimic RNA.
  • the amplicon with primers screen-Pf + screen-Nr on viral genome is too large (8388 bp) to interfere with the analysis.
  • Changes in the ratio of mimic to wildtype virus also during production in cell culture is indicative of the desired interference of the artificial construct with replication of the pathogen.

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Abstract

La présente invention concerne le traitement de maladies provoquées par un nidovirus par administration d'une molécule d'acide nucléique synthétique. Ce traitement consiste à aider le système immunitaire à combattre le nidovirus et, par conséquent, à prévenir et à atténuer les symptômes de maladies provoquées par un nidovirus.
PCT/EP2021/066890 2020-06-23 2021-06-21 Molécule d'acide nucléique pour le traitement de maladies provoquées par un nidovirus WO2021259883A1 (fr)

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WO2002095040A1 (fr) * 2001-05-21 2002-11-28 Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. Deletions des replicons de l'arterivirus

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Publication number Priority date Publication date Assignee Title
WO2002095040A1 (fr) * 2001-05-21 2002-11-28 Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. Deletions des replicons de l'arterivirus

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