WO2012175735A1 - Inhibiteurs d'a20 pour le traitement d'infections virales respiratoires - Google Patents

Inhibiteurs d'a20 pour le traitement d'infections virales respiratoires Download PDF

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WO2012175735A1
WO2012175735A1 PCT/EP2012/062227 EP2012062227W WO2012175735A1 WO 2012175735 A1 WO2012175735 A1 WO 2012175735A1 EP 2012062227 W EP2012062227 W EP 2012062227W WO 2012175735 A1 WO2012175735 A1 WO 2012175735A1
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pneumonia
viral
mice
cells
virus
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PCT/EP2012/062227
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Rudi Beyaert
Jonathan MAELFAIT
Geert VAN LOO
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Vib Vzw
Universiteit Gent
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/19—Omega peptidases (3.4.19)
    • C12Y304/19012—Ubiquitinyl hydrolase 1 (3.4.19.12)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to the field of medicine, more particularly to the field of viral pneumonia.
  • the invention provides inhibitors of A20 and their use for the treatment of viral infections, more particularly viral lung infections such as for example influenza virus.
  • Pneumonia is a common illness that continues to be the major killer of young children in developing countries and elderly people in developed countries. Many microorganisms are associated with pneumonia, and recently attention is turning to the importance of viruses as pathogens. About 200 million cases of viral community-acquired pneumonia occur every year— 100 million in children and 100 million in adults. Molecular diagnostic tests have greatly increased our understanding of the role of viruses in pneumonia, and findings indicate that the incidence of viral pneumonia has been underestimated. In children, respiratory syncytial virus, rhinovirus, human metapneumovirus, human bocavirus, and parainfluenza viruses are the agents identified most frequently in both developed and developing countries. Dual viral infections are common, and a third of children have evidence of viral-bacterial co-infection.
  • viruses are the putative causative agents in a third of cases of community-acquired pneumonia, in particular influenza viruses, rhinoviruses, and coronaviruses.
  • influenza viruses Apart from neuraminidase inhibitors for pneumonia caused by influenza viruses, there is no clear role for use of specific antivirals to treat viral community-acquired pneumonia.
  • Influenza vaccines are the only available specific preventive measures. In the USA alone, the economic burden of community-acquired pneumonia has been estimated to be more than US$17 billion annually.
  • Viruses are a class of highly diverse pathogens which depend on the host cell for their replication.
  • PRR pattern recognition receptors
  • TLRs Endosomal Toll like receptors
  • RLR cytoplasmic RNA helicase RIG-l-like receptors
  • NLRs Nod-like receptors
  • TLR7, TLR8, Nod2 viral single stranded RNA
  • TLR3, RIG-I, MDA5 viral single stranded RNA
  • Intracellular DNA sensors that mediate antiviral immune responses to DNA viruses include TLR9, DAI 4 and the PYHIN domain containing proteins AIM2 5"7 and IFI 16 8 .
  • TLR mediated antiviral responses are restricted to specialized type-l IFN producing plasmacytoid dendritic cells (DC), while most other cell types, including conventional DC, macrophages and fibroblasts, depend on the cytosolic RNA and DNA sensors for the production of antiviral proteins 9 .
  • DC plasmacytoid dendritic cells
  • Influenza A virus is the etiological agent of a contagious acute respiratory disease that causes considerable mortality, which is generally believed to be due to an excessive host inflammatory response. Emergence of drug-resistant strains of influenza viruses with pandemic potential underscores the importance of developing novel antiviral strategies. In this context, understanding of the mechanisms that regulate lAV-induced immune responses is critical. IAV infection leads to the exposure in the host cell of single-stranded genomic RNA and double stranded RNA, the latter being an intermediate of viral replication. Both TLR3 and RIG-I have been implicated as sensors of IAV infection 10"12 .
  • RIG-I contains a C-terminal DExD/H box helicase domain, which is required for ligand recognition, and two N-terminal CARD domains.
  • the CARD domains of RIG-I associate with the CARD domain of the MAVS adaptor protein, which subsequently translocates to and inserts in the outer mitochondrial membrane via its C-terminal transmembrane domain 17"20 .
  • Signaling downstream of MAVS requires the action of various ubiquitin modifying enzymes, which both positively and negatively regulate RLR mediated signal transduction.
  • K63-specific ubiquitin ligases such as TRIM25 21 and Riplet 22"24 , have been shown to directly promote RIG-I activation.
  • well characterized ubiquitin ligases such as TRAF3 25, 26 and TRAF6 27 mediate respectively NF-kappaB and IRF3 activation upon RIG-I stimulation.
  • deubiquitinating enzymes such as DUBA 28 , CYLD 29 ' 30 and OTUB1/2 31 have been shown to negatively regulate RLR signaling by specifically removing K63-linked polyubiquitin chains from several signaling molecules.
  • various K48-specific ubiquitin ligases such as AIP4 32 and TRIAD3A 33 mark respectively MAVS and TRAF3 for proteasome mediated degradation, thus inhibiting further downstream signaling.
  • the attachment of K48-specific polyubiquitin chains to the IRF3 and IRF7 transcription factors by E3s such as RAUL 34 , TRIM21 35 and RBCK1 36 further dampens antiviral signal transduction.
  • A20 is an ubiquitin-editing enzyme belonging to the OTU-domain family of DUBs. Interestingly, A20 also harbors atypical zinc finger dependent K48-specific ubiquitin ligase activity. Both DUB and E3 activities of A20 were previously shown to be involved in the negative regulation of proinflammatory signaling in response to TNF receptor I 37, 38 , TLR4 39, 40 , IL-1 R 41 and NOD2 42 . The anti-inflammatory role of A20 is clearly demonstrated by the fact that A20 deficient mice die early after birth due to severe multi-organ inflammation and cachexia 37 .
  • A20 myel"KO mice myeloid cell specific A20 knockout mice (A20 myel"KO ) that were recently generated in our lab and primary cells derived of these mice, we here provide evidence that A20 is a crucial negative regulator of lAV-induced pro-inflammatory and antiviral signaling in macrophages.
  • A20 myel"KO mice show enhanced survival and reduced morbidity in response to IAV lung infection compared to wild type mice.
  • mice are resistant to influenza A infection, and demonstrate that this protection does not result from an improved viral clearance or hyperresponsive innate immune response, but results from an accumulation of CD1 1 b- resident macrophages involved in the suppression of cytotoxic CD8+ T cell (CTL) responses during later stages of infection.
  • CTL cytotoxic CD8+ T cell
  • bone marrow derived macrophages deficient in A20 also restrict vesicular stomatitis virus (VSV) and herpes simplex virus-1 (HSV-1 ) replication.
  • VSV vesicular stomatitis virus
  • HSV-1 herpes simplex virus-1
  • Figure 1 A20 inhibits NF-kappaB and IRF3 activation in response to RIG-I stimulation.
  • HEK293T cells were transfected with NF-kappaB (left), ISRE (middle) or IFNalpha4 reporter (right) plasmids, together with plasmids expressing RIG-I (2CARD), IRF7 (right) and increasing amounts of A20. Numbers are averages +/- SD of 3 samples per set-up.
  • A20 deficient (A20 myel”KO ) and wild-type control (A20 myel"WT ) BMDM were transfected with LMW poly(l:C) to stimulate RIG-I and analysed at different time-points (minutes) after the start of transfection by immunoblotting of total cell extracts with the indicated antibodies.
  • C Similar to (B), showing either cytoplasmic or nuclear lysates.
  • D Bioassay (IL-6) and ELISA (TNF and IFNbeta) performed on cell culture supernatant of A20 myel"WT and A20 myel"KO BMDM stimulated with LMW poly(l:C) for the indicated time-points (min). Data are representative of 2 independent experiments.
  • FIG. 2 A20 negatively regulates lAV-induced gene expression in BMDM.
  • A20 myel"WT and A20 mye i -Ko BMDM were j n f ec ted with IAV X-47 (moi 1 ). At different hours post infection (hours p.i.) cells were lyzed and IL-6, IFNbeta and A20 mRNA expression was analyzed by qPCR.
  • B Similar to (A), 18 hours post infection cell culture supernatant was analyzed for TNF and IFNbeta protein levels.
  • C Alveolar macrophages were mock treated or infected with IAV X-47 (moi 1 ) for 18 hours. TNF, IFNbeta and IL-6 protein levels were determined in cell culture supernatant. Error bars represent mean values (+/- SD) of 3 samples. Results are representative for 2 independent experiments.
  • FIG. 3 A20 deficiency in myeloid cells protects against IAV lung infection.
  • a - E A20 myel"WT and A20 myel"KO mice were infected intranasally with a sublethal dose of X-47 and weight loss (A) was monitored.
  • B At day 4 and 10 post infection (p.i.) viral titers in the lung were measured and expressed as mean TCID 50 .
  • BAL was isolated from X-47 infected mice at 4 and 10 days p.i. and KC, MCP-1 , IFNalpha and IL-6 protein levels were analyzed.
  • FIG. 4 A20 deficiency in myeloid cells protects against lethal IAV infection.
  • FIG. 5 A20 AEC"KO mice are protected from influenza A infection.
  • D Pulmonary viral titers measured by TCID 50 following sublethal X-47 challenge.
  • Figure 7 A20 deficiency in BMDM reduces proinflammatory cytokine and type-l IFN expression in response to VSV.
  • A20 myel"WT and A20 myel"KO BMDM were infected with VSV at a moi 1. At different hours post infection (hours p.i.), TNF, IL-6, A20, IFNbeta and IP10 mRNA expression was analyzed by qPCR.
  • B A20 myel"WT and A20 myel"KO BMDM were infected with VSV at a moi 1 . At different hours p.i. culture supernatant was analyzed for IFNbeta protein levels. Values represent the mean (+/- SD) of 3 samples. Results are representative for 2 independent experiments.
  • FIG. 8 A20 deficiency in BMDM increases HSV-1 -induced proinflammatory cytokine and IFNbeta expression.
  • A20 myel"WT and A20 myel"KO BMDM were infected with HSV-1 at a moi 1 .
  • At different hours post infection (hours p.i.), IL-6, IFNbeta and A20 mRNA expression was analyzed by qPCR.
  • B A20 myel"WT and A20 myel"KO BMDM were infected with HSV-1 at a moi 1.
  • culture supernatant was analyzed for TNF and IFNbeta protein levels. Values represent the mean (+/- SD) of 3 samples. Results are representative for 2 independent experiments.
  • A20 is known in the art as an essential negative regulator of NF-kappaB signaling, and A20 deficient mice die prematurely due to massive multi-organ inflammation triggered by infiltrating intestinal bacteria.
  • A20 conditional knockout mice we have generated mice which specifically lack A20 in myeloid cells and in the respiratory epithelium, in order to assess its role in the protection against viral-induced antiviral immunity.
  • airway epithelial cell-specific A20 deficiency and myeloid cell-specific A20 deficiency protects mice from influenza A-induced morbidity and lethality.
  • the present invention shows that inhibitors of A20 can be used for the treatment of viral lung infections.
  • the present invention provides for an inhibitor of A20 wherein said inhibitor is a molecule selected from the list comprising of a ribozyme with a specificity for the A20 gene, a siRNA with a specificity for the A20 gene, an artificial microRNA with a specificity for the A20 gene, an antisense nucleic acid construct with a specificity for the A20 gene, a peptide with a specificity for the A20 protein or an antibody with a specificity for the A20 protein for use in the treatment of pneumonia.
  • said inhibitor is a molecule selected from the list comprising of a ribozyme with a specificity for the A20 gene, a siRNA with a specificity for the A20 gene, an artificial microRNA with a specificity for the A20 gene, an antisense nucleic acid construct with a specificity for the A20 gene, a peptide with a specificity for the A20 protein or an antibody with a specificity for the A20 protein for use in the treatment of pneumonia.
  • said pneumonia is viral pneumonia.
  • Viral pneumonia is a pneumonia wherein the infection is caused by a virus. Viral pneumonia is equivalent to the term 'respiratory viral infections'.
  • Viruses which are linked to pneumonia in humans and animals comprise respiratory syncytial virus, rhinovirus, influenza A, B and C viruses, human metapneumovirus, parainfluenza viruses types 1 , 2, 3 and 4, human bocavirus, vesicular stomatitis virus, coronavirus types 229E, OC43, NL63, HKU1 and SARS, adenovirus, enteroviruses, varicella-zoster virus, hantavirus, parechoviruses, Epstein-Barr virus, Human herpesvirus 6 and 7, Herpex Simplex virus, mimivirus, cytomegalovirus and measles.
  • said pneumonia is bacterial pneumonia.
  • Bacterial pneumonia is a pneumonia wherein the infection is caused by a bacterium. Such a pneumonia is typically designated as a 'respiratory bacterial infection'.
  • Infectious bacteria include, but are not limited to, gram negative and gram positive bacteria.
  • Gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species.
  • Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species.
  • infectious bacteria include but are not limited to: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M.
  • avium M. intracellular, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria nmeningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clo
  • the A20 protein (or the human tumor necrosis factor alpha inducible protein A20) is also designated in the general literature as TNFAIP3, OTU domain-containing protein 7C; TNFA1 P2; MGC104522; MGC138687 and MGC138688
  • SEQ ID NO: 1 The amino acid sequence of human A20 is depicted in SEQ ID NO: 2.
  • Polypeptide, peptide, or peptide mimetic A20 inhibitors include fragments or amino acid sequence variants of native polypeptide or peptide components of A20 which when bind to
  • A20 inhibit the function (or the activity) of A20.
  • A20 The activity of A20 is known in the art as a sequential de-ubiquitination and ubiquitination of the TNF Receptor-interacting protein (RIP) thereby targeting RIP to proteasomal degradation.
  • RIP TNF Receptor-interacting protein
  • the function of A20 can be conveniently measured by monitoring the inhibition of the NF- kappaB signalling while the inhibition of A20 upregulates the NF-kappaB signalling. Methods for measuring the NF-kappaB signalling are described in the art.
  • antibody is used in the broadest sense and specifically covers, for example, polyclonal antibodies, monoclonal antibodies (including antagonist and neutralizing antibodies), antibody compositions with polyepitopic specificity, single chain antibodies, camelid antibodies (nanobodies®) and fragments of antibodies, provided that they exhibit the desired biological or immunological activity.
  • an “isolated antibody” is one which has been identified and separated and/ or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.
  • An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures.
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • Monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries. High affinity (nM range) human antibodies can be generated by chain shuffling, as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries.
  • the DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides.
  • the monoclonal antibodies used herein include "chimeric" antibodies in which a portion of the heavy and/ or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • the anti-A20 antibodies of the invention may comprise humanized antibodies or human antibodies.
  • a "humanized antibody” is an antibody that has been modified using recombinant DNA techniques to circumvent the problem of a human's immune system reacting to an antibody as a foreign antigen.
  • the standard procedure of producing monoclonal antibodies produces mouse antibodies.
  • murine antibodies are very similar to human ones, there are differences. Consequently, the human immune system recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects.
  • "Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain a reduced percentage of sequence derived from the non-human antibody.
  • Various forms of humanized anti-A20 are contemplated.
  • Humanized antibodies may be intact antibodies, such as intact lgG1 antibodies, antibody chains or fragments thereof (such as Fv, Fab, Fab, F(ab) 2/ or other antigen-binding subsequences of antibodies).
  • Humanized antibodies include human antibodies (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human antibody are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human antibody and all or substantially all of the FR regions are those of a human antibody consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an antibody constant region (Fc), typically that of a human antibody.
  • Fc antibody constant region
  • human antibodies can be generated.
  • transgenic animals e.g mice
  • phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
  • V immunoglobulin variable
  • Antibody fragments comprise a portion of an antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2 f and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • the inhibitor of A20 is a peptide or peptide mimetic.
  • peptide mimetic and “peptidomimetic” are used interchangeably.
  • a peptide inhibitor is a peptide that binds specifically to A20 and neutralizes the function of A20.
  • Peptide inhibitors may be chemically synthesized using known peptide synthesis methodology or may be prepared and purified using recombinant technology.
  • the preferred length of peptide inhibitors of A20 is from about 10 amino acid residues to about 50 amino acid residues. It is contemplated that longer peptides may prove useful.
  • Peptide inhibitors may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening peptide libraries for peptides that are capable of specifically binding to a polypeptide target are well known in the art.
  • one preferred variation involves peptides that have been modified to comprise an intramolecular bond between two non-adjacent amino acid residues of the primary sequence, thereby forming a cyclic peptide.
  • the peptide comprises a pair of cysteine residues, such as amino-and carboxy-terminal cysteines, and the intramolecular bond comprises a disulfide bond between the cysteines.
  • organic chemists and peptide chemists are capable of synthesizing intramolecular bonds between a wide variety of amino acids using conventional techniques.
  • protein interference as described in the patent application WO2007071789 can be used to downregulate the A20 protein.
  • the latter technology is a knockdown technology which in contrast to RNAi acts at the post-translational level (i.e. it works directly on the protein level by inducing a specific protein aggregation of a chosen target).
  • Protein aggregation is essentially a misfolding event which occurs through the formation of intermolecular beta-sheets resulting in a functional knockout of a selected target.
  • a dedicated algorithm it is possible to accurately predict which amino acidic stretches in a chosen target protein sequence have the highest self-associating tendency (Fernandez- Escamilla A. M.
  • the A20-protein can be specifically targeted by inducing its irreversible aggregation and thus its functional knockout.
  • the inhibitor of the A20 gene is an antisense molecule that reduces transcription and/ or translation of A20, thereby reducing A20 activity.
  • the antisense molecule comprises RNA or DNA prepared using antisense technology, where, for example, an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to reduce or block expression of A20 and thus the A20 activity.
  • oligonucleotides can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of A20.
  • Inhibitors of A20 activity include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences.
  • a fragment generally comprises about 10 to 40 nucleotides in length, preferably at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
  • Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar- phosphodiester backbones that are resistant to endogenous nucleases, or are covalently linked to other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, or intercalating agents to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
  • small Interfering RNA siRNA with a specificity for A20 can be used in the context of the present invention.
  • siRNA refers to a small interfering RNA(s), which also has been referred to in the art as short interfering RNA and silencing RNA, among others.
  • siRNAs generally are described as relatively short, often 20-25 nucleotide-long, double-stranded RNA molecules that are involved in RNA interference (RNAi) pathway(s).
  • RNAi RNA interference
  • siRNAs are, in part, complementary to specific mRNAs (such as the A20 mRNA) and mediate their down regulation (hence, "interfering"). siRNAs thus can be used for down regulating the expression of specific genes and gene function in cells and organisms.
  • siRNAs also play a role in related pathways. The general structure of most naturally occurring siRNAs is well established.
  • siRNAs are short double-stranded RNAs, usually 21 nucleotides long, with two nucleotides single stranded "overhangs" on the 3 of each strand. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group.
  • the structure results from processing by the enzyme "dicer,” which enzymatically converts relatively long dsRNAs and relatively small hairpin RNAs into siRNAs.
  • the term siNA refers to a nucleic acid that acts like a siRNA, as described herein, but may be other than an RNA, such as a DNA, a hybrid RNA:DNA or the like. siNAs function like siRNAs to down regulate expression of gene products.
  • RNA interference which also has been designated as "RNA mediated interference” and refers to the cellular processes by which RNA (such as siRNAs) down regulate expression of genes; i.e., down regulate or extinguish the expression of gene functions, such as the synthesis of a protein encoded by a gene.
  • RNA interference pathways are conserved in most eukaryotic organisms. It is initiated by the enzyme dicer, which cleaves RNA, particularly double-stranded RNA, into short double-stranded fragments 20-25 base pairs long.
  • RNA-induced silencing complex RISC
  • the thus incorporated guide strand serves as a recognition sequence for binding of the RISC to nucleic acids with complementary sequences. Binding by RISC to complementary nucleic acids results in their being "silenced.”
  • the best studied silencing is the binding of RISCs to RNAs resulting in post-transcriptional gene silencing. Regardless of mechanism, interfering nucleic acids and RNA interference result in down regulation of the target gene or genes that are complementary (in pertinent part) to the guide strand.
  • a polynucleotide can be delivered to a cell to express an exogenous nucleotide sequence, to inhibit, eliminate, augment, or alter expression of an endogenous nucleotide sequence, or to affect a specific physiological characteristic not naturally associated with the cell.
  • the polynucleotide can be a sequence whose presence or expression in a cell alters the expression or function of cellular genes or RNA.
  • polynucleotide-based expression inhibitors of A20 which may be selected from the group comprising siRNA, microRNA, interfering RNA or RNAi, dsRNA, ribozymes, antisense polynucleotides, and DNA expression cassettes encoding siRNA, microRNA, dsRNA, ribozymes or antisense nucleic acids.
  • MicroRNAs miRNAs
  • Antisense polynucleotides comprise a sequence that is complimentary to a gene or mRNA.
  • Antisense polynucleotides include, but are not limited to: morpholinos, 2'-0-methyl polynucleotides, DNA, RNA and the like.
  • the polynucleotide-based expression inhibitor may be polymerized in vitro, recombinant, contain chimeric sequences, or derivatives of these groups.
  • the polynucleotide-based expression inhibitor may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited.
  • Polynucleotides may contain an expression cassette coded to express a whole or partial protein, or RNA.
  • An expression cassette refers to a natural or recombinantly produced polynucleotide that is capable of expressing a sequence.
  • the cassette contains the coding region of the gene of interest along with any other sequences that affect expression of the sequence of interest.
  • An expression cassette typically includes a promoter (allowing transcription initiation), and a transcribed sequence.
  • the expression cassette may include, but is not limited to, transcriptional enhancers, non-coding sequences, splicing signals, transcription termination signals, and polyadenylation signals.
  • An RNA expression cassette typically includes a translation initiation codon (allowing translation initiation), and a sequence encoding one or more proteins.
  • the expression cassette may include, but is not limited to, translation termination signals, a polyadenosine sequence, internal ribosome entry sites (IRES), and non- coding sequences.
  • the polynucleotide may contain sequences that do not serve a specific function in the target cell but are used in the generation of the polynucleotide. Such sequences include, but are not limited to, sequences required for replication or selection of the polynucleotide in a host organism.
  • siRNA molecules with the ability to knock-down A20 activity can be obtained by chemical synthesis or by hairpin siRNA expression vectors.
  • There are numerous companies that provide the supply of costumer-designed siRNAs on a given RNA sequence e.g. Ambion, Imgenex, Dharmacon.
  • the A20 siRNAs of the invention may be chemically modified, e.g.
  • siRNAs may also be conjugated to small molecules or peptides, such as membrane-permeant peptides or polyethylene glycol (PEG).
  • siRNA conjugates which form part of the present invention include cholesterol and alternative lipid-like molecules, such as fatty acids or bile-salt derivatives.
  • the present invention also relates to an expression vector comprising any of the above described polynucleotide sequences encoding for example an A20 siRNA molecule in a manner that allows expression of the nucleic acid molecule, and cells containing such vector.
  • the polynucleic acid sequence is operably linked to regulatory signals (promoters, enhancers, suppressors etc.) enabling expression of the polynucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs.
  • regulatory signals promoters, enhancers, suppressors etc.
  • a variety of viral-based systems are available, including adenoviral, retroviral, adeno-associated viral, lentiviral, herpes simplex viral vector systems. Selection of the appropriate viral vector system, regulatory regions and host cell is common knowledge within the level of ordinary skill in the art.
  • A20 siRNA molecules of the invention may be delivered by known gene delivery methods, e.g. as described in US20030143732, including the use of naked siRNA, synthetic nanoparticles composed of cationic lipid formulations, liposome formulations including pH sensitive liposomes and immunoliposomes, or bioconjugates including siRNAs conjugated to fusogenic peptides.
  • the inhibitor of A20 comprises nucleic acid molecules in triple-helix formation.
  • Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single- stranded and composed of deoxynucleotides.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription. The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • the inhibitor of A20 is a ribozyme that reduces transcription of A20.
  • a "ribozyme” is an enzymatic RNA molecule capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques.
  • the inhibitor of A20 is a small molecule.
  • a "small molecule” is defined herein to have a molecular weight below about 2000 daltons, and preferably below about 500 Daltons.
  • Potential inhibitors of A20 include small molecules that bind to A20 thereby blocking the normal biological activity of A20.
  • small molecules include, but are not limited to, synthetic non-peptidyl organic or inorganic compounds. Small molecule inhibitors of A20 may be identified without undue experimentation using known techniques and chemically synthesized using known methodology. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are known in the art.
  • the invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising a molecule selected from the list comprising of a ribozyme against the gene of A20, a siRNA with a specificity for the gene of A20, an artificial microRNA with a specificity for the gene of A20, an antisense nucleic acid construct with a specificity for the gene of A20, a small molecule inhibiting the function of the A20 protein, a peptide with a specificity for the A20 protein or an antibody with a specificity for the A20 protein and at least one pharmaceutically acceptable carrier for use in the treatment of viral pneumonia.
  • the pharmaceutical composition is used for the treatment of viral pneumonia.
  • said viral pneumonia is caused by an influenza virus, a respiratory syncytial virus, a herpes simplex virus or a vesicular stomatitis virus.
  • composition of the invention is used in intratracheal delivery in the treatment of viral pneumonia.
  • the pharmaceutical composition of the invention is used in intranasal delivery in the treatment of viral pneumonia.
  • pulmonary delivery refers to delivery methods wherein the pharmaceutical delivery reaches the lungs, examples for pulmonary delivery are intratracheal and intranasal delivery methods.
  • a pharmaceutical composition is one which is suitable for administration to humans.
  • a veterinary composition is one that is suitable for administration to animals.
  • compositions used in the methods of the invention may comprise one or more pharmaceutically acceptable carriers and optionally other therapeutic agents.
  • Each carrier, diluent, adjuvant and/or excipient must be pharmaceutically "acceptable”.
  • pharmaceutically acceptable carrier is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected active agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • a “pharmaceutically acceptable” salt or ester of an inhibitor of A20-activity is a salt or ester which is not biologically or otherwise undesirable.
  • a “pharmaceutical carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the agent to the subject.
  • the carrier may be liquid or solid and is selected with the planned manner of administration in mind.
  • Each carrier must be pharmaceutically "acceptable” in the sense of being not biologically or otherwise undesirable i.e. the carrier may be administered to a subject along with the agent without causing any or a substantial adverse reaction.
  • compositions of the invention may be administered orally, intranasally, topically, or parenterally in formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles
  • preferred routes of administration for the treatment of viral pneumonia are intranasal and intra-tracheal delivery methods.
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or antibodies; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, PLURONICSTM or PEG.
  • buffers such as phosphate, citrate and other organic acids
  • antioxidants including ascorbic acid
  • sustained-release compositions may be prepared for pulmonary delivery.
  • Several techniques are known in the art to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • Suitable examples of sustained-release compositions include semipermeable matrices of solid hydrophobic polymers containing a particular inhibitor of A20 as herein described, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl- methacrylate), or poly -vinylalcohol, polylactides, copolymers of L- glutamic acid and v-ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers.
  • microencapsulation of the inhibitor is contemplated.
  • the A20-inhibitor may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and polymethylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles, and nanocapsules) or in macroemulsions .
  • the pulmonary pharmaceutical composition may also be administered as a spray, mist or fine dry powder emitted from a suitable delivery device such as a nebulizer etc.
  • composition may also be delivered intranasally using a suitable delivery device such as nebulizer.
  • the composition e.g. an antibody composition
  • the composition may be formulated for and suitably delivered in a particle size effective for reaching the lower airways of the lung.
  • Any of a variety of inhalation or nasal delivery devices known in the art for administration of a therapeutic agent may be employed.
  • Such devices are capable of depositing aerosolized formulations in the alveoli of a patient or animal and include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like.
  • Such devices dispense the formulation as an aerosol. Aerosols can be comprised of solutions (aqueous or non-aqueous), suspensions or solid particles.
  • Some nebulizers produce aerosols from solutions, whilst metered dose inhalers, dry powder inhalers, etc.
  • Formulations suitable for use with a sprayer or nebulizer may include, for example, the active ingredient (i.e. the inhibitor) in an aqueous solution at a concentration of about 0.1 mg to about 100 mg per milliliter.
  • the formulation can include agents such as an excipient, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
  • the formulation can also include an excipient or agent for stabilization of the A20-inhibitor composition, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • an excipient or agent for stabilization of the A20-inhibitor composition such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • Bulk proteins useful in formulating such compositions include albumin, protamine, or the like.
  • Typical carbohydrates useful in formulating the compositions include sucrose, mannitol, lactose, trehalose, glucose, or the like.
  • the surfactant can reduce or prevent surface-induced aggregation of the active agent, such as an antibody, caused by atomization of the solution in forming an aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 14% by weight of the formulation.
  • Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan mono-oleate, polysorbate 80, polysorbate 20, or the like.
  • the surfactant can be chosen to stabilize the active agent as a suspension in the propellant, to protect the active agent against chemical degradation, and the like.
  • Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like. In some cases solution aerosols are preferred using solvents such as ethanol.
  • the inhalation device can deliver particles of size less than about 10 ⁇ , preferably in the range of about 1 ⁇ to about 5 ⁇ , and most preferably about 2 ⁇ to about 3 ⁇ , for good respirability.
  • a formulated spray including the composition can be produced by forcing a suspension or solution of the composition through a nozzle under pressure.
  • the nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size.
  • An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed.
  • composition may also be delivered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
  • a nebulizer such as jet nebulizer or an ultrasonic nebulizer.
  • a compressed air source is used to create a high-velocity air jet through an orifice.
  • a low-pressure region is created, which draws a solution of the composition through a capillary tube connected to a liquid reservoir.
  • the liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol.
  • a range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer.
  • high-frequency electrical energy is used to create vibrational, mechanical energy, typically employing a piezoelectric transducer.
  • This energy is transmitted to the formulation either directly or through a coupling fluid, creating an aerosol including the A20-inhibitor composition.
  • Dry powder inhalers use breath-actuation of a mixed powder (see, e.g. U.S. 4,668,218, EP 237507, WO 97/25086, WO 94/08552, U.S. 5,458,135, WO94/06498).
  • Metered dose inhalers typically use a propellant gas and require actuation during inspiration (see, e.g., WO 94/16970, WO 98/35888).
  • a propellant, the A20-inhibitor and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol.
  • the desired aerosol particle size can be obtained by employing a formulation of the composition produced by various methods known to those of skill in the art, including jet- milling, spray drying, critical point condensation, or the like.
  • Formulations for use with a metered dose inhaler will generally include a finely divided powder containing the active agent as a suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant.
  • the propellant can be any conventional material employed for this purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1 , 1 , 1 , 2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227) or the like.
  • the propellant is a hydrofluorocarbon.
  • internalizing antibodies are preferred, i.e. to be able to inhibit the intracellular target A20.
  • lipofections or liposomes can also be used to deliver the antibody, antibody fragment, or other inhibitor into cells.
  • a "liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • PEG-PE PEG- derivatized phosphatidylethanolamine
  • antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • antibody fragments can be designed that retain the ability to bind the target protein sequence.
  • Such antibody fragments can be synthesized chemically and/ or produced by recombinant DNA technology.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride
  • lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, growth factors and inert gases and the like.
  • therapeutically useful agents such as anti-viral compounds may optionally be included in or administered simultaneously or sequentially with the inhibitor of A20.
  • Administration "in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • the invention contemplates administration of an inhibitor as described herein in combination with a complementary inhibitor as known or described elsewhere in the literature.
  • the invention includes administration of an inhibitor as described herein in combination with a therapeutic agent that targets the risk condition or trigger event that is associated with development of pulmonary edema.
  • the second agent or medicament to be administered will depend upon the identity of the risk condition that is associated with the particular viral infective agent.
  • the inhibitor of A20 may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. It is especially advantageous to formulate the veterinary or pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units, such as a container, pack, or dispenser, suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Instructions for administration may also be included.
  • Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. When in vivo administration of an inhibitor of A20 is employed, normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 ⁇ g kg day to 10 mg/kg/day, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.
  • compositions of the present invention are preferably administered into the lung by inhalation of an aerosol containing one or more A20- inhibitors as described herein before, or by intranasal or intratracheal instillation of said compositions.
  • pulmonary delivery of pharmaceutical compositions see Weiss et al., Human Gene Therapy 1999. 10:2287-2293; Densmore et al, Molecular therapy 1999. 1 :180-188; Gautam et al, Molecular Therapy 2001 . 3:551 -556; and Shahiwala & Misra, AAPS PharmSciTech 2004. 24;6(3):E482-6.
  • respiratory formulations for siRNA are described in U.S. Patent Application Publication No. 2004/0063654.
  • A20 inhibits RIG-l-induced NF-kappaB and IRF3 activation
  • RIG-I signaling induces the activation of NF-kappaB, IRF3 and IRF7 transcription factors, which promote the expression of proinflammatory cytokines and type-l IFNs that restrict further viral propagation.
  • Previous studies have shown that ectopically expressed A20 negatively regulates NF-kappaB and IRF3 activation upon RIG-I stimulation 48"50 .
  • Crossing these mice with transgenic mice expressing Cre recombinase under control of the lysozyme M promoter leads to specific deletion in myeloid cells 55 and allowed us to generate myeloid cell specific A20 knockout mice (Matmati et al., unpublished).
  • A20 deficient bone-marrow derived macrophages (A20 myel"KO BMDM) derived from these mice grew normally and displayed no enhanced basal cytokine production.
  • A20 myel"KO BMDM To stimulate the RIG-I receptor, we transfected A20 myel"KO BMDM and wild type control cells with minimal amounts of low molecular weight (LMW) poly(l:C), which is known to preferentially bind and activate RIG-I rather than MDA5 56 .
  • LMW low molecular weight
  • this concentration of poly(l:C) was not able to induce significant TLR3 dependent NF-kappaB and IRF3 activation or cytokine production (data not shown).
  • A20 deficient BMDM (figure 1 C).
  • NF-kappaB controls the expression of IL-6 and TNF
  • NF- kappaB and IRF3 control the expression of IFNbeta.
  • A20 myel"KO BMDM secreted increased amounts of IL-6, TNF and IFNbeta (figure 1 D). Similar results were obtained using peritoneal macrophages (data not shown). Together, these results demonstrate that A20 plays an important role in the negative regulation of RIG-l-induced NF-kappaB and IRF3 activation in primary macrophages. 2.
  • A20 negatively regulates lAV-induced gene expression in BMDM
  • A20 deficient and control BMDM with IAV X-47 H3N2
  • A20 mRNA levels were rapidly induced in wild-type BMDM, but not in A20 deficient BMDM, upon viral infection (figure 2A).
  • A20 myel"KO BMDM show enhanced expression of IL-6 and IFNbeta mRNA after IAV infection compared to control cells (figure 2A).
  • cell culture supernatant collected from these cells contained higher levels of TNF and IFNbeta (figure 2B).
  • IAV alveolar macrophages Upon host infection with IAV alveolar macrophages are an important source of cytokines and chemokines, attracting innate immune cells to the lung during the primary phase of infection.
  • A20 directly controls lAV-induced cytokine and chemokine production in these cells
  • A20 deficiency in myeloid cells protects against lethal lAV infection
  • KC is the murine orthologue of IL-8 and serves as chemo-attractant for neutrophils.
  • MCP-1 is mainly known as a chemoattractant for monocytes, which eventually develop into macrophages upon entering the alveolar lumen 59 .
  • the number of resident alveolar macrophages was slightly higher in A20 myel"KO mice but did not differ significantly between lAV infected or mock infected mice (figure 3D). Elimination of lAV infected cells has been shown to depend on the clonal expansion of virus specific cytotoxic CD8+ T (CTL) cells 60" 62 . To test whether A20 expression in myeloid cells regulates the antiviral CTL response, CD8+ T cells were measured in BAL of A20 myel"WT and A20 myel"KO mice.
  • CTL cytotoxic CD8+ T
  • mice are protected from Influenza A morbidity and lethality
  • A20 floxed mice (A20 FL/FL ) were crossed with a double transgenic mouse line that expresses the reverse tetracycline transactivator controlled by the rat CCSP promoter and the Cre recombinase under control of the (TetO) 7 CMV operator, generating A20 FL/FL /CCSP-rTA/(tetO) 7 -Cre triple transgenic mice (hereafter termed A20 AEC"KO mice), enabling the inducible depletion of A20 in AEC of the proximal airways.
  • A20 AEC airway epithelial cells
  • A20 aec"ko mice were born at Mendelian ratios and displayed no developmental defects or signs of spontaneous pulmonary inflammation.
  • WT wild-type
  • AEC only represent a minor fraction of total lung tissue
  • AEC immunoblot analysis of protein extracts from purified AEC revealed absence of A20 in AEC from A20 AEC"KO mice.
  • A20 is expressed at low levels in most cell types, we induced its expression in AEC by intratracheal instillation of LPS, a TLR4 ligand known to promote A20 expression in these cells.
  • A20 AEC"KO mice and control littermates were intranasally infected with a lethal dose of the mouse-adapted influenza A laboratory strain X-47 (H3N2), and monitored for survival and morbidity in terms of weight loss up to 3 weeks post-infection.
  • H3N2 mouse-adapted influenza A laboratory strain X-47
  • A20 AEC"KO animals showed an increased survival response and reduced body weight loss upon lethal X-47 infection compared to wild type littermates (figure 5 A, B). Similar results were obtained when infecting A20 aec"ko mice with a sublethal dose of X-47.
  • Viral titers in lungs from A20' mice were only slightly lower at day 2 and at day 5 post-infection compared to viral titers in lungs from control littermate mice, and no infectious viral particles could be isolated from lungs in both groups 8 days post-infection (figure 5D), indicating that viral clearance is similar in both groups.
  • IFNalpha protein levels known to efficiently inhibit viral replication, were similar in BAL fluid isolated from A20 AEC"KO and A20 AEC"WT animals at day 5 post-infection (figure 5E).
  • A20 deficiency restricts VSV replication and VSV-induced cytokine expression
  • VSV single stranded RNA virus vesicular stomatitis virus
  • virus replication as measured by the synthesis of viral M- and L-RNA transcripts was efficiently inhibited in A20 deficient BMDM compared to wild type cells (figure 6B), indicating that A20 deficiency restricts VSV replication (figure 6B).
  • A20 deficient BMDM may result from an enhanced antiviral immune response in these cells. Therefore we determined the expression of different pro-inflammatory genes and type-l I FN upon VSV infection. As expected, A20 mRNA expression was induced in wild type but not in A20 deficient BMBM following VSV infection. To our surprise we found decreased mRNA expression of TNF, IL-6, IFNbeta and IP10 at different time points after infection in A20 deficient BMDM compared to wild type cells. Although the initial secretion of IFNbeta (6 hours p.i.) was not hampered in A20 deficient BMDM, we observed lower levels of IFN in cell culture supernatant of these cells at 12 hours p.i. These results demonstrate that restricted VSV replication in A20 deficient BMDM is not associated with the enhanced expression of proinflammatory cytokines or IFNbeta.
  • TLR9 can detect some viral DNAs in plasmacytoid dendritic cells (pDC), whereas IFI16 and AIM2 were recently identified as DNA sensors in other cell types 4 .
  • pDC plasmacytoid dendritic cells
  • IFI16 and AIM2 were recently identified as DNA sensors in other cell types 4 .
  • the IFI 16 protein Upon sensing double stranded DNA, the IFI 16 protein induces the activation of NF-kappaB and IRF3 whereas the AIM2 protein forms an inflammasome, which promotes the secretion of IL-1 beta.
  • HSV-1 prototypical DNA virus Herpes simplex virus 1
  • siRNA oligofectamine (Invitrogen) complexes are prepared as follows: oligofectamine is diluted 1 :1 in PBS and incubated for 10 min at room temperature (RT). siRNA and diluted oligofectamine are mixed in a 2:3 ratio and incubated for 20 min at RT.
  • each mouse receives 50 ⁇ (final concentration of 20 ⁇ g siRNA) siRNA oligofectamine through the intranasal route respectively 1 day before or 2 days after infection, in v/Vo-jetPEI (polyplus-transfection SA) is an alternative approach for siRNA delivery.
  • siRNA is mixed with in v/Vo-jetPEI (dissolved in 5% glucose) at an N/P ratio (number of nitrogen residues of in v/Vo-jetPEI per nucleic acid phosphate) of 6-8 according to manufacturer's instructions.
  • Mice will undergo intranasal instillation with 50-100 ⁇ of this mixture (final concentration of 20 ⁇ g siRNA) respectively 1 day before or 2 days after infection (Ge Q ef al (2004).
  • mice For each treatment, groups of 6 C57BI/6 mice are infected intranasally with mouse adapted 1 xLD 50 influenza A PR/8 (H 1 N1 ) diluted in 50 ⁇ PBS.
  • mice For intranasal siRNA administration and viral challenges, mice are lightly anesthetisized with isoflurane. Upon viral challenge, morbidity (in terms of body weight loss) and survival are monitored for 3 weeks.
  • morbidity in terms of body weight loss
  • survival At day 5 post infection mice are sacrificed and pulmonary viral titers are determined by TCID 50 . For this, lungs are harvested and homogenized with a Polytron homogenizer (Kinematica) in PBS.
  • Polytron homogenizer Polytron homogenizer
  • TCID 50 titers are calculated according to the method of Reed and Muench (Reed LJ and Muench H (1937) Am J Hyg 27: 493-497). Table. A20-specific RNAi sequences.
  • mice were generated as previously described. A20 l/ l mice were crossed with LysMCre mice (provided by I. Forster, Institute of Genetics, University of Cologne, Germany) and will be described in detail elsewhere (Matmati et al., submitted). Mice were housed in individually ventilated cages at the VIB Department of Molecular Biomedical Research in specific pathogen-free animal facilities. All experiments on mice were conducted according to institutional, national, and European animal regulations. Animal protocols were approved by the ethics committee of Ghent University.
  • mice were anesthetized by i.p. injection with ketamine (12 mg/kg) and xylazine (60 mg/kg) and 50 ⁇ X-47 diluted in PBS was administered intranasally.
  • mice received respectively 2-LD 50 or 0,05-LD 50 X-47.
  • TCID 50 median tissue culture infectious dose
  • HEK293T and MDCK cells were grown in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.
  • HEK293T cells were transfected using the calcium phosphate precipitate transfection method with specific expression vectors (pCAGGSEhA20 (LMBP 3778), pCAGGS-E-RIG-l-CARD (LMBP 6517), pEF-HA-IRF-7 (kindly provided by T.
  • BMDM bone marrow cells were cultured 7 days in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate, antibiotics and 40ng/ml recombinant M-CSF.
  • BMDM were of >95% purity.
  • the trachea was canulated and the lungs were flushed 4 times with HBSS with 1 mM EDTA.
  • Alveolar macrophages were cultured in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L- glutamine, 0.4 mM sodium pyruvate and antibiotics.
  • BMDM bone marrow cells were cultured 7 days in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate, antibiotics and 40ng/ml recombinant M-CSF.
  • BMDM were of >95% purity.
  • VSV Indiana; ATCC VR-1415
  • HSV-1 KOS; ATCC VR-1493
  • BMDM were infected with IAV, VSV or HSV-1 for 1 h under serum-free conditions, after which cells were washed and fresh medium was added to the cells. 4.
  • lysis buffer 200 mM NaCI, 1 % NP-40, 10 mM Tris-HCI pH 7.5, 5 mM EDTA, 2 mM DTT
  • protease and phosphatase inhibitors nuclear and cytoplasmic lysates were prepared by resuspending cells in B1 (10mM Hepes pH 7.5, 10mM KCI, 1 mM MgCI 2 , 5% glycerol, 0.5mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C.
  • NP-40 detergent was added to a final concentration of 0.65% and cells were centrifuged at 500g for 5 min.
  • the nuclear fraction containing pellet was lyzed in B2 (20mM Hepes pH 7.5, 1 % NP-40, 400mM NaCI, 10mM KCI, 1 mM MgCI2, 20% glycerol, 0.5mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C.
  • the lysates were subsequently separated by SDS-PAGE and analyzed by western blotting and ECL detection (Perkin Elmer Life Sciences).
  • Immunoblots were revealed with anti-A20, anti-lkappaBa, anti- p65, and anti-H1 (Santa Cruz), anti-IRF3 (Invitrogen), anti-phospho-IRF3 and anti-phospho- IkappaBa (Cell Signaling) and anti-actin (MP Biomedicals).
  • Lungs were dissected and incubated with collagenase type IV (1 mg/ml) and DNAse (100U/ml) at 37°C for 30 min. Subsequently, samples were filtered through a 70 ⁇ and 40 ⁇ nylon mesh. For the preparation of BAL, trachea were canulated and airway lumen was flushed 4 times with HBSS with 1 mM EDTA.
  • TNF ELISA 96-well plates were coated with TNF coating (TN3-19, eBioscience) and detection (R4-6A2, eBioscience) antibodies. IFNalpha and IFNbeta protein levels were determined with an ELISA kit (PBL Biomedical Laboratories).
  • IFNgamma ELISA 96-well plates were coated with IFNgamma coating (XMG1 .2) and detection (R4-6A2) antibodies (eBiosciences). Detection of MCP-1 , KC and IL-6 in BAL fluid was performed using Bioplex (BioRad) technology according to the manufacturer's instructions.
  • qPCR was performed by using SYBR Green I master mix I (Roche) in the Lightcycler 480 detection system (Roche) with the following primers: HPRT: 5' AGTGTTG GATACAG G CCAG AC-3 ' (SEQ ID NO: 7) and 5'CGTGATTCAAATCCCTGAAGT- 3' (SEQ ID NO: 8); IL-6: 5'-GAGGATACCACTCCCAACAGACC-3' (SEQ ID NO: 9) and 5'- AAGTGCATCATCGTTGTTCATACA-3' (SEQ ID NO: 10); IFNbeta: 5'- TCAGAATGAGTGGTGGTTGC-3' (SEQ ID NO: 1 1 ) and 5'- GACCTTTCAAATGCAGTAGATTCA-3' (SEQ ID NO: 12);
  • Results are expressed as the mean ⁇ SEM. Statistical significance between experimental groups was assessed using an unpaired two-sample Student's i-test. Statistical significance of differences between survival rates was analyzed by comparing Kaplan-Meier curves using the log-rank test.
  • IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol 1 1 , 997-1004 (2010).
  • Meylan, E. et al. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1 167-1 172 (2005).
  • REUL is a novel E3 ubiquitin ligase and stimulator of retinoic-acid- inducible gene-l.
  • TRAF6 establishes innate immune responses by activating NF-kappaB and IRF7 upon sensing cytosolic viral RNA and DNA.
  • tumour suppressor CYLD is a negative regulator of RIG-I- mediated antiviral response. EMBO Rep 9, 930-936 (2008).
  • A20 is a potent inhibitor of TLR3- and Sendai virus-induced activation of NF-kappaB and ISRE and IFN-beta promoter. FEBS Lett 576, 86-90 (2004).
  • RNA helicase RIG-I has an essential function in double- stranded RNA-induced innate antiviral responses. Nat Immunol 5, 730-737 (2004).

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Abstract

La présente invention se rapporte au domaine de la médecine, et plus particulièrement au domaine de la pneumonie virale. L'invention montre que l'inhibition de l'A20 dans l'épithélium respiratoire, de même que l'inhibition de l'A20 dans les macrophages alvéolaires, accélère la clairance de poumons infectés par des virus. Ainsi, l'invention propose des inhibiteurs de l'A20 et leur utilisation pour le traitement d'infections virales, et plus particulièrement pour le traitement d'infections pulmonaires virales telles que la grippe.
PCT/EP2012/062227 2011-06-23 2012-06-25 Inhibiteurs d'a20 pour le traitement d'infections virales respiratoires WO2012175735A1 (fr)

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