WO2012175735A1 - A20 inhibitors for the treatment of respiratory viral infections - Google Patents

Abstract

The present invention relates to the field of medicine, more particularly to the field of viral pneumonia. The invention shows that inhibition of A20 in respiratory epithelium and also the inhibition of A20 in alveolar macrophages results in an accelerated clearance of virus infected lungs. Accordingly, the invention provides for inhibitors of A20 and their use for the treatment of viral infections, more particularly viral lung infections such as influenza.

Description

A20 INHIBITORS FOR THE TREATMENT OF RESPIRATORY VIRAL INFECTIONS

Field of the invention

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.

Introduction of the invention

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. In adults, viruses are the putative causative agents in a third of cases of community-acquired pneumonia, in particular influenza viruses, rhinoviruses, and coronaviruses. 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. The initiation of a protective innate antiviral immune response involves the action of specialized pattern recognition receptors (PRR), which detect conserved molecular structures of the invading pathogen. Triggering of PRRs induces the production of host proinflammatory cytokines (e.g. TNF, IL-6 and IL-1 ) and type-l interferons (e.g. interferon-alpha (IFN-alpha) and IFN-beta) through activation of downstream signaling pathways that control various transcription factors such as NF-kappaB, AP-1 , IRF3 and IRF7^{1, 2}. The presence of viral nucleic acids, such as viral RNA and DNA, viral replication intermediates and viral transcription products, can be sensed by specific intracellular PRRs³. Endosomal Toll like receptors (TLRs) and cytoplasmic RNA helicase RIG-l-like receptors (RLR) or Nod-like receptors (NLRs) detect the presence of viral single stranded RNA (TLR7, TLR8, Nod2) or double stranded RNA (TLR3, RIG-I, MDA5). Intracellular DNA sensors that mediate antiviral immune responses to DNA viruses include TLR9, DAI⁴ and the PYHIN domain containing proteins AIM2^5"7 and IFI 16⁸. 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 proteins⁹.

Influenza A virus (IAV) 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. Both receptors contribute to the proinflammatory response to IAV, but the initiation of the innate antiviral immune response largely depends on RIG-I mediated signaling¹³. Interestingly, RIG-I deficient mice are highly susceptible to IAV^{14, 15}, whereas TLR3 deficient mice have a survival advantage to acute infection¹⁶. These results indicate that an imbalance between the beneficial and harmful effects of mediators released by immune cells is likely to contribute to the pathogenesis of influenza. RIG-I contains a C-terminal DExD/H box helicase domain, which is required for ligand recognition, and two N-terminal CARD domains. Upon ligand binding, 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 (E3s), such as TRIM25²¹ and Riplet^22"24, have been shown to directly promote RIG-I activation. In addition, well characterized ubiquitin ligases such as TRAF3^{25, 26} and TRAF6²⁷ mediate respectively NF-kappaB and IRF3 activation upon RIG-I stimulation. On the other hand, deubiquitinating enzymes (DUBs), such as DUBA²⁸, CYLD²⁹' ³⁰ and OTUB1/2³¹ have been shown to negatively regulate RLR signaling by specifically removing K63-linked polyubiquitin chains from several signaling molecules. Furthermore, various K48-specific ubiquitin ligases, such as AIP4³² and TRIAD3A³³ mark respectively MAVS and TRAF3 for proteasome mediated degradation, thus inhibiting further downstream signaling. Additionally, the attachment of K48-specific polyubiquitin chains to the IRF3 and IRF7 transcription factors by E3s such as RAUL³⁴, TRIM21³⁵ and RBCK1³⁶ 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 R⁴¹ and NOD2⁴². 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 cachexia³⁷. More recently, gene targeting of A20 in specific cell types was shown to be associated with autoimmunity and chronic inflammation^43"46, further illustrating that A20 is an important brake on the inflammatory response. The relevance of these findings for human disease has recently been illustrated by the fact that polymorphisms in the A20 locus are associated with several autoimmune diseases and chronic inflammation⁴⁵. In contrast to its well established function in the regulation of pro-inflammatory responses, the role of A20 in the regulation of antiviral immune responses is less well described and limited to a number of in vitro studies using overexpression or silencing in specific cell lines, indicating that A20 may regulate RIG-I- and TLR3-induced signaling to NF-kappaB and IRF-3^47"50. However, the precise role of A20 in vivo remains to be clarified. Using 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. Surprisingly, A20^myel"KO mice show enhanced survival and reduced morbidity in response to IAV lung infection compared to wild type mice. These results are in contradiction to what is described in the art and challenge the general view that an excessive pro-inflammatory response is associated with lAV-induced lethality. Protection against IAV in A20^myel"KO mice is associated with increased cytokine production, augmented recruitment of innate immune cells, and enhanced viral clearance. These results illustrate that inhibition of NF-kappaB and IRF-3 by A20 is disadvantageous to the host defense mechanism against IAV infection. In a second model we generated mice in which A20 expression was specifically deleted in airway epithelial cells (AEC), which are the primary target cells for influenza A and main producers of infectious viral progeny. In this second model we here also show that these 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. Last but not least, we have shown that bone marrow derived macrophages deficient in A20 also restrict vesicular stomatitis virus (VSV) and herpes simplex virus-1 (HSV-1 ) replication. The invention shows that the inhibition of A20, by local administration of A20 inhibitory compounds in the lung, is a promising therapeutic strategy against viral pneumonia such pneumonia caused by influenza infection. Figures

Figure 1 : A20 inhibits NF-kappaB and IRF3 activation in response to RIG-I stimulation. (A) 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. (B) 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.

Figure 2: A20 negatively regulates lAV-induced gene expression in BMDM. (A) A20^myel"WT and _A20myeⁱ-Ko _{BMDM were} j_nf_ected 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.

Figure 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 TCID₅₀. (C) 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. (D) Absolute numbers of resident alveolar macrophages, recruited macrophages and neutrophils in BAL were analyzed by FACS 4 and 10 days p.i. (E). Absolute numbers of CD8+ T cells and IFNgamma protein levels were measured in BAL fluid at 4 and 10 p.i. Numbers are averages +/- SEM of 5 mice per group and are representative of at least 2 independent experiments. ^*p<0,05; ^**p<0,01 ; ^***p<0,001 .

Figure 4: A20 deficiency in myeloid cells protects against lethal IAV infection. A20^myel"WT (n = 1 1 ) and A20^myel"KO (n = 1 1 ) mice were intranasally infected with a lethal dose of X/47 and survival (p<0.001 ) (A) and weight loss +/- SEM (B) was monitored for respectively 21 and 15 days post infection. Data are representative of 2 independent experiments.

Figure 5: A20^AEC"KO mice are protected from influenza A infection. (A) Survival curves of A20^AEC"KO (n=30) and control littermates (n=28) upon intranasal (i.n.) administration of a lethal dose of X-47 . (B) Body weight curves of A20^aec"ko (n=30) and control littermates (n=28) following lethal X-47 infection. (C) Body weight curves of A20^AEC"KO mice (n=6) and control littermates (n=8) following sublethal X-47 infection. (D) Pulmonary viral titers measured by TCID₅₀ following sublethal X-47 challenge. (E) IFNalpha protein levels in BAL fluid 5 days after sublethal X-47 challenge, as measured by ELISA. Data are representative of at least 2 independent experiments. Data are shown as mean (figure 1 C) SEM. ^*, p<0.05. ^***, p<0.001 . Figure 6: A20 deficiency restricts VSV replication in BMDM. (A) A20^myel"WT and A20^myel"KO BMDM were infected with VSV at a moi 1. At 12 hours post infection (hours p.i.), cytolytic effects were analyzed using light microscopy (B) A20^myel"WT and A20^myel"KO BMDM were infected with VSV at a moi 1 . At different hours p.i. expression of M- and L-transcripts was analyzed by qPCR. Values represent the mean (+/- SD) of 3 samples. Results are representative for 2 independent experiments.

Figure 7: A20 deficiency in BMDM reduces proinflammatory cytokine and type-l IFN expression in response to VSV. (A) 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.

Figure 8: A20 deficiency in BMDM increases HSV-1 -induced proinflammatory cytokine and IFNbeta expression. (A) 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. At 18 hours p.i. 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.

Detailed description of the invention

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. To study the function of A20 in specific cell types, in the present invention, we have generated A20 conditional knockout mice, and 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. We have surprisingly found that 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.

Accordingly 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.

In a particular embodiment 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.

In yet another particular embodiment 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. Specific examples of 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, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Citrobacter, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema palladium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelii. 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

To avoid confusion the complete coding sequence of the human A20 gene is depicted in 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.

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.

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.

The person skilled in the art will appreciate that there exist many mechanisms for inhibiting

A20 activity. Some examples will be described below, but such a list is not intended to be limiting.

Antibody Inhibitors of the A20 protein

The term "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. Generally, 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. Once isolated, 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. Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies. 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.

Human and Humanized Antibodies

The anti-A20 antibodies of the invention may comprise humanized antibodies or human antibodies. Generally, 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. Although 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. In some instances, 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. In general, 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. Various humanization strategies have been described in the prior art and it is envisaged that practice of the invention extends to the use of both known humanization strategies and any new strategies to be developed in the future. Examples of known humanization strategies include those described by Studnicka (US 5,869,619) and Padlan (1991 , Molec. Immunol., 28, 489-498), Winter (US 5,225,539) and Jones et al (1986, Nature, 321 , 522-525), Queen et al. (US 5,693,761 ) and Foote (US 6,881 ,557) . As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. Alternatively, 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. Phage display can be performed in a variety of formats. Several sources of V-gene segments can be used for phage display.

"Antibody fragments" comprise a portion of an antibody, preferably the antigen binding or variable region of the intact antibody. Examples of 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.

In another embodiment, the inhibitor of A20 is a peptide or peptide mimetic. As used herein, "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. For any of the foregoing peptides, 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. For example, in one variation, 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. However, organic chemists and peptide chemists are capable of synthesizing intramolecular bonds between a wide variety of amino acids using conventional techniques. In yet another embodiment protein interference as described in the patent application WO2007071789 (means and methods for mediating protein interference) 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. Through the use of 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. et al (2004) Nat Biotec nol 22(10): 1302-6. By expressing these specific peptides in for example the lung epithelial or lung alveolar macrophages the A20-protein can be specifically targeted by inducing its irreversible aggregation and thus its functional knockout.

Nucleic Acid Molecules which are able to inhibit the A20 gene

In yet another embodiment, 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.

Such 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. Such 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. In a specific embodiment small Interfering RNA (siRNA) with a specificity for A20 can be used in the context of the present invention. The term "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). Generally, 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. Generally, 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. In vivo, 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. The term "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. Typically, double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. 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. One strand of the double-stranded RNA (called the "guide strand") is part of a complex of proteins called the 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.

In addition, the present invention contemplates 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) are small noncoding polynucleotides, about 22 nucleotides long, 7that direct destruction or translational repression of their mRNA targets. 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. Optionally, 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. Optionally, 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.

Based on the RNA sequence of A20, 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. as described in US20030143732, by phosphorothioate internucleotide linkages, 2'-0-methyl ribonucleotides, 2'-deoxy-2'fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation. The sense strand of A20 siRNAs may also be conjugated to small molecules or peptides, such as membrane-permeant peptides or polyethylene glycol (PEG). Other siRNA conjugates which form part of the present invention include cholesterol and alternative lipid-like molecules, such as fatty acids or bile-salt derivatives.

In particular embodiments 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. 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.

As gene delivery and gene silencing techniques improve, the selective deletion of A20, in particular in lung tissues may prove useful in order to limit the impact of protein deletion to a particular system under study. The 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.

In a specific embodiment, 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.

In a related embodiment, 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. In a further embodiment, 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. Examples of 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. In another embodiment the invention provides for 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.

In yet another embodiment the pharmaceutical composition is used for the treatment of viral pneumonia.

In yet another embodiment said viral pneumonia is caused by an influenza virus, a respiratory syncytial virus, a herpes simplex virus or a vesicular stomatitis virus.

In yet another embodiment the pharmaceutical composition of the invention is used in intratracheal delivery in the treatment of viral pneumonia.

In yet another embodiment the pharmaceutical composition of the invention is used in intranasal delivery in the treatment of viral pneumonia. In the present application the term "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.

The pharmaceutical or veterinary 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". By "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. Similarly, a "pharmaceutically acceptable" salt or ester of an inhibitor of A20-activity is a salt or ester which is not biologically or otherwise undesirable.

As used herein, 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.

Although the pharmaceutical compositions of the invention may be administered orally, intranasally, topically, or parenterally in formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles, the 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 TWEEN™, PLURONICS™ or PEG.

In particular embodiments 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. Examples of 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.

Where sustained-release administration of an inhibitor of A20 is desired in a composition with release characteristics suitable for the treatment of pulmonary viral infections requiring administration of an A20-inhibitor, 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.

The composition may also be delivered intranasally using a suitable delivery device such as nebulizer.

For pulmonary administration, the composition, e.g. an antibody 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. generate small particle aerosols. There are several desirable features of an inhalation device for administering the active agent, such as an antibody. For example, delivery by the inhalation device is advantageously reliable, reproducible and accurate. 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. 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. Advantageously, the inhalation device can deliver particles of size less than about 10 μηι, preferably in the range of about 1 μηι to about 5 μηι, and most preferably about 2 μηη to about 3 μηι, 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.

The composition may also be delivered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer.

Typically, in a jet nebulizer, a compressed air source is used to create a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, 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. In an ultrasonic 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). In a metered dose inhaler, 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. Preferably the propellant is a hydrofluorocarbon. In particular embodiments when antibodies are used as inhibitors, internalizing antibodies are preferred, i.e. to be able to inhibit the intracellular target A20. However, 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.

Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable- region sequences of an antibody, 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. Examples of 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.

In yet another embodiment other 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. In particular embodiments, the invention contemplates administration of an inhibitor as described herein in combination with a complementary inhibitor as known or described elsewhere in the literature. Alternatively, in a preferred embodiment, 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 μg 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.

As mentioned herein before the therapeutic 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. For further information on 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. Additionally, respiratory formulations for siRNA are described in U.S. Patent Application Publication No. 2004/0063654.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any- other element or integer or group of elements or integers. It must also be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

Examples

1. 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. Similarly, we show that A20 overexpression in HEK293T cells prevents NF-kappaB- and IRF3-dependent luciferase reporter gene activation induced by transfection of a truncated constitutive active form of RIG- I⁵¹, expressing only the two N-terminal CARD domains of RIG-I [RIG-I (2 CARD)] (figure 1A, left and middle graph). We next investigated whether A20 also inhibits IRF7 activation. Unlike IRF3, IRF7 is not or weakly expressed under naive conditions and IRF7 protein levels are rapidly upregulated upon virus-induced IRF3 activation^{52, 53}. To determine the effect of A20 on IRF7 activation, we therefore transfected minor amounts of an IRF7 expression plasmid together with plasmids encoding RIG-I (2CARD), A20 and an IRF7-specific IFNa4 luciferase reporter construct. RIG-I (2CARD) expression in the absence of IRF7 co-expression showed negligible IFNalpha4 promoter activation (grey bar, figure 1A, right graph), whereas significant reporter gene expression was observed in the presence of IRF7. Similar to its inhibitory effect on NF-kappaB and IRF3 activation, A20 also prevented RIG-l-induced IRF7 activation (figure 1A, right graph). These results demonstrate the potential of A20 overexpression to inhibit RIG- l-induced NF-kappaB and IRF3/7 activation.

To study the effect of endogenously expressed A20 on RIG-l-induced signaling in a more immunological relevant context, we performed further experiments in A20 deficient primary macrophages. Since A20 full knockout mice die prematurely as a result of severe multi-organ inflammation³⁷, we generated mice carrying a conditional A20 allele in which exon IV and exon V were flanked by two loxP sites⁵⁴. Crossing these mice with transgenic mice expressing Cre recombinase under control of the lysozyme M promoter leads to specific deletion in myeloid cells⁵⁵ 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. 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 MDA5⁵⁶. Of note, 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). As expected, poly(l:C) transfection induced the rapid expression of A20 in wild type, but not in A20^myel"KO BMDM (figure 1 B, upper panel). At early time points, solely slower migrating forms of A20 were observed, indicating that A20 undergoes a yet unknown modification upon RIG-I stimulation. Compared to wild-type BMBM, A20 deficient cells showed enhanced NF-kappaB activation as indicated by increased phosphorylation and sustained degradation of l-kappaBalpha(figure 1 B). Furthermore, nuclear translocation of the p65 NF-kappaB subunit was enhanced (especially at 90 min) in RIG-l-stimulated A20^myel"KO BMDM compared to wild-type cells (figure 1 C). IRF3 is known to be activated upon phosphorylation of a series of carboxyl terminal serine residues by the IKK-like kinases TBK1 and IKKkappa⁵⁷, leading to its dimerization and subsequent translocation to the nucleus⁵⁸. Using immunoblotting with an antibody directed against phosphorylated Ser396, maximum IRF3 phosphorylation was detected at earlier time-points in A20^myel"KO BMDM compared to wild- type BMDM (figure 1 B). Similar to p65, IRF3 nuclear translocation was accelerated in A20 deficient BMDM (figure 1 C). NF-kappaB controls the expression of IL-6 and TNF, and NF- kappaB and IRF3 control the expression of IFNbeta. In line with the enhanced activation of NF- kappaB and IRF3 as described above, 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

To investigate the role of A20 in the lAV-induced proinflammatory and antiviral innate immune responses, we infected 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). Furthermore, A20^myel"KO BMDM show enhanced expression of IL-6 and IFNbeta mRNA after IAV infection compared to control cells (figure 2A). In accordance with these data, cell culture supernatant collected from these cells contained higher levels of TNF and IFNbeta (figure 2B).

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. To test whether A20 directly controls lAV-induced cytokine and chemokine production in these cells, we isolated alveolar macrophages from lungs of A20^myel"KO and control littermates and determined in vitro cytokine production upon IAV X-47 infection. Uninfected cells already secreted detectable amounts of TNF, IFNbeta and IL-6, which was further increased upon infection (figure 2C). Similar to our observations with BMDM, we could detect an increased production of TNF, IFNbeta and IL-6 in alveolar macrophages isolated from A20^myel"KO mice compared to cells derived from wild type animals. Taken together these results demonstrate that A20 negatively regulates lAV-induced pro-inflammatory and antiviral gene expression, consistent with the inhibitory effect of A20 seen on RIG-l-induced NF-kappaB and IRF3 activation.

3. A20 deficiency in myeloid cells protects against lethal lAV infection

To determine the role of A20 expression in myeloid cells during an lAV infection in vivo, we intranasally inoculated both A20^myel"KO mice and control littermates with a sublethal dose of the mouse adapted lAV X-47 (H3N2) strain and monitored morbidity in terms of weight loss. A20^myel"KO mice showed reduced weight loss compared to wild type control littermates and recovered faster from the viral challenge (figure 3A). Next, we measured pulmonary viral titers at 4 and 10 days post infection. No differences in viral titers were observed in A20^myel"KO mice versus wild type mice at day 4 post infection. However, after 10 days, almost no virus could be detected in the lungs of A20^myel"KO mice while abundant infectious viral particles could still be isolated from lungs of wild type mice (figure 3B). This indicates that loss of A20 in myeloid cells does not affect early viral replication but contributes to viral clearance at later stages during infection. To verify if A20 deficiency in myeloid cells affects lAV-induced gene expression in the lung, we analyzed the levels of several chemokines and cytokines in the bronchoalveolar lavage (BAL) at day 4 and day 10 following infection. The levels of KC and MCP-1 chemokines, as well as IL-6 and IFNalpha cytokines were significantly higher in BAL from lAV infected A20^myel"KO mice compared to lAV infected wild type mice (figure 3C). KC is the murine orthologue of IL-8 and serves as chemo-attractant for neutrophils. While MCP-1 is mainly known as a chemoattractant for monocytes, which eventually develop into macrophages upon entering the alveolar lumen⁵⁹. Consistent with the higher KC and MCP-1 levels in A20^myel"KO mice compared to wild type mice, the number of neutrophils (CD1 1 b+ Gr1 + F4/80-) and macrophages (autofluorescent+ CD1 1 c'° F4/80+ CD1 1 b+) that were recruited in the bronchoalveolar spaces upon lAV infection was also clearly higher in A20^myel"KO mice compared to control animals (figure 3D). The number of resident alveolar macrophages (autofluorescent+ CD1 1 c^hi F4/80+ CD1 1 b-) 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. A clear CTL response could be detected at day 10 post infection, but no differences were observed between A20^myel"KO and wild type mice (figure 3E). Together, these data indicate that mechanisms other than antiviral CD8+ T cells, such as increased IFNbeta production and accelerated influx of neutrophils and macrophages, contribute to the increased viral clearance observed in A20^myel"KO mice.

It is generally believed that lAV-induced mortality is due to an excessive proinflammatory response in the lung. We therefore analyzed whether the increased proinflammatory cytokine production and infiltration of proinflammatory cells in A20^myel"KO mice affects mortality induced by intranasal infection with a lethal dose of IAV X-47. Surprisingly, almost all A20^myel"KO mice survived (10/1 1 ), while all control mice succumbed (0/11 ) within 16 days after infection (figure 4A). A20^myel"KO mice still showed significant weight loss during the course of infection but were able to recover, in contrast to wild type mice that succumbed (figure 4B). Collectively these data indicate that A20 deficiency in myeloid cells is associated with protection against a lethal IAV infection.

4. A20 mice are protected from Influenza A morbidity and lethality

To study the role of A20 in airway epithelial cells (AEC), 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)₇CMV 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. To ensure proper deletion of A20 in AEC at all times, breeding pairs and offspring were continuously maintained on a doxycycline-containing diet. A20^aec"ko mice were born at Mendelian ratios and displayed no developmental defects or signs of spontaneous pulmonary inflammation. In order to determine Cre-mediated recombination of the floxed A20 allele in AEC of A20^AEC"KO mice, we performed Southern blot analysis on genomic DNA isolated from lungs of A20^AEC"KO mice and control wild-type (WT) littermates. As expected, recombination was only observed in lungs, and not in spleens, of A20^AEC"KO mice. Since AEC only represent a minor fraction of total lung tissue, only partial recombination was observed in total lung tissue from A20^AEC"KO mice. In addition, immunoblot analysis of protein extracts from purified AEC revealed absence of A20 in AEC from A20^AEC"KO mice. As 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. Finally, semi-quantitative PCR analysis on genomic DNA isolated from various tissues of A20^aec"ko mice, confirmed the specific deletion of A20 in lung tissue.

In a next step we investigated the AEC-specific role of A20 in Influenza A antiviral immunity. 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. Surprisingly, 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. In contrast to wild type mice, which lost up to 15 percent of their initial body weight peaking at day 9 post infection, A20^aec"ko displayed only mild weight loss, stabilizing around 5 percent of their original body weight (figure 5C). This ■AEC-KO

reduced morbidity in A20' animals could be a consequence of the enhanced intrinsic capacity of A20 deficient AEC to inhibit viral replication. Therefore we determined pulmonary

■AEC-KO

viral titers at different times post-infection. 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. Furthermore, 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). Together, these data suggest that, although the initial mechanisms leading to viral clearance are not affected, A20 depletion in AEC is beneficial for the host survival.

5. A20 deficiency restricts VSV replication and VSV-induced cytokine expression

To assess whether the inhibitory effect of A20 could be extended to other viruses than IAV, we tested the effect of abrogated A20 expression in BMDM infected with the single stranded RNA virus vesicular stomatitis virus (VSV). Similar to IAV, the antiviral immune response in macrophages against VSV is mainly initiated by RIG-I. VSV is known to exert a cytolytic replication cycle, and infection of wild-type BMDM with VSV resulted in significant cell death 12 hours post infection. In contrast, A20^myel"KO BMDM were found relatively resistant to VSV- induced cytolytic effects (figure 6A). Moreover, 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).

We hypothesized that the restricted VSV replication in 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.

6. A20 deficiency increases HSV-1 -induced cytokine production

In contrast to viral RNA sensing, knowledge of intracellular signaling and its regulation in response to DNA viruses such as herpes viruses is much more limited. TLR9 can detect some viral DNAs in plasmacytoid dendritic cells (pDC), whereas IFI16 and AIM2 were recently identified as DNA sensors in other cell types⁴. 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. To test whether A20 also regulates gene expression in response to DNA viruses, we infected A20 deficient BMDM and control cells with the prototypical DNA virus Herpes simplex virus 1 (HSV-1 ). Similar to cells infected with IAV, A20 deficient BMDM infected with HSV-1 displayed enhanced mRNA and protein expression of IL-6, TNF and IFNbeta, suggesting that A20 also negatively regulates the innate immune response against DNA viruses.

7. Prophylactic and therapeutic treatment against influenza A infection through intranasal delivery of A20-specific siRNA

A20 targeting and control siRNAs (see Table 1 ) are purchased from Dharmacon (Thermo Scientific). siRNAs are resuspended according to manufacturer's instructions to a final concentration of 1 mg/ml. As previously described (Tompkins SM et al (2004) Proc. Natl. Acad. Sci USA 101 : 8682-8686), 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. For prophylactic or therapeutic treatment, each mouse receives 50μΙ (final concentration of 20μg 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 μΙ of this mixture (final concentration of 20 μg siRNA) respectively 1 day before or 2 days after infection (Ge Q ef al (2004).

For each treatment, groups of 6 C57BI/6 mice are infected intranasally with mouse adapted 1 xLD₅₀ influenza A PR/8 (H 1 N1 ) diluted in 50μΙ PBS. 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. At day 5 post infection mice are sacrificed and pulmonary viral titers are determined by TCID₅₀. For this, lungs are harvested and homogenized with a Polytron homogenizer (Kinematica) in PBS. Ten-fold serial dilutions of lung homogenates are incubated on MDCK cells for 5 days in DMEM supplemented with trypsin (^g/ml), 2mM L-glutamine, 0.4mM sodium pyruvate and antibiotics. For read-out, 1 % chicken red blood cells are added and end-point dilution of haemagglutination is monitored. TCID₅₀ 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.

Materials and methods

1. Mice

A20 ^{l/ l} 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.

2. Viral infection and determination of viral titers

Mouse adapted IAV X-47 (H3N2) was propagated in MDCK cells. For viral inoculation, mice were anesthetized by i.p. injection with ketamine (12 mg/kg) and xylazine (60 mg/kg) and 50μΙ X-47 diluted in PBS was administered intranasally. For lethal and sublethal infection, mice received respectively 2-LD₅₀ or 0,05-LD₅₀ X-47. To determine pulmonary viral titers, median tissue culture infectious dose (TCID₅₀) was measured as follows: lungs were homogenized with a Polytron homogenizer (Kinematica ) in PBS. Ten-fold serial dilutions of lung homogenates were incubated on MDCK cells for 5 days in DMEM supplemented with trypsin (^g/ml), 2 mM L-glutamine, 0,4 mM sodium pyruvate and antibiotics. For read-out, 1 % chicken red blood cells were added and end-point dilution of hemagglutination was monitored. TCID₅₀ titers were then calculated according to the method of Reed and Muench⁸¹.

3. Cells and transfection 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. Taniguchi, Graduate School of Medicine and Faculty of Medicine, University of Tokyo⁸²)), NF-kappaB, IRF3, IRF7 reporter plasmids (respectively pConLuc (LMBP3248), pISRE-luc (LMBP401 1 ), pGL3-IFNalpha4-luc (kindly provided by J. Hiscott, McGill University, Montreal, Quebec, Canada⁸³)), and pACTbetagal (LMBP4341 ) for transfection efficiency normalization. Details of plasmids are presented along with detailed sequence maps at the BCCM-LMBP plasmid databank http://bccm.belspo.be/index.php. For the generation of 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. For the isolation of alveolar macrophages, 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.

Cells and viruses

For the generation of 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) and HSV-1 (KOS; ATCC VR-1493) were grown in Vero cells. 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. Western blotting

For total lysates, cells were lysed at 4°C for 15 min in lysis buffer (200 mM NaCI, 1 % NP-40, 10 mM Tris-HCI pH 7.5, 5 mM EDTA, 2 mM DTT) supplemented with protease and phosphatase inhibitors. Nuclear and cytoplasmic lysates were prepared by resuspending cells in B1 (10mM Hepes pH 7.5, 10mM KCI, 1 mM MgCI₂, 5% glycerol, 0.5mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. Next, 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).

5. Flow cytometry

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 70μηι and 40μΙ nylon mesh. For the preparation of BAL, trachea were canulated and airway lumen was flushed 4 times with HBSS with 1 mM EDTA. Cells were stained with monoclonal antibodies directed against MHC- II (l-A/l-E) FITC (M5/1 14.15.2), CD1 1 c PerCPCy5.5 (N418) CD8alpha PECy7 (53-6.7), F4/80 APC (BM8), B220 PECy7 (RA3-6B2) from eBiosciences and CD45 Horizon v450 (30-F1 1 ), CD3epsilon Horizon V500 (500A2), Gr1 PE (RB6-8C5), CD1 1 b APCCy7 (M1/70) and CD16/32 (2.4G2) from BD Pharmingen. Samples were acquired on a LSRII Cytometer and analyzed using FACSDiva software (BD Biosciences). 6. Cytokine quantification

For 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). For 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.

7. RNA isolation, cDNA synthesis and qPCR

Total RNA was extracted using Aurum Total RNA mini kit (BioRad) and reverse transcribed into cDNA with iScript cDNA synthesis kit (BioRad) 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); A20:

5'AAACCAATGGTGATGGAAACTG-3' (SEQ ID NO: 13) and 5'- GTTGTCCCATTCGTCATTCC-3' (SEQ ID NO: 14). 8. Statistics

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.

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Claims

Claims

1 . A molecule selected from the list consisting of a ribozyme with a specificity for the A20 gene, a siRNA with a specificity for the A20 gene and/or an antisense nucleic acid construct with a specificity for the A20 gene for use in the treatment of pneumonia.

2. A molecule for use according to claim 1 wherein said pneumonia is viral pneumonia.

3. A molecule for use according to claim 1 wherein said pneumonia is bacterial pneumonia.

4. A molecule for use according to claims 1 or 2 wherein said viral pneumonia is caused by an influenza virus, a respiratory syncytial virus, a herpes simplex virus or a vesicular stomatitis virus.

5. A molecule for use according to claims 1 or 2 wherein said viral pneumonia is caused by an influenza A virus.

6. A molecule for use according to claims 1 or 3 wherein said bacterial pneumonia is caused by Pasteurella species, Staphylococcus species and Streptococcus species.

7. A pharmaceutical composition comprising a molecule selected from the list consisting of a ribozyme with a specificity for the A20 gene, a siRNA with a specificity for the A20 gene and/or an antisense nucleic acid construct with a specificity for the A20 gene and at least one pharmaceutically acceptable carrier for use in the treatment of pneumonia.

8. A pharmaceutical composition according to claim 7 wherein said pneumonia is viral pneumonia.

9. A pharmaceutical composition according to claim 7 wherein said pneumonia is bacterial pneumonia.

10. A pharmaceutical composition according to claims 7 or 8 wherein said viral pneumonia is caused by an influenza virus, a respiratory syncytial virus, a herpes simplex virus or a vesicular stomatitis virus.

1 1 . A pharmaceutical composition according to claim 10 wherein said viral pneumonia is caused by an influenza A virus.

12. A pharmaceutical composition according to any one of claims 7-1 1 for use in intratracheal delivery in the treatment of pneumonia.

13. A pharmaceutical composition according to any one of claims 7-1 1 for use in intranasal delivery in the treatment of pneumonia.