Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the present invention relates to the use of the A20-binding inhibitor of NF- ⁇ B activation (ABIN) to protect against TNF-induced liver failure, such as viral hepatitis and alcoholic liver disease. More particularly, it relates to the prevention of the toxic effects of said diseases, including lethality, by over expressing ABIN.
• ABIN A20-binding inhibitor of NF- ⁇ B activation
• Acute liver failure is a clinical syndrome that results from massive necrosis and apoptosis of liver cells leading to hepatic encephalopathy and severe impairement of hepatic function. It is caused by different kinds of diseases, such as viral hepatitis (A, B, C, ...), drugs, intoxication, auto-immune hepatitis, etc.
• diseases such as viral hepatitis (A, B, C, 7), drugs, intoxication, auto-immune hepatitis, etc.
• TNF plays a central role in liver disease. TNF is produced mainly by activated macrophages but is also produced in smaller amounts by several other cell types. TNF exerts a variety of effects on different cell types and is implicated as an important mediator in various physiological and pathophysiological conditions.
• TNF is an important mediator of apoptosis (programmed cell death).
• TNF was originally identified by its capacity to induce hemorrhagic necrosis of tumors in mice. Attempts to use TNF for systemic anti-cancer therapy have failed due to the appearance of severe side effects before therapeutic doses could be reached.
• One of the side effects of TNF treatment was an elevation in serum levels of transaminases and bilirubin levels, indicating a direct cytotoxic effect of TNF on human hepatocytes.
• TNF may be involved in viral hepatitis, alcoholic liver disease, and fulminant hepatic failure (Muto et al., 1988; Bird et al., 1990; Gonzalez-Amaro et al., 1994; Diehl et al., 1994; Larrea et al., 1996).
• TNF serum levels are clearly elevated in patients with fulminant hepatitis (Muto et al., 1988).
• serum TNF levels were significantly higher in patients who died than in patients who survived (Bird et al., 1990).
• a role for TNF in the pathogenesis of chronic hepatitis B and C viral infection has been suggested.
• the serum levels of soluble TNF-R2 correlate closely with the extent of inflammation and hepatocyte death in the liver.
• the response and the increase in transaminases are associated with an increase in soluble TNF-R2 serum levels.
• interferon treatment clears the virus and reduces TNF levels to normal in responsive patients (Larrea et al., 1996).
• pretreatment levels of TNF were higher in unresponsive compared with responsive patients (Larrea et al., 1996).
• Hepatitis C proteins interact with the TNF receptor, although whether this interaction promotes or prevents apoptosis is not clear (Ray et al., 1998).
• TNF liver injury
• TNF-R1 TNF-R2
• TNF-R2 TNF/Galactosamine
• TNF is administered in combination with D-(+)-galactosamine (GalN), a hepatotoxin, that selectively blocks transcription in hepatocytes by depleting uridine nucleotides (Dekker and Keppler, 1974), inducing lethality, activation of caspases and subsequent hepatocyte apoptosis (Leist et al., 1995; Van Molle et al., 1999; Tiegs et al., 1989).
• TNF-R1 knockout mice are resistant to TNF/GalN treatment, demonstrating the essential role of TNF-R1 in this apoptosis model (Leist et al., 1995).
• NF- ⁇ B is an essential transcription factor that is ubiquitously expressed in all cell types and whose activity is modulated by a wide range of inducers, including cytokines and bacterial or viral products. Many of the NF- ⁇ B responsive genes play a key role in the regulation of inflammatory and immune responses. Deregulation of NF- ⁇ B activity is often observed in several chronic inflammatory diseases such as rheumatoid arthritis, asthma and inflammatory bowel disease, as well as in acute diseases such as septic shock.
• NF- ⁇ B serves to protect against apoptosis and supports cell cycle progression.
• the first indication that NF- ⁇ B activation may modulate hepatocyte responses relevant to liver injury was the finding that knockout mice deficient in the p65/Rel-A subunit of NF- ⁇ B were nonviable because of massive hepatocyte apoptosis during embryogenesis (Beg et al., 1995).
• Recent reports from several laboratories have now demonstrated that NF- ⁇ B activation regulates hepatocyte proliferation and apoptosis in vivo and in vitro.
• NF- B-dependent gene product(s) that protects hepatocytes against TNF-induced injury remains to be identified.
• Possible candidate genes are iNOS and interleukin-6, since they are regulated by NF- ⁇ B and their gene products may have hepatoprotective effects. It also remains to be determined whether NF- ⁇ B activation inhibits hepatotoxicity from injurious agents other than TNF.
• treatment with a nontoxic concentration of the superoxide generator menadione protected against subsequent toxic doses of menadione or H2O 2 by an NF- ⁇ B dependent mechanism (Chen and Cederbaum, 1997).
• NF- ⁇ B activation may therefore stimulate a defense mechanism specific for the TNF death pathway.
• the possibility that NF- ⁇ B activation in hepatocytes is protective following liver injury points to the complexity of events following global activation of NF- ⁇ B in all cell types in the liver.
• cytokines act by binding to specific cell surface receptors, which in turn initiate the recruitment of a number of specific adaptor proteins, and the activation of a kinase complex that phosphorylates the NF- ⁇ B inhibitor l ⁇ B.
• the latter retains NF- ⁇ B in the cytoplasm in an inactive dimeric form.
• l ⁇ B is marked for ubiquitination and subsequent degradation by the proteasome, allowing the nuclear translocation of NF- ⁇ B.
• A20 is a potent inhibitor of NF- ⁇ B activation in response to TNF, IL-1 , LPS and CD-40 (reviewed in Beyaert et al., 2000).
• A20 also exerts an anti-apoptotic function in a number of cell lines. A20 is only expressed upon NF- ⁇ B activation, and is involved in the negative feedback regulation of NF- ⁇ B activation.
• A20 deficient mice were recently shown to be defective in the termination of NF- B activation, leading to strong inflammatory responses and cachexia (Lee et al., 2000).
• the underlying mechanisms responsible for the inhibition of NF- ⁇ B dependent gene expression by A20 is still unclear.
• A20 interacts with the l ⁇ B kinase complex, as well as with TRAF2 and TRAF6, which are part of the l ⁇ B kinase activation cascade initiated by TNF and IL-1/LPS, respectively.
• TRAF2 and TRAF6 which are part of the l ⁇ B kinase activation cascade initiated by TNF and IL-1/LPS, respectively.
• three novel A20-binding proteins (ABIN, ABIN-2 and ABIN-3) were recently isolated.
• the present invention relates to the surprising finding that overexpression of ABIN prevents TNF-induced lethal hepatitis in mice.
• FIG. 1 Effect of AdABIN on TNF/GalN-stimulated degradation of l ⁇ B (upper panel) and DNA-binding of NF- ⁇ B (lower panel) in vivo.
• mice were killed and liver homogenates were prepared.
• FIG. 1 Effect of AdABIN on TNF-mediated cell death in vitro.
• AdABIN, AdRR ⁇ or mock infected BWTG3 cells were seeded in 96 well plates and stimulated with a serial dilution of mTNF in the absence (upper) or presence of CHX (lower) for 8 hours.
• Figure 4 Effect of AdABIN on TNF/GalN-induced lethality.
• FIG. 5 Effect of AdABIN on TNF/GalN induced alanine aminotransferase (ALT) release in serum.
• AdABIN or AdRR ⁇ infected mice, challenged with a lethal dose of ⁇ TNF/GalN were bled 8 hours after the injection. Serum-was prepared and serum ALT values were measured (in IL).
• FIG. 6 Effect of AdABIN on TNF/GalN induced DNA-fragmentation in the liver. Livers of AdABIN or AdRR ⁇ infected mice were isolated 8 hours after a challenge with a lethal dose of TNF/GalN. DNA fragmentation was measured by ELISA, and is 0 expressed as % of control mice (AdRR ⁇ ).
• FIG. 7 Effect of AdABIN on TNF/GalN induced caspase activity in liver homogenates.
• 30 ⁇ g of liver homogenate was tested for its proteolytic activity on Ac-DEVD.AMC.
• Proteolytic activity is expressed as the increase in AMC fluorescence as a function of ⁇ time ( ⁇ F/min).
• FIG. 8 Effect of AdABIN on TNF/GalN induced cleavage of caspase-3 in liver homogenates.
• Liver homogenates were prepared, proteins were separated by 1 ⁇ % SDS-PAGE, and immunoblotted using a polyclonal anti-caspase-3 0 antibody.
• Inactive pro-caspase-3 as well as the p20 subunit of caspase-3 that is proteolytically released are indicated by an arrow.
• Figure 10 Effect of AdABIN on anti-Fas induced lethality. Mice were injected (i.v.) with
• Figure 11 Effect of AdABIN on TNF/ActD-induced lethality. Mice were injected (i.v.) with
• a first aspect of the invention is the use of ABIN, as represented in SEQ ID N°2, or a functional fragment or variant thereof for the preparation of a medicament for the treatment of TNF-induced liver failure.
• the term 'ABIN' relates to ABIN, ABIN-2 and ⁇ ABIN-3 as disclosed in Beyaert et al., 2000; Heyninck et al., 1999; Van Huffel et ah, 2001, Van Huffel et al. (unpublished; AJ320 ⁇ 34) and WO 99/ ⁇ 7133.
• ABIN relates to any polypeptide that comprises the consensus amino acid sequence(s) as depicted in SEQ ID N° 4 and/or SEQ ID N° ⁇ which are also disclosed in WO 99/ ⁇ 7133 that is hereby incorporated by reference.
• a second aspect of the 0 invention is the use of a nucleotide sequence encoding ABIN, as represented in SEQ ID N°1, or for a functional fragment or a variant thereof, for the manufacture of a medicament for the treatment of TNF-induced liver failure.
• a functional fragment of ABIN is a polypeptide that is still able to interact with protein A20 and/or capable of modulating NF- ⁇ B activation.
• said modulation is an inhibition of NF- ⁇ B ⁇ activation.
• Functional fragments are, as a non limiting example, fragments that comprise at least amino acids 420-647 of SEQ ID N°2, preferably at least amino acids 390-647, more preferably at least 54-647 (SEQ ID N° 3).
• Preferentially said fragment is essentially consisting of at least amino acids 420-647 of SEQ ID N°2, preferably at least amino acids 390-647, more preferably at least ⁇ 4-647 (SEQ ID N° 3).
• Variants are 0 polypeptides with at least 6 ⁇ % identity on amino acid level, preferably 70% identity, as measured by BLAST (Altschul ef al., 1997).
• Variants have common characteristics, such as biological activity, immunological reactivity, conformation etc.
• Naf1 alpha protein AJ01189 ⁇
• Naf1 beta protein AJ011896)
• virion- associated nuclear shuttling protein AY0121 ⁇
• a further aspect is the use of an ABIN inducing and/or activating compound for the preparation of a medicament for the treatment of TNF-induced liver failure.
• phytohemagglutinin PHA
• PHA phytohemagglutinin
• LPS induces expression of this protein in THP1 ⁇ monocytes.
• Said TNF-induced liver failure is, as a non-limiting example, viral hepatitis such as hepatitis A, B or C, fulminant hepatitis and/or alcoholic liver disease.
• a nucleic acid is used, said medicament is preferably intended for delivery of said nucleic acid into the cell, in a gene therapy treatment.
• a large number of delivery methods are well 0 known to those of skill in the art.
• the nucleic acids are administered for in vivo or ex vivo gene therapy uses.
• Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
• Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
• Methods of non-viral 6 delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in, e.g., US Pat. No. ⁇ .049,386, US Pat No. 4,946,787; and US Pat. No. 4,897,3 ⁇ and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
• Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Flegner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues ⁇ in vivo administration).
• the preparation of lipid: nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, 199 ⁇ ; ⁇ Blaese ef al., 199 ⁇ ; Behr, 1994; Remy et al., 1994; Gao and Huang, 199 ⁇ ; U.S. Pat. Nos.
• RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
• Viral 0 vectors can be administered directly to patients ⁇ in vivo) or they can be used to treat, cells in vitro and the modified cells are administered to patients (ex vivo).
• viral based systems for the delivery of nucleic acids could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
• Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long- term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues, ⁇ In cases where transient expression of the nucleic acid is preferred, adenoviral based systems, including replication deficient adenoviral vectors are typically used.
• Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple 0 system.
• Adeno-associated virus (“AAV") vectors including recombinant adeno- associated virus vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures ⁇ see, e.g., U.S. Patent No. 4,797,368; WO 93/24641 ; Kotin, 1994; Muzyczka.
• the construction of recombinant AAV vectors is described in a number of ⁇ publications, including U.S. Pat. No. 6,173,414; Hermonat & Muzyczka, 1984; Samulski et al., 1989).
• Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitbneal, intramuscular, subdermal, or intracranial infusion) or topical application.
• vectors can be 0 delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
• Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
• cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient).
• a nucleic acid gene or cDNA
• Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., 1994) and the references cited therein for a discussion of how to isolate and culture 0 cells from patients).
• the invention provides a method for the production or manufacture of a medicament or a pharmaceutical composition comprising ABIN or a functional fragment or variant thereof and further more mixing said polypeptide with a pharmaceutically acceptable carrier.
• the administration of said pharmaceutical composition may be by way of oral, inhaled or parenteral administration.
• the active compound may be administered alone or preferably formulated as a pharmaceutical composition.
• a unit dose will normally contain 0.01 to 60 mg for example 0.01 to 10 mg, or O.O ⁇ to 2 mg of compound or a ⁇ pharmaceutically acceptable salt thereof.
• Unit doses will normally be administered once or more than once a day, for example 2, 3, or 4 times a day, more usually 1 to 3 times a day, such that the total daily dose is normally in the range of 0.0001 to 1 mg/kg; thus a suitable total daily dose for a 70 kg adult is 0.01 to 60 mg, for example 0.01 to 10 mg or more usually O.O ⁇ to 10 mg. It is greatly preferred that the compound or a 0 pharmaceutically acceptable salt thereof is administered in the form of a unit-dose composition, such as a unit dose oral, parenteral, or inhaled composition.
• a unit-dose composition such as a unit dose oral, parenteral, or inhaled composition.
• compositions are prepared by admixture and are suitably adapted for oral, inhaled or parenteral administration, and as such may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable ⁇ and infusable solutions or suspensions or suppositories or aerosols.
• Tablets and capsules for oral administration are usually presented in a unit dose, and contain conventional excipients such as binding agents, fillers, diluents, tabletting agents, lubricants, disintegrants, colourants, flavourings, and wetting agents.
• the tablets may be coated according to well-known methods in the art.
• Suitable fillers for use include 0 cellulose, mannitol, lactose and other similar agents.
• Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycollate.
• Suitable lubricants include, for example, magnesium stearate.
• Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate.
• Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other 0 suitable vehicle before use.
• Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p- hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
• suspending agents for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monoole
• Oral formulations also include conventional sustained release formulations, ⁇ such as tablets or granules having an enteric coating.
• compositions for inhalation are presented for administration to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose.
• the particles of active compound suitably have diameters of less than ⁇ O microns, preferably less than 10 0 microns, for example between 1 and ⁇ microns, such as between 2 and ⁇ microns.
• a favored inhaled dose will be in the range of O.O ⁇ to 2 mg, for example O.O ⁇ to O. ⁇ mg, 0.1 to 1 mg or O. ⁇ to 2 mg.
• fluid unit dose forms are prepared containing a compound of the present invention and a sterile vehicle.
• the active compound depending on the vehicle and the concentration, can be either 6 suspended or dissolved.
• Parenteral solutions are normally prepared by dissolving the compound in a vehicle and filter sterilising before filling into a suitable vial or ampoule and sealing.
• adjuvants such as a local anaesthetic, preservatives and buffering agents are also dissolved in the vehicle.
• the composition can be frozen after filling into the vial and the water removed under vacuum.
• Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilised by exposure to ethylene oxide before suspending in the sterile vehicle.
• a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active compound.
• bronchodilators for example sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine; xanthine derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACTH may be included.
• sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine
• xanthine derivatives such as theophylline and aminophylline
• corticosteroids such as prednisolone and adrenal stimulants such as ACTH
• ACTH adrenal stimulants
• protein transduction domains PTDs
• TAT protein from human immunodeficiency virus (HIV-1)
• HIV-1 human immunodeficiency virus
• VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells (reviewed in Ford et al., ⁇ 2001).
• Protein delivery based on a short amphipathic peptide carrier, Pep-1 is efficient for delivery of a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling (Morris et al., 2001).
• the capacity of VP22 chimeric proteins to spread from the primary transduced cell to surrounding cells can improve gene therapy approaches (Zender et 0 al., 2002).
• Liposomes have been used as vehicles for drug delivery and gene therapy and they have been shown to have substantial potential in the targeting of specific cell types of the liver. Thus, the use of liposomes may improve targeting efficacy in the treatment of a variety of liver diseases (Wu and Zerm, ⁇ 1999).
• Nucleotide sequence as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the 0 primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occuring nucleotides with an analog.
• Overexpression as used here means that the transformed cells do produce more of the overexpressed protein that the untransformed control, when kept under the same ⁇ condition. Preferably, overexpression is obtained by placing the coding sequence downstream a constitutive promoter.
• Coding sequence is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation 0 start codon at the ⁇ '-terminus and a translation stop codon at the 3'-terminus.
• a coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
• Protein A20 means the TNF induced zinc finger protein, described by Dixit et al., 1990; Opipari et al., 1990 and Tewari et al., 1995, or an active fragment thereof, such as the zinc finger containing part (amino acids 387-790 of human A20, amino acids 369-775 of murine A20).
• the terms protein and polypeptide as used in this appliction are interchangeable. Polypeptide refers to a polymer of amino acids and does not refer to a specific lenth of the molecule: This term also includes post-translational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation.
• IKB superrepressor (l ⁇ B s ) means a nondegradable mutant form of B- ⁇ , with S32A 0 and S36A mutations, that locks NF- ⁇ B in a cytosolic protein complex, preventing its nuclear action.
• Example 1 Generation of the ABIN adenovirus 6
• AdABIN replication-deficient E1-deleted adenovirus type ⁇
• AdRR ⁇ replication-deficient E1-deleted adenovirus type ⁇
• the infectious unit titer was determined in a plaque assay that was performed on confluent HEK293 cells with different virus dilutions. The plaques of lysed cells were counted and calculated as 0 plaque forming units (pfu) per ml virus stock.
• AdABIN was tested for the expression of the transgene in the BWTG3 hepatoma cell line (Szpirer and Szpirer, 1976). Infection with AdABIN was performed at a multiplicity of infection (moi) 100:1. Cells were incubated with virus in a minimal volume of serum- free medium for 2 hours, after which serum containing medium was added for overnight incubation. For controlling the expression of ABIN, cells were lysed 24 hours after infection and analyzed by SDS-PAGE and immunoblotting with HRP-coupled anti E-tag 5 antibodies (Amersham). Infection with AdABIN resulted in clear expression of ABIN (data not shown).
• coverslips were mounted ⁇ with Vectashield (Vector Laboratories), and analysed with a Leica DM-IL microscope. This revealed that the efficiency of infection was more than 90 %, and that ABIN was exclusively localized in the cytoplasm (data not shown).
• Example 3 Inhibition of TNF-induced NF- ⁇ B dependent gene expression in vitro 0 by AdABIN
• Example 4 ABIN does not significantly inhibit TNF mediated cell death.
• AdABIN had an effect on TNF mediated cell death
• AdRR ⁇ or mock infected BWTG3 cells were incubated with TNF, or combinations of TNF and cycloheximide (CHX). More specifically, 24 hours after infection, cells were seeded in 96 wel plates at a density of 4 x 10 4 cells per well. Another 24 hours later, cells were ⁇ stimulated with dilutions of mTNF alone, or with a combination of dilutions of TNF and a constant concentration (10 ⁇ g/ml) CHX.
• Example 5 Expression of ABIN in the liver of AdABIN infected mice 5
• AdABIN was tested for the expression of the transgene and its biological activity in vivo by injecting C57BL/6 mice with 2.5x10 9 pfu AdABIN into the tail vein. 1-6 days after infection, mice were sacrificed and livers were isolated. One third of the liver was cut in small pieces and homogenized by douncing in lysis buffer (1 % NP-40, 200 mM NaCl, 10 mM Tris-CI pH 7.5, 5 mM EDTA, 10 % glycerol) supplemented with 0.1 mM 0 aprotinine, 1 mM PMSF, and 1 mM gluthation.
• lysis buffer (1 % NP-40, 200 mM NaCl, 10 mM Tris-CI pH 7.5, 5 mM EDTA, 10 % glycerol
• Example 6 Inhibition of TNF/GalN-induced NF- ⁇ B activation in vivo by AdABIN and byAd-l ⁇ B s
• mice were killed and liver homogenates were prepared.
• l ⁇ B ⁇ expression was analyzed by SDS-PAGE and immunoblotting with polyclonal anti- IKBOC antibody (Santa Cruz) (Fig. 1 , upper panel).
• l ⁇ B ⁇ was almost complete degraded after 0.5 h TNF/GalN treatment, and reappeared after 1.5 h. This reappearance is most likely due to de novo synthesis of l ⁇ B ⁇ in response to TNF. Strong l ⁇ B ⁇ signals were visible in the Ad Bs infected mice, in which the expression of the transgene masked the expression of the endogenous gene.
• Pieces of murine liver were homogenized by Douncing in 1 ml of swelling buffer (10 mM Hepes pH 7.5, 10 mM KCl, 1 mM MgCI2, 5% glycerol, O. ⁇ mM EDTA pH7.5, 0.1 mM EGTA pH 7.5, 2 mM Pefablock, 0.5 mM DTT, 0.15 lU/ml aprotinin). After 15 min incubation on ice, 65 ⁇ l of a 10% NP-40 solution was added, followed by centrifugation at maximum speed in an eppendorff centrifuge for 15 min.
• swelling buffer 10 mM Hepes pH 7.5, 10 mM KCl, 1 mM MgCI2, 5% glycerol, O. ⁇ mM EDTA pH7.5, 0.1 mM EGTA pH 7.5, 2 mM Pefablock, 0.5 mM DTT, 0.15 lU/ml aprotinin.
• the pellet was resuspended in 100 ⁇ l of nuclear extraction buffer (20 mM Hepes pH 7.5, 1% NP-40, 1 mM MgCI2, 400 mM NaCl, 10 mM KCL, 20% glycerol, 0.5 mM EDTA pH7. ⁇ , 0.1 mM EGTA pH 7.6, 2 mM Pefabloc, 0.5 mM DTT, 0.15 lU/ml aprotinin). After centrifugation for 15 min at maximum speed in an eppendorff centrifuge, supernatants were stored at -70 until use.
• nuclear extraction buffer 20 mM Hepes pH 7.5, 1% NP-40, 1 mM MgCI2, 400 mM NaCl, 10 mM KCL, 20% glycerol, 0.5 mM EDTA pH7. ⁇ , 0.1 mM EGTA pH 7.6, 2 mM Pefabloc, 0.5 mM DTT, 0.15 lU/m
• AdABIN as well as Adl ⁇ B s strongly prevented TNF/GalN-induced nuclear translocation and DNA-binding of NF- ⁇ B (Fig. 1, lower panel). From this, we can conclude that adenoviral infection with AdABIN or AdkB ⁇ inhibits NF- ⁇ B TNF/GalN-induced NF- B activation in mouse liver.
• Example 7 Inhibition of TNF/GalN-induced lethal hepatitis by ABIN
• AdRR ⁇ mice showed a strong decrease in body temperature
• animals were sacrificed for histology and biochemistry studies.
• Blood was collected from AdRR ⁇ and AdABIN mice, and livers were prepared for further analysis.
• the concentration of alanine aminotransferase (ALT) in the blood after TNF/GalN injection was determined using an enzymatic/colorimetric kit (Sigma), and served as a parameter for liver necrosis (Reutter et al., 1968).
• liver homogenates were added and a biotinylated detection Ab specific for the nucleosome subparticle of histones H2A, H2B, and DNA was administered. Detection was performed with alkaline phosphatase-conjugated streptavidin (Sanvertech, Boechout, Belgium) and substrate (Sigma). Signals obtained with samples from TNF/GalN-treated mice were set as 100%. These experiments show that TNF/GalN- induced DNA fragmentation is significantly reduced in AdABIN infected animals (Fig. 6). Caspase activation was revealed by the hydrolysis of Ac-DEVD-amc upon incubation with liver cell extracts.
• liver homogenate was incubated in 200 ul cell free system buffer (10 mM Hepes pH 7.5, 220 mM Mannitol, 68 mM Sucrose, 2 mM NaCl, 2 mM MgCI 2 , 2.5 mM KH 2 PO 4 , 10 mM DTT) in the presence of 50 ⁇ M Ac- DEVD.amc (Peptide Institute; Osaka, Japan), for 60 min at 30 degrees.
• 200 ul cell free system buffer (10 mM Hepes pH 7.5, 220 mM Mannitol, 68 mM Sucrose, 2 mM NaCl, 2 mM MgCI 2 , 2.5 mM KH 2 PO 4 , 10 mM DTT
• TNF/GalN-induced lethal hepatitis is associated with total tissue destruction of the parenchymal tissue, influx of erythrocytes (hemorrhage) at the site of the sinusoids and apoptosis and necrosis of the hepatocytes.
• erythrocytes hemorrhage
• a massive influx of macrophages and neutrophils in the liver can be observed.
• Livers of AdABIN pretreated mice show better preservation of the tissue integrity and nearly no hemorrhage.
• hepatocyte cell death, and hemorrhage infiltration of white blood cells was only partially reduced by in vivo expression of ABIN.
• adenoviral administration of a dominant IKBA superrepressor does not protect against TNF/GalN induced lethality.
• adenoviral administration of ABIN completely protected the mice (Fig. 9).
• ABIN in contrast to l ⁇ B , inhibits NF- ⁇ B activation upstream of the IKK complex.
• Fas is an apoptosis-signalling cell surface molecule that triggers cell death upon specific ligand or antibody binding.
• Treatment of mice with an anti-Fas antibody causes fulminant hepatic failure due to massive apoptosis (Ogasawara et al., 1993).
• anti-Fas does not lead to NF- ⁇ B activation and an inflammatory response in the liver, but rather induces induces a direct apoptotic response.
• mice were injected with AdRR ⁇ or AdABIN as described above, and three days later (i.v.) injected with 10 ⁇ g anti-Fas (Pharmingen). Both AdRR ⁇ and AdABIN pretreated mice died within 3-5 hours following administration of anti-Fas (Fig. 10). This demonstrates that ABIN does not significantly influence the signalling pathway of Fas-mediated apoptosis.
• G562-G561. Dixit V.M., Green, S., Sarma, V., Holzman, L.B., Wolf, F.W., O'Rourke, K., Ward,

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