WO2001047530A1

WO2001047530A1 - A method of therapy and prophylaxis of inflammation

Info

Publication number: WO2001047530A1
Application number: PCT/AU2000/001610
Authority: WO
Inventors: Mimi Lai-Kuan Tang
Original assignee: Murdoch Childrens Research Institute
Priority date: 1999-12-28
Filing date: 2000-12-28
Publication date: 2001-07-05
Also published as: AU2493301A

Abstract

The present invention relates generally to a method for the treatment and/or prophylaxis of inflammation and, more particularly, to a method for reducing, retarding or otherwise inhibiting inflammation or the risk of developing inflammation. The present invention is particularly directed to the use of oligonucleotides to inhibit or otherwise down-regulate the level of pro-inflammatory cytokines whereby inflammation or the risk of developing inflammation is reduced, retarded or otherwise inhibited. The method of the present invention is particularly useful in the treatment and/or prophylaxis of disease conditions such as allergic airways inflammation.

Description

A method of therapy and prophylaxis of inflammation

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to in this specification are collected alphabetically at the end of the description.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

Allergic airways disease is a major cause of morbidity and mortality. Inflammation of the airway lining is the central feature in the pathogenesis of allergic airways diseases such as asthma and correlates with disease severity and activity. Currently, treatment for allergic airways disease relies upon the use of non-specific anti-inflammatory agents such as corticosteroids. Corticosteroids function by reducing inflammation via the non-specific inhibition of T cell functions. Such therapy is only partially effective in severe or chronic disease conditions and administering high doses of these agents is often associated with significant systemic side effects including growth suppression, osteoporosis, cataracts and other features of Cushing syndrome.

Accordingly, there is a need to develop therapeutic strategies which more selectively target inflammatory conditions such as asthma. In work leading up to the present invention, the inventors have determined that the administration of nucleic acid molecules, such as antisense oligonucleotides directed to pro-inflammatory cytokines, which act to reduce the level and/or functional activity of pro-inflammatory cytokines, achieves a more selective anti-inflammatory response, thereby minimising the incidence of side effects which are experienced when non-specific anti-inflammatory agents, such as corticosteroids, are used.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The subject specification contains nucleotide sequence information prepared using the programme Patentln Version 2.0, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210> 1, <210>2, etc). The length, type of sequence (DNA, etc) and source organism for each nucleotide sequence are indicated by information provided in the numeric indicator fields <211 > , <212> and < 213 > , respectively. Nucleotide sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (e.g. <4001 > 1, <400> 2, etc).

One aspect of the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof for a time and under conditions sufficient to reduce, inhibit or otherwise down-regulate the level and/or functional activity of a cytokine or derivative, mimetic, analogue or homologue thereof.

Another aspect of the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue. or homologue thereof for a time and under conditions sufficient to reduce, inhibit or otherwise down-regulate the level and/or functional activity of a pro- inflammatory cytokine or derivative, mimetic, analogue or homologue thereof.

Still another aspect of the present invention more particularly provides a method from the treatment and/or prophylaxis of inflammation in a mammal said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof for a time and under conditions sufficient to reduce, inhibit or otherwise down-regulate the level and/or functional activity of IL-4, IL-5 and/or IL-13 or derivative, mimetic, analogue or homologue thereof.

Still yet another aspect of the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a second nucleic acid molecule which is translatable to all or part of an amino acid sequence of a cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or its derivative, mimetic, analogue or homologue. Yet another aspect of the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a RNA molecule which is translatable to all or part of an amino acid sequence of a cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or derivative, mimetic, analogue or homologue.

A further aspect of the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with an RNA molecule which is translatable to all or part of an amino acid sequence of a cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or its derivative, mimetic, analogue or homologue.

Another further aspect of the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with an RNA molecule which is translatable to all or part of an amino acid sequence of IL-4, IL-5 and/or IL-13 or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said IL-4, IL-5 and/or IL-13 whereby said interaction facilitates a reduction in the level and/or functional activity of said IL-4, IL-5 and/or IL-13 or its derivative, mimetic, analogue or homologue. Yet another further aspect of the present invention provides a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a mRNA molecule which is translatable to all or part of an amino acid sequence of a pro- inflammatory cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or its derivative, mimetic, analogue or homologue.

Still yet another further aspect of the present invention provides a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a mRNA molecule which is translatable to all or part of an amino acid sequence of a pro- inflammatory cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or its derivative, mimetic, analogue or homologue.

Another aspect of the present invention provides a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of a C-5 propyne phosphorothioate oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a mRNA molecule which is translatable to all or part of an amino acid sequence of a pro-inflammatory cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates the reduction in the level and/or functional activity of said pro-inflammatory cytokine or its derivative, mimetic, analogue or homologue. Yet another aspect of the present invention contemplates a method for the treatment and/or prophylaxis of an allergic condition in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a second nucleic acid molecule which is translatable to all or part of an amino acid sequence of a pro-inflammatory cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates reduction in the level and/or functional activity of a pro-inflammatory cytokine or its derivative, mimetic, analogue or homologue.

Still another aspect of the present invention provides a composition for use in reducing, retarding or otherwise inhibiting inflammation in a mammal said composition comprising nucleic acid molecules or derivatives, chemical equivalents, mimetics, analogues or homologues thereof which nucleic acid molecule reduces, inhibits or otherwise down- regulates the level and/or functional activity of a pro-inflammatory cytokine or derivative, mimetic, analogue or homologue thereof and one or more pharmaceutically acceptable carriers and/or diluents. The composition may also comprise two or more different oligonucleotides or one or more oligonucleotides and another compound.

The present invention further extends to the use of the subject nucleic acid molecules in the manufacture of a medicament for the treatment or prophylaxis of inflammation in a mammal.

Another aspect of the present invention provides antisense oligonucleotides or derivatives, chemical equivalents, mimetics, analogues or homologues thereof, which oligonucleotides are directed to RNA sequences encoding one or more pro-inflammatory cytokines or derivatives, mimetics, analogues or homologues thereof, capable of reducing, retarding or otherwise inhibiting inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation of the airway reactivity responses in mice treated with IL-4 antisense oligonucleotides, control oligonucleotides or OVA alone.

Figure 2 is an image of IL4 and β-actin mRNA expression in 3T3 cells treated with IL4 antisense ODN or control. 3T3 cells were incubated with GSV alone (lanes 1 and 6), 500nM IL4 antisense ODN in 2.5 μg/ml GSV (lanes 2 and 7), 500nM antisense control in 2.5 μg/ml GSV (lanes 3 and 8). Expression of IL4 mRNA (lanes 1-3) and β-actin mRNA (lanes 6-8) were assessed by RT/PCR. Negative (lane 4) and positive IL4 cDNA (lane 5) controls are also shown. Incubation of 3T3 cells with antisense ODN inhibited IL4 mRNA expression without affecting β-actin expression. IL4 mRNA and β-actin mRNA expression were not altered by the antisense control ODN.

Figure 3 is an image of cytokine mRNA expression in splenocytes treated with IL4 antisense ODN or control. Splenocytes from C57BL/6 mice were isolated with incubated (2xl06/ml) for 6 hours in the presence of GSV alone (lanes 1-5), 500nM IL4 antisense ODN in GSV (lanes 6-10) or 500nM control ODN (lanes 11-15). Expression of IL4 mRNA (lanes 1, 6, 11), IL5 mRNA (lanes 2, 7, 12), IL2 mRNA (lanes 3,8, 13), IFNγ mRNA (lanes 4, 9, 14) and β-actin mRNA (lanes 5, 10, 15) were assessed by RT-PCR. IL4 mRNA expression was inhibited in the presence of IL4 antisense ODN but not control ODN.

Figure 4 is a graphical representation of the effect of in vivo IL4 antisense therapy on airway hyperresponsiveness in an animal model of asthma. C57BL/6 mice were sensitised (i.p.) with 1 μg ovalbumin/alum and then challenged with nebulised ovalbumin (1 % w/v in saline) daily for 20 min from days 14-20. Mice were treated with IL4 antisense ODN (0.5 mg/dose), control ODN (0.5 mg/dose) or saline alone twice daily on days 14-20. On day 21, airway hyperresponsiveness was assessed by whole body plethysmography (Buxco system). Airway hyperresponsiveness was significantly inhibited by treatment with IL4 antisense ODN but not control ODN. DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the observation that anti-inflammatory treatment can be more selectively targeted when the level of pro-inflammatory cytokines is down-regulated via the administration of nucleic acid molecules, such as antisense oligonucleotides directed to the RNA molecules encoding these cytokines, which act to reduce the level and/or functional activity of pro-inflammatory cytokines. Furthermore, the inventors have investigated the aerosol administration of antisense oligonucleotides and have sought to show that this is effective in reducing inflammation of the airways. In one particular example, the inventors have shown delivery of aerosol nebulisation of oligonucleotide into the lymphocyte regions of the submucosa.

Accordingly, one aspect of the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof for a time and under conditions sufficient to reduce, inhibit or otherwise down-regulate the level and/or functional activity of a cytokine or derivative, mimetic, analogue or homologue thereof.

More particularly, the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof for a time and under conditions sufficient to reduce, inhibit or otherwise down-regulate the level and/or functional activity of a pro- inflammatory cytokine or derivative, mimetic, analogue or homologue thereof.

Reference to a "nucleic acid molecule" should be understood as a reference to any molecule comprising a nucleotide sequence which molecule either directly or indirectly facilitates reduction, inhibition or other form of down-regulation of the level and/or functional activity of a cytokine, and more particularly of a pro-inflammatory cytokine. Examples of nucleic acid molecules which fall within the scope of this definition include, but are not limited to:

oligonucleotides, such as antisense oligonucleotides, which interact with a nucleic acid sequence encoding a cytokine, a cytokine receptor, a transcription or translation regulatory factor or a promoter thereby inhibiting expression of the cytokine, cytokine receptor, the transcription or translation regulatory factor or the functioning of the promoter.

- nucleic acid molecules which encode all or part of a cytokine, cytokine receptor or a transcription or translation regulatory factor wherein co- ^{~ "} suppression of the expression of the cytokine, cytokine receptor or transcription or translation regulatory factor is induced.

- ribozymes, the hybridisation component of which interacts with a nucleic acid molecule encoding a cytokine, cytokine receptor, or transcription or translation regulatory factor or a promoter and the catalytic component of which cleaves this nucleic acid molecule.

These nucleic acid molecules may function directly to down-regulate the level and/or functional activity of a cytokine by interacting with a cytokine gene or the transcription product of that gene. Alternatively, the nucleic acid molecule may function indirectly to down-regulate the level and/or functional activity of a cytokine by modulating the level and/or functional activity of a molecule other than the cytokine, which molecule thereby either directly or indirectly down-regulates the level and/or functional activity of the cytokine. For example, down-regulating the translation of IL-13 mRNA by introducing an antisense IL-13 oligonucleotide or a ribozyme is an example of direct down-regulation of a cytokine. Down-regulating the translation of IL-13 receptor mRNA utilising an antisense IL-13 receptor oligonucleotide or ribozyme is an example of indirect down-regulation of IL-13 functional activity since in the absence of cell surface IL-13 receptor expression, IL- 13 is unable to execute its biological function. It should be understood that reference to a reduction in the "level" of a cytokine is a reference to a reduction in the level of functionally active cytokine. Accordingly, where the action of a nucleic acid molecule, such as an antisense oligonucleotide, results in the translation of a partial amino acid sequence of the cytokine (as opposed to completely inhibiting the production of a cytokine translation product), where that partial amino acid sequence lacks pro-inflammatory functional activity, the level of said cytokine will have been effectively reduced. Similarly, where a ribozyme cleaves a cytokine mRNA molecule and partial translation products are nevertheless produced, where that partial translation product lacks pro-inflammatory functional activity, the level of the cytokine will have been reduced. The production of a partial but non-functional cytokine sequence therefore falls within the scope of the meaning of a reduction in the level of the cytokine.

Reference to the "functional activity" of a cytokine should be understood as a reference to any one or more of the pro-inflammatory activities which that cytokine either directly or indirectly induces, enhances or otherwise facilitates. The down-regulation of a cytokine 's functional activity may be achieved either by modulating the cytokine molecule itself or by modulating some other molecule, such as a receptor, which thereby inhibits the cytokine from transducing its biological action.

Reference to a "pro-inflammatory" cytokine should be understood as a reference to any soluble molecule such as, but not limited to, a cytokine, chemokine or hormone, which induces, enhances or otherwise facilitates any one or more aspects of an inflammatory response. For example, reference to "pro-inflammatory cytokine" includes, but is not limited to, reference to cytokines which facilitate the differentiation of ThO cells to Th2 cells, or which otherwise facilitate a Th2 response. Said cytokine may mediate its functional activity either by direct or indirect means. For example, where a cytokine binds to a cell surface receptor and thereby induces the release of an inflammatory mediator, such as histamine, its activity is direct. Where the cytokine acts on a cell to induce the production of other regulatory molecules, such as other cytokines, which other cytokines induce, for example, the release of an inflammatory mediator, the functional activity is indirect. The method of the present invention should be understood to extend to - l i ¬

the reduction, inhibition or other down-regulation of the level and/or functional activity of cytokines which are both directly and indirectly pro-inflammatory. Preferably, said cytokines are IL-4, IL-5 and/or IL-13.

Accordingly the present invention more particularly provides a method from the treatment and/or prophylaxis of inflammation in a mammal said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof for a time and under conditions sufficient to reduce, inhibit or otherwise down-regulate the level and/or functional activity of IL-4, IL-5 and/or IL-13 or derivative, mimetic, analogue or homologue thereof.

In a preferred embodiment of the present invention, the nucleic acid molecule is an oligonucleotide, such as, but not limited to, an antisense oligonucleotide which reduces, inhibits or otherwise down-regulates the level and/or functional activity of a cytokine. Without limiting the invention in any way the oligonucleotide acts by interacting with and thereby interfering with the expression of either the cytokine mRNA or the mRNA of molecules which directly or indirectly facilitate cytokine expression and/or functional activity such as, but not limited to, the cytokine promoter, transcription or translation regulatory factors or the cytokine receptor.

Accordingly, the present invention preferably contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a second nucleic acid molecule which is translatable to all or part of an amino acid sequence of a cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or its derivative, mimetic, analogue or homologue. More preferably, the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a RNA molecule which is translatable to all or part of an amino acid sequence of a cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or derivative, mimetic, analogue or homologue.

Still more preferably, the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with an RNA molecule which is translatable to all or part of an amino acid sequence of a cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or its derivative, mimetic, analogue or homologue.

In accordance with this embodiment of the present invention, reference to an "oligonucleotide" should be understood as a reference to any molecule comprising a sequence of nucleotides, or derivative, mimetic, equivalent, homologue or analogue thereof, the functional activity of which includes the interaction of at least part of said nucleotide sequence with one or more regions of a target RNA sequence wherein the level of the amino acid sequence encoded by said RNA sequence is reduced. Reference to a reduction in the level of said amino acid sequence should be understood as including reference to the prevention of the initial synthesis of said amino acid sequence. The oligonucleotides may comprise any number of nucleotides, however, 2 to 70 nucleotides is preferred, more preferably between 2 and 50 nucleotides and still more preferably between 5 and 25 nucleotides. The oligonucleotides of the present invention may also comprise non-nucleic acid components. For example, the oligonucleotide may comprise a non- nucleic acid detection tag or some other non-nucleic acid component which facilitates the functioning of the oligonucleotide. The nucleotides comprising the oligonucleotide may be deoxyribonucleotides and/or ribonucleotides. The antisense oligonucleotides of the present invention may be of any origin including naturally occurring, recombinantly produced or synthetically produced.

Without limiting the invention to any one theory or mode of action, it is thought that the oligonucleotides of the present invention function by hybridising to the RNA sequence encoding the pro-inflammatory cytokine, thereby preventing translation of the RNA sequence to an amino acid polypeptide. In this regard, oligonucleotides of the type disclosed in the present invention are often referred to as "antisense oligonucleotides".

The cytokines to which the method of the present invention is directed are preferably pro- inflammatory cytokines such as, but not limited to, IL-4, IL-5 and/or IL-13. In this regard, the molecules which facilitate the expression and/or functional activity of IL-4, IL- 5 and/or IL-13 include the IL-4, IL-5 and IL-13 receptors and the IL-4, IL-5 and IL-13 promoters.

According to this preferred embodiment, the present invention contemplates a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with an RNA molecule which is translatable to all or part of an amino acid sequence of IL-4, IL-5 and/or IL-13 or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said IL-4, IL-5 and/or IL-13 whereby said interaction facilitates a reduction in the level and/or functional activity of said IL-4, IL-5 and/or IL-13 or its derivative, mimetic, analogue or homologue. Reference to "RNA sequences" should be understood as a reference to any molecule comprising ribonucleotides. Accordingly, the phrase "RNA sequences" is intended to encompass, but is not limited to, both primary RNA transcripts and messenger RNA. Preferably, said RNA sequence is messenger RNA (referred to herein as "mRNA").

Accordingly, there is provided a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a mRNA molecule which is translatable to all or part of an amino acid sequence of a pro-inflammatory cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or its derivative, mimetic, analogue or homologue.

Preferably, said pro-inflammatory cytokine is IL-4, IL-5 and/or IL-13.

Accordingly, the present invention preferably provides a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a mRNA molecule which is translatable to all or part of an amino acid sequence of IL-4, IL-5 and/or IL-13 or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said IL-4, IL-5 and/or IL-13 or its derivative, mimetic, analogue or homologue.

Reference to "functional derivatives, chemical equivalents, mimetics, analogues or homologues" of a cytokine or nucleic acid molecule should be understood to include reference to fragments, parts, portions, chemical equivalents, mutants and mimetics from natural, synthetic or recombinant sources exhibiting any one or more of the functional activities of a cytokine or nucleic acid molecule, respectively . Derivatives may be derived from insertion, deletion or substitution of amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein, for example, random insertion. Deletional variants are characterised by the removal of one or more amino acids from the sequence.

Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intra sequence insertion of single or multiple amino acids. In addition to cytokine derivatives which may be naturally expressed in vivo, the method of the present invention also contemplates recombinantly produced cytokine derivatives, the encoding nucleic acid sequence of which may have been introduced into the body for the treatment or prophylaxis of an unrelated disease condition for example, the side effects of which treatment include airway inflammation. In this situation, localised treatment in the airways, according to the method of the present invention, is required to reduce or prevent the development of inflammation.

Derivatives, chemical equivalents, mimetics, analogues or homologues should also be understood to include fragments having particular parts of the molecule fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. Analogues contemplated herein include, but are not limited to, modification to side chains incorporating of a natural amino acids and/or the derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogue.

Derivatives of nucleic acid sequences may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules or non-nucleic acid molecules. The derivatives of said nucleic acid molecules include fragments having particular epitopes or parts of the nucleotide or nucleic acid sequence fused to other proteinaceous or non-proteinaceous molecules. Analogues contemplated herein include, but are not limited to, modifications to the nucleotide or nucleic acid molecules such as modifications to its chemical makeup or overall conformation. This includes, for example, modification to the manner in which the nucleotide or nucleic acid molecule interacts with other nucleotides or nucleic acid sequences, for example, at the level of backbone formation or complementary base pair hybridisation. The term "functional derivatives" should be understood to encompass molecules exhibiting any one or more of the functional activities of a cytokine or nucleic acid molecule, such as for example, molecules which are obtained following natural product screening.

Reference to "interacting" should be understood as a reference to any form of interaction such as ^"Hybridisation between complementary nucleic acid base pairs or some other form of interaction such as the formation of bonds between any portion of the oligonucleotide with any portion of the RNA molecule. Said interaction may occur via the formation of bonds such as covalent bonds, hydrogen bonds, van der Waals forces or via any other mechanism of interaction.

Without limiting the present invention to any one theory or mode of action, antisense therapy is a tool which can be used to specifically and selectively inhibit the production of a protein. If the nucleic acid sequence of a gene encoding a protein of interest is known, specific oligonucleotides can be designed to interact with the corresponding mRNA thereby inhibiting the production of protein. Antisense oligonucleotides may inhibit protein expression by any one or more of a variety of mechanisms including, but not limited to, translational arrest, inhibition of RNA processing and/or promotion of RNA degradation by RNase H. The more potent antisense effects are obtained when RNase H is the mechanism of action rather than non-cleaving steric blockade of RNA processing and translation.

Still without limiting the present invention to any one mode of action, binding of antisense oligonucleotides to their target mRNA is related to the binding affinity of an oligonucleotide, its length and RNA accessibility. While most regions of the mRNA can be targeted by antisense oligonucleotides, some sites may not be accessible because of RNA secondary structure or may not impart sufficient binding affinity to support oligonucleotide hybridisation. Therefore, several sites of a mRNA molecule should be examined to locate an active oligonucleotide and the sequence of an effective oligonucleotide can be determined by screening potential sequences. Suitable techniques for achieving these aims would be well known to those skilled in the art. In this regard, in a preferred embodiment, the antisense oligonucleotides are preferably targeted to a region of the IL-4 mRNA, IL-5 mRNA, IL-13 mRNA, the promoter region of the IL-4, IL-5 or IL-13 gene or the IL-4, IL-5 or IL-13 receptor mRNAs.

Modification of the heterocycle of oligonucleotides to incorporate C-5 propynyl pyrimidiϊies produces C-5 propyne phosphorothioate oligonucleotides which have increased mRNA affinity and antisense effects without interfering with RNase H activity. This increased antisense activity reduces the number of sequences that need to be screened to identify an active antisense oligonucleotide, and reduces the minimal oligonucleotide length required for specificity of binding. Assuming a random distribution of sequences in RNA, any 13 residue sequence should occur only once in a cellular RNA population, and if the non-random nature of mammalian RNA sequences is taken into account, an 1 lmer oligonucleotide should identify and bind to a unique sequence. Accordingly, the oligonucleotides of the present invention are preferably C-5 propyne phosphorothioate oligonucleotide.

Accordingly, the present invention provides a method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of a C-5 propyne phosphorothioate oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a mRNA molecule which is translatable to all or part of an amino acid sequence of a pro-inflammatory cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates the reduction in the level and/or functional activity of said pro-inflammatory cytokine or its derivative, mimetic, analogue or homologue.

Preferably said cytokine is IL-4, IL-5 and/or IL-13.

In one preferred embodiment of the present invention said antisense oligonucleotides interact with IL-4 mRNA and even more preferably with the 5' region of IL-4 mRNA

In another preferred embodiment said oligonucleotides interact with IL-5 mRNA and even more preferably with the 5' region of the IL-5 mRNA.

In another ^"preferred embodiment said oligonucleotides interact with IL-13 mRNA and even more preferably with the 5' region of the IL-5 mRNA.

Reference to the treatment or prophylaxis of "inflammation" should be understood as a reference to the treatment of any disease or other condition, the symptoms, cause or side effects of which include inflammation or the occurrence of one or more components or steps of an inflammatory pathway but which may not have yet resulted in inflammation occurring. This includes, for example, inflammatory conditions which occur as a side effect of a treatment regime for an unrelated disease condition. Examples of inflammatory conditions includes, but is not limited to, an allergic condition, eczema, bronchitis, bronchiolitis, acute respiratory distress syndrome, cystic fibrosis or allergic rhinitis (hay fever). Preferably, said inflammation is an allergic condition such as an allergic airways disease or an allergic skin disorder such as eczema and most preferably an allergic airways disease such as extrinsic or intrinsic asthma or allergic bronchopulmonary aspergillasis.

According to this preferred embodiment, the present invention contemplates a method for the treatment and/or prophylaxis of an allergic condition in a mammal, said method comprising administering to said mammal an effective amount of an oligonucleotide or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a second nucleic acid molecule which is translatable to all or part of an amino acid sequence of a pro-inflammatory cytokine or functional derivative, mimetic, analogue or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates reduction in the level and/or functional activity of a pro-inflammatory cytokine or its derivative, mimetic, analogue or homologue.

More preferably said allergic condition is an allergic airways disease. Even more preferably said cytokines are IL-4, IL-5 and/or IL-13.

Most preferably said second nucleic acid molecule is mRNA.

The method of the present invention facilitates the subject inflammation being reduced, retarded or otherwise inhibited. Reference to "reduced, retarded or otherwise inhibited" should be understood as a reference to inducing or facilitating the partial or complete inhibition of any one or more components of the inflammatory response. In this regard, it should be understood that an inflammatory response is a complex response comprising numerous physiological events which often occur simultaneously such as vasodilation, inflammatory mediator synthesis, chemotaxis, complement activation, mucus secretion, development of oedema or bronchial spasm (where the inflammatory condition is inflammation of the airways). Said inhibition may occur by either direct or indirect mechanisms. For example, the inhibition of a given cytokine may result in the inhibition of vasodilation. Alternatively, the inhibition of a given cytokine may directly result in the inhibition of the synthesis of a further molecule which thereby results in the inhibition of vasodilation.

Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or prevent or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity of onset of a particular condition. "Treatment" may also reduce the severity of an existing condition or the frequency of acute attacks (for example, reducing the frequency of acute asthma attacks).

The subject of the treatment or prophylaxis is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig) companion animal (e.g. dog, cat) laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster) captive wild animal (e.g. fox, deer). Preferably the mammal is a human or primate. Most preferably the mammal is a human. Although the present invention is exemplified utilising a murine model, this is not intended as a limitation on the application of the method of the present invention to other species, in particular, humans.

In accordance with the method of the present invention, more than one type of oligonucleotide may be administered (that is, oligonucleotides directed to more than one cytokine) or the oligonucleotide may be co-administered with another compound such as, but not limited to, a compound which facilitates the inhibition of mRNA translation or a compound used to treat other conditions which the mammal may be simultaneously experiencing. For example, the oligonucleotide may be co-administered with an RNase H degradation molecule. Although oligonucleotides may inhibit protein expression by a variety of mechanisms, without limiting the present invention in any way, one of the more potent oligonucleotide effects is obtained when RNase H degradation of mRNA is the mechanism of action. Accordingly, the co-administration of an oligonucleotide with an RNase H degradation molecule may be useful. Alternatively, the oligonucleotide may be co-administered with, in the case of airway inflammation, a compound which treats acute symptoms such as a bronchodilator (beta 2 agonists or anticholinergic agents) or uncolytic. By "co-administered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the various types of oligonucleotides or the oligonucleotide and the other compound. These molecules may be administered in any order.

Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeally, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via IN drip patch and implant. Preferably, the route of administration is a route which permits directed delivery of the antisense oligonucleotide. For example, aerosol administration (such as by nebulisation) into the airways or delivery to the nose via an aqueous or aerosol spray permits directed delivery to the airways region, in contrast to systemic delivery which results in delivery to the whole body. Nevertheless, systemic delivery may be required under certain circumstances. Where the condition being treated is a skin condition, oligonucleotides can be delivered, for example, via topical cream application.

Where the inflammation which is the subject of treatment or prophylaxis is an allergic airways disease, delivery of the oligonucleotide to the airway, for example as an aerosol via nebulisation, is an ideal approach since this maximises delivery to the airway where the inflammation has occurred and minimises systemic delivery which may be associated with side effects. Delivery of oligonucleotides to the airway is thought to be facilitated by the presence of surfactant which lines the airway epithelium. A component of the surfactant promotes the entry of DNA into the cells. Accordingly, oligonucleotides can penetrate airway epithelium and enter the nuclei of cells in the surrounding tissue. In this regard, the oligonucleotides of the present invention are preferably delivered as an aerosol where the disease condition being treated is asthma.

The term "aerosol" is used in its most general sense to include any formulation capable of administration via nasal, pharyngeal, tracheal, bronchial or oral passages. Aerosols generally comprise particles of liquid or solid suspended in a gas or vapour. Conveniently, the aerosol is a colloidal system such as a mist in which the dispersion medium is a gas. The method of administering the aerosol formulation is not critical and may be achieved using a nasal spray hand pump, electric pump, pressurised dispenser, nasal drip or other convenient means. Alternatively, the formulation may be administered in a dry powder delivery system. It should be understood that the method of the present invention extends to direct application of said formulations to intra nasal surfaces. In a particularly preferred embodiment, the aerosol is delivered at a rate of from about 1 to about 20 litres/min. and preferably from about 2 to about 15 litres/min. at a droplet size of from about 0.1 to about 10 μm and more preferably from about 0.1 to about 6 μm. Conveniently, a stock solution of material is prepared at a concentration of from about 0.5 to about 20 mg/ml or more preferably from about 1.0 to about 10 mg/ml of carrier solution. It will also be understood by those of ordinary skill in the art that in some circumstances, it may be more appropriate to use the minimum dose required to maintain a therapeutic effect in order to minimise any potential side effects. The lowest effective dose may be determined by starting with a maximum dose and then 'back titrating' the dose according to sound medical judgment to the lowest dose at which a clinical effect can still be maintained.

The formulation is administered in a therapeutically effective amount. A therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular conditions being treated, the severity of the condition and individual patient parameters including age, physical conditions, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.

Generally, daily nebulised doses of formulation will be from about 0.01 μg/kg per day to 1000 mg/kg per day. Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localised delivery route) may be employed to the extent patient tolerance permits. A single dose may be administered or multiple doses may be required on an hourly, daily, weekly or monthly basis. Effective amounts of formulation vary depending on the individual but may range from about 0.1 μg to about 20 mg, alternatively from about 1 μg to about 10 mg and more preferably from about 1 μg to 5 mg per dose.

Accordingly, another aspect of the present invention provides a composition for use in reducing, retarding or otherwise inhibiting inflammation in a mammal said composition comprising nucleic acid molecules or derivatives, chemical equivalents, mimetics, analogues ^"or homologues thereof which nucleic acid molecule reduces, inhibits or otherwise down-regulates the level and/or functional activity of a pro-inflammatory cytokine or derivative, mimetic, analogue or homologue thereof and one or more pharmaceutically acceptable carriers and/or diluents. The composition may also comprise two or more different oligonucleotides or one or more oligonucleotides and another compound.

Compositions suitable for use according to this aspect of the present invention include aerosols, as hereinbefore described, together with compositions suitable for use according to any other method of administration. For example, the composition may comprise a lipsomal carrier or other compound which facilitates oligonucleotide entry into cells such as, but not limited to, TSX50, Lipofectamine or Superfect.

Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. They must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by, for example, filter sterilization or sterilization by other appropriate means. Dispersions are also contemplated and these may be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, a preferred method of preparation includes vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution.

When the active ingredients are suitably protected, they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1 % by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0. lng and 2000 mg of active compound. The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or other flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl, and propylparabens as preservatives, a dye and flavouring. Any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

The present invention also extends to forms suitable for topical application such as creams, lotions and gels. In such forms, the antisense oligonucleotides may need to be modified to permit penetration of the surface barrier.

Pharmaceutically acceptable carriers and/or diluents include any and all liposomal carriers, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding such an active.

Effective amounts of antisense oligonucleotides contemplated by the present invention will vary depending on the severity of the inflammation and the health and age of the recipient. In general terms, effective amounts for systemic delivery may vary from 0.01 ng/kg body weight to about 100 mg/kg body weight. Alternative amounts include for about 0.1 ng/kg body weight about 100 mg/kg body weight or from 1.0 ng/kg body weight to about 1000 mg/kg 6bdy weight. Effective amounts may also vary from 5 mg/m² to 100 mg/m².

With respect to the identification of active antisense oligonucleotides, if the nucleic acid sequence of a gene encoding a protein of interest is known, specific C5 phosphorothioate antisense oligonucleotide (ODNs) can be designed to hybridize to the corresponding messenger RNA (mRNA) thereby inhibiting the production of protein. The C5 phosphorothioate antisense oligonucleotide would preferably be 15mer but could be as short as llmer while still retaining potency and specificity (Flanagan et al. , 1996). The antisense oligonucleotide may be targeted against any portion of the mRNA molecule, although some sites may not be accessible because of RNA secondary structure or may not impart sufficient binding affinity to support ODN hybridisation (Chiang et al., 1991; Monia et al. , 1996; Bennett et al. , 1994). Therefore, several sites of a mRNA need to be examined to locate an active ODN and the sequence of an effective ODN must be determined by screening potential sequences. For a 1 -base C-5 propyne phosphorothioate ON, screening a minimum number of 6 sequences should yield 2 ODNs (Stepkowski et al. , 1994).

ODN sequences with maximal antisense activity against the mRNA of interest can be identified by screening panels of ODNs which hybridise to different regions of the mRNA. Nonsense ODNs with varying GC contents should also be generated using a random numbers table and used as control ODNs. The GC content of an ODN can influence binding affinity to mRNA and high GC content has been associated with nonspecific binding. Therefore, the GC contents of controls should be selected to match those of the antisense constructs. The antisense activity of each ODN would be examined over a range of concentrations to generate dose response curves. An optimal antisense construct can be identified by demonstrating inhibition of both cytokine mRNA expression and protein secretion in vitro using a cell line or any other cells that produce the protein of interest. The most potent antisense ODN can then be selected for use.

The specificity of antisense activity must also be confirmed by demonstrating that levels of other mRNA and protein products are not effected by the antisense ODN in question. For example^~iή the case of an antisense IL4 ODN, levels of other cytokine mRNA and protein such as IL5 and IL2 mRNA and protein should be demonstrated to be unaffected by the presence of antisense IL4 ODN. Specificity of antisense activity for the selected cytokine may also be confirmed by demonstrating that the target cytokine mRNA and protein levels are unaffected by 2 mismatch control ODNs for each antisense ODN examined. Mismatch control ODNs will have scrambled nucleotide sequences of identical base composition to that of the specific antisense construct in question.

Preferably, said nucleic acid molecules are antisense oligonucleotides directed to mRNA encoding IL-4, IL-5 and/or and/or IL-13.

Even more preferably said inflammation is an allergic airways disease.

Another aspect of the present invention provides antisense oligonucleotides or derivatives, chemical equivalents, mimetics, analogues or homologues thereof, which oligonucleotides are directed to RNA sequences encoding one or more pro-inflammatory cytokines or derivatives, mimetics, analogues or homologues thereof, capable of reducing, retarding or otherwise inhibiting inflammation.

The present invention is further described by the following non-limiting examples.

EXAMPLE 1

OVALBUMIN MODEL OF ALLERGEN-INDUCED

ALLERGIC AIRWAYS DISEASE

Ovalbumin-induced allergic airways inflammation in the mouse is an ideal model for evaluation of antisense therapy in asthma. Mice sensitized to ovalbumin (OVA) and challenged with OVA aerosols develop both an immediate IgE-dependent reaction and a late phase reaction with increased specific IgE and increased numbers activated T cells and eosinophils in bronchoalveolar lavage fluid (Kung et al., 1995). Histologically, there is epithelial shedding, mucosal oedema, eosinophil and lymphocyte infiltration, and goblet cell hyperplasia in the airways. These clinical and histologic changes are all characteristic features- of asthma in humans. This model is also an excellent model for chronic asthma since multiple challenges with antigen can reproduce subepithelial fibrosis which is a common histologic feature of chronic asthma (Kung et al., 1995). Evaluation of antisense IL-4 and IL-5 oligonucleotide therapy in this model allows the examination of multiple endpoints which are all physiologically relevant to asthma in humans.

EXAMPLE 2 DESIGN OF ANTISENSE OLIGONUCLEOTIDES (ODNs)

Antisense ODNs (15mer C-5 propyne phosphorothioate, Beckman) which have increased mRNA affinity and preserved RNase H activity are used. To identify ODN sequences with maximal antisense activity against IL-4 and IL-5 mRNA, panels of ODNs which hybridise to different regions of IL-4 and IL-5 mRNA are generated. Eighteen ODNs are designed to hybridise to different regions on murine IL-5 mRNA. Specifically, 6 sequences are directed to the 5' UTR region, 6 to the 3' UTR region and 6 to the coding regions of IL-5 mRNA. Screening this number of sequences should yield at least 2 ODNs which inhibit IL-5 protein expression. Twelve ODNs are designed to hybridise to different regions on murine IL-4 mRNA. This smaller number is selected since several antisense IL-4 ODNs which are effective in vitro have been identified previously

(Hassuneh et al., 1997; Haruna et al., 1993; Salern et al., 1995) and are included in our panel of ODNs to be screened. Three sequences are directed to the 5' UTR region, 3 to the 3' UTR, 3 to coding regions of IL-4 mRNA and 3 incorporate the previously published antisense IL-4 sequences. Screening this number of ODNs yields at least 2 ODNs, in addition to those previously reported, which inhibit IL-4 protein production (Wagner, 5 1994). Four nonsense ODNs with varying GC contents are generated using a random numbers table. The GC content of an ODN can influence binding affinity to mRNA and high GC content has been associated with nonspecific binding. Therefore, the GC contents of controls is selected to match those of the antisense constructs.

0 EXAMPLE 3

EXAMINATION OF LIPOSOMAL DELIVERY MECHANISMS

To evaluate antisense activity of ODNs in vitro and in vivo it is essential to confirm effective penetration into cells and nuclei. The ability of liposome preparations to deliver 5 antisense ODNs into cells in culture may vary for different cell types. Most studies have examined transfection of cell lines or tumor cells and circulating lymphocytes and tissue lymphocytes may have different requirements for successful transfection. Furthermore, ODNs may access activated lymphocytes with greater success than resting lymphocytes in vitro. The optimal liposome formulation for delivery of antisense constructs to murine 0 lymphocytes in vitro is therefore examined. Two formulations are evaluated -

DOTMA/DOPE (lipofectin) and cytofectin GSV. Entry of ODNs into the cytoplasm and nuclei of cells is determined using fluorescein labelled ODNs (Beckman). Fluorescein labelled ODNs are suspended in lipofectin or cytofectin GSV and incubated with resting or activated lymphocytes. Penetration of fluorescein labelled ODNs into cells is examined by 5 fluorescence microscopy. EXAMPLE 4

SELECTION OF OPTIMAL ANTISENSE IL-4 AND IL-5

OLIGONUCLEOTIDES BY In vitro ANALYSIS

Oligonucleotides with maximal antisense activity against the mRNA concerned are identified by 2 approaches. Firstly, antisense IL-4 ODNs are screened for antisense activity using an IL-4 producing cell line. 3T3 cells which have been transduced with a MFG retroviral vector (Dranoff et al., 1997; Jaffee et al., 1997) expressing the IL-4 gene and which produce IL-4 constitutively are available (D Ashley, Royal Children's Hospital). Antisense activity against IL-4 and IL-5 mRNA is also examined using in vitro generated Th2 lymphocytes which produce both IL-4 and IL-5. Th2 lymphocytes are generated as previously described (Austrup et al., 1997). Briefly, spleens of donor mice are harvested and CD4 cells isolated by magnetic bead separation. CD4 lymphocytes are placed in 24 well plates (lxl0⁶/well) coated with antiCD3 antibody (1 microgram/well) and stimulated with IL-2 50 U/ml and IL-4 10 ng/ml for 2 days. Cells are then transferred to uncoated fresh wells without changing media and left for a further 4 days. At day 6, cells are harvested and washed before incubating in media alone for 24 hours. Cell lines or Th2 lymphocytes are cultured in the presence of specific antisense or control ODNs suspended in a liposome formulation. Four nonsense ODNs with varying GC contents are used as controls.

Antisense activity is demonstrated by (i) measurement of mRNA levels using Northern blot analysis or RNase protection assay and (ii) measurement of cytokine levels in supernatants by ELISA and/or bioassay. mRNA levels are measured after 6 hr of culture since peak IL-4 and IL-5 mRNA expression in cultured lymphocytes is at this time. Supernatants are collected at 24 hours for measurement of IL-4 and IL-5 protein levels. The antisense activity of each ODN is examined over a range of concentrations to generate dose response curves. Where an optimal antisense construct acts via an RNase H mediated mechanism, it is identified by demonstrating inhibition of both cytokine mRNA expression and protein secretion. An RNase H mechanism of action also allows greater ease of monitoring antisense activity in vivo since mRNA analysis is more sensitive than protein measurement of cytokines in the in vivo setting. The most potent antisense IL-4 and IL-5 ODNs are then selected for use in further studies.

EXAMPLE 5 CHARACTERIZATION OF ANTISENSE ACTIVITY In Vitro

Further studies to characterise the in vitro activity of selected antisense IL-4 and antisense IL-5 oligonucleotides are undertaken using in vitro generated Th2 lymphocytes.

Specificity of antisense activity is confirmed by several approaches. Levels of mRNA and protein for two cytokines other than the target cytokine are examined to confirm that these are unaffected by the antisense ODN in question. In the case of the antisense IL-4 ODN, levels of IL-5 and IL-2 mRNA and protein will be measured by Northern blot analysis or RNase protection assay and ELISA assay respectively. For the antisense IL-5 ODN, levels of IL-4 and IL-2 mRNA and protein will be measured by Northern blot analysis or RNase protection assay and ELISA. Specificity of antisense activity for the selected cytokine is also confirmed by demonstrating that the target cytokine mRNA and protein levels are unaffected by 2 mismatch control ODNs for each antisense ODN examined. Mismatch control ODNs have scrambled nucleotide sequences of identical base composition to that of the specific antisense construct in question.

Time course of antisense activity in vitro is also examined using in vitro generated Th2 lymphocytes. mRNA and protein levels of IL-4 or IL-5 in cultured Th2 lymphocytes are measured at various time points in the presence of specific antisense or control mismatch ODNs suspended in cytofectin GSV or other liposome formulation. EXAMPLE 6 CHARACTERIZATION OF ANTISENSE IL-4 AND IL-5 ODN ACTIVITY In Vivo

The ability of ODNs to enter lymphocytes in the airway and lung is examined using fluorescein labelled ODNs. Fluorescein labelled ODNs (0-20mg) suspended in 1 ml saline are nebulised with an ultrasonic nebuliser (0.5 ml/min) as described (Nyce and Metzger, 1997). Bronchioalveolar lavage lymphocytes and cryostat sections of lung tissue are examined by fluorescence microscopy to determine if ODNs have entered cells effectively. BAL is performed on anaesthetised mice as described (Brusselle et al., 1994). Cytospin BAL cell preparations are then examined by fluorescence microscopy. Immediately after performing BAL, mice are euthanised. Lungs are dissected and frozen in OCT mounting solution. Five micron cryostat sections are stained with a phycoerythrin-conjugated antibody against mouse CD3 (Pharmingen) to identify T lymphocytes as previously described (Tang et al., 1997). Penetration of fluorescein labelled ODNs into T lymphocytes within the lung are then determined by two colour fluorescence microscopy. Cryostat sections are examined within 24 hours of harvesting tissues to ensure fluorescence activity. If nebulised delivery of ODNs to airway and lung lymphocytes is suboptimal in the absence of a liposomal carrier, oligonucleotides are suspended in cytofectin GSV or other liposome formulation and penetration of cells evaluated as above.

In vivo antisense activity of oligonucleotides is confirmed by demonstrating inhibition of IL-4 and IL-5 mRNA and protein in BAL and lung lymphocytes following nebulisation of antisense IL-4 and IL-5 oligonucleotides. To demonstrate antisense activity in vivo, it is necessary to first induce an in vivo Th2 response. This is achieved by using an OVA-induced model of allergic airways disease (Brusselle et al., 1994). Briefly, mice are sensitised i.p. with ovalbumin on day 0. Groups of 8-10 mice are then challenged with nebulised ovalbumin once daily from day 14 through day 21. Antisense IL-4, antisense IL-5 or mismatch control ODNs are delivered by nebulisation twice daily for 2 days on days 20 and 21 as described (Nyce and Metzger, 1997). On the morning of the day 22, BAL is performed and cells isolated by centrifugation. Mice are then euthanised for lung dissection and isolation of lung lymphocytes. Cytokine mRNA in BAL cells is assessed using semiquantative RT/PCR or RNASE protection assay. This is necessary since insufficient RNA is available from the small number of BAL cells isolated to perform Northern blot analysis. Briefly, RNA is extracted and reverse transcribed to cDNA before amplification by PCR using sequence specific primers for IL-4 and IL-5. Semiquantitative measurement of mRNA is achieved by comparing the density of specific IL-4 and IL-5 bands with that of known quantities of control oligonucleotide amplified using the same sets of primers. IL-4 and IL-5 mRNA in the lung is quantitated by Northern blot analysis or RNase protection assay using total lung RNA. Protein production in BAL and lung lymphocytes is examined by immunofluorescent staining of intracellular cytokines and fluorescence cytometry analysis (Assenmacher et al., 1994; Ferrick et al. , 1995). This method has been used to identify Thl and Th2 cells ex vivo after in vivo activation previously (Ferrick et al., 1995). Briefly, following in vivo activation, ex vivo lymphocytes are cultured with brefeldin A, an inhibitor of intracellular transport, which results in accumulation of cytokine within the Golgi complex. Cells are then fixed, permeabilised and stained with specific antibodies directed against IL-4 or IL-5 (Cytofix/Cytoperm plus Cytostain kit, PharMingen). If antisense activity in vivo is less than optimal we will pursue several approaches to increase efficacy can be increased by methods including modifications of the delivery vehicle or the oligonucleotide itself to increase entry into cells and mRNA binding affinity.

EXAMPLE 7 ALLERGEN INDUCED AIRWAY INFLAMMATION MODEL

A model of OVA-induced allergic airways disease is carried out as described (Brusselle et al. , 1994). Briefly, mice are injected i.p. with OV A/alum on day 0. Mice are then exposed to nebulised OVA challenges for 30min daily from day 14 to 21. EXAMPLE 8 EVALUATION OF AIRWAY INFLAMMATION

Airway inflammation is determined by cell counts and differential smears in BAL, and by morphometric and immunohistochemical analysis of lymphocytes in the lungs. On day 22, BAL is performed on anaesthetised mice and cells isolated by centrifugation. Mice are then euthanised and lungs dissected. A portion is frozen in OCT mounting media for immunohistochemistry, and the remainder are fixed in 4% paraformaldehyde and mounted in paraffin for routine histologic analysis.

EXAMPLE 9 ^r PROTOCOL FORDELIVERY OF ANTISENSE THERAPY

To evaluate the efficacy of antisense IL-4 and IL-5 oligonucleotides in preventing the onset of airway inflammation, antisense cytokine (IL-4 or IL-5) ODNs or mismatch control ODNs are administered twice daily on the day prior to i.p. OVA sensitisation and twice daily from day 14 to 21 when OVA challenges take place. To evaluate the efficacy of antisense IL-4 and IL-5 oligonucleotides in treating established airway inflammation, antisense cytokine (IL-4 or IL-5) ODNs or mismatch control ODNs are administered twice daily for 2 days on day 20 and 21 of the ovalbumin challenge.

Briefly, mice are sensitised and challenged with OVA as described. Antisense cytokine (IL-4 or IL-5) ODNs or mismatch control ODNs (0-20mg/ml) are delivered by ultrasonic nebulisation (0.5ml/min) to groups of 8-10 mice held in an exposition chamber.

EXAMPLE 10 CHARACTERISATION OF THE IMMUNE RESPONSE TO OVA

Where treatment with antisense oligonucleotides inhibits or prevents airway inflammation, it is important to characterise the nature of the immune response to OVA and to confirm that mice were sensitised to OVA. This is especially relevant since IL-4 is involved both in the initial stages of inducing T cell differentiation towards a Th2 profile and in the later amplification of IgE and mast cell activity in the allergic response. Without limiting the theory or mode of action of the present invention antisense IL-4 therapy may reduce airway inflammation by preventing the generation of a Th2 response and diverting T cells towards a Thl profile. Alternatively, antisense IL-4 therapy may inhibit allergic inflammation by inhibiting IgE and mast cell activity without influencing the underlying Th2 response to OVA. To confirm that mice are sensitised to OVA, levels of IgE and OVA-specific IgE in the serum are quantitated by ELISA as described (Brusselle et al., 1994). If IgE levels are not elevated, it is possible that a Thl response has been induced. Ovalbumin-specific T lymphocyte responses are therefore examined by standard in vitro tritiated thymidine incorporation assays. ^'-^"

EXAMPLE 11

To investigate the systemic effects on broader aspects of immune function, detailed evaluation of B and T lymphocyte function in vivo are performed.

Evaluation ofB lymphocyte function. In vivo B lymphocyte function is assessed by immunising mice receiving antisense or control oligonucleotide therapy with DNP-KLH a T dependent and T independent antigen. At day 7 and 14 mice are bled from the cavernous sinus and serum collected for measurement of specific antibody. Specific IgG and IgM antibodies are quantitated by ELISA.

Evaluation of T lymphocyte function.

In vivo T lymphocyte function is assessed by examining in vivo contact hypersensitivity responses. Briefly, mice are sensitised with topically applied oxazolone on the flank region. Mice are challenged with oxazolone applied to the ear on day 7 and 8. Challenge of the contralateral ear with saline serves as a control. Swelling is measured at 24 and 48 hr using a specialised microcaliper. In vitro T lymphocyte proliferative responses are examined. Splenocytes are isolated and cultured with mitogens (PHA and ConA). Tritiated thymidine incorporation is measured after 3 days of culture.

EXAMPLE 12

Materials and Methods (i) Reagents

Ovalbumin (OVA, Grade V), Aluminum Potassium Sulphate (alum, ACS grade), BSA (Fraction V) were purchased from Sigma-Aldrich Pty Ltd, Castle Hill, Australia. Nembutal (Pentobarbitone Sodium, Boehringer Ingleheim Pty Ltd, Artarmon, Australia), chromium-51 (⁵¹Cr, Amersham Pharmacia Batik UK Ltd, England). RPMI-1640 with L-glutamine media, FCS and penicillin-streptomycin solution were purchased from GIBCO BRL, Melbourne, Australia. 2-mercaptoethanol (BDH Chemicals, Poole, England). Cytofectin GSV (GS2888 cytofectin, Glenn Research Laboratories). 3T3 cells transduced with a MGF retroviral vector expressing the IL-4 gene and which produce IL-4 constitutively were kindly provided by Dr David Ashley (Royal Children's Hospital, Melbourne, Australia).

(ii) Animals

C57BL/6 mice aged 6-10 weeks were purchased from The Walter and Eliza Hall Institute (Melbourne, Australia). Mice were maintained in a standard animal care facility. All procedures were approved by The Royal Children's Hospital Animal Experimentation Ethics Committee (Melbourne, Australia).

(Hi) Ovalbumin (OVA) induced allergic airway disease model

Mice were sensitised i.p. on day 0 with 10μg OVA/lmg alum in 0.5ml saline. Mice were then challenged with OVA from days 14 to 20 by daily exposure to an aerosol of 1 % (w/v) OVA in saline for 30 minutes. Control mice were sensitised with alum alone and challenged with saline. Aerosol challenge was carried out on groups of up to 12 mice in a closed-system chamber (24 X 37.5 X 15cm) attached to an ultrasonic nebuliser (NE-U07, Omron Corporation, Japan) with an output of lml/min and l-5μm particle size. 5

(iv) Measurement of airway hyperreactivity

Airway reactivity was assessed by measuring changes in airway resistance following methacholine challenge. Measurements of airway resistance were carried out on

10 unrestrained mice by barometric, whole body plethysmography and expressed as an "enhanced pause" (Penh) (Buxco Electronics Inc. , Troy, New York, USA) as recently described (Handmann et al., 1997). "Penh" is a dimensionless value representing a function of the proportion of maximal expiratory to maximal inspiratory box pressure signals and of the timing of inspiration (Handmann et al., 1997). Penh has been shown

15 to accurately represent airway resistance (Handmann et al., 1997). In brief, the bio- system was first calibrated with a rapid injection of 1ml of air into the mouse chambers. Mice were then placed in individual chambers and allowed to settle for approximately 10 to 15 min. Baseline Penh readings were taken and averaged for 5 min. Aerosolised methacholine of increasing concentrations (1.5mg/ml, 3mg/ml, 6mg/ml, 25mg/ml,

20 50mg/ml and lOOmg/ml in saline) (Methacholine Chloride desiccate, ICN Biomedicals Inc., Aurora, Ohio, USA) were then nebulised into the chambers for 2 min. Penh readings were taken for 6 min after each dose challenge and averaged.

(v) Antisense oligonucleotides (ODN)

25

Antisense therapy is used to specifically and selectively inhibit the production of a protein. If the nucleic acid sequence of a gene encoding a protein of interest is known, the specific antisense oligonucleotides can be designed to hybridize to the corresponding mRNA thereby inhibiting the production of the protein. Antisense

30 oligonucleotides may inhibit protein expression by multiple mechanisms, however, the most potent antisense effects are obtained when Rnase H degradation of mRNA is the mechanism of action (Wagner, 1995). The sequence of an active antisense oligonucleotide can be determined by screening of sequences which bind to different regions of an mRNA molecule. Most regions of an mRNA molecule can be targeted (Wagner, 1995). C5 propyne phosphorothioate oligonucleotides are modified oligonucleotides with greatly enhanced mRNA binding affinity, increased resistance to nucleases, and have the ability to recruit RNase H cleavage. These properties provide greatly enhanced specificity and potency of antisense effects. C5 propyne phosphorothioate oligonucleotides reduce the number of sequences that need to be screened; screening 6 15mer C5 propyne phosphorothioate oligonucleotides yielded 2 oligonucleotides with specific and potent antisense activity against 3 genes (Wagner, 1995). C5 propyne phosphorothioate oligonucleotides as short as l lmer can induce specific antisense effects (Flannagan et al., 1996).

A 15mer C5 propyne phosphorothioate antisense oligonucleotides complementary to the 5' region of murine IL-4 mRNA was generated with the sequence: CTG GGG GTT GAG ACC ( <400> 1). A control antisense oligonucleotide with scrambled base sequence was also generated: GTG ACG GTG CGT ACG ( <400>2). Antisense oligonucleotides were purchased from Oligos etc, Inc. (Wilsonville, OR).

Oligonucleotides have poor permeability through cell membranes in vitro and specialised delivery methods are required to ensure effective entry of oligonucleotides into cells in vitro. Liposome-mediated delivery is the most reliable method for efficient oligonucleotide entry into cells in culture (Wagner, 1995). A novel liposome preparation, cytofectin GSV (GS2888 cytofectin, Glenn Research Laboratories) transfects oligonucleotides and plasmids into a wide variety of cells with high efficiency and low toxicity, and markedly enhances antisense potency in vitro (Wagner, 1995). Cytofectin GSV promotes accumulation of oligonucleotides in the nucleus and facilitates antisense effects at low oligonucleotide concentrations, thereby reducing nonspecific effects related to binding with proteins which occurs at high oligonucleotide concentrations (Wagner, 1995). Oligonucleotides have a greater ability to penetrate cells in vivo and liposomes may not be required for in vivo activity (Momia et al. , 1996). Specifically, the airway and lung are exception targets for topical delivery of oligonucleotides, since there is a very large absorptive surface and the lung surface is lined with surfactant which can facilitate the intracellular uptake of oligonucleotides. A major component of surfactant, dipalmitoylphosphatidylcholine, is a weak cationic lipid at physiologic pH, and cationic lipids can enhance cellular uptake of oligonucleotides (Wagner, 1995). Surfactant based DNA delivery systems have been described and direct intratracheal delivery of plasmid DNA results in high levels of DNA expression by airway cells, with equivalent expression achieved when DNA is administered alone or complexed with cationic liposomes (Meyer et al., 1995).

(vi) . Selection of cytokines that can be targeted by antisense oligonucleotide therapy

Cytokines that can be targeted by antisense oligonucleotide therapy to prevent or inhibit the development of airway inflammation asthma may be identified by several approaches.

Potential targets for antisense oligonucleotide therapy aimed at preventing or reducing airway inflammation in asthma include those cytokines known to promote the development of T helper type 2 (Th2) responses and the Th2 cytokines themselves. Th2 lymphocytes and the cytokines they secrete, particularly IL-4 and IL-5, are critical for the generation and propagation of allergic inflammation. IL-4 is essential for Ig production by B lymphocytes and promotes mast cell growth and differentiation, thus regulation key factors in the initiation of the allergic response (Ryan, 1997). IL-4 also promotes the differentiation of naive T helper cells towards a Th2 profile, thereby perpetuating allergic inflammation (Ryan, 1997). Eosinophils are important effector cells in allergic inflammation, causing tissue injury by release of potent cytotoxic mediators (Frigas et al., 1986). IL-5 is necessary for eosinophil proliferation, is an eosinophil chemotactic factor, and activates eosinophils to release toxic mediators (Frigas et al., 1986). Other targets for antisense oligonucleotide therapy aimed at preventing or reducing the development of allergic airway inflammation include cytokines that are expressed by airway or lung leukocytes or other cells within the lung during an acute asthma attack, during ongoing airway inflammation or following antigen challenge. These cytokines can be identified by examining cytokine expression in BAL leukocytes or lung during an acute asthma attack, during ongoing airway inflammation or following antigen challenge

Using a model of OVA-induced allergic airway disease, the kinetics of cytokine expression following antigen challenge was examined. C57BL/6 mice (aged 6 wk) were sensitised i.p. with OV A/alum on day 0 and challenged with nebulised OVA (1 % in saline) on days 14-20. Mice were euthanised on days 14, 15, 16, 17, 18, 19 and 20 of the challenge protocol and lungs harvested for mRNA isolation. IL-4, IL-5, IL-2 and IFNg mRNA expression was assessed by RT/PCR. Mice sensitised and challenged with saline alone were used as controls. Saline sensitised and challenged mice expressed low levels of IL-2 and IL-5 mRNA and significant levels of IFNg mRNA. IL-4 mRNA was undetectable in the majority of control mice. In contrast, in mice sensitised and challenged with OVA, IL-4, IL-5 and IL-2 mRNA was detected in high levels within 2 hr of the first OVA antigen challenge. Expression of IL-4, IL-5 and IL- 2 increased over the first 3 days of OVA challenge (days 14, 15, 16) before reducing rapidly to become undetectable by the fifth day of OVA challenge (day 18). In contrast, levels of IFNγ mRNA decreased from the third day after antigen challenge. Levels of IL-4, IL-5 and IL-2 mRNA in lungs from OVA sensitised and challenged mice was markedly higher than those seen in saline sensitised and challenged control mice. Levels of IFNγ mRNA were significantly lower than that seen in saline sensitised and challenged control mice from the third day of antigen challenge. Thus IL-4 and IL-5 represent targets for antisense oligonucleotide therapy aimed at reducing airway inflammation in asthma. Results

(i) Antisense activity in vitro

Activity and specificity of the IL-4 antisense oligonucleotide was evaluated in vitro using an IL-4 producing cell line (3T3-IL-4 kindly provided by Dr David Ashley, Royal Children's Hospital, Melbourne, Australia) and PHA stimulated splenocytes. 3T3-IL-4 cells were confirmed to constitutively express murine IL-4 mRNA. 3T3-IL-4 cells were grown to confluence, harvested and then cultured in DMEM in the presence of IL-4 antisense oligonucleotide or control oligonucleotide (500nM) suspended in the lipid carrier cytofectin GSV (2.6μg/ml). After 6 hr, cells were washed twice in DMEM media, mRNA extracted and IL-4 mRNA expression assessed by RT/PCR. Expression of β-actin mRNA served as an internal control. IL-4 mRNA expression was inhibited by — 50% in the presence of IL-4 antisense oligonucleotide but not control oligonucleotide. Expression of β-actin was equivalent in all cultures.

Activity of IL-4 antisense oligonucleotide was also examined using PHA-activated splenocyte cultures. C57BL/6 mice (aged 6 week) were sensitised with OV A/alum on day 0 and challenged with nebulised OVA (1 % in saline) on days 14-20. On day 21, mice were euthanised and splenocytes harvested by density gradient centrifugation. Splenocytes from OVA sensitised challenged mice were cultured in complete RPMI media containing 10% FCS and activated with PHA (lμg/ml) in the presence of IL-4 antisense oligonucleotide (500nM) suspended in cytofectin GSV (2.6μg/ml), control oligonucleotide (500nM) suspended in cytofectin GSV (2.6μg/ml), or cytofectin GSV alone ( 2.6μg/ml). After 6hr, splenocytes were harvested, mRNA extracted and IL-4 mRNA expression examined by RT/PCR. Expression of β-actin served as an internal control. PHA-activated splenocytes from OVA sensitised and challenged mice were shown to express IL-4 mRNA in the presence of GSV alone. IL-4 mRNA expression was almost completely inhibited by IL-4 antisense oligonucleotide but not control oligonucleotide. Expression of β-actin was equivalent in all cultures. (ii) In vivo expression ofIL4 mRNA in airway /lung

The kinetics of IL-4 mRNA expression within the lung following antigen challenge was assessed using an animal model of OVA-induced allergic airway disease. C57BL/6 mice (aged 6 wk) were sensitised i.p. with OV A/alum on day 0 and challenged with nebulised OVA (1 % in saline) on days 14-20. Mice were euthanised on days 14, 15, 16, 17, 18, 19 and 20 of the challenge protocol and lungs harvested for mRNA isolation. IL-4 mRNA expression was assessed by RT/PCR. IL-4 mRNA was detected within 2 hr of the first OVA challenge on day 14. IL-4 mRNA then increased on the second and third days of OVA challenge (day 15 and 16) before reducing rapidly to become undetectably by the fifth day of OVA challenge (day 18). This indicates that once a subject is sensitised to an antigen, re-exposure to that antigen rapidly induces IL- 4 production and that in the presence of continued antigen exposure IL-4 production continues for several days. Thus any therapy aimed at reducing IL-4 mRNA expression would be most effective when given either before or early after antigen exposure.

(Hi) Efficacy of IL-4 antisense ODN therapy in prevention or treatment of airway inflammation in asthma

The efficacy of IL-4 antisense oligonucleotide therapy in the prevention of airway inflammation in asthma was assessed. C57BL/6 mice (aged 6 wk) were sensitised i.p. with OV A/alum on day 0 and challenged with nubulised OVA (1 % in saline) on days 14-20. On days 14-17, mice were treated with nebulised IL-4 antisense oligonucleotide or control oligonucleotide (0.5mg/dose, 250'¹g/ml nebulised at lml/min for 2 mins) delivered 12 hours for a total of 6 doses. On day 21, 24hr after the last OVA challenge, airway inflammation was assessed by performing cell counts and differential smears of BAL cells, and by evaluation of airway hyperreactivity.

Mice treated with IL-4 antisense oligonucleotide demonstrated reduced BAL cell counts and reduced airway reactivity as compared with mice treated with control oligonucleotide or OVA alone. Airway reactivity responses in mice treated with IL-4 antisense oligonucleotide, control oligonucleotide or OVA alone are show in Figure 1.

Application to Human Disease

Antisense oligonucleotide that targets a cytokine or cytokines involved in the generation of allergic airway inflammation is effective in both preventing and inhibiting the development of allergic airway inflammation in asthma. Antisense oligonucleotide can be delivered directly to the airway and lung as suspended particles. Examples of such delivery methods include nebulisation, aerosol inhaler, dry powder inhaler or direct instillation into the trachea where access is available via endotracheal intubation or tracheostomy or other medical or surgical device or manipulation. The timing of delivery may be prior to or shortly after exposure to known triggers for asthma. Antisense oligonucleotide can also be used as a prophylactic therapy in a similar manner to which inhaled corticosteroids are presently used. IL-4 antisense oligonucleotide therapy or other cytokine antisense oligonucleotide therapy provides an effective means of controlling allergic airway inflammation and provides a superior alternative to the currently available inhaled and oral corticosteroid preparations used in the management of asthma.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY:

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Kung, T.T., Stelts, D.M., Zurcher, J.A., Adams, G.K., Egan, R.W., Kreutner, W., Watnick, A.S., Jones, H. and Chapman, R.W. (1995) Am. J. Resp. Cell and Mol. Biol. 13:360

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Claims

CLAIMS:

1. A method for the treatment and/or prophylaxis of inflammation in a mammal said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof for a time and under conditions sufficient to reduce, inhibit or otherwise down-regulate the level and/or functional activity of a cytokine or derivative, homologue, analogue or mimetic thereof.

2. The method according to claim 1 wherein said cytokine is a pro-inflammatory cytokine.

3. The method according to claim 2 wherein said pro-inflammatory cytokine is one or more of IL-4, IL-5 and/or IL-13 or derivative, homologue, analogue or mimetic thereof.

4. The method according to any one of claims 1 to 3 wherein said inflammation is associated with allergic airways disease.

5. The method according to claim 4 wherein said allergic airways disease is asthma.

6. The method for the treatment and/or prophylaxis of inflammation in a mammal, said method comprising administering to said mammal an effective amount of a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof which is capable of interacting with a second nucleic acid molecule which is translatable to all or part of an amino acid sequence of a cytokine or derivative, analogue, mimetic or homologue thereof or a molecule which facilitates the expression and/or functional activity of said cytokine whereby said interaction facilitates a reduction in the level and/or functional activity of said cytokine or its derivative, homologue, analogue or mimetic thereof.

7. The method according to claim 6 wherein said inflammation is associated with allergic airways disease.

8. The method according to claim 7 wherein said allergic airways disease is asthma.

9. The method according to any one of claims 6 to 8 wherein said second nucleic acid molecule is a RNA molecule.

10. The method according to claim 9 wherein said first nucleic acid molecule is an antisense oligonucleotide.

11. The method according to claim 10 wherein said cytokine is a pro-inflammatory cytokine.

12. The method according to claim 11 wherein said pro-inflammatory cytokine is one or more of IL-4, IL-5 and/or IL-13 or functional equivalents thereof.

13. The method according to claim 12 wherein said RNA is mRNA.

14. The method according to claim 13 wherein said antisense oligonucleotide interacts with the 5' region of said mRNA.

15. A composition for use in reducing, retarding or otherwise inhibiting inflammation in a mammal said composition comprising nucleic acid molecules or derivatives, chemical equivalents, mimetics, analogues or homologues thereof which nucleic acid molecule reduces, inhibits or otherwise down-regulates the level and/or functional activity of a cytokine or derivative, homologue, analogue or mimetic thereof and one or more pharmaceutically acceptable carriers and/or diluents.

16. A composition according to claim 15 wherein said cytokine is a pro- inflammatory cytokine.

17. Use of a nucleic acid molecule or derivative, chemical equivalent, mimetic analogue or homologue thereof which nucleic acid molecule is directed to a second nucleic acid molecule which is translatable to all or part of an amino acid

. sequence of a cytokine in the manufacture of a medicament for the treatment or prophylaxis of inflammation in a mammal.

18. Use according to claim 17 wherein said cytokine is a pro-inflammatory cytokine.

19. Use according to claim 18 wherein said nucleic acid molecule is an antisense oligonucleotide and said second nucleic acid molecule is mRNA encoding IL-4, IL-5 and/or IL-13 or functional equivalent thereof.

20. Use according to any one of claims 17 to 19 wherein said inflammation is associated with allergic airways disease.

21. A composition comprising a nucleic acid molecule or derivative, chemical equivalent, mimetic, analogue or homologue thereof which nucleic acid molecule is directed to a second nucleic acid molecule encoding one or more cytokines or derivatives homologue, analogue or mimetic thereof, capable of reducing, retarding or otherwise inhibiting inflammation.

22. A composition according to claim 21 wherein said cytokine is a pro- inflammatory cytokine.

3. A composition according to claim 22 wherein said nucleic acid molecule is an antisense oligonucleotide and said second nucleic acid molecule is mRNA encoding IL-4, IL-5 and/or IL-13 or functional equivalents thereof.