WO2012092645A1 - Novel antisense oligonucleotides - Google Patents

Abstract

A purified and isolated antisense molecule capable of binding to a selected target site to induce exon skipping and/or translation blocking in the granulocyte macrophage colony-stimulating factor gene (GM-CSF) selected from the antisense molecules of SEQ ID NO: 1 to 17. Methods of treating conditions associated with abnormal levels of GM- CSF such as asthma, rheumatoid arthritis, acute myeloid leukaemia are also disclosed.

Description

Novel Antisense Oligonucleotides

Field of the Invention

[0001] The present invention relates to novel antisense compounds and compositions suitable for facilitating exon skipping and blockage of translation and protein synthesis of the GM-CSF gene. It also provides methods for inducing exon skipping and suppressing translation using the novel antisense compounds as well as therapeutic compositions adapted for use in the methods of the invention.

Background Art

[0002] The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

[0003] Significant effort is currently being expended into researching methods for suppressing pathways associated with diseases and pathological conditions such as abnormal or excessive inflammation. Antisense technologies are being developed using a range of chemistries to affect gene expression at a variety of different levels (transcription, splicing, stability, translation). Much of that research has focused on the use of antisense compounds to correct or compensate for abnormal or disease-associated expression of genes in a myriad of different conditions.

[0004] Antisense molecules are able to inhibit gene expression with exquisite specificity and, because of this, many research efforts concerning oligonucleotides as modulators of gene expression have focused on inhibiting the expression of targeted genes such as oncogenes or viral genes. The antisense oligonucleotides are directed either against RNA (sense strand) or against DNA where they form triplex structures inhibiting transcription by RNA polymerase II.

[0005] To achieve a desired effect in specific gene down-regulation, the oligonucleotides must either promote the decay of the targeted mRNA or block translation of that mRNA, thereby effectively preventing de novo synthesis of the undesirable target protein.

Asthma

[0006] Asthma is a chronic immunologic inflammatory disease of the airways characterized by hyper-reactive airway responses to various stimuli, with allergic asthma being the most common form of asthma. In allergic asthma, allergens can be processed by immature dendritic cells (DC) residing in the airway lumen or submucosa. In the presence of cytokines, activated DCs initiate T and B lymphocyte stimulation and immunoglobulin E (IgE) production in regional lymph nodes. Subsequently, IgE is distributed systemically and binds to the FCDRI receptors on resident mast cells. When re-exposed to antigen, resident mast cells degranulate and secrete cytokines, chemokines, proteases, histamine and phospholipid metabolites, which lead to vasodilation, an increase in vascular permeability and recruitment of innate immune cells (including neutrophils, eosinophils, lymphocytes and basophils). In chronic disease states, airway inflammation is aggravated by cytokines released from innate immune cells and the epithelium-muscle tissue unit, leading to remodeling of the airways.

Rheumatoid arthritis

[0007] Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. The pathology of the disease process often leads to the destruction of articular cartilage and ankylosis of the joints. RA is characterized by synovial tissue infiltration with macrophages, dendritic cells (DCs) and lymphocytes, and accumulation of neutrophils in synovial fluid. In RA, the key inflammatory factor involved is the overproduction and overexpression of tumour necrosis factor (TNF), which drives both synovial inflammation and joint destruction. Interactions between T and B lymphocytes, synovial-like fibroblasts, and macrophages may lead to TNF overproduction and to the overproduction of many cytokines such as interleukin(IL)-6, IL-8 and GM-CSF, which also drives persistent inflammation and joint destruction. RA can also produce diffuse inflammation in the lungs, pericardium, pleura, and sclera, and also nodular lesions, most common in subcutaneous tissue under the skin.

Acute Myeloid Leukaemia

[0008] Acute myeloid leukaemia (AML), also known as acute myelogenous leukaemia, is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. AML is the most common acute leukaemia affecting adults, and its incidence increases with age. Although AML is a relatively rare disease, its incidence is expected to increase as the population ages. AML leukaemic cells generate significant amounts of GM-CSF that can act in an autocrine fashion to stimulate growth of more leukaemic cells. Furthermore, AML leukaemic cells have increased GM-CSF receptor expression and, in theory, may be more sensitive to GMCSF stimulation.

Granulocyte macrophage colony stimulating factor (GM-CSF)

[0009] GM-CSF is known to play a significant role in granulocyte and macrophage maturation and was initially identified by its capacity to stimulate bone marrow cells to form in vitro colonies of myeloid cells. GM-CSF also plays a role in allergic inflammation by promoting growth, migration, maturation and survival of eosinophils, which play a pivotal role in the underlying pathophysiology of asthma and neutrophils which have an important role in rheumatoid arthritis. In asthma patients, the proportion of cells expressing GM-CSF mRNA in bronchoalveolar lavage fluid (BALF) is much higher than is found in healthy controls. When asthmatics were challenged with antigen the level of GM-CSF increased and correlated with the number and percentage of eosinophils in the BALF.

[00010] The genomic structure of GM-CSF is provided in Figure 1 , which shows the four exons encoding 38 kDa protein. Studies of ligand-receptor intermolecular interactions demonstrated the functional domains of GM-CSF, with exon 3 being reported to be essential for receptor ligand formation. Cyclic peptides were subsequently discovered to be potent GM-CSF neutralizing agents. [00011] Administration of anti-GM-CSF antibody has shown to result in reduced mucus production and attenuated airway inflammation in murine models of asthma when stimulated with diesel exhaust particles and house dust mite antigen. Clinical trials of humanized monoclonal antibodies as a treatment for asthma are being conducted.

[00012] The present invention seeks to provide an alternative method for the regulation and control of GM-CSF expression in diseases such as AML, and inflammatory conditions such as asthma and rheumatoid arthritis.

Summary of the Invention

[00013] The present invention provides antisense molecule compounds and compositions suitable for binding to GM-CSF RNA motifs involved in the splicing and translation of pre-mRNA that are able to induce specific and efficient exon skipping and/or specifically reduce translation of GM-CSF protein and a method for their use as a therapeutic.

[00014] The choice of target selection plays a crucial role in the efficiency of exon skipping and hence its subsequent application as a potential therapy. Simply designing antisense molecules to target regions of pre-mRNA presumed to be involved in splicing is no guarantee of inducing efficient and specific exon skipping. The most obvious or readily defined targets for splicing intervention are the donor and acceptor splice sites, although there are also less defined or conserved motifs including exonic splicing enhancers, silencing elements and branch points. The acceptor and donor splice sites have consensus sequences of about 16 and 8 bases respectively (see Figure 2 for schematic representation of motifs and domains involved in exon recognition, intron removal and the splicing process).

[00015] According to a first aspect, the invention provides antisense molecules capable of binding to a selected target in the GM-CSF RNA to induce exon skipping or blocking of translation. Preferably, such antisense molecules are able to prevent or at least reduce production of the functional GM-CSF protein. [00016] For example, if antisense molecules are used to induce skipping of one of GM-CSF's four exons (for example exon 3), the normal RNA splicing process would be interrupted, leading to the production of non-functional transcripts or a reduction in translation.

[00017] In a further example, it is possible to combine two or more antisense oligonucleotides of the present invention together to induce more efficient exon skipping. A combination or "cocktail" of antisense oligonucleotides are directed at exons to induce efficient exon skipping.

[00018] According to a second aspect, the present invention provides antisense molecules selected and or adapted to aid in the prophylactic or therapeutic treatment of a condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF comprising at least an antisense molecule in a form suitable for delivery to a patient.

[00019] According to a third aspect, the invention provides a method for treating a patient suffering from a condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF, comprising the steps of: (a) selecting an antisense molecule in accordance with the methods described herein; and (b) administering the molecule to a patient in need of such treatment.

[00020] The invention also addresses the use of purified and isolated antisense oligonucleotides of the invention, for the manufacture of a medicament for treatment of a condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF.

[00021] The invention further provides a method of treating a condition characterized by a condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF, which method comprises administering to a patient in need of treatment an effective amount of an appropriately designed antisense oligonucleotide of the invention. Further, the invention provides a method for prophylactically treating a patient to prevent or at least minimise a condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF, comprising the step of: administering to the patient an effective amount of an antisense oligonucleotide or a pharmaceutical composition comprising one or more of these biological molecules.

[00022] The invention also provides kits for treating a condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF, which kits comprise at least an antisense oligonucleotide of the present invention, packaged in a suitable container and instructions for its use.

[00023] Other aspects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing description, which proceeds with reference to the following figures.

Brief Description of the Drawings

Figure 1 Schematic representation of the GM-CSF gene, mature transcript and protein. The position of some of the AOs of the present invention is indicated on the gene/pre-mRNA.

Figure 2 Schematic representation of splicing motifs involved in recognition of exon 3 of human GM-CSF.

Figure 3 Graph of the level of supernatant GM-CSF measured 48 hour post

LPS stimulation of 16HBE cells.

Figure 4 An agarose gel showing levels of human GM-CSF mRNA after treatment with 2OMeAOs targeting splicing motifs on exon 3 of the GM-CSF pre-mRNA (FL - full length GM-CSF transcript, Δ3 - GM- CSF transcript lacking exon 3) and stimulated with LPS; 18s was used as an internal control gene for quantification.

Figure 5 An agarose gel showing levels of human GM-CSF mRNA after treatment with combinations of 2OMeAOs targeting splicing motifs on exon 3 of the GM-CSF pre-mRNA (FL - full length GM-CSF transcript, Δ3 - GM-CSF transcript lacking exon 3) and stimulated with LPS; 18s was used as an internal control gene for quantification.

Figure 6 An agarose gel showing levels of human GM-CSF mRNA after treatment with titrated combinations of 20MeAOs targeting splicing motifs on exon 3 of the GM-CSF pre-mRNA (FL - full length GM- CSF transcript, Δ3 - GM-CSF transcript lacking exon 3) and stimulated with LPS; GAPDH was used as an internal control gene for quantification.

Figure 7 (a) A Western blot gel showing the level of expression of intracellular GM-CSF protein after transfection with 200 nM and 400nM of the 20MeAO (c+d) combination and stimulated with LPS; β- tubulin was used as an internal control for quantification; (b) Graph showing the densitometric analysis of the level of expression of intracellular GM-CSF protein in 16HBE transfected with either the 20MeAO (c+d) combination or a control AO (Ctr A) and stimulated with LPS; (c) Graph showing the level of supernatant GM-CSF in cells after transfection with 200 nM and 400nM of the 20MeAO (c+d) combination or control AO (Ctr A) and stimulated with LPS.

Figure 8 An agarose gel showing the level of expression of GM-CSF transcript on 16HBE cells transfected with 20MeAO (I, p or q) at 150nM, 300nM or 600nM or untreated 16HBE cells and stimulated with LPS (FL-GM-CSF - full length GM-CSF transcript; GAPDH was used as an internal control.

Figure 9 A graph showing the level of supernatant GM-CSF in 16HBE cells treated with 5 different 20MeAO combinations at 900 nM and stimulated with LPS compared to three control AOs: Ctr A, Ctr B and Ctr C. Figure 10 (a) An agarose gel of RT PCR showing the level of expression of GM-CSF mRNA in 16HBE cells treated with 20MeAO (h, i, j or k) at 300 nM or 600n and stimulated with LPS; (b) the level of supernatant GM-CSF in 16HBE cells after treatment with 20MeAO (k) and stimulated with LPS. The control AO used was a non-specific Ctr A.

Figure 11 A graph showing the level of GM-CSF in the supernatant of 16HBE cells treated with 20MeAO (c+d+k) combination and stimulated with LPS. The control used was a non-specific (Ctr A and Ctr B).

Figure 12 (a) A graph showing the level of GM-CSF in supernatant of 16HBE cells treated for 5 days with PMO (c+d) or PMO (k) and stimulated with LPS compared to a control comprising a Ctr PMO E and Ctr PMO F-treated sample; (b) A graph showing the level of GM-CSF in 16HBE cells treated for 7 days with PMO (c+d) and stimulated with LPS compared to untreated control 16HBEs.

Figure 13 Stimulation protocols A and B for cells stimulated with LPS1 and

LPS2.

Figure 14 Inhibition of human GM-CSF transcript after treatment with single (a) and combination (b) 20Me AOs targeting splicing motifs on exon 3 of the GM-CSF pre-mRNA (FL - full length GM-CSF transcript); GAPDH was used as an internal control, n=2.

Figure 15 Inhibition of human GM-CSF transcript after treatment with combinations of 20Me AOs targeting splicing motifs on exon 3 (c+d) and targeting exon 1 (k) of the GM-CSF pre-mRNA (FL - full length GM-CSF transcript); n=4.

Figure 16 Inhibition of human GM-CSF protein after treatment with combinations of 20Me AOs targeting splicing motifs on exon 3 (c+d) and targeting exon 1 (k) of the GM-CSF pre-mRNA; GAPDH and B- tubulin were used as RT-PCR and Western internal control, respectively, n=4.

Figure 17 Inhibition of LPS-induced release of GM-CSF in 16HBEs supernatants following treatment with (c+d) combination of PMO AO.

Figure 18 Inhibition of intracellular unstimulated (a) and LPS-stimulated release (b) of GM-CSF by 16HBE with combination (c+d) PMO. B- tubulin was used as the Western internal control, n=4.

Figure 19 Protocol for intranasal delivery of therapeutic PMOs.

Figure 20 Distribution of fluorescein-labelled PMO in mouse lung; n=4.

Detailed Description of the Invention

General

[00024] 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 the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[00025] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

[00026] Sequence identity numbers (SEQ ID NO:) containing nucleotide and amino acid sequence information included in this specification are collected at the end of the description and have been prepared using the programme Patentln - i n version 3.0. Each nucleotide or amino acid 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 and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211> <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (e.g. <400>1 , <400>2, etc.).

[00027] An antisense molecule nomenclature system was proposed and published to distinguish between the different antisense molecules (see Mann et al., (2002) J Gen Med 4, 644-654). This nomenclature became especially relevant when testing several slightly different antisense molecules, all directed at the same target region, as shown below:

H # A D (x y).

The first letter designates the species (e.g. H: human, M: murine, C: canine) "#" designates target GM-CSF exon number.

"A D" indicates acceptor or donor splice site at the beginning and end of the exon, respectively.

(x y) represents the annealing coordinates where "-" or "+" indicate intronic or exonic sequences respectively. As an example, A(-6+18) would indicate the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. The closest splice site would be the acceptor so these coordinates would be preceded with an "A". Describing annealing coordinates at the donor splice site could be D(+2-18) where the last 2 exonic bases and the first 18 intronic bases correspond to the annealing site of the antisense molecule. Entirely exonic annealing coordinates that would be represented by A(+65+85), that is the site between the 65^th and 85^th nucleotide from the start of that exon. [00028] The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates.

[00029] As used herein the term "derived" and "derived from" shall be taken to indicate that a specific integer may be obtained from a particular source albeit not necessarily directly from that source.

[00030] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[00031] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

Detailed Description

[00032] When antisense molecule(s) are targeted to nucleotide sequences involved in splicing in exons within pre-mRNA sequences, normal splicing of the exon may be inhibited, causing the interruption of normal RNA splicing and the induction of non-functional transcripts and/or the blocking of translation.

[00033] A preferred aim of a therapy based on antisense molecules is to get maximum exon skipping by providing the lowest possible concentration of the antisense molecule. Generally, an antisense molecule may cause strong, robust exon skipping; weak, sporadic exon skipping or no exon skipping at all. It is preferable to develop antisense molecules (alone or in combination) which can deliver strong, robust consistent exon skipping at a low therapeutic dose. Antisense Molecules

[00034] According to a first aspect of the invention, there is provided antisense molecules capable of binding to a selected target to induce exon skipping. To induce exon skipping in exons of the GM-CSF gene transcript, the antisense molecules are preferably selected from the group of antisense molecules shown in Table 1.

Table 1

[00035] There is also provided a combination of two or more antisense molecules capable of binding to a selected target to induce exon skipping. To induce exon skipping in exons of the GM-CSF gene transcript, the antisense combinations are preferably selected from the following combinations (see Table 1 for sequences): c+d, c+d+k, b+d. Most preferably, the combination of antisense oligonucleotides selected is c+d.

[00036] The preferred target site(s) of the GM-CSF RNA are those involved in mRNA splicing (i.e. splice donor sites, splice acceptor sites, or exonic splicing enhancer elements). Splicing branch points and exon recognition sequences or splice enhancers are also potential target sites for modulation of mRNA splicing.

[00037] Oligonucleotides, DNA and RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridisable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense molecule need not be 100% complementary to that of its target sequence to be specifically hybridisable. An antisense molecule is specifically hybridisable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA enough to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[00038] The length of an antisense molecule may vary so long as it is capable of binding selectively to the intended location within the pre-mRNA molecule. The length of such sequences can be determined in accordance with selection procedures described herein. Generally, the antisense molecule will be from about 10 nucleotides in length up to about 50 nucleotides in length. However, it will be appreciated that any length of nucleotides within this range may be used in the method. Preferably, the length of the antisense molecule is between 17 to 30 nucleotides in length. Surprisingly, it has been found that longer antisense molecules are often more effective at inducing exon skipping. Thus, most preferably the antisense molecule is between 24 and 30 nucleotides in length. [00039] Once the antisense molecules to be tested have been identified, they are prepared according to standard techniques known in the art. The most common method for producing antisense molecules is the methylation of the 2' hydroxyribose position and the incorporation of a phosphorothioate backbone. This produces molecules that superficially resemble RNA but that are much more resistant to nuclease degradation.

[00040] To avoid degradation of pre-mRNA during duplex formation with the antisense molecules, the antisense molecules used in the method may be adapted to minimise or prevent cleavage by endogenous RNase H. This property is highly preferred, as the presence of unmethylated RNA oligonucleotides in an intracellular environment or in contact with crude extracts that contain RNase H will lead to degradation of the pre-mRNA: antisense oligonucleotide duplexes. Any form of modified antisense molecules that are capable of by-passing or not inducing such degradation may be used in the present method. The nuclease resistance may be achieved by modifying the antisense molecules of the invention so that it comprises partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups including carboxylic acid groups, ester groups, and alcohol groups.

[00041] An example of antisense molecules which, when duplexed with RNA, are not cleaved by cellular RNase H are 2'-O-methyl derivatives. 2'-O- methyl-oligoribonucleotides (2OME) are very stable in a cellular environment and in animal tissues, and their duplexes with RNA have higher Tm values than their ribo- or deoxyribo- counterparts. Alternatively, the nuclease resistant antisense molecules of the invention may have at least one of the last 3'-terminus nucleotides fluoridated. Still alternatively, the nuclease resistant antisense molecules of the invention have phosphorothioate bonds linking between at least two of the last 3-terminus nucleotide bases, preferably having phosphorothioate bonds linking between the last four 3'-terminal nucleotide bases.

[00042] Antisense molecules that do not activate RNase H can be made in accordance with known techniques (see, e.g., U.S. Pat. 5,149,797). Such antisense molecules, which may be deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modification which sterically hinders or prevents binding of RNase H to a duplex molecule containing the oligonucleotide as one member thereof, which structural modification does not substantially hinder or disrupt duplex formation. Because the portions of the oligonucleotide involved in duplex formation are substantially different from those portions involved in RNase H binding thereto, numerous antisense molecules that do not activate RNase H are available. For example, such antisense molecules may be oligonucleotides wherein at least one, or all, of the inter-nucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Preferred antisense molecules are phosphorodiamidate morpholino oligonucleotides (morpholino antisense oligonucleotides - PMOs). In one aspect, every second internudeotide bridging phosphate residues may be modified as described.

[00043] In another non-limiting example, the antisense molecules are molecules wherein at least one, or all, of the nucleotides contain a 2' lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). In one aspect, every second nucleotide may be modified as described.

[00044] While antisense oligonucleotides are a preferred form of the antisense molecules, the present invention incorporates other oligomeric antisense molecules, including but not limited to oligonucleotide mimetics such as are described below.

[00045] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural inter-nucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their inter-nucleoside backbone can also be considered to be oligonucleosides.

[00046] In other preferred oligonucleotide mimetics, both the sugar and the inter-nucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomenc compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleo-bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

[00047] Modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. Certain nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2°C and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.

[00048] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di- hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-0-hexadecyl-rac-glycero-3-H- phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety.

[00049] In order to enhance transfection rates, antisense molecules such as PMOs may be annealed to 'leashes' as previously reported in (Gebski 2003) to form, for example, PMCdeash lipoplexes. Since antisense molecules such as PMOs are neutral molecules, they do not readily diffuse across cell membrane nor can they be transfected using standard transfecting agents. To overcome this limitation, complementary DNA molecules (leashes) are annealed to the antisense molecules. These leashes facilitate delivery of the antisense molecules into the cells.

[00050] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[00051] The present invention also includes antisense compounds that are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are antisense molecules, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the increased resistance to nuclease degradation, increased cellular uptake, and an additional region for increased binding affinity for the target nucleic acid.

Methods of Manufacturing Antisense Molecules

[00052] The antisense molecules used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesising oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.

[00053] Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. In one such automated embodiment, diethyl- phosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

[00054] The antisense molecules of the invention are synthesised in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The molecules of the invention may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical, inhaled or other formulations, for assisting in uptake, distribution and/or absorption. Most preferably, if the condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF is asthma, a formulation which may be inhaled is preferred.

Therapeutic Agents

[00055] The present invention also can be used as a prophylactic (preventative) or therapeutic (treatment), which may be utilised for the purpose of treatment of a condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF. Conditions which may be treated or prevented by application of the antisense molecules of the present invention include asthma, rheumatoid arthritis and AML.

The antisense molecules of the present invention may be used to treat the condition associated with abnormal levels of GM-CSF, whereby the terms "treat", "treating" and "treatment" and derivatives used herein have the meaning to affect a subject, tissue or cell to produce a desired pharmacological and/or physiological effect. The treatment may be therapeutic in terms of: preventing progression of the condition; relieving or ameliorating the effects of the condition; or causing a partial or complete cure and/or regression of the condition. "Preventing" or "prevention" and derivative terms relate to the partial or complete prevention of development of the condition or its symptoms in a subject who: has not yet been diagnosed with a disorder; has the condition in some but not all of their tissues (e.g. joints in relation to rheumatoid arthritis) and wishes to prevent it developing on new regions; has had the condition and may be in remission and wishes to prevent re-occurrence; or has been diagnosed as being at risk of developing an condition. The condition associated with abnormal levels of GM-CSF may be a disease such as AML or may be an inflammatory condition such as asthma or rheumatoid arthritis.

[00056] Accordingly, in one embodiment the present invention provides antisense molecules that bind to a selected target in the GM-CSF pre-mRNA to induce efficient and consistent exon skipping described herein in a therapeutically effective amount admixed with a pharmaceutically acceptable carrier, diluent, or excipient.

[00057] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similarly untoward reaction, such as gastric upset and the like, when administered to a patient. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA, (1990). [00058] In a more specific form of the invention there are provided pharmaceutical compositions comprising therapeutically effective amounts of an antisense molecule together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCI, acetate, phosphate), pH and ionic strength and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The material may be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 that are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilised form.

[00059] It will be appreciated that pharmaceutical compositions provided according to the present invention may be administered by any means known in the art. Generally, the pharmaceutical compositions for administration may be administered by the pulmonary, or nasal route, or by injection or orally. The antisense molecules are more preferably delivered by inhalation, or by intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous routes of administration. The route of administration will depend largely on the disease to be treated, with antisense molecules used in the treatment of asthma preferably being delivered via the pulmonary route and antisense molecules used in the treatment of rheumatoid arthritis preferably being delivered via parenteral or oral routes of delivery.

[00060] Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition comprising the antisense oligonucleotide within the dispersion can reach the lung where it can, for example, be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.

[00061] Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations; administration by inhalation may be oral and/or nasal. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. A composition comprising an antisense oligonucleotide may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.

[00062] Examples of pharmaceutical devices for aerosol delivery include metered dose inhalers (MDIs), dry powder inhalers (DPIs), and air-jet nebulizers. Exemplary delivery systems by inhalation which can be readily adapted for delivery of the subject antisense oligonucleotides are described in, for example, U.S. Pat. Nos. 5,756,353; 5,858,784; and PCT applications W098/31346; WO98/10796; WO00/27359; WO01/54664; WO02/060412. Other aerosol formulations that may be used for delivering the antisense oligonucleotide agents are described in U.S. Pat. Nos. 6,294,153; 6,344,194; 6,071 ,497, and PCT applications WO02/066078; WO02/053190; WO01/60420; WO00/66206. Methods for delivering antisense oligonucleotides by inhalation are also described in Templin et al., Antisense Nucleic Acid Drug Dev, 2000, 10:359-68; Sandrasagra et al., Expert Opin Biol Ther, 2001 , 1 :979-83; Sandrasagra et al., Antisense Nucleic Acid Drug Dev, 2002, 12:177-81. [00063] The delivery of the inventive agents may also involve the administration of so called "pro-drugs", i.e. formulations or chemical modifications of a therapeutic substance that require some form of processing or transport by systems innate to the subject organism to release the therapeutic substance, preferably at the site where its action is desired. For example, the human lungs can remove or rapidly degrade hydrolytically cleavable deposited aerosols over periods ranging from minutes to hours. In the upper airways, ciliated epithelia contribute to the "mucociliary excalator" by which particles are swept from the airways toward the mouth. Pavia, D., "Lung Mucociliary Clearance," in Aerosols and the Lung: Clinical and Experimental Aspects, Clarke, S. W. and Pavia, D., Eds., Butterworths, London, 1984. In the deep lungs, alveolar macrophages are capable of phagocytosing particles soon after their deposition. Warheit et al. Microscopy Res. Tech., 26: 412-422 (1993); and Brain, J. D., "Physiology and Pathophysiology of Pulmonary Macrophages," in The Reticuloendothelial System, S. M. Reichard and J. Filkins, Eds., Plenum, New York, pp. 315-327, 1985.

[00064] In preferred embodiments, particularly where systemic dosing with the antisense molecule is desired, the aerosoled antisense molecules are formulated as microparticles. Microparticles having a diameter of between 0.5 and 10 microns can penetrate the lungs, passing through most of the natural barriers. A diameter of less than 10 microns is required to bypass the throat; a diameter of 0.5 microns or greater is required to avoid being exhaled.

[00065] If the route of delivery is parenteral, for example for the treatment of rheumatoid arthritis or AML, then suspensions of the antisense molecules as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, e. g., ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol and/or dextran.

Antisense molecule based therapy [00066] Also addressed by the present invention is the use of antisense molecules of the present invention, for manufacture of a medicament for modulation of a condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF. Conditions which may be modulated by the antisense molecules of the present invention include AML and inflammatory conditions such as asthma or rheumatoid arthritis.

[00067] The delivery of a therapeutically useful amount of antisense molecules may be achieved by methods previously published. For example, intracellular delivery of the antisense molecule may be via a composition comprising an admixture of the antisense molecule and an effective amount of a block copolymer. An example of this method is described in US patent application US 20040248833·.

[00068] Other methods of delivery of antisense molecules to the nucleus are described in Mann CJ et al., (2001 ) ["Antisense-induced exon skipping and the synthesis of dystrophin in the mdx mouse". Proc, Natl. Acad. Science, 98(1) 42- 47] and in Gebski et al., (2003). Human Molecular Genetics, 12(15): 1801 -1811.

[00069] A method for introducing a nucleic acid molecule into a cell by way of an expression vector either as naked DNA or complexed to lipid carriers, is described in US patent US 6,806,084.

[00070] It may be desirable to deliver the antisense molecule in a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome formulations.

[00071] Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. These formulations may have net cationic, anionic or neutral charge characteristics and are useful characteristics with in vitro, in vivo and ex vivo delivery methods. It has been shown that large unilamellar vesicles (LUV), which range in size from about 50-500 nm, can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA and DNA can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981).

[00072] In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the antisense molecule of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotech niques, 6:682, 1988).

[00073] The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[00074] Alternatively, the antisense molecule may be combined with other pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition may be formulated for pulmonary, parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration. Most preferably, the composition is formulated for pulmonary delivery.

[00075] The routes of administration described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and any dosage for any particular animal and condition.

[00076] The antisense molecules of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such pro-drugs, and other bioequivalents.

[00077] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[00078] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, (including by nebulizer, intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-0-methoxyethyl modification are believed to be particularly useful for oral administration. [00079] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Kits of the Invention

[00080] The invention also provides kits for treatment of a patient with a condition, such as an inflammatory condition, associated with abnormal levels of GM-CSF which kit comprises at least an antisense molecule, packaged in a suitable container, together with instructions for its use.

[00081] In a preferred embodiment, the kits will contain at least one antisense molecule as shown in Table 1, or a cocktail of antisense molecules and corresponding Leashes as shown in Table 2. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

[00082] The contents of the kit can be lyophilized and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized components. Individual components of the kit would be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[00083] When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example a sterile aqueous solution. For in vivo use, the expression construct may be formulated into a pharmaceutically acceptable syringeable composition. In this case the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an affected area of the animal, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit.

[00084] The components of the kit may also be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. Irrespective of the number or type of containers, the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.

[00085] Those of ordinary skill in the field should appreciate that applications of the above method has wide application for identifying antisense molecules suitable for use in the treatment of a range of inflammatory conditions associated with abnormal levels of GM-CSF. Conditions which may be modified by application of the antisense molecules of the present invention include AML and inflammatory conditions such as asthma or rheumatoid arthritis.

Examples

[00086] The following Examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these Examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. The references cited herein are expressly incorporated by reference.

[00087] Any methods of molecular cloning, immunology and protein chemistry that are not explicitly described in the following examples are reported in the literature and are known by those skilled in the art. General texts that describe conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, include, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); Glover ed., DNA Cloning: A Practical Approach, Volumes I and II, MRL Press, Ltd., Oxford, U.K. (1985); and Ausubel, F., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. Current Protocols in Molecular Biology. Greene Publishing Associates/Wiley Intersciences, New York (2002).

Determining Induced Exon Skipping in GM-CSF

[00088] Attempts by the inventors to develop a rational approach in antisense molecules design were not completely successful as there did not appear to be a consistent trend that could be applied to all exons. As such, the identification of the most effective and therefore most therapeutic antisense molecule compounds have been the result of empirical studies.

[00089] These empirical studies involved the use of computer programs to identify motifs potentially involved in the splicing process. Other computer programs were also used to identify regions of the pre-mRNA which may not have had extensive secondary structure and therefore potential sites for annealing of antisense molecules. Neither of these approaches proved completely reliable in designing antisense oligonucleotides for reliable and efficient induction of exon skipping.

• Example 1

Methods

Antisense Oligonucleotides (AOs) and primers

[00090] AO nomenclature is based on that described by Mann et al., 2002. The sequences of the AOs investigated are provided in Table 1. AOs were designed to anneal to either exonic sequences or exon/intron junctions of human GM-CSF exons 1 , 2, 3 or 4, or the ribosomal entry site or translation start site.

[00091] 20Methyl AOs (20MeAO) were synthesized on an Expedite 8909 Nucleic Acid synthesizer using the 1 μιηοΐβ thioate synthesis protocol. Morpholino AOs (phosphorodiamidate morpholino oligonucleotides - PMOs) were synthesized by gene-tools (Philomath, OR, USA).

[00092] In order to enhance transfection rates, PMOs were annealed to 'leashes' as previously reported in (Gebski 2003) to form PMO:leash lipoplexes. Since PMOs are neutral molecules, they do not readily diffuse across cell membrane nor can they be transfected using standard transfecting agents. To overcome this limitation, complementary DNA molecules (leashes) are annealed to PMOs. These leashes facilitate delivery of PMOs into the cells. The leashes for PMO transfection and the primers for RT-PCR were synthesized by Geneworks (Adelaide, Australia) and are listed in Table 2.

Table 2

[00093] All PMO:leash working solutions were prepared at a final concentration of 50 μΜ of both PMO and respective leash at a 1 :1 molar ratio. To make 50 μΙ of working solution, 12.5 μΙ of 10X phosphate saline buffer (PBS) (pH 7.4), 5 μΙ of PMO stock solution (500 μΜ), appropriate volume of leash (depending on the individual leash concentration) and water was added, resulting in a final concentration of 2.5XPBS and 50 μΜ of both PMO and respective leash. Tubes with the annealing mix were incubated in a thermal cycler according to the following temperature profile: 95°C for 5 min, 85°C for 1 min, 75°C for 1 min, 65°C for 5 min, 55°C for 1 min, 45°C for 1 min, 35°C for 5 min, 25°C for 1 min and 15°C for 1 min. Working stocks were stored at 4°C.

Cell culture and transfection

[00094] Immortalized human bronchial epithelial cells (16HBEs) and Human foetal lung fibroblasts (HFL-1 ) were propagated in 10 ml of 10% foetal bovine serum in Dulbecco minimum eagle media (DMEM) with Glutamine supplemented with penicillin (10 U/ml) and streptomycin (lOpg/ml). 2 x 104 or 1 .25 x 105 16HBE cells were seeded into 24-well or 6-well plate respectively and subsequently incubated for 24 hours before transfection. HFL-1 cells were seeded at 1 .5x104 cells in 24-well plate, or 1 x 105 cells in 6-well plate 24 hours prior transfection.

[00095] Cells were then transfected, using Lipofectin (Invitrogen), with either 20MeAOs or PMCdeash lipoplexes (simply termed PMOs from hereon) at a ratio of 2: 1 Lipofectin to AO.

[00096] Briefly, Lipofectin was mixed with DMEM (Invitrogen) to a final volume of 100 μΙ and incubated for 15 minutes at room temperature. The AO (either 20MeAO or PMO), which had been diluted to a volume of 100 μΙ in DMEM, was then combined with the Lipofectin/DMEM mixture and incubated for a further 25 minutes. The mixture was then made up to a final volume of 1 ml using antibiotic-free 1.5% FCS DMEM. 500 μΙ aliquots of this mixture were added to each well of 24-well plate containing 16HBE/ HFL-1 cells and incubated for 48 hours. For 6-well plates, Lipofectin/AO/DMEM mix was made up to a final volume of 1.5 ml with antibiotic-free 1.5% FCS DMEM and then added to wells containing cells and incubated for 48 hours.

[00097] In the transfection experiments, a variety of non-specific 20MeAOs or PMOs were used as controls, including AOs to antisense strand of Mouse GM- CSF (Control A), intronic sense strand of human Chloride channel receptor gene (Control B), and intronic sense strand of human survival motor neuron gene (Control C, Control E and Control F ), as presented in Table 3. All had no effect. Table 3

[00098] After this time, lipopolysaccharide (LPS) (Escherichia coli serotype 055: B5; Sigma) stimulations were added dependent upon the stimulation protocol as outlined below.

LPS stimulation

[00099] Various doses of LPS, between 1 pg/ml and 0.0128 ng/ml were used to stimulate cultured 16HBE cells 24 hours after seeding to determine normal levels of GM-CSF expression. Supernatants were collected after 12, 24 and 48 hours and stored at -80°C until analysis by Enzyme linked immunosorbent assay (ELISA) to determine the levels of GM-CSF expression.

[000100] Titration study of LPS stimulation on 16HBE showed that LPS at between 8 to 1 .6ng/ml is ample to elicit a high level of supernatant GM-CSF in culture media 48 hours after stimulation (Figure 3).

[000101 ] Two stimulation/tranfection protocols were used to assess the efficiency of AOs (either 2OMeAO or PMO) at inhibiting supernatant GM-CSF production.

[000102] In protocol A (see Figure 13), 16HBE cells were transfected with AOs, according to the protocol described above, at 0 hour, and incubated for 48 hours. This transfection is named transfection 1 (TF1 ). The 16HBE cells were then stimulated with a first LPS stimulation (LPS1 ) for 3 hours. The supernatant was then collected for analysis and the 16HBEs were washed with 1XPBS. The second transfection (TF2) was performed, and the transfection mix was then dispensed onto the cells and incubated for 2 hours. After this, a second LPS stimulation (LPS2) was added and incubated for 24 hours. The supernatant was again collected for analysis, along with the 16HBE cells.

[000103] In protocol B (see Figure 13), 16HBEs were transfected with AOs for 48 hours, and LPS was added at 48 hour post transfection. Supernatant and cells were collected at 24 hours, 5 days (5D) or 7 days (7D) after LPS stimulation for RT-PCR and ELISA.

RNA extraction and RT-PCR

[000104] Total RNA was harvested from 16HBE cells using RNeasy Mini kit (Qiagen Doncaster, Vic, Australia), according to the manufacturer's protocol.

[000105] One-step RT-PCR was undertaken using 150 ng of total RNA as template, in a 12.5 μΙ reaction for 40 cycles of amplification. After the reverse transcription step at 55°C for 30 minutes, the reaction was heated to 94°C for 2 minutes before the primary thermal cycling rounds of 94°C for 40 seconds, 55°C for 1 minute, and 68°C for 1 minute for the amplification of GM-CSF transcript.

[000106] Cycling conditions for PCR to detect 18srRNA (Forward primer: 5' egg eta cca cat cca agg aa 3' (SEQ ID NO: 26); Reverse primer: 5' tgc tgg cac cag act tgc etc 3' (SEQ ID NO: 27)) and GAPDH (Forward primer: 5' aca gtc age cgc ate ttc tt 3' (SEQ ID NO: 28); Reverse primer: 5' acg ace aaa tec gtt gac tc 3' (SEQ ID NO: 29)) transcript started with a reverse transcription step similar to the GM-CSF RT-PCR amplification described above. After heating to 94°C for 2 minutes, the reaction was subjected to 25 rounds of thermal cycling of 94°C for 40 seconds and 60°C for 1 minute.

[000107] PCR products were separated on 2% agarose gels in TAE buffer and the images captured on a CHEMISMART-3000 (Vilber Lourmat, Marne-la- vallee, France) gel documentation system.

Western blotting [000108] 16HBEs were harvested and 4.5 mg of cell pellet was lysed using 100 μΙ lysing buffer (125 mM Tris-HCL pH 6.8, 15% SDS, 10% Glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 15mM dithiothreitol with 3% protease inhibitor mix (Sigma, Missoury, USA)).

[000109] Protein extracts were fractionated by electophoresis on a 4-12% SDS gradient gel (Novex, Invitrogen, Carlsbad, USA)).

[000110] Proteins were transferred from the gel to nitrocellulose membranes (Amersham Biosciences, Castle Hill, Australia) overnight at 4°C, at 290mA.

[000111] GM-CSF was visualised using anti-GM-CSF polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, USA), β-tubulin protein was. employed to standardise the amount of loading protein. Images were captured on a Vilber Lourmat Chemi-Smart 3000 gel documentation system (Vilber Lourmat, Marne-la- vallee, France).

Enzyme linked immunosorbent assay (ELISA) for measuring supernatant GM-CSF

[000112] Supernatants were harvested and kept at -80°c until the ELISA tests were performed.

[000113] GM-CSF production was quantified by sandwich enzyme-linked immunosorbent assay (ELISA) (DuoSet ELISA kit; R&D Systems, Minneapolis, USA), according to the protocol provided by the company. 100 [it of assay diluent was added into each well of a 96-well plate, along with 100 μί of standard, control, or sample. The mixtures were incubated for 2 hours at room temperature. After rigorous washes, 200 μ L of GM-CSF conjugate was added into each well and incubated for 1 hour at room temperature. Substrate solution was subsequently added to each well to develop the colour in proportion to the amount of GM-CSF bound in the initial step. The optical density of each well was determined by using a EnVision multilabel plate reader 2102 (Perkin Elmer, Waltham, USA) at 450 nm. Results

Identification of individual and combinations of 20MeAO treatments for suppression of GM-CSF expression at the transcript level using human lung fibroblasts

[000114] Six 20MAOs ((a-e) - see Table 1) were designed to target splice motifs on the pre-mRNA of human GM-CSF exon 3. HFL-1 cells treated with either 20MAO (a) or (b) showed 5-10% induction of exon 3 skipping, compared to the full-length transcript, while AO (e) had no effect (Figure 4). However, treatment with either 20MeAO (c) or (d) led to a substantial reduction in the full- length GM-CSF transcript. In particular, AO (d) induced 80-90% and AO (c) induced 100% knockdown of wildtype transcript.

[000115] Several combinations of 20MeAOs (a+d, a+e, b+d, b+e, c+d, c+e) were evaluated and appeared to induce transcripts missing exon 3. These combinations reduced the intact transcript at three different 20MeAO concentrations (600, 300 and 150 nM) (Figure 5).

[000116] Further titration studies of selected combinations showed that some 20MeAO combinations can decrease the level of intact transcript when transfected with concentrations as low as 12.5 nM in a dose dependent manner (Figure 6). The (c+d) combination of 20MeAOs was chosen for further evaluation studies

Evaluating efficiency of 20MeAOs in inhibiting the production of GM-CSF

[000117] The efficiency of a combination of 20MeAOs (c+d) to reduce transcription of GM-CSF was evaluated.

[000118] The (c+d) 20MeAO combination was transfected at concentrations of 400 and 200 nM into 16HBEs. The level of full-length GM-CSF transcript was substantially down-regulated as determined by RT-PCR and Western blotting (Figure 7a). An approximately 40% decrease in GM-CSF protein expression at AO concentration at 200nM was demonstrated the in the 20MeAO (c+d) treated sample, as measured by densitometric analysis of Westem blotting of GM-CSF normalised against β-tubulin using Vilber Lourmat Chemi-Smart 3000 gel documentation system (Vilber Lourmat, Marne-la-vallee, France) and Biol D scanning software; Vilber Lourmat, Marne-la-vallee, France) (Figure 7b). A 50% reduction in the production of GM-CSF in the supernatant was detected (Figure 7c) as determined by ELISA at both 200 nM and 400 nM AOs.

[000119] Similarly, after 16HBE cells were treated with 600 nM of 20MeAOs (c+d) and stimulated with 5 ng/ml of LPS (stimulation protocol B), the production of GM-CSF transcript was ablated and an approximately 40% decrease in supernatant GM-CSF was observed.

20MeAO optimisation targeting other exons of GM-CSF transcript to enhance GM-CSF suppression.

[000120] Six further 20MeAOs were designed to target either donor splice site of exons 1 , 2 or acceptor splice site of exons 2 and 4 to induce nonproductive transcripts.

[000121] In unstimulated 16HBE, transfection with 20MeAOs (I, p and q) (Figure 8) or (m, n, or o) (results not shown) failed to either reduce intact GM-CSF transcript or induce transcript missing the targeted exon. However, combinations of 20MeAOs (o+c, p+c, l+c, n+c) targeting multiple splice motifs were evaluated in unstimulated 16HBEs, and substantial reductions in GM-CSF transcript were noted (Figure 9).

[000122] Four new combinations of 20MeAOs (o+c, p+c, l+c, n+c) at a concentration of 900nM were compared to the initial AO-cocktail c+d in inhibiting GM-CSF production on 16HBEs stimulated with 5ng/ml (LPS1) and 1 ng/ml (LPS2) using stimulation protocol A. The combination of 20MeAOs c+d was the most effective at reducing GM-CSF production compared to other combinations (Figure 9). [000123] Four 20MeAOs (h, i, j, k) were designed to mask internal ribosome entry with the aim of interfering with the translation process. Whilst the four 20MeAOs did not change the level of GM-CSF RNA (Figure 10a), transfection with 600 nM 20MeAO (k) led to 15% reduction in GM-CSF production, as measured by EL ISA, in 16HBE treated with 1ug/ml of LPS (Figure 10b).

[000124] A triple combination of 20MeAOs (c+d+k) at 1200nM was transfected into 16HBE stimulated with 10ng/ml of LPS1 and 1ng/ml of LPS2 using stimulation protocol A. A significant reduction in the production of GM-CSF was noted compared to control 20MeAO (Ctr A or Ctr B) - treated 16HBE cells (Figure 11). This result was further improved when compared with the inhibition induced when two AOs (c+d) were used in combination.

Evaluating efficiency of AO on PMOs backbone in inhibiting the production of supernatant GM-CSF

[000125] In order to determine if alternative backbones for the AOs would increase efficacy and/or decrease AO degradation, we determined the inhibitory effect of AO cocktails using a different oligomer backbone, PMO, on GM-CSF expression.

[000126] 16HBEs transfected with PMO (c+d) at a final concentration of 600 nM were stimulated with 1ng/ml of LPS for either 5 or 7 days (Stimulation protocol B). A significant decrease in GM-CSF protein expression at 5 and 7 days was observed compared to control PMO AO (Ctr PMO E or F) - treated 16HBE cells as measured by ELISA (Figure 12).

[000127] The efficacy of PMO (k), (which was previously shown to inhibit GM- CSF production in the form of 20MeAO (k)), was evaluated by ELISA (Figure 12), and led to a significant 50% inhibition of GM-CSF production.

• Example 2

Methods Antisense Oligonucleotides (AOs) and primers

[000128] The AO synthesis and stock solution generation were performed as shown in Example 1.

Cell culture and transfection

[000129] Immortalized human bronchial epithelial cells (16HBEs) were propagated in Dulbecco minimum eagle media (DMEM) with 10% foetal bovine serum (FBS), glutamine, penicillin (10 U/ml) and streptomycin (10pg/ml). 1.25 x 105 16HBE cells were seeded into 6-well plates and incubated for 24 hours before transfection. Cells were transfected using Lipofectin (Invitrogen) with either 20Me AOs or PMO:DNA leash lipoplexes (simply termed PMOs from hereon) at a ratio of 2:1 Lipofectin to AO. Briefly, Lipofectin was mixed with DMEM (Invitrogen) in a final volume of 100 μΙ and incubated for 15 minutes at room temperature. The AO (either 20Me AO or PMO), ^"which had been diluted to 100 μΙ in DMEM, was then combined with the Lipofectin/DMEM mixture and incubated for a further 25 min at room temperature. The mixture was then made up to a final volume of 1.5ml (6-well plate) using antibiotic-free DMEM/1.5% FBS and 500 μΙ aliquots added to each well and incubated for 48 hrs. Unrelated 20Me AOs or PMOs, used in transfection experiments as control AOs, are listed in Table 1a.

LPS stimulation

[000130] Following 48hr incubation post transfection, 5ng/ml lipopolysaccharide (LPS) (Escherichia coli serotype 055: B5; Sigma) stimulation was performed. 16HBE cells were stimulated with LPS to obtain maximal GM- CSF expression. Cells were stimulated for 48hrs when using 20Me AO and 5 days when using PMO AO. Supernatants were collected and stored at -80°C.

RNA extraction and RT-PCR analysis

[000131] Total RNA was harvested from 16HBE cells using RNeasy Mini kit (Qiagen Doncaster, Vic, Australia), according to the manufacturer's protocol. One-step RT-PCR (Invitrogen) was performed using 150 ng of total RNA as template, in a 12.5 μΙ reaction for 40 cycles of amplification, using Superscript RT. After the reverse transcription step at 55 °C for 30 min, the reaction was heated to 94°C for 2 min before the primary thermal cycling rounds of 94°C for 40 sec, 55°C for 1 min, and 68°C for 1 min for the amplification of GM-CSF transcript.

[000132] Cycling conditions for PCR to detect GAPDH (Forward primer: 5' aca gtc age cgc ate ttc tt 3' (SEQ ID NO: 28) Reverse primer: 5' acg acc aaa tec gtt gac tc 3' (SEQ ID NO: 29)) transcript started with a reverse transcription step similar to the GM-CSF RT-PCR amplification described above. After heating to 94°C for 2 minutes, the reaction was subjected to 25 rounds of thermal cycling of 94°C for 40 seconds and 60°C for 1 minute.

[000133] PCR products were separated on 2% agarose gels in TAE buffer and the images captured on a CHEMISMART-3000 (Vilber Lourmat, Marne-la- vallee, France) gel documentation system.

Western blotting

[000 34] Western Blotting was performed as shown in Example 1.

Enzyme linked immunosorbent assay (ELISA) for measuring supernatant GM-CSF

[000135] GM-CSF ELISA was performed as shown in Example 1. Intranasal delivery of PMO in the mouse lung

[000136] To demonstrate a delivery method for therapeutic PMOs into the lung via intranasal route, fluorescently labelled PMOs were administered in the mouse model of allergen challenged lung inflammation (Figure 20). Two days after the allergen sensitisation, mice were treated with 5 mg/kg of fluorescent PMO or the saline solution. 4 hours later, mice were euthanized (pentobarbitone sobium, 160 mg/kg) and lungs and tracheas collected, fixed, dehydrated in the sucrose and snap frozen in liquid nitrogen. Lungs were cryosectioned and the intensity of the fluorescence was evaluated using fluorescent microscope. Results

[000137] 2'-0-Methyl modified anti-oligonucleotides (20Me AOs) targeting exon 3 of human GM-GSF (a, b, c, d, e) can suppress baseline gene expression.

[000138] Five 20Me AOs (a-e) were designed to target splice motifs in exon 3 of human GM-CSF pre-mRNA (Figure 1). When antisense molecule(s) are targeted to nucleotide sequences involved in splicing in exons within pre-mRNA sequences, normal splicing of the exon may be inhibited, causing the interruption of normal RNA splicing and the induction of non-functional transcripts and/or the blocking of translation.

[000139] 16HBE cells treated with 20Me AO (a) or (d) showed induction of exon 3 skipping at 600nM, compared to the full-length transcript (sham), while AO (b) or (e) had no effect (Figure 14a). However, treatment with 150-600nM 20Me AO (c) led to a substantial reduction in the full-length GM-CSF transcript with an induced near complete knockdown of wildtype transcript (Figure 14a).

[000140] Several combinations of 20Me AOs (a+d, a+e, b+d, b+e, c+d, c+e) were evaluated (Figure 14b). Only the 20Me AOs (c+d) combination showed an induction of exon 3 skipping. This effect was dose dependent and observed at the very low concentration of 25 nM (Figure 14b). This (c+d) combination of 20Me AOs was chosen for further evaluation studies.

[000141] 20Me AOs targeting the acceptor of donor sites of exon 1, 2 or 4 (I, n, o, p; Figure 1) did not show any suppression of GM-CSF expression (data not shown).

[000142] 2'-0-Methyl modified anti-oligonucleotides (20Me AOs) targeting the translation start site of human GM-GSF to prevent translation did not suppress baseline gene expression.

[000143] 20Me AOs (f, g, h, i, j, k) were designed to mask internal ribosome entry with the aim of interfering with the translation process. None of these individual 20Me AOs changed the production of GM-CSF mRNA or protein (Figures 15 for (k) and data not shown for (f,g,h,i,j)).

[000144] Not surprisingly a combination of the (k) AO with the 20Me AOs (c+d), targeting exon 3, did not further induce knockdown of the wildtype transcript at the mRNA or protein level (Figure 15). However, the 20Me AOs (c+d) combination, targeting exon 3, elicited a significant reduction in both mRNA and intracellular (p=0.0017) GM-CSF levels (Figure 15 and 16).

[000145] The combination of (c+d) 20MeAOs was transfected into 16HBE and stimulated with 5ng/ml LPS for 48hr. A significant reduction in GM-CSF mRNA and secreted GM-CSF was noted compared to sham 20MeAO (Figure 16).

[000146] The effectiveness of phosphorodiamidate morpholino oligomers (PMOs) anti-oligonucleotide backbone, at baseline and LPS stimulation was investigated to determine if alternative backbones for the AOs would increase efficacy and/or decrease AO degradation. The combination of (c+d) PMO significantly reduced unstimulated intracellular protein GM-CSF (Fig 18a) and LPS-induced secreted GM-CSF protein (Fig 18b), compared to sham PMO.

[000147] Modifications of the above-described modes of carrying out the various embodiments of this invention will be apparent to those skilled in the art based on the above teachings related to the disclosed invention. The above embodiments of the invention are merely exemplary and should not be construed to be in any way limiting.

Claims

The Claim Defining the Invention is as Follows:

1. A purified and isolated antisense molecule capable of binding to a selected target site to induce exon skipping and/or translation blocking in the GM-CSF gene selected from the antisense molecules of SEQ ID NO: 1 to 17.

2. The antisense molecule of claim 1 that is SEQ ID NO: 3 or SEQ ID NO: 4.

3. A combination of two or more purified and isolated antisense molecules capable of binding to a selected target site to induce exon skipping and/or translation blocking in the GM-CSF gene selected from the antisense molecules of SEQ ID NO: 1 to 17.

4. The combination of claim 3 comprising SEQ ID NO: 3 and SEQ ID NO: 4.

5. The antisense molecules of claim 1 or 3 that is a morpholino or 2'-0-methyl derivative.

6. An antisense molecule according to claim 1 or 3 further comprising a leash.

7. Use of the antisense molecules of claim 1 or 3 for the induction of exon skipping and/or reduction in translation of GM-CSF.

8. A method for treating a patient suffering from a condition associated with abnormal levels of GM-CSF, comprising the steps of: (a) selecting an antisense molecule according to claim 1 or 3; and (b) administering the antisense molecule and a pharmaceutically acceptable carrier to a patient in need of such treatment.

9. A method for prophylactically treating a patient to prevent or at least minimise a condition associated with abnormal levels of GM-CSF, comprising the steps of: (a) selecting an antisense molecule according to claim 1 or 3; and (b) administering to the patient an effective amount of the antisense molecule and a pharmaceutically acceptable carrier to a patient in need of such treatment.

10. Use of an antisense molecule of claim 1 for the manufacture of a medicament for treatment or prophylaxis of a condition associated with abnormal levels of GM-CSF.

11. A kit for treating a condition associated with abnormal levels of GM-CSF, which kit comprises an antisense molecule according to claim 1 or 3, packaged in a suitable container and instructions for use of the antisense molecule.

12. The method of claims 7 or 8, or the use of claim 9 wherein the condition is selected from the following: asthma, rheumatoid arthritis, acute myeloid leukaemia.