AU753972B2 - Low adenosine oligonucleotide, composition, kit and methods for obtaining oligonucleotide and for treatment of airway disease(s) - Google Patents

Description

LOW ADENOSINE OLIGONUCLEOTIDE, COMPOSITION, KIT METHODS FOR OBTAINING OLIGONUCLEOTIDE FOR TREATMENT OF AIRWAY DISEASE(S) Field of the Invention This application concerns a method of administering antisense oligonucleotides essentially free of adenosine as a treatment for lung diseases.

Background of the Invention Antisense oligonucleotides have received considerable theoretical consideration as potentially useful pharmacologic agents in human disease. R. Wagner, Nature 372, 333-335 (1994). However, practical applications of these molecules in actual models of human 15 disease have been elusive. One important consideration in the pharmacologic application of these molecules is route of administration. Most experiments utilizing antisense oligonucleotides in vivo have involved direct application to limited regions of the brain (see C.

20 Wahlestedt, Trends in Pharmacological Sciences 15, 42-46 (1994); J. Lai et al., Neuroreport 5, 1049-1052 (1994); K. Standifer et al., Neuron 12, 805-810 (1994); A.

Akabayashi et al., Brain Research 21, 55-61 (1994)), or to spinal fluid (see e.g. L. Tseng et al., European J.

Pharmacol. 258, R1-3 (1994); R. Raffa et al., European J. Pharmacol. 258, R5-7 (1994); F. Gillardon et al., European J. Neurosci. 6, 880-884 (1994)). Such applications have limited clinical utility due to their invasive nature.

The systemic administration of antisense oligonucleotides also poses significant problems with respect to pharmacologic application, not the least of which is the difficulty in targeting disease-involved tissues. In contrast, the lung is an excellent potential target for antisense oligonucleotide application since it may be approached noninvasively and in a tissue-specific manner. Additionally, the lung represents an exceptional target for antisense ODN therapeutics ascompared to other in vivo target organs or tissues, possibly because the lung is lined with surfactant which consists primarily of cationic lipids, well known to enhance cellular uptake of ODNs in other systems. However, the technology involved in delivering antisense agents to the lung remains relatively undeveloped, and potential problems related to the application of antisense agents to the lung remain unexplored.

Adenosine, a purine which contributes to intermediary metabolism and participates in the regulation of physiological activity, is a recognized neuromodulator. This nucleoside is involved in many local regulatory mechanisms, in particular at synapses in the CNS and at neuroeffector junctions in the periphery.

In the CNS adenosine is known to inhibit the release of 25 a variety of neurotransmitters (noradrenaline, serotonin, GABA, acetylcholine, dopamine, glutamate, etc.), to inhibit neurotransmission, depress neuronal firing, induce spinal analgesia, and to possess anxiolytic properties Ben-Soreket al., Archives of Internal Medicine 153, 2701-2702 (1993)). In the heart, adenosine •is known to slow atrioventricular (AV) conduction, suppress pacemaker activity, possess antiarrhythmic effects, modulate autonomic control, and to trigger the synthesis and release of prostaglandins. M.K. Church et 35 al., J. Allergy Clinical Immunology 92, 190-194 (1993). It also possesses potent vasodilatory effects and modulates vascular tone. S.T. Holgate et al., Annals -3of the New York Academy of Sciences 629, 227-236 (1991).

As a therapeutic agent, adenosine has achieved considerable recent success as an antiarryhthmic agent in the treatment of supraventricular tachycardia. See C.G.

DeGroff and M.J. Silka, Journal of Pediatrics 125, 822- 823 (1994); I. Drake et al., Human and Exp. Toxicol. 13, 263-265 (1994). However, many adverse effects of adenosine treatment have been reported in the literature.

See, A. Aggarwal, et al., Anesthesiology 79, 1132- 1135 (1993); K.K. Burkhart, American J. Emergency Med.

11, 249-250 (1993); S.K. Srinivasan and P.J. Iversen, J.

Clin. Lab. Analysis 9, 129- 137 (1995); C.A. Stein et al., Pharmacology Therapeutics 52, 365-384 (1991); B.B.

Fredholm et al., Pharmacological Reviews 46, 143-156 (1994); H. Saito, et al., Blood 66, 1233-1240 (1985). In particular, asthmatic individuals show an extreme sensitivity to adenosine and adenosine monophosphate.

See, J.H. Butterfield et al., Leukemia Res. 12, 345-355 (1988); CLONETICS: Normal Human Cell Systems Manual (1995); R.W. Wagner, Nature 372, 333-335 (1994).

SSerious, near-fatal induction of bronchospasm has occurred in asthmatic individuals administered adenosine for supraventricular tachycardia. See, S. Tabor, in: 25 Current Protocols in Molecular Biology, Vol. 1, Section 3.10.2 (John Wiley Sons, 1987); J.H. Weiss, Id., at Section 6.2.2.

Similarly, asthmatic rabbits produced using the dust mite allergic rabbit model of human asthma also were shown to respond to aerosolized adenosine with marked bronchoconstriction, while non asthmatic rabbits showed no response. S. Ali et al., Agents Actions 37, 165-176 (1992). Recent work using this model system has suggested S"that adenosine-induced bronchoconstriction and bronchial hyperresponsiveness in asthma are mediated primarily through the stimulation of adenosine receptors. S. Ali et.

4 al., J. Pharmacol, Exp. Ther. 268, 1328-1334 (1994); S.

Ali et al., Am. J. Physiol 266, L271-277 (1994).

Accordingly, adenosine is contraindicated in the lungs of asthmatics (who represent 10% of the adult and of the paediatric population in the United States).

Since antisense ODNs are typically composed of all four base pairs, adenine, guanine, cytosine and thymidine, their breakdown products will produce free deoxyadenosine monophosphate in these hyperresponsive airways.

Deoxyadenosine monophosphate differs from adenosine monophosphate only by the loss of an oxygen atom on the 3' carbon of the sugar moiety.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", means "including but not limited to" and its not intended to exclude other additives, components, integers or steps.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the 25 references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does 30 not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

SUMMARY OF THE INVENTION 9 .99 9 The present invention provides a method of treating airway disease in a subject in need of such H:\RBell\Keep\71749-00.doc 10/09/02 4a treatment. The method comprises administering an antisense oligonucleotide containing three or fewer adenosines per 21 nucleotides to the lungs of the subject in an amount effective to treat the airway disease.

The present invention further provides a pharmaceutical composition, comprising a pharmaceutically or veterinarily acceptable carrier or diluent, and an aerosol of an oligonucleotide(s) (oligo(s)) which is (are) effective for alleviating bronchoconstriction, lung inflammation or allergy(ies), wherein the oligo contains no more than 3 adenosines or, if at least 21 nucleotides long, no more than 3 A per every 21 nucleotides and is antisense to a target selected from the group consisting of the initiation codon, the coding region or the 5'-end and the 3'-end of a gene encoding a polypeptide associated with lung airway dysfunction or anti-sense to the corresponding mRNA(s), combinations of the oligos and mixtures of the oligos.

The present invention further provides the use of an antisense oligonucleotide containing three or fewer adenosines per 21 nucleotides for the preparation of a Smedicament for treating airway disease in a subject in 25 need of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1-4 demonstrate that antisense 30 oligonucleotides can be utilised as effective agents in the H:\RBell\Keep\71749-00.doc 10/09/02 5 treatment or prevention of airway diseases.

Figure 1 illustrates the effects of Ai adenosine receptor antisense oligonucleotides and mismatch control antisense oligonucleotides on the dynamic compliance of the bronchial airway in a rabbit model.

Figure 2 illustrates the specificity of Ai adenosine receptor antisense oligonucleotides as indicated by the Ai and A 2 adenosine receptor number present in Ai adenosine receptor antisense oligonucleotide-treated airway tissue.

Figure 3 is a graphical representation illustrating that aerosolized deoxyadenosine monophosphate is a potent bronchoconstrictor in asthmatic pathways of allergic rabbits. Further, the figure shows that the effect of deoxyadenosine monophosphate is equipotent to that observed for adenosine monophosphate.

Figure 4 is a graphical representation illustrating that bronchoconstrictor effects occur with aerosolized phosphorothioate oligodeoxynucleotides containing adenosine, but not with oligodeoxynucleotides that are free of adenosine.

DETAILED DESCRIPTION OF THE INVENTION Nucleotide sequences are presented herein by 25 single strand only, in the 5' and 3' direction, from left to right. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by three letter code, in accordance with 37 CFR §1.822 and 30 established usage. See, PatentIn User Manual, 99-102 (Nov. 1990) Patent and Trademark Office, Office of the Assistant Commissioner for Patents, Washington, D.C.

20231); U.S. Patent No. 4,871,670 to Hudson et al. at Col.

S3 lines 20-43. The relevant sections of the disclosures of this and all other patents and other references cited in this patent are incorporated herein by reference).

The method of .the present invention may be used 6 to treat airway disease in a subject for any reason, with the intention that adenosine content of antisense compounds be eliminated or reduced so as to prevent its liberation upon antisense degradation. Such liberation may cause serious, even life-threatening, bronchoconstriction in patients with hyperreactive airways. Examples of airway diseases that may be treated by the method of the present invention include cystic fibrosis, asthma, chronic obstructive pulmonary disease, bronchitis, and other airway diseases characterised by an inflammatory response.

Antisense oligonucleotides to the Ai and A 3 receptor are shown to be effective in the downregulation of Ai or A 3 in the cell. One novel feature of this treatment, as compared to traditional treatments for adenosine-induced bronchoconstriction, is its direct administration to the lungs. The present treatment, additionally selectively reduces the amount or level of a receptor protein itself as is the case with treatments where the drug merely interacts with the receptor. The selective characteristic of the present oligonucleotide results in a reduction in toxicity.

Other proteins that may be targeted with antiserse agents for the treatment of lung conditions include, but are not S.limited to: human A2a adenosine receptor, human A2b adenosine receptor, human IgE receptor i, human Fcepsilon receptor CD23 antigen, human histidine decarboxylase, human beta tryptase, human tryptase-I, human prostaglandin D synthase, human cyclooxygenase-2, human eosinophil cationic protein, human eosinophil derived neurotoxin, human eosinophil peroxidase, human intercellular adhesion molecule-1 (ICAM-1), human vascular cell adhesion molecule 1 (VCAM-1), human endothelial leukocyte adhesion molecule (ELAM-1), human P selectin, human endothelial monocyte activating factor, o•* 6a human IL-3, human IL-4, human IL-5, human IL-6, human IL- 8, human monocyte-derived neutrophil chemotactic factor, human neutrophil elastase, human neutrophil oxidase factor, human cathepsin G, human defensin 1, human defensin 3, human macrophage inflammatory protein-1alpha, human muscarinic acetylcholine receptor HM1, human muscarinic acetylcholine receptor HM3, human fibronectin, human GM-CSF, human tumor necrosis factor a, human e -7leukotriene C4 synthase, human major basic protein, and human endothelin 1. In these latter targets, and. in target genes in general, it is particularly imperative to eliminate or reduce the adenosine content of the corresponding antisense oligonucleotide to prevent their breakdown products from liberating adenosine.

As used herein, the term "treat" or "treating" a lung disease refers to a treatment which decreases the likelihood that the subject administered such treatment will manifest symptoms of the lung disease. The term "downregulate" 'refers to inducing a decrease in production, secretion or availability (and thus a decrease in concentration) of the targeted intracellular protein.

The present invention is concerned primarily with the treatment of human subjects but may also be employed for the treatment of other mammalian subjects, such as dogs and cats, for veterinary purposes. Targeted proteins are preferably mammalian and more preferably of the same species as the subject being treated.

In general, "antisense" refers to the use of small, synthetic oligonucleotides, resembling singlestranded DNA, to inhibit gene expression by inhibiting the function of the target messenger RNA (mRNA).

Milligan, J.F. et al., J. Med. Chem. 36(14), 1923-1937 (1993). In the present invention, inhibition of gene expression of the A, or A 3 adenosine receptor is desired.

Gene expression is inhibited through hybridization to coding (sense) sequences in a specific messenger RNA (mRNA) target by hydrogen bonding according to Watson- Crick base pairing rules. The mechanism of antisense inhibition is that the exogenously applied oligonucleotides decrease the mRNA or protein levels of the target gene or cause changes in the growth 35 characteristics or shapes of the cells. Id. See also Helene, C. and Toulme, Biochim. Biophys. Acta 1049, 99-125 (1990); Cohen, Ed., Oligodeoxynucleotides as -8- Antisense Inhibitors of Gene Expression; CRC Press:Boca Raton, FL (1987).

As used herein, "antisense oligonucleotide" is defined as a short sequence of synthetic nucleotides that hybridizes to any coding sequence in an mRNA which codes for the targeted protein, according to hybridization conditions described below, and upon hybridization causes a decrease in gene expression of the targeted protein.

The mRNA sequence of the A, or A 3 adenosine receptor is derived from the DNA base sequence of the gene expressing either the A, or A 3 adenosine receptor.

The sequence of the genomic human A, adenosine receptor is known and is disclosed in U.S. Patent No. 5,320,963 to G.

Stiles et al. The A 3 adenosine receptor has been cloned, sequenced and expressed in rat (see F. Zhou et al., Proc.

Nat'1 Acad. Sci. USA 89:7432 (1992)) and human (see M.A.

Jacobson et al., U.K. Patent Application No. 9304582.1 (1993)). Thus, antisense oligonucleotides that downregulate the production of the Ai or A 3 adenosine receptor may be produced in accordance with standard techniques.

One aspect of this invention is an antisense oligonucleotide having a sequence capable of binding specifically with any sequence of an mRNA molecule which encodes an airway disease-associated protein so as to prevent translation of the mRNA molecule.

Chemical analogs of oligonucleotides oligonucleotides in which the phosphodiester bonds have been modified, to the methylphosphonate, the phosphotriester, the phosphorothioate, the phosphorodithioate, or the phosphoramidate, so as to render the oligonucleotide more stable in vivo) are also an aspect of the present invention. The naturally 35 occurring phosphodiester linkages in oligonucleotides are susceptible to degradation by endogenously occurring cellular nucleases, while many analogous linkages are -9highly resistant to nuclease degradation. See Milligan et al., and Cohen, supra. Protection from degradation can be achieved by use of a "3'-end cap" strategy by which nuclease-resistant linkages are substituted for phosphodiester linkages at the 3' end of the oligonucleotide. See Tidd, D.M. and Warenius, H.M., Br. J. Cancer 60, 343-350 (1989); Shaw, J.P. et al., Nucleic Acids Res. 19, 747-750 (1991). Phosphoramidates, phosphorothioates, and methylphosphonate linkages all function adequately in this manner. More extensive modification of the phosphodiester backbone has been shown to impart stability and may allow for enhanced affinity and increased cellular permeation of oligonucleotides. See Milligan, et al., supra. Many different chemical strategies have been employed to replace the entire phosphodiester backbone with novel linkages. Id. Backbone analogues include phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, boranophosphate, phosphotriester, formacetal, 3'-thioformacetal, 5'-thioformacetal, thioether, carbonate, 5'-N-carbamate, sulfate, sulfonate, s" sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methylimino) (MMI) or methyleneoxy(methylimino) (MOMI) linkages.

25 Phosphorothioate and methylphosphonate-modified oligonucleotides are particularly preferred due to their availability through automated oligonucleotide synthesis.

Id. Where appropriate, the antisense oligonucleotides may be administered in the form of their pharmaceutically acceptable salts.

Antisense oligonucleotides may be of any suitable length from about 10 to 60 nucleotides in length), depending on the particular target beingbound *and the mode of delivery thereof. Preferably the S" 35 antisense oligonucleotide is directed to an mRNA region containing a junction between intron and exon. Where the antisense oligonucleotide is directed to an intron/exon junction, it may either entirely overlie the junction or may be sufficiently close to the junction to inhibit splicing out of the intervening exon during processing of precursor mRNA to mature mRNA with the 3' or terminus of the antisense oligonucleotide being is positioned within about, for example, 10, 5, 3, or 2 nucleotides of the intron/exon junction).

When practicing the present invention, the antisense nucleotides administered may be related in origin to the species to which it is administered. When treating humans, human antisense may be used if desired.

Pharmaceutical compositions comprising an antisense oligonucleotide as given above effective to reduce expression of an A, or A 3 adenosine receptor by passing through a cell membrane and binding specifically with mRNA encoding an A, or A 3 adenosine receptor in the cell so as to prevent its translation are another aspect of the present invention. Such compositions are provided in a suitable pharmaceutically acceptable carrier sterile pyrogen-free saline solution). The antisense oligonucleotides may be formulated with a hydrophobic carrier capable of passing through a cell membrane in a liposome, with the liposomes carried in a pharmaceutically acceptable aqueous carrier). The 25 oligonucleotides may also be coupled to a substance which inactivates mRNA, such as a ribozyme. Such oligonucleotides may be administered to a subject to inhibit the activation of A, or A 3 adenosine receptors, which subject is in need of such treatment for any of the reasons discussed herein.. Furthermore, the pharmaceutical formulation may also contain chimeric molecules comprising antisense oligonucleotides attached to molecules which are known to be internalized by cells.

.These oligonucleotide conjugates utilize cellular uptake 35 pathways to increase cellular concentrations of oligonucleotides. Examples of macromolecules used in this manner include transferrin, asialoglycoprotein -11- (bound to oligonucleotides via polylysine) and streptavidin.

In the pharmaceutical formulation the antisense compound may be contained within a lipid particle or vesicle, such as a liposome or microcrystal. The particles may be of any suitable structure, such as unilamellar or plurilamellar, so long as the antisense oligonucleotide is contained therein. Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,Ntrimethyl-ammoniumethylsulfate, or "DOTAP," are particularly preferred for such particles and vesicles.

The preparation of such lipid particles is well known.

See, U.S. Patent Nos. 4,880,635 to Janoff et al.; 4,906,477 to Kurono et al.; 4,911,928 to Wallach; 4,917,951 to Wallach; 4,920,016 to Allen et al.;4,921,757 to Wheatley et al.; etc.

Subjects may be administered the active composition by any means which transports the antisense nucleotide composition to the lung. The antisense compounds disclosed herein may be administered to the lungs of a patient by any suitable means, but are preferably administered by generating an aerosol comprised of respirable particles, the respirable particles comprised of the antisense compound, which 25 particles the subject inhales. The respirable particles may be liquid or solid. The particles may optionally contain other therapeutic ingredients.

Particles comprised of antisense compound for practicing the present invention should include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about .5 to microns in size are respirable. Particles of non- 35 respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is preferably -12minimized. For nasal administration, a particle size in the range of 10-500 Am is preferred to ensure retention in the nasal cavity.

Liquid pharmaceutical compositions of active compound for producing an aerosol can be prepared by combining the antisense compound with a suitable vehicle, such as sterile pyrogen free water. Other therapeutic compounds may optionally be included.

Solid particulate compositions containing respirable dry particles of micronized antisense compound may be prepared by grinding dry antisense compound with a mortar and pestle, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprised of the antisense compound may optionally contain a dispersant which serves to facilitate the formation of an aerosol. A suitable dispersant is lactose, which may be blended with the antisense compound in any suitable ratio a 1 to 1 ratio by weight). Again, other therapeutic compounds may also be included.

The dosage of the antisense compound administered will depend upon the disease being treated, the condition of the subject, the particular formulation, the route of administration, the timing of administration to a subject, etc. In general, intracellular concentrations of the oligonucleotide of from .05 to AM, or more particularly .2 to 5 AM, are desired. For administration to a subject such as a human, a dosage of from about .01, or 1 mg/Kg up to 50, 100, or 150 mg/Kg or more is typically employed. Depending on the solubility of the particular formulation of active compound administered, the daily dose may be divided Samong one or several unit dose administrations.

Administration of the antisense compounds may be carried out therapeutically as a rescue treatment) or prophylactically.

-13- Aerosols of liquid particles comprising the antisense compound may be produced by any suitable means, such as with a nebulizer. See,. U.S. Patent No.

4,501,729. Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient comprising up to w/w of the formulation, but preferably less than w/w. the carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride.

Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactants.

Aerosols of solid particles comprising the active compound may likewise be produced with any solid particulate medicament aerosol generator. Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration.

One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff.

In the insufflator, the powder a metered dose thereof effective to carry out the treatments described 35 herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn -14through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 to 150 Al, to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation -may additionally contain one or more cosolvents, for example, ethanol, surfactants, such as S. oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents.

The aerosol, whether formed from solid or 25 liquid particles, may be produced by the aerosol generator at a rate of from about 10 to 150 liters per minute, more preferably from about 30 to 150 liters per minute, and most preferably about 60 liters per minute.

Aerosols containing greater amounts of medicament may be administered more rapidly.

The following examples are provided to illustrate the present invention, and should not be construed as limiting thereon. In these examples, AM means micromolar, mL means milliliters, Am means S. 35 micrometers, mm means millimeters, cm means centimeters, OC means degrees Celsius, Ag means micrograms, mg means milligrams, g means grams, kg means kilograms, M means molar, and h means hours.

EXAMPLE 1 Design and synthesis of antisense oligonucleotides The design of antisense oligonucleotides against the Ai and A 3 adenosine receptors may require the solution of the complex secondary structure of the target A, receptor mRNA and the target A 3 receptor mRNA. After generating this structure, antisense nucleotides are designed which target regions of mRNA which might be construed to confer functional activity or stability to the mRNA and which optimally may overlap the initiation codon. Other target sites are readily usable. As a demonstration of specificity of the antisense effect, other oligonucleotides not totally complementary to the target mRNA, but containing identical nucleotide compositions on a w/w basis, are included as controls in antisense experiments.

Adenosine A, receptor mRNA secondary structure was analyzed and used as described above to design a phosphorothioate antisense oligonucleotide. The antisense oligonucleotide which was synthesized was designated HAdAlAS and had the following sequence: 5'-GAT GGA GGG CGG CAT GGC GGG-3' (SEQ ID NO:1) As a control, a mismatched phosphorothioate antisense nucleotide designated HAdA1MM was synthesized with the following sequence: 5'-GTA GCA GGC GGG GAT GGG GGC-3' (SEQ ID NO:2) Each oligonucleotide had identical base content and 30 general sequence structure. Homology searches in GENBANK .i (release 85.0) and EMBL (release 40.0) indicated that the antisense oligonucleotide was specific for the human and rabbit adenosine A, receptor genes, and that the -16mismatched control was not a candidate for hybridization with any known gene sequence.

Adenosine

A

3 receptor mRNA secondary structure was similarly analyzed and used as described above to design two phosphorothioate antisense oligonucleotides.

The first antisense oligonucleotide (HAdA3AS1) synthesized had the following sequence: GTT GGG CAT CTT GCC-3' (SEQ ID NO:3) As a control, a mismatched phosphorothioate antisense oligonucleotide (HAdA3MM1) was synthesized, having the following sequence: CTT GCG GAT CTA GGC-3' (SEQ ID NO:4) A second phosphorothioate antisense oligonucleotide (HAdA3AS2) was also designed and synthesized, having the following sequence: GGC CTA GCT CTC GCC-3' (SEQ ID Its control oligonucleotide (HAdA3MM2) had the sequence: 5'-GTC GGG GTA CCT GTC GGC-3' (SEQ ID NO:6) Phosphorothioate oligonucleotides were synthesized on an Applied Biosystems Model 396 Oligonucleotide Synthesizer, and purified using NENSORB chromatography (DuPont, MD).

EXAMPLE 2 Testing of Al-Adenosine Receptor 25 Antisense Oliconucleotides in vitro The antisense oligonucleotide against the human AI receptor (SEQ ID NO:1) described above was tested for -17efficacy in an in vitro model utilizing lung adenocarcinoma cells HTB-54. HTB-54 lung adenocarcinoma cells were demonstrated to express the A, adenosine receptor using standard northern blotting procedures and receptor probes designed and synthesized in the laboratory.

HTB-54 human lung adenocarcinoma cells (106/100 mm tissue culture dish) were exposed to 5.0 AM HAdAlAS or HAdA1MM for 24 hours, with a fresh change of media and oligonucleotides after 12 hours of incubation. Following 24 hour exposure to the oligonucleotides, cells were harvested and their RNA extracted by standard procedures.

A 21-mer probe corresponding to the region of mRNA targeted by the antisense (and therefore having the same sequence as the antisense, but not phosphorothioated) was synthesized and used to probe northern blots of RNA prepared from HAdAlAS-treated, HAdA1MM-treated and nontreated HTB-54 cells. These blots showed clearly that HAdAlAS but not HAdA1MM effectively reduced human adenosine receptor mRNA by This result showed that HAdAlAS is a good candidate for an anti-asthma drug since it depletes intracellular mRNA for the adenosine A, receptor, which is involved in asthma.

EXAMPLE 3 25 Efficacy of A,-Adenosine Receptor Antisense Oligonucleotides in vivo A fortuitous homology between the rabbit and human DNA sequences within the adenosine A, gene

S

overlapping the initiation codon permitted the use of the 30 phosphorothioate antisense oligonucleotides initially designed for use against the human adenosine. A receptor in a rabbit model.

Neonatal New Zealand white Pasteurella-free rabbits were immunized intraperitoneally within 24 hours of birth with 312 antigen units/mL house dustmite (D.

farinae) extract (Berkeley Biologicals, Berkeley, CA), -18mixed with 10% kaolin. Immunizations were repeated weekly for the first month and then biweekly for the next 2 months. At 3-4 months of age, eight sensitized rabbits were anesthetized and relaxed with a mixture of ketamine hydrochloride (44 mg/kg) and acepromazine maleate (0.4 mg/kg) administered intramuscularly.

The rabbits were then laid supine in a comfortable position on a small molded, padded animal board and intubated with a 4.0-mm intratracheal tube (Mallinkrodt, Inc., Glens Falls, NY). A polyethylene catheter of external diameter 2.4 mm with an attached latex balloon was passed into the esophagus and maintained at the same distance (approximately 16 cm) from the mouth throughout the experiments. The intratracheal tube was attached to a heated Fleisch pneumotachograph (size 00; DOM Medical, Richmond, VA), and flow was measured using a Validyne differential pressure transducer (Model DP-45161927; Validyne Engineering Corp., Northridge, CA) driven by a Gould carrier amplifier (Model 11-4113; Gould Electronic, Cleveland, OH). The esophageal balloon was attached to one side of the differential pressure transducer, and the outflow of the intratracheal tube was connected to the opposite side of the pressure transducer to allow 25 recording of transpulmonary pressure. Flow was integrated to give a continuous tidal volume, and measurements of total lung resistance (RL) and dynamic compliance (Cdyn) were calculated at isovolumetric and flow zero points, respectively, using an automated respiratory analyzer (Model 6; Buxco, Sharon, CT).

Animals were randomized and on Day 1 pretreatment values for PC50 were obtained for aerosolized adenosine. Antisense (HAdAlAS) or mismatched control (HAdAIMM) oligonucleotides were dissolved in 35 sterile physiological saline at a concentration of 5000 ug (5 mg) per 1.0 ml. Animals were subsequently administered the aerosolized antisense or mismatch -19oligonucleotide via the intratracheal tube (approximately 5000 Ag in a volume of 1.0 ml), twice daily for two days.

Aerosols of either saline, adenosine, or antisense or mismatch oligonucleotides were generated by an ultrasonic nebulizer (DeVilbiliss, Somerset, PA), producing aerosol droplets 80% of which were smaller than 5 Am in diameter.

In the first arm of the experiment, four randomly selected allergic rabbits were administered antisense oligonucleotide and four the mismatched control oligonucleotide. On the morning of the third day, values (the concentration of aerosolized adenosine in mg/ml required to reduce the dynamic compliance of the bronchial airway 50% from the baseline value) were obtained and compared to PC50 values obtained for these animals prior to exposure to oligonucleotide.

Following a 1 week interval, animals were crossed over, with those previously administered mismatch control oligonucleotide now administered antisense oligonucleotide, and those previously treated with antisense oligonucleotide now administered mismatch control oligonucleotide. Treatment methods and measurements were identical to those employed in the first arm of the experiment. It should be noted that in six of the eight animals treated with antisense 25 oligonucleotide, adenosine-induced bronchoconstriction could not be obtained up to the limit of solubility of adenosine, 20 mg/ml. For the purpose of calculation, values for these animals were set at 20 mg/ml. The values given therefore represent a minimum figure for 30 antisense effectiveness. Actual effectiveness was higher. The results of this experiment are illustrated in both Figure 1 and Table 1.

TABLE 1. EFFECTS OF ADENOSINE A, RECEPTOR ANTISENSE OLIGONUCLEOTIDE UPON PC50 VALUES IN ASTHMATIC RABBITS.

Mismatch Control A, receptor Antisense oligonucleotide Pre Post Pre Post oligonucleotide oligonucleotide oligonucleotide oligonucleotide 3.56 1.02 5.16 1.93 2.36 0.68 >19.5 0.34** Results are presented as the mean (N 8) SEM. Significance was determined by repeatedmeasures analysis of variance (ANOVA), and Tukey's protected t test. **Significantly different from all other groups, P 0.01.

In both arms of the experiment, animals receiving the antisense oligonucleotide showed an order of magnitude increase in the dose of aerosolized adenosine required to reduce dynamic compliance of the lung by 50%. No effect of the mismatched control oligonucleotide upon PC50 values was observed. No toxicity was observed in any animal receiving either antisense or control inhaled oligonucleotide.

These results show clearly that the lung has exceptional potential as a target for antisense oligonucleotide-based therapeutic intervention in lung disease. They further show, in a model system which closely resembles human asthma, that downregulation of the adenosine A, receptor largely eliminates adenosineinduced bronchoconstriction in asthmatic airways.

25 Bronchial hyperresponsiveness in the allergic rabbit model of human asthma is an excellent endpoint for antisense intervention since the tissues involved in this response lie near to the point of contact with aerosolized oligonucleotides, and the model closely simulates an important human disease.

-21- EXAMPLE 4 Specificity of A,-adenosine receptor Antisense oligonucleotide At the conclusion of the crossover experiment of Example 3, airway muscle from all rabbits was quantitatively analyzed for adenosine A, receptor number.

As a control for the specificity of the antisense oligonucleotide, adenosine A 2 receptors, which should not have been affected, were also quantified.

Airway smooth muscle tissue was dissected from each rabbit and a membrane fraction prepared according to described methods Kleinstein and H. Glossmann, Naunyn-Schmiedeberg's Arch. Pharmacol. 305, 191-200 (1978), with slight modifications. Crude plasma membrane preparations were stored at 70 0 C until the time of assay. Protein content was determined by the method of Bradford Bradford, Anal. Biochem. 72, 240-254 (1976)). Frozen plasma membranes were thawed at room temperature and were incubated with 0.2 U/ml adenosine deaminase for 30 minutes at 37 0 C to remove endogenous adenosine. The binding of 3 H]DPCPX receptorspecific) or 3 H]CGS-21680

(A

2 receptor-specific) was measured as previously described. S. Ali et al., J.

Pharmacol. Exp. Ther. 268, 1328-1334 (1994); S. Ali et 25 al., Am. J. Physiol 266, L271-277 (1994).

As illustrated in both Figure 2 and Table 2, animals treated with adenosine Al antisense oligonucleotide in the crossover experiment had a nearly 75% decrease in Ai receptor number compared to controls, as assayed by specific binding of the A,-specific antagonist DPCPX. There was no change in adenosine

A

2 receptor number, as assayed by specific binding of the A 2 receptor-specific agonist 2- [p-(2-carboxyethyl)adenosine

(CGS-

35 21680).

**i -22- TABLE 2. SPECIFICITY OF ACTION OF ADENOSINE A, RECEPTOR ANTISENSE

OLIGONUCLEOTIDE.

Mismatch Control A, Antisense oligonucleotide oligonucleotide A,-Specific Binding 1105 293 18

A

2 -Specific Binding 302 22 442 171 Results are presented as the mean (N 8) SEM. Significance was determined by repeatedmeasures analysis of variance (ANOVA), and Tukey's protected t test. **Significantly different from mismatch control, P 0.01.

The above demonstrates the effectiveness of antisense oligonucleotides in treating airway diseases. Since the antisense oligonucleotides described above eliminate the receptor systems responsible for adenosine-mediated bronchoconstriction, it may be less imperative to eliminate adenosine from them. However, it would be preferable to eliminate adenosine from even these oligonucleotides. Examples of such adenosine-free oligonucleotides are provided below in Example EXAMPLE The method of the present invention is also practiced with the following antisense oligonucleotides targeted to their corresponding proteins, in essentially the same manner as given above, for the treatment of various conditions in the lungs. Described below is a series of antisense oligonucleotides targetting the mRNA of proteins involved in inflammation. Adenosine has been eliminated from their nucleotide content to prevent its liberation during degradation.

In the following, the first sequence provided after the name of the targeted inflammation-involved protein is the antisense sequence that targets the initiation codon, wherein the naturally-occurring adenosine is substituted by one of the following: a S. universal base that is not adenosine; a adenosine analog that lacks the ability to bind to the adenosine Al -23and/or A3-receptors; or a "spacer." Any one of these three is represented in the sequence as the letter recognized by the IUPAC-IUB Nomenclature Commission as "not-A." See Patentin User Manual, p.99 (November 1990).

Listed following the antisense sequence targeted against the initation codon are additional antisense oligonucleotide sequences directed against other portions of the mRNA of the targeted protein. These additional sequences are the "des-adenosine antisense sequences," in that they do not contain adenosine within the sequence.

Fragments of the following sequences that are at least ten, and more preferably at least twelve, nucleotides in length are also an aspect of the presnet invention and are useful in carrying out the present invention. Fragments set forth below that span multiple lines of test indicate at the beginning thereof, and at the end thereof.

Human Al adenosine receptor: GGC CTG GBB.BGC TGB GBT GGB GGG CGG CBT GGC GGG CBC BGG CTG GGC-3' des-adenosine antisense sequences: TTT TCC TTC CTT TGT CTC TCT TC GCT CCC GGC TGC CTG CTC GGC CGT GCG GCT CTG TCG CTC CCG GT CCG CCG CCC TCC GGG GGG TC TGC TGC CGT TGG CTG CCC CTT CTG CGG GTC GCC GG TGC TGG GCT TGT GGC GGC CTC TCT TCT GGG 30 CCT GGT CCC TCC GT GGT GGC TCC TCT GC GCT TGG TCC TGG GGC TGC TGC TCT CCT CTC CTT -24- Human A2a-adenosine receptor: GTBCBCCGBGGBGCCCBTGBTGGGCBTGCCB CBGB CGB CBGGC des-adenosine antisense sequences: HSA2ARECAS1: TGC TTT TCT TTT CTG GGC CTC (SEQ ID NO:7) HSA2ARECAS2: TGT GGT CTG TTT TTT TCT G HSA2ARECAS3: GCC CTG CTG GGG CGC TCT CC HSA2ARECAS4: GCC GCC CGC CTG GCT CCC GGB GCC CBT GBT GGG CBT GCC HSA2ARECAS6: GTG GTT CTT GCC CTC CTT TGG CTG HSA2ARECAS7: CCG TGC CCG CTC CCC GGC HSA2ARECAS8: CTC CTG GCG GGT GGC CGT TG HSA2ARECAS9: GGC CCG TGT TCC CCT GGG HSA2ARECAS1O: GCC TGG GGC TCC CTT CTC TC HSA2ARECAS11: GCC CTT CTT GCT GGG CCT C HSA2ARECAS12: TGC TGC TGC TGG TGC TGT GGC CCCC Human A2b adenosine receptor:

-BCBGCGCGTCCTGTGTCTCCBGCBGCBTGGCC

GGGCCBGCTGGGCCCC -3' des-adenosine antisense sequences: HSA2BRECAS1: 5'-GGC GCC GTG CCG CGT CTT GGT GGC GGC GG-3' (SEQ ID NO:8) HSA2BRECAS2: 5'-GTT CGC GCC CGC GCG GGG CCC CTC CGG TCC-31 HSA2BRECAS3: 5'-TTG GCC CGC GCG CCC GCC CGT CTC GGG CTG GGC GG-3' HSA2BRECAS4: CGG GTC GGG GCC CCC CGC GGC C 5'-GCC TCG GGG CTG GGG CGC TGG TGG CCG GG-3' HSA2BRECAS6: CCG CGC CTC CGC CTG CCG CTT CTG HSA2BRECAS7: GCT GGG CCC CGG GCG CCC CCT HSA2BRECAS8: CCC CTC TTG CTC GGG TCC CCG TG adenosine receptor -BCB GBG CBG TGC TGT TGT TGG GCB TCT TGC CTT CCC BGG G-31 des-adenosine antisense oligonucleo tides: CCC TTT TCT GGT GGG GTG GTG CTG TTG TTG GGC TTT CTT CTG TTC CC Hutman A3 *5 40 Hulmain IgE receptor 16: 5' -BTTTGCTCTCCTBTTBCTTTCTGTGTCCBTTTTTT CBTTBBCCGBGCTGT- 3' des-adenosine antisense sequences: HUMIgEo3rAS1: TTT CCC CTG GGT CTT CC (SEQ ID 45 NO:9) HEJMIgEjgrAS2: CTC CTG CTC TTT TTT C Human Fc-epsilon receptor CD23 antigen (IgE receptor):

-TCTCTGBBTBTTGBCCTTCCTCCBTGGCGGTCCTGCTT

GGBTTCTCCCGB-3' des-adenosine antisense sequences: HUMIgErCD23AS1: GCC TGT GTC TGT CCT CCT (SEQ ID NO:1O) HUMIgErCD23AS2: GCT TCG TTC CTC TCG TTC HUNIgErCD23AS3: CTG CTT GGT GCC CTT GCC G HUMIgErCD23AS4: GTC CTG CTC CTC CGG GCT GTG G HUMIgErCD23AS5: 5'-GTC GTG GCC CTG GCT CCG GCTGGT GGG CTC CCC TGG-31 HUMIgErCD23AS6: CCT TCG CTG GCT GGC GGC GTG C HUI4IgErCD23AS7: GGG TCT TGC TCT GGG CCT GGC TGT HUMIgErCD23AS8: GGC CGT GGT TGG GGG TCT TC is HUMIgErCD23AS9:.GCT GCC TCC GTT TGG GTG GC Human IgE receptor, a subunit:

-BCBGTBGBGTBGGGGBTTCCBTGGCBGGBGCCBTC

TTCTTCBTGGBCTCC- 3' and 51-TTC BBG GBG BCC TTB GGT TTC TGB GGG BCT GCT BBC BCG CCB TCT GGB GC-31 des-adenosine antisense sequences: HUMIgEraAS1: GCCTTTCCTGGTTCTCTT (SEQ ID NO:1l) GTT GTT TTT GGG GTT TGG CTT Human IgE receptor, Fc epsilon R: 51 -GBT CTC TGB BTB TTGB CCT TCC BTG GCG GTC CTG CTT GGB-31 des-adenosine antisense sequences: V HSJGEBFRAS1: GCC TGT GTC TGT CCT CCT (SEQ ID NO:12) HSJGEBFRAS2: GCT TCG TTC CTC TCG TTC HSJGEBFRAS3: CTG CTT GGT GCC CTT GCC G *HSJGEBFRAS4: GTC CTG CTC CTC CGG GCT GTG G 5'-GTC CTC GCC CTG GCT CCG GCT GGT GGG CTC CCC TGG-3' HSJGEBFRAS6: CCT TCG CTG GCT GGC GGC GTG C HSJGEBFRAS7: CCC BGB BCG BGB CCC GGB CCG BCB HSJGEBFRAS8: GGC CGT GGT TGG GGG TCT TC HSJGEBFRAS9: GCT GCC TCC GTT TGG GTG GC 40 Human histidine decarboxylase: 5'-CTC TGT CCC TCT CTC TCT GTB CTC CTC BGG CTC CBT CBT CTC CCT TGG GC-3' des-adenosine antisense sequences: HUMHDCAS1: TCT CCC TTG GGC TCT GGC TCC TTC TC 4 (SEQ ID NO:13) -26- HUMHDCAS2: TCT CTC TCC CTC TCT CTC TGT HUMHDCAS3: CGCCTCCGCCCTGGCTGCTGGGGTGGTGGTGC HtJMHDCAS4: TTT TGT TCT TCC TTG CTG CC GCC CCG CTG CTT GTC TTC CTC G Human beta tryptase: CCT GGC CTG GGG CBG GGG CCG CGT BGG CGC GGC TCG CCB GGB CGG GCB GCG CCB GCB GCB GCB GBT TCB GCB TCC TGG-3' des-adenosine antisense sequences: HUNMBTRYPAS1: CTTGCTCCTGGGGGCCTCCTG (SEQ ID NO:14) HUMBTRYPAS2: GTC CCT CCG GGT GTT CCC GGC Human tryptase-I: 51-CCT GGB CTG GGG CBG GGG CCG CGT BGG CGC GGC TCG CCB GGB CGG GCB GCG CCB GCB GCB GCB GGC TCB GCB TCC TGG CCB CGG BBT TCC-31 des-adenosine antisense sequences: HUIMTRYAS1: CTTGCTCCTGGGGGCCTCCTG (SEQ ID NO: HUMTRYAS2: GTC CCT CTG GCT G TT CCC GGC Human prostaglandin D synthase: -CCC CBG CBG GBC CBG TCC CBT CCB CBG CGT GTG BTG BGT BGC CBT TCT CCT GCB GCC GBG-31 des-adenosine antisense sequences: HUMPROSYNASi GGTGTGCGGGGCCTGGTGCC (SEQ ID NO: 16) HUMPROSYNAS 2: CCT GGG CCT CGG GTG CTG CCT GT HUMPROSYNAS 3: GCG CTG CCT TCT TCT CCT GG HtJMPROSYNAS 4: 5'-GTC CTC GCC GGG GCC CTT GCT GCC CTG.GCT GT -3' HUNPROSYNAS 5: GCC CTG GGG GTC TGG GTT CGGCTGT Human cyclooxygenase-2: -TGB GCG CCB GGB CCG CGC BCB GCB GCB GGG CGC GGG CGB GCB TCG CBG CGG CGG GCB GGG-31 des-adenosine antisense sequences: HUNCYCLOXASi: GGGCGCGGGCGBGCBTCGC (SEQ ID NO: 17) HUMCYCLOXAS2: TTT GGG CTT TTC TCC TTT GGT T Human easinophil cationic protein: BCB BBT TTG GGB BGT GBB CBG TTT TGG BBC CBT GTT TCC CBG TCT CTG BGC TGT GGC-31 des-adenosine antisense sequences: HSECPAS1: CCTCCTTCC TGG TCT GTC TGC (SEQ.- ID NO: 18) Human eosinopbhil derived neurotoxin: 5'-CCC CBB CBG BBG BBG CBG BCB BBT TTG GGB BGT GEB CBG TTT TGG BBC CBT GTT TCC TGT-31 -27des-adenosine antisense sequences: ISEOSDNAS1: GCC CTG CTG CTC TTT CTG CT (SEQ ID NO: 19) HSEOSDNAS 2: TCC CTT GGT GGG TTG GGC C HSEOSDNAS 3: GCT GGT TGT TCT GGG GTT C HSEOSDNAS 4: TTG CTG CCC CTT CTG TCC C HSEOSDNAS 5: TGT TTG CTG GTG TCT GCG C Human eosinophil major basic protein: GGG GGB GTT TCB TCT TGG CTT T des-adenosine antisense sequences: TCT CCC CTT GTT CCT CCC C TCT CCT GCT CTG GTG TCT CCT C TTC CCT CCC TCC CCT GCC GTG TTG TCT GTG GGT GTC C GTT TCG CTC TTG TTG CCC TGG GCC CTT CCC TGC TGG Human eosinophil peroxidase: -GCB CCG TCC BGT GBT GGT GCG GTB CTT GTC GCT GCB GCG CTC GGC CTG GTC CCG GBG BGC-3' des-adenosine antisense sequences: HSEPASi: GCGCTCGGCCTGGTCCCGG (SEQ ID HSEPAS2: GGG TCT CCT CTT GTT GTT GC HSEPAS3: TTG CGC CTC CTG CTG GGG GT CC HSEPAS4: CTC TGT TCT TGT TTT GGG GGC HSEPAS5: GGG CCC GGC CGT TGT CTT G HSEPAS6: GTT TGG GGG TTT CCG TTG HSEPAS7: GGG TTC TCC TGG CCC GGG CCT TGC CC HSEPAS8: GGC CGT GGT CCC GGC TTC GTT GC HSEPAS9: CCT GTC TCC GTC TCG GCT CTT CTG HSEPAS10: GGG CCT TGC GCT GTC TTT GGT G Human intercellular adhesion molecule-i (CAM-i): CGG BGC CTC CCC GGG GCB GGB TGB CTT TTG BGG GGG BCB CBG BTG TCT GGG CBT TGC CBG GTC CTG GGB BCB GBG CCC CGB GCB GGB CCB GGB GTG CGG GCB GCG CGG GCC GGG GGC TGC TGG GBG CCB TBG CGB GGC TGB G-3f des-adenosine antisense sequences§: HSICAM1AS1: GCGCGGGCCGGGGGCTGCTGGG (SEQ ID NO:21) HSICAMlAS2: GGT TGG CCC GGG GTG CCC C HSICAMlAS3: GCC GCT GGG TGC CCT CGT CCTCTGCGGTC *HSICAM1AS4: GTG TCT CCT GGC TCT GGT TCC CC 5'-GCT GCG CCC GTT GTC CTC TGG GGT Ar, GGCCTTC-31 -28- HSICAMlAS6: GCT CCC GGG TCT GGT TCT TGT GT HSICAMlAS7: TGG GGG TCC CTT TTT GGG CCT GTT GT HSICAMlAS8: GGC GTG GCT TGT GTG TTC GGT TTC HSICAMlAS9: TGC CCT GTC CTC CGG CGT CCC Human vascular cell adhesion molecule 1 (VCAM-1): EGC BBG BTB TCT BGB TTC TGG GGT GGT CTC GET TTT BB GCT TGB GBE GCT GCB BEC BTT ETC CBB EGT BTB TTT GBG GCT CCB EGG ETC BCG BCC BTC TTC CCB GGC ETT TTB EGT TGC TGT CGT -3' des -adenosirie an tisense sequences: HSVCAMlAS1: CCTCTTTTCTGTTTTTCCC (SEQ ID NO:22) H-SVCAM1AS2: CTC TGC CTT TGT TTG GGT TCG HSVCAMlAS3: CTT CCT TTC TGC TTC TTC C HSVCAMlAS4: CTGTGTCTCCTGTCTCCGCTTTTTTCTTC is HSVCAM1AS5: GTC TTT GTT GTT TTC TCT TCC TTG Human endothelial leukocyte adhesion molecule (ELAM-l): TGE GEG CTG EGE GEE ECT GTG BEG CEE TCE TGB CTT CEE GEG TTC TTT TCEB CCC -3' des-adenosine antisense sequences: HUMELAMlAASl: GTTCTTGGCTTCTTCTGTC (SEQ ID NO: 23) HUMELAMlAAS2: CGT TGG CTT CTC GTT GTC CC HUNELAAMlAAS3: TGT GGG CTT CTC GTT GTC CC HtJMELAMlAAS4: CCC TTC GGG GGC TGG TGG HtJMELAMlAASS: GGC CGT CCT TGC CTG CTG G Human P Selectin: des-adenosine antisense sequences: HUNPSELECTASl: CTCTGCTGGT TTTCTGCCTT CTGCCC (SEQ ID NO:24) Human endothelial monocyte activating factor: des-adenosine antisense sequences: HUNEMAPIIASl: 5'-TTT TCT CTT TCG CTT TCT TTT CGTCTCCTGTTCCTCCTTTT-3' (SEQ ID HUMEMAPIIAS2: 5'-TTG CTG TTT TTT CTC CTT CTT CTC TCC TTT CTT TTC -3' Human 1L3: 5' -GGCGGBCCBGGBGTTGGBGCBGGBGCEGGECGGGCE GGCGGCTCBTGTTTGGBTCGGCBGGBGGCBCTC -3' des-adenosine antisense sequences: HUMIL3AASl: 5'-CTC TGT CTT GTT.CTG GTC CTT CGT GGG GCT CTG (SEQ ID NO:26) -3' HUMIL3AAS2: TGT CGC GTG G GTG CGG CCG TGG CC Human 1L3 receptor: 5' -GCBGGEGBCEGGGCEGGGCGBTCBGGBGCBGCGT 45 GBGCCBBBGGBGGBCCBTCGGGBBCGCEGCTCCG -29des-adenosine antiserise sequences:

TCTGGGGTGTCCTG

GCCTTCGTGGTTCC

TCTTCCTTCGTTTGC

CGTCCGCGGGGGCCCCCGGGCCT

GGCTGCGCTCCTGCCCCGC

CTCTTTCCCGGGCTCTT

GCGCTGGGGGGTGCTCC

CGTGTGTTTGCGCCCTCCTCCTGGTCGC

GCTTGTCGTTTTGG

GGCCGGCTTTGCCCGCCTCCC

GGCGCCTGGCCCGGCC

TTCCTGGGCTGCGTGCGC

GTTCTGTTCTTCTTCCTGGC

Human 1L4 Human 1L4

-GCCGGCBCBTGCTBGCBGGBBGBBCBGBGGGGGB

BGCBGTTGGGBGGTGBGBCCCBTTBBTBGGTGTCGB -3' des-adenosirie antisense sequences: HUMIL4AS1: CTC TGG TTG GCT TCC TTC-3' (SEQ ID NO:27) receptor: 5' -GTTCCCBGBGCTTGCCBCCTGCB(GCBGB

CCBGGCBGCTC

BCBGGGBBCBGGBGCCCBGBGCBBBGCCBCCCCBTTGGGBG

BTGCCBBGGCBCCBGGCTG-3' des-adenosine-ant isense sequences:

TCTGCGCGCCCCTGCTCC

CGCCCGGCTTCTCT

CGTGTGGGCTTCGG

CCCCGCGCCTCCGTTGTTCTC

TGCTCGCTGGGCTTG

GGTTTCCTGGGGCCCTGGGTTTC

TCTGCCGGGTCGTTTTC

GGGTGCTGGCTGCG

CTTGGTGCTGGGGCTCC

GGCGGCTGCGGGCTGGGTTGGG

CTTGGCTGGTTCCTGGCCTCGGG

CCTCCTCCTCCTCCTC

GCTCCCTTTTTCTTCCTCT

TCCCTGCTGCTCTC

TGCCCTCCCTTCCCTCCTGG

GGTGCCTCCTTGGGCCCTGC

GGCTGCTCCTTGCCCC

CTCTGGGTCGGGCTGGC

GGGGCGTCTCTGTGC

CTGGCCTGGGTGCC

GCCTCTCCTGGGGG

GGTGGCTCCCTGTCC

CCTTTTCCCCCGGCTCC

GTGGGGGCTTTGGC

GGGGGTCTGTGGCCTGCTCCTGGGG

AGGGGTCTGGGGCCCTC

****TTTTGGGGGTCTGGCTTG

GCCTGGCTGCCTTCC

GGGGCCTGCCGTGGGGC

TGTCCTCTGTTGCTCCCCTT

TGCCTGCTGTCTGG

GGTTCCCGCCTTCCCT

Human I L

-GTGGGBBTTTCTGTGGGGBTGGCBTBCBCGTBGGCB

GCTCCBBGBGCTBGCBBBCTCBBBTGCBGBBGCBTC

.:CTCBTGGCTCTGBBBCG -3' -31des-adenosine antisense sequences: TCC CTG TTT CCC CCC TTT (SEQ ID NO: 28) HUMIL5AS2: CGT TCT GCG TTT GCC TTT GGC HTJMIL5AS3: GTT TTT TGT TTG TTT TCT HUMIL5AS4: CTC TCC GTC TTT CTT CTC C CCT CCT GCC TGT GTC CCT GCT CCC C HUMIL5AS6: GAG GGT TTC TGG CTT CCT CTC T HUMIL5AS7: TGT CTC TCT GTC CTT TTG TT HUMIL5AS8: 5'-TGT TGT GCG GCC TGG TGC TGC CCT GCCCCG GG-31 Human IL5 receptor antisense oligonucleotide

-CTCBGTGGCCCCCBBBBGGBT

GBGTBBTB CBTGCGCCBCGBT GBTCBTBTCCTTTTTBCTBTGBGG- 3' des -adenosine antisense sequences:

CCGTGTCTGTCGTGTCT

TTCCTTTGCTCTTG

GTGTGTCTTTGCTGT

GCCCTGCCTCTCTGC

Human ILG:

-CTCCTGGGGGTBCTGGGGCBGGGBB

GGCBGCEGGCBBCBCCBGGBGCBGC

CCCBGGGBGBBGGCBBCTGGBCCGB

BGGCGCTTGTGGBGBBGGBGTTCBT

BGCTGGGCTCCTGGBGGGGBGBTBGBGC -3' Human IL6 des-adenosine antisense sequence: HUMIL6AS1: GCT TCT CTT TCG TTC CCG GTG GGC TCG (SEQ ID NO:29) HUNIL6AS2: GTG GCT GTC TGT GTG GGG CGG CT HLUMIL6AS3: GTG CCT CTT TGC TGC TTT C HtJMIL6AS4: GAT TCT TTG CCT TTT TCT GC receptor antisense oligonucleotides

-GCBCGCCTCTTGCCBCCTCCTGCGCBGGGCB

GCGCCTTGGGGCCBGCGCCGCTCCCGGCGCG

GCCBGCBGGGCBGCCBGCBGCGCGCBGCCGB

CGGCCBGCBTGCTTCCTCCTCGGCTBCCBCT

CCBTGGTCCCGCBGBGGCGGBCBGGC- 3' des-adenosine antisense sequences:,'

GGGGGTGGCTTCCTGCC

GCGTCTCTGGGCCGTCCC

0

S*

0*

GTCCCTCGGCCCCGCGCCGCGCTCGGCTCCTCTCCC

TCTGGCCCGGCTC

-32-

GGGGCGGGGCGGGGCGGTGGGCGGGC

GGCGCTGCCCTGCGC

GCGGCGCTGGCCCC

TGCTGGCCGTCGGCTGCGCGCTGCTGGCTGCCCT

GCTGGCCGCGCCGGG

GCCTGTCCGCCTCTGCGGG

CGCTGTCTCCTGGC

TTGTCTTCCGGCTCT

TCTGCTGGGGTGGG

GCTGGGCGGCCGGCCCGGT

GCTGGGGCTCCTCGGGGGG

GGGGGCTCTTCCGG

GCTGTCTCCCTCCGGG

GCGGGGGTTTCTGGCC

GTGGGGGTCTTGCC

TGGCCTCCGGGCTCC

TGCTTGTCTTGCCTTCCTTC

000:00TCTGGTCGGTTGTGGCTCG

GGGCTCCGTGGGTCCCTGGC

S. :**egoo

GCCCGTTTGTGTTTTGTC

TTTTCCCCTGGCGT

CCCTGTGCCCCTCTCCTCTCCTTCCTCTGCTTCTC

GCTCTCCTTTGTGGG

GCCCTCCCTGCTGCT

CTTGGTTTTGGGCT

TTTTTTCTCTTCCTCCTTTTTC

GTGCGTGGGCCTCC

-33- Human monocyte-derived neutrophil chemotactic factor: -GGGGTGGBBBGGTTTGGBGTBTGTCTTTBTGCB

CTGB

CBTCTBBGTTCTTTBGCBCTCCTTGGCBBBBCTGCB

C

CTTCBCBCBGBGCTGCBGBBBTCBGGBBGGCTGCCBB

GBGBGCCBCGGCCBGCTTGGBBGTCBTGTTTBCBCBC

BGTGBGBTGGTTCCTTCCGG- 3' des-adenosine antisense sequences: HSMDNCFAS1: GCT TGT GTG CTC TGC TGT CTC T (SEQ ID HSMDNCFAS2: 5'-TGG TTC CTT CCG GTG GTT TCT TCC TGG CTC TTG TCC T -3' HSMDNCFAS3: TTC TCT TGG CCC TTG GC Human neutrophil elastase (medullasin):

-GGGCTCCCGCCGCGBGBGGTTBTGGGCTCCCBGGBCCBC

CCGCBCCGCGCGGBCGTTTBCBTTCGCCBCGCBGTGCGC

GGCCGB CBTGBCGBBGTTGGGCGCBBTCBGGGTGGCGCC

GCBGBBGTGGCCTCCGCGCBGCTGCBGGGBCBCCBTGBB

GGGCCBCGCGTGGGGCCGCGCTCGCCGGCCCCCCBCBBT

CTCCGBGGCCBGCGCGGTGCCCCCCBGCBGCBBGGCCGG

CBGGBCBCBGGCGBGGBGBCBCGCGBGTCGGCGGCCGBG

GGTCBTGGTGGGGCTGGGGCTCCGGGGTCTCTGCCCCTC

CGTGC-31 des-adenosine antisense oligonuclec tides: HSMEDURAS1: 5'-TGG TGG GGC TGG GGC TCC GGG GTC TCT GCC CCT CCG TGC-3' (SEQ ID NO:31) HSMEDURAS2: CGC GTG GGG CCG CGC TCG CCG GCCCCCC HSMEDUTRAS3': CCT GCC GGG TGG GCT CCC GCC GCG HSMEDURAS4: CGC CGG CCT GCC GGC CCC TC 5'-GTG GGT CCT GCT GGC CGG GTC CGG GTC CCG GGG GTG GGG-3' HSMEDURAS6: CGC GBG TCG GCG GCC GBG GGT C Human neutrophil oxidase factor:

-CGGGBGTGGGGGTCCTGGBCGGCBCTGBBGGCBTCCBGGG

CTCCCTTCCBGTCCTTCTTGTCCGCTGCCBGCBCCCCTTC

TBGBBBCTBGGBGGCC-31 des-adenosine antisense sequence: *HUMNOXFAS1: GGC CTC CBC CBG GGB CBT G (SEQ ID NO:32) HUMNOXFAS2: GTC CTT CTT GTC CGC TGC C HUMVNOXFAS3: TCT CTG GGG TTT TCG GTC TGG GTG G HUMNOXFAS4: GCT TTC CTC CTG GGG CTG CTG CTG 5'-GGC TCT TCT TTT TGT TTC TGG CCT GGTG-31 HIMNOXFAS6: CTC TCT CGT GCC CTT TCC **HUMNOXFAS7: CTT GGG TGT CTT GTT TTT GT HUMNOXFAS8: 5' -GGCCTCCBCCBGGGBCBTGGTCCTTCTT GTCCGCTGCC -3' -34- Human cathepsin G:

-CCCTCCBCBTCTGCTCTGBCCTGCTGGBCTCTG

GBTCTGBBGBTBCGCCBTGTBGGGGCGGGBGTG

GGGCCTGCTCTCCCGGCCTCCGBTGBTCTCCCCT

GCCTCBGCCCCBGTGGGTBGGBGBBBGGCCBGCB

GBBGCBGGBGTGGCTGCBTCTTTCCTG -3' des-adenosine antisense sequences: IIUMCTHGAS1: GTG GGG CCT GCT CTC CCG GCC TCC G (SEQ ID NO:33) HIUMCTHGAS2: TGTGTTGCTGGGTGTTTTCCCGTCTCTGG I{UMCTHGAS3: TCT GCC TTC GGG GGT CGT Human defensin 1:

-CCGGGGCTGCBGCBBCCTCBTCBGCTCTTGCCT

GGBGTGGCTCBGCCTGGGCCTGCBGGGCCBCCB

GGBGBBTGGCBGCBBGGBTGGCGBGGGTCCTCB

TGGCTGGGGTCBCBGBTCCTCTBGCTBGGCBGG

GTGBCCBGBGBGGGC- 3' des-acienosine antisense sequences: HtJMDEF1AAAS1: GGG TCC TCB TGG CTG GGG (SEQ ID NO:34) HtJMDEF1AAAS2: GCC TGG GCC TGC BGG GCC HUMDEF1AAAS3: GCT CTT GCC TGG BGT GOC TC HUMDEF1AAAS4: GCC CBG BGT OTT CCC TGG T Human defensin 3: 5' -CGCTGCBBTCTGCTCCGGGGCTGCBGCBBCCTCBTC

BGCTCTTGCCTGGBGTGGCTCBGCCTGGGCCTGCBG

GGCCBCCBGGBGBBTGGCBGCBBGGBTGGCGBGGGT

CCTCBTGGCTGGGGTCBCCTGGBGGBGGGBGBGCBGG- 3' des-adenosine antisense sequences: HtJMNTRIIIAS1: GGG TCC TCB TGG CTG GGG TC (SEQ HUMNTRIIIAS2: COT CTC TOO CGT CCT **Human macrophage inflammatory protein-i1-alpha: RANTES

RECEPTOR

5' -GBGGGGGOBGOBGTTGGGCCOBBBGGOOOTOTOGT

TCBCCTTCTGGCBCGGBGTTGCBTCCCCBTBGTCBB

BCTCTGTGGTCGTGTCBTBGTCCTCTGTGGTGTTTG

GBGTTTCCBTCCCGGCTTCTCTCTGGTTCCBBGGGB -3' des-adenosine antisense sequences: HUMRANTESA.Sl: GTO TTT GTT TCT GGG CTC GTG CC~ (SEQ ID NO:36) HUMRANTESAS2: COB TCC CGG CTT CTC TCT GGT TCC HUJMRANTESAS3: GTC CTCTGT GGT GTT TGG .:HUMRANTESAS4: 5,-COO TGC TTC CTT TTG CCT GTT TCTTTGTTT CTGGGCTCGT GOC -3'

RANTES:

-GGGCBCGGGGCBGTGGGCGGGCBBTGTBGGC

BBBGCBGCBGGGTGTGGTGTCCGBGGBBTBTGGG

GBGGCBGBTGCBGGBGCGCBGBGGGCBGTBGCBB

TGBGGBTGB CBGCGBGGCGTGCCGCGGBGBCCTTC BTGGTBCCTGTGGBGBGGCTGTCGGBGG- 3' des-adenosine antisense sequences:

GGGTGTGGTGTCCG

CTTGGCGGTTCTTTCGGGTG

TTTCTTCTCTGGGTTGGC

CTGCTGCTCGTCGTGGTC

GCTCCGCTCCCGGGTTC

GTCTCGCTCTGTCGCCC

CTTCCTTCCTTGTC

GTGTTCCTCCCTTCCTTGCCTCT

Human muscarinic acetylcholine receptor HM1: des -adenosine antisense sequences: HSHM1AS1: GTT CBT GGT GGC TBG GTG GGG C (SEQ ID NO: 37) HSHM1AS2: GCT GCC CGG CGG GGT OTO CGC TTG GC HSHM1AS3: GCTCCCGTO CTC GOT TCT CTG TCTCCCGGT HSHM1AS4: CCC CCT TTG CCT GOC GTC TCG 0 GCC TTC GTC CTC TTC CTC TTC TTC CTTCC HSHM1AS6: 5'-OCT CCG TGG GGG CTG CTTGGTGGG GGCCTG TOC CTC GGG GTC C-31 *HSHM1AS7: CGG GGC TTC TOG CCC TTG CC V 35 Human muscarinic acetylcholine receptor HM3: des-adenosine antisense sequences: *HSHM3AS1: GGG GTG GGT BGG CCG TGT CTG GGG (SEQ ID NO:38) HSHM3AS2: GTT GGC CBT OTT GOT TGC C HSHM3AS3: TCT TGG TGG TGC GCC 000 C HSHM3AS4: 5'-GCO TCT TGG CTT-TCT TCT CCT TCG GGC CCT CGG 0CC GOT GCT TGT GG-3' 5'-OCT CCT CCC GGG CGG-CCT CCC CGG GCG GGG GCT TCT TG-31 HSHM3AS6: GCG CTG OCG-000 GGG CCT CCT CC HSHM3AS7: 5'-GCT CTG TGO CTG GOC GTT CCT TGG **TGT TCT GOG TGG C-31 HSHM3AS8: TOG CGG GCG TGG TGG CCT CTG TGG TGG HSHM3AS9: GGG CCC GCG GCT GCB GGG G HSHIM3AS1O: TTG CCT GTC TGC TTC GTC HSHM3A-S11: CTT TGC GCT CCC 000 CCG CC -36- Human fibronectin: des-adenosine aritiserise sequences: HUMFNA/HSFIBlAS1: ID NO:39) HMFNA/HSFIB1AS2: HUMFNA/HSFIB1AS3: HUMFNA/HSFIB1AS4: HUMFNA/HSFIBlASG: HUTMFNA/HSFIBlAS7: HUNMFNA/HSFIB1AS9: HUMFNA/HSFIB1AS1O: 1UMFNA/HSFIB1AS 11: HUMFNA/HSFIB1AS12: HUMFNA/HSFIB1AS13: HUNMFNA/HSFIB1AS14: HUTMFNA/HSFIB1AS15: HUMFNA/HSFIB1AS 16: HUTMFNA/HSFIBlAS17: HUMFNA/HSFIBlAS18: HUMFNA/HSFIB1AS19: HUMFNA/HSFIB1AS2O: HtJMFNA/HSFIBlAS21: HUMFNA/HSFIB1AS22: HUMFNA/HSFIB1AS23: IUMFNA/HSFIB1AS24: HUMFNA/HSFIB1AS25: HUMFNA/HSFIB1AS26: HUMFNA/HSFIB1AS27: HUMFNA/HSFIB1AS28: HUMFNA/HSFIB1AS29: HUMFNA/HSFIBlAS3O: CGG TTT CCT TTG CGG TC (SEQ TTG GCC CGG GCT CCG GGT G CCC GCC CGC CCG CCG GCC GCCGC 5' -CCC GCC GGG CTG TCC CCG CCC CGC CCC-3' GGC CCG GGG CGC GGG GG CGG CCC TCC CGC CCC TCT GG GCC GGC GCG GGC GTC GG 5' -CCG CTC GCG CCT GGG GTT CCC TCT CCT CCCCCTGTGC-3' GCC TGC CTC TTG CTC TTC TGC GTC CGC TGC CTT CTC CC CTC TCC TCG GCC GTT GCCTGTGC 5'-TGT CCG TCC TGT CGC CCT TCC GTG GTG C-3' TGT TGT CTC TTC TGC CCT C GGT GTG CTG GTG CTGGTGGTGGTG CCT CTG CCC GTG CTC GCC CTG CCT GGG CTG GCCTCTTCGGGT 5'-GTG GCT TTG GGG CTC TCT TGG TTG CCC TTT-3' 5'-CTT CTC GTG GTG CCT CTC CTC CCT GGC TTG GTC GT-3' TGT CTG GGG TGG TGCTCCTCTCCC TTT CCC TGC TGG CCG TTT GT CCT GTT TTC TGT CTT CCT CT TTC CTC CTG TTT CTC CGT 5' -TTG GCT TGC TGC TTG CGG GGC TGT CTC C-3' CTT GCC CCT GTG GGC TTT CCC TGG TCC GGT CTTCTCCTTGGGGGTC GCC CTT CTT GGT GGG CTG GCT CGT CTG TCT TTT TCC TTCC 5'-TGG GGG TGG CCG TTG TGG GCG GTG TGG TCC GCC T-3' TGC CTC TGC TGG TCT TTC 40 Human interleukin 8: 5' -GBTGTTTGTTBCCBBBGCBTCBBGBBTBGCTTTGC

TBTCTBBGGBTCBCBTTTBGBCBTBGGBBBBCGC

TGTBGGTCBGBBBGBTGTGCTTBCCTTCBCBCBG

BGCTGCBGBBBTCBGGBBGGCTGCCBBGBGBGCC

BCGGCCBGCTTGGBGTCBTGTTTBCBCBCBGTGBG-3' des-adenosine antisense sequences,-' HUMIL8AAS1: GTG CTC CGG TGG CTT TTT (SEQ ID, NO: HUMIL8AAS2: GCT TGT GTG CTC TGC TGT CTC TG 50 }IUMIL8AAS3: 5'-TTC CTT CCG GTG GTT TCT TCC TGG CTC TTG TCC T-3' HUMIL8AAS4: TTC TCT TGG CCC TTG GCC C -37- Human IL-B receptor-alpha

-BCBGGGGCTGTBBTCTTCBTCTGCBGGTGGCB

TGCCBGTGBBBTTTBGBTCBTCBBBBTCCCBCBT

CTGTGGBTCTGTBBTBTTTGB CBTGTCCTCTTC BGTTTCBGCBBTGGTTTGBTCTBB CTGBBGCBCCG GCCBGG-3' des-aderiosine antisense sequences:

TGGCTCGGTGCTTCTGCC

TGTTGTTGCGGCGCTC

GGTTGGTGTGGCCCCTG

TGGTGCTTCGTTTCC

CCCTCTTTCTCTTTGTTC

GGGGGTTCTTGTGGC

GGGCTGCTTGTCTCGTTCC

Human GM-CSF:

-CTTGBGOBGGBBGOTCTGGGGOBGGGBGCTGGCBG

GGCCCBGGGGGGTGGCTTCCTGCBCTGTCCBGBGT

GCBOTGTGCCBCBGCBGCBGOTGCBGGGCCBTCBG

CTTCBTGGGOTCTGGGTGGCBGGTCCBGCCBTGG

GTCTGGGTGGGGCTGGGCTGOBGGCTOOGGGO -3' des-adenosine antisense sequences: HUMGCSFAS1:GGT COB GCC BTG GGT CTG GG (SEQ ID NO: 41) HUMGCSFAS2:GGO TGG GOT GOB GGC TCC GG HUMGOSFAS3: GCG GGC GGG TGC GGG OTG CGT GCT GGG HUMGCSFAS4: GGC TGC COO GCA GGC CCT GO Hulman tumor necrosis factor a: 5' -CBOCGCCTGGBGOOCTGGGGCCCOOTGTOTTOTTGGG

GBGCGCCTCCTCGGCCBGCTCCBCGTCCCGGBTCBTGCTTT

CBGTGCTCBTGGTGTOCTTTOOBGGGGBGBGBGGG-3' des-adenosine antisense sequences *HSTNFAAS1: GCT GGT OCT OTG CTG TCC TTG CTG (SEQ ID NO:42) HSTNFAAS2: GTG OTO BTG GTG TOC TTT CO HSTNFAAS3: GCC CTG GGG COO COO TGT CTT CTT GGGG *HSTNFAAS4: COT CTT CCC TOT GGG GGC. OG TOT OTO TOO OTO TOT TGO GTO TOT C HSTNFAAS6: TOT TTO TOT OTO TOT OTT COO 0 *HSTNFAAS7: TTT 000 GOT OTT TOT GTO TO *45 HSTNFAAS8: GGT GTC TGG TTT TOT OTO TOO HSTNFAAS9: GOT GGC TGO CTG TOT GGO OTG OGO TCTT GGO CTG TGO TGT TOO TOO HSTNFAAS11: TOO GGT TOO TGT OCT OTO TGT OTG TO HSTNFAAS12: GOC COO TOT GGG GTO TOO OTO TGG C so HSTNFAAS13: GTG GTG GTC TTG TTG OTT -38- HSTNFAAS14: GGG CTG GGC TCC GTG TCT C CBG TGC TCB TGG TGT CC HSTNFAAS16: GCT GBG GGB GCG TCT GCT GGC Human leukotriene C4 synthase: 5' -CTCGGTBGBCGCGCTCGBBCTCGGGTGGGCCGGTGGTG

BGCGGCGGCGBCBCGCGGBBGGCCCTGCGCGCCGBGBTCBC

CTGCBGGGBGBBGTBGGCTTGCBGCBGGBCTCCCBGGBGGG

TGBCBGCBGCCBGTBGBGCTBCCTCGTCCTTCBTGGTBCCG

TCGGTGTGGTGGCBCGGGCTGTGTGTGBBGGCGBGCTGG -3' des-adenosine antiserise sequences: HSU11552AS1:GCC CCG TCT GCT GCT CCT CGT GCC G (SEQ ID NO:43) HSU11552AS2: 5'-CCT CGT CCT TCA TGG TAC CGT CGGTGT GGT GGC-3' HStJ11552AS3: CTC GGG TGG GCC GGT GGT G HSU11552AS4: GGG CGC GCG CGC TCG CGT H-SU11552AS5: 5'-GGC TCC GGC TCT TCT TTC CCG GCTCCG TCG GCC CGG GGG CCTTGGTCTC-3' HStJ11551AS6:CCT CGT CCT TCB TGG TBC CG Human Endothelin-1: -B CCGGCGGBGCCGCCBGGGTGGBCTGGGBGTGGGTT

TCTCCCCGCCGTTCTCBCCCBCCGCGCTGBGCTCBGCGC

CTBBGBCTGCTGTTTCTGGBGCTCCTTGGCBBGCCBCBB

BCBGCBGBGBGBBBBTCBTGBGCBBBTBBTCCBTTCTGB

BBBBBBGGGBTCBBBBBCCTCCCGT-31 des-adenosine antiserise sequences:

CCCGTTCGCCTGGCGC

GCGCTGCGGGTTCCtC

GTGGGTTTCTCCCCGCCGTTCTC

CGGTCTGTTGCCTTTGTGGG

CTTCTTGTCTTTTTGGCT

GTTCTTTTCCTGCTTGGC

GTCTTTTCCTTTCTT

TGTGCTCGGTTGTGGGTC

CGCTGGTCCTTTGCC

CTGTGTGTTTCTGCTG

Endothelin receptor ET-B antisense oligonucleotides 5' -GCCCTGTCGGGCGGGBBGCCTCTCTCCTCTCCCCBG

BTCCGCGBCBGGCCGCBGGCBBGBBCCBGCGCBBCCBGG

GCGCGTCCGCBCBGBCTTGGBGGCGGCTGCBTGCTGCTB

CCTGCTCCBGBBGCGTCCGGTGGCCGCCGC- 3' -39- .des-adenosine antisense sequences:

GCGTCCGGTGGCCGCCGC

GCCTCTCTCCTCTCCCC

GTGGCCCTGTCGGGCGGG

TCCTGCCGTCCTGTCTCCTTT

TCTTTTGCTGTCTTGT

CTTCCCGTCTCTGCTTT

Endothelin ETA receptor antisense oligonucleotides

-CBTCCBCBTGBTTGCTTBGBTTTGTGCTGTBTCTCTCB

GGBTTBTCBCTGBTTBCBCBTCCBBCCBGTGCCBGCCBBBB

GGBTGCCCTGBGGCBBBGGGTTTCCBTCTTGBGGCBBBTTT

GBGGB-31 des-adenosine antisense sequences:

GTCTGTCCTCCCCGTCTCCTCCC

ACTGCTTCTCCCGGGG

GCTTCCCCGGCTTC

GGGTGGCCGGTGTCCCGGGCTCCGGCGCGGCGGC

GGCTTCGGCTGC

GGGTGGGTGGCGCGG

GCTGCCGGGTCCGCGCGGCGCCTGGGCC

CTTGTGCTGCTTTT

TGCTTGTTCCGTTC

TGGCTGCTCCGGTCTGTGTTGTGGTTGTTTTG

TTTCTTCTTGGGTGTGGG

CCTTGCGGTTTTGG

CTGTGGGCCCTTTG

GGGCCTTGGCTTCTGGCTC

Substance P antisense oligonucleotide 5' -CTGCTGBGGCTTGGGTCTCCGGGCGBTTCTCTGCBGBBGBT

GCTCBBBGGGCTCCGGCBGTTCCTCCTTGBTCTGGTCGCTGTCG

TBCCBGTCGGBCCBGTBBTTCBGBTCBTCBTTGGCTCCTBTTTC

TTCTGCBBBCBGCTGBGTGGBGBCBBGBBBBBBGBCTGCCBBGG

CCBCGBGGBTTTTCBTGTTGGBTTTTGCGBCGGBCBGTCCCGCG

GGGTGCTGAGTTTCTCTGGTTCCTCCGBGCGCB -3' des -adenosine an tisense sequences:

CGTGGTCGCTCCGC

TTTCTCTGGTTCCTCCG

GTCCCGCGGGGTGCTG

TCTGGTCGCTGTCGT

GGCTTGGGTCTCCGGGCG

GTTTCCTTCCTTTTCCGC

Substance P receptor antisense oligonucleotide

-GGCTBBGBTGBTCCBCBTCBCTBCCBCGTTGCCCBCCBCB

GBGGTCBCCBCBBTGB CCGTGTBGGCBGCTGCCCBBBGGB CBB

TTTGCCBGGCTGGTTGCBCGBBCTGBTTGGGTTCCGBGGTGTT

BGTGGBGBTGTTTGGGGBGBGGTCTGBGTCCB

CCGGGBGGBCG

TTBTCCBTTTCGBBGCTBGGCGGTBBBGCCCTBCTBTCTGTBC

BCBBCCCCCCTCTGCBGCBGBGTCCTGTCGTGGCGCCTGGGC

TCBGGGTCC-31 des-adenosine antisense sequences:

GTCCTGTCGTGGCGCCTGGGGCTC

TTCTTTTGTGGGCT

CTTTGGTGGCTGTGGCTG

TGGTCTCTGTGGTTG

CTGCCCTGGGTCTGG

Chymase

GGGTGTGGCCTTGGGGCCGTCCTCTGGCTCCTCCTCGTGGGCCCCC

-GGBGCTGBTBCTGCBGATTTCBGBGGGBBGBBCCCT

GBTBCTCBCCBGCTTCBGCTCTGGBGCBCBBGBGBBBGB

GCBGCBGGGGGBGBGGBBGBBGCBGCBTCTTCCCBGBGB

GGCTGCCTGBGCBBBTGCTGGTTTTCCTTTCCBGTCTTG

GGTTTTBTBBCTCCCBGBBGGCBBGBGBGGGGCBBGG- 3' des-adenosine antisense sequences:

CGTTTTCTTCTCTC

TGCTGGTTTTCCTTTCC

TGGCAGTGGGTGGGGGTGGGGGTGGGGTGGC

TTCCTTGTTCCTGGGGGTGTCCT

CTTGCTCTGGGCTTTTCT

CCCCTTTTCCTTCC

TGTCTGTTTTCCTGGGG

-41-

CTCTCCTCTGTCTCTGTGT

CCTTGCCCTGGCCC

TCTTCCCTCTCCTGTCTCCTGT

CCCTGTGTTCCGCCC

GTCTTCCCTCTCCTG

ACCTCCTTTTCCTCCG

CTGGGTGGGGCCCTG

CCTGTTCTCTGCTCCC

TGGCTTGGGGTTTCTTCTG

TGTGTCTTCTTCCTCTGTT

GGCTGGCTTTCTCCTTC

TTTTGTCTTCCTGGG

TGCCCCTTCTTCCTTTCTTGGG

TCCTTGGTGCTTGGGCTGGG

Endothelial nitric oxide synthase

-GCGTCTTGGGGTGCBGGGCCCBTCCTGCTGCGCCTGGGCG

CTGBGGGTGTCBTBGGTGBTGCTCCCCBCCTCCCBGTTCTTCB

GCTCGGGGGGCTGTGTTCTGGCGCTGGTGGGBGTBGGGBTGCT

GGGGCCCGGCTGGGCTCBGGGGCCGGGGTGGCTGGGCCCTGCT

TGCCGCBCBGCCCBBGGCCCBGCCCCBGCCCCBGGCCGCBGGG

TGGCCCBGGCTCCTGGGCCB CGCTCTTCBBGTTGCCCBTGTTB CTGTGCGTCCGTCTGCTGGBGCBGGCBGCBGBGTGGGBBTTC-3' des-adenosine antisense sequences:

CTGTGCGTCCGTCTGCTGG

GGGGCCGGGGTGGCTGGGCCCTGCTTGCCGC

ACGACCCCGGGCCGACCCGAG

GCTCGGGGGGCTGTGTTCTGGCGCTGGTGGG

CTTGGGCCCCTCTGGGGGCTGGGTT

a. TCCTGCTGCGCCTGGGCGCTG

GCGTCTTGGGGTGC

GGGGCCGGGGGGCCGGGGG

GCCGCTGTTCGTGGGCCTGGG

-42-

GGTGCCTGTGGCTGCC

GGTTGCCCCGGTTGGTGGC

GCCGTCCTGCTGCCGGT

CGTTGGCTGGGTCCCCCCGC

CCGTTTCCTGGGGTCC

GCGTGGGGTGCTCC

GGTTCCTCGTGCCG

CTGCTGCCTTGTCTTTCC

GGCCGTGGCGGCGTGGTGGTCC

GCCCCCCCTGGCCTTCTGCTC

GGGGTCTGGCTGGT.

TGCCGGTGCCCTTGGCGGC

GGTCTTCTTCCTGGTG

GCTCTGGGCCCGGCCGGTCTCGG

GCGTCTCGTGTTCG

CTCTTGTGCTGTTCCGGCCG

*CTCCTTCCTCTTCCGCCGCC

GCCGCTCCCCGCCC

GCTCGTCGCCCTGGCCC

:*tooGGCCTCCTCCTGGCCGC

TGTCTCGGGCGGCGGCCTTGGC

o

GCTCCGTTTGGGGCTG

CCTCTGGCGCTTCC

a 25 GGCCCTCGGCCTGGGCGCTC

TCTTCCGCCTGTGC

S. TGGTGGCCCTCGTGG 55 GCCCCTCCTGGCCTCCGGTGTCC

TGTGGTCCCCCGGCTGGT

-43-

GGCCGGGCCGGTTGGGCGGGC

GTGGGCGCCGGCGGGTCCTCC

GGGCTGCCCTTCTCC

GCCGGGGGTCCCGC

GCTCCTGCTGTTCCCTGGGCTCTTCTGCC

TCTCTCCTGGGTGGGTGCTGGGTGCCG

LO GGGTCTCCGGGCTTG

CCCCGCGCTGCTGGGCGTTCTGC

GGTCTTGGGGTTGTC

TGTGGCCCCGCTCG

TGTCGCCCTCCGTCGCC

CGTCGCCGGCCTCGTCC

CCTCCTGGGTGCGC

GGCGGGCTGGTCCT

GGCGTTTTGCTCCTTCCTGG

Inducible nitric oxide synthase

-CTGCCCCBGTTTTTGBTCCTCBCBTGCCGTGGGGBGGB

CBBTGGGGTTGCBTCCBGCTTGBCCBGBGBTTCTGGBG

30 BCTTCTTTCCCGTCTCCBCGBGGGGCTGCGGGGBCTCB

TTCTGCTGCTTGCTGBGGTTGTGBTBCTGBGGTCBTCC

TGTGTCBCTGGBCTGGBGGTGGCBCBGGGGGCTTTCTC

CBCBTTGTTGTTGBTGTCTTTTTCCCCBTTCBTTGCBT

BCTGGTGGBBTTTGGTCTTGBBCBGBBBTTTCCBBGGB

CBGGCCBTCTCTBTGGCTTTBCBBBGCBGGTCBCTTBT

GTCBCTTBTCTGGBTTTGBGCTCBGBTGTTCTTCB

CTG

TGGGGCTTGCBGCTGGCTGCBCTGCCTCCCCGGGGTB -3' Human major basic protein: GTTTCATCTT GGCTTTATCC (SEQ ID NO:44) 40 EXAMPLE 6 Turning now to Figure 3, two asthmatic rabbits were adminstered adenosine, and two rabbits were adminstered dAMP, at the indicated concentrations, by inhalation as described above in Example 3. The results (shown in Figure 3 as change in compliance) indicate that dAMP, a breakdown product of antisense -44oligodeoxynucleotides containing adenosine, is as potent in the induction of bronchoconstriction as adenosine in the hyperresponsive airways of asthmatic rabbits.

EXAMPLE 7 An aerosolized phosphorothioate 21-mer antisense ODN consisting of 50% adenosine and 50% guanine plus cytosine in a random configuation was found to produce potent bronchoconstrictor effects in hyperreactive airways of asthmatic rabbits, as illustrated in Figure 4. The control molecule used in this study, a phosphorothioate 21-mer antisense ODN consisting of 50% guanine and 50% thymidine plus cytosine (des-adenosine ODN) produced no bronchoconstrictor or any other effect in these same animals.

In this study, bronchoconstrictor effects were measured as a percentage change in bronchial compliance.

Each group consisted of two allergic rabbits, and data shown are for the period following the second of two daily administrations of 5 mg aerosolized ODN by nebulizer.

These results indicate that antisense oligonucleotides, even when modified to slow degradation, produce adenosine metabolites capable of potent bronchoconstriction when adminstered in asthmatic airways.

The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

The entire disclosure in the complete specification of our Australian Patent Application No.

60959/96 is by this cross-reference incorporated into the present specification.

45 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Nyce, Jonathan W.

(ii) TITLE OF INVENTION: METHOD OF TREATMENT FOR LUNG DISEASES

USING

ANTISENSE OLIGONUCLEOTIDES (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Kenneth D. Sibley STREET: Post Office Drawer 34009 CITY: Charlotte STATE: North Carolina COUNTRY: USA ZIP: 28234 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: September 29, 1997 CLASSIFICATION: Unknown (viii) ATTORNEY/AGENT INFORMATION: NAME: Sibley, Kenneth D.

35 REGISTRATION NUMBER: 31,665 REFERENCE/DOCKET NUMBER: 5128-32 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (919) 881-3140 S 40 TELEFAX: (919)881-3175 TELEX: 575102 INFORMATION FOR SEQ ID NO:1: 45 SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GATGGAGGGC GGCATGGCGG G 21 INFORMATION FOR SEQ ID No:2: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 46 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GTAGCAGGCG GGGATGGGGG C 21 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 GTTGTTGGGC ATCTTGCC 18 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: S 40 GTACTTGCGG ATCTAGGC 18 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: 45 LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 50 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GTGGGCCTAG CTCTCGCC 18 e INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 47 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GTCGGGGTAC CTGTCGGC 18 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGCTTTTCTT TTCTGGGCCT C 21 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: S* GGCGCCGTGC CGCGTCTTGG TGGCGGCGG 29 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: 40 LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTTCCCCTGG GTCTTCC 17 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: 55 LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GCCTGTGTCT CTCCTCCT 18 48 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GCCTTTCCTG GTTCTCTT 18 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GCCTGTGTCT GTCCTCCT 18 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: S* LENGTH: 26 base pairs 35 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear i (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TCTCCCTTGG GCTCTGGCTC CTTCTC 26 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CTTGCTCCTG GGGGCCTCCT G 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single 49 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CTTGCTCCTG GGGGCCTCCT 21 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCGCTCGGCC TGGTCCCGG 21 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GGGCGCGGGC GAGCATCGC 19 INFORMATION FOR SEQ ID NO:18: 40 SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CCTCCTTCCT GGTCTGTCTG C 21 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 50 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GCGCTCGGCC TGGTCCCGG 19 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCGCGGGCCG GGGGCTGCTG GG 22 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CCTCTTTTCT GTTTTTCCC 19 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GTTCTTGGCT TCTTCTGTC 19 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: 51 LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CTCTGCTGGT TTTCTGCCTT CTGCCC 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID TTTTCTCTTT CGCTTTCTTT TCGTCTCCTG TTCCTCCTTT T 41 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 33 ase pairs TYPE: nucleic acid 35 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CTCTGTGTTG TTCTGGTCCT TCGTGGGGCT CTG 33 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CTCTGGTTGG CTTCCTTC 18 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid 52 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: TCCCTGTTTC CCCCCTTT 18 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GCTTCTCTTT CGTTCCCGGT GGGCTCG 27 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 35 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID 40 GCTTGTGTGC TCTGCTGTCT CT 22 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TGGTGGGGCT GGGGCTCCGG GGTCTCTGCC CCTCCGTGC 39 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 53 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GTCCTTCTTG TCCGCTGCC 19 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GTGGGGCCTG CTCTCCCGGC CTCCG 18 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii).MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GGGTCCTCAT GGCTGGGG 18 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid S.r. STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GGGTCCTCAT GGCTGGGGTC S(2) INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 54 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GTCTTTGTTT CTGGGCTCGT GCC 23 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GTTCATGGTG GCTAGGTGGG GC 22 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) S: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: GGGGTGGGTA GGCCGTGTCT 24 INFORMATION FOR SEQ ID NO:39: f SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single 45 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CGGTTTCCTT TGCGGTC 17 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID 55 GTGCTCCGGT GGCTTTTT 18 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: GGTCCAGCCA TGGGTCTGGG INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GCTGGTCCTC TGCTGTCCTT GCTG 24 INFORMATION FOR SEQ ID NO:43: 40 SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

I

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: GCCCCGTCTG CTGCTCCTCG TGCCG INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 56 GTTTCATCTT GGCTTTATCC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 900 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID

GATOGAGGGC

TTTTCT'FITc

TTCCGCCTGT

TCCCTTGGGC

CTCCTGCGCT

CTGCGCCCTG

CTGGGCCTCT

CTTCTGCCCT

TTCTGGTCCT

C'ITCTCITTC

GGCTCCGGGG

CCCGGCCTCC

TGGGCTCGTG

C ITTGCGGTC

CTGTCCTTGC

GGCATGGCGG

TGGGCCTCGG

GTCTCTCCTC

TCTGGCTCCT

CGGCCTGGTC

CTGCTCTTTC

T'rTCTGITTTT TTTCTCT'PrC

TCGTGGGGCT

G'ITCCCGGTG

TCTCTGCCCC

GGGGTCCTCA

CCGTTCATGG

GTGCTCCGGT

TGGCCCCGTC

GGTTGTTGGG

CGCCGTGCCG

CTGCCTTTCC

TCTCCTTGCT

CCGGGGGCGC

TGCTGCGCTC

TCCCGTTCTT

GCTTTCTTTT

CTGCTCTGGT

GGCTCGGCTT

TCCGTGCGTC

TGGCTGGGGG

TGGCTAGGTG

GGCTTTTTGG

TGCTGCTCCT

CATCTTGCCG

CGTCTTGGTG

TGGTTCTCTT

CCTGGGGGCC

GGGCGAGCAT

GGCCTGGTCC

GGCTTCITCT

CGTCTCCTGT

TGGCTTCCTT

GTGTGCTCTG

CTTCTTGTCC

GGTCCTC!ATG

GGGCGGGGTG

TCCAGCCATG

CGTGCCGGTT

TGGGCCTAGC

GCGGCGGTTT

GCCTGTGTCT

TCCTGCTTGC

CGCCCTCCTT

CGGGCGCGGG

GTCCTCTGCT

TCCTCCTTTT

CTCCCTGTTT

CTGTCTC TTG

GCTGCCGTGG

GCTGGGGTCG

GGTAGGCCGT

GGTCTGGGGC

TCATCTTGGC

TCTCGCCTGC

CCCCTGGGTC

GTCCTCCTTC

TCCTGGGGGC

CCTGGTCTGT

CCGGGGGCTG

GGTTTTCTGC

CTCTGTGTTG

CCCCCCTTTG

GTGGGGCTGG

GGCCTGCTCT

TC ETTGTTTC

TCTCGGTTTC

T GGTCCTCTG T TTATCC 120 180 240 300 360 420 480 540 600 660 720 780 840 900 0

Claims (48)

1. A pharmaceutical composition, comprising a pharmaceutically or veterinarily acceptable carrier or diluent, and an aerosol of an oligonucleotide(s) (oligo(s)) which is (are) effective for alleviating bronchoconstriction, lung inflammation or allergy(ies), wherein the oligo contains no more than 3 adenosines (A) or, if at least 21 nucleotides long, no more than 3 A per every 21 nucleotides and is antisense to a target selected from the group consisting of the initiation codon, the coding region or the 5'-end and the 3'-end of a gene encoding a polypeptide associated with lung airway dysfunction or anti-sense to the corresponding mRNA(s), combinations of the oligos and mixtures of the oligos.

2. A composition according to claim 1, wherein the oligo is A-free. o

3. A composition according to claim 1 or claim 2, wherein the target is selected from the group consisting of the initiation codon, the coding region and the 5'-end and the 3'-end of an oncogene(s) and a gene(s) encoding a polypeptide(s) associated with lung airway dysfunction or anti-sense to the corresponding mRNA(s), combinations of the oligos and mixtures of the oligos; and wherein the polypeptide(s) is(are) selected from .the group consisting of peptide factors, peptide transmitters, antibodies, cytokines, chemokines, enzymes, binding proteins, adhesion molecules, receptors for the peptide factors, peptide transmitters, antibodies, cytokines, chemokines, enzymes, binding proteins, adhesion molecules and non-peptide transmitters, and malignancy associated proteins.

4. A composition according to claim 3, wherein the target is selected from the group consisting of the initiation codon, the coding region and the 5'-end and the E: \Karen\Keep\P40151-EAST-CAROLINA. 1 .doc 21/11/00 58 3'-end of an oncogene(s) and a gene(s) encoding a polypeptide(s) associated with lung airway dysfunction or to the corresponding mRNA(s), combinations of the oligos, and mixtures of the oligos; wherein the polypeptides are selected from the group consisting of adenosine receptors, immunoglobulin receptors, enzymes, factors, antigens, neurotoxins, interleukins, interleukin receptors, acetylcholine receptors and muscarinic receptors.

5. A composition according to claim 4, wherein the target is selected from the group consisting of the initiation codon, the coding region and the 5'-end and the 3'-end of a gene(s) encoding a polypeptide(s) associated with lung airway dysfunction or to the oncogene mRNA and the corresponding mRNA, combinations of the oligos, and mixtures of the oligos; wherein the polypeptide(s) is(are) selected from the group consisting of adenosine Ai receptor, adenosine A2a receptor, adenosine A2b receptor, adenosine A 3 receptor, IgE receptor P, Fc-epsilon receptor CD23 antigen, IgE receptor a subunit, IgE receptor Fce, histidine decarboxylase, beta tryptase, tryptase-I, prostaglandin D synthase, cyclooxygenase-2, eosinophil cationic protein, eosinophil derived neurotoxin, eosinophil peroxidase, P selectin, endothelial monocyte activating factor interleukin-3 interleukin-5 interleukin-6 monocyte-derived neutrophil chemotactic factor, neutrophil elastase (medullasin), neutrophil oxidase factor, cathepsin G, defensin 1, defensin 3, macrophage inflammatory protein-i-a, muscarinic acetylcholine receptor HM1, muscarinic acetylcholine receptor HM3, fibronectin, interleukin-8 GM-CSF, tumor necrosis factor a, leukotriene C4 synthase and major basic protein.

6. A composition according to any one of claims 1 to wherein the mononucleotide(s) in the oligo(s) is(are) linked or is(are) modified by one or more of E: \Karen\Keep\P40151-EAST-CAROLINA.l.doc 21/11/00 59 phosphorothioate, methylphosphonate, phosphotriester, thioformacetal, phosphorodithioate, phosphoramidate, formacetal boranophosphate, 3'-thioformacetal, thioether, carbonate, 5'-N-carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite., sulfoxide, sulfide, hydroxylamine, methylene(methylimino) (MMI) or methyleneoxy(methylimino) (MOMI) linkages, phosphorothioate or methylphosphonate.

7. A composition according to claim 6, wherein the mononucleotide residues are linked by phosphorothioate residues.

8. A composition according to any one of claims 1 to 7, wherein the anti-sense oligo comprises about 7 to about mononucleotides.

9. A composition according to any one of claims 1 to 7, wherein the anti-sense oligo comprises a fragment selected from the group consisting of SEQ. ID NO:1, SEQ. ID NO:3, SEQ. ID NO:5, SEQ. ID NO:7 to SEQ. ID NO:44. A composition according to any one of claims 1 to 9, wherein the anti-sense oligo is operatively linked to, or complexed with, an agent selected from the group consisting of cell internalized or up-taken agents and cell targeting agents.

11. A composition according to any one of claims 1 to 10, wherein the cell internalized or up-taken agent is S. selected from the group consisting of transferrin, asialoglycoprotein and streptavidin.

12. A composition according to any one of claims 1 to 11, wherein the oligo is operatively linked to a vector that is a prokaryotic or eukaryotic vector. E:\Karen\Keep\P40151-EAST-CAROLINA..doc 21/11/00 1. .1 60

13. A composition according to any one of claims 1 to 12, wherein the oligo(s) is(are) hybridized to a ribonucleic acid.

14. A cell, carrying a composition according to any one of claims 1 to 13. A composition according to any one of claims 1 to 13, wherein the carrier or diluent is selected from the group consisting of gaseous, liquid, and solid carriers or diluents.

16. A composition according to claim 15, further comprising an agent selected from the group consisting of other therapeutic compounds, surfactants, flavoring and coloring agents, fillers, volatile oils, buffering agents, dispersants, RNA-inactivating agents, anti-oxidants, flavoring agents, propellants and preservatives. 20 17. A composition according to claim 16, comprising one or more oligo(s), a surfactant, and a carrier or diluent for the oligo and the surfactant.

18. A composition according to claim 16, wherein the agent is an RNA-inactivating agent which comprises an enzyme, optionally an ribozyme.

19. A composition according to any one of claims 1 to 18, wherein the anti-sense oligo is present in an amount of about 0.01 to about 99.99 w/w of the composition.

20. A composition according to any one of claims 1 to 19, which is a systemic formulation.

21. A composition according to any one of claims 1 to 19, which is an inhalable, nasal or respirable formulation. E: \Karen\Keep\P40151-EAST-CAROLINA.1.doc 21/11/00 11 111 61

22. A composition according to claim 21, wherein the formulation is a nasal formulation comprising particles about 10 to about 500 pm in size.

23. A composition according to claim 21, wherein the formulation is a respirable or inhalable formulation comprising particles about 0.5 to about 10 pm in size.

24. A composition according to any one of claims 1 to 23, which is provided in a capsule or a cartridge. A composition according to any one of claims 1 to 24, wherein the carrier is selected from the group consisting of aqueous and alcoholic solutions and suspensions, oily solutions and suspensions and oil-in- water and water-in-oil emulsions.

26. A composition according to claim 21, wherein the carrier comprises a hydrophobic carrier.

27. A composition according to claim 26, wherein the carrier comprises lipid vesicles, and optionally liposomes, or particles, and optionally microcrystals.

28. A composition according to claim 27, wherein the carrier comprises liposomes, and the liposomes comprise the anti-sense oligo.

29. A composition according to claim 21, wherein the formulation is an aerosol formulation. 0 30. A composition according to any one of claims 1 to 29, in single- or multi-unit dose form.

31. A composition according to any one of claims 1 to 29, in bulk. E:\Karen\Keep\P40151-EAST-CAROLINA.1.doc 21/11/00 62

32. A composition according to any one of claims 1 to 31, comprising an oligo, a carrier and a surfactant.

33. A composition according to claim 32, further comprising a propellant.

34. A composite, comprising, in separate containers, a delivery device, a composition according to any one of claims 1 to 33; and instructions for adding a carrier and for use of the composition. A composite according to claim 34, wherein the formulation is a respirable formulation and the delivery device comprises a nebulizer, which delivers single metered doses of the formulation.

36. A composite according to claim 35, wherein the nebulizer comprises an insufflator and the composition is provided in a piercable or openable capsule or cartridge.

37. A composite according to claim 36, wherein the delivery device comprises a pressurized inhaler and the composition comprises a suspension, solution or dry formulation of the oligo.

38. A composite according to claim 37, further comprising, in a separate container, an agent selected from the group consisting of other therapeutic agents, surfactants, anti-oxidants, flavoring agents, fillers, volatile oils, dispersants, antioxidants, propellants, preservatives, buffering agents, RNA inactivating agents, cell-internalized or up-taken agents and coloring agents.

39. A composite according to claim 38, comprising, in separate containers, one or more oligos, and one or more selected from the group consisting of surfactants, propellants, a carrier or diluent, and therapeutic E: \Karen\Keep\P40151-EAST-CAROLINA.1.doc 21/11/00 1 4b 63 compounds other than the oligo(s). An in vivo method of delivering an oligonucleotide(s) (oligo(s)) that is anti-sense to one or more target polydeoxyribonucleotide(s), comprising administering into airways of a subject an aerosol comprising one or more oligo(s) that are anti-sense to a target polydeoxyribonucleotide(s)encoding a polypeptide associated with high sensitivity to or high levels of adenosine, bronchoconstriction, inflammation, allergy or asthma, in an amount effective to reach and hybridize to the target polydeoxyribonucleotide(s), and reduce the production or availability, or to increase the degradation, of mRNA corresponding to the polydeoxyribonucleotide(s), or to reduce the amount of the target polypeptide present in the lungs.

41. An in vivo method of delivering an anti-sense oligonucleotide (oligo) to a target polydeoxyribonucleotide 20 associated with high sensitivity to or high levels of adenosine, bronchoconstriction, lung inflammation, allergy(ies) or asthma, comprising administering to a 'subject a composition according to any one of claims 1 to 33, that comprises a therapeutic amount of the oligo(s).

42. A method according to claim 41, wherein the amount of the oligo(s) is effective to reach and hybridize to the target polynucleotide(s), and reduce or inhibit the polynucleotide(s)' transcription and/or expression and, thereby, alleviating high sensitivity to adenosine or to high levels of adenosine, bronchoconstriction, lung inflammation, allergy(ies) and/or asthma.

43. A method according to claim 41 or claim 42, wherein the composition is administered nasally, by inhalation or into the respiratory airways or the lung (s) of the subject. E: \Karen\Keep\P40151-EAST-CAROLINA.1.doc 21/11/00 *r 64

44. A method according to any one of claims 41 to 43, wherein the administered composition comprises an amount of the oligo(s) and is administered under conditions effective to reduce the production or availability, or to increase the degradation, of the target mRNA or to reduce the amount of the target polypeptide present in the lungs. A method according to any one of claims 41 to 44, for the treatment of asthma.

46. A method according to any one of claims 41 to 44, for the treatment of bronchoconstriction.

47. A method according to any one of claims 41 to 44, wherein the pulmonary obstruction, and/or bronchoconstriction and\or lung inflammation and/or allergy(ies) are associated with high sensitivity to or high levels of adenosine.

48. A method according to any one of claims 41 to 47, o. wherein the subject is a non-human mammal.

49. A method according to any one of claims 41 to 47, 25 wherein the subject is a human. A method according to any one of claims 41 to 49, wherein the oligo is administered in amount of about 0.005 to about 150 mg/kg body weight. S. 51. A method of treating a disease or condition afflicting lung airways, comprising conducting a method according to any one of claims 41 to

52. A method according to claim 51, wherein the amount of oligo(s) administered is (are) effective to reduce the production or availability, or to increase the E:\Karen\Keep\P40151-EAST-CAROLINA. .doc 21/11/00 65 degradation, of the mRNA, or to reduce the amount of the polypeptide present in the lungs.

53. A composition according to any one of claims 1 to 33, wherein the oligo(s) is(are) anti-sense to the initiation codon, the coding region or the 5'-end or the 3'-end of a gene(s) encoding an adenosine Al, A2a, A2b and/or A3 receptor, or anti- sense to the adenosine Al, A2a, A2b and/or A3 receptor mRNA.

54. A composition according to claim 53, wherein all nucleotide linking residues are phosphorothioates. A composition according to claim 1, wherein the oligo is a DNA.

56. A composition according to claim 1, wherein the oligo is an RNA.

57. A composition according to claim 1, wherein the oligo comprises about 7 to up to about 60 mononucleotides.

58. Use of a composition according to any one of claims 1 to 33 in the manufacture of a medicament used to treat a disease or a condition afflicting lung airways.

59. A composition according to claim 1, substantially as hereinbefore described with reference to any one of the examples. 30 60. An in vivo method of delivering an aligonucleotides according to claim 40, substantially as herein described with reference to the examples and figures. Dated this 10 th day of September 2002 o. 35 EAST CAROLINA UNIVERSITY By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\RBell\Keep\71749-00.doc 10/09/02