Methods to minimize the effects of smoke exposure

Abstract

The present invention relates to compositions, methods, and kits for treating or preventing the effects of smoke exposure. In particular, the compositions, methods, and kits are useful for treating or preventing acute smoke exposure, but are not limited thereto. In one embodiment, the present invention provides a method for treating or preventing smoke-related or smoke-induced obstructive pulmonary disease in a subject requiring such treatment or prevention, the method comprising administering an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE to the subject, thereby treating or preventing smoke-related or smoke-induced obstructive pulmonary disease in the subject.

Description

Technical Field

[0001] The present invention relates to compositions, methods, and kits for treating or preventing the effects of smoke exposure. In particular, the compositions, methods, and kits are useful for, but not limited to, treating or preventing acute smoke exposure.

[0002] Related Applications This application claims the benefit of Australian Provisional Application No. 2023 / 901559, filed May 19, 2023, the entire disclosure of which is incorporated herein by reference.

Background Art

[0003] Smoke exposure causes inflammation in the airways, causing the migration of neutrophils and monocytes into the lungs among other inflammatory cells, and releasing cytotoxic enzymes and other oxidants. The accumulation of these toxic substances is a major factor in damage to the airway epithelium and the endothelium of the lung parenchyma. As a result, gas exchange is impaired by damaged alveoli, causing a series of obstructive lung diseases.

[0004] The most common obstructive lung disease associated with smoke exposure is chronic obstructive pulmonary disease (COPD), which affects approximately 30 million people in the United States and hundreds of millions worldwide. The most common cause of COPD is smoking, and it often progresses even after smoking cessation. COPD encompasses two lung pathologies, chronic bronchitis and emphysema, which together constitute the disease. Chronic bronchitis involves the large and small airways of the lungs, causing epithelial hypertrophy, increased mucus production, and increased airway sclerosis. Emphysema is accompanied by a loss of elasticity in the alveoli, creating "dead space" in the lungs and preventing adequate respiration of fresh air. Many patients have some combination of these two pathologies. COPD is medically managed with bronchodilators, beta-agonists, and anti-inflammatory agents, and in advanced cases, oxygen. These treatments do not have a significant impact on the long-term decline in lung function characteristic of this disease, and there is no cure for it.

[0005] There is a need for new or improved methods for treating and / or preventing obstructive pulmonary disease associated with or caused by smoke exposure.

[0006] Any reference to prior art in this specification does not constitute an endorsement or suggestion that such prior art forms part of common general knowledge in any jurisdiction, or that such prior art can be reasonably expected to be understood, considered relevant, and / or combined with other prior art by those skilled in the art. [Overview of the project]

[0007] In one embodiment, the present invention provides a method for treating or preventing obstructive pulmonary disease associated with or caused by smoke exposure in a subject requiring such treatment or prevention, the method comprising administering an antisense oligonucleotide (AON) to the subject that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE), thereby treating or preventing obstructive pulmonary disease associated with or caused by smoke exposure in the subject.

[0008] In another aspect, the present invention provides a method for alleviating or improving symptoms of smoke-related obstructive pulmonary disease or obstructive pulmonary disease caused by smoke exposure in a subject requiring such alleviation or improvement, the method comprising administering an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE) to a subject requiring such alleviation or improvement of symptoms of smoke-related obstructive pulmonary disease or obstructive pulmonary disease caused by smoke exposure in the subject.

[0009] In another aspect, the present invention also provides the use of antisense oligonucleotides (AONs) that promote the production of endogenous soluble RAGE and / or reduce the production of membrane-bound RAGE (mRAGE) in the manufacture of pharmaceuticals for treating or preventing obstructive pulmonary disease associated with or caused by smoke exposure in subjects requiring such treatment or prevention.

[0010] In another aspect, the present invention also provides the use of antisense oligonucleotides (AONs) that promote the production of endogenous soluble RAGE and / or reduce the production of membrane-bound RAGE (mRAGE) in the manufacture of pharmaceuticals for subjects requiring the alleviation or improvement of symptoms of obstructive pulmonary disease associated with or caused by smoke exposure.

[0011] In another aspect, the present invention also provides an antisense oligonucleotide (AON) for use in a method of doing so in a subject requiring treatment or prevention of smoke-related obstructive pulmonary disease or obstructive pulmonary disease caused by smoke exposure, wherein the method comprises administering the antisense oligonucleotide (AON) to a subject requiring it, thereby treating or preventing smoke-related obstructive pulmonary disease or obstructive pulmonary disease caused by smoke exposure in the subject.

[0012] In another aspect, the present invention also provides an antisense oligonucleotide (AON) for use in a method to do so in a subject that requires relief or improvement of symptoms of obstructive pulmonary disease associated with smoke exposure or obstructive pulmonary disease caused by smoke exposure, the AON promoting the production of endogenous soluble RAGE and / or reducing the production of membrane-bound RAGE (mRAGE), wherein the AON is administered to a subject requiring it, thereby relieving or improving symptoms of obstructive pulmonary disease associated with smoke exposure or obstructive pulmonary disease caused by smoke exposure in the subject.

[0013] In another aspect, the present invention relates to a method for treating or preventing obstructive pulmonary disease associated with or caused by exposure to smoke in a person who requires such treatment or prevention, wherein the method is - A step of identifying subjects who have obstructive pulmonary disease associated with exposure to smoke or obstructive pulmonary disease caused by exposure to smoke, - The step of administering an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE) to a subject in need thereof, This provides a method for treating or preventing obstructive pulmonary disease associated with or caused by smoke exposure in a subject.

[0014] In another aspect, the present invention relates to an antisense oligonucleotide (AON) for use in a method of treating or preventing obstructive pulmonary disease associated with or caused by smoke exposure, which promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE), wherein the method - A step of identifying subjects who have obstructive pulmonary disease associated with exposure to smoke or obstructive pulmonary disease caused by exposure to smoke, - The step of administering an antisense oligonucleotide (AON) to a subject requiring it, This provides an antisense oligonucleotide (AON) for treating or preventing obstructive pulmonary disease associated with or caused by smoke exposure in a subject.

[0015] In one embodiment, the present invention provides a method for treating or preventing respiratory inflammation associated with or caused by smoke exposure in a subject requiring such treatment, the method comprising administering an antisense oligonucleotide (AON) to the subject that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE), thereby treating or preventing respiratory inflammation associated with or caused by smoke exposure in the subject.

[0016] In another aspect, the present invention provides a method for alleviating or improving symptoms of respiratory inflammation associated with or caused by smoke exposure in subjects requiring such treatment, the method comprising administering an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE) to subjects requiring such treatment, thereby alleviating or improving symptoms of respiratory inflammation associated with or caused by smoke exposure.

[0017] In another aspect, the present invention also provides the use of antisense oligonucleotides (AONs) that promote the production of endogenous soluble RAGE and / or reduce the production of membrane-bound RAGE (mRAGE) in the manufacture of pharmaceuticals for treating or preventing respiratory inflammation associated with or caused by smoke exposure in subjects requiring such treatment or prevention.

[0018] In another aspect, the present invention also provides the use of antisense oligonucleotides (AONs) that promote the production of endogenous soluble RAGE and / or reduce the production of membrane-bound RAGE (mRAGE) in the manufacture of pharmaceuticals for subjects requiring the alleviation or improvement of symptoms of respiratory inflammation associated with or caused by smoke exposure.

[0019] In another aspect, the present invention also provides an antisense oligonucleotide (AON) for use in a method of treating or preventing respiratory inflammation associated with smoke exposure or caused by smoke exposure in a subject requiring such treatment or prevention, wherein the method comprises administering the antisense oligonucleotide to a subject requiring such treatment or prevention of respiratory inflammation associated with smoke exposure or caused by smoke exposure in the subject.

[0020] In another aspect, the present invention also provides an antisense oligonucleotide (AON) for use in a method to alleviate or improve symptoms of respiratory inflammation associated with smoke exposure or caused by smoke exposure, the AON promoting the production of endogenous soluble RAGE and / or reducing the production of membrane-bound RAGE (mRAGE), wherein the method comprises administering the antisense oligonucleotide to a subject requiring it, thereby alleviating or improving symptoms of respiratory inflammation associated with smoke exposure or caused by smoke exposure.

[0021] In another aspect, the present invention relates to a method for treating or preventing respiratory inflammation associated with or caused by exposure to smoke in a person who requires such treatment or prevention. - identifying a subject having respiratory inflammation associated with exposure to smoke or respiratory inflammation caused by exposure to smoke; - administering to a subject in need thereof an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE); and thereby providing a method of treating or preventing respiratory inflammation associated with exposure to smoke or respiratory inflammation caused by exposure to smoke in the subject.

[0022] In another aspect, the invention provides an antisense oligonucleotide (AON) for use in a method of treating or preventing respiratory inflammation associated with exposure to smoke or respiratory inflammation caused by exposure to smoke in a subject in need thereof, the method comprising: - identifying a subject having respiratory inflammation associated with exposure to smoke or respiratory inflammation caused by exposure to smoke; - administering an antisense oligonucleotide (AON) to a subject in need thereof; and thereby treating or preventing respiratory inflammation associated with exposure to smoke or respiratory inflammation caused by exposure to smoke in the subject.

[0023] In any aspect, the AON that promotes the production of endogenous soluble RAGE is administered directly to the airway and / or lung.

[0024] For any aspect of the present invention, administration to the airway and lungs is via any route that allows an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE) to contact the airway, lungs, or a part thereof. For example, an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE) can be administered via any route such that there is an improvement in the symptoms of the disease or condition being treated or prevented. Preferably, the route of administration allows exposure to the airway or lung parenchyma, such as alveolar tissue having respiratory bronchioles, alveolar ducts, and terminal bronchioles. Preferably, an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE is formulated for inhalation or intranasal administration.

[0025] In any aspect or embodiment, an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE) is administered via inhalation or intranasal administration.

[0026] In another aspect, the present invention provides a kit or article of manufacture comprising any antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE), and / or a pharmaceutical composition comprising an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE).

[0027] In any aspect or embodiment of the present invention, the obstructive lung disease is chronic obstructive pulmonary disease (COPD), emphysema, combined pulmonary fibrosis and emphysema (CPFE), bronchitis, respiratory bronchiolitis / respiratory bronchiolitis-interstitial lung disease, bronchiectasis, asthma, lung cancer, tracheal cancer, or smoking-related interstitial lung disease (SR-ILD).

[0028] In any embodiment of the present invention, chronic obstructive smoking-related interstitial lung disease (SR-ILD) is selected from pulmonary Langerhans cell histiocytosis, exfoliative interstitial pneumonia, and acute eosinophilic pneumonia.

[0029] In any embodiment, the methods or uses of the present invention may promote the production of endogenous soluble RAGE and / or reduce the production of membrane-bound RAGE (mRAGE) in a subject, particularly in the airway of the subject. In any embodiment, the methods of the present invention may promote the production of endogenous soluble RAGE in one or more tissues of the airway, for example, one or more of the airway tissues described herein.

[0030] In any embodiment, AON may promote the production of endogenous soluble RAGE by promoting the inclusion of exon 9b and / or the exclusion (e.g., skipping) of exon 10. Thus, in any embodiment, AON may promote splicing in RAGE premRNA, resulting in the inclusion of exon 9b and / or the skipping of exon 10. For example, AON may result in an increase in the levels of RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19 mRNA in one or more tissues of the airway, preferably an increase in the levels of RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19 mRNA.

[0031] In any configuration, AON is administered to the whole airway, upper airway, or lower airway.

[0032] As used herein, the upper respiratory tract may include one or more of the following areas: the nose and nasal cavity, the sinuses, the pharynx, and the larynx above the vocal cords. Typically, the lower respiratory tract includes one or more of the following areas: the larynx below the vocal cords, the trachea, the bronchi, and the bronchioles. The lungs may be included in the lower respiratory tract and include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0033] In any embodiment, AON is administered as an aerosol, dry powder, or nasal spray. In any embodiment, AON may be administered using a nasal spray pump, intranasal drip, intratracheal drip, medium-dose inhaler (MDI), dry powder inhaler (DPI), nebulizer (jet, ultrasonic mesh, or vibrating mesh), or soft mist inhaler (SMI).

[0034] In any embodiment, the AON is a 10-50 nucleotide AON containing a target sequence complementary to a region near or within an intron of the RAGE premRNA. Alternatively, the AON is a 10-50 nucleotide AON containing a target sequence complementary to or adjacent to a splice site of the RAGE premRNA.

[0035] Factors such as RNA secondary structure, competition between AON and SR proteins, heteronuclear ribonucleoproteins (hnRNPs), and / or other components constituting the splicosome may affect the action of AON; therefore, AON directed to a critical acceptor or donor splice site does not necessarily alter splicing. As a result, in any aspect of the present invention, the AON is a 10-50 nucleotide containing a target sequence complementary to or adjacent to a cis-acting RNA element in the pre-mRNA of RAGE. The cis-acting RNA element acts as an enhancer or silencer that modulates splicing of nearby exons when a splicosome component (e.g., protein splicing factor, uRNA, lncRNA) binds to it.

[0036] In any embodiment, the AON is a 10-50 nucleotide sequence containing a target sequence complementary to the RAGE premRNA, which modulates the secondary structure of the mRNA and influences the selection of the splice site.

[0037] In any embodiment, AON is isolated or purified AON for inducing the exclusion (also known as skipping) of one or more exon sequences in the RAGE gene transcript or a portion thereof.

[0038] In any embodiment, AON is isolated or purified AON for inducing retention of an intron sequence in the RAGE gene transcript or a portion thereof.

[0039] In any embodiment, AON comprises at least one modified nucleotide. Typically, AON is chemically modified to prevent degradation of the premRNA-AON complex. Chemical modifications include, but are not limited to, phosphorodiamidate morpholino oligomers (PMOs), 2'-O-methylphosphorothioate oligonucleotides (2OMe PSs), and 2'-O-methoxyethyl phosphorothioate oligonucleotides (2'-MOE PSs), locked nucleic acid (LNA) modified AONs, heat-stable twist-intercalating nucleic acids (TINAs), and peptide nucleic acids (PNAs).

[0040] In any embodiment, the AON comprises at least one modified nucleotide selected from the group consisting of phosphorodiamidate morpholino oligomers (PMOs), 2'-O-methyl oligonucleotides (2OMe), 2'-O-methoxyethyl oligonucleotides (2'-MOEs), phosphorothioate oligonucleotides, locked nucleic acid (LNA) modified AONs, heat-stable twist-intercalating nucleic acids (TINAs), and peptide nucleic acids (PNAs).

[0041] In any embodiment, AON may be conjugated to a portion comprising, but not limited to, a cell-permeable peptide (CPP), vivo-morpholino (VMO), or peptide phosphorodiamidate morpholino oligomer (PPMO) to improve their delivery.

[0042] In any embodiment, the antisense oligonucleotide contains, essentially consists of, or comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target region of exon 10 of the RAGE premRNA throughout the antisense oligonucleotide. Preferably, the most 5' nucleotide of the AON is at nucleotide position 88 or 114 of exon 10, or is located at nucleotide positions 88-114 of exon 10.

[0043] In any embodiment, the AON may be 8 to 40 nucleotides long, 15 to 25 nucleotides long, or 18 nucleotides long.

[0044] In any embodiment, AON is selected from the group including sequences shown in any of Tables 1a to 1d. Preferably, AON is selected from the list including SEQ ID NOs: 1 to 31 and 33, for example, AON is SEQ ID NOs: 11, 18, 19, or 20, or a nucleotide sequence that is at least 85%, 90%, or 95% identical thereto.

[0045] In any embodiment, the AON may be selected to be an AON capable of binding to a selected target site, the target site being a putative mRNA splicing site selected from a splice donor site, a splice acceptor site, a splice enhancer sequence, a splice silencer sequence, or a site that modulates the secondary structure of premRNA. If a donor or acceptor splice site is targeted, the target site may also include several adjacent intron sequences.

[0046] More specifically, AON may be selected from the group comprising one or more of SEQ ID NOs: 1-31 and 33 and / or sequences shown in any of Tables 1a-1d, as well as combinations or cocktails thereof. More preferably, AON is SEQ ID NOs: 11, 18, 19, or 20. A combination of AON is preferably SEQ ID NOs: 11 and 10, or SEQ ID NOs: 11 and 13. This includes sequences that can hybridize to such sequences under harsh hybridization conditions, complementary sequences, sequences containing modified bases, modified backbones, and functional cleavage or extensions thereof that have or regulate premRNA processing activity in the RAGE gene transcript.

[0047] In certain embodiments, the AON may be 100% complementary to the target sequence, or it may contain mismatches to accommodate variants, for example, as long as the heteroduplex formed between the oligonucleotide and the target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation that may occur in vivo. Thus, a particular oligonucleotide may have about or at least about 70% sequence complementarity between the oligonucleotide and the target sequence, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity.

[0048] The methods and uses of the present invention also extend to combinations of two or more AONs that can bind to a selected target and modulate alternative splicing of RAGE premRNA, and include constructs comprising two or more such AONs. The constructs may be used together in AON-based combination therapies. The combination of AONs is preferably the combination of SEQ ID NOs 11 and 10, or SEQ ID NOs 11 and 13.

[0049] As used herein, unless otherwise specified in the context, the term “comprise,” and its variations such as “comprising,” “comprises,” and “comprised,” are not intended to exclude further additives, ingredients, elements, or steps.

[0050] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, which is given by example and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0051] [Figure 1] Experimental timeline used in a mouse model study of smoke-induced inflammation. [Figure 2] Mouse body weight. Percentage change relative to day 0. Mean ± 95% CI, n=8 mice / group. Statistical significance of % body weight change between groups was determined by one-way ANOVA without correction for multiple comparisons. [Figure 3] Preliminary BALF cell count. Group 1 is represented by red data points, and Group 2 is represented by blue data points. Data are mean ± 95% CI, n=8 animals / group. Statistical significance of % body weight change between groups was determined by one-way ANOVA without correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4] Acute CSE causes acute pneumonia. Mice were exposed to air or CSE (9 cigarettes / day for 4 days). Cell count of the fractionated bronchoalveolar lavage fluid (BALF). Group 1 is represented by red data points, and Group 2 is represented by blue data points. Data are mean ± 95% CI, n=8 mice / group. Statistical significance of % body weight change between groups was determined by two-way ANOVA without correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001. [Figure 5]RAGE_v1 mRNA expression in the left lung after a single dose of ASO6713 or saline vehicle. C57BL / 6 mice were intratracheally administered ASO6713 (3 mg / kg), ASO6713 (10 mg / kg), or saline. RAGE_v1 mRNA levels (encoding esRAGE) were measured in samples collected from the left lung at 11, 14, and 17 days post-administration. Data are shown mean ± SEM; *p<0.05 (compared to control). [Figure 6] Treatment with AON-m79 prevents airway inflammation, lung damage, and experimental COPD in mice exposed to cigarette smoke for 8 weeks. Lung function was assessed in terms of (A) cell count in bronchoalveolar lavage fluid, (B) airway epithelial thickness, (C) collagen deposition under the small airway epithelium, (D) alveolar diameter, and (E) total lung volume. Data are shown mean ± SEM; *p<0.05 (compared to control), #p<0.05 (compared to smoke group). [Figure 7] RAGE exon 10 mRNA expression in the left lung after a single dose of ASO6713 or saline vehicle. C57BL / 6 mice were intratracheally administered ASO6713 (3 mg / kg), ASO6713 (10 mg / kg), or saline. RAGE exon 10 mRNA levels (encoding membrane-bound RAGE (mRAGE)) were measured in samples collected from the left lung on days 11, 14, and 17 post-administration. Data are shown mean ± SEM; *p<0.05 (compared to control). [Modes for carrying out the invention]

[0052] It will be understood that the present invention, as disclosed and defined herein, encompasses all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

[0053] Herein, specific embodiments of the present invention will be described in detail. While the present invention will be described in conjunction with the embodiments, it should be understood that the invention is not intended to be limited to those embodiments. Rather, the present invention is intended to cover all alternatives, modifications, and equivalents that may fall within the scope of the invention as defined by the claims.

[0054] Those skilled in the art will recognize many methods and materials similar to or equivalent to those described herein that can be used in carrying out the present invention. The present invention is by no means limited to the methods and materials described herein. It will be understood that the present invention disclosed and defined herein encompasses all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

[0055] All patents and publications referenced herein are incorporated in their entirety by reference.

[0056] For the purposes of interpreting this specification, terms used in the singular form also include their plural forms, and vice versa.

[0057] This invention is based on the remarkable discovery that administration of antisense oligonucleotides (AONs) that promote the production of endogenous soluble RAGE and / or reduce the production of membrane-bound RAGE (mRAGE) can prevent or treat smoke-induced inflammation. In particular, in a preclinical model of acute smoke exposure, administration of AONs that promote the production of endogenous soluble RAGE and / or reduce the production of membrane-bound RAGE (mRAGE) into the airways was able to suppress inflammation.

[0058] Indications The present invention relates to a method for treating or preventing obstructive pulmonary (lung) disease associated with or caused by exposure to smoke. Preferably, the exposure is acute exposure to smoke.

[0059] Such obstructive disorders may result from narrowing of the smaller and larger bronchi of the lungs and are generally characterized by inflamed and easily collapsing airways, airflow obstruction, difficulty breathing, and frequent clinic visits and hospitalizations.

[0060] Examples of obstructive pulmonary diseases that can be treated or prevented according to this method include chronic obstructive pulmonary disease (COPD), including cigarette smoke-induced COPD; emphysema; emphysema-associated pulmonary fibrosis (CPFE); bronchitis; respiratory bronchiolitis / respiratory bronchiolitis-interstitial lung disease; bronchiectasis; asthma; lung cancer; tracheal cancer; or smoking-related interstitial lung disease (SR-ILD). In any embodiment of the present invention, chronic obstructive smoking-related interstitial lung disease (SR-ILD) is pulmonary Langerhans cell histiocytosis, exfoliative interstitial pneumonia, or acute eosinophilic pneumonia.

[0061] Those skilled in the art will be familiar with the various classifications of obstructive pulmonary disease. COPD is also known as chronic obstructive airway disease (COAD) or chronic airflow limitation (CAL). Those skilled in the art will understand that, in common use, the term COPD encompasses emphysema and chronic bronchitis. Chronic bronchitis is typically diagnosed when a patient has suffered from chronic cough, mucus production, or both for at least three months over at least two consecutive years, after excluding other causes of chronic cough. In chronic bronchitis, airway obstruction is caused by chronic and excessive secretion of abnormal airway mucus, inflammation, and bronchospasm. Often, chronic bronchitis is aggravated by frequent or chronic infections.

[0062] Emphysema involves the destruction of elastin in the terminal bronchioles, leading to the reconstruction, destruction, and eventual collapse of the airway walls. Patients with emphysema gradually lose expiratory capacity, leading to elevated levels of waste gases (such as carbon dioxide) in the blood, decreased blood oxygen levels, and a general deterioration of the patient's physical strength and overall health. A characteristic feature of emphysema is the permanent loss of alveoli. Reconstruction leads to permanent dilation of the air spaces distal to the terminal bronchioles and destruction of the terminal bronchiolar walls, but without fibrosis. Emphysema is progressive and has a poor prognosis. Treatment is generally symptomatic and persistent, as there are no known methods to repair elastin or regenerate alveoli. Most patients with COPD have both emphysema and chronic bronchitis.

[0063] Bronchiectasis refers to the abnormal and irreversible dilation of the bronchi caused by destructive and inflammatory changes in the airway walls. There are three main anatomical patterns of bronchiectasis: columnar bronchiectasis, varicose bronchiectasis, and cystic bronchiectasis.

[0064] Chronic bronchitis is defined as chronic cough and sputum production present for at least three months within a two-year period, in the absence of other diseases causing sputum production. Epidemiologically, chronic bronchitis involves chronic inflammation of the bronchial epithelium, accompanied by hypertrophy of mucous glands and an increase in goblet cells. Cilia are also destroyed, significantly reducing the efficiency of the mucociliary escalator. Mucus viscosity and mucus production increase, making expectoration difficult. Mucus accumulation leads to increased susceptibility to infection. Microscopically, there is infiltration of the airway wall by inflammatory cells. Following inflammation, scarring and reconstruction occur, thickening of the airway wall and narrowing of the airway. As chronic bronchitis progresses, squamous metaplasia (abnormal changes in the tissue lining the inside of the airway) and fibrosis (further thickening and scarring of the airway wall) occur. As a result of these changes, airflow is restricted. Repeated infections and inflammation over time can lead to irreversible structural damage and scarring of the airway walls, resulting in narrowing and deformation of smaller peripheral airways.

[0065] Asthma is defined as a chronic inflammatory state of the airways, causing widespread and variable airway obstruction, which is reversible spontaneously or with treatment. In some patients with chronic asthma, the disease progresses and leads to irreversible airway obstruction, especially if the asthma is undiagnosed or uncontrolled and therefore untreated, or if it is particularly severe. Inflammation of the airways in asthma over time can lead to airway remodeling through increased smooth muscle, destruction of the surface epithelium, increased collagen deposition, and thickening of the basement membrane.

[0066] The symptoms of obstructive airway diseases described above include airway obstruction, inflammation, cough, wheezing, mucus gland hyperplasia and hypersecretion, excessive mucus production, wet cough, hemoptysis (coughing up blood or bloody mucus from the bronchi, larynx, trachea, or lungs), and dyspnea (shortness of breath).

[0067] As used herein, “obstructive pulmonary disease” “associated with” exposure to smoke includes any obstructive pulmonary disease caused by, resulting from, or suspected to be caused by exposure to smoke.

[0068] As used herein, “exposure” includes short-term (acute) exposure to an environment characterized by high levels of aerosolized smoke particles. Exposure may include inhalation and respiration of aerosolized particles such that the aerosolized particles enter the airways of an individual and cause damage. Thus, in the context of the present invention, obstructive pulmonary disease associated with acute smoke exposure includes obstructive pulmonary disease caused (or suspected to be caused) by inhalation (passive or active) of smoke particles.

[0069] The “individuals” or “subjects” requiring treatment or prevention according to the present invention include mammals. Mammals may be humans, or livestock, zoo animals, or companion animals. While the methods of the present invention are intended to be particularly suitable for the medical treatment of humans, they are also applicable to veterinary treatments, including the treatment of companion animals such as dogs and cats, as well as livestock such as horses, cattle, and sheep, or zoo animals such as felines, canines, bovines, and ungulates.

[0070] Those skilled in the art will be familiar with various methods for identifying individuals requiring treatment for obstructive airway disease, including, for example, determining that the FEV1 / FVC ratio (the ratio of forced expiratory volume per second (FEV1) to forced vital capacity (FCV)) is less than 0.7 (i.e., the inability to exhale 70% of the air in one minute). For example, in COPD, there is an increase in airway resistance, indicated by a decrease in forced expiratory volume per second (FEV1) as measured by vital capacity measurement. COPD is sometimes defined as a ratio of forced expiratory volume per second to forced vital capacity (FEV1 / FVC) of less than 0.7. In COPD, residual volume (i.e., the amount of air remaining in the lungs after complete exhalation) is often increased, and total lung volume increases as well, but vital capacity remains relatively normal. The increase in total lung volume (hyperinflation) may result in the clinical feature of a "barrel chest," which is a large anteroposterior diameter chest that occurs in some individuals with COPD. Hyperinflation may be observed on chest X-rays as flattening of the diaphragm.

[0071] Those skilled in the art will be familiar with the various instruments, methods, and guidelines available for performing pulmonary function tests. An example of such guidelines is the GOLD guideline for determining pulmonary diffusion capacity (Rabe et al., 2007, Am J Respir Crit Care Med, 176:532-555). Vital capacity measurements and peak flow meters can also be used to record variations in airflow limitation.

[0072] Other methods for diagnosing obstructive airway disease include the use of chest X-rays (to look for hyperinflation in the case of COPD) or CT scans (to determine the presence of emphysema).

[0073] The term "therapeutic effective dose" refers to the amount of AON that can treat, prevent, or alleviate obstructive pulmonary disease. The therapeutic effective dose may be determined empirically and in a conventional manner in relation to the treatment of obstructive pulmonary conditions.

[0074] Furthermore, it should be understood that the present invention includes methods in which an individual may have previously been exposed to the aerosol for a long period (long-term chronic exposure) or a short period (acute exposure), even if there is no imminent risk of exposure to the aerosol in question. Therefore, those skilled in the art will understand that there may be a period of time between exposure to the aerosol and the onset of symptoms of obstructive pulmonary disease and the subsequent need for treatment. In some embodiments, the period between aerosol exposure and treatment is one day, one week, one to six months, one year, or longer. Preferably, the period between aerosol exposure and treatment is less than one year.

[0075] It should be understood that some of the obstructive pulmonary diseases described above cause significant damage to the respiratory system. For example, in the case of emphysema, those skilled in the art will understand that emphysema is irreversible, that is, once the bronchioles are destroyed, they cannot be restored. Therefore, the present invention also aims to provide a method for preventing the occurrence of such irreversible damage to lung tissue.

[0076] As used herein, “preventing,” “prevention,” “preventative,” or “prophylactic” means preventing, hindering, defending against, or protecting against the occurrence of a condition, disease, disorder, or phenotype, including any abnormality or symptom. Subjects requiring prevention are those who are more susceptible to developing the condition.

[0077] Prevention may include immediate preventive steps aimed at mitigating damage to lung tissue resulting from recent exposure to aerosols. For example, the present invention includes the use of AON as a prophylactic agent for use to reduce or prevent inflammation immediately after smoke exposure, preferably acute smoke exposure. Such prevention may include administration of AON to an individual immediately after inhaling smoke from a cigarette (as either a smoker or a passive smoker), administration immediately before a workday (in the case of an individual exposed to occupational aerosols), or administration immediately before or after a prolonged stay in an area with air pollution or industrial pollution (e.g., a visit to an industrial city). For example, prevention may also include administration of AON in the morning before going out and before exposure to air pollution.

[0078] The term "effective for prevention" refers to the amount of nuclease that can prevent the onset of obstructive pulmonary disease.

[0079] Those skilled in the art will understand that both short-term and long-term approaches can be used to determine the success of treating an individual using the method of the present invention. First, considering the acute, short-term use of AON to treat obstructive pulmonary disease as defined herein, those skilled in the art may expect immediate improvement in symptoms after treatment. For example, those skilled in the art would look for signs of improved airflow, reduced airway inflammation, reduced cough, reduced wheezing, reduced mucosal gland hyperplasia and hypersecretion, reduced mucus production, and reduced occurrence of wet cough as indicators of the success of treating an individual using the method of the present invention.

[0080] Furthermore, those skilled in the art may perform standard tests to determine whether there is improvement or decrease in the levels of certain inflammatory markers associated with the presence, development, or risk of obstructive pulmonary disease. Examples of inflammatory markers evaluated to determine the success of treatment or prevention include C-reactive protein (CRP), serum IgE and IgG, IL-8, sICAM, soluble tumor necrosis factor receptor (sTNFR) 1, alpha-1 antitrypsin, total white blood cell count and neutrophil count, as well as neutrophil percentage, osteoprotegerin, neutrophil-activating peptide 2, CXCL16, and monocyte chemotactic protein 4 levels. Those skilled in the art will be familiar with methods for measuring the levels of these inflammatory markers in individuals receiving treatment according to the present invention, including the use of ELISA, flow cytometry, and other methods for measuring inflammatory markers in plasma samples from patients.

[0081] When the methods of the present invention are used over a long period, including as a preventive method, those skilled in the art will notice improvements in lung function (measured by the conventional methods described herein) and a reduction in the progression of lung disease as indicators of the success of treating or preventing obstructive pulmonary disease caused by aerosols.

[0082] smoke Acute exposure to smoke can refer to temporary and intermittent exposure to pulmonary toxins contained in the smoke. The pathophysiological pathways activated in the lungs after acute exposure to cigarette smoke lead to a progressive decline in lung function. In particular, a crucial underlying mechanism is airway inflammation, which has been observed in mice after acute and repeated acute exposure to cigarette smoke, leading to bronchial wall thickening and pulmonary emphysema.

[0083] Chronic obstructive pulmonary disease (COPD) is the cumulative result of repeated exposure to inhaled toxins, primarily from cigarette and other types of tobacco and marijuana smoke (e.g., pipes, cigars, water pipes, bidis, and kreteks), emissions from heated tobacco products and smokeless tobacco (e.g., vaping), and air pollution (e.g., burning of wood and other biomass fuels, particulate matter, chemicals, haze, surface ozone, nitrogen or sulfur oxides, heavy metals, passive smoking). Currently, there is evidence that most smokers develop some form of respiratory impairment due to smoking, and that their annual decline in lung function (FEV1) is faster than that of non-smokers. Cigarette smoke-induced COPD (COPD-C) is the cumulative result of repeated "acute" exposure to cigarette smoke.

[0084] Smoke can be an aerosol or particulate matter from the combustion of one or more of the following: cigarettes, including electronic cigarettes; tobacco or marijuana; coal; wood; oil; gas; or other biomass. The above are examples of smoke-related aerosols that can cause damage or injury to the lungs, and include aerosolized carbon particles (which may be found in smoke from burnt organic matter, coal dust, smog, exhaust fumes, and other industrial pollutants).

[0085] One of the main causes of obstructive pulmonary disease, particularly COPD, is the inhalation of cigarette smoke. However, those skilled in the art will understand that other types of smoke or injuries can also cause obstructive airway disease. Examples of such smoke include, generally speaking, smoke from tobacco (e.g., cigars, tobacco pipes) or other “smoking” items inhaled by individuals for recreational purposes (e.g., marijuana joints). It should also be understood that obstructive pulmonary disease can be caused by “passive smoking” (i.e., non-smokers inhaling tobacco smoke produced by smokers). It has also been reported that maternal smoking during pregnancy increases the risk of COPD in infants. As used herein, “smoker” typically refers to an individual who smokes more than 10 packets of cigarettes per year. A “non-smoker” typically refers to a person who smokes fewer than 10 packets of cigarettes per year.

[0086] Smoke that causes obstructive airway disease can also be smoke from combustion. This includes burning petroleum, burning wood (e.g., that used for cooking or wildfires), burning grass, burning coal, burning tires, or burning biomass (such as burning animal manure as fuel for cooking in developing countries).

[0087] In the context of environmental pollutants, relevant aerosols include soot, automobile exhaust (including diesel exhaust), and smog in industrial areas. For example, individuals living in highly industrialized areas or areas affected by severe pollution or air pollution are at higher risk of developing obstructive pulmonary disease and are candidates for treatment / therapy according to the present invention.

[0088] The subjects requiring this are those currently exposed to smoke, those who have been exposed to smoke, or those who will be exposed to smoke (preferably acute exposure to smoke). For example, a subject may currently or in the past smoke cigarettes, currently or in the past be exposed to passive smoking, currently or in the past be exposed to smoke from burning wood or forests, and / or currently or in the past be exposed to gaseous or particulate natural or artificial air pollutants. A subject does not have to be a cigarette smoker, or may have smoked less than one pack a day in the previous year but have been or will be exposed to acute levels of smoke.

[0089] Those skilled in the art will understand that, in the context of the present invention, there are certain individuals who are more susceptible (higher risk) to developing obstructive pulmonary disease associated with aerosol exposure. For example, such susceptible individuals may have underlying conditions such as asthma or other autoimmune diseases. Alternatively, individuals at higher risk of obstructive pulmonary disease may have multiple airway injuries (e.g., prolonged infections or poor health).

[0090] Alternative splicing of RAGE premRNA Alternative splicing is recognized as a crucial layer of post-transcriptional gene regulation of the advanced glycation end product receptor (RAGE). While most RAGE is expressed in its full-length isoform, several different coding isoforms are generated by alternative splicing (also known as splicoforms), including splicoforms that retain N-terminal cleavage, C-terminal cleavage, and intron sequences. These different splicoforms may function as potential regulators of the full-length RAGE receptor by competitive ligand binding or by replacing the full-length protein from its binding partner. More than 20 splicoforms have been identified in different tissues, including the lung, liver, kidney, smooth muscle, endothelial cells, and brain.

[0091] Different RAGE gene splice variants are named RAGE, RAGE_v1~RAGE_v19 according to the Human Gene Nomenclature Committee and are described in Hudson et al., (2008) The FASEB Journal, 22:1572-1580 (the entire content is incorporated therein).

[0092] As used herein, “endogenous soluble RAGE” refers to a polypeptide lacking any signaling element and / or the transmembrane domain of full-length RAGE. Endogenous soluble RAGE is encoded by alternatively spliced ​​RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 (e.g., skipping), which can then be translated, resulting in premature termination and complete loss of the transmembrane and cytoplasmic domains. For example, endogenous soluble RAGE may be encoded by one or more of RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19 mRNA, preferably RAGE_v1. Therefore, it will be understood that any AON described herein that promotes splicing in RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 (e.g., skipping) may be used in the present invention to promote the production of “endogenous soluble RAGE” in the airways and blood of the subject.

[0093] For example, “endogenous soluble RAGE” as described herein may be endogenous secretory RAGE (esRAGE) encoded by RAGE_v1 mRNA. esRAGE constitutes approximately 5% of circulating RAGE in humans. The skipping of exon 10 in esRAGE-type splicing is due to the limitations of intron length in higher eukaryotes. The 5' splice site and branching point must be approximately 45 nucleotides apart, and the minimum distance between the branching point and the 3' splice site appears to be approximately 18 nucleotides, respectively. Therefore, introns shorter than 70 nucleotides are very rare in mammals and cannot be efficiently spliced ​​away. If an esRAGE 5' splice site is selected in intron 9, the distance between this site and the 3' splice site bordering exon 10 is 46 nucleotides, which is considerably shorter than the lower limit of intron length. Therefore, the use of the esRAGE 5' splice site downstream of intron 9 and the inclusion of exon 10 would be mutually exclusive. Among the known splice variants analyzed, all variants using the esRAGE 5' splice site downstream of intron 9 skipped exon 10. In contrast, all variants using the RAGE 5' splice site upstream of intron 9 included exon 10. Therefore, the available evidence indicates that the selection of one of the two alternative 5' splice sites in intron 9 results in the inclusion or exclusion of exon 10. No modulo or external means for modulating this splicing have been previously known.

[0094] Membrane-bound RAGE can be wild-type or full-length RAGE, or a protein resulting from normal splicing. In other words, membrane-bound RAGE contains a transmembrane domain and a short (42-amino acid) cytoplasmic tail. Furthermore, membrane-bound RAGE may be encoded by post-splicing mature RNA that does not contain exon 9b and / or contains exon 10.

[0095] Soluble splice form of the Advanced Glycation End Product Receptor (RAGE) Alternative splicing is recognized as a crucial layer of post-transcriptional gene regulation of the advanced glycation end product receptor (RAGE). While most RAGE is expressed in its full-length isoform, several different coding isoforms are generated by alternative splicing (also known as splicoforms), including splicoforms that retain N-terminal cleavage, C-terminal cleavage, and intron sequences. These different splicoforms may function as potential regulators of the full-length RAGE receptor by competitive ligand binding or by replacing the full-length protein from its binding partner. More than 20 splicoforms have been identified in different tissues, including the lung, liver, kidney, smooth muscle, endothelial cells, and brain.

[0096] Different RAGE gene splice variants are named RAGE, RAGE_v1 to RAGE_v19 according to the Human Gene Nomenclature Committee and are described in Hudson et al., (2008) The FASEB Journal, 22:1572-1580 (the entire content is incorporated therein). For example, retention of intron 9 (exon 9b) (runon) results in early termination, and complete loss of the transmembrane and cytoplasmic domains generates a C-terminally cleaved splice form. For example, RAGE_v1 is a C-terminally cleaved splice form produced by skipping exon 10 and retaining intron 9 (exon 9b). Alternatively, in another example, the AON described herein may increase RAGE_v10 mRNA levels in tissue or specimen by promoting splicing of RAGE premRNA resulting in skipping of exons 10 and 11.

[0097] The C-terminally cleaved splice forms described herein lack any signaling elements or transmembrane domains and translate C-terminally cleaved RAGE, such as endogenous secretory RAGE (esRAGE), which function as decoy receptors and can compete with full-length RAGE for ligands or increase ligand clearance. In particular, esRAGE accounts for approximately 5% of circulating RAGE in humans.

[0098] Elevated circulating levels of esRAGE are associated with improved health outcomes and longevity, while decreased esRAGE are associated with a wide range of disease conditions, including but not limited to atherosclerosis, diabetes, metabolic syndromes, cardiovascular mortality, anemia, autism, and various tumorigenic states. Treatment of diabetic mice with recombinant esRAGE reduces atherosclerosis, vasculitis, and renal and retinal damage.

[0099] Abnormal splicing of RAGE (and therefore dysfunctional RAGE signaling) has been reported in diabetes, certain cancers, and Alzheimer's disease.

[0100] The skipping of exon 10 in esRAGE splicing is due to the limitations of intron length in higher eukaryotes. The 5' splice site and branching point must be approximately 45 nucleotides apart, and the minimum distance between the branching point and the 3' splice site is thought to be approximately 18 nucleotides, respectively. Therefore, introns shorter than 70 nucleotides are very rare in mammals and cannot be efficiently spliced ​​out. If an esRAGE 5' splice site is selected in intron 9, the distance between this site and the 3' splice site bordering exon 10 is 46 nucleotides, which is considerably shorter than the lower limit of intron length. Therefore, the use of an esRAGE 5' splice site downstream of intron 9 and the inclusion of exon 10 are likely mutually exclusive. Among the known splice variants analyzed, all variants using an esRAGE 5' splice site downstream of intron 9 skipped exon 10. In contrast, all variants using the RAGE 5' splice site upstream of intron 9 included exon 10. Therefore, the available evidence indicates that the selection of either one of the two alternative 5' splice sites in intron 9 involves the inclusion or exclusion of exon 10. No modulo or external means for modulating this splicing have been previously known.

[0101] Antisense oligonucleotides (AONs) Antisense oligonucleotides (AONs) are short synthetic antisense modification chains of DNA or RNA that selectively hybridize to preRNA / mRNA via Watson-Crick base pairing and can selectively regulate the function of target RNA.

[0102] The terms "AON" and "ASO" are both abbreviations for the term "antisense oligonucleotide" and are used interchangeably herein.

[0103] Antisense oligonucleotides (AONs) are short synthetic antisense modification chains of DNA or RNA that selectively hybridize to preRNA / mRNA via Watson-Crick base pairing and can selectively regulate the function of target RNA.

[0104] When AONs are used to modulate alternative splicing of mRNA, they are often referred to as splice-switching oligonucleotides (SSOs). In this invention, the terms AON and SSO may be used interchangeably. SSOs disrupt the normal splicing repertoire of the transcript by base-pairing with premRNA and blocking RNA-RNA base pairing or protein-RNA binding interactions that occur between the components of the splicing mechanism and premRNA. SSOs can modulate the translational product by inducing "skipping" of selected exons and / or retention of intron sequences. This can be achieved by directly targeting splice sites, or by targeting cis-acting sequences involved in splicing enhancement or silencing by modulating the binding of specific proteins or altering the secondary structure of premRNA.

[0105] Therapeutic SSOs may be used to treat genetic disorders and, as a therapy, may skip defective or misaligned portions to enable the production of internally deleted but functional proteins.

[0106] For use in the method of the present invention, AON selectively manipulates the alternative splicing pattern of RAGE premRNA, resulting in the generation of either a non-functional or innate RAGE mRNA spliceform that acts as a decoy receptor antagonizing ligand-dependent activation and ligand-independent transactivation of full-length RAGE. Preferably, AON also reduces the production of membrane-bound RAGE.

[0107] Notably, there are no common RAGE polymorphisms in these splice sites. RAGE sequences are highly conserved. Therefore, unlike the management of hereditary disorders using exon skipping techniques, individualized or personalized sequence modifications are not required.

[0108] According to any embodiment of the method of the present invention, the AON described herein can bind to a selected target on the RAGE gene transcript to modulate the splicing of premRNA in the RAGE gene transcript or a portion thereof. Generally, AON can be isolated or purified AON. "Isolated" means material that is substantially or essentially free of components normally associated with it in its natural state. For example, as used herein, "isolated polynucleotide" or "isolated oligonucleotide" may refer to a polynucleotide that has been purified or extracted from a sequence adjacent to it in its naturally occurring state, e.g., a DNA fragment extracted from a sequence adjacent to a fragment in a genome. The term "isolate," when related to cells, refers to the purification of cells (e.g., fibroblasts, lymphoblasts) from a source subject (e.g., a subject with polynucleotide recurrent disease). In the context of DNA, mRNA, or proteins, "isolate" refers to the recovery of DNA, mRNA, or proteins from a source (e.g., cells).

[0109] According to any embodiment of the method of the present invention, the AON described herein promotes splicing in RAGE premRNA, resulting in the inclusion of exon 9b and / or exclusion of exon 10 (e.g., skipping), leading to premature termination and complete loss of the transmembrane and cytoplasmic domains. Accordingly, the AON described herein can increase the levels of RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19 mRNA in tissue or sample, preferably increasing the levels of RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19 mRNA. For example, the AON described herein may increase RAGE_v1 mRNA levels in a tissue or sample by promoting splicing in RAGE premRNA, resulting in the skipping of exon 10 and retention of intron 9 (exon 9b). In another example, the AON described herein may increase RAGE_v10 mRNA levels in a tissue or sample by promoting splicing of RAGE premRNA, resulting in the skipping of exons 10 and 11.

[0110] AON can be said to be “directed to” or “targeted to” the target sequence to hybridize. In certain embodiments, the target sequence includes a region of the pre-treated mRNA containing a 3' or 5' splice site, a branch point, or other sequences involved in the regulation of splicing, including splice enhancers and splice silencers, as well as sites that determine the RNA secondary structure affecting splicing. The target sequence may span within an exon or an intron, or across an intron / exon junction.

[0111] In any embodiment, the antisense oligonucleotide contains, essentially consists of, or comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target region of exon 10 of the RAGE premRNA throughout the antisense oligonucleotide. Preferably, the most 5' nucleotide of the AON is nucleotide position 88 or 114 of exon 10, or is located at nucleotide positions 88-114 of exon 10. Typically, the nucleotide positions may be identical or equivalent to SEQ ID NO: 32, where the first nucleotide is at position 1. [Table 1]

[0112] In certain embodiments, the AON has sufficient sequence complementarity to the target RNA (i.e., the RNA whose splice site selection is regulated) and effectively blocks the region of the target RNA (e.g., premRNA). In exemplary embodiments, such blockade of RAGE premRNA functions to regulate splicing by masking the binding sites of spliceosome proteins that would otherwise regulate splicing and / or by altering the structure of the target RNA. In some embodiments, the target RNA is a target premRNA (e.g., RAGE gene premRNA).

[0113] An AON with sufficient sequence complementarity to the target RNA sequence for regulating target RNA splicing means that the AON has a sequence sufficient to induce masking of the binding site of a native protein that would otherwise regulate splicing and / or alter the three-dimensional structure of the target RNA.

[0114] The selected AON can be made shorter (e.g., about 12 bases) or longer (e.g., about 50 bases), and may contain a small number of mismatches, as long as the sequence is sufficiently complementary to the target sequence to result in splice regulation during hybridization, and optionally forms a heteroduplex with RNA having a Tm of 45°C or higher.

[0115] Preferably, AON is selected from the group including SEQ ID NOs. 1-31 and 33 and / or from any of the sequences shown in Tables 1a-1d. More preferably, AON is SEQ ID NOs. 11, 18, 19, or 20.

[0116] In certain embodiments, the degree of complementarity between the target sequence and the AON is sufficient to form a stable double helix. The complementary region of the AON with the target RNA sequence may be as short as 8–11 nucleotides, but can be 12–15 nucleotides or more, for example, 10–50 nucleotides, 10–40 nucleotides, 12–30 nucleotides, 12–25 nucleotides, 15–25 nucleotides, 12–20 nucleotides, or 15–20 nucleotides (including all integers between these ranges). An AON of about 16–17 nucleotides is generally long enough to have a unique complementary sequence. In certain embodiments, as discussed herein, a minimum length of complementary nucleotides may be required to achieve the desired binding Tm.

[0117] In certain embodiments, oligonucleotides up to 50 bases in length may be preferred, with at least a minimum number of bases (e.g., 10–12 bases) being complementary to the target sequence. However, generally, enhanced or active uptake in cells is optimized for oligonucleotide lengths of less than approximately 30 bases. In the case of phosphorodiamidate morpholino oligomers (PMOs) AONs further described herein, the optimal balance between binding stability and uptake is generally obtained at lengths of 18–25 bases. It contains AONs consisting of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 bases (e.g., PMO, PMO-X, PNA, LNA, TINA, 2'-OMe).

[0118] In certain embodiments, the AON may be 100% complementary to the target sequence, or it may contain mismatches to accommodate variants, for example, as long as the heteroduplex formed between the oligonucleotide and the target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation that may occur in vivo. Thus, a particular oligonucleotide may have about or at least about 70% sequence complementarity between the oligonucleotide and the target sequence, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity.

[0119] Mismatches, when present, typically cause less destabilization towards the terminal region of the hybrid double helix than towards the central region. The acceptable number of mismatches will depend on the length of the oligonucleotide, the G:C base pair ratio in the double helix, and the location of the mismatch(s) in the double helix, according to well-understood principles of double helix stability. Such AONs do not necessarily need to be 100% complementary to the target sequence, but are effective in binding stably and specifically to the target sequence so that splicing of the target preRNA is regulated.

[0120] The stability of the double helix formed between the AON and the target sequence depends on the binding Tm and the sensitivity of the double helix to cellular enzymatic cleavage. The Tm of the oligonucleotide to the complementary sequence RNA can be measured by conventional methods, such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or as described in Miyada CG and Wallace RB, 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107. In certain embodiments, the AON may have a binding Tm with respect to the complementary sequence RNA that is higher than body temperature, preferably higher than about 45°C or 50°C. Tms in the range of 60-80°C or higher are also included.

[0121] Further examples of variants include AONs having approximately or at least approximately 70% sequence identity or homology over the entire length of any of sequence numbers 1-31 and 33 and / or any of the sequences shown in Tables 1a-1d, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or homology. More preferably, the AON is sequence number 11, 18, 19, or 20.

[0122] Pre-mRNA splicing modifications preferably induce "skipping," or the removal of one or more exons, or the retention of introns in the mRNA. The resulting protein is preferably shorter in length than the parent full-length RAGE protein, either due to internal cleavage or premature termination. Preferably, the resulting protein has a C-terminal cleavage. These cleaved RAGE proteins may be called splice forms of the full-length RAGE protein.

[0123] The remaining exons of the generated mRNA may be in-frame, producing a shorter protein with a sequence similar to that of the parent full-length protein, except that it has an internal cleavage in the region between the original 3' and 5' ends. Alternatively, exon skipping may induce a frameshift, resulting in a protein where the first portion is substantially identical to the parent full-length protein, but the second portion has a different sequence (e.g., a nonsense sequence) due to the frameshift. Alternatively, exon skipping may induce the production of prematurely terminated proteins due to the disruption of the reading frame and the presence of premature termination of translation. Prematurely terminated proteins may be the result of prematurely terminated mRNA (e.g., skipping of exons 10 and / or 11), or the result of run-on to an intron (e.g., RAGE9b), or may be missense skipping, providing mRNA containing exon 10 and / or 11 mRNA but not providing expression of the protein encoded by these exons.

[0124] Skipping individual exons from exons 1-9 is preferable because it disrupts the reading frame of the RAGE transcript. This will result in increased RNA degradation via nonsense-mediated degradation.

[0125] Skipping individual exons 1-11 is preferable to keep the reading frame intact. This would preferably result in translation to an internally cleaved protein. The cleaved protein or RAGE mRNA splice form may be completely deficient in function, have reduced function, or act as a decoy receptor.

[0126] Preferably, these cleaved, nonsense, or prematurely terminated proteins lack one or more functional domains involved in the induction of intracellular signaling pathways by RAGE ligands or non-ligand-dependent transactivation of RAGE by co-localized GPCRs. For example, exon 10 encodes a transmembrane domain, and removal of this exon may generate a soluble RAGE protein, which may act as a soluble decoy or competitive antagonist for RAGE-mediated ligand-induced signaling. Cleaved, nonsense, or prematurely terminated proteins may further lack attachment or binding sites to other factors, and their removal may lead to reduced interaction between the RAGE protein and associated signaling pathways.

[0127] Alternatively, the removal of one or more exons may lead to misfolding of the RAGE protein and a decrease in the protein's ability to be transported correctly across the membrane.

[0128] The presence of internally cleaved proteins (i.e., proteins lacking amino acids encoded by one or more exons) is preferable. When RAGE proteins are inhibited, the body attempts to compensate for the decrease in the total amount of RAGE proteins, which can lead to an increase in RAGE transcription. In contrast, the presence of internally cleaved proteins (preferably lacking one or more of the features of the complete RAGE protein) is sufficient to prevent the increase in transcription, but also provides therapeutic benefits due to the reduction in the total amount of functional RAGE proteins.

[0129] Exon skipping induced by AON as described herein does not need to completely or even substantially eliminate the function of the RAGE protein. Preferably, the modulation of alternative splicing via the exon skipping process results in a decrease or deficiency of the RAGE protein's functionality.

[0130] Different isoforms of RAGE produced using different skipping strategies may result in proteins with lost or reduced signaling activity. These can preferably be used to treat or prevent different diseases associated with RAGE activity, such as neurodegenerative diseases, cancer, lung disorders, or inflammatory diseases. Alternative splicing strategies may produce cleaved or deactivated proteins. These can preferably be used to treat specific aspects, forms, or progressions of diseases associated with RAGE expression and activity.

[0131] A skipping process using AON can either skip individual exons or result in the skipping of two or more exons at once.

[0132] Skipping processes using AONs may involve preservation of intron sequences, with or without direct skipping of one or more exons.

[0133] The AONs for use in the method of the present invention may be a combination of two or more AONs capable of binding to a selected target and inducing exon exclusion in the RAGE gene transcript. This combination may be a cocktail of two or more AONs and / or a construct containing two or more AONs bound together. [Table 2] [Table 3] [Table 4] [Table 5]

[0134] More specifically, the AON for use in the method of the present invention may be selected from those shown in any of Tables 1a to 1d. The sequence is preferably selected from the group consisting of one or more of SEQ ID NOs: 1 to 31 and 33, and combinations or cocktails thereof. More preferably, the AON is SEQ ID NOs: 11, 18, 19, or 20. The combination of AONs is preferably SEQ ID NOs: 11 and 10, or a combination of SEQ ID NOs: 11 and 13. This includes sequences that can hybridize to such sequences under harsh hybridization conditions, complementary sequences, sequences containing modified bases, modified backbones, and functional cleavage or extensions that have or regulate premRNA processing activity in the RAGE gene transcript.

[0135] In the context of AONs for use in the methods of the present invention, those skilled in the art will recognize that both U and T residues can bind to A, and therefore are interchangeable in any of the AON sequences disclosed herein. Accordingly, in any AON sequence disclosed herein, any T may be U, and / or any U may be T. These U-to-T substitutions or T-to-U substitutions generate equally potent AONs for use in the methods of the present invention.

[0136] Oligomers and DNA, cDNA, or RNA are complementary if a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen-bond to one another. Therefore, “specifically hybridizable” and “complementary” are terms used to indicate a sufficient degree of complementarity or pairing between the oligomer and the DNA, cDNA, or RNA target, resulting in stable and specific binding. In the art, it should be understood that the sequence of an AON does not need to be 100% complementary to its target sequence in order to be specifically hybridizable. An AON is specifically hybridizable if the binding of the compound to the target DNA or RNA molecule would interfere with the normal function of the target DNA or RNA product, and is complementary enough to avoid nonspecific binding of the AON to non-target sequences under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays.

[0137] Selective hybridization may occur under low, moderate, or highly severe conditions, but is preferably under highly severe conditions. Those skilled in the art will recognize that the severity of hybridization is influenced by conditions such as salt concentration, temperature, or organic solvent, in addition to the base composition, complementary chain length, and the number of nucleotide base mismatches between the nucleic acids to hybridize. Severe temperature conditions generally include temperatures above 30°C, typically above 37°C, preferably above 45°C, preferably at least 50°C, typically 60°C to 80°C or higher. Severe salt conditions are usually below 1000 mM, typically below 500 mM, preferably below 200 mM. However, the combination of parameters is far more important than the measurement of any single parameter. An example of severe hybridization conditions is 65°C and 0.1 × SSC (1 × SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Therefore, the AON used in the method of the present invention may include oligomers that selectively hybridize to any of the sequences provided in Tables 1a to 1d or SEQ ID NOs: 1 to 31 and 33. More preferably, the AON is SEQ ID NOs: 11, 18, 19, or 20.

[0138] It should be understood that the codon sequences at the ends of exons in structural proteins are not necessarily cleaved at the codon ends, and therefore, it may be necessary to remove multiple exons from premRNA to ensure in-frame reading of mRNA. In such situations, it may be necessary to select multiple AONs by the method of the present invention, each directed to a different region that plays a role in inducing the inclusion of the desired exon and / or intron. Given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to complementary polynucleotides. Such hybridization can occur with "near" or "substantial" complementarity of the AON to the target sequence, as well as with exact complementarity.

[0139] Typically, selective hybridization will occur when there is at least about 55% identity, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%, 95%, 98%, or 99% identity between the AON nucleotides and a stretch of at least about 14 nucleotides. The length of the homology comparison may extend over longer stretches as described, and in certain embodiments, it will often extend over stretches of at least about 9 nucleotides, usually at least about 12 nucleotides, more usually at least about 20 nucleotides, often at least about 21, 22, 23, or 24 nucleotides, at least about 25, 26, 27, or 28 nucleotides, at least about 29, 30, 31, or 32 nucleotides, and at least about 36 or more nucleotides.

[0140] Therefore, AONs for use in the sequencing method of the present invention preferably have at least 75%, more preferably at least 85%, more preferably at least 86%, 87%, 88%, 89%, or 90% homology with the sequences shown in the sequence listings herein. More preferably, they have at least 91%, 92%, 93%, 94%, or 95%, more preferably at least 96%, 97%, 98%, or 99% homology. In general, the shorter the length of the AON, the greater the homology required to obtain selective hybridization. Therefore, if the AON consists of fewer than about 30 nucleotides, the percentage identity is preferably greater than 75%, preferably greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to the AONs shown in the sequence listings herein. Nucleotide homology comparisons may be performed using a sequence comparison program such as the GCG Wisconsin Bestfit program or GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of similar or substantially different lengths as those cited herein can be compared by inserting gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0141] The AON used in the method of the present invention may have regions with reduced homology to the target sequence and regions with exact homology to the target sequence. The oligomer does not need to have exact homology over its entire length. For example, the oligomer may have a stretch of at least 4 or 5 bases identical to the target sequence, preferably a stretch of at least 6 or 7 bases identical to the target sequence, and more preferably a stretch of at least 8 or 9 bases identical to the target sequence. The oligomer may have a stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 bases identical to the target sequence. The remaining stretch of the oligomer sequence may be intermittently identical to the target sequence; for example, the remaining sequence may have identical bases followed by non-identical bases followed by identical bases. Alternatively (or similarly), the oligomeric sequence may have several stretches of the same sequence (e.g., 3, 4, 5, or 6 bases) interspersed with stretches of less than complete homology. Such sequence mismatches preferably result in little to no loss of splice-switching activity.

[0142] The terms “modulate” or “modulates” include, at the discretion of any choice, “increasing” or “decreasing” one or more quantifiable parameters by a defined amount and / or a statistically significant amount. The terms “increase” or “increasing,” “enhance” or “enhancing,” or “stimulate” or “stimulating” generally refer to the ability of one or more AONs or compositions to produce or induce a greater physiological response (i.e., downstream effect) in a cell or subject in response to a response induced by either the AON or the control compound. The terms “decreasing” or “decrease” generally refer to the ability of one or more AONs or compositions to produce or induce a reduced physiological response (i.e., downstream effect) in a cell or subject in response to a response induced by either the AON or the control compound.

[0143] The associated physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include increased exclusion of specific exons in the premRNA encoding RAGE, decreased amount of premRNA encoding RAGE, or decreased expression of the functional RAGE protein in cells, tissues, or subjects that require it. The “increased” or “enhanced” amount is typically a statistically significant amount and may include increases of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 times or more (e.g., 500, 1000 times) (including all integers greater than or equal to 1 and decimals between them, e.g., 1.5, 1.6, 1.7, 1.8 times). The terms “reduce” or “inhibit” may generally relate to the ability of one or more AONs or compositions to “reduce” relevant physiological or cellular responses, such as symptoms of the diseases or conditions described herein, as measured according to the usual techniques of the diagnostic field. The relevant physiological or cellular responses (in vivo or in vitro) will be obvious to those skilled in the art and may include a reduction in symptoms or pathologies of diseases such as cancer, neurodegenerative diseases, lung diseases, and other inflammatory diseases. A “decrease” in response may be statistically significant compared to the response not produced by AON or the control composition, and may include decreases of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers in between).

[0144] The length of the AON can vary as long as it can selectively bind to the intended position within the premRNA molecule. The length of such a sequence can be determined by the selection procedure described herein. Generally, the AON will be about 10 nucleotides to a maximum of about 50 nucleotides in length. However, it will be understood that nucleotides of any length within this range may be used in the present method. Preferably, the length of the AON is 10-40, 10-35, 15-30 nucleotides, or 20-30 nucleotides, and most preferably about 25-30 nucleotides. For example, the oligomer may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0145] As used herein, “AON” refers to a linear sequence of nucleotides or nucleotide analogs that enables nucleic acid bases to hybridize to a target sequence in RNA by Watson-Crick base pairing, thereby forming an oligonucleotide:RNA heteroduplex within the target sequence. The terms “AON,” “AON,” “oligomer,” and “antisense compound” may be used interchangeably to refer to oligonucleotides. The cyclic subunit may be based on ribose or another pentose sugar, or, in certain embodiments, a morpholino group (see the description of morpholino oligonucleotides below). Also intended are peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and 2'-O-methyl oligonucleotides, 2'-O-methoxyethyl oligonucleotides, as well as other antisense agents known in the art.

[0146] The materials include AONs or "oligonucleotide analogs" that do not exist in nature, which include (i) modified skeletal structures, e.g., skeletal structures other than the standard phosphodiester bonds found in naturally occurring oligo and polynucleotides, and / or (ii) modified sugar moieties, e.g., AONs or oligonucleotides having a morpholino moiety rather than a ribose or deoxyribose moiety. Oligonucleotide analogs support bases that can hydrogen bond to standard polynucleotide bases by Watson-Crick base pairing, and the analog skeleton presents the bases in such a sequence-specific manner that such hydrogen bonding between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA) is possible. Preferred analogs are those having a substantially uncharged phosphorus-containing skeleton.

[0147] One method for generating AON is methylation of the 2' hydroxyribose position, and the incorporation of a phosphorothioate backbone produces a molecule that is superficially similar to RNA but far more resistant to nuclease degradation; however, those skilled in the art of the present invention will recognize other suitable forms of backbone that may be usable for the purposes of the present invention.

[0148] Increased splice switching can also be achieved with alternative oligonucleotide chemistry. For example, AONs include phosphoramides or phosphorodiamidate morpholino oligomers (PMOs), PMO-X, PPMOs, peptide nucleic acids (PNAs), locked nucleic acids (LNAs) and derivatives (including alpha-L-LNA, 2'-amino-LNA, 4'-methyl-LNA, and 4'-O-methyl-LNA), ethylene-bridged nucleic acids (ENAs) and their derivatives, phosphorothioate oligomers, tricycloDNA oligomers (tcDNAs), tricyclophosphorothioate oligomers, 2'- The following can be selected from the list, including O-methyl modified oligomers (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-fluoro,2'-fluoroarabino (FANA), unlocked nucleic acids (UNA), heat-stable twist-intercalating nucleic acids (TINA), hexitol nucleic acids (HNA), cyclohexenyl nucleic acids (CeNA), 2'-amino (2'-NH2), 2'-O-ethyleneamine, or any combination of the above as mixmers or gapmers. To further improve delivery efficacy, the above-mentioned modified nucleotides are often conjugated with fatty acids / lipids / cholesterol / amino acids / carbohydrates / polysaccharides / nanoparticles, etc., to the sugar or nucleic acid base portion. These conjugated nucleotide derivatives can also be used to construct exon-skipping AONs. Antisense oligonucleotide-induced splice modifications of human RAGE gene transcripts generally use one of the following on a phosphorothioate backbone: oligoribonucleotide, PNA, 2OMe, or MOE-modified base. When alternative chemistry is used to produce AON for use in the method of the present invention, uracil (U) in the sequences provided herein may be replaced with thymine (T).

[0149] AONs for use in the methods of the present invention include non-naturally occurring oligomers or "oligonucleotide analogs," which include oligomers having (i) a modified skeletal structure, e.g., a skeletal structure other than the standard phosphodiester bond found in naturally occurring oligos and polynucleotides, and / or (ii) a modified sugar moiety (e.g., a morpholino moiety rather than a ribose or deoxyribose moiety). The oligomer analogs support bases that can be hydrogen-bonded to standard polynucleotide bases by Watson-Crick base pairing, and the analog skeleton presents the bases in such a sequence-specific manner that such hydrogen bonding between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Preferred analogs are those having a substantially uncharged phosphorus-containing skeleton.

[0150] Antisense oligonucleotides that do not activate RNase H can be prepared according to known techniques (see, for example, U.S. Patent No. 5,149,797). Such AONs may be deoxyribonucleotides or ribonucleotide sequences and merely include any structural modifications that sterically interfere with or prevent the binding of RNase H to a double-chain molecule containing an oligomer as one of its members, such structural modifications do not substantially interfere with or disrupt double-chain formation. Since the oligomeric portion involved in double-chain formation is substantially different from the portion involved in the binding of RNase H, a number of RNase H-nonactivating AONs are available. For example, such AONs may be oligomers in which at least one or all of the internucleotide crosslinking phosphate residues are modified phosphates, such as methylphosphonate, methylphosphorothioate, phosphoromolholide, phosphoropiperadate, boranophosphate, amide bond, and phosphoramidate. For example, all other internucleotide crosslinking phosphate residues may be modified as described. In another non-limiting example, such an AON is a molecule in which at least one or all of the nucleotides contain a 2' lower alkyl moiety (e.g., C1-C4 linear or branched, saturated or unsaturated alkyl such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, all other nucleotides may be modified as described.

[0151] Preferred specific examples of AONs for use in the methods of the present invention include oligomers containing a modified skeleton or unnatural internucleoside bonds. As defined herein, oligomers having a modified skeleton include oligomers that retain a phosphorus atom in the skeleton and oligomers that do not have a phosphorus atom in the skeleton. For the purposes of this specification and as sometimes referred to in the art, modified oligomers that do not have a phosphorus atom in their internucleoside skeletons can also be considered AONs.

[0152] In other preferred oligomeric mimics, both the sugar and nucleoside bonds (i.e., the backbone) of the nucleotide unit are replaced with novel groups. The base unit is maintained for hybridization with a suitable nucleic acid target compound. One such oligomeric compound, an oligomeric mimic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligomer is replaced with an amide containing a backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bonded directly or indirectly to the aza nitrogen atom of the amide portion of the backbone.

[0153] Another preferred chemistry is phosphorodiamidate morpholino oligomer (PMO) compounds that are not degraded by known nucleases or proteases. These compounds are uncharged and, when bound to RNA chains, do not activate RNase H activity and have been shown to exert sustained splice regulation after in vivo administration (Summerton and Weller, Antisense Nucleic Acid Drug Development, 7, 187-197).

[0154] Modified oligomers may also contain one or more substituted sugar moieties. Oligomers may also contain modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). Certain nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds described herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, and 5-methylcytosine substitutions). These have been shown to increase the stability of nucleic acid double helix by 0.6–1.2°C, and stability is further increased, especially when combined with 2'-O-methoxyethyl sugar modification.

[0155] The activity of AONs and their variants can be assayed according to the usual art of the field. For example, the splice morphology and expression levels of the RNA and protein investigated can be evaluated by any of the various well-known methods for detecting splice morphology and / or the expression of transcribed nucleic acids or proteins. Non-limiting examples of such methods include RT-PCR of the spliced ​​morphology of RNA followed by size separation of the PCR product, nucleic acid hybridization methods (e.g., Northern blotting) and / or the use of nucleic acid arrays, nucleic acid amplification methods, immunological methods for protein detection, protein purification methods, and protein function or activity assays.

[0156] RNA expression levels can be assessed by preparing mRNA / cDNA (i.e., transcribed polynucleotides) from cells, tissues, or organisms, and by hybridizing the mRNA / cDNA with a reference polynucleotide or fragment thereof that is a complement to the nucleic acid being assayed. The cDNA may optionally be amplified using any of the various polymerase chain reaction or in vitro transcription methods before hybridization with the complementary polynucleotide, but preferably it is not amplified. The expression of one or more transcripts can also be detected using quantitative PCR to assess the level of transcript expression.

[0157] AON for use in the method of the present invention provides induction of splice switching of RAGE gene transcripts, clinically relevant oligomeric chemistry, and a delivery system, thereby bringing RAGE splice manipulation to a therapeutic level and achieving enhancement of RAGE mRNA splice coforms (e.g., RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19 mRNA), which are non-signaling decoy receptors, and thus can be used to treat RAGE-related respiratory (lung) diseases or disorders.

[0158] AONs for use in the method of the present invention can be readily prepared by well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several vendors, including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesizing oligomers on a modified solid support is described in U.S. Patent No. 4,458,066.

[0159] Any other means known in the art for such synthesis may be used additionally or alternatively. It is well known to use similar techniques to prepare oligomers such as phosphorothioates and alkylated derivatives. In one such automated embodiment, diethyl phosphoramidite may be used as a starting material and synthesized as described in Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

[0160] The AON used in the method of the present invention is synthesized in vitro and does not include antisense compositions of biological origin or gene vector constructs designed to direct the in vivo synthesis of AON.

[0161] Administration, dosage, and formulation In any embodiment, the method of the present invention may be used to prevent or treat diseases associated with or caused by exposure to smoke. Preferably, diseases associated with or caused by acute smoke exposure.

[0162] The term "respiratory system" refers to the process by which oxygen is taken into the body and carbon dioxide is expelled through a bodily system that includes the nose, throat, larynx, trachea, bronchi, and lungs.

[0163] As used herein, the upper respiratory tract may include the following areas: the nose and nasal cavity, the sinuses, the pharynx, and the portion of the larynx above the vocal cords. Typically, the lower respiratory tract includes one or more of the following areas: the portion of the larynx below the vocal cords, the trachea, the bronchi, and the bronchioles. The lungs may be included in the lower respiratory tract and include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0164] The term “respiratory disease” or “respiratory condition” refers to one of several diseases that involve inflammation and affect the components of the respiratory system, including the upper respiratory tract (including the nasal cavity, pharynx, and larynx) and the lower respiratory tract (including the trachea, bronchi, and lungs).

[0165] Symptoms of respiratory illness may include cough, excessive sputum production, shortness of breath, or chest tightness accompanied by audible wheezing. Exercise capacity may be severely limited. In asthma, FEV1.0 (forced expiratory volume in one second) as a percentage predicted by a nomograph based on weight, height, and age may be reduced, and maximal expiratory flow during forced exhalation may also be reduced. In COPD, FEV1.0 as a percentage of FVC typically falls to less than 0.7. The impact of each of these conditions can be measured by the number of days absent from work / school, sleep disturbances, need for bronchodilators, and need for glucocorticoids, including oral glucocorticoids.

[0166] The presence, improvement, treatment, or prevention of respiratory disease may be determined by any clinically or biochemically relevant method of biopsy of or from the subject. For example, parameters measured may include the presence or degree of lung function, signs and symptoms of obstruction, exercise tolerance, nocturnal awakenings, days absent from school or work, use of bronchodilators, inhaled corticosteroid (ICS) dose, use of oral (glucocorticoid) GC, need for other medications, need for medical treatment, and hospitalization.

[0167] As used herein, “prevention” or “prevention” is intended to mean reducing the likelihood of at least one risk (or susceptibility) to developing a disease or disorder (i.e., preventing at least one clinical symptom of a disease in patients who may be exposed to the disease or are predisposed to the disease but have not yet experienced or presented with symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are well known to physicians.

[0168] The terms “treatment” or “to treat” include the application or administration of any AON described herein, aimed at delaying, slowing, stabilizing, curing, restoring, reducing, alleviating, altering, correcting, preventing exacerbation, improving, enhancing, or modulating a disease or condition, symptoms of a disease or condition, or the risk (or susceptibility) to a disease or condition. The term “to treat” means any sign of success in treating or improving a disease, pathology, or condition, including objective or subjective parameters such as reduction, remission, slowing the rate of exacerbation, reducing the severity of the disease, stabilizing, decreasing symptoms, or making the injury, pathology, or condition acceptable to the subject, slowing the rate of degeneration or decline, making the final stage of degeneration less debilitating, or improving the subject’s physical or mental health.

[0169] The terms “improve” or “reform” refer to a reduction, mitigation, or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom. Subjects requiring treatment may already have the condition, be prone to developing the condition, or should be prevented from developing the condition.

[0170] The AONs described herein may be adapted in a form suitable for delivery to patients to assist in the prophylactic or therapeutic treatment, prevention, or improvement of symptoms of diseases such as RAGE-related disorders or pathologies.

[0171] A positive response to therapy may also indicate prevention or reduction of exacerbation of respiratory symptoms (e.g., asthma symptoms) after smoke exposure. This can be assessed by comparing the mean change in disease scores from baseline to the end of the study period based on the Juniper Asthma Management Questionnaire (ACQ-6), and by assessing lower respiratory tract symptom scores (LRSS - symptoms of chest tightness, wheezing, shortness of breath, and cough). Changes from baseline lung function (maximum expiratory flow (PEF)) can also be assessed, and a positive response to therapy may indicate a significant reduction in the decline of PEF. For example, the placebo group may show a significant 15% decrease in morning PEF from baseline at peak, while the treatment group may show a non-significant decrease of less than 15% in PEF.

[0172] The method of the present invention can be applied to humans, but it is also useful for therapeutic veterinary purposes. The present invention is useful for livestock or farm animals such as cattle, sheep, horses, and poultry, companion animals such as cats and dogs, and zoo animals.

[0173] In certain embodiments, the AONs described herein can be delivered via the pulmonary or nasal route (e.g., via aerosolized saline containing the AON). The highest endogenous expression of RAGE mRNA in healthy human tissue is found in the lungs, which are accessible via the airways. Inhaled oligonucleotides represent a novel mode of treatment for respiratory diseases. The airways are intrinsically lined with pulmonary surfactant, which is composed primarily of amphoteric lipids. These surfactant lipids have cationic properties at the pH of the airways. It is hypothesized that when anionic oligonucleotides are inhaled, they are readily adsorbed by the surfactant, resulting in particle reconstitution, which is then efficiently taken up by bronchial and alveolar epithelial cells into lung cells. Notably, AONs have been shown to withstand the aerosolization process.

[0174] The AONs described herein may be compositions formulated for administration to the lower respiratory tract only. This limitation to the lower respiratory tract can be achieved by the amount (in particular, volume) of the composition that would otherwise be administered to the upper respiratory tract, and by the form of the composition, i.e., particle size, physical form (whether dry powder or solution droplets). Alternatively, the AONs described herein may be administered via a device that ensures retention only in the lower respiratory tract.

[0175] The AONs described herein may be formulated for intranasal administration, including as dry powders, sprays, mists, or aerosols. This may be particularly preferable for the treatment of respiratory infections.

[0176] A preferred formulation, for example, includes an aqueous or oily solution of the active ingredient when the carrier is liquid for administration as a nasal spray or nasal drop. Alternatively, the AONs described herein are provided as a dry powder and may be administered to the upper respiratory tract only as defined herein.

[0177] The selection of an appropriate carrier depends on the specific type of administration intended. For administration via the upper respiratory tract, e.g., the nasal mucosa surface, the compound may be formulated as a solution, such as water, or buffered or unbuffered isotonic saline, or as a suspension, for intranasal administration as droplets or sprays. Preferably, such a solution or suspension is isotonic with respect to nasal secretions and has approximately the same pH (e.g., in the range of about pH 4.0 to about pH 7.4 or pH 6.0 to pH 7.0). The buffer must be physiologically compatible and includes, as mere examples, phosphate buffer. For example, a typical nasal congestion reliever buffered to about pH 6.2 is described (Remington's, ibid., p. 1445). Of course, those skilled in the art can easily determine suitable saline content and pH of a harmless aqueous carrier for nasal and / or upper respiratory tract administration.

[0178] Other components, including preservatives, colorants, lubricating or viscous mineral or vegetable oils, natural or synthetic plant extracts such as fragrances and aromatic oils, and wetting agents and viscosity enhancers such as glycerol, known in the art, can be used to provide the formulation with additional viscosity, moisture retention, and a pleasant texture and scent. For nasal administration of solutions or suspensions by the method of the present invention, various devices for generating drops, droplets, and sprays are available in the art. For example, the AON described herein can be administered intranasally by a simple dropper (or pipette) containing a glass, plastic, or metal dispensing tube. The contents of the dropper (or pipette) are dispensed drop by drop by air pressure provided by a manual pump (e.g., a flexible rubber valve) attached to one end.

[0179] Pharmaceutical compositions may be formulated as inhalation formulations including sprays, mists, or aerosols. This may be particularly preferred for the treatment of airway or lung conditions involving smoke exposure as described herein. Inhalation formulations may be intended for application to the upper respiratory tract (including the nasal cavity, pharynx, and larynx) and the lower respiratory tract (including the trachea, bronchi, and lungs). For inhalation formulations, the compositions or combinations provided herein may be delivered via any inhalation method known to those skilled in the art. Such inhalation methods and devices include, but are not limited to, metered-dose inhalers containing propellants such as HFA or physiologically and environmentally acceptable propellants. Other suitable devices include respiratory inhalers, high-dose dry powder inhalers, and aerosol nebulizers. Aerosol formulations for use in the subject matter typically contain a propellant, a surfactant, and a co-solvent and may be filled into conventional aerosol containers closed by a suitable metering valve. Depending on whether it is applied to the upper respiratory tract (including the nasal cavity, pharynx, and larynx) or the lower respiratory tract (including the trachea, bronchi, and lungs), different devices and excipients may be used and can be determined by those skilled in the art. Furthermore, the processes for micronization and nanoparticle formation for the preparation of AONs described herein for use in inhalers such as dry powder inhalers are also known to those skilled in the art.

[0180] The inhalation composition may include a liquid or powder composition containing an active ingredient suitable for spray and intrabronchial use, or an aerosol composition administered via an aerosol unit dispensing metered doses. A suitable liquid composition contains the active ingredient in an aqueous, pharmaceutically acceptable inhalation solvent, such as isotonic saline or bacteriostatic water. The solution is administered by a pump or squeeze-operated spray dispenser, or by any other conventional means to cause the required dose of the liquid composition to be inhaled into the patient's lungs or to enable inhalation. Suitable formulations include, for example, an aqueous or oily solution of the active ingredient when the carrier is liquid for administration as a nasal spray or nasal drop. Examples of inhalation drug delivery devices are described in Ibrahim et al. Medical Devices: Evidence and Research 2015:8 131-139, which are intended for use in the present invention.

[0181] The delivery of therapeutically useful amounts of AON can be achieved by previously published methods. For example, intracellular delivery of AON may be via a composition comprising a mixture of AON and an effective amount of block copolymer. An example of this method is described in U.S. Patent Application No. 2004 / 0248833. Other methods for delivering AON to the nucleus are described in Mann CJ et al. (2001) Proc, Natl. Acad. Science, 98(1) 42-47 and Gebski et al. (2003) Human Molecular Genetics, 12(15): 1801-1811. A method for introducing nucleic acid molecules into cells by either an expression vector as naked DNA or an expression vector complexed to a lipid carrier is described in U.S. 6,806,084.

[0182] The AON for use in the methods and uses of the present invention is administered in an effective dose. The terms “therapeutic effective dose” or “effective dose” generally refer to the amount of AON, pharmaceutically acceptable salts, polymorphs, or prodrugs described herein that (i) treat a particular disease, condition, or disorder; (ii) reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. Undesirable effects, such as side effects, may occur along with the desired therapeutic effect. Therefore, practitioners should balance the potential benefits with the potential risks when determining what the appropriate “effective dose” is.

[0183] The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, as well as the method of administration. Therefore, it may not be possible to specify an exact "effective dose." However, the appropriate "effective dose" in any individual case can be determined by those skilled in the art using only standard experiments. In one embodiment, the dose administered to the subject is any dose that reduces the viral load.

[0184] The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically acceptable and, typically, do not cause allergic reactions or similar adverse reactions (e.g., nausea) when administered to a patient. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle on which a compound is administered. Such pharmaceutically acceptable carriers can be sterile liquids such as water and oils (including petroleum, animal, plant, or synthetic sources such as peanut oil, soybean oil, mineral oil, and sesame oil). Water or saline solutions, as well as aqueous dextrose and glycerol solutions, are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutically acceptable carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA, (1990).

[0185] Treatment may be monitored, for example, by common indicators of diseases known in the art. The efficacy of in vivo administered AON for use in the method of the present invention may be determined from biological samples (tissue, blood, urine, etc.) taken from the subject before, during, and after administration of AON. Assays of such samples include (1) monitoring for the presence or absence of heteroduplex formation with target and non-target sequences using procedures known to those skilled in the art, e.g., electrophoretic gel mobility assays, and (2) monitoring the amount of mutant mRNA relative to reference normal mRNA or protein, as determined by standard techniques such as RT-PCR, Northern blotting, ELISA, or Western blotting.

[0186] The method of the present invention also extends to combinations of two or more AONs that can bind to a selected target and induce exon exclusion in the RAGE gene transcript. This combination may be two or more AONs, two or more AONs linked together for use in AON-based therapies, or a cocktail of constructs containing two or more AONs. The combination of AONs is preferably the combination of SEQ ID NOs 11 and 10, or SEQ ID NOs 11 and 13.

[0187] Also provided are kits for use in the methods of the present invention, particularly for treating, preventing, or improving diseases or conditions associated with the manifestation of RAGE in patients. The kits include at least the AONs described herein, along with instructions for use, the instructions for use, and the methods of the present invention.

[0188] The contents of the kit can be freeze-dried, and the kit may further contain a solvent suitable for reconstituting the freeze-dried components. Each component of the kit is packaged in a separate container, and in connection with such container, may be a notice in the form prescribed by the government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, the notice reflecting the approval by the agency for manufacture, use, or sale for human administration.

[0189] As used herein, the terms “derived from” and “derived from” shall be interpreted as indicating that a particular element may be obtained from a particular source, but not necessarily directly from that source.

[0190] As used herein, the singular forms "a," "an," and "the" also include plural references unless the context clearly indicates otherwise.

[0191] Unless otherwise indicated in the operating examples or elsewhere, all numbers used herein and in the claims to represent quantities of components, reaction conditions, etc., should be understood in all cases to be modified by the term “approximately.” Therefore, unless otherwise indicated, the numerical parameters shown herein and in the claims are approximations that may vary depending on the desired properties to be obtained by the method of the present invention. Thus, “approximately 80%” means “approximately 80%” and also “80%.” At a minimum, each numerical parameter should be interpreted in light of significant figures and the usual rounding approach.

[0192] Although the numerical ranges and parameters representing the broad scope of the present invention are approximations, the numerical values ​​shown in the specific examples are reported as accurately as possible. However, any numerical value will inevitably contain certain errors arising from the standard deviation found in each test measurement.

[0193] Other definitions of the selected terms used herein are found in the detailed description of the invention and may apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains.

[0194] It will be understood that the present invention, as disclosed and defined herein, encompasses all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

[0195] It will be understood that the present invention, as disclosed and defined herein, encompasses all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention. [Examples]

[0196] Example 1 - Study design of a mouse model of cigarette smoke-induced inflammation the purpose To determine the anti-inflammatory activity of a single intratracheal dose of fully modified antisense oligonucleotides in mice exposed to cigarette smoke for 4 days.

[0197] Protocol Overview Three solutions were provided, labeled as physiological saline, ASO6713 (3 mg / kg), and ASO6713 (10 mg / kg) (SEQ ID NO: 33). Upon receipt, the materials were immediately stored at -20°C in a temperature-controlled freezer until needed. The solutions were thawed 30 minutes before use and used at room temperature.

[0198] Before the start of the experiment, mice were trained for one week. After training, mice were anesthetized using a ketamine / xylazine mixture (90 mg / 10 mg / kg body weight via intraperitoneal injection) and administered the solution after the reflex response (limb withdrawal to the hind paw) had disappeared. The tracheal opening was directly visualized using an otoscope / mouth opener carefully positioned in the mouth. The allocated 50 microliters of sterile solution were administered directly into the trachea via an aerozoizer-type microsprayer (Penn-Century IA-1C) connected to a high-pressure syringe (Penn-Century FMJ-250). The microspray and syringe were flushed between each solution. As shown in Table 2, groups of mice were administered either ASO6713 (3 mg / kg) or ASO6713 (10 mg / kg).

[0199] One week after intratracheal administration, mice were exposed to cigarette smoke extract (CSE) for 4 days using a whole-body unrestrained exposure chamber method. Specifically, mice were placed in an 18 L Perspex chamber in a standard fume hood cabinet and exposed to either air or Winfield Red Cigarettes (total particulate matter of approximately 419 mg m-3, tar of approximately 16 mg, nicotine of approximately 1.2 mg, and CO of approximately 15 mg; Philip Morris). CSE mice were exposed to 9 whole cigarettes per day. Mice were exposed to either air or CSE three times a day with a 2-hour break between smoking sessions. After the exposure period, mice were humanely killed and the following endpoints were evaluated as outlined in Figure 1.

[0200] Animal Specific pathogen-free male C57Bl / 6 mice (obtained at approximately 8 weeks of age) were obtained from Animal Resource Centre Pty. Ltd. (Perth, Australia). Mice were randomly assigned to two batches of four groups (4 mice / group) for each endpoint and evaluated at three endpoints (Table 2). Animals were housed in sterilized passive microisolation cages at a constant temperature of 20°C on a 12-hour light / dark cycle, fed irradiated Barastoc mouse feed, and allowed free access to irradiated tap water. Mouse body weights were monitored throughout the experiment.

Table 6

[0201] Plasma collection Mice were bled by collecting blood from the inferior vena cava into Microvette lithium / heparin tubes (Sarstedt, Aust.) using a 19G syringe coated with lithium heparin. Blood was maintained at room temperature for at least 15 minutes and centrifuged at 2000 g for 5 minutes at 20°C. Plasma was transferred to 1.5 mL snap-lock Eppendorf tubes, rapidly frozen in liquid nitrogen, and stored at -80°C.

[0202] Bronchoalveolar lavage and differential cell count Bronchoalveolar lavage (BAL) was performed via surgical tracheotomy and washed in situ with 0.4 mL of cold PBS followed by an additional 0.3 mL PBS aliquot until approximately 1 mL of BAL fluid (BALF) was collected. Cytocentrifugation spots were prepared by centrifuging the BALF at 400×g for 10 minutes. The cell spots were stained with the Shandon Kwik-Diff kit (registered trademark) and Merck Hemacolor as described above. A differential count was performed, counting a total of 200 cells and identifying macrophages + monocytes, neutrophils + eosinophils, and lymphocytes using standard morphological criteria.

[0203] Tissue collection The lungs were perfused to remove circulating blood. A 20G needle was inserted into the right ventricle and 5 - 10 mL of cold sterile PBS was administered at a rate of 300 μl / second. The lungs were then excised as described above. The left lobe was divided into upper, lower left (LL), and lower right (LR) portions and collected in sterile 1.5 mL snap-lock Eppendorf tubes. The right upper lobe, right lower lobe, and posterior vena cava lobe were collected in sterile 1.7 mL snap-lock Eppendorf tubes. All lung tissues were immediately frozen in liquid nitrogen. The rapidly frozen tissues were transferred to -80°C for storage.

[0204] Measurement of RAGE_v1 mRNA expression levels in lung tissue To measure the effect of interventions on alternative splicing of RAGE premRNA, the expression of RAGE mRNA splice variants was determined using real-time RT-qPCR. This was performed using the TaqMan system based on previously used real-time detection of accumulated fluorescence (ABI Prism 7700, Perkin-Elmer Inc, PE Biosystems, Foster City, CA, USA) (43). Frozen lung tissue from the left lower lobe was used for in vivo experiments. RNA extraction and cDNA synthesis were performed using Trizol. Gene expression was estimated by RT-qPCR. This was performed using either the Taqman or SYBR Green (Sigma) system based on real-time detection of accumulated fluorescence (Applied Biosystems Q3 and Q5). Gene expression was normalized to 18S mRNA and reported as a fold change compared to the expression level in control RNA-treated cells (any value was assigned 1). To define the expression of RAGE mRNA splice variants retaining exon 9b, probes were designed to span exon 9b. To define the expression of RAGE mRNA splice variants that retain exon 10, we designed PCR primers / probes spanning exons 8–10. To define the expression of all RAGE mRNA, we designed PCR probes / primers spanning exons 8 and 11. Reduced signaling of cytoplasmic tail exons 8–10 relative to exons 8–11 indicates less full-length RAGE transcript (signal-ready) produced by the cell.

[0205] Example 2 - Analysis of bronchoalveolar lavage fluid (BALF) cell count in a smoke-induced inflammation mouse model In this experiment, mice (8 per group) were administered saline or ASO6713 (3 mg / kg or 10 mg / kg) into the left lobe of their lungs using an aerosolizing microsprayer, and one week later were exposed to cigarette smoke as described above. The mice were humanely euthanized on day 11, day 14, or day 17 after saline or ASO6713 treatment. Bronchoalveolar lavage fluid (BALF) was collected from the left lobe of the mouse lungs for analysis.

[0206] All mice exposed to the smoke experienced slight acute weight loss, consistent with the appetite-suppressing effect of nicotine, but fully recovered after the smoke exposure was discontinued (Figure 2).

[0207] Treatment with low-dose (3 mg / kg) and high-dose (10 mg / kg) ASO6713 increased RAGE_v1 mRNA expression (encoding the esRAGE protein) in cigarette smoke-exposed mice, as measured on days 11, 14, and 17 after ASO6713 treatment (Figure 5). Consistent with the splice-switching activity of ASO6713, RAGE mRNA levels, including exon 10, were found to decrease on days 11, 13, and 17 after ASO6713 treatment (Figure 7).

[0208] On day 11, the total number of cells in BALF in the lungs of mice increased after smoke exposure compared to the control group. However, the initial high dose of ASO6713 (10 mg / kg) before smoke exposure significantly reduced the increase in BALF cell count (Figure 3). The BALF cell count was observed to decrease slowly in the smoke-untreated mouse group from day 11 to 14, and there was no significant difference compared to the non-smoke-exposed group on day 14 (data not shown).

[0209] Example 3 - Analysis of immune cell subtypes in BALF samples collected from mice exposed to smoke. Next, the inventors performed a cell fraction count of immune cells within the BALF to determine whether the increase in the number of BALF cells was due to an increase in the number of inflammatory immune cells present in the lungs.

[0210] As expected, the smoke-exposed group showed increased infiltration of interstitial (non-alveolar) macrophages and neutrophils on day 11 compared to the non-smoke-exposed group (Figure 4). Consistent with the above findings, high-dose ASO6713 treatment was able to suppress the infiltration of these inflammatory cells. Low-dose ASO6713 treatment was also shown to significantly suppress neutrophil infiltration into the left lung.

[0211] Example 4 - Design for an experimental study of cigarette smoke-induced COPD animal All animal rearing and experimental procedures were carried out in accordance with local guidelines and approved by the Animal Ethics Committee of the Sydney Local Health District (NSW, Australia). Female wild-type C57BL / 6 mice were obtained from Australian BioResources Ltd (Mossvale, NSW, Australia), and the groups (5 mice per cage) were housed at the Centenary Institute (NSW, Australia) under conditions free of specific pathogens. The light / dark cycle was 12 / 12 hours, the temperature range was 22–26°C, and the humidity range was 30–70% relative humidity. The mice were randomly numbered and then divided into experimental groups. All tissues and samples were processed using this number as an identifier. Assays were performed without knowing which number belonged to which group. After the assay was completed, this number was used to identify the experimental group to which the sample belonged.

[0212] Protocol Overview Female C57BL / 6 mice (6-8 weeks old) were exposed to cigarette smoke from 12 research-grade cigarettes (3R4F, University of Kentucky, Lexington, KY, USA; each cigarette was smoked for 5 minutes) or normal air for 75 minutes twice daily for 8 weeks. The cigarette smoke was delivered to the mice using a custom-designed, dedicated nasal-only, directional inhalation and smoke exposure system (CH Technologies, Westwood, NJ, USA).

[0213] To test the therapeutic potential of AONs that have their most 5' nucleotide at position 88 or 114 of exon 10, or that are located at nucleotide positions 88–114 of exon 10, AON-m79 was selected. Mice were treated with AON-m79 (3 mg / kg, once weekly) or vehicle control via intranasal drops. The initial treatment was performed on the day of first exposure to cigarette smoke and continued weekly throughout the study period.

[0214] Bronchoalveolar lavage and cell fractionation counting Airway inflammation was assessed by counting the total leukocyte count and specific inflammatory cell types in the BAL fluid. BAL fluid was obtained by washing the lungs twice with 400 μL PBS aliquots at room temperature. Total cell count was assessed using trypan blue exclusion. Cytospin was performed on the cells in the remaining BAL fluid, and cell fraction counting was performed based on morphology.

[0215] Staining of lung tissue Paraffin-embedded, formalin-fixed sections (4 μm thick) of inflated lung tissue were placed on microscope slides and stained with hematoxylin and eosin. Epithelial thickness was evaluated by measuring the difference in area between the basement membrane and the lumen of the epithelial cells. For this purpose, photographs of the stained lung sections were taken and visualized using ImageJ software (version 1.47).

[0216] Small airway-associated fibrosis was assessed by measuring peri-airway collagen deposition stained with Masson's Trichrome Blue in formalin-fixed lung sections. Stained lung sections were photographed on slides, and collagen volume was calculated by a blinded observer using ImageJ software (version 1.47) to assess peri-airway deposition areas less than 1 mm in diameter. The formula (Wct / Pbm) was used for the calculation (Wct=AoAi, inner collagen region=Ai, outer collagen region=Ao, and basement membrane length=Pbm).

[0217] Paraffin-embedded, formalin-fixed sections (4 μm thick) of inflated lung tissue were placed on microscope slides and stained with hematoxylin and eosin. Emphysema-like alveolar dilation was evaluated using the mean linear intersection (MLI) technique, a standard method for assessing alveolar size and emphysema in mice. A standardized template including horizontal lines was placed on micrographs of the lung sections. The number of alveolar intercepts was counted, and the resulting mean number of intercepts was determined.

[0218] Evaluation of total lung volume Total lung volume (TLC) was assessed on the endpoint day. Mice were anesthetized with ketamine and xylazine hydrochloride in PBS. Tracheostomies were then performed, cannulas were inserted and connected to a flexiVent FX2 system (SCIREQ Scientific Respiratory Equipment Inc., Montreal, Canada), operated with flexiWare version 8 software. Mice were placed on ventilators for lung volume manipulation. This involved degassing the lungs with 100% oxygen to allow inflation to begin from zero volume. The lungs were then inflated using three full-range pressure-volume loop ramp inflation and deflation cycles to calculate TLC.

[0219] statistical analysis The data presented are mean ± SEM (n=6-8). All datasets were determined to be normally distributed based on previous experiments and were tested using the Kolmogorov-Smirnov test (α=0.05). The data were analyzed using one-way ANOVA with Tukey's multiple comparison test, using PRISM software (V6.0d, GraphPad, La Jolla, CA, USA). P<0.05 was considered statistically significant.

[0220] Example 4 - The AONm79 treatment mitigates the characteristics of smoke-induced experimental chronic obstructive pulmonary disease (COPD). To determine whether RAGE splice-switching oligonucleotides can be used to prevent smoke-induced lung injury, female C57BL / 6 mice were exposed to cigarette smoke from 12 3R4F reference cigarettes twice daily, five times a week, for eight consecutive weeks. This exposure is roughly equivalent to a human smoking one pack per day and is known to induce pathophysiological features consistent with human COPD, including the induction of persistent airway inflammation predominantly driven by macrophages and neutrophils, which drive the expression of pathological and disease characteristics.

[0221] Intranasal treatment with AON-m79 (3 mg / kg / once weekly) significantly inhibited the increase in inflammatory cells in bronchoalveolar (BAL) fluid associated with smoke exposure (Figure 6A). In fact, the treatment completely suppressed macrophage influx in normal air exposure controls, bringing it back to baseline levels. Furthermore, after treatment with AON-m79, lymphocytes (cigarette smoke (CS); mean = 425 cells / mL (in BAL fluid) vs. CS + AON-m79; mean = 189.8 cells / mL, p = 0.05) and eosinophils (CS; mean = 839.57 cells / mL vs. CS + AON-m79; mean = 78.56 cells / mL, p = 0.05) in BAL fluid decreased. In this 8-week model, smoke-induced lung injury was associated with airway remodeling accompanied by epithelial thickening (Figure 6B), emphysema-like alveolar dilation (Figure 6C), and peri-small airway fibrosis (Figure 6D). These are characteristic pathological features of early human COPD. Together, these changes were associated with decreased lung function (Figure 6E). Treatment with AON-m79 substantially prevented each of these chronic features specific to human COPD: airway remodeling, emphysema, and changes in lung function.

[0222] These results, taken together, suggest that treatment with RAGE splice-switching oligonucleotides may protect the lungs from increased inflammation and characteristic features associated with the development of COPD.

Claims

1. A pharmaceutical composition for use in subjects requiring treatment or prevention of obstructive pulmonary disease associated with or caused by smoke exposure, comprising an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE).

2. A pharmaceutical composition for use in subjects requiring relief or improvement of symptoms of obstructive pulmonary disease associated with or caused by smoke exposure, comprising an antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE).

3. The pharmaceutical composition for use according to claim 1 or 2, wherein the AON, which promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE), is administered directly to the airway and / or lungs.

4. The pharmaceutical composition for use according to claim 1 or 2, wherein the administration of AON promotes the production of endogenous soluble RAGE in the whole airway, lower airway, or upper airway, and / or reduces the production of membrane-bound RAGE (mRAGE).

5. A pharmaceutical composition for use according to claim 1 or 2, wherein the use promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE) in the lower respiratory tract, preferably the lung.

6. A pharmaceutical composition for use according to claim 1 or 2, wherein the antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE) is administered by inhalation.

7. A pharmaceutical composition for use according to claim 1 or 2, wherein the antisense oligonucleotide (AON) that promotes the production of endogenous soluble RAGE and / or reduces the production of membrane-bound RAGE (mRAGE) is administered via intranasal administration.

8. The pharmaceutical composition for use according to claim 1 or 2, wherein the obstructive pulmonary disease is chronic obstructive pulmonary disease (COPD), emphysema, emphysema-associated pulmonary fibrosis (CPFE), bronchitis, respiratory bronchiolitis / respiratory bronchiolitis-interstitial lung disease, bronchiectasis, asthma, lung cancer, tracheal cancer, or smoking-related interstitial lung disease (SR-ILD), and preferably, the chronic obstructive smoking-related interstitial lung disease (SR-ILD) is pulmonary Langerhans cell histiocytosis, exfoliative interstitial pneumonia, or acute eosinophilic pneumonia.

9. (i) The AON promotes the production of the endogenous soluble RAGE by promoting the inclusion of exon 9b and / or the exclusion of exon 10 (e.g., skipping); (ii) AON promotes splicing in RAGE premRNA, resulting in the inclusion of exon 9b and / or skipping of exon 10; or (iii) The pharmaceutical composition for use according to claim 1 or 2, wherein administration of AON results in an increase in the level of RAGE_v1, preferably the level of RAGE_v1 mRNA, in one or more tissues of the airway.

10. (i) The AON is administered to the whole airway, the upper airway, or the lower airway; (ii) The AON is administered as an aerosol; or (iii) The pharmaceutical composition for use according to claim 1 or 2, wherein the AON is administered in a single dose.

11. (i) The AON is a 10 to 50 nucleotide AON containing a target sequence complementary to a region near or within an intron of the RAGE premRNA, and optionally, the AON is a 10 to 50 nucleotide AON containing a target sequence complementary to or adjacent to a splice site of the RAGE premRNA; (ii) The AON is 10 to 50 nucleotides containing a target sequence complementary to the RAGE premRNA, which modulates the secondary structure of the mRNA and influences the selection of the splice site; (iii) The AON is isolated or purified AON for inducing the exclusion (also known as skipping) of one or more exon sequences in the RAGE gene transcript or a portion thereof; or (iv) The pharmaceutical composition for use according to claim 1 or 2, wherein the AON is isolated or purified AON for inducing the retention of an intron sequence in the RAGE gene transcript or a portion thereof.

12. (i) The AON comprises at least one modified nucleotide; (ii) The AON is chemically modified to prevent degradation of the premRNA-AON complex, preferably the chemical modification is selected from the group consisting of phosphorodiamidate morpholino oligomers (PMOs), 2'-O-methylphosphorothioate oligonucleotides (2OMe), and 2'-O-methoxyethyl phosphorothioate oligonucleotides (2'-MOE), locked nucleic acid (LNA) modified AONs, thermally stable twist intercalating nucleic acids (TINAs), and peptide nucleic acids (PNAs); or (iii) The AON is conjugated with a portion, preferably a cell-permeable peptide (CPP), to increase its delivery; or (iv) The pharmaceutical composition for use according to claim 1 or 2, wherein the AON is vivomorpholino (VMO) or peptide phosphorodiamidate morpholino oligomer (PPMO).

13. The pharmaceutical composition for use according to claim 1 or 2, wherein the AON contains, essentially consists of, or consists of, a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target region of exon 10 of RAGE premRNA throughout the antisense oligonucleotide, preferably the most 5' nucleotide of the AON is at nucleotide position 88 or 114 of exon 10, or is located at nucleotide positions 88-114 of exon 10.

14. The pharmaceutical composition for use according to claim 1 or 2, wherein the AON is 8 to 40 nucleotides long, 15 to 25 nucleotides long, or 18 nucleotides long.

15. (i) The AON is selected from the group including the sequences shown in any of Tables 1a to 1d; (ii) The AON includes a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 31 and 33, or a nucleotide sequence that is at least 85%, 90%, or 95% identical thereto; or (iii) The pharmaceutical composition for use according to claim 1 or 2, wherein the AON comprises the nucleotide sequence shown in SEQ ID NO: 11, 18, 19, or 20, or a nucleotide sequence that is at least 85%, 90%, or 95% identical thereto.

16. The pharmaceutical composition for use according to claim 1 or 2, wherein the smoke is an aerosol or particulate matter from the combustion of one or more of the following: cigarettes, tobacco or marijuana, coal, wood, oil, gas or other biomass.

17. The pharmaceutical composition for use according to claim 1 or 2, wherein the smoke is cigarette smoke.

18. The pharmaceutical composition for use according to claim 17, wherein the cigarette smoke is "passive smoking".

19. (i) The smoke is from burnt organic matter, coal dust, smog, exhaust gases, and other industrial pollutants; or (ii) The pharmaceutical composition for use according to claim 1 or 2, wherein the subject is exposed to smoke from burning wood or forest.

20. The pharmaceutical composition for use according to claim 1 or 2, wherein the subject has experienced acute exposure to smoke.

21. The pharmaceutical composition for use according to claim 1 or 2, wherein the obstructive pulmonary disease is smoke-induced COPD.

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