How to monitor splicing

Abstract

The present invention relates to a method for identifying splicing of RAGE premRNA in a subject that has received or is receiving antisense oligonucleotides that modulate RAGE premRNA splicing, resulting in the inclusion of exon 9b and / or exclusion of exon 10 in that subject. In one embodiment, the present invention provides a method for determining the level of endogenous soluble advanced glycation end product receptor (RAGE) in the lung of a subject. The method comprises determining the presence of endogenous soluble RAGE in a blood sample from a subject that has received or is receiving treatment administered to the airway, wherein the treatment increases endogenous soluble RAGE, and the level of endogenous soluble RAGE in the blood sample correlates with the level of endogenous soluble RAGE in the lung of the subject.

Description

Technical Field

[0001] The present invention relates to a method for determining the level of RAGE isoforms, particularly endogenous soluble RAGE isoforms, or a portion thereof, in a subject who has received or has received treatment with an antisense oligonucleotide that regulates the pre-mRNA splicing of the receptor for advanced glycation end products (RAGE).

[0002] Related Applications This application claims the benefit of Australian Provisional Application No. 2023 / 901363, filed on May 5, 2023, the entire disclosure of which is incorporated herein by reference.

Background Art

[0003] The receptor for advanced glycation end products (RAGE) is a multivalent type I transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. The human RAGE (Ager) gene is located within the major histocompatibility complex class III region on chromosome 6. It contains 11 exons and 10 introns, as well as a 5' flanking region that regulates its transcription. The transcribed RAGE mRNA is approximately 1.4 kb and has a short 3' UTR.

[0004] The glycosylated RAGE protein of 50-55 kDa is constitutively expressed in a limited range of cells (e.g., vascular endothelial cells, type I lung cells, leukocytes), but the expression of RAGE can be induced in most cell types and tissues after injury, stress, hypoxia, or inflammation, providing a pathway for pro-inflammatory and pro-proliferative signaling. As a result, the expression of RAGE is upregulated in inflammatory and metabolic disorders including, but not limited to, neurodegenerative diseases, cancer, cardiovascular diseases, diabetes, autoimmunity, and ischemic injury, and the involvement of RAGE in their onset and progression has also been suggested.

[0005] In a healthy state, RAGE expression in the lungs is the highest of all tissues. Upregulation of RAGE signaling in other cells and other sites of the lungs has been suggested to be involved in a variety of lung disorders, including chronic obstructive pulmonary disease (COPD) / emphysema, asthma, tobacco smoking / contamination damage, acute lung injury / acute respiratory distress syndrome, and pulmonary fibrosis.

[0006] Alternative splicing of RAGE is also important for regulating RAGE activity through the generation of RAGE isoforms with altered ability to be activated by ligand-dependent and ligand-independent signaling pathways. Alternative splicing of RAGE is altered in disease states, including malignancies, diabetes, and Alzheimer's disease.

[0007] Alternative splicing of RAGE can be modulated by targeting premRNA RAGE using antisense oligonucleotides (AONs). These AONs may be administered systemically or topically to modify the expression and / or activity of RAGE isoforms.

[0008] When AON is administered to the target airway, a minimally invasive approach is needed to determine alternative splicing of RAGE premRNA.

[0009] 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]

[0010] In one aspect, the present invention relates to a method for determining the level of endogenous soluble advanced glycation end product receptor (RAGE) in a target lung, wherein the method is - Determining the presence of endogenous soluble RAGE in blood samples from subjects who have received or are receiving treatment administered to the airways, including determining whether the treatment increases endogenous soluble RAGE. This invention provides a method for correlating the level of endogenous soluble RAGE in a blood sample with the level of endogenous soluble RAGE in the lung of a subject.

[0011] In one embodiment, the present invention relates to a method for determining the presence or level of endogenous soluble advanced glycation end product receptor (RAGE) in a target blood sample, wherein the method is - Determining the presence or level of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving treatment administered to the airway, including determining whether the treatment increases endogenous soluble RAGE. This provides a method for determining the presence or level of endogenous soluble RAGE in a target blood sample.

[0012] In one embodiment, the present invention relates to a method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method is - Determining the presence of endogenous soluble RAGE in blood samples from subjects who have received or are receiving antisense oligonucleotides (AONs) in the airway, which promote splicing in RAGE premRNA and result in the inclusion of exon 9b and / or exclusion of exon 10, This invention provides a method in which the presence of endogenous soluble RAGE in a blood sample indicates splicing of advanced glycation end product receptor (RAGE) premRNA, resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

[0013] In another aspect, the present invention relates to a method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method - To provide a blood sample from a subject who has received or is receiving an antisense oligonucleotide (AON) in the airway, wherein the AON promotes splicing in RAGE premRNA, resulting in the inclusion of exon 9b and / or exclusion of exon 10. - Measuring the level of endogenous soluble RAGE in a blood sample, The present invention provides a method in which the level of endogenous soluble RAGE in a blood sample indicates splicing of RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

[0014] In another aspect, the present invention relates to a method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method - To provide blood samples from subjects who have received or are receiving antisense oligonucleotides (AONs) in the airway, which promote splicing in RAGE premRNA and result in the inclusion of exon 9b and / or exclusion of exon 10. - Measuring the level of endogenous soluble RAGE in a blood sample, The present invention provides a method in which the level of endogenous soluble RAGE in a blood sample exceeding a threshold level indicates splicing of RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

[0015] In another aspect, the present invention relates to a method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method - To provide blood samples from subjects before receiving an antisense oligonucleotide (AON) into the airway, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. - To provide blood samples from subjects who have received AON in the airway, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. - This includes measuring the level of endogenous soluble RAGE in each blood sample, This method provides evidence that a higher level of endogenous soluble RAGE in a blood sample from a subject after receiving an AON in the airway, compared to the level of endogenous soluble RAGE in a blood sample from a subject before receiving an AON in the airway, indicates splicing of RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

[0016] In another aspect, the present invention relates to a method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method - Obtain blood samples from subjects before they receive an antisense oligonucleotide (AON) in their airways, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. - Obtain blood samples from subjects who have received AON in the airway, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. - This includes measuring the level of endogenous soluble RAGE in each blood sample, This method provides evidence that a higher level of endogenous soluble RAGE in a blood sample from a subject after receiving an AON in the airway, compared to the level of endogenous soluble RAGE in a blood sample from the subject before receiving an AON, indicates splicing of RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

[0017] In another aspect, the present invention relates to a method for determining whether a treatment / AON can increase endogenous soluble RAGE levels in the airway, wherein the method is - Administer experimental treatment / AON to the target airway. - obtaining a blood sample from the subject after administration, providing a method wherein the presence or increased level of endogenous soluble RAGE in the blood sample indicates that the test treatment / AON increases the level of endogenous soluble RAGE in the airway.

[0018] In any aspect or embodiment herein, the blood sample comprises or consists of whole blood, plasma or serum.

[0019] In any aspect or embodiment, one or more or all steps of the method of the invention are performed in vitro or ex vivo.

[0020] In any aspect, the blood sample is obtained from the subject at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, or at least about 28 days after the subject has received an antisense oligonucleotide (AON) that promotes splicing in RAGE pre-mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10 in the airway.

[0021] In any aspect, the blood sample is obtained at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, or at least 28 days after the subject has received an antisense oligonucleotide (AON) that promotes splicing in RAGE pre-mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10 into the airway.

[0022] In any aspect, the blood sample is obtained 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days after the subject has received an antisense oligonucleotide (AON) that promotes splicing in RAGE pre-mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10 into the airway.

[0023] In any aspect, the AON can 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 aspect, the AON can promote splicing in RAGE pre-mRNA, resulting in the inclusion of exon 9b and / or the skipping of exon 10. For example, the AON can 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.

[0024] In any embodiment, endogenous soluble RAGE may be a polypeptide encoded by one or more of RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19, preferably RAGE_v1 (i.e., esRAGE).

[0025] In any configuration, AON is administered to the whole airway, upper airway, or lower airway.

[0026] 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.

[0027] 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).

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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), and 2'-O-methoxyethyl phosphorothioate oligonucleotides (2MOE), locked nucleic acid (LNA) modified AONs, heat-stable twist-intercalating nucleic acids (TINAs), and peptide nucleic acids (PNAs).

[0034] 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).

[0035] 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.

[0036] 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.

[0037] In any embodiment, the AON may be 8 to 40 nucleotides long, 15 to 25 nucleotides long, or 18 nucleotides long.

[0038] 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, 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.

[0039] The AON for use in the method of the present invention 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.

[0040] More specifically, AON may be selected from the group comprising one or more sequences from SEQ ID NOs: 1 to 31 and / or sequences shown in any of Tables 1a to 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.

[0041] 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.

[0042] The methods of the present invention also include combinations of two or more AONs capable of binding to a selected target and modulating 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.

[0043] 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.

[0044] 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]

[0045] [Figure 1] Sections of the mouse lung as described in the examples of this specification. [Figure 2]Outline of experimental design: Time-dependent pharmacodynamic activity of a single intratracheal treatment with ASO m79 (3 mg / kg) in male C57BL / 6 mice. [Figure 3] esRAGE protein levels in the lung correlate with plasma esRAGE concentrations measured 14 days after a single intratracheal treatment with ASO m79. Male C57BL / 6 mice were administered either a control oligonucleotide or ASO m79 (3 mg / kg) on ​​day 0, and esRAGE levels in left lung tissue and plasma were measured on day 14. Data are shown mean ± SEM; *p<0.05 (compared to control oligonucleotide). [Figure 4] Outline of experimental design: Time-dependent pharmacodynamic activity of a single aerosolized dose of 18mer ASO(ASO2) (3 mg / kg or 0.3 mg / kg) in male C57BL / 6 mice. [Figure 5] esRAGE protein in the lungs correlates with plasma esRAGE concentrations measured after a single aerosol treatment with ASO2. Male C57BL / 6 mice were administered either a vehicle, 0.3 mg / kg ASO2, or 3 mg / kg ASO2 on day 0, and esRAGE levels were measured in left lung tissue and plasma on days 10(a) and 14(b). Both 0.3 mg / kg ASO2 and 3 mg / kg ASO2 levels showed a statistically significant increase in esRAGE levels compared to the vehicle. Spearman correlation, p<0.05. [Figure 6] Outline of experimental design: Dose-dependent pharmacodynamic activity of single-dose inhalation therapy with ASO2 (delivered by microspray) in male C57BL / 6 mice. [Figure 7] esRAGE protein in the lungs correlates with plasma esRAGE concentrations measured 7 days after a single inhalation treatment with ASO2. Male C57BL / 6 mice were administered either a vehicle, 3 mg / kg ASO2, or 10 mg / kg ASO2 on day 0, and esRAGE levels were measured in left lung tissue and plasma on day 7. Both 3 mg / kg ASO2 and 10 mg / kg ASO2 levels showed a statistically significant increase in esRAGE levels compared to the vehicle-only Spearman correlation (p<0.05). [Modes for carrying out the invention]

[0046] 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.

[0047] 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.

[0048] 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.

[0049] All patents and publications referenced herein are incorporated in their entirety by reference.

[0050] For the purposes of interpreting this specification, terms used in the singular form also include their plural forms, and vice versa.

[0051] 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.

[0052] 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).

[0053] 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 increase “endogenous soluble RAGE” in the airways and plasma of the subject.

[0054] 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.

[0055] sample In any aspect or embodiment of this specification, the blood sample includes or consists of whole blood, plasma, or serum.

[0056] The samples used herein may be obtained from the subject or components of the subject (e.g., cells). The sample may also be a “clinical sample” derived from a patient. In one embodiment, the method of the present invention is not performed on a human or animal body, and for example, the measurement of endogenous soluble RAGE levels may be determined by analyzing a previously obtained blood sample.

[0057] In one embodiment, the blood sample may be decontaminated of red blood cells (e.g., in the form of plasma, serum, or untreated whole blood). In one embodiment, when endogenous soluble RAGE is measured in the blood sample, red blood cells are either absent or absent at significant levels. In another embodiment, red blood cells are present at normal levels, i.e., they are not decontaminated from the blood sample.

[0058] In any embodiment, the blood sample is obtained at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, or at least about 28 days after the subject has received an antisense oligonucleotide (AON) into the airway, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10.

[0059] In any embodiment, the blood sample is obtained at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or at least 28 days after the subject has received an antisense oligonucleotide (AON) into the airway, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10.

[0060] In any embodiment, the blood sample is obtained 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days after the subject received an antisense oligonucleotide (AON) into the airway, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10.

[0061] In any embodiment, the blood sample is obtained approximately 7 to 28 days, 7 to 27 days, 7 to 26 days, 7 to 25 days, 7 to 24 days, 7 to 23 days, 7 to 22 days, 7 to 21 days, 7 to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, or 7 to 14 days after the subject has received an antisense oligonucleotide (AON) into the airway that promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or the exclusion of exon 10.

[0062] In any embodiment, the blood sample is obtained 7–28 days, 7–27 days, 7–26 days, 7–25 days, 7–24 days, 7–23 days, 7–22 days, 7–21 days, 7–20 days, 7–19 days, 7–18 days, 7–17 days, 7–16 days, 7–15 days, or 7–14 days after the subject has received an antisense oligonucleotide (AON) into the airway, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10.

[0063] Alternatively, in any embodiment, the blood sample is obtained within 28 days of the subject receiving an antisense oligonucleotide (AON) into the airway, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or the exclusion of exon 10. For example, the blood sample is obtained within 7 days, 14 days, or 28 days of the subject receiving an antisense oligonucleotide (AON) into the airway, which promotes splicing in RAGE premRNA and results in the inclusion of exon 9b and / or the exclusion of exon 10.

[0064] In any embodiment, a blood sample may be obtained and then frozen before being used in the method of the present invention. For example, a blood sample may be obtained and frozen for a certain period of time and under such storage conditions that does not substantially reduce the detectable level of endogenous soluble RAGE present in the blood sample before freezing.

[0065] In any embodiment, umbilical cord blood erythrocytes are depleted by ammonium chloride lysis, density gradient technique, hypotonic lysis, immunomagnetic cell separation or sedimentation, flow cytometry sorting, or equivalent methods as understood by those skilled in the art.

[0066] Detection and measurement of endogenous soluble RAGE The provided method is based on the unexpected discovery that the level or presence of endogenous soluble RAGE in a blood sample promotes splicing of advanced glycation end product receptor (RAGE) premRNA, resulting in the inclusion of exon 9b and / or exclusion of exon 10 after administration of AON to the airway. Therefore, endogenous soluble RAGE levels in the blood can be used to monitor and determine the expression of alternative splicing of RAGE in the airway.

[0067] In any embodiment, the present method of the present invention includes detecting the presence of endogenous soluble RAGE as described herein in a blood sample or measuring its level.

[0068] In any embodiment, the level of endogenous soluble RAGE in the blood sample is higher or increased after AON administration compared to the level of endogenous soluble RAGE in the blood sample from the subject before AON administration to the airway.

[0069] As used herein, the terms “presence” or “level” refer to the occurrence or change of a signal that can be detected directly or indirectly by observation or instrumentation. Typically, a detectable response is the occurrence of a signal in which a fluorophore essentially emits fluorescence. Alternatively, a detectable response is an optical response resulting from a change in wavelength distribution pattern or absorbance or fluorescence intensity, or from light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.

[0070] It is understood that the level or presence of endogenous soluble RAGE in a sample can be measured by any suitable method known in the art. The measurement of the expression level of endogenous soluble RAGE may be direct or indirect.

[0071] For example, the level or presence of endogenous soluble RAGE may be measured using a labeled reagent, such as an antibody that specifically binds to endogenous soluble RAGE. As used herein, the term “labeled” refers to a chemical moiety or protein that, when bound to an antibody, is detectable directly or indirectly (for example, due to its spectral characteristics, conformation, or activity).

[0072] Antibodies that specifically bind to endogenous soluble RAGE, such as esRAGE, are known in the art and can be purchased commercially. For example, esRAGE antibodies can be purchased from Sigma-Aldrich in Australia (product number: MAB5328).

[0073] The detection label conjugated to the reagent can be any label that enables separate detection and quantification by flow cytometry. For example, a fluorescent dye. Suitable fluorescent labels are known in the art, including fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinine chlorophyll protein (PerCP), allophycocyanin (APC), Alexa fluor 488, Alexa fluor 647, Alexa fluor 710, Alexa fluor 405, cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), pacific blue (PacB), horizon violet 450 (HV450), pacific orange (PacO), horizon V500 (HV500), Krome Orange, Brilliant Violet 421 (BV421), Brilliant Violet 510 (BV510), Brilliant Violet 605 (BV605), Brilliant Violet 650 (BV650), and Brilliant This includes Violet 711 (BV711), Brilliant Violet 785 (BV785), Brilliant Ultraviolet 395 (BUV395), Brilliant Ultraviolet 496 (BUV496), Brilliant Ultraviolet 737 (BUV737), Orange Cytognos (OC) 515, quantum dots and their conjugates coupled with PE, APC, or PerCP (e.g., PE / Cy5, PE / Cy5.5, PE / Cy7, PerCP / Cy5.5, APC / Cy7, APC-H7, APC-Alex750, PE-Texas Red, PE-Dazzle, PE-CF594), or additional compatible fluorescent dyes or tandem fluorescent dyes.

[0074] For example, the antibodies include: (1) Pacific Blue (PacB), Brilliant Violet 421 (BV421), or Horizon V450; (2) Pacific Orange (PacO), Horizon V500 (HV500), BV510, Khrome Orange (KO), or OC515; (3) Horizon BB515, Fluorescein Isothiocyanate (FITC), or Alexa488; (4) Phycoerythrin (PE); (5) Peridinine Chlorophyll Protein / Cyanine 5.5 (PerCP-Cy5.5), PerCP, or PE-TexasRed; (6) Phycoerythrin / Cyanine 7 (PE-Cy7); (7) Allophycocyanin (APC), or Alexa647; and (8) Allophycocyanin / Hilite It can be conjugated to 7 (APC-H7), APC-Cy7, Alexa680, APC-A750, APC-C750, or Alexa700.

[0075] In another example, the antibodies are conjugated to (1) brilliant violet 421, (2) brilliant violet 510 (BV510), (3) brilliant violet 650 (BV650), (4) brilliant violet 786 (BV786), (5) fluorescein isothiocyanate (FITC), (6) peridinine chlorophyll protein / cyanine 5.5 (PerCP-Cy5.5), (7) phycoerythrin (PE), (8) phycoerythrin / cyanine 7 (PE-Cy7), (9) allophycocyanin (APC), and (10) allophycocyanin / H7 (APC-H7), APC-C750, or APC-Alexa750.

[0076] Suitable labels can be directly or indirectly linked to the reagent through the use of suitable tags. In a preferred embodiment, the detectable label is linked to streptavidin.

[0077] The method of the present invention further comprises contacting a blood sample with a reagent that enables the identification of endogenous soluble RAGE. Typically, the molecule is either bound to a detectable label or is a detectable label itself. For example, the molecule may be a fluorescent dye, antibody, or enzyme that leads to the generation of a substrate. Alternatively, the reagent is linked to a tag that facilitates binding to the detectable label. For example, the tag may be non-covalently bound to the detectable label or form a covalent interaction with the detectable label. Suitable tags are known in the art and are described herein.

[0078] For example, the reagent may be an antibody for detecting endogenous soluble RAGE, and the detectable label may be a fluorescent dye. Suitable fluorescent dyes are known in the art and are described herein.

[0079] Preferably, the reagent is an antibody that specifically binds to the c-terminal domain of the endogenous soluble RAGE as defined herein, but does not significantly bind to other RAGE isoforms. Due to the intrinsic cleavage of the transmembrane domain and intracellular signaling domain, the endogenous soluble RAGE proteins described herein possess unique conformational epitopes not found in other RAGE isoforms, which may be targeted to provide specific detection / measurement of endogenous soluble RAGE levels. Such antibodies and kits for detecting endogenous soluble RAGE (e.g., esRAGE) are commercially available, for example, the human esRAGE ELISA kit (HUFI04782; Assay Genie).

[0080] Detection of reagents linked to detectable labels that bind to endogenous soluble RAGE can be performed by biochemical techniques such as antibody conjugation, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell analysis (FACS). These methods are carried out as is known in the art. Suitable ELISA kits for the detection of endogenous soluble RAGE, such as esRAGE, are commercially available, for example, from mybiosource.com (catalog #MBS015358).

[0081] If the sample is bound to a fluorescent dye-selective reagent, the presence or level of reagent-labeled endogenous soluble RAGE can be detected using flow cytometry or microscopy. Such methods are carried out as are known in the art.

[0082] In any embodiment, the level of endogenous soluble RAGE (e.g., esRAGE) measured in a blood sample from an subject who has received or is receiving the AON described herein at a value of at least about 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 20 times, 100 times, or more is compared to the level of endogenous soluble RAGE measured in a blood sample from the same subject before receiving the AON.

[0083] The level of endogenous soluble RAGE in a blood sample from a subject may refer to the concentration, volume, level, relative concentration, relative volume, or activity of endogenous soluble RAGE. Generally, the level of endogenous soluble RAGE indicates splicing of RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject (preferably within airway cells).

[0084] In any embodiment, measuring the level or presence of endogenous soluble RAGE involves performing an in vitro assay. In any embodiment, the in vitro assay is an immunoassay, an aptamer-based assay, or a histological or cytological assay. In some cases, the value of one or more biomarkers is measured by enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry assay, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, inhibition assay, or avidity assay.

[0085] One technique for comparing protein expression levels from two different samples involves subjecting each sample to 2D gel electrophoresis separately. Alternatively, each sample is labeled differently, and both samples are loaded onto the same 2D gel. See, for example, Unlu et al. Electrophoresis, 1997;18:2071-2077 (at least with respect to its teaching of a method for evaluating and comparing protein expression levels, which is incorporated herein by reference). The same protein in each sample is identified by its relative position within the pattern of proteins separated by 2D electrophoresis. The expression level of the protein in the first sample is then compared to the expression level of the same protein in the second sample, thereby enabling the identification of proteins (e.g., biomarkers) that are expressed differently between the two samples. This comparison is preferably performed on the subject before and after undergoing AON, which promotes splicing of RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10.

[0086] In another technique, the expression level of endogenous soluble RAGE is expressed as a percentage of the total expressed protein in a single sample. This evaluated expression level is compared to an existing reference standard, thereby enabling the identification of endogenous soluble RAGE that is expressed differently in the sample compared to the reference standard.

[0087] In any embodiment, increased endogenous soluble RAGE may be compared to a reference standard. The reference standard may be from one or more subjects that (a) have or do not have RAGE-related disease or disease, and / or (b) have not been administered an AON that splices RAGE as described herein. Thus, if the expression level of endogenous soluble RAGE in a blood sample from a subject that has received or is receiving an AON is increased or higher, preferably statistically significant, compared to the expression level of endogenous soluble RAGE in the reference standard, it is determined that the subject has increased splicing of RAGE premRNA, which preferably results in the inclusion of exon 9b and / or exclusion of exon 10 in airway cells. In some embodiments, a statistically significant increase is two or more standard deviations above the reference standard (exempic values ​​of the reference standard are described below). Alternatively, if the expression level of endogenous soluble RAGE in a sample from a subject that has received or is receiving AON is the same as, or not statistically significant to, the expression level of endogenous soluble RAGE in a reference standard, it is determined that the subject does not have an increase in RAGE premRNA splicing resulting in the inclusion of exon 9b and / or exclusion of exon 10, preferably in airway cells. Therefore, in any method or use of the present invention, if it is determined that the subject does not have an increase in RAGE premRNA splicing resulting in the inclusion of exon 9b and / or exclusion of exon 10, the method or use further comprises the step of administering another dose of AON as described herein.

[0088] As used herein, the threshold level for endogenous soluble RAGE may be a level determined from one or more control subjects who have not received treatment to increase endogenous soluble RAGE. In one embodiment, these control subjects may be healthy subjects, or otherwise normal subjects who do not have a RAGE-mediated condition or respiratory condition or disease as defined herein and have not received treatment to increase endogenous soluble RAGE. In another embodiment, these control subjects may have a RAGE-mediated condition or respiratory condition or disease as defined herein and have not received treatment to increase endogenous soluble RAGE. An exemplary threshold level or reference standard may be 250 ± 25 pg / mL (mean ± SD).

[0089] To demonstrate that AON treatment results in splicing of RAGE premRNA in the subject, leading to inclusion of exon 9b and / or exclusion of exon 10, endogenous soluble RAGE data may be analyzed by various methods to identify endogenous soluble RAGE, and the statistical significance of the difference in observed levels of endogenous soluble RAGE between the test expression profile and the reference expression profile in blood samples taken before and after receiving the AON described herein may be determined.

[0090] Antisense oligonucleotides (AONs) Antisense oligonucleotides (AONs) are short synthetic antisense modification strands of DNA or RNA that selectively hybridize to preRNA / mRNA via Watson-Crick base pairing and can selectively regulate the function of target RNA.

[0091] The terms "AON" and "ASO" are both abbreviations for the term "antisense oligonucleotide" and are used interchangeably herein.

[0092] 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.

[0093] 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.

[0094] 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 non-functional or innate RAGE mRNA spliceforms that act as decoy receptors antagonizing ligand-dependent activation and ligand-independent transactivation of full-length RAGE.

[0095] 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 targeted sequence modifications are not required.

[0096] 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).

[0097] 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.

[0098] 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.

[0099] 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]

[0100] 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).

[0101] 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.

[0102] 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.

[0103] Preferably, AON is selected from the group including SEQ ID NOs. 1 to 31 and / or from any of the sequences shown in Tables 1a to 1d. More preferably, AON is SEQ ID NOs. 11, 18, 19, or 20.

[0104] 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.

[0105] 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).

[0106] 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.

[0107] 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.

[0108] 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, and may also include Tms of 60-80°C or higher.

[0109] 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 to 31 and / or any of the sequences shown in Tables 1a to 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.

[0110] 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.

[0111] 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.

[0112] Skipping target 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.

[0113] Skipping the target exons 1-11 preferably keeps the reading frame intact. This will 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] A skipping process using AON may exclude (skip) the target exon, or it may result in the skipping of two or more exons at once.

[0120] Skipping processes using AONs may involve preservation of intron sequences, with or without direct skipping of one or more exons.

[0121] 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]

[0122] 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 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 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.

[0123] 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.

[0124] 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 sequences provided in any of Tables 1a to 1d or SEQ ID NOs: 1 to 31. More preferably, the AON is SEQ ID NOs: 11, 18, 19, or 20.

[0125] 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.

[0126] 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 over 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, or at least about 36 or more nucleotides.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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).

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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).

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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).

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] Administration, dosage, and formulation In any embodiment, the subject is a treatment administered to the airway that increases endogenous soluble RAGE, which may or may not have been treated. The treatment may be a treatment for respiratory (lung) disorders or diseases associated with or caused by RAGE, selected from the following groups: acute upper respiratory tract infections, rhinitis, nasopharyngitis, sinusitis, laryngitis, influenza and pneumonia, acute bronchitis, acute bronchiolitis, asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, emphysema, chronic lung disease due to exogenous factors, acute respiratory distress syndrome (ARDS), pulmonary eosinophilia, and pleurisy, lung trauma, and recovery from lung injury, trauma, and surgery.

[0149] 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.

[0150] 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.

[0151] 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).

[0152] 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, or need for glucocorticoids, including oral glucocorticoids.

[0153] 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.

[0154] The methods of the present invention may provide the AON described herein, adapted in a form suitable for delivery to a patient, to assist in the prophylactic or therapeutic treatment, prevention or improvement of symptoms of diseases such as RAGE expression-related diseases or pathologies.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] The AONs described herein may be formulated for intranasal administration, including dry powders, sprays, mists, or aerosols. This may be particularly preferable for the treatment of respiratory infections.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] AON for use in the method of the present invention may be 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 thereof 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.

[0164] 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, an appropriate "effective dose" in any given subject case can be determined by a person 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.

[0165] In any embodiment, the AON described herein is administered in an amount and manner effective to produce a maximum blood concentration of at least 200–400 nM of AON. Typically, one or more doses of AON are administered at regular intervals over a period of about 1–2 weeks.

[0166] In any embodiment, AON is administered in an amount of 0.024 mg / kg or more. In any embodiment, AON is administered in an amount of 0.24 mg / kg or more. In any embodiment, AON is administered in an amount of 0.5 mg / kg or less.

[0167] Medication depends on the severity and responsiveness of the disease condition being treated, and the duration of treatment ranges from several days to several months, or continues until a cure is achieved or the disease condition is reduced. The optimal medication schedule can be calculated from measurements of drug accumulation in the patient's body. Those skilled in the art can easily determine the optimal dosage, method of administration, and frequency of administration. The optimal dosage may vary depending on the relative potency of the target oligomer and can generally be estimated based on the EC50, which has been found to be effective in in vitro and in vivo animal models.

[0168] 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.

[0169] Also provided is a kit for use in the method of the present invention, in particular for measuring the level of endogenous soluble RAGE as described herein in a blood sample obtained from a subject. The kit includes at least one probe for detecting said endogenous soluble RAGE, along with instructions for use, the instructions for use describing the method of the present invention. The kit may further include the AON described herein for use in the method of the present invention.

[0170] The contents of the kit can be freeze-dried, and the kit may further contain a solvent suitable for reconstituting the freeze-dried components. The components of the kit are packaged in separate containers, and in connection with such containers 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.

[0171] 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.

[0172] As used herein, the singular forms "a," "an," and "the" also include plural references unless the context clearly indicates otherwise.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] It will be understood that the present invention, as disclosed and defined herein, encompasses all of two or more alternative combinations of subject matter features referred to or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention. [Examples]

[0177] Example 1 - Materials and Method animal A study to explore the shelf life of antisense oligonucleotides targeting mouse RAGE preRNA was conducted using healthy, pathogen-free male C57Bl / 6 mice (8-10 weeks old). The animals were housed in sterile passive microisolators at a constant temperature of 20°C in a 12-hour light / dark cycle, fed irradiated Barastoc mouse feed, and given free access to irradiated tap water. Mouse body weight was monitored throughout the experiment. The animals had free access to mouse feed and water throughout the study period. All experiments were approved by the local animal ethics committee and conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" (NIH Publication No. 85-23, revised 1996) issued by the National Institutes of Health.

[0178] Endotracheal treatment Mice were anesthetized with a ketamine / xylazine mixture (90 mg / 10 mg / kg body weight via intraperitoneal injection) and tracheal treatment was performed 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. A allocated 50 microliters of sterile solution was administered directly into the trachea via a blunt needle and high-pressure syringe. After the procedure, the mice were returned to their home cages and allowed to recover with a warm blanket. The mice were monitored until full recovery was observed, after which they were provided with moisture in the form of moistened tissue and moistened food pellets, with dry food pellets placed on the cage floor, and returned to the rearing room. The mice were then followed for 3, 7, 14, or 28 days and euthanized humanely using CO2 anesthesia.

[0179] Aerosol therapy Mice were anesthetized with a ketamine / xylazine mixture (90 mg / 10 mg / kg body weight via intraperitoneal injection) and tracheal treatment was performed 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. A allocated 50 microliters of sterile solution was administered directly into the trachea via an aerosolizer-type microsprayer (Penn-Century IA-1C) connected to a high-pressure syringe (Penn-Century FMJ-250). Between each administration of solution, the microsprayer and syringe were flushed three times with 250 μl of sterile ultrapure water and once with 250 μl of sterile saline. After the procedure, the mice were returned to their home cages, allowed to recover on paper towels, and kept warm by placing half of the cage on a heat pad. The mice were monitored until full recovery was observed, after which they were provided with moisture in the form of moistened tissue and moistened food pellets, and dry food pellets were placed on the bottom of the cage before being returned to the rearing room. The mice were then followed for 7 days, at which point they were humanely euthanized.

[0180] Sample collection and recovery Immediately after humane euthanasia, all mice were excised by collecting blood from the inferior vena cava using a lithium heparin-coated 19G syringe into a Microvette lithium / heparin tube (Sarstedt, Aust.). The blood was maintained at room temperature for at least 15 minutes and centrifuged at 2000g at 20°C for 5 minutes. The plasma was transferred to a 1.5 mL snap-lock Eppendorf tube, rapidly frozen in liquid nitrogen, and stored at -80°C. The lungs were perfused to remove circulating blood. A 20G needle was inserted into the right ventricle, and 5–10 mL of chilled sterile phosphate-buffered saline was administered at a rate of 300 μl / second. The lungs were then resected as shown in Figure 1. The left lobe was divided into upper, lower left (LL), and lower right (LR) portions and frozen in liquid nitrogen.

[0181] Measurement of esRAGE levels in plasma and 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.

[0182] The levels of extracellular soluble RAGE (esRAGE) in mouse plasma and the right lower right lobe of the lung, extracted with RIPA buffer, were estimated using a commercially available ELISA kit for mouse esRAGE (ELISA kit-MBS7606654-mybiosource). For lung tissue, the results were standardized to protein content (pg / 60ug protein) using a BCA assay (Pierce). Plasma results were standardized to volume (pg / ml plasma).

[0183] statistical analysis The data presented are mean ± SEM. 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.

[0184] Example 2 - Plasma esRAGE correlates with lung tissue esRAGE after endotracheal treatment with ASO m79. In this experiment, mice (6-8 per group) were treated with an intratracheal microspray of 50 µl of AON m79 (also known as ASO m79) (3 mg / kg; SEQ ID NO: 27) or a control oligonucleotide (GCAGUUGGCCCCUCCUC; SEQ ID NO: 33). ASO m79 is a 25-mer mouse antisense oligonucleotide that, upon administration to cells, promotes RAGE_v1 mRNA expression and therefore esRAGE production. AON m79 is an exemplary AON, having its most 5' nucleotide at position 88 or 114 of exon 10, or located at nucleotide positions 88-114 of exon 10. esRAGE levels in left lung tissue and plasma were measured on day 14 (Figure 2).

[0185] Plasma esRAGE on day 14 was strongly correlated with lung tissue esRAGE. In contrast, the control group showed a significant correlation between plasma and lung tissue esRAGE levels (Figure 3).

[0186] This data suggests that plasma esRAGE levels can be used as a biomarker to determine the effectiveness of ASO m79 treatment in the lungs.

[0187] Example 3 - Plasma esRAGE correlates with lung tissue esRAGE after intratracheal treatment with ASO2. In this experiment, mice were treated with an intratracheal microspray of 50 µl of ASO2 (0.3 or 3.0; 8 mice per group) or physiological saline (vehicle; 4 mice per group) (Figure 4). ASO2 is a human 18-mer antisense oligonucleotide with modified nucleotide bases, and upon administration to cells, it promotes the expression of RAGE_v1 mRNA and, therefore, the production of esRAGE. esRAGE levels in lung tissue and plasma were measured on days 10 and 14 after treatment with ASO2.

[0188] Plasma esRAGE on days 10 and 14 after a single dose of 3 mg / kg or 0.3 mg / kg of ASO2 strongly correlated with lung tissue esRAGE (p<0.01) (Figure 5).

[0189] Similar statistically significant correlations were observed when ASO2 was administered at doses of 10 mg / kg and 3 mg / kg using an intratracheal microsprayer (Figures 6 and 7).

[0190] These results, taken together, suggest that plasma esRAGE can be used as a biomarker to determine the level of esRAGE in the lung after treatment with AONs that modulate premRNA RAGE splicing.

Claims

1. A method for determining the level of endogenous soluble advanced glycation end product receptors (RAGEs) in the lung of a subject, wherein the method is Determining the presence of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving treatment administered to the airway, including determining whether the treatment increases endogenous soluble RAGE. A method wherein the level of endogenous soluble RAGE in the blood sample correlates with the level of endogenous soluble RAGE in the lung of the subject.

2. A method for determining the level of endogenous soluble advanced glycation end product receptor (RAGE) in a target blood sample, wherein the method is Determining the presence of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving treatment administered to the airway, including determining whether the treatment increases endogenous soluble RAGE. A method for determining endogenous soluble RAGE in the blood sample of the subject.

3. A method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method is This includes determining the presence of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving an antisense oligonucleotide (AON) in the airway that promotes splicing in the RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10, A method wherein the presence of the endogenous soluble RAGE in the blood sample indicates splicing of the advanced glycation end product receptor (RAGE) premRNA, resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

4. A method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method is To provide a blood sample from a subject who has received or is receiving an antisense oligonucleotide (AON) in the airway, which promotes splicing in the RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. This includes measuring the level of endogenous soluble RAGE in the blood sample, A method wherein the level of endogenous soluble RAGE in the blood sample indicates splicing of the RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

5. A method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method is To provide a blood sample from a subject who has received or is receiving an antisense oligonucleotide (AON) in the airway, which promotes splicing in the RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. This includes measuring the level of endogenous soluble RAGE in the blood sample, A method wherein a level of endogenous soluble RAGE in the blood sample exceeding a threshold level indicates splicing of the RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

6. A method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method is To provide a blood sample from a subject before receiving an antisense oligonucleotide (AON) into the airway, which promotes splicing in the RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. To provide a blood sample from the subject after receiving AON in the airway, which promotes splicing in the RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. This includes measuring the level of endogenous soluble RAGE in each of the aforementioned blood samples, A method in which a higher level of endogenous soluble RAGE in a blood sample from a subject after receiving the AON into the airway, compared to the level of endogenous soluble RAGE in the blood sample from the subject before receiving the AON into the airway, indicates splicing of RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

7. A method for determining splicing of an advanced glycation end product receptor (RAGE) premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in a subject, wherein the method is Obtain a blood sample from a subject before receiving an antisense oligonucleotide (AON) into the airway, which promotes splicing in the RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. Obtain a blood sample from the subject after receiving AON in the airway, which promotes splicing in the RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10. This includes measuring the level of endogenous soluble RAGE in each of the aforementioned blood samples, A method in which a higher level of endogenous soluble RAGE in a blood sample from a subject after receiving the AON in the airway compared to the level of endogenous soluble RAGE in the blood sample from the subject before receiving the AON indicates the splicing of RAGE premRNA resulting in the inclusion of exon 9b and / or exclusion of exon 10 in the subject.

8. A method for determining whether treatment / AON can increase endogenous soluble RAGE levels in the airway, wherein the method is Investigative treatment / administer AON to the aforementioned airway, This includes obtaining a blood sample from the subject after the administration, A method in which the presence or increase in the level of endogenous soluble RAGE in the blood sample indicates that the test treatment / AON increases the level of endogenous soluble RAGE in the airway.

9. The method according to any one of claims 1 to 8, wherein the blood sample includes or consists of whole blood, plasma, or serum.

10. The method according to any one of claims 2 to 9, wherein the blood sample is obtained at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, or at least about 28 days after the subject has received an antisense oligonucleotide (AON) in the airway that promotes splicing in the RAGE premRNA and results in the inclusion of exon 9b and / or exclusion of exon 10.

11. The method according to any one of claims 2 to 10, wherein the AON promotes the production of endogenous soluble RAGE by promoting the inclusion of exon 9b and / or the exclusion (e.g., skipping) of exon 10.

12. The method according to any one of claims 2 to 11, wherein the AON results 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.

13. The method according to any one of claims 2 to 12, wherein the AON is administered to the whole airway, the upper airway, or the lower airway.

14. The method according to claim 13, wherein the AON is administered to the lower respiratory tract.

15. The method according to claim 14, wherein the AON is administered to one or more of the following areas: the larynx below the vocal cords, the trachea, the bronchi, and the bronchioles.

16. The method according to claim 14, wherein the AON is administered to the lungs.

17. The method according to any one of claims 2 to 16, wherein the AON is administered as an aerosol.

18. The method according to any one of claims 2 to 17, wherein the AON is 10 to 50 nucleotides containing a target sequence complementary to a region near or within the intron of the RAGE premRNA.

19. The method according to any one of claims 2 to 17, wherein the AON is 10 to 50 nucleotides comprising a target sequence complementary to or adjacent to the splice site of the RAGE premRNA.

20. The method according to any one of claims 2 to 17, wherein the AON is a cis-acting RNA element in the premRNA of RAGE, and comprises 10 to 50 nucleotides including a target sequence complementary to or adjacent to the cis-acting RNA element, which acts as an enhancer or silencer that modulates the splicing of nearby exons when a spliceosome component binds to it.

21. The method according to any one of claims 2 to 17, wherein the AON is 10 to 50 nucleotides containing a target sequence complementary to the RAGE premRNA, and the secondary structure of the mRNA is modified to influence the selection of a splice site.

22. The method according to any one of claims 2 to 21, wherein the AON is isolated or purified AON for inducing the exclusion of one or more exon sequences in the RAGE gene transcript or a portion thereof.

23. The method according to any one of claims 2 to 22, wherein the AON is isolated or purified AON for inducing the retention of an intron sequence in the RAGE gene transcript or a part thereof.

24. The method according to any one of claims 2 to 23, wherein the AON comprises at least one modified nucleotide.

25. The method according to any one of claims 2 to 24, wherein the AON is chemically modified to prevent degradation of the premRNA-AON complex.

26. The method according to any one of claims 2 to 25, wherein the AON comprises one or more nucleotide modifications selected from the group consisting of phosphorodiamidate morpholino oligomer (PMO), 2'-O-methylphosphorothioate oligonucleotide (2OMe), and 2'-O-methoxyethyl phosphorothioate oligonucleotide (2MOE), locked nucleic acid (LNA) modified AON, heat-stable twist intercalating nucleic acid (TINA), and peptide nucleic acid (PNA).

27. The method according to any one of claims 2 to 26, wherein the AON is conjugated into at least one portion to increase their delivery.

28. The method according to claim 27, wherein the portion is one or more of a cell-permeable peptide (CPP), vivo-morpholino (VMO), and peptide phosphorodiamidate morpholino oligomer (PPMO).

29. The method according to any one of claims 2 to 28, 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.

30. The method according to claim 29, wherein the nucleotide furthest 5' of AON is nucleotide position 88 or 114 of exon 10, or is located at nucleotide positions 88 to 114 of exon 10.

31. The method according to claim 30, wherein the nucleotide position of exon 10 correlates with the nucleotide sequence of sequence number 32.

32. The method according to any one of claims 2 to 31, wherein the AON is 8 to 40 nucleotides long, 15 to 25 nucleotides long, or 18 nucleotides long.

33. The method according to any one of claims 2 to 32, wherein the AON is selected from the group including the sequences shown in any one of Tables 1a to 1d.

34. The method according to claim 33, wherein the AON is selected from a list containing sequence numbers 1 to 31, or nucleotide sequences that are at least 85%, 90%, or 95% identical thereto.

35. The method according to claim 34, wherein the AON is a nucleotide sequence identical to sequence number 11, 18, 19, or 20, or at least 85%, 90%, or 95% thereof.

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