Method for monitoring splicing

Abstract

The present invention relates to a method for identifying a RAGE precursor mRNA splice that results in inclusion of exon 9b and / or exclusion of exon 10 in a subject that has received or is receiving an antisense oligonucleotide that modulates the RAGE precursor mRNA splice. In one aspect, the invention provides a method for determining an endogenous soluble advanced glycosylation end product receptor (RAGE) level in a lung of a subject, the method comprising: determining the presence of an endogenous soluble RAGE in a blood sample from the subject that has received or is receiving a treatment administered to a respiratory tract thereof, wherein the treatment increases endogenous soluble RAGE, and wherein the level of endogenous soluble RAGE in the blood sample is associated with the level of endogenous soluble RAGE in the lung of the subject.

Description

Technical Field

[0001] This invention relates to a method for determining the levels of receptor for advanced glycation end products (RAGE), particularly endogenous soluble RAGE isotypes or portions thereof, in subjects who are receiving or have received treatment that regulates RAGE precursor mRNA splicing (e.g., antisense oligonucleotides).

[0002] Related applications This application claims priority to Australian Provisional Application No. 2023901363, filed on 5 May 2023, and International Application No. PCT / AU2023 / 050959, filed on 5 October 2023, the entire disclosures of which are incorporated herein by reference. Background Technology

[0003] The receptor for advanced glycation end products (RAGE) is a multivalent type I transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. Human RAGE ( Ager The gene is located in 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] 50 kDa-55 kDa glycosylated RAGE proteins are constitutively expressed in a limited range of cells (e.g., vascular endothelium, type I lung cells, leukocytes), although RAGE expression 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. Therefore, RAGE expression is upregulated in inflammatory and metabolic diseases, including but not limited to neurodegenerative diseases, cancer, cardiovascular diseases, diabetes, autoimmune diseases, and ischemic injuries, in which RAGE is also associated with the development and progression of these diseases.

[0005] Under healthy conditions, RAGE expression is highest in the lungs among all tissues. Upregulation of RAGE signaling in other lung cells and other sites is associated with a range of lung diseases, including: chronic obstructive pulmonary disease (COPD) / emphysema; asthma; damage from smoking / pollution; acute lung injury / acute respiratory distress syndrome; and pulmonary fibrosis.

[0006] Alternative splicing of RAGE is also important for regulating RAGE activity by generating RAGE isoforms with the ability to be altered 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] Antisense oligonucleotides (AONs) can be used to modulate alternative splicing of RAGE by targeting precursor mRNA RAGE. These AONs can be administered systemically or locally to alter the expression and / or activity of RAGE isotypes.

[0008] When administering AON to a subject's respiratory tract, a minimally invasive method is needed to determine the alternative splicing of RAGE precursor mRNA.

[0009] References to any prior art in the specification do not imply an admission or implication that such prior art forms part of common general knowledge in any jurisdiction, or that such prior art could reasonably be expected to be understood, considered relevant, and / or combined with other prior art by a person skilled in the art. Summary of the Invention

[0010] In one aspect, the present invention provides a method for determining the level of endogenous soluble receptor for advanced glycation end products (RAGE) in the lungs of a subject, the method comprising: - Determine the presence of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving treatment administered to their respiratory tract that increases endogenous soluble RAGE. The level of endogenous soluble RAGE in the blood sample was correlated with the level of endogenous soluble RAGE in the subject's lungs.

[0011] In one aspect, the present invention provides a method for determining the presence or level of endogenous soluble receptor for advanced glycation end products (RAGE) in a blood sample of a subject, the method comprising: - Determine the presence or level of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving treatment administered to their respiratory tract that increases endogenous soluble RAGE. This allows us to determine the presence or level of endogenous soluble RAGE in the subject's blood sample.

[0012] In one aspect, the present invention provides a method for determining splicing of precursor mRNA of receptor for advanced glycosylation (RAGE), the splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10 in a subject, the method comprising: - Identify the presence of endogenous soluble RAGE in blood samples from subjects who have received or are receiving antisense oligonucleotides (AONs) via the respiratory tract. These AONs promote splicing in RAGE precursor mRNA, leading to the inclusion of exon 9b and / or the exclusion of exon 10. The presence of endogenous soluble RAGE in the blood sample indicates that splicing of the receptor for advanced glycation end products (RAGE) precursor mRNA resulted in the inclusion of exon 9b and / or the exclusion of exon 10 in the subject.

[0013] In another aspect, the present invention provides a method for determining splicing of receptor for advanced glycation end products (RAGE) precursor mRNA in a subject, the splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10, the method comprising: - Provide a blood sample from a subject who has received or is receiving an antisense oligonucleotide (AON) via the respiratory tract, wherein the AON promotes splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. - Measure the level of endogenous soluble RAGE in this blood sample. The level of endogenous soluble RAGE in the blood sample indicates splicing of RAGE precursor mRNA in the subject, which results in the inclusion of exon 9b and / or the exclusion of exon 10.

[0014] In another aspect, the present invention provides a method for determining splicing of receptor for advanced glycation end products (RAGE) precursor mRNA in a subject, the splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10, the method comprising: - Provide a blood sample from a subject who has received or is receiving antisense oligonucleotides (AONs) via the respiratory tract. These AONs promote splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. - Measure the level of endogenous soluble RAGE in this blood sample. The presence of endogenous soluble RAGE levels above a threshold in the blood sample indicates splicing of RAGE precursor mRNA in the subject, which results in the inclusion of exon 9b and / or the exclusion of exon 10.

[0015] In another aspect, the present invention provides a method for determining splicing of receptor for advanced glycation end products (RAGE) precursor mRNA in a subject, the splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10, the method comprising: - Provide a blood sample from the subject prior to receiving an antisense oligonucleotide (AON) via the respiratory tract. This AON promotes splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. - Provide a blood sample from the subject after receiving AON via the respiratory tract. AON promotes splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. -Measure the endogenous soluble RAGE level in each of the blood samples. The levels of endogenous soluble RAGE in blood samples from subjects who received AON via the respiratory tract were higher than those in blood samples from subjects who received AON via the respiratory tract, indicating splicing of RAGE precursor mRNA in the subjects, which resulted in the inclusion of exon 9b and / or the exclusion of exon 10.

[0016] In another aspect, the present invention provides a method for determining splicing of receptor for advanced glycation end products (RAGE) precursor mRNA in a subject, the splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10, the method comprising: - A blood sample was obtained from the subject prior to receiving an antisense oligonucleotide (AON) via the respiratory tract. This AON promotes splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. - Blood samples were obtained from subjects after receiving AON via the respiratory tract. This AON promotes splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. -Measure the endogenous soluble RAGE level in each of the blood samples. The levels of endogenous soluble RAGE in blood samples from subjects who received AON via the respiratory tract were higher than those in blood samples from subjects who received AON before receiving AON, indicating splicing of RAGE precursor mRNA in the subjects, which resulted in the inclusion of exon 9b and / or the exclusion of exon 10.

[0017] In another aspect, the present invention provides a method for determining whether treatment / AON can increase the level of endogenous soluble RAGE in the respiratory tract, the method comprising: - Administer the experimental treatment to the subject's respiratory tract / AON, - Obtain a blood sample from the subject after administration. The presence or increase in the level of endogenous soluble RAGE in the blood sample indicates that the experimental treatment / AON increases the level of endogenous soluble RAGE in the respiratory tract.

[0018] In any aspect or implementation thereof, the blood sample includes or consists of whole blood, plasma, or serum.

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

[0020] In any respect, a blood sample is obtained at least approximately 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 has received an antisense oligonucleotide (AON) in the respiratory tract, and the AON promotes splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0021] In any respect, a blood sample is obtained at least 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 has received an antisense oligonucleotide (AON) in the respiratory tract, and the AON promotes splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0022] In any respect, when blood samples were 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 had received an antisense oligonucleotide (AON) in the respiratory tract, the AON promoted splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0023] In any respect, AON can promote the production of endogenous soluble RAGE by facilitating the inclusion of exon 9b and / or the exclusion (e.g., skipping) of exon 10. Therefore, in any respect, AON can promote splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the skipping of exon 10. For example, AON can lead to 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 respiratory tract, 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 respect, the endogenous soluble RAGE can 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 a polypeptide encoded by RAGE_v1 (i.e. esRAGE).

[0025] In any case, AON is applied to the entire respiratory tract, the upper respiratory tract, or the lower respiratory tract.

[0026] As used herein, the upper respiratory tract may include one or more of the following areas: the nose and nasal cavity, paranasal sinuses, pharynx, and the larynx above the vocal cords (vocal folds). The lower respiratory tract typically includes one or more of the following areas below the vocal cords: the larynx, trachea, bronchi, and bronchioles. The lungs may be classified as part of the lower respiratory tract and include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0027] In any respect, AON is administered as an aerosol, dry powder, or nasal drops. In any implementation, AON may be administered using a nasal spray pump, intranasal device, intratracheal instillation, metered-dose inhaler (MDI), dry powder inhaler (DPI), nebulizer (jet, ultrasonic mesh, or vibrating mesh), or soft fog inhaler (SMI).

[0028] In any respect, an AON is an AON of 10 to 50 nucleotides containing a targeting sequence complementary to a region near or within an intron of the RAGE precursor mRNA. Alternatively, an AON is an AON of 10 to 50 nucleotides containing a targeting sequence complementary to or adjacent to a splice site of the RAGE precursor mRNA.

[0029] Because factors such as RNA secondary structure, competition between AON and SR proteins, heterologous ribonucleoproteins (hnRNPs), and / or other elements constituting the spliceosome can affect the function of AON, AON targeting key receptor or donor splicing sites does not always alter splicing. Therefore, in any aspect of the invention, the AON is 10 to 50 nucleotides containing a target sequence complementary to or adjacent to a cis-acting RNA element in the precursor mRNA of RAGE that acts as an enhancer or silencer, and which regulates the splicing of nearby exons when bound to elements of the spliceosome (e.g., protein splicing factors, uRNA, lncRNA).

[0030] In any respect, AON is 10 to 50 nucleotides containing a targeting sequence complementary to the RAGE precursor mRNA, which regulates the secondary structure of the mRNA to influence splice site selection.

[0031] In any respect, AON is isolated or purified AON used to induce the exclusion (also known as skipping) of one or more exon sequences in the RAGE gene transcript or a portion thereof.

[0032] In any respect, AON is used to induce isolated or purified AON that retains intron sequences in the RAGE gene transcript or a portion thereof.

[0033] In any respect, AON contains at least one modified nucleotide. Typically, AON is chemically modified to prevent degradation of the precursor mRNA-AON complex, including but not limited to phosphodiamidomorpholino oligomers (PMO), 2'-O-methylphosphothioester oligonucleotides (2OMe) and 2'-O-methoxyethylphosphothioester oligonucleotides (2MOE), locked nucleic acid (LNA) modified AONs, thermally stable twisted intercalated nucleic acids (TINA), and peptide nucleic acids (PNA).

[0034] In any respect, AON comprises at least one modified nucleotide selected from the following: phosphodiamidomorpholino oligomer (PMO), 2'-O-methyl oligonucleotide (2OMe), 2'-O-methoxyethyl oligonucleotide (2'-MOE), phosphate thioester oligonucleotide, locked nucleic acid (LNA) modified AON, thermally stable twisted intercalated nucleic acid (TINA), and peptide nucleic acid (PNA).

[0035] In any respect, AON can be conjugated with certain moieties to enhance their delivery, including but not limited to cell-penetrating peptides (CPP), in vivo morpholino oligomers (VMO), or peptide phosphodiesteromorpholino oligomers (PPMO).

[0036] In any respect, the antisense oligonucleotide comprises, is substantially composed of, or is composed of the following nucleotide sequences: 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 precursor mRNA throughout the antisense oligonucleotide. Preferably, the 5' nucleotide of the AON is the 88th or 114th nucleotide of exon 10, or between the 88th and 114th nucleotides of exon 10. Preferably, the 5' nucleotide of the target region is the 88th or 114th nucleotide of exon 10, or between the 88th and 114th nucleotides of exon 10.

[0037] In any implementation, the 5' nucleotide of the target region is either position 88 or 113 of exon 10 of the RAGE precursor mRNA, or between positions 88 and 113.

[0038] In any implementation, the 5' nucleotide of the target region is the 90th or 113th exon 10 of the RAGE precursor mRNA, or between the 90th and 113th nucleotides.

[0039] In any implementation, the 5' nucleotide of the target region is the 88th or 108th exon 10 of the RAGE precursor mRNA, or between the 88th and 108th nucleotides.

[0040] In any implementation, the 5' nucleotide of the target region is the 90th or 108th exon 10 of the RAGE precursor mRNA, or between the 90th and 108th nucleotides.

[0041] In any implementation, the 5' nucleotide of the target region is the 88th or 95th exon 10 of the RAGE precursor mRNA, or between the 88th and 95th nucleotides.

[0042] In any implementation, the target region is located at nucleotide positions 88 through 137 of exon 10 of the RAGE precursor mRNA.

[0043] In any implementation, the target region is derived from nucleotides 88 through 107 of exon 10 of the RAGE precursor mRNA.

[0044] In any implementation, the target region is derived from nucleotides 90 through 102 of exon 10 of the RAGE precursor mRNA.

[0045] In any implementation, the target region is derived from nucleotides 95 through 119 of exon 10 of the RAGE precursor mRNA.

[0046] In any implementation, the target region is between nucleotides 108 and 132 of exon 10 of the RAGE precursor mRNA.

[0047] In any implementation, the target region is between nucleotides 113 and 137 of exon 10 of the RAGE precursor mRNA.

[0048] In any respect, the length of an AON can be 8 to 40 nucleotides, 15 to 25 nucleotides, or 18 nucleotides.

[0049] In any embodiment, AON is selected from sequences listed in any one of Tables 1a to 1d. Preferably, AON is selected from SEQ ID NO: 1-31, for example, AON is a nucleotide sequence of SEQ ID NO: 11, 18, 19 or 20 or having at least 85%, 90% or 95% identity with it.

[0050] The AON used in the method of the present invention can be selected as an AON capable of binding to a selected target site, wherein the target site is a putative mRNA splicing site selected from splice donor sites, splice acceptor sites, splice enhancer sequences, splice silencing sequences, or sites regulating the secondary structure of precursor mRNA. When a donor or acceptor splice site is targeted, the target site may also include some flanking intron sequences.

[0051] More specifically, the AON may be selected from any one or more of SEQ ID NO: 1-31 and / or the sequences listed in any one of Tables 1a to 1d, as well as combinations or mixtures thereof. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. Combinations of AON are preferably combinations of SEQ ID NO: 11 and 10 or SEQ ID NO: 11 and 13. This includes sequences that can hybridize with such sequences under strict hybridization conditions, sequences complementary to them, sequences containing modified bases, modified backbones, and sequences with functional truncated or extended portions that have or regulate precursor mRNA processing activity in RAGE gene transcripts.

[0052] In some implementations, the AON may be 100% complementary to the target sequence, or may include mismatches, such as to accommodate variants, provided that the heteroduplex formed between the oligonucleotide and the target sequence is sufficiently stable to withstand the action of cellular nucleases and other degradation modes that may occur in vivo. Thus, certain oligonucleotides may have about or at least about 70% sequence complementarity with 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.

[0053] The method of the present invention is also extended to combinations of two or more AONs capable of binding to selected targets to modulate alternative splicing of RAGE precursor mRNA, including constructs containing two or more such AONs. These constructs can be used together for AON-based combination therapies. The combination of AONs is preferably a combination of SEQ ID NO: 11 and 10 or SEQ ID NO: 11 and 13.

[0054] As used herein, unless the context otherwise requires, the term “comprising” or variations thereof, such as “including” and “containing”, is not intended to exclude additional additives, components, whole or steps.

[0055] Other aspects of the invention and other embodiments of the various aspects described in the foregoing paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Attached Figure Description

[0056] Figure 1 Segmentation of mouse lungs as described in the embodiments of this article.

[0057] Figure 2 Overview of experimental design. Time-dependent pharmacokinetic activity of a single intratracheal administration of ASO m79 (3 mg / kg) in male C57BL / 6 mice.

[0058] Figure 3 Lung esRAGE protein levels were correlated with plasma esRAGE concentrations measured 14 days after a single endotracheal treatment with ASO m79. Male C57BL / 6 mice were administered either control oligonucleotides 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 presented as mean ± SEM; *p < 0.05 compared to control oligonucleotides.

[0059] Figure 4Overview of the experimental design. Time-dependent pharmacokinetic activity of a single nebulized dose (3 mg / kg or 0.3 mg / kg) of 18-mercury ASO (ASO2) in male C57BL / 6 mice.

[0060] Figure 5 Lung esRAGE protein levels were correlated with plasma esRAGE concentrations measured after a single nebulization with ASO2. Male C57BL / 6 mice were administered the solvent, 0.3 mg / kg ASO2, or 3 mg / kg ASO2 on day 0, and esRAGE levels in left lung tissue and plasma were measured on days 10 (a) and 14 (b). Compared to the solvent, both 0.3 mg / kg and 3 mg / kg ASO2 levels showed a statistically significant increase in esRAGE levels (Spilman correlation p < 0.05).

[0061] Figure 6 Overview of experimental design. Dose-dependent pharmacokinetic activity of a single inhalation treatment with ASO2 (delivered via a microneedle) in male C57BL / 6 mice.

[0062] Figure 7 Lung esRAGE protein levels were correlated with plasma esRAGE concentrations measured 7 days after a single ASO2 inhalation treatment. Male C57BL / 6 mice were administered the solvent, 3 mg / kg ASO2, or 10 mg / kg ASO2 on day 0, and esRAGE levels in left lung tissue and plasma were measured on day 7. Compared with the solvent, both 3 mg / kg and 10 mg / kg ASO2 levels showed a statistically significant increase in esRAGE levels (Spilman correlation p < 0.05). Detailed Implementation

[0063] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned in or clearly visible from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0064] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with embodiments, it should be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined by the claims.

[0065] Those skilled in the art will recognize that many methods and materials are similar to or equivalent to those described and materials herein, and that can be used in the practice of this invention. This invention is by no means limited to the methods and materials described. It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned in or clearly apparent from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0066] All patents and publications mentioned in this article are incorporated in full by way of citation.

[0067] For the purposes of interpreting this specification, terms used in the singular will also include the plural forms, and vice versa.

[0068] Alternative splicing of RAGE precursor mRNA Alternative splicing is considered a crucial aspect of posttranscriptional gene regulation of receptors for advanced glycosylation (RAGE). Although most RAGEs are expressed in their full-length isoform, many different coding isoforms arise through alternative splicing (also known as splice forms), including splice forms with N-terminal and C-terminal truncations, and splice forms that retain intron sequences. These different splice forms can act as potential regulators of the full-length RAGE receptor by competitive ligand binding or by replacing the full-length protein with its binding partner. More than twenty splice forms have been identified in various tissues, such as the lung, liver, kidney, smooth muscle, endothelial cells, and brain.

[0069] Different RAGE gene splicing variants are named RAGE, RAGE_v1 to RAGE_v19 according to the Human Genome Nomenclature Committee, and described in Hudson et al., (2008) The FASEB Journal, Vol. 22: pp. 1572-1580, the contents of which are incorporated herein by reference in full.

[0070] As used herein, “endogenous soluble RAGE” refers to a polypeptide lacking any signaling elements and / or transmembrane domains of a full-length RAGE. Endogenous soluble RAGEs can be encoded by alternatively spliced ​​RAGE precursor mRNAs, which are subsequently translated, resulting in the inclusion of exon 9b and / or the exclusion (e.g., skipping) of exon 10, leading to premature termination and complete loss of both transmembrane and cytoplasmic domains. For example, endogenous soluble RAGEs can be encoded by one or more of the following mRNAs: RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19, with RAGE_v1 being preferred. Therefore, it should be understood that any AON that promotes splicing in RAGE precursor mRNA as described herein, resulting in the inclusion of exon 9b and / or the exclusion (e.g., skipping) of exon 10, can be used in the methods of the present invention to increase “endogenous soluble RAGE” in the respiratory tract and plasma of the subject.

[0071] For example, “endogenous soluble RAGE” as described herein can be endogenous secretory RAGE (esRAGE) encoded by RAGE_v1 mRNA. esRAGE accounts for approximately 5% of circulating RAGE in the human body. The skipping of exon 10 in esRAGE-type splicing is attributed to the limitation of intron length in higher eukaryotes. There must be approximately 45 nucleotides between the 5' splice site and the branching point, and the minimum distance between the branching point and the 3' splice site appears to be approximately 18 nucleotides. Therefore, introns shorter than 70 nucleotides are extremely rare in mammals and cannot be spliced ​​efficiently. When the esRAGE 5' splice site in intron 9 is chosen, the distance between this site and the 3' splice site of the adjacent exon 10 is 46 nucleotides, which is much shorter than the lower limit of intron length. Therefore, using the esRAGE 5' splice site downstream of intron 9 and containing exon 10 would be mutually exclusive. Among the known splicing variants analyzed, all variants using the downstream esRAGE 5' splice site in intron 9 skip exon 10; conversely, all variants using the upstream RAGE 5' splice site in intron 9 include exon 10. Therefore, available evidence suggests that the selection of either of the two alternative 5' splice sites in intron 9 is related to the inclusion or exclusion of exon 10. This mode of splicing regulation, or external control, was previously unknown.

[0072] sample In any aspect or implementation thereof, the blood sample includes or consists of whole blood, plasma, or serum.

[0073] As used herein, samples may be obtained from a subject or from components of a subject, such as cells. Samples may be “clinical samples,” i.e., samples derived from a patient. In one embodiment, the method of the present invention is not performed on a human or animal body; for example, measurements of endogenous soluble RAGE levels may be determined by analyzing previously obtained blood samples.

[0074] In one embodiment, the blood sample may be free of red blood cells (e.g., in the form of plasma, serum, or untreated whole blood). In one embodiment, red blood cells are absent or not present at significant levels when measuring endogenous soluble RAGE in the blood sample. In another embodiment, red blood cells are present at normal levels, i.e., they have not been removed from the blood sample.

[0075] In any respect, a blood sample is obtained at least approximately 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 has received an antisense oligonucleotide (AON) in the respiratory tract, and the AON promotes splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0076] In any respect, a blood sample is obtained at least 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 has received an antisense oligonucleotide (AON) in the respiratory tract, and the AON promotes splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0077] In any respect, when blood samples were 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 had received an antisense oligonucleotide (AON) in the respiratory tract, the AON promoted splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0078] In any respect, when a blood sample is obtained approximately 7 to 28 days, approximately 7 to 27 days, approximately 7 to 26 days, approximately 7 to 25 days, approximately 7 to 24 days, approximately 7 to 23 days, approximately 7 to 22 days, approximately 7 to 21 days, approximately 7 to 20 days, approximately 7 to 19 days, approximately 7 to 18 days, approximately 7 to 17 days, approximately 7 to 16 days, approximately 7 to 15 days, or approximately 7 to 14 days after the subject receives an antisense oligonucleotide (AON) via the respiratory tract, the AON promotes splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0079] In any respect, when blood samples were obtained 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 received antisense oligonucleotide (AON) in the respiratory tract, the AON promoted splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0080] Alternatively, in any respect, a blood sample may be obtained no more than 28 days after the subject receives an antisense oligonucleotide (AON) via the respiratory tract, which promotes splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. For example, a blood sample may be obtained no more than 7 days, no more than 14 days, or no more than 28 days after the subject receives an antisense oligonucleotide (AON) via the respiratory tract, which promotes splicing in the RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0081] In any respect, the blood sample can be obtained prior to use in the method of the present invention and then frozen. For example, a blood sample can be obtained and frozen for a period of time under storage conditions that substantially do not reduce the detectable level of endogenous soluble RAGE present in the blood sample before freezing.

[0082] In any implementation, umbilical cord blood erythrocytes are removed by ammonium chloride lysis, density gradient technology, hypotonic lysis, immunomagnetic cell separation or sedimentation, flow cytometry sorting, or equivalent methods understood by those skilled in the art.

[0083] Detection and measurement of endogenous soluble RAGE The method provided is based on the unexpected finding that the level or presence of endogenous soluble RAGE in blood samples indicates splicing of receptor for advanced glycation end products (RAGE) mRNA following administration of an AON that promotes splicing in RAGE precursor mRNA (resulting in the inclusion of exon 9b and / or the exclusion of exon 10), which leads to the inclusion of exon 9b and / or the exclusion of exon 10. Therefore, the level of endogenous soluble RAGE in blood can be used to monitor and determine the expression of alternative splicing of RAGE in the respiratory tract.

[0084] In any respect, the methods of the present invention relate to detecting the presence or measuring the level of endogenous soluble RAGE in a blood sample as described herein.

[0085] In any respect, the level of endogenous soluble RAGE in blood samples after respiratory administration of AON was higher or increased compared to the level of endogenous soluble RAGE in blood samples from subjects before AON administration.

[0086] 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 instruments. Typically, a detectable response is the presence of a signal in which the fluorophore itself exhibits fluorescence. Alternatively, a detectable response is an optical response that results in a change in wavelength distribution pattern or absorbance or fluorescence intensity, or a change in light scattering, fluorescence lifetime, fluorescence polarization, or a combination of these parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioactive isotopes, magnetic attraction, and electron density.

[0087] It should be 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 endogenous soluble RAGE expression levels can be direct or indirect.

[0088] In one instance, a labeling reagent (e.g., an antibody that specifically binds to endogenous soluble RAGE) can be used to measure the level or presence of endogenous soluble RAGE. As used herein, the term "label" refers to a chemical moiety or protein that, for example, when attached to an antibody, can be detected directly or indirectly (e.g., due to its spectral properties, conformation, or activity).

[0089] Antibodies that specifically bind to endogenous soluble RAGE (e.g., esRAGE) are known in the art and are commercially available. For example, esRAGE antibodies are available from Sigma-Aldrich, Australia (product number: MAB5328).

[0090] The detection marker conjugated with the reagent can be any marker that allows for individual detection and quantification by flow cytometry. For example, a fluorescent dye. Suitable fluorescent labels are known in the art and include fluorescein isothiocyanate (FITC), phycoerythrin (PE), polydinophyll-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), horizontal violet 450 (HV450), pacific orange (PacO), horizontal-V500 (HV500), Krome Orange, Brilliant Violet 421 (BV421), Brilliant Violet 510 (BV510), Brilliant Violet 605 (BV605), Brilliant Violet 650 (BV650), Brilliant Violet 711 (BV711), and 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 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 any other compatible fluorescent dye or tandem fluorescent dye, etc.

[0091] In one instance, the antibody was conjugated to (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, FITC or Alexa488, (4) phycoerythrin (PE), (5) polydiophytoxanthophyll chlorophyll protein / anthocyanin 5.5 (PerCP-Cy5.5), PerCP or PE-TexasRed, (6) phycoerythrin / anthocyanin 7 (PE-Cy7), (7) allophycocyanin (APC) or Alexa647, and (8) allophycocyanin / hilite7 (APC-H7), APC-Cy7, Alexa680, APC-A750, APC-C750 or Alexa700.

[0092] In another example, antibodies were 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) polydinophyte chlorophyll protein / anthocyanin 5.5 (PerCP-Cy5.5), (7) phycoerythrin (PE), (8) phycoerythrin / anthocyanin 7 (PE-Cy7), (9) allophycocyanin (APC) and (10) allophycocyanin / H7 (APC-H7), APC-C750 or APC-Alexa750.

[0093] Suitable markers can be attached to reagents directly or indirectly using appropriate labels. In a preferred embodiment, the marker can be detected by attaching to streptavidin.

[0094] The method of the present invention further includes contacting a blood sample with a reagent that allows for the identification of endogenous soluble RAGEs. Typically, the molecule is either bound to or is itself a detectable marker. For example, the molecule may be a fluorescent dye, an antibody, or an enzyme that causes substrate production. Alternatively, the reagent is linked to a tag that promotes binding to the detectable marker. For example, the tag may bind nonvalently to the detectable marker or form a covalent interaction with it. Suitable tags are known in the art and have been described herein.

[0095] For example, the reagent could be an antibody for detecting endogenous soluble RAGE, and the detectable label could be a fluorescent dye. Suitable fluorescent dyes are known in the art and have been described herein.

[0096] Preferably, the reagent is an antibody that specifically binds to the C-terminal domain of endogenous soluble RAGE as defined herein, but does not significantly bind to other RAGE isotypes. Due to the unique truncation of transmembrane and intracellular signaling domains, endogenous soluble RAGE proteins as described herein possess unique conformational epitopes not found on other RAGE isotypes, which can 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, such as the Human esRAGE ELISA Kit (HUFI04782; Assay Genie).

[0097] The binding of reagents linked to detectable labels to endogenous soluble RAGEs can be detected using biochemical techniques such as antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). These methods are performed as known in the art. ELISA kits suitable for detecting endogenous soluble RAGEs (e.g., esRAGE) are commercially available, for example, from mybiosource.com (catalog number MBS015358).

[0098] When a sample is combined with 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 performed as are known in the art.

[0099] In any respect, the level of endogenous soluble RAGE (e.g., esRAGE) measured in blood samples from subjects who have received or are receiving AON as described herein is at least about 1.1, 1.2, 1.5, 2, 3, 4, 5, 7, 10, 20, 100, and higher than the level of endogenous soluble RAGE measured in blood samples from the same subject prior to receiving AON.

[0100] The level of endogenous soluble RAGE in a blood sample from a subject can refer to the concentration, amount, level, relative concentration, relative amount, or activity of endogenous soluble RAGE. Typically, the level of endogenous soluble RAGE indicates the splicing of RAGE precursor mRNA in a subject (preferably within respiratory cells) that results in the inclusion of exon 9b and / or the exclusion of exon 10.

[0101] In any respect, measuring the level or presence of endogenous soluble RAGE includes performing an in vitro assay. In any embodiment, this 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), Western blotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry, surface plasmon resonance (SPR), chemiluminescence assay, lateral immunoassay, inhibition assay, or affinity assay.

[0102] In one technique for comparing protein expression levels from two different samples, each sample is subjected to 2D gel electrophoresis separately. Alternatively, each sample is differently labeled, and both samples are loaded onto the same 2D gel. See, for example, Unlu et al., Electrophoresis, 1997, Vol. 18: pp. 2071-2077, whose teachings on methods for assessing and comparing protein expression levels are incorporated herein by reference. The same proteins in each sample are identified by their relative positions within the protein map resolved by 2D electrophoresis. The expression levels of the proteins in the first sample are then compared with the expression levels of the same proteins in the second sample, thereby allowing the identification of proteins (e.g., biomarkers) that are expressed differently between the two samples. Preferably, this comparison is performed before and after the subject receives an AON that promotes splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

[0103] In another technique, the expression level of endogenous soluble RAGE in a single sample is expressed as a percentage of the total expressed protein. This assessed expression level is compared to a pre-existing reference standard, allowing the identification of endogenous soluble RAGE that is differentially expressed in the sample relative to the reference standard.

[0104] In any respect, increased endogenous soluble RAGE may involve comparison with a reference standard. The reference standard may be derived from one or more subjects who (a) may or may not have RAGE-related diseases or conditions and / or (b) have not been administered RAGE splicing AON as described herein. Therefore, if the expression level of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving AON is increased or higher than the expression level of endogenous soluble RAGE in the reference standard, preferably statistically significantly increased, then the subject is determined to have an increase in RAGE precursor mRNA splicing leading to the inclusion of exon 9b and / or the exclusion of exon 10, preferably in respiratory cells. In some embodiments, a statistically significant increase is equal to or greater than two standard deviations above the reference standard (example values ​​for the reference standard are described below). Alternatively, if the expression level of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving AON is the same as or not statistically significant compared to the expression level of endogenous soluble RAGE in reference standards, it is determined that the subject does not have an increase in RAGE precursor mRNA splicing leading to exon 9b inclusion and / or exon 10 exclusion, preferably in respiratory cells. Therefore, in any method or use of the invention, if it is determined that the subject does not have an increase in RAGE precursor mRNA splicing leading to exon 9b inclusion and / or exon 10 exclusion, the method or use further includes the step of administering another dose of AON as described herein.

[0105] As used herein, the threshold level for endogenous soluble RAGE can be determined from one or more control subjects who have not received treatment to increase endogenous soluble RAGE. In one embodiment, those control subjects may be healthy or otherwise normal subjects who do not have RAGE-mediated conditions or respiratory conditions or diseases as defined herein, and who have not received treatment to increase endogenous soluble RAGE. In another embodiment, those control subjects may have RAGE-mediated conditions or respiratory conditions or diseases as defined herein, and who have not received treatment to increase endogenous soluble RAGE. An exemplary threshold level or reference standard may be 250 pg / mL ± 25 pg / mL (mean ± SD).

[0106] Endogenous soluble RAGE data can be analyzed using various methods to identify endogenous soluble RAGE and determine the statistical significance of the observed differences in endogenous soluble RAGE levels between the test expression profile and the reference expression profile, for example in blood samples obtained before and after receiving AON as described herein, to demonstrate that AON treatment has led to the splicing of RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10 in the subject.

[0107] Antisense oligonucleotides (AON) Antisense oligonucleotides (AONs) are short, synthetic, antisense, modified DNA or RNA chains that can selectively hybridize with precursor RNA / mRNA through Watson-Crick base pairing and selectively regulate the function of target RNA.

[0108] The terms “AON” and “ASO” are both abbreviations for “antisense oligonucleotide” and are used interchangeably in this document.

[0109] When AONs are used to regulate alternative splicing of mRNA, they are often referred to as splice-conversion oligonucleotides (SSOs). In this invention, the terms AON and SSO are used interchangeably. SSOs base-pair with precursor mRNA and disrupt the normal splicing library of the transcript by blocking RNA-RNA base-pairing or protein-RNA binding interactions between components of the splicing mechanism and the precursor mRNA. SSOs can induce the “skipping” of selected exons and / or the retention of intron sequences to regulate the translation product. This can be achieved by directly targeting splice sites or by targeting cis-acting sequences that are involved in enhancing or silencing splicing by regulating the binding of specific proteins or altering the secondary structure of the precursor mRNA.

[0110] Therapeutic SSOs can be used to treat genetic disorders by bypassing defective or misaligned parts, allowing the production of proteins that are internally missing but now functional, as a treatment.

[0111] The AON used in the method of the present invention selectively manipulates the alternative splicing pattern of RAGE precursor mRNA, resulting in the production of native RAGE mRNA splice forms that are nonfunctional or act as decoy receptors to antagonize ligand-dependent activation and ligand-independent transactivation of full-length RAGE.

[0112] It is noteworthy that common RAGE polymorphisms were not observed at these splicing sites. RAGE sequences are highly conserved. Therefore, unlike managing genetic diseases using exon skipping techniques, personalized or subject-specific sequence modifications are not required.

[0113] According to any aspect of the method of the invention, the AON described herein is capable of binding to selected targets on the RAGE gene transcript to regulate precursor mRNA splicing in the RAGE gene transcript or a portion thereof. In a broad sense, the AON can be isolated or purified. "Isolated" means material that is substantially or substantially free of components normally associated with it in its natural state. For example, as used herein, "isolated polynucleotide" or "isolated oligonucleotide" can refer to a polynucleotide that has been purified or removed from a sequence flanking it in its natural state, such as a DNA fragment removed from a sequence adjacent to that fragment in the genome. The term "isolation," when referring to cells, means purification of cells (e.g., fibroblasts, lymphoblasts) from a source subject (e.g., a subject with a polynucleotide duplication disease). In the context of DNA, mRNA, or protein, "isolation" means recovery of DNA, mRNA, or protein from a source (e.g., cells).

[0114] According to any aspect of the method of the invention, AON as described herein promotes splicing in RAGE precursor mRNA that results in the inclusion of exon 9b and / or the exclusion (e.g., skipping) of exon 10, thereby leading to premature termination and complete loss of transmembrane and cytoplasmic domains. Thus, AON as 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, preferably increasing the levels of RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19 mRNA in tissues or samples. For example, AON as described herein can increase the level of RAGE_v1 mRNA in tissues or samples by promoting splicing in RAGE precursor mRNA, which leads to the skipping of exon 10 and the retention of intron 9 (exon 9b). In another instance, as described herein, AON can increase the level of RAGE_v10 mRNA in a tissue or sample by promoting splicing in RAGE precursor mRNA, which leads to the skipping of exons 10 and 11.

[0115] AON can be referred to as a "target" or "targeted" sequence to which it hybridizes. In some implementations, the target sequence includes a region containing a 3' or 5' splice site, branch point, or other sequence involved in splice regulation of the pretreated mRNA, including splice enhancers and splice silencers, as well as sites that determine RNA secondary structures that can affect splicing. The target sequence can be within an exon, within an intron, or across an intron / exon junction.

[0116] In any respect, the antisense oligonucleotide comprises, is substantially composed of, or consists of the following nucleotide sequences: 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 precursor mRNA throughout the antisense oligonucleotide. Preferably, the 5' nucleotide of AON is the 88th or 114th nucleotide of exon 10, or between the 88th and 114th nucleotides of exon 10. Preferably, the 5' nucleotide of the target region is the 88th or 114th nucleotide of exon 10, or between the 88th and 114th nucleotides of exon 10. Typically, the nucleotide positions may be the same as or equivalent to SEQ ID NO: 32, wherein the first nucleotide is position 1.

[0117]

[0118] In some embodiments, the AON has sufficient sequence complementarity with the target RNA (i.e., the RNA that regulates splice site selection) to effectively block regions of the target RNA (e.g., precursor mRNA). In exemplary embodiments, this blocking of the RAGE precursor mRNA regulates splicing by masking the binding sites of spliceosome proteins that would normally regulate splicing and / or by altering the structure of the target RNA. In some embodiments, the target RNA is a target precursor mRNA (e.g., RAGE gene precursor mRNA).

[0119] An AON having a sequence sufficiently complementary to the target RNA sequence to regulate target RNA splicing means that the AON has a sequence sufficient to trigger the masking of the natural protein binding site (which would normally regulate splicing) and / or alter the three-dimensional structure of the target RNA.

[0120] The selected AON can be shorter (e.g., about 12 bases) or longer (e.g., about 50 bases) and include a small number of mismatches, provided that the sequence is sufficiently complementary to enable splicing regulation when hybridizing with the target sequence, and optionally form a heteroduplex with RNA having a Tm of 45°C or higher.

[0121] Preferably, AON is selected from the sequences listed in SEQ ID NO: 1-31 and / or Tables 1a to 1d. More preferably, AON is SEQ ID NO: 11, 18, 19 or 20.

[0122] In some implementations, the complementarity between the target sequence and the AON is sufficient to form a stable double strand. The complementary region between the AON and the target RNA sequence can be as short as 8-11 bases, but can be 12-15 bases or more, such as 10-50 bases, 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers between these ranges. An AON of approximately 16-17 bases is generally long enough to have a unique complementary sequence. In some implementations, a minimum length of complementary bases may be required to achieve the necessary binding Tm, as discussed herein.

[0123] In some implementations, oligonucleotides up to 50 bases in length may be suitable, wherein at least a minimum number of bases (e.g., 10-12 bases) are complementary to the target sequence. However, in general, promoted or active uptake in the cell is optimal when the oligonucleotide length is less than about 30 bases. For the phosphodiamidomorpholino oligomer (PMO) AON further described herein, the optimal balance between binding stability and uptake typically occurs at a length of 18-25 bases. AONs consisting of about 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) are included.

[0124] In some implementations, the AON may be 100% complementary to the target sequence, or may include mismatches, such as to accommodate variants, provided that the heteroduplex formed between the oligonucleotide and the target sequence is sufficiently stable to withstand the action of cellular nucleases and other degradation modes that may occur in vivo. Thus, certain oligonucleotides may have about or at least about 70% sequence complementarity with 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.

[0125] Mismatches (if present) are generally less likely to cause instability at the terminal regions of a hybrid duplex than in the middle regions. According to well-known principles of duplex stability, the permissible number of mismatches will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the location of one or more mismatches within the duplex. Although such AONs are not necessarily 100% complementary to the target sequence, they bind efficiently, stably, and specifically to the target sequence, thereby regulating the splicing acceptance of the target precursor RNA.

[0126] The stability of the duplex formed between the AON and the target sequence depends on the binding Tm and the sensitivity of the duplex to enzymatic cleavage by the cell. The Tm of the oligonucleotide relative to the complementary RNA sequence can be measured by conventional methods, such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or those described in Miyada CG and Wallace RB, 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154, pp. 94-107. In some embodiments, the AON may have a binding Tm relative to the complementary RNA that is greater than body temperature and preferably greater than about 45°C or 50°C. Tm in the range of 60°C-80°C or greater is also included.

[0127] Other examples of variants include AONs having approximately or at least approximately 70% sequence identity or homology (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) over the entire length of any of the sequences listed in any of the tables 1a to 1d. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

[0128] Modifications to the splicing of precursor mRNA preferably induce the "skipping" or removal of one or more exons of the mRNA or the retention of introns. Due to internal truncation or premature termination, the resulting protein preferably has a shorter length compared to the parental full-length RAGE protein. Preferably, the resulting protein has a C-terminal truncation. These truncated RAGE proteins can be referred to as spliced ​​forms of the full-length RAGE protein.

[0129] The remaining exons of the resulting mRNA can remain within the reading frame and produce a shorter protein with a sequence similar to the full-length parental protein, except that the shorter protein has an internal truncation in the region between the original 3' and 5' ends. Alternatively, exon skipping can induce a frameshift, resulting in a protein whose first portion is substantially identical to the full-length parental protein, but whose 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 reading frame disruption and the presence of premature translation termination. These prematurely terminated proteins can result from premature mRNA termination (e.g., skipping of exons 10 and / or 11), or from readthrough to introns (e.g., RAGE 9b) or missense skipping, thus providing mRNA containing exons 10 and / or 11, but not from the expression of the protein encoded by those exons.

[0130] Skipping the target exons 1 through 9 will preferentially disrupt the reading frame of the RAGE transcript. This will lead to accelerated RNA degradation via nonsense-mediated decay.

[0131] Skipping the target exons 1 through 11 will preferably preserve the reading frame intact. This will preferably result in translation into an internally truncated protein. The truncated protein, or the RAGE mRNA splice form, may have complete loss of function, reduced function, or act as a decoy receptor.

[0132] Preferably, these truncated, nonsense, or prematurely terminated proteins lack one or more functional domains that are involved in RAGE ligand-induced intracellular signaling pathways or in the non-ligand-dependent transactivation of RAGE by co-localized GPCRs. For example, exon 10 encodes a transmembrane domain, and removal of this exon can produce a soluble RAGE protein that can potentially act as a soluble decoy or competitive antagonist for ligands involved in RAGE-induced signaling. Truncated, nonsense, or prematurely terminated proteins may also lack attachment or binding sites for other factors, the removal of which could lead to reduced interactions between the RAGE protein and related signaling pathways.

[0133] Alternatively, the removal of one or more exons may lead to misfolding of RAGE proteins and a reduced ability of proteins to be successfully transported across the membrane.

[0134] Preferably, there is an internally truncated protein (i.e., a protein lacking the amino acids encoded by one or more exons). If RAGE proteins are suppressed, there may be problems with increased RAGE transcription as the body attempts to compensate for the reduction in the total amount of RAGE proteins. Conversely, the presence of an internally truncated protein (preferably lacking one or more features of the complete RAGE protein) should be sufficient to prevent increased transcription, while still providing a therapeutic advantage due to the reduction in the total amount of functional RAGE proteins.

[0135] As described herein, AON-induced exon skipping does not require complete or even substantial elimination of RAGE protein function. Preferably, regulation of alternative splicing via the exon skipping process results in reduced or impaired RAGE protein function.

[0136] Different isoforms of RAGE generated using different skipping strategies can produce proteins with eliminated or reduced signal transduction activity, which can preferably be used to treat or prevent various diseases associated with RAGE activity, such as neurodegenerative diseases, cancer, lung diseases, or inflammatory diseases. Alternative splicing strategies can form truncated proteins or proteins with reduced function, which can preferably be used as a treatment for specific aspects, forms, or progressions of diseases associated with RAGE expression and activity.

[0137] Using the skipping process of AON can exclude (skip) the target exon, or can result in skipping two or more exons at once.

[0138] The skipping process using AON can include preserving the intron sequence while skipping one or more exons directly or without skipping them directly.

[0139] The AON used in the method of the present invention can be a combination of two or more AONs capable of binding to a selected target to induce exon exclusion in RAGE gene transcripts. This combination can be a mixture of two or more AONs and / or a construct containing two or more AONs linked together.

[0140] Table 1a: AON sequences used to regulate alternative splicing in human RAGE exon 9

[0141] Table 1b. Sequences of AONs used to regulate alternative splicing in human RAGE exon 10

[0142] Table 1c. Sequences of AONs used to regulate selective splicing in human RAGE intron 9.

[0143] Table 1d. Sequences of AONs used to regulate selective splicing in mouse RAGE

[0144] More specifically, the AON used in the method of the present invention may be selected from those AONs listed in any one of Tables 1a to 1d. These sequences are preferably selected from any one or more of SEQ ID NO: 1-31, as well as combinations or mixtures thereof. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. Combinations of AONs are preferably combinations of SEQ ID NO: 11 and 10 or SEQ ID NO: 11 and 13. This includes sequences that can hybridize with such sequences under strict hybridization conditions, sequences complementary to such sequences, sequences containing modified bases, modified backbones, and sequences with functional truncated or extended portions that have or regulate precursor mRNA processing activity in RAGE gene transcripts.

[0145] Oligomers are complementary to DNA, cDNA, or RNA when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen-bond with each other. Therefore, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or pairing that results in stable and specific binding between the oligomer and its DNA, cDNA, or RNA target. It should be understood in the art that the sequence of an AON does not need to be 100% complementary to the sequence of its specifically hybridizable target sequence. An AON is specifically hybridizable when the binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA product, and when sufficient complementarity exists under conditions requiring specific binding (i.e., physiological conditions for in vivo assays or therapeutic treatments, and in the case of in vitro assays, under the conditions under which the assay is performed) to avoid non-specific binding of the AON to non-target sequences.

[0146] Selective hybridization can be performed under low, medium, or high stringency conditions, but is preferably performed under high stringency conditions. Those skilled in the art will recognize that, in addition to the base composition, the length of the complementary strand, and the number of nucleotide base mismatches between the hybridized nucleic acids, the stringency of hybridization will also be affected by conditions such as salt concentration, temperature, or organic solvents. Stringent temperature conditions will typically include temperatures exceeding 30°C, typically exceeding 37°C, and preferably exceeding 45°C, preferably at least 50°C, and typically 60°C–80°C or higher. Stringent salt conditions will typically be below 1000 mM, typically below 500 mM, and preferably below 200 mM. However, the combination of parameters is far more important than the measurement of any single parameter. An example of stringent 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 methods of the present invention can comprise oligomers that selectively hybridize with sequences provided in Tables 1a to 1d or SEQ ID NO: 1-31. More preferably, AON is SEQ ID NO: 11, 18, 19 or 20.

[0147] It should be understood that the codon alignment at the exon ends in structural proteins may not always break at the codon ends, thus requiring the deletion of more than one exon from the precursor mRNA to ensure in-reading within the mRNA's reading frame. In such cases, it may be necessary to select multiple AONs using the method of this invention, where each AON targets a different region responsible for inducing the desired exon and / or intron. At a given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes with the complementary polynucleotide. This hybridization can occur when the AON is "near" or "substantially" complementary to the target sequence, or even under conditions of precise complementarity.

[0148] Typically, selective hybridization occurs when the nucleotides of AON share at least about 55% identity with each other over a segment of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%, 95%, 98%, or 99%. As described, the length of the homology comparison can span a relatively long segment, and in some embodiments will typically span a segment of at least about 9 nucleotides, typically at least about 12 nucleotides, more typically at least about 20 nucleotides, typically 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.

[0149] Therefore, the AON sequence used in the method of the present invention preferably has at least 75%, more preferably at least 85%, even more preferably at least 86%, 87%, 88%, 89%, or 90% homology with the sequence shown in the sequence listing herein. More preferably, it has at least 91%, 92%, 93%, 94%, or 95%, even more preferably at least 96%, 97%, 98%, or 99% homology. Generally, the shorter the AON, the greater the homology required to obtain selective hybridization. Therefore, when the AON consists of fewer than about 30 nucleotides, it is preferable that the percentage of identity with the AON shown in the sequence listing herein is greater than 75%, preferably greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Nucleotide homology comparisons can be performed using sequence comparison programs such as GCG Wisconsin Bestfit or GAP (Deveraux et al., 1984, Nucleic Acids Research, Vol. 12, pp. 387-395). In this way, sequences similar in length or significantly different from those cited herein can be compared by inserting gaps in the alignment, which are determined, for example, by the comparison algorithm used in GAP.

[0150] The AON used in the method of the present invention can have regions of reduced homology to the target sequence and regions of complete homology. The oligomer need not be completely homologous along its entire length. For example, the oligomer can have a continuous segment of at least 4 or 5 bases identical to the target sequence, preferably a continuous segment of at least 6 or 7 bases identical to the target sequence, more preferably a continuous segment of at least 8 or 9 bases identical to the target sequence. The oligomer can have a segment 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 segments of the oligomer sequence can be intermittently identical to the target sequence; for example, the remaining sequence can have the same bases, followed by different bases, followed by the same bases. Alternatively (or similarly), the oligomer sequence may have several identical sequence segments (e.g., 3, 4, 5, or 6 bases), interspersed with incompletely homologous segments. Such sequence mismatches will preferably have no loss of splice conversion activity or very little loss of splice conversion activity.

[0151] The term “modulation” includes, optionally, a quantity that “increases” or “decreases” one or more quantifiable parameters by definition and / or is statistically significant. The terms “increase,” “enhance,” or “stimulate” generally refer to the ability of one or more AONs or compositions to produce or evoke a greater physiological response (i.e., a downstream effect) in cells or a subject compared to a response evoked by the absence of an AON or control compound. The terms “decrease” or “reduce” generally refer to the ability of one or more AONs or compositions to produce or evoke a reduced physiological response (i.e., a downstream effect) in cells or a subject compared to a response evoked by the absence of an AON or control compound.

[0152] The relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include increasing the exclusion of specific exons in RAGE-encoded precursor mRNA, decreasing the amount of RAGE-encoded precursor mRNA, or decreasing the expression of functional RAGE proteins in cells, tissues, or subjects in need. The amount of “increase” or “enhancement” 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, or more times (e.g., 500, 1000 times) (including all integers and decimals of 1 and above, e.g., 1.5, 1.6, 1.7, 1.8) in the amount produced without AON (the reagent is absent) or control compounds. The terms “decrease” or “inhibition” may generally refer to the ability of one or more AONs or compositions to “reduce” relevant physiological or cellular responses (e.g., symptoms of the disease or condition described herein), as measured according to conventional techniques in the diagnostic field. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in symptoms or pathology of a disease (e.g., cancer, neurodegenerative diseases, lung diseases, and other inflammatory diseases). A “reduction” in response may be statistically significant compared to a response produced by the composition without AON or a control composition and may include reductions 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 therein.

[0153] The length of the AON can vary, as long as it selectively binds to the desired location within the precursor mRNA molecule. The length of such sequences can be determined according to the selection procedure described herein. Typically, the length of the AON will range from about 10 nucleotides to at most about 50 nucleotides. However, it should be understood that nucleotides of any length within this range can be used in this method. Preferably, the length of the AON is 10 to 40, 10 to 35, 15 to 30 nucleotides, or 20 to 30 nucleotides, with about 25 to 30 nucleotides being most preferred. For example, the length of the oligomer can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0154] As used herein, “AON” refers to a linear sequence of a nucleotide or nucleotide analog that allows nucleobases to hybridize with a target sequence in RNA via Watson-Crick base pairing to form an oligonucleotide:RNA heteroduplex within the target sequence. The terms “AON,” “AON,” “oligomer,” and “antisense compound” are used interchangeably to refer to oligonucleotides. The cyclic subunit may be based on ribose or another pentose, or in some embodiments on a morpholino group (see the description of morpholino oligonucleotides below). Peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and 2'-O-methyl oligonucleotides, 2'-O-methoxyethyl oligonucleotides, and other antisense agents known in the art are also considered.

[0155] This includes non-naturally occurring AONs or "oligonucleotide analogs," including AONs or oligonucleotides having (i) a modified backbone structure (e.g., a backbone different from the standard phosphodiester bonds found in naturally occurring oligonucleotides and polynucleotides) and / or (ii) a modified sugar moiety (e.g., a morpholino moiety instead of a ribose or deoxyribose moiety). Oligonucleotide analogs support bases capable of hydrogen bonding with standard polynucleotide bases via Watson-Crick base pairing, wherein the analog backbone presents the bases in a manner that allows such hydrogen bonding between the oligonucleotide analog molecule and bases in the standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA) in a sequence-specific manner. Preferred analogs are those having a substantially uncharged phosphorus-containing backbone.

[0156] One method for generating AON is the methylation of the 2' hydroxyribose position, and the introduction of a thiophosphate backbone produces a molecule that is superficially similar to RNA but more resistant to nuclease degradation. However, those skilled in the art will know other suitable backbones that can be used for the purposes of the methods of the present invention.

[0157] The increased splicing conversion can also be achieved through optional oligonucleotide chemistry. For example, AON can be selected from the following list, which includes: phosphoramide or phosphodiamid morpholino oligomers (PMO); PMO-X; PPMO; peptide nucleic acid (PNA); locked nucleic acid (LNA) and its derivatives, including α-L-LNA, 2'-aminoLNA, 4'-methylLNA and 4'-O-methylLNA; ethylene-bridged nucleic acid (ENA) and its derivatives; thiophosphate oligomers; tricyclic-DNA oligomers (tcDNA); tricyclic thiophosphate oligomers; 2'-O-methyl modified oligomers (2'-OMe); 2'-O-methoxyethyl (2'-MOE); 2'-fluoro, 2'-fluoroarabinose (FANA); non-locked nucleic acid (UNA); thermally stable twisted intercalated nucleic acid (TINA), hexitol nucleic acid (HNA); cyclohexenyl nucleic acid (CeNA); 2'-amino (2'-NH2); 2'-O-ethyleneamine, or any combination of the foregoing substances as a mixture or interstitial. To further improve delivery efficiency, the modified nucleotides mentioned above are typically conjugated to sugar or nucleobase moieties with fatty acids / lipids / cholesterols / amino acids / carbohydrates / polysaccharides / nanoparticles, etc. These conjugated nucleotide derivatives can also be used to construct exon-skipping AONs. Antisense oligonucleotide-induced splicing modifications of human RAGE gene transcripts typically use oligonucleotides, PNA, 2OMe, or MOE-modified bases on a phosphate thioester backbone. When using alternative chemicals to generate AONs for the methods of this invention, the uracil (U) in the sequences provided herein can be replaced with thymine (T).

[0158] Non-naturally occurring oligomers or "oligonucleotide analogs" are included within the AON used in the methods of the present invention. These non-naturally occurring oligomers or "oligonucleotide analogs" include oligomers having: (i) a modified backbone structure, for example, a backbone different from the standard phosphodiester bonds found in naturally occurring oligonucleotides and polynucleotides, and / or (ii) a modified sugar moiety, such as a morpholino moiety instead of a ribose or deoxyribose moiety. The oligomeric analogs support bases capable of hydrogen bonding with standard polynucleotide bases via Watson-Crick base pairing, wherein the analog backbone presents the bases in a manner that allows such hydrogen bonding to occur in a sequence-specific manner between the oligomeric analog molecule and the bases in the standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Preferred analogs are those having a substantially uncharged phosphorus-containing backbone.

[0159] Antisense oligonucleotides that do not activate RNase H can be prepared according to known techniques (see, for example, U.S. Patent 5,149,797). Such AONs can be deoxyribonucleotide or ribonucleotide sequences containing only any structural modifications that spatially impede or prevent the binding of RNase H to a duplex molecule containing the oligomer as one of its members, and such structural modifications do not substantially impede or disrupt duplex formation. Because the portions of the oligomer involved in duplex formation are significantly different from those involved in RNase H binding, many RNase H-inactive AONs are available. For example, such AONs can be oligomers in which at least one or all of the internucleotide bridging phosphate residues are modified phosphate esters, such as methylphosphonates, methylthiophosphates, phosphorylmorpholinates, phosphorylpiperazine esters, borophosphates, amide bonds, and phosphoramides. For example, the internucleotide bridging phosphate residues can 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., a C1-C4 straight-chain or branched saturated or unsaturated alkyl group, such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other nucleotide may be modified as described.

[0160] Specific examples of preferred AONs used in the methods of the present invention include oligomers containing a modified backbone or non-natural internucleotide links. As defined in this specification, oligomers having a modified backbone include oligomers that retain phosphorus atoms in the backbone and oligomers that do not have phosphorus atoms in the backbone. For the purposes of this specification, and as sometimes cited in the art, modified oligomers that do not have phosphorus atoms in their internucleotide backbone may also be considered AONs.

[0161] In other preferred oligomer mimics, the sugar and nucleoside links (i.e., the backbone) of the nucleotide units are replaced by novel groups. Base units are retained to allow hybridization with suitable nucleic acid target compounds. One such oligomer compound (which has shown excellent hybridization properties) is called peptide nucleic acid (PNA). In PNA compounds, the oligomer's sugar backbone is replaced by an amide-containing backbone (particularly an aminoethylglycine backbone). Nucleobases are retained and bind directly or indirectly to the nitrogen atoms of the amide moiety of the backbone.

[0162] Another preferred chemical is phosphodiamidomorpholino oligomer (PMO) oligomers, which are not degraded by any known nucleases or proteases. These compounds are uncharged, do not activate RNase H activity when bound to RNA chains, and have been shown to exert sustained splicing regulatory effects after in vivo administration (Summerton and Weller, Antisense Nucleic Acid Drug Development, Vol. 7, pp. 187-197).

[0163] Modified oligomers may also contain one or more substituted sugar moieties. Oligomers may also include nucleobase (generally referred to in the art simply as "bases") modifications or substitutions. Certain nucleobases are particularly useful for increasing the binding affinity of oligomers as described herein. These nucleobases 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), which have been shown to improve the stability of nucleic acid duplexes by 0.6 °C–1.2 °C, even more particularly in combination with 2'-O-methoxyethyl sugar modifications.

[0164] The activity of AON and its variants can be determined using conventional techniques in the art. For example, the splicing form and expression level of the investigated RNA and protein can be assessed by any of a variety of well-known methods for detecting the spliced ​​form and / or expression of transcribed nucleic acids or proteins. Non-limiting examples of such methods include RT-PCR for RNA splicing followed by size separation of the PCR products; nucleic acid hybridization methods, such as the use of RNA blotting and / or nucleic acid arrays; nucleic acid amplification methods; immunological methods for protein detection; protein purification methods; and protein function or activity assays.

[0165] 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, which is a complement to the nucleic acid or fragment thereof being measured. The cDNA may optionally be amplified using any of a variety of polymerase chain reactions or in vitro transcription methods prior to hybridization with the complementary polynucleotide; preferably, the cDNA is not amplified. Quantitative PCR can also be used to detect the expression of one or more transcripts to assess transcript expression levels.

[0166] The AON used in the method of the present invention can provide induced splicing conversion of RAGE gene transcripts, clinically relevant oligomer chemistry, and delivery systems to guide RAGE splicing manipulation to a therapeutic level. It can promote the splicing of non-signaling decoy receptor RAGE mRNA forms (e.g., RAGE_v1, RAGE_v6, RAGE_v8, RAGE_v9, RAGE_v10, RAGE_v15, RAGE_v18, and RAGE_v19 mRNA) and can therefore be used to treat RAGE-related respiratory (lung) diseases or conditions.

[0167] The AON used in the method of this invention can be conveniently prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, California). A method for synthesizing oligomers on a modified solid support is described in U.S. Patent 4,458,066.

[0168] Alternatively or in addition, any other method known in the art for this synthesis may be used. Similar techniques are well known to be used to prepare oligomers, such as thiophosphates and alkylated derivatives. In one such automated embodiment, diethylphosphonamide is used as a starting material and can be synthesized as described in Beaucage et al., (1981) Tetrahedron Letters, Vol. 22: pp. 1859-1862.

[0169] The AON used in the method of the present invention is synthesized in vitro and does not include biologically derived antisense compositions or genetic vector constructs designed to guide the in vivo synthesis of AON.

[0170] Application, dosage and preparation In any respect, the subject may have received or may be receiving treatment applied to their respiratory tract that increases endogenous soluble RAGE. Treatment may target respiratory (lung) conditions or diseases associated with or caused by RAGE, selected from: acute upper respiratory tract infection, rhinitis, nasopharyngitis, sinusitis, laryngitis, influenza and pneumonia, acute bronchitis, acute bronchiolitis, asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, emphysema, chronic lung disease caused by external factors, acute respiratory distress syndrome (ARDS), pulmonary eosinophilia and pleurisy, lung trauma, and recovery from lung injury, trauma, and surgery.

[0171] The term "breathing" refers to the process of inhaling oxygen and exhaling carbon dioxide through the body's systems, including the nose, pharynx, larynx, trachea, bronchi, and lungs.

[0172] As used herein, the upper respiratory tract may include the following areas: the nose and nasal passages, paranasal sinuses, pharynx, and the larynx above the vocal cords (vocal folds). The lower respiratory tract typically includes one or more of the following areas: the larynx below the vocal cords, the trachea, bronchi, and bronchioles. The lungs may be classified as part of the lower respiratory tract and include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0173] The term “respiratory disease” or “respiratory condition” refers to any of several diseases involving inflammation that affect 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).

[0174] Symptoms of respiratory illnesses can include cough, excessive sputum production, shortness of breath, or chest tightness accompanied by audible wheezing. Exercise capacity may be quite limited. In asthma, FEV1.0 (forced expiratory volume in one second) as a percentage of FEV1.0 predicted by a nomogram based on weight, height, and age can be reduced, as can the maximum expiratory flow rate during forced exhalation. In COPD, the ratio of FEV1.0 to FVC typically drops to less than 0.7. The impact of each of these conditions can also be measured by the number of days of work / school absence, disrupted sleep, need for bronchodilators, or need for glucocorticoids (including oral glucocorticoids).

[0175] The term “treatment” for a subject includes the application or administration of an AON as described herein, with the aim of delaying, slowing, stabilizing, curing, healing, reducing, alleviating, altering, remedying, reducing worsening, improving, modifying, or influencing the disease or condition, symptoms of the disease or condition, or the risk (or susceptibility) to the disease or condition. The term “treatment” means any indication of successful treatment or improvement of an injury, pathology, or condition, including any objective or subjective parameter such as slowing, alleviating, or reducing the rate of worsening; reducing the severity of the disease; stabilizing, weakening symptoms, or making the injury, pathology, or condition more tolerable for the subject; slowing the rate of degeneration or decline; making the endpoint of degeneration less debilitating; or improving the physical or mental health of the subject.

[0176] The methods of the present invention can provide AONs as described herein, which are adapted to aid in the preventive or therapeutic treatment, prevention or improvement of symptoms of diseases (e.g., diseases or pathologies associated with RAGE expression) in a form suitable for delivery to patients.

[0177] While the method of this invention is applicable to humans, it is also applicable to veterinary treatment. This invention is applicable to livestock or farm animals, such as cattle, sheep, horses, and poultry; to companion animals, such as cats and dogs; and to zoo animals.

[0178] In some implementations, AONs, as described herein, can be delivered via the lungs or nose (e.g., via nebulized saline incorporating AON). The highest endogenous expression of RAGE mRNA in healthy human tissues is found in the lungs and can be accessed via the airways. Inhaled oligonucleotides are an emerging therapeutic approach for respiratory diseases. The airways are uniquely lined with pulmonary surfactant, which is primarily composed of zwitterionic lipids. These surfactant lipids have cationic properties at the pH of the respiratory tract. When anionic oligonucleotides are inhaled, they tend to be adsorbed by surfactant, resulting in reformulated particles that are presumably efficiently absorbed into lung cells by bronchial and alveolar epithelial cells. Notably, AONs have been shown to withstand nebulization.

[0179] AON as described herein can be formulated into compositions for application only to the lower respiratory tract. Restriction to the lower respiratory tract can be achieved by the amount of the composition (particularly its volume and form, i.e., particle size, physical form (dry powder or solution drops)) which would otherwise be applied to the upper respiratory tract. Alternatively, AON as described herein can be administered via a device that ensures retention only in the lower respiratory tract.

[0180] As described herein, AON can be formulated for intranasal application, including as dry powder, spray, nebulizer, or aerosol. This may be particularly preferred for the treatment of respiratory infections.

[0181] Suitable formulations for application (where the carrier is a liquid, such as a nasal spray or nasal drops) comprise an aqueous or oily solution of the active ingredient. Alternatively, AON as described herein may be provided as a dry powder and, as defined herein, be applied only to the upper respiratory tract.

[0182] The choice of a suitable carrier depends on the specific type of administration considered. For administration via the upper respiratory tract (e.g., the nasal mucosa), the compound can be formulated as a solution (e.g., buffered or unbuffered water or isotonic saline) or as a suspension (for intranasal administration as drops or sprays). Preferably, such solutions or suspensions are isotonic relative to nasal secretions and have approximately the same pH, ranging, for example, from about pH 4.0 to about pH 7.4 or from pH 6.0 to pH 7.0. The buffer should be physiologically compatible and, by way of example only, includes phosphate buffers. For example, a representative nasal decongestant is described as buffered to about 6.2 (Remington's, Id., p. 1445). Of course, those skilled in the art can readily determine the appropriate saline concentration and pH for a harmless aqueous carrier used for nasal and / or upper respiratory tract administration.

[0183] Other ingredients may also be included, such as preservatives, colorants, lubricating or viscous mineral or vegetable oils, fragrances, natural or synthetic plant extracts (e.g., aromatic oils), humectants, and viscosity enhancers (e.g., glycerin), thereby providing the formulation with additional viscosity, moisturizing properties, and a pleasant texture and odor. For the nasal administration of solutions or suspensions according to the methods of the invention, various devices in the art are available for producing drops, liquids, and sprays. For example, AON as described herein can be administered into the nasal passage via a simple dropper (or pipette) comprising a glass, plastic, or metal dispensing tube from which contents are dispensed drop by drop by air pressure provided by a manual pump (e.g., a flexible rubber bulb) attached to one end.

[0184] Delivery of therapeutically effective amounts of AON can be achieved by previously disclosed methods. For example, intracellular delivery of AON can be carried out via a composition comprising AON and an effective amount of a block copolymer. An example of this method is described in U.S. Patent Application US20040248833. Other methods for delivering AON to the nucleus are described in Mann CJ et al. (2001), Proc, Natl. Acad. Science, Vol. 98, No. 1: pp. 42-47, and Gebski et al. (2003), Human Molecular Genetics, Vol. 12, No. 15: pp. 1801-1811. Methods for introducing nucleic acid molecules into cells as naked DNA or as expression vectors complexed with lipid carriers are described in US 6,806,084.

[0185] The AON used in the methods of this invention can be administered in an effective amount. The phrase “therapeutic effective amount” or “effective amount” generally refers to an amount of AON, its pharmaceutically acceptable salt, polymorph, or prodrug as described herein, which (i) treats a particular disease, condition, or disorder, (ii) alleviates, improves, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder as described herein. Undesirable effects (e.g., side effects) sometimes occur alongside desired therapeutic effects; therefore, practitioners balance potential benefits with potential risks when determining an appropriate “effective amount.”

[0186] The exact amount required will vary from subject to subject, depending on the subject's species, age and general condition, method of administration, etc. Therefore, it may be impossible to specify an exact "effective amount." However, those skilled in the art can determine the appropriate "effective amount" for any subject using only routine experiments. In one respect, the dose administered to the subject is any dose that reduces viral load.

[0187] In any implementation, AON, as described herein, is administered in a dose and manner that effectively results in peak blood concentrations of at least 200 nM-400 nM AON. Typically, one or more doses of AON are administered at regular intervals over a period of approximately one to two weeks.

[0188] In any embodiment, AON is administered at a dose equal to or greater than 0.024 mg / kg. In any embodiment, AON is administered at a dose equal to or greater than 0.24 mg / kg. In any embodiment, AON is administered at a dose equal to or less than 0.5 mg / kg.

[0189] Dosing depends on the severity and responsiveness of the disease state being treated, where the treatment process can last from days to months, or until a cure or remission of the disease state is achieved. The optimal dosing regimen can be calculated from measurements of drug accumulation in the patient. Ordinary technicians can readily determine the optimal dose, method of administration, and repetition rate. The optimal dose can vary depending on the relative potency of the oligomer in the subject and is often estimated based on the EC50, which has proven effective in in vitro and in vivo animal models.

[0190] The method of the present invention is also extended to combinations of two or more AONs capable of binding to selected targets to induce exon exclusion in RAGE gene transcripts. This combination may be a mixture of two or more AONs, comprising constructs of two or more AONs linked together for use in AON-based therapies. The combination of AONs is preferably a combination of SEQ ID NO: 11 and 10 or SEQ ID NO: 11 and 13.

[0191] A kit for use with the method of the present invention is also provided, particularly for measuring the level of endogenous soluble RAGE as described herein in blood samples obtained from subjects. This kit contains at least one probe for detecting said endogenous soluble RAGE, along with instructions for use describing the method of the present invention. The kit may further contain an AON as described herein for use with the method according to the present invention.

[0192] The contents of this kit can be lyophilized, and the kit may additionally contain a suitable solvent for reconstituted lyophilized components. The target components of the kit will be packaged in separate containers, and accompanied by a notification, which may be in the form prescribed by a government agency regulating the manufacture, use, or sale of a drug or biological product, reflecting that agency’s approval for its manufacture, use, or sale for human administration.

[0193] As used in this article, the terms “source” and “derived from” should be understood to indicate that a particular whole can be obtained from a specific source, although not necessarily directly from that source.

[0194] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “a,” and “the” include plural references.

[0195] Except as provided in the operational examples or otherwise, all figures used in the specification and claims to indicate the amount of ingredients, reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters listed in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained according to the method of the invention. Thus, "about 80%" means both "about 80%" and "80%". At a minimum, each numerical parameter should be interpreted according to the number of significant figures and common rounding methods.

[0196] Although the numerical ranges and parameters illustrating the broad scope of the methods of the present invention are approximations, the numerical values ​​described in the specific embodiments are reported as precisely as possible. However, any numerical value inherently contains some errors, which are necessarily caused by the standard deviation found in its corresponding test measurement.

[0197] Other definitions of the selected terms used herein can be found in the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0198] It should be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more target features mentioned in or clearly visible from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0199] Example Example 1 – Materials and Methods animal This study investigated the duration of efficacy of antisense oligonucleotides targeting mouse RAGE precursor RNA in healthy, pathogen-free male C57Bl / 6 mice (8–10 weeks old). Animals were housed in sterile, passively isolated enclosures at a constant 20°C with a 12-hour day / night cycle, fed irradiated Barastoc mouse diet, and provided with irradiated tap water for free access. Mouse weight was monitored throughout the experiment. Animals had free access to mouse diet and water throughout the study. All experiments were approved by local animal ethics committees and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, 1996 revision).

[0200] Endotracheal treatment Mice were anesthetized with a mixture of ketamine / toluidine (90 mg / 10 mg / kg body weight, via intraperitoneal injection), and endotracheal intervention was performed when no reflex response (limb withdrawal in response to hind paw stimulation) was observed. An otoscope / endoscopic device was carefully placed in the mouth to directly observe the tracheal opening. 50 μL of sterile solution was administered directly into the trachea using a blunt needle and a high-pressure injector. Postoperatively, mice were returned to their cages and placed on warm blankets for recovery. Mice were monitored until full recovery was observed, after which they were provided with liquid in the form of wet wipes and wet food pellets, as well as dry food pellets on the cage floor, before being returned to their rearing room. Mice were then followed up for 3, 7, 14, or 28 days, at which point they were humanely euthanized under CO2 anesthesia.

[0201] Atomization process Mice were anesthetized with a mixture of ketamine / toluidine (90 mg / 10 mg / kg body weight, via intraperitoneal injection) and endotracheal procedures were performed when no reflex response (limb withdrawal in response to hind paw stimulation) was observed. An otoscope / endoscopic device was carefully placed in the mouth to directly examine the tracheal opening. A 50 μL dose of sterile solution was administered directly to the trachea via a nebulizer microneedler (Penn-Century IA-1C) connected to a high-pressure injector (Penn-Century FMJ-250). Between each solution change, the microneedler and injector were rinsed three times with 250 μL of sterile ultrapure water and once with 250 μL of sterile saline. Postoperatively, mice were returned to their cages to recover on paper towels, and warmth was provided by placing half of the cage on a heating pad. Mice were monitored until full recovery was observed, after which they were provided with liquid in the form of wet paper towels and wet food pellets, as well as dry food pellets on the cage floor, before being returned to their rearing room. The mice were then followed up for 7 days, at which point they were humanely euthanized.

[0202] Sample collection and harvest Immediately after humane euthanasia, all mice were bled from the inferior vena cava into Microvette lithium / heparin tubes (Sarstedt, Aust.) using a lithium heparin-lined 19G syringe. The blood was kept at room temperature for at least 15 minutes and centrifuged at 2000 g for 5 minutes at 20°C. The plasma was transferred to 1.5 mL snap-on Eppendorf tubes, 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 cooled, sterile phosphate-buffered saline was administered at a rate of 300 μL / s. The lungs were then removed, as shown in the image. Figure 1 As shown. The left lung lobe was divided into an upper part, a lower left part (LL), and a lower right part (LR), and then flash-frozen in liquid nitrogen.

[0203] Measuring esRAGE levels in plasma and lung tissue To measure the effect of intervention on alternative splicing of RAGE precursor mRNA, expression of RAGE mRNA splice variants was determined using real-time RT-qPCR performed on a previously used TaqMan system (ABI Prism 7700, Perkin-Elmer Inc., PE Biosystems, Foster City, CA, USA) based on cumulative fluorescence real-time detection (43). For in vivo experiments, frozen lung tissue from the lower lobe of the left lung was used. RNA extraction and cDNA synthesis were performed using Trizol. Gene expression was assessed by RT-qPCR using a TaqMan or SYBR Green (Sigma) system based on cumulative fluorescence real-time detection (Applied Biosystems Q3 and Q5). Gene expression was normalized to 18S mRNA and reported as a fold change compared to expression levels in control RNA-treated cells, which were assigned an arbitrary value of 1. To determine the expression of RAGE mRNA splice variants that retain exon 9b, probes were designed to span exon 9b. To determine the expression of RAGE mRNA splice variants that retain exon 10, PCR primers / probes spanning exons 8–10 were designed. To determine the expression of all RAGE mRNAs, PCR probes / primers spanning exons 8 and 11 were designed. The reduced signal at exons 8–10 of the cytoplasmic tail relative to exons 8–11 indicates that cells produce fewer full-length RAGE transcripts (capable of signal transduction).

[0204] The levels of mouse extracellular soluble RAGE (esRAGE) in plasma and in the right lower lobe of the left lung extracted in RIPA buffer were estimated using a commercial ELISA kit for mouse esRAGE (ELISA kit-MBS7606654-mybiosource). For lung tissue, protein content results were normalized using the BCA assay (Pierce) (pg / 60 µg protein). Plasma results were normalized to volume (pg / mL plasma).

[0205] Statistical analysis The data shown are mean ± SEM. Based on previous experiments, all datasets were determined to be normally distributed, and this was further verified using the Kolmogorov–Smirnov test (α=0.05). Data analysis was performed using PRISM software (V6.0d, GraphPad, La Jolla, CA, USA) via one-way ANOVA and Tukey's multiple comparison test. P < 0.05 was considered statistically significant.

[0206] Example 2 — Correlation between plasma esRAGE and lung tissue esRAGE after endotracheal treatment with ASO m79 In this experiment, mice (n=6–8 per group) were treated with intratracheal microneedle 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, when administered to cells, promotes RAGE_v1 mRNA expression, thereby promoting esRAGE production. AON m79 is an exemplary AON complementary to a precursor mRNA target region having a 5' nucleotide at position 88 or 114 of exon 10, or between positions 88 and 114 of exon 10. esRAGE levels in left lung tissue and plasma were measured on day 14. Figure 2 ).

[0207] On day 14, plasma esRAGE levels were strongly correlated with lung tissue esRAGE levels. In contrast, the control group did show a significant correlation between plasma and lung tissue esRAGE levels. Figure 3 ).

[0208] This data suggests that plasma esRAGE levels can be used as a biomarker to determine the efficacy of ASO m79 treatment in the lungs.

[0209] Example 3 — Correlation between plasma esRAGE and lung tissue esRAGE after endotracheal treatment with ASO2 In this experiment, mice were treated with intratracheal microneedling of 50 μL ASO2 (0.3 or 3.0; 8 mice per group) or physiological saline (solvent; 4 mice per group). Figure 4 ASO2 is a human 18-mer antisense oligonucleotide with modified nucleotide bases that, when applied to cells, promotes RAGE_v1 mRNA expression and thus promotes esRAGE production. esRAGE levels in lung tissue and plasma were measured on days 10 and 14 following ASO2 treatment.

[0210] On days 10 and 14 following a single dose of ASO2 at 3 mg / kg or 0.3 mg / kg, plasma esRAGE was strongly correlated with lung tissue esRAGE (p < 0.01). Figure 5 ).

[0211] Similar statistically significant correlations were also observed when ASO2 was administered using an intratracheal microneedle at doses of 10 mg / kg and 3 mg / kg. Figure 6 and Figure 7 ).

[0212] In summary, these results suggest that plasma esRAGE can be used as a biomarker to determine the level of esRAGE in the lungs after treatment with AON that regulates precursor mRNARAGE splicing.

Claims

1. A method for determining the level of endogenous soluble receptor for advanced glycation end products (RAGE) in the lungs of a subject, the method comprising: The presence of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving treatment administered to their respiratory tract that increases endogenous soluble RAGE is determined. The level of endogenous soluble RAGE in the blood sample was correlated with the level of endogenous soluble RAGE in the lungs of the subject.

2. A method for determining the level of endogenous soluble receptor for advanced glycation end products (RAGE) in a blood sample of a subject, the method comprising: The presence of endogenous soluble RAGE in a blood sample from a subject who has received or is receiving treatment administered to their respiratory tract that increases endogenous soluble RAGE is determined. This allows for the determination of endogenous soluble RAGE in the blood sample of the subject.

3. A method for determining splicing of receptor for advanced glycation end products (RAGE) precursor mRNA in a subject, said splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10, said method comprising: The presence of endogenous soluble RAGE in blood samples from subjects who have received or are receiving antisense oligonucleotides (AONs) via the respiratory tract was determined. These AONs promote splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. The presence of endogenous soluble RAGE in the blood sample indicates splicing of the receptor for advanced glycation end products (RAGE) precursor mRNA in the subject, which results in the inclusion of exon 9b and / or the exclusion of exon 10.

4. A method for determining splicing of receptor for advanced glycation end products (RAGE) precursor mRNA in a subject, said splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10, said method comprising: Provide a blood sample from a subject who has received or is receiving antisense oligonucleotides (AONs) via the respiratory tract. These AONs promote splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. The level of endogenous soluble RAGE in the blood sample was measured. The level of endogenous soluble RAGE in the blood sample indicates splicing of the RAGE precursor mRNA in the subject, which results in the inclusion of exon 9b and / or the exclusion of exon 10.

5. A method for determining splicing of receptor for advanced glycation end products (RAGE) precursor mRNA in a subject, said splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10, said method comprising: Provide a blood sample from a subject who has received or is receiving antisense oligonucleotides (AONs) via the respiratory tract. These AONs promote splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. The level of endogenous soluble RAGE in the blood sample was measured. The level of endogenous soluble RAGE in the blood sample above a threshold level indicates splicing of the RAGE precursor mRNA in the subject, resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

6. A method for determining splicing of receptor for advanced glycation end products (RAGE) precursor mRNA in a subject, said splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10, said method comprising: Provide a blood sample from the subject prior to receiving an antisense oligonucleotide (AON) via the respiratory tract, the AON promoting splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. A blood sample from the subject following administration of AON via the respiratory tract is provided. AON promotes splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. The endogenous soluble RAGE level was measured in each of the blood samples. The higher level of endogenous soluble RAGE in blood samples from subjects after receiving the AON via the respiratory tract, compared to the level of endogenous soluble RAGE in blood samples from subjects prior to receiving the AON via the respiratory tract, indicates splicing of the RAGE precursor mRNA in the subjects, which results in the inclusion of exon 9b and / or the exclusion of exon 10.

7. A method for determining splicing of receptor for advanced glycation end products (RAGE) precursor mRNA in a subject, said splicing resulting in the inclusion of exon 9b and / or the exclusion of exon 10, said method comprising: Blood samples were obtained from subjects prior to receiving antisense oligonucleotides (AONs) via the respiratory tract. These AONs promote splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. Blood samples were obtained from the subjects after receiving AON via the respiratory tract. The AON promotes splicing in RAGE precursor mRNA, resulting in the inclusion of exon 9b and / or the exclusion of exon 10. The endogenous soluble RAGE level was measured in each of the blood samples. The higher level of endogenous soluble RAGE in blood samples from the respiratory tract of the subjects after receiving the AON, compared to the level of endogenous soluble RAGE in blood samples from the subjects prior to receiving the AON, indicates splicing of the RAGE precursor mRNA in the subjects, which results in the inclusion of exon 9b and / or the exclusion of exon 10.

8. A method for determining whether treatment / AON can increase the level of endogenous soluble RAGE in the respiratory tract, the method comprising: Administer the experimental treatment / AON to the respiratory tract of the subjects. Blood samples were obtained from the subjects after administration. The presence or increase in the level of endogenous soluble RAGE in the blood sample indicates that the experimental treatment / AON increases the level of endogenous soluble RAGE in the respiratory tract.

9. The method according to any one of claims 1 to 8, wherein the blood sample comprises whole blood, plasma, or serum, or is composed 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 antisense oligonucleotides (AON) in the respiratory tract, wherein the AON promotes splicing in the RAGE precursor mRNA, thereby resulting in the inclusion of exon 9b and / or the exclusion of exon 10.

11. The method according to any one of claims 2 to 10, wherein the AON promotes the generation 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, 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 in one or more tissues of the respiratory tract.

13. The method according to any one of claims 2 to 12, wherein the AON is applied to the entire respiratory tract, the upper respiratory tract, or the lower respiratory tract.

14. The method of any one of claims 13, wherein the AON is applied to the lower respiratory tract.

15. The method of claim 14, wherein the AON is applied to any one or more of the following regions: the larynx, trachea, bronchi, and bronchioles located below the vocal cords.

16. The method of claim 14, wherein the AON is applied to the lungs.

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

18. The method according to any one of claims 2 to 17, wherein the AON is 10 to 50 nucleotides comprising a targeting sequence complementary to a region near or within an intron of the RAGE precursor mRNA.

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 splicing site of the RAGE precursor mRNA.

20. 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 a cis-acting RNA element in the RAGE precursor mRNA that acts as an enhancer or silencer, the target sequence regulating splicing of nearby exons when the spliceosome element binds.

21. The method according to any one of claims 2 to 17, wherein the AON is 10 to 50 nucleotides comprising a targeting sequence complementary to the RAGE precursor mRNA, the targeting sequence regulating the secondary structure of the mRNA to influence splice site selection.

22. The method according to any one of claims 2 to 21, wherein the AON is an isolated or purified AON used to induce 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 an isolated or purified AON used to induce the retention of intron sequences in the RAGE gene transcript or a portion 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 precursor mRNA-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 phosphodiamidomorpholino oligomer (PMO), 2'-O-methylphosphothioester oligonucleotide (2OMe) and 2'-O-methoxyethylphosphothioester oligonucleotide (2MOE), locked nucleic acid (LNA) modified AON, thermally stable twisted intercalated 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 with at least one portion to increase their delivery.

28. The method of claim 27, wherein the portion is one or more of the following: cell-penetrating peptide (CPP), in vivo morpholino oligomer (VMO), and peptide phosphodiesteramide morpholino oligomer (PPMO).

29. The method according to any one of claims 2 to 28, wherein the AON comprises, is substantially composed of, or is composed of the following nucleotide sequences: The entire antisense oligonucleotide contains 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 precursor mRNA.

30. The method of claim 29, wherein the 5' nucleotide of the target region is the 88th or 114th nucleotide of exon 10, or between the 88th and 114th nucleotides of exon 10.

31. The method of claim 30, wherein the nucleotide position of exon 10 is associated with the nucleotide sequence of SEQ ID NO:

32.

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

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

34. The method of claim 33, wherein the AON is selected from: SEQ ID NO: 1-31 or a nucleotide sequence having at least 85%, 90% or 95% identity with it.

35. The method of claim 34, wherein the AON is SEQ ID NO: 11, 18, 19 or 20, or a nucleotide sequence having at least 85%, 90% or 95% identity with it.

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