Modulators and modulation of receptor for advanced glycation end-products RNA

Abstract

To provide splice-switching antisense oligonucleotides (AONs) for use in the manufacture of a medicament to treat, prevent, or ameliorate the effects of a disease associated with receptor for advanced glycation end-products (RAGE) expression, and to provide methods to treat, prevent, or ameliorate the effects of a disease associated with RAGE expression.SOLUTION: The present invention provides an isolated or purified AON for modifying pre-mRNA splicing in the Receptor for Advanced Glycation End-products (RAGE) to modulate splicing of the RAGE gene transcript or part thereof. In one aspect of the invention, there is provided an AON of 10 to 50 nucleotides comprising a targeting sequence complementary to a region near or within an intron of the RAGE pre-mRNA.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for regulating the alternative splicing of pre-mRNA encoding the receptor for advanced glycation end products (RAGE) or a part thereof, and modifying the expression and / or activity of RAGE isoforms using splicing-switching antisense oligonucleotides (AONs), and a method for treating RAGE-related disorders using said modulator.

Background Art

[0002] The receptor for advanced glycation end products (RAGE) is a multivalent type I transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. The human RAGE (Ager) gene is located within the major histocompatibility complex class III region on chromosome 6. It contains 11 exons and 10 introns, as well as the 5' flanking region that controls its transcription. The transcribed RAGE mRNA is approximately 1.4 kb and has a short 3' UTR.

[0003] The 50-55 kDa glycated RAGE protein is constitutively expressed in a limited range of cells (e.g., vascular endothelium, type I lung cells, leukocytes), but RAGE expression is induced following injury, stress, hypoxia or inflammation in most cell types and tissues, and can lead to conduits in pro-inflammatory and pro-proliferative signaling. As a result, RAGE expression is upregulated in inflammatory and metabolic disorders including, but not limited to, neurodegenerative diseases, cancer, cardiovascular diseases, diabetes, autoimmune and ischemic disorders in which RAGE is also involved in expression and progression.

[0004] RAGE is involved in Alzheimer's disease; amyotrophic lateral sclerosis; Huntington's disease; Creutzfeldt-Jakob disease; neurodegenerative conditions such as diabetic neuropathy, familial amyloid polyneuropathy, Charcot neuroarthropathy and vasculitic neuropathy; neuropathic pain; the expression and progression of gliomas; various brain disorders including ischemic brain injury / stroke, and multiple sclerosis.

[0005] RAGE is involved in many aspects of tumor biology, including tumor cell proliferation, migration, and invasion. Many cancers express higher levels of RAGE (examples include breast cancer, colon cancer, kidney cancer, and gastric cancer). An exception is lung cancer, where RAGE expression is reduced as lung cells differentiate and become more malignant, resulting in the loss of RAGE.

[0006] In C6 glioma cells, tumor volume significantly decreases within tumors composed of cells in which RAGE has been blocked. In contrast, tumors overexpressing wild-type RAGE grew rapidly and invaded the surrounding tissue very efficiently. It would be desirable to have therapeutic agents for blocking RAGE signaling as cancer treatments for many common cancers such as glioblastoma / multiforme; pancreatic cancer; melanoma; prostate cancer; breast cancer; liver cancer / hepatic tumors; and colon cancer.

[0007] Under healthy conditions, RAGE expression in the lung is the highest among all tissues. However, RAGE expression in the lung is usually only observed within type I lung cells. Upregulation of RAGE signaling in other cells and at other sites in the lung is involved in various lung disorders, including chronic obstructive pulmonary disease (COPD) / emphysema; asthma; damage caused by tobacco smoking / pollution; acute lung injury / acute respiratory distress syndrome; and pulmonary fibrosis.

[0008] RAGE is also critically involved in several inflammatory conditions such as inflammatory arthritis; osteoarthritis; retinal diseases; atherosclerosis; vascular calcification; ischemic heart disease / heart remodeling / fibrosis; heart failure; diabetic and non-diabetic kidney diseases; inflammatory bowel disease; preeclampsia; polycystic ovary syndrome; fatty liver, fibrosis, ischemic and non-ischemic liver injury; muscular dystrophy; spinal cord injury; skin inflammation and aging; and keratitis.

[0009] Human RAGE is composed of an immunoglobulin-like ectodomain, a single transmembrane domain, and a short (42 amino acid) cytoplasmic tail. The ectodomain of RAGE (also known as the extracellular domain) contains three immunoglobulin-like regions: an N-terminal V-type domain followed by two C-type domains (C and C’ or alternatively designated C1 and C2).

[0010] Binding of advanced glycation end products (AGEs) and non-AGE ligands to the ectodomain of RAGE activates intracellular signaling cascades involved in inflammation, injury, and cell growth and differentiation. Activation of RAGE also induces a positive feedback loop such that the RAGE ligand-receptor interaction enhances the expression of RAGE via NFκB activation, thereby enhancing subsequent RAGE-induced cell activation. In fact, the only means by which the inventors are aware of to potently downregulate RAGE expression is to reduce the activation of RAGE. This situation is in contrast to other receptors where an increase in ligand levels reduces receptor expression.

[0011] In humans, the cytoplasmic tail of RAGE is 43 amino acids in length (residues 362–404). This cytoplasmic tail has motifs that are crucial for RAGE-dependent cell activation, unlike ligand binding. The cytoplasmic tail of RAGE can be trans-activated following activation by their cognate ligands of co-localized G protein-coupled receptors in the absence of AGE binding of non-AGE ligands to the RAGE ectodomain, which results in activation of the same pathway (also known as ligand-independent activation of RAGE) (Pickering et al. J Clin Invest 2019;129:406–421). Activation of the RAGE cytoplasmic tail by specific co-localized activated G protein-coupled receptors appears to be an important pathway when RAGE is activated in vivo.

[0012] In both ligand-dependent activation of RAGE and ligand-independent activation of RAGE, intracellular signal transduction is mediated by the cytoplasmic domain of RAGE, which interacts with various signal transduction partners.

[0013] Alternative splicing of RAGE is also important for the regulation of RAGE activity through the generation of RAGE isoforms that have an altered ability to be activated by ligand-dependent and ligand-independent signal transduction pathways. Alternative splicing of RAGE is altered in pathological conditions including cancer, diabetes, and Alzheimer's disease. However, while alternative splicing appears to be important for the regulation / dysregulation of RAGE, no methods have been identified that specifically target this mechanism.

[0014] It is against this background that the present method using splice-switching AONs for regulating RAGE splicing towards the preferential generation of the cytoprotective RAGE isoform over full-length RAGE is described.

Summary of the Invention

Problems to be Solved by the Invention

[0015] The above disclosure of the background art is intended only to facilitate the understanding of the present invention. Such discussion is not an admission or recognition that any of the materials cited was part or a part of common general knowledge at the priority date of the present application.

Means for Solving the Problems

[0016] Broadly, according to one embodiment of the present invention, there is provided an isolated or purified AON used for regulating the alternative splicing of a pre-mRNA gene transcript encoding the receptor for advanced glycation end products (RAGE) or a part thereof.

[0017] In one aspect of the present invention, an AON of 10 to 50 nucleotides is provided that contains a targeting sequence complementary to a region near or within an intron of RAGE pre-mRNA.

[0018] In one aspect of the present invention, an AON of 10 to 50 nucleotides is provided that contains a targeting sequence complementary to or adjacent to a splice site of RAGE pre-mRNA.

[0019] Since elements such as RNA secondary structure, competition between AON and SR proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), and / or other elements that form splice isoforms can affect the action of AON, an AON directed to a definitive acceptor or donor splice site does not always modify splicing. As a result, in one aspect of the present invention, an AON of 10 to 50 nucleotides is provided that contains a targeting sequence complementary to or adjacent to a cis-acting RNA element in the pre-mRNA of RAGE that acts as an enhancer or silencer and regulates the splicing of a neighboring exon when bound by an element of the spliceosome (e.g., a protein splicing factor, uRNA, lncRNA).

[0020] In one aspect of the present invention, an AON of 10 to 50 nucleotides is provided that contains a targeting sequence complementary to RAGE pre-mRNA and regulates the secondary structure of the mRNA so as to affect splice site selection.

[0021] In one form of the present invention, an isolated or purified AON is provided for inducing the removal (also known as skipping) of one or more exon sequences in a RAGE gene transcript or a portion thereof.

[0022] In one form of the present invention, an isolated or purified AON is provided for inducing the retention of an intron sequence in a RAGE gene transcript or a portion thereof.

[0023] In one embodiment of the invention, the AON is chemically modified to prevent the degradation of the pre-mRNA-AON complex and includes, but is not limited to, phosphorodiamidate morpholino oligomers (PMOs), 2'-O-methyl phosphorothioate oligonucleotides (2OMe), and 2'-O-methoxyethyl phosphorothioate oligonucleotides (2OMe), locked nucleic acid (LNA)-modified AONs, thermally stable twisted intercalating nucleic acids (TINA), and peptide nucleic acids (PNAs).

[0024] In one embodiment of the invention, the AONs are conjugated to moieties for enhancing their delivery, including, but not limited to, cell-penetrating peptides (CPPs), vivo morpholinos (VMO), or peptide phosphorodiamidate morpholino oligomers (PPMO).

[0025] Preferably, the AON is selected from the group consisting of sequences shown in any of Tables 3a - 3d. Preferably, the AON is selected from the list consisting of SEQ ID NOs: 1 - 31. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

[0026] The AON of the invention may be selected to be an AON capable of binding to a selected target site, where the target site is a putative mRNA splicing site selected from a splice donor site, a splice acceptor site, a splice enhancer sequence, a splice silencer sequence, or a site that modulates the secondary structure of the pre-mRNA. The target site may also include some adjacent intron sequences when the donor or acceptor splice site is targeted.

[0027] More specifically, the AON may be selected from the group consisting of any one or more of SEQ ID NOs: 1 to 31 and / or the sequences shown in any of Tables 3a to 3d, and combinations or cocktails thereof. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. The combination of AONs is preferably a combination of SEQ ID NO: 11 and 10, or SEQ ID NO: 11 and 13. This includes sequences that can hybridize to such sequences under stringent hybridization conditions, sequences complementary thereto, modified bases, modified backbones, and sequences having functional cleavage or extension portions thereof (possessing or regulating the pre-mRNA processing activity in the RAGE gene transcript).

[0028] In certain embodiments, the AONs may be 100% complementary to the target sequence, or may contain mismatches as long as the heteroduplex formed between the oligonucleotide and the target sequence is stable enough to resist the action of cellular nucleases and other modes of degradation that may occur in vivo, for example, to accommodate variants. Thus, certain oligonucleotides may have approximately or at least about 70% sequence complementarity between the oligonucleotide and the target sequence, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity.

[0029] The invention is also extended to combinations of two or more AONs, such as constructs comprising two or more such AONs, having the ability to bind to a selected target for regulating the alternative splicing of RAGE pre-mRNA. The constructs may be used together, intended for a therapy based on the combined AONs. The combination of AONs is preferably a combination of SEQ ID NO: 11 and 10, or SEQ ID NO: 11 and 13.

[0030] According to a further aspect of the present invention, it extends to cDNA or a cloned copy of the AON sequence of the present invention, and a vector having the AON sequence of the present invention. The present invention further extends to cells having such sequences and / or vectors.

[0031] A method for manipulating the splicing of RAGE gene transcripts, comprising: a) providing one or more of the AONs as described herein and enabling the oligomer to bind to the target nucleic acid site is also provided.

[0032] A pharmaceutical, prophylactic, or therapeutic composition for treating, preventing, or alleviating the effects of a disease associated with RAGE expression in a patient, comprising: a) one or more AONs as described herein; and b) one or more pharmaceutically acceptable carriers and / or diluents is also provided.

[0033] The composition may contain each of the desired AONs of the present invention at about 1 nM to 1000 nM. Preferably, the composition may contain each of the AONs of the present invention between about 10 nM to 500 nM, most preferably between 1 nM to 10 nM.

[0034] A method for treating, preventing, or alleviating the effects of a disease associated with RAGE expression, comprising: a) administering to a patient in an effective amount one or more AONs as described herein or a pharmaceutical composition comprising one or more AONs is also provided.

[0035] Also provided is the use of the purified and isolated AONs as described herein in the manufacture of a medicament for treating, preventing, or alleviating the effects of a disease associated with RAGE expression and / or activity.

[0036] A kit for treating, preventing, or alleviating the effects of a disease associated with RAGE expression in a patient, the kit comprising at least an AON and combinations or cocktails thereof as described herein, packaged together with instructions for use in a suitable container.

[0037] Preferably, the disease associated with RAGE expression in the patient is a neurodegenerative disease, cancer, lung disorder, or inflammatory disease.

[0038] The subject having a disease associated with RAGE expression may be a mammal including a human.

[0039] Further aspects of the invention will be described herein with reference to the accompanying non-limiting examples and drawings.

[0040] Further features of the invention will be more fully described in the following description of some non-limiting embodiments thereof. This description is included solely for the purpose of exemplifying the invention. It should not be understood as a limitation on the general summary, disclosure, or description of the invention as set forth above. The description will be made with reference to the following accompanying drawings.

Brief Description of the Drawings

[0041]

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Mode for Carrying Out the Invention

[0042] An antisense oligonucleotide (AON) is a short modified synthetic antisense strand of DNA or RNA that can selectively hybridize to pre-RNA / mRNA through Watson-Crick base pairing and selectively regulate the function of the target RNA.

[0043] When AONs are used to regulate the selective splicing of mRNA, they are often referred to as splice-switching oligonucleotides (SSOs). In the present invention, the terms AON and SSO may be used interchangeably. SSOs base pair with pre-mRNA and disrupt the normal splicing repertoire of transcripts by blocking RNA-RNA base pairs or protein-RNA binding interactions that occur between components of the splicing machinery and pre-mRNA. SSOs can induce the "skipping" of selected exons and / or the retention of intron sequences in order to regulate the products of translation. This can be achieved by directly targeting splice sites, or by targeting cis-acting sequences involved in enhancing or silencing splicing by regulating the binding of specific proteins or modifying the secondary structure of pre-mRNA.

[0044] Therapeutic SSOs are used to treat genetic disorders and may enable the production of a protein that functions as a therapeutic at this stage, even if it skips incomplete or misaligned exons and is internally deleted.

[0045] Alternative splicing is recognized as an important layer of post-transcriptional gene regulation in the receptor for advanced glycation end products (RAGE). Although most RAGE is expressed as its full-length isoform, several different coding isoforms are generated through alternative splicing (also known as spliceoforms), such as spliceoforms with N-terminal truncation, C-terminal truncation, and spliceoforms that retain intron sequences. These different spliceoforms may act as potential regulators of the full-length RAGE receptor, either by competitive ligand binding or displacement of the full-length protein from binding partners. Over 20 spliceoforms have been identified in different tissues such as lung, liver, kidney, smooth muscle, endothelial cells, and brain.

[0046] Different RAGE gene splice variants are named RAGE, RAGE_v1 to RAGE_v19 according to the Human Gene Nomenclature Committee. For example, retention of intron 9 (exon 9b) (run-on) results in premature termination and complete deletion of the transmembrane and cytoplasmic domains that generate the C-cleaved soluble splice form (RAGE_v1, endogenous secreted RAGE or esRAGE) that constitutes approximately 5% of circulating RAGE in humans. In the absence of any signaling element or transmembrane domain, esRAGE can act as a decoy receptor that competes with full-length RAGE for ligands or enhances ligand clearance. While higher circulating levels of esRAGE are associated with improved health outcomes and longevity, lower esRAGE is associated with many pathologies including, but not limited to, atherosclerosis, diabetes, metabolic syndrome, cardiovascular mortality, anemia, autism and various neoplastic states. Treatment of diabetic mice with recombinant esRAGE reduces atherosclerosis, vascular inflammation, kidney injury and retinal injury.

[0047] RAGEΔ (also known as DN RAGE or RAGEv20) lacks 16 amino acids of the intracellular domain but retains the ligand-binding and transmembrane domains, acting as a dominant-negative inhibitor of cell surface RAGE. Furthermore, splicing events that result in changes in the extracellular domain may affect the ligand-binding domain by insertion, deletion, or removal of part or all of the Ig-V domain of RAGE. For example, N-RAGE does not have the signal peptide or V-domain required for ligand binding by starting at an alternative start site in exon 3.

[0048] Aberrant splicing of RAGE (and thus dysfunctional RAGE signaling) has been reported in diabetes, some cancers and Alzheimer's disease.

[0049] Skipping of exon 10 in esRAGE splicing is due to the restriction of intron length in higher eukaryotes. Approximately 45 nucleotides need to be separated between the 5' splice site and the branch point site, and the minimum distance between the branch point site and the 3' splice site seems to be approximately 18 nucleotides each. Thus, introns shorter than 70 nucleotides are extremely rare in mammals and cannot be efficiently spliced out. When the 5' splice site of esRAGE in intron 9 is selected, the distance between this site and the 3' splice site bordering exon 10 is 46 nucleotides, which is significantly shorter than the lower limit of intron length. Therefore, downstream, the use of the 5' splice site of esRAGE in intron 9 and the inclusion of exon 10 are mutually exclusive. Among the known splice variants analyzed, all mutants that use the 5' splice site of esRAGE in the downstream intron 9 skip exon 10; in contrast, all mutants that use the 5' splice site of RAGE in the upstream intron 9 include exon 10. Therefore, available evidence indicates that the selection of either one of the two alternative 5' splice sites in intron 9 is associated with the inclusion or removal of exon 10. Means for regulating this splicing or external means for regulation have not been previously known.

[0050] In the present invention, SSOs are used to selectively manipulate the alternative splicing pattern of RAGE pre-mRNA and to result in the generation of any native RAGE mRNA spliceform that acts as a decoy receptor to antagonize either non-functional or ligand-dependent activation and ligand-independent transactivation of full-length RAGE.

[0051] In particular, no RAGE polymorphisms common to these splice sites are observed. The RAGE sequence is highly conserved. Thus, personalized or individualized sequence modifications are not required, unlike the management of genetic disorders by exon skipping technology.

[0052] The present invention provides an alternative method for the treatment, prevention or remission of the effects of diseases in which RAGE is involved in expression or progression, including but not limited to neurodegenerative diseases, cancer, lung disorders, or inflammatory diseases, by developing AONs that regulate the alternative splicing of RAGE pre-mRNA or a part thereof.

[0053] Broadly, according to one aspect of the present invention, there is provided an isolated or purified AON for regulating pre-mRNA splicing in the receptor for advanced glycation end products (RAGE) gene transcript or a part thereof. Preferably, there is provided an isolated or purified AON for inducing exon removal and / or intron retention in RAGE pre-mRNA or a part thereof.

[0054] The present invention provides an AON that binds to a selected target on the receptor for advanced glycation end products (RAGE) gene transcript and has the ability to regulate pre-mRNA splicing in the RAGE gene transcript or a part thereof.

[0055] For example, in one aspect of the present invention, there is provided a 10-50 nucleotide AON comprising a targeting sequence complementary to a region of RAGE pre-mRNA or a part thereof that is related to the binding of a protein involved in the regulation of alternative splicing of mRNA.

[0056] [Table 1]

[0057] [Table 2]

[0058] [Table 3]

[0059] Unlike other AON-based therapies, the present invention does not induce enhanced degradation of RNA via the recruitment of ribonuclease H, where ribonuclease H preferentially binds to and degrades RNA that is double-stranded bound to the DNA of the RAGE gene. Regulating the amount of RAGE protein produced by interfering with normal functions such as replication, transcription, translocation, and translation does not depend on the hybridization of AON to RAGE genomic DNA or the binding of AONs to mRNA. Rather, AONs are used to regulate pre-mRNA splicing in the RAGE gene transcript or a part thereof, and to induce exon "skipping" or retention of intron sequences (run-on). The method preferably reduces the expression of RAGE spliceoforms capable of mediating RAGE-dependent signaling and / or enhances the production of RAGE spliceoforms lacking functional domains capable of mediating RAGE-dependent signaling.

[0060] According to a first aspect of the present invention, there are provided AONs that bind to a selected target on the RAGE gene transcript and are capable of regulating pre-mRNA splicing in the RAGE gene transcript or a part thereof. Broadly, there are provided isolated or purified AONs for inducing targeting of exon removal / intron retention in the RAGE gene transcript or a part thereof.

[0061] "Isolated" means a material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polynucleotide" or "isolated oligonucleotide" as used herein refers to a polynucleotide that has been purified or removed from the sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from the sequences flanking it in a DNA fragment within the genome. The term "isolating" when it relates to a cell refers to the purification from a source subject of cells (e.g., fibroblasts, lymphocytes), e.g., a subject having a polynucleotide repeat disease. "Isolating" in the context of DNA, mRNA, or protein refers to the recovery from a source, e.g., a cell, of DNA, mRNA, or protein.

[0062] AON can be described as being "induced" or "targeted" to the target sequence of the partner it hybridizes to. In certain embodiments, the target sequence includes regions that include the 3' or 5' splice site of the preprocessed mRNA, the branch point site, or other sequences involved in the regulation of splicing, such as splice enhancers and silencers and sites that determine the secondary structure of the RNA that affects splicing. The target sequence may be within an exon or an intron, or may span an intron / exon junction.

[0063] In certain embodiments, the AON has sufficient sequence complementarity to the target RNA (e.g., pre-mRNA) to block a region of the target RNA in an effective manner. In an exemplary embodiment, such blocking of the RAGE pre-mRNA functions to regulate splicing by masking the binding site for a splicing protein that would otherwise regulate splicing and / or by altering the structure of the targeted RNA. In some embodiments, the target RNA is the target pre-mRNA (e.g., RAGE gene pre-mRNA).

[0064] To regulate the splicing of the target RNA, an AON having sufficient sequence complementarity to the target RNA sequence means that the AON has a sequence sufficient to induce masking of the binding site for a natural protein that would otherwise regulate splicing and / or to alter the three-dimensional structure of the targeted RNA.

[0065] The selected AONs can be shortened, e.g., about 12 bases or more, e.g., about 50 bases, and are sufficiently complementary to the sequence to effect splice regulation upon hybridization to the target sequence and optionally contain a few mismatches as long as they form a heteroduplex with the RNA having a Tm of 45°C or higher.

[0066] Preferably, the AON is selected from the group consisting of the sequences shown in any of SEQ ID NOs: 1-31 and / or Tables 3a-3d. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

[0067] In certain embodiments, the degree of complementarity between the target sequence and the AON is sufficient to form a stable duplex. The region of complementarity between the AONs and the target RNA sequence may be as short as about 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). AONs of about 16-17 bases are generally long enough to have a unique complementary sequence. In certain embodiments, the minimum length of the complementary bases may be required to achieve the required binding Tm as discussed herein.

[0068] In certain embodiments, oligonucleotides about 50 bases in length may be suitable when at least a minimum number of bases, such as 10-12 bases, are complementary to the target sequence. However, generally, facilitated or active uptake in cells is optimized with oligonucleotide lengths of less than about 30 bases. For the phosphorodiamidate morpholino oligomers (PMO) AONs further described herein, the optimal balance of binding stability and uptake generally occurs at 18-25 bases in length. AONs (e.g., PMOs, PMO-X, PNAs, LNAs, TINA, 2'-OMe) consisting of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 bases are included.

[0069] In certain embodiments, the AONs may be 100% complementary to the target sequence or may contain mismatches as long as the heteroduplex formed between the oligonucleotide and the target sequence is stable enough to resist the action of cellular nucleases and other modes of degradation that may occur in vivo, for example, to accommodate variants. Thus, certain oligonucleotides may have a sequence complementarity of approximately or at least about 70% between the oligonucleotide and the target sequence, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0070] When present, mismatches typically do not destabilize more towards the middle than towards the terminal regions of the hybrid duplex. The number of tolerated mismatches will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatches within the duplex, according to well-understood principles of duplex stability. Such AONs, while not necessarily 100% complementary to the target sequence, are effective in stably and specifically binding to the target sequence, thereby regulating the splicing of the target pre-RNA.

[0071] The stability of the double-strand formed between the AON and the target sequence is a function of the binding Tm and sensitivity of the double-strand to cellular enzyme cleavage. The Tm of the oligonucleotide for the complementary sequence RNA may be measured by conventional methods, such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or as described in Miyada C.G. and Wallace R.B., 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107. In certain embodiments, the AONs may have a binding Tm for the complementary sequence RNA that exceeds body temperature, preferably exceeds about 45°C or about 50°C. Also included are cases where the Tm is in the range of 60-80°C or higher.

[0072] As further examples of variants, AONs having sequence identity or homology of approximately or at least about 70% over the full length of the sequences shown in any of SEQ ID NOs: 1-31 and / or any of Tables 3a-3d, such as 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 are included. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

[0073] More specifically, provided are AONs that are capable of binding to a selected target site and modifying pre-mRNA splicing in the RAGE gene transcript or a portion thereof. The AON is preferably selected from the sequences shown in any of SEQ ID NOs: 1-31 and / or any of Tables 3a-3d. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

[0074] Modification of pre-mRNA splicing preferably induces "skipping" or removal of one or more exons or retention of introns of the mRNA. The resulting protein is preferably shorter in length compared to the parental full-length RAGE protein due to either internal cleavage or premature termination. These truncated RAGE proteins may be referred to as splice isoforms of the full-length RAGE protein.

[0075] The remaining exons of the generated mRNA are in-frame and may produce a shorter protein with a sequence similar to that of the parental full-length protein, except for having an internal cleavage within the region between the original 3' and 5' ends. In another possibility, exon skipping may induce a frameshift that results in a protein where the first part of the protein is substantially identical to the parental full-length protein, but the second part of the protein has a different sequence (e.g., a nonsense sequence) due to the frameshift. Alternatively, exon skipping may result in the disruption of the reading frame and the presence of premature termination of translation, thereby inducing the production of a prematurely terminated protein. The prematurely terminated protein may be the result of prematurely terminated mRNA (e.g., skipping of exons 10 and / or 11), or it may be the result of readthrough into an intron (e.g., RAGE 9b) or missense skipping (providing mRNA having exons 10 and / or 11 mRNA but not providing the expression of the proteins encoded by these exons).

[0076] Skipping of each of exons 1-9 will preferably disrupt the reading frame of the RAGE transcript. This will result in enhanced degradation of the RNA through nonsense-mediated decay.

[0077] Skipping of each of exons 1 to 11 of Exxon will preferably result in maintaining the reading frame intact. This will preferably result in translation into an internally truncated protein. The truncated protein or RAGE mRNA splice isoform may have a completely lost function, may have a reduced function or may act as a decoy receptor.

[0078] Preferably, these truncated nonsense or immature termination proteins lack one or more functional domains involved in the induction of intracellular signaling pathways by RAGE ligands or the ligand-independent transactivation of RAGE by the arranged GPCRs. For example, exon 10 encodes a transmembrane domain, and removal of this exon may generate a soluble RAGE protein that may potentially act as a soluble decoy or competitive antagonist of ligand-induced signaling via RAGE. The truncated nonsense or immature termination proteins may further lack attachment or binding sites for other factors, and their removal may cause a decrease in the interaction with the signaling pathways associated with the RAGE protein.

[0079] Alternatively, removal of one or more exons may cause misfolding of the RAGE protein and a decrease in the ability of the protein to be efficiently transported across the membrane.

[0080] The presence of an internally truncated protein (i.e., a protein lacking amino acids encoded by one or more exons) is preferred. When the RAGE protein is inhibited, problems associated with enhanced RAGE transcription may occur because the body attempts to compensate for the reduced total amount of the RAGE protein. In contrast, the presence of an internally truncated protein (preferably lacking one or more characteristics of the full RAGE protein) needs to be sufficient to provide a therapeutic benefit due to the reduced total amount of the functional RAGE protein, even if it blocks enhanced transcription.

[0081] The AON-induced exon skipping of the present invention needs to substantially abolish the function of the RAGE protein, rather than completely. Preferably, the regulation of alternative splicing through the exon skipping process results in a decrease or loss of the functionality of the RAGE protein.

[0082] For different isoforms of RAGE generated using different skipping methods, preferably, proteins with lost or reduced signaling activity can be obtained, which can be used for treating or preventing different diseases related to RAGE activity, such as neurodegenerative diseases, cancers, lung disorders, or inflammatory diseases. The alternative splicing method may form truncated proteins or proteins with reduced function that can preferably be used as treatments for specific aspects, forms, or progressions of diseases related to RAGE expression and activity.

[0083] The skipping process of the present invention using AONs may remove (skip) each exon or may result in the simultaneous skipping of two or more exons.

[0084] The skipping process of the present invention using AONs may include retaining intron sequences in the presence or absence of direct skipping of one or more exons.

[0085] The AONs of the present invention may be a combination of two or more AONs capable of binding to a selected target and inducing exon removal in the RAGE gene transcript. The combination may be a cocktail of two or more AONs and / or a construct comprising two or more AONs linked together.

[0086]

Table 4

[0087]

Table 5

[0088]

Table 6

[0089]

Table 7

[0090] The present invention is a method for regulating alternative splicing in RAGE gene transcripts, providing one or more of the AONs as described herein, and enabling the oligomer to bind to the target nucleic acid site further providing a method comprising.

[0091] According to yet another aspect of the present invention, a nucleic acid sequence target for regulating alternative splicing of RAGE pre-mRNA, a DNA equivalent of a nucleic acid sequence selected from the group consisting of the sequences shown in any of SEQ ID NOs: 1 to 31 and / or Tables 3a to 3d, and a nucleic acid sequence target comprising a sequence complementary thereto is provided. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. The AON may be a combination of AONs, preferably a combination of SEQ ID NO: 11 and 10, or SEQ ID NO: 11 and 13.

[0092] By designing AONs to completely mask the consensus splice site, it is not necessarily necessary to cause a change in the splicing of the targeted exon. Furthermore, the inventors have discovered that the size or length of the AON itself is not always a major factor when designing AONs. Using some targets, AONs as short as about 20 bases were able to induce some exon inclusion more efficiently than other longer (eg 25 bases) oligomers specific for the same exon in certain cases.

[0093] The inventors have also discovered that there does not appear to be any standard motifs that can be blocked or masked by AONs for redirecting splicing. It has been found that AONs need to be designed for each gene target and their individual effectiveness needs to be empirically evaluated.

[0094] More specifically, the AON may be selected from those shown in any of Tables 3a - 3d. The sequences are preferably selected from the group consisting of any one or more of SEQ ID NOs: 1 - 31, and combinations or cocktails thereof. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. Combinations of AONs are preferably the combination of SEQ ID NO: 11 and 10, or the combination of SEQ ID NO: 11 and 13. This includes sequences that can hybridize to such sequences under stringent hybridization conditions, sequences complementary thereto, modified bases, modified backbones, and sequences having functional cleavage or extension portions thereof (having or regulating pre - mRNA processing activity in the RAGE gene transcript).

[0095] Oligomers and DNA, cDNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen - bond to each other. Thus, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or pairing such that a stable and specific binding occurs between the oligomer and the DNA, cDNA or RNA target. It is understood in the art that the sequence of an AON need not be 100% complementary to the sequence of its target sequence to which it is specifically hybridizable. 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 has a sufficient degree of complementarity to avoid non - specific binding to non - target sequences of the AON under the conditions where specific binding is desired, i.e., in the case of in vivo assays or therapeutic treatments, and under physiological conditions in the case of in vitro assays, the conditions under which the assay is performed.

[0096] Selective hybridization may be under low, medium or high stringency conditions, but is preferably under high stringency. One of ordinary skill in the art will understand that the stringency of hybridization is affected by conditions such as salt concentration, temperature, or organic solvent, in addition to the base composition, length of the complementary strand, and the number of nucleotide base mismatches between hybridizing nucleic acids. Stringent temperature conditions will generally include temperatures above 30°C, typically above 37°C, and preferably above 45°C, preferably at least 50°C, and typically 60°C - 80°C or higher. Stringent salt conditions will usually be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is far more important than any single parameter scale. 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). Accordingly, the AONs of the present invention may include oligomers that selectively hybridize to the sequences provided in Tables 3a - 3d, or any of SEQ ID NOs: 1 - 31. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

[0097] It will be understood that the codon sequence at the end of an exon in a structural protein does not always disrupt at the end of the codon, and as a result, it may be necessary to delete two or more exons from the pre - mRNA to ensure in - frame reading of the mRNA. In such circumstances, it may be necessary to select multiple AONs by the method of the present invention, where each is specific for a different region involved in inducing the inclusion of the desired exon and / or intron. Tm is the temperature at which 50% of the target sequence hybridizes to the complementary polynucleotide at a given ionic strength and pH. Such hybridization may occur with "approximate" or "substantial" complementarity, as well as exact complementarity, to the target sequence of the AON.

[0098] Typically, selective hybridization will occur when there is at least about 55% identity, preferably at least about 65%, more preferably at least about 75% and most preferably at least about 90%, 95%, 98% or 99% identity over a stretch of at least about 14 nucleotides with the nucleotides of the AON. As noted above, the length of the homology comparison may extend to longer stretches and in certain embodiments is at least about 9 nucleotides, usually at least about 12 nucleotides, more usually at least about 20 nucleotides, often at least about 21, 22, 23 or 24 nucleotides, at least about 25, 26, 27 or 28 nucleotides, at least about 29, 30, 31 or 32 nucleotides, often at least about 36 or more nucleotides.

[0099] Accordingly, the AON sequences of the present invention preferably have at least 75%, more preferably at least 85%, more preferably at least 86%, 87%, 88%, 89% or 90% homology to the sequences shown in the Sequence Listing herein. More preferably, there is at least 91%, 92%, 93%, 94% or 95% homology, more preferably at least 96%, 97%, 98% or 99% homology. Generally, as the length of the AON is shortened, the homology required to obtain selective hybridization increases. As a result, when the AON of the present invention consists of less than about 30 nucleotides, the percentage identity is preferably greater than 75%, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% greater than the AONs shown in the Sequence Listing herein. Nucleotide homology comparisons may be performed by sequence comparison programs such as the GCG Wisconsin Bestfit program or GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this method, sequences of similar or substantially different lengths as cited herein are comparable by insertion into the alignment of gaps, and such gaps can be determined by the comparison algorithm used, for example, by GAP.

[0100] The AON of the present invention may have regions with reduced homology to the target sequence and regions with exact homology. It is not necessary for the oligomer to have exact homology throughout its entire length. For example, the oligomer may have a continuous stretch of at least 4 or 5 bases that are identical to the target sequence, preferably a continuous stretch of at least 6 or 7 bases that are identical to the target sequence, more preferably a continuous stretch of at least 8 or 9 bases that are identical to the target sequence. The oligomer may have a stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 bases that are identical to the target sequence. The remaining stretches of the oligomer sequence may be intermittently identical to the target sequence; for example, the remaining sequence may have an identical base, followed by a non-identical base, followed by an identical base. Alternatively (or similarly), the oligomer sequence may have several stretches (e.g., 3, 4, 5 or 6 bases) of identical sequences with stretches of homology that are not fully satisfied scattered therein. Such sequence mismatches will preferably have no or only a very slight reduction in splicing switching activity.

[0101] The terms "modulate" or "modulates" include increasing or decreasing, optionally defined and / or by a statistically significant amount, one or more quantifiable parameters. The terms "increase" or "increasing", "enhance" or "enhancing", or "stimulate" or "stimulating" generally refer to the ability of one of the AONs or compositions to produce or cause a greater physiological response (i.e., downstream effect) in a cell or subject as compared to the response caused by either no AON or a control compound. The terms "decreasing" or "decrease" generally refer to the ability of one of the AONs or compositions to produce or cause a reduced physiological response (i.e., downstream effect) in a cell or subject as compared to the response caused by either no AON or a control compound.

[0102] The relevant physiological or cellular response (in vivo or in vitro) will be apparent to those skilled in the art and may include an increase in the removal of a particular exon in the pre-mRNA encoding RAGE, a decrease in the amount of pre-mRNA encoding RAGE, or a decrease in the expression of functional RAGE protein in the cells, tissues, or subjects requiring it. An "increased" or "enhanced" amount is typically a statistically significant amount and is a multiple of 1.1-fold, 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points greater than 1 and less than 2, e.g., 1.5, 1.6, 1.7, 1.8) of the amount produced without an AON (in the absence of the agent) or by a control compound. The terms "reduce" or "inhibit" may generally relate to the ability of one or more AONs or compositions, as a measurement according to ordinary techniques in diagnostic techniques, to "reduce" the relevant physiological or cellular response, such as the symptoms of the diseases or conditions described herein. 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 the symptoms or pathology of diseases such as cancer, neurodegenerative diseases, lung disorders, and other inflammatory diseases. A "reduction" in the response may be statistically significant compared to the response brought about by an AON-free or control composition and may include a reduction 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 therebetween).

[0103] The length of the AON may vary as long as it has the ability to selectively bind to the intended position within the pre-mRNA molecule. The length of such a sequence can be determined according to the selection procedures described herein. Generally, the AON will range from about 10 nucleotides in length to a maximum of about 50 nucleotides in length. However, it will be understood that any length of nucleotides within this range may be used in the method. Preferably, the length of the AON is between 10 and 40, between 10 and 35, 15 to 30 nucleotides in length or 20 to 30 nucleotides in length, most preferably about 25 to 30 nucleotides in length. For example, the oligomer may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

[0104] As used herein, "AON" refers to a linear sequence of nucleotides, or nucleotide analogs, in which the nucleobases hybridize to a target sequence in RNA by Watson-Crick base pairing and are capable of forming an oligonucleotide:RNA heteroduplex within the target sequence. The terms "AON", "AON", "oligomer" and "antisense compound" may be used interchangeably to refer to oligonucleotides. The cyclic subunits may be based on ribose or another pentose sugar or, in certain embodiments, a morpholino group (see description of morpholino oligonucleotides below). Additionally, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and 2'-O-methyl oligonucleotides are contemplated among other antisense agents known in the art.

[0105] Non-naturally occurring AONs, or "oligonucleotide analogs," include, for example, (i) modified backbone structures, such as backbones other than the standard phosphodiester bond found in oligonucleotides and polynucleotides, and / or (ii) modified sugar moieties, such as morpholino moieties rather than ribose or deoxyribose moieties. Oligonucleotide analogs support bases having the ability to hydrogen bond by Watson-Crick base pairing with standard polynucleotide bases, where the analog backbone presents bases such that such hydrogen bonding occurs in a sequence-specific manner between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Preferred analogs are those having a substantially uncharged phosphorus-containing backbone.

[0106] One method for generating AONs is methylation at the 2'-hydroxyl ribose position, which, by incorporation of a phosphorothioate backbone, generates molecules that are outwardly similar to RNA but are far more resistant to nuclease degradation. Those skilled in the art of the present invention will understand other forms of suitable backbones that may be usable in the context of the present invention.

[0107] To avoid degradation of pre-mRNA during double-strand formation with AONs, the AONs used in this method may be adapted to minimize or prevent cleavage by endogenous ribonuclease H. This property is highly preferred because treatment of RNA with unmethylated oligomers, either intracellularly or in a crude extract containing ribonuclease H, causes degradation of the pre-mRNA:AON duplex. Any form of modified AONs capable of bypassing or not inducing such degradation may be used in this method. Nuclease resistance may be achieved by modifying the AONs of the present invention to include a partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups including carboxylic acid groups, ester groups, and alcohol groups.

[0108] Examples of AONs that are not cleaved by cellular ribonuclease H when duplexed with RNA are 2'-O-methyl derivatives. Such 2'-O-methyl-oligoribonucleotides are stable in the cellular environment and in animal tissues, and their duplexes with RNA have higher Tm values than their ribo- or deoxyribo counterparts. Alternatively, the nuclease-resistant AONs of the invention may have at least one fluorine-added final 3'-terminal nucleotide. Further alternatively, the nuclease-resistant AONs of the invention have phosphorothioate linkages connecting at least two of the final 3-terminal nucleotide bases, preferably phosphorothioate linkages connecting between the final four 3'-terminal nucleotide bases.

[0109] Enhanced splice switching may also be achieved by alternative oligonucleotide chemistries. For example, AONs may be selected from the list including phosphoramidates or phosphorodiamidate morpholino oligomers (PMO); PMO-X; PPMO; peptide nucleic acids (PNA); locked nucleic acids (LNA) and derivatives including α-L-LNA, 2'-amino LNA, 4'-methyl LNA and 4'-O-methyl LNA; ethylene-bridged nucleic acids (ENA) and their derivatives; phosphorothioate oligomers; tricyclic DNA oligomers (tcDNA); tricyclic phosphorothioate oligomers; 2'O-methyl modified oligomers (2'-OMe); 2'-O-methoxyethyl (2'-MOE); 2'-fluoro, 2'-fluoroarabinonucleic acid (FANA); unlocked nucleic acids (UNA); thermally stable kinked intercalating nucleic acids (TINA), hexitol nucleic acids (HNA); cyclohexenyl nucleic acids (CeNA); 2'-amino (2'-NH2); 2'-O-ethyleneamine, or any combination of the foregoing as mixmers or gapmers. To further improve delivery efficacy, the modified nucleotides described above are often conjugated to sugars or nucleobase moieties with fatty acids / lipids / cholesterol / amino acids / carbohydrates / polysaccharides / nanoparticles etc. These conjugated nucleotide derivatives can also be used to construct exon-skipping AONs. Antisense oligonucleotide-induced splice modifications of human RAGE gene transcripts generally use either oligoribonucleotides, PNAs, 2OMe or MOE modified bases on a phosphorothioate backbone. For oligonucleotide design, 2OMeAOs are used because of their efficient uptake in vitro when delivered as cationic lipoplexes, but these compounds are susceptible to nuclease degradation and are not considered ideal for in vivo or clinical applications. When alternative chemistries are used to make the AONs of the present invention, the uracil (U) of the sequences provided herein may be replaced with thymine (T).

[0110] Non-naturally occurring oligomers, or "oligonucleotide analogs," such as (i) modified backbone structures, e.g., backbones other than the standard phosphodiester bond found in naturally occurring oligonucleotides and polynucleotides, and / or (ii) modified sugar moieties, e.g., morpholino moieties rather than ribose or deoxyribose moieties, are included in the AONs of the present invention. Oligomer analogs support bases having the ability to hydrogen bond by Watson-Crick base pairing with standard polynucleotide bases, where the analog backbone presents bases such that sequence-specific hydrogen bonding between the oligomer analog molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA) is tolerated. Preferred analogs are those having a substantially uncharged phosphorus-containing backbone.

[0111] Antisense oligonucleotides that do not activate ribonuclease H can be prepared according to known techniques (see, for example, U.S. Patent No. 5,149,797). Such AONs may be deoxyribonucleotide or ribonucleotide sequences, and simply have any structural modification that sterically hinders or prevents the binding of ribonuclease H to a double-stranded molecule having an oligomer as one member, and has a structural modification that does not substantially interfere with or disrupt double-strand formation. Since the portion of the oligomer involved in double-strand formation is substantially different from the portion involved in its binding to ribonuclease H, a very large number of AONs that do not activate ribonuclease H are available. For example, such AONs may be oligomers in which at least one or all of the internucleotide bridging phosphate residues are modified phosphates, such as methylphosphonate, methylphosphorothioate, phosphoromorpholidate, phosphoropiperazidate, boranophosphate, amide bond, and phosphoroamidate. For example, all other internucleotide bridging phosphate residues may be modified as described above. In another non-limiting example, such AONs are molecules in which at least one or all of the nucleotides have a 2'-lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl, etc.). For example, all other nucleotides may be modified as described above.

[0112] While the above AONs are preferred forms of the AONs of the present invention, the present invention includes other oligomeric antisense molecules, including, but not limited to, oligomer mimics such as the following.

[0113] Specific examples of preferred AONs useful in the present invention include oligomers having a modified backbone or non-natural internucleoside linkages. As defined herein, oligomers having a modified backbone include those that retain a phosphorus atom within the backbone and those that do not have a phosphorus atom within the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified oligomers that do not have a phosphorus atom within their internucleoside backbone can also be considered AONs.

[0114] In other preferred oligomer mimics, both the sugar of the nucleotide unit and the internucleoside linkage, i.e., the backbone, are replaced with novel groups. The base units are maintained in hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an oligomer mimic that has been shown to have superior hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligomer is replaced with an amide having a backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone.

[0115] Another preferred chemistry is an oligomeric compound of phosphorodiamidate morpholino oligomers (PMOs) that are not degraded by any known nuclease or protease. These compounds are uncharged and have been shown not to activate ribonuclease H activity when bound to an RNA strand and to exhibit persistent splice modulation after in vivo administration (Summerton and Weller, Antisense Nucleic Acid Drug Development, 7, 187 - 197).

[0116] The modified oligomers may also contain one or more substituted sugar moieties. The oligomers may further include modifications or substitutions of nucleobases (often simply referred to as "bases" in the art). Certain nucleobases are particularly useful for enhancing the binding affinity of the oligomeric compounds of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6 substituted purines, such as 2-aminopropyladenine, 5-propenyluracil and 5-propynylcytosine. 5-Methylcytosine substitution has been shown to enhance the duplex stability of nucleic acids by 0.6 - 1.2 °C when combined more particularly with 2'-O-methoxyethyl sugar modification.

[0117] Another modification of the oligomers of the present invention involves chemically linking one or more moieties or conjugates of the oligomer that enhance the activity, cellular distribution or cellular uptake of the oligomer. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholic acid, thioethers, such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains, such as dodecanediol or undecyl residues, phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantaneacetic acid, palmitoyl moieties, myristyl, or octadecylamine or hexylamino-carbonyl-oxysterol moieties.

[0118] To enhance cellular uptake and nuclear localization, cell-penetrating peptides have been added to phosphorodiamidate morpholino oligomers. As shown by Jearawiriyapaisarn et al. (2008), Mol. Ther. 16 9, 1624 - 1629, different peptide tags have been shown to affect the efficiency of uptake and target tissue specificity.

[0119] It is not necessary that all positions in a given compound be uniformly modified; indeed, two or more of the above modifications may be incorporated into a single nucleoside within a single compound or even within an oligomer. The invention also includes AONs that are chimeric compounds. "Chimeric" AONs or "chimeras" are, in the context of the present invention, AONs, particularly oligomers, having two or more chemically distinct regions, each of which is composed of at least one monomer unit, i.e., a nucleotide in the case of an oligomeric compound. These oligomers typically have at least one region where the oligomer is modified to confer additional regions for enhanced nuclease degradation resistance, enhanced cellular uptake, and enhanced binding affinity for a target nucleic acid, relative to the oligomer or AON.

[0120] The activities of AONs and their variants are assayable according to conventional techniques in the art. For example, the splice forms and expression levels of the RNAs and proteins being investigated may be evaluated by any of a variety of well-known methods for detecting the splice forms and / or expression of transcribed nucleic acids or proteins. Non-limiting examples of such methods include RT-PCR of RNA splice forms followed by size separation of the PCR products, nucleic acid hybridization methods such as the use of Northern blots and / or nucleic acid arrays; nucleic acid amplification methods; immunological methods for protein detection; protein purification methods; and protein function or activity assays.

[0121] RNA expression levels can be evaluated by preparing mRNA / cDNA (i.e., transcribed polynucleotide) from a cell, tissue, or organism and hybridizing the mRNA / cDNA with a reference polynucleotide that is a complement of, or a fragment of, the nucleic acid being assayed. The cDNA can optionally be amplified using any of a variety of polymerase chain reaction or in vitro transcription methods prior to hybridization with the complementary polynucleotide; preferably, it is not amplified. Further, the expression of one or more transcripts can be detected using quantitative PCR to evaluate the level of expression of the transcript.

[0122] The present invention provides an AON-induced splicing switch of RAGE gene transcripts, a clinically important oligomer chemistry, and a delivery system for inducing RAGE splicing manipulation to therapeutic levels. A substantial decrease in the amount of full-length RAGE mRNA from RAGE gene transcription, and thus RAGE protein, a) (i) Experimental evaluation of the intron enhancer target motif, (ii) development of AON length and oligomer cocktails, (iii) selection of chemistry, and (iv) addition of a cell-penetrating peptide (CPP) to enhance oligomer delivery, through the use of fibroblast cell lines for in vitro oligomer purification; and b) detailed evaluation of a novel approach for generating RAGE transcripts with one or more deleted exons is achieved.

[0123] As such, it is demonstrated herein that the alternative splicing of RAGE pre-mRNA can be regulated by specific AONs. In this way, a functionally significant decrease in the amount of full-length (signal-transducing) RAGE protein can be obtained, and / or an increase in the non-signal-transducing decoy receptor RAGE mRNA spliceform (RAGE_v1) or other decoy receptors can be achieved, thereby reducing the severe pathologies associated with diseases such as neurodegenerative diseases, cancer, lung disorders, and other inflammatory diseases.

[0124] The AONs used in accordance with the present invention may be conveniently prepared through well-known techniques related to solid-phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesizing oligomers on a modified solid support is described in U.S. Patent No. 4,458,066.

[0125] Any other means for such synthesis known in the art may additionally or alternatively be utilized. It is well known to use similar techniques for preparing oligomers such as phosphorothioates and alkylated derivatives. In one such automated embodiment, diethyl phosphoramidite may be used as a starting material and synthesized as described in Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

[0126] The AONs of the present invention are synthesized in vitro and do not contain antisense compositions of biological origin, or gene vector constructs are designed to induce in vivo synthesis of the AONs. The molecules of the present invention may also be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds, such as liposomes, receptor targeting molecules, oral, rectal, topical, or other formulations, for example, to assist in uptake, distribution, and / or absorption.

[0127] The AONs of the present invention may be utilized for the treatment of diseases and can also be used as prophylactic or therapeutic agents. Thus, in one embodiment, the present invention provides AONs that bind to selected targets in RAGE pre-mRNA to induce efficient and consistent exon skipping as described herein, in a therapeutically effective amount, mixed with a pharmaceutically acceptable carrier, diluent, or excipient.

[0128] Accordingly, the present invention is a medicament, prophylactic, or therapeutic composition for treating, preventing, or alleviating the effects of diseases associated with RAGE expression in a patient, a) one or more AONs as described herein, and b) one or more pharmaceutically acceptable carriers and / or diluents comprising a composition.

[0129] Preferably, the diseases associated with RAGE expression are selected from the list including neuropathy, cancer; cardiovascular disorders; digestive disorders; respiratory disorders, musculoskeletal, connective tissue disorders, kidney disorders, genital disorders, skin disorders, eye diseases and endocrine disorders.

[0130] In one embodiment of the present invention, the RAGE-related disorder is a cardiovascular disorder selected from the group consisting of atherosclerosis, ischemic heart disease, myocarditis, endocarditis, cardiomyopathy, acute rheumatic fever, chronic rheumatic heart disease, cerebrovascular disease / stroke, heart failure, vascular calcification, peripheral vascular disease, and lymphangitis.

[0131] In one embodiment of the present invention, the RAGE-related disorder is a digestive system disorder selected from the group consisting of periodontitis, esophagitis, gastritis, gastric / duodenal ulceration, Crohn's disease, ulcerative colitis, ischemic colitis, enteritis and pancolitis, peritonitis, alcoholic liver disease, hepatitis, toxic liver disease, biliary cirrhosis, liver fibrosis / cirrhosis, non-alcoholic fatty liver disease / non-alcoholic steatohepatitis (NAFLD / NASH), liver injury, trauma or hepatic trauma from surgery and recovery.

[0132] In one embodiment of the present invention, the RAGE-related disorder is cancer selected from the group consisting of malignant neoplasms of the lip, oral cavity and pharynx, malignant neoplasms of the digestive organs, malignant neoplasms of the respiratory and intrathoracic organs, malignant neoplasms of the bone and articular cartilage, melanoma and other malignant neoplasms of the skin, malignant neoplasms of the mesothelium and soft tissues, malignant neoplasms of the breast, malignant neoplasms of the female genital organs, malignant neoplasms of the male genital organs, malignant neoplasms of the urinary tract, malignant neoplasms of the eye, brain and other parts of the central nervous system, malignant neoplasms of the thyroid and other endocrine glands, malignant neoplasms of the lymph, hematopoietic and related tissues, secondary and / or unspecified site malignant neoplasms not clearly defined.

[0133] In one embodiment of the present invention, the RAGE-related disorder is a neurological disorder, selected from the group consisting of inflammatory diseases of the central nervous system, mainly systemic atrophy affecting the central nervous system, extrapyramidal and movement disorders, Parkinson's disease, demyelinating diseases of the central nervous system, Alzheimer's disease, focal cerebral atrophy, Lewy body disease, epilepsy, migraine, neuropathic pain, diabetic neuropathy, multiple neuropathies, the expression and progression of gliomas, spinal cord injury, and ischemic brain injury / stroke, brain injury, brain trauma from trauma or surgery, and recovery.

[0134] In one embodiment of the present invention, the RAGE-related disorder is a musculoskeletal disorder, selected from the group consisting of muscular dystrophy, congenital and storage myopathies, polymyositis, myasthenia gravis, dermatomyositis, inclusion body myositis, muscle atrophy, and muscle injury.

[0135] In one embodiment of the present invention, the RAGE-related disorder is a mental disorder, selected from the group consisting of dementia, Alzheimer's disease, vascular dementia, addiction, schizophrenia, major affective disorder, depression, mania, bipolar disorder, and anxiety disorder.

[0136] In one embodiment of the present invention, the RAGE-related disorder is a respiratory (lung) disorder, selected from the group consisting of acute upper respiratory tract infection, rhinitis, pharyngitis, sinusitis, laryngitis, influenza and pneumonia, acute bronchitis, acute bronchiolitis, asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, pulmonary emphysema, chronic lung diseases caused by topical agents, acute respiratory distress syndrome (ARDS), pulmonary eosinophilia, and pleurisy, lung injury, lung trauma from trauma or surgery, and recovery.

[0137] In one embodiment of the present invention, the RAGE-related disorder is a connective tissue disorder and is selected from the group consisting of osteoarthritis, infectious arthritis, rheumatoid arthritis, psoriatic and inflammatory bowel arthritis, juvenile arthritis, gout and other crystal arthropathies, diabetic arthropathy, polyarteritis nodosa, Churg-Strauss, mucocutaneous lymph node syndrome [Kawasaki], hypersensitivity vasculitis, Goodpasture syndrome, thrombotic microangiopathy, Wegener's granulomatosis, aortic arch syndrome [Takayasu], giant cell arteritis, polymyalgia rheumatica, microscopic polyangiitis, hypocomplementaemic vasculitis, systemic lupus erythematosus, dermatomyositis, polymyositis, systemic sclerosis, CREST syndrome, Sjogren's syndrome, mixed connective tissue disease, Behcet's disease, traumatic muscle injury, contusion, muscle contusion, and fracture.

[0138] In one embodiment of the present invention, the RAGE-related disorder is a renal disorder and is selected from the group consisting of glomerulonephritis, nephritis, diabetic kidney disease, interstitial nephritis, obstructive and reflux nephropathy, acute renal failure, and chronic kidney disease.

[0139] In one embodiment of the present invention, the RAGE-related disorder is a genital disorder and is selected from the group consisting of prostatitis, prostatic hyperplasia, prostatic dysplasia, salpingitis, oophoritis, pelvic inflammatory disease (PID), polycystic ovary syndrome, cervicitis, cervical dysplasia, vaginitis, vulvitis.

[0140] In one embodiment of the present invention, the RAGE-related disorder is a skin disorder selected from the group consisting of dermatitis, eczema, pemphigus / pemphygoid, psoriasis, pityriasis rosea, lichen planus, urticaria, erythema multiforme, erythema nodosum, sunburn, keratosis, photoaged skin ulcer, superficial skin injury, and open wound.

[0141] In one embodiment of the present invention, the RAGE-related disorder is an eye disorder selected from the group consisting of keratitis, conjunctivitis, retinitis, glaucoma, scleritis, episcleritis, choroiditis, diabetic retinopathy, macular edema, retinopathy of prematurity, and optic neuritis, eye injury, ocular trauma from injury or surgery and recovery.

[0142] In one embodiment of the present invention, the RAGE-related disorder is an endocrine disorder selected from the group consisting of diabetes, insulin resistance, impaired glucose tolerance, and thyroiditis.

[0143] The composition may contain each of the desired AONs of the present invention at about 1 nM to 1000 nM. Preferably, the composition contains each of the AONs of the present invention at approximately 1 nM to 500 nM, 10 nM to 500 nM, 50 nM to 750 nM, 10 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, and most preferably between 1 nM and 10 nM.

[0144] The composition may contain each of the desired AONs of the present invention at about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 50 nm, about 75 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm.

[0145] The present invention further provides one or more AONs adapted to contribute to the prophylactic or therapeutic treatment, prevention, or remission of symptoms of diseases such as RAGE-expression related diseases or pathologies in a form suitable for delivery to a patient.

[0146] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and typically produce no allergic or similarly adverse reaction, such as excessive pruritus, when administered to a patient. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered together. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Martin, Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA, (1990).

[0147] In a more specific form of the present invention, a pharmaceutical composition is provided that comprises a therapeutically effective amount of one or more AONs of the present invention together with a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions include diluents of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, and additives such as clarifying agents and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and filling substances (e.g., lactose, mannitol). The materials may be incorporated into particulate formulations of polymeric compounds such as polylactic acid, polyglycolic acid, or into liposomes. Hyaluronic acid may also be used. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the protein and derivatives. See, for example, Martin, Remington’s Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, which is incorporated herein by reference. The composition may be prepared in liquid form or may be a dry powder, such as a lyophilized form.

[0148] It will be understood that the pharmaceutical compositions provided in accordance with the present invention may be administered by any means known in the art. Preferably, the pharmaceutical composition intended for administration is administered orally, by topical injection, or by the pulmonary or nasal route. The appropriate route may be determined by one of ordinary skill in the art as appropriate for the condition of the subject being treated.

[0149] In certain embodiments, the AONs of the present disclosure can be delivered by the pulmonary or nasal route (e.g., incorporating the AONs via nebulized saline). The highest endogenous expression of RAGE mRNA in healthy human tissues is found in the lung and is accessible via the airway. Inhaled oligonucleotides are an emerging therapeutic modality in respiratory diseases. The airway is exclusively lined with pulmonary surfactant consisting mainly of zwitterionic lipids. These surfactant lipids have cationic properties at airway pH. When anionic oligonucleotides are inhaled, they tend to be adsorbed by the surfactant, resulting in reformulated particles that are hypothesized to be efficiently taken up by lung cells by bronchial and alveolar epithelial cells. Notably, AONs have been shown to be resistant to the nebulization process.

[0150] In certain embodiments, the AONs are more preferably delivered by intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous administration routes. The vasculature or extravascular circulation, blood or lymphatic system, and cerebrospinal fluid are some non-limiting sites where the AON may be introduced.

[0151] In certain embodiments, direct CNS delivery may be utilized, e.g., ventricular or intrathecal administration may be used as the administration route.

[0152] Formulations for topical administration include those in which the oligomers of the present disclosure are mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Lipids and liposomes include neutral (e.g., dioleoyl phosphatidylethanolamine DOPE, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), negative (e.g., dimyristoyl phosphatidylglycerol DMPG), and cationic (e.g., dioleoyl tetramethylaminopropyl DOTAP and dioleoyl phosphatidylethanolamine DOTMA). For topical or other administration, the oligomers of the present disclosure may be encapsulated within liposomes or may form complexes therewith, particularly with cationic liposomes. Alternatively, the oligomers may form complexes with lipids, particularly cationic lipids. Fatty acids and esters, their pharmaceutically acceptable salts, and their uses are further described in U.S. Patent No. 6,287,860 and / or U.S. Patent Application No. 09 / 315,298 (filed May 20, 1999).

[0153] In certain embodiments, the AONs of the present disclosure can be delivered by a transdermal method (e.g., via incorporation into an emulsion, such as an emulsion optionally packaging the AONs in liposomes having such AONs). Such transdermal and emulsion / liposome-mediated delivery methods are described, for example, in U.S. Patent No. 6,965,025 for the delivery of AONs in the art.

[0154] The AONs described herein may also be delivered via an implantable device. The design of such devices is a process approved in the art, such as by a synthetic implant design, as described, for example, in U.S. Patent No. 6,969,400.

[0155] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids or binders may be desirable. Oral formulations are those in which the oligomers of the present disclosure are administered in combination with one or more permeation enhancers, surfactants and chelating agents. Surfactants include fatty acids and / or their esters or salts, bile acids and / or their salts. Bile acids / salts and fatty acids and their use are further described in U.S. Patent No. 6,287,860. In some embodiments, the present disclosure provides combinations of permeation enhancers, such as combinations of fatty acid / salts and bile acid / salts. An exemplary combination is the sodium salts of lauric acid, capric acid and UDCA. Further permeation enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The oligomers of the present disclosure may be orally delivered in a granular form including spray-dried particles or complexed to form microparticles or nanoparticles. Oligomer complexing agents and their use are further described in U.S. Patent No. 6,287,860. Oral formulations and their preparation in oligomers are detailed in U.S. Patent No. 6,887,906, U.S. Patent Application No. 09 / 315,298 (filed May 20, 1999), and / or U.S. Patent Application Publication No. 20030027780.

[0156] Compositions and formulations for parenteral, intrathecal or intracerebroventricular administration may include a sterile aqueous solution, which may also contain buffers, diluents and other suitable additives, such as, but not limited to, permeation enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0157] Delivery of therapeutically useful amounts of AONs may be achieved by methods published previously. For example, intracellular delivery of AONs may be via a composition comprising a mixture of an AON and an effective amount of a block copolymer. An example of this method is described in U.S. Patent Application Publication No. 20040248833. Other methods of delivering AONs to the nucleus are described in Mann CJ et al. (2001) Proc, Natl. Acad. Science, 98(1)42-47, and Gebski et al. (2003) Human Molecular Genetics, 12(15):1801-1811. Methods for introducing nucleic acid molecules into cells using expression vectors either as naked DNA or complexed to lipid carriers are described in U.S. Patent No. 6,806,084.

[0158] It may be desirable to deliver AONs in a colloidal dispersion system. Colloidal dispersion systems include polymer complexes, nanocapsules, microparticles, beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome formulations. These colloidal dispersion systems can be used in the manufacture of therapeutic pharmaceutical compositions.

[0159] Liposomes are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. These formulations may have characteristics of net cationic, anionic, or neutral charge and may have useful characteristics in in vitro, in vivo, and ex vivo delivery methods. It has been shown that large unilamellar liposomes can encapsulate in substantial percentages an aqueous buffer containing large macromolecules. RNA and DNA can be encapsulated within the aqueous interior and delivered to cells in a bioactive form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981).

[0160] Since liposomes are efficient gene delivery vehicles, the following characteristics need to be presented: (1) encapsulation of the AON of interest with high efficiency but without compromising its biological activity; (2) preferential and substantial binding to target cells compared to non-target cells; (3) efficient delivery of the aqueous contents of the vesicles to the cytoplasm of target cells; and (4) accurate and effective expression of the genetic information (Mannino, et al., Biotechniques, 6:682, 1988). Liposome compositions are usually combinations of phospholipids, especially phospholipids with high phase transition temperatures, usually in combination with steroids, especially cholesterol. Additionally, other phospholipids or other lipids may be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Cationic liposomes are positively charged liposomes that are thought to interact with negatively charged DNA molecules and form stable complexes. pH-sensitive or negatively charged liposomes are thought to encapsulate DNA but not form complexes with it. Both cationic and non-cationic liposomes are used to deliver DNA to cells.

[0161] As used herein, the term "sterically stabilized" liposomes includes liposomes that are one or more specialized lipids that, when incorporated into a liposome, result in an increased circulation lifetime compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid moiety of the liposome contains one or more glycolipids or is a derivative with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Liposomes and their use are further described in U.S. Patent No. 6,287,860.

[0162] Furthermore, the AONs described herein may be delivered via an implantable device. The design of such a device is a process approved in the art, such as that described in U.S. Patent No. 6,969,400 (the contents of which are hereby incorporated by reference in their entirety), for example, in a synthetic implant design.

[0163] Antisense oligonucleotides can be introduced into cells using techniques approved in the art (e.g., transfection, electroporation, fusion, liposomes, colloidal polymeric particles, and viral and non-viral vectors, as well as other means known in the art). The delivery method selected will depend at least on the cells to be treated and the location of the cells and will be apparent to those skilled in the art. For example, localization can be achieved by liposomes having specific markers on their surface to induce liposomes, direct injection into the tissue containing the target cells, specific receptor-mediated uptake, and the like.

[0164] As is known in the art, AONs can be delivered, for example, by liposome-mediated uptake, lipid conjugates, polylysine-mediated uptake, nanoparticle-mediated uptake, and receptor-mediated endocytosis, as well as additional non-endocytotic modes of delivery, such as microinjection, permeabilization (e.g., streptolysin-O permeabilization, anionic peptide permeabilization), methods involving electroporation, and various non-invasive non-endocytotic methods of delivery known in the art (see Dokka and Rojanasakul, Advanced Drug Delivery Reviews 44, 35-49 (incorporated herein by reference in its entirety)).

[0165] Furthermore, the AON may be combined with other pharmaceutically acceptable carriers or diluents to prepare a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, such as phosphate-buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, or transdermal administration.

[0166] The described routes of administration are intended merely as a guide, since those skilled in the art can readily determine the optimal route of administration and any dosage for any particular animal and condition.

[0167] Multiple techniques have been attempted to introduce functional new genetic material into cells both in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These techniques include the incorporation of the gene to be expressed into a modified retrovirus (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.:5074S-5079S); incorporation into a non-retroviral vector (Rosenfeld, et al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434); or delivery of the transgene linked to a heterologous promoter enhancer element via liposomes (Friedmann(1989), supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-311; Nabel, et al. (1990) Science, 249:1285-1288; Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); conjugation with a ligand-specific cation-based transport thread (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624) or the use of naked DNA, expression vectors (Nabel et al. (1990), supra); Wolff et al. (1990) Science, 247:1465-1468). Direct injection of the transgene into tissues results in only localized expression (Rosenfeld (1992) supra; Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra).The group of Brigham et al. (Am. J. Med. Sci. (1989) 298:278-311 and Clinical Research (1991) 39 (abstract)) reported in vivo transfection limited to the lungs of mice after either intravenous or intratracheal administration of DNA-liposome complexes. Examples of review papers on human gene therapy procedures include Anderson, Science (1992) 256:808-813; Barteau et al. (2008), Curr Gene Ther; 8(5):313-23; Mueller et al. (2008). Clin Rev Allergy Immunol; 35(3):164-78; Li et al. (2006) Gene Ther., 13(18):1313-9; Simoes et al. (2005) Expert Opin Drug Deliv; 2(2):237-54.

[0168] The AONs of the present invention include any pharmaceutically acceptable salt, ester, or salt of such ester, or any other compound capable of (directly or indirectly) providing a bioactive metabolite or residue thereof upon administration to an animal, including a human. Thus, by way of example, the present disclosure also focuses on prodrugs and pharmaceutically acceptable salts of the compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents.

[0169] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention that are physiologically and pharmaceutically acceptable: i.e., salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects thereto. In the case of oligomers, preferred examples of pharmaceutically acceptable salts include, but are not limited to, (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine; (b) hydrochloride addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid; (c) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid; and (d) salts formed from anionic elements such as chlorine, bromine, and iodine. The pharmaceutical compositions of the present invention may be administered in several ways depending on whether local or systemic treatment is desired and on the area to be treated. Administration may be local (including ocular and mucosal, as well as rectal delivery), for example, by inhalation of powders or aerosols or insufflation of gases (e.g., by nebulizer, intratracheal, intranasal, topical, and transdermal) to the lung, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, for example, intrathecal or intraventricular administration. Oligomers with at least one 2'-O-methoxyethyl modification are considered to be particularly useful for oral administration. Preferably, the AON is delivered via a subcutaneous or intravenous route.

[0170] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing the active ingredient into association with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0171] In one embodiment, the AON is administered in an amount and manner effective to provide a peak blood concentration of at least 200-400 nM AON. Typically, one or more doses of AON are administered, generally at regular intervals for about 1-2 weeks. A preferred dose for oral administration is about 1 mg to 1000 mg of oligomer / 70 kg. In some cases, a dose of oligomer greater than 1000 mg per patient may be required. For intravenous administration, a preferred dose is about 0.5 mg to 1000 mg of oligomer / 70 kg. For intravenous or subcutaneous administration, the AON may be administered daily or weekly at a dose of about 120 mg / kg.

[0172] The AON may be administered at regular intervals for a short period, e.g., daily for up to 2 weeks. However, in some cases, the oligomer is administered intermittently over a longer period. Administration may be followed by or concurrent with the administration of antibiotics or other therapeutic treatments. The treatment plan may be adjusted as directed based on the results of immunological measurements, other biochemical tests, and physiological tests of the subject being treated (dose, frequency, route, etc.).

[0173] Administration depends on the severity and responsiveness of the condition being treated, with a treatment course lasting from several days to several months, or until cure is effected or a reduction in the disease state is achieved. The optimal dosing schedule can be calculated from measurements of drug accumulation in the patient's body. A person skilled in the art can readily determine the optimal dosage, method of administration and rate of repetition. The optimal dosage may vary according to the relative potency of each oligomer and can generally be evaluated based on the EC50 found to be effective in in vitro and in vivo animal models. Generally, the dosage is from 0.01 μg to 100 g / kg body weight and may be administered once or more per day, per week, per month or per year, or once more every 2 to 20 years. A person skilled in the art can readily estimate the rate of repetition in administration based on the residence time and concentration of the drug in the measured body fluid or tissue. After successful treatment, it may be desirable to subject the patient to maintenance therapy to prevent recurrence of the condition, where the oligomer is administered at a maintenance dose in the range of 0.01 μg to 100 g / kg body weight, from once or more per day to once every 20 years.

[0174] An effective in vivo treatment plan using the AONs of the present invention may vary according to the duration, dosage, frequency and route of administration, and the condition of the subject during treatment (i.e., prophylactic administration and administration according to local or systemic infection). Thus, such in vivo therapy often requires tests suitable for a particular type of disorder being treated and monitoring by adjustment corresponding to the dosage or treatment plan to achieve optimal treatment results.

[0175] Treatment may be monitored, for example, by common indicators of diseases known in the art. The efficacy of the in vivo-administered AONs of the present invention may be determined from biological samples (such as tissues, blood, urine, etc.) taken from the subject before, during, and after administration of the AON. Assays of such samples include: (1) monitoring the presence or absence of heteroduplex formation with target and non-target sequences using procedures known to those skilled in the art, such as electrophoretic gel mobility assays; (2) monitoring the amount of mutant mRNA relative to reference normal mRNA or protein as a measurement by standard techniques such as RT-PCR, Northern blotting, ELISA, or Western blotting.

[0176] Nuclear oligomer delivery is a major issue for AONs. Different cell-penetrating peptides (CPPs) localize PMOs to varying degrees in different conditions and cell lines, and novel CPPs have been evaluated by the inventors for their ability to deliver PMOs to target cells. The term CPP or "peptide moiety that enhances cell uptake" is used interchangeably and refers to cationic cell-penetrating peptides, also referred to as "transport peptides", "carrier peptides", or "peptide transduction domains". The peptide has the ability to induce cell permeability within approximately or at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells of a given cell culture population, as shown herein, and enables macromolecule translocation in multiple tissues in vivo upon systemic administration. CPPs are well-known in the art and are disclosed, for example, in U.S. Patent Application Publication No. 2010 / 0016215, which is incorporated herein by reference in its entirety.

[0177] Accordingly, the present invention provides the AONs of the present invention in combination with cell-penetrating peptides for making therapeutic pharmaceutical compositions.

[0178] According to a further aspect of the invention, there is provided one or more AONs as described herein, which are intended for use in a therapy based on AON. Preferably, the therapy is directed to a condition associated with RAGE expression. More preferably, the therapy for a condition associated with RAGE expression is a therapy for a disease selected from cancer, neurodegenerative diseases, lung disorders, and inflammatory diseases.

[0179] More specifically, the AON may be selected from the group consisting of any one or more of the AONs listed in Tables 3a - 3d and / or any one of SEQ ID NOs: 1 - 31, and combinations or cocktails thereof. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. This includes sequences that can hybridize to such sequences under stringent hybridization conditions, sequences complementary thereto, modified bases, modified backbones, and sequences having functional cleavage or extension portions thereof (which possess or regulate the pre - mRNA processing activity in the RAGE gene transcript).

[0180] The invention is also extended to combinations of two or more AONs that have the ability to bind to a selected target in order to induce exon removal in the RAGE gene transcript. The combination may be a cocktail of two or more AONs, a construct comprising two or more AONs, or two or more AONs linked together, which are intended for use in a therapy based on AON. The combination of AONs is preferably the combination of SEQ ID NO: 11 and 10, or the combination of SEQ ID NO: 11 and 13.

[0181] The invention is a method for treating, preventing, or alleviating the effects of a disease associated with RAGE expression, comprising: a) administering to a patient in an effective amount one or more AONs as described herein or a pharmaceutical composition comprising one or more AONs and provides a method.

[0182] Furthermore, the invention is a method for treating, preventing, or alleviating the effects of cancer, neurodegenerative diseases, lung disorders, and inflammatory diseases, comprising: a) administering to a patient in an effective amount a pharmaceutical composition comprising one or more AONs as described herein or one or more AONs A method is provided that includes:

[0183] Preferably, the treatment is used to reduce the level of functional RAGE protein via an exon skipping method. The reduction in the level of RAGE is preferably achieved by reducing the transcript level by modifying pre-mRNA splicing in the RAGE gene transcript or a portion thereof.

[0184] The reduction in RAGE will preferably result in a decrease in the amount, duration or severity of symptoms of RAGE-related pathologies or pathologies, such as neurodegenerative diseases, cancer, lung disorders, and inflammatory diseases.

[0185] As used herein, "treatment" of a subject (e.g., a mammal, e.g., a human) or a cell is any type of intervention used in an attempt to alter the natural course of an individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition and may be carried out prophylactically or following the initiation of a pathogenic event or exposure by a pathogen. Further included are "preventive" treatments that can be induced to reduce the rate of progression of a disease or pathology during treatment, delay the onset of a disease or pathology, or reduce the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of a disease or pathology, or its associated symptoms.

[0186] According to another aspect of the invention, there is provided the use of one or more AONs as described herein in the manufacture of a medicament for modulating or controlling a disease associated with RAGE expression.

[0187] The invention also provides the use of purified and isolated AONs as described herein in the manufacture of a medicament for treating a disease associated with RAGE expression.

[0188] Provided is the use of the purified and isolated AONs as described herein in the manufacture of a medicament for treating, preventing, or alleviating the effects of a disease associated with RAGE expression.

[0189] Preferably, the RAGE-related pathology or disease is a neurodegenerative disease, cancer, lung disorder, or inflammatory disease.

[0190] According to a further aspect of the invention, it extends to cDNA or a cloned copy of the AON sequence of the invention, and to vectors having the AON sequence of the invention. The invention further extends to cells having such sequences and / or vectors.

[0191] The AONs of the invention may be co-administered with another therapeutic molecule. For example, the AON may be administered with a second therapeutic agent that is a compound capable of modulating the RAGE cytoplasmic tail, such as a peptide. Such compounds include IQGAP-1, diaphanous-1, protein kinase C zeta (PKCζ), Dock7, MyD88, TIRAP, ERK1 / 2, olfactory receptor 2T2, ADP / ATP translocase 2, protein phosphatase 1G, IRAK4, protein DJ-1 (PARK7), calponin 3, drebrin, filamin B, Ras-related protein Rab-13, radixin / ezrin / moesin, proteolipid protein 2, coronin, S100A11, succinyl-CoA ligase [GDP-forming] subunit alpha, Hsc70 interacting protein, apoptosis inhibitor 5, neuropilin, cleavage stimulating factor, growth factor receptor-bound protein 2, sec61 beta subunit, or Nck1.

[0192] The invention provides a kit for treating, preventing, or alleviating a disease or condition associated with RAGE expression in a patient, the kit comprising at least an isolated or purified AON for modifying pre-mRNA splicing in a RAGE gene transcript or a portion thereof, packaged together with instructions for use in a suitable container.

[0193] In a preferred embodiment, the kit will have at least one AON as described herein, any one or more of SEQ ID NOs: 1-31 and / or the sequences shown in any of Tables 3a-3d, or a cocktail of AONs, as described herein. The kit may further have peripheral reagents such as buffers, stabilizers, etc. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

[0194] Accordingly, there is provided a kit for treating, preventing, or alleviating a disease or condition associated with RAGE expression in a patient, the kit comprising at least one AON as described herein, any one or more of SEQ ID NOs: 1-31 and / or the sequences shown in any of Tables 3a-3d, and combinations or cocktails thereof, packaged together with instructions for use in a suitable container. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. The combination of AONs is preferably a combination of SEQ ID NO: 11 and 10, or SEQ ID NO: 11 and 13.

[0195] Preferably, the disease or condition is selected from the list including cancer, neurodegenerative disease, lung disorder, or inflammatory disease.

[0196] The contents of the kit are lyophilizable, and the kit may further contain a solvent suitable for reconstitution of the lyophilized components. Each component of the kit will be packaged in a separate container, and the notice associated with such container may be in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, and the notice reflects approval by the same agency for manufacture, use, or sale for human administration.

[0197] When the components of the kit are provided in one or more solutions, the solution can be an aqueous solution, such as a sterile aqueous solution. For in vivo use, the expression construct may be formulated into a pharmaceutically acceptable injectable composition. In this case, the container means itself may be an inhaler, syringe, pipette, eyedropper, or other devices, and the formulation therefrom may be applied to the affected part of the animal, such as the lung, injected into the animal, or further applied to and mixed with other components of the kit.

[0198] The components of the kit may further be provided in a dry or lyophilized form. When the reagent or component is provided in a dry form, it is generally reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may be provided in yet another container means. Irrespective of the number or type of containers, the kit of the present invention may include, or be packaged with, a device for assisting in the injection / administration or placement of the final composite composition into an animal body. Such a device may be an inhaler, syringe, pipette, forceps, measuring spoon, eyedropper or any such medically approved delivery medium.

[0199] One skilled in the art should understand that the application of the above method has a wide range of applications for identifying AONs suitable for use in the treatment of many other diseases.

[0200] The AONs of the present invention may further be used in combination with alternative therapies, such as drug therapy.

[0201] Accordingly, the present invention provides a method for treating, preventing, or alleviating the effects of a disease or condition associated with RAGE expression, wherein the AONs of the present invention are administered over time or simultaneously with another alternative therapy associated with treating, preventing, or alleviating the effects of a disease or condition associated with RAGE expression. Preferably, the disease or condition is selected from the list including neurodegenerative diseases, cancer, lung disorders, or inflammatory diseases.

[0202] Summary Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention encompasses all such variations and modifications. The invention further includes all steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of steps or features.

[0203] The invention should not be limited to the specific embodiments described herein which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are within the scope of the invention as described herein.

[0204] The entire disclosure content of all publications (including patents, patent applications, journal articles, experimental manuals, books, or other documents) cited herein is incorporated herein by reference. It is not admitted that any of the references constitutes prior art or is part of the common general knowledge in the field related to the present invention.

[0205] Each document, reference, patent application, or patent cited in the text is hereby expressly incorporated by reference in its entirety and is to be read and considered by the reader as part of the text. For the sake of brevity, the documents, references, patent applications, or patents cited in the text are not repeated in the text.

[0206] The use instructions, specifications, product specifications, and product sheets of any product mentioned in this specification or of any manufacturer in any document incorporated herein by reference are hereby incorporated herein by reference and may be used in the practice of the present invention.

[0207] As used herein, the terms "derived from" and "derived of" shall be construed to indicate that a particular integer may be obtained from a particular source, whether directly or not directly from that source.

[0208] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0209] Throughout this specification, unless the context requires otherwise, the terms "comprise", "comprises", "comprising", and variations thereof mean that the stated integer or group of integers is included, but do not exclude any other integer or group of integers.

[0210] Except where otherwise indicated in the working examples or where otherwise specified, all numbers expressing amounts of ingredients, reaction conditions, etc. used in this specification and the claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and the claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Therefore, "about 80%" means "about 80%" as well as "80%". At the very least, each numerical parameter should be construed in light of the number of significant figures and the ordinary rounding method.

[0211] Although the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors resulting from the standard deviation found in their respective testing measurements.

[0212] Other definitions for selected terms used herein may be found in the detailed description of the invention and may apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0213] Sequence identification numbers ("SEQ ID NOs") containing nucleotide and amino acid sequence information included in this specification are summarized at the end of the description and were created using the program Patentln version 3.0. Each nucleotide or amino acid sequence is identified in the sequence listing by the numerical indicator <210> followed by a sequence identifier (e.g., <210>1, <210>2, etc.). The length of the sequence, the type, and the source organism for each nucleotide or amino acid sequence are indicated by the respective information provided in the numerical indicator fields <211>, <212>, and <213>.

[0214] An antisense oligomer nomenclature system has been proposed and published (see Mann et al., (2002) J Gen Med 4, 644 - 654) to distinguish between different antisense oligomers. This nomenclature has become particularly important when testing several slightly different antisense oligomers, all targeting the same target region, as shown below: H#A / D(x:y) The first letter indicates the species (e.g., H: human, M: mouse) "#" indicates the target exon number "A / D" indicates the acceptor or donor splice site at the start / end of the exon, respectively (x y) represents the annealing coordinates when "-" or "+" indicates an intron or exon sequence, respectively. As an example, for A(-6+18), it indicates the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. For the closest splice site, these coordinates will be the acceptor preceded by "A". When describing the annealing coordinates for a donor splice site, it could be D(+2-18) if the last 2 exon bases and the first 18 intron bases correspond to the annealing site of the antisense oligomer. Overall, for exon annealing coordinates represented by A(+65+85), it is the site between the 65th nucleotide (including the start) and the 85th nucleotide from the start of that exon.

[0215] The following examples more fully illustrate modes of using the above invention and set forth the best mode contemplated for carrying out various aspects of the invention. These methods are presented for purposes of illustration only and are not intended to limit the scope of the invention.

Example

[0216] In each of the following examples, the following general materials and methods apply, unless the context dictates otherwise.

[0217] Human microvascular endothelial cells (HMEC1) were cultured in MCDB131 medium (10% FCS with 10 mM glutamine, EGF, and hydrocortisone). Human lung epithelial cells (A549) (cancer) were cultured in F-12K medium (10% FBS with 2 mM glutamine). Primary murine aortic endothelial cells (PMAEC) were isolated from the aortas of (wild-type) C57bl6 mice and AGER KO mice and cultured in Dulbecco's Modified Eagle Medium (DMEM) / F12 medium supplemented with endothelial cell growth supplement (ECGS).

[0218] For transfection of AONs, epithelial or endothelial cells were seeded in 48-well plates for 24 or 48 hours and then transfected with either AONs targeting exon 9 of human AGER in (HMEC1 and A549) and murine AGER in murine PMAEC cells or each control using Lipofectamine 3000 reagent (0.15 ul / well of Lipofectamine 3000; 0.4 ul / well of P3000 / well) (Tables 3a - 3d). Cells were incubated with AON / cationic lipoplexes at 37°C for 24 hours, and at subsequent time points, cells were lysed, RNA was extracted, and cDNA was prepared using either the TRIZOL technique or the Cells to CT method.

[0219] To measure the effect on the alternative splicing of human RAGE pre-mRNA, real-time RT-PCR primers were used to measure the expression of the 11th exon of RAGE, exon 9b, or in the range of exons 8 - 10, which respectively denote the expression (mRNA) retained among all RAGE mRNA splice variants, the retention of exon 9b, or the expression of the RAGEv1 isoform encoding the transmembrane cytoplasmic tail. A decrease in the PCR signal of exons 8 - 10 of the cytoplasmic tail compared to exon 11 indicates that the RAGE mRNA splice isoform has little exon 10 and that the full-length RAGE protein isoform (with signaling ability) is not produced much by the cells. In mouse cells and tissues, primers spanning exons 10 - 11 of mouse RAGE mRNA were used to represent the expression of RAGE mRNA splice isoforms having exon 10.

[0220] The medium was also collected and the level of soluble RAGE was measured by ELISA. The medium (6 ml) was concentrated using a molecular weight cut-off filter and then tested by ELISA.

[0221] Chinese hamster ovary (CHO) cells are an ideal system for examining the regulation of pre-mRNA splicing with modified protein secretion as an endpoint because they express recombinant proteins at high levels. As a model system to more fully explore the effect of AONs on the splicing of RAGE, CHO cells grown in F12 medium (supplemented with 10% FBS) were transfected with Lipofectamine 2000 with a plasmid encoding the human genomic AGER (gRAGE) sequence (excluding the native 5' and 3' untranslated regions) together with AONs. After 2 days, the medium was collected, concentrated using a molecular weight cut-off filter, and tested by Western blot using an anti-hRAGE antibody (R&D systems).

[0222] To determine the exact sequences of the RAGE variants detected after AON administration, RNA was extracted using the Trizol method, cDNA was prepared using oligo dT primers, and then PCR was performed using primers specific for RAGE against the 5’ and 3’ UTR sequences of RAGE. Semi-nested PCR was carried out to amplify the mixture of RAGE sequences. The gel-purified RAGE bands were cloned into a TA cloning vector and then transformed into E. coli Top10 cells. More than 50 colonies were selected and characterized using a MultiNA sequence analyzer using primers spanning exons 8 - 11 to determine the size of the RAGE insert. Five clones of each size were sent for Sanger sequencing to confirm the RAGE sequence splice isoforms corresponding to the band sizes.

[0223] To measure the functional effects of interfering with ligand-dependent and ligand-independent activation of RAGE, cells were exposed to the AT1R cognate ligand angiotensin II (1 μM) or the RAGE ligand S100A8 / A9 (0.6 μg / mL) for 4 hours. Next, the cells were frozen and stored until mRNA extraction, extracted, and cDNA was synthesized using the Cells to Ct method. Changes in gene expression of the NFκB subunit, p65 (RelA), or NFκB-activated target genes (e.g., ICAM-1) were evaluated by quantitative real-time RT-PCR performed using the TaqMan system based on real-time detection of accumulated fluorescence (ABI Prism 7700, Perkin-Elmer Inc, PE Biosystems, Foster City, CA, USA). Gene expression was normalized to 18S mRNA and reported as fold change compared to the level of expression in untreated control mice / cells, which was assigned an arbitrary value of 1.

[0224] Example 1. Regulation of alternative splicing of RAGE in human cells using antisense oligonucleotides targeting a cis-acting RNA element in exon 10 This example shows that the splicing of RAGE pre-mRNA can be regulated using an AON that targets a cis-acting RNA element in exon 10 of RAGE pre-mRNA.

[0225] RAGE pre-mRNA undergoes natural alternative splicing to generate various mRNA splice forms. Most of these RAGE splice forms have both exon 10 and 11, which each encode the transmembrane and cytoplasmic domains of RAGE, and the protein isoforms, and thus can be activated by RAGE ligands and trans-activated by GPCRs located and arranged, and pro-inflammatory and pro-proliferative signal transduction is induced. Alternative 5' splice site selection in exon 9 can also lead to an 83-nucleotide extension of exon 9 (exon 9B; Figure 1a). Since the distance between this alternative splice site and the 3' (acceptor) splice site at the start of exon 10 is only 46 nucleotides, which is much shorter than the lower limit of intron length in eukaryotes, exon 10 is consequently skipped, and the 3' (acceptor) site at the start of exon 11 is selected instead. This alternative splicing also introduces a premature termination codon, but since it is in the immediate vicinity (29 bp) of the final exon-exon junction, it does not undergo nonsense-mediated decay. Therefore, the mature mRNA (RAGE_v1, Figure 1a) obtained from this alternative splicing encodes a protein isoform lacking the elements required for each of the membrane retention and cytoplasmic signal transduction encoded by exons 10 and 11, and is secreted and found naturally in circulation, bronchoalveolar, and cerebrospinal fluid. This esRAGE can act as a decoy receptor that competes with full-length RAGE on the cell surface for ligands or enhances ligand clearance. Furthermore, the novel 17-amino acid C-terminus encoded by exon 9b may have an action independent of RAGE signal transduction or multimerization.

[0226] Transfection of 10 nM AONs targeting exon 10 into human lung epithelial cells (A549) resulted in a change in the expression of the RAGE mRNA spliceform having exon 9b (Figure 1b). Some AONs, particularly AON3779, enhanced the expression of the RAGE mRNA spliceform having exon 9b, as measured by real-time RT-PCR, compared to control (scrambled RNA-treated) cells. Other AONs that also target exon 10, particularly AON3777 and AON3781, had no effect.

[0227] Transfection with 10 nM concentrations of AONs targeting exon 10 into human lung epithelial cells (A549) also resulted in a change in the expression of the RAGE mRNA spliceform, such that some AONs, particularly AON3779, AON3780, AON3781, AON82, AON83, and AON87, decreased the expression of the RAGE mRNA spliceform having exon 10 (i.e., encoding the signaling element of the cytoplasmic tail), as measured by real-time RT-PCR, compared to control (scrambled RNA-treated) cells (Figure 1c and 1d). Other AONs that also target exon 10, particularly AON3777, AON84, and AON85, had no effect.

[0228] Next, all RAGE mRNA spliceforms expressed within human lung epithelial cells (A549) were isolated in the presence and absence of selected AONs targeting exon 10 and cloned into Escherichia coli (E.Coli) Top10 cells. Primers spanning between exon 8 and exon 11 were used to determine the size of each RAGE insert. Treatment with selected AONs targeting exon 10, particularly AON3779 and AON3778, resulted in an increased expression of the RAGE mRNA spliceform having a 250 kB fragment (Figure 1e), which implies retention of exon 9b.

[0229] Furthermore, all AONs targeting exon 10 decreased the expression of the percentage of mRNA clones representing the 300 kB band, which means the complete expression of the signaling elements contained within exons 8 - 10. In particular, AON3779 resulted in a 49% decrease in the RAGE mRNA spliceform with a fragment size of 300 kB (Figure 1f).

[0230] Furthermore, all AONs targeting exon 10 induced the new expression of RAGE mRNA spliceforms with exon 8 completely deleted (confirmed by Sanger sequencing and shown by the empty lane on the MultiNA analyzer indicated by the arrow on the gel; Figure 1f) and represented by the "bandless" fragment (Figure 1e).

[0231] Furthermore, transfection of AONs targeting exon 10, particularly AON3777 and AON3778, into human lung epithelial cells (A549) resulted in the new expression of RAGE mRNA spliceforms with fragments sized 276 kB and 430 kB (Figure 1e), which means the atypical removal of exon 9 and the retention of exons 9, 9b, and 10, respectively.

[0232] Furthermore, transfection of 10 nM AONs targeting exon 10, particularly AON3779, into human lung epithelial cells (A549) resulted in a change in the number of copies of the RAGE mRNA spliceform with exon 10 as measured by digital PCR compared to control (scrambled RNA-treated) cells (Figure 1g).

[0233] Furthermore, transfection of 10 nM AONs targeting exon 10, particularly AON3779, into human lung epithelial cells (A549) enhanced the release of endogenous soluble RAGE into the medium (Figure 1h), which is consistent with the increased expression of the RAGE mRNA spliceform with exon 9b.

[0234] Transfection of a plasmid encoding the genomic sequence (gDNA) of human RAGE into CHO cells resulted in alternative splicing of the gene product, including a small amount of endogenous RAGE (red arrow) secreted into the cell culture medium, as detected by Western blot, which was consistent with the normal splicing pattern of RAGE (Figure 1i). The amount of endogenous soluble RAGE (esRAGE) secreted into the medium increased when these CHO cells expressing genomic human RAGE were co-transfected with AON3779 or AON87 (Figure 1i). At the same time, the full-length RAGE (white arrow) expressed and retained intracellularly decreased when CHO cells expressing genomic human RAGE were co-transfected with AON3779 or AON87. The cells also had an increased amount of C-cleaved RAGE intracellularly.

[0235] Furthermore, the quantitative amount of soluble RAGE as measured by ELISA in CHO cells expressing genomic human RAGE increased after transfection with selected AONs targeting exon 10, such as AON3779, AON82, AON83, AON84, and AON85 (Figure 1j). Among these, AON3779 had the greatest effect.

[0236] Furthermore, the quantitative amount of soluble RAGE as measured by ELISA increased by an amount similar to AON3779 (10 nM) by some other AONs targeting exon 10 within a region similar to AON3779, including AON90 and 93 (Figure 1k). The quantitative amount of soluble RAGE released from CHO cells expressing genomic human RAGE increased in a dose-dependent manner after transfection with AON3779 (Figure 1l).

[0237] Furthermore, transfection of 10 nM AONs targeting exon 10, particularly AON3779, into human lung epithelial cells (A549) regulated the induction of pro-inflammatory signaling after activation of RAGE by the RAGE ligand S100A8 / A9 (0.6 μg / mL), which induces ICAM-1 expression (Figure 1m).

[0238] Furthermore, transfection of 10 nM AONs targeting exon 10, particularly AON3779, AON3780, AON81, AON82, and AON83, into human aortic endothelial cells (HAEC) blocked the induction of ICAM-1 expression after exposure to the RAGE ligand S100A8 / A9 (0.6 μg / mL; Figure 1m).

[0239] Furthermore, transfection of 10 nM AONs targeting exon 10, particularly AON3779, into human lung epithelial cells (A549) regulated the induction of pro-inflammatory signaling after trans-activation of RAGE following activation of the AT1 receptor by angiotensin II (1 μM), which induces the induction of ICAM-1 expression (Figure 1o).

[0240] Furthermore, transfection of 10 nM AONs targeting exon 10, particularly AON3777, AON3779, AON3780, AON3780, AON3782, and AON3783, into human aortic endothelial cells (HAEC) regulated the induction of pro-inflammatory signaling, including the induction of ICAM-1 expression after ligand-independent trans-activation of RAGE following activation of the AT1 receptor by angiotensin II (1 μM; Figure 1p). Other AONs targeting exon 10, particularly AON3778 and AON84, had no effect.

[0241] Transfection of 10 nM AONs targeting exon 10, particularly AON3779, AON3780, AON82, AON83, and AON87, into human microvascular endothelial cells (HMEC1) resulted in changes in the expression of RAGE mRNA splice forms, including a decrease in the expression of the RAGE mRNA splice form having exon 10 (Figure 1q). Furthermore, transfection of 10 nM AONs targeting exon 10, particularly AON3779 and AON87, into human microvascular endothelial cells (HMEC1) induced an increase in the expression of the RAGE mRNA splice form having exon 9b (Figure 1r).

[0242] Morpholino oligonucleotides covalently linked to octaguanidine dendrimer (known as vivo morpholino) can be used in vitro and in vivo without the need for transfection reagents. Treatment of human aortic endothelial cells (HAECs) with the vivo morpholino formulation (1 μM) of AON3779 resulted in changes in the expression of RAGE mRNA splice forms, for example, an increase in the RAGE mRNA splice form having exon 9b as measured by RT-PCR (Figure 1s). Furthermore, the expression of esRAGE in cell culture medium was also enhanced compared to cells treated with the control morpholino AON (Figure 1t). Treatment with AON79 in the absence of transfection reagent had no effect. Transfection of AON3779 with a transfection reagent is shown as a positive control.

[0243] Furthermore, treatment of CHO cells expressing genomic human RAGE with the vivo morpholino formulation (1 μM) of AON3779 enhanced the expression of soluble RAGE as measured by ELISA (Figure 1u).

[0244] Poly(G) stretches, particularly GGGG motifs, can act as transcriptional silencers and are known to be frequently bound by hnRNP H and F (Sohail et al., BMC Genomics. 2014). RAGE splicing is hypothesized to be partially regulated by a G-rich cis element within intron 9 / exon 9b and heterogeneous nuclear ribonucleoprotein H, which is required for the preferential utilization of the upstream RAGE 5’ splice site. Poly(G) stretches support exon skipping when continuous G stretches are required for hnRNP H binding. AONs generally have poor affinity and reduced efficacy within G-rich regions. Exon 10 has three GGGG motifs, one of which is recognized as an hnRNPF binding target and flanked by AON3779, AON3787, and AON3782 (Figure 1v). However, mutating this region (RAGE mutant M3) had no effect on exon splicing or response to AON3779 (Figure 1w), suggesting that we are modulating a novel silencer of RAGE splicing by targeting these regions with AONs, which could not have been predicted from the prior art.

[0245] Example 2. Regulation of alternative splicing of RAGE in human cells using antisense oligonucleotides targeting cis-acting RNA elements in exon 9 This example shows that alternative splicing of RAGE pre-mRNA can be regulated using AONs that target cis-acting RNA elements in exon 9 of the RAGE pre-mRNA (Figure 2a).

[0246] Transfection of some AONs at a concentration of 10 nM targeting exon 9, particularly AON3668, AON3669, AON3670, AON4103, AON4104, AON4105, into human lung epithelial cells (A549) resulted in a decrease in the expression of RAGE mRNA splice forms with exon 10, as measured by real-time RT-PCR, compared to control (scrambled RNA-treated) cells (Figure 2b). Another AON, particularly AON4106, targeting exon 9 had no effect. However, after treatment with all AONs targeting exon 9, the overall expression of RAGE mRNA was slightly enhanced.

[0247] All RAGE mRNA splice forms expressed in human lung epithelial cells (A549) in the presence and absence of selected AONs targeting exon 9 were isolated and cloned into E. coli Top10 cells. Primers spanning exons 8 - 11 were used to determine the size of each RAGE insert (Figure 2c). Treatment with all AONs targeting exon 9 decreased the expression in terms of the percentage of mRNA clones representing the 300 kB band (Figure 2d), which means sufficient expression of signaling elements contained in exons 10 and 11. In particular, AON3670 brought about a decrease in the RAGE mRNA splice form with a fragment of size 300 kB (Figure 2d).

[0248] Furthermore, transfection of AONs targeting exon 9, particularly AON3669, AON3670, AON4103, into human lung epithelial cells (A549) induced the new expression of RAGE mRNA splice forms in which exon 8 was skipped (represented by empty lanes in the multiNA analyzer; Figure 2c).

[0249] Furthermore, transfection of AONs targeting exon 9, particularly AON3669, AON3670, AON4103, and AON4105, into human lung epithelial cells (A549) resulted in the novel expression of an RAGE mRNA splice form with a 276 kB fragment, which means the removal of exon 9 from the splice form (Figure 2d).

[0250] In CHO cells transfected with a plasmid having the genomic sequence of human RAGE (gRAGE), the amount of soluble RAGE secreted into the medium was moderately increased by several AONs targeting the RAGE splice site in exon 9, specifically AON3669 and AON3671, as measured by ELISA, compared to control (scrambled RNA-treated) cells (Figure 2e).

[0251] Furthermore, induction of pro-inflammatory signaling was inhibited by transfection of different concentrations of AONs targeting exon 9, particularly AON3669, AON3670, and AON4103, into human lung epithelial cells (A549) after activation of RAGE with the RAGE ligand S100A8 / A9 (0.6 μg / mL), which includes induction of TLR4 gene expression (Figure 2f). Other AONs targeting exon 9, particularly AON3668 and AON4104, had no effect.

[0252] Furthermore, transfection of 10 nM AONs targeting exon 9, particularly AON3669, AON3670, and AON4104, into human lung epithelial cells (A549) regulated the induction of inflammatory signaling via ligand-independent transactivation of RAGE after activation of the AT1 receptor by angiotensin II (1 μM; Figure 2g). Other AONs targeting exon 9, particularly AON3668, AON3671, and AON4103, had no effect.

[0253] Furthermore, transfection of 10 nM AONs targeting exon 9, particularly AON3669 and AON3670, into human microvascular endothelial cells (HMECs) decreased the expression of endogenous RAGE mRNA splice forms having exon 10 (Figure 2h).

[0254] Example 3. Regulation of alternative splicing of RAGE in human cells using antisense oligonucleotides targeting cis-acting RNA elements in exon 9B This example shows that alternative splicing of RAGE can be regulated using AONs that target RNA elements within exon 9b in the RAGE pre-mRNA.

[0255] Exon 9b is removed by alternative splicing in most RAGE isoforms (Figure 1e). This removal potentially requires the interaction of the spliceosome with motifs in exon 9b. We hypothesized that alternative splicing of the RAGE pre-mRNA could also be regulated by modulating these binding factors using AONs.

[0256] Transfection of 10 nM AONs targeting exon 9b into human lung epithelial cells (A549) resulted in changes in the mRNA expression of RAGE mRNA splice forms. After treatment with both AONs targeting exon 9b, the overall expression of RAGE was slightly enhanced (Figure 3a). Furthermore, the expression of the RAGE mRNA splice form having exon 10 was moderately decreased as measured by real-time RT-PCR after transfection with AON88, compared to scramble control-treated cells (Figure 3a). Additionally, the expression of the RAGE mRNA splice form having exon 9b was moderately increased by AON88 and AON89 (Figure 3b). Transfection of AON3779 targeting exon 10 is shown as a positive control.

[0257] Furthermore, transfection of human microvascular endothelial cells (HMECs) with 10 nM concentrations of AONs targeting exon 9b resulted in a decrease in the expression of the RAGE mRNA spliceform having exon 10 (Figure 3c) and an increase in the expression of the RAGE mRNA spliceform having exon 9b (Figure 3d). Furthermore, the overall expression of RAGE mRNA was slightly enhanced after treatment with both AONs targeting exon 9b (Figure 3c). Transfection of AON3779 targeting exon 10 is shown as a positive control.

[0258] In CHO cells transfected with a plasmid having the genomic sequence of human RAGE (gRAGE), the amount of soluble RAGE secreted into the medium was moderately increased by some AONs targeting exon 9b, specifically AON89, as measured by ELISA, compared to control RNA (Figure 3e). Transfection of AON3779 targeting exon 10 is shown as a positive control.

[0259] The human RAGE sequence of intron 9 and the complementary targets for AON88 and 89 are shown in Figure 3f.

[0260] Example 4. Regulation of alternative splicing of mouse RAGE using antisense oligonucleotides targeting cis-acting RNA elements in RAGE pre-mRNA This example shows that the alternative splicing of RAGE pre-mRNA can also be regulated using specific AONs targeting cis-acting RNA elements in exons 9, 9B and 10 in the pre-mRNA of mouse RAGE, and that this regulation is demonstrable in vitro, ex vivo and in vivo.

[0261] To test the effect of AONs on the alternative splicing of mouse RAGE, CHO cells were transfected with a plasmid having the genomic sequence of mouse RAGE. This resulted in the secretion of a small amount of esRAGE. Transfection with AONs targeting mouse RAGE, particularly AON m3779 (designed to target exon 10 of the mouse RAGE sequence within the equivalent sequence targeted by AON3779 in the human RAGE sequence detailed in Example 1) and AON m101, increased the secretion of esRAGE into the cell culture medium (Figure 4a).

[0262] Primary mouse aortic endothelial (PMAEC) cells were transfected with AONs (50 mM) targeting exon 9 of RAGE (Table 3d). This treatment resulted in a moderate change in the expression of RAGE mRNA splice isoforms compared to their respective negative controls. In particular, AON3674 and AON3675 were able to reduce the expression of the RAGE mRNA splice isoform having exon 10 as measured by real-time RT-PCR (Figure 4b).

[0263] However, transfection of these AONs into PMAECs at 10 nM did not show a significant effect on the expression of RAGE mRNA splice isoforms (Figure 4c). The levels of soluble RAGE secreted into the medium moderately increased 24 hours after transfection of 50 nM AON3674 and AON3675 into cultured PMAECs (Figure 4d).

[0264] Transfection of m3779 (10 nM) into CHO cells expressing genomic mouse RAGE decreased the expression of the RAGE mRNA splice isoform having exon 10 as evaluated by real-time RT-PCR (Figure 4e), and increased the secretion of esRAGE compared to control (scrambled RNA-treated) cells (Figure 4f). Furthermore, AON3779 targeting the human RAGE sequence increased esRAGE secretion, but not as much as AON m3779.

[0265] When AONs designed to target exon 10 of the mouse RAGE sequence were transfected from a plasmid into CHO cells expressing the genomic sequence of human RAGE, AON m3779 (designed to target mouse RAGE exon 10) also induced an increase in soluble human RAGE in the cell culture medium (Figure 4g). However, AON3779 (specifically targeting human RAGE exon 10) was more effective than AON m3779 in regulating genomic human RAGE. This data indicates that AONs targeting specific sequences in exon 10 can be effective in regulating RAGE splicing despite non-identical regions.

[0266] To test the effect of AON targeting RAGE in mouse tissues, precision-cut lung slices (PCLS) were obtained from mice and then cultured ex vivo. Briefly, mice were humanely killed, agarose was injected into their explanted lungs, cylindrical cores were harvested, and then cut with a tissue slicer to produce lung slices of uniform diameter and thickness. Next, the PCLS were immersed in the medium in a multi-well plate under tissue culture conditions. After 24 hours, the PCLS were then transfected with vivo morpholino AON3779 or a non-target control for 48 hours. Treatment with AON3779 resulted in a significant increase in the expression of RAGE mRNA having exon 9b and a decrease in the expression of the RAGe mRNA spliceform having exon 10 (Figure 4h), and the maximum effect on exon 9b was observed 72 hours after administration (Figure 4i).

[0267] To verify the in vivo relevance of AONs targeting RAGE splicing, male C57bl6 mice were subcutaneously injected with AON m3779 (1 mg / kg / day) for 7 days. The expression of the RAGE mRNA spliceform having exon 9b increased in the lungs of the mice, and the spliceform having exon 10 decreased after 1 week of subcutaneous treatment with AON m3779 (Figure 4j).

[0268] To verify its potential as an inhaled therapeutic agent, surviving mice were anesthetized and then 30 μL of 11 μM vivo morpholino AON m3779 or (as a negative control) a non-targeting vivo morpholino control (ribonuclease-free water) was delivered to the lungs via the trachea using a cannula or water alone. The anesthesia was reversed and the mice were killed 48 hours later. Intratracheal treatment with m3779 resulted in a significant decrease in the lung expression of the RAGE mRNA spliceform having exon 10 compared to water alone, which had no significant effect. Furthermore, the expression of the RAGE mRNA isoform having exon 9b was increased (Figure 4k). A similar decrease in the expression of the RAGE mRNA spliceform having exon 10 was observed after intratracheal delivery of 30 μL of 11 μM 2-0’Me-phosphorothioate AON m3779 compared to media (sterile water) alone (Figure 4l). In addition, an increase in circulating soluble RAGE was further observed after treatment of mice with AON m3779 compared to sterile water or an ineffective AON 4105 targeting human RAGE (Figure 4m).

[0269] Example 5. Regulation of alternative splicing of RAGE using combinations of antisense oligonucleotides targeting RAGE pre-mRNA This example shows that alternative splicing of RAGE can be regulated using combinations of AONs that target different cis-acting RNA elements in the pre-mRNA of RAGE.

[0270] The definition of exon is thought to be regulated by the assembly of a multi-component "cross-exon" recognition complex that promotes target retention or removal. After AON3779 binds to exon 10 in the pre-mRNA of RAGE and enhances the production of esRAGE, it is speculated that other regulatory targets in exon 10, including but not limited to the predicted splice sites, may become more decisive. Furthermore, in the presence of AON3779, by selectively targeting these cis-acting RNA elements in exon 10 in the pre-mRNA of RAGE using other AONs, any avoidance of exon removal is blocked and RAGE splicing is further regulated towards the production of the preferred esRAGE.

[0271] The secondary structure of exon 10 of RAGE is predicted to have two hairpin loops linked by a central hinge region. Targeting the exon enhancer sequence in the 3' hairpin using AON3779 is speculated to increase the importance of the target in the alternative 5' hairpin, including the upstream 5' splice site in exon 10.

[0272] To test this hypothesis, CHO cells carrying a plasmid encoding genomic human RAGE were transfected with AON3779 (10 nM) in combination with other AONs (also 10 nM) targeting the predicted 5’ (acceptor) splice site in exon 10. This combined treatment resulted in a change in the expression of RAGE mRNA splice forms, where several combinations, particularly AON3779 + AON3777, AON3779 + AON3778, significantly increased the expression of the RAGE mRNA isoform with exon 9b as measured by real-time RT-PCR compared to AON3779 alone or control scrambled RNA-treated cells (Figure 5a). Further, this combination increased the production of esRAGE protein as measured by ELISA in the medium (Figure 5b). Additionally, the combination of AON3997 and AON3778 was more effective at lower doses (5 nM + 5 nM). In particular, AON3777 and AON3778 had little or no effect on their own at 10 nM.

[0273] In another experiment, CHO cells carrying a plasmid encoding genomic human RAGE were transfected with AON3779 in combination with other AONs targeting either the 3’ or 5’ splice site in exon 10. Further, several combinations resulted in a change in the expression of RAGE mRNA splice forms, where several combinations, particularly AON3779 + AON3778, AON3779 + AON3781, and AON3779 + AON3778 + AON3781, significantly increased the expression of soluble RAGE as measured by ELISA compared to AON3779 alone or scrambled control-treated cells (Figure 5c). This increase was similar when using AON93 or AON87 instead of AON3779, but the combination with AON3779 (represented in boxes) was overall the strongest and most consistent.

[0274] Other combinations of AONs targeting adjacent regions in exon 10 of exon 10, particularly the transfection of AON3779+AON3782, AON3779+AON3784, AON3779+AON3785 into human lung epithelial cells (A549), had no significant effect on the expression of the RAGE isoform with exon 9b, exceeding that of AON3779 alone (Figure 5d). In particular, these AONs also targeted the 5' hairpin (similar to AON3779) and / or the central hinge region, which is different from AON3777 or AON3778, which were effective in combinations targeting exon enhancers on other (3') hairpins.

[0275] CHO cells having a plasmid encoding the genomic sequence of mouse RAGE were transfected with AON m3779 combined with AONs targeting either the 3' or 5' splice site in exon 10. This combined treatment resulted in changes in the expression of RAGE mRNA splice isoforms, where several combinations, particularly AON m3779+AON m102, significantly increased the secretion of soluble RAGE into the cell culture medium as measured by ELISA, compared to AON m3779 alone (Figure 5e).

Claims

1. An isolated or purified AON for modifying pre-mRNA splicing in the receptor for advanced glycation end products (RAGE) and regulating the splicing of the RAGE gene transcript or a part thereof.

2. a) SEQ ID NOs: 1-31; b) Tables 3a-3d; and / or c) SEQ ID NO: 11, 18, 19, or 20 The AON according to claim 1, selected from the list comprising.

3. The AON according to claim 1, having one or more nucleotide positions that undergo alternative chemistry or modification selected from the list comprising (i) a modified sugar moiety; (ii) resistance to ribonuclease H; and / or (iii) oligomer mimetic chemistry.

4. The antisense oligomer according to claim 1, further modified by (i) chemical conjugation to a moiety; and / or (ii) labeling with a cell-permeable peptide.

5. The AON according to claim 1, wherein when uracil is present in the AON, the uracil (U) of the AON is replaced with thymine (T).

6. The AON according to claim 1, wherein the modification of pre-mRNA splicing results in the skipping of one or more exon sequences of the RAGE pre-mRNA.

7. The AON according to claim 1, wherein the modification of pre-mRNA splicing results in the retention (run-on) of one or more intron sequences of the RAGE RNA.

8. A pharmaceutical, prophylactic, or therapeutic composition for treating, preventing, or alleviating the effects of a disease associated with RAGE expression in a patient, a) one or more AONs according to any one of claims 1-8, and b) one or more pharmaceutically acceptable carriers and / or diluents A composition comprising.

9. A method for manipulating splicing in a RAGE gene transcript, a) providing one or more of the AONs according to any one of claims 1-7 and enabling the oligomer to bind to a target nucleic acid site A method comprising.

10. A method for regulating the expression, concentration, or activity of a RAGE isoform, a) administering to the patient in an effective amount a pharmaceutical composition comprising one or more AONs according to any one of claims 1-7 or one or more AONs A method comprising.

11. A method for treating, preventing, or alleviating the effects of a disease associated with RAGE expression, a) administering to the patient, in an effective amount, a pharmaceutical composition comprising one or more AONs according to any one of claims 1 to 7 or one or more AONs A method comprising.

12. Use of the purified and isolated AONs according to any one of claims 1 to 7 in the manufacture of a medicament for treating, preventing or alleviating the effects of a disease associated with RAGE expression.

13. A kit for treating, preventing or alleviating the effects of a disease associated with RAGE expression in a patient, comprising, together with instructions for use, at least one AON according to any one of claims 1 to 7 and its combinations or cocktails, packaged in a suitable container.

14. The composition according to claim 8, the method according to any one of claims 9 to 11, the use according to claim 12 or the kit according to claim 13, wherein the disease associated with RAGE expression is selected from the list comprising neurodegenerative diseases, cancer, lung disorders and inflammatory diseases.

15. The composition according to claim 8, the method according to any one of claims 9 to 11, the use according to claim 12 or the kit according to claim 13, wherein the AON is administered in combination with a second therapeutic agent selected from the list comprising soluble RAGE isoforms, compounds capable of modulating the RAGE cytoplasmic tail.

16. The composition according to claim 8, the method according to any one of claims 9 to 11, the use according to claim 12 or the kit according to claim 13, wherein the AON is a combination of AONs.

17. The composition, method, use or kit according to claim 16, wherein said combination of AONs is selected from the combination of SEQ ID NO: 11 and 10, or the combination of SEQ ID NO: 11 and 13.

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