RU2820247C2 - Deep glycation end products receptor modulators and modulation - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: present invention relates to antisense oligonucleotides and can be used in medicine. Invention discloses a novel antisense oligonucleotide for modifying pre-mRNA splicing at the receptor of advanced glycation end products (RACE). Antisense oligonucleotides according to the present invention are capable of selectively reducing the expression of pro-inflammatory transmembrane RAGE (mRAGE; RAGE containing exon 10), simultaneously increasing expression of extracellular soluble RAGE (esRAGE; exon 10 skipping leads to preservation of intron 9b).

EFFECT: invention can be used in medicine for treating, preventing or relieving the effects of the disease associated with RAGE expression.

36 cl, 5 dwg, 3 tbl, 5 ex

Description

FIELD OF THE INVENTION

The present invention relates to a method for modulating alternative splicing of pre-mRNA encoding the receptor for advanced glycation end products (RAGE) or a portion thereof using splice-switching antisense oligonucleotides (AON) to modify the expression and/or activity of RAGE isoforms and methods for treating RAGE-related disorders using the specified modulators.

BACKGROUND ART

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

The 50-55 kDa glycosylated RAGE protein is constitutively expressed in a limited range of cells (eg, vascular endothelium, type I pneumocytes, leukocytes), although RAGE expression can be induced in most cell types and tissues following injury, stress, hypoxia, or inflammation, providing a proinflammatory and proproliferative signaling. RAGE expression is therefore up-regulated in inflammatory and metabolic disorders, including but not limited to neurodegenerative disease, cancer, cardiovascular disease, diabetes, autoimmune and ischemic diseases, in which RAGE is also implicated in development and progression.

RAGE has been linked to a range of brain disorders, including 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; development and progression of glioma; ischemic brain damage/stroke and multiple sclerosis.

RAGE has been linked to many aspects of tumor biology, including tumor cell growth, migration, and invasion. Many cancers express high levels of RAGE (breast, colon, kidney and stomach cancers are illustrative examples). An exception is lung cancer, in which RAGE expression is decreased because RAGE is lost as lung cells differentiate and become more malignant.

In C6 glioma cells, tumor volume is markedly reduced in tumors composed of cells in which RAGE has been blocked. In contrast, tumors overexpressing wild-type RAGE grew rapidly and invaded surrounding tissue very efficiently. It would be desirable to have therapeutic agents to block RAGE signaling as a cancer treatment for many common cancers such as glioma/medulloblastoma polymorpha; pancreas cancer; melanoma; prostate cancer; mammary cancer; liver cancer/hepatoma and colon cancer.

In health, pulmonary RAGE expression is the highest of all tissues. However, RAGE expression in the lung is typically observed only in type I pneumocytes. Up-regulation of RAGE signaling in the lung in other cells and at other sites has been associated with a variety of pulmonary disorders, including the following: chronic obstructive pulmonary disease (COPD)/emphysema; asthma; damage due to cigarette smoking/pollution; acute lung injury/acute respiratory distress syndrome and pulmonary fibrosis.

RAGE is also critically involved in a range of inflammatory conditions such as inflammatory arthritis; osteoarthritis; retinal disease; atherosclerosis; vascular calcification; coronary heart disease/cardiac remodeling/fibrosis; heart failure; diabetic and non-diabetic kidney disease; inflammatory bowel disease; preeclampsia; polycystic ovary syndrome; fatty liver degeneration, fibrosis, ischemic and non-ischemic liver damage; muscular dystrophy; spinal cord injury; inflammation and aging of the skin; and keratitis.

Human RAGE consists of an immunoglobulin-like ectodomain, a single transmembrane domain, and a short (42 amino acids) cytosolic tail. This ectodomain (also known as the extracellular domain) of RAGE includes three immunoglobulin-like regions: an N-terminal V-type domain followed by two C-type domains (referred to as C and C or alternatively C1 and C2).

Binding of advanced glycation end products (AGEs) and non-AGE ligands to the RAGE ectodomain activates intracellular signal transduction cascades involved in inflammation, wounding, cell proliferation and differentiation. Activation of RAGE also initiates a positive feedback loop in which RAGE ligand-receptor interaction increases RAGE expression through activation of NFκB (nuclear factor kappa bi), thereby increasing subsequent RAGE-induced cellular activation. In fact, the only way known to the inventors to strongly down-regulate RAGE expression is to reduce RAGE activation. This situation is in contrast to other receptors, for which increased ligand levels reduce receptor expression.

In humans, the cytosolic tail of RAGE is 43 amino acids long (from residue 362 to residue 404). This cytosolic tail contains motifs that are critical for RAGE-dependent cellular activation but not for ligand binding. The cytosolic tail of RAGE can be trans-activated following activation of colocalized G protein-coupled receptors by their cognate ligands without binding of AGE or non-AGE ligands to the RAGE ectodomain (also known as ligand-independent activation of RAGE), resulting in activation the same pathways (Pickering et al. J Clin Invest 2019;129:406-421). Ligand-independent RAGE activation of the cytosolic tail of RAGE through specific colocalized activated G protein-coupled receptors appears to be an important pathway by which RAGE is activated in vivo.

In both ligand-dependent RAGE activation and ligand-independent RAGE activation, intracellular signaling is mediated by the cytosolic domain of RAGE, which interacts with a variety of signaling partners.

Alternative splicing of RAGE is also important for regulating RAGE activity through the generation of RAGE isoforms that have an altered ability to be activated by ligand-dependent and ligand-independent signaling pathways. Alternative splicing of RAGE is altered in disease conditions including cancer, diabetes, and Alzheimer's disease. But while alternative splicing appears to be important for RAGE regulation/misregulation, there has been no way to specifically target this mechanism.

It is with respect to this prior art that the present method of using a splice switching AON (antisense oligonucleotide) to modulate RAGE splicing toward the preferential generation of cytoprotective RAGE isoforms along the entire length of RAGE is described.

The above discussion of the prior art is intended only to facilitate understanding of the present invention. This discussion is not an admission or assumption that any material referred to constitutes or was part of ordinary general knowledge as of the priority date of this application.

SUMMARY OF THE INVENTION

Broadly, one form of the present invention provides an isolated or purified AON that is used to modulate alternative splicing of a pre-mRNA transcript of a gene encoding the receptor for advanced glycation end products (RAGE) or a portion thereof.

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

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

Due to factors such as RNA secondary structure, competition between AON and SR proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), and/or other elements that make up the spliceosome and may influence AON action, AONs are targeted to key splice donor and acceptor sites , will not always change splicing. Therefore, one aspect of the present invention provides an AON of 10-50 nucleotides containing a targeting sequence complementary to cis-acting RNA elements or adjacent regions in the RAGE pre-mRNA that act as enhancers or silencers, which, when bound by spliceosomal elements ( for example, splicing factor proteins, uRNA, IncRNA), modulates the splicing of a nearby exon.

In one aspect of the present invention, there is provided a 10-50 nucleotide AON containing a targeting sequence complementary to the RAGE pre-mRNA that modulates the secondary structure of said mRNA to influence splice site selection.

In one form of the present invention, an isolated or purified AON is provided to induce exclusion (also known as skipping) of one or more exonic sequences in a RAGE gene transcript or a portion thereof.

In one form of the present invention, an isolated or purified AON is provided to induce retention of intronic sequences in a RAGE gene transcript or a portion thereof.

In one form of the present invention, AON is chemically modified to prevent degradation of the pre-mRNA-AON complex, including phosphorodiamidate morpholino oligomers (PMOs), 2'-O-methylphosphorothioate oligonucleotides (2OMe) and 2'-O-methoxyethyl phosphorothioate oligonucleotides (2OMe), AON , modified locked nucleic acids (LNA), thermostable twisted intercalating nucleic acid (TINA), and peptide nucleic acids (PNA), but not limited to.

In one form of the present invention, AONs are conjugated to moieties to enhance their delivery, including, but not limited to, cell penetrating peptides (CPPs), vivo morpholines (VMOs), or peptide phosphodiamidate morpholino oligomers (PPMOs).

Preferably, the AON is selected from the group containing the sequences set forth in any of Tables 3a-3d. Preferably, the AON is selected from the list containing SEQ ID NO: 1-31. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

The AON of the invention may be selected as an AON capable of binding to a selected target site, wherein the target site is a putative mRNA splice site selected from a splice donor site, splice acceptor sites, splice enhancer sequences, splice silencer sequences, or sites , which modulate the secondary structure of pre-mRNA. This target site may also include some flanking intronic sequences when targeting splice donor or acceptor sites.

More specifically, the AON may be selected from the group consisting of any one or more than one SEQ ID NO: 1-31 and/or the sequences set forth in any of Tables 3a-3d, and combinations or mixtures thereof. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. The AON combination is preferably the 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, their complementary sequences, sequences containing modified bases, modified backbones and their functional truncations or extensions, which have pre-mRNA processing activity or modulate the pre-mRNA processing activity in the RAGE gene transcript.

In some embodiments, AONs may be 100% complementary to the target sequence or may include mismatches, for example, to accommodate variants, provided that the heteroduplex formed between the oligonucleotide and the target sequence is sufficiently stable to withstand the action of cellular nucleases and other methods degradation that can be observed in vivo. Therefore, certain oligonucleotides may have about or at least about 70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75% , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or 100% sequence complementarity between the oligonucleotide and the target sequence.

The present invention also extends to a combination of two or more than two AONs capable of binding to a selected target to modulate alternative splicing of RAGE pre-mRNA, including a construct containing two or more such AONs. These constructs can be used together for AON-based combination therapy. The AON combination is preferably a combination of SEQ ID NOs: 11 and 10 or SEQ ID NOs: 11 and 13.

The present invention extends, in yet another aspect, to cDNAs or cloned copies of the AON sequences of the invention, as well as vectors containing the AON sequences of the invention. The present invention also further extends to cells containing such sequences and/or vectors.

A method for manipulating the splicing of the RAGE gene transcript is also proposed, including the stage:

a) providing one or more than one AON, as described herein, and causing the oligomer(s) to bind to a target nucleic acid site.

Also proposed is a pharmaceutical, prophylactic or therapeutic composition for treating, preventing or reducing the intensity of disease effects associated with RAGE expression in a patient, containing:

a) one or more than one AON, as described herein; And

b) one or more than one pharmaceutically acceptable carrier and/or diluent.

The composition may contain from about 1 nM to 1000 nM of each of the desired AONs of the invention. Preferably, the composition may contain from about 10 nM to 500 nM, most preferably from 1 nM to 10 nM, of each of the AONs of the invention.

A method for treating, preventing or reversing the effects of a disease associated with RAGE expression is also proposed, including the step of:

a) administering to a patient an effective amount of one or more than one AON or a pharmaceutical composition containing one or more than one AON, as described herein.

Also provided is the use of purified and isolated AONs, as described herein, for the manufacture of a medicament for the treatment, prevention or reversal of the effects of a disease associated with RAGE expression and/or activity.

Also provided is a kit for treating, preventing or reversing the effects of a disease associated with RAGE expression in a patient, which contains at least one AON as described herein, and combinations or mixtures thereof, packaged in a suitable container, together with instructions for its use .

Preferably, the disease associated with RAGE expression in the patient is a neurodegenerative disease, cancer, pulmonary disorder or inflammatory disease.

The subject with the disease associated with RAGE expression may be a mammal, including a human.

Other aspects of the present invention will now be described with reference to the accompanying non-limiting examples and drawings.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Other characteristics of the present invention are more fully described in the following description of its several non-limiting embodiments. This description is included solely for the purpose of illustrating the present invention by example. It is not to be construed as limiting the broad summary, disclosure, or description of the invention as set forth above. This description will be made with reference to the accompanying graphic materials, in which:

In FIG. Figure 1a shows the alternative splicing of RAGE that generates RAGE and RAGE_v1. Exons are shown as rectangles. The coding sequences are black and the non-coding sequence is white. Lines with angles indicate splicing events. S - premature termination codon.

In FIG. Figure 1b shows the fold change in expression of any RAGE mRNA splice form (total human RAGE) and a RAGE mRNA splice form containing the human RAGE 9b sequence in A579 cells as detected by real-time RT-PCR followed by treatment with selected AONs targeting exon 10 or control cells (treated with scrambled RNA).

In FIG. Figure 1c shows the fold change in mRNA expression of any RAGE mRNA splice form (total human RAGE) and a human RAGE mRNA splice form containing exon 10 as detected by real-time RT-PCR followed by treatment of A579 cells with selected AONs targeting exon 10 or control cells ( treated with scrambled RNA).

In FIG. Figure 1d shows the fold change in the expression of any RAGE splice form (total human RAGE) and a human RAGE splice form containing exon 10 as detected by real-time RT-PCR, followed by treatment of A579 cells with selected AONs also targeting exon 10 adjacent to the putative hnRNPF binding site, or control cells (treated with scrambled RNA).

In FIG. Figure 1e shows the percentage of RAGE clones encoding constructs containing fragments of exons 8–10 of different sizes, indicating the relative expression of different splice forms of RAGE mRNA after treatment of A579 cells with specific AONs targeting exon 10 or control cells (treated with scrambled RNA). For example, band 300 indicates a splice form containing exons 8, 9, 10, and 11 (i.e., signaling-competent splice forms), while band 250 indicates a splice form containing RAGE 9b, and no band indicates a RAGE mRNA splice form associated with loss exon 8.

Fig. 1f is a representative gel image of PCR DNA products spanning exons 8–11, with bands of varying sizes or no bands (arrows) indicating the presence of other splice forms.

In FIG. 1g shows the copy number of RAGE mRNA, which contains exon 10, as measured by digital PCR after treatment of A579 cells with AON 3779 compared to control cells (treated with scrambled RNA).

In FIG. 1h shows the concentration of endogenous soluble RAGE protein detected in the cell environment after treatment of A579 cells with selected AONs targeting exon 10, compared to control cells (treated with scrambled RNA).

In FIG. Figure 1i shows RAGE protein expression in cell media (left) and cell lysate (right) following transfection of CHO (Chinese hamster ovary) cells using DNA encoding the human RAGE genomic sequence (gRAGE), with or without AON3779 or AON87, or control (CTL ) RNA by Western blotting with detection using an antibody specific for RAGE. Lane M is a marker, lane 2 is a vector control (no endogenous RAGE expressed), lane 3 is gRAGE plus control (scrambled RNA), lane 4 is gRAGE plus AON3779, and lane 5 is gRAGE plus AON87. Arrows indicate the size of smaller soluble RAGE in cell media (red) and larger full-length RAGE in cell lysate (white).

In FIG. 1j shows the expression of soluble RAGE protein in the cell environment after transfection of CHO cells using DNA encoding the genomic sequence of human RAGE (gRAGE), with or without transfection of AONs targeting exon 10, as measured by ELISA (enzyme-linked immunosorbent assay).

In FIG. 1k shows expression of soluble RAGE protein in the medium following transfection of CHO cells using DNA encoding the genomic sequence of human RAGE (gRAGE), with or without selected AONs targeting exon 10 adjacent to or overlapping with a site also targeted by AON 3779, with ELISA measurement.

In FIG. 1l shows the dose-dependent induction of soluble RAGE expression in the medium following transfection of CHO cells using DNA encoding the genomic sequence of human RAGE, with or without AON 3779, as measured by ELISA. The dotted line represents the expression of soluble RAGE in CHO cells transfected with scrambled RNA (10 nM).

In FIG. 1m shows the induction of ICAM-1 gene expression after treatment of A549 cells with the RAGE ligand - S100A8/9 (0.6 μg/ml) and its modulation by pre-transfection with selected AONs targeting exon 10, compared with control cells (treated with scrambled RNA) .

In FIG. 1n shows the induction of ICAM-1 gene expression following treatment of primary human aortic endothelial cells (HAECs) with RAGE-S100A8/9 ligand (0.6 μg/ml) and its modulation by pre-transfection with selected AONs targeting exon 10, compared to controls cells (treated with scrambled RNA).

In FIG. Figure 1o shows the induction of ICAM-1 gene expression after treatment of A549 cells with Ang II, which is capable of inducing RAGE transactivation. Remarkably, this induction of ICAM-1 gene expression was also modulated by pretransfection of A579 cells with selected AONs targeting exon 10 compared with control cells (treated with scrambled RNA).

In FIG. Figure 1p shows the induction of ICAM-1 gene expression following treatment of primary human aortic endothelial cells (HAECs) with angiothesin II (1 μM) and its modulation by pretransfection with selected AONs targeting exon 10, compared with control cells (treated with scrambled RNA).

In FIG. Figure 1q shows the fold change in expression of any splice form of RAGE mRNA (total human RAGE) and splice forms of human RAGE mRNA containing exon 10 as detected by real-time PCR after treatment with HMEC AONs specifically targeting exon 10 adjacent to the putative hnRNP- binding site. F/H1, or control cells (treated with scrambled RNA).

In FIG. 1r shows the fold change in mRNA expression of RAGE mRNA splice forms containing the human RAGE 9b sequence following treatment of HMEC AONs targeting exon 10 adjacent to the putative hnRNPF binding site or control cells (treated with scrambled RNA).

In FIG. Figure 1s shows the effect of in vivo morpholino treatment of primary human aortic endothelial cells (HAECs) with AON 3779 (0.1-1 μM) on the expression of RAGE mRNA splice forms containing exon 9b, compared to non-targeted morpholino AON controls.

In FIG. 1t shows the effect of in vivo morpholino treatment of primary human aortic endothelial cells (HAECs) with AON 3779 (0.1-1 μM) on soluble RAGE protein expression in the culture medium compared to non-morpholino AON-targeted control.

In FIG. 1u shows the effect of treating CHO cells expressing genomic human RAGE with the in vivo morpholino drug AON 3779 (1 μM) on the expression of soluble RAGE protein in the culture medium compared to a non-morpholino AON-targeted control.

In FIG. 1v shows the DNA sequence of human RAGE, including and delimiting exon 10, indicating the different AONs used in the above experiments and their complementary targets. The magenta box indicates the putative polyG binding target of hnRNPF.

In FIG. Figure 1w shows the lack of effect after mutation of the putative polyC hnRNP target on alternative splicing of expressed human RAGE (gRAGE M2 mutant), on the observed increase in soluble RAGE in response to AON 3779 transfection.

In FIG. 2a shows the genetic sequence of human AGER, including and delimiting exon 9, indicating the different AONs used below and their complementary targets on the RAGE pre-mRNA.

In FIG. Figure 2b shows the expression of any RAGE mRNA splice forms (total human RAGE) and human RAGE mRNA splice forms containing exon 10 as detected by real-time PCR after treatment of A579 cells with selected AONs targeting exon 9 or control cells (scrambled RNA treated) .

Fig. Figure 2c is a representative gel image of PCR DNA products spanning exon 8-11, with or without bands of different sizes, indicating the presence of different splice forms.

In FIG. Figure 2d shows the percentage of RAGE mRNA clones encoding constructs containing exon 8–11 fragments of different sizes, indicating the relative expression of different mRNA splice forms after treatment of A579 cells with selected AONs targeting exon 10 or control scrambled RNA (CTL). For example, band 300 indicates the RAGE mRNA splice form containing exons 8, 9, 10, and 11 (i.e., signaling-competent splice forms), while band 250 indicates the RAGE 9b-containing splice form, and no band indicates the RAGE mRNA splice form associated with loss of exon 8.

In FIG. 2e shows the expression of soluble RAGE protein in the medium following transfection of CHO cells with DNA encoding the genomic sequence of human RAGE, with or without selected AONs targeting exon 9, as measured by ELISA, control cells (treated with scrambled RNA). ND - not detected.

In FIG. 2f shows the induction of TLR-4 gene expression after treatment with RAGE ligand - S100A8/9 (0.6 μg/ml) and its modulation after transfection with selected AONs targeting exon 9, compared to control cells (treated with scrambled RNA).

In FIG. Figure 2g shows the induction of ICAM-1 gene expression after treatment with Ang II (1 μM), which is capable of inducing RAGE transactivation. Remarkably, this induction of ICAM-1 gene expression was modulated following transfection of A579 cells with selected AONs targeting exon 9 compared with control cells (treated with scrambled RNA).

In FIG. Figure 2h shows the fold change in expression of any RAGE mRNA splice forms (total human RAGE) and human RAGE mRNA splice forms containing exon 10 as detected by real-time PCR after treatment of HMEC1 cells with selected AONs targeting exon 9 or control cells (treated with scrambled RNA).

In FIG. Figure 3a shows the fold change in the expression of any splice forms of RAGE mRNA (total RAGE) and human splice forms of RAGE mRNA containing exon 10, as detected by real-time PCR after treatment of A579 cells with selected AONs targeting intron 9, AON 3779 (as a positive control ) or control cells (treated with scrambled RNA).

In FIG. Figure 3b shows the fold change in expression of RAGE mRNA splice forms containing the exon 9b sequence as detected by real-time PCR after treatment of A579 cells with selected AONs targeting exon 9, AON 3779 (as a positive control) or control cells (treated with scrambled RNA). .

In FIG. Figure 3c shows the expression of any RAGE mRNA splice forms (total human RAGE) and human RAGE mRNA splice forms containing exon 10 as detected by RT-PCR after treatment of HMEC1 cells with selected AONs targeting exon 9, AON 3779 (as a positive control). or control cells (treated with scrambled RNA).

In FIG. Figure 3d shows the fold change in expression of RAGE mRNA splice forms containing exon 9b as detected by real-time PCR after treatment of HMEC1 cells with selected AONs targeting exon 9, AON 3779 (as a positive control) or control cells (treated with scrambled RNA).

In FIG. 3e shows the concentration of soluble RAGE as detected by ELISA in the medium after treatment of A579 cells expressing genomic human RAGE with specific AONs targeting intron 9, AON 3779 (as a positive control) or control cells (treated with scrambled RNA).

In FIG. 3f shows the DNA sequence of human RAGE, including and delimiting intron 9, with AONs and their complementary targets designated.

In FIG. 4a shows the concentration of soluble RAGE as detected by ELISA in the medium after treatment of CHO cells expressing genomic mouse RAGE, AON targeting RAGE pre-mRNA, or control cells (treated with scrambled RNA).

In FIG. Figure 4b shows the fold change in expression of any RAGE mRNA splice forms (total mouse RAGE) and RAGE mRNA splice forms containing exons 10 and 11 by real-time PCR after PMAEC treatment with selected AONs (50 nM) targeting exon 9 of mouse RAGE, or control cells (treated with scrambled RNA).

In FIG. Figure 4c shows the fold change in expression of any RAGE mRNA splice forms (total mouse RAGE) and RAGE mRNA splice forms containing exons 10 and 11 after treatment of PMAEC with selected AONs (10 nM) targeting exon 9 of mouse RAGE, or control cells (treated with scrambled RNA). .

In FIG. 4d shows the concentration of soluble RAGE protein in the medium after treatment of PMAEC cells with specific AONs (10 nM) targeting mouse RAGE exon 9 or control cells (treated with scrambled RNA).

In FIG. Figure 4e shows the fold change in expression of any RAGE mRNA splice forms (total mouse RAGE) and RAGE mRNA splice forms containing exons 10 and 11 by RT-PCR after treatment of PMAEC AON m3779 (10 nM) or control cells (treated with scrambled RNA).

In FIG. 4f shows the concentration of soluble RAGE protein in the medium after treatment of CHO cells expressing genomic mouse RAGE with AON 3779 (10 nM), AON m3779 or control cells (treated with scrambled RNA).

In FIG. 4g shows the concentration of soluble RAGE protein in the medium after treatment of CHO cells expressing genomic human RAGE with AON 3779 (10 nM), AON m3779 or control cells (treated with scrambled RNA).

In FIG. Figure 4h shows the expression of any RAGE mRNA splice forms (total RAGE) and RAGE mRNA splice forms containing exons 10 and 11 by RT-PCR after treatment of finely cut lung pieces from mice with AON m3779 or non-targeting AON morpholino for 48 hours.

In FIG. Figure 4i shows the time-dependent expression of any RAGE mRNA splice forms (total RAGE) and RAGE mRNA splice forms containing exon 9b by RT-PCR after treatment of finely sliced lung pieces from AON m3779 mice or vehicle (saline).

In FIG. Figure 4j shows the fold change in pulmonary expression of any RAGE mRNA splice forms (total mouse RAGE) and mouse RAGE mRNA splice forms containing exon 9b or exon 10 by RT-PCR one week after subcutaneous injection of AON m3779 in C57BI6 mice compared to control in in the form of an untargeted AON.

In FIG. Figure 4k shows the fold change in pulmonary expression of any splice forms of RAGE mRNA (total mouse RAGE) and mouse splice forms of RAGE mRNA containing exon 10 or 11 by RT-PCR 48 hours after intratracheal instillation of the morpholine drug AON m3779 in C57BI6 mice compared to control in the form of an untargeted AON.

In FIG. Figure 4l shows the fold change in pulmonary expression of any RAGE mRNA splice forms (total mouse RAGE) and mouse RAGE mRNA splice forms containing exon 10 or 11 by real-time RT-PCR 48 hours after intratracheal instillation of 2-O'Me drug AON m3779 in C57BI6 mice compared to vehicle control. * denotes p equals 0.01 relative to baseline.

In FIG. Figure 4m shows the fold change (%) of soluble RAGE in the circulation compared to blood sampling at the initial time point two days after intratracheal injection of AON m3779, AON 4105 or sterile water control into C57BI6 mice.

In FIG. Figure 5a shows the expression of exon 9b-containing RAGE mRNA splice forms as measured by real-time RT-PCR after transfection of CHO cells with DNA encoding the human RAGE genomic sequence (gRAGE), with or without combinations of selected AONs targeting different regions of exon 10, or controls cells (treated with scrambled RNA).

In FIG. 5b shows the expression of soluble RAGE protein in the medium following transfection of CHO cells with DNA encoding the genomic sequence of human RAGE (gRAGE), with or without combinations of selected AONs targeting the 5' splice site of exon 10, or a control (treated with scrambled RNA) as measured by ELISA.

In FIG. 5c shows the expression of soluble RAGE protein in the medium following transfection of CHO cells with DNA encoding the genomic sequence of human RAGE (gRAGE), combinations or no combinations of selected AONs targeting the 3' and 5' splice site of exon 10, or control RNA as measured by ELISA. Boxes indicate treatments involving AON 3779.

In FIG. Figure 5d shows the expression of RAGE mRNA splice forms containing exon 9b following transfection of CHO cells with DNA encoding the genomic sequence of human RAGE, combinations and no combinations of selected AONs targeting adjacent regions of exon 10, or control cells (treated with scrambled RNA) as measured by RT-PCR in real time.

In FIG. 5e shows the expression of soluble RAGE protein in the medium following transfection of CHO cells with DNA encoding the mouse RAGE genomic sequence, with and without combinations of selected AONs, or control (scrambled RNA) as measured by ELISA.

DESCRIPTION OF THE INVENTION

Detailed Description of the Invention

Antisense oligonucleotides (AONs) are short, synthetic, antisense, modified DNA or RNA strands that can selectively hybridize to pre-RNA/mRNA through Watson-Crick base pairing and selectively modulate the function of the target RNA.

When AONs are used to modulate alternative splicing of mRNAs, they are often referred to as splice switch 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 transcript splicing repertoire by blocking RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery and pre-mRNA. SSOs can induce skipping of selected exons and/or retention of intronic sequences to podulate the translation product. This can be achieved by directly targeting splice sites or by targeting cis-acting sequences involved in splicing enhancement or silencing through modulation of specific protein binding or alteration of pre-mRNA secondary structure.

Therapeutic SSOs can be used to treat genetic disorders by skipping spoiled or misaligned stretches, providing an intrinsically deleted but now functional protein as a therapy.

Alternative splicing is considered an important level of post-transcriptional gene regulation in relation to the receptor for advanced glycation end products (RAGE). Although most RAGE is expressed in its full-length isoform, alternative splicing generates a number of different coding isoforms (also known as splice forms), including splice forms with N-terminal truncations, C-terminal truncations, and splice forms that retain intronic sequences. These different splice forms may act as possible regulators of the full-length RAGE receptor, either through competitive ligand binding or by displacing the full-length protein from binding partners. Over twenty splice forms have been identified in different tissues, such as lung, liver, kidney, smooth muscle, endothelial cells and brain.

The different splice variants of the RAGE gene have been named RAGE, RAGE_v1 to RAGE_v19 by the Committee on Human Gene Nomenclature. For example, (triggering) retention of intron 9 (exon 9b) results in premature arrest (translation) and complete loss of the transmembrane and cytoplasmic domains, generating a C-truncated soluble splice form (RAGE_v1 - endogenous secretory RAGE or esRAGE), which accounts for approximately 5% of RAGE in human circulatory system. In the absence of any signaling elements or transmembrane domain, esRAGE is able to act as a decoy receptor, competing with full-length RAGE for ligands or increasing the clearance of ligands. Higher levels of esRAGE in the circulatory system are associated with improved health outcomes and longevity, while lower levels of esRAGE are associated with many disease states, including atherosclerosis, diabetes, metabolic syndrome, cardiovascular mortality, anemia, autism and various oncogenic conditions. but not limited to them. Treatment of diabetic mice with recombinant esRAGE reduces atherosclerosis, vascular inflammation, and renal and retinal damage.

RAGEΔ (also known as DN RAGE or RAGEv20) lacks 16 amino acids in the intracellular domain but retains ligand binding and transmembrane domains such that it acts as a dominant negative inhibitor of cell surface RAGE. Splicing events leading to changes in the extracellular domain can also affect the ligand binding domain through insertion, deletion, or removal of some or all of the Ig-V RAGE domain. For example, N-RAGE begins at an alternative translation initiation site in exon 3 such that it lacks the signal peptide or V domain required for ligand binding.

Impaired RAGE splicing (and thus impaired RAGE signaling) has been described in diabetes, some cancers, and Alzheimer's disease.

Skipping of exon 10 by esRAGE splicing has been attributed to intron length limitation in higher eukaryotes. About 45 nucleotides must separate the 5' splice site and the branch point, and the minimum distance between the branch point and the 3' splice site appears to be approximately 18 nucleotides, respectively. Consequently, introns shorter than 70 nucleotides are extremely rare in mammals and cannot be efficiently spliced. When selecting the 5' splice site of esRAGE, the distance between this site and the 3' splice site that bounds exon 10 is 46 nucleotides, which is significantly shorter than the lower limit of intron length. Therefore, use of the downstream 5′ esRAGE splice site of intron 9 and inclusion of exon 10 would be mutually exclusive. Among the known splice variants analyzed, all variants that used the downstream 5' esRAGE splice site in intron 9 skipped exon 10; in contrast, all variants that used the upstream RAGE 5' splice site in intron 9 involved exon 10. Thus, the available evidence indicates that the choice of either one of the two alternative 5' splice sites in intron 9 is associated with the inclusion or exclusion of exon 10. Methods of regulation of this splicing or external methods of modulation were not previously known.

The present invention uses SSOs to selectively manipulate the alternative splicing pattern of RAGE pre-mRNA, resulting in the generation of one of two natural splice forms of RAGE mRNA that are either non-functional or that act as a decoy receptor with an antagonistic effect on ligand-dependent activation and ligand-independent transactivation of full-length RAGE .

It is noteworthy that these splice sites do not contain common RAGE polymorphisms. The RAGE sequence is highly conserved. Therefore, no personalization or customized sequence modification is required, unlike the management of genetic disorders using exon skipping technologies.

The present invention provides an alternative method for treating, preventing or reversing the effects of diseases in which RAGE is involved in the development or progression, including, but not limited to, neurodegenerative diseases, cancer, pulmonary disorders or inflammatory diseases, through the development of AONs that modulate alternative splicing of pre- RAGE mRNA or parts thereof.

In a broad sense, one aspect of the present invention provides an isolated or purified AON to modulate pre-mRNA splicing in a receptor for advanced glycation end products (RAGE) gene transcript or a portion thereof. Preferably, an isolated or purified AON is provided to induce exon exclusion and/or intron retention in RAGE pre-mRNA or a portion thereof.

The present invention provides an AON capable of binding to a selected target on a receptor for advanced glycation end products (RAGE) gene transcript to modulate pre-mRNA splicing in the RAGE gene transcript or a portion thereof.

For example, in one aspect of the present invention there is provided an AON of 10-50 nucleotides containing a targeting sequence complementary to a region of the RAGE pre-mRNA or a portion thereof associated with the binding of a protein involved in the regulation of alternative splicing of mRNA.

Unlike other AON-based therapies, the present invention does not induce increased RNA degradation through the recruitment of RNase H, where RNase H preferentially binds and degrades RNA duplexed to the RAGE gene DNA. It does not rely on the hybridization of AON with RAGE genomic DNA, nor on the binding of AON to mRNA to modulate the amount of RAGE protein produced by interfering with normal functions such as replication, transcription, translocation and translation. Rather, these AONs are used to modulate pre-mRNA splicing in the RAGE gene transcript or part thereof and induce exon skipping or (triggering) retention of intronic sequences. This strategy preferentially reduces the expression of RAGE mRNA splice forms capable of mediating RAGE-dependent signaling and/or increases the generation of RAGE mRNA splice forms that lack functional domains capable of mediating RAGE-dependent signaling.

According to a first aspect of the present invention, AONs are provided that are capable of binding to a selected target on a RAGE gene transcript to modulate pre-mRNA splicing in the RAGE gene transcript or a portion thereof. In a broad sense, an isolated or purified AON is provided to induce targeted exon exclusion/intron retention in a RAGE gene transcript or a portion thereof.

By “isolated” is meant a substance which does not contain substantially or substantially the components that normally accompany it in its natural state. For example, the terms "isolated polynucleotide" or "isolated oligonucleotide" as used herein may refer to a polynucleotide that has been purified or removed from sequences that flank it in a naturally occurring state, such as a fragment DNA that is removed from sequences that are adjacent to a given fragment in the genome. The term "isolating" as it relates to cells refers to the purification of cells (eg, fibroblasts, lymphoblasts) from a source subject (eg, a subject with a repeat polynucleotide disease). In the context of DNA, mRNA or protein, the term "isolating" refers to the extraction of DNA, mRNA or protein from a source, such as cells.

An AON can be said to be "directed toward" or "targeted against" the target sequence to which it hybridizes. In some embodiments, the target sequence includes a region including a 3' or 5' preprocessing mRNA splice site, a branch point, or other sequences involved in the regulation of splicing, including splicing enhancers and splicing silencers, and RNA secondary structure determining sites that influence splicing. The target sequence may be within an exon or within an intron, or span an intron/exon junction.

In some embodiments, the AON has sufficient sequence complementarity with the target RNA (ie, the RNA to which splice site selection is modulated) to effectively block a region of the target RNA (eg, pre-mRNA). In typical embodiments, such blocking of RAGE pre-mRNA serves to modulate splicing either by masking a spliceosomal protein binding site that would otherwise modulate splicing and/or by altering the structure of the target RNA. In some embodiments, the target RNA is a target pre-mRNA (eg, RAGE gene pre-mRNA).

The phrase "AON having sufficient sequence complementarity with a target RNA sequence to modulate splicing of the target RNA" means that the AON has sufficient sequence to trigger masking of the binding site of a native protein that would otherwise modulate splicing and/or alter the three-dimensional structure of the target RNA.

Selected AONs can be made shorter, e.g., about 12 bases, or longer, e.g., about 50 bases, and include a small number of mismatches, provided that the sequence is sufficiently complementary to effect splicing modulation when hybridized to the target sequence and possibly forms with RNA heteroduplex having a Tm (melting temperature) of 45°C or more.

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

In some embodiments, the degree of complementarity between the target sequence and the AON is sufficient to form a stable duplex. The region of complementarity of the AON with the target RNA sequence can be as short as 8-11 bases, but can be 12-15 bases or longer, e.g. 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 intervals. An AON of about 16-17 bases is usually long enough to have a unique complementary sequence. In some embodiments, a minimum length of complementary bases may be required to achieve the required Tm binding, as discussed herein.

In some embodiments, oligonucleotides 50 bases in length may be suitable, where at least a minimum number of bases, for example 10-12 bases, are complementary to the target sequence. In general, however, facilitated or active uptake into cells is optimized for oligonucleotide lengths less than about 30 bases. For the AON phosphoamidate morpholino oligomer (PMO) described later herein, the optimal balance of binding stability and uptake is typically observed at a length of 18-25 bases. Included are AONs (e.g., PMO, PMO-X, PNA, LNA, TINA, 2'-OMe), which consist of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 bases.

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

Mismatches, when present, are typically less destabilizing toward the end regions of the hybrid duplex than toward the middle. The net mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex according to well-understood principles of duplex stability. Although such an AON is not necessarily 100% complementary to the target sequence, it is effective in stably and specifically binding to the target sequence such that splicing of the target pre-RNA is modulated.

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

Additional examples of variants include AONs having about or at least about 70% sequence identity or homology, e.g., 70%, 71%, 72%, 73%, 74%, 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or 100% sequence identity or homology throughout the entire length of any of SEQ ID NO: 1-31 and/or the sequences set forth in any of Tables 3a-3d. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

More specifically, an AON capable of binding to a selected target site is provided to modify pre-mRNA splicing in a RAGE gene transcript or a portion thereof. The AON is preferably selected from SEQ ID NO: 1-31 and/or the sequences set forth in any of Tables 3a-3d. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

Pre-mRNA splicing modification preferably involves skipping or deleting one or more exons or retaining introns of the mRNA. The resulting protein is preferentially shorter in length compared to the parent full-length RAGE protein due to either internal truncation or premature termination. These truncated RAGE proteins may be referred to as splice forms of the full-length RAGE protein.

The remaining exons of the generated mRNA may be in frame and may produce a shorter protein with a sequence that is similar to that of the parent full-length protein except that it is internally truncated in the region between the original 3' and 5' ends. Alternatively, exon skipping may induce a frameshift that results in a protein in which the first part of the protein is substantially identical to the parent full-length protein, but in which the second part of the protein has a different sequence (eg, nonsense sequence) due to the shift reading frames Alternatively, exon skipping may induce the production of a prematurely terminated protein due to disruption of the reading frame and the presence of premature translation termination. A prematurely terminated protein may result from an mRNA that terminates prematurely (eg, skipping exons 10 and/or 11), or may result from continuation into an intron (eg, RAGE 9b) or a nonsense skip that produces an mRNA that contains exon 10 and/or or 11 mRNA, but which does not provide expression of the protein encoded by these exons.

Skipping of individual exons - exons 1-9 - will preferentially disrupt the reading frame of the RAGE transcript. This will lead to increased RNA degradation through nonsense-mediated decay.

Skipping individual exons—exons 1–11—would preferentially maintain an intact reading frame. This will preferentially result in translation into an internally truncated protein. A given truncated protein or splice form of RAGE mRNA may have completely eliminated function, may have reduced function, or may act as a decoy receptor.

Preferably, these truncated, nonsense or prematurely terminated proteins do not have one or more than one functional domain involved in the induction of intracellular signaling pathways through RAGE ligands or ligand-independent transactivation of RAGE through colocalized GPCRs (G protein-coupled receptors). For example, exon 10 encodes a transmembrane domain, and deletion of this exon could generate a soluble RAGE protein that could potentially act as a soluble decoy or competitive antagonist of ligand-induced signaling through RAGE. Truncated, nonsense, or prematurely terminated proteins may also lack attachment or binding sites for other factors, the removal of which may result in decreased interaction of the RAGE protein with relevant signaling pathways.

Alternatively, deletion of one or more than one exon may result in misfolding of the RAGE protein and a decrease in the ability of the protein to be successfully transported across the membrane.

The presence of internally truncated proteins (ie proteins that do not have amino acids encoded by one or more than one exon) is preferred. When RAGE protein is inhibited, there may be problems with increasing RAGE transcription because the body tries to compensate for the decrease in the total amount of RAGE protein. In contrast, the presence of an internally truncated protein (preferably lacking one or more elements of the complete RAGE protein) should be sufficient to prevent increased transcription but still provide a therapeutic benefit due to the reduction in the total amount of functional RAGE protein.

AON-induced exon skipping of the present invention does not necessarily have to completely or even substantially eliminate the function of the RAGE protein. Preferably, modulation of alternative splicing through the exon skipping method results in reduced or impaired functionality of the RAGE protein.

Different RAGE isoforms produced using different skipping strategies could result in proteins with eliminated or reduced signaling activity that could advantageously be used to treat or prevent various diseases associated with RAGE antigens, such as neurodegenerative diseases, cancer, pulmonary disorders, or inflammatory diseases. Alternative splicing strategies can generate truncated proteins or proteins with attenuated functions, which can advantageously be used as treatments for specific aspects, forms or progression of diseases associated with RAGE expression and activity.

The skipping method of the present invention using AON may exclude (skip) an individual exon or may result in skipping two or more than two exons at once.

The skipping method of the present invention using AON may involve retaining intronic sequences with or without directly skipping one or more than one exon.

The AONs of the present invention may be a combination of two or more than two AONs capable of binding to a selected target to induce exon exclusion in a RAGE gene transcript. This combination may be a mixture of two or more than two AONs and/or a design containing two or more than two AONs associated with each other.

According to this invention, there is additionally provided a method for modulating alternative splicing in a RAGE gene transcript, comprising the step of:

providing one or more than one AON, as described herein, and causing the given oligomer(s) to bind to a target nucleic acid site.

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

Designing an AON to completely mask consensus splice sites may not necessarily generate a splicing change in the target exon. In addition, the present inventors have discovered that the size or length of the AON itself is not always the primary factor in the design of the AON. With some targets, AONs as short as 20 bases could induce some exon inclusion, in some cases more efficiently than other longer (e.g., 25 bases) oligomers targeting the same exon.

The present inventors have also discovered that there does not appear to be any standard motif that can be blocked or masked by AON to redirect splicing. We found that AONs must be designed for each target gene and their individual effectiveness must be empirically assessed.

More specifically, the AONs may be selected from the AONs set forth in any of Tables 3a-3d. The sequence data is preferably selected from the group consisting of any one or more than one SEQ ID NO: 1-31 and combinations or mixtures thereof. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. The AON combination is preferably the 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, their complementary sequences, sequences containing modified bases, modified backbones and their functional truncations or extensions, which have or modulate pre-mRNA processing activity in the RAGE gene transcript.

An oligomer 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 be hydrogen bonded to each other. Thus, “specifically hybridizable” and “complementary” are terms that are used to indicate a sufficient degree of complementarity or pairing such that stable and specific binding occurs between the oligomer and the DNA, cDNA or RNA target. It is understood in the art that an AON sequence does not need to be 100% complementary to its target sequence in order to be specifically hybridizable. An AON is specifically hybridizable when binding of a compound to a target DNA or RNA molecule interferes with the normal function of that target DNA or RNA product, and there is a sufficient degree of complementarity to avoid nonspecific binding of the AON to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatments, and in the case of in vitro assays under the conditions under which the assays are performed.

Selective hybridization can be carried out under low, moderate or high stringency conditions, but preferably under high stringency conditions. Those skilled in the art will appreciate that hybridization stringency will be influenced by conditions such as salt concentration, temperature, or organic solvents, in addition to base composition, complementary strand length, and the number of nucleotide base mismatches between the nucleic acids being hybridized. Severe temperature conditions will typically include temperatures greater than 30°C, typically greater than 37°C, and preferably greater than 45°C, preferably at least 50°C, and typically 60°C-80°C or higher. Harsh salt conditions will typically be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, it is the combination of parameters that is much more important than the measure of any single parameter. An example of stringent hybridization conditions is 65°C and 0.1 SSC (1 SSC - 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Thus, the AONs of the present invention may include oligomers that selectively hybridize to the sequences shown in any of Tables 3a-3d or SEQ ID NO: 1-31. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

It will be appreciated that codon organizations at the end of exons in structural proteins may not always be terminated at the end of the codon, therefore, it may be necessary to remove more than one exon from the pre-mRNA to ensure that the mRNA is read in frame. Under such circumstances, it may be necessary to select a range of AONs by the method of the invention, each targeting a different region responsible for inducing inclusion of the desired exon and/or intron. At a given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to the complementary polynucleotide. Such hybridization can occur with “close” or “substantial” AON complementarity with the target sequence, as well as runoff complementarity.

Typically, selective hybridization will occur when there is at least about 55% identity over a span of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%, 95%, 98%, or 99% identity with AON nucleotides. The length of the homology comparison, as described, can be made over longer lengths, and in some embodiments will often be made over a length of at least about nine nucleotides, typically at least about 12 nucleotides, more typically about 20, 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, at least about 36 or more nucleotides.

Thus, the AON sequences of the invention preferably have at least 75%, more preferably at least 85%, more preferably at least 86, 87, 88, 89 or 90% homology with the sequences shown in sequence listings in this document. More preferably there is at least 91, 92, 93, 94 or 95%, most preferably at least 96, 97, 98 or 99% homology. Generally, the shorter the length of the AON, the greater the homology required to obtain selective hybridization. Therefore, where the AON of the invention consists of less than about 30 nucleotides, it is preferred that the percentage identity is greater than 75%, preferably greater than 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95%, 96, 97, 98%, or 99% compared to the AONs set forth in the sequence listings herein. Nucleotide homology comparisons can be made by sequence comparison programs such as the GCG Wisconsin Bestfit program or GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). This method could compare sequences of similar or substantially different lengths to sequences cited herein by inserting gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

AONs of the present invention may have regions of reduced homology and regions of exact homology to the target sequence. An oligomer does not need to have exact homology along its entire length. For example, a given oligomer may have contiguous stretches of at least 4 or 5 bases that are identical to the target sequence, preferably contiguous stretches of at least 6 or 7 bases that are identical to the target sequence, more preferably contiguous stretches of at least at least 8 or 9 bases that are identical to the target sequence. The oligomer may have stretches 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 portions of the oligomer sequence may be intermittently identical to the target sequence; for example, the rest of the sequence may have an identical base followed by a non-identical base followed by an identical base. Alternatively (or also), the oligomer sequence may have multiple stretches of identical sequence (eg, 3, 4, 5, or 6 bases) interspersed with stretches of less than perfect homology. Such sequence mismatches will preferably have no or very little loss of splicing switch activity.

The term “modulate” or “modulates” includes “increasing” or “decreasing” one or more than one quantifiable parameter, possibly by a specific and/or statistically significant amount. The terms “increase” or “augmenting”, “amplifying” or “enhancing”, or “stimulating” or “stimulant” generally refer to the ability of one or more than one AON or composition to produce or cause a greater physiological response (e.g., effects below ) in a cell or subject relative to the response elicited by either no AON or a control compound. The terms “reducing” or “reducing” generally refer to the ability of one or more AONs or compositions to produce or cause a lesser physiological response (eg, lower effects) in a cell or subject relative to the response caused by either no AON or a control compound.

Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include increases in exclusion of specific exons in the RAGE pre-mRNA, decreases in the amount of RAGE pre-mRNA, or a decrease in the expression of functional RAGE protein in the cell. , tissue or from the subject in need. An "increased" or "increased" amount typically represents a statistically significant amount and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15. 20, 30, 40, 50 or more times (e.g., 500, 1000 times) (including all whole numbers and tenths in between and greater than 1, e.g., 1.5; 1.6; 1.7; 1.8) relative to the amount produced in the absence of AON (no agent) or in the presence of a control compound. The term “reduce” or “inhibit” may generally refer to the ability of one or more than one AON or composition to “reduce” a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, when measured according to conventional techniques in the field of diagnostics. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include reductions in symptoms or pathology of a disease such as cancer, neurodegenerative diseases, pulmonary disorders and other inflammatory diseases. The “reduction” in response may be statistically significant compared to the response produced without AON or control composition and may include a decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9 %, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, including all integers in between.

The length of the AON can vary, provided that it is capable of selective binding to its intended location within the pre-mRNA molecule. The length of such sequences can be determined according to the selection techniques described herein. In general, an AON will be from about 10 nucleotides in length up to about 50 nucleotides in length. However, it will be understood that any length of nucleotides within this range can be used in this method. Preferably, the length of the AON is from 10 to 40, from 10 to 35, from 15 to 30 nucleotides in length, or from 20 to 30 nucleotides in length, most preferably from about 25 to 30 nucleotides in length. For example, a given oligomer may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

The term "AON" as used herein refers to a linear sequence of nucleotides or nucleotide analogues that allows nucleotide bases to hybridize to a target sequence in RNA through Watson-Crick base pairing to form an oligonucleotide heteroduplex: RNA within the target sequence. The terms "AON", "oligomer" and "antisense compound" can be used interchangeably to name an oligonucleotide. The cyclic subunits may be based on ribose or other pentose sugar, or, in some embodiments, on a morpholino group (see description of morpholino oligonucleotides below). Also contemplated are peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and 2'-O-methyl oligonucleotides, among other antisense agents known in the art.

Included are non-naturally occurring AONs or "oligonucleotide analogues", including AONs or oligonucleotides having (i) a modified backbone structure, e.g., a backbone different from the standard phosphodiester linkage found in naturally occurring oligo- and polynucleotides, and/or ( ii) modified sugar moieties, eg morpholino moieties rather than ribose or deoxyribose moieties. Oligonucleotide analogues support bases capable of hydrogen bonding by forming Watson-Crick pairs with bases of standard polynucleotides, wherein the analogue backbone represents bases in such a way as to allow such hydrogen bonding in a sequence-specific manner between the oligonucleotide analogue molecule and the bases in standard polynucleotide (for example, single-stranded RNA or single-stranded DNA). Preferred analogs are those having a substantially uncharged phosphorus backbone.

One method of producing AON is to methylate the 2'-hydroxyribose position, and the inclusion of a phosphorothioate backbone produces molecules that have superficial similarities to RNA but which are significantly more resistant to nuclease degradation, although those skilled in the art will be aware of other forms of suitable backbones that may be applicable for the purposes of this invention.

To avoid degradation of pre-mRNA during duplex formation with AON, the AON used in this method can be tailored to minimize or prevent degradation by endogenous RNase H. This property is highly advantageous since treatment of RNA with unmethylated oligomers, either intracellular, or in crude extracts that contain RNase H, leads to the degradation of pre-mRNA:AON duplexes. Any form of modified AON that is capable of bypassing or not inducing such degradation can be used in the present method. Nuclease resistance can be achieved by modifying the AONs of the invention such that they contain a partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups, including carboxyl groups, ester groups and alcohol groups.

An example of AONs that, when found in a duplex with RNA, are not cleaved by cellular RNase H, are 2'-O-methyl derivatives. Such 2'-O-methyl oligoribonucleotides are stable in cells and animal tissues, and their RNA duplexes have higher Tm values than their ribo- or deoxyribo-analogues. Alternatively, the nuclease-resistant AONs of the invention may have at least one of the last 3' terminal nucleotides fluorinated. In addition, alternatively, the nuclease-resistant AONs of the invention have phosphorothioate bonds linking between at least two of the last 3'-terminal nucleotide bases, preferably having phosphorothioate linkages linking between the last four 3'-terminal nucleotide bases.

Enhanced splicing switching can also be achieved using alternative oligonucleotide chemistries. For example, the AON may be selected from a list containing: phosphoramidate or phosphorodiamidate morpholino oligomer (PMO); RMO-X; RRMO; peptide nucleic acid (PNA); locked nucleic acid (LNA) and derivatives including alpha-L-LNA, 2'-amino-LNA, 4'-methyl-LMA and 4'-O-methyl-LNA; ethylene-linked nucleic acids (ENAs) and their derivatives; phosphorothioate oligomer; tricyclo-DNA oligomer (tsDNA); tricyclophosphorothioate oligomer; 4'-O-methyl-modified oligomer (2'-OMe); 2-O-methoxyethyl (2'-MOE); 2'-fluoro-, 2'-fluoroarabino (FANA); unlocked nucleic acid (UNA); thermostable twisted intercalating nucleic acid (TINA); hexitol nucleic acid (HNA); cyclohexenyl nucleic acid (CeNA); 2'-amino (2'-NH2); 2'-O-ethyleneamine or any combination of the above in the form of mixers or in the form of gapmers. To further improve the delivery efficiency, the above-mentioned modified nucleotides are often conjugated with fatty acids/lipid/cholesterol/amino acids/carbohydrates/polysaccharides/nanoparticles, etc. with a sugar group or a nucleic base group. These conjugated nucleotide derivatives can also be used to construct AONs for exon skipping. Antisense oligonucleotide-induced splicing modification of human RAGE gene transcripts has typically used one of the oligoribonucleotides, PNA, 2OMe- or MOE-modified bases on a phosphorothioate backbone. Although 2OMeAO is used for the design of oligonucleotides, due to its efficient in vitro uptake when delivered as cationic lipoplexes, these compounds are sensitive to nuclease degradation and are not considered ideal for in vivo or clinical applications. When using alternative chemistries to produce the AON of the present invention, uracil (U) of the sequences proposed herein can be replaced by thymine (T).

Included within the scope of the AON of the present invention are non-naturally occurring oligomers or "oligonucleotide analogues", including oligomers having (i) a modified backbone structure, for example, a backbone different from the standard phosphodiester linkage found in naturally occurring oligo- and polynucleotides, and/or (ii) modified sugar moieties, eg morpholino moieties rather than ribose or deoxyribose moieties. Oligomer analogues support bases capable of forming hydrogen bonds by forming Watson-Crick pairs with standard polynucleotide bases, where the analogue backbone represents the bases in such a way as to allow such hydrogen bonding in a sequence-specific manner between the oligomeric analogue molecule and the bases in standard polynucleotide (for example, single-stranded RNA or single-stranded DNA). Preferred analogues are those having an essentially unchanged phosphorus-containing backbone.

Antisense oligonucleotides that do not activate RNase H can be prepared according to known techniques (see, for example, US Pat. No. 5,149,797). Such AONs, which may be deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modification that sterically interferes with or prevents RNase H from binding to a duplex molecule containing an oligomer as one member thereof, the structural modification of which does not substantially interfere with or impair duplex formation. Because the portions of the oligomer involved in duplex formation are substantially different from those involved in RNase H binding to it, numerous AONs are available that do not activate RNase H. For example, such AONs may be oligomers in which at least one or All of the internucleotide-bridged phosphate residues are modified phosphates, such as methylphosphonates, methylphosphorothioates, phosphoromorpholidates, phosphoropiperazidates, boranophosphates, amide linkages and phosphoramidates. For example, every second of the phosphate residues forming internucleotide bridges can be modified as described above. In another non-limiting example, such AONs are molecules in which at least one or all of the nucleotides contain a 2' lower alkyl moiety (such as, for example, C ₁ -C ₄ linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl and isopropyl). For example, every second of the nucleotides may be modified as described.

While the AONs described above represent the preferred form of the AONs of the present invention, the present invention includes other oligomeric antisense molecules, including oligomeric mimetics, such as those described below, but not limited to.

Specific examples of preferred useful AONs in this invention include oligomers containing modified backbones or non-natural internucleoside linkages. As defined herein, oligomers having modified backbones include oligomers that retain a phosphorus atom in the backbone and oligomers that do not have a phosphorus atom in the backbone. For purposes of this specification, and as is sometimes taught in the art, modified oligomers that do not have a phosphorus atom in their internucleoside backbone may also be considered AONs.

In other preferred oligomeric mimetics, both the sugar and the internucleoside linkage, e.g. the backbone of nucleotide units are replaced by new groups. The base units are retained for hybridization with a suitable target nucleic acid compound. One such oligomeric compound, an oligomeric mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligomer is replaced by an amide backbone, in particular an aminoethylglycine backbone. Nucleic bases are retained and bind directly or indirectly to the aza nitrogen atoms of the amide part of the backbone.

Another preferred chemistry is oligomeric compounds based on phosphorodiamidate morpholino oligomer (PMO), which are not degraded by any known nuclease or protease. These compounds are uncharged, do not activate RNase H activity upon binding to the RNA strand, and have been shown to have long-lasting modulation of splicing after administration in vivo (Summerton and Weller, Antisense Nucleic Acid Drug Development, 7, 187-197).

Modified oligomers may also contain one or more substituted sugar moieties. Oligomers may also include a nucleic acid base (often referred to in the art simply as a "base"), modifications or substitutions. Certain nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. Substitutions with 5-methylcytosine have been shown to increase the stability of the nucleic acid duplex by 0.6-1.2°C, even more specifically when combined with 2'-O-methoxyethyl sugar modifications.

Another modification of the oliomers of the invention involves chemically linking to the oligomer one or more than one moiety or conjugate that increases the activity, cellular distribution or cellular uptake of the oligomer. Such moieties include lipid moieties such as cholesterol moiety, cholinic acid, thioester such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chain such as dodecanediol or undecyl moieties, phospholipid such as di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chain, or adamantaneacetic acid, palmityl group, myristyl or octadecylamine or hexylamino-carbonyl-oxycholesterol group, but are not limited to them.

Cell-penetrating peptides were added to phosphorodiamidate morpholino oligomers to enhance cell uptake and nuclear localization. It has been shown that different peptide tags influence uptake efficiency and target tissue specificity, as demonstrated by Jearawiriyapaisarn et al. (2008), Mol. Ther. 16 9, 1624-1629.

In a given compound, it is not necessary to modify all positions uniformly, and, in fact, more than one of the above modifications can be included in a single compound or even a single nucleoside within an oligomer. The present invention also includes AONs, which are chimeric compounds. "Chimeric" AONs or "chimeras" in the context of this invention are AONs, in particular oligomers, which contain two or more than two chemically distinct regions, each composed of at least one monomer unit, i.e. nucleotide in the case of an oligomeric compound. These oligomers typically contain at least one region where the oligomer is modified to provide the oligomer or AON with increased resistance to nuclease degradation, increased cellular uptake, and an additional region for increased binding affinity for a target nucleic acid.

The activity of AONs and variants thereof can be assessed according to techniques conventional in the art. For example, the splice forms and expression levels of the RNAs and proteins of interest can be analyzed by any of a wide variety of well-known methods to detect splice forms and/or expression of the transcribed nucleic acid or protein. Non-limiting examples of such methods include RT-PCR (reverse transcription polymerase chain reaction) of RNA splice forms followed by size separation of PCR products, nucleic acid hybridization methods such as Northern blotting and/or the use of nucleic acid chips; methods for amplification of nucleic acids; immunological methods for identifying proteins; protein purification methods; and protein function or activity assays.

RNA expression levels can be assessed by obtaining mRNA/cDNA (ie, a transcribed polynucleotide) from a cell, tissue or organism and by hybridizing this mRNA/cDNA with a reference polynucleotide that is complementary to the nucleic acid or fragment thereof being analyzed. The cDNA may optionally be amplified using any of a variety of polymerase chain reaction or in vitro transcription techniques prior to hybridization with a complementary polynucleotide; preferably it is not amplified. Expression of one or more transcripts can also be detected using quantitative PCR to assess the expression level of the transcript(s).

The present invention provides an AON that induces RAGE gene transcript splicing switch, clinically relevant oligomer chemistries, and delivery systems for manipulating RAGE splicing to therapeutic levels. Significant reductions in the amount of full-length RAGE mRNA and therefore RAGE protein from RAGE gene transcription are achieved by:

a) in vitro oligomer refinement using fibroblast cell lines through experimental evaluation of (i) intronic enhancer targeting motifs, (ii) AON length and development of oligomer mixtures, (iii) chemical compound selection, and (iv) addition of cell penetrating peptides (CPPs) ) to enhance oligomer delivery; And

b) a detailed evaluation of a new approach for generating RAGE transcripts with one or more than one missing exon.

As such, this paper demonstrates that alternative splicing of RAGE pre-mRNA can be modulated by specific AONs. This method can produce functionally significant reductions in the amount of full-length (signaling-capable) RAGE protein and/or can achieve increases in the non-signaling splice form of RAGE decoy receptor mRNA (RAGE_v1) or other decoy receptors, thereby reducing the severity of the pathology associated with such diseases such as neurodegenerative diseases, cancer, pulmonary disorders and other inflammatory diseases.

The AONs used in this invention can be conveniently prepared by well known solid phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). One method for performing the synthesis of oligomers on a modified solid support is described in US Pat. No. 4,458,066.

Additionally or alternatively, any other methods for such synthesis may be used. The use of similar techniques is well known for the preparation of oligomers such as phosphorothioates and alkylated derivatives. In one such automated embodiment, diethyl phosphoramidites are used as starting materials and can be synthesized as described by Beaucage, et al., (1981) Tetrahedron Letters, 22: 1859-1862.

The AONs of the invention are synthesized in vitro and do not include biologically derived antisense compositions or genetic vector constructs designed to direct AON synthesis in vivo. The molecules of this invention may also be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds, such as, for example, liposomes, receptor-targeted molecules, oral, rectal, topical, or other formulations, to assist in absorption, distribution and/or absorption.

The AON of the present invention can also be used as a prophylactic or therapeutic agent, which can be used for the purpose of treating a disease. Accordingly, in one embodiment, the present invention provides AONs that bind to a selected target in RAGE pre-mRNA to induce effective and predictive exon skipping, as described herein, in a therapeutically effective amount, admixed with a pharmaceutically acceptable carrier, diluent or excipient.

The present invention therefore provides a pharmaceutical, prophylactic or therapeutic composition for treating, preventing or reversing the disease effects associated with RAGE expression in a patient, comprising:

a) one or more than one AON, as described herein, and

b) one or more than one pharmaceutically acceptable carrier and/or diluent.

Preferably, the disease associated with RAGE expression is selected from the list comprising: neurological disorders, cancer; cardiovascular disorders; digestive disorders; respiratory disorders, musculoskeletal disorders, connective tissue disorders, renal disorders, sexual disorders, skin disorders, eye disorders and endocrine disorders.

In one form of the present invention, the RAGE-related disorder is a cardiovascular disorder selected from the following group: atherosclerosis, coronary artery disease, myocarditis, endocarditis, cardiomyopathy, acute rheumatic fever, chronic rheumatic heart disease, cerebrovascular disease/stroke, heart failure , vascular calcification, peripheral vascular disease and lymphangitis.

In one form of the present invention, the RAGE-related disorder is a digestive system disorder selected from the following group: periodontitis, esophagitis, gastritis, gastric and duodenal ulcers, Crohn's disease, ulcerative colitis, ischemic colitis, enteritis and enterocolitis, peritonitis, alcoholic liver disease , hepatitis, toxic liver poisoning, biliary cirrhosis, liver fibrosis/cirrhosis, non-alcoholic liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), liver injury and recovery from liver injury, trauma or surgery.

In one form of the present invention, the RAGE-associated disorder is a cancer selected from the following group: malignancies of the lip, oral cavity and pharynx, digestive malignancies, respiratory and intrathoracic malignancies, bone and articular cartilage malignancies, melanoma, and others malignant neoplasms of the skin, malignant neoplasms of the mesothelial and soft tissue, 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 gland and other endocrine glands, malignant neoplasms of lymphoid, hematopoietic and related tissue, malignant neoplasms in certain, secondary and/or nonspecific places.

In one form of the present invention, the RAGE-related disorder is a neurological disorder and is selected from the following group: inflammatory diseases of the central nervous system, systemic atrophies affecting primarily the central nervous system, extrapyramidal and movement disorders, Parkinson's disease, demyelinating diseases of the central nervous system, Alzheimer's disease, Pick's syndrome, dementia with Lewy bodies, epilepsy, migraine, neuropathic pain, diabetic neuropathy, polyneuropathy, development and progression of glioma, spinal cord injury, ischemic brain injury/stroke, brain injury and recovery from injury, brain injury or surgery.

In one form of the present invention, the RAGE-related disorder is a musculoskeletal disorder and is selected from the following group: muscular dystrophy, congenital and acquired myopathy, polymyositis, myasthenia gravis, dermatomyositis, inclusion body myositis, muscular atrophy, and muscle damage.

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

In one form of the present invention, the RAGE-related disorder is a respiratory (pulmonary) disorder, and is selected from the following group: acute upper respiratory tract infections, rhinitis, nasopharyngitis, sinusitis, laryngitis, influenza and pneumonia, acute bronchitis, acute bronchiolitis, asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, emphysema, chronic lung disease due to external agents, acute respiratory distress syndrome (ARDS), pulmonary eosinophilia, pleural, pulmonary trauma and recovery from lung injury, trauma or surgery.

In one form of the present invention, the RAGE-related disorder is a connective tissue disorder and is selected from the following group: osteoarthritis, infectious arthritis, rheumatoid arthritis, psoriatic and enteropathic arthropathy, chronic polyarthritis in children, gout and other crystalline arthropathy, diabetic arthropathy, nodular polyarteritis, Churg-Strauss syndrome, mucocutaneous lymph node syndrome [Kawasaki], hypersensitivity angiitis, Goodpasture's syndrome, thrombotic microangiopathy, Wegener's granulomatosis, aortic arch syndrome [Takayasu], giant cell arteritis, polymyalgia rheumatica, microscopic polyangiitis, vasculitis with hypocomplementemia, systemic red lupus, dermatopolymyositis, polymyositis, systemic sclerosis, CR(E)ST syndrome, Sicca [Sjögren's] syndrome, mixed connective tissue disease, Behçet's disease, traumatic muscle injury, ligament sprain, tendon sprain and fracture.

In one form of the present invention, the RAGE-related disorder is a renal disorder and is selected from the following group: glomerulonephritis, nephritis, diabetic kidney disease, interstitial nephritis, obstructive and reflux nephropathy, acute kidney failure, and chronic kidney disease.

In one form of the present invention, the RAGE-related disorder is a sexual disorder and is selected from the following group: prostatitis, prostatic hypertrophy, prostatic dysplasia, salpingitis, oophoritis, pelvic inflammatory disease (PID), polycystic ovary disease, cervicitis, cervical dysplasia, vaginitis , vulvitis.

In one form of the present invention, the RAGE-related disorder is a skin disorder selected from the following group: dermatitis, eczema, pemphigus vulgaris/pemphigoid, psoriasis, pityriasis rosea, Wilson's pityriasis, urticaria, erythema multiforme, erythema nodosum, sunburn, keratosis, formation skin ulcers due to photoaging, superficial skin damage and open wound.

In one form of the present invention, the RAGE-related disorder is an ocular disorder selected from the following group: keratitis, conjunctivitis, retinitis, glaucoma, scleritis, episcleritis, chorioretinal inflammation, diabetic retinopathy, macular edema, retinopathy of prematurity, optic neuritis, ocular trauma, and recovery from eye damage, trauma or surgery.

In one form of the present invention, the RAGE-related disorder is an endocrine disorder selected from the following group: diabetes mellitus, insulin resistance, impaired glucose tolerance, and thyroiditis.

The composition may contain from about 1 nM to 1000 nM of each of the desired AONs of the invention. Preferably, the composition may contain from about 1 nM to 500 nM, from 10 nM to 500 nM, from 50 nM to 750 nM, from 10 nM to 500 nM, from 1 nM to 100 nM, from 1 nM to 50 nM, from 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, most preferably 1 nM to 10 nM of each of the AONs of the invention.

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

The present invention further provides one or more AONs adapted to assist in the prophylactic or therapeutic treatment, prevention or amelioration of symptoms of a disease, such as a disease or pathology associated with RAGE expression, in a suitable form for delivery to a patient.

The phrase "pharmaceutically acceptable" refers to chemical compounds and compositions that are physiologically tolerable and typically do not produce an allergic or similar adverse reaction, such as stomach upset and the like, when administered to a patient. The term "carrier" refers to the diluent, adjuvant, excipient or vehicle with which the compound is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including liquids of petroleum, animal, vegetable or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Preferably, water or saline solutions, aqueous dextrose and glycerol solutions are used as carriers, in particular for injectable solutions. Suitable pharmaceutical carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA, (1990).

In a more specific form, the present invention provides pharmaceutical compositions containing therapeutically effective amounts of one or more AONs of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of varying buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, and additives such as detergents and solubilizers (e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (eg thiomersal, benzyl alcohol) and bulking agents (eg lactose, mannitol). This substance can be included in preparations in the form of particles of polymer compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid can also be used. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the present proteins 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. These compositions may be provided in liquid form or may be in dry powder form, such as lyophilized form.

It will be understood that the pharmaceutical compositions provided by the present invention can be administered by any means known in the art. Preferably, the pharmaceutical compositions for administration are administered by injection, orally, topically, or via the pulmonary or nasal route. A suitable route can be determined by one of ordinary skill in the art according to the condition of the subject being treated.

In some embodiments, the AONs of this disclosure may be delivered by the pulmonary or nasal routes (eg, through a nebulized saline solution including the AON). The highest endogenous expression of RAGE mRNA in healthy human tissue is found in the lung and is accessible through the respiratory tract. Inhalable oligonucleotides represent a promising therapeutic option against respiratory diseases. The airways are uniquely lined with pulmonary surfactants, which consist primarily of zwitterionic lipids. These surfactant lipids have cationic properties at the pH of the respiratory tract. When anionic oligonucleotides are inhaled, they tend to be absorbed by surfactants, resulting in particles in the new formulation that were expected to be efficiently taken up by bronchial and alveolar epithelial cells in the lung. Interestingly, AONs have been shown to withstand the spraying process.

In some embodiments, AONs are more preferably delivered via intravenous, intra-arterial, intraperitoneal, intramuscular or subcutaneous routes of administration. The vascular or extravascular circulation, circulatory or lymphatic system, and cerebrospinal fluid are some non-limiting sites where AON may be administered.

In some embodiments, direct delivery to the CNS may be used, for example, intracerebral, ventricular or intrathecal administration may be used as routes of administration.

Formulations for topical administration include those in which the oligomers of this disclosure are in admixture with a topical delivery vehicle such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelators and surfactants. Lipids and liposomes include neutral (eg, dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine), negatively charged (eg, dimyristoylphosphatidylglycerol (DMPG)) and cationic (eg, dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). For topical or other administration, the oligomers of this disclosure can be encapsulated in or complexed with liposomes, in particular with cationic liposomes. Alternatively, the oligomers can be complexed with lipids, in particular cationic lipids. Fatty acids and esters, their pharmaceutically acceptable salts and their uses are further described in US Pat. No. 6,287,860 and/or US Patent Application Ser. No. 09/315298, filed May 20, 1999.

In some embodiments, the AONs of this disclosure may be delivered by transdermal routes (eg, by incorporating the AON, eg, in an emulsion, possibly with such AON packaged in liposomes). Such transdermal and emulsion/liposome-mediated delivery methods are described in the art for AON delivery, for example, in US Pat. No. 6,965,025.

The AONs described herein can also be delivered via an implantable device. The construction of such a device is a method known in the art, for example, the construction of a synthetic implant, described, for example, in US patent No. 6969400.

Compositions and preparations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Oral formulations are those in which the oligomers of this disclosure are administered in combination with one or more penetration enhancers, surfactants and chelators. Surfactants include fatty acids and/or their esters or salts, bile acids and/or their salts. Bile acids/salts and fatty acids and their uses are further described in US Pat. No. 6,287,860. In some embodiments, the present disclosure provides combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A typical combination is the sodium salt of lauric acid, capric acid and UDCA (ursodeoxycholic acid). Other penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The oligomers of this disclosure may be delivered orally, in granular form, including spray-dried particles, or complexed to form micro- or nanoparticles. Oligomer complexing agents and their uses are further described in US Pat. No. 6,287,860. Oral preparations for oligomers and their preparation are described in detail in US 6887906, 09/315298, filed May 20, 1999, and/or US 20030027780.

Compositions and preparations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions, which may also contain, but are not limited to, buffers, diluents and other suitable additives such as penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Delivery of a therapeutically useful amount of AON can be achieved by previously published methods. For example, intracellular delivery of AON can be accomplished through a composition containing a mixture of AON and an effective amount of a block copolymer. An example of this method is described in US patent application US 20040248833. Other methods for delivering AON to the nucleus are described in Mann CJ et al. (2001) Proc, Natl. Acad. Science, 98 (1) 42-47 and in Gebski et al. (2003) Human Molecular Genetics, 12(15): 1801-1811. A method for introducing a nucleic acid molecule into a cell via an expression vector, either in the form of naked DNA or in complex with lipid carriers, is described in US 6,806,084.

It may be desirable to deliver AON in a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome preparations. These colloidal dispersion systems can be used in the manufacture of therapeutic pharmaceutical compositions.

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

For a liposome to be an effective gene transfer vehicle, the following characteristics must be present: (1) encapsulate the AONs of interest with high efficiency without interfering with their biological activity; (2) preferential and significant binding to the target cell compared to non-target cells; (3) delivery of the aqueous contents of the vesicle into the cytoplasm of the target cell with high efficiency; and (4) accurate and efficient expression of genetic information (Mannino, et al., Biotechniques, 6: 682, 1988). The liposome composition is typically a combination of phospholipids, in particular high phase transition temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also 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 to form a stable complex. Liposomes, which are pH-sensitive or negatively charged, are thought to capture DNA rather than form a complex with it. Both cationic and non-cationic liposomes have been used to deliver DNA into cells.

Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposomes, result in increased shelf life. circulatory system relative to liposomes devoid of such specialized lipids. Examples of sterically stabilized liposomes are liposomes in which the vesicle-forming lipid portion of the liposome contains one or more one glycolipid or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in US 6,287,860.

The AONs described herein can also be delivered via an implantable device. Constructing such a device is a method known in the art, using, for example, a synthetic implant design described, for example, in US Pat. No. 6,969,400, the contents of which are incorporated herein in its entirety.

Antisense oligonucleotides can be introduced into cells using techniques known in the art (eg, transfection, electroporation, fusion, liposomes, colloidal polymer particles, and viral and non-viral vectors, as well as other methods known in the art). The delivery method chosen will depend at least on the cells to be treated and the location of the cells, and will be apparent to one skilled in the art. For example, localization can be achieved by liposomes with specific markers on the surface to guide the liposome, direct injection into tissue containing target cells, uptake mediated by a specific receptor, or the like.

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

AON may also be combined with other pharmaceutically acceptable carriers or diluents to form 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.

The routes of administration described are intended as a guide only, as a qualified practitioner will readily be able to determine the optimal route of administration and any dosage for any particular animal and condition.

Many approaches have been attempted to introduce new functional genetic material into cells both in vitro and in vivo (Friedmann (1989) Science, 244: 1275-1280). These approaches include integration of the gene to be expressed into modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51 (18), suppl.: 5074S-5079S); integration into non-retroviral vectors (Rosenfeld, et al. (1992) Cell, 68: 143-155; Rosenfeld, et al. (1991) Science, 252: 431-434); or delivery of a 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; . (USA), 84: 7851-7855); binding to ligand-specific cation-based transport systems (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 transgenes into tissue produces only localized expression (Rosenfeld (1992) supra); Rosenfeld et al. (1991) above; Brigham et al. (1989) above; Nabel (1990) supra; and Hazinski et al. (1991) above). The group of Brigham et al. (Am. J. Med. Sci. (1989) 298: 278-311 and Clinical Research (1991) 39 (abstract)) described in vivo transfection of only mouse lungs after either intravenous or intratracheal administration of a DNA-liposome complex. An example of a review article on human gene therapy techniques is: 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.

The AONs of the invention include any pharmaceutically acceptable salts, esters or salts of such esters, or any other compound which, when administered to an animal, including a human, is capable of providing (directly or indirectly) a biologically active metabolite or residue thereof. Accordingly, by way of example, this disclosure is also directed to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of this invention: i.e. salts that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects to it. For oligomers, preferred examples of pharmaceutically acceptable salts include (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids, such as, for example, 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, naphthalene sulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, polygalacturonic acid and the like; and (d) salts formed from elemental anions such as, but not limited to, chlorine, bromine and iodine. The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area being treated. Administration may be local (including ophthalmic and mucosal as well as rectal delivery), pulmonary, such as by inhalation or insufflation of powders or aerosols (including by nebulizer, intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. Oligomers with at least one 2'-O-methoxyethyl modification are considered to be particularly useful for oral administration. Preferably, AON is delivered via the subcutaneous or intravenous route.

The pharmaceutical preparations of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with pharmaceutical carrier(s) or excipient(s). In general, these preparations are prepared by bringing the active ingredients into uniform and intimate association with liquid carriers or finely divided solid carriers, or both, and then, if necessary, molding the product.

In one embodiment, AON is administered in an effective amount and in an effective manner to result in a peak blood concentration of at least 200-400 nM AON. Typically one or more doses of AON are administered, usually at equal intervals, over a period of about one to two weeks. Preferred dosages for oral administration range from about 1 mg to 1000 mg of oligomer per 70 kg. In some cases, doses greater than 1000 mg oligomer/patient may be necessary. For i.v. administration, preferred doses are from about 0.5 mg to 1000 mg of oligomer per 70 kg. For intravenous or subcutaneous administration, AON can be administered at a dosage of approximately 120 mg/kg daily or weekly.

AON may be administered at regular intervals over a short period of time, such as daily for two weeks or less. However, in some cases, this oligomer is administered periodically over a longer period of time. Administration may be followed or concurrently administered with an antibiotic or other therapeutic treatment. The treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated based on the results of immunoassays, other biochemical tests, and physiological examination of the subject being treated.

Dosage depends on the severity and susceptibility of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is achieved or relief of the disease state is achieved. Optimal dosing regimens can be calculated from measurements of drug accumulation in the patient's body. Ordinary practitioners can easily determine optimal dosages, dosing methodologies, and repetition rates. Optimal dosages may vary, depending on the relative potency of the individual oligomers, and can usually be estimated based on the EC50 (half maximal effective concentration) found to be effective in in vitro and in vivo animal models. In general, the dosage ranges from 0.01 mcg to 100 g per kg body weight, and can be given one or more times daily, weekly, monthly or yearly, even once every 2 to 20 years. Those of ordinary skill in the art can readily determine repetition rates for dosing based on measured residence times and drug concentrations in body fluids or tissues. After successful treatment, it may be desirable that the patient undergoes maintenance therapy to prevent recurrence of the disease state, where the oligomer is administered in maintenance doses ranging from 0.01 μg to 100 g per kg body weight, from one or more times per day to once every 20 years.

An effective in vivo treatment regimen using the AON of the present invention may vary according to the duration, dose, frequency and route of administration, as well as the condition of the subject being treated (ie, prophylactic administration versus administration in response to a localized or systemic infection). Accordingly, such in vivo therapy will often require monitoring through appropriate assays for the specific type of disorder being treated and appropriate adjustments in dosage or treatment regimen in order to achieve optimal therapeutic outcome.

Treatment can be monitored, for example, by general disease indicators known in the art. The effectiveness of the AONs of the invention administered in vivo can be determined from biological samples (tissue, blood, urine, etc.) collected from a subject before, during and after administration of the AON. Analyzes of such samples include (1) monitoring the presence or absence of heteroduplex formation with target and non-target sequences using techniques known to those skilled in the art, such as gel electrophoretic mobility assays; (2) monitoring the amount of mutant mRNA relative to a control normal mRNA or protein as determined by standard techniques such as RT-PCR, Northern blotting, ELISA (enzyme-linked immunosorbent assay), or Western blotting.

Intranuclear delivery of the oligomer is a major challenge for AON. Different cell penetrating peptides (CPPs) localize PMO to varying degrees in different conditions and cell lines, and new CPPs have been evaluated by the present inventors for their ability to deliver PMO to target cells. The terms CPP or “cellular uptake enhancing peptide moiety” are used interchangeably and refer to cationic cell penetrating peptides, also referred to as “transport peptides,” “carrier peptides,” or “transduction domain peptides.” These peptides, as shown herein, have the ability to induce cell entry in about or at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the cells in a given culture population cells and provide translocation of macromolecules within many tissues in vivo when administered systemically. CPPs are well known in the art and are disclosed, for example, in US Application No. 2010/0016215, which is incorporated by reference in its entirety.

The present invention therefore provides the AONs of the present invention in combination with cell penetrating peptides for the manufacture of therapeutic pharmaceutical compositions.

According to yet another aspect of the present invention, one or more AONs are provided, as described herein, for use in AON-based therapy. Preferably, the therapy is for a condition associated with RAGE expression. More preferably, therapy for a condition associated with RAGE expression is therapy against a disease selected from cancer, neurodegenerative diseases, pulmonary disorders and inflammatory diseases.

More specifically, a given AON may be selected from the group consisting of any one or more than one AON listed in any of Tables 3a-3d and/or SEQ ID NO: 1-31, and combinations or mixtures 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, sequences containing modified bases, modified backbones and functional truncations thereof, or extensions that possess or modulate pre-mRNA processing activity in the RAGE gene transcript.

The present invention also extends to a combination of two or more than two AONs capable of binding to a selected target to induce exon exclusion in a RAGE gene transcript. The combination may be a mixture of two or more than two AONs, a construct comprising two or more than two AONs connected to each other for use in AON-based therapy. This AON combination is preferably the combination of SEQ ID NOs: 11 and 10 or SEQ ID NOs: 11 and 13.

According to this invention, a method is provided for treating, preventing or reversing the effects of a disease associated with the expression of RAGE, comprising the step of:

a) administering to a patient an effective amount of one or more than one AON, or a pharmaceutical composition containing one or more than one AON, as described herein.

In addition, according to this invention, there is provided a method for treating, preventing or reversing the effects of cancer, neurodegenerative diseases, pulmonary diseases and inflammatory diseases, comprising the step of:

a) administering to a patient an effective amount of one or more than one AON, or a pharmaceutical composition containing one or more than one AON, as described herein.

Preferably, this therapy is used to reduce levels of functional RAGE protein through an exon skipping strategy. Reducing RAGE levels is preferably achieved by reducing transcript levels through modification of pre-mRNA splicing in the RAGE gene transcript or part thereof.

Reducing RAGE will preferably result in a reduction in the number, duration, or severity of symptoms of a RAGE-related condition or pathology, such as neurodegenerative diseases, cancer, pulmonary disorders, and inflammatory diseases.

The term "treatment" of a subject (eg, a mammal, such as a human) or cell, as used herein, is any type of intervention used in an attempt to change the natural course of an individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be administered either prophylactically or after the initiation of a pathogenic event or exposure. Also included are “preventative” treatments, which may be aimed at reducing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. “Treatment” or “prevention” does not necessarily indicate complete elimination, cure, or prevention of a disease or condition, or symptoms associated therewith.

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

The present invention also provides the use of purified or isolated AONs as described herein for the manufacture of a medicament for the treatment of a disease associated with RAGE expression.

The use of purified or isolated AONs as described herein is provided for the manufacture of a medicament for the treatment, prevention or reversal of the effects of a disease associated with RAGE expression.

Preferably, the RAGE-associated pathology or disease is a neurodegenerative disease, cancer, pulmonary disorder or inflammatory disease.

The present invention extends, in yet another aspect, to cDNAs or cloned copies of the AON sequences of the invention, as well as vectors containing the AON sequences of the invention. The present invention further also extends to cells containing such sequences and/or vectors.

The AON of the present invention may be co-administered with another therapeutic molecule. For example, a given AON may be administered with a second therapeutic agent that is a compound, such as a peptide, that is capable of modulating the cytosolic tail of RAGE. Such compounds include the following: IQGAP-1, transparent-1, protein kinase C zeta (PKCζ), Dock7, MyD88, TIRAP, ERK1/2, olfactory receptor 2T2, ADP/ATP translocase 2, protein phosphatase 1G, IRAK4, DJ-1 protein ( PARK7), calponin-3, drebrin, filamin B, Ras-related protein Rab-13, radixin/ezrin/moesin, proteolipid protein 2, coronin, S100 A11, succinyl-CoA ligase alpha subunit [GDP-forming], protein interacting with Hsc70, inhibitor of apoptosis 5, neuropilin, cleavage promoting factor, growth factor receptor associated protein 2, beta subunit sec61 or Nck1.

The present invention also provides kits for the treatment, prevention or relief of a disease or condition associated with RAGE expression in a patient, which comprise at least an isolated or purified AON for modifying pre-mRNA splicing in a RAGE gene transcript or a portion thereof, packaged in a suitable container , along with instructions for its use.

In a preferred embodiment, these kits will contain at least one AON as described herein, any one or more than one SEQ ID NO: 1-31 and/or the sequences set forth in any of Tables 3a-3d, or a mixture of AONs, as described in this document. These kits may also contain minor reagents such as buffers, stabilizers, etc. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20.

Accordingly, a kit is provided for treating, preventing or reversing a disease or condition associated with RAGE expression in a patient, which comprises at least the AON described herein, any one or more of SEQ ID NO: 1-31 and/or sequences , set out in any of Tables 3a to 3d, and combinations or mixtures thereof, packaged in a suitable container, along with instructions for its use. More preferably, the AON is SEQ ID NO: 11, 18, 19, or 20. This AON combination is preferably a combination of SEQ ID NO: 11 and 10 or SEQ ID NO: 11 and 13.

Preferably, the disease or condition is selected from the list consisting of: cancer, neurodegenerative diseases, pulmonary disorders or inflammatory diseases.

The contents of this kit may be lyophilized, and the kit may further contain a suitable solvent for dissolving the lyophilized components. The individual components of this kit would be packaged in separate containers, and such containers may be associated with a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects that agency's approval of the manufacture, use, or sale. for administration to humans.

When the kit components are provided in one or more than one liquid solution, the liquid solution may be an aqueous solution, such as a sterile aqueous solution. For in vivo use, the expression construct can be formulated in a pharmaceutically acceptable syringeable composition. In this case, the container means itself may be an inhaler, syringe, pipette, eye dropper or other such device, from which the drug can be applied to the affected area of the animal, such as the lungs, injected into the animal, or even applied or mixed with other components of the set.

The components of this kit may also be provided in dried or lyophilized forms. When providing reagents or components in dried form, dissolution is usually accomplished by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container device. Regardless of the number or type of containers, the kits of the invention may also contain or be packaged with a device to assist in the injection/administration or placement of the final complex composition within the body of the animal. Such a device may be an inhaler, syringe, dropper, tweezers, measuring spoon, eye dropper, or any such medically approved delivery device.

Those of ordinary skill in the art will appreciate that applications of the above method have broad application in identifying suitable AONs for use in the treatment of many other diseases.

The AONs of the present invention can also be used in combination with alternative therapies, such as drug therapies.

The present invention therefore provides a method of treating, preventing or reducing the effects of a disease or condition associated with RAGE expression, wherein the AON of the present invention is administered sequentially or concomitantly with another alternative therapy associated with treating, preventing or reducing the effects of the disease or condition , associated with RAGE expression. Preferably, the disease or condition is selected from the list consisting of: neurodegenerative diseases, cancer, pulmonary disorders or inflammatory diseases.

General

Those skilled in the art will appreciate that the invention described herein is subject to variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications. This invention also includes all of the steps, features, compositions and compounds referred to or set forth in the specification, individually or collectively, and any and all combinations or any two or more than two steps or features.

The present invention is not limited to the scope of the specific embodiments described herein, which are intended for purposes of illustration by example only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books or other documents) cited herein are hereby incorporated by reference. No assumption is made that any of these references constitute prior art or constitute part of the ordinary general knowledge of those working in the field to which this invention relates.

Each document, reference, patent application or patent cited in this text is expressly incorporated herein in its entirety by reference, which means that it is intended to be read and considered part of this text by the reader. That a document, reference, patent application or patent cited in this text is not repeated in this text is simply for the sake of brevity.

Any instructions, descriptions, manufacturer's product specifications and product descriptions for any products mentioned herein or in any document incorporated by reference herein are hereby incorporated by reference herein and may be used in the practice of this invention.

The terms "derived from" and "derived from" as used herein should be taken to indicate that a particular whole may be derived from a particular source, although not necessarily a directly published source.

Singular forms as used herein include plural references unless the context clearly dictates otherwise.

Throughout this specification, unless the context otherwise requires, it will be understood that the word “comprise” or variations such as “comprises” or “comprising” is intended to include the claimed whole or group of wholes, but not to exclude any other whole or group of wholes. whole groups.

Unlike the working example, or where otherwise indicated, all numbers expressing amounts of ingredients, reaction conditions, etc., used in the specification and claims are to be understood in all cases as modified by the term “about.” Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be achieved by the present invention. Therefore "about 80%" means "about 80%" and also "80%". At the very least, each numeric parameter should be interpreted in light of the number of significant numbers and normal rounding practices.

Although the numerical ranges and parameters setting out the broad scope of this invention are approximations, the numerical values set forth in the specific examples are given as accurately as possible. Any numeric value; however, by their nature they contain certain errors necessarily arising from the standard deviation found in their respective experimental measurements.

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

Sequence identification numbers (“SEQ ID NO:”), which contain nucleotide and amino acid sequence information included in this specification, are collected at the end of this specification and were obtained using PatentIn Version 3.0. Each nucleotide or amino acid sequence is identified in the sequence listing by a numeric indicator <210> followed by a sequence identifier (eg, <210> 1, <210> 2, etc.). The length, sequence type, and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numerical indicator fields <211>, <212>, and <213>, respectively.

An antisense oligomer nomenclature system has been proposed and published to distinguish between different antisense oligomers (see Mann et al., (2002) J Gen Med 4, 644-654). This nomenclature becomes particularly relevant when analyzing several slightly different antisense oligomers, all directed to the same target region, as shown below:

N # A/D (x:y)

with the first letter indicating the species (for example, H: human, M: mouse)

"#" denotes target exon number

“A/D” indicates an acceptor or donor splice site at the beginning/end of the exon, respectively

(xy) represents the annealing coordinates, where “-” or “+” indicate intronic or exonic sequences, respectively. As an example, A(-6+18) would indicate the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. The nearest splice site would be an acceptor such that the given coordinates would be preceded by an "A". The description of the annealing coordinates at the donor splice site could be D(+2-18), where the last 2 exonic bases and the first 18 intronic bases correspond to the annealing site of the antisense oligomer. Fully exonic annealing coordinates, which would be represented by A(+65+85), that is, the site between the 65th and 85th nucleotides inclusive from the beginning of a given exon.

The following examples more fully describe the method of application of the above invention, and also set forth the best ways to carry out various aspects of the present invention. It should be understood that these methods do not limit the scope of the present invention, but rather are presented for illustrative purposes.

EXAMPLES

In each of the following examples, the following general materials and methods are used unless the context requires otherwise.

Human microvascular endothelial cells (HMEC1) were cultured in MCDB 131 medium (10% FCS (fetal calf serum) with 10 mM glutamine, EGF (epidermal growth factor) and hydrocortisone). Human lung epithelial cells (A549) (carcinoma) were cultured in F-12K medium (10% FBS with 2 mM glutamine). Primary aortic endothelial cells (PMAEC) were isolated from the aorta of C57bI6 (wild type) and AGER knockout mice and cultured in Dulbecco's modified Eagle's medium (OMEM)/F12 medium supplemented with endothelial cell growth supplement (ECGS).

For AON transfection, epithelial or endothelial cells were seeded in 48-well plates for 24 or 48 hours and then transfected using Lipofectamine 3000 reagent (0.15 μl/well Lipofectamine 3000; 0.4 μl/well P3000/well) with one of the AONs , targeting exon 9 of human AGER (for HMEC1 and A549) and mouse AGER for mouse PMAEC cells or corresponding controls (Tables 3a-3d). Cells were incubated with AON/cationic lipoplexes at 37°C for 24 hours, after which the cells were lysed, RNA was extracted, and cDNA was prepared using either the TRIzol method or the cell-in-CT method.

To measure the effect on alternative splicing of human RAGE pre-mRNA, expression was determined using real-time RT-PCR primers to one of exon 11, exon 9b, or spanning exons 8–10 of RAGE, noting the expression (mRNA) conserved across variants splicing of RAGE mRNA, retention of exon 9b, or expression of the RAGEv1 isoform encoding the transmembrane-cytosolic tail, respectively. The decrease in the PCR signal of exon 8-10 of the cytosolic tail relative to exon 11 indicates that fewer splice forms of RAGE mRNA contain exon 10, and fewer isoforms of full-length RAGE protein (capable of signaling) are produced by the cell. In mouse cells and tissues, primers spanning exon 10-11 of mouse RAGE mRNA were used to target the expression of RAGE mRNA splice forms containing exon 10.

Media were also collected and soluble RAGE levels were determined by ELISA. Media (6 ml) were concentrated using a molecular weight cutoff filter before analysis by ELISA.

Chinese hamster ovary (CHO) cells express recombinant proteins at high levels, making them an ideal system for studying the regulation of pre-mRNA splicing with altered protein secretion as an expected outcome. As a model system to better study the effect of AON on RAGE splicing leading to the expression and secretion of extracellular soluble RAGE, CHO cells grown in F12 medium (supplemented with 10% FBS) were transfected with a plasmid encoding the human genomic AGER (gRAGE) sequence (excluding natural 5' and 3' untranslated regions), along with AON, using Lipofectamine 2000. After 2 days, media were collected, concentrated using a molecular weight cutoff filter, and checked by Western blotting using an anti-hRAGE antibody (R&D systems).

To determine the exact sequence of RAGE variants identified following AON administration, RNA was isolated using the Trizol method and cDNA was prepared using oligo dT primers before PCR using RAGE-specific primers to the 5' and 3' UTR sequences of RAGE and half-nested PCR for amplification mixtures of RAGE sequences. Gel purified RAGE bands were cloned into TA cloning vectors before transformation into E. coli Top10 cells. More than 50 colonies were selected for characterization 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 submitted for Sanger sequencing to confirm the sequence of the RAGE splice forms corresponding to the band sizes.

To measure the functional impact of interfering with ligand-dependent and ligand-independent RAGE activation, cells were exposed to AT1R cognate ligand, Ang II (1 μM) or RAGE ligand - S100A8/A9 (0.6 μg/ml cells) for 4 hours. The cells were then kept frozen until the mRNA was extracted, and cDNA was synthesized using the cell-to-Ct method. Changes in NFκB subunit gene expression, p65 (ReIA), or NFκB-activated target genes (eg, ICAM-1) were assessed by quantitative real-time RT-PCR performed using a 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 relative to the expression level of untreated control mice/cells, which is given an arbitrary value of 1.

EXAMPLE 1. MODULATION OF ALTERNATIVE RAGE SPLICING IN HUMAN CELLS USING ANTISENSE OLIGONUCLEOTIDES TARGETED TO CIS-ACTING RNA ELEMENTS IN EXON 10

This example demonstrates that RAGE pre-mRNA splicing can be modulated using AONs targeting cis-acting RNA elements in exon 10 of the RAGE pre-mRNA.

RAGE pre-mRNA undergoes alternative splicing in vivo, generating a range of mRNA splice forms. Most of these splice forms contain both exon 10 and 11, encoding the transmembrane and cytosolic domains of RAGE, respectively, and the protein isoforms that are produced are therefore capable of activation by RAGE ligands and transactivation by colocalizing GPCRs to induce proinflammatory and proproliferative signaling. Selection of an alternative 5' splice site in exon 9 can also result in an 83-nucleotide extension of exon 9 (exon 9B; Fig. 1a). Because the distance between this alternative splice site and the 3' splice (acceptor) site at the beginning of exon 10 is only 46 nucleotides, which is significantly shorter than the lower bound on the length of introns in eukaryotes, exon 10 is therefore skipped and the 3' site is selected instead (acceptor) at the beginning of exon 11. This alternative splicing also introduces a premature termination codon, but does not undergo nonsense-mediated decay due to its close (29 bp) proximity to the last exon-exon contact. The mature mRNA resulting from this alternative splicing (RAGE_v1, Fig. 1a) therefore encodes a protein isoform that lacks elements required for membrane retention and cytoplasmic signaling, encoded by exons 10 and 11, respectively, and is thus secreted and is naturally found in the bloodstream, broncho-alveolar and cerebrospinal fluids. This esRAGE may act as a decoy receptor by competing with full-length cell surface RAGE for ligands or increasing ligand clearance. The novel 17 amino acid C terminus encoded by exon 9b may also have an independent effect on RAGE signaling or multimerization.

Transfection of human lung epithelial cells (A549) with 10 nM AON targeting exon 10 resulted in changes in the expression of RAGE mRNA splice forms containing exon 9b (Fig. 1b). Some AONs, particularly AON 3779, increased the expression of RAGE mRNA splice forms containing exon 9b compared to control (scrambled RNA-treated) cells when measured by real-time RT-PCR. Other AONs also targeting exon 10, particularly AONs 3777 and 3781, had no effect.

Transfection of human lung epithelial cells (A549) with 10 nM concentrations of AONs targeting exon 10 also resulted in changes in the expression of RAGE mRNA splice forms such that some AONs, particularly AONs 3779, 3780, 3781, 82, 83, and 87, had reduced expression splice forms of RAGE mRNA containing exon 10 (i.e. encoding cytosolic tail signaling elements) compared with control (scrambled RNA-treated) cells as measured by real-time RT-PCR (Figures 1c and 1d). Other AONs also targeting exon 10, particularly AONs 3777, 84, and 85, had no effect.

All RAGE mRNA splice forms expressed in human lung epithelial cells (A549) in the presence and absence of selected AONs targeting exon 10 were then isolated and cloned into E. coli Top10 cells. Using primers spanning the region between exon 8 and exon 11, the size of each RAGE insert was determined. Treatment with selected AONs targeting exon 10, specifically AONs 3779 and 3778, resulted in increased expression of RAGE mRNA splice forms containing 250-kb fragments. (Fig. 1e), noting conservation of exon 9b.

All AONs targeting exon 10 also reduced the percentage of expression of mRNA clones expressing the 300-kb band denoting the full expression of signaling-competent elements contained in exons 8–10. Specifically, AON 3779 resulted in a 49% reduction in the level of RAGE mRNA splice forms containing 300-kb fragments. (Fig. 1f).

All AONs targeting exon 10 also induced de novo expression of RAGE mRNA splice forms lacking exon 8 completely (confirmed by Sanger sequencing and indicated by an empty band on the MultiNA analyzer, indicated by arrows on the gel; Fig. 1f) and a “no band” fragment ( Fig. 1e).

Transfection of human lung epithelial cells (A549) with AONs targeting exon 10, specifically AONs 3777 and 3778, also resulted in de novo expression of RAGE mRNA splice forms containing 276- and 430-kb fragments. (Fig. 1e), indicating atypical deletion of exon 9 and retention of exon 9, 9b and exon 10, respectively.

Transfection of human lung epithelial cells (A549) with 10 nM AON targeting exon 10, specifically AON 3779, also resulted in copy number changes in RAGE mRNA splice forms containing exon 10, in contrast to control cells (scrambled RNA treated), when measured by digital PCR (Fig. 1g).

Transfection of human lung epithelial cells (A549) with 10 nM AON targeting exon 10, specifically AON 3779, also increased the release of endogenous soluble RAGE into the medium (Fig. 1h), consistent with increased expression of the RAGE mRNA splice form containing exon 9b.

Transfection of CHO cells with a plasmid encoding the human RAGE genomic sequence (gDNA) resulted in alternative splicing of the gene product, including a small amount of endogenous RAGE secreted into the cell media (red arrow), when detected by Western blotting, consistent with the normal RAGE splicing pattern (Fig. .1i). The amount of endogenous soluble RAGE (esRAGE) secreted into the medium was increased when these CHO cells expressing human genomic RAGE were cotransfected with AON 3779 or AON 87 (Figure 1i). At the same time, full-length RAGE expressed and maintained in the cells (white arrow) was reduced when CHO cells expressing human genomic RAGE were cotransfected with AON 3779 or AON 87. The cells also contained intracellularly increased amounts of C-truncated RAGE.

The quantification of soluble RAGE as measured by ELISA was also increased in CHO cells expressing human genomic RAGE following transfection with selected AONs targeting exon 10, including AON 3779, AON 82, 83, 84 and 85 (Fig. 1j). Of these, AON 3779 had the greatest effect.

The quantification of soluble RAGE when measured by ELISA was also increased by several other AONs targeting exon 10 in a region similar to AON 3779, including AON 90 and 93 (Fig. 1k), by a similar amount to AON 3779 (10 nM). The amount of soluble RAGE released from CHO cells expressing human genomic RAGE increased in a dose-dependent manner following transfection with AON 3779 (Fig. 11).

Transfection of human lung epithelial cells (A549) with 10 nM exon 10-targeted AONs, specifically AON 3779, also modulated the induction of proinflammatory signaling following RAGE activation by RAGE ligand S100A8/A9 (0.6 μg/ml), leading to induction of ICAM expression -1 (Fig. 1m).

Transfection of human aortic endothelial cells (HAECs) with 10 nM AONs targeting exon 10, specifically AONs 3779, 3780, 81, 82, and 83, also prevented the induction of ICAM-1 expression following exposure to RAGE ligand S100A8/A9 (0.6 μg /ml; Fig. 1m).

Transfection of human lung epithelial cells (A549) with 10 nM AONs targeting exon 10, specifically AON 3779, also modulated the induction of proinflammatory signaling following RAGE transactivation following AT1 receptor activation by angiotensin II (1 μM), leading to induction of ICAM-1 expression ( Fig. 1o).

Transfection of human aortic endothelial cells (HAECs) with 10 nM AONs targeting exon 10, specifically AONs 3777, 3779, 3780, 3780, 3782, and 3783, also modulated the induction of proinflammatory signaling, including induction of ICAM-1 expression following ligand-independent transactivation RAGE following activation of the AT1 receptor by angiotensin II (1 μM; Fig. 1p). Other AONs targeting exon 10, particularly AONs 3778 and 84, had no effect.

Transfection of human microvascular endothelial cells (HMEC1) with 10 nM AONs targeting exon 10, specifically AONs 3779, 3780, 82, 83, and 87, resulted in changes in the expression of RAGE mRNA splice forms, including a decrease in the expression of RAGE mRNA splice forms containing exon 10 ( Fig. 1q). Transfection of human microvascular endothelial cells (HMEC1) with 10 nM AONs targeting exon 10, specifically AONs 3779 and 87, also induced an increase in the expression of RAGE mRNA splice forms containing exon 9b (Fig. 1r).

Morpholino oligonucleotides with a covalently linked 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) in vivo with AON 3779 (1 μM) resulted in changes in the expression of RAGE mRNA splice forms, including an increase in the level of RAGE mRNA splice forms containing exon 9b, as measured by RT-PCR (Fig. 1s). In addition, esRAGE expression in the cell environment was also increased compared to cells treated with the control AON morpholino (Figure 1t). Treatment with AON 79 without transfection reagents had no effect. Transfection of AON 3779 with transfection reagents is indicated as a positive control.

Treatment of CHO cells expressing genomic human RAGE with the vivo morpholino drug AON 3779 (1 μM) also increased the expression of soluble RAGE as measured by ELISA (Fig. 1u).

It is known that poly(G) stretches, especially GGGG motifs, can act as expression silencers, and they are often bound by hnRNP H and F (Sohail et al., BMC Genomics. 2014). It is proposed that RAGE splicing is regulated in part by G-rich cis elements and heterogeneous nuclear ribonucleoprotein H within intron 9/exon 9b, which are required for preferential use of the upstream 5' RAGE splice site. Poly(G) stretches favor exon skipping because hnRNP H binding requires a contiguous G stretch. AONs typically have poor affinity and reduced efficiency in G-rich regions. Exon 10 contains three GGGG motifs, one of which is recognized as a target for hnRNPF binding and is flanked by AON 3779, 3787 and AON 3782 (Fig. 1v). However, mutating this region (RAGE M3 mutant) had no effect on exon splicing or response to AON 3779 (Figure 1w), suggesting that we modulate the novel RAGE splicing silencer by targeting these AON regions to modulate RAGE splicing. which could not have been predicted from the prior art.

EXAMPLE 2. MODULATION OF ALTERNATIVE RAGE SPLICING IN HUMAN CELLS USING ANTISENSE OLIGONUCLEOTIDES TARGETED CIS-ACTING RNA ELEMENTS IN EXON 9

This example demonstrates that alternative splicing of RAGE pre-mRNA can be modulated using AONs targeting cis-acting RNA elements in exon 9 of RAGE pre-mRNA (Figure 2a).

Transfection of human lung epithelial cells (A549) with 10 nM concentrations of several AONs targeting exon 9, specifically AONs 3668, 3669, 3670, 4103, 4104, 4105, reduced the expression of RAGE mRNA splice forms containing exon 10 compared to control cells (treated with scrambled RNA) when measured by real-time RT-PCR (Fig. 2b). Other AONs targeting exon 9, particularly AON 4106, had no effect. However, overall RAGE mRNA expression was slightly increased after treatment with all exon 9-targeting AONs.

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. Using primers spanning exons 8–11, the size of each RAGE insert was determined (Fig. 2c). All AONs targeting exon 9 reduced the percentage of expression of mRNA clones expressing the 300-kb band. (Fig. 2d), indicating full expression of signaling-competent elements contained in exons 10 and 11. Specifically, AON 3670 resulted in decreased levels of RAGE mRNA splice forms containing 300 kb fragments. (Fig. 2d).

Transfection of human lung epithelial cells (A549) with AONs targeting exon 9, specifically AONs 3669, 3670, 4103, also induced de novo expression of RAGE mRNA splice forms in which exon 8 was skipped (indicated by empty bars on the multiNA analyzer; Fig. 2c).

Transfection of human lung epithelial cells (A549) with AONs targeting exon 9, specifically AONs 3669, 3670, 4103 and 4105, also resulted in de novo expression of RAGE mRNA splice forms containing 276 kb fragments, denoting exon deletion 9 from splice forms (Fig. 2d).

In CHO cells transfected with a plasmid containing the human RAGE genomic sequence (gRAGE), the amount of soluble RAGE secreted into the medium as measured by ELISA was moderately increased by certain AONs targeting the RAGE splice site in exon 9, particularly AONs 3669 and 3671 ( Fig. 2e) compared to control cells (treated with scrambled RNA).

Transfection of human lung epithelial cells (A549) with varying concentrations of AONs targeting exon 9, specifically AONs 3669, 3670, and 4103, also inhibited the induction of proinflammatory signaling following RAGE activation by RAGE ligand S100A8/A9 (0.6 μg/ml), including induction of TLR-4 gene expression (Fig. 2f). Other AONs targeting exon 9, particularly AONs 3668 and 4104, had no effect.

Transfection of human lung epithelial cells (A549) with 10 nM AONs targeting exon 9, specifically AONs 3669, 3670, and 4104, also modulated the induction of proinflammatory signaling through ligand-independent transactivation of RAGE following activation of the AT1 receptor by angiotensin II (1 μM; Fig. 2g). Other AONs targeting exon 9, particularly AONs 3668, 3671, and 4103, had no effect.

Transfection of human microvascular endothelial cells (HMECs) with 10 nM AONs targeting exon 9, specifically AONs 3669 and 3670, also reduced the expression of endogenous RAGE mRNA splice forms containing exon 10 (Fig. 2h).

EXAMPLE 3. MODULATION OF ALTERNATIVE RAGE SPLICING IN HUMAN CELLS USING ANTISENSE OLIGONUCLEOTIDES. TARGETED TO CIS-ACTING RNA ELEMENTS IN EXON 9B

This example demonstrates that RAGE alternative splicing can be modulated using AONs targeting RNA elements within exon 9b of RAGE pre-mRNA.

Exon 9b is removed in most RAGE isoforms by alternative splicing (Fig. 1e). This deletion potentially requires interaction of the spliceosome with motifs in exon 9b. We hypothesized that modulation of these binding elements using AON could also modulate alternative splicing of RAGE pre-mRNA.

Transfection of human lung epithelial cells (A549) with 10 nM concentrations of AON targeting exon 9b resulted in changes in mRNA expression of RAGE mRNA splice forms. Overall RAGE expression was slightly increased following treatment with both AONs targeting exon 9b (Fig. 3a). In addition, the expression of exon 10-containing RAGE mRNA splice forms was slightly reduced following AON 88 transfection compared with control scrambled RNA-treated cells when measured by real-time RT-PCR (Fig. 3a). Expression of RAGE mRNA splice forms containing exon 9b was also moderately increased by AON 88 and 89 (Fig. 3b). Transfection with AON 3779 targeting exon 10 is indicated as a positive control.

Transfection of human microvascular endothelial cells (HMECs) with 10 nM concentrations of AON targeting exon 9b also resulted in decreased expression of RAGE mRNA splice forms containing exon 10 (Figure 3c) and increased expression of RAGE mRNA splice forms containing exon 9b (Figure 3d ). Again, overall RAGE mRNA expression was slightly increased following treatment with both AONs targeting exon 9b (Fig. 3c). Transfection with AON 3779 targeting exon 10 is indicated as a positive control.

In CHO cells transfected with a plasmid containing the genomic sequence of human RAGE (gRAGE), the amount of soluble RAGE secreted into the medium, as measured by ELISA, was moderately increased by some AONs targeting exon 9b, particularly AON 89 (Figure 3e). compared to control RNA. Transfection with AON 3779 targeting exon 10 is indicated as a positive control.

The human RAGE intron 9 sequence and complementary targeting for AONs 88 and 89 are shown in FIG. 3f.

EXAMPLE 4. MODULATION OF ALTERNATIVE SPLICING OF MOUSE RAGE USING ANTISENSE OLIGONUCLEOTIDES. TARGETTING CIS-ACTING RNA ELEMENTS IN RAGE PRE-mRNA

This example demonstrates that alternative splicing of RAGE pre-mRNA can also be modulated using specific AONs targeting cis-acting RNA elements in exons 9, 9B and 10 of mouse RAGE pre-mRNA, and that this modulation can be demonstrated in vitro , ex vivo and in vivo.

To analyze the effect of AON on alternative splicing of mouse RAGE, CHO cells were transfected with a plasmid containing the genomic sequence of mouse RAGE. This resulted in the secretion of small amounts of esRAGE. Transfection of AONs targeting murine RAGE, specifically AON m3779 (AON designed to target exon 10 of the murine RAGE sequence at a comparable sequence to that targeted by AON 3779 in the human RAGE sequence detailed in Example 1) and AON m101 , increased the secretion of esRAGE into the cell environment (Fig. 4a).

Primary mouse aortic endothelial cells (PMAECs) were transfected with AON (50 mM) targeting RAGE exon 9 (Table 3d). This treatment resulted in modest changes in the expression of RAGE mRNA splice forms compared to their corresponding negative control. In particular, AON 3674 and 3675 could reduce the expression of RAGE mRNA splice forms containing exon 10 when measured by real-time RT-PCR (Fig. 4b).

However, transfection of PMAEC with these AONs, used at a concentration of 10 nM, did not show a significant effect on the expression of RAGE mRNA splice forms (Fig. 4c). Levels of soluble RAGE secreted into the medium increased modestly 24 hours after transfection of cultured PMAEC AON 3674 and 3675 at a concentration of 50 nM (Fig. 4d).

Transfection of CHO cells expressing mouse genomic RAGE with m3779 (10 nM) reduced the expression of exon 10-containing RAGE mRNA splice forms as assessed by real-time RT-PCR; Fig. 4f) and increased secretion of esRAGE compared to control cells (treated with scrambled RNA) (Fig. 4f). AON 3779, targeting human RAGE sequences, also increased esRAGE secretion, but not as strongly as AON m3779.

When AONs engineered to target exon 10 of the mouse RAGE sequence were transfected into CHO cells expressing the human RAGE genomic sequence from a plasmid, AON m3779 (engineered to target exon 10 of the mouse RAGE) also induced an increase in the level of soluble human RAGE in the cell media (Fig. .4g). However, AON 3779 (specifically targeting exon 10 of human RAGE) was more effective in modulating human genomic RAGE than AON m3779. These data demonstrate that AONs targeting a specific sequence in exon 10 can be effective in modulating RAGE splicing despite regions of lack of identity.

To analyze the effect of AON targeting RAGE in mouse tissue, precision cut lung pieces (PCLS) were obtained from mice and then cultured ex vivo. Briefly, mice were humanely sacrificed, their lung explants were infused with agarose, cylindrical rods were collected, and then knife-cut into tissue sections, yielding lung sections of uniform diameter and thickness. PCLS were then immersed in culture medium in multiwell plates under tissue culture conditions. After 24 h, PCLS were transfected with in vivo morpholino AON 3779 or nontargeting control for 48 h. Treatment with AON 3779 resulted in a significant increase in the expression of exon 9b-containing RAGE mRNA and decreased expression of exon 10-containing RAGE mRNA splice forms (Figure 4h), with the greatest effect on exon 9b observed at 72 hours post-dosing (Figure 4i).

To confirm 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 RAGE mRNA splice forms containing exon 9b was increased in mouse lungs, and the level of splice forms containing exon 10 was decreased after subcutaneous treatment with AON m3779 for one week (Fig. 4j).

To confirm their potential as an inhaled therapeutic agent, live mice were anesthetized before delivery of 30 μL of 11 μM Vivo-Morpholino-AON m3779 or an untargeted Vivo-Morpolino control (as a negative control) in RNase-free water to the lung via the trachea using cannula or water alone. The anesthetic was reversed and mice were sacrificed after 48 h. Intratracheal treatment with m3779 resulted in a significant reduction in pulmonary expression of RAGE exon 10-containing splice forms of RAGE mRNA compared with water alone, which had no significant effect. Expression of RAGE mRNA isoforms containing exon 9b was also increased (Fig. 4k). A similar decrease in the expression of RAGE mRNA splice forms containing exon 10 was observed after intratracheal delivery of 30 μl of 11 μM 2-O'-Me-phosphorothioate AON m3779 compared to vehicle alone (sterile water) (Fig. 41). Additionally, an increase in soluble RAGE levels in the circulation was also observed following treatment of mice with AON m3779 compared to sterile water or the ineffective human RAGE-targeting AON 4105 (Figure 4m).

EXAMPLE 5. MODULATION OF ALTERNATIVE RAGE SPLICING USING A COMBINATION OF ANTISENSE OLIGONUCLEOTIDES TARGETED TO RAGE PRE-MRNA

This example demonstrates that RAGE alternative splicing can be modulated using combinations of AONs targeting different cis-acting RNA elements in the RAGE pre-mRNA.

It is believed that exon detection is regulated by the assembly of a multicomponent “cross-exon” recognition complex that facilitates target retention or exclusion. By binding AON 3779 to exon 10 of RAGE pre-mRNA to increase esRAGE formation, we propose that other regulatory targets in exon 10 may become more critical, including, but not limited to, predicted splice sites. Additionally, in the presence of AON 3779, by selectively targeting these cis-acting RNA elements in exon 10 in the RAGE pre-mRNA using other AONs, the present inventors would prevent any escape from exon exclusion and further modulate RAGE splicing towards the preferred direction esRAGE education.

The secondary structure of RAGE exon 10 is predicted to contain two hairpin loops connected by a central hinge region. It is predicted that targeting exonic enhancer sequences in the 3' hairpin using AON 3779 would increase the importance of targets in the alternative 5' hairpin, including the upstream 5' splice site in exon 10.

To confirm this hypothesis, CHO cells containing a plasmid encoding human genomic RAGE were transfected with AON 3779 (10 nM) in combination with other AONs targeting the predicted 5′ (acceptor) splice site in exon 10 (also 10 nM). This combination treatment resulted in changes in the expression of RAGE mRNA splice forms such that some combinations, notably AON 3779 plus 3777, 3779 plus 3778, significantly increased the expression of RAGE mRNA isoforms containing exon 9b compared to AON 3779 alone or control cells. treated with scrambled RNA, as measured by real-time RT-PCR (Fig. 5a). This combination also increased esRAGE protein production measured in the medium by ELISA (Fig. 5b). The combination of AON 3997 and AON 3778 was also more effective at the low dose (5 nM plus 5 nM). Interestingly, AON 3777 and AON 3778 had little or no effect on their own at a concentration of 10 nM.

In another experiment, CHO cells containing a plasmid encoding human genomic RAGE were transfected with AON 3779 in combination with other AONs targeting either the 3' or 5' splice sites in exon 10. Some combinations also resulted in changes in the expression of RAGE mRNA splice forms such such that certain combinations, particularly AON 3779 plus 3778, 3779 plus 3781, and 3779 plus 3778 plus 3781, significantly increased soluble RAGE expression compared to AON 3779 alone or control scrambled RNA-treated cells when measured by ELISA (Figure 5c). This increase was similar when using AON 93 or AON 87 instead of 3779, although overall combinations with AON 3779 (shown in boxes) were the strongest and had the most consistent effect.

Transfection of human lung epithelial cells (A549) with other AON combinations also targeting adjacent regions in exon 10, specifically AON 3779 plus 3782, 3779 plus 3784, 3779 plus 3785, had no significant effect on the expression of RAGE isoforms containing exon 9b other than one AON 3779 (Fig. 5d). Notably, these AONs also target the 5' hairpin (similar to AON 3779) and/or the central hinge region, unlike AON 3777 or AON 3778, which were effective in combinations that target exonic enhancers at the other (3') stiletto heel

CHO cells containing a plasmid encoding the genomic sequence of mouse RAGE were transfected with AON m3779 in combination with AON targeting either the 3' or 5' splice sites in exon 10. This combination treatment resulted in changes in the expression of RAGE mRNA splice forms such that that some combinations, particularly AON m3779 plus m102, significantly increased the secretion of soluble RAGE into the cell media when measured by ELISA compared to AON m3779 alone (Figure 5e).

--->

LIST OF SEQUENCES

<110> Monash University

Murdoch University

<120> MODULATORS AND MODULATION OF RNA RECEPTOR OF DEEP END PRODUCTS

GLYCATION

<130>N423236EP

<140> 20806464.2

<141> 2020-05-07

<150>AU2019901641

<151> 2019-05-14

<150>AU2019902095

<151> 2019-06-17

<150>AU2019902772

<151> 2019-08-02

<150>AU2019903900

<151> 2019-10-16

<160> 77

<170> Patent In version 3.5

<210> 1

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3668

<400> 1

ccuccucgcc ugguucugga agaca 25

<210> 2

<211> 20

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3669

<400> 2

uuggccccuc cucgccuggu 20

<210> 3

<211> 20

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3670

<400> 3

cugcaguugg ccccuccucg 20

<210> 4

<211> 20

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3671

<400> 4

ucaaaccccu caccugcagu 20

<210> 5

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 4103

<400> 5

ccugcaguug gccccuccuc gccug 25

<210> 6

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 4104

<400> 6

ccccucaccu gcaguuggcc ccucc 25

<210> 7

<211> 17

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 4105

<400> 7

gcaguuggcc ccuccuc 17

<210> 8

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 4106

<400> 8

caccugcagu uggccccucc ucgcc 25

<210> 9

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3777

<400> 9

auccucccac agagccugua cggag 25

<210> 10

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3778

<400> 10

cccugauccu cccacagagc cugua 25

<210> 11

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3779

<400> 11

ggcguugccg ccuuugccac aagau 25

<210> 12

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3780

<400> 12

cucaccucuc cucuccgg cguug 25

<210> 13

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3781

<400> 13

cucaccucac cucuccuc cucgg 25

<210> 14

<211> 20

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 82

<400> 14

agcagggcgg cuguccccag 20

<210> 15

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 83

<400> 15

gccacaagau gaccccaaug agcag 25

<210> 16

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 84

<400> 16

ccuuugccac aagaugaccc caaug 25

<210> 17

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223>AON 85

<400> 17

ugccgccuuu gccacaagau gaccc 25

<210> 18

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 87

<400> 18

ucccucggcgu ugccgccuuu gccac 25

<210> 19

<211> 13

<212>RNA

<213> Artificial Sequence

<220>

<223>AON 90

<400> 19

uuugccacaa gau 13

<210> 20

<211> 20

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 93

<400> 20

ccgccuuugc cacaagauga 20

<210> 21

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223>AON 88

<400> 21

uuucuuguug accauccccc caguc 25

<210> 22

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 89

<400> 22

ccauccccccc agucacaugu guugg 25

<210> 23

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3672

<400> 23

ccuccucgcc ugguucugga agaca 25

<210> 24

<211> 20

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3673

<400> 24

cuggccccuc aucgccgguu 20

<210> 25

<211> 20

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3674

<400> 25

cuucagcugg ccccucaucg 20

<210> 26

<211> 20

<212>RNA

<213> Artificial Sequence

<220>

<223> AON 3675

<400> 26

uccagucccu caccuucagc 20

<210> 27

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223> AON m3779

<400> 27

uggguugucg uuuucgccac aggau 25

<210> 28

<211> 23

<212>RNA

<213> Artificial Sequence

<220>

<223>AON m97

<400> 28

acuacuccca ggccucccag gau 23

<210> 29

<211> 22

<212>RNA

<213> Artificial Sequence

<220>

<223>AON m98

<400> 29

gcagggcuac uacucccagg ca 22

<210> 30

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223>AON m101

<400> 30

ucucacucac cucuccucac gccug 25

<210> 31

<211> 25

<212>RNA

<213> Artificial Sequence

<220>

<223>AON m102

<400> 31

ucucacucac cucuccucac gccug 25

<210> 32

<211> 320

<212> DNA

<213> Homo sapiens

<400> 32

gataaagtca gggaagcaga agatagcccc caacacatgt gactgggggg atggtcaaca 60

agaaaggaat ggtgagtggt ggtggctgtg ctctcaattt tccctgtctc cgtacaggct 120

ctgtgggagg atcagggctg ggaactctag ccctggccct ggggatcctg ggaggcctgg 180

ggacagccgc cctgctcatt ggggtcatct tgtggcaaag gcggcaacgc cgaggagagg 240

agaggtgagt ggagaaagcc agacccctca gacctagggc ttccaggcag caagcgaaga 300

ggggtcgggg ggtggaacga 320

<210> 33

<211> 320

<212> DNA

<213> Homo sapiens

<400> 33

tcgttccacc ccccgacccc tcttcgcttg ctgcctggaa gccctaggtc tgaggggtct 60

ggctttctcc actcacctct cctctcctcg gcgttgccgc ctttgccaca agatgacccc 120

aatgagcagg gcggctgtcc ccaggcctcc caggatcccc agggccaggg ctagagttcc 180

cagccctgat cctcccacag agcctgtacg gagacaggga aaattgagag cacagccacc 240

accactcacc attcctttct tgttgaccat ccccccagtc acatgtgttg ggggctatct 300

tctgcttccc tgactttatc 320

<210> 34

<211> 53

<212>PRT

<213> Homo sapiens

<400> 34

Ile Lys Ser Gly Lys Gln Lys Ile Ala Pro Asn Thr Cys Asp Trp Gly

1 5 10 15

Asp Gly Gln Gln Glu Arg Asn Gly Glu Trp Trp Trp Leu Cys Ser Gln

20 25 30

Phe Ser Leu Ser Pro Tyr Arg Leu Cys Gly Arg Ile Arg Ala Gly Asn

35 40 45

Ser Ser Pro Gly Pro

50

<210> 35

<211> 6

<212>PRT

<213> Homo sapiens

<400> 35

Ser Gln Gly Ser Arg Arg

15

<210> 36

<211> 45

<212>PRT

<213> Homo sapiens

<400> 36

Pro Pro Thr His Val Thr Gly Gly Met Val Asn Lys Lys Gly Met Val

1 5 10 15

Ser Gly Gly Gly Cys Ala Leu Asn Phe Pro Cys Leu Arg Thr Gly Ser

20 25 30

Val Gly Gly Ser Gly Leu Gly Thr Leu Ala Leu Ala Leu

35 40 45

<210> 37

<211> 13

<212>PRT

<213> Homo sapiens

<400> 37

Asp Lys Val Arg Glu Ala Glu Asp Ser Pro Gln His Met

1 5 10

<210> 38

<211> 10

<212>PRT

<213> Homo sapiens

<400> 38

Leu Gly Gly Trp Ser Thr Arg Lys Glu Trp

1 5 10

<210> 39

<211> 24

<212>PRT

<213> Homo sapiens

<400> 39

Val Val Val Ala Val Leu Ser Ile Phe Pro Val Ser Val Gln Ala Leu

1 5 10 15

Trp Glu Asp Gln Gly Trp Glu Leu

20

<210> 40

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic DNA

<400> 40

gctatcttct gcttccctga c 21

<210> 41

<211> 160

<212> DNA

<213> Homo sapiens

<400> 41

caaaagtgaa actccatctc aaaaaaaaaa agaaagggaa agactccact ggggctccca 60

ctaaataacc ctctctcaac ccgaagtctt cctttctgac tggatccaac tttgtcttcc 120

agaaccaggc gaggaggggc caactgcagg tgaggggttt 160

<210> 42

<211> 160

<212> DNA

<213> Homo sapiens

<400> 42

aaacccctca cctgcagttg gcccctcctc gcctggttct ggaagacaaa gttggatcca 60

gtcagaaagg aagacttcgg gttgagagg ggttatttag tgggagcccc agtggagtct 120

ttccctttct tttttttttt gagatggagt ttcacttttg 160

<210> 43

<211> 143

<212> DNA

<213> Homo sapiens

<400> 43

agaagatagc ccccaacaca tgtgactggg gggatggtca acaagaaagg aatggtgagt 60

ggtggtggct gtgctctcaa ttttccctgt ctccgtacag gctctgtggg aggatcaggg 120

ctgggaactc tagccctggc cct 143

<210> 44

<211> 143

<212> DNA

<213> Homo sapiens

<400> 44

agggccaggg ctagagttcc cagccctgat cctcccacag agcctgtacg gagacaggga 60

aaattgagag cacagccacc accactcacc attcctttct tgttgaccat ccccccagtc 120

acatgtgttg ggggctatct tct 143

<210> 45

<211> 50

<212> DNA

<213> Homo sapiens

<400> 45

aggactcttg tcccaaaggc atgaattcct agcattccct gtgacaagac 50

<210> 46

<211> 154

<212> DNA

<213> Homo sapiens

<400> 46

gactgaaaga tgggggctgg agagagggtg caggccccac ctaggcgga ggccacagca 60

gggagagggg cagacagagc caggaccctg gaaggaagca ggatggcagc cggaacagca 120

gttggagcct gggtgctggt cctcagtctg tggg 154

<210> 47

<211> 25

<212> DNA

<213> Homo sapiens

<400> 47

gtgagccact ccctcaaccc cactg 25

<210> 48

<211> 25

<212> DNA

<213> Homo sapiens

<400> 48

cctctaccat ggtgctatct cccag 25

<210> 49

<211> 107

<212> DNA

<213> Homo sapiens

<400> 49

gggcagtagt aggtgctcaa aacatcacag cccggattgg cgagccactg gtgctgaagt 60

gtaagggggc ccccaagaaa ccaccccagc ggctggaatg gaaactg 107

<210> 50

<211> 25

<212> DNA

<213> Homo sapiens

<400> 50

gtaagcgggg ctcctgttgc agcct 25

<210> 51

<211> 25

<212> DNA

<213> Homo sapiens

<400> 51

ttaggccctg cttctctgct tctag 25

<210> 52

<211> 196

<212> DNA

<213> Homo sapiens

<400> 52

aacacaggcc ggacagaagc ttggaaggtc ctgtctcccc agggaggagg cccctgggac 60

agtgtggctc gtgtccttcc caacggctcc ctcttccttc cggctgtcgg gatccaggat 120

gaggggattt tccggtgcca ggcaatgaac aggaatggaa aggagaccaa gtccaactac 180

cgagtccgtg tctacc 196

<210> 53

<211> 25

<212> DNA

<213> Homo sapiens

<400> 53

gtaagaattc cagggtcttc tccaa 25

<210> 54

<211> 25

<212> DNA

<213> Homo sapiens

<400> 54

tctgactgga tttttcctcc ttcag 25

<210> 55

<211> 65

<212> DNA

<213> Homo sapiens

<400> 55

agattcctgg gaagccagaa attgtagatt ctgcctctga actcacggct ggtgttccca 60

ataag 65

<210> 56

<211> 25

<212> DNA

<213> Homo sapiens

<400> 56

gtagtggaag aaagcaggag aagta 25

<210> 57

<211> 25

<212> DNA

<213> Homo sapiens

<400> 57

tctgaggtca ccactctttc cccag 25

<210> 58

<211> 88

<212> DNA

<213> Homo sapiens

<400> 58

gtggggacat gtgtgtcaga gggaagctac cctgcaggga ctcttagctg gcacttggat 60

gggaagcccc tggtgcctaa tgagaagg 88

<210> 59

<211> 25

<212> DNA

<213> Homo sapiens

<400> 59

gtgagtccta aggtgccccc caagc 25

<210> 60

<211> 25

<212> DNA

<213> Homo sapiens

<400> 60

aatttgtctt atcctcccat catag 25

<210> 61

<211> 183

<212> DNA

<213> Homo sapiens

<400> 61

gagtatctgt gaaggaacag accaggagac accctgagac agggctcttc acactgcagt 60

cggagctaat ggtgacccca gcccggggag gagatccccg tcccaccttc tcctgtagct 120

tcagcccagg ccttccccga caccggggcct tgcgcacagc ccccatccag ccccgtgtct 180

ggg 183

<210> 62

<211> 25

<212> DNA

<213> Homo sapiens

<400> 62

gtgagcatag gtggggaggg cccca 25

<210> 63

<211> 25

<212> DNA

<213> Homo sapiens

<400> 63

acctcaaaac ccttccaact cccag 25

<210> 64

<211> 131

<212> DNA

<213> Homo sapiens

<400> 64

agcctgtgcc tctggaggag gtccaattgg tggtggagcc agaaggtgga gcagtagctc 60

ctggtggaac cgtaaccctg acctgtgaag tccctgccca gccctctcct caaatccact 120

ggatgaaggat 131

<210> 65

<211> 25

<212> DNA

<213> Homo sapiens

<400> 65

gtgagtgacc tggagagagg ggctg 25

<210> 66

<211> 25

<212> DNA

<213> Homo sapiens

<400> 66

gtctcctctc cccttccccc accag 25

<210> 67

<211> 142

<212> DNA

<213> Homo sapiens

<400> 67

ggtgtgccct tgccccttcc ccccagccct gtgctgatcc tccctgagat agggcctcag 60

gaccagggaa cctacagctg tgtggccacc cattccagcc acgggcccca ggaaagccgt 120

gctgtcagca tcagcatcat cg 142

<210> 68

<211> 25

<212> DNA

<213> Homo sapiens

<400> 68

gtgagacctc tccccaagcc ctaca 25

<210> 69

<211> 25

<212> DNA

<213> Homo sapiens

<400> 69

gactggatcc aactttgtct tccag 25

<210> 70

<211> 27

<212> DNA

<213> Homo sapiens

<400> 70

aaccaggcga ggaggggcca actgcag 27

<210> 71

<211> 25

<212> DNA

<213> Homo sapiens

<400> 71

gtgaggggtt tgataaagtc aggga 25

<210> 72

<211> 25

<212> DNA

<213> Homo sapiens

<400> 72

ctcaattttc cctgtctccg tacag 25

<210> 73

<211> 127

<212> DNA

<213> Homo sapiens

<400> 73

gctctgtggg aggatcaggg ctgggaactc tagccctggc cctggggatc ctgggaggcc 60

tggggacagc cgccctgctc attggggtca tcttgtggca aaggcggcaa cgccgaggag 120

aggagag 127

<210> 74

<211> 25

<212> DNA

<213> Homo sapiens

<400> 74

gtgagtggag aaagccagac ccctc 25

<210> 75

<211> 25

<212> DNA

<213> Homo sapiens

<400> 75

cattcccccc aatctttctc ctcag 25

<210> 76

<211> 272

<212> DNA

<213> Homo sapiens

<400> 76

gaaggcccca gaaaaccagg aggaagagga ggagcgtgca gaactgaatc agtcggagga 60

acctgaggca ggcgagagta gtactggagg gccttgaggg gccccacagac agatcccatc 120

catcagctcc cttttctttt tcccttgaac tgttctggcc tcagaccaac tctctcctgt 180

ataatctctc tcctgtataa ccccaccttg ccaagctttc ttctacaacc agagcccccc 240

acaatgatga ttaaacacct gacacatctt gc 272

<210> 77

<211> 50

<212> DNA

<213> Homo sapiens

<400> 77

tcttgtgtgt ctgtgtgtgt gtatgagaca caacctcacc cctataccct 50

<---

Claims (84)

1. Antisense oligonucleotide to manipulate receptor for advanced glycosylation end products (RAGE) pre-mRNA splicing resulting in exon 10 skipping, containing: a nucleotide sequence that is at least 80% complementary to the target region of exon 10 of the RAGE pre-mRNA along the entire length of the antisense oligonucleotide, wherein the 5'most nucleotide of the target region is nucleotide at position 88 or 114 or is between nucleotide positions 88 and 114 of exon 10 of the RAGE pre-mRNA, where the numbering of nucleotide positions of exon 10 of the RAGE pre-mRNA corresponds to SEQ ID NO: 73, wherein said antisense oligonucleotide is from 8 to 40 nucleotides in length, and wherein said antisense oligonucleotide contains at least one modified nucleotide. 2. Antisense oligonucleotide according to claim 1, where the extreme 5'-nucleotide of the target region is: a) at position 88 or 113, or between nucleotide positions 88 to 113 of exon 10 of RAGE pre-mRNA; b) at position 90 or 113, or between nucleotide positions 90 to 113 of exon 10 of RAGE pre-mRNA; c) at position 88 or 108, or between nucleotide positions 88 to 108 of exon 10 of RAGE pre-mRNA; d) at position 90 or 108, or between nucleotide positions 90 to 108 of exon 10 of RAGE pre-mRNA; or e) at position 88 or 95, or between nucleotide positions 88 to 95 of exon 10 of the RAGE pre-mRNA. 3. Antisense oligonucleotide according to claim 1 or 2, where the target region is: a) between positions 88 and 113 of exon 10 of RAGE pre-mRNA; b) between nucleotide positions from 90 to 113 of exon 10 of the RAGE pre-mRNA; c) between nucleotide positions from 88 to 108 of exon 10 of the RAGE pre-mRNA; d) between nucleotide positions from 90 to 108 of exon 10 of the RAGE pre-mRNA; or e) between nucleotide positions 88 to 95 of exon 10 of the RAGE pre-mRNA. 4. Antisense oligonucleotide according to any one of paragraphs. 1-3, containing a nucleotide sequence that is at least 90% complementary to the target region of exon 10 of the RAGE pre-mRNA. 5. Antisense oligonucleotide according to any one of paragraphs. 1-4, containing a nucleotide sequence that is at least 95% complementary to the target region of exon 10 of the RAGE pre-mRNA. 6. Antisense oligonucleotide according to any one of paragraphs. 1-5, containing a nucleotide sequence that is 100% complementary to the target region of exon 10 of the RAGE pre-mRNA. 7. Antisense oligonucleotide according to any one of paragraphs. 1-6, the length of which ranges from 15 to 25 nucleotides. 8. Antisense oligonucleotide according to any one of paragraphs. 1-7, which is 18 nucleotides long. 9. Antisense oligonucleotide according to any one of paragraphs. 1-7, the nucleotide sequence of which is SEQ ID NO: 11 or a nucleotide sequence that is at least 85%, 90% or 95% identical thereto. 10. Antisense oligonucleotide according to any one of paragraphs. 1-7, the nucleotide sequence of which is SEQ ID NO: 19 or a nucleotide sequence that is at least 85%, 90% or 95% identical thereto. 11. Antisense oligonucleotide according to any one of paragraphs. 1-7, the nucleotide sequence of which is SEQ ID NO: 20 or a nucleotide sequence that is at least 85%, 90% or 95% identical thereto. 12. Antisense oligonucleotide according to any one of paragraphs. 1-11, where the modified nucleotide contains one or more of the following: a morpholino group, a modified linkage and a modified sugar moiety. 13. The antisense oligonucleotide of claim 12, wherein the modified sugar moiety includes methylation at the 2' position of the hydroxyribose. 14. The antisense oligonucleotide according to claim 12, wherein the modified linkage is a phosphorothioate linkage. 15. Antisense oligonucleotide according to any one of paragraphs. 1-14, where if uracil is present in the antisense oligonucleotide, then uracil (U) of the antisense oligonucleotide is replaced by thymine (T). 16. A method for manipulating splicing in pre-mRNA of the receptor for advanced glycosylation end products (RAGE), leading to skipping of exon 10, including: bringing the cell into contact with one or more antisense oligonucleotides containing a nucleotide sequence that is at least 80% complementary to the target region of exon 10 of the RAGE pre-mRNA along the entire length of the antisense oligonucleotide, wherein the 5'most nucleotide of the target region is nucleotide at position 88 or 114 or is between nucleotide positions 88 and 114 of exon 10 of the RAGE pre-mRNA, where the numbering of nucleotide positions of exon 10 of the RAGE pre-mRNA corresponds to SEQ ID NO: 73, wherein said antisense oligonucleotide is from 8 to 40 nucleotides in length, and wherein said antisense oligonucleotide contains at least one modified nucleotide; And manipulation of splicing in the RAGE pre-mRNA resulting in exon 10 skipping occurs through the binding of one or more antisense oligonucleotides to the target region of exon 10 in the RAGE pre-mRNA. 17. A method for treating a disease associated with RAGE expression, comprising: administering to the patient an effective amount of an antisense oligonucleotide containing a nucleotide sequence that is at least 80% complementary to the target region of exon 10 of the RAGE pre-mRNA along the entire length of the antisense oligonucleotide, wherein the 5'most nucleotide of the target region is nucleotide at position 88 or 114 or is between nucleotide positions 88 and 114 of exon 10 of the RAGE pre-mRNA, where the numbering of nucleotide positions of exon 10 of the RAGE pre-mRNA corresponds to SEQ ID NO: 73, wherein said antisense oligonucleotide is from 8 to 40 nucleotides in length, and wherein said antisense oligonucleotide contains at least one modified nucleotide, which constitutes a treatment for the patient. 18. The method according to claim 16 or 17, where the extreme 5'-nucleotide of the target region is: a) at position 88 or 113, or between nucleotide positions 88 to 113 of exon 10 of RAGE pre-mRNA; b) at position 90 or 113, or between nucleotide positions 90 to 113 of exon 10 of RAGE pre-mRNA; c) at position 88 or 108, or between nucleotide positions 88 to 108 of exon 10 of RAGE pre-mRNA; d) at position 90 or 108, or between nucleotide positions 90 to 108 of exon 10 of RAGE pre-mRNA; or e) at position 88 or 95, or between nucleotide positions 88 to 95 of exon 10 of the RAGE pre-mRNA. 19. Method according to any one of paragraphs. 16-18, wherein the antisense nucleotide has a nucleotide sequence that is at least 90%, at least 95%, or 100% complementary to the target region of exon 10 of the RAGE pre-mRNA. 20. Method according to any one of paragraphs. 16-19, where the length of the antisense oligonucleotide is from 15 to 25 nucleotides or 18 nucleotides. 21. Method according to any one of claims 16-20, where the antisense oligonucleotide has the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 85%, 90% or 95% identical thereto; or the antisense oligonucleotide has the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence that is at least 85%, 90% or 95% identical thereto; or the antisense oligonucleotide has a nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence that is at least 85%, 90% or 95% identical thereto. 22. The method according to any one of claims 16 to 21, where the modified nucleotide contains one or more of the following: a morpholino group, a modified linkage and a modified sugar moiety. 23. The method according to claim 22, where the modified sugar moiety contains methylation at the 2'-position of hydroxyribose. 24. The method of claim 22, wherein the modified bond is a phosphorothioate bond. 25. Method according to any one of paragraphs. 16-24, where if uracil is present in the antisense oligonucleotide, then uracil (U) of the antisense oligonucleotide is replaced by thymine (T). 26. Method according to any one of paragraphs. 16-25, wherein the disease associated with RAGE expression is selected from the group consisting of neurodegenerative diseases, cancer, pulmonary disorders, inflammatory diseases, cardiovascular diseases; digestive disorders; respiratory diseases, musculoskeletal diseases, connective tissue diseases, kidney diseases, genital diseases, skin diseases, eye diseases and endocrine diseases. 27. The use of an effective amount of an antisense oligonucleotide in the manufacture of a medicament for the treatment of a disease associated with the expression of RAGE, where the antisense oligonucleotide contains: a nucleotide sequence that is at least 80% complementary to the target region of exon 10 of the RAGE pre-mRNA along the entire length of the antisense oligonucleotide, wherein the 5'most nucleotide of the target region is nucleotide at position 88 or 114 or is between nucleotide positions 88 and 114 of exon 10 of the RAGE pre-mRNA, where the numbering of nucleotide positions of exon 10 of the RAGE pre-mRNA corresponds to SEQ ID NO: 73, wherein said antisense oligonucleotide is from 8 to 40 nucleotides in length, and wherein said antisense oligonucleotide contains at least one modified nucleotide. 28. Use according to claim 27, where the extreme 5'-nucleotide of the target region is: a) at position 88 or 113, or between nucleotide positions 88 to 113 of exon 10 of RAGE pre-mRNA; b) at position 90 or 113, or between nucleotide positions 90 to 113 of exon 10 of RAGE pre-mRNA; c) at position 88 or 108, or between nucleotide positions 88 to 108 of exon 10 of RAGE pre-mRNA; d) at position 90 or 108, or between nucleotide positions 90 to 108 of exon 10 of RAGE pre-mRNA; or e) at position 88 or 95, or between nucleotide positions 88 to 95 of exon 10 of the RAGE pre-mRNA. 29. Use according to claim 27 or 28, wherein the nucleotide sequence of said antisense oligonucleotide is at least 90%, at least 95%, or 100% complementary to the target region of exon 10 of the RAGE pre-mRNA. 30. Application according to any one of paragraphs. 27-29, where the length of the antisense oligonucleotide is from 15 to 25 nucleotides or 18 nucleotides. 31. Application according to any one of paragraphs 26-30, where the antisense oligonucleotide has the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 85%, 90% or 95% identical thereto; or the antisense oligonucleotide has the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence that is at least 85%, 90% or 95% identical thereto; or the antisense oligonucleotide has a nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence that is at least 85%, 90% or 95% identical thereto. 32. Application according to any one of paragraphs. 27-31, wherein said modified nucleotide contains one or more of the following: a morpholino group, a modified linkage, and a modified sugar moiety. 33. Use according to claim 32, wherein the modified sugar moiety contains methylation at the 2' position of the hydroxyribose. 34. Use according to claim 32, wherein the modified linkage is a phosphorothioate linkage. 35. Application according to any one of paragraphs. 27-34, where if uracil is present in the antisense oligonucleotide, then uracil (U) of the antisense oligonucleotide is replaced by thymine (T). 36. Application according to any one of paragraphs. 27-35, wherein the disease associated with RAGE expression is selected from the group consisting of neurodegenerative diseases, cancer, pulmonary disorders, inflammatory diseases, cardiovascular diseases; digestive disorders; respiratory diseases, musculoskeletal diseases, connective tissue diseases, kidney diseases, genital diseases, skin diseases, eye diseases and endocrine diseases.