Treatment of psoriasis

Abstract

An isolated or purified antisense oligomer (ASO) for modifying pre-mRNA splicing or mRNA translation of one or more of the following proteins encoded by a corresponding gene transcript or portion thereof: S100A8; S100A9; and / or IL1RL2.

Description

Technical Field

[0001] This invention relates to antisense oligomers (ASOs) for promoting the protein synthesis of S100 alarm proteins S100A8 and S100A9 and the IL1RL2 subunit (IL1RL2) of the IL36 receptor. The invention also provides methods for treating, preventing, or mitigating the effects of these proteins on psoriasis by administering ASOs targeting the S100 alarm proteins and the IL1RL2 subunit of the IL36 receptor, and therapeutic compositions comprising said ASOs. Background Technology

[0002] Psoriasis is a chronic autoimmune skin disease with a complex etiology involving multiple genetic and environmental factors. It is characterized by inflammation and abnormal epidermal differentiation, accompanied by inflammatory cell infiltration in the epidermis and dermis, as well as proliferation and dilation of superficial dermal blood vessels, resulting in erythematous plaques and scaling. According to published studies, the global prevalence of psoriasis ranges from 0.09% to 11.4%, but in most developed countries, the prevalence is between 1.5% and 5%.

[0003] Nearly 90% of psoriasis patients have the most common type of the disease, called psoriasis vulgaris or plaque psoriasis, characterized by well-defined, erythematous plaques covered with rough scales. Other major subtypes of psoriasis include pustular, guttate, and erythrodermic psoriasis.

[0004] It is a chronic (long-term) condition, and there is currently no way to completely cure it. However, existing treatments can control psoriasis, thereby reducing or eliminating symptoms for a period of time.

[0005] Current management of psoriasis and psoriatic arthritis (PsA) lacks precision and relies heavily on clinical assessment. However, a more precise approach, which comprehensively considers patient differences in genes, proteins, environment, and lifestyle, will provide deeper insights to help design precision therapies for better treatment outcomes. Antisense oligonucleotides (ASOs) represent a promising new class of therapies.

[0006] There is a need to provide a more precise method (taking into account the differences among patients in terms of genes, proteins, environment, and lifestyle), or at least to provide alternatives for the control and treatment of psoriasis as a complement to existing treatments. This invention aims to provide an improved or alternative treatment for psoriasis.

[0007] The preceding discussion of the background art is intended only to facilitate understanding of the invention. This discussion is not an admission or endorsement that any material mentioned was or was part of common general knowledge at the priority date of this application. Summary of the Invention

[0008] In general, according to one aspect of the invention, an isolated or purified antisense oligomer (ASO) is provided for modifying precursor mRNA splicing or mRNA translation of one or more proteins encoded by the corresponding gene transcript or a portion thereof: i.S100A8; ii.S100A9; and / or iii.IL1RL2.

[0009] Preferably, an isolated or purified antisense oligomer is provided, said antisense oligomer being used to induce: the production of truncated proteins, the production of proteins lacking functional regions, or a reduction in the total amount of proteins produced.

[0010] Preferably, the antisense oligomer is phosphoryl diamine morpholino oligomer (PMO) or 2'-O-methyl thiophosphate oligomer (2'-OMePS).

[0011] Preferably, the antisense oligomer is selected from the group comprising the sequences shown in Table 1. Preferably, the antisense oligomer is selected from the list comprising SEQ ID NO: 1-17. More preferably, the antisense oligomer used in this invention is SEQ ID NO: 2, 3, 5 or 12.

[0012] According to another aspect of the invention, the present invention relates to cDNA or clonal copies of the antisense oligomer sequence of the present invention, and vectors containing the antisense oligomer sequence of the present invention. The invention further relates to cells containing such sequences and / or vectors.

[0013] A method for manipulating splicing factor binding in target protein gene transcripts is also provided, the method comprising the following steps: Provide one or more antisense oligomers as described herein, and enable the oligomers to bind to target nucleic acid sites.

[0014] Also provided is a preventive or therapeutic composition of a medicament for treating, preventing, or improving the effects of psoriasis in a subject, said composition comprising: One or more antisense oligomers as described herein, and One or more pharmaceutically acceptable carriers and / or diluents.

[0015] Subjects with psoriasis can be mammals, including humans.

[0016] A method for treating, preventing, or improving the effects of psoriasis is also provided, the method comprising the steps of: Administer to a subject an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers as described herein.

[0017] The use of purified and isolated antisense oligomers, as described herein, in the preparation of medicaments for the treatment, prevention, or improvement of the effects of psoriasis is also provided.

[0018] Kits for treating, preventing, or improving the effects of psoriasis in subjects are also provided, the kits comprising at least one antisense oligomer as described herein and combinations or mixtures thereof, the kits being packaged in suitable containers and accompanied by instructions for use.

[0019] Other aspects of the invention will now be described with reference to the accompanying non-limiting embodiments and drawings. Attached Figure Description

[0020] Other features of the invention are described more fully in the following description of several non-limiting embodiments thereof. This description is included only as an example of the invention and should not be construed as limiting the scope of the broad overview, disclosure, or description of the invention as described above. The description will be made with reference to the accompanying drawings, in which: Figure 1 shows the Western blot of S100A8 protein in Hacat cells (keratinocytes) after treatment with translation-blocking PMO. Figure 1A S100A8 H2A(+11+35), Figure 1B : S100A8H2A(+01+25). Both PMOs were treated at 100 μm and 200 μm for 24–72 hours. Compared with untreated (UN), GTC, and ZAP, significantly lower protein levels were observed at 100 μm and 200 μm after treatment with both PMOs.

[0021] Figure 2 shows the results of IL36R-exon 9-transmembrane domain L3K transfection: preliminary screening of six ASOs at concentrations of 50 nM and 200 nM. Highly efficient hopping (~77%) was achieved at the 50 nM concentration.

[0022] Figure 3 shows the titration curves for IL1RL2-9 exon transmembrane domain L3K transfection at low concentrations (1, 3, 10, 30, and 100 nm). At a low concentration of 30 nM, efficient hopping rates of 81–91% were achieved (for different ASOs).

[0023] Figure 4 shows the L3K transfection of the IL1RL2-exon 10-TIR domain: preliminary screening of seven ASOs at concentrations of 50 nM and 200 nM. Efficient hopping (~73%) was achieved at the 50 nM concentration.

[0024] Figure 5 shows the titration curves for IL1RL2-exon 10 transmembrane domain L3K transfection at low concentrations (1, 3, 10, 30, and 100 nm). At a low concentration of 30 nM, efficient hopping of 72–90% was achieved (for different ASOs).

[0025] Figure 6 Western blot of S100A9 protein in Hacat cells (keratinocytes) after treatment with translation-blocking PMOs; S100A9 H2A (+01+25), S100A8 H2A (+16+40). Both PMOs were applied at 100 μm and 200 μm for 24–72 hours. Significantly lower protein levels were observed at 100 μm and 200 μm after treatment with both PMOs compared to untreated (UN), GTC, and ZAP.

[0026] Implementation Detailed Implementation

[0027] Excessive epidermal proliferation caused by enhanced inflammation and immune response is a major characteristic of psoriasis and a primary cause of the typical appearance of psoriatic lesions. Transcriptomic analysis of skin samples from psoriatic areas (both lesion and non-lesion areas) has identified potential alternativesplicing variants of different genes involved in inflammation and immune responses.

[0028] This invention is based on the surprising discovery that modifying the precursor mRNA splicing or translation of S100 alarm proteins (S100A8 and S100A9) and the IL1RL2 subunit of the IL36 receptor using isolated or purified antisense oligomers (ASO) can lead to a reduction in the production of functional target proteins. This can be achieved by producing truncated proteins, proteins lacking functional regions, or reducing the total amount of protein produced.

[0029] In this invention, antisense oligomers are also referred to as antisense oligonucleotides, ASO, and AON-, and these terms are interchangeable.

[0030] target protein

[0031] S100A8 / S100A9

[0032] Antimicrobial peptides (AMPs), such as S100 proteins, are a family of small calcium-binding proteins that play a crucial role in skin immunity by helping the epithelial surface control infection and mediating innate and adaptive immune responses. AMPs not only function as natural antibiotics controlling microbial growth but also modulate the host's inflammatory response by acting as angiogenic factors, chemokines, and cell proliferation regulators. High expression of AMPs has been found in psoriatic lesions. Cellular stress or inflammation induces the release of these proteins, and these genes are highly upregulated in psoriatic skin.

[0033] Calgranulin genes are members of the S100 gene family of calcium-binding proteins. These proteins have known functions in immune responses and act as damage-associated molecular pattern (DAMP) proteins, signaling via the receptor for advanced glycation end products (RAGE), and as ligands for endogenous Toll-like receptor 4 (TLR4). The S100 proteins that serve as drug targets in this invention are calgranulin A / S100A8 and calgranulin B / S100A9. These proteins are also referred to as: S100A8-migration inhibitor-(MIF)-related protein-8 or MRP8; and S100A9-migration inhibitor-(MIF)-related protein-14 or MRP14.

[0034] S-100 alarm proteins (calprotective proteins) S100A8 and S100A9 induce a strong immune response in the skin and keratinocytes, leading to uncontrolled proliferation of keratinocytes, which in turn forms hyperproliferative plaques on the skin and causes tissue deformation.

[0035] The S100A8 / 9 genes consist of three exons of different lengths (33, 164, and 211 bp for S100A8, and 28, 165, and 380 bp for S100A9), separated by two introns of different lengths (484 and 150 bp for S100A8, and 292 bp and approximately 2 kb for S100A9). In each gene, exon 1 encodes the untranslated region. The protein is encoded by the sequence in exon 2, which encodes the N-terminal 47 amino acids of S100A8 and the N-terminal 50 amino acids of S100A9. Exon 3 encodes the C-terminal 46 amino acids of S100A8 and the C-terminal 64 amino acids of S100A9.

[0036] IL36 receptor (IL1RL2 subunit)

[0037] Psoriasis possesses a strong immune component associated with its disease flare-ups. Immune dysfunction in psoriasis involves crosstalk of cytokines between immune and non-immune cells. Several important types of immune cells have been identified as playing a role in the pathogenesis of psoriasis, including Th1, Th17, and regulatory T cells. The inflammatory response in psoriasis is driven by pro-inflammatory cytokines, primarily: interleukin-17, interleukin-23, tumor necrosis factor (TNF), interferon-γ, the IL-1 cytokine family (including IL-36 cytokine), IL-36 receptor (IL36R), and receptor antagonists (IL36Ra / IL36RN).

[0038] Pro-inflammatory IL-36 cytokines (IL-36α, IL-36β, and IL-36γ) and their two antagonists (IL36RN or IL36Ra and IL-38) all utilize the same receptor complex, IL36R. IL36R is a member of the interleukin-1 receptor family and is a heterodimeric complex composed of the IL1RL2 subunit and the IL-1RAcP co-receptor. The agonistic binding of pro-inflammatory cytokines (IL-36α, IL-36β, and IL-36γ) to the IL1RL2 subunit activates the inflammatory pathway. Alternative names for the IL1RL2 subunit of the IL36 receptor include IL-1Rrp2, IL-36R, IL1R-rp2, IL1RRP2, and interleukin-1 receptor-like 2.

[0039] The IL1RL2 subunit of IL36R contains a transmembrane domain in exon 9 and a Toll-interleukin-1 receptor (TIR) ​​homologous domain in exon 10, which mediates protein-protein interactions between Toll-like receptors (TLRs) and signal transduction components.

[0040] Stimulation of the IL1RL2 subunit of the IL36 receptor induces the production of IL-17 and IL-22, both known drivers of psoriatic cytopathology. Th17 lymphocytes produce IL-17 and IL-22, which play crucial roles in host defense against microbes and in the development of chronic inflammatory diseases by recruiting neutrophils and inducing the production of antimicrobial peptides. IL-17 primarily induces inflammatory tissue responses and is involved in the pathogenesis of various autoimmune diseases, while IL-22 is primarily protective and regenerative. Blocking the IL1RL2 receptor inhibits the production of these Th17 cytokines.

[0041] Unbound by any theoretical framework, this invention is based on the understanding that downregulating the expression of one or more of the invention's target proteins (S100A8 and S100A9, and IL1RL2) leads to reduced inflammation, decreased keratinocyte proliferation, and increased cell death. Blocking S100A8 and S100A9 halts the inflammatory cascade in the skin. Blocking the IL1RL2 subunit of the IL36 receptor inhibits the production of IL-17 and IL-22. These factors subsequently lead to a reduction in psoriasis symptoms.

[0042] This invention provides the ASOs listed in Table 1 for regulating the expression of S100A8, S100A9, and IL1RL2 proteins.

[0043] Table 1: Antisense Oligomers of the Invention

[0044] The downregulation of one or more proteins in this invention is preferably achieved by the following: i. Translation blockade; ii. Inducing exon skipping (by increasing the production of truncated, nonsense, or prematurely terminated proteins, such as proteins with premature stop codons); and / or iii. Downregulate gene expression through RNase-H-dependent cleavage of target mRNA.

[0045] Any of these techniques can lead to a reduction in the production of functional target proteins.

[0046] Downregulation can also be achieved by inducing exon skipping, resulting in the absence of transmembrane domains in mature proteins, thereby converting the membrane-bound form of the protein into a soluble form. This strategy may lead to the soluble form of the protein forming a decoy receptor.

[0047] This invention may not affect the overall expression of the target protein, for example, by blocking or removing all RNA transcripts. Instead, it may be designed to increase the yield of truncated, nonsense, or prematurely terminated proteins. The total yield of target protein RNA molecules may not change significantly (although some variation is possible). Preferably, the truncated, nonsense, or prematurely terminated proteins lack one or more functional domains involved in the activity of the target protein.

[0048] Preferably, there is an internally truncated protein (i.e., a protein lacking one or more amino acids encoded by one or more exons). If the target protein is knocked out, there may be a problem of increased target protein RNA transcription levels, as the body attempts to compensate for the reduction in the total amount of the target protein. Conversely, the presence of an internally truncated protein (preferably lacking one or more features of the complete target protein) should be sufficient to prevent increased transcription levels, but still provide a therapeutic advantage due to the reduction in the total amount of functional target protein.

[0049] In this invention, exon skipping induced by antisense oligomers does not require complete or even substantial elimination of the target protein's function. Preferably, the exon skipping process results in a reduction or impairment of the target protein's functionality.

[0050] Preferably, the manipulation of protein expression of the present invention induced by antisense oligomers leads to the following results: S100A8: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); S100A9: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); IL1RL2: Exon 9 skipping (targeting the transmembrane domain, exon skipping); IL1RL2: Exon 10 or 11 skipping (targeting the Toll-interleukin-1 receptor (TIR) ​​homologous domain, exon skipping).

[0051] The steric hindrance of the second exon of S100A8 and / or S100A9 preferably leads to the blocking of translation initiation sites and inhibits translation by blocking the movement of translation factors, regulatory factors and ribosomes along the mRNA chain.

[0052] Skipping of exon 9 of IL1RL2 preferentially results in a soluble form of the protein rather than a membrane-bound form. This soluble form can also act as a bait receptor to bind pro-inflammatory IL-36 cytokines (IL-36α, IL-36β, and IL-36γ) and antagonists (IL36RN or IL36Ra and IL-38).

[0053] Skipping of exons 10 / 11 of IL1RL2 preferentially results in a truncated protein lacking the complete TIR homology domain. The TIR domain interacts with receptors and plays a crucial role in activating cellular signaling pathways in response to inflammation, stress, and cytokines. Skipping the TIR domain affects receptor binding and subsequent signal transduction pathways.

[0054] Preferably, the antisense oligomer targets the splicing site in the target protein gene. The target site may also include some flanking sequences surrounding the splicing site.

[0055] Antisense oligomers can also, or alternatively, bind to polyadenylation sites. The target site is also located near the polyadenylation site but does not overlap with it; that is, it can instead cover sequences upstream or downstream of the polyadenylation site, and in these cases, the antisense oligomer can non-specifically cover the polyadenylation site. Location near the polyadenylation site may be sufficient to disrupt the ability of the cleavage factor to bind to the polyadenylation site.

[0056] According to a first aspect of the invention, an antisense oligomer is provided that is capable of binding to selected targets on a target protein gene transcript to modify the splicing of precursor mRNA in the target protein gene transcript or a portion thereof.

[0057] For example, in one aspect of the invention, an antisense oligomer of 10-50 nucleotides is provided, comprising a targeting sequence complementary to a region near or within a splicing site and / or polyadenylation site of a target protein precursor mRNA.

[0058] Alternatively, the present invention can induce an increase in RNA degradation by recruiting RNase H, wherein RNase H preferentially binds to and degrades double-stranded RNA formed by DNA binding to target protein genes. RNase-H recognizes the DNA-RNA heteroduplex formed between mRNA and genomic DNA and cleaves the RNA strand. The antisense oligomers designed as substrates for RNase-H are inhibitors of intermediate metabolism of precursor and spliced ​​mRNA.

[0059] The terms “antisense oligomer” and “antisense compound” and “antisense oligonucleotide,” “AON” and “ASO” are used interchangeably and refer to the sequence of cyclic subunits, each having a base-pairing portion linked by an intersubunit bond. This intersubunit bond allows the base-pairing portion to hybridize with a target sequence in a nucleic acid (typically RNA) via Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence. The cyclic subunit is based on ribose or another pentose, or, in a preferred embodiment, on a morpholino group (see description of morpholino oligomers below). The oligomer may have precise or near sequence complementarity with the target sequence; sequence variation near the oligomer terminus is generally preferred over internal variation. The terms “precursor RNA” and “precursor mRNA” are used interchangeably.

[0060] "Separated" means that the material substantially or substantially does not contain the components that are normally present in its natural state. For example, "separated polynucleotides" or "separated oligonucleotides" as used herein can refer to purified polynucleotides or polynucleotides removed from sequences flanking them in their natural state, such as DNA fragments removed from sequences adjacent to that fragment in the genome. The term "separated" when referring to cells means purifying cells (e.g., fibroblasts, lymphoblasts) from a source subject (e.g., a subject with psoriasis). In the context of mRNA or protein, "separated" means recovering mRNA or protein from a source such as cells.

[0061] Antisense oligomers can be described as "targeting" or "confronting" the target sequence with which they hybridize. In some embodiments, the target sequence includes a region and surrounding region containing a splice site and / or a polyadenylation site. Target sequences typically include the AUG start codon of mRNA, a translation-suppressing oligomer, or a splice site of pre-processed mRNA, or a region of a splice-suppressing oligomer (SSO). Target sequences for splice sites may include mRNA sequences with their 5' end located 1 to 25 base pairs downstream of the normal splice acceptor junction in the pre-processed mRNA. Preferred target sequences are any region of pre-processed mRNA that includes a splice site or is entirely contained within an exon coding sequence or spans a splice acceptor or donor site. When oligomers target the target nucleic acid in the manner described above, they are more generally "targeting" biologically relevant targets such as proteins, viruses, or bacteria.

[0062] As used herein, “sufficient length” means that the antisense oligonucleotide is complementary to at least 8, more typically 8–30 consecutive nucleotides in the target protein precursor mRNA. In some embodiments, the sufficient length of the antisense comprises at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleotides in the target protein precursor mRNA. In other embodiments, the sufficient length of the antisense comprises at least 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides in the target protein precursor mRNA. The sufficient length of the antisense oligonucleotide has at least a minimum number of nucleotides to enable specific hybridization with exon 2. Preferably, the sufficiently long oligonucleotide is about 10 to about 50 nucleotides in length, including oligonucleotides of 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, and 40 or more nucleotides. In one embodiment, the sufficiently long oligonucleotide is about 10 to about 30 nucleotides in length. In another embodiment, the sufficiently long oligonucleotide is about 15 to about 25 nucleotides in length. In another embodiment, the sufficiently long oligonucleotide is about 20 to 30 or 20 to 50 nucleotides in length. In yet another embodiment, the sufficiently long oligonucleotide is about 22 to 28, 25 to 28, 24 to 29, or 25 to 30 nucleotides in length.

[0063] In some embodiments, the antisense oligomer has sufficient sequence complementarity with the target RNA (i.e., the RNA that regulates the selection of its splicing factor binding sites) to effectively block regions of the target RNA (such as precursor mRNA). In exemplary embodiments, this blocking of the target protein precursor mRNA is used to regulate or modify splicing by masking the binding sites of native proteins that would normally regulate splicing and / or by altering the structure of the targeted RNA. In some embodiments, the target RNA is a target precursor mRNA (e.g., a target protein gene precursor mRNA).

[0064] The antisense oligomer having sufficient sequence complementarity with the target RNA sequence to regulate the binding of the target RNA splicing factor means that the antisense oligomer has a sequence sufficient to trigger the masking of the natural protein binding site, otherwise the natural protein would cause the target protein to be truncated and / or alter the three-dimensional structure of the target RNA.

[0065] The selected antisense oligomer can be shorter, for example, about 12 bases, or longer, for example, about 50 bases, and includes a small number of mismatches, as long as the sequence is sufficiently complementary to the target sequence when hybridizing to affect the regulation of splicing factor binding, and optionally forms a heteroduplex with the RNA antisense oligomer at a Tm of 45°C or higher.

[0066] Preferably, the antisense oligomer is selected from the group comprising the sequences shown in Table 1. Preferably, the antisense oligomer is selected from the list comprising SEQ ID NO: 1-17. Preferably, the antisense oligomer used in this invention is selected from the list comprising SEQ ID NO: 2, 3, 5 or 12. Preferably, the manipulation of protein expression of this invention induced by the antisense oligomer results in the following results: S100A8: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); S100A9: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); IL1RL2: Exon 9 skipping (targeting the transmembrane domain, exon skipping); IL1RL2: Exon 10 or 11 skipping (targeting the Toll-interleukin-1 receptor (TIR) ​​homologous domain, exon skipping).

[0067] In some embodiments, the complementarity between the target sequence and the antisense oligomer is sufficient to form a stable duplex. The complementary region of the antisense oligomer to the target RNA sequence can be as short as 8-11 bases, but can be 12-15 bases or more, such as 10-50 bases, 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers between these ranges. Antisense oligomers of about 16-17 bases are generally long enough to have a unique complementary sequence. In some embodiments, as discussed herein, a minimum length of complementary bases may be required to achieve the necessary binding Tm. In some embodiments, oligonucleotides up to 50 bases may be suitable, where at least a minimum number of bases, such as 10-12 bases, are complementary to the target sequence. However, generally, oligonucleotides shorter than about 30 bases exhibit optimal intracellular uptake or active uptake. For the phosphoryldiamine morpholino oligomer (PMO) antisense oligomers further described herein, the balance between binding stability and uptake is typically optimal in the length range of 18–25 bases. This includes antisense oligomers consisting of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 bases (e.g., PMO, PMO-X, PNA, LNA, 2'-OMe).

[0068] In some implementations, the antisense oligomer may be 100% complementary to the target sequence, or may contain mismatches, such as to accommodate variants, provided that the heteroduplex formed between the antisense oligomer and the target sequence is sufficiently stable to withstand the action of cellular nucleases and other degradation modes that may occur in vivo. Thus, certain oligonucleotides may have about or at least about 70% sequence complementarity, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity between the oligonucleotide and the target sequence.

[0069] Mismatches (if present) are generally less unstable at the terminal regions of the hybrid duplex than at the intermediate regions. According to well-known principles of duplex stability, the permissible number of mismatches will depend on the length of the antisense oligomer, the percentage of G:C base pairs in the duplex, and the location of the mismatch within the duplex. Although this antisense oligomer does not need to be 100% complementary to the target sequence, it can effectively, stably, and specifically bind to the target sequence, thereby regulating the binding of splicing factors to target precursor mRNA.

[0070] The stability of the duplex formed between the antisense oligomer and the target sequence is a function of the binding Tm and the duplex's susceptibility to enzymatic cleavage by the cell. The Tm of the oligonucleotide relative to the complementary RNA sequence can be measured by conventional methods, such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or as described in Miyada C. G. and Wallace RB, 1987, Oligonucleotide Hybridization Techniques, MethodsEnzymol. Vol. 154, pp. 94-107. In some embodiments, the antisense oligomer may have a binding Tm relative to the complementary RNA that is above body temperature, and preferably above about 45°C or 50°C. Tm ranges of 60-80°C or higher are also included.

[0071] Other examples of variants include antisense oligomers having about or at least about 70% sequence identity or homology over the full length of any of SEQ ID NOS: 1-126, such as 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or homology.

[0072] More specifically, an antisense oligomer is provided that can bind to a selected target site to regulate or modify splicing in a target protein gene transcript or a portion thereof. The antisense oligomer is preferably selected from those provided in Table 1. Preferably, the antisense oligomer is selected from the list containing SEQ ID NO: 1-17. Preferably, the antisense oligomer used in this invention is selected from the list containing SEQ ID NO: 2, 3, 5, or 12.

[0073] In this invention, splicing factor blocking induced by antisense oligomers does not require complete or even substantial reduction in the amount of target protein produced.

[0074] How to use

[0075] The present invention also provides a method for manipulating the binding of splicing factors in the transcript of a target protein gene, the method comprising the following steps: a) Provide one or more antisense oligomers as described herein, and enable the oligomers to bind to target nucleic acid sites.

[0076] According to another aspect of the invention, a splicing factor-binding modified target nucleic acid sequence of a target protein gene is provided, comprising a DNA equivalent of a nucleic acid sequence selected from SEQ ID NO: 1-2, 3, 5, or 12 and its complementary sequence. Preferably, the manipulation of protein expression of the invention induced by antisense oligomers results in the following: S100A8: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); S100A9: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); IL1RL2: Exon 9 skipping (targeting the transmembrane domain, exon skipping); IL1RL2: Exon 10 or 11 skipping (targeting the Toll-interleukin-1 receptor (TIR) ​​homologous domain, exon skipping).

[0077] Designing antisense oligomers to completely mask splice sites and / or polyadenylation sites may not be necessary for changes in the proportion of truncated, nonsense, or prematurely terminated proteins. Furthermore, the inventors have found that the size or length of the antisense oligomer itself is not always a primary consideration when designing it. For certain targets, antisense oligomers as short as 20 bases can induce cleavage modifications, and in some cases are even more efficient than other, longer (e.g., 25 bases) oligomers targeting the same region.

[0078] More specifically, the antisense oligomer may be selected from those listed in Table 1. The sequence is preferably selected from any one or more of SEQ ID NO: 1-17 and combinations thereof or mixtures thereof. This includes sequences that hybridize to these sequences under strict hybridization conditions, sequences complementary to them, sequences containing modified bases, modified backbones, and sequences that are functionally truncated or extended, which have or regulate RNA processing activity in the transcripts of target protein genes. Preferably, the ASO used in this invention is selected from the list containing SEQ ID NO: 2, 3, 5, or 12.

[0079] Antisense oligomers are complementary to DNA, cDNA, or RNA when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can be hydrogen-bonded together. Therefore, "specific hybridization" and "complementarity" are terms used to describe sufficient complementarity or pairing to allow stable and specific binding between the oligomer and its DNA, cDNA, or RNA target. It should be understood in the art that the sequence of an antisense oligomer does not need to be 100% complementary to the sequence of its target sequence to achieve specific hybridization. Antisense oligomers are specifically hybridizable when the binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA product, and possess sufficient complementarity to avoid non-specific binding of the antisense oligomer to non-target sequences under conditions requiring specific binding (physiological conditions for in vivo assays or therapy; conditions for in vitro assays).

[0080] Selective hybridization can be performed under low, medium, or high stringency conditions, but high stringency conditions are preferred. Those skilled in the art will recognize that, in addition to base composition, complementary strand length, and the number of nucleotide base mismatches between hybridized nucleic acids, the stringency of hybridization will also be affected by conditions such as salt concentration, temperature, or organic solvents. Stringent temperature conditions generally include temperatures exceeding 30°C, typically exceeding 37°C, preferably exceeding 45°C, and preferably at least 50°C. C, usually 60 C-80 C or higher. Strict salt conditions are typically below 1000 mM, typically below 500 mM, and preferably below 200 mM. However, the combination of parameters is far more important than the measurement of any single parameter. An example of strict hybridization conditions is 65°C and 0.1xSSC (1x SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Therefore, the antisense oligomers of the present invention can comprise oligomers that selectively hybridize with the sequences SEQ ID NO: 1-126 provided in Table 1.

[0081] At a given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes with the complementary polynucleotide. This hybridization can occur when the antisense oligomer is "near" or "substantially" complementary to the target sequence, or when the antisense oligomer is completely complementary to the target sequence.

[0082] Typically, selective hybridization occurs when there is at least about 55% identity with the antisense oligomer nucleotides in a stretch 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. As described, the length of the homology comparison can be on a longer stretch, and in some embodiments, typically on a stretch of at least about 9 nucleotides, typically at least about 12 nucleotides, more typically at least about 20 nucleotides, typically at least about 21, 22, 23, or 24 nucleotides, at least about 25, 26, 27, or 28 nucleotides, at least about 29, 30, 31, or 32 nucleotides, at least about 36 or more nucleotides.

[0083] Therefore, the antisense oligomer sequence of the present invention preferably has at least 75%, more preferably at least 85%, more preferably at least 86, 87, 88, 89, or 90% homology with the sequences shown in the sequence listing herein. More preferably, it has at least 91, 92, 93, 94, or 95%, more preferably at least 96, 97, 98%, or 99% homology. Generally, the shorter the length of the antisense oligomer, the greater the homology required to obtain selective hybridization. Therefore, when the antisense oligomer of the present invention consists of fewer than about 30 nucleotides, the percentage of identity with the antisense oligomers listed in the sequence listing herein is preferably greater than 75%, preferably greater than 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96, 97, 98%, or 99%. Nucleotide homology comparisons can be performed using sequence comparison programs such as GGWisconsin Bestfit or GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). This allows for the comparison of sequences similar in length or substantially different from those cited herein by inserting gaps in the alignment, which can be determined using comparison algorithms such as those employed by GAP.

[0084] The antisense oligomers of the present invention can have regions of reduced homology and regions precisely homologous to the target sequence. The oligomer need not have precise homology along its entire length. For example, the oligomer can have a continuous segment of at least 4 or 5 bases identical to the target sequence, preferably a continuous segment of at least 6 or 7 bases identical to the target sequence, more preferably a continuous segment of 8 or 9 bases identical to the target sequence. The oligomer can have a segment of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 bases identical to the target sequence. The remaining segments of the oligomer sequence can intermittently correspond to the target sequence; for example, the remaining sequence can have identical bases followed by dissimilar bases, followed by identical bases. Alternatively (or similarly), the oligomeric sequence may have several segments of homologous sequence (e.g., 3, 4, 5, or 6 bases) interspersed with segments that are not entirely homologous. Such sequence mismatches preferably result in little or no loss of cleavage modification activity.

[0085] The term “regulation (and its various parts of speech)” includes an optionally defined and / or statistically significant amount that “increases” or “decreases” one or more measurable parameters. The terms “increase (and its various parts of speech),” “enhance (and its various parts of speech),” or “stimulate (and its various parts of speech)” generally refer to the ability of one or more antisense oligomers or compositions to produce or induce a stronger physiological response (i.e., a downstream effect) in cells or subjects relative to the response induced by the absence of the antisense oligomer or control compound.

[0086] "Enhancement (and its various parts of speech)" or "increase (and its various parts of speech)" or "stimulation (and its various parts of speech)" generally refers to the ability of one or more antisense compounds or compositions to produce or evoke a stronger physiological response (i.e., a downstream effect) in cells or a subject compared to a response evoked by the antisense compound or control compound. Measurable physiological responses may include increased expression of the functional form of the target protein, as well as other responses that are apparent from the understanding in the art and from the description herein. The amount of "increase" or "enhancement" is generally a "statistically significant" amount and may include increases of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more (e.g., 500, 1000 times) compared to the amount produced by the antisense compound (in the absence of the reagent) or control compound (including all integers and decimals between 1 and the above values ​​and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0087] The term “reduction (and its various parts of speech)” generally refers to the ability of one or more antisense oligomers or compositions to produce or evoke a weakened physiological response (i.e., a downstream effect) in cells or a subject, relative to a response induced by the absence of the antisense oligomer or control compound. The terms “weaken” or “inhibit” can generally refer to the ability of one or more antisense compounds of the present invention to “reduce” associated physiological or cellular responses (e.g., symptoms of the disease or condition described herein), as measured according to conventional techniques in the diagnostic field. Associated physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in psoriasis symptoms or pathology. The “reduction” in the reaction compared to the reaction produced by the non-antense compound or the control composition can be statistically significant and can include reductions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all integers therein. The associated physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in the expression level of the target protein. The “increased” or “enhanced” amount is generally a statistically significant amount and may include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000 times) compared to the amount produced by the antisense oligomer (in the absence of the reagent) or control compound (including all integers and decimals between 1 and the above values ​​and greater than 1, such as 1.5, 1.6, 1.7, 1.8). The terms “attenuated” or “inhibited” may generally refer to the ability of one or more antisense oligomers or compositions to “reduce” associated physiological or cellular responses (e.g., symptoms of the disease or condition described herein), as measured according to conventional techniques in the diagnostic field. The associated physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in psoriasis symptoms or pathology. The “reduction” in the reaction compared to the reaction produced by the non-antense oligomer or the control composition can be statistically significant and can include reductions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all integers in between.

[0088] The length of the antisense oligomer can vary, as long as it can selectively bind to the desired location within the precursor mRNA molecule. The length of such sequences can be determined according to the selection procedure described herein. Typically, the length of the antisense oligomer is from about 10 nucleotides to a maximum of about 50 nucleotides. However, it should be understood that nucleotides of any length within this range can be used in this method. Preferably, the length of the antisense oligomer is 10 to 40, 10 to 35, 15 to 30 nucleotides, or 20 to 30 nucleotides, most preferably about 25 to 30 nucleotides. For example, the length of the oligomer can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0089] As used herein, “antisense oligomer,” “AON,” or “ASO” refers to a linear sequence of a nucleotide or nucleotide analog that allows its nucleobases to hybridize with a target sequence in RNA via Watson-Crick base pairing, thereby forming an oligonucleotide:RNA heteroduplex within the target sequence. The cyclic subunit may be based on ribose or another pentose, or in some embodiments, on a morpholino group (see description of morpholino oligomers below). Also considered were aminophosphate or phosphoryl diamine morpholine oligomers (PMO); PMO-X; PPMO; thiophosphoramide morpholine oligomers (TMO); peptide nucleic acids (PNA); locked nucleic acids (LNA) and their derivatives, including α-L-LNA, 2'-aminoLNA, 4'-methylLNA and 4'-O-methylLNA; ethylene-bridged nucleic acids (ENA) and their derivatives; thiophosphate oligomers; tricyclic DNA oligomers (tcDNA); tricyclic thiophosphate oligomers; 2'O-methyl modified oligomers (2'-OMe); 2'-O-methoxyethyl (2'-MOE); 2'-O-methyl thiophosphate oligomers (2'-OMePS); 2'-fluorine... 2'-Fluoroarabinonucleotide (FANA); nonlocked nucleic acid (UNA); hexitol nucleic acid (HNA); cyclohexene nucleic acid (CeNA); 2'-amino(2'-NH2); 2'-O-ethyleneamine; or any combination thereof as a mixer or gapmer, and other antisense reagents known in the art.

[0090] In some implementations, the antisense oligonucleotide has the chemical composition of a naturally occurring nucleic acid molecule, meaning that the antisense oligonucleotide does not include modified or substituted bases, sugars, or inter-subunit bonds.

[0091] In a preferred embodiment, the antisense oligonucleotide of the present invention is a non-naturally occurring nucleic acid molecule, or an "oligonucleotide analog." For example, a non-naturally occurring nucleic acid may include one or more non-natural bases, sugars, and / or inter-subunit bonds, such as bases, sugars, and / or bonds that have been modified or substituted relative to those found in naturally occurring nucleic acid molecules. Exemplary modifications are described below. In some embodiments, the non-naturally occurring nucleic acid includes more than one type of modification, such as sugar and base modifications, sugar and bond modifications, base and bond modifications, or base, sugar, and bond modifications. For example, in some embodiments, the antisense oligonucleotide contains non-natural (e.g., modified or substituted) bases. In some embodiments, the antisense oligonucleotide contains non-natural (e.g., modified or substituted) sugars. In some embodiments, the antisense oligonucleotide contains non-natural (e.g., modified or substituted) inter-subunit bonds. In some embodiments, the antisense oligonucleotide contains more than one type of modification or substitution, such as non-natural bases and / or non-natural sugars, and / or non-natural inter-subunit bonds.

[0092] Therefore, this includes non-naturally occurring antisense oligomers having (i) a modified backbone structure, such as a backbone different from the standard phosphodiester bonds found in naturally occurring oligonucleotides and polynucleotides, and / or (ii) a modified sugar moiety, such as a morpholino moiety instead of a ribose or deoxyribose moiety. Oligonucleotide analogs support bases capable of hydrogen bonding with standard polynucleotide bases via Watson-Crick base pairing, wherein the analog backbone presents bases in a manner that allows such hydrogen bonding between the oligonucleotide analog molecule and bases in the standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA) in a sequence-specific manner. Preferred analogs are those having a substantially uncharged phosphorus-containing backbone.

[0093] One method for generating antisense oligomers is to methylate the 2'-hydroxyribose position and incorporate a thiophosphate backbone, resulting in molecules that superficially resemble RNA but are more resistant to nuclease degradation, although those skilled in the art will know of other suitable backbones that can be used for the purposes of this invention.

[0094] To avoid degradation of precursor RNA during duplex formation with antisense oligomers, the antisense oligomers used in this method can be modified to minimize or prevent cleavage by endogenous ribonuclease (RNase) H. Antisense molecules that do not activate RNase H can be prepared using known techniques (see, for example, U.S. Patent No. 5,149,797). Such antisense molecules can be deoxyribonucleotide or ribonucleotide sequences containing only any structural modifications that spatially impede or prevent the binding of RNase H to a duplex molecule containing that oligonucleotide as a member, and which substantially do not impede or disrupt duplex formation. Because the oligonucleotide moiety involved in duplex formation is significantly different from the oligonucleotide moiety involved in RNase H binding, a wide variety of antisense molecules that do not activate RNase H can be obtained. This characteristic is highly preferred because treatment of RNA (whether intracellular or in crude extracts containing RNase H) with unmethylated oligomers leads to degradation of the precursor mRNA:antisense oligomer duplex. Any modified antisense oligomer capable of bypassing or not inducing such degradation can be used in this method. Nuclease resistance can be achieved by modifying the antisense oligomer of the present invention to include a partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups, including carboxylic acid groups, ester groups, and alcohol groups.

[0095] An example of an antisense oligomer that is not cleaved by cellular RNase H when double-stranded with RNA is a 2'-O-methyl derivative. These 2'-O-methyl oligonucleotides are stable in cellular environments and animal tissues, and their double-stranded RNA counterparts have a higher Tm value than their ribose or deoxyribose counterparts. Alternatively, at least one of the last few nucleotides at the 3' end of the nuclease-resistant antisense oligomer of the present invention is fluorinated. Alternatively, the nuclease-resistant antisense oligomer of the present invention has a phosphate thioester bond connecting at least two of the last 3'-terminal nucleotide bases, preferably a phosphate thioester bond connecting the last four 3'-terminal nucleotide bases.

[0096] Modified or regulated RNA splicing can also be achieved via alternative oligonucleotide chemistry (see, for example, U.S. Patent No. 5,149,797). For example, the antisense oligomer may be selected from a list comprising: aminophosphate or phosphorylated diamine morpholino oligomers (PMO); PMO-X; PPMO; thiophosphoramide morpholino oligomers (TMO); peptide nucleic acids (PNA); locked nucleic acids (LNA) and their derivatives, including α-L-LNA, 2'-aminoLNA, 4'-methylLNA, and 4'-O-methylLNA; ethylene-bridged nucleic acids (ENA) and their derivatives; thiophosphate oligomers; tricyclic DNA oligomers. (tcDNA); tricyclic thiophosphate oligomer; 2'O-methyl modified oligomer (2'-OMe); 2'-O-methoxyethyl (2'-MOE); 2'-fluoro, 2'-fluoroarabinonucleotide (FANA); nonlocked nucleic acid (UNA); hexitol nucleic acid (HNA); cyclohexene nucleic acid (CeNA); 2'-amino (2'-NH2); 2'-O-ethyleneamine; or any combination thereof as a mixmer or as a gapmer.

[0097] To further improve delivery efficiency, the modified nucleotides described above are typically conjugated to sugar or nucleobase moieties with fatty acids / lipids / cholesterol, amino acids, carbohydrates / polysaccharides, nanoparticles, etc. These conjugated nucleotide derivatives can also be used to construct antisense oligomers to modify cleavage factor binding. Antisense oligomer-induced modification of target protein gene transcript splicing factor binding is typically achieved using oligonucleotides, PNA, 2'OMe, or MOE-modified bases located on a phosphate thioester backbone. Although 2'OMe ASOs have been used in oligomer design due to their efficient in vitro uptake when delivered as cationic lipid complexes, these compounds are susceptible to nuclease degradation and are not considered ideal for in vivo or clinical applications. When using alternative chemical methods to generate the antisense oligomers of this invention, the uracil (U) in the sequence provided herein can be replaced with thymine (T).

[0098] For example, such antisense molecules can be oligonucleotides in which at least one or all of the phosphate ester residues bridging the nucleotides are modified phosphate esters, such as methylphosphonate, methyl phosphorothioate, phosphoromorpholidate, phosphoropiperazidate, and phosphor amidate. For example, every other phosphate ester residue bridging the nucleotides can be modified as described. In another non-limiting example, such antisense molecules are molecules in which at least one or all of the nucleotides contain a 2' lower alkyl moiety (e.g., Ci-C4, straight-chain or branched, saturated or unsaturated alkyl group, such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other nucleotide can be modified as described.

[0099] Specific examples of antisense oligonucleotides that can be used in this invention include oligonucleotides containing a modified backbone or non-natural subunit bonds.

[0100] Oligonucleotides with modified backbones include oligonucleotides that retain phosphorus atoms in their backbone and oligonucleotides that do not have phosphorus atoms in their backbone. Modified oligonucleotides that do not have phosphorus atoms in their internucleotide backbone can also be considered oligonucleotides.

[0101] In other antisense molecules, the sugar and nucleoside internucleotide bonds of the nucleotide units, i.e., the backbone, are replaced by new groups. Base units are retained to allow hybridization with suitable nucleic acid target compounds. One such oligomer, an oligonucleotide mimic that has shown excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleobases are retained and directly or indirectly bound to the nitrogen atom of the amide moiety of the backbone.

[0102] Modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides may also contain nucleobase (generally referred to in the art simply as "bases") modifications or substitutions. Oligonucleotides containing modified or substituted bases include oligonucleotides in which one or more of the most common purine or pyrimidine bases in the nucleic acid are replaced by less common or non-natural bases.

[0103] Purine bases consist of a pyrimidine ring fused to an imidazole ring; adenine and guanine are the two most common purine nucleobases in nucleic acids. These purines can be substituted by other naturally occurring purines, including but not limited to N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

[0104] Pyrimidine bases contain a six-membered pyrimidine ring; cytosine, uracil, and thymine are the most common pyrimidine bases in nucleic acids. These can be substituted by other naturally occurring pyrimidines, including but not limited to 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotide described herein contains a thymine base in place of uracil.

[0105] Other modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g., 2-thiouracil, 2-thiothymidine), G-clamp and its derivatives, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, SuperT), 7-deazoguanine, 7-deazoadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazoguanine, 8-aza-7-deazoadenine, 8-aza-7-deazo-2,6-diaminopurine, Super G, Super A and N4-ethylcytosine or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP) and N2-propyl-2-aminopurine (Pr-AP), pseudouracil or derivatives thereof; and degenerate or universal bases, such as 2,6-difluorotoluene, or base deletions such as abase desaturation sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives wherein the epoxide has been nitrogen-substituted (azaribose)). Examples of derivatives of Super A, Super G and Super T can be found in U.S. Patent 6,683,173 (Epoch Biosciences). Studies have shown that when cPent-G, cPent-AP and Pr-AP are incorporated into siRNA, their immunostimulatory effects are reduced (Peacock H. et al., J. Am. Chem. Soc. 2011, 133, 9200). Pseudorazine is a naturally occurring isomerized form of uracil, possessing a C-glycoside instead of the N-glycoside found in uridine. Synthetic mRNAs containing pseudouridine may have improved safety profiles compared to uridine-containing mPvNAs (see WO 2009127230).

[0106] Certain modified or substituted nucleobases are particularly useful for increasing the binding affinity of the antisense oligonucleotides of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6–1.2 °C and is currently the preferred base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0107] In some embodiments, modified or substituted nucleobases can be used to facilitate the purification of antisense oligonucleotides. For example, in some embodiments, the antisense oligonucleotide may contain three or more (e.g., 3, 4, 5, 6, or more) consecutive guanine bases. In some antisense oligonucleotides, a series of three or more consecutive guanine bases can lead to oligonucleotide aggregation, complicating purification. In such antisense oligonucleotides, one or more consecutive guanine bases can be substituted with inosine. Inosine substitution of one or more guanine bases in a series of three or more consecutive guanine bases can reduce the aggregation of antisense oligonucleotides, thereby facilitating purification.

[0108] In one embodiment, another modification of the antisense oligonucleotide involves chemically linking one or more moieties or conjugates to the oligonucleotide to enhance its activity, cellular distribution, or cellular uptake. Such moieties include, but are not limited to: lipid moieties such as cholesterol moieties, bile acids, thioethers such as hexyl-5-triphenylmethylthiol, thiocholesterol, aliphatic chains such as dodecyl glycol or undecyl residues, phospholipids such as di-hexadecyl-racemic-glycerol or triethylammonium 1,2-di-O-hexadecyl-racemic-glycero-3-H-phosphonate, polyamine or polyethylene glycol chains, or adamantaneacetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0109] In another non-limiting example, such an antisense oligomer is a molecule in which at least one or all of the nucleotides contain a 2' lower alkyl moiety (e.g., C1-C4, straight-chain or branched, saturated or unsaturated alkyl group, such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other nucleotide can be modified as described.

[0110] While the above-described antisense oligomers are preferred forms of the antisense oligomers of the present invention, the present invention includes other oligo-antisense molecules, including but not limited to oligomimetics such as those described below.

[0111] Another preferred chemical is phosphoryldiamine morpholino oligomer (PMO) oligomers, which are not degraded by any known nucleases or proteases. These compounds are uncharged, do not activate RNase H activity when bound to RNA chains, and have been shown to exert sustained cleavage factor binding regulation after in vivo administration (Summerton and Weller, Antisense Nucleic Acid Drug Development, 7, 187-197).

[0112] Modified oligomers may also contain one or more substituted sugar moieties. Oligomers may also include nucleobase (generally referred to in the art simply as "base") modifications or substitutions. Certain nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 °C, especially when combined with 2'-O-methoxyethyl sugar modifications. In one embodiment, at least one pyrimidine base of the oligonucleotide comprises a 5-substituted pyrimidine base, wherein the pyrimidine base is selected from cytosine, thymine, and uracil. In one embodiment, the 5-substituted pyrimidine base is 5-methylcytosine. In another embodiment, at least one purine base of the oligonucleotide comprises an N-2, N-6 substituted purine base. In one embodiment, the N-2, N-6 substituted purine bases are 2,6-diaminopurine.

[0113] In one embodiment, the antisense oligonucleotide comprises only one or more 5-methylcytosine substitutions, or comprises one or more 5-methylcytosine substitutions combined with another modification such as a 2'-O-methoxyethyl sugar modification. In yet another embodiment, the antisense oligonucleotide contains one or more 2,6-diaminopurine substitutions, which may be present alone or in combination with other modifications.

[0114] In some embodiments, the antisense oligonucleotide is chemically linked to one or more moieties (e.g., polyethylene glycol moieties) or conjugates (e.g., arginine-rich cell-penetrating peptides) to enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide. In one exemplary embodiment, the arginine-rich polypeptide is covalently coupled at its N-terminal or C-terminal residue to the 3' or 5' end of the antisense compound. Furthermore, in one exemplary embodiment, the antisense compound consists of an inter-unit bond between a morpholino subunit and a phosphorus-containing subunit, the bond linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit.

[0115] On the other hand, the present invention provides an expression vector that integrates the aforementioned antisense oligonucleotides, such as the antisense oligonucleotides of SEQ ID NO: 1-126. In some embodiments, the expression vector is a modified retroviral or non-retroviral vector, such as an adeno-associated virus vector.

[0116] Another modification of the oligomers of the present invention involves chemically linking one or more moieties or conjugates to the oligonucleotide to enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. These moieties include, but are not limited to, lipid moieties such as cholesterol moieties, bile acids, thioethers such as hexyl-S-triphenylmethylthiol, thiocholesterol, aliphatic chains such as dodecyl glycol or undecyl residues, phospholipids such as di-hexadecyl-racemic-glycerol or triethylammonium 1,2-di-O-hexadecyl-racemic-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantaneacetic acid, palmityl moieties, myristyl or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0117] Cell-penetrating peptides have been added to phosphorylated diamine morpholino oligomers to enhance cellular uptake and nuclear localization. Different peptide tags have been shown to influence uptake efficiency and target tissue specificity, as demonstrated by Jearawiriyapaisarn et al., (2008), Mol. Ther. 16 9, 1624–1629. The terms “cell-penetrating peptide” and “CPP” are used interchangeably to refer to cationic cell-penetrating peptides, also known as transport peptides, carrier peptides, or peptide transduction domains. As shown herein, the peptides possess the ability to induce cell penetration within 100% of a given cell culture population and allow for the translocation of macromolecules in a variety of tissues in vivo after systemic administration.

[0118] It is not necessary to uniformly modify all positions in a given compound, and in fact, more than one of the above modifications can be incorporated into a single compound or even into a single nucleoside within an oligomer. The invention also includes antisense oligomers as chimeric compounds. In the context of this invention, a "chimeric" antisense oligomer or "chimer" is an antisense oligomer, particularly an oligomer, containing two or more chemically distinct regions, each region consisting of at least one monomeric unit, i.e., a nucleotide in the case of oligomeric compounds. These oligomers typically contain at least one modified region that imparts enhanced resistance to nuclease degradation and enhanced cellular uptake to the oligomer or antisense oligomer, and include an additional region that enhances affinity for binding to target nucleic acids.

[0119] The activity of antisense oligomers and their variants can be determined using conventional techniques in the art. For example, the isoform and expression levels of the investigated RNA and protein can be assessed using any of a variety of known methods for detecting isoforms and / or expression of transcribed nucleic acids or proteins. Non-limiting examples of these methods include RT-PCR for RNA isoforms, followed by size separation of PCR products; nucleic acid hybridization methods such as Northern blotting and / or the use of nucleic acid arrays; fluorescence in situ hybridization for detecting intracellular RNA transcripts; nucleic acid amplification methods; immunological methods for detecting proteins; protein purification methods; and assays of protein function or activity.

[0120] RNA expression levels can be assessed by preparing RNA / cDNA (i.e., transcribed polynucleotides) from cells, tissues, or organisms and hybridizing the RNA / cDNA with a reference polynucleotide (which is the complementary sequence of the nucleic acid being measured) or a fragment thereof. Prior to hybridization with the complementary polynucleotide, the cDNA can optionally be amplified using any of a variety of polymerase chain reactions or in vitro transcription methods; preferably, it is not amplified. The expression level of transcript 1 can also be assessed by detecting the expression of one or more transcripts using quantitative PCR.

[0121] This invention provides splicing factor binding, clinically relevant oligomer chemistry, and delivery systems for antisense oligomer modification of target protein gene transcripts to guide the reduction of full-length target protein gene transcripts to therapeutic levels. This is achieved through a substantial alteration in the amount of target protein RNA: 1) Optimize in vitro oligomers using cell lines, experimentally evaluating the following aspects: (i) modification of splicing factor binding to target motifs, (ii) development of antisense oligomer lengths and oligomer cocktails, (iii) selection of chemical types, and (iv) addition of cell-penetrating peptides (CPPs) to enhance oligomer delivery; and 2) Detailed evaluation of a novel approach to reduce target protein gene transcripts.

[0122] Therefore, this paper demonstrates that the processing of target protein RNA can be manipulated using specific antisense oligomers. In this way, the amount of target protein can be significantly reduced functionally, thereby alleviating the pathological conditions of psoriasis.

[0123] The antisense oligomers used according to the present invention can be conveniently prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, California). A method for synthesizing oligomers on a modified solid support is described in U.S. Patent No. 4,458,066.

[0124] Any other method known in the art for this synthesis may be used, either alternatively or as a substitute. The preparation of oligomers such as thiophosphates and alkylated derivatives using similar techniques is well known. In one such automated embodiment, diethylphosphamide is used as a starting material and can be synthesized as described by Beaucage et al. (1981) Tetrahedron Letters, 22:1859-1862.

[0125] The antisense oligomers of the present invention are synthesized in vitro and do not include biologically derived antisense compositions or genetic vector constructs designed to guide the in vivo synthesis of antisense oligomers. The molecules of the present invention can be mixed, encapsulated, conjugated, or otherwise associated with mixtures of other molecules, molecular structures, or compounds to form, for example, liposomes, receptor-targeting molecules, orally, rectally, topically, or other formulations to aid in uptake, distribution, and / or absorption.

[0126] This also includes vector delivery systems capable of expressing the target sequences of the oligomers of the present invention, such as vectors expressing polynucleotide sequences comprising any one or more of SEQ ID NO: 1-17, as described herein. “Vector” or “nucleic acid construct” refers to a polynucleotide molecule, preferably derived from DNA molecules such as plasmids, bacteriophages, yeast, or viruses, into which polynucleotides can be inserted or cloned. The vector preferably contains one or more unique restriction sites and is capable of autonomous replication within a defined host cell comprising a target cell or tissue or progenitor cell or tissue thereof, or can be integrated into the genome of a defined host such that the cloned sequence is reproducible. Thus, the vector can be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as linear or closed circular plasmids, extrachromosomal elements, mini-chromosomes, or artificial chromosomes. The vector may contain any means to ensure self-replication. Alternatively, the vector can be such that, when introduced into a host cell, it is integrated into the genome and replicates along with the chromosome into which it is integrated.

[0127] Treatment

[0128] The antisense oligomers of the present invention can also be used as preventative or therapeutic agents for the purpose of treating diseases. Therefore, in one embodiment, the present invention provides antisense oligomers that are mixed with pharmaceutically acceptable carriers, diluents, or excipients to bind a therapeutically effective amount to selected targets in target protein RNA to modify RNA splicing as described herein.

[0129] "Effective dose" or "therapeutic effective dose" refers to the amount of a therapeutic compound (such as an antisense oligomer) administered to a mammalian subject in a single dose or a portion of a series of doses that effectively produces the desired therapeutic effect.

[0130] Therefore, the present invention provides a preventive or therapeutic composition for treating, preventing, or improving the effects of psoriasis in a subject, the composition comprising: a) One or more antisense oligomers as described herein, and b) One or more pharmaceutically acceptable carriers and / or diluents.

[0131] Preferably, the antisense oligomers used in this invention are selected from a list including the following: SEQ ID NO: 1-17; or SEQ ID NO: 2, 3, 5 or 12.

[0132] Preferably, the manipulation of protein expression of the present invention induced by antisense oligomers leads to the following results: S100A8: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); S100A9: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); IL1RL2: Exon 9 skipping (targeting the transmembrane domain, exon skipping); IL1RL2: Exon 10 or 11 skipping (targeting the Toll-interleukin-1 receptor (TIR) ​​homologous domain, exon skipping).

[0133] The composition may comprise about 1 nM to 1000 nM of each desired antisense oligomer of the present invention. Preferably, the composition may comprise about 1 nM to 500 nM, 10 nM to 500 nM, 50 nM to 750 nM, 10 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, and most preferably 1 nM to 10 nM of each antisense oligomer of the present invention.

[0134] The composition may comprise each desired antisense oligomer of the present invention at a wavelength of 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.

[0135] The present invention also provides one or more antisense oligomers that, in a form suitable for delivery to a subject, assist in the preventive or therapeutic treatment, prevention or improvement of symptoms of psoriasis.

[0136] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and generally do not produce allergic reactions or similar adverse reactions, such as gastrointestinal upset, when administered to subjects. The term "carrier" refers to a diluent, adjuvant, excipient, or solvent administered with a compound. Such drug carriers can be sterile liquids, such as water and oils, including oils derived from petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water or saline solutions, as well as aqueous solutions of glucose and glycerol, are preferred as carriers, especially for injectable solutions. Suitable drug carriers are described in Martin's... Remington's Pharmaceutical Sciences , 18th edition, Mack Publishing Co., Easton, PA, (1990).

[0137] Pharmaceutical Composition

[0138] In one form of the invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of one or more antisense oligomers of the invention, along with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions include diluents with varying buffer contents (e.g., Tris-HCl, acetates, phosphates), pH, and ionic strength, as well as additives such as detergents and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol), and fillers (e.g., lactose, mannitol). This material can be incorporated into particulate formulations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Hyaluronic acid may also be used. These compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the proteins and derivatives of the invention. See, for example, Martin, Remington's Pharmaceutical Sciences, 18th edition (1990, Mack Publishing Co., Easton, PA 18042), pp. 1435-1712, incorporated herein by reference. The composition may be prepared in fluid form or may be a dry powder, such as a lyophilized form.

[0139] It should be understood that the pharmaceutical compositions provided according to the present invention can be administered in any manner known in the art. Preferably, the pharmaceutical compositions for administration are administered by injection, oral administration, topical administration, or via the pulmonary or nasal route. The antisense oligomers can be delivered via intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous administration. A suitable route can be determined by those skilled in the art based on the condition of the subject receiving treatment. Vascular or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are some non-limiting sites where the antisense oligomers can be introduced. Direct CNS delivery can be employed; for example, intraventricular or intrathecal administration can be used as a route of administration.

[0140] Since the ASO of the present invention is used to treat psoriasis, a local delivery route is preferred.

[0141] Formulations for topical application include those in which the oligomers of this disclosure are mixed with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine, myristoylphosphatidylcholine DMPC, distearylphosphatidylcholine), anion-positive (e.g., myristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropylDOTAP and dioleoylphosphatidylethanolamine DOTMA). For topical or other applications, the oligomers of this disclosure may be encapsulated in liposomes or may form complexes therewith, particularly cationic liposomes. Alternatively, the oligomers may be complexed with lipids, particularly with cationic lipids. Fatty acids and esters, their pharmaceutically acceptable salts, and their uses are further described in U.S. Patent No. 6,287,860 and / or U.S. Patent Application Serial No. 09 / 315,298, filed May 20, 1999.

[0142] In some embodiments, the antisense oligomers of this disclosure can be delivered percutaneously (e.g., by incorporating the antisense oligomers into, for example, an emulsion, wherein such antisense oligomers are optionally packaged into liposomes). Descriptions of such percutaneous and emulsion / liposome-mediated delivery methods for delivering antisense oligomers can be found in the art, for example, in U.S. Patent No. 6,965,025.

[0143] The antisense oligomers described herein can also be delivered via implantable devices. The design of such devices is a well-established process in the art, for example, the synthetic implant design described in U.S. Patent No. 6,969,400.

[0144] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, gel capsules, sachet powders, tablets, or microtablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersants, or binders may be desired. Oral formulations are those in which the oligomers of this disclosure are administered in combination with one or more penetration enhancers, surfactants, and chelating agents. Surfactants include fatty acids and / or their esters or salts, bile acids and / or their salts. Bile acids / salts and fatty acids and their uses are further described in U.S. Patent No. 6,287,860. In some embodiments, this disclosure provides combinations of penetration enhancers, such as combinations of fatty acids / salts with bile acids / salts. Exemplary combinations are sodium salts of lauric acid, decanoic acid, and UDCA. Other penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The oligomers of this disclosure may be delivered orally in particulate form (including spray-dried particles) or complexed to form micron or nanoparticles. Oligomeric complexes and their uses are further described in U.S. Patent No. 6,287,860. Oral formulations of oligomers and their preparation are described in detail in US 6,887,906 and / or US 20030027780.

[0145] Compositions and formulations intended for parenteral, intrathecal, or intracardiac administration may include sterile aqueous solutions and may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0146] Delivery of therapeutically useful amounts of antisense oligomers can be achieved by previously disclosed methods. For example, intracellular delivery of antisense oligomers can be carried out by a composition comprising an antisense oligomer and an effective amount of block copolymer. An example of this method is described in U.S. Patent Application US20040248833. Other methods for delivering antisense oligomers to the cell nucleus are described in Mann CJ et al., (2001) Proc, Natl. Acad. Science, 98(1) 42-47, and Gebski et al., (2003) Human Molecular Genetics, 12(15): 1801-1811. Methods for introducing nucleic acid molecules into cells in the form of naked DNA or complexed with a lipid carrier via an expression vector are described in US 6,806,084.

[0147] It may be necessary to deliver antisense oligomers in colloidal dispersions. Colloidal dispersions include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, liposomes, and liposome formulations. These colloidal dispersions can be used to prepare therapeutic pharmaceutical compositions.

[0148] Liposomes are artificial membrane vesicles used as delivery solvents in vitro and in vivo. These formulations can be characterized by net cations, anions, or neutral charges and possess useful features for in vitro, in vivo, and ex vivo delivery methods. It has been shown that large monolayer vesicles can encapsulate a considerable percentage of aqueous buffer solutions containing macromolecules. RNA and DNA can be encapsulated within an aqueous interior and delivered to cells in their biologically active form (Fraley et al., Trends Biochem. Sci. 6:77, 1981).

[0149] For liposomes to be effective gene transfer media, they should possess the following characteristics: (1) efficient encapsulation of target antisense oligomers without impairing their biological activity; (2) preferential and substantially binding to target cells compared to non-target cells; (3) efficient delivery of the aqueous contents of vesicles to the cytoplasm of target cells; and (4) precise and efficient expression of genetic information (Mannino et al., Biotechniques, 6:682, 1988). Liposome compositions are typically combinations of phospholipids, particularly high phase transition temperature phospholipids, often combined with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical properties 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 stable complexes. pH-sensitive or negatively charged liposomes are thought to capture DNA rather than complex with it. Both cationic and non-cationic liposomes have been used for DNA delivery to cells.

[0150] Liposomes also include “sterically stable” liposomes, a term herein referring to liposomes containing one or more specific lipids that, when incorporated into the liposome, result in an increased cycle life compared to liposomes lacking such specific lipids. Examples of sterically stable liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome contains one or more glycolipids or is derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) portions. Liposomes and their uses are further described in U.S. Patent No. 6,287,860.

[0151] The antisense oligomers described herein can also be delivered via implantable devices. The design of such devices is a well-established practice in the art, for example, synthetic implant designs are described in US 6,969,400, the contents of which are incorporated herein by reference in their entirety.

[0152] Antisense oligomers can be introduced into cells using techniques well-established in the art, such as transfection, electroporation, fusion, liposomes, colloidal polymer particles, and viral and nonviral vectors, as well as other methods known in the art. The chosen delivery method will depend at least on the cells to be treated and their location, and will be apparent to those skilled in the art. For example, localization can be achieved by liposomes with specific markers on their surface to guide the liposomes, direct injection into tissues containing target cells, specific receptor-mediated uptake, etc.

[0153] As is known in the art, antisense oligomers can be delivered using methods involving, for example, liposome-mediated uptake, lipid conjugates, polylysine-mediated uptake, nanoparticle-mediated uptake, and receptor-mediated endocytosis, as well as other non-endocytic delivery modalities such as microinjection, permeation (e.g., streptococcal hemolysin-O permeation, anionic peptide permeation), electroporation, and various non-invasive non-endocytic delivery methods known in the art (see Dokka and Rojanasakul’s review, Advanced Drug Delivery Reviews 44, 35-49, the full text of which is incorporated herein by reference).

[0154] Antisense oligomers can also be combined with other pharmaceutically acceptable carriers or diluents to prepare pharmaceutical compositions. Suitable carriers and diluents include isotonic saline solutions, such as phosphate-buffered saline. The composition can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, or transdermal administration.

[0155] The described routes of administration are for guidance only, as skilled practitioners will be able to easily determine the optimal route of administration and any dosage for any particular animal and condition.

[0156] Various methods have been explored for introducing novel functional genetic materials into cells in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These methods include integrating the gene to be expressed into a modified retrovirus (Friedmann (1989), ibid.; Rosenberg (1991) Cancer Research 51(18), Supplement: 5074S-5079S); integrating it into a non-retroviral vector (Rosenfeld et al., (1992) Cell, 68:143-155; Rosenfeld et al., (1991) Science, 252:431-434); or delivering the transgene linked to a heterologous promoter-enhancer element via liposomes (Friedmann (1989), ibid.; Brigham et al., (1989) Am. J. Med. Sci., 298:278-281; ​​Nabel et al., (1990) Science, 249:1285-1288; Hazinski et al., (1991) Am. J. Resp. Cell). Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); coupled to ligand-specific, cation-based transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624) or using naked DNA, expression vectors (Nabel et al., (1990), supra); Wolff et al., (1990) Science, 247:1465-1468). Brigham et al.'s group (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (Abstract)) reported in vivo transfection in mouse lungs only after intravenous or intratracheal administration of DNA liposome complexes. Examples of review articles on human gene therapy procedures include: Anderson, Science (1992) 256:808-813; Barteau et al., (2008), Curr Gene Ther; 8(5):313-23; Mueller et al., (2008).Clin Rev Allergy Immunol; 35(3):164-78; Li et al., (2006) Gene Ther., 13(18):1313-9; Simoes et al., (2005) Expert Opin Drug Deliv; 2(2):237-54.

[0157] The antisense oligomers of the present invention encompass any pharmaceutically acceptable salt, ester, or salt of such esters, or any other compound that, upon administration to animals, including humans, is capable of providing (directly or indirectly) its biologically active metabolites or residues. Therefore, by way of example, this disclosure also relates to prodrugs and pharmaceutically acceptable salts of the compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0158] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of the compounds of the present invention: that is, a salt that retains the desired biological activity of the parent compound and does not impart undesirable toxicological effects to the parent compound. Preferred examples of pharmaceutically acceptable salts for oligomers include, but are not limited to, (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, and polyamines such as spermine and spermidine; (b) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid; (c) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid; and (d) salts formed by elemental anions such as 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 required and the area to be treated. Administration can be local (including ocular and mucous membrane delivery, as well as rectal delivery), pulmonary (e.g., by inhalation or blowing of powders or aerosols (including via nebulizer, intratracheal, intranasal, epidermal, and percutaneous)), oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. Oligomers having at least one 2'-O-methoxyethyl modification are considered particularly useful for oral administration. Preferably, antisense oligomers are delivered via subcutaneous or intravenous routes.

[0159] The pharmaceutical formulations of the present invention can be conveniently presented in unit dosage forms and can be prepared according to conventional techniques known in the pharmaceutical industry. These techniques include the step of combining the active ingredient with a drug carrier or excipient. Generally, the preparation of the formulation involves uniformly and tightly combining the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0160] application

[0161] Antisense oligomers can be administered at regular intervals over short periods, such as daily, for two weeks or less. However, in some cases, oligomers are administered intermittently over longer periods. Antibiotics or other therapeutic treatments may be given after administration, or administered concurrently with antibiotics or other therapeutic treatments. The treatment regimen (dosage, frequency, route, etc.) may be adjusted as needed based on the results of immunoassays, other biochemical tests, and physiological examinations of the treated subject.

[0162] The dosage depends on the severity and responsiveness of the disease state being treated, and treatment may last from days to months, or until a cure or remission is achieved. The optimal dosing regimen can be calculated by measuring the accumulation level of the drug in the subject. Those skilled in the art can readily determine the optimal dose, method of administration, and frequency of repetition. The optimal dose may vary depending on the relative potency of the individual oligomers and is typically estimated based on EC50 values ​​effectively measured in in vitro and in vivo animal models. Generally, the dose may be administered once or more daily, weekly, monthly, or annually, or even once every 2 to 20 years. Those skilled in the art can readily estimate the frequency of repetition based on measured residence time and concentration of the drug in body fluids or tissues. Following successful treatment, maintenance therapy may be expected to prevent relapse of the disease state, wherein the oligomer is administered at a maintenance dose, once or more daily to once every 20 years.

[0163] Effective in vivo treatment regimens using the antisense oligomers of the present invention can vary depending on the duration of treatment, dosage, frequency, route of administration, and the condition of the treated subject (i.e., prophylactic administration versus administration in response to local or systemic infection). Therefore, such in vivo therapy typically requires monitoring with appropriate diagnostic methods tailored to the specific type of condition being treated, and adjustments to the dosage or treatment regimen accordingly to achieve optimal therapeutic outcomes.

[0164] Treatment can be monitored, for example, by common indicators of diseases known in the art. As used herein, “treatment” for a subject (e.g., a mammal, such as a human) or cell means any type of intervention designed to attempt to alter the natural processes of that individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and may be administered preventively or after the initiation of a pathological event or exposure to a pathogen. Treatment includes any desired effect on psoriasis symptoms or pathological processes and may include minor changes or improvements, such as in one or more measurable markers of the treated psoriasis. It also includes “preventive” treatments, which may be designed to reduce the rate of progression of the treated psoriasis, delay the onset of psoriasis, or reduce the severity of its onset. “Treatment” or “prevention” does not necessarily imply the complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0165] As used herein, "subject" includes any animal exhibiting or at risk of exhibiting symptoms that can be treated with the antisense compounds of this invention, or any symptoms associated with the condition (e.g., psoriasis). Suitable subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and domesticated animals or pets (e.g., cats or dogs). Non-human primates are included, with human subjects being preferred.

[0166] The therapeutic effect of the antisense oligomers administered in vivo according to the present invention can be determined by measuring biological samples (tissues, blood, urine, etc.) collected from subjects before, during, and after administration of the antisense oligomers. The analysis of these samples includes (1) monitoring the presence or absence of heteroduplex formation between the target and non-target sequences using methods known to those skilled in the art, such as electrophoretic gel mobility assays; and (2) monitoring the amount of mutant RNA relative to reference normal RNA or protein by standard techniques such as RT-PCR, Northern blotting, ELISA, or Western blotting.

[0167] Intranuclear oligomer delivery is a major challenge for antisense oligomers. Different cell-penetrating peptides (CPPs) localize PMOs to varying degrees under different conditions and cell lines, and the inventors have evaluated the ability of novel CPPs to deliver PMOs to target cells. The term CPP or “peptide portion that enhances cellular uptake” is used interchangeably herein to refer to a cationic cell-penetrating peptide, also known as a “transport peptide,” “carrier peptide,” or “peptide transduction domain.” As shown herein, the peptide has the ability to induce cell penetration in about or at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in a given cell culture population and allows for the translocation of macromolecules in various tissues in vivo after systemic administration. CPPs are well known in the art and are disclosed, for example, in US 20100016215, which is incorporated herein by reference in its entirety. Therefore, the present invention provides a combination of the antisense oligomers of the present invention with cell-penetrating peptides for the preparation of therapeutic pharmaceutical compositions.

[0168] According to another aspect of the invention, one or more antisense oligomers described herein are provided for use in antisense oligomer-based therapies. Preferably, the therapy is used to treat psoriasis.

[0169] More specifically, the antisense oligomer may be selected from any one or more of SEQ ID NO: 1-17, or combinations thereof or mixtures thereof. This includes sequences that hybridize to these sequences under strict hybridization conditions, sequences complementary to them, sequences containing modified bases, modified backbones, and sequences that are functionally truncated or extended, which have or regulate precursor RNA processing activity in the transcripts of target protein genes. More preferably, the antisense oligomer used in this invention is selected from the list containing SEQ ID NO: 2, 3, 5, or 12.

[0170] Preferably, the manipulation of protein expression of the present invention induced by antisense oligomers leads to the following results: S100A8: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); S100A9: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); IL1RL2: Exon 9 skipping (targeting the transmembrane domain, exon skipping); IL1RL2: Exon 10 or 11 skipping (targeting the Toll-interleukin-1 receptor (TIR) ​​homologous domain, exon skipping).

[0171] This invention also relates to combinations of two or more antisense oligomers capable of binding to selected targets to modify the splicing of target protein gene transcripts. The combinations may be mixtures of two or more antisense oligomers, or constructs comprising two or more antisense oligomers linked together, for use in antisense oligomer-based therapies.

[0172] This invention provides a method for treating, preventing, or improving the effects of psoriasis, comprising the steps of: a) Administering to a subject an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers as described herein.

[0173] The therapy can be used to generate nonfunctional, truncated, or nonsense target proteins, or to reduce target protein expression. The reduction in target protein levels is preferably achieved by: using exon skipping to reduce full-length transcript levels by binding to splicing sites, altering the binding of precursor mRNA splicing factors, and / or altering mRNA translation in or within the target protein gene transcript.

[0174] Alternatively, the present invention can induce an increase in RNA degradation by recruiting RNase H, wherein the RNase H preferentially binds to and degrades RNA that forms a double-stranded bond with the DNA of the target protein gene.

[0175] A reduction in target proteins will preferably lead to a decrease in the number, duration, or severity of psoriasis symptoms.

[0176] According to another aspect of the invention, the use of one or more antisense oligomers as described herein in the preparation of medicaments for regulating or controlling psoriasis is provided.

[0177] The present invention also provides the use of the purified and isolated antisense oligomers described herein in the preparation of a medicament for treating psoriasis.

[0178] The use of purified and isolated antisense oligomers as described herein in the preparation of medicaments for the treatment, prevention or improvement of the effects of psoriasis is provided.

[0179] Preferably, the antisense oligomers used in this invention are selected from the list including the following: SEQ ID NO: 1-17; or SEQ ID NO: 2, 3, 5 or 12.

[0180] Preferably, the manipulation of protein expression of the present invention induced by antisense oligomers leads to the following results: S100A8: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); S100A9: Spatial steric hindrance of exon 2 (targeting the translation initiation complex, translation blocking); IL1RL2: Exon 9 skipping (targeting the transmembrane domain, exon skipping); IL1RL2: Exon 10 or 11 skipping (targeting the Toll-interleukin-1 receptor (TIR) ​​homologous domain, exon skipping).

[0181] According to another aspect of the invention, the present invention relates to cDNA or clonal copies of the antisense oligomer sequence of the present invention, and vectors containing the antisense oligomer sequence of the present invention. The invention further relates to cells containing such sequences and / or vectors.

[0182] The present invention also provides a kit for treating, preventing or improving psoriasis in subjects, the kit comprising at least isolated or purified antisense oligomers for modifying precursor mRNA splicing factor binding or mRNA translation in a target protein gene transcript or a portion thereof, the kit being packaged in a suitable container and accompanied by instructions for use.

[0183] In a preferred embodiment, the kit will contain at least one antisense oligomer (SEQ ID NO: 1-17) or a mixture of antisense oligomers as described herein or in Table 1, as described herein. The kit may also contain peripheral reagents, such as buffers, stabilizers, etc.

[0184] Therefore, the present invention provides a kit for treating, preventing or improving psoriasis in subjects, the kit comprising at least the antisense oligomers described herein or the antisense oligomers in SEQ ID NO: 117, Table 1, and combinations or mixtures thereof, the kit being packaged in a suitable container and accompanied by instructions for use.

[0185] The present invention also provides a kit for treating, preventing or improving psoriasis in subjects, the kit comprising at least one or more antisense oligomers selected from SEQ ID NO: 1-17 and combinations or mixtures thereof, the kit being packaged in a suitable container and accompanied by instructions for use.

[0186] The kit components may also be provided in dry or lyophilized form. When reagents or components are provided in dry form, they are typically reconstituted by adding a suitable solvent. The kit may additionally contain a suitable solvent for reconstituted lyophilized components. The individual components of the kit may be packaged in separate containers. Regardless of the number or type of containers, the kit of the present invention may also contain or package an instrument for assisting in the injection / administration or placement of the final composite composition in an animal. Such an instrument may be an inhaler, syringe, pipette, forceps, measuring spoon, eye dropper, or any such medically approved delivery tool.

[0187] The kit may contain a notification in a format prescribed by a government agency that regulates the production, use, or sale of pharmaceuticals or biological products, reflecting that agency’s approval for the production, use, or sale for human use.

[0188] When the components of the kit are provided as one or more liquid solutions, the liquid solutions may be aqueous solutions, such as sterile aqueous solutions. For in vivo applications, the expression constructs may be formulated into pharmaceutically acceptable injectable compositions. In this case, the container device itself may be an inhaler, syringe, pipette, eye dropper, or other similar device from which the formulation may be applied to a diseased area of ​​the animal, such as the skin, injected into the animal, or even applied to and mixed with other components of the kit.

[0189] Those skilled in the art should understand that the above methods have a wide range of applications and can be used to identify antisense oligomers suitable for the treatment of a variety of other diseases.

[0190] The antisense oligomers of the present invention can also be used in combination with alternative therapies such as drug therapy.

[0191] Therefore, the present invention provides a method for treating, preventing or improving the effects of psoriasis, wherein the antisense oligomer of the present invention is administered sequentially or simultaneously with another alternative therapy associated with the treatment, prevention or improvement of psoriasis.

[0192] Alternative treatments for psoriasis may include topical therapies selected from the list containing the following: corticosteroids, vitamin D analogs, retinoids, calcineurin inhibitors, salicylic acid, coal tar, or dithranol. Alternative treatments for psoriasis may include phototherapy selected from the list containing the following: sunlight, broadband UVB, narrowband UVB, psoralen combined with long-wave ultraviolet radiation (PUVA), or excimer laser. Alternative treatments for psoriasis may include oral therapies selected from the list containing the following: steroids, retinoids, methotrexate, cyclosporine, and biologics such as monoclonal antibodies. Alternative treatments for psoriasis may be a combination of topical therapy, phototherapy, and / or oral therapy.

[0193] Overview

[0194] Those skilled in the art will understand that, in addition to those specifically described, the invention described herein is susceptible to variation and modification. This invention includes all such variations and modifications. The invention also includes all steps, features, formulations, and compounds individually or collectively mentioned or indicated in this specification, and any and all combinations of any two or more of said steps or features.

[0195] Every document, reference, patent application, or patent cited in this article is explicitly incorporated herein by reference in its entirety, meaning that the reader should read and consider it as part of this article. Documents, references, patent applications, or patents cited in this article are not repeated here solely for the sake of brevity.

[0196] Manufacturer's instructions, descriptions, product specifications, and product manuals for any product mentioned herein or referenced in any document herein are incorporated herein by reference and may be used in the practice of this invention.

[0197] The scope of this invention is not limited to any of the specific embodiments described herein. These embodiments are for illustrative purposes only. As described herein, functionally equivalent products, formulations, and methods are obviously within the scope of this invention.

[0198] The invention described herein may include one or more numerical ranges (e.g., dimensions, displacements, and field strengths). A numerical range will be understood to include all values ​​within that range, including the value defining the range and values ​​adjacent to the range that result in the same or substantially the same result as the value immediately adjacent to the boundary defining the range. Therefore, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximate values ​​and may vary depending on the desired properties sought to be obtained by the invention. Thus, “about 80%” means both “about 80%” and “80%”. At a minimum, each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods.

[0199] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations thereof such as “comprises” or “comprising” will be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes. It should also be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as “comprise” and its various parts of speech may have the meanings conferred upon them by U.S. patent law; for example, they may mean “comprising” and its various parts of speech, etc.; and terms such as “consisting essentially of” and “consists essentially of” also have the meanings conferred upon them by U.S. patent law, for example, they allow the inclusion of elements not expressly listed, but exclude elements present in the prior art or affecting the essential or novel features of the invention.

[0200] Other definitions of the selected terms used herein can be found in specific embodiments of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The term "activator" may refer to one activator or may include two or more activators.

[0201] The following examples are provided to more fully describe the ways in which the invention is used, and to illustrate the best mode contemplated for carrying out various aspects of the invention. It should be understood that these methods are not intended to limit the true scope of the invention, but are merely illustrative.

[0202] References

[0203] Koks, S. et al. Psoriasis-Specific RNA Isoforms Identified by RNA-Seq Analysis of 173,446 Transcripts. Front Med (Lausanne), 2016.3: Page 46.

[0204] Tsoi, LC et al. Large scale meta-analysis characterizes genetics architecture for common psoriasis associated variants. Nat Commun, 2017.8: Page 15382.

[0205] Example

[0206] Other features of the invention will be described in more detail in the following non-limiting embodiments. This description is included merely as an example and should not be construed as limiting the scope of the general description of the invention above.

[0207] Example 1

[0208] Antisense oligonucleotide-mediated translation blockade of S100A8 protein

[0209] To downregulate the expression of S100A8 protein, a steric hindrance strategy to block protein translation initiation using ASO was employed.

[0210] Antisense oligonucleotide design and synthesis: Antisense oligonucleotides targeting the ATG site at exon 2 of S100A8 (translation initiation complex) were designed (Table 2). These were purchased as phosphoryldiamine morpholino oligomers (PMO) from Gene Tools (Philomath, OR, USA).

[0211] Transfection using PMO – Neon transfection: Following the manufacturer's protocol, ASO (concentrations of 100 μm and 200 μm) was delivered into HACAT cells using the Neon transfection system and 10 μl of the kit. In short, cells were digested with trypsin, counted, washed with PBS, and centrifuged at 1 rcf for 3 min. The washed cells were resuspended in resuspension buffer R, with 3 × 10⁶ cells in 10 μl buffer per treatment. 5 Cells were collected. In a separate tube, 10 µl of the cell mixture was mixed with ASO and then aspirated into a 10 µl Neon® pipette tip using a Neon® pipette. The pipette and tip were then placed into a Neon® tube containing 3 ml of electrolysis buffer E and electroporated using a protocol optimized for HACAT, consisting of single pulses at 1700 volts and 10 ms intervals. The transfected cells were plated into 6-well plates containing Optimem and 1 ng / ml M5 cytokines (IL17A, TNFα, oncogene, IL22, and IL1α). Cells were collected at different time points (24–72 hours) for protein extraction and Western blotting.

[0212] Protein extraction and Western blotting: Cell pellets were lysed in lysis buffer containing 25% SDS, 1.25M Tris-HCl, glycerol, benzyl sulfonyl fluoride (PMSF), and a protease inhibitor. Samples were sonicated 6 times per 1-second pulse, heated to 94°C for 5 minutes, centrifuged at 14,000g for 2 minutes, and rapidly frozen on dry ice. Protein concentration was quantified using the BCA assay. 15 µg of total protein from each sample was loaded onto a NUPAGE Novex 4-12% Bis / Tris gel (Life Technologies) and electrophoresed at 200V for 55 minutes in 1X NuPAGE MOPS buffer. 3 μL of LiGacic Mark (Life Technologies) and 7 μL of Kaleidoscape (Biorad, NSW, Australia) molecular weight markers were loaded to monitor protein separation in the gel. Proteins were transferred to PVDF membranes using the P0 program on an iBlot 2 dry transfer system (Thermo Fisher Scientific) according to the manufacturer's instructions. The membrane was blocked for one hour in freshly prepared TBS-T containing 5% skim milk and incubated with primary antibodies against S100A8 and S100A9, as well as the corresponding secondary antibodies. Proteins were detected using an enhanced chemiluminescence substrate (Immobilon HRP chemiluminescence, Milipore) and incubated in the dark for 5 minutes. Images were captured using Fusion Capture Advance FX7 software on a Vilber Lourmat Fusion FX system (Vilber, Collegien, France).

[0213] Table 2: Translation-blocking ASOs and their sequences used for S100A8

[0214] result

[0215] Protein levels in ASO-treated cells at a concentration of 100 μM over 24 hours were compared with those in untreated (UN), gene tool control (GTC-negative control), and ZAP-treated (UN). Figure 1A -S100A8 H2A(+11+35) Figure 1B A significant contrast between -S100A8 H2A(+01+25)).

[0216] Example 2

[0217] Antisense oligonucleotide-mediated translation blockade of S100A9 protein

[0218] To downregulate S100A9 protein expression, a steric hindrance strategy was used to block protein translation initiation using ASO.

[0219] Antisense oligonucleotide design and synthesis: Antisense oligonucleotides targeting the ATG site at exon 2 of S100A9 (translation initiation complex) were designed (Table 2). These were purchased as phosphoryldiamine morpholino oligomers (PMO) from Gene Tools (Philomath, OR, USA).

[0220] Transfection using PMO – Neon transfection: Following the manufacturer's protocol, ASO (concentrations of 100 μm and 200 μm) was delivered into HACAT cells using the Neon transfection system and 10 μl of the kit. In short, cells were digested with trypsin, counted, washed with PBS, and centrifuged at 1 rcf for 3 min. The washed cells were resuspended in resuspension buffer R, with 3 × 10⁶ cells in 10 μl buffer per treatment. 5 Cells were collected. In a separate tube, 10 µl of the cell mixture was mixed with ASO and then aspirated into a 10 µl Neon® pipette tip using a Neon® pipette. The pipette and tip were then placed into a Neon® tube containing 3 ml of electrolysis buffer E and electroporated using a protocol optimized for HACAT, consisting of single pulses at 1700 volts with 10 ms intervals. The transfected cells were plated into 6-well plates containing Optimem and 12 ng / ml M5 cytokines (IL17A, TNFα, oncogene, IL22, and IL1α). Cells were collected at different time points (24–72 hours) for protein extraction and Western blotting.

[0221] Protein extraction and Western blotting: Cell pellets were lysed in lysis buffer containing 25% SDS, 1.25M Tris-HCl, glycerol, benzyl sulfonyl fluoride (PMSF), and a protease inhibitor. Samples were sonicated 6 times per 1-second pulse, heated to 94°C for 5 minutes, centrifuged at 14,000g for 2 minutes, and rapidly frozen on dry ice. Protein concentration was quantified using the BCA assay. 15 µg of total protein from each sample was loaded onto a NUPAGE Novex 4-12% Bis / Tris gel (Life Technologies) and electrophoresed at 200V for 55 minutes in 1X NuPAGE MOPS buffer. 3 μL of LiGacic Mark (Life Technologies) and 7 μL of Kaleidoscape (Biorad, NSW, Australia) molecular weight markers were loaded to monitor protein separation in the gel. Proteins were transferred to PVDF membranes using the P0 program on an iBlot 2 dry transfer system (Thermo Fisher Scientific) according to the manufacturer's instructions. The membrane was blocked for one hour in freshly prepared TBS-T containing 5% skim milk and incubated with the primary antibody against S100A9 and the corresponding secondary antibody. Protein detection was performed using an enhanced chemiluminescence substrate (Immobilon HRP chemiluminescence, Milipore) and incubated in the dark for 5 minutes. Images were captured using Fusion Capture Advance FX7 software on a Vilber Lourmat Fusion FX system (Vilber, Collegien, France).

[0222] Table 3: Translation-blocking ASOs and their sequences for S100A9

[0223] result

[0224] Protein levels in ASO-treated cells at a concentration of 100 μM over 24 hours were compared with those in untreated (UN), gene tool control (GTC-negative control), and ZAP-treated (UN). Figure 6 A striking contrast.

[0225] Example 3

[0226] Knockout of the transmembrane domain of the IL1RL2 subunit of the interleukin-36 receptor mediated by antisense oligonucleotides

[0227] To downregulate IL36R receptor expression, an ASO exon skipping strategy was employed to skip the transmembrane domain of the IL1RL2 subunit of IL36R and convert the membrane-bound form of IL1RL2 into a soluble form, thereby making it a decoy receptor.

[0228] Antisense oligonucleotide design and synthesis: Antisense oligonucleotides (AOs) were designed to anneal to receptor and donor splicing sites and potential splicing factor motifs predicted by computer simulations using SpliceAid. For exon hopping across the transmembrane domain, 2′-O-methylphosphothioester RNA (2′-OMePS)ASO was designed targeting exon 9 (transmembrane domain) of the IL1R2 subunit (Table 4).

[0229] use OMe-PS AO transfection: Initially screened cells were transfected in Opti-MEM™-reduced serum using Lipofectamine® 3000 transfection reagent. OMe-PS AO was delivered as a cationic liposome complex. The reagent was used according to the manufacturer's instructions. In short, 3 μl of Lipofectamine 3000 reagent and sufficient ASO (for initial screening) at final concentrations of 50 and 200 nM were incubated in 100 μl of Opti-MEM for 15 minutes to form the complex. The pre-transfection mixture was then diluted to 1 ml with Opti-MEM. The final transfection mixture was added to fibroblasts pre-seeded in 24-well plates 24 hours prior. Transfection was performed in duplicate, with 500 μl of the transfection mixture added to each well. The cells were then incubated again at 37°C and 5% CO2 for 24 hours.

[0230] RNA Extraction and RT-PCR Analysis: Total RNA was extracted using the MagMax™ Nucleic Acid Isolation Kit (ThermoFisher Scientific, Melbourne, Australia) according to the manufacturer's protocol. Total RNA concentration and purity were determined using an Implen nanophotometer. Transcripts were amplified using a one-step SuperScript® III reverse transcriptase assay with 50 ng of total RNA as a template. To observe exon 9 and 10 skipping, IL1RL2 transcripts were amplified using a forward primer for exon 7 and a reverse primer for exon 12 (Integrated DNA Technologies, IDT, NSW, Australia). Cycling conditions were as follows: 55°C for 30 min, 94°C for 2 min, followed by 29 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 1 min. PCR products were separated in Tris-acetate-EDTA buffer using a 2% agarose gel. Images were analyzed using ImageJ (version 1.8.0_112) imaging software (NIH, Bethesda, MD, USA) 156 to determine the relative density of amplicon.

[0231] Table 4: Exon skipping ASOs and their sequences for exon 9 of IL1RL2

[0232] result

[0233] ASO for skipping exon 9 showed that efficient skipping (approximately 77%) was achieved at a concentration of 50 nm. Figure 2A ).

[0234] Titration curves were plotted by transfecting cells with low concentrations (1, 3, 10, 30, and 100 nm) of ASO. At a concentration of 30 nM, approximately 91% hopping was achieved. Figure 3A ).

[0235] Example 4

[0236] Toll-interleukin-1 receptor of the IL1RL2 subunit of the antisense oligonucleotide-mediated interleukin-36 receptor (TIR) ​​homology domain knockout

[0237] To downregulate IL36R receptor expression, an ASO exon skipping strategy was used to skip the Toll-interleukin-1 receptor (TIR) ​​homologous domain of the IL1RL2 subunit of IL36R.

[0238] Antisense oligonucleotide design and synthesis: Antisense oligonucleotides (AOs) were designed to anneal to receptor and donor splicing sites and potential splicing factor motifs predicted by computer simulations using SpliceAid. For exon hopping across the transmembrane domain, 2′-O-methylphosphothioester RNA (2′-OMePS)ASOs were designed targeting exons 10 and 11 (transmembrane domain) of the IL1R2 subunit (Table 5).

[0239] use OMe-PS AO transfection: Initially screened cells were transfected in Opti-MEM™-reduced serum using Lipofectamine® 3000 transfection reagent. OMe-PS AO was delivered as a cationic liposome complex. The reagent was used according to the manufacturer's instructions. In short, 3 μl of Lipofectamine 3000 reagent and sufficient ASO (for initial screening) at final concentrations of 50 and 200 nM were incubated in 100 μl of Opti-MEM for 15 minutes to form the complex. The pre-transfection mixture was then diluted to 1 ml with Opti-MEM. The final transfection mixture was added to fibroblasts pre-seeded in 24-well plates 24 hours prior. Transfection was performed in duplicate, with 500 μl of the transfection mixture added to each well. The cells were then incubated again at 37°C and 5% CO2 for 24 hours.

[0240] RNA Extraction and RT-PCR Analysis: Total RNA was extracted using the MagMax™ Nucleic Acid Isolation Kit (ThermoFisher Scientific, Melbourne, Australia) according to the manufacturer's protocol. Total RNA concentration and purity were determined using an Implen nanophotometer. Transcripts were amplified using a one-step SuperScript® III reverse transcriptase assay with 50 ng of total RNA as a template. To observe exon 9 and 10 skipping, IL1RL2 transcripts were amplified using a forward primer for exon 7 and a reverse primer for exon 12 (Integrated DNA Technologies, IDT, NSW, Australia). Cycling conditions were as follows: 55°C for 30 min, 94°C for 2 min, followed by 29 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 1 min. PCR products were separated by 2% agarose gel electrophoresis in Tris-acetate-EDTA buffer. Images were analyzed using ImageJ (version 1.8.0_112) imaging software (NIH, Bethesda, MD, USA) 156 to determine the relative density of amplicon.

[0241] Table 5: Exon skipping ASOs and their sequences used for exon 10 of IL1RL2

[0242] result

[0243] The TIR domain ASO designed for exon 10 (TIR domain) of the IL1R2 subunit exhibits a hopping efficiency of approximately 60%–73% at a concentration of 50 nM. Figure 4A ).

[0244] Titration curves plotted by transfecting cells with lower concentrations (1, 3, 10, 30, and 100 nM) of ASO showed that at a concentration of 30 nM, the skipping efficiency was mainly 70-90%. Figure 4B At least a portion of the TIR domain was hopped.

Claims

1. An isolated or purified antisense oligomer (ASO) for modifying precursor mRNA splicing or mRNA translation of one or more proteins encoded by the corresponding gene transcript or a portion thereof: S100A8; S100A9; and / or IL1RL2。 2. The antisense oligomer of claim 1, wherein the modified precursor mRNA splicing or mRNA translation results in one or more of the following outcomes: The production of truncated proteins; The production of proteins lacking functional regions; and / or The total amount of protein produced is reduced.

3. The antisense oligomer according to claim 1, wherein the antisense oligomer is phosphoryl diamine morpholino oligomer (PMO) or 2'-O-methyl phosphate thioester oligomer (2'-OMePS).

4. The antisense oligomer of claim 1, wherein the antisense oligomer is selected from the group consisting of the following sequences: i. The sequences shown in Table 1; ii. A list containing SEQ ID NO: 1-17; or iii. A list containing SEQ ID NO: 2, 3, 5 or 12.

5. A vector containing the antisense oligomer sequence of claim 1.

6. Cells containing the antisense oligomer sequence of claim 1 and / or the vector of claim 5.

7. A method for manipulating the binding of splicing factors in the transcript of a target protein gene, the method comprising the steps of: a) Provide one or more antisense oligomers according to claim 1, and bind the oligomers to a target nucleic acid site.

8. A medicament composition for treating, preventing, or improving the effects of psoriasis in a subject, the composition comprising: a) one or more antisense oligomers according to claim 1; and b) One or more pharmaceutically acceptable carriers and / or diluents.

9. A method for treating, preventing, or improving the effects of psoriasis on a subject, the method comprising the steps of: a) Administering to a subject an effective amount of one or more antisense oligomers according to claim 1 or a pharmaceutical composition according to claim 8.

10. Use of one or more of the purified and isolated antisense oligomers according to claim 1 in the preparation of a medicament for treating, preventing or improving the effects of psoriasis.

11. A kit for treating, preventing, or improving the effects of psoriasis in a subject, said kit comprising... b) one or more antisense oligomers according to claim 1; and c) Instruction manual.

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