Method for targeting kit with splice switching oligonucleotides to induce apoptosis of mast cells

Abstract

To provide compositions and methods for modulating Kit biological activities.SOLUTION: Provided are antisense oligomers having 10 to 50 linked nucleotides, wherein the antisense oligomer is targeted to a region of a Kit-encoding pre-mRNA, and wherein the targeted region includes sequences involved in splicing of the Kit-encoding pre-mRNA. Also provided are expression vectors encoding the antisense oligomers, morpholino oligomer derivatives of the antisense oligomers, and pharmaceutical compositions comprising the antisense oligomers, the expression vectors, and / or the morpholino oligomers. Further provided are methods for modulating splicing of a Kit pre-mRNA in a cell and / or a tissue, including induction of apoptosis in mast cells, and treating diseases, disorders, and / or conditions associated with Kit expression.SELECTED DRAWING: Figure 1A

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This invention claims priority to U.S. Provisional Patent Application No. 62 / 723,326, filed August 27, 2018. and is incorporated herein by reference in its entirety. This invention was made in part through support of the U.S. Government under Grant No. ES025128 awarded by the National Institutes of Health. This invention was made with support from the United States Government. As such, the United States Government has certain rights in this invention. [Technical Field]

[0002] In some embodiments, the present invention provides a method for inducing apoptosis of mast cells. Splice-switching oligonucleotides were used to isolate the genomic locus of the Kit gene. targeting the Kit gene product, including, but not limited to, the RNA transcripts In some embodiments, the present invention relates to the treatment of mast cells and Kit-associated tumors. Applications include selective targeting of disease. [Background technology]

[0003] c-Kit is a proto-oncogene that is highly conserved among various species and is a receptor-type tyrosine kinase. Kit (CD117), a cytokine kinase, and / or the mast cell growth factor receptor (SCFR Kit encodes the blastocysts (blastocysts) that encode ... It plays a role in the proliferation, survival and differentiation of bone marrow-derived hematopoietic stem cells (Pittoni et al. (Reviewed by , 2011). Kit expression is lost in most hematopoietic cells during differentiation. However, mast cells (MCs) maintain Kit expression throughout their lifespan, and Kit signaling is It remains essential for the survival and proliferation of mast cells (MCs) (Tsai M et al., 1991; Mekori et al ., 1993; Iemura et al., 1994; Galli et al., 1995). , whose viral counterpart, v-Kit, mediates transformation of Hardy-Zuckerman IV feline sarcoma virus. It was first recognized when it was found to be involved in the activity of Subsequently, activating gain-of-function c-Kit mutations are involved in the initiation and progression of several human malignancies. It is associated with gastrointestinal stromal tumors (GIST) and other inflammatory diseases such as mastocytosis and mast cell (MC) leukemia. The incidence is particularly high in MC proliferative disorders (reviewed by Cruse et al., 2014). (Yew).

[0004] Mastcytosis is a rare disease characterized by the abnormal expansion and accumulation of mast cells (MCs). It is a heterogeneous group of neoplastic conditions in humans (Valent et al., 2017). Treatment is challenging due to the wide biological heterogeneity of disease subtypes, all of which are associated with obesity. It is characterized by neoplastic proliferation of MC cells (MCs), but shows clear clinical symptoms and therapeutic responses (Valent et al. l., 2017). Current frontline treatments for mastocytosis and Kit-driven tumors include chemotherapy and These include tyrosine kinase inhibitors (TKIs) (Gleixner et al., 2006; Gotlib et al., 201 6) However, these treatments are not always effective in advanced disease and do not provide complete relief or improve the prognosis. Furthermore, nonspecific responses to chemotherapy and tyrosine kinase inhibitors (TKIs) are rare. Due to their nature, there is a high risk of off-target toxicity (Jensen et al., 2008). A Kit-specific therapeutic approach with a better safety profile in the field There is still a need for development.

[0005] The Kit gene product, which is strongly associated with cancer development and abnormal mast cell (MC) growth, is promising. Human c-Kit, located on human chromosome 4q12 (Giebel et al., 1992), is a challenging therapeutic target. It contains 10 exons and is susceptible to a range of mutations. The Kit ligand, stem cell factor ( This is a gain-of-function mutation that allows tyrosine kinase activity independent of SCF (Cruse et al. (Reviewed by Arock et al., 2015; Arock et al., 2014). Wild-type Kit has an SCF-binding extracellular domain. It has several distinct domains, including a transmembrane domain, an autoinhibitory juxtamembrane domain, and an autoantibody domain. It consists of two intracellular tyrosine kinase domains capable of autophosphorylation (Cruse et al. l., 2014; Arock et al., 2015). Under normal circumstances, mast cell (MC) survival, proliferation, and differentiation are Kit-SCF interaction is required for activation, but the presence of activating mutations abolishes SCF dependence, Oncogenic Kit signaling promotes the growth of neoplastic mast cells (MCs).

[0006] Mutations are known to occur throughout the Kit gene, but most of these occur in They are concentrated in hot spots and are associated with various diseases (Cruse et al., 2014). For example, mutations in exon 11, which encodes the juxtamembrane domain of Kit, are commonly found in gastrointestinal stromal tumors (GI stromal tumors). ST) (Taniguchi et al., 1999; Miettinen et al., 2002), but the second kinase The D816V mutation in exon 17, which encodes the main nucleotide polymorphism, is responsible for the majority of patients with systemic mastocytosis. (Arock et al., 2015). The type of mutation and the occurrence of further mutations are It determines the success of treatment for IT-related malignancies, assesses the prognosis of the disease, and identifies mutations in patients. Identifying obesity is important for determining treatment approaches, especially for adult generalized obesity. The missense mutation D816V, found in over 80% of cases of cytosis (Arock et al., 2015), is a Ki This induces a conformational change in Kit, which inactivates the ATP-binding site of Kit. Conformation-targeting imatinib mesylate (Gleevec; Ma et al., 2002; Parda It is resistant to tyrosine kinase inhibitors (TKIs) such as erythropoietin (L.Nani et al., 2003).

[0007] Heterogeneity of c-Kit mutations in Kit-associated malignancies and some embodiments In some cases, the occurrence of mutations that confer resistance to the TKIs of the present invention has led to In embodiments, described herein are methods for targeting Kit expression as a therapeutic approach. The compositions and methods disclosed herein are based on standard siRNA approaches to silencing Kit. However, it is inefficient in vivo (especially in vivo) (Wu et al., 2012; Yang et al., 2013). In some embodiments of the present invention, aberrant splicing of pre-mRNA (exon Chemically stable antisense oligonucleotides (ASOs) that induce nucleotide skipping and other nucleotide-dependent ... is employed to alter the expression of the c-Kit gene product. In this study, exon-skipping oligonucleotides (ESOs) called KitSop were used. , which inhibits c-Kit expression by introducing a frameshift into the mature c-Kit mRNA transcript. KitStop targets the expression of the in vivo downregulates both wild-type and mutant Kit expression in mast cells (MCs) in vitro This prevents proliferation and induces rapid cell death. When administered to the skin, it significantly reduces the number of mast cells (MCs) in these tissues in vivo. These data suggest that KitStop ESO can deplete mast cells (MCs) in animals. This demonstrates that the antibody can be used to treat Kit-associated malignancies, serving as proof-of-principle for therapeutic utility. Recently, there has been interest in the treatment of Duchenne muscular dystrophy (DMD; Dowling, 2016; Syed, 2016). ESO eteplirsen has been approved by the Food and Drug Administration for the treatment of In view of this, the present invention provides therapeutic methods that target Kit. Summary of the Invention [Problem to be solved by the invention]

[0008] The state of the art is the first to develop Kit-specific therapeutic approaches with a better safety profile. There is still a need for development of approaches. [Means for solving the problem]

[0009] This specification lists several embodiments of the invention, and in many cases, these embodiments Various variations and permutations are listed. This specification is merely illustrative of the many different embodiments. Mention of one or more representative features of a given embodiment is also exemplary. Such embodiments may generally exist with or without the recited features. Similarly, these features may be incorporated into the present invention whether or not they are described in this specification. The present invention is applicable to other embodiments of the present invention. This does not illustrate or suggest all possible combinations of such features.

[0010] In some embodiments, the present invention provides an amphoteric nucleotide sequence comprising 10 to 50 linked nucleotides. an antisense oligomer comprising a pre-mRNA encoding Kit; The target region is a region of the pre-mRNA encoding Kit. In some embodiments, antisense oligomers are provided that contain sequences involved in isolating. In this manner, the antisense oligomer binds to the pre-mRNA encoding Kit. Hybridization alters the splicing of the pre-mRNA. In some embodiments, the antisense oligomer is directed to a pre-mRNA encoding Kit. Hybridization of the nucleotides reduces the expression of the Kit protein. In the method, the Kit protein whose expression is reduced is a wild-type Kit protein or In some embodiments, the target region is a mutant Kit protein. intron sequences, exon sequences, sequences containing intron / exon junctions, splice sequences splice donor sequence, splice acceptor sequence, splice enhancer sequence, splice fragment a polynucleotide sequence selected from the group consisting of a branch point sequence, or a polypyrimidine tract In some embodiments, the polynucleotide sequence comprises at least a portion of: exon 4 splice donor sequences. The pre-mRNA encoding Kit is transcribed from the c-Kit gene. In some embodiments, the Kit protein is a human Kit protein, a mouse Kit protein, or a Kit protein, canine Kit protein, feline Kit protein, and equine protein. In some embodiments, the c-antisense oligomer is selected from the group consisting of: Hybridization to Kit pre-mRNA lacks at least part of exon 4 In some embodiments, the amplified c-Kit mRNA molecule is generated. Hybridization of antisense oligomers to c-Kit pre-mRNA resulted in the identification of truncated forms of the In some embodiments, this results in the production of an mRNA molecule encoding the Kit protein. and the oligonucleotide is pre-assembled so that it specifically hybridizes to the target sequence. The 10 to 50 linked nucleotides are linked to a target nucleus in the pre-mRNA encoding Kit. In some embodiments, the nucleic acid sequence includes a target-directed nucleic acid sequence that is sufficiently complementary to the nucleic acid sequence. Hybridization of the antisense oligomer to the pre-mRNA encoding Kit In some embodiments, the addition alters the splicing of the pre-mRNA. hybridization of the antisense oligomer to the pre-mRNA encoding Kit; In some embodiments, the pre-transfection reduces the expression of the Kit protein. The Kit protein whose expression is reduced is a wild-type Kit protein or a mutant Kit protein. It is a protein.

[0011] In some embodiments, the target-directed sequence comprises at least 6 of the target sequence. It contains at least six consecutive nucleobases that are perfectly complementary to two consecutive nucleobases. In some embodiments, the target-directed sequence is a sequence that is identical in length to the target sequence. is at least 80% complementary to a similarly sized stretch of consecutive nucleobases of In some embodiments, the target region comprises an intron sequence, an exon sequence, ... Sequences containing intron / exon junctions, splice donor sequences, and splice acceptors -sequence, splice enhancer sequence, splice branch point sequence, or polypyrimidine tran Some of the polynucleotide sequences include at least a portion of a polynucleotide sequence selected from the group consisting of: In one embodiment, the polynucleotide sequence comprises: (exon 4 splice donor sequence Kit exons 2 to 20, including, but not limited to, In some embodiments, the Kit-encoding pre-m The RNA is transcribed from the c-Kit gene. The proteins are human Kit protein, mouse Kit protein, and canine Kit protein. , feline Kit protein, and equine protein.

[0012] In some embodiments, the target sequence is SEQ ID NO: 18-22 (optionally, SEQ ID NO: Nos. 23 to 60) In some embodiments, the portion is at least 10 contiguous nucleotides. In some embodiments, the target sequence is SEQ ID NO: 18-22 (optionally, The sequence contains a sequence that is at least 90% identical to a partial sequence of SEQ ID NOs: 23 to 60. In one embodiment, the target sequence is selected from the group consisting of the reverse complements of SEQ ID NOs: 1, 2, and 23-60. In some embodiments, the targeting sequence comprises a sequence selected from the sequence In the column, from the group consisting of SEQ ID NO: 1, 2 and the reverse complement of one of SEQ ID NOs: 23 to 60 At least one nucleic acid sequence identical to at least 10 consecutive nucleic acid bases in the selected sequence In some embodiments, the targeting sequence comprises 10 consecutive nucleobases. , a reverse of at least a part of a partial sequence of SEQ ID NOs: 18 to 22 (optionally SEQ ID NOs: 23 to 60) In some embodiments, the sequence is at least 80% complementary to the complement. The target-directed sequence is a reverse complement of one of SEQ ID NOs: 1, 2 and 23 to 60. are at least 80% identical over the entire length of a sequence selected from the group consisting of: In an embodiment, the targeting sequences are selected from the group consisting of SEQ ID NOs: 1, 2 and 23-60. and the reverse complement of one of the following:

[0013] In some embodiments, the c-Kit transcript is selected from SEQ ID NOs: 8, 10, 12, 1 4 and 16, or any open reading frame present therein. In some embodiments, the antisense oligomer is an antisense RNA molecule. In some embodiments, the antisense RNA molecule is a nucleotide Modifications, internucleotide modifications, sugar modifications, sugar-nucleotide bond modifications, and combinations thereof In some embodiments, the antisense oligonucleotide comprises a modification selected from the group consisting of: The oligomer is a morpholino oligomer. In some embodiments, the present invention provides a method for producing an antisense oligomer comprising coating the antisense oligomers disclosed herein. In some embodiments, the present invention provides an expression vector for encoding the antisense oligomers, expression vectors disclosed herein, and / or morpholinos disclosed herein Pharmaceutical compositions containing the oligomers are provided. In some embodiments, the present invention provides a method for detecting a signal encoding Kit in cells and / or tissues. In some embodiments, methods for regulating splicing of a pre-RNA are provided. The present invention relates to a method for treating the cells and / or tissues with an antisense oligomer disclosed herein, contacting the cells with an expression vector disclosed herein and / or a morpholino oligomer disclosed herein; A method is provided that includes the steps of:

[0014] In some embodiments, the present invention provides a method for inducing apoptosis in mast cells. In some embodiments, the present invention provides a method for the treatment of mast cells. The disclosed antisense oligomers, the expression vectors disclosed herein, and / or the contacting the antibody with a morpholino oligomer. In some embodiments of the invention, the method is performed in an individual. In some embodiments, the present invention provides a method for detecting a disease, disorder, or condition associated with Kit expression in an individual. In some embodiments, the method comprises: administering to the individual an antisense oligomer disclosed herein, an expression vector disclosed herein, and and / or administering a morpholino oligomer disclosed herein. wherein the disease, disorder, or condition associated with Kit expression is cancer or mastocytosis. In some embodiments, the cancer is gastrointestinal stromal tumor or leukemia. In some embodiments, the individual is an animal, optionally a mammal. wherein the individual is a human, mouse, dog, cat or horse. Therefore, an object of the present invention is to provide a splice switch for inducing apoptosis of mast cells. US201301226633A1 - Method for targeting Kit gene products using linking oligonucleotides and mast cells - Google Patents and to provide a method for selectively targeting Kit-associated neoplastic diseases. The object of the present invention is to provide a method for treating a variety of conditions, including the use of a steroid hormone, which is a hormone that is produced by the steroid hormone and which ... The present invention is fully or partially accomplished as best described below in connection with the accompanying drawings. It will become clear as time goes on. [Brief explanation of the drawings]

[0015] The accompanying drawings, which are incorporated by reference and constitute a part of this specification, illustrate the disclosed Several representative embodiments of the present invention are illustrated, along with descriptions illustrating compositions and methods that utilize the present invention. do. [Figure 1]Figure 1A shows exon-skipping oligonucleotide (ESO)-mediated alternative splicing of exon 4 in c-Kit pre-mRNA. In Figure 1A, KitStop ESO was designed to target the donor splice site of exon 4, leading to spliceosome-mediated exon 4 exclusion. This is predicted to introduce a premature stop codon, resulting in a truncated mRNA transcript, even in the presence of the G560V and D816V activating mutations located downstream of the KitStop target site. Boxes in the figure represent exons, and thick, black bars represent introns. "FL-c-Kit" indicates full-length c-Kit, and "tc-Kit" indicates truncated c-Kit. Figure 1B and 1C show gel electrophoresis data demonstrating splice switching of wild-type and mutant c-Kit by KitStopESO compared with a standard control antisense oligonucleotide (ASO) (Stndcon) in HMC-1.2 (Figure 1B) and LAD2 cells (Figure 1C), respectively, as assessed by analysis of total RNA by RT-PCR. Black arrows indicate full-length c-Kit, and gray arrows indicate alternatively spliced c-Kit. [Figure 2]Transfection of KitStop ESO results in loss of wild-type Kit expression. LAD2 cells were transfected with 10 μM of a standard control ASO (Stndcon) or KitStop ESO, followed by flow cytometry to assess Kit expression. Figure 2A-B shows flow cytometry histograms of surface (A) and total (B) wild-type Kit expression 2 days (upper panel) and 7 days (lower panel) after transfection with KitStop ESO. Figure 2C-D shows average flow cytometry data for surface (C) and total (D) Kit expression, calculated from the geometric MFI and expressed as a percentage of the standard control antisense oligonucleotide (Stndcon). Data are means ± SEM from three independent experiments. *** p < 0.001, **** p < 0.0001 by analysis of variance (ANOVA) with Sidak's post-hoc test. Figure 2EF shows a representative flow cytometry gating strategy and plots for LAD2 cells labeled with anti-human CD117 antibody. After transfecting LAD2 cells with 10 μM KitStop ESO, Kit expression in LAD2 cells was assessed by flow cytometry. Representative density plots and gates for live (E) and fixed and permeabilized (F) LAD2 cells labeled with APC-conjugated anti-human CD117 antibody are shown. In the figure, "Stndcon" indicates the standard control ASO, "I" indicates the isotype, "S" indicates Stndcon, and "K" indicates KitStop. [Figure 3]This figure shows that transfection of KitStop ESO abolishes mutant Kit expression. HMC-1.2 cells were transfected with 10 μM of standard control ASO (Stndcon) or KitStop ESO, and then Kit expression was assessed by flow cytometry. Figure 3A shows flow cytometry histograms of total Kit expression 24 hours (upper panel), 48 hours (middle panel), and 72 hours (lower panel) after transfection with KitStop ESO. Figure 3B shows the mean flow cytometry data for total Kit expression calculated from the geometric mean fluorescence intensity (MFI). Figure 3C shows the mean flow cytometry data for total Kit expression expressed as a percentage (%) of the standard control (Stndcon) ASO. Data are means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by analysis of variance (ANOVA) with Sidak's post-hoc test. Figure 3D-E shows a representative flow cytometry gating strategy and plots for HMC-1.2 cells labeled with anti-human CD117 antibody. Figure 3D-E shows a representative density plot and gate for fixed and permeabilized HMC-1.2 cells transfected with 10 μM KitStop ESO and labeled with APC-conjugated anti-human CD117 antibody after 24 hours (D), 48 hours (E), and 72 hours (E). KitStop-treated cells exhibit decreased Kit expression, decreased forward scatter, and cell shrinkage. "Stndcon" indicates standard control ASO, "I" indicates isotype, "S" indicates Stndcon, and "K" indicates KitStop. [Figure 4]KitStop reduces constitutive KIT signaling in HMC-1.2 cells. Figure 4A shows immunoblots of constitutive KIT and ERK phosphorylation in HMC-1.2 cells 24 hours after KitStop treatment, with data from three independent experiments from left to right. Figures 4B and 4C show combined phosphorylation data normalized to control cell phosphorylation for pKIT (B) and pERK (C), respectively, after correction for β-actin or total ERK. Data are means ± SEM from three independent experiments. *p < 0.05 using Student's paired t-test. [Figure 5]This figure shows that transfection of KitStop ESO increases apoptosis in HMC-1.2 cells. HMC-1.2 cells were transfected with 10 μM of standard control ASO (Stndcon) or KitStop ESO, and then stained with Annexin V-FITC to assess apoptosis in HMC-1.2 cells by flow cytometry. Figure 5A is a histogram showing the shift in Annexin V-positive staining in KitStop-transfected cells compared to Stndcon and unstained cells. Figure 5B shows the combined Annexin V flow cytometry data expressed as geometric MFI. Figures 5C-D show the percentage of HMC-1.2 cells within the non-apoptotic (C), early apoptotic (D), and late apoptotic (E) gates at each time point. Data are means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by analysis of variance (ANOVA) with Sidak's post-hoc test. Figure 5F-G shows a representative flow cytometry gating strategy and plots of HMC-1.2 cells stained with Annexin V-FITC. Figure 5F shows the gating strategy and representative density plots of viable, untreated HMC-1.2 cells, either unstained or stained with Annexin V-FITC. Figure 5G shows flow cytometry density plots of Annexin V staining intensity of HMC-1.2 cells at various time points after transfection with a standard control ASO (Stndcon) or KitStopESO. Apoptotic cells are smaller as measured by forward scatter (X-axis) and Annexin V positive (Y-axis). "U" indicates no label, "S" indicates Stndcon, and "K" indicates KitStop. [Figure 6]This figure shows that transfection of KitStop exon-skipping oligonucleotides (ESOs) reduces the viability of HMC-1.2 cells. HMC-1.2 cells were transfected with 10 μM of a standard control ASO (Stndcon) or KitStop ESO, and then stained with propidium iodide (PI) to assess viability by flow cytometry. Figures A–D show the percentages of PI-negative and -positive cells at 24 h (A), 48 h (B), and 72 h (C–D). Figure 6E shows flow cytometry histograms of HMC-1.2 cells stained with LIVE / DEAD Green Dead Cell stain at 24 h (left), 48 h (center), and 72 h (right). The combined geometric MFI of LIVE / DEAD staining of HMC-1.2 cells at each time point is shown. Data are means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by analysis of variance (ANOVA) with Sidak's post-hoc test. Figure 6F shows a representative flow cytometry gating strategy and plot for HMC-1.2 cells stained with propidium iodide. Figure 6F shows a representative density plot for HMC-1.2 cells stained with propidium iodide (PI). Figure 6GH shows flow cytometry density plots of PI staining intensity for HMC-1.2 cells at various time points after transfection with standard control ASO (Stndcon) (G) or KitStop ESO (H). Figure 6IJ shows the flow cytometry gating strategy for HMC-1.2 cells using LIVE / DEAD staining. The figure shows representative density plots showing gating for viable HMC-1.2 cells and single cell populations (I) and gating for untreated cells after LIVE / DEAD Green Dead Cell staining (J). "U" indicates no label, "S" indicates Standcon, and "K" indicates Kit Stop. [Figure 7]This figure shows that transfection of KitStop ESO reduces HMC-1.2 cell proliferation. HMC-1.2 cells were transfected with 10 μM of a standard control ASO (Stndcon) or KitStop ESO, and proliferation was assessed by cell counting and flow cytometry. Figure 7A shows the total number of viable HMC-1.2 cells cultured under normal conditions, as assessed by trypan blue counting. Figure 7B shows representative density plots of HMC-1.2 cells loaded with CellTrace and stained with LIVE / DEAD Green Dead Cell stain 24 hours (left), 48 hours (center), and 72 hours (right) after transfection with Stndcon ASO (top panel) or KitStop ESO (bottom panel). Stndcon-transfected cells show a loss of CellTrace fluorescence intensity due to dye dilution between daughter cells, indicating cell proliferation. In contrast, KitStop-transfected cells retained CellTrace fluorescence and showed increased LIVE / DEAD staining intensity, except for a small population after 72 hours. Figure 7C shows the percentage of total cells in the lower left quadrant (CellTrace low; LIVE / DEAD negative), corresponding to proliferating cells. Figure 7D shows the percentage of total cells in the upper right and lower right quadrants, corresponding to non-proliferating cells (CellTrace high). Figure 7E shows the percentage of total cells in the upper left and upper right quadrants (LIVE / DEAD+), corresponding to non-viable cells. Data are means ± SEM from three independent experiments. * p < 0.05, *** p < 0.001, **** p < 0.0001 by ANOVA with Sidak's post-hoc test. [Figure 8]KitStop ESO reduces wild-type Kit expression in mouse mast cells in vitro and in vivo. Figure 8A shows RT-PCR demonstrating splice switching of wild-type c-Kit by mouse KitStop ESO in bone marrow-derived mast cells (BMMCs) compared with the standard control ASO (Stndcon). Black arrows indicate full-length c-Kit, and gray arrows indicate alternatively spliced c-Kit. Figure 8B shows flow cytometry histograms of surface wild-type Kit expression in bone marrow-derived mast cells (BMMCs) after transfection with KitStop ESO. Data are representative of three independent experiments in separate mice. Figure 8C shows the average flow cytometry data for surface Kit expression calculated from geometric MFI and expressed as a percentage of Stndcon ASO. Data are means ± SEM from three independent experiments in separate mice. *p <0.01 by paired t-test. Figure 8D shows the timeline of intraperitoneal delivery of KitStop VivoMorpholinos. Figure 8E shows RT-PCR of c-Kit expression in cells collected by peritoneal lavage, with evidence of splice switching. Black arrows indicate full-length c-Kit; gray arrows indicate alternatively spliced c-Kit. Figures 8F-G show representative flow cytometry density plots of peritoneal cells collected from mice treated with Stndcon ASO (F) or KitStop ESO (G) VivoMorpholinos. Figure 8H shows the percentage of mast cells identified as Kit and FcεRI double-positive cells (upper right quadrants of Figures 8F and 8G) collected by peritoneal lavage after treatment with Stndcon ASO or KitStop ESO VivoMorpholinos. Each data point represents a different mouse, and data are combined from two independent experiments (p values from unpaired t-tests). "I" indicates isotype, "S" indicates Stndcon, and "K" indicates KitStop. [Figure 9]KitStop ESO reduces the number of cutaneous mast cells in mice in vivo. Figure 9A shows the timeline of cutaneous injections of KitStop VivoMorpholinos. In Figure 9B, representative sections from standard control ASO (Stndcon) and KitStop ESO-treated groups show easily identifiable mast cells by the presence of positively stained metachromatic granules (see arrowheads), imparting a deep purple hue. In Figure 9C, hematoxylin and eosin (H&E)-stained skin histology from Stndcon ASO- and KitStop ESO-treated mice showed normal morphology and no signs of pathology. Figure 9D shows a plot of the number of dermal mast cells per mm2 from toluidine blue-stained skin sections taken from skin treated with Stndcon ASO or KitStop ESO. Figure 9E shows a plot of the number of mast cells per mm2 from full-thickness skin sections, including the dermis and subcutaneous adipose tissue surrounding the adnexal structures. Each dot represents the average mast cell count across specimens. P values are from unpaired t-tests. [Figure 10] This figure shows that systemic delivery of KitStop ESO inhibits tumor growth in a humanized xenograft mast cell tumor model. Figure 10A shows a schematic diagram of the xenograft model protocol used. Figure 10B shows measurements of tumor volume over time during treatment. Arrows indicate days when KitStop or vehicle control was administered. Figure 10C shows the weight of tumors excised after euthanasia of mice on day 14. Figure 10D shows a photograph of tumors after euthanasia of mice on day 14. Measurements are in centimeters. Figure 10E shows spleen weights at the end of the experiment. Figure 10F shows liver weights at the end of the experiment. Figure 10G shows that mouse weights were monitored over the course of the experiment. No significant differences were observed over the course of the experiment. Data are mean ± SEM from five mice per group. *p <0.05, **p <0.01, ***p <0.001. Analysis of variance (ANOVA) used Dunnett's post-hoc test (FIG. 10B) or unpaired t-test (FIGS. 10C and 10E). A brief description of arrays

[0016] SEQ ID NO: 1 is an exemplary human c-Kit gene product (GENBANK® Biosequence Database) KitStop ES designed to target exon 4 of NM_000222.2) O, which targets a region within the splice donor site. Number 1 is represented by the GENBANK® Biosequence Database accession number NM_000222.2 The human KIT gene product contains 6 nucleotides at the 3' end of exon 4 and 19 nucleotides in the downstream intron. SEQ ID NO: 1 is designed to specifically hybridize to the nucleotide sequence of ... It corresponds to the reverse complement of nucleotides 41,835 to 41,859 of sequence number 18. SEQ ID NO: 2 is an exemplary mouse c-Kit gene product (GENBANK® Biosequence Database The exon skipping oligonucleotide (ESO) ) is the nucleotide sequence of SEQ ID NO: 2, which is an accession number of the GENBANK® Biosequence Database. The 3' end of exon 4 of the mouse Kit gene product, represented by session number NM_001122733.1 It specifically hybridizes to 6 nucleotides of the first intron and 19 nucleotides of the downstream intron. Therefore, SEQ ID NO: 2 is designed to contain nucleotides 35,953 to 35,954 of SEQ ID NO: 19. It corresponds to the reverse complement of 77.

[0017] SEQ ID NO:3 has the same chemical properties as KitSop ESO, but does not contain any known gene encoding the A standard control antisense oligonucleotide (AS) that does not induce axon skipping 0) is the nucleotide sequence of SEQ ID NOs: 4 and 5 can be used together to amplify exon 4 of human c-KIT mRNA. The nucleotide sequences of the forward and reverse primers are shown in Table 1. SEQ ID NOs: 6 and 7 can be used together to amplify exon 4 of mouse c-kit mRNA. nucleotide sequences of the forward and reverse primers. SEQ ID NOs: 8 and 9 are the nucleotide and amino acid sequences, respectively, of an exemplary human c-KIT gene product. This nucleotide sequence is an amino acid sequence. This amino acid sequence corresponds to session number NM_000222.2 and is available from the GENBANK® Biosequence Database. The present invention corresponds to the database accession number NP_000213.1. The transmembrane domain corresponds to amino acids 525 to 545 of SEQ ID NO: 9 and is encoded within exon 10. Any truncation before this may result in some embodiments in the defective protein. In some embodiments, this may result in the production of proteins and / or cell membranes. localization to the

[0018] SEQ ID NOs: 10 and 11 are the nucleotide sequences of exemplary mouse c-Kit gene products, respectively. The nucleotide sequence and amino acid sequence are listed in the GENBANK® Biosequence Database. This amino acid sequence corresponds to the accession number NM_001122733.1 in the GENBANK® database. This corresponds to the Io sequence database accession number NP_001116205.1. SEQ ID NOs: 12 and 13 are the nucleotide sequences of exemplary feline c-Kit gene products, respectively. The nucleotide sequence is the nucleotide and amino acid sequence of the nucleotide ... This amino acid sequence corresponds to the accession number NM_001009837.3 in the GENBANK® database. This corresponds to the sequence database accession number NP_001009837.3. SEQ ID NOs: 14 and 15 are the nucleotide sequences of exemplary canine c-Kit gene products, respectively. The nucleotide sequence is the nucleotide and amino acid sequence of the nucleotide ... This amino acid sequence corresponds to the accession number NM_001003181.1 of the GENBANK® Biosequencer. Corresponds to column database accession number NP_001003181.1. SEQ ID NOs: 16 and 17 are the nucleotide and sequence sequences, respectively, of an exemplary equine c-Kit gene product. The nucleotide sequence is an amino acid sequence. This amino acid sequence corresponds to the accession number NM_001163866.2 and is available from the GENBANK® BioSequence Database. This corresponds to database accession number NP_001157338.2.

[0019] SEQ ID NO: 18 is an exemplary genomic sequence of the human c-Kit locus. ) Nucleotide 54,657,928 of BioSequence Database accession number NC_000004.12 Corresponds to ~54,740,715. SEQ ID NO: 19 is an exemplary genomic sequence of the mouse c-kit locus. (R) Nucleotide 75,574,987 of BioSequence Database accession number NC_000071.6 Corresponds to ~75,656,745. SEQ ID NO: 20 is an exemplary genomic sequence of the feline c-Kit locus. ) Nucleotide 163,952,966 of BioSequence Database accession number NC_018726.3 It corresponds to the reverse complement of 164,039,337. SEQ ID NO: 21 is an exemplary genomic sequence of the canine c-Kit locus. ) Nucleotide 47,108,504 of BioSequence Database accession number NC_006595.3 Corresponds to 47,190,020. SEQ ID NO: 22 is an exemplary genomic sequence of the equine c-Kit locus. ) Nucleotide 79,538,697 of BioSequence Database accession number NC_009146.3 It corresponds to the reverse complement of 79,618,653.

[0020] SEQ ID NOs: 23-60 are exemplary human c-KIT gene products (GENBANK® BioSequence Data It targets one of exons 2 to 20 of the base accession number NM_000222.2. Additional exemplary exon skipping oligonucleotides (ESOs) designed as follows: A target nucleotide sequence that contains a splice donor site or a splice acceptor site. Regions containing SEQ ID NOs: 23 and 24 can be targeted. See Table 3. Using the nucleotide sequence shown in Figure 1, exon 2 can be skipped, resulting in a sequence encoding SEQ ID NO: 61. SEQ ID NOs: 25 and 26 are used to generate the in-frame deleted nucleotide sequence. Axon 3 can be skipped, resulting in an in-frame sequence encoding SEQ ID NO:62. The deleted nucleotide sequence is obtained. SEQ ID NOs: 27 and 28 are used to skip exon 4. nucleotide sequence encoding SEQ ID NO:63. Sequences are obtained. SEQ ID NOs: 29 and 30 can be used to skip exon 5. resulting in a frameshifted nucleotide sequence encoding SEQ ID NO:64. SEQ ID NOs: 31 and 32 can be used to skip exon 6, resulting in a sequence A frameshift nucleotide sequence encoding sequence number 65 is obtained. 34 can be used to skip exon 7, resulting in coding sequence SEQ ID NO: 66. Using SEQ ID NOs: 35 and 36, a frameshift nucleotide sequence is obtained. Exon 8 can be skipped, resulting in a frame sequence encoding SEQ ID NO:67. The nucleotide sequence is obtained using SEQ ID NOs: 37 and 38 to skip exon 9. nucleotide sequence encoding SEQ ID NO:68. Sequences obtained are shown in Table 1. SEQ ID NOs: 39 and 40 can be used to skip exon 10. As a result, a frameshifted nucleotide sequence encoding SEQ ID NO: 69 is obtained, Because Son 10 encodes a transmembrane domain, in some embodiments, signal transduction SEQ ID NOs: 41 and 42 can be used to generate soluble receptors that cannot be expressed in vivo. Sequence 11 can be skipped, resulting in a frameshift that encodes SEQ ID NO:70 The nucleotide sequence was obtained and exon 11 contains the amino acids just C-terminal to the transmembrane domain. In some embodiments, the enzyme encodes a dominant-negative protein that inhibits kinase death. Proteins can be generated by skipping exon 12 using SEQ ID NOs: 43 and 44. resulting in an in-frame deleted nucleotide sequence encoding SEQ ID NO:71. SEQ ID NOs: 45 and 46 can be used to skip exon 13, The result is an in-frame deleted nucleotide sequence encoding SEQ ID NO:72. Using columns 47 and 48, exon 14 can be skipped, resulting in the sequence A frameshift nucleotide sequence encoding SEQ ID NO: 73 is obtained. 0 can be used to skip exon 15, resulting in the coding sequence for SEQ ID NO: 74. The frameshift nucleotide sequence is obtained using SEQ ID NOs: 51 and 52. Axon 16 can be skipped, resulting in a frameshifted sequence encoding SEQ ID NO:75. The nucleotide sequence is obtained by skipping exon 17 using SEQ ID NOs: 53 and 54. resulting in an in-frame deleted nucleotide sequence encoding SEQ ID NO:76 SEQ ID NOs: 55 and 56 can be used to skip exon 18, This results in a frameshifted nucleotide sequence encoding SEQ ID NO: 77. SEQ ID NO: 57 and 58 can be used to skip exon 19, resulting in SEQ ID NO: 7 A frameshift nucleotide sequence encoding 8 is obtained. 60 can be used to skip exon 20, resulting in the coding sequence for SEQ ID NO: 79. A frameshift nucleotide sequence corresponding to the nucleotide sequence is obtained.

[0021] SEQ ID NOs: 61 to 79 are sequences corresponding to the exon skipping oligonucleotides of SEQ ID NOs: 23 to 60. Amino acid sequence of human Kit polypeptide resulting from exon skipping using ESO In particular, SEQ ID NO: 61 is the predicted amino acid sequence of the exon 2 skipped protein. SEQ ID NO: 62 is the predicted amino acid sequence of the exon 3 skipped protein, and SEQ ID NO: No. 63 is the predicted amino acid sequence of the severely truncated exon 4 skipped protein, SEQ ID NO: 64 is the predicted amino acid sequence of the severely truncated exon 5 skipped protein. and SEQ ID NO: 65 is the predicted amino acid sequence of a severely truncated exon 6 skipped protein. SEQ ID NO: 66 is the predicted sequence of a severely truncated exon 7 skipped protein. SEQ ID NO: 67 is the amino acid sequence of the severely truncated exon 8 skipped protein. The predicted amino acid sequence, SEQ ID NO: 68, corresponds to the severely truncated exon 9 skipping protein. SEQ ID NO: 69 is the predicted amino acid sequence of the protein, which contains a severely truncated exon 10 skip. The predicted amino acid sequence of the protein, SEQ ID NO: 70, corresponds to the severely truncated exon 11 sequence. SEQ ID NO: 71 is the predicted amino acid sequence of the exon 12 skip protein. SEQ ID NO: 72 is the predicted amino acid sequence of the exon 13 skipped protein. SEQ ID NO: 73 is the predicted amino acid sequence of the exon 14 skipped protein. SEQ ID NO: 74 is the predicted amino acid sequence of the exon 15 skipped protein , SEQ ID NO: 75 is the predicted amino acid sequence of the exon 16 skipped protein, SEQ ID NO: 76 is the predicted amino acid sequence of the exon 17 skipped protein, and SEQ ID NO: 77 is the exon 17 skipped protein. SEQ ID NO: 78 is the predicted amino acid sequence of the exon 19 skipping protein. SEQ ID NO: 79 is the predicted amino acid sequence of the exon 20 skip protein. The predicted amino acid sequence of the protein. BEST MODE FOR CARRYING OUT THE INVENTION

[0022] Activating mutations in the proto-oncogene c-Kit cause malignancies such as systemic mastocytosis and mast cell (MC) leukemia. c-Kit is involved in the proliferation of mast cells (MCs) and regulates the viability of mast cells (MCs). Kit (CD117); obesity / stem cell growth factor receptor (S) are receptor tyrosine kinases required for cell survival. Therefore, treatments that block Kit signaling may be effective in preventing mast cell ( MC) is a major strategy for treating proliferative diseases. However, despite some success, current treatments have off-target effects and Some patients are unable to achieve complete sedation or improved survival. Typically, it is due to c-kit mutations that confer resistance to tyrosine kinase inhibitors. In some embodiments of the present invention, disclosed herein are wild-type or mutant A frameshift was introduced into the mature mRNA of either type of c-Kit, resulting in the exon-skipping oligonucleotide. Kit expression was specifically targeted using an oligonucleotide (ESO) to downregulate Kit expression. stimulation, inhibition of neoplastic mast cell (MC) proliferation, and induction of mast cell (MC) apoptosis. Furthermore, administration of ESO has been shown to induce the growth of mast cells in vivo. (MC) depletion, demonstrating therapeutic potential. The therapeutic potential of exon-skipping oligonucleotides (ESOs) lies in their stability and bioavailability. It has been significantly enhanced by chemical modifications that improve its bioavailability. In genetic diseases caused by loss-of-function frameshift mutations, such as dystrophia, ESO has clear clinical applications, restoring the reading frame of mature mRNA transcripts and This results in the translation of an alternatively spliced but partially functional protein. In some embodiments, the present invention achieves the opposite, loss of function in the mature mRNA. disrupting the open reading frame of the oncogenic transcript resulting in a frameshift, Some embodiments of the present invention provide enzyme-skipping oligonucleotides (ESOs). Targeting Kit induces rapid and efficient mast cell (MC) death in vivo. and mast cell (MC) numbers, providing an unmet clinical need for Kit-related malignancies. We present Kit-targeted therapeutics to address the need.

[0023] I. Definition All technical and scientific terms used herein are defined as follows unless otherwise defined below: , are intended to have the same meaning as commonly understood by those skilled in the art. References to techniques used in the specification include variations of those techniques or equivalent techniques that would be apparent to those skilled in the art. The following terms are intended to refer to techniques commonly understood in the art, including substitutions: Although it is believed that the present invention will be well understood by those skilled in the art, the following is provided to facilitate explanation of the present invention. Here is the definition: The following terms are believed to be well understood by those of ordinary skill in the art, but are provided to facilitate the description of the present invention. To make this possible, the following definitions are provided: As used in this specification and the appended claims, the singular forms "a" and "one" are used interchangeably. "An" and "the" refer to plural referents unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. The terms "one," "an," "one or more," and "at least one" have the same meaning. Similarly, "comprising," "including," and " The terms "having" and "having" can be used interchangeably. Please note that ranges may be stated excluding optional elements. The disclosure does not address the use of "solely," "only," or "indirectly" in connection with the recitation of claim elements or the use of "negative" limitations. It is intended to serve as a pre-declaration of which exclusive terms to use.

[0024] Ranges may be expressed herein as from "about" one particular value to "about" another particular value. When such a range is expressed, some embodiments may include any one particular value. and / or to another specified value. Similarly, values may be further specified by use of the antecedent "about." When expressed as approximations, it is understood that the particular values form embodiments. Furthermore, each endpoint of a range is relative to and It should be understood that the points are significant independently of each other. Many values of the present invention are disclosed, Each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Also, as will be appreciated by those skilled in the art, when a value is designated as "less than or equal to" that value, It is also understood that when a value is mentioned, "greater than or equal to" and the possible range between those values is also disclosed. For example, if the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" should also be disclosed. Throughout this application, data is provided in several different formats. The data represent endpoints and starting points in some embodiments, and It is also understood that the embodiments represent ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, In this case, between 10 and 15, as well as greater than, equal to, or greater than 10 and 15 , equal or lesser, and equivalents are also considered to be disclosed. It should also be understood that each unit between two specified units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 should also be disclosed. It has been done.

[0025] When the term "and / or" is used in the context of a list of several events, it Refers to the existence of events alone or in combination. As used herein, the terms "optional" and "optionally" refer to the events described thereafter. events, circumstances, elements, and / or methods, steps may or may not occur, and / or There may be a description of the event, circumstance, element, method, or step that occurs. This indicates that the example contains examples that are present or absent. Before the compounds, compositions, articles, devices, and / or methods of the present invention are disclosed and described, These may not be derived from specific synthetic methods or specific recombinant biological methods, or Unless otherwise specified, the methods are not limited to particular reagents, which may, of course, vary. It should be understood that the terminology used herein is for describing particular embodiments only. It should also be understood that this is for illustrative purposes only and is not intended to be limiting.

[0026] II. General considerations Human malignancies associated with activating c-KIT mutations include mast cell proliferative disorders, gastrointestinal stromal These include gastrointestinal stromal tumors (GIST), and rarely melanoma and acute myeloid leukemia (AML). Increased expression of KIT occurs in cells that do not normally express KIT and are exposed to an environment rich in stem cell factor (SCF). It may also contribute to the formation of solid lung cancer tumors from small lung cells. Gastrointestinal stromal tumors (GISTs) are thought to originate from interstitial cells of Cajal and are the most common type of sporadic GIST. Approximately 80% have at least 17 different activating mutations involving exons 8, 11, 13, or 17 of c-Kit Similarly, approximately 90% of adults with the disease abnormal mast cell proliferation (mastocytosis) % of the c-KIT(D816V) catalytic domain is involved in the conversion of aspartic acid to valine. consisting of substitutions that make it constitutively active and / or result in other mutations in c-KIT; It has a point mutation.

[0027] Therefore, therapies that block KIT activity are a major approach to treating this patient population. and to some extent, treatment of some of these malignancies, either alone or in combination, is While successful, complete sedation or improved survival is rare elsewhere. The success rate is based on the type of mutation and / or the presence of additional mutations in c-KIT or other proto-oncogenes. Pharmacological targeting of KIT catalytic activity blocks KIT-mediated responses. The limiting factor in the treatment of KIT-related malignancies is the lack of mesylate Imatinib (Imatinib, STI571, Gleevec or Gleevec) and its derivatives (Nilo Anti-KIT proteins, such as quinib (AMN107) and PD180970, are designed to conformate the inactive ATP-binding site of KIT. Inhibitors targeting this conformation block the active, extended conformation of KIT. Therefore, the most aggressive mastocytosis patients have a failure to Mutations that stabilize the active conformation (mainly including the common D816V mutation) Kinase domain mutations) are resistant to this drug. A further drawback of imatinib is the obvious Acquisition of other mutations in the kinase domain of the receptor that are insensitive to imatinib ( Secondary resistance to the drug may develop in some patients, due in part to the Therefore, patients initially responded to imatinib, which eliminated imatinib-sensitive cells. However, imatinib-resistant clonal proliferation may recur.

[0028] KIT-associated neoplastic diseases are often nonspecific and therefore have many off-target Imatinib is a drug that fits into the kinase pocket of KIT. It is the drug of choice because it is more specific to KIT signaling than other drugs. Many patients, especially those with the most severe disease, are resistant to imatinib, resulting in widespread and often severe More common inhibitors are used which have severe side effects. In some embodiments, the present invention targets KIT, making it a commonly used Compared to existing kinase inhibitors, it has minimal off-target effects. Other compounds, such as dasatinib (BMS-354825), have catalytic activity associated with D816V and other KD mutations. Dasatinib inhibits Kit autophosphorylation, and human mast cell line (HMC)-1.1, which expresses the V560G mutation or the V560G and D816V mutations, respectively. It has been reported that α-glucan inhibits the proliferation of both HMC-1.2 human mast cell line and HMC1.2 mast cell line. The cell lines harbor mutations in the kinase domain that confer resistance to imatinib and other inhibitors. However, dasatinib and its derivatives have broader specificity and inhibit Src kinases. , Tec kinase, Bruton's tyrosine kinase (Btk), mitogen-activated protein kinase and several kinases, including AKT, and NF-κB, in mast cells and other cell types. Affects other receptor kinases important for other functions. Thus, various pathways While potentially more effective at acting on the also need to be taken into consideration.

[0029] The present invention provides a method for treating Kit-mediated responses by eliminating Kit expression in mast cells. , proliferation of transformed HMC1.2 mast cell leukemia cells harboring the V560G and D816V mutations. These results in the loss of ATP, induction of apoptosis, and rapid cell death. The cells are used to test new drugs for mastocytosis, and they express constitutively The active conformation of Kit makes it resistant to many kinase inhibitors. kills these cells within hours and effectively kills cells harboring any activating Kit mutations. In some embodiments, the present invention provides a chemically stable spirochete. Using a sequence-switching oligonucleotide, exon 4 (very early mRNA) Splicing of Kit pre-mRNA induces exon skipping of the exon The open read sequence was altered to result in an immediate STOP codon, thus eliminating receptor expression. Introduces a frameshift in the

[0030] The present invention can be used for Kit-related cancers and tumor diseases, and the administration of this drug It specifically depletes mast cells in the tissues and can therefore be applied to mast cell-related diseases. Depleting the cells can lead to hypersensitivity to mast cell-driven allergies. In addition, the number of mast cells in the tissues of allergy sufferers can be reduced. Although Ki increases, this administration may deplete these cells. The expression of t is restricted to a few cell types, which is the target of the present invention. Mast cell depletion can also be used as a research tool to identify mast cell-containing tissues and their role in obesity. If it were possible to compare mast cell function with tissues lacking these cells, it would be possible to study mast cell function. Kit-deficient mice have been used in these studies, but these mice lack Kit in the bone marrow. Finally, the present invention provides a method for treating dogs (and to some extent cats) with steroids that may have other immune problems associated with their function. The present invention provides a method for treating mast cell tumors, the most common tumor in humans. Given its high potency in hematologic cells, its use to kill mast cell tumor cells can be done.

[0031] III. Oligonucleotides and Related Methods Antisense technology has proven to be an effective method for altering the expression levels of gene products. It has been demonstrated (e.g., U.S. Pat. No. 8,765,703, U.S. Pat. No. 8,946,183 and U.S. Pat. Pub. No. See Patent Publication No. 2015 / 0376615, which is incorporated herein by reference in its entirety. Antisense technology interferes with well-known steps in the normal processing of mRNA. Simply put, RNA molecules are transcribed from genomic DNA within the nucleus of a cell. These newly synthesized mRNA molecules are called primary mRNA or pre-mRNA and are synthesized by the ribosome. It must be processed before it can be transported to the cytoplasm for translation into protein. The process involves the addition of a 5' methylated cap and a poly(A) tail to the 3' end of the mRNA. do.

[0032] In mammals, 90-95% of mRNA maturation occurs through splicing. A transgene (or intervening sequence) is a sequence in the primary transcript (or mRNA) that is not included in the coding sequence of the mature mRNA. exons (expressed sequences) are regions of the DNA that encode the gene. It is the region of the primary transcript (or the DNA encoding it) that remains in the mature mRNA when During the splicing process, multiple exons in a pre-mRNA molecule are spliced together. The mature mRNA sequence is formed by splicing. The cellular machinery determines which sequences are removed and where the ends to be joined are opened. This is used to determine when to start and stop the The sequence 5' of this junction is called the 5' splice site or splice donor site. The sequence 3' of this junction is called the "3' splice site" or "splice access site." During splicing, the 3' end of the upstream exon is connected to the downstream exon at a site called the "scepter site." The unspliced RNA (or pre-mRNA) is then attached to the 5' end of the mRNA. , exon / intron junction at the 5' end of the intron and Contains intron / exon junctions. After intron removal, mature mRNA exon / exon junctions or boundaries, Cryptic splice sites are less commonly used than common splice sites. may be used if other factors are blocked or unavailable. The use of different combinations of exons in a gene allows for multiple mRNA transcripts from a single gene. It may bring things.

[0033] In one application of antisense technology, antisense oligonucleotides (AONs) By binding to mRNA molecules transcribed from the gene of interest and increasing their degradation and / or by preventing translation or translocation of mRNA by steric hindrance. The end result is that the expression of the corresponding gene (i.e., The ultimate production of the protein encoded by the gene is reduced or eliminated. Alternatively, antisense technology can be used to affect the splicing of gene transcripts. In this application, the antisense oligonucleotide can be pre-plated. It binds to isolated RNA molecules (also called pre-messenger RNA or pre-mRNA) and redirects the cellular splicing machinery, resulting in the exons of spliced mRNA molecules This results in an altered content of the protein encoded by the altered mRNA. The target sequence is the unspliced protein (i.e., the complete protein) translated from the mRNA. The protein translated from this altered mRNA is different from the full-length wild-type protein. , truncated, or may be missing important sequences necessary for proper function. Typically, compounds used to affect splicing are targeted mRNAs. The oligonucleotide is or contains a base sequence complementary to NA. In the specification, such oligonucleotides are referred to as "antisense oligonucleotides" (AO N). The present invention provides a method for regulating splicing of pre-mRNA encoding the Kit protein. antisense technology for the purpose of inhibiting the expression of Kit protein expressed by cells The method of the present invention provides a technique for causing a reduction in the amount or "level" of Then, cells expressing Kit transcripts were treated with antisense oligonucleotides targeting a region of the Kit pre-mRNA. Such contacting can be achieved by contacting the oligomer with the Uptake of antisense oligomers by cells, hybridization of oligomers to Kit mRNA This results in the regulation of the splicing of the Kit pre-mRNA and subsequent splicing of the Kit pre-mRNA. In some embodiments of the method, the splicing of such Kit mRNA is The node reduces Kit expression.

[0034] The present invention is not limited to the particular embodiments described herein and as such may vary. Furthermore, the terminology used herein is intended to describe particular embodiments only. It is not intended to limit the invention. As used herein, the term "Kit" refers to a gene that is involved in the induction of cancer in animals (e.g., mammals). The protogene c-Kit, also known as the tyrosine protein kinase Kit, and CD117 The gene locus that encodes the obesity / stem cell factor receptor (SCFR) is involved in the development of Kit proteins. Kit is a receptor tyrosine kinase. The Kit gene and gene product have been identified in many species. In certain species, there are generally multiple genes encoded by the Kit locus. There are different gene products (e.g. RNA). Exemplary, non-limiting examples of Kit gene products present in the GENBANK® Biosequence Database include: A summary is provided in Table 1. Table 1 lists specific exemplary sequences in the GENBANK® Biosequence Database. The Kit gene product of other species is also included. t gene products, all of which are included within the scope of the present invention. It is understood that the species listed and other species not expressly listed may be It is possible to have additional Kit gene products, and all of these additional Kit gene products are also Therefore, all Kit orthologs are within the scope of the compositions and methods of the present invention. It should be clearly stated that this is included in the

[0035] [Table 1]

[0036] Similarly, a Kit coding sequence refers to a nucleic acid sequence that encodes at least a portion of the Kit protein. This part can be a fragment of a protein (e.g., a fragment from any part of this whole protein). 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive amino acid segments), It may be a domain or a membrane domain (e.g., a transmembrane domain), or any splice. The c-Kit gene or the c-Kit protein of the present invention may also refer to the entire protein including isolating variants. The coding sequence may be derived from any mammal that possesses this gene or coding sequence. By way of example and not limitation, the c-Kit gene, coding sequence, and / or gene product may be derived from a human, It may be of murine, canine, feline, equine, or other mammalian origin. In some cases, a c-Kit transcript is an RNA molecule transcribed from the c-Kit locus. In embodiments, the c-Kit transcript targeted by the oligomers of the invention is the primary transcript or is a pre-mRNA molecule. As used herein, primary mRNA or pre-mRNA are mRNA transcripts that have not yet been spliced, whereas mature mRNA molecules is a spliced mRNA molecule.

[0037] As used herein, the term "antisense oligomer" refers to a polymer comprising nucleobases. refers to a hybridization molecule that hybridizes to the sequence of a nucleic acid molecule, such as an mRNA molecule. As used herein, the term "nucleobase" refers to the heterocyclic base portion of a nucleoside. Generally, a nucleobase is any group containing one or more atoms or groups of atoms. The nucleoside base is a loop that can hydrogen bond to the base of another nucleoside. Denine (A) and guanine (G), and the pyrimidine nucleobases thymine (T) and cytosine (C) In addition to "unmodified" or "natural" nucleobases such as uracil (U) and uracil (U), Modified nucleobases or nucleobase mimetics that contain nucleobases are also within the scope of the present invention. refers to a nucleobase that is structurally similar to the parent nucleobase, e.g., 7-deazapurine, 5- These include nucleobase mimetics such as methylcytosine, G-clamps, or tricyclic phenoxazines. Methods for preparing these modified nucleobases are known to those skilled in the art. As mentioned above, a nucleoside is a combination of a base and a sugar. The base moiety is typically a heterocyclic base (e.g., a nucleobase or simply a "base"). The two most common classes of heterocyclic bases are the purines and pyrimidines. A nucleoside is a nucleoside that further contains a phosphate group covalently linked to the sugar portion of the nucleoside. In the case of nucleosides containing a pentofuranosyl sugar, the phosphate group may be located on the 2', 3', or 5' hydroxyl group of the sugar. When forming an oligonucleotide, this phosphate can be attached to the hydroxyl moiety. The group covalently links consecutive nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups generally form the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester bond. It is a combination.

[0038] It is well known in the art that RNA molecules often have short half-lives, limiting their use as therapeutic agents. Therefore, it is necessary to use oligonucleotides to alter their activity. Chemical modifications of peptides are often used. Chemical modifications can be used to enhance the antisense oligonucleotides to their target RNA. Increasing the affinity of oligomers, nuclease resistance (e.g., ribosomal enzymes such as RNase H) nuclease resistance) and / or improve the pharmacokinetics of the oligomer (e.g. The activity of the oligomer can be altered by, for example, changing its half-life. For example, the sugar, nucleobase, and / or internucleoside linkages may be hybridized to the oligomer's target sequence. Desired properties for oligomers (e.g., resistance to degradation) while maintaining the ability to hybridize The compound can be substituted with groups that confer resistance, increased half-life, etc. These compounds are sometimes called analogues (e.g., sugar analogues, nucleobase analogues, etc.). -When using analogs to replace internucleoside bond combinations, the selected target The nucleobases are maintained for hybridization to the glycomimetics. include, but are not limited to, cyclohexene or morpholino. Representative examples of mimics of inter-oxodiazole bond combinations include those linked by uncharged achiral bonds. These include, but are not limited to, ligated peptide nucleic acids (PNAs) and morpholino groups. In some cases, analogs are used in place of nucleobases. Acid-base mimetics are well known in the art and include tricyclic phenoxazine analogs and universal salts. groups (see, e.g., Berger et al., 2000). (These are incorporated herein by reference.) Such sugars, nucleotides, and Examples of nucleobase mimetics are disclosed in U.S. Patent Nos. 8,765,703 and 8,946,183, These are incorporated herein by reference. Synthesis of Sugar, Nucleotide, and Nucleobase Mimetics The methods, as well as the use of such mimetics to generate oligonucleotides, are well known to those skilled in the art. It is well known.

[0039] The term "oligomer" refers to an oligonucleotide, an oligonucleoside, an oligonucleotide, a These include oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations thereof. Such molecules are generally known to those skilled in the art. The oligomers of the present invention include primers, promoters, and the like. Antisense compounds, antisense oligonucleotides, external guide sequence (EGS) These include, but are not limited to, oligonucleotides, alternative splicers, and siRNAs. Therefore, these compounds can be introduced in single-stranded, double-stranded, circular, branched, or hairpin form. The present invention can include structural elements such as internal or terminal bulges or loops. The oligomers are administered to cells or individuals to modulate the splicing of mRNA molecules. For example, the length of the antisense oligomer of the present invention may be any length suitable for comprises about 10 to about 50 nucleosides (i.e., about 10 to about 50 linked nucleosides). Those skilled in the art will recognize that this may include 0, 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, embodied antisense oligomers of 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases In some embodiments, the antisense The oligomer may comprise 10 to 30 nucleobases, or 10 to 25 nucleobases, or Determining the appropriate length of the antisense oligomer of the present invention The method will be apparent to those of skill in the art upon review of this disclosure.

[0040] As used herein, the terms "targeted to" and "targeted" and the like refer to an A transsense oligomer is designed to be specific for a desired nucleic acid molecule, such as a desired mRNA molecule. The term "hybridize" refers to the process of hybridizing a molecule to another molecule. The terms "hybridization," "hybridize to," and the like are understood in the art. The term refers to a combination of multiple oligonucleotides (e.g., antisense oligomers and mRNA The pairing of nucleic acid bases in a complementary strand of a molecule (a target sequence) by a specific mechanism. The most common mechanism of pairing, but not limited to, is the pairing of complementary nucleosides or nucleoside pairs. This involves hydrogen bonds between bases (nucleobases), which are For example, the natural base adenine has It is complementary to the natural nucleobases thymidine and uracil and pairs with them through the formation of hydrogen bonds. Similarly, the natural base guanine is complementary to the natural bases cytosine and 5-methylcytosine. In the context of the present invention, the phrase "specifically hybridizes" refers to the The transsense oligomer encodes a protein unrelated to the Kit protein structure. Rather than binding to RNA, it binds to the mRNA encoding the Kit protein (e.g., pre-mRNA). It also refers to the ability to preferentially bind to target sequences (e.g., RNA). The oligomers are linked to the mRNA encoding the Kit protein (Kit pre-mRNA). mRNAs encoding proteins that hybridize with, but are not structurally related to, the Kit protein. NA means no significant hybridization. Hybridization refers to, for example, the binding of the oligomer of the present invention to the structure of the Kit protein. Antisense oligomers exert their desired effect on mRNAs encoding unrelated proteins. This refers to binding with a high enough affinity or avidity to interfere with the ability of a molecule to achieve its intended function. Examples of desired effects include modulation of Kit pre-mRNA splicing, Kit protein expression, and and reducing or inhibiting Kit-related symptoms in an individual. Thus, upon reviewing this disclosure, it is understood that specific binding may occur under conditions where specific binding is desired (i.e., Under physiological conditions in the case of in vivo assays or therapeutic treatments, and in vitro assays Antisense oligos to non-target nucleic acid sequences (or, if applicable, under conditions under which the assay is performed) The linear sequence and target sequence of the nucleobases in the antisense oligomer are determined to avoid significant binding of the nucleotides. If there is a sufficient degree of complementarity between the linear sequence of nucleobases in the target sequence and the The ligomer is specific (e.g., hybridizable, e.g., hybridizable to) the target sequence. It is thought that this is due to the following reasons:

[0041] As used herein, the terms "complementary," "complementarity," "complement," and the like refer to: The ability to form precise pairing between nucleic acid bases in an oligomer and nucleic acid bases in a target sequence Thus, a nucleobase (e.g., adenine) at a particular position in the oligomer is selected to be a target nucleic acid. can hydrogen bond with a nucleobase (e.g., uracil) at a specific position in the target sequence If the oligomer has a complementary structure, the hydrogen bond positions between the oligomer and the target nucleic acid are considered to be complementary positions. Generally, the term "complementary," "complementarity," "complementary," etc., refers to a single base pair. between bases, but not between bases, for a defined number of consecutive nucleic acids in a first nucleic acid molecule (e.g., an oligomer). a nucleotide and a similar number of contiguous nucleotides in a second nucleic acid molecule (e.g., an mRNA molecule) For example, if the antisense oligomer is 25 nucleotides, When the length of the oligomer is 0.1, complementarity with the target sequence is usually determined over the entire oligomer or a defined portion thereof. The sequence of the mRNA is determined by comparing the sequence of the mRNA with a certain number of consecutive nucleotides in the mRNA molecule. The oligomer and the target sequence are determined so that a sufficient number of corresponding positions in each molecule are water-soluble to each other. If a position is occupied by a nucleobase that can bond with another nucleotide, they are complementary to each other. The bases in a nucleic acid are spatially arranged so that, if complementary, the bases will form hydrogen bonds. In one example, the sequence of the oligomer corresponds to the sequence of the target sequence. When compared to sequences of similar size, the first nucleotide in this oligomer is the same as the target sequence. The second nucleotide in the oligomer is then compared to the selected nucleotide at the start of the sequence. The nucleotide (3' to the first nucleotide) is 3' to the selected starting nucleotide. This process is then repeated for each nucleotide along the length of the oligomer. Thus, the terms "specifically hybridizable" and "complementary" are used interchangeably. "Suitable" means that the antisense compound is sufficiently stable to form a stable and specific binding site between the antisense compound and the target nucleic acid. used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of consecutive nucleobases will be done.

[0042] Hybridization, in which a first nucleic acid molecule specifically hybridizes to a second nucleic acid molecule. These stringent hybridization conditions are generally referred to in the art as stringent hybridization conditions. Those skilled in the art will appreciate that stringent hybridization conditions are sequence dependent. It is understood that this may be different in different circumstances. Stringent conditions under which an oligomer specifically hybridizes to a target sequence are defined as the oligomer's specific hybridization conditions. Determined by the complementarity of the target sequence to the mer sequence and the nature of the assay in which they are being investigated. Those skilled in the relevant art will appreciate that for a given assay or for a given application, A complementary sequence can be designed that will specifically hybridize to the sequence.

[0043] The process of designing antisense oligomers that target nucleic acid molecules typically involves determining their expression. The method begins with identifying the target nucleic acid to be modulated and determining the sequence of the target nucleic acid molecule. When used, the terms "target nucleic acid," "nucleic acid encoding a Kit protein," and the like refer to: For example, DNA encoding the Kit protein, RNA transcribed from such DNA (pre-mRNA and and mRNA), and cDNA derived from such RNA. are cellular genes (or pre-mRNAs transcribed therefrom) whose expression is associated with a particular disease or pathology. Thus, in some embodiments, useful target nucleic acids are In some embodiments, the target nucleic acid encodes the c-Kit protein. In some embodiments, the target nucleic acid is a c-Kit pre-mRNA. Once a target nucleic acid is identified, the targeting process begins with the identification of a small number of sites where antisense interactions occur. determining at least one target region, thereby controlling splicing of the target nucleic acid; As used herein, a target region includes at least one identifiable A target region is defined as a portion of a target nucleic acid that has a structure, function, or characteristic. Exemplary target regions include: These are regions containing sequences involved in splicing of pre-mRNA molecules. Examples of structures, functions, or characteristics include at least a portion of an intron or exon, / Exon junctions, splice donor sites, splice acceptor sites, splice fragments These include, but are not limited to, branch points or splice enhancer sites. In some embodiments, the target region is at least one of an intron or an exon. Some splice donor sites, splice acceptor sites, splice branch points, and / or or a splice enhancer site. In some embodiments, the target region , intron or exon, splice donor site, splice acceptor site, splice Contains Rice branch points and / or splice enhancer sites.

[0044] Following identification of the target region, the target sequence within the target region can be identified. When used, the target sequence is a sequence to which the antisense oligomer of the invention specifically hybridizes. An exemplary target sequence is a nucleic acid sequence within a target region that is targeted for splicing of a pre-mRNA. It is something that is involved. Once the target sequence is identified, antisense oligomers are introduced into the target Contains a nucleobase sequence sufficiently complementary to the target sequence so as to specifically hybridize to the nucleic acid. More specifically, the nucleotide sequence of the antisense oligomer is The antisense oligomer is selected from the target sequence so that it specifically hybridizes to the target nucleic acid. The oligomer is designed to contain a region of consecutive nucleotides that is sufficiently complementary to the sequence. Such a region of consecutive complementary nucleotides is called an "antisense sequence" or "target sequence." It's called a "row."

[0045] The higher the degree of complementarity between two nucleic acid sequences, the stronger the hybridization interaction. It is well known in the art that the most potent and most specific Differential hybridization can also occur between two perfectly complementary nucleic acid molecules. As used herein, the term perfectly complementary refers to each nucleic acid in a nucleic acid sequence. A situation in which a base can hydrogen bond with a nucleobase at a corresponding position in a second nucleic acid molecule. In some embodiments, the targeting sequence is perfectly complementary to the target sequence. In some embodiments, the targeting sequence is complementary to at least one of the target sequences. at least six consecutive nucleobases that are perfectly complementary to each other in a region of six consecutive nucleobases In some embodiments, the targeting sequence comprises a base region. at least eight consecutive nucleic acids that are perfectly complementary to at least eight consecutive nucleic acid base sequences In some embodiments, the targeting sequence comprises a small number of bases in the target sequence. A sequence of at least 10 contiguous nucleic acid bases that is perfectly complementary to the sequence of at least 10 contiguous nucleic acid bases. In some embodiments, the targeting sequence comprises a nucleobase sequence at least 12 sequences that are completely complementary to at least 12 consecutive nucleic acid base sequences in In some embodiments, the targeting sequence comprises a contiguous nucleobase sequence. at least one nucleic acid sequence that is perfectly complementary to a sequence of at least 14 consecutive nucleic acid bases in the target sequence; In some embodiments, the targeting sequence comprises four consecutive nucleobases. is perfectly complementary to a sequence of at least 16 consecutive nucleic acid bases in the target sequence. In some embodiments, the target-directed The sequence is perfectly complementary to a sequence of at least 18 consecutive nucleic acid bases in the target sequence. In some embodiments, the target comprises a sequence of at least 18 consecutive nucleic acid bases. The target sequence is perfectly complementary to a sequence of at least 20 consecutive nucleic acid bases in the target sequence. The nucleic acid sequence comprises at least 20 consecutive nucleic acid bases

[0046] Those skilled in the art will appreciate that this targeting sequence constitutes the entire antisense oligomer of the present invention. or which may form part of the antisense oligomer of the invention. For example, an oligomer consisting of 20 of 30 nucleotides In the Alternatively, for example, 20 consecutive nucleosides in the oligomer can be Only the nucleotides are complementary to the 20 consecutive nucleotide target sequence, and this oligomer The remaining 10 nucleotides in the sequence were mismatched with nucleotides outside the target sequence. In some embodiments, the oligomer of the invention comprises at least 10 nucleic acids. bases, at least 11 nucleobases, at least 12 nucleobases, at least 13 nucleobases group, at least 14 nucleobases, at least 15 nucleobases, at least 16 nucleobases , at least 17 nucleobases, at least 18 nucleobases, at least 19 nucleobases, At least 20 nucleobases, at least 21 nucleobases, at least 22 nucleobases, At least 23 nucleobases, at least 24 nucleobases, at least 25 nucleobases, at least at least 26 nucleobases, at least 27 nucleobases, at least 28 nucleobases, at least Both have targeting sequences of 29 nucleobases or at least 30 nucleobases in length.

[0047] Those skilled in the art will appreciate that the inclusion of mismatches between the target-directed sequence and the target sequence is essential for the synthesis of oligomers. It is understood that this is possible without eliminating the activity of the target gene (e.g., regulation of splicing). Furthermore, such mismatches may prevent the antisense oligomer from binding to the target nucleic acid molecule. As long as the target sequence can specifically hybridize to the target, the alignment between the target sequence and the target sequence is acceptable. The antisense interaction can occur anywhere within the antisense oligomer of the present invention. As long as the antisense oligomer specifically hybridizes to the target sequence, The oligomer is then allowed to react with the target sequence, with the mismatched nucleotides disrupting base pairing. In some embodiments, the antisense oligomer may comprise about 20% The mismatch is set to 20% or less, about 15% or less, about 10% or less, about 5% or less, or about 3% or less. In some embodiments, the antisense strands involved in the pairing are There are no mismatches between the nucleotides in the oligomer and the complementary target sequence. In some embodiments, the mismatches do not occur at consecutive positions. For example, three mismatches In antisense oligomers containing nucleotide sequences, in some embodiments, these sequences are The mismatch position is a sequence of consecutive nucleotides ( They can be separated by multiple connections (e.g., 3, 4, 5, etc.).

[0048] Use of the expression "percent identical" defines the number of mismatches between two nucleic acid sequences. For example, two sequences with the same nucleic acid base pairing ability are identified by a 10 0% identical. Furthermore, both uracil and thymidine bind to adenine. It is necessary to understand that, as a result, one has a uracil at position x and the other has the corresponding Two molecules that have a thymidine at position x but are otherwise identical in sequence will The percent identity is determined over the entire length of the oligomeric compound or over the entire length of the oligomeric compound. may be calculated over only a portion of the oligomer, e.g., a nucleic acid molecule containing the target sequence. To determine the ability of an oligomer containing a target-directed sequence to bind to the target, The percent identity of the sequence directed to this target can be calculated. In this embodiment, the target-directed sequence is at least 80% of the length of the target sequence in the target nucleic acid molecule. Identical, at least 85% identical, at least 90% identical, at least 95% identical, at least Some examples are 97% identical, at least 98% identical, or at least 99% identical. In an embodiment, the targeting sequence is fused over its entire length to the target sequence in the target nucleic acid molecule. Those skilled in the art will recognize that the antisense oligomers are identical to the antisense oligomers described herein. It should be noted that the oligomer sequence does not need to be completely identical to that of the oligomer of the present invention in order to function similarly to the oligomer. It will be understood that shortened versions of the antisense oligomers taught herein, Non-identical versions of the antisense oligomers taught herein are also within the scope of the present invention. This non-identical version is one in which every base is 100% identical to the antisense oligomer of the invention. Alternatively, non-identical versions are versions that have different pairing activities. It can contain at least one base replaced with a different base that has the same For example, replace G with C, A, or T).

[0049] Percent identity is the number of identical bases in the corresponding oligomers being compared. The non-identical bases are either adjacent to each other or dispersed throughout the oligomer. For example, the same sequence as nucleobases 2-17 of a 20-mer may be used. A 16-mer with a sequence that is 80% identical to a 20-mer, or four nuclei that are not identical to a 20-mer The 20-mer containing the acid and bases is also 80% identical to the 20-mer. The 18-mer has the same sequence as nucleobases 1-14. is 78% identical to the 18-mer. Such calculations are within the capabilities of one of ordinary skill in the art. Therefore, the antisense oligomer of the present invention is Any suitable modification of the sequences disclosed herein may be used as long as it is capable of regulating the splicing of a desired mRNA molecule. At least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical 1. At least 94% identical, at least 96% identical, or at least 98% identical oligonucleotides Contains an octide sequence.

[0050] The antisense oligomers of the present invention can regulate the splicing of mRNA molecules. As used herein, "modulation" of splicing refers to exon skipping (or exon inclusion), deletion of one or more exons, or spliced mRNA splicin resulting from the addition of unusual sequences (e.g., intron sequences) The pre-mRNA transcript is then sequenced so that the resulting mRNA molecule contains the desired combination of exons. This refers to the ability of antisense oligomers to affect processing, e.g., splicing. Regulation of splicing is determined by whether the spliced mRNA (mature mRNA) contains at least one exon. Deletion of part or the entire Kit pre-mRNA affects splicing. In some embodiments, the spliced mRNA contains exon 4. A truncated isoform of the Kit protein is present in the cell, Such truncation is due to a truncation in exon 4 of the mRNA encoding the Kit protein. Therefore, for purposes of describing the present invention, the shortened Kit tag Effect of antisense oligomers to generate truncated mRNAs encoding proteins Splicing of Kit pre-mRNA by ATP can be called alternative splicing. Furthermore, the deletion of at least a part or the whole of exon 4 due to the influence of antisense oligomers Kit mRNA transcripts are products of alternative splicing. In this study, splicing regulation induces alternative splicing of Kit pre-mRNA molecules. This reduces the level of mRNA molecules containing the entire exon 4 and It can refer to increasing the level of an mRNA molecule that lacks at least a portion.

[0051] The present disclosure relates to compositions targeting exon 4 of the human or murine Kit gene. and methods are exemplified, but other exons of both the human and murine Kit genes may also be used. It should be understood that the present invention can be targeted in cats, dogs, and mice. Exon 4 or any other exon may also be targeted in mice, horses, or other animals. and therefore, the specific examples of targeting strategies disclosed herein are understood to be illustrative only. A summary of the genomic organization of the Kit locus in certain exemplary animals is shown in Table 2.

[0052] [Table 2]

[0053] Table 2 lists the positions of the exons of SEQ ID NOs: 18 to 22. It should be understood that the nucleotides between are intron sequences. For example, human c-KI The T exon sequence is nucleotides 1-154, 37585-37854, 40357-40638, 4170 of SEQ ID NO: 18. 3-41839, 45797-45965, 49171-49360, 51497-51612, 65657-65771, 67930-68123, 69291- 69397, 69489-69615, 69896-70000, 70084-70194, 71408-71558, 73401-73492, 73944-74 Showing 071, 75143-75265, 78571-78682, 78794-78893, 79248-79353, and 80502-82788 Since SEQ ID NO: 18 is a genomic sequence, the human c-KIT intron sequence is SEQ ID NO: 18 Nucleotides 155-37584, 37855-40356, 40639-41702, 41840-45796, 45966-49170, 493 61-51496, 51613-65656, 65772-67929, 68124-69290, 69398-69488, 69616-69895, 70001 -70083, 70195-71407, 71557-73400, 73493-73943, 74072- 75142, 75266-78570, 78683- Equivalent to 78793, 78894-79247, and 79354-80501.

[0054] In designing exon skipping oligonucleotides (ESOs), several embodiments In this study, ESO spans exon / intron or intron / exon boundaries. This is because the ESO is both 5' and 3' of the splice donor or splice acceptor. The number of nucleotides in the exon that are part of the exon. The number of nucleotides in the ESO that are part of the adjacent intron may vary, but In some embodiments, at least five exonic nucleotides and at least five By way of example and not limitation, ESO may have SEQ ID NO: 1 and If the design has 25 nucleotides, as in the case of 2, some implementations In embodiments, the ESOs are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, Contains 18, 19, or 20 contiguous exonic nucleotides and includes 10, 19, 18, or 20 immediately adjacent exonic nucleotides. 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 consecutive intron nucleotides In other words, for all of SEQ ID NOs: 18 to 22, ESO includes several In one embodiment, 15, 16, 17, 18, 19, 20, 21, 22, or any one of SEQ ID NOs: 18 to 22 Contains 23, 24, 25 or more consecutive nucleotides, 15 to 25 or more consecutive The nucleotides contain at least one splice donor or splice acceptor sequence. Includes ESO lengths 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more pre-mRNA sequences. The sequence can be extended in the 5' and / or 3' directions so that it can be designed as follows:

[0055] Table 3 lists exon skipping sequences for use in targeting the human c-KIT gene product. Exemplary target sequences that can be used to design oligonucleotides (ESOs) are shown. Target sequence of the ESO used to target the c-Kit gene product in non-human animals can be used for this purpose, but is based on the approach taken for the example ESO in Table 3. Please note that:

[0056] [Table 3-1]

[0057] [Table 3-2] *ESOs targeting these sequences will be the reverse complement of the sequences shown or subsequences thereof . **"Acceptor" refers to the target sequence containing the splice acceptor, and "Donor" refers to the The target sequence includes the splice donor of the targeted exon. In the table, intron sequences are shown in lower case and exon sequences are shown in upper case.

[0058] In some embodiments of the present invention, an antisense oligonucleotide containing 10 to 50 linked nucleosides is A sense oligomer that targets a region of an RNA molecule encoding the Kit protein. In some embodiments, the oligomer comprises an RNA molecule. Hybridization to the nucleotide sequence of the present invention regulates splicing of this RNA molecule. In some embodiments, the antisense oligomer specifically hybridizes to the target sequence. Kit mRNA transcripts to regulate splicing. Antisense comprising a nucleic acid sequence sufficiently complementary to a target sequence in a target region of an mRNA molecule These antisense oligomers consist of 10-50 linked oligomers. It may comprise, consist essentially of, or consist of nucleosides These antisense oligomers can contain 15 to 35 linked nucleotides. These antisense oligomers consist essentially of 15 to 35 linked nucleosides. These antisense oligomers can consist of, or consist solely of, 10-linked nucleosides, 11-linked nucleosides, 12-linked nucleosides, 13-linked nucleosides 14 linked nucleosides, 15 linked nucleosides, 16 linked nucleosides, 17 linked Linked nucleosides, 18-linked nucleosides, 19-linked nucleosides, 20-linked nucleosides , 21 linked nucleosides, 22 linked nucleosides, 23 linked nucleosides, 24 linked nucleosides 25 linked nucleosides, 26 linked nucleosides, 27 linked nucleosides, 28 Linked Nucleosides, 29 Linked Nucleosides, 30 Linked Nucleosides, 31 Linked Nucleosides 32 linked nucleosides, 33 linked nucleosides, 34 linked nucleosides, 35 linked nucleosides Nucleosides, 36 linked nucleosides, 37 linked nucleosides, 38 linked nucleosides, 39 40-linked nucleosides, 41-linked nucleosides, 42-linked nucleosides 43 linked nucleosides, 44 linked nucleosides, 45 linked nucleosides, 46 linked Nucleosides, 47 linked nucleosides, 48 linked nucleosides, 49 linked nucleosides, or 50 linked nucleosides, This mRNA molecule can be derived from any mammal that produces the Kit protein. Examples of such mammals include humans, mammals, and mammalian species. These include, but are not limited to, mice, dogs, cats, and horses. and the mRNA has at least 80%, at least 85%, or At least 90%, at least 95%, at least 97%, at least 99%, or 10 In some embodiments, the mRNA contains a nucleotide sequence that is 0.001 to 0.001% identical to the sequence of the mRNA. Column number 9 or 11 and at least 80%, at least 85%, at least 90%, Proteins containing amino acid sequences that are 95%, at least 97%, or at least 99% identical to each other In some embodiments, the mRNA encodes SEQ ID NO: 9 or 11. The RNA molecule may be a Kit transcript. In some embodiments, the RNA molecule is a Kit mRNA molecule. In embodiments, the RNA molecule is a Kit pre-mRNA.

[0059] The target region targeted by this antisense oligomer is the splicing region of this RNA molecule. This "splicing" may be any region of this RNA molecule that is functionally involved in splicing. "Functionally involved in splicing" refers to the interaction of a target region with a target protein to effect splicing of an mRNA molecule. The sequence of this region is spliced by the cellular splicing machinery (e.g., the spliceosome or its components). Examples of such regions include regions containing intron sequences, Regions containing exon sequences, regions containing intron / exon junctions, splice donor sites Regions containing sequences, regions containing splice acceptor site sequences, splice enhancers A region containing a site sequence, a region containing a branch point sequence, and a region containing a polypyrimidine tract are Such sequences include, but are not limited to, those known to those skilled in the art. Suitable sequences are also disclosed herein. Thus, in some embodiments, the target region is exon sequences, intron sequences, sequences containing exon / intron junctions, splice donors -site sequence, splice acceptor site sequence, splice enhancer site sequence, branching At least a portion of a sequence selected from the group consisting of a polypyrimidine tract, In the context of the present invention, "at least a portion" means a portion whose length is at least 5 nucleotides long. nucleosides, at least 6 nucleosides, at least 7 nucleosides, at least 8 nucleosides Nucleosides, at least 9 nucleosides, at least 10 nucleosides, at least 11 At least 12 nucleosides, at least 13 nucleotides, At least 14 nucleosides, at least 15 nucleosides, at least 16 nucleosides , at least 17 nucleosides, at least 18 nucleosides, at least 19 nucleosides In some embodiments, this "at least 20 nucleosides" refers to a "At least a portion" refers to known splice donor site sequences, splice acceptor site sequences, and site sequence, splice enhancer site sequence, branch point sequence, or polypyrimidine tract At least 10%, at least 25%, at least 50%, at least 75% of the sequence, Contains at least 90%, at least 90%, at least 95%, or at least 97% The splice donor site sequence, splice acceptor site sequence, and splice enhancer sequence are The sensor site sequence, branch point sequence, or polypyrimidine sequence was obtained from Kit pre-mRNA. It may also be the case.

[0060] In some embodiments, the target region comprises at least a portion of a Kit sequence of the present invention. which is any of SEQ ID NOs: 8, 10, 12, 14 and 16, or SEQ ID NO: 18 In some embodiments, this "at least a portion" may be any of the above. " is at least 10%, at least 25%, at least 50% of the Kit sequence of the present disclosure, At least 75%, at least 80%, at least 90%, at least 95%, or less In some embodiments, this "at least a portion" comprises at least 97% of the total protein. " is at least 80%, at least 90%, at least 90% identical to a portion of the Kit sequence of the present disclosure. In some embodiments, the polynucleotide sequences are at least 5%, or at least 97% identical. In this case, the target region is SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: Sequence number 16, sequence number 18, sequence number 19, sequence number 20, sequence number 21 or sequence number 2 2. At least 80%, at least 9 0%, at least 95%, at least 97%, at least 99%, or 100% identical In some embodiments, the target region comprises the nucleotide sequence of SEQ ID NO: 8, Sequence number 10, sequence number 12, sequence number 14, sequence number 16, sequence number 18, sequence number 19 20, SEQ ID NO: 21, or SEQ ID NO: 22. Some of these are also included.

[0061] In some embodiments, the antisense oligomer is a target of the Kit pre-mRNA. In some embodiments, the target is a region or sequence involved in splicing. The antisense oligomers of Kit target the Kit intronic sequence, Kit exon sequence, and Kit splice sequence. Rice donor site sequence, Kit splice acceptor site sequence, Kit splice end sequence Targeting the Hanser site sequence, Kit branchpoint sequence, or Kit polypyrimidine tract In some embodiments, the antisense oligomer is a target of the Kit pre-mRNA. In some embodiments, the antisense oligomer targets exon 4. , Kit exon 4 splice donor sequence, Kit exon 4 splice acceptor sequence In some embodiments, the gene targeting the Kit exon 4 splice enhancer sequence is targeted. In this embodiment, the antisense oligomer is SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 or a subsequence thereof, At least 80%, at least 90%, at least 95%, at least 97%, at least 99% In some embodiments, the target molecule comprises a sequence identical to the target molecule of interest, or a sequence identical to the target molecule of interest. The antisense oligomers are SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:1 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 or a subsequence thereof. In some embodiments, the antisense oligomer comprises SEQ ID NO: 8, SEQ ID NO: SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 or a subsequence thereof. At least 80%, at least 90%, at least 95%, at least 97% , targeting sequences that are at least 99%, or 100% identical.

[0062] In some embodiments, the antisense oligomer is derived from Kit mRNA. splice donor site sequence, splice acceptor site sequence, splice enhancer sequence, Perfectly complementary to at least a portion of the sensor site sequence, branch point sequence, or polypyrimidine sequence At least 80%, at least 90%, at least 95%, at least 97% In some embodiments, the target sequence is at least 99% identical to the target sequence. The transsense oligomer contains the splice donor site sequence, splice donor site sequence, derived from Kit mRNA. splice acceptor site sequence, splice enhancer site sequence, branch point sequence, or polypeptide In some embodiments, the target sequence is perfectly complementary to at least a portion of the amino acid sequence. In this case, the antisense oligomer is SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: Sequence number 14, sequence number 16, sequence number 18, sequence number 19, sequence number 20, sequence number 21 and at least a portion of a sequence selected from the group consisting of SEQ ID NO: 22 or a subsequence thereof. a sequence that is completely complementary to the This portion (a portion of the fragment) contains at least 97%, at least 99%, or 100% identical target sequence. The length of the fragment is, in some embodiments, at least 10 nucleotides. Antisense oligomers may regulate splicing of Kit pre-mRNA molecules. stomach.

[0063] In some embodiments, the target region includes the Kit splice donor site sequence, Kit Splice acceptor site sequence, Kit splice enhancer site sequence, Kit branch The kit comprises at least a portion of a sequence selected from a Kit polypyrimidine sequence, a Kit polypyrimidine sequence, and a Kit polypyrimidine sequence. In some embodiments, this "at least a portion" is a Kit splice donor site sequence. Column, Kit splice acceptor site sequence, Kit splice enhancer site sequence, Kit branch point sequence or Kit polypyrimidine sequence. %, at least 50%, at least 75%, at least 90% or at least 90% The Kit splice donor site sequence, Kit splice acceptor site sequence, and K Kit splice enhancer site sequence, Kit branch point sequence, or Kit polypyrimidine sequence The sequence may be derived from exon 4 of the Kit pre-mRNA. The antisense oligomer may be at least 10 nucleotides in length. may regulate the splicing of Kit pre-mRNA molecules.

[0064] In some embodiments, the complementary nucleic acid sequence contained in the antisense oligomer (i.e., the "targeting sequence") is a splice donor site sequence derived from Kit mRNA. sequence, splice acceptor site sequence, splice enhancer site sequence, branch point sequence, or at least 80% to a sequence that is completely complementary to at least a portion of the polypyrimidine sequence; At least 90%, at least 95%, at least 97%, or at least 99% identical In some embodiments, the complementary nucleic acid contained in the antisense oligomer The sequences are SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 or parts thereof a sequence that is completely complementary to at least a portion of a sequence selected from the group consisting of At least 80%, at least 90%, at least 95%, at least 97%, at least 99% %, or 100% identical sequences. This portion (part) is at least 10 The antisense oligomer may be a nucleotide. The splicing of the offspring may be regulated. In some embodiments, the antisense oligomer consists of SEQ ID NOs: 1 and 2 at least 80% identical, at least 85% identical, at least 90% identical, to a sequence selected from the group % identical, at least 95% identical, at least 97% identical, or at least 99% identical In some embodiments, the antisense oligomer comprises a sequence similar to SEQ ID NO: 1 and a sequence similar to SEQ ID NO: 2. and 2. It consists of.

[0065] In some embodiments of the present invention, a gene encoding an antisense oligomer of the present invention is used. As used herein, an "expression vector" refers to a vector containing a promoter. A nucleic acid molecule comprising operably linked polynucleotide sequences, such that the polymerase Transcription of this polynucleotide sequence by the enzyme results in the production of the antisense oligomers of the present invention. An exemplary expression vector is a nucleic acid molecule into which a polynucleotide is inserted. or cloned, e.g., plasmid, bacteriophage, yeast or virus ( (e.g., adenovirus, adeno-associated virus, lentivirus, retrovirus, etc.) and in some embodiments, DNA molecules derived from Suitable expression vectors are known to those skilled in the art. In some embodiments, the present invention provides an antisense oligomer or expression vector of the present invention. The present invention provides pharmaceutical compositions comprising the antisense oligonucleotides described herein. The present invention therefore provides a method for the therapeutic delivery of one or more oligomers or expression vectors. A therapeutically effective amount of the antisense oligomer or expression vector of the present invention is contained in one or more A pharmaceutical composition formulated together with the above pharmaceutically acceptable carriers (additives) and / or diluents. The antisense oligomer or expression vector of the present invention may be administered alone. However, in some embodiments, the antisense oligomer or expression vector of the invention , administered as a pharmaceutical composition. The pharmaceutical compositions of the present invention may be prepared for administration in solid or liquid form, comprising: (1) Oral administration, e.g., liquid medicine (aqueous or non-aqueous solution or suspension) liquid), tablet (e.g., buccal, sublingual), and systemic absorption, bolus, powder, granule, applied to the tongue (2) as a paste for parenteral administration, e.g., as a sterile solution or suspension or sustained-release preparation; (3) as a topical application, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; For example, as a cream, ointment, or sustained-release patch or spray applied to the skin; (4) Intravaginally or rectally, for example, as a pessary, cream, or foam; (5) sublingually; (6) ophthalmic; (7) transdermal; (8) pulmonary, e.g., by nebulizer or aerosol inhaler. or (9) nasally. Examples of suitable carriers, excipients, and diluents are described in U.S. Patent Publication No. 2015 / 033494. No. 61428, which are incorporated herein by reference in their entireties.

[0066] As described above, the antisense oligomer of the present invention can reduce the expression of Kit. Such a decrease is due to the splicing of the mRNA molecule encoding the Kit protein. Thus, in some embodiments of the present invention, A method for detecting Kit mRNA expression in a cell, comprising contacting the cell with an antisense oligomer of the present invention. The present invention provides a method for regulating the splicing of NA. The cell is characterized in that it encodes a Kit mRNA molecule. The cell may be any cell that expresses the gene. Thus, the cell may be a cell in culture or an individual. It may be a cell in the body. In an exemplary embodiment, the cell is a mast cell. In some embodiments of the present invention, mast cells are treated with the antisense oligomers of the present invention. A method of inducing apoptosis in mast cells is provided, comprising the step of contacting: Thus, in some embodiments of the present invention, the antisense oligomers of the present invention are administered In some embodiments, a method of treating a Kit-related disease in an individual is provided, the method comprising administering a Kit-related disease to an individual. In some embodiments, the Kit-associated disease is cancer or mastocytosis. In some cases, the cancer is a gastrointestinal stromal tumor or leukemia. Mastocytosis is caused when an individual has too many mast cells and mast cell precursors. This is a rare mast cell activation disorder caused by the inflammatory bowel disease. Mast cells are involved in atopic reactions, so Therefore, individuals suffering from mastocytosis may experience hives, itching, and anaphylactic shock. Thus, one method of the present invention involves administering an antisense antibody of the present invention to an individual in need of treatment. A method of treating an individual suffering from mastocytosis comprising administering an oligomer This individual exhibited symptoms of mastocytosis, including itching, hives, and anaphylaxis. In some embodiments, the antisense oligomer is administered to individuals at risk of developing symptoms of mastocytosis.

[0067] This mRNA has at least 80% identity with SEQ ID NO: 8, 10, 12, 14 or 16, at least 85%, at least 90%, at least 95%, at least 97%, at least 9 In some embodiments, the nucleotide sequence may be 9% or 100% identical. and the mRNA has at least 85% identity to SEQ ID NO: 9, 11, 13, 15 or 17. At least 90%, at least 95%, at least 97%, or at least 99% identical In some embodiments, this mRNA encodes a protein comprising the amino acid sequence. encodes a protein consisting of SEQ ID NO: 9, 11, 13, 15 or 17. In some embodiments, the antisense oligomer is a target of the Kit pre-mRNA. In some embodiments, the target region is hybridized to a target region involved in splicing. , this antisense oligomer is a Kit splice donor site sequence, Kit splice Acceptor site sequence, Kit splice enhancer site sequence, Kit branch point sequence, and Kit polypyrimidine sequences. A. This Kit splice donor site sequence, Kit splice Splice acceptor site sequence, Kit splice enhancer site sequence, Kit branch point The Kit polypyrimidine sequence is derived from exon 4 of the Kit pre-mRNA. So that's fine. In some embodiments of the invention, a cell is contacted with an antisense oligomer of the invention. and a method for reducing Kit protein expression in a cell, the method comprising: In an embodiment of the present invention, the cell is any cell that expresses the Kit protein. Thus, the cells may be cells in culture (e.g., tissue culture) or in vivo in an individual. In some embodiments, the amount of Kit expressed by the cell. At least 50%, at least 60%, at least 70%, at least 80%, reduced by at least 90%, at least 95%, at least 97%, or at least 99% In an exemplary embodiment, the cell is a mast cell.

[0068] The antisense oligomers of the present invention can be administered to any individual that expresses the Kit protein. As used herein, the terms individual, subject, patient, etc. include humans, mice, and / or mammals expressing the Kit protein, such as, but not limited to, mammals such as horses, cats, dogs, or horses. The terms "individual," "subject," and "animal" are intended to encompass any mammal. The term "patient" does not refer to a specific age, gender, race, etc. Individuals of any age, male or female, are intended to be subjects of the present invention. The methods of the present invention may be used on, for example, Caucasians, African Americans (Blacks), Native Americans, Native Hawaiians, Hispanics, Latinos, Asians, and Europeans. In some embodiments of the present invention, this Such characteristics may be salient. In such cases, salient characteristics (such as age, sex, race, etc.) ) are presented. Furthermore, the term "individual" refers to both human and non-human animals. Suitable non-human animals to which the antisense oligomers of the present invention may be administered include: This includes companion animals (i.e., pets), food animals, working animals, or zoo animals. Exemplary animals include, but are not limited to, cats, dogs, horses, ferrets, and other including, but not limited to, mustelids, cattle, sheep, pigs, and rodents .

[0069] The antisense oligomers of the present invention can be administered to an individual by any suitable route of administration. Examples of such routes include oral and parenteral routes (e.g., intravenous (IV), cutaneous). intraperitoneal (IP), and intramuscular), inhalation (e.g., nebulization and inhalation), and transdermal delivery (e.g., These include, but are not limited to, topical administration, local administration, etc. Any method effective for delivery into the bloodstream of an individual is also contemplated in these methods. For example, transdermal delivery of antisense oligomers can be achieved by using pharmaceutically acceptable carriers adapted for topical administration. This can be achieved by using a carrier. They can be administered in the absence of other molecules such as proteins or lipids, or they can be administered in the presence of other molecules such as proteins or lipids. It can be administered in a complex with other molecules, such as proteins or lipids. The use of cationic lipids to encapsulate ligomers is disclosed in U.S. Pat. No. 8,569,252. No. 6,806,084, which are incorporated herein by reference in their entireties. Similarly, peptide-linked morpholino antisense oligonucleotides Methods of use are disclosed in U.S. Patent Publication No. 2015 / 0238627, which is incorporated by reference in its entirety. The body is incorporated herein. The antisense oligomers of the present invention can reduce Kit-mediated responses, Such antisense oligomers can be used to treat allergic conditions. Thus, in some embodiments of the present invention, an individual in need of treatment can be administered a therapeutic agent of the present invention. Treating the individual's allergic symptoms by administering an antisense oligomer. The allergic symptoms to be treated are mediated by a pathway involving Kit. This may be any condition. Such conditions include asthma, food allergies, allergic reactions, These include, but are not limited to, conjunctivitis and atopic dermatitis. [Example]

[0070] The following examples will enable one of ordinary skill in the art to easily understand the claimed compounds, compositions, products, devices, and / or The method is presented to provide a complete disclosure and description of how it is constructed and evaluated. These are purely illustrative and are not intended to limit the invention. No effort has been made to ensure accuracy with respect to values (amounts, temperatures, etc.). Although efforts have been made, some errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, temperature is in °C or is ambient temperature, and pressure is atmospheric. pressure or close to it.

[0071] Materials and methods used in the examples Mast cell culture The HMC-1.2 human mast cell (MC) line was purchased from MilliporeSigma (Burlington, Massachusetts, US A) and cultured according to the manufacturer's instructions. The human mast cell (MC) line LAD2 was used as an allergen. Dr. Kirschenbaum and Dr. Meyer of the National Institute of Advanced Industrial Science and Technology (NIAID, NIH, Bethesda, Maryland, USA) Obtained from Dr. Tocalfe and as described in the literature (Kirshenbaum et al., 2003). Bone marrow-derived mast cells (BMMCs) were obtained and cultured as described previously (Jensen et al., 2006). Experiments on mice were performed at the University of North Carolina at Raleigh, North Carolina. The study was conducted under a protocol approved by the Institutional Animal Care and Use Committee of the University of California, San Diego, CA, USA. . Transfection of mast cells (MCs) with antisense oligonucleotides (ASOs) Mast cells (MCs) were transfected as described previously (Cruse et al., 2013). Receptor expression Receptor expression was analyzed by flow cytometry as described previously (Cruse et al., 2013). I looked it up. Apoptosis and viability assays Apoptosis was measured using FITC-Annexin V (eBioscience, a division of Thermo Fisher Scientific) The survival rate was assessed using a tific Inc. (Waltham, Massachusetts, USA). , cells were stained with LIVE / DEAD Green Dead Cell Stain (Invitrogen, Carlsbad, California, USA). The cells were stained with HCl according to the manufacturer's instructions or stained with propidium iodide. Cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, California) , USA). Proliferation assay Proliferation was assessed using CellTrace Far Red (Invitrogen) according to the manufacturer's instructions. Cells were also stained with LIVE / DEAD Green DeadCellStain and trypan blue. Viable cell counts were assessed using the ELISA kit. Intraperitoneal injection and peritoneal lavage The protocol was approved by the Institutional Animal Care and Use Committee at North Carolina State University. Peritoneal lavage was performed after intraperitoneal injection of ASO. Mast cells were identified by flow cytometry. They were identified by Lee as Kit and IgE receptor positive cells. Intradermal injection and skin histology The protocol was approved by the Institutional Animal Care and Use Committee at North Carolina State University. ASO was injected into the skin by ID injection, and skin sections were collected. Mast cells (MCs) Toluidine blue staining was used to identify and count the cells, and H&E was used to evaluate tissue morphology. It was.

[0072] Supplementary Materials and Methods for the Examples Mast cell culture: The HMC-1.2 human mast cell (MC) line was purchased from MilliporeSigma (Burlington, Massachusetts, US A) and diluted in Iscove's modified Dulbecco's medium (IMDM; MilliporeSigma) at 10% (vol / vol ) Fetal bovine serum (FBS) and penicillin (100 U / mL) / streptomycin (100 μg / mL) The human mast cell (MC) line LAD2 was cultured in medium supplemented with 100% ethanol according to the manufacturer's instructions. obtained from Dr. Kirschenbaum and Dr. Metcalfe at the Institute for Disease Research, and StemPro-34 Dietary supplements, L-glutamine (2 mM), penicillin (100 U / mL) / streptomycin (1 00 μg / mL) (Gibco; Gaithersburg, Maryland, USA) in StemPro-34 medium containing 100 ng / m Recombinant human stem cell factor (SCF) (R&D Systems, Minneapolis, Minnesota, USA) was added. The cells were cultured in a medium supplemented with stem cell factor ( Half of the medium supplemented with SCF was replaced once a week. Bone marrow-derived mast cells (BMMCs) were cultured as described in the literature (Je C57BL / 6J mice (The Jackson Laboratory, Bar Harbor) were cultured as described (Bernsen et al., 2006). or, Maine, USA) and was developed from bone marrow obtained from the femur of a mouse recombinant SCF and IL-3 (R&D Systems) 20 ng / mL. Experiments in mice were performed at the North Carolina State University facility. The study was conducted under a protocol approved by the Animal Care and Use Committee. Transfection of mast cells (MCs) with antisense oligonucleotides (ASOs) When transfecting mast cells (MCs) with ASOs, , 2x10 6 of human mast cells (MCs) or 3x10 6 Mouse bone marrow-derived mast cells (BMMC) were used. Transfection was performed using Nucleofector II and CellLine Kit V (Lonza). Transfection was performed as described previously (Cruse et al., 2013). Transfection efficiency was measured at 10 Binding of μM FITC was measured using a standard control 25-mer ASO (Gene Tools LLC). Program T-0 30 was used for LAD2 cells and HMC-1.2 cells, and Program X-001 was used for mouse bone marrow-derived mast cells (BMM C). Cell viability was monitored during the experiment, and no significant difference was observed between ASO transfection and the control group. There was no evidence of cytotoxicity. The standard control ASO and KitStop ESO (exon skipping) used in the experiment It was not bound to the phosphodiesterase (peptide oligonucleotide).

[0073] Antisense oligonucleotide (ASO) design KitStop ESO was used to identify exon 4 of human c-KIT (accession number 10 ... It was designed to target the splice donor site (accession number NM_000222.2). The region was targeted using the sequence of human c-KIT, 5'-GAATGAAGCGATTCACTCACCTGAC-3' (SEQ ID NO: 1). In the case of mouse c-kit, the splice donor site of mouse c-Kit (GENBANK® biosynthesis) was targeted. The region containing the sequence 5'- AGGA All oligonucleotides were targeted with the sequence CTTAAACAGCACTCACCTGAG-3' (SEQ ID NO: 2). The non-binding standard control ASO was provided by Gene Tools LLC. , which does not match any known sequence in mammalian genomes, and contains the sequence 5'-CCTCTTACCTCAGTTACAATTTATA-3' (sequence The standard control ASO was KitStop ESO (exon-skipping oligonucleotides). It has the same chemical properties as ribonucleotides (ribonucleotides), but does not induce exon skipping in known genes. For in vivo studies, octaguanidinium dendrimers (Vivo M) were synthesized via the terminal 3'-N. A morpholino ESO (exon skipping oligonucleotide) linked to a morpholino ESO (exon skipping oligonucleotide) was It was purchased from Gene Tools, LLC (Philomath, Oregon, USA).

[0074] RT-PCR LAD2 cells, HMC-1.2 cells, and mouse bone marrow-derived mast cells (BMMCs) were transfected as described above. For LAD2 and HMC-1.2 cells, RNAeasy plus mini-kit (Qiagen, German Town, Maryland, USA) according to the manufacturer's instructions, including the QIAShredder step. For mouse bone marrow-derived mast cells (BMMCs), the same protocol was used. Total RNA was collected after 24 hours according to the method described in the literature. RT-PCR was performed using a Thermo Fisher Scientific Verso 1-St RT-PCR was performed using the Eppendorf kit according to the manufacturer's instructions. The primers used were exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon KitStopESO amplifies exons 4 and its surrounding exons, and amplifies multiple exons of c-Kit mRNA. For human c-KIT mRNA, the following primers were used: Used: forward: 5'-GAAGCCTCTTCCCAAGGACT-3' (SEQ ID NO: 4), reverse: 5'-GTGTTCAGG TTTGGGGAATG-3' (SEQ ID NO: 5). For mouse c-Kit mRNA, the following primers were used: Forward: 5'-TCATCGAGTGTGATGGGAAA-3' (SEQ ID NO: 6), Reverse: 5'-TCACAGGGGAGATGTTGA TG-3' (SEQ ID NO: 7).

[0075] Receptor expression LAD2 and HMC-1.2 cells were transfected as described above with standard control ASO or KitStop ESO. The transfected cells were incubated in complete medium for 2–7 days and 24–72 hours, respectively. Infected bone marrow-derived mast cells (BMMCs) were incubated in complete medium for 48 hours. For cells, flow cytometry was performed at each time point to examine surface and total Kit expression. APC-conjugated anti-human CD117 monoclonal antibody (clone 104D2) and APC-conjugated mouse IgG1, κ, performed using an isotype control antibody (Biolegend, San Diego, California, USA). For mouse cells, use flow cytometry to identify surface Fcε using the following antibodies: Expression of RIα and Kit was assessed using PE-conjugated anti-mouse FcεRIα (clone MAR-1) monoclonal antibody. Antibodies and PE-conjugated IgG isotype controls (eBioscience via Thermo Fisher Scientific, Waltham, Massachusetts) and FITC-conjugated anti-mouse CD117 (clone 2B8) monoclonal antibody and and FITC-conjugated IgG2b, κ, and isotype control antibodies (BD Biosciences, San Jose, California, USA).

[0076] Apoptosis and viability assays HMC-1.2 cells (2 × 10 6 ) were transfected with standard control ASO or KitStop ESO as described above. The cells were then cut into 100 ml pieces and incubated in complete IMDM for 24–72 hours. At each time point, the cells were pelleted and FI Apoptosis was examined using TC-Annexin V (eBioscience). Cells were stained with LIVE / DEAD Green Dead Cell Stain (Invitrogen) or iodine according to the manufacturer's instructions. The cells were stained with propridium chloride using a CytoFLEX flow cytometer (Beckman Coulter). Flow cytometry was performed using

[0077] Proliferation assay Before transfection, HMC-1.2 cells were washed once with PBS. The cells were treated with 1 μM Cel 1x10 cells were cultured using lTrace Far Red (Invitrogen). 6 Cells / mL concentration according to manufacturer's instructions Briefly, cells were stained for 20 min at 37°C, then washed with 5 volumes of medium for 5 min. Cells were pelleted, resuspended in complete medium, and incubated for 10 min at 37 °C. The stained cells were then incubated with standard counter- measures as described above. HMC-1.2 cells were transfected with the control ASO or KitStopESO and cultured at 37°C for 24–72 hours. At each indicated time point, cells were stained with LIVE / DEAD Green Dead Cell Stain, which was used to detect dead cells. Proliferating cells were identified and removed using an additional set of gates for single cell populations. Flow cytometry was performed using a CytoFLEX flow cytometer. Trypan blue was used to assess the number of viable cells at various time points.

[0078] Intraperitoneal injection and peritoneal lavage Female BALB / c mice aged 4 to 8 weeks were purchased from Jackson Laboratory and allowed to acclimate for at least 1 week. This was done under the supervision of the Institutional Animal Care and Use Department at North Carolina State University. To prevent trauma and blood contamination during injection, gas anesthesia is used. Treatment groups received 50 μl of 0.1% guanosine monophosphate (GU) by intraperitoneal injection using a 29-gauge needle on days 0 and 3 under anesthesia. 5 mM KitStop Vivo Morpholino ESO or control Vivo Morpholino ASO were administered. Euthanasia was performed according to animal health guidelines, and peritoneal fluid was collected via peritoneal lavage. 5 mL of ice-cold PBS containing A was injected through the abdominal muscles. The abdomen was gently massaged for 30 seconds. Then, 3-4 mL of ascites mixed with the lavage fluid was collected by aspirating it into a syringe and placed on ice in a conical container. The cells were transferred to a tube and transported. 1 x 10 6 Flow cytometry of the cells revealed that the surface Total mast cell numbers were determined by Kit and IgE receptor expression. For RT-PCR, 5x10 6 Individual cells The cells were pelleted and flash frozen in liquid nitrogen. Total RNA was collected and RT-PCR was performed as described above. Ta.

[0079] Intradermal injection and skin histology Female BALB / c mice aged 4 to 8 weeks were purchased from Jackson Laboratory and allowed to acclimate for at least 1 week. This was done using the Institutional Animal Care and Use Facility at North Carolina State University. Approved by the committee. Gas anesthesia was administered on days 0, 7, and 14 to allow for precise injection. Under sedation, the mice were incised with 29-gauge needles into separate sites of the skin overlying the dorsum, 1.5 cm lateral to the midline. By repeatedly injecting the KitStop Vivo morpholinoESO and the control VivomorpholinoASO, On day 16, euthanasia was performed according to AVMA-approved guidelines, and 1 cm x 1 cm skin sections were excised and fixed in 10% neutral buffered formalin for at least 48 hours and then 5 microns thickened. The skin was sectioned at 100 mm intervals and stained with toluidine blue or hematoxylin and eosin. Microscopic examination of sections was performed by two ACVP board-certified pathologists, one of whom was independent of the treatment group. The toluidine blue-stained skin sections were stained with 100% ethanol. Nuclei containing intact mast cells were counted. Two 5-micron-thick pairs representing opposite halves of a cross section through phosphorus-fixed, paraffin-embedded skin tissue The count was taken along the entire length of the fabric section.

[0080] Humanized xenograft mast cell tumor model Six- to eight-week-old NSG mice (NOD-scidIL2R) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). gamma null ) on the right flank, 1 x 10 6 HMC-1.2 tumor mast cells (MCs) were injected subcutaneously. The size was measured with a Mitutoyo IP65 caliper. 3 When it reaches for systemic administration, intravenously (iv) or subcutaneously at the tumor site every 3 days for up to 14 days. For intravenous (iv) injection, 8.33 mg / kg KitStop ESO was injected, and for subcutaneous (sc) injection, For the 100% ovarian tumors, 3.33 mg / kg KitStop was injected. Tumor size was measured daily and tumors were measured to determine whether they were 100% ovarian tumors. Mice were euthanized when they reached 0.5 cm or on day 14.

[0081] [Example 1] The field in mast cells (MCs) treated with exon skipping oligonucleotides (ESOs) Wild-type and mutant c-Kit exon 4 skipping Based on the results of propidium iodide and Live / Dead staining (Cruse et al., 2016), Exon-skipping oligonucleotides produce >95% of the desired activity in mouse mast cells (MCs) Transfection efficiency of human c-KIT protein (ESO) was achieved without evidence of cytotoxicity. Analysis of the mRNA sequence revealed that ESO-induced skipping of exon 4 resulted in the formation of a mature This is predicted to introduce a frameshift into the open reading frame of the mature mRNA. The protein predicted from this mRNA contains a premature stop codon. Therefore, ESO-induced frameshifting induces premature termination of c-Kit mRNA translation, resulting in the production of the protein. significantly shortening the quality of the mRNA or inducing nonsense-mediated mRNA decay (NMD) (Fig. 1A). Either outcome would eliminate the expression of the Kit receptor. A morpholino ESO of mer inhibits the donor splice of exon 4 of c-Kit pre-mRNA, called KitStop. The splice site is located early in the mRNA transcript. Therefore, when the production of a truncated protein occurs, the protein is significantly shortened. HMC-1.2 cells expressing the G560V and D816V mutations were transfected with LAD2 cells expressing wild-type Kit (Fig. 1A). As shown by RT-PCR, the transfection was performed with an equivalent 25-mer standard control ASO. Compared with transfected cells, KitStop showed a significant difference in the expression of mutant c-Kit (Fig. 1B) and wild-type (Fig. 1C) It induced exon skipping in both mRNAs.

[0082] [Example 2] Loss of Kit expression by c-Kit exon skipping Introduction of a frameshift into wild-type or mutant c-Kit mRNA prevents expression of the Kit protein To investigate whether this is the case, we used flow cytometry to measure the expression of β-glucan in LAD2 and HMC-1.2 cells. Kit expression was measured in 100% HO cells (see Figures 2E and 2F for gating strategy). Mast cells (MCs) express wild-type Kit on the cell surface, but Kit is expressed only by its ligand They are rapidly internalized following activation by stem cell factor (SCF), a cytoplasmic receptor. After activation, Kit signals within intracellular compartments before degradation (Wiley & Burke, 2014). 01). Therefore, both surface (Figures 2A and 2C) and total Kit (Figures 2B and 2D) expression KitStop reduced surface Kit expression by 94.5±1.4% after 48 hours and 7.5±1.4% after 7 hours. After 48 hours, total Kit expression in the same cells was reduced by 99.0 ± 0.6% (Fig. 2C). After 7 days, the decrease was 3.5 ± 0.9%, and after 7 days, it was 95.6 ± 1.8% (Figure 2D). In human mast cells (MCs) treated with p, the expression of surface and wild-type Kit was almost completely suppressed. It indicates that it is completely lost. In neoplastic mast cells (MCs), such as the HMC-1.2 cell line, oncogenic Kit signaling is mediated by SCF occurs within intracellular compartments independent of K (Obata et al., 2014). The total expression level of it was measured in HMC-1.2 cells (see Figures 3D-3F for gating strategy). HMC-1.2 cells proliferate significantly more than LAD2 cells, and therefore KIT expression is higher. Short time points were assessed (Figure 3A), and KitStop was found to significantly reduce total mutant KIT expression. KIT expression was partially reversed over 72 hours in HMC-1.2 cells (Fig. 3B and 3C). The recovery was observed in the LAD2 cells (Fig. 3B and 3C), but not in the KitSto cells (Fig. 2D). The total number of viable cells in HMC-1.2 cells treated with p was 2% compared to cells treated with the standard control ASO. Transfection efficiency was approximately 95%. Therefore, it can be efficiently transfected with exon-skipping oligonucleotides (ESOs). A small fraction of untransfected cells remained after 72 hours, while efficiently transfected cells It is believed that the expression of Kit was lost and consequently decreased during the experiment.

[0083] [Example 3] KitStop reduces constitutive KIT signaling in HMC-1.2 cells The KIT D816V mutation results in constitutive KIT signaling, which is critical for the survival and proliferation of HMC-1.2 cells. This provides further evidence that the effect of KitStop on mitochondrial function is due to KIT-dependent signaling. We then examined the phosphorylation of KIT and the downstream kinase ERK. ERK was chosen because it is involved in the Ras-R It is a downstream kinase in the af-MEK-ERK pathway, which is downstream of both KIT signaling pathways. This is because K Both IT and ERK were constitutively phosphorylated (Fig. 4A). KitStop treatment reduced the phosphorylation of both KIT (Fig. 4B) and ERK (Fig. 4C) equally. Rather than inhibiting KIT phosphorylation, it effectively suppresses the expression of functional KIT protein. This reduction in KIT phosphorylation is associated with the expression of constitutively active KIT protein. However, KitStop showed a decrease in the expression of β-actin compared to β-actin. , did not affect ERK expression (Fig. 4A), so downstream effects on the ERK pathway were not related to phosphorylation This is due to a decrease in KIT protein, possibly a direct result of the decrease in KIT protein. can be.

[0084] [Example 4] Frameshifted c-Kit induces the death of neoplastic mast cells (MCs) Non-transformed mast cells (MCs) express apoptosis in vitro in the absence of exogenous supportive cytokines. apoptosis occurs rapidly (Mekori et al., 1993; Yanagida et al., 1995; Asai et al. , 2001), but in contrast, activating c-Kit mutations such as D816V are not constitutive Kit In neoplastic mast cell (MC) disease cell lines such as HMC-1.2, constitutive Kit expression is Activation of SCF allows for the continued survival and growth of mast cells (MCs) in an SCF-independent manner. Therefore, KitStop treatment inhibited the imatinib-insensitive activity of c-Kit mutations in tumorigenic HMC-1.2 cells. We first investigated whether the pro-survival effect of activation could be eliminated by activating HMC-1.2 cells. The activity of the standard control ASO was assessed using FITC-conjugated annexin V (Figures 5A, 5F, and 5G). Compared to transfected cells, KitStop-treated cells showed significantly higher levels of apoptosis over 72 hours. KitStop showed increased Nexin V-FITC staining over 72 hours (Figures 5A and 5B). The number of apoptotic cells was reduced (Fig. 5C), and early (Fig. 5D) and late (Fig. 5E) apoptotic cells were The number of cytosed cells increased correspondingly. Stained with propidium chloride (PI) (Figures 6A, 6F-6H). In contrast to standard control ASOs, Furthermore, KitStop caused an increase in PI staining over 72 hours (Figures 6A-6C). Similar staining patterns were observed in color (Figures 6D, 6I, and 6J), with no significant difference in the number of dead cells at each measured time point. In summary, these findings suggest that exon skipping oligonucleotides may be involved in the regulation of genomic DNA synthesis. ESO-mediated frameshifting of c-Kit transcripts in neoplastic mast cells (MCs) We show that it rapidly induces apoptosis and cell death in

[0085] [Example 5] c-Kit frameshifting inhibits proliferation of neoplastic mast cells (MCs) Activating c-Kit mutations expressed by HMC-1.2 cells promote SCF-independent growth. Considering this ( Longley et al., 1999 ; Chan et al., 2013 ), we investigated the role of MC in tumor proliferation. We examined the effect of c-Kit frameshifting on cells transfected with a standard control ASO. In contrast, trypan blue cell counting showed an increase in viable cell numbers. As shown in Figure 7A, KitStop prevented the proliferation of HMC-1.2 cells. Proliferation assays were performed using CellTrace dilution and LIVE / DEAD staining. Similarly, the population of live KitStop-treated cells was significantly higher than that of the CellTrace dye, as shown by LIVE / DEAD staining. While the CellTrace fluorescence of standard control ASO-treated cells remained constant, it became increasingly diluted at each time point. Both the standard control ASO and KitStop-treated cells produced viable, non-proliferating cells (Figures 7B and 7C). The percentage of viable non-proliferating cells decreased over time (Fig. 7D). The decrease in the number of cells was due to an increase in nonviable cells rather than proliferating cells (Fig. 7 B and 7E). Taken together, these findings support the role of ESO-induced c-Kit filamentation. We demonstrated that this phenotype significantly suppresses mutant Kit-mediated tumor cell growth and survival. As a result, the frameshifting of c-Kit-targeting oligonucleotides is associated with a significant increase in the in vivo We present an efficient approach to reduce mast cell (MC) burden in mice.

[0086] [Example 6] Depletion of peritoneal mast cells (MCs) by KitStopESO in vivo c-Kit frameshift oligonucleotide expression in neoplastic human mast cells (MCs) in vitro Given the results with otide, we investigated this approach in vivo to demonstrate its therapeutic utility. We evaluated the efficacy of this approach by using mouse bone marrow-derived mast cells (BMMCs) to express mouse c-Kit. We tested the targeting KitStopESO by RT-PCR (Figure 8A) and found a significant reduction in Kit protein expression. As shown by the small number of exons in the mouse c-Kit mRNA (Fig. 8B and 8C), KitStop inhibited the exons of the mouse c-Kit mRNA. Skipping was induced in vivo by two intraperitoneal injections followed by peritoneal lavage. The effectiveness of KitStop was tested (Figure 8D). Exon skipping of c-Kit mRNA was observed in the peritoneal Although not always evident in lavage cells, c-Kit mRNA levels were significantly elevated in the majority of mice. The peritoneal Kit + This suggests a decrease in cells. In treated mice, flow cytometry of peritoneal lavage cells showed that peritoneal mast cells (MCs) were significantly increased by approximately 50%. (Figures 8F-8H). Taken together, these data suggest that KitS top also functions in vivo and is a viable method for depleting mast cells (MCs) in other tissues. This suggests that this may be an approach.

[0087] [Example 7] Topical ESO administration reduces the number of cutaneous mast cells (MCs) in vivo As further evidence of the therapeutic potential of KitStop for depletion of mast cells (MCs) in tissues The efficacy of KitStop was investigated by intradermal (ID) injection into the dorsal skin. effectively downregulates mast cell (MC) function in response to IgE signaling, It has been shown to provide a model to assess ESO activity on mast cells (MCs) in vivo Therefore, we evaluated the efficacy of KitStop in the skin. is long-lived and abundant in the skin, so the administration protocol has been expanded from peritoneal studies. After injecting KitStop or a standard control ASO into the dorsal skin of mice three times over a two-week period, The skin histology was evaluated (Figure 9A). The skin sections were stained with toluidine blue (Figure 9B) or H&E (Figure 9C). Mast cells (MCs) were identified and examined by staining with either HCl or HCl (9C). Two board-certified pathologists assessed mast cell (MC) counts and tissues, one blinded and one unblinded. Although both pathologists examined tissue sections, the number and conclusions reached were comparable. Furthermore, the area of skin treated with KitStop was free of obvious lesions from the same mice. Dermal mast cells (MCs) when compared to standard control ASO-treated skin areas harvested (Fig. 9D) and total dermal mast cells (MC) (Fig. 9E) were reduced by approximately 50%. The data show that KitStop depletes mast cells (MCs) present in mature tissues in vivo. This provides further evidence that

[0088] [Example 8] Systemic administration of KitStop ESO inhibits proliferation of neoplastic mast cells (MCs) in vivo We confirmed that KitStopESO functions effectively both in vitro and in vivo. KitStop was tested in a humanized in vivo model relevant to the treatment of aggressive tumors. Tumor-forming HMC-1.2 cells were inoculated into NSG mice. Tumor volumes were approximately 50 mm 3 reached (day 0 ) followed by intravenous (IV) or subcutaneous (SC) injection of KitStop once daily for every 3 days. Using this conservative dosing protocol, tumor progression over time was significantly reduced compared to control (Figure 10A). Tumor volume was assessed and measured to demonstrate that systemic IV administration of KitStop significantly reduced tumor growth. On day 14 of treatment, the mice were euthanized, and the tumors were excised and weighed. The tumors in the KitStop intravenous (IV) injection group were measured (Fig. 10C and 10D). The tumors were significantly smaller than those in the control group and the KitStop subcutaneous (SC) injection group. The liver showed a tendency to decrease in size in the KitStopIV group (Fig. 10E), but this tendency was not observed during the course of the experiment. There were no differences in liver weight (Fig. 10F) or mouse weight (Fig. 10G) over the entire study. Taken together, these data support the efficacy of KitStop in an aggressive humanized mast cell tumor model. This supported the conclusion that systemic (IV) administration of KitStop significantly inhibited tumor growth. The treatment was well tolerated and had little effect on the body weight of the mice.

[0089] Discussion of Examples The role of constitutive Kit signaling in abnormal mast cell (MC) proliferation is unclear. , and targeting for the resulting clonal disease. This disease varies depending on the patient's age, organ system, and The heterogeneity and treatment challenges stem from differences in the involvement of Notoriously (Valent et al., 2017). However, the underlying mechanisms of oncogenic Kit signaling are unclear. The diversity of Kit mutations makes receptor targeting proportionally more difficult. Among these mutations: The D816V mutation, present in the majority of patients with systemic mastocytosis, is particularly problematic. By inducing a conformational change in the tyrosine kinase domain of t, It confers resistance to imatinib mesylate (Gleevec), a kinase inhibitor (TKI) (Ant onescu et al., 2005; Theoharides et al., 2015). D816V + New treatments for systemic mastocytosis Therapy is midostaurin, a multikinase inhibitor that has shown high response rates. Even after treatment with midostaurin, there is a risk of disease progression and transformation to leukemia (Gotlib et al., 2016; Valent et al., 2017). Other neoplastic diseases such as gastrointestinal stromal tumors (GISTs) It can be treated with imatinib, but similar to mastocytosis, it acquires additional c-Kit mutations over time. As a result, imatinib sensitivity may be lost (Heinrich et al., 2006; Tamborini Other neoplastic diseases, such as GIST, commonly involve somatic gain-of-function mutations in c-Kit. and predicts the risk of metastasis (Heinrich et al., 2006; Ito et al., 2014). Combinations of multiple tyrosine kinase inhibitors (TKIs) have been shown to inhibit the growth of neoplastic mast cells (MCs). These compounds may have synergistic inhibitory effects, potentially offering therapeutic benefit. However, acquired resistance to tyrosine kinase inhibitors (TKIs) remains a risk (Gleixner et al., 2007; Gallogly et al., 2017). This results in abnormal mast cell (MC) proliferation. Given the strong association between proliferation and survival with activating c-Kit mutations, A desirable approach is to identify receptors that are compatible with the many possible mutations and conformations of the receptor. The first approach would be to specifically target c-Kit with high efficacy.

[0090] Antisense oligonucleotide (ASO) technology holds promise for selectively targeting c-Kit This is a promising approach. Advances in new generation chemical modifications improve efficacy, efficacy, and delivery (Juliano, 2016; Godfrey et al., 2019). 17; McClorey & Banerjee, 2018) is an exciting advancement in this ASO technology. They are versatile and, depending on their design, can be used for a variety of genetic engineering applications. For example, some applications of ASOs include blocking protein translation or exon unpacking. This is because it promotes the inclusion of ribosomal proteins and induces exon skipping in mature mRNA. In the examples, the latter is achieved using exon skipping oligonucleotides (ESOs). This introduced a premature stop codon into the c-Kit mRNA, allowing the expression of both wild-type and mutant forms of the c-Kit mRNA. Kit expression is inhibited and Kit-dependent proliferation of neoplastic mast cells (MCs) is inhibited in vitro. In tumorigenic HMC-1.2 cells, KitStop inhibited intracellular Kit expression and significantly suppressed cell viability. down-regulated Kit expression, but the D816V mutation abolished the requirement for Kit surface expression This is particularly important because oncogenic Kit signaling is not mediated by ligand binding. This occurs intracellularly independently (Obata et al., 2014), and therefore, the absence of mutant Kit expression on the surface This makes antibody-based therapeutic approaches ineffective. In vivo administration of Kit Stop increased the number of peritoneal mast cells (MCs) and skin mast cells in mouse tissues. It has been shown that KitStop reduces the number of mast cells (MCs). Therefore, KitStop reduces the burden of mast cells (MCs). KitStop reduced the number of mast cells (MC) in the peritoneal cavity by approximately 50% after 4 days and after 3 treatments. A similar decrease in mature cutaneous mast cells was observed after treatment. It is common for skin mast cells (MCs) to have a long lifespan, and ex vivo cutaneous mast cell (MC) MC) are resistant to growth factors and the absence of growth factor receptors (SCFR) (Hazzan et al. (t al., 2017), the effect of KitStop on skin mast cell (MC) numbers has been shown to be related to diseases such as mastocytosis. This is particularly important to demonstrate the potential effectiveness of KitStop in

[0091] Rather than targeting c-Kit using traditional siRNA approaches, we targeted the pre-mRNA splice. A typical reason for choosing to alter the signaling is to alter the c-Ki expressed by mast cells (MCs). The abundance of t transcripts and the availability of siRNA approaches to mast cells (MCs), especially in vivo The two main reasons for this are the inefficiency of KitStop in targeting c-Kit mRNA and the inefficiency of RT-PCR. Apparently inefficient expression in human and mouse mast cells (MCs) as determined by PCR. This inefficient exon skipping leads to the Stoichiometry of c-Kit mRNA transcripts compared to exon skipping oligonucleotides (ESOs) The efficiency of exon skipping depends on the number of exons. This could be improved by testing different ESOs targeting other splicing regulatory sites. However, even with the apparently inefficient exon skipping, KitStop was able to produce wild-type and It efficiently reduces the expression of the mutant Kit protein, resulting in loss of proliferation, apoptosis, and cell death. KitStop significantly induced death. Does KitStop result in the production of severely truncated C-terminal proteins? The CRISPR / Cas system, which introduces a frameshift into a transcript, may This leads to sense-mediated mRNA decay (NMD), but NMD is often inefficient and difficult to detect. This results in the generation of C-terminal truncated proteins (Reber et al., 2018). The traditional siRNA approach or RNA-induced silencing of transcripts via the RNA-induced silencing complex (RISC) In contrast to the degradation pathway, the inefficiency of saturable mRNA degradation using ESO limits the efficiency of knockdown. rather than loss of the protein, resulting in the production of a non-functional or dominant-negative protein. While not wishing to be bound by a particular theory of operation, it is possible that Regarding high copy number of undesired transcripts, this phenomenon plays a role in therapeutic success. However, ESO is a reversible manipulation of transcripts and does not alter the genome, The production of a severely truncated protein is transient, and therefore, Compared to the CRISPR / Cas system, which must overcome internal regulatory processes, the present invention It makes targeted changes to elephant genes, which has advantages for certain therapeutic applications (Haap aniemi et al., 2018; Ihry et al., 2018).

[0092] In summary, the in vitro and in vivo data disclosed above support the conclusion that mast cell (MC) tumorigenesis Proof of concept for ESO-induced frameshifting of c-Kit as a therapeutic treatment for disease These experiments with wild-type mice are a promising tool for evaluating the therapeutic potential of KitStop. This was a proof-of-concept step. As previously discussed (Cruse et al., 2016), Skipping oligonucleotides (ESOs) represent a reversible therapy and therefore Avoid irreversible changes in the phenotype. This study provides promising approaches for the treatment of mast cell (MC) tumors and gastrointestinal stromal tumors (GIST). This represents a Kit-specific therapeutic strategy with translational potential for clinical application. Furthermore, c-Kit inhibits Kit-driven cancers, such as certain melanomas, and tumors such as small cell lung cancer and brain tumors. Cancers in which secondary c-Kit mutations are acquired and subsequently contribute to tumor progression (Pittoni et al. (see review in [End Page 111], 2011). It also plays a role in other types of cancer. In the treatment of ESO, which is used to alter subtype variants and protein isoforms The possible risk factors are hypercholesterolemia and cardiovascular disease (Disterer et al., 2013), breast cancer ( It has already been emphasized in diseases such as cerebrospinal fluid (Wan et al., 2009) and allergies (Cruse et al., 2016). Improvements can be made for ESO's specific delivery platform, but Latest results of ESO eteplirsen for the treatment of Duchenne muscular dystrophy (DMD) Recent approvals (Dowling, 2016; Syed, 2016) have highlighted the potential of other ESOs as personalized targeted therapies. This indicates a high probability of occurrence.

[0093] References In addition to all references cited in the above disclosure, all patents listed below licenses, patent applications and publications, scientific journal articles, and database entries (e.g., GENB ANK® database entries and all annotations available therein), but these All references, including but not limited to, the methodologies, techniques, and / or compositions, to the extent that they supplement, explain, provide background to, or teach the subject matter herein. is incorporated herein by reference. Antonescu et al. (2005) Clin Cancer Res 11(11): 4182-4190. Arock et al. (2015) Leukemia 29(6): 1223-1232. Asai et al. (2001) Immunity 14(6): 791-800. Berger et al. (2000) Nuc Acid Res 28:2911-2914. Besmer et al. (1986) Nature 320(6061): 415-421. Chan et al. (2013) Clin Exp Dermatol 38(5): 538-544. Cruse et al. (2013) Immunity 38(5): 906-917. Cruse et al. (2014) Immunol Allergy Clin North Am 34(2): 219-237. Cruse et al. (2016) Proc Natl Acad Sci U S A 113(49): 14115-14120. Disterer et al. (2013) Mol Ther 21(3): 602-609. Dowling (2016) Nat Rev Neurol 12(12): 675-676. Galli et al. (1995) Int Arch Allergy Immunol 107(1-3): 51-53. Gallogly et al. (2017) Ther Adv Hematol 8(9): 245-261. Giebel et al. (1992) Oncogene 7(11): 2207-2217. Gleixner et al. (2006) Blood 107(2): 752-759. Gleixner et al. (2007) Haematologica 92(11): 1451-1459. Godfrey et al. (2017) EMBO Mol Med 9(5): 545-557. Gotlib et al. (2016) N Engl J Med 374(26): 2530-2541. Haapaniemi et al. (2018) Nat Med 24(7):927-930. Hazzan et al. (2017) Cell Death Discov 3: 17048. Heinrich et al. (2006) J Clin Oncol 24(29): 4764-4774. Iemura et al. (1994) Am J Pathol 144(2): 321-328. Ihry et al. (2018) Nat Med 24(7):939–946. Ito et al. (2014) Int J Clin Exp Pathol 7(11): 8024-8031. Jensen et al. (2006) Curr Protoc Immunol Chapter 3: Unit 3.23. Jensen et al. (2008) Br J Pharmacol 154(8): 1572-1582. Juliano (2016) Nucleic Acids Res 44(14): 6518–6548. Kirshenbaum et al. (2003) Leuk Res 27(8): 677-682. Longley et al. (1999) Proc Natl Acad Sci USA 96(4): 1609-1614. Ma et al. (2002) Blood 99(5): 1741-1744. McClorey & Banerjee (2018) Biomedicines 6(2): 10.3390 / biomedicines6020051. Mekori et al. (1993) J Immunol 151(7): 3775-3784. Miettinen et al. (2002) Eur J Cancer 38 Suppl 5:S39-51. Obata et al. (2014) Nat Commun 5: 5715. Pardanani et al. (2003) Lancet 362(9383): 535-536. Pittoni et al. (2011) Oncogene 30(7): 757-769. Reber et al. (2018) Mol Biol Cell 29(2): 75-83. Syed (2016) Drugs 76(17): 1699-1704. Tamborini et al. (2006) Oncogene 25(45): 6140-6146. Taniguchi et al. (1999) Cancer Res 59(17): 4297-4300. Theoharides et al. (2015) N Engl J Med 373(19): 1885-1886. Tsai M et al. (1991) Proc Natl Acad Sci USA 88(14): 6382-6386. US Patent Nos. 6,806,084; 8,569,256; 8,765,703; 8,946,183. US Patent Publication Nos. 2015 / 0238627; 2015 / 0376615. Valent et al. (2017) Cancer Res 77(6): 1261-1270. Wan et al. (2009) Int J Cancer 124(4): 772-777. Wiley & Burke (2001) Traffic 2(1): 12-18. Wu et al. (2012) Int J Mol Med 30(1):63-68. Yanagida et al. (1995) Blood 86(10): 3705-3714. Yang et al. (2015) Am J Transl Res 7(9):1499-1509.

[0094] Various details of the invention may be changed without departing from the scope of the invention disclosed herein. It should be understood that the foregoing description is intended to be illustrative only. and is not intended to be limiting.

Claims

1. An antisense oligomer comprising 10 to 50 linked nucleotides, The oligomer targets a region of the pre-mRNA encoding Kit, and the target region is K Antisense oligonucleotides containing sequences involved in splicing of the pre-mRNA encoding it Ligomar.

2. Hybridization of the antisense oligomer to the pre-mRNA encoding Kit 2. The antisense oligonucleotide of claim 1, wherein the splicing of the pre-mRNA alters the splicing of the pre-mRNA. Ins oligomer.

3. Hybridization of the antisense oligomer to the pre-mRNA encoding Kit 2. The antisense oligonucleotide of claim 1, wherein the oligonucleotide reduces the expression of Kit protein. Gomer.

4. The Kit protein whose expression is reduced is a wild-type Kit protein or a mutant Kit protein.

4. The antisense oligomer of claim 3, which is a t protein.

5. The target region may include an intron sequence, an exon sequence, an intron / exon junction, a sequence containing a splice donor sequence, a splice acceptor sequence, a splice enhancer sequence, -sequence, splice branch point sequence, or polypyrimidine tract. The antibody according to any one of claims 1 to 4, comprising at least a portion of a polynucleotide sequence. Antisense oligomers.

6. The polynucleotide sequence may be a sequence encoding a nucleotide ... Kit exons 2 to 20 (not specified) The antisense oligomer of claim 5 .

7. The pre-mRNA encoding Kit is transcribed from the c-Kit gene.

7. An antisense oligomer according to any one of claims 6 to 6.

8. The Kit protein is selected from the group consisting of human Kit protein, mouse Kit protein, canine Kit protein, and the like. protein, feline Kit protein, and equine Kit protein. The antisense oligomer according to any one of claims 1 to 7.

9. hybridization of the antisense oligomer to c-Kit pre-mRNA, resulting in the production of a mature c-Kit mRNA molecule lacking at least a portion of exon 4. The antisense oligomer according to claim 7.

10. hybridization of the antisense oligomer to c-Kit pre-mRNA, 9. The method of claim 8, wherein the method results in the production of an mRNA molecule encoding a truncated Kit protein. Antisense oligomers.

11. The 10 oligonucleotides are arranged so that the oligonucleotides specifically hybridize to the target sequence. 50 linked nucleotides of the target nucleic acid sequence in the pre-mRNA encoding Kit 10. The antisense oligomer of claim 1, comprising a target-directed nucleic acid sequence sufficiently complementary to -.

12. Hybridization of the antisense oligomer to the pre-mRNA encoding Kit 12. The antibody of claim 11, wherein the nucleotide sequence alters the splicing of the pre-mRNA. Sense oligomer.

13. Hybridization of the antisense oligomer to the pre-mRNA encoding Kit 12. The antisense oligonucleotide of claim 11, wherein the oligonucleotide reduces the expression of Kit protein. Ligomar.

14. The Kit protein whose expression is reduced is a wild-type Kit protein or a mutant Kit protein. t protein.

15. The target-directed sequence perfectly matches at least six consecutive nucleic acid bases in the target sequence.

14. The antisense oligonucleotide of claim 13, comprising at least six consecutive complementary nucleobases. Gomer.

16. The target-directed sequence is composed of consecutive similarly sized fragments of the target sequence over its entire length. The antisense oligonucleotide of claim 11, which is at least 80% complementary to the nucleobase linkages. Sense oligomer.

17. The target region may include an intron sequence, an exon sequence, an intron / exon junction, a sequence containing a splice donor sequence, a splice acceptor sequence, a splice enhancer sequence, -sequence, splice branch point sequence, or polypyrimidine tract. The method according to any one of claims 11 to 16, comprising at least a portion of a polynucleotide sequence Antisense oligomers.

18. The polynucleotide sequence may be a sequence encoding a nucleotide ... Kit exons 2 to 20 (not specified) The antisense oligomer of claim 17 .

19. 11. The pre-mRNA encoding Kit is transcribed from the c-Kit gene.

19. The antisense oligomer according to any one of claims 1 to 18.

20. The Kit protein is selected from the group consisting of human Kit protein, mouse Kit protein, canine Kit protein, and the like. protein, feline Kit protein, and equine Kit protein. The antisense oligomer according to any one of claims 11 to 19.

21. The target sequence is a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 18-22 and (optionally, the target sequence is selected from the group consisting of SEQ ID NOs: 23-60) 21. The antibody of claim 20, comprising at least a portion of a selected polynucleotide sequence. Sense oligomer.

22. 22. The amino acid sequence of claim 21, wherein the portion is at least 10 consecutive nucleotides. Chisense oligomer.

23. the target sequence comprises a sequence at least 90% identical to a partial sequence of SEQ ID NOs: 18-22; (Optionally, the target sequence is at least 90% identical to one of SEQ ID NOs: 23-60. The antisense oligomer of claim 21 , comprising the sequence

24. The target sequence is preferentially selected from the group consisting of SEQ ID NOs: 1, 2, and 23-60.

22. The antisense oligomer of claim 21 which hybridizes to

25. The target-directed sequence has in its sequence the sequence of SEQ ID NO: 1, 2 and SEQ ID NO: 23 to 60 at least 10 consecutive nucleobases in a sequence selected from the group consisting of a reverse complement of 21. The nucleic acid of claim 20, comprising at least 10 consecutive nucleic acid bases identical in sequence to Antisense oligomers.

26. The target-directed sequence corresponds to the reverse complement of at least a portion of a partial sequence of SEQ ID NOs: 18 to 60.

21. The antisense oligomer of claim 20, comprising a sequence that is at least 80% complementary to 。

27. The targeting sequence is the reverse complement of one of SEQ ID NOs: 1, 2 and 23-60.

2. A sequence selected from the group consisting of:

10. The antisense oligomer according to claim 0.

28. The targeting sequence is the reverse complement of one of SEQ ID NOs: 1, 2 and 23-60.

21. The antisense oligomer of claim 20, selected from the group consisting of:

29. The c-Kit transcript is any one of SEQ ID NOs: 8, 10, 12, 14 and 16, or The antisense oligonucleotide according to claim 2, comprising an open reading frame present in Gomer.

30. 30. The method of claim 1, wherein the antisense oligomer is an antisense RNA molecule. The antisense oligomer according to any one of claims 1 to 4.

31. The antisense RNA molecule may comprise a nucleotide modification, an internucleotide modification, a sugar modification, a sugar- internucleotide bond modifications, and combinations thereof 31. The antisense oligomer of claim 30.

32. An expression vector encoding the antisense oligomer according to any one of claims 1 to 31. -.

33. Any of claims 1 to 31, wherein the antisense oligomer is a morpholino oligomer. The antisense oligomer according to claim 1.

34. The antisense oligomer according to any one of claims 1 to 31, the antisense oligomer according to claim 32 34. A pharmaceutical composition comprising the present vector or the morpholino oligomer of claim 33.

35. Methods for modulating splicing of Kit pre-RNA in cells and / or tissues The cells and / or tissues are treated with an antisense oligonucleotide according to any one of claims 1 to 31.

34. The oligomer, the expression vector of claim 32, or the morpholino oligonucleotide of claim 33. A method comprising contacting a soluble fiber with a soluble fiber.

36. 1. A method for inducing apoptosis in mast cells, comprising administering the mast cells to a mammalian subject comprising administering to the mammalian subject a mammalian subject apoptosis inhibitor, ... The antisense oligomer according to any one of claims 1 to 31, and the expression vector according to claim 32. or the morpholino oligomer of claim 33.

37. 37. The method of any one of claims 35 and 36, wherein the method is performed in an individual.

38. 1. A method for treating a disease, disorder, or condition associated with Kit expression in an individual, comprising: The individual is administered an antisense oligomer according to any one of claims 1 to 31, or or the morpholino oligomer of claim 33. Methods including:

39. 38. The method of claim 37, wherein the disease, disorder, or condition associated with Kit expression is cancer or mastocytosis. The method described below.

40. 40. The method of claim 39, wherein the cancer is a gastrointestinal stromal tumor or leukemia.

41. 49. The method according to any one of claims 40 to 48, wherein the individual is an animal (optionally a mammal). The method described.

42. 42. The method of claim 41, wherein the individual is a human, mouse, dog, cat, or horse.

Images