EA045399B1 - OLIGONUCLEOTIDES INTERPRETER THE EXPRESSION OF PROTEIN ASSOCIATED WITH FRAGILE X SYNDROME AND METHODS OF THEIR APPLICATION - Google Patents

Description

Cross reference to related applications

This application claims benefit and priority to U.S. Provisional Patent Application No. 62/810697, filed February 26, 2019, the disclosure of which is incorporated herein by reference in its entirety in all respects.

BACKGROUND OF THE INVENTION RNA binding proteins (RBPs) are involved in virtually all stages of the post-transcriptional regulatory process, determining the fate and function of each transcript in the cell and ensuring cellular homeostasis. RBPs establish highly dynamic interactions with proteins and with coding and noncoding RNAs, creating functional units called ribonucleoprotein complexes that regulate RNA splicing, polyadenylation, stability, localization, translation, and degradation.

It is now clear that RBP is dysregulated in different types of cancer, affecting the expression and function of protumorigenic and tumor suppressor proteins and inflammatory mediators. Several studies have provided evidence that RBPs are abnormally expressed in cancer compared with adjacent normal tissues, and their expression correlates with patient prognosis. In recent years, fragile X mental retardation protein (FMRP) has become recognized as critically important in controlling the development and growth of many different types of human cancer. Mutations or absence of FMRP cause fragile X syndrome (FXS), the most common form of inherited mental retardation in humans. FMRP is an RBP involved in several steps of RNA metabolism. In the brain, its functional absence causes impairment of synaptic plasticity due to defects in cytoskeletal organization and receptor mobility at synapses. Depending on the type of target mRNA, the presence of noncoding RNA, and/or the cellular environment, FMRP can act as a negative regulator of translation, modulate mRNA stability, regulate mRNA transport, or influence RNA editing. Notably, FMRP-regulated mRNAs are involved in several mechanisms that control cancer progression and metastasis.

Converging evidence highlights the involvement of FMRP in different cancer types: the FMR1 gene, which encodes FMRP, is expressed in different tissues and cancer cell types; an autosomal paralog and partner of FMR1, FXR1, was recently identified as a predictor of distant metastasis in triple-negative breast cancer; and several FMRP target mRNAs have been implicated in cancer progression. Moreover, compared with rates in the general population, the standardized incidence rate for cancer in patients with FSX is significantly lower, and patients with FSX may be protected against certain forms of cancer.

Colon and rectal cancer (CRC) is one of the most common forms of cancer worldwide, killing more than half a million people every year. The CRC tumorigenesis model involves several genetic alterations that are required for cancer initiation and progression. These changes are largely due to changes in oncogenic and/or tumor suppressor signaling pathways that are responsible for the transition of normal mucosa to adenomatous polyp and then to carcinoma. It was found that colon tumor cells use post-transcriptional mechanisms that allow them to quickly and robustly regulate protein expression levels in response to internal and extracellular signals, leading to cell adaptation to the local microenvironment.

Epidemiological/genetic studies have also documented the frequent development of immune-related disorders (eg, thyroiditis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, and multiple sclerosis) in women who have premutated FMR1 alleles containing an extended trinucleotide (CGG) repeat. element. Although the underlying mechanism by which such FMRP gene alterations confer susceptibility to immune system pathologies remains unknown, two recent studies have documented altered cytokine profiles in children with FSX.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that affects approximately 1.4 million patients in the United States. It is one of the five most common gastrointestinal diseases in the United States, with total healthcare costs of more than $1.7 billion. Each year in the United States, IBD accounts for more than 700,000 physician visits, 100,000 hospitalizations, and disability in 119,000 patients. There is currently no cure, so lifelong surveillance is required to manage the disease.

The two most common forms of IBD are Crohn's disease and ulcerative colitis. Although Crohn's disease can affect the entire gastrointestinal tract, it primarily affects the ileum (the distal or lower part of the small intestine) and the large intestine. Ulcerative colitis primarily affects the colon and rectum. The etiology of inflammatory bowel disease is not fully understood, although both environmental and genetic factors are thought to play a role in the development of the disease. Environmental components may include changes in intestinal flora, which are influenced by the foods and medications consumed.

IBD is associated with abdominal pain, vomiting, diarrhea, rectal bleeding, severe cramps, muscle spasms, weight loss, malnutrition, fever and anemia. Patients with IBD may also suffer from skin lesions, joint pain, eye inflammation and liver problems, and children with ulcerative colitis may suffer from growth problems. Although these symptoms are rarely fatal, they reduce the quality of life of patients.

Thus, there is an urgent need to develop treatments for intestinal diseases such as CRC and IBD. There is also a need to identify treatments that provide effective and consistent symptomatic relief in a wide range of patients and that are not associated with side effects or cycles of remission and/or inflammation.

Brief Disclosure of the Present Invention

Disclosed herein are antisense oligonucleotides that inhibit the expression of fragile X syndrome-associated protein (FMRP) and methods for their use. In some embodiments, the present disclosure provides a method of treating intestinal disease in a patient in need thereof, comprising administering to the patient an effective amount of an antisense oligonucleotide that inhibits FMRP expression. In some embodiments, the bowel disease may be colorectal cancer or inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. In some embodiments, the antisense oligonucleotide induces necroptosis.

In some embodiments, the present disclosure provides a method of treating a solid tumor, tumor invasion, or tumor metastasis in a patient in need thereof, comprising administering to the patient an effective amount of an antisense oligonucleotide that inhibits FMRP expression. In some embodiments, the present disclosure relates to a method of preventing or eliminating tumor invasion or metastasis of a tumor. In certain embodiments, the present disclosure relates to a method of preventing or eliminating tumor invasion in colorectal cancer or tumor metastasis in colorectal cancer.

In some embodiments, an antisense oligonucleotide that inhibits FMRP expression comprises a sequence selected from the group consisting of

5'-CCACCACCAGCTCCTCCA-3' (SEQ ID NO: 1),

5'-CTTCCACCACCAGCTCCT-3' (SEQ ID NO: 2),

5'-TCCACCACCAGCTCCTCC-3' (SEQ ID NO: 3),

5'-CTTCCACCACCAGCTCC-3' (SEQ ID NO: 4) and

5'-TCACCCTTATCATCCTC-3' (SEQ ID NO: 5) or their complementary strand.

In further embodiments, an antisense oligonucleotide that inhibits FMRP expression comprises a sequence selected from the group consisting of 5'-TCCACCACCAGCTCCTCCAT-3' (SEQ ID NO: 6), 5'-ACTTCCACCACCAGCTCCTC-3' (SEQ ID NO: 7) , 5'-TTCCACCACCAGCTCCTCCA-3' (SEQ ID NO: 8), 5'-ACTTCCACCACCAGCTCCT-3' (SEQ ID NO: 9) and 5'-CTCACCCTTTATCATCCTCA-3' (SEQ ID NO: 10) or their complementary strand.

In some embodiments, an antisense oligonucleotide that inhibits FMRP expression consists of a sequence selected from the group consisting of

5'-TCCACCACCAGCTCCTCCAT-3' (SEQ Ш NO: 6),

5'-ACTTCCACCACCAGCTCCTC-3' (SEQ Ш NO: 7),

5'-TTCCACCACCAGCTCCTCCA-3' (SEQ ID NO: 8), 5-ACTTCCACCACCAGCTCCT-3' (SEQ ID NO: 9) and 5-CTCACCCTTTATCATCCCTCA-3' (SEQ ID NO: 10) or their complementary strand.

In some embodiments, the antisense oligonucleotide may be an antisense oligonucleotide wherein at least one internucleoside linkage of the sequence is a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylenephosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphomorpholidate linkage, phosphopiperazidate

- 2 045399 bond, aminoalkylphosphoramidate bond, thiophosphoramidate bond, thionoalkylphosphonate bond, thionoalkylphosphotriester bond, thiophosphate bond, selenophosphate bond or boranophosphate bond. In a specific embodiment, at least one internucleoside linkage of the antisense oligonucleotide sequence is a phosphorothioate linkage. In some embodiments, all of the internucleoside linkages in the antisense oligonucleotide sequence are phosphorothioate linkages. In some embodiments, at least one nucleoside linkage of the sequence is a methylphosphonate linkage.

The number of nucleotides included in the FMRP antisense oligonucleotides described herein may vary. For example, in some embodiments, the antisense oligonucleotide is between 20 and 40 nucleotides in length. In some embodiments, the antisense oligonucleotide is between 20 and 24 nucleotides in length.

In some embodiments, an antisense oligonucleotide for FMRP induces necroptosis through activation of the receptor-interacting protein kinase 1 (RIP1 or RIPK1)-receptor-interacting protein kinase 3 (RIP3 or RIPK3)-mixed lineage kinase domain-like protein (MLKL) complex. For example, an antisense oligonucleotide for FMRP may increase RIPK1 expression.

In some embodiments, the antisense oligonucleotide for FMRP comprises one or more ribonucleotides, one or more deoxyribonucleotides, or a mixture of ribonucleotides and deoxyribonucleotides.

In some embodiments, the antisense oligonucleotide for FMRP contains one or more modified nucleosides, for example, 5-methylcytidine, 5-methyl-2'deoxycytidine, deoxycytidine, 5-methyl-2'-deoxycytidine-5'-monophosphate, or 3-methyl-2 'deoxycytidine-6'-monophosphorothioate. In certain embodiments, the antisense oligonucleotide for FMRP contains one or more modified nucleosides, for example, 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methylthymidine, 2'-O-methyluridine, or 2'-Omethyladenosine. In some embodiments, the antisense oligonucleotide for FMRP contains one or more modified nucleotides, such as 5-methylcytosine or 5methylguanine. In some embodiments, antisense oligonucleotides for FMRP contain one or more modified nucleotides, such as 2'-O-(2-methoxyethyl)nucleosides, 2'-deoxy-2'-fluoro-nucleosides, or 2'-fluoro-β-D- arabinonucleosides.

In some embodiments, the antisense oligonucleotide for FMRP comprises bridged nucleic acids, locked nucleic acids (LNA), conformationally constrained nucleoside ethyl analog (cET) nucleic acids, tricyclo-DNA (tcDNA), 2'-O,4'- nucleic acids. C-ethylene bridge (ENA) or peptide nucleic acids (PNA).

In some embodiments, the antisense oligonucleotide for FMRP is an siRNA for FMRP or a pharmaceutically acceptable salt thereof.

In some embodiments, the antisense oligonucleotide for FMRP is administered enterally or parenterally to a patient. For example, in some embodiments, an antisense oligonucleotide for FMRP is administered to a patient orally, sublingually, gastrointestinally, or rectally. In other embodiments, administration of the antisense oligonucleotide for FMRP to a patient is intravenous, intratumoral, jejunal, ileal, colon, or rectal.

In specific embodiments, the antisense oligonucleotide for FMRP of the present disclosure is for the treatment of an intestinal disease in a human patient.

Also described herein is a pharmaceutically acceptable composition comprising an FMRP antisense oligonucleotide described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is suitable for oral, sublingual, gastric or rectal administration. In other embodiments, the pharmaceutical composition is suitable for intravenous, intratumoral, jejunal, ileal, colonic, or rectal administration.

Also described herein is the use of an antisense oligonucleotide for FMRP in the manufacture of a medicament for the treatment of an intestinal disease. In some embodiments, the bowel disease is CRC or IBD, such as Crohn's disease or ulcerative colitis. In other embodiments, the drug is administered enterally or parenterally to the patient. For example, in some embodiments, the drug formulation is suitable for oral, sublingual, gastric, or rectal administration. In some embodiments, the drug formulation is suitable for intravenous, intratumoral, jejunal, ileal, colonic, or rectal administration. In some embodiments, the medicament is for treating an intestinal disease in a human.

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In some embodiments, the present disclosure relates to the use of an antisense oligonucleotide for FMRP in the manufacture of a medicament for the treatment of a solid tumor, tumor invasion, or tumor metastasis.

Brief description of drawings

In fig. 1A is an immunohistology image showing the results of FMRP staining in human CRC samples (IgG = isotype control; NC = normal colon control; P = tumor surrounding tissue sample; T = tumor sample). Fig. 1B is a line graph showing data on FMRP mRNA expression levels in tumor surrounding tissue (P) and CRC tumor (T) samples as determined by RT-PCR analysis (**P<0.01). Fig. 1C is a blot plot showing FMRP protein expression in paired tumor surrounding tissue (P) and CRC tumor (T) samples from CRC patients. Fig. 1D is a graph showing FMRP protein expression relative to β-actin expression in paired tumor-surrounding tissue (P) and CRC-tumor (T) samples as measured by quantitative densitometry from Western blot analysis (relative expression is shown in relative units (r.u.).

Fig. 2A is an endoscopic photograph of the colon of untreated wild-type (WT) and FMR1 knockout (KO) mice or mice 21 weeks after intraperitoneal injection of azoxymethane (AOM). Fig. 2B are bar graphs showing tumor burden (left) and tumor size (right) in the colon of WT and FMR1-K0 mice 21 weeks after intraperitoneal injection of AOM (*p<0.05, **p<0, 01). Fig. 2C is a graph showing the % survival of WT and FMR1-K0 mice administered AOM by intraperitoneal injection. Fig. 2D are histological images showing the results of hematoxylin and eosin (H&E) staining of samples obtained from AOM-induced tumors in WT and FMR1-K0 mice. Fig. 2E is a blot plot showing FMRP and β-actin protein expression (loading control) in colon samples obtained from untreated WT mice and AOM-treated mice (P = tumor surrounding tissue sample; T = CRC tumor sample). Fig. 2F are histological images showing the results of TUNEL staining of samples obtained from AOM-induced tumors in WT and FMR1-K0 mice. Fig. 2G is an immunohistology image showing the results of Ki67 staining in AOM-induced tumor samples from WT and FMR1-K0 mice.

Fig. 3A is a representative blot showing FMRP and β-actin protein expression (loading control) in the human colon cancer cell lines DLD-1 and HCT-116 and the non-cancer human colon epithelial cell line HCEC-1ct. Fig. 3B is a graph showing FMRP protein expression data relative to β-actin expression in the DLD-1, HCT-116, and HCEC-1ct cell lines as determined by densitometry from the Western blot analyzes described in FIG. 3A (relative expression shown in relative units (r.u.)) (**p<0.01, ***p<0.001). Fig. 3C is immunofluorescence images showing DLD-1, HCT-116 and HCEC-1ct cells stained for FMRP expression. Fig. 3D, 3G, and 31 are blotgrams showing FMRP and β-actin protein expression (loading control) in DLD-1 cells, HCT-116 cells, and HCEC-1ct cells, respectively, treated with sense or antisense oligonucleotides for FMRP (U = untreated; S = sense; AS = antisense) for 48 hours. In FIG. 3E, 3H, and 3J show dot plots of flow cytometry data for DLD-1 cells, HCT-116 cells, and HCEC-1ct cells, respectively, treated with sense or antisense oligonucleotides for FMRP (Neg = negative staining control; U = untreated; S = sense ; AS = antisense) and stained with propidium iodide (PI) and annexin V (AnnV). In fig. 3F and 3K show bar graphs of the corresponding flow cytometry data from the dot plots of FIG. 3E and 3J, respectively (**p<0.01).

Fig. 4A is histograms of flow cytometry data showing caspase 8 and caspase 3 staining results in untreated CRC cells (U), CRC cells treated with sense (S) oligonucleotide for FMRP, CRC cells treated with antisense (AS) oligonucleotide for FMRP, or CRC cells treated with staurosporine (Stauro). Fig. 4B is a bar graph of the corresponding flow cytometry data from FIG. 4A (***p<0.001). Fig. 4C is a bar graph showing relative cell death as determined by flow cytometry analysis for PI- and AnnV-stained CRC cells. CRC cells were left unpretreated or pretreated with a caspase inhibitor (Cas in) before treatment with FMRP sense (S) oligonucleotide, FMRP antisense (AS) oligonucleotide, or staurosporine (Stauro). Fig. 4D is dot plots of flow cytometry data showing the results of Brdu and PI staining of untreated DLD-1 cells (U) or DLD-1 cells treated with smyle (S) oligonucleotide for FMRP or antisense (AS). oligonucleotide for FMRP. Fig. 4E is a bar graph showing the corresponding flow cytometry data from the dot plot of FIG. 4D. Fig. 4F is a bar graph showing proliferation data of HCT-116 cells assessed by the BrdU incorporation assay (U = untreated; S = sense; AS = antisense) over 36 hours. FIG. 4G is a bar graph showing the cell cycle phase distribution of HCT-116 cells treated with sense or antisense oligonucleotides for FMRP (U = untreated; S = sense; AS = antisense) for 36 hours.

In fig. Figure 5 shows the results of RNA immunoprecipitation for human CRC cell lysates. Fig. 5A is a blotgram confirming the specificity of the anti-FMRP antibody used to immunoprecipitate RNA for human CRC cell lysates. Fig. 5B is a bar graph showing the enrichment of FMRP-coprecipitated mRNA as determined by RT-PCR analysis using primers specific for actin, E-cadherin, RIPK3, and RIPK1. Fig. 5C is a bar graph showing the enrichment of FMRP-coprecipitated mRNA from CRC cell line lysates as determined by RT-PCR analysis using primers specific for actin, vimentin, RIPK3, and RIPK1.

Fig. 6A is a representative blot showing protein/phosphorylated protein expression for FMRP, phospho-RIPKl (pRIPKl), RIPK1, phospho-RIPK3 (pRIPK3), RIPK3, phospho-MLKL (pMLKL), MLKL, and β-actin (load control ) in an untreated human colon cell line (U) or a human colon cell line treated with a sense (S) oligonucleotide for FMRP, or a human colon cell line treated with an antisense (AS) oligonucleotide for FMRP. Fig. 6B, 6C, and 6D are bar graphs showing the expression levels of pRIPK1, pRIPK3, and pMLKL, respectively, as determined by densitometry from the Western blot analyzes described in FIG. 6A (**p<0.01, ***p<0.001, ****p<0.0001). Fig. 6E and 6F are bar graphs showing relative cell death as determined by flow cytometry analysis for PI- and AnnV-stained human CRC cell lines treated with a RIPKl-specific inhibitor (NEC1) or an MLKL-specific inhibitor ( NSA) respectively, and either a sense (S) oligonucleotide for FMRP or an antisense (AS) oligonucleotide for FMRP. Fig. 6G is a representative blot showing protein/phosphorylated protein expression for FMRP, pRIPKl, RIPK1, pRIPK3, RIPK3, pMLKL, MLKL and β-actin (loading control) in untreated HCEC-lct cells, sense-treated HCEC-lct cells (S) oligonucleotide for FMRP, or HCEC-lct cells treated with antisense (AS) oligonucleotides for FMRP.

Fig. 7A is a line graph showing RT-PCR analysis of CREB mRNA expression levels in tumor surrounding tissue (P) and CRC tumor (T) samples (***p<0.001). Fig. 7B is a representative blot showing CREB, FMRP, and β-actin (loading control) protein expression in paired tumor surrounding tissue (P) and CRC tumor (T) samples from CRC patients. Fig. 7C is a bar graph showing CREB protein expression relative to β-actin expression as determined by densitometry from the Western blot analyzes described in FIG. 7B (**p<0.01). Fig. 7D is a representative blot showing the protein expression of CREB, FMRP, and βactin (loading control) in untreated CRC cell lines (U), CRC cell lines treated with sense (S) oligonucleotide for FMRP, or CRC cell lines treated with antisense ( AS) oligonucleotides for FMRP. Fig. 7E and 7F are bar graphs showing CREB and FMRP protein expression data, respectively, relative to βactin expression as determined by densitometry from the Western blot analysis described in FIG. 7D (*p<0.05, **p<0.01, ***p<0.001).

Fig. 8A is microscopy images showing cell migration of HCT-116 cells treated with sense or antisense oligonucleotides for FMRP (U = untreated; S = sense; AS = antisense) and seeded on each side of a cell culture insert ibidi®, at the time of removal of the cell culture insert (CC) and after 24 and 48 hours of incubation (T24 and T48, respectively). Fig. 8B is a bar graph showing the percentage of cell covered area corresponding to the microscopy images of FIG. 8A (T24: U versus AS, **p<0.01; S versus AS *p<0.05; T48: U versus AS and S versus AS, ***p<0.001 ). Fig. 8C and 8D are microscopy images and corresponding bar graph of cell count data, respectively, for HCT-116 cells treated with sense or antisense oligonucleotides for FMRP (U = untreated; S = sense; AS = antisense) that migrated through Matrigel-coated ® culture inserts

- 5,045,399 Transwell® cells, after 48 hours of incubation (***p<0.001, ****p<0.0001). Fig. 8E is a representative blot showing protein expression of FMRP, E-cadherin, β-catenin, and β-actin (loading control) in HCT-116 cells treated with sense or antisense oligonucleotides for FMRP (U = untreated; S = sense; AS = antisense) for 48 hours. FIG. 8F and 8G are bar graphs showing E-cadherin and β-catenin protein expression data, respectively, relative to β-actin expression in HCT-116 cells, as determined by densitometry from the Western blot analyzes described in FIG. . 8E (**p<0.01, ***p<0.001).

Fig. 9A is a representative blot showing protein expression of FMRP, MCC, and β-actin (loading control) in untreated HCT-116 cells (U) or HCT116 cells treated with sense (S) oligonucleotide or antisense (AS) oligonucleotide for FMRP in within 48 hours. Fig. 9B is a bar graph showing MCC protein expression relative to β-actin expression in HCT-116 cells as determined by densitometry from the Western blot analysis described in FIG. 9A. Fig. 9C is a representative blot showing the protein expression of MCC, FMRP, E-cadherin, β-catenin and β-actin (loading control) in untreated HCT-116 cells (U) or HCT116 cells treated with sense (S) oligonucleotide, antisense (AS) oligonucleotide for FMRP and/or siRNA as control (control siRNA) or MCC-specific siRNA (MCC siRNA) for 48 hours. FIG. 9D and 9E are bar graphs showing E-cadherin and β-catenin protein expression data, respectively, relative to β-actin expression in HCT-116 cells, as determined by densitometry from the Western blot analyzes described in FIG. 9C (*p<0.05, **p<0.01). Fig. 9F is microscopy images showing cell migration of HCT-116 cells treated with sense or antisense oligonucleotides for FMRP (U = untreated; S = sense; AS = antisense), and/or control siRNA or siRNA for MCC, and seeded on each side of the ibidi® cell culture insert, at the time of removal of the cell culture insert (TC) and after 24 and 48 hours of incubation (T24 and T48, respectively). Fig. 9G is a bar graph showing the percentage of cell covered area corresponding to the microscopy images of FIG. 9F (T24: HCT-116 cells transfected with antisense oligonucleotide for FMRP and siRNA for MCC, compared with HCT-116 cells transfected with antisense oligonucleotide for FMRP alone, ***p<0.001; T48: HCT-116 cells transfected with antisense oligonucleotide for FMRP and siRNA for MCC, compared with HCT-116 cells transfected with antisense oligonucleotide for FMRP alone, ****p<0.0001).

Detailed Disclosure of the Present Invention Antisense Oligonucleotides for FMRP

An antisense oligonucleotide as used herein refers to a short synthetic oligonucleotide sequence complementary to messenger RNA (mRNA) that encodes a target protein (eg, FMRP). Antisense oligonucleotide sequences hybridize to the mRNA to form a double-stranded molecule, which can lead to the activation of nucleases that recognize and cleave the double-stranded molecule, thereby preventing translation of the mRNA. Antisense oligonucleotides may include single-stranded DNA oligonucleotides, short hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), and modified antisense oligonucleotides, which include, but are not limited to, 2'-O-alkyl modified nucleic acid, peptide nucleic acid (PNA). ), locked nucleic acid (LNA) and morpholine oligomers.

In some embodiments, the antisense oligonucleotide may be a single-stranded nucleic acid molecule containing a nucleotide sequence that is complementary to the target mRNA (eg, FMRP). For example, the antisense oligonucleotide may be a single-stranded DNA oligonucleotide having a sequence complementary to the FMRP mRNA. Hybridization of an antisense oligonucleotide for FMRP with the target mRNA results in the formation of a double-stranded DNA/RNA hybrid, which leads to the activation of ubiquitous nucleases such as RNase H, which recognize and cleave the DNA/RNA hybrid strands, thereby preventing translation of the target protein (e.g., FMRP ).

Alternatively, the antisense oligonucleotide may be double-stranded. A double-stranded antisense oligonucleotide may consist of a single oligonucleotide having self-complementary sense and antisense regions. In other embodiments, a double-stranded antisense oligonucleotide may consist of two separate oligonucleotides, wherein one oligonucleotide is the sense strand and the other oligonucleotide is the antisense strand, and wherein the antisense strand has a nucleotide sequence that is complementary to the target mRNA (e.g., FMRP).

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Antisense oligonucleotides can be designed such that the targeted portion of the included nucleotide sequence of each antisense oligonucleotide is completely or nearly completely complementary to the FMRP mRNA sequence. The inclusion of such complementary or near-complementary nucleotide sequences allows the design of antisense oligonucleotides with a high degree of specificity for a given target. Specificity can be assessed by measuring parameters such as the dissociation constant, or by other criteria such as changes in protein or RNA expression levels, or by other assays that measure the activity or expression level of FMRP.

The present disclosure relates to methods that involve administering to a patient an antisense oligonucleotide for FMRP capable of targeting FMRP mRNA for cleavage, disrupting mRNA splicing, or preventing gene expression or translation of the FMRP protein. The FMRP antisense oligonucleotides of the present disclosure can target different regions of the human FMRP mRNA for binding. Human FMRP mRNA has an NCBI reference sequence number: NM_001185075 (SEQ ID NO: 18), NM_001185076 (SEQ ID NO: 19), NM_001185081 (SEQ ID NO: 20), NM_001185082 (SEQ ID NO: 21) or NM_002024 ( SEQ ID NO: 22).

An antisense oligonucleotide for FMRP, such as disclosed herein, may be an oligonucleotide sequence 5 to 100 nucleotides in length, e.g., 10 to 40 nucleotides in length, e.g., 14 to 40 nucleotides in length, e.g., 10 to 30 nucleotides in length , for example, from 14 to 30 nucleotides in length, for example, from 14 to 25 nucleotides in length, for example, from 15 to 22 oligonucleotides in length, for example, from 18 to 40 nucleotides in length, for example, from 18 to 24 nucleotides in length, for example, from 20 to 40 nucleotides or, for example, 20 to 24 nucleotides long. In some embodiments, the length of the antisense oligonucleotide for FMRP may be, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. An antisense oligonucleotide for FMRP may comprise an oligonucleotide sequence complementary to one or more portions of the FMRP mRNA sequence.

In some embodiments, the antisense oligonucleotides for FMRP of the present disclosure may be, but are not limited to, short hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), morpholine oligomers, microRNAs, and compositions that include such compounds, e.g., compositions that include pharmaceutically acceptable excipient.

In some embodiments of the present disclosure, an antisense oligonucleotide targeting FMRP comprises a sequence or portion of a sequence (e.g., contains a sequence having 90, 95, or 99% length identity) selected from any of the following:

5'-CCACCACCAGCTCCTCCA-3' (SEQ Ш NO: 1),

5'-CTTCCACCACCAGCTCCT-3' (SEQ Ш NO: 2), 5'-TCCACCACCAGCTCCTCC-3' (SEQ ID NO: 3), 5'-CTTCCACCACCAGCTCC-3' (SEQ ID NO: 4) and 5'-TCACCCCTTTATCATCCTC- 3' (SEQ ID NO: 5) or their complementary strand.

In further embodiments, the antisense oligonucleotide targeting FMRP comprises a sequence or portion of a sequence (e.g., contains a sequence having 90, 95, or 99% length identity) selected from any of the following:

5'-TCCACCACCAGCTCCTCCAT-3' (SEQ ID NO: 6),

5'-ACTTCCACCACCAGCTCCTC-3' (SEQ ID NO: 7), 5'-TTCCACCACCAGCTCCTCCA-3' (SEQ ID NO: 8), 5'-ACTTCCACCACCAGCTCCT-3' (SEQ ID NO: 9) and 5'-CTCACCCTTTATCATCCTCA- 3' (SEQ ID NO: 10) or their complementary strand.

In some embodiments, the antisense oligonucleotide for FMRP of the present disclosure comprises one or more ribonucleotides, deoxyribonucleotides, or a mixture of ribonucleotides and deoxyribonucleotides.

In some embodiments, the antisense oligonucleotide for FMRP of the present disclosure contains one or more modified nucleosides selected from the group consisting of 5-methylcytidine, 5-methyl-2'-deoxycytidine, deoxycytidine, 5-methyl-2'deoxycytidine-5'- monophosphate and 3-methyl-2'-deoxycytidine-6'-monophosphorothioate.

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In certain embodiments, the antisense oligonucleotide for FMRP of the present disclosure contains one or more modified nucleosides selected from the group consisting of 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methylthymidine, 2'-Omethyluridine, and 2'-O-methyladenosine.

In some embodiments, the antisense oligonucleotide for FMRP of the present disclosure contains one or more modified nucleotides selected from the group consisting of 5-methylcytosine and 5-methylguanine.

In some embodiments, the antisense oligonucleotide for FMRP of the present disclosure contains one or more modified nucleosides selected from 2'O-(2-methoxyethyl)nucleosides, 2'-deoxy-2'-fluoro-nucleosides, and 2'-fluoro-β- D-arαbinonucleosides.

In certain embodiments, the antisense oligonucleotide for FMRP of the present disclosure comprises one or more of the group selected from bridged nucleic acids, locked nucleic acids (LNA), conformationally constrained nucleoside ethyl analogue (cET) nucleic acids, tricyclo-DNA (tcDNA), nucleic acids with a 2'O,4'-C-ethylene bridge (ENA) and peptide nucleic acids (PNA).

In some embodiments, at least one internucleoside linkage of the disclosed antisense oligonucleotide for FMRP may have a modified linkage, such as a phosphorothioate linkage, phosphorodithioate linkage, phosphotriester linkage, alkylphosphonate linkage, aminoalkylphosphotriester linkage, alkylenephosphonate linkage, phosphinate linkage, phosphoramidate linkage, phosphomorpholidate linkage , phosphopiperazidate linkage and aminoalkylphosphoramidate linkage, thiophosphoramidate linkage, thionoalkylphosphonate linkage, thionoalkylphosphotriester linkage, thiophosphate linkage, selenophosphate linkage and/or boranophosphate linkage. For example, in some embodiments, one, two, or more, for example, all of the internucleoside linkages of a disclosed antisense oligonucleotide for FMRP may be phosphorothioate linkages. In other embodiments, one, two, or more, for example, all of the internucleoside linkages of a disclosed antisense oligonucleotide for FMRP may be methylphosphonate linkages.

For example, in one embodiment, the antisense oligonucleotide for FMRP is a phosphorothioate antisense oligonucleotide against FMRP comprising the sequence 5'-CCACCACCAGCTCCTCCA-3' (SEQ ID NO: 1), wherein each internucleotide linkage of the antisense oligonucleotide is a phosphorothioate linkage. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence

5'-CCACCACCAGCTCCTCCA-3' (SEQ ID NO: 1), _Where one or more than one internucleoside linkage of the antisense oligonucleotide is a phosphorothioate linkage.

In another embodiment, the antisense oligonucleotide for FMRP is a phosphorothioate antisense oligonucleotide against FMRP comprising the sequence 5-CCACCACCAGCTCCTCCA-3' (SEQ ID NO: 2), wherein each internucleotide linkage of the antisense oligonucleotide is a phosphorothioate linkage. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence

-CTTCCACCACCAGCTCCT-3 (SEQ ID NO. 2), wherein one or more of the internucleoside linkages of the antisense oligonucleotide is a phosphorothioate linkage.

In another embodiment, the antisense oligonucleotide for FMRP is a phosphorothioate antisense oligonucleotide against FMRP comprising the sequence 5'-TCCACCACAGCTCCTCC-3' (SEQ ID NO: 3), wherein each internucleotide linkage of the antisense oligonucleotide is a phosphorothioate linkage. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence

5-TCCACCACCAGCTCCTCC-3'(SEQ ID NO: 3), wherein one or more of the internucleoside linkages of the antisense oligonucleotide is a phosphorothioate linkage.

In another embodiment, the antisense oligonucleotide for FMRP is a phosphorothioate antisense oligonucleotide against FMRP comprising the sequence 5'-CTTCCACCACCAGCTCC-3'(SEQIDNO: 4), wherein each internucleotide linkage of the antisense oligonucleotide is a phosphorothioate linkage. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence 5'-CTTCCACCACCAGCTCC-3' (SEQ ID NO: 4), wherein one or more of the internucleoside linkages of the antisense oligonucleotide is a phosphorothioate linkage.

In another embodiment, the FMRP antisense oligonucleotide is a phosphorothioate anti-FMRP antisense oligonucleotide containing the sequence

- 8 045399 5'-TCASSTTTATSATSSTC-3'(SEQ ID _NO : 5), _where each internucleotide bond of the antisense oligonucleotide is a phosphorothioate bond. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence

5-TCASSSTTTTATSCATSSTS-3' (SEQ ID NO: 5), wherein one or more of the internucleoside linkages of the antisense oligonucleotide is a phosphorothioate linkage.

In another embodiment, the antisense oligonucleotide for FMRP is a phosphorothioate antisense oligonucleotide against FMRP comprising the sequence 5'-TCCACCACCAGCTCCTCCAT-3' (SEQ ID NO: 6), wherein each internucleotide linkage of the antisense oligonucleotide is a phosphorothioate linkage. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence

5-TCCACCACAGCTCCTCCAT-3' (SEQ ID NO: 6), wherein one or more of the internucleoside linkages of the antisense oligonucleotide is a phosphorothioate linkage.

In another embodiment, the antisense oligonucleotide for FMRP is a phosphorothioate antisense oligonucleotide against FMRP comprising the sequence 5'-ACTTCCACCACCAGCTCCTC-3' (SEQ ID NO: 7), wherein each internucleotide linkage of the antisense oligonucleotide is a phosphorothioate linkage. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence

5'-ACTTCCACCACCAGCTCCTC-3' (SEQ ID NO: 7), wherein one or more of the internucleoside linkages of the antisense oligonucleotide is a phosphorothioate linkage.

In another embodiment, the antisense oligonucleotide for FMRP is a phosphorothioate antisense oligonucleotide against FMRP comprising the sequence 5'-TTCCACCACCAGCTCCTCCA-3' (SEQ ID NO:8), wherein each internucleotide linkage of the antisense oligonucleotide is a phosphorothioate linkage. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence

5'-TTCCACCACCAGCTCCTCCA-3'(SEQ ID NO: 8), wherein one or more of the internucleoside linkages of the antisense oligonucleotide is a phosphorothioate linkage.

In another embodiment, the antisense oligonucleotide for FMRP is a phosphorothioate antisense oligonucleotide against FMRP comprising the sequence 5'-ACTTCCACCACCAGCTCCT-3'(SEQ ID NO:9), wherein each internucleotide linkage of the antisense oligonucleotide is a phosphorothioate linkage. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence

5'-ACTTCCACCACCAGCTCCT-3' (SEQ ID NO: 9), wherein one or more of the internucleoside linkages of the antisense oligonucleotide is a phosphorothioate linkage.

In another embodiment, the antisense oligonucleotide for FMRP is a phosphorothioate antisense oligonucleotide against FMRP comprising the sequence 5'-CTCASSTTTATSATSSCTCA-3' (SEQ ID NO: 10), wherein each internucleotide linkage of the antisense oligonucleotide is a phosphorothioate linkage. In some embodiments, the antisense oligonucleotide against FMRP is a phosphorothioate antisense oligonucleotide comprising the sequence

5'-STCASSTTTATCATSCSTCA-3'(SEQ ID NO: 10), wherein one or more of the internucleoside linkages of the antisense oligonucleotide is a phosphorothioate linkage.

In some embodiments, the antisense oligonucleotide for FMRP is an siRNA that contains the nucleotide sequence of any of SEQ ID NOs: 1-10, or a pharmaceutically acceptable salt thereof. For example, siRNA for FMRP may contain the sequence of any of

- 9 045399

5'-CCACCACCAGCTCCTCCA-3' (SEQ Ш NO: 1), 5'-CTTCCACCACCAGCTCCT-3' (SEQ ID NO: 2), 5'-TCCACCACCAGCTCCTCC-3' (SEQ Ш NO: 3), 5'-CTTCCACCACCAGCTCC- 3' (SEQ ID NO: 4), 5'-TCACCCCTTTATCATCCTC-3' (SEQ Ш NO: 5), 5'-TCCACCACCAGCTCCTCCAT-3' (SEQ ID NO: 6), 5'-ACTTCCACCACCAGCTCCTC-3' (SEQ ID NO: 7), 5'-TTCCACCACCAGCTCCTCCA-3' (SEQ ID NO: 8), 5'-ACTTCCACCACCAGCTCCT-3' (SEQ ID NO: 9) and 5'-CTCACCCTTTATCATCCTCA-3'(SEQ ID NO: 10), or a pharmaceutically acceptable siRNA salt for FMRP may be provided comprising the sequence of any of SEQ ID NOs: 1-10.

In some embodiments, the siRNA for FMRP contains at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylenephosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphomorpholidate linkage, a phosphopiperazidate linkage , aminoalkylphosphoramidate bond, thiophosphoramidate bond, thionoalkylphosphonate bond, thionoalkylphosphotriester bond, thiophosphate bond, selenophosphate bond and boranophosphate bond. In certain embodiments, at least one siRNA internucleoside linkage for FMRP is a phosphorothioate linkage. For example, in some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 1-5, 1- 10, 1-14, 1-15, 1-16, 1-19, 5-10, 5-14, 5-15, 5-19, 10-14, 10-15 or 10-19 siRNA internucleoside linkages for FMRP are phosphorothioate bonds. In some embodiments, all of the siRNA internucleoside linkages for FMRP are phosphorothioate linkages.

In various embodiments, the disclosed antisense oligonucleotide for FMRP (e.g., siRNA for FMRP) may optionally have at least one modified nitrogen base, e.g., 5-methylcytosine and/or at least one methylphosphonate nucleotide in the sequence, which is, for example, either only at one of the 5' or 3' ends, or at both the 5' and 3' ends, or along the oligonucleotide sequence.

Antisense oligonucleotides for FMRP (eg, siRNA for FMRP) may optionally include at least one modified sugar. For example, the sugar moiety of at least one nucleotide constituting the oligonucleotide may be ribose, in which the 2-OH group may be replaced by any one selected from the group consisting of OR, R, R'OR, SH, SR, NH2, NR2 , N ₃ , CN, F, Cl, Br and I (wherein R is alkyl or aryl and R' is alkylene).

In some embodiments, certain disclosed nucleotides may be modified or have variations, for example, certain cytidines in a disclosed FMRP antisense oligonucleotide (e.g., FMRP siRNA) may be, for example, 5-methyl2'-deoxycytidine, including but not limited to 5-methyl -2'-deoxycytidine-5'-monophosphate and 5-methyl-2'deoxycytidine-5'-monophosphorothioate.

In certain embodiments, antisense oligonucleotides for FMRP (e.g., siRNA for FMRP) may include chemically modified nucleosides, e.g., 2'O-methyl (2'-OMe) ribonucleosides, e.g., 2'-O-methylcytidine, 2'-O- methylguanosine, 2'-Omethylthymidine, 2'-O-methyluridine and/or 2'-O-methyladenosine. Antisense oligonucleotides for FMRP (eg, siRNA for FMRP) described herein may also include one or more chemically modified bases, including 5-methylpyrimidine, such as 5-methylcytosine, and/or 5-methylpurine, such as 5-methylguanine. In some embodiments, antisense oligonucleotides for FMRP (e.g., siRNA for FMRP) may include one or more 2'-O-(2-methoxyethyl) (2'-MOE)-nucleosides, 2'-deoxy-2'-fluoronucleosides, 2 '-fluoro-β-0 arabinonucleosides, bridged nucleic acids, locked nucleic acids (LNA), nucleic acids with conformationally constrained ethyl nucleoside analogues (sET), tricyclo-DNA (tcDNA), nucleic acids with 2'-O,4'-C -ethylene bridge (ENA) and/or peptide nucleic acids (PNA).

In some embodiments, at least one of the internucleotide linkages of the provided antisense oligonucleotide (eg, siRNA for FMRP) is an O,O-phosphorothioate linkage. For example, each of the internucleotide linkages of SEQ ID NO: 1-10 may be an O,O-phosphorothioate linkage. In some embodiments, compositions disclosed herein may include a pharmaceutically acceptable salt (e.g., the sodium salt of a disclosed sequence antisense oligonucleotide that

- 10 045399 may optionally include from 1 to 24 or more O,O-phosphorothioate internucleotide linkages.

Provided salts of oligonucleotides include those that are completely neutralized, for example, each phosphorothioate bond is associated with an ion such as Na ⁺ . Oligonucleotides may include naturally occurring nitrogenous bases, sugars, and covalent internucleoside (within the backbone) bonds, as well as non-naturally occurring moieties.

Also provided herein are isotopologues of the disclosed antisense oligonucleotides, pharmaceutical compositions containing them, and methods of using them. For example, in some embodiments, provided herein are deuterated antisense oligonucleotides of SEQ ID NO: 1-10, including a plurality of hydrogen atoms (H), wherein one or more hydrogen atoms of the plurality of hydrogen atoms are replaced by deuterium (D).

Intestinal diseases

The present disclosure relates to oligonucleotides for the treatment and/or prevention of intestinal diseases. As used herein, bowel disease refers to any disease, disorder and/or syndrome affecting the part of the digestive tract after the stomach, i.e. small intestine, large intestine, large intestine and rectum. For example, bowel diseases may include, but are not limited to, colon cancer, inflammatory bowel disease, familial adenomatous polyposis, Gardner's syndrome, Turco's syndrome, Lynch syndrome, celiac disease, gastrointestinal carcinoid tumors, small bowel cancer, duodenal cancer, small bowel cancer, and stromal tumors of the gastrointestinal tract. For example, provided herein are methods of treating a patient suffering from an intestinal disease comprising administering to the patient an effective amount of a disclosed antisense oligonucleotide.

Colon and rectal cancer

Colon and rectal cancer, as used herein, refers to any cancer affecting the colon and/or rectum. Forms of colorectal cancer can be caused by any one or more than one environmental and genetic factor that causes the progressive accumulation of genetic and/or epigenetic changes that cause the suppression of tumor suppressor genes and the activation of oncogenes in epithelial cells of the colon or rectum. Colon and rectal neoplasms are often associated with loss of genomic and/or epigenomic stability, which accelerates malignant transformation.

There is significant heterogeneity in the specific gene mutations present in colorectal cancer and includes, but is not limited to, alterations in APC, CTNNB1, KRAS, BRAF, SMAD4, TGFBR2, TP53, PIK3CA, ARID1A, SOX9, FAM123B, and ERBB2. Colon and rectal cancers are often initiated by mutations that lead to dysregulation of the Wnt signaling pathway, and tumors progress with further dysregulation of other signaling pathways, including the RAS-RAF-MAPK, TGFP, and PI3K-AKT pathways. The disclosed sequences can carry out post-transcriptional regulation of the expression of oncogenes, tumor suppressors and key signaling proteins participating in the Wnt, RAS-RAF-MAPK, TGFP and PI3K-AKT pathways. Disclosed herein are methods of treating patients suffering from colorectal cancer comprising administering to the patients a disclosed antisense oligonucleotide.

Inflammatory bowel disease

Inflammatory bowel disease, as used herein, refers to a number of chronic inflammatory diseases, including Crohn's disease, ulcerative colitis, gastroduodenal Crohn's disease, Crohn's (granulomatous) colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behçet's disease, microscopic colitis, ulcerative proctitis, proctosigmoiditis, jejunoileitis, left-sided colitis, pancolitis, enterocolitis, ileitis and unspecified colitis. Crohn's disease and ulcerative colitis are the two most common forms of inflammatory bowel disease. Inflammatory bowel disease is an autoimmune disease of the digestive system. Crohn's disease can be located in any part of the gastrointestinal tract, including the terminal ileum, and can affect all cell types of the gastrointestinal tract. Ulcerative colitis is localized in the colon and rectum and affects only the cells of the mucous membrane. Provided herein are methods of treating patients suffering from inflammatory bowel disease by administering to the patient a disclosed antisense oligonucleotide.

Inflammatory bowel disease is associated with symptoms including abdominal pain, vomiting, diarrhea, rectal bleeding, severe cramps, muscle spasms, weight loss, malnutrition, fever, anemia, skin lesions, joint pain, eye inflammation, liver problems, arthritis , prioderma gangrenosum, primary sclerosing cholangitis and pseudothyroid dysfunction syndrome, and also in one embodiment, treatment of these symptoms using the disclosed antisense compound is provided, for example, treatment of a child suffering from ulcerative colitis, who may also suffer from growth disorders. In some embodiments, provided herein are methods of, for example, eliminating or treating such symptoms by administering to a patient an effective amount of a disclosed antisense oligonucleotide.

Necroptosis, as used herein, refers to regulated caspase-independent cell death, which may be an alternative means of killing apoptosis-resistant cancer cells. The major pathway of necroptosis involves the receptor-interacting protein kinase 1 (RIP1 or RIPK1)-receptor-interacting protein kinase 3 (RIP3 or RIPK3) complex and mixed lineage kinase domain-like protein (MLKL), also called the necrosome. The necrosome initiates downstream effector functions such as release of reactive oxygen species (ROS), permeabilization of the plasma membrane, and reduction of cytosolic ATP, which further drives the mechanisms causing irreversible necroptosis. Provided herein are methods of treating patients by modulating necroptosis by administering the disclosed antisense oligonucleotide to the patient.

A patient in need of treatment as used herein refers to a patient suffering from any of the symptoms or manifestations of a bowel disease, a patient who may suffer from any of the symptoms or manifestations of a bowel disease, or any patient who may benefit from a method of treating a bowel disease by this disclosure. The patient in need of treatment may include a patient who has been diagnosed as at risk of developing a bowel disease, a patient who has suffered from a bowel disease in the past, or a patient who has previously been treated for a bowel disease. Particularly suitable are individuals who suffer from intestinal disease associated with increased levels of FMRP expression or activity.

Terms treat, treatment, treating, etc. are used herein to generally refer to obtaining the desired pharmacological and/or physiological effect. The effect may be prophylactic in the context of complete or partial prevention of a disease or symptom thereof and/or may be therapeutic in the context of partial or complete cure of a disease and/or an undesirable effect attributed to the disease. The term treatment as used herein includes any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease in a subject who may be predisposed to developing the disease but has not yet been diagnosed; (b) inhibition of disease, i.e. preventing an increase in the severity or extent of the disease; (c) reducing the severity of the disease, i.e. partial or complete elimination of the disease or (d) prevention of relapse of the disease, i.e. preventing the disease from returning to an active state after previous successful treatment of the symptoms of the disease or treatment of the disease.

An effective amount as used herein refers to an amount of an agent that is sufficient to at least partially treat or eliminate the symptoms of a condition when administered to a patient. The effective amount will vary depending on the severity of the condition, route of administration of the component and age, weight, etc. the patient being treated. Accordingly, an effective amount of a disclosed FMRP antisense oligonucleotide is the amount of FMRP antisense oligonucleotide needed to treat an intestinal disease in a subject such that administration of the agent to a patient prevents the onset of an intestinal disease in the subject, prevents the progression of an intestinal disease (e.g., prevents the onset or increase in severity symptoms of bowel disease such as rectal bleeding, anemia or gastrointestinal inflammation) or provides relief or complete elimination of all associated symptoms of bowel disease, i.e. provides regression of the disease.

In some embodiments, the disclosed methods comprise administering to a patient at least 1 μg, at least 5 μg, at least 10 μg, at least 20 μg, at least 30 μg, at least 40 μg, at least 50 μg , at least 60 μg, at least 70 μg, at least 80 μg, at least 90 μg, or at least 100 μg of antisense oligonucleotide. In some embodiments, the methods of the present disclosure include administering to a patient 35 to 500 mg, 1 to 10 mg, 10 to 20 mg, 20 to 30 mg, 30 to 40 mg, 40 to 50 mg, 50 to 60 mg, 60 to 70 mg, 70 to 80 mg, 80 to 90 mg, 90 to 100 mg, 100 to 150 mg, 150 to 200 mg, 200 to 250 mg, 250 to 300 mg , from 300 to 350 mg, from 350 to 400 mg, from 400 to 450 mg, from 450 to 500 mg, from 500 to 600 mg, from 600 to 700 mg, from 700 to 800 mg, from 800 to 900 mg, from 900 to 1 g, 1 to 50 mg, 20 to 40 mg, or 1 to 500 mg antisense oligonucleotide.

The effectiveness of treatment can be determined by evaluation of overt symptoms associated with intestinal disease, histological analysis of tissue, biochemical analysis, imaging techniques such as, for example, magnetic resonance imaging, or other known methods. For example, the effectiveness of treatment can be assessed by analyzing the overt symptoms of the disease, such as changes in abdominal pain, vomiting, diarrhea, rectal bleeding, seizures, muscle spasms, weight loss, malnutrition, fever, anemia or other aspects of gross pathology associated with intestinal disease following administration of the disclosed antisense oligonucleotide for FMRP to a patient suffering from intestinal disease.

- 12 045399

The effectiveness of treatment can also be assessed at the tissue or cellular level, for example, by obtaining tissue biopsies (eg, tumor or gastrointestinal tissue biopsies) and assessing macroscopic tissue or cell morphology or staining characteristics. Biochemical assays that examine protein or RNA expression can also be used to evaluate the effectiveness of treatment. For example, FMRP, caspases (eg, caspase 3 or caspase 8), RIPK1, Phospho-RIPKI, RIPK3, phospho-RIPK3, MLKL, phospho-MLKL, CREB, IL-6, IL-8, TNF-alpha or levels of another protein or gene product indicative of the presence of colorectal cancer, necroptosis, inflammatory bowel disease, or the production of inflammatory cytokines, by immunocytochemistry, immunohistochemistry, Western blotting or Northern blotting, or methods suitable for assessing RNA levels, such as quantitative or semiquantitative polymerase chain reaction. The presence or expression level of useful biomarkers found in feces, plasma or serum can also be assessed to assess disease status and treatment effectiveness.

When assessing the effectiveness of a treatment, appropriate controls can be selected to ensure a reliable assessment. For example, symptoms assessed in a patient with bowel disease after administration of a disclosed antisense oligonucleotide for FMRP may be compared with those in the same patient before treatment or earlier in the course of treatment, or in another patient who has not been diagnosed with bowel disease. . Alternatively, the results of biochemical or histological analysis of intestinal tissue after administration of the disclosed antisense oligonucleotide for FMRP to a patient can be compared with those of intestinal tissue from the same patient or from an individual who has not been diagnosed with intestinal disease, or from the same patient before administration. patient antisense oligonucleotide for FMRP. In addition, samples of blood, serum, cells, or stool after administration of an antisense oligonucleotide for FMRP to a patient can be compared with matched samples from an individual who has not been diagnosed with bowel disease, or from the same patient before administration of an antisense oligonucleotide for FMRP to the patient.

The validity of FMRP inhibition can be determined by directly or indirectly assessing levels of FMRP expression or activity. For example, biochemical assays that measure FMRP protein or RNA expression can be used to assess overall FMRP inhibition. For example, FMRP protein levels in intestinal tissue can be measured using Western blot to assess total FMRP levels. FMRP mRNA levels can also be measured using Northern blot or quantitative polymerase chain reaction to determine overall FMRP inhibition. It is also possible to assess FMRP protein levels or levels of another protein indicative of FMRP activity/expression in dissociated cells, undissociated tissue or feces by immunocytochemical or immunohistochemical methods.

FMRP inhibition can also be assessed indirectly by measuring parameters such as increased RTPK1 expression and/or activation of the RIPK1-RIPK3-MLKL complex, which are associated with necroptosis. For example, one can measure the levels of PhosPho-RIPKI, phospho-RIPK3, or phospho-MLKL in intestinal tissue using Western blot.

It is also possible to indirectly assess the negative regulation of FMRP by measuring parameters such as CREB expression. For example, biochemical assays that measure CREB protein or RNA expression can be used to assess overall FMRP inhibition. For example, it is possible to measure CREB protein levels in intestinal tissue using Western blotting. CREB mRNA levels can also be measured using Northern blot or quantitative polymerase chain reaction to determine overall FMRP inhibition. CREB protein levels or levels of another protein indicative of CREB activity/expression can also be assessed in dissociated cells, undissociated tissue or feces by immunocytochemical or immunohistochemical methods.

The present disclosure relates to methods of treating colon cancer. Treatment of colon cancer is intended to provide one or more of the following: for example, complete elimination of the disease; reduction in the number and/or grade of tumors; reduction of metastasis; reduction in the number of reappearances; and reducing the incidence or severity of symptoms (eg, diarrhea, constipation, bloody stools, rectal bleeding, abdominal pain, weakness, fatigue, and weight loss).

The present disclosure also relates to methods of treating IBD (eg, Crohn's disease and ulcerative colitis). Treatment for IBD is intended to provide one or more of the following: for example, complete elimination of the disease; decreased inflammation, including decreased production of inflammatory cytokines and intestinal infiltration of immune cells; restoration of intestinal/mucosal architecture; reduction in the number of reappearances; and reducing the incidence or severity of symptoms (eg, diarrhea, constipation, bloody stools, bleeding, abdominal pain, weakness, fatigue, and weight loss). Inflammatory cytokine production refers to the expression of cytokines that initiate and/or stimulate cytokine-mediated

- 13 045399 inflammatory response. Cytokine-mediated inflammatory response refers to an immune response that may be characterized by granulocyte recruitment, lymphocyte recruitment, systemic inflammation (especially of the gastrointestinal tract or part or parts thereof), fever, tissue destruction, shock and/or death. The cytokine-mediated inflammatory response can be characterized by the binding of individual cytokines to their cognate cell surface receptor and subsequent intracellular signal transduction cascades that lead to changes in cell function and gene expression. Inflammatory cytokines include, but are not limited to, IL-1, IL-6, IL-8 and TNFa. Expression of inflammatory cytokines may occur, for example, in macrophages, monocytes, mononuclear cells of the lamina propria or other cells of the gastrointestinal tract or cells of the immune system. Methods of inhibiting the production of inflammatory cytokines include methods that reduce the expression levels of some or all inflammatory cytokines in a patient suffering from inflammatory bowel disease. Methods of inhibiting the production of inflammatory cytokines also include methods that reduce the expression levels of some or all inflammatory cytokines in cells of a patient suffering from an inflammatory disease.

The present disclosure also relates to methods of inhibiting FMRP in cells of a patient suffering from an intestinal disease. FMRP can be inhibited in any cell in which FMRP expression or activity occurs, including gastrointestinal, immune and blood cells. Cells of the gastrointestinal tract (including cells of the stomach, duodenum, jejunum, ileum, colon, rectum and anal canal) include columnar epithelial cells, mucosal epithelial cells, zymogen cells, cervical mucous cells, parietal cells, G- cells, goblet cells, Paneth cells, oligomucosal cells and villous absorptive cells. Cells of the immune system include white blood cells, phagocytes (eg, macrophages, neutrophils, and dendritic cells), monocytes, mast cells, eosinophils, basophils, natural killer cells, innate immune cells, lymphocytes, B cells, and T cells. Blood cells include red blood cells (erythrocytes) and white blood cells (leukocytes, monocytes and platelets).

Tumor invasion and tumor metastasis

Tumor invasion refers to the proliferation of cancer cells and increase in tumor size, leading to the expansion, passage, penetration and spread of cancer cells into surrounding tissues. Tumor metastasis occurs when cancer cells break away from the primary site of the tumor, move with the blood or lymph flow and form a new tumor site in other organs and tissues of the body.

In some embodiments, an effective amount of an antisense oligonucleotide for FMRP of the present disclosure can be administered to a patient in need thereof to treat a solid tumor, tumor invasion, or tumor metastasis. In some embodiments, an effective amount of an antisense oligonucleotide for FMRP of the present disclosure can be used to prevent or eliminate tumor invasion or metastasis of a tumor. In certain embodiments, an effective amount of an antisense oligonucleotide for FMRP of the present disclosure can be used to prevent or eliminate tumor invasion in colorectal cancer or tumor metastasis in colorectal cancer.

Pharmaceutical compositions and routes of administration

The present disclosure also relates to methods of treating an intestinal disease by administering to a patient a pharmaceutical composition containing the disclosed antisense oligonucleotide for FMRP. In another aspect, the present disclosure relates to a pharmaceutical composition for use in the treatment of an intestinal disease. The pharmaceutical composition may consist of a disclosed antisense oligonucleotide that targets FMRP and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutical composition means, for example, a mixture containing a specified amount of a therapeutic compound, for example, an effective amount of a therapeutic compound in a pharmaceutically acceptable carrier, to be administered to a mammal, such as a human, for the purpose of treating an intestinal disease. In some embodiments, provided herein are pharmaceutical compositions comprising a disclosed antisense oligonucleotide for FMRP and a pharmaceutically acceptable carrier. In another aspect, the present disclosure relates to the use of the disclosed antisense oligonucleotide for FMRP in the manufacture of a medicament for the treatment of an inflammatory disease. A drug as used herein has essentially the same meaning as the term pharmaceutical composition.

As used herein, a pharmaceutically acceptable carrier means buffers, carriers and excipients suitable for use involving contact with tissues of humans and animals without undue toxicity, irritation, allergic reaction or other problem or complication, consistent with a reasonable benefit/risk ratio. Media(s)

- 14 045399 must be acceptable in the sense that it is compatible with the other ingredients of the formulations and is not harmful to the recipient. Pharmaceutically acceptable carriers include buffers, diluents, dispersing media, coatings, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. The use of such media and means for pharmaceutically active substances is known in the art. In one embodiment, the pharmaceutical composition is administered orally to a patient and includes an enteric coating suitable for regulating the site of absorption of the encapsulated substances in the digestive system or intestines. For example, the enteric coating may include a copolymer of ethyl acrylate and methacrylic acid.

In some embodiments, the disclosed antisense oligonucleotide for FMRP and any pharmaceutical composition based thereon can be administered to a patient by one or more routes, including enteral or parenteral delivery or intratumoral injection. As used herein, enteral or intraintestinal administration or delivery refers to the administration of a disclosed antisense oligonucleotide for FMRP to a patient via the gastrointestinal tract and may include oral, sublingual, gastric and rectal delivery. As used herein, parenteral administration refers to the administration of a disclosed antisense oligonucleotide for FMRP to a patient via routes other than the gastrointestinal tract and includes, without limitation, intravenous, intratumoral, intranasal, transdermal, subcutaneous, intramuscular, intraperitoneal, intestinal injections or infusions (e.g., into the jejunum colon, ileum), injection or infusion into the colon or rectum.

For example, the disclosed antisense oligonucleotide for FMRP can be administered subcutaneously to a patient. In certain examples, the disclosed antisense oligonucleotide for FMRP can be administered orally to a subject. In various examples, the disclosed antisense oligonucleotide for FMRP can be administered to a patient directly into the gastrointestinal tract or into specific regions of the gastrointestinal tract (eg, jejunum, ileum, colon, or rectum) via parenteral administration.

Pharmaceutical compositions containing the disclosed antisense oligonucleotide for FMRP, such as those disclosed herein, may be in unit dosage form and may be prepared by any suitable method. The pharmaceutical composition must be formulated so that it is compatible with the intended route of administration. Suitable compositions can be prepared using methods well known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

Pharmaceutical formulations, for example, are sterile. Sterilization can be accomplished, for example, by filtration through sterile filter membranes. If the composition is lyophilized, filter sterilization can be carried out before or after lyophilization and reconstitution.

Parenteral administration

The pharmaceutical compositions of the present disclosure may be formulated for parenteral administration, for example, they may be formulated for injection via intravenous, intratumoral, intramuscular, subcutaneous, intralesional, intestinal (eg, jejunal, ileal), colon, or rectal routes. , or via the intraperitoneal route. The preparation of an aqueous composition, such as an aqueous pharmaceutical composition, containing the disclosed antisense oligonucleotide for FMRP will be known to those skilled in the art in light of the present disclosure. Typically, such compositions can be prepared as injectable preparations, either as liquid solutions or suspensions; it is also possible to obtain solid forms suitable for use in preparing solutions or suspensions after adding liquid before injection; and drugs can also be emulsified.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that it can be easily drawn up with a syringe. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. It is also possible to obtain dispersions in glycerin, liquid polyethylene glycols and their mixtures, as well as in oils. In addition, sterile fixed oils can be used as a solvent or suspending medium. For this purpose, any soft, fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injectable preparations. The sterile injectable preparation may also be a sterile injectable solution, suspension

- 15 045399 or an emulsion in a non-toxic diluent or solvent acceptable for parenteral administration, for example, as a solution in 1,3-butanediol. Acceptable bases and solvents that can be used are water, Ringer's solution, U.S.?. and isotonic sodium chloride solution. In one embodiment, the disclosed antisense oligonucleotide for FMRP can be suspended in a carrier liquid containing 1% w/v sodium carboxymethylcellulose and 0.1% v/v TWEEN™ 80. Under normal conditions of storage and use, these formulations contain a preservative , preventing the growth of microorganisms.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated in accordance with the prior art using suitable dispersing or wetting agents and suspending agents. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile base that contains the basic dispersion medium and the required other ingredients from those listed above herein. Sterile injectable solutions of the present disclosure can be prepared by incorporating the disclosed antisense oligonucleotide for FMRP into a required amount of an appropriate solvent with varying amounts of the other ingredients listed above, if necessary, followed by filtration sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques, which result in a powder of the active ingredient and any additional required ingredient from a previously sterilized solution based on filtration. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter.

It is also envisaged to obtain larger quantities or highly concentrated solutions for intramuscular injection. In this regard, the use of DMSO as a solvent is preferred since it will provide very rapid penetration, delivering high concentrations of the disclosed FMRP antisense oligonucleotide to a small area.

Suitable preservatives for use in such a solution include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like. Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate 10, sodium acetate, sodium diphosphate, and the like, in amounts sufficient to maintain the pH in the range of about pH 6 to pH 8 , and, for example, from about pH 7 to pH7.5. Suitable tonicity agents include dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, etc., with the sodium chloride solution equivalent being in the range of 0.9 plus or minus 0.2%. Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfate, thiourea and the like. Suitable wetting and brightening agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suitable thickeners include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose and the like.

Enteral administration

In some embodiments, provided herein are compositions suitable for oral, sublingual, gastric, or rectal delivery of a disclosed antisense oligonucleotide for FMRP.

For example, compositions containing a disclosed FMRP antisense oligonucleotide may be suitable for oral delivery, e.g., tablets that include an enteric coating, e.g., a gastric resistant coating, such that the compositions can deliver the FMRP antisense oligonucleotide, e.g. the patient's gastrointestinal tract. For example, such administration to a patient may provide a local effect, essentially by locally applying an antisense oligonucleotide for FMRP directly to the affected portion of the patient's gastrointestinal tract. In some embodiments, such administration to the patient essentially avoids unwanted systemic absorption of the FMRP antisense oligonucleotide.

For example, there is provided a tablet for oral administration that contains beads (e.g., formed at least in part by beads) that include a disclosed antisense oligonucleotide for FMRP, e.g., the antisense oligonucleotide represented by any of SEQ ID NOs: 1-10, and pharmaceutically acceptable excipients substances. Such a tablet may be enteric-coated. The tablets provided may include pharmaceutically acceptable excipients such as fillers, binders, disintegrants and/or lubricants, as well as colorants, release regulators, coating agents, sweeteners, flavoring agents such as wintergreen, orange, xylitol, sorbitol, fructose and maltodextrin. , and flavoring agents, preservatives and/or antioxidants.

In some embodiments, the pharmaceutical compositions provided include an intragranular phase that includes a disclosed antisense oligonucleotide for

- 16 045399

FMRP, for example, an antisense oligonucleotide represented by any of SEQ ID NO: 1-10, a pharmaceutically acceptable salt and/or a pharmaceutically acceptable excipient. For example, the disclosed FMRP antisense oligonucleotide and excipient can be mixed together, optionally with other excipients, and formed into beads. In some embodiments, the intragranular phase can be formed using wet granulation, for example, a liquid (eg, water) is added to a mixed FMRP oligonucleotide compound and excipient, and then the combination is dried, ground, and/or screened to form granules. One skilled in the art will appreciate that other processes can be used to produce the intragranular phase.

In some embodiments, the compositions provided include an extragranular phase, which may include one or more pharmaceutically acceptable excipients, and which can be mixed with the intragranular phase to form the disclosed composition.

The disclosed composition may include an intragranular phase that includes a filler. Exemplary fillers include, but are not limited to, cellulose, gelatin, calcium phosphate, lactose, sucrose, glucose, mannitol, sorbitol, microcrystalline cellulose, pectin, polyacrylates, dextrose, cellulose acetate, hydroxypropyl methylcellulose, partially pregelatinized starch, calcium carbonate, etc., including their combinations.

In some embodiments, the disclosed composition may include an intragranular phase and/or an extragranular phase that includes a binder that can generally hold the ingredients of the pharmaceutical formulation together. Exemplary binders of the present disclosure may include, without limitation, the following: starches, sugars, cellulose or modified cellulose such as hydroxypropylcellulose, lactose, pre-gelatinized maize starch, polyvinylpyrrolidone, hydroxypropylcellulose, hydroxypropylmethylcellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, methylcellulose, ethylcellulose , sugar alcohols, etc., including combinations thereof.

Formulations, for example, which include an intragranular phase and/or an extragranular phase may include a disintegrant such as, but not limited to, starch, cellulose, cross-linked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethylcellulose, alginates, corn starch, croscarmellose sodium, cross-linked carboxymethylcellulose, hydroxypropylcellulose with a low degree of substitution, gum arabic, etc., including combinations thereof. For example, the intragranular phase and/or the extragranular phase may include a disintegrant.

In some embodiments, the composition provided includes an intragranular phase containing a disclosed antisense oligonucleotide for FMRP and excipients selected from the following: mannitol, microcrystalline cellulose, hydroxypropyl methylcellulose and sodium starch glycolate or combinations thereof, and an extragranular phase containing one or more of the following: microcrystalline cellulose , sodium starch glycolate and magnesium stearate or mixtures thereof.

In some embodiments, the composition may include a lubricant, for example, the extragranular phase may contain a lubricant. Lubricants include, but are not limited to, talc, silica, fats, stearin, magnesium stearate, calcium phosphate, silicon dioxide, calcium silicate, calcium phosphate, colloidal silicon dioxide, metal stearates, hydrogenated vegetable oil, corn starch, sodium benzoate, polyethylene glycols, sodium acetate, calcium stearate, sodium lauryl sulfate, sodium chloride, magnesium lauryl sulfate, talc and stearic acid.

In some embodiments, the pharmaceutical composition comprises an enteric coating. Typically, enteric coatings provide a barrier to an oral drug that controls the site of drug absorption in the gastrointestinal tract. Enteric coatings may include a polymer that degrades at different rates depending on pH. Enteric coatings may include, for example, cellulose acetate phthalate, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, methyl methacrylate-methacrylic acid copolymers, ethyl acrylate-methacrylic acid copolymers, methacrylic acid copolymer type C, polyvinyl acetate phthalate, and ace phthalate tata pulp.

Exemplary enteric coatings include Opadry® AMB, Acryl-EZE®, Eudragit® brands. In some embodiments, the enteric coating may be from about 5 to about 10%, from about 5 to about 20%, from 8 to about 15%, from about 8 to about 20%, from about 10 to about 20%, or from about 12 to about 20% or about 18% of the intended tablet by weight. For example, enteric coatings may include a copolymer of ethyl acrylate and methacrylic acid.

For example, according to the provided embodiment, a tablet is provided that

- 17 045399 contains or consists essentially of from about 0.5% to about 70%, for example from about 0.5% to about 10% or from about 1% to about 20% by weight of the disclosed antisense oligonucleotide for FMRP or its pharmaceutically acceptable salt. Such a tablet may include, for example, from about 0.5% to about 60% by weight mannitol, for example from about 30% to about 50% by weight mannitol, for example about 40% by weight mannitol; and/or from about 20% to about 40% by weight microcrystalline cellulose or from about 10% to about 30% by weight microcrystalline cellulose. For example, the opened tablet may contain an intragranular phase that includes from about 30% to about 60%, such as from about 45% to about 65% by weight, or, alternatively, from about 5 to about 10% by weight of the disclosed antisense oligonucleotide for FMRP, from about 30% to about 50% or, alternatively, from about 5% to about 15% by weight of mannitol, from about 5% to about 15% microcrystalline cellulose, from about 0% to about 4%, or from about 1% to about 7% hydroxypropyl methylcellulose; and about 0% to about 4%, such as about 2% to about 4% sodium starch glycolate, by weight.

In various embodiments, a pharmaceutical tablet formulation for oral administration of the disclosed FMRP antisense oligonucleotide comprises an intragranular phase, wherein the intragranular phase comprises the disclosed FMRP antisense oligonucleotide or a pharmaceutically acceptable salt thereof (such as a sodium salt) and a pharmaceutically acceptable excipient, and may also include an extragranular phase, which may include a pharmaceutically acceptable excipient such as a disintegrant.

The extragranular phase may include components selected from microcrystalline cellulose, magnesium stearate, and mixtures thereof. The pharmaceutical composition may also include an enteric coating comprising from about 12 to 20% by weight of the tablet. For example, a pharmaceutically acceptable tablet for oral use may contain from about 0.5 to 10% by weight of a disclosed FMRP antisense oligonucleotide, e.g., a disclosed FMRP antisense oligonucleotide or a pharmaceutically acceptable salt thereof, from about 30 to 50% by weight of mannitol, from approximately 10 to 30% by weight microcrystalline cellulose and an enteric coating containing a copolymer of ethyl acrylate and methacrylic acid.

In some examples, a pharmaceutically acceptable tablet for oral use may contain an intragranular phase containing from about 5 to about 10% by weight of a disclosed FMRP antisense oligonucleotide, e.g., a disclosed FMRP antisense oligonucleotide or a pharmaceutically acceptable salt thereof, about 40% by weight mannitol, about 8% by weight microcrystalline cellulose, about 5% by weight hydroxypropyl methylcellulose and about 2% by weight sodium starch glycolate; an extragranular phase containing approximately 17% by weight microcrystalline cellulose, approximately 2% by weight sodium starch glycolate, approximately 0.4% by weight magnesium stearate; and an enteric coating covering the tablet containing a copolymer of ethyl acrylate and methacrylic acid.

In some embodiments, the pharmaceutical composition may comprise an enteric coating containing about 13 or about 15, 16, 17 or 18% by weight, for example, Acryl-EZE® (see, for example, PCT Publication No. WO 2010/054826, which is included incorporated herein by reference in its entirety).

The rate at which the coating dissolves and the active ingredient is released is the dissolution rate. In some embodiments, the tablet may have a dissolution profile, for example, when tested on a USP/EP Type 2 (paddle) apparatus at 100 rpm and 37° C. in phosphate buffer pH 7.2, of from about 50% to about 100 % release of antisense oligonucleotide for FMRP after a period of time ranging from about 120 to about 240 minutes, for example after 180 minutes. In certain embodiments, the tablet may have a dissolution profile, for example, when tested on a USP/EP Type 2 (paddle) meter at 100 rpm and 37°C in dilute HCl pH 1.0, such that after 120 min the antisense oligonucleotide for Almost no FMRP is released. In some embodiments, the tablet may have a dissolution profile, for example, when tested on a USP/EP Type 2 (paddle) apparatus at 100 rpm and 37° C. in phosphate buffer pH 6.6, providing from about 10% to about 30 % or no more than 50% release of antisense oligonucleotide for FMRP after 30 min.

Formulations, such as tablets, in some embodiments, when administered orally to a patient, can provide a minimum concentration of antisense oligonucleotide for FMRP in the patient's blood plasma. In another embodiment, the disclosed compositions, when administered orally, provide local delivery to the colon or rectum of a patient,

- 18 045399 for example, to a part of the patient’s body that is affected or damaged due to a disease.

Administration of the disclosed antisense oligonucleotide for FMRP to a patient can be done rectally. Pharmaceutical compositions for rectal administration include foams, solutions (enemas), and suppositories (Block, chapter 87 in: Remington's Pharmaceutical Sciences, 18th ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990).

In some embodiments, the methods provided herein may further include administering to the patient at least one other agent that is directed at treating the diseases and disorders disclosed herein. In some embodiments, the other agents provided may be administered to the patient together (eg, sequentially or simultaneously).

For example, such agents include, but are not limited to, chemotherapeutic agents such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, ezorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosourea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisourea. zones , hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosourea, nitrogen mustards, melphalan, cyclophosphamide, 6mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacite idine, hydroxyurea, deoxycoformycin, 4hydroxyperoxycyclophosphoramide , 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES).

Agents also include immunosuppressive agents, including glucocorticoids, cytostatics, antibodies, immunophilin agents, interferons, opioids, TNF-binding proteins, mycophenolate and small molecule biological agents. For example, contemplated immunosuppressive agents include, but are not limited to, tacrolimus, cyclosporine, pimecrolimus, sirolimus, everolimus, mycophenolic acid, fingolimod, dexamethasone, fludarabine, cyclophosphamide, methotrexate, azathioprine, leflunomide, teriflunomide, anakinra, antithymocyte globulin, antilymphocyte ny globulin, muromonab-CD3, afutuzumab , rituximab, teplizumab, efalizumab, daclizumab, basiliximab, adalimumab, infliximab, certolizumab pegol, natalizumab and etanercept. Other remedies include antibiotics, antidiarrheals, laxatives, pain relievers, dietary supplements containing iron, and dietary supplements containing calcium or vitamin D or B-12.

Dosage and frequency of administration

Exemplary formulations include dosage forms that include or consist essentially of the disclosed antisense oligonucleotide for FMRP in an amount of from about 35 to about 500 mg. For example, formulations provided herein include approximately 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, or 250 mg of disclosed antisense oligonucleotide for FMRP. In one embodiment, the formulation may include about 40 mg, 80 mg, or 160 mg of the disclosed antisense oligonucleotide for FMRP. In some embodiments, the formulation may include at least 100 μg of a disclosed antisense oligonucleotide for FMRP. For example, formulations may include about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg of the disclosed antisense oligonucleotide for FMRP. The amount administered to a patient will depend on variables such as the type and severity of the disease or symptom being treated, the general health and size of the patient, the in vivo activity of the FMRP antisense oligonucleotide, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased from the upper level in order to quickly achieve the desired blood level or tissue level. The initial dosage may be lower than optimal and the dosage may be gradually increased over the course of treatment. Dosing in humans can be optimized, for example, in a standard phase I dose-escalation study involving a 40 to 160 mg step-up design. Dosing frequency may vary depending on factors such as route of administration, dosage amount, and disease being treated. Exemplary dosing frequencies include once daily, once weekly, and once every two weeks. In some embodiments, dosing is performed once daily for 7 days.

Diagnostic methods

The present disclosure also relates to a method for diagnosing an intestinal disease in a patient that is based on detecting signal levels indicative of FMRP expression in one or more biological samples from the patient. As used herein, the term FMRP expression signal may refer to any indication of expression of the FMR1 gene, FMR1 gene products, or FMRP activity. FMR1 gene products include RNA (eg, mRNA), peptides, and proteins (eg, FMRP, variants, analogs and/or parts thereof). Measures of FMR1 gene expression that can be assessed include, but are not limited to, FMR1 gene interaction

- 19 045399 or the FMR1 gene as part of chromatin with cellular components that regulate gene expression, expression levels of the FMR1 gene product (for example, FMRP RNA expression levels, FMRP protein expression levels) or the interaction of FMRP RNA or protein with transcriptional, translational or post-translational processing apparatus. Measures of FMRP activity include, but are not limited to, assessments of RIPK/MLKL signaling pathway activation (eg, assessment of RIPK1, RIPK3, and/or MLKL phosphorylation) and CREB expression (eg, mRNA and protein expression).

Detection of signal indicative of FMRP expression can be performed using in vivo, in vitro or ex vivo methods. In a preferred embodiment, the methods of the present disclosure can be carried out in vitro. Methods of detection may include detection in the patient's blood, serum, stool, tissue or cells. Detection can be accomplished by measuring the signal indicative of FMRP expression in whole tissue, tissue explants, cell cultures, dissociated cells, cell extract or biological fluids, including blood or serum. Specified detection methods include assays that measure expression levels of the FMR1 gene product, such as Western blotting, flow cytometry, ELISA, other quantitative binding assays, cell or tissue growth assays, Northern blot, quantitative or semiquantitative polymerase chain reaction, medical imaging (eg, MRI) or immunostaining techniques (eg, immunohistochemistry or immunocytochemistry).

Examples

The present disclosure is further illustrated by the following examples. The examples are presented for illustrative purposes only and should not be construed as limiting in any way the scope or content of this disclosure.

Example 1 FMRP expression level is increased in human colon and rectal cancer.

To study the role of FMRP in CRC tumorigenesis, FMRP mRNA levels and protein levels were analyzed in human CRC samples. In this example, the presence of increased levels of FMRP expression in human CRC samples was demonstrated.

Patients and samples

Paired samples of human CRC tumors and adjacent macroscopically uninvolved areas were collected from 48 patients undergoing colon resection for sporadic CRC at the Tor Vergata University Teaching Hospital (Rome, Italy). None of the patients received radiation therapy or chemotherapy before surgery.

Immunohistochemical analysis

Immunohistochemical analysis was performed using formalin-fixed, paraffin-embedded normal tissue sections and paired samples of tumor and tumor-surrounding tissue from patients with CRC. Colon sections were dewaxed and dehydrated using xylene and ethanol and antigen retrieval was performed in Tris-EDTA-citrate buffer (pH 7.8) for 30 min in a thermostatic bath at 98°C (Dako Agilent Technologies, Glostrup, Denmark). Immunohistochemical staining was performed using a monoclonal anti-human FMRP antibody (final dilution 1:500, LifeSpan BioSciences, Inc.) incubated at room temperature for 1 hour, followed by a biotin-free detection technique using HRP polymer and 3,3'-diaminobenzidine (DAB) as a chromogen (MACH 4™ Universal HRP-Polymer kit, Biocare Medical, Pacheco, CA). Sections were counterstained with hematoxylin, dehydrated, and mounted. IgG isotype control-stained sections were prepared under conditions identical to the immunohistochemical analysis described above, with the primary antibody replaced by a purified control normal mouse IgG antibody (R&D Systems, Minneapolis, MN).

FMRP expression was analyzed in sections (n=40) taken from paired human CRC tumor samples and adjacent areas and in samples taken from normal control (NC) colon tissues. As shown in FIG. 1A, the expression level of FMRP protein was significantly increased in CRC tumor samples (T) compared with that in the tumor surrounding areas (P) and NC. FMRP was expressed at high levels in approximately 61% of CRC samples (24/40), compared with adjacent non-tumor areas and NC, in which FMRP was expressed at relatively low levels.

FMRP mRNA expression

RNA was extracted from paired samples of human CRC tumors and adjacent macroscopically unaffected areas using the PureLink® mRNA Mini kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. A constant amount of RNA (1 μg per sample) was reverse transcribed to form complementary DNA (cDNA) and amplified using the following conditions: denaturation for 1 min at 95°C; annealing for 30 s at 59°C for human FMRP, 60°C for human/mouse β-actin; 30 s elongation at 72°C. B-actin was used as a housekeeping gene product. Gene expression was calculated using the AACt algorithm. The primer sequences were as follows: FMRP

- 20 045399 human (direct 5'- GTTGAGCGGCCGAGTTTGTCAG-3' (SEQ ID NO: 11); reverse 5'CCCACTGGGAGAGGATTATTTGGG-3' (SEQ ID NO: 12)), human and mouse β-actin (direct 5'AAGATGACCCAGATCATGTTTGAGACC-3 ' (SEQ ID NO: 13); reverse 5'-AGCCAGTCCAGACGCAGGAT-34

SEQ ID NO: 14)).

As can be seen in FIG. 1B, RT-PCR results confirmed the immunohistochemical data and demonstrated that FMRP mRNA expression levels were significantly increased in human CRC tumor samples (T) compared with those in tumor-surrounding tissue samples (P) that were taken from grossly unaffected tumors. region level from the same patients.

FMRP protein expression

Total protein was extracted from human colon tissue and lysed on ice in a buffer containing 10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM ethylene glycol-bis-(in -aminoethyl ester)-N,N,N',N'-tetraacetic acid (EGTA) and 0.5% Nonidet P40, supplemented with 1 mM dithiothreitol (DTT), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na ₃ VO ₄ and 1 mM NaF. Lysates were clarified by centrifugation and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Blot membranes were incubated with anti-FMRP antibodies (Cell Signaling, Danvers, MA) followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Dako, Milan, Italy). After analysis, each blot membrane was stripped and incubated with mouse monoclonal anti-human β-actin antibody (Sigma-Aldrich) to ensure equivalence of lane loading.

As can be seen in FIG. 1C and 1D, the results of Western blot analysis confirmed the data from immunohistochemistry and RT-PCR and demonstrated that the expression level of FMRP protein was significantly increased in human CRC tumor samples (T) compared with that in tumor tissue samples (P ), which were taken from macroscopically unaffected areas from the same patients.

Example 2: FMR1 knockout mice are resistant to azoxymethane-induced tumorigenesis.

To elucidate the role of FMRP in CRC, a mouse model of sporadic CRC induced by azoxymethane (AOM) was used. This example describes the tumor-resistant phenotype of FMR1 knockout mice in a model of AOM-induced CRC.

Induction of CRC by AOM

Wild-type (WT) and FMR1 knockout (KO) mice were treated by intraperitoneal injection with the alkylating agent, AOM (10 mg/kg; Sigma Aldrich, Milan, Italy), once a week for 5 weeks to induce tumor formation. Mice were monitored for tumor formation and screened endoscopically using a high-resolution endoscopic system 7 days before sacrifice. At week 22, mice were sacrificed by cervical dissection and colonic tissues were collected for analysis.

Colonoscopy procedures were performed in a blinded manner to monitor tumor formation using a high-resolution mouse endoscopic system. Tumors were detected during an endoscopic examination performed at week 21. After completion of treatment, tumors were counted to determine the total number of lesions in the colon. All tumors were assessed based on their size and counted using the protocol described in Becker C. et al., Gut. 2005; 54(7):9504., incorporated herein by reference in its entirety. Briefly, tumors were staged as follows: stage 1 (very small but detectable tumor), stage 2 (tumor covering up to one-eighth of the circumference of the colon), stage 3 tumor covering up to a quarter of the circumference of the colon), stage 4 (tumor , covering up to half the circumference of the colon) and stage 5 (tumor covering more than half the circumference of the colon).

As can be seen in FIG. 2A and 2B, AOM-treated WT mice developed several large tumors, whereas the number and size of tumors in AOM-treated FMR1-KO mice were significantly reduced. These results were confirmed by direct assessment of tumors in mice sacrificed at week 22 (data not shown). As shown in FIG. 2C, AOM-treated WT mice had a 20% reduction in viability compared to AOM-treated FMR1-KO animals.

Immunohistochemical analysis

Histological analysis was performed on frozen sections of mouse colon obtained from WT and FMR1-KO mice from tumor and surrounding tumor areas after hematoxylin and eosin (H&E) staining. Colon sections were deparaffinized and dehydrated using xylene and ethanol and antigen retrieval was performed in Tris-EDTA-citrate buffer (pH 7.8) for 30 min in a thermostatic bath at 98°C (Dako Agilent Technologies, Glostrup, Denmark) Immunohistochemistry staining was performed using a monoclonal antibody to human FMRP (final dilution 1:500, LifeSpan BioSciences, Inc.) incubated at room temperature for 1 hour, followed by a biotin-free detection technique using HRP polymer and 3.3 '-diaminobenzidine (DAB) as chromogen (MACH 4™ Universal HRP-Polymer kit, Biocare Medical, Pacheco, CA). Sections were counterstained with hematoxylin, dehydrated, and mounted. IgG isotype control-stained sections were prepared under conditions identical to the immunohistochemical analysis described above, with the primary antibody replaced by a purified control normal mouse IgG antibody (R&D Systems, Minneapolis, MN).

In the absence of AOM treatment, intestinal tissues from FMRP-KO mice were normal and no macroscopic abnormalities were observed compared with WT mice (data not shown). However, as shown in FIG. 2D, tumors excised 22 weeks after AOM treatment indicated that WT mice developed tumors with pervasive dysplasia, whereas FMR1-KO animals maintained normal mucosal architecture adjacent to dysplastic areas.

FMRP protein expression

Total protein was extracted from mouse colon tissue and lysed on ice in a buffer containing 10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM ethylene glycol-bis-(in -aminoethyl ester)-N,N,N',N'-tetraacetic acid (EGTA) and 0.5% Nonidet P40, supplemented with 1 mM dithiothreitol (DTT), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na ₃ VO ₄ and 1 mM NaF. Lysates were clarified by centrifugation and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Blot membranes were incubated with anti-FMRP antibodies (Cell Signaling, Danvers, MA) followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Dako, Milan, Italy). Following analysis, each blot membrane was stripped and incubated with mouse monoclonal anti-human β-actin antibody (Sigma-Aldrich) to ensure equivalence of lane loading.

As shown in FIG. 2E, consistent with human CRC samples, the level of FMRP protein expression was increased in mucosal tumors (T) of AOM-treated WT mice compared with that in adjacent tumor-surrounding areas (P) of the same animals.

TUNEL and Ki67 staining

To determine whether the reduced tumorigenesis observed in fMRI-KO animals was due to increased cell death or decreased tumor cell proliferation, TUNEL staining and Ki67 immunohistochemical staining were performed. In frozen sections of colon taken from WT and FMR1-KO mice, apoptotic cells were detected using the TUNEL in situ cell death detection kit (Roche Applied Science) according to the manufacturer's instructions. 3-amino-9-ethylcarbazole was used as a chromogen, and sections were counterstained with hematoxylin. The nuclei of apoptotic cells appeared as red-colored structures on a blue-violet background.

Sections for immunocytochemical analysis were also incubated with mouse monoclonal anti-mouse Ki67 antibody (clone MIB-5, final dilution 1:100, DaKO, Agilent, Santa Clara, CA, USA) at room temperature for 30 min, followed by the biotin-free method detection using HRP polymer (Ultravision detection system, Thermo Scientific, Waltham, MA, USA) and 3,3'-diaminobenzidine as a chromogen (DaKO, Agilent). Sections were counterstained with hematoxylin, dehydrated, and mounted.

As shown in FIG. 2F, TUNEL staining revealed that AOM-treated FMR1-KO mice had increased levels of DNA fragmentation compared with those of AOM-treated WT animals. However, as can be seen in FIG. 2G, no differences in the number of Ki67 positive cells were observed. These results collectively suggest that FMRP is associated with increased tumor resistance to cell death.

Example 3 Antisense Oligonucleotides for FMRP Induce Cell Death in CRC Cell Lines

To determine how FMRP affects CRC survival and cell death, human CRC epithelial cell lines were treated with a specific antisense oligonucleotide for FMRP. This example demonstrates that antisense oligonucleotides for FMRP can induce cell death in CRC cell lines through an apoptosis-independent mechanism.

Expression of FMRP in colon and rectal cancer cell cultures

Human CRC cell lines, DLD-1 and HCT-116, were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in RPMI1640 medium (DLD-1) and McCoy 5A medium (HCT-116), respectively. All media were supplemented with 10% fetal calf serum, 1% penicillin/streptomycin (both from Lonza, Verviers, Belgium). A normal human colon epithelial cell line (HCEC-1ct) was obtained from EVERCYTE GmbH (Vienna, Austria) and cultured in ColoUp® medium (EVERCYTE GmbH). Cells were maintained in a fully humidified incubator set at 37°C, 5% CO ₂ . Cell line lysates were prepared and analyzed by SDS-polyacrylamide gel electrophoresis/immunoblotting using

- 22 045399

[5'-ATGGAGGAGCTGGTGGTGGA-3' (SEQ III NO: 15)] agents described previously in example 2. For immunofluorescent staining, cell lines were fixed with 3.7% formaldehyde for 10 min at 4°C, permeabilized with 0.1% Triton for 10 min at room temperature and blocked (1% bovine serum albumin, Tween 0.1%, glycerol 2%) for 1 h at room temperature. Fixed and blocked cells were incubated with anti-FMRP monoclonal antibody (1:500, Cell Signaling, Danvers, MA) overnight at 4°C. After washing with PBS, Alexa 488-conjugated goat anti-rabbit IgG secondary antibody (1:2000, A11008; Invitrogen) was added for 1 h at room temperature. Slides were washed with PBS, mounted with the fluorescence antifade reagent Prolong gold® with 4',6diamidino-2-phenylindole (P36931; Invitrogen) and analyzed with a Leica DMI4000 B microscope using the Leica software package (V4.6.2).

As shown in FIG. 3A-3C, FMRP was expressed at higher levels in human CRC cell lines (DLD-1 and HCT-116) compared to HCEC-1ct epithelial cells (ie, normal colon cells).

Knockdown of FMRP in colon and rectal cancer cell cultures

Phosphorothioate single-stranded antisense oligonucleotides complementary to human FMRP [5'-TCCACCACCAGCTCCTCCAT-3' (SEQ ID NO: 6)] and single-stranded sense oligonucleotides were synthesized

CRC cell lines and HCEC-1ct cells were transfected with either antisense (AS) oligonucleotide for FMRP (final concentration 0.5–100 nM) or sense (S) oligonucleotide for FMRP (final concentration 100 nM) for 24 and 48 h using Opti-MEM medium and Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

As can be seen in FIG. 3D, 3G, and 31, treatment of DLD-1 cells (Fig. 3C), HCT-116 cells (Fig. 3G), and HCEC-1ct cells (Fig. 31) with an antisense (AS) oligonucleotide for FMRP resulted in significant inhibition of FMRP expression , and no significant inhibition was observed in cells treated with the sense oligonucleotide.

Cell death in cells treated with antisense oligonucleotide for FMRP

Cells were transfected with either antisense (AS) oligonucleotide for FMRP (final concentration 0.5 nM and 100 nM) or sense oligonucleotide (S) for FMRP (final concentration 100 nM). After 24 h (Figs. 3E, 3F, 3J, and 3K) or 48 h (Fig. 3H), cells were harvested, washed twice in Annexin V (AV) labeling buffer, and stained with FITC-Annexin V (final dilution 1:100 ; Immunotools, Friesoite, Germany) according to the manufacturer's instructions and incubated with 5 mg/ml propidium iodide (PI) for 30 min at 4°C. Cells were also transfected with either sense (S) oligonucleotide for FMRP or AS oligonucleotide for FMRP (100 nM final concentration) for 36 h and assayed for activated caspase 3 and caspase 8. As positive and negative controls, cells were treated with staurosporine, respectively. (final concentration 1 μM, Sigma-Aldrich, Milan, Italy) or Q-VD-OPh (caspase inhibitor; final concentration 1 μM) (R&D Systems, Inc, Minneapolis, MN). Additionally, 36 h (Figures 4F and 4G) or 48 h posttransfection (Figures 4D and 4E), cells were stimulated with 10 M bromodeoxyuridine for 60 min, fixed in 70% chilled ethanol, and stored at -20°C for up to for at least 3 h. Cells were then disrupted in 2 M HCl and stained with a monoclonal anti-bromodeoxyuridine antibody (Immunotech, Marseille, France) followed by a fluorescein isothiocyanate-conjugated secondary antibody against immunoglobulin G (Molecular Probes, Milan, Italy) and 100 g/ml PL fluorescence was measured using a Gallios flow cytometer (Beckman Coulter, Life Sciences, Pasadena, CA, USA) and analyzed using Kaluza software (Beckman Coulter). Living cells were considered AV7PI cells.

As shown in FIG. 3E and 3F, treatment of DLD-1 cells with an antisense oligonucleotide for FMRP resulted in increased numbers of annexed V+ or annexin V+PI+ cells, indicating that these CRC cells underwent spontaneous apoptosis or necroptosis. However, as shown in FIG. 3H, 3J, and 3K, neither HCT-116 nor HCRC-1ct cells treated with an antisense oligonucleotide for FMRP exhibited increased numbers of annexed V+ or annexed V+PI+ cells. Similar results were observed when FMRP-specific siRNA was used to transfect cell lines (data not shown).

To elucidate the mechanisms of cell death induced by an antisense oligonucleotide for FMRP, the effect of FMRP knockdown on the activation of proapoptotic caspase 8 and caspase 3 was analyzed. As shown in FIG. 4A and 4B, treatment of CRC cells with an antisense oligonucleotide for FMRP did not change the percentage of activated caspase 3-positive cells or activated caspase 8-positive cells. As expected, staurosporine (Stauro) significantly increased the percentage of activated caspase 3-positive cells.

As can be seen in FIG. 4C, pretreatment of cells with a caspase inhibitor (Q-VD-OPh; Cas in) did not lead to a change in antisense oligonucleotide-induced cell death

- 23 045399 leotide for FMRP. Moreover, to test whether cell death induced by an antisense oligonucleotide for FMRP is secondary to cell growth arrest, the cell cycle progression of DLD-1 cell lines was analyzed. As shown in FIG. 4D-4G, an antisense oligonucleotide for FMRP, did not affect the relative percentage of cells in G2/M, S, or G0/G1 cell cycle phases before induction of cell death. Taken together, these data suggest that FMRP influences CRC cell death through an apoptosis-independent pathway that does not affect the cell cycle.

Example 4 Antisense oligonucleotides for FMRP induce necroptosis.

Cancer cells have evolved a variety of mechanisms to avoid programmed cell death. Necroptosis is a regulated caspase-independent cell death pathway that is an alternative mechanism for eliminating apoptosis-resistant cells. This example demonstrates that antisense oligonucleotides for FMRP induce cell death among CRC cells through activation of the necroptosis pathway.

FMRP associates with necroptosis pathway-associated mRNA

FMRP from human CRC samples and CRC cell lines was immunoprecipitated along with its associated RNA using an FMRP-specific antibody (Fig. 5A) to identify associated transcripts by real-time PNR. β-Actin was used as a negative control, and E-cadherin and vimentin were used as positive controls. As shown in Figures 5B and 5C, RIPK3 mRNA did not coimmunoprecipitate with FMRP in human CRC samples or CRC cell lines. In contrast, RIPK1 mRNA coimmunoprecipitated with FMRP at significant levels compared with IgG isotype control.

To confirm the association of FMRP with components of the necroptosis pathway, cells were left untreated or transfected with either an antisense oligonucleotide for FMRP (final concentration of 0.5 and 100 nM) or a sense oligonucleotide for FMRP (final concentration of 100 nM); incubated with pMLKL inhibitor (necrosulfonamide, NSA; final concentration 1 μM) (Calbiochem); or pRIPK1 inhibitor (necrostatin-1, NEC1; final concentration 10 μM) (Cayman Chemical, Ann Arbor, MI, USA). After 24 hours, cells were collected and analyzed by Western blot or stained with FITC-Annexin V and PI for analysis using flow cytometry as previously described in Examples 1-3.

As shown in FIG. 6A-6D, treatment with an antisense oligonucleotide for FMRP resulted in increased expression levels of total RIPK1 protein in human CRC cell lines. Treatment with an antisense oligonucleotide for FMRP further resulted in increased phosphorylation of RIPK1, RIPK3, and MLKL. However, as can be seen in FIG. 6G, a similar increase in phosphorylation was not observed in HCEC-1ct cells treated with an antisense oligonucleotide for FMRP.

As shown in FIG. 6E and 6F, human CRC cell lines incubated with the RIPK1-specific inhibitor, necrostatin-1 (NEC1), and the MLKL-specific inhibitor, necrosulfonamide (NSA), were protected from cell death induced by an antisense oligonucleotide for FMRP. Taken together, these results suggest that FMRP abolishes necroptosis in CRC cells by inhibiting the signaling cascade involving RIPK1, thereby suppressing necroptosis.

Example 5: FMRP expression is regulated by CREB.

Several intracellular protein kinases, such as mTOR and MAPK, and transcription factors, such as CREB, have been reported to positively regulate FMRP expression. This example demonstrates that FMRP expression is positively regulated by the transcription factor CREB.

CREB mRNA expression and protein expression

To assess whether CREB regulates FMRP expression, CRC cell lines were transfected with either an antisense (ASc) oligonucleotide for CREB (5'-GCATCTCCACTCTGCTGGTT-3') (SEQ ID NO: 16) or a sense (Ss) oligonucleotide for (5'- AACCAGCAGAGTGGAGATGC-3') (SEQ ID NO: 17) (200 nM final concentration) for 24 or 48 h. The mRNA AND total protein lysate were prepared and analyzed by RT-PCR and Western blot, respectively, as previously described in examples 1-3.

As shown in FIG. 7A, human CRC tumors (T) had significantly higher levels of CREB mRNA transcripts compared with levels in tumor-surrounding tissue samples (P). Likewise, as shown in FIG. 7B and 7C, the expression level of CREB protein is significantly higher in human CRC tumors (T) compared to that in tumor tissue samples (P).

To examine whether CREB regulates FMRP expression in CRC cell lines, CREB expression was inhibited using a specific AS oligonucleotide. As shown in FIG. 7D-7F, human CRC cell lines treated with an antisense oligonucleotide for CREB had reduced levels of CREB protein expression compared with that of untreated cells or cells treated with a sense oligonucleotide as a control.

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In addition, treatment with an antisense oligonucleotide for CREB resulted in a concomitant decrease in FMRP protein expression levels. These data suggest that in human CRC, CREB positively regulates FMRP expression.

Example 6: FMRP levels influence CRC migration and invasion.

In vitro models of wound surface and cell invasion were used to study the role of FMRP in CRC migration and invasion. This example describes that FMRP regulates proteins involved in cell migration and invasion, including the following: E-cadherin, β-catenin, and colorectal cancer mutated protein (MCC), a tumor suppressor whose gene is silenced by methylation promoter in colorectal cancer and, in particular, in patients with increased lymph node metastasis.

FMRP levels influence cell migration and invasion in CRC by regulating E-cadherin and βcatenin.

To study the role of FMRP in tumor cell migration and invasion, HCT-116 cells were seeded on each side of an ibidi® cell culture insert and grown to confluency, then left untreated (U) or transfected with a sense (S) oligonucleotide for FMRP (final concentration 0 .5 nM) or antisense (AS) oligonucleotide for FMRP (final concentration 0.5 nM). Additionally, HCT-116 cells were seeded onto Transwell® cell culture inserts pre-coated with Matrigel® and either left untreated (U) or transfected with sense (S) oligonucleotide (100 nM final concentration) or antisense (AS) oligonucleotide for FMRP (final concentration 0.5 nM) for 48 hours.

As shown in FIG. 8A and 8B, transfection of HCT-116 cells with an antisense oligonucleotide for FMRP produced a significant reduction in the percentage of cells closing the pseudo-wound compared to untreated cells or cells transfected with the sense oligonucleotide. The graph shows the average percentage of area covered by cells ± S.D. for 3 separate experiments.

Likewise, as shown in FIG. 8C and 8D, HCT-116 cells transfected with the antisense oligonucleotide for FMRP had significantly reduced migration through the Matrigel®-coated Transwell® cell culture insert compared to untreated cells or cells transfected with the sense oligonucleotide. The graph shows the average number of migrating cells ± S.D. for 3 independent experiments.

To determine whether FMRP regulates the levels of E-cadherin and/or β-catenin, key proteins associated with cell adhesion, cytoskeletal remodeling, and inhibition of tumor migration and invasion, cells were left untreated (U) or transfected with either a sense (S) oligonucleotide for FMRP (final concentration 0.5 nM), or antisense (AS) oligonucleotide for FMRP (final concentration 0.5 nM) for 48 h.

As shown in FIG. 8E-8G, transfection of HCT-116 cells with an antisense oligonucleotide for FMRP resulted in a significant increase in the expression levels of E-cadherin and β-catenin compared with untreated cells or cells transfected with a sense oligonucleotide. In fig. 8F and 8G values are expressed in relative units (r.u.), and they indicate the mean ±S.D. for 3 separate experiments.

FMRP suppresses the expression of MCC, a protein that regulates the expression of Ecadherin and β-catenin.

To examine whether FMRP regulates MCC, a protein known to interact with the E-cadherin/β-catenin complex, HCT-116 cells were left untreated (U) or transfected with a sense (S) oligonucleotide for FMRP (final concentration 0.5 nM) or antisense (AS) oligonucleotide for FMRP (final concentration 0.5 nM) for 48 h.

As shown in FIG. 9A and 9B, HCT-116 cells transfected with an antisense oligonucleotide for FMRP had significantly increased levels of MCC expression compared to untreated cells or cells transfected with a sense oligonucleotide for FMRP.

Recent studies have described a novel tumor suppressor function of MCC in the regulation of intercellular adhesion mediated by the E-cadherin/β-catenin complex in colon and rectal cancer cells. To examine whether MCC mediates the observed increase in Ecadherin and β-catenin expression in HCT-116 cells transfected with an antisense oligonucleotide for FMRP (e.g., Figures 8E-8G), cells were left untreated (U) or transfected with a sense oligonucleotide for FMRP (final concentration 0.5 nM), or antisense oligonucleotide for FMRP (final concentration 0.5 nM), and/or siRNA as control (control siRNA), or siRNA specific for MCC (MCC siRNA) for 48 h.

As shown in a representative Western blot (Fig. 9C) and corresponding quantitative assays (Figs. 9D and 9E), the presence of siRNA for MCC abolished the positive regulation of Ecadherin and β-catenin expression in cells transfected with an antisense oligonucleotide for FMRP from

Claims (8)

efficient. To determine whether MCC regulation of the E-cadherin/β-catenin complex is also required for the observed inhibition of migration and invasion in a colon cancer cell line (e.g., Figures 8A and 8B), HCT-116 cells were plated on each side of the insert for culture of ibidi cells and grown to confluency, then left untreated (U) or transfected with sense (S) oligonucleotide (final concentration 0.5 nM) or antisense (AS) oligonucleotide (final concentration 0.5 nM) and/or control siRNA or siRNA for MCC. As shown in FIG. 9F and 9G, the presence of MCC-specific siRNA resulted in a significant reduction in FMRP antisense oligonucleotide-mediated inhibition of migration and invasion in a colon and rectal cancer cell line. Taken together, these data suggest that FMRP controls cell-cell adhesion in colon cancer cells by regulating the MCC/E-cadherin/p-catenin complex. Incorporation by reference The entire disclosure of each of the patent documents and scientific articles cited herein is incorporated by reference in all respects. Equivalents The present disclosure may be embodied in other specific forms without departing from its essential characteristics. Accordingly, the foregoing embodiments should be considered illustrative and not limiting of the disclosure described herein. The scope of the present disclosure is determined by the appended claims and not by the preceding description, and it is intended that all modifications that fall within the meaning and range of the claims are covered by them. CLAIM 1. An oligonucleotide for inhibiting the expression of protein associated with fragile X syndrome (FMRP), containing a sequence selected from the group consisting of 5'-CCACCACCAGCTCCTCCA-3' (SEQ ID NO: 1), 5'-CTTCCACCACCAGCTCCT-3' (SEQ ID NO: 2), 5'-TCCACCACCAGCTCCTCC-3' (SEQ ID NO: 3), 5'-CTTCCACCACCAGCTCC-3' (SEQ ID NO: 4), 5'-TCACCCTTATCATCCTC-3' (SEQ ID NO: 5), 5'-TCCACCACCAGCTCCTCCAT-3' (SEQ ID NO: 6), 5'-ACTTCCACCACCAGCTCCTC-3' (SEQ ID NO: 7), 5'-TTCCACCACCAGCTCCTCCA-3' (SEQ ID NO: 8), 5'-ACTTCCACCACCAGCTCCT-3' (SEQ ID NO: 9) and 5'-CTCACCCTTTATCATCCCTCA-3' (SEQ ID NO: 10) or their complementary strand. 2. The oligonucleotide of claim 1, wherein the oligonucleotide contains ribonucleotides, deoxyribonucleotides, or a combination of ribonucleotides and deoxyribonucleotides. 3. The oligonucleotide according to claim 1, wherein the oligonucleotide is an antisense oligonucleotide or part of an siRNA, or a pharmaceutically acceptable salt thereof. 4. Oligonucleotide according to claims 1-3, where the oligonucleotide contains one or more chemically modified nucleosides selected from the group consisting of 5-methylcytidine, 5-methyl-2'deoxycytidine, deoxycytidine, 5-methyl-2'-deoxycytidine-5 '-monophosphate and 5-methyl-2'deoxycytidine-5'-monophosphorothioate, 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methylthymidine, 2'-O-methyluridine, 2'-O- methyladenosine, 5-methylcytosine and 5-methylguanine, 2'-O-(2methoxyethyl)nucleosides, 2'-deoxy-2'-fluoronucleosides and 2'-fluoro-β-D-arabinonucleosides, bridged nucleic acids, locked nucleic acids (LNA ), nucleic acids with conformationally constrained ethyl nucleoside analogues (sET), tricyclo-DNA (tcDNA), nucleic acids with a 2'-O,4'-C-ethylene bridge (ENA) and peptide nucleic acids (PNA). 5. An oligonucleotide according to any one of claims 1 to 4, where the oligonucleotide contains at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage bond, phosphomorpholidate bond, phosphopiperazidate bond, aminoalkylphosphoramidate bond, thiophosphoramidate bond, thionoalkylphosphonate bond, thionoalkylphosphotriester bond, thiophosphate bond, selenophosphate bond and boranophosphate bond. 6. Oligonucleotide according to claim 5, where the oligonucleotide contains at least one phosphorothioate bond. 7. The oligonucleotide according to claim 5, wherein all internucleoside bonds of the oligonucleotide are phosphorothioate bonds. 8. Oligonucleotide according to any one of claims 1 to 7, where the oligonucleotide is capable of inducing necroptosis. -