Smad7 inhibitory antisense oligonucleotides (ASO) for treating pouchitis and methods of using the same

Abstract

Provided herein are methods for treating or preventing pouchitis comprising administering a SMAD7 antisense oligonucleotide or pharmaceutical formulations comprising the SMAD7 antisense oligonucleotide.

Description

SMAD7 INHIBITORY ANTISENSE OLIGONUCLEOTIDES (ASO) FOR TREATING POUCHITIS AND METHODS OF USING THE SAMECROSS-REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 492,755, filed March 28, 2023, which is incorporated by reference in its entirety herein.SUMMARY

[0002] Provided herein are methods for treating or preventing pouchitis in an individual in need thereof comprising administering a therapeutically effective amount of a SMAD7 antisense oligonucleotide or pharmaceutical formulations comprising the SMAD7 antisense oligonucleotide.

[0003] Described herein, in certain embodiments, are methods of treating pouchitis in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5’-GTXGCCCCTTCTCCCXGCAGC-3’). In some embodiments, all internucleoside linkages are 0,0-linked phosphorothioate linkages and X is 5-methyl 2’ -deoxy cytidine. In some embodiments, the SMAD7 antisense oligonucleotide is administered enterally. In some embodiments, the enteral administration is oral, sublingual, gastric, or rectal. In some embodiments, the enteral administration is rectal. In some embodiments, the SMAD7 antisense oligonucleotide is administered parenterally. In some embodiments, the parenteral administration is intravenous, intratumoral, intrajejunal, intraileal, intracolonic, intrarectal, or intrapouch. In some embodiments, the parenteral administration is intrapouch. In some embodiments, the therapeutically effective amount of the SMAD7 ASO comprises a dose of about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, the therapeutically effective amount of the SMAD7 ASO comprises a dose of about 20 mg to about 500 mg. In some embodiments, the therapeutically effective amount of the SMAD7 ASO is administered about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, about every 72 hours, everyday, two-times a week, once in 2 weeks, or once a month. In some embodiments, the therapeutically effective amount of the SMAD7 ASO comprises a dose of about 160 mg daily. In some embodiments, the SMAD7 ASO down-regulates SMAD7 mRNA and / or protein in a cell. In some embodiments, the down-regulation of SMAD7 protein expression is more than 40% compared to an untreated cell. In some embodiments, at least one sample from the individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis. In some embodiments, the pouchitis is acute pouchitis. In some embodiments, the pouchitis is chronic pouchitis. In some embodiments, the individual is determined as having pouchitis using a modified pouchitis disease activity index (mPDAI) score of at least 5.

[0004] Described herein, in certain embodiments, are methods of treating pouchitis in an individual in need thereof, comprising administering to the individual a pharmaceutical formulation comprising: a) a SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5’-GTXGCCCCTTCTCCCXGCAGC-3’); and b) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation comprises 100 millimoles to 5 moles of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises 100 millimoles to 2 moles, 100 millimoles to 900 millimoles, 300 millimoles to 5 moles, 300 millimoles to 2 moles, 300 millimoles to 900 millimoles, 900 millimoles to 5 moles, or 900 millimoles to 2 moles of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises about 300 millimoles of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises about 900 millimoles of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises about 2 moles of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation comprises greater than 300 millimoles, greater than 900 millimoles, or greater than 2 moles of the SMAD7 ASO. In some embodiments, the pharmaceutical formulation is administered orally. In some embodiments, the pharmaceutical formulation comprises: about 0.5% to about 30% by weight of the SMAD7 ASO; about 20% to about 50% by weight mannitol; about 10% to about 30% by weight microcrystalline cellulose; and an enteric coating comprising an ethylacrylate-methacrylic acid copolymer. In some embodiments, the pharmaceutical formulation comprises: a) an intra-granular phase comprising: i) about 5% to about 10% by weight of the SMAD7 ASO; ii) about 40% by weight mannitol; iii) about 8% by weight microcrystalline cellulose; iv) about 5% by weight hydroxypropyl methylcellulose; andv) about 2% by weight sodium starch glycolate; b) an extra-granular phase comprising: i) about 17% by weight microcrystalline cellulose; ii) about 2% by weight sodium starch glycolate, iii) about 0.4% by weight magnesium stearate; and c) an enteric coating comprising an ethylacrylate-methacrylic acid copolymer, wherein the percentages by weight are the weights of the ingredients compared to the total weight of the pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises: about 5% to about 30% by weight of the SMAD7 ASO; about 20% to about 50% by weight mannitol; about 10% to about 30% by weight microcrystalline cellulose, about 0.5% to about 10% by weight hydroxypropyl methylcellulose; about 0.5% to about 10% by weight sodium starch glycolate; about 0.05% to about 1% by weight magnesium stearate; about 0.5% to about 10% by weight of a moisture barrier film coating; and about 5% to about 20% by weight of a polymer coating. In some embodiments, the pharmaceutical formulation comprises: about 8.5% by weight of the SMAD7 ASO; about 40% by weight mannitol; about 25% by weight microcrystalline cellulose, about 5% by weight hydroxypropyl methylcellulose; about 4% by weight sodium starch glycolate; about 0.4% by weight magnesium stearate; about 4% by weight of a moisture barrier film coating; and about 10% to about 15% by weight of a polymer coating. In some embodiments, the pharmaceutical formulation comprises: about 23% by weight of the SMAD7 ASO; about 28% by weight mannitol; about 25% by weight microcrystalline cellulose, about 5% by weight hydroxypropyl methylcellulose; about 4% by weight sodium starch glycolate; about 0.4% by weight magnesium stearate; about 4% by weight of a moisture barrier film coating; and about 7% to about 12% by weight of a polymer coating In some embodiments, the pharmaceutical formulation down-regulates SMAD7 mRNA and / or protein in a cell. In some embodiments, the down-regulation of SMAD7 protein expression is more than 40% compared to an untreated cell. In some embodiments, at least one sample from the individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis. In some embodiments, the pouchitis is acute pouchitis. In some embodiments, the pouchitis is chronic pouchitis. In some embodiments, the individual is determined as having pouchitis using a modified pouchitis disease activity index (mPDAI) score of at least 5. A method for predicting pouchitis in an individual in need thereof, comprising determining a level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level is predictive of pouchitis, wherein the known control level is the SMAD7 level in a samplecollected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

[0005] Described herein, in certain embodiments, are methods of identifying an individual at risk of pouchitis, comprising: determining the level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level identifies the individual as being at risk for pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis. In some embodiments, the methods comprise: if the SMAD7 level is elevated relative to the known control level, then administering to the individual a pharmaceutical formulation comprising a SMAD7 ASO; or if the SMAD7 level is not elevated relative to the known control level, then determining the level of SMAD7 in a second sample from the individual. In some embodiments, the second sample is collected immediately after, about 1 hour after, about 3 hours after, about 6 hours after, about 12 hours after, about 1 day after, about 3 days after, about 1 week after, about 2 weeks after, about 1 month after, about 2 months after, about 3 months after, about 4 months after, about 5 months after, about 6 months after, about 7 months after, about 8 months after, about 9 months after, about 10 months after, about 11 months after, or about 12 months after the first sample.

[0006] Described herein, in certain embodiments, are methods for treating pouchitis in an individual in need thereof, comprising determining a level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level is predictive of pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis. In some embodiments, the SMAD7 ASO comprises a sequence selected from any one of SEQ ID NOs: 1-6 or a pharmaceutically acceptable salt thereof. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO: 1 (5’-GTXGCCCCTTCTCCCXGCAGC-3’). In some embodiments, all internucleoside linkages are O,O-linked phosphorothioate linkages and X is 5-methyl 2’- deoxycytidine. In some embodiments, the individual is human.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to understand the disclosure and to demonstrate how it may be carried out in practice, embodiments are now described, by way of non-limiting example only, with reference to the accompanying drawings in which:

[0008] FIGs. 1A-1C depict representative images showing single and doubleimmunofluorescence staining of fresh mucosal biopsy samples analyzed for the expression of SMAD7 (green), and DAPI (blue). FIG. 1A shows staining of a section of healthy ileum. FIG. IB shows staining of a section of an uninflamed pouch. FIG. 1C shows staining of a section of an inflamed pouch. The scale bars shown in all panels are 50 pm. The figure is representative of three separate experiments in which similar results were obtained.

[0009] FIGs. 2A-2B depict representative Western blot images showing SMAD7 and P- actin in total proteins extracted from mucosal biopsy samples and a quantitative graphical analysis of the SMAD7 / p-actin ratio from Western blotting as measured by densitometry scanning. FIG. 2A shows Western blots (left) probed with SMAD7 (top panel) and P-actin loading control (bottom panel) of total proteins extracted from mucosal samples of the inflamed pouch (pouchitis, n=10), normal, pre-pouch ileum (n=5) of the same patient with pouchitis, uninflamed pouch (n=3), and normal controls (healthy ileum, n=4). Right panel of FIG. 2A shows the quantitative analysis of SMAD7 / p-actin ratio as measured by densitometry scanning of the Western blots. Values are expressed in arbitrary units (a.u.) and indicate mean ± SEM of all samples (* p < 0.05, ** p < 0.01, *** p < 0.001). ). FIG. 2B shows Western blots (left) probed with SMAD7 (top panel) and P-actin loading control (bottom panel) of total proteins extracted from mucosal samples of the inflamed pouch (pouchitis, n=10), inflamed ileum (prepouch ileitis, n=3) of the same patients with pouchitis, uninflamed pouch (n=5), and normal controls (healthy ileum, n=4). The right panel of FIG. 2B shows the quantitative analysis of SMAD7 / p-actin ratio as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units (a.u.) and indicate mean ± SEM of all samples (* p < 0.05, ** p < 0.01).

[0010] FIGs. 3A-3B depict representative images showing single and doubleimmunofluorescence stainings. FIG. 3A shows images of fresh mucosal biopsy samples from a patient with chronic pouchitis analyzed for the expression of SMAD7 (green), CD3 (red), and DAPI (blue). FIG. 3B shows images of fresh mucosal biopsy samples from a patient with chronic pouchitis analyzed for the expression of SMAD7 (green), HLA-DRII (red), and DAPI (blue). The scale bars shown in all panels are 75 pm. The figure is representative of three separate experiments in which similar results were obtained.

[0011] FIGs. 4A-4B show the relative expression levels of various inflammatory and antiinflammatory molecules in ex vivo organ cultures transfected (10 pg / ml) with SMAD7 sense oligonucleotide (S) or SMAD7 antisense oligonucleotide (AS) for 24 hours. FIG. 4A shows the relative expression of SMAD 7 (graph y-axis) by evaluating SMAD7 RNA transcripts by real-time polymerase chain reaction. Levels were normalized to P-actin. Values indicate the mean ± SD of three experiments. Differences were analyzed using a two-tailed Student's t-test (*p<0.005). FIG. 4B shows the analysis of cell-free culture supernatants analyzed using a commercial protein array kit. Right panel Heatmap showing the protein differential expression based on densitometry scanning of array blots. Log2 (fold change) of AS vs S transfected ex vivo organ cultures.DETAILED DESCRIPTION

[0012] Pouchitis is inflammation of the lining of a pouch that is surgically created in the treatment of inflammatory bowel diseases such as ulcerative colitis. Pouchitis is the most frequent long-term complication of ileal pouch-anal anastomosis. Symptoms of pouchitis include increased bowel frequency, abdominal cramping or bloating, lower abdominal pain, or sometimes blood in the stool. Current therapies for pouchitis are limited and generally include antibiotics.

[0013] Improved therapies for treating or preventing pouchitis are needed. Described herein are methods of treating or preventing pouchitis by administering an oligonucleotide (e.g., SMAD7 antisense oligonucleotide).Definitions

[0014] The term “oligonucleotide” refers to short DNA or RNA molecules. Oligonucleotides can be composed of 2'-deoxyribonucleotides (oligodeoxyribonucleotides), which can be modified at the phosphate backbone or at the 2’ sugar position.

[0015] “Antisense oligonucleotide,” (“ASO”) as used herein, refers to a short synthetic oligonucleotide sequence complementary to the messenger RNA (mRNA) that encodes a target protein (e.g., SMAD7). Without being bound to a particular theory, antisense oligonucleotide sequences can hybridize to a complementary region in an mRNA molecule thereby producing a double-stranded hybrid that can lead to the activation of ubiquitous catalytic enzymes, such as RNase H, which degrade DNA / RNA hybrid strands thus preventing protein translation. Without being bound by theory, an antisense oligonucleotide provided herein can hybridize to its target sequence as RNA or DNA. Thus, even if a DNA sequence is provided as a target, the corresponding RNA sequence (including uracil instead of thymine) is included as an ASO. “Phosphorothioate antisense oligonucleotides” or “PS ASOs” are antisense oligonucleotides that are modified to have a phosphorothioate backbone.

[0016] As used herein, “Mothers against decapentaplegic homolog 7” ( “SMAD7,” also known as CRCS3, FLJ16482, MADH7, MADH8, MAD (mothers against decapentaplegic,Drosophila) homolog 7, MAD homolog 8, SMAD, mothers against DPP homolog 7, mothers against DPP homolog 8) means the human protein or any of the mRNA transcripts encoded by the gene identified by Entrez GenelD No. 4092 and allelic variants thereof.

[0017] As used herein, “SMAD7 antisense oligonucleotide” or “SMAD7 ASO” is understood to refer to an oligonucleotide comprising a nucleic acid sequence that is complementary to a nucleic acid sequence in an mRNA molecule transcribed from the SMAD7 gene. More specifically, such an oligonucleotide can be complementary to the nucleic acid sequence in the coding region of such an mRNA.

[0018] The term “pouchitis,” as used herein refers to a disease or disorder characterized by inflammation of the pouch. The term “pouch,” as used herein, can refer to an artificial rectum surgically created out of ileum (the last section of the small intestine) in individual that have undergone a proctocolectomy or total colectomy (removal of the colon and rectum) to manage diseases such as ulcerative colitis, indeterminate colitis, familial adenomatous polyposis, cancer, or other colitides. An individual may be identified as having pouchitis using the Pouchitis Disease Activity Index (PDAI) or modified Pouchitis Disease Activity Index (mPDAI) scoring systems. Individuals assessed with an total PDAI score of 7 or higher are classified as having pouchitis. Individuals assessed with a total mPDAI score of 5 or higher are classified as having pouchitis.

[0019] The term “acute pouchitis” refers to pouchitis where symptoms last less than or equal to four weeks and with disease responding to 2-week courses of antibiotics.

[0020] The term “chronic pouchitis” refers to pouchitis where symptoms last four weeks or more and / or despite a standard antibiotic course of treatment.

[0021] The terms “recipient,” “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and in some embodiments, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the mammal is human. None of these terms require the supervision of medical personnel.

[0022] The terms “disease,” “disorder,” and “condition” are used interchangeably herein.

[0023] As used herein, the terms “treatment,” “treating,” and the like refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of affecting a partial or complete cure for a disease and / orsymptoms of the disease. “Treatment,” as used herein, may include treatment of a disease or disorder (e.g., pouchitis) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease); (b) inhibiting the disease, / .< ., arresting its development; and (c) relieving the disease, / .< ., causing regression of the disease. Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms; or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term “treating” includes the administration of the compounds or agents of the present disclosure to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with diseases (e.g., pouchitis). The term “therapeutic effect” refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0024] As used herein, “preventing” or “prevent” describe reducing or eliminating the onset of the symptoms or complications of the disease, condition or disorder. The term “preventing,” when used in relation to a condition, such as inflammation or the recurrence of pouchitis, is art- recognized, and refers to the ability of a formulation, composition, and / or device to reduce the frequency of, or delay the onset of, signs and / or symptoms of a medical condition in a subject relative to a subject which does not receive the formulation, composition, and / or device. Insofar as the methods of the present disclosure are directed to preventing disorders, it is understood that the term “prevent” does not require that the disease state be completely thwarted.

[0025] The term “therapeutically effective amount” or “therapeutic amount” generally refers to an amount of an antibody or a drug effective to “treat” a disease or disorder in a subject or mammal. In some embodiments, a composition described herein is administered to a subject in an amount that is effective for producing some desired therapeutic effect by inhibiting a disease or disorder as described herein at a reasonable benefit / risk ratio applicable to any medical treatment. A therapeutically effective amount is an amount that achieves at least partially a desired therapeutic or prophylactic effect in an organ or tissue. The amount of an antibody or drug necessary to bring about prevention and / or therapeutic treatment of a disease or disorder is not fixed per se. In some embodiments, the amount of an antibody or drug administered varieswith the type of disease, extensiveness of the disease, and size of the mammal suffering from the disease or disorder. When used in conjunction with therapeutic methods involving administration of a therapeutic agent after the subject presents symptoms of a disease or disorder, the term “therapeutically effective” means that, after treatment, one or more signs or symptoms of the disease or disorder is ameliorated or eliminated.

[0026] As used herein, singular forms “a”, “and,” and “the” include plural referents unless the context clearly indicates otherwise.

[0027] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.

[0028] “About” a number, as used herein, refers to range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.

[0029] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as ’’comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0030] “Percent (%) identity” refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988;Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present specification will control. Although methods andmaterials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.SMAD7 Oligonucleotides

[0032] Described herein, in certain embodiments, are methods for treating or preventing pouchitis by administering a therapeutically effective amount of a SMAD7 oligonucleotide (e.g., a SMAD7 antisense oligonucleotide (ASO)) or a pharmaceutical formulation comprising the SMAD7 oligonucleotide. In some embodiments, the SMAD7 oligonucleotide is a SMAD7 antisense oligonucleotide (ASO).

[0033] In some embodiments, the SMAD7 ASO comprises DNA. In some embodiments, the SMAD7 ASO comprises RNA.

[0034] In some embodiments, the SMAD7 ASO has a sequence at least 90% identical to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'), in which X is 5-methyl 2’- deoxycytidine. In some embodiments, the SMAD7 ASO has a sequence at least 95% identical to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'), in which X is 5-methyl 2’- deoxycytidine. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO: 1 (5'-GTXGCCCCTTCTCCCXGCAGC-3'), in which X is 5-methyl 2’ -deoxy cytidine.

[0035] In some embodiments, the SMAD7 ASO has a sequence at least 90% identical to SEQ ID NO: 2 (5'-GTXGCCCCTTCTCTCXGCAGC-3') in which X is 5-methyl 2’- deoxycytidine. In some embodiments, the SMAD7 ASO has a sequence at least 95% identical to SEQ ID NO: 2. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO: 2.

[0036] In some embodiments, the SMAD7 ASO has a sequence at least 90% identical to SEQ ID NO: 3 (5'-GTXYCCCCTTCTCCCXYCAG-3'), in which X is a nucleotide including a nitrogenous base of cytosine, 5-methylcytosine, or a 2’-(9-methylcytosine and Y is a nucleotide including a nitrogenous base of guanine, 5-methylguanine, or a 2’-(9-methylguanine, provided that at least one of X and Y includes a methylated nitrogenous base. In some embodiments, the SMAD7 ASO has a sequence at least 95% identical to SEQ ID NO: 3. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO: 3.

[0037] In some embodiments, the SMAD7 ASO has a sequence at least 90% identical to SEQ ID NO: 4 (5'-GTXGCCCCTTCTCCCXGCAG-3'), in which X is 5-methyl 2’- deoxycytidine. In some embodiments, the SMAD7 ASO has a sequence at least 95% identical to SEQ ID NO: 4. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO: 4.

[0038] In some embodiments, the SMAD7 ASO has a sequence at least 90% identical to SEQ ID NO: 5 (5'-GTC*GCCCCTTCTCCCC*YCAGC-3’), in which C* is 5-methyl-2’- deoxycytidine and Y is a nucleotide including a nitrogenous base of guanine, 5-methylguanine, or a 2’-O-methylguanine. In some embodiments, the SMAD7 ASO has a sequence at least 95% identical to SEQ ID NO: 5. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO: 5.

[0039] In some embodiments, the SMAD7 ASO has a sequence at least 90% identical to SEQ ID NO: 6 (5'-GTC*GCCCCTTCTCTCC*YCAGC -3’), in which C* is 5-methyl-2’- deoxycytidine and Y is a nucleotide including a nitrogenous base of guanine, 5-methylguanine, or a 2’-O-methylguanine. In some embodiments, the SMAD7 ASO has a sequence at least 95% identical to SEQ ID NO: 6. In some embodiments, the SMAD7 ASO has a sequence according to SEQ ID NO: 6.

[0040] In some embodiments, the SMAD7 ASO includes naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages as well as non-naturally occurring portions. In some embodiments, the SMAD7 ASO of the present disclosure, for example, the SMAD7 ASO of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, include nucleotides including deoxycytidine and / or 5-methyl 2’- deoxy cytidine, including, but not limited to, 5-methyl-2’ -deoxy cytidine 5 ’-monophosphate and 5-methyl-2’ -deoxy cytidine 5 ’ -monophosphorothioate.

[0041] In some embodiments, the SMAD7 ASO (e.g., the SMAD7 ASO of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6) comprises one or more methylated cytosine. In some embodiments, any cytosine of the SMAD7 ASO is methylated. In some embodiments, at least one cytosine of the SMAD7 ASO is replaced with 5- m ethylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO: 1 comprises one or more methylated cytosine. In some embodiments, any cytosine of the SMAD7 ASO of SEQ ID NO: 1 is methylated. In some embodiments, at least one cytosine of the SMAD7 ASO of SEQ ID NO: 1 is replaced with 5-methylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO: 2 comprises one or more methylated cytosine. In some embodiments, any cytosine of the SMAD7 ASO of SEQ ID NO: 2 is methylated. In some embodiments, at least one cytosine of the SMAD7 ASO of SEQ ID NO: 2 is replaced with 5-methylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO: 3 comprises one or more methylated cytosine. In some embodiments, any cytosine of the SMAD7 ASO of SEQ ID NO: 3 is methylated. In some embodiments, at least one cytosine of the SMAD7 ASO of SEQ ID NO: 3 is replaced with 5- m ethylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO: 4 comprises one ormore methylated cytosine. In some embodiments, any cytosine of the SMAD7 ASO of SEQ ID NO: 4 is methylated. In some embodiments, at least one cytosine of the SMAD7 ASO of SEQ ID NO: 4 is replaced with 5-methylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO: 5 comprises one or more methylated cytosine. In some embodiments, any cytosine of the SMAD7 ASO of SEQ ID NO: 5 is methylated. In some embodiments, at least one cytosine of the SMAD7 ASO of SEQ ID NO: 5 is replaced with 5-methylcytidine. In some embodiments, the SMAD7 ASO of SEQ ID NO: 6 comprises one or more methylated cytosine. In some embodiments, any cytosine of the SMAD7 ASO of SEQ ID NO: 6 is methylated. In some embodiments, at least one cytosine of the SMAD7 ASO of SEQ ID NO: 6 is replaced with 5- methylcytidine.

[0042] In some embodiments, the SMAD7 ASO is chemically modified. In some embodiments, the SMAD7 ASO comprises one or more inter-nucleoside linkers modified from the natural phosphodiester. In some embodiments, all of the inter-nucleoside linkers of the SMAD7 ASO, or contiguous nucleotide sequence thereof, are modified. For example, in some embodiments, the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.

[0043] In some embodiments, the SMAD7 ASO comprises modifications to a ribose sugar or nucleobase. In some embodiments, the SMAD7 ASO comprises one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the SMAD7 ASO comprises one or more LNAs. In some embodiments, the sugar-modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids. In some embodiments, the modified nucleosides comprise nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example peptide nucleic acids (PNA) or morpholino nucleic acids.

[0044] In some embodiments, the SMAD7 ASO comprises one or more modified sugars. In some embodiments, the sugar modifications comprise modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’ -OH group naturally found in DNA and RNA nucleosides. In some embodiments, substituents are introduced at the 2’, 3’, 4’, or 5’ positions, or combinations thereof. In some embodiments,nucleosides with modified sugar moieties comprise 2’ modified nucleosides, e.g., 2’ substituted nucleosides. A 2’ sugar modified nucleoside, in some embodiments, is a nucleoside that has a substituent other than -H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradical, and comprises 2’ substituted nucleosides and LNA (2’ -4’ biradical bridged) nucleosides. Examples of 2 ’-substituted modified nucleosides comprise, but are not limited to, 2’-O-alkyl, 2’-O-methyl, 2’-alkoxy, 2’ -O-m ethoxy ethyl (MOE), 2’-amino, 2’-Fluoro, and 2’-F- ANA nucleosides. In some embodiments, the modification in the ribose group comprises a modification at the 2’ position of the ribose group. In some embodiments, the modification at the 2’ position of the ribose group is selected from the group consisting of 2’-O-methyl, 2’ -fluoro, 2’ -deoxy, and 2 ’-O-(2 -methoxy ethyl). In some embodiments, the SMAD7 ASO comprises one or more nucleosides with a 2’-O-methyl (2’-0Me). In some embodiments, the SMAD7 ASO comprises one or more nucleosides with a 2’ -O-m ethoxy ethyl (MOE). In some embodiments, the SMAD7 ASO comprises one or more nucleosides with a 2’-O-ethyl (cEt).

[0045] In some embodiments, the SMAD7 ASO comprises 2'-deoxyribonucleotides (oligodeoxyribonucleotides). In some embodiments, the SMAD7 ASO comprises 2'- deoxyribonucleotides (oligodeoxyribonucleotides), which are modified at the phosphate backbone or at the 2’ sugar position. In some embodiments, the SMAD7 ASO is a phosphorothioate (PS) oligonucleotide wherein one of the non-bridging oxygen atoms in the phosphate backbone is replaced with a sulfur atom. In some embodiments, the SMAD7 ASO has at least one intemucleoside linkage that is an O,O-linked phosphorothioate linkage (i.e., a phosphorothioate linkage). In some embodiments, the SMAD7 ASO has all internucleoside linkages being O,O-linked phosphorothioate linkages (i.e., a phosphorothioate linkage).

[0046] Phosphorothioate (PS) linkages in oligonucleotides are chiral, with right-handed (Rp) and left-handed (Sp) isomers termed diastereomers. These two PS diastereomers can differentially impact the physicochemical and biological properties of oligonucleotides, such as nuclease sensitivity, thermodynamic stability of double-stranded structures, and interaction with various enzymes involved in the antisense and RNAi pathways. In some embodiments, the SMAD7 ASO has one or more diastereomerically pure internucleoside linkages with respect to the chiral linkage phosphorus. In some embodiments, the SMAD7 ASOs are prepared by using stereoselective oligonucleotide synthesis, to form one or more pre-designed diastereomerically pure internucleoside linkages with respect to the chiral linkage phosphorus.

[0047] In some embodiments, a pharmaceutically acceptable salt of the SMAD7 ASO is provided herein. In some embodiments, a sodium salt of the SMAD7 ASO of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, thatoptionally includes 1 to 20 ( ,0-linked phosphorothioate internucleoside linkages (i.e. phosphorothioate bonds) is provided herein. In some embodiments, the SMAD7 ASO is an antisense oligonucleotide comprising the free acid form, the salt form, or the anionic form without a counterion of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, wherein each of the 20 internucleoside linkages is an O,O-linked phosphorothioate linkage. In some embodiments, the phosphorothioate backbone of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 are fully or partially protonated to form an acidic form of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. Contemplated salts of oligonucleotides include those that are fully neutralized, e.g., each phosphorothioate linkage is associated with an ion such as Na+. In some embodiments, the salt of the SMAD7 ASO of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 is only partially neutralized, e.g., less than all phosphorothioate linkages are associated with an ion (e.g., less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, or less than 1% are neutralized).

[0048] In some embodiments, a SMAD7 ASO of the disclosure (e.g., SEQ ID NO: 1) or a pharmaceutical formulation disclosed herein (e.g, a composition comprising of SEQ ID NO: 1) down-regulates SMAD7 expression. In some embodiments, the SMAD7 expression is SMAD7 protein and / or mRNA expression. In some embodiments, the SMAD7 protein expression is down-regulated in a cell by more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 55%, more than about 60%, or more than about 65% compared to an untreated cell. In some embodiments, the SMAD7 protein expression is down-regulated in a cell by more than about 40% compared to an untreated cell.

[0049] In some embodiments, a SMAD7 ASO possesses the inherent functional property of targeting the SMAD7 gene, its RNA or protein products, or another molecular entity whose activity or expression impinges upon the activity or expression of SMAD7 or its products either exclusively or with a high degree of specificity. In some embodiments, a SMAD7 ASO reduces the expression of SMAD7 when introduced into a cell (e.g, an epithelial cell, a lamina propria mononuclear cell, or an immune cell, such as a PBMC, dendritic cell, or B-cell). In some embodiments, a SMAD7 ASO reduces expression of an mRNA transcribed from the gene. In some embodiments, a SMAD7 ASO reduces expression of a protein encoded by the gene. Insome embodiments, a SMAD7 ASO reduces secretion of a protein encoded by the gene from the cell into which the SMAD7 ASO was introduced.

[0050] In some embodiments, a SMAD7 ASO modulates inflammation. In some embodiments, a SMAD7 ASO modulates expression or activity of an inflammatory gene. In some embodiments, a SMAD7 ASO reduces expression or activity of an inflammatory gene. In some embodiments, a SMAD7 ASO modulates expression or activity of ENA-78, G-CSF, GM- CSF, GRO, GRO-alpha, 1-309, IL-lalpha, IL-lbeta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 10, IL-12, IL-13, IL-15, IFN-gamma, MCP-1, MCP-2, MCP-3, M-CSF, MDC, MIG, MIP-1- delta, RANTES, SCF, SDF-1, TARC, TGF-beta 1, TNF-alpha, TNF-beta, EGF, IGF-1, Angiogenin, Oncostatin M, TPO VEGF, PDGF-BB, Leptin, or combinations thereof. In some embodiments, a SMAD7 ASO reduces expression or activity of ENA-78, G-CSF, GM-CSF, GRO-alpha, IL-lbeta, IL-10, IL-12, IL-13, IL-15, IFN-gamma, MIG, MIP-l-delta, RANTES, SCF, SDF-1, TARC, TGF-beta 1, TNF-alpha, angiogenin, Oncostatin M, TPO, VEGF, PDGF- BB, Leptin, or combinations thereof. In some embodiments, a SMAD7 ASO increases expression or activity of GRO, 1-309, IL-lalpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, MCP-1, MCP-2, MCP-3, M-CSF, MDC, TNF-beta, EGF, IGF-1, or combinations thereof.Methods of Treating Pouchitis

[0051] Described herein, in some embodiments, are methods of treating pouchitis in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a SMAD7 antisense oligonucleotide (ASO). Further described herein, in some embodiments, are methods of treating pouchitis in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5’- GTXGCCCCTTCTCCCXGCAGC-3’). Further described herein, in some embodiments, are methods of treating pouchitis in an individual in need thereof, comprising administering to the individual a pharmaceutical composition comprising: a) a SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5’-GTXGCCCCTTCTCCCXGCAGC- 3’); and b) a pharmaceutically acceptable carrier.

[0052] In some embodiments, the individual is determined as having pouchitis using a pouchitis disease activity index (PDAI) score of at least 7. In some embodiments, the individual is determined as having pouchitis using a modified pouchitis disease activity index (mPDAI) score of at least 5.

[0053] In some embodiments, the individual has acute pouchitis. In some embodiments, the individual has chronic pouchitis.

[0054] Further described herein, in some embodiments, are methods for predicting pouchitis in an individual in need thereof, comprising determining a level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level is predictive of pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis. Further described herein, in some embodiments, are methods of identifying an individual at risk of pouchitis, comprising: determining the level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level identifies the individual as being at risk for pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

[0055] In some embodiments, if the SMAD7 level is elevated relative to the known control level, then the individual is administered a pharmaceutical composition comprising a SMAD7 ASO. In some embodiments, if the SMAD7 level is not elevated relative to the known control level, then the level of SMAD7 in a second sample from the individual is determined. In some embodiments, the second sample is collected immediately after, about 1 hour after, about 3 hours after, about 6 hours after, about 12 hours after, about 1 day after, about 3 days after, about 1 week after, about 2 weeks after, about 1 month after, about 2 months after, about 3 months after, about 4 months after, about 5 months after, about 6 months after, about 7 months after, about 8 months after, about 9 months after, about 10 months after, about 11 months after, or about 12 months after the first sample.

[0056] Further described herein, in some embodiments, are methods for treating pouchitis in an individual in need thereof, comprising determining a level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level is predictive of pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

[0057] In some embodiments, the individual having pouchitis has at least about a 5%, about a 10%, about a 20%, about a 30%, about a 40% or even about a 50% or more reduction in the pouchitis disease activity index (PDAI) score or the modified pouchitis disease activity index (mPDAI) score after administering a SMAD7 antisense oligonucleotide of SEQ ID NO: 1, 2, 3,4, 5, or 6 or a pharmaceutically acceptable salt thereof, after e.g., 1 day, 2 days, 1 week, 1 month, or 6 months, or more. In some embodiments, administering a SMAD7 antisense oligonucleotide is on, e.g., at least a daily basis. In some embodiments, the delay of clinical manifestation of pouchitis in an individual as a consequence of administering a SMAD7 antisense oligonucleotide is at least, e.g., 6 months, 1 year, 18 months or even 2 years or more as compared to an individual who is not administered a SMAD7 ASO.Pharmaceutical Formulations

[0058] Described herein, in some embodiments, are pharmaceutical formulations containing an oligonucleotide, such as a SMAD7 ASO described herein, that are presented in a dosage unit form and are prepared by any suitable method. A pharmaceutical formulation should be formulated to be compatible with its intended route of administration. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0059] In some embodiments, the pharmaceutical formulation (e.g. an intrapouch formulation) comprises an aqueous injectable composition comprising an antisense oligonucleotide described herein (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof). For example, an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation and liquid solution or suspension are blended together. In some embodiments, solid forms of the pharmaceutical formulation suitable for using to prepare solutions or suspensions upon addition of a liquid prior to injection are prepared; and the preparations are emulsified. In some embodiments, the pharmaceutical formulations suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the pharmaceutical formulations suitable for injectable use include sesame oil, peanut oil or aqueous propylene glycol. In some embodiments, the pharmaceutical formulations suitable for injectable use include sterile powders.

[0060] In some embodiments, an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation (e.g., an intrapouch formulation) comprises an aqueous injectable composition that includes a surfactant (e.g., hydroxypropylcellulose).

[0061] In some embodiments, an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation (e.g., intrapouch formulation) comprises dispersions prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.

[0062] In some embodiments, the pharmaceutical formulation of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a rectal pharmaceutical formulation (e.g., suppositories, enemas, gels, and foams). In some embodiments, the rectal pharmaceutical formulation comprises an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof).

[0063] In some embodiments, the pharmaceutical formulation (e.g., a rectal pharmaceutical formulation) of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a suppository. In some embodiments, the suppository comprises a lipophilic base excipient (e.g., cocoa butter, coconut oil, hydrogenated vegetable oils, hard fats). In some embodiments, the suppository comprises a hydrophilic base excipient (e.g., glycerinated gelatin and polyethylene glycols). In some embodiments, the suppository includes surfactants (e.g., polysorbate 80, Tween 20, Span 60).

[0064] In some embodiments, the pharmaceutical formulation (e.g., a rectal pharmaceutical formulation) of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is an enema. In some embodiments, the enema is a solution. In some embodiments, the enema is a suspension. In some embodiments, the enema is an emulsion.

[0065] In some embodiments, the pharmaceutical formulation (e.g., a rectal pharmaceutical formulation) of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a gel. In some embodiments, the gel includes cosolvents (e.g., glycerin and propylene glycol). In some embodiments, the gel includes electrolytes.

[0066] In some embodiments, the gels have gel-like consistencies but are sufficiently flowable so as to be capable of local or regional administration through a catheter, needle, syringe, or other comparable means of local or regional administration (e.g., to the rectum).

[0067] In some embodiments, the concentration of a thickener used in a gel formulation is in an amount or concentration suitable to achieve a desired thickness or viscosity, e.g., from about 0.05% to about 10% by weight. In some embodiments, the concentration of the thickener used in such gel formulations ranges from about 0.05% to about 8% by weight. In some embodiments, the concentration of the thickener used in such gel formulations ranges from about 0.05% to about 7% by weight. In some embodiments, the concentration of the thickener used in such gel formulations ranges from about 0.05% to about 6% by weight. In some embodiments, the concentration of the thickener used in such gel formulations ranges from about 0.05% to about 5% by weight. In some embodiments, the concentration of the thickener used in such gelformulations ranges from about 0.05% to about 4% by weight. In some embodiments, the concentration of the thickener used in such gel formulations ranges from about 0.05% to about 3% by weight. In some embodiments, the concentration of the thickener used in such gel formulations ranges from about 0.05% to about 2% by weight. In some embodiments, the concentration of the thickener used in such gel formulations ranges from about 0.05% to about 1% by weight. In some embodiments the gel formulation includes methyl cellulose having a concentration from about 0.05% to about 2%, while in other embodiments the gel formulation includes methyl cellulose having a concentration of about 1%.

[0068] In some embodiments, the pharmaceutical formulation (e.g., a rectal pharmaceutical formulation) of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a foam. In some embodiments, the foam formulation comprises a surfactant / emulsifier with foaming properties and a propellant (e.g., a propellant gas). In some embodiments, the foam formulation also comprises one or more of the following: a suspending / solubilizing agent, a thickener, a preservative, a chelating agent, a buffer, an antioxidant, a tonicity modifiers, and a spreading agent. In some embodiments, surfactants / emulsifiers include, by way of non-limiting example, non-ionic surfactants, anionic surfactants, cationic surfactants, and combinations thereof. In some embodiments, foam formulations are filled in pressurized containers prior to administration (e.g., rectal administration). In some embodiments the pressurized container is a can. In some embodiments, propellants used herein include, by way of non-limiting example, hydrocarbons (such as isobutane, N-butane or propane), fluorocarbons (e.g. dichlorodifluoromethane and dichlorotetrafluoroethane), chlorofluorocarbons, dimethyl ether, hydrofluorocarbons, compressed gases, freon (such as freon 12, freon 114), hydrochlorofluorocarbons, hydrofluorocarbons or mixtures thereof.

[0069] In some embodiments, the maximum amount of propellant used is determined by its miscibility with other components in the composition to form a mixture, such as a homogeneous mixture. In some embodiments, the minimal level of propellant used in the composition is determined by the desired foam characteristics, and its ability to substantially or completely evacuate the container.

[0070] In some embodiments, the propellant concentration used in such foam formulations is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 50%, 55% to about 60% (w / w).

[0071] In some embodiments, foams are formed upon administration (e.g., rectal administration), wherein the dispensing valve of the can allows rapid expansion of thepropellant, triggering the foaming action of the surfactant and resulting foam forms within the rectum and colon. In some embodiments, the foams used for administration of the compositions described herein are formed within the dispensing container prior to administration.

[0072] In some embodiments, the pharmaceutical formulation of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is an oral pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises an intra-granular phase comprising an antisense oligonucleotide described herein (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable filler. For example, an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) and a filler are blended together, with optionally other excipients, and formed into granules. In some embodiments, the intragranular phase is formed using wet granulation, e.g., a liquid (e.g., water) is added to the blended antisense compound and filler, and then the combination is dried, milled and / or sieved to produce granules. Other processes in the art may be used to achieve an intragranular phase.

[0073] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises an extra-granular phase, which includes one or more pharmaceutically acceptable excipients, and which are blended with the intragranular phase to form a disclosed formulation.

[0074] In some embodiments, an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation includes 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, hydroxypropylmethyl cellulose, partially pregelatinized starch, calcium carbonate, and others including combinations thereof.

[0075] In some embodiments, an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation (e.g., an oral pharmaceutical formulation) includes an intragranular phase and / or an extra-granular phase that includes a binder to hold the ingredients of the pharmaceutical formulation together. Exemplary binders include, but are not limited to, the following: starches, sugars, cellulose or modified cellulose such as hydroxypropyl cellulose, lactose, pregelatinized maize starch, polyvinyl pyrrolidone, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, low substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, sugar alcohols and others including combinations thereof.

[0076] In some embodiments, pharmaceutical formulations (e.g., an oral pharmaceutical formulation) described herein that include an intragranular phase and / or an extra-granular phase, further include a disintegrant such as but not limited to, starch, cellulose, crosslinked polyvinyl pyrrolidone, sodium starch glycolate, sodium carboxymethyl cellulose, alginates, corn starch, croscarmellose sodium, crosslinked carboxymethyl cellulose, low substituted hydroxypropyl cellulose, acacia, and others including combinations thereof. For example, an intragranular phase and / or an extra-granular phase includes a disintegrant.

[0077] In some embodiments, an oligonucleotide described herein (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation includes an intra-granular phase including a disclosed antisense oligonucleotide and excipients chosen from: mannitol, microcrystalline cellulose, hydroxypropylmethyl cellulose, and sodium starch glycolate or combinations thereof, and an extra-granular phase including one or more of: microcrystalline cellulose, sodium starch glycolate, and magnesium stearate or mixtures thereof.

[0078] In some embodiments, an oligonucleotide described herein (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) formulation includes a lubricant, e.g., an extra-granular phase contains a lubricant. Lubricants include but are not limited to talc, silica, fats, stearin, magnesium stearate, calcium phosphate, silicone dioxide, calcium silicate, calcium phosphate, colloidal silicon dioxide, metallic 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.

[0079] In some embodiments, pharmaceutical formulations (e.g., an oral pharmaceutical formulation) of the present disclosure include an enteric coating. Generally, enteric coatings create a barrier for the oral medication that controls the location at which the drug is absorbed along the digestive tract. In some embodiments, enteric coatings include a polymer that disintegrates at different rates according to pH. In some embodiments, enteric coatings include, for example, cellulose acetate phthalate, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxylpropylmethyl cellulose phthalate, methyl methacrylate-methacrylic acid copolymers, ethylacrylate-methacrylic acid copolymers, methacrylic acid copolymer type C, polyvinyl acetate-phthalate, and cellulose acetate phthalate.

[0080] In some embodiments, the enteric coating includes an anionic, cationic, or neutral copolymer based on methacrylic acid, methacrylic / acrylic esters or their derivatives. In certain embodiments, the enteric coating includes an ethylacrylate-methacrylic acid copolymer. Commercially available enteric coatings include Opadry® AMB, ethylacrylate-methacrylic acid copolymers (e.g., Acryl-EZE®), dimethylaminoethyl methacrylate-butyl methacrylate- methylmethacrylate copolymer (2:1:1), or poly(methacrylic acid-co-methyl-methacrylate) 1:1 and poly(methacrylic acid-co-methyl-methacrylate) 1:2 copolymers (e.g., Eudragit®) grades. In some embodiments, the enteric coating makes up about 5% to about 10%, about 5% to about 20%, about 8 to about 15%, about 8% to about 18%, about 10% to about 12%, or about 12% to about 16%, of a contemplated pharmaceutical formulation (e.g., a tablet) by weight. In some embodiments, the enteric coating comprises an ethylacrylate-methacrylic acid copolymer that makes up about 8 to about 15% of the pharmaceutical formulation (e.g., tablet) by weight. In some embodiments, the enteric coating comprises an ethylacrylate-methacrylic acid copolymer that makes up about 12% of the pharmaceutical formulation (e.g., tablet) by weight. In some embodiments, the enteric coating comprises an ethylacrylate-methacrylic acid copolymer that makes up about 10% of the pharmaceutical formulation (e.g., tablet) by weight.

[0081] In some embodiments, the pharmaceutical formulation (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) comprises about 0.5% to about 70%, e.g., about 0.5% to about 30%, about 1% to about 20%, or about 5% to about 30% by weight of an antisense oligonucleotide or a pharmaceutically acceptable salt thereof. Such a pharmaceutical formulation includes, in some embodiments, about 0.5% to about 60% by weight of mannitol, e.g., about 20% to about 50% by weight mannitol, e.g., about 40% or about 28% by weight mannitol; and / or about 20% to about 40% by weight of microcrystalline cellulose, or about 10% to about 30% by weight of microcrystalline cellulose. In some embodiments, the pharmaceutical formulation includes an intragranular phase that includes about 30% to about 60%, about 45% to about 65% by weight, or alternatively, about 5% to about 10% by weight of a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, about 30% to about 50%, or alternatively, about 5% to about 15% by weight mannitol, about 5% to about 15% microcrystalline cellulose, about 0% to about 4%, or about 1% to about 7% hydroxypropyl methylcellulose, and about 0% to about 4%, e.g., about 2% to about 4% sodium starch glycolate by weight. In some embodiments, the pharmaceutical formulation includes about 5% to about 10% or about 10% to about 30% by weight of an oligonucleotide of SEQ ID NO: 1, about 20% to about 50% by weight mannitol, about 10% to about 30% by weight microcrystalline cellulose, about 0.5% to about 10% by weight hydroxypropyl methylcellulose, and about 0.5% to about 10% by weight sodium starch glycolate by weight. In some embodiments, the pharmaceutical formulation is in a form of a tablet.

[0082] Exemplary pharmaceutical formulations include dosage forms that include or consist essentially of about 10 mg to about 500 mg or about 20 mg to about 500 mg of aSMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, for example, tablets that include about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg of a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, are contemplated herein. In some embodiments, pharmaceutical formulations described herein are formulated as tablets comprising about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg of an oligonucleotide of SEQ ID NO: 1. In some embodiments, pharmaceutical formulations described herein are formulated as tablets comprising about 40 mg of an oligonucleotide of SEQ ID NO: 1. In some embodiments, pharmaceutical formulations described herein are formulated as tablets comprising about 160 mg of an oligonucleotide of SEQ ID NO: 1.

[0083] In certain embodiments, pharmaceutical formulations described herein are formulated as tablets for oral use, including: about 0.5% to about 30% by weight of the oligonucleotide; about 20% to about 50% by weight mannitol; and about 10% to about 30% by weight microcrystalline cellulose.

[0084] In some embodiments, a pharmaceutically acceptable tablet for oral administration is provided that includes about 5% to about 30% by weight of an oligonucleotide of SEQ ID NO: 1, 20% to about 50% by weight of mannitol, about 10% to about 30% by weight of microcrystalline cellulose, about 0.5% to about 10% by weight of hydroxypropyl methylcellulose, and about 0.5% to about 10% by weight of sodium starch glycolate, about 0.5% to about 10% by weight of magnesium stearate, about 0.5% to about 10% by weight Opadry® AMB, and about 5% to about 20% by weight Acryl-EZE®. In some embodiments, a pharmaceutically acceptable tablet for oral administration is provided that includes about 8.5% by weight of an oligonucleotide of SEQ ID NO: 1, 40% by weight of mannitol, about 25% by weight of microcrystalline cellulose, about 5% by weight ofhydroxypropyl methylcellulose, about 4% by weight of sodium starch glycolate, about 0.4% by weight of magnesium stearate, about 4% by weight Opadry® AMB, and about 10% to about 15% by weight Acryl-EZE®. In some embodiments, a pharmaceutically acceptable tablet for oral administration is provided that includes about 23% by weight of an oligonucleotide of SEQ ID NO: 1, 28% by weight of mannitol, about 25% by weight of microcrystalline cellulose, about 5% by weight of hydroxypropyl methylcellulose, about 4% by weight of sodium starch glycolate, about 0.4% by weight of magnesium stearate, about 4% by weight Opadry® AMB, and about 7% to about 12% by weight Acryl-EZE®. In some embodiments, a pharmaceutically acceptable tablet for oral administration is formulated according to Table 1 and Table 2.Table 1 Table 2

[0085] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises an intra-granular phase that includes about 50% by weight of a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, about 11.5% by weight mannitol, about 10% by weight microcrystalline cellulose, about 3% by weight hydroxypropyl methylcellulose, and about 2.5% by weight sodium starch glycolate; and an extra-granular phase includes about 20% by weight microcrystalline cellulose, about 2.5% by weight sodium starch glycolate, and about 0.5% by weight magnesium stearate. In some embodiments, the pharmaceutical formulation further comprises an enteric coating.

[0086] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises or consists essentially of: an intra-granular phase thatincludes about 5% to about 10%, e.g., about 8% by weight of a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof (e.g., a sodium salt), 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; and an extra-granular phase that includes about 17% by weight microcrystalline cellulose, about 2% by weight sodium starch glycolate, and about 0.4% by weight magnesium stearate.

[0087] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises an enteric coating comprising about 13%, about 14%, about 15%, about 16%, or about 17% by weight of an enteric coating, e.g., ethyl aery late- methacrylic acid copolymers (e.g., AcrylEZE®).

[0088] For example, the pharmaceutical formulation is in the form of a pharmaceutically acceptable tablet for oral use including an intra-granular phase and extra-granular phase, in which for example, the intra-granular phase includes about 5% to about 10%, by weight (for example about 8% by weight) of an oligonucleotide represented by SEQ ID NO: 1, 2, 3, 4, 5, or 6, 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, and for example, the extra-granular phase includes about 17% by weight microcrystalline cellulose, about 2% by weight sodium starch glycolate, and about 0.4% by weight magnesium stearate, where the tablet further includes an enteric coating.

[0089] Contemplated formulations, in some embodiments, when orally administered to the individual result in minimal plasma concentration of the SMAD7 ASO in the individual. In some embodiments, contemplated formulations, when orally administered to an individual, topically deliver to the pouch of an individual, e.g., to an affected or diseased pouch of an individual. In some embodiments, formulations suitable for oral administration are in the form of capsules, cachets, pills, tablets, lozenges (using, e.g., a flavored basis such as sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of a subject composition thereof as an active ingredient. In some embodiments, formulations are administered as a bolus, electuary, or paste.

[0090] In some embodiments, pharmaceutical formulations described herein comprise an intra-granular phase, wherein the intra-granular phase includes an antisense oligonucleotide such as a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable saltthereof e.g., a sodium salt), and a pharmaceutically acceptable filler. In some embodiments, pharmaceutical formulations described herein comprise an extra-granular phase comprising pharmaceutically acceptable excipient such as a disintegrant. In some embodiments, the extra- granular phase comprises components including microcrystalline cellulose, magnesium stearate, and mixtures thereof. In some embodiments, the pharmaceutical formulation further comprises an enteric coating of about 12% to 16% by weight of the pharmaceutical formulation.

[0091] In some embodiments, pharmaceutical formulations (e.g., an oral pharmaceutical formulation) described herein comprise about 0.5% to about 10% by weight of an antisense oligonucleotide, e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, about 30% to 50% by weight mannitol, about 10% to 30% by weight microcrystalline cellulose, and an enteric coating including an ethylacrylate-methacrylic acid copolymer.

[0092] In some embodiments, pharmaceutical formulations (e.g., an oral pharmaceutical formulation) described herein comprise an intra-granular phase, including about 5% to about 10% by weight of an antisense oligonucleotide, e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, about 40% by weight mannitol, about 8% by weight microcrystalline cellulose, about 5% by weight hydropropylmethyl cellulose, and about 2% by weight sodium starch glycolate; an extra-granular phase including about 17% by weight microcrystalline cellulose, about 2% by weight sodium starch glycolate, about 0.4% by weight magnesium stearate; and an enteric coating over the tablet including an ethylacrylatemethacrylic acid copolymer.

[0093] In some embodiments, the pharmaceutical formulation (e.g., an oral pharmaceutical formulation) comprises an enteric coating including about 13%, about 15%, about 16%, about 17% or about 18% by weight, e.g., AcyrlEZE® (see, e.g., PCT Publication No.WO20 10 / 054826).

[0094] The rate at which point the coating dissolves and the active ingredient is released is its dissolution rate. In some embodiments, the pharmaceutical formulation (e.g., in tablet form) has a dissolution profile, e.g., when tested in a USP / EP Type 2 apparatus (paddle) at 100 rpm and 37 °C in a phosphate buffer with a pH of 7.2, of about 50% to about 100% of the oligonucleotide releasing after about 120 minutes to about 240 minutes, for example after 180 minutes. In some embodiments, the pharmaceutical formulation (e.g., in tablet form) has a dissolution profile, e.g., when tested in a USP / EP Type 2 apparatus (paddle) at 100 rpm and 37 °C in diluted HC1 with a pH of 1.0, where substantially none of the oligonucleotide is released after 120 minutes. In some embodiments, the pharmaceutical formulation (e.g., intablet form) has a dissolution profile, e.g. when tested in USP / EP Type 2 apparatus (paddle) at 100 rpm and 37 °C in a phosphate buffer with a pH of 6.6, of about 10% to about 30%, or not more than about 50%, of the oligonucleotide releasing after 30 minutes.

[0095] In some embodiments, pharmaceutical formulations described herein when administered to the individual results in minimal plasma concentration of the oligonucleotide in the individual. In some embodiments, disclosed formulations, when orally administered to an individual, topically deliver to the colon or rectum of an individual, e.g., to an affected or diseased site of an individual.

[0096] In some embodiments, pharmaceutical formulations described herein are sterile. In some embodiments, sterilization is accomplished, for example, by filtration through sterile filtration membranes. In some embodiments, where the composition is lyophilized, filter sterilization is conducted prior to or following lyophilization and reconstitution.

[0097] In some embodiments, the pharmaceutical formulations comprising of SEQ ID NO: 1 comprise greater than 50 millimoles, greater than 100 millimoles, greater than 200 millimoles, greater than 300 millimoles, greater than 400 millimoles, greater than 500 millimoles, greater than 600 millimoles, greater than 700 millimoles, greater than 800 millimoles, greater than 900 millimoles, greater than 1 mole, greater than 1.5 moles, greater than 2 moles, greater than 2.5 moles, greater than 3 moles, greater than 4 moles, or greater than 5 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulations comprising of SEQ ID NO: 1 comprise greater than 2 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises less than 5 moles, less than 4 moles, less than 3 moles, less than 2.5 moles, less than 2 moles, less than 1.5 moles, less than 1 moles, less than 900 millimoles, less than 850 millimoles, less than 800 millimoles, less than 750 millimoles, less than 700 millimoles, less than 650 millimoles, less than 600 millimoles, less than 550 millimoles, less than 500 millimoles, less than 450 millimoles, less than 400 millimoles, less than 350 millimoles, less than 300 millimoles, less than 250 millimoles, less than 200 millimoles, less than 150 millimoles, or less than 100 millimoles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises greater than 100 millimoles to about 5 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises greater than 100 millimoles to about 2 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises greater than 300 millimoles to about 5 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises greater than 300 millimoles to about 2 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises greater than 900 millimoles to about 5 moles of SEQ ID NO: 1. In some embodiments, the pharmaceuticalformulation comprises greater than 900 millimoles to about 2 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises greater than 300 millimoles to about 900 millimoles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises greater than 300 millimoles to about 5 moles, greater than 300 millimoles to about 4 moles, greater than 300 millimoles to about 3 moles, greater than 300 millimoles to about 2.5 moles, greater than 300 millimoles to about 2 moles, greater than 300 millimoles to about 1.5 moles, greater than 300 millimoles to about 1 mole, greater than 300 millimoles to about 900 millimoles, greater than 300 millimoles to about 800 millimoles, greater than 300 millimoles to about 700 millimoles, greater than 300 millimoles to about 600 millimoles, greater than 300 millimoles to about 500 millimoles, greater than 300 millimoles to about 400 millimoles, greater than 400 millimoles to about 5 moles, greater than 400 millimoles to about 4 moles, greater than 400 millimoles to about 3 moles, greater than 400 millimoles to about 2.5 moles, greater than 400 millimoles to about 2 moles, greater than 400 millimoles to about 1.5 moles, greater than 400 millimoles to about 1 mole, greater than 400 millimoles to about 900 millimoles, greater than 400 millimoles to about 800 millimoles, greater than 400 millimoles to about 700 millimoles, greater than 400 millimoles to about 600 millimoles, greater than 400 millimoles to about 500 millimoles, greater than 500 millimoles to about 5 moles, greater than 500 millimoles to about 4 moles, greater than 500 millimoles to about 3 moles, greater than 500 millimoles to about 2.5 moles, greater than 500 millimoles to about 2 moles, greater than 500 millimoles to about 1.5 moles, greater than 500 millimoles to about 1 mole, greater than 500 millimoles to about 900 millimoles, greater than 500 millimoles to about 800 millimoles, greater than 500 millimoles to about 700 millimoles, greater than 500 millimoles to about 600 millimoles, greater than 600 millimoles to about 5 moles, greater than 600 millimoles to about 4 moles, greater than 600 millimoles to about 3 moles, greater than 600 millimoles to about 2.5 moles, greater than 600 millimoles to about 2 moles, greater than 600 millimoles to about 1.5 moles, greater than 600 millimoles to about 1 mole, greater than 600 millimoles to about 900 millimoles, greater than 600 millimoles to about 800 millimoles, greater than 600 millimoles to about 700 millimoles, greater than 700 millimoles to about 5 moles, greater than 700 millimoles to about 4 moles, greater than 700 millimoles to about 3 moles, greater than 700 millimoles to about 2.5 moles, greater than 700 millimoles to about 2 moles, greater than 700 millimoles to about 1.5 moles, greater than 700 millimoles to about 1 mole, greater than 700 millimoles to about 900 millimoles, greater than 700 millimoles to about 800 millimoles, greater than 800 millimoles to about 5 moles, greater than 800 millimoles to about 4 moles, greater than 800 millimoles to about 3 moles, greater than 800 millimoles to about 2.5 moles, greater than 800 millimoles toabout 2 moles, greater than 800 millimoles to about 1.5 moles, greater than 800 millimoles to about 1 mole, greater than 800 millimoles to about 900 millimoles, greater than 900 millimoles to about 5 moles, greater than 900 millimoles to about 4 moles, greater than 900 millimoles to about 3 moles, greater than 900 millimoles to about 2.5 moles, greater than 900 millimoles to about 2 moles, greater than 900 millimoles to about 1.5 moles, greater than 900 millimoles to about 1 mole, greater than 1 mole to about 5 moles, greater than 1 mole to about 4 moles, greater than 1 mole to about 3 moles, greater than 1 mole to about 2.5 moles, greater than 1 mole to about 2 moles, greater than 1 mole to about 1.5 moles, greater than 1.5 moles to about 5 moles, greater than 1.5 moles to about 4 moles, greater than 1.5 moles to about 3 moles, greater than 1.5 moles to about 2.5 moles, greater than 1.5 moles to about 2 moles, greater than 2 moles to about 5 moles, greater than 2 moles to about 4 moles, greater than 2 moles to about 3 moles, greater than 2 moles to about 2.5 moles, greater than 2.5 moles to about 5 moles, greater than 2.5 moles to about 4 moles, greater than 2.5 moles to about 3 moles, greater than 3 moles to about 5 moles, greater than 3 moles to about 4 moles, or greater than 4 moles to about 5 moles of SEQ ID NO: 1. In some embodiments, the pharmaceutical formulation comprises about 1 moles, about 900 millimoles, about 850 millimoles, about 800 millimoles, about 750 millimoles, about 700 millimoles, about 650 millimoles, about 600 millimoles, about 550 millimoles, about 500 millimoles, about 450 millimoles, about 400 millimoles, about 350 millimoles, about 300 millimoles, about 250 millimoles, about 200 millimoles, about 150 millimoles, or about 100 millimoles of SEQ ID NO: 1.Routes of AdministrationEnteral Administration

[0098] In some embodiments, the SMAD7 ASO described herein is administered enterally. In some embodiments, the enteral administration is oral, sublingual, gastric, or rectal. In some embodiments, the enteral administration is rectal.

[0099] In some embodiments, the SMAD7 ASO described herein is suitable for rectal delivery (e.g., suppositories, enemas, gels, and foams). In some embodiments, the SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) is administered rectally.

[0100] In some embodiments, the formulation of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a rectal pharmaceutical formulation. In some embodiments, the rectal pharmaceutical formulation includes an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1 or a pharmaceuticallyacceptable salt thereof). In some embodiments, the rectal pharmaceutical formulation includes a SMAD7 ASO (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof).

[0101] In some embodiments, the SMAD7 ASO described herein is suitable for oral delivery (e.g, capsules, tablets, caplets, pills, troches, lozenges, powders, and granules). In some embodiments, the SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof) is administered orally.

[0102] In some embodiments, the formulation of an oligonucleotide (e.g, a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is an oral pharmaceutical formulation. In some embodiments, the oral pharmaceutical formulation includes an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof). In some embodiments, the oral pharmaceutical formulation includes a SMAD7 ASO (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof).

[0103] In some embodiments, the pharmaceutical formulation of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is a tablet or capsule. In some embodiments, the tablet or capsule is enteric-coated.

[0104] In some embodiments, the tablet of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is formulated as a minitablet, granulate, micropellet, or a microtablet. In some embodiments, the tablet of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is formulated as a minitablet. In some embodiments, the tablet of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is formulated as a granulate. In some embodiments, the tablet of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is formulated as a micropellet. In some embodiments, the tablet of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is formulated as a microtablet. In some embodiments, the minitablet, granulate, micropellet, or a microtablet is enteric-coated.

[0105] In some embodiments, the oral pharmaceutical formulation of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof) is not enteric-coated. In certain embodiments, the oral pharmaceutical formulation of an oligonucleotide (e.g., an oligonucleotide of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof) is not enteric-coated.

[0106] In some embodiments, the oral pharmaceutical formulation comprises a gastro- resistant coating, such that the formulations deliver the compound to, e.g, the gastrointestinal tract of an individual. Such administration, in some embodiments, result in a topical effect, for example, by substantially topically applying the antisense compound directly to an affected portion of the gastrointestinal tract of a subject. Such administration, in some embodiments, substantially avoid unwanted systemic absorption of the antisense compound.

[0107] In some embodiments, the pharmaceutical formulations described herein (e.g., oral pharmaceutical formulation and rectal pharmaceutical formulation) comprise a pharmaceutically acceptable excipient. Exemplary pharmaceutical acceptable excipients include, but are not limited to, fillers, binders, disintegrants, and / or lubricants, as well as coloring agents, release agents, coating agents, sweetening, flavoring such as wintergreen, orange, xylitol, sorbitol, fructose, and maltodextrin, and perfuming agents, preservatives and / or antioxidants.Parenteral Administration

[0108] The SMAD7 ASOs and pharmaceutical formulations of the disclosure, in some embodiments, are formulated for parenteral administration, e.g, formulated for injection via the intravenous, intratumoral, intramuscular, subcutaneous, intralesional, intraintestinal (e.g, intrajejunal, intraileal), intracolonic, or intrarectal, intrapouch, or intraperitoneal routes. In some embodiments, the SMAD7 ASO or the pharmaceutical formulations described herein are administered intrapouch. In some embodiments, the SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or pharmaceutically acceptable salts thereof is administered intrapouch.

[0109] In some embodiments, the preparation of an aqueous composition, such as an aqueous pharmaceutical formulation containing a disclosed SMAD7 ASO, will be known to those of skill in the art in light of the present disclosure. In some embodiments, such compositions are prepared as injectables, either as liquid solutions or suspensions. In some embodiments, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection are prepared; and the preparations are emulsified.

[0110] In some embodiments, the pharmaceutical formulations suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the pharmaceutical formulations suitable for injectable use include sesame oil, peanut oil or aqueous propylene glycol. In some embodiments, the pharmaceutical formulations suitable for injectable use sterile powders. In some embodiments, the pharmaceutical formulations are sterile and fluid to the extent that easy syringability exists. In some embodiments, the pharmaceutical formulations are stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0111] In some embodiments, solutions of active compounds as free base or pharmacologically acceptable salts are prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. In some embodiments, dispersions are prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. In some embodiments, sterile, fixed oils are employed as a solvent or suspending medium. For this purpose, in some embodiments, bland fixed oil is employed including synthetic mono- or diglycerides. In some embodiments, fatty acids such as oleic acid are used in the preparation of injectables. In some embodiments, the sterile injectable preparation is a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. In some embodiments, the acceptable vehicles and solvents employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In some embodiments, a disclosed SMAD7 ASO is suspended in a carrier fluid comprising 1% (w / v) sodium carboxymethylcellulose and 0.1% (v / v) TWEEN™ 80. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0112] In some embodiments, injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. In some embodiments, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In some embodiments, sterile injectable solutions of the disclosure are prepared by incorporating a disclosed SMAD7 ASO in the required amount of the appropriate solvent with various amounts of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, in some embodiments, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. In some embodiments, the injectable formulations are sterilized, for example, by filtration through a bacteria-retaining filter.

[0113] In some embodiments, the preparation of more, or highly concentrated solutions for intramuscular injection is also contemplated. In some embodiments, the use of DMSO as solvent is preferred as this will result in extremely rapid penetration, delivering high concentrations of the disclosed SMAD7 ASO to a small area.

[0114] In some embodiments, suitable preservatives for use in such a solution include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like. In some embodiments, suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium 10 carbonate, sodium acetate, sodium biphosphate and the like, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, and for example, between about pH 7 and pH 7.5. In some embodiments, suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, and the like, such that the sodium chloride equivalent of the solution is in the range 0.9 plus or minus 0.2%. In some embodiments, suitable antioxidants and stabilizers include sodium bisulfite, sodium metabi sulfite, sodium thiosulfite, thiourea and the like. In some embodiments, suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suitable viscosity -increasing agents include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin, methylcellulose , petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose and the like.Dosage Regimen

[0115] In some embodiments, the pharmaceutical formulation of an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6) or a pharmaceutically acceptable salt thereof, provided in the present disclosure is administered or is suitable for administration before and / or after symptoms of pouchitis are developed.

[0116] In some embodiments, the pharmaceutical formulation of the present disclosure (including e.g., SEQ ID NO: 1, 2, 3, 4, 5, or 6 or a pharmaceutically acceptable salt thereof) is administered or is suitable for administration about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, about every 72 hours, every day, two-times a week, once in 2 weeks, or once a month.

[0117] Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. In certain embodiments, dosing is once per day for 7 days.

[0118] In some embodiments, the pharmaceutical formulations include dosage forms that comprise or consist essentially of about 10 mg to about 500 mg or about 20 mg to about 500 mg of an oligonucleotide (e.g., a SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or a pharmaceutically acceptable salt thereof). For example, formulations that include about 20 mg,about 30 mg, about 35 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg of an oligonucleotide (e.g., a SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or a pharmaceutically acceptable salt thereof) are contemplated herein. In certain embodiments, a formulation includes about 40 mg, about 80 mg, or about 160 mg of an oligonucleotide such as a SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In certain embodiments, a formulation includes at least about 100 pg of an oligonucleotide such as a SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. For example, formulations includes about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg of an oligonucleotide such as a SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6.

[0119] The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health and size of the individual, the in vivo potency of the oligonucleotide, the pharmaceutical formulation, and the route of administration. In some embodiments, the initial dosage is increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level. In some embodiments, the initial dosage is smaller than the optimum, and the dosage is progressively increased during the course of treatment. In some embodiments, human dosage is optimized, e.g., in a conventional Phase I dose escalation study designed to run from 40 mg to 160 mg.

[0120] In some embodiments, an individual having pouchitis will be administered an initial dose of an SMAD7 ASO, for instance, a SMAD7 ASO of SEQ ID NO: 1. As used herein, “initial dose” refers to a dose of an SMAD7 ASO administered to an individual having pouchitis, in a series of doses. In some embodiments, a series of doses includes one or more doses. For instance, a series of doses includes a single dose of an SMAD7 ASO or more than a single dose of an SMAD7 ASO. In some embodiments, an initial dose is a dose of an SMAD7 ASO administered to an individual prior to any later dose administered to the individual. For instance, an initial dose is, but is not limited to, the first dose of an SMAD7 ASO administered to a treatment-naive individual. In some embodiments, an initial dose is a first dose in any treatment cycle of the SMAD7 ASO. For example, an initial dose is the first dose of a first treatment cycle, of a second treatment cycle, or of any subsequent treatment cycles.

[0121] In some embodiments, an individual having pouchitis is administered a subsequent dose of a SMAD7 ASO, for instance, a SMAD7 ASO of SEQ ID NO: 1. As used herein, “subsequent dose” refers to a dose of an SMAD7 ASO administered to an individual having pouchitis, after administration of a prior dose, for example, an initial dose. In some embodiments, a subsequent dose is administered to an individual having pouchitis, in a series of doses including two or more doses. In some embodiments, the amount of a subsequent dose is calibrated with respect to an initial dose or a prior dose, such that a subsequent dose is greater, equal to, or lesser than a prior dose. In some embodiments, a subsequent dose is a dose administered to an individual having pouchitis, after a first dose, for instance, an initial dose, of an SMAD7 ASO administered to the individual. In some embodiments, a subsequent dose is a dose administered after a prior dose of an SMAD7 ASO administered to an individual having pouchitis, for instance, a dose administered after a prior dose in the same round of treatment or a different round of treatment, for instance, a previous round of treatment. In some embodiments, a subsequent dose subsequent dose is a subsequent dose with respect to any prior dose, for instance, a prior dose immediately preceding the subsequent dose or a prior dose followed by one or more doses administered prior to administration of the subsequent dose.

[0122] The composition or formulation or method the present disclosure provides administration of an oligonucleotide (e.g., a SMAD7 oligonucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6) or a pharmaceutically acceptable salt thereof, to a subject who is refractory to a first therapy.

[0123] In some embodiments, a subject who is refractory to the first treatment is treated with an oligonucleotide (e.g., a SMAD7 ASO of SEQ ID NO: 1, 2, 3, 4, 5, or 6) or a pharmaceutically acceptable salt thereof, concurrently or subsequent to the first therapy.

[0124] In some embodiments, methods provided herein further include administering at least one other agent that is directed to treatment of diseases and disorders disclosed herein (e.g., pouchitis). In some embodiments, contemplated other agents are co-administered (e.g., sequentially or simultaneously).EXAMPLES

[0125] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of the disclosure in any way.Example 1: Patient mucosal sample preparation

[0126] The mucosal samples prepared and used in the following examples (i.e., Examples 2- 5) were taken from the inflamed pouch of 16 patients with chronic pouchitis (CP). The modified pouchitis disease activity index (mPDAI), which was evaluated using clinical and endoscopic findings, was used for the diagnosis of pouchitis, with pouchitis defined as mPDAI > 5. Samples used as experimental controls include mucosal biopsy samples taken from the uninflamed pouch of patients with no clinical / endoscopic evidence of pouchitis and ileal samples taken from normal / inflamed pre-pouch of patients with chronic pouchitis and normal controls who underwent colonoscopy for colorectal carcinoma screening programs at the Tor Vergata University Hospital (Rome, Italy).Example 2: SMAD7 is increased in chronic pouchitis

[0127] This Example assesses SMAD7 expression in various mucosal samples via immunofluorescent staining.

[0128] Briefly, patient mucosal samples were prepared as in Example 1. Sections of the fresh mucosal biopsy samples were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% TritonX-100 for 20 min at room temperature. Sections were then blocked for 1 hour at room temperature (BSA 1%, Tween 0.1%, glycine 2%), and then incubated overnight at 4°C with mouse primary antibody against SMAD7 (1 : 150). After overnight incubation, the sections were washed with PBS IX, and the secondary antibody goat anti-mouse Alexa488 (1 : 1000) was applied for 1 hour at room temperature. Slides were then washed with PBS IX, mounted using the prolonged gold antifade reagent with DAPI, and analyzed by microscopy.

[0129] Data is seen in FIGs. 1A-1C that show sections of the fresh patient mucosal biopsy samples analyzed for the expression of SMAD7 (green), and DAPI (blue). The figure (FIG. 1 A- 1C) is representative of three separate experiments in which similar results were obtained. Exemplary results are shown for single and double-immunofluorescence staining of sections of healthy ileum (FIG. 1A), uninflamed pouch (FIG. IB), and inflamed pouch (FIG. 1C). Scale bars are 50 pm.

[0130] The data demonstrate that expression of SMAD7, visualized as green immunofluorescent signal, is increased in sample sections of inflamed pouch when compared to expression of SMAD7 in sections of healthy ileum or uninflamed pouch.Example 3: SMAD7 protein expression is increased in chronic pouchitis

[0131] This Example assesses SMAD7 protein expression in various mucosal samples via Western blotting, with corresponding quantitative analysis of Western blot results by densitometry scanning.

[0132] Briefly, mucosal biopsy samples used for Western blotting were lysed on ice in a buffer containing 10 mM HEPES (pH 7.9), 10 mM potassium chloride (KC1), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM ethylene glycol-bis (P-aminoethyl ether)- 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 phenylmethyl sulphonyl fluoride (PMSF), 1 mM Na3 VO4, and 1 mM sodium fluoride (NaF). Lysates were clarified by centrifugation and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The blots were incubated with antibodies against SMAD7 (1 : 1000) or P-actin (1 :5000), followed by a secondary antibody conjugated to horseradish peroxidase (1 :20000). Computer-assisted scanning densitometry was used to analyze the intensity of the immunoreactive bands.

[0133] The Western blotting results depicted in FIG. 2A-2B show SMAD7 and P-actin protein expression in total proteins extracted from fresh mucosal biopsy samples and include a corresponding analysis of the SMAD7 / p-actin ratio as measured by densitometry scanning of the Western blot.

[0134] The Data seen in FIGs. 2A-2B show Western blot detection of SMAD7 and P-actin in total proteins extracted from mucosal biopsy samples, and includes corresponding quantitative graphical analysis, as measured by densitometry scanning, of the SMAD7 / p-actin ratio from Western blotting results. In FIG. 2A analyzed total protein extracts were isolated from mucosal samples of the inflamed pouch (pouchitis), normal pre-pouch ileum of the same patient with pouchitis, uninflamed pouch, and healthy ileum (normal controls). In FIG. 2B analyzed total protein extracts were isolated from mucosal samples of the inflamed pouch (pouchitis), inflamed ileum (pre-pouch ileitis) of the same patients with pouchitis, and healthy ileum (normal controls).

[0135] The data demonstrate that SMAD7 protein expression was increased in inflamed prepouch and inflamed pouch mucosal samples when compared to the levels of SMAD7 protein expression that occurred in uninflamed and normal, healthy ileum mucosal samples.Example 4: SMAD7 co-localizes with CD3 and HLA-DRII in pouchitis tissue

[0136] This Example assesses the co-localization of SMAD7 with CD3 and HLA-DRII in pouchitis tissue samples using immunofluorescent staining.

[0137] Briefly, sections of the fresh mucosal biopsy samples were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% TritonX-100 for 20 min at room temperature. Sections were then blocked for 1 hour at room temperature (BSA 1%, Tween 0.1%, glycine 2%), and then incubated overnight at 4°C with either a rabbit primary antibody against CD3 (1 : 100) or rabbit primary antibody anti -HL A Class II (1 : 100), and a mouse primary antibody against SMAD7 (1 : 150). After washing with PBS IX, the secondary antibody goat anti-rabbit Alexa488 (1 : 1500) or the goat anti-mouse Alexa488 (1 : 1000) was applied for 1 hour at room temperature. Slides were then washed with PBS IX, mounted using the prolonged gold antifade reagent with DAPI, and analyzed by microscopy.

[0138] Data is seen in FIGs. 3A-3B that show single and double-immunofluorescence staining of sections of the fresh mucosal biopsy samples taken from a patient with chronic pouchitis analyzed for the expression of SMAD7 (green), CD3 (red; Fig. 3A), HLA-DRII (red; Fig. 3B) and DAPI (blue). The figure (FIG. 3A-3B) is representative of three separate experiments in which similar results were obtained. Scale bars are 75 pm.

[0139] The data in FIG. 3A demonstrate that SMAD7 expression was co-localized with CD3 in pouchitis tissue, and FIG. 3B demonstrates that SMAD7 expression was co-localized with HLA DRII in pouchitis tissue.Example 5: SMAD7 knockdown in ex vivo organ cultures alters the production of several inflammatory and anti-inflammatory molecules

[0140] This example assesses the expression level SMAD7 and of various inflammatory and anti-inflammatory molecules in ex vivo organ cultures transfected with SMAD7 sense oligonucleotide (S) or SMAD7 antisense oligonucleotide (AS). Exemplary results are shown in FIGs. 4A-4B

[0141] Briefly, ex vivo organ cultures were prepared from mucosal samples taken from the inflamed pouch and were placed on steel grids in an organ culture chamber at 37°C in a 5% CO2 / 95% 02 atmosphere and cultured in RPMI 1640 medium. Ex vivo organ cultures were transfected with SMAD7 sense oligonucleotide or SMAD7 AS (lOpg / ml) for 24 h using Opti- MEM medium and Lipofectamine 3000 reagent . The phosphorothioate single-stranded oligonucleotide matching the region 107-128 (5'-GCTGCGGGGAGAAGGGGCGAC-3') of the human SMAD7 complementary DNA sequence was synthesized in the sense and antisense orientation

[0142] For the real-time polymerase chain reaction results shown in FIG. 4A, total RNA was extracted and a constant amount of RNA (1 pg / sample) was retrotranscribed intocomplementary DNA (cDNA) using Oligo(dT) primers and M-MLV-reverse transcriptase. This was amplified using the following conditions: denaturation for 1 minute at 95°C; annealing for 30 seconds at 59°C for human SMAD7 and at 60°C for human P-actin; 30 seconds of extension at 72°C. RNA expression was calculated relative to the P-actin gene using the AACt algorithm. Primer sequences were as follows: SMAD7 Fw 5'-GCCCGACTTCTTCATGGTGT-3', Rev 5'- TGCCGCTCCTTCAGTTTCTT-3'; p-actin FW 5’- AAGATGACCCAGATCATGTTTGAGACC-3’, Rev 5’-AGCCAGTCCAGACGCAGGAT-3’.

[0143] For the human cytokine expression array assay results shown in FIG. 4B, a Human Cytokine Array C3 was used. Briefly, supernatants of inflamed pouch samples transfected respectively with SMAD7 sense oligonucleotide or SMAD7 AS (10 pg / ml) were added to membranes and incubated at 4°C overnight. The membranes then were incubated with biotin- conjugated antibodies for 2 h, and then with HRP-conjugated streptavidin for 30 minutes.Unbound reagents were removed by washing, and the bound antibodies on the membranes were visualized using an ECL system. The quantitative analysis was performed by densitometry scanning of array blots.

[0144] For statistical analyses, differences between groups were compared using the student’s t-test. Correlation between SMAD7 and Modified Pouchitis Disease Activity Index (mPDAI) was made using Pearson’s correlation coefficient.

[0145] The data in FIG. 4A demonstrate that ex vivo organ cultures transduced with SMAD7 antisense oligonucleotide show reduced SMAD7 RNA expression when compared to cultures transduced with SMAD7 sense oligonucleotide. The data in FIG. 4B demonstrate that ex vivo organ cultures transfected with SMAD7 antisense oligonucleotide displayed altered expression profiles for several inflammatory and anti-inflammatory molecules when compared to cultures transfected with SMAD7 sense oligonucleotide. Together, these data indicate that SMAD7 ASO down-regulates SMAD7 RNA production and alters production of several inflammatory and anti-inflammatory molecules in organ cultures derived from inflamed pouch tissue.EQUIVALENTS

[0146] The disclosure can be embodied in other specific forms with departing from the essential characteristics thereof. The foregoing embodiments therefore are to be considered illustrative rather than limiting on the disclosure described herein. The scope of the disclosure is indicated by the appended claims rather than by the foregoing description, and all changes thatcome within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of treating pouchitis in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5 ’-GTXGCCCCTTCTCCCXGC AGC- 3’)-2. The method of claim 1, wherein all internucleoside linkages are O,O-linked phosphorothioate linkages and X is 5-methyl 2’ -deoxy cytidine.

3. The method of any one of claims 1-2, wherein the SMAD7 antisense oligonucleotide is administered enterally.

4. The method of claim 3, wherein the enteral administration is oral, sublingual, gastric, or rectal.

5. The method of claim 3, wherein the enteral administration is rectal.

6. The method of any one of claims 1-2, wherein the SMAD7 antisense oligonucleotide is administered parenterally.

7. The method of claim 6, wherein the parenteral administration is intravenous, intratumoral, intrajejunal, intraileal, intracolonic, intrarectal, or intrapouch.

8. The method of claim 7, wherein the parenteral administration is intrapouch.

9. The method of any one of claims 1-8, wherein the therapeutically effective amount of the SMAD7 ASO comprises a dose of about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg.

10. The method of any one of claims 1-8, wherein the therapeutically effective amount of the SMAD7 ASO comprises a dose of about 20 mg to about 500 mg.

11. The method of any one of claims 1-8, wherein the therapeutically effective amount of the SMAD7 ASO comprises about 160 mg.

12. The method of any one of claims 1-11, wherein the therapeutically effective amount of the SMAD7 ASO is administered about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, about every 72 hours, every day, two-times a week, once in 2 weeks, or once a month.

13. The method of any one of claims 1-12, wherein the therapeutically effective amount of the SMAD7 ASO comprises a dose of about 160 mg daily.

14. The method of any one of claims 1-13, wherein the SMAD7 ASO down-regulates SMAD7 mRNA and / or protein in a cell.

15. The method of claim 14, wherein the down-regulation of SMAD7 protein expression is more than 40% compared to an untreated cell.

16. The method of any one of claims 1-15, wherein at least one sample from the individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

17. The method of any one of claims 1-16, wherein the pouchitis is acute pouchitis.

18. The method of any one of claims 1-16, wherein the pouchitis is chronic pouchitis.

19. The method of any one of claims 1-18, wherein the individual is determined as having pouchitis using a modified pouchitis disease activity index (mPDAI) score of at least 5.

20. A method of treating pouchitis in an individual in need thereof, comprising administering to the individual a pharmaceutical formulation comprising: a) a SMAD7 antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 1 (5’-GTXGCCCCTTCTCCCXGCAGC-3’); and b) a pharmaceutically acceptable carrier.

21. The method of claim 20, wherein the pharmaceutical formulation comprises 100 millimoles to 5 moles of the SMAD7 ASO.

22. The method of claim 20, wherein the pharmaceutical formulation comprises 100 millimoles to 2 moles, 100 millimoles to 900 millimoles, 300 millimoles to 5 moles, 300 millimoles to 2 moles, 300 millimoles to 900 millimoles, 900 millimoles to 5 moles, or 900 millimoles to 2 moles of the SMAD7 ASO.

23. The method of claim 20, wherein the pharmaceutical formulation comprises about 300 millimoles of the SMAD7 ASO.

24. The method of claim 20, wherein the pharmaceutical formulation comprises about 900 millimoles of the SMAD7 ASO.

25. The method of claim 20, wherein the pharmaceutical formulation comprises about 2 moles of the SMAD7 ASO.

26. The method of claim 20, wherein the pharmaceutical formulation comprises greater than 300 millimoles, greater than 900 millimoles, or greater than 2 moles of the SMAD7 ASO.

27. The method of any one of claims 20-26, wherein the pharmaceutical formulation is administered orally.

28. The method of claim 27, wherein the pharmaceutical formulation comprises: about 0.5% to about 30% by weight of the SMAD7 ASO; about 20% to about 50% by weight mannitol; about 10% to about 30% by weight microcrystalline cellulose; and an enteric coating comprising an ethylacrylate-methacrylic acid copolymer.

29. The method of any one of claim 27, wherein the pharmaceutical formulation comprises: a) an intra-granular phase comprising: i) about 5% to about 10% by weight of the SMAD7 ASO; ii) about 40% by weight mannitol; iii) about 8% by weight microcrystalline cellulose; iv) about 5% by weight hydroxypropyl methylcellulose; and v) about 2% by weight sodium starch glycolate; b) an extra-granular phase comprising: i) about 17% by weight microcrystalline cellulose; ii) about 2% by weight sodium starch glycolate, iii) about 0.4% by weight magnesium stearate; and c) an enteric coating comprising an ethylacrylate-methacrylic acid copolymer, wherein the percentages by weight are the weights of the ingredients compared to the total weight of the pharmaceutical formulation.

30. The method of claim 27, wherein the pharmaceutical formulation comprises: about 5% to about 30% by weight of the SMAD7 ASO; about 20% to about 50% by weight mannitol; about 10% to about 30% by weight microcrystalline cellulose, about 0.5% to about 10% by weight hydroxypropyl methylcellulose; about 0.5% to about 10% by weight sodium starch glycolate; about 0.05% to about 1% by weight magnesium stearate; about 0.5% to about 10% by weight of a moisture barrier film coating; and about 5% to about 20% by weight of a polymer coating.

31. The method of claim 27, wherein the pharmaceutical formulation comprises: about 8.5% by weight of the SMAD7 ASO; about 40% by weight mannitol; about 25% by weight microcrystalline cellulose,about 5% by weight hydroxypropyl methylcellulose; about 4% by weight sodium starch glycolate; about 0.4% by weight magnesium stearate; about 4% by weight of a moisture barrier film coating; and about 10% to about 15% by weight of a polymer coating.

32. The method of claim 27, wherein the pharmaceutical formulation comprises: about 23% by weight of the SMAD7 ASO; about 28% by weight mannitol; about 25% by weight microcrystalline cellulose, about 5% by weight hydroxypropyl methylcellulose; about 4% by weight sodium starch glycolate; about 0.4% by weight magnesium stearate; about 4% by weight of a moisture barrier film coating; and about 7% to about 12% by weight of a polymer coating33. The method of any one of claims 20-32, wherein the pharmaceutical formulation down- regulates SMAD7 mRNA and / or protein in a cell.

34. The method of claim 33, wherein the down-regulation of SMAD7 protein expression is more than 40% compared to an untreated cell.

35. The method of any one of claims 20-34, wherein at least one sample from the individual has an elevated SMAD7 level relative to a known control level, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

36. The method of any one of claims 20-35, wherein the pouchitis is acute pouchitis.

37. The method of any one of claims 20-35, wherein the pouchitis is chronic pouchitis.

38. The method of any one of claims 20-37, wherein the individual is determined as having pouchitis using a modified pouchitis disease activity index (mPDAI) score of at least 5.

39. A method for predicting pouchitis in an individual in need thereof, comprising determining a level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level is predictive of pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

40. A method of identifying an individual at risk of pouchitis, comprising: determining the level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 levelrelative to a known control level identifies the individual as being at risk for pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

41. The method of claim 39 or 40, comprising:(i) if the SMAD7 level is elevated relative to the known control level, then administering to the individual a pharmaceutical formulation comprising a SMAD7 ASO; or(ii) if the SMAD7 level is not elevated relative to the known control level, then determining the level of SMAD7 in a second sample from the individual.

42. The method of claim 41, wherein the second sample is collected immediately after, aboutI hour after, about 3 hours after, about 6 hours after, about 12 hours after, about 1 day after, about 3 days after, about 1 week after, about 2 weeks after, about 1 month after, about 2 months after, about 3 months after, about 4 months after, about 5 months after, about 6 months after, about 7 months after, about 8 months after, about 9 months after, about 10 months after, aboutI I months after, or about 12 months after the first sample.

43. A method for treating pouchitis in an individual in need thereof, comprising determining a level of SMAD7 in a first sample from the individual, wherein an elevated SMAD7 level relative to a known control level is predictive of pouchitis, wherein the known control level is the SMAD7 level in a sample collected from the individual prior to or during the treatment for pouchitis or the SMAD7 level in a sample collected from a healthy individual without pouchitis.

44. The method of any one of claims 39-42, wherein the SMAD7 ASO comprises a sequence selected from any one of SEQ ID NOs: 1-6 or a pharmaceutically acceptable salt thereof.

45. The method of any one of claims 39-42, wherein the SMAD7 ASO has a sequence according to SEQ ID NO: 1 (5’-GTXGCCCCTTCTCCCXGCAGC-3’).

46. The method of claim 45, wherein all internucleoside linkages are O,O-linked phosphorothioate linkages and X is 5-methyl 2’ -deoxy cytidine.

47. The method of any one of claims 1-46, wherein the individual is human.

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