Therapeutic Uses and Methods

Abstract

A method for treating muscular dystrophy in a subject, or for improving muscle function or delaying the decline of muscle function in a subject with muscular dystrophy, comprises administering to the subject a pharmaceutical composition comprising an antisense oligonucleotide against VLA-4 for a time and under conditions sufficient to improve one or more markers, signs or symptoms of muscular dystrophy or dystrophic muscle fibers, or to delay the progression of muscular dystrophy in the subject. In one embodiment, the method uses a low dose of the antisense oligonucleotide, less than about 50 mg per dose. In one embodiment, the antisense oligonucleotide treatment is used as an adjunctive therapy with corticosteroid treatment in a subject with muscular dystrophy, such as DMD.

Description

[Technical field]

[0001] Enabled herein are compositions and methods for treating muscular disorders, such as muscular dystrophies. [Background technology]

[0002] Bibliographic details of the references to the subject specification are also provided at the end of the specification. The reference to prior art in this specification is not intended to, and should not be construed as, an admission that this prior art is part of the common general knowledge in any country, or as any form of suggestion.

[0003] Muscular dystrophies (MD) are a group of disorders characterized by progressive weakening and wasting of certain muscle tissues (myonecrosis) and replacement of skeletal muscle with fibrous, bony, or fatty tissue. There are several different forms of muscular dystrophies that affect either men or both men and women, many of which appear from infancy and childhood to middle age and beyond. Form and severity vary, particularly with age of onset, with younger subjects often experiencing acute progressive disease.

[0004] The most common forms of MD are Duchenne muscular dystrophy (DMD), Limb-girdle muscular dystrophy (LGMD), Becker muscular dystrophy (BMD), congenital muscular dystrophies (CMD, Fukuyama congenital MD and congenital MD with myosin deficiency, facioscapulohumeral, oculopharyngeal, Emery-Dreifuss and distal types, etc.). Almost all forms of MD result from mutations in a single gene.

[0005] DMD and BMD involve defects in the dystrophin gene on the X chromosome. The dystrophin protein serves to link the contractile machinery (actin filaments) and cytoskeleton of muscle cells (sarcomeres) with the extracellular matrix (ECM) where collagen transmits muscle force (Grounds MD, 2008). The ECM is known to play a complex role in muscle function and muscle regeneration. Dystrophic muscle fibrosis is associated with necrosis, inflammation and fibrosis. The exact sequence of events that lead to progressive disease as a result of dystrophin deficiency is not understood at the molecular level. Children with DMD have muscles that are deficient in dystrophin, making them more susceptible to contraction, causing damage to the muscle and triggering the immune system to exacerbate muscle damage (summarized in a publication by Direct of the FDA CDER (Rosen et al, 2015)). The ongoing loss of muscle strength affects the lower limbs, causing movement disorders, and also affects the upper limbs, causing further loss of function and self-care abilities. While gene therapy and exon skipping approaches would be ideal, researchers are also focused on understanding the nature of the disease in order to develop strategies and agents that can ameliorate its severity and slow its progression. The Mdx mouse model has been widely adopted to investigate preclinical mechanisms and interventions. Grounds MD, 2008, identified the need for a two-phase approach targeting both the chronic and acute phases of the disease in mdx mice.

[0006] DMD is a devastating condition that primarily affects boys, occurring in approximately 1:3,500 births. Boys lose the ability to walk at an early age and typically become wheelchair bound after puberty, with death from cardiopulmonary failure often occurring between the ages of 20 and 29. BMD is similar to DMD but is much milder.

[0007] Current treatment with corticosteroids aims to reduce the severity of the disease by reducing inflammation and preserving muscle mass and function for a period of time. Corticosteroids have an acute anti-inflammatory effect that may be of short duration, and their mechanism of action is not understood. Corticosteroids are not optimal because their side effects severely limit their use and may cause atrophy. Prednisolone (0.75 mg / kg / day) and deflazacort (0.9 mg / kg / day) are the standard treatment for ambulatory DMD patients, but once boys become nonambulatory, there is no consensus on the benefits of CS, and boys may be on the fixed dose they were on when they became nonambulatory, and may continue treatment at a reduced dose (mg / kg / day) or may be taken off CS treatment. Edasalonexent, an anti-inflammatory NF-κB drug, is being developed as a monotherapy in young ambulatory boys with DMD. There are several drugs in clinical trials targeting different aspects of the dystrophy. For example, tamoxifen targets fibrosis and respiratory function, and idebenone ataluren for stop codon skipping is undergoing clinical trials in MD. Oligonucleotide therapy for DMD by inducing targeted exon skipping of the dystrophin gene is being evaluated with mixed results. Eteplirsen, a morpholino oligonucleotide, advanced to confirmatory studies in 13% of DMD children with a gene stop codon mutation in exon 51 suitable for exon 51 skipping, but drisapersen, a 2'-O-methyl phosphorothioate oligonucleotide for exon 51 skipping, failed to obtain activity and was not approved by the FDA.

[0008] These deficiencies in current therapies suggest that additional therapeutic approaches are needed. Summary of the Invention [Problem to be solved by the invention]

[0009] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0010] As used herein, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to a "composition" includes a single composition, as well as two or more compositions, reference to an "agent" includes one agent as well as two or more agents, reference to a "disclosure" includes single and multiple aspects of the disclosure, and so forth.

[0011] In one embodiment, the disclosure provides a method for treating muscular dystrophy in a subject, the method comprising administering an inhibitory oligonucleotide to human CD49d (alpha 4 chain of VLA-4).

[0012] In one embodiment, exemplary inhibitory oligonucleotides include isolated or synthetic antisense RNA or DNA, siRNA or siDNA, miRNA, miRNA mimics, shRNA or DNA, and antisense DNA or RNA or DNA:RNA hybrids.

[0013] In one embodiment, the disclosure provides a method of treating muscular dystrophy in a subject in need thereof, comprising periodically administering to the subject a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of an oligonucleotide comprising the following structure: 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me CMe U-3' During the ceremony, a) each of the 19 internucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at positions 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; c) the nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides; d) the nucleotides at positions 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; e) All cytosines are 5-methylcytosines ( Me C) or or a pharma- ceutically acceptable salt thereof.

[0014] In one embodiment, administration is for a time and under conditions sufficient to ameliorate one or more markers, signs or symptoms of muscular dystrophy, or slow the progression of muscular dystrophy in the subject.

[0015] In one embodiment, administration is in combination with standard corticosteroid therapy.

[0016] In one embodiment, the corticosteroid is administered in a low dose. References to low dose corticosteroid include ⅔, ½, ¼, and ⅓ of the standard dose.

[0017] In one embodiment, administration of the antisense oligonucleotide is therapeutically effective in the presence of standard or low doses of corticosteroids.

[0018] In one embodiment, administration of the antisense oligonucleotide is therapeutically effective in the absence of corticosteroids.

[0019] In one embodiment, administration of the antisense oligonucleotide is therapeutically effective in the absence of corticosteroids and the subject is ambulatory.

[0020] In one embodiment, administration of the antisense oligonucleotide is therapeutically effective in the absence of corticosteroids and the subject is non-ambulatory.

[0021] In another embodiment, the description enables a method for improving muscle function or slowing the decline of muscle function in a subject with muscular dystrophy, the method comprising periodically administering to the subject a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of an oligonucleotide comprising the following structure: 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me C Me U-3' During the ceremony, a) each of the 19 internucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at positions 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; c) the nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides; d) the nucleotides at positions 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; e) All cytosines are 5-methylcytosines ( Me C) or or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0022] In one embodiment, the oligonucleotide is an RNA-DNA hybrid.

[0023] In one embodiment, the subject is non-ambulatory due to MD.

[0024] In one embodiment, the subject is post-pubertal.

[0025] In one embodiment, the method includes monitoring CD4+ and / or CD8+ T cell levels. In one embodiment, the method includes monitoring a decrease in CD4+ and / or CD8+ T cell levels. In one embodiment, the method includes monitoring a decrease in M1 macrophages or HLADR1 cells. + Monitoring monocytes. In one embodiment, the method comprises monitoring the reduction of M1 macrophages or HLADR+ monocytes.

[0026] In one embodiment, the method includes determining the level or presence of one or more markers of MD or dystrophic muscle fibers, including the level or number of immune cells or immune regulators produced thereby, the level of a marker of inflammation, or the level of a marker of fibrosis, or the level of a marker of muscle condition.

[0027] In one embodiment, the one or more markers of MD or MD progression or dystrophic muscle fibers include the level or number of immune cells or immune regulators produced thereby, the level of a marker of inflammation or the level of a marker of fibrosis or the level of a marker of muscle condition.

[0028] Markers of muscle status include, but are not limited to, markers of exercise muscle function, markers indicative of muscle fibrosis or its absence, markers indicative of muscle degeneration or regeneration, markers of cardiac function, and markers of pulmonary function.

[0029] In one embodiment, improvement in one or more symptoms of MD or dystrophic muscle fibers includes improvement in limb function, body muscle function, cardiac function, and / or pulmonary function.

[0030] In one embodiment, the method includes determining the level or presence of one or more indicia of MD or dystrophic muscle fibers. Illustrative indicia include limb function, body muscle function, cardiac function, and pulmonary function.

[0031] In one embodiment, the one or more symptoms of MD or dystrophic muscle fibers include quality of life factors such as energy level, well-being, perceived ease of walking, and upper extremity functional activity.

[0032] In one embodiment, subjects in need thereof include those with a genetic and / or clinical diagnosis of MD and relatively low levels of dystrophic muscle fibers and inflammatory markers.

[0033] In one embodiment, the subject exhibits normal or only slightly elevated levels of inflammatory cells, including T cells (CD4, CD8), B cells (CD-19), and granulocytes (neutrophils, basophils, and eosinophils).

[0034] In one embodiment, the subject exhibits normal or only slightly elevated levels of CD49d cells.

[0035] In one embodiment, the subject exhibits normal or only slightly elevated levels of CD49d T cells.

[0036] In one embodiment, the subject exhibits normal or slightly elevated levels of immune cell markers such as CD3, CD4, CD8, CD49d, CD29 and HLA-DR.

[0037] Suitable methods for markers such as cell or protein / nucleic acid or lipid analysis are known in the art and include, but are not limited to, flow cytometry, bead technology and ELISA-based methods, chromatography and / or MS methods, hybridization or sequencing-based methods, etc.

[0038] In further embodiments, subjects diagnosed with MD exhibit significantly elevated or acute levels of severely dystrophic muscle fibers accompanied by severe muscle necrosis and inflammation.

[0039] In one embodiment, the subject exhibits significantly elevated levels of CD49d T cells compared to normal healthy controls.

[0040] In one embodiment, the form of MD in the subject is selected from the group consisting of Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy (LGMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD such as Fukuyama congenital MD, congenital MD with myosin deficiency), facioscapulohumeral, oculopharyngeal, Emery-Dreifuss, and distal muscular dystrophy.

[0041] In one embodiment, the subject has DMD or BMD and is non-ambulatory.

[0042] In one embodiment, the subject has DMD or BMD and is post-pubertal.

[0043] In another aspect of the disclosure, embodiments are contemplated to be directed to pharmaceutical compositions when used in the methods or uses described herein, to the use of the compositions described herein in the manufacture of a medicament for the treatment or prevention of muscular dystrophy in a subject, and to pharmaceutical compositions for use in the methods described herein.

[0044] Thus, in one embodiment, the present disclosure provides a compound comprising the following structure: 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me C Me U-3' During the ceremony, a) each of the 19 internucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at positions 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; c) the nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides; d) the nucleotides at positions 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; e) All cytosines are 5-methylcytosines ( Me C) or or a pharma- ceutically acceptable salt thereof.

[0045] In another embodiment, the description provides for an oligonucleotide comprising the following structure for use in treating or preventing, or slowing the progression of, muscular dystrophy in a subject: 5- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me C Me U-3' During the ceremony, a) Each of the 19 internucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester. b) The nucleotides 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides. c) The nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides. d) The nucleotides 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides. e) All cytosines are 5-methylcytosines ( Me C) or or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0046] In one embodiment, the disclosure enables a method of treating muscular dystrophy in a subject in need thereof, comprising periodically administering to the subject a therapeutically effective amount of an inhibitory oligonucleotide against human CD49d to ameliorate one or more markers, signs or symptoms of muscular dystrophy or slow the progression of muscular dystrophy in the subject.

[0047] In one embodiment, administration of the antisense oligonucleotide is in combination with or as an adjunct to standard or low dose corticosteroid treatment.

[0048] In one embodiment, the corticosteroid is administered in a low dose.

[0049] In one embodiment, administration of the antisense oligonucleotide is effective in the absence of corticosteroid therapy.

[0050] In another embodiment, the use of an inhibitory oligonucleotide against human CD49d in the preparation of a medicament for ameliorating one or more markers, signs or symptoms of muscular dystrophy or for slowing the progression of muscular dystrophy in a subject having muscular dystrophy is disclosed.

[0051] In one embodiment, there is provided a pharmaceutical composition comprising an inhibitory oligonucleotide against human CD49d for use in treating or slowing the progression of muscular dystrophy in a subject.

[0052] The above summary is not, and should not be, viewed as an exhaustive listing of all embodiments of the present disclosure.

[0053] Many modifications will be apparent to those of ordinary skill in the art without departing from the scope of the disclosure.

[0054] It will be appreciated by those skilled in the art that many variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive. Reading the sequence listing

[0055] SEQ ID NO:1 Human α4 integrin antisense compound (ATL1102) SEQ ID NO:2 Mouse α4 integrin antisense compound (ISIS348574) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The subject disclosure is not limited to the specific screening procedure of the agent, the specific formulation of the agent, and various medical methodologies, which may vary as such.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Any materials and methods similar or equivalent to those described herein can be used to carry out or test the present disclosure. Practitioners are particularly directed to Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; Colowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986; Remington's Pharmaceutical Sciences (18th ed., Mack Easton, Pa. (1990)) for definitions and terms of skill in the art, as well as other methods known to those of skill in the art.

[0057] The term "subject" includes a human subject or individual diagnosed with MD, or a clinical research animal model.

[0058] For example, DMD is often diagnosed clinically when an infant's motor milestones are delayed at 18 months of age. Early features of muscle weakness include broad-based gait, hyperlordosis of the spine with toe walking, frequent falls, muscle hypertrophy in the calves, deltoids, quadriceps, and tongue biting, difficulty rising, and arm weakness. Loss of ambulation typically occurs between 7 and 13 years of age in DMD, whereas later ambulation is characteristic of BMD. Cardiopulmonary impairment may also be evident. Fatigue and language development may also be delayed. However, no upper motor neuron signs or muscle fasciculations are observed.

[0059] The diagnosis of DMD can be confirmed by dystrophin immunofluorescence and / or immunoblot showing dystrophin deficiency, as well as clinical picture consistent with typical DMD. Alternatively, gene deletion results in a positive (lack of one or more exons) in the dystrophin gene, and the reading frame can be predicted as "out of frame", showing clinical picture consistent with typical DMD. In one embodiment, complete dystrophin gene sequencing can show point mutations, duplications, or other mutations resulting in stop codon mutations that can be reliably associated with DMD. A positive family history of DMD in a sibling or maternal uncle confirmed by one of the above criteria is also useful. Evaluation of characteristic clinical symptoms or signs of DMD (e.g., proximal muscle weakness, Gowers' manoeuvre, elevated serum creatine kinase level) is also used.

[0060] Suitable markers of MD or dystrophic muscle fibers, amelioration of signs and symptoms, and improvement of muscle function will be known to those of skill in the art.

[0061] Suitable tests include tests of increases in exercise, muscle, heart, blood flow and lung function over time during treatment.

[0062] In subjects with preclinical cardiomyopathy, cardiac efficacy can be determined based on serum biomarker responses. This can be accomplished by determining the levels of one or more markers, such as myostatin ratio, cardiac troponin, cardiac BNP, and changes in eGFR can also be monitored. Other cardiac function can be assessed by telemetry, or rhythm abnormalities assessed by continuous mobile telemetry monitoring.

[0063] Further tests include examination of muscle oxygenation parameters and mitochondrial phenotype.

[0064] The reduction in fibrosis can be assessed by MRI, muscle fat reduction, reduction in cardiac fibrosis, increased pinch strength, grip strength, improved cardiac and pulmonary function, and other assessments show a reduced rate of decline in the above functions.

[0065] Quality of life questionnaires are very useful in determining the effectiveness of treatment.

[0066] Clinical outcomes may involve, for example, determining the percentage change in normalized upper limb reachable surface area, the percentage change in cardiac circumferential strain by MRI to assess the strain of the lateral and posterior cardiac walls. Another useful test is to measure forced vital capacity, delayed loss of respiratory function, e.g., change in FVC5p from baseline by spirometry.

[0067] Motor function tests included the mean change in four standard stair climbing tests before and after treatment, floor rise time, magnetic resonance spectroscopy to determine the mean change in the fat fraction of the vastus lateralis by MRS, quadriceps muscle testing, peak torque measurements of the knee extensors, and ultrasound muscle microvascular blood supply to the forearm.

[0068] Important clinical assessments include time to walk / run 6 or 10 meters, time to ascend 4 flights of stairs, time to descend 4 flights of stairs, and time to rise from a supine position. Changes in weight, height, and BMI may also be assessed.

[0069] Alternatively, or in addition, biomarkers from muscle biopsy assessment, pharmacodynamic markers measuring changes in plasma biomarker panels measured by ELISA or proteomics, or changes in circulating immune cell markers are assessed.

[0070] The term "antisense compound" as used herein refers to an oligomeric compound that hybridizes to a nucleic acid molecule encoding the α4 integrin chain of VLA-4 (α4β1) and / or α4β7 integrin. The human α4 integrin chain is CD49d. Antisense compounds may disrupt expression of CD49d, β1 integrin and / or β7 integrin.

[0071] As used herein, the term "nucleic acid molecule encoding an α4 integrin" includes DNA encoding the α4 integrin chain of VLA-4 or α4β7 integrin, RNA transcribed from such DNA (such as pre-mRNA and mRNA or portions thereof), and also cDNA derived from such RNA.

[0072] The term "VLA-4" as used herein refers to a heterodimer of α4 integrin and β1 integrin. VLA-4 is expressed at significant levels on normal peripheral blood B and T cells, thymocytes, monocytes, other cells, and hematopoietic stem and progenitor cells. VLA-4 is also expressed on mesenchymal and endothelial progenitor cells, as well as mesenchymal and possibly endothelial stem cells. Ligands for VLA-4 include vascular cell adhesion molecule-1 (VCAM-1) and CS-1, an alternatively spliced ​​domain within the HepII region of fibronectin.

[0073] The term "α4β7 integrin" as used herein refers to a heterodimer of α4 integrin and β7 integrin. α4β7 integrin identifies a subset of memory T cells with tropism for the gut. α4β7 integrin is also expressed on mast cells, lymphocyte cells, and a subset of NK progenitor cells. α4β7 integrin is expressed on some stem and progenitor cells. Ligands for α4β7 integrin include MAdCam-1 and VCAM-1. nucleic acid

[0074] The present disclosure encompasses the use of various oligonucleotides, also referred to as nucleic acids. Exemplary nucleic acids include DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA)), RNA (e.g., message RNA (mRNA), short hairpin RNA (shRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, DNA or RNA analogs (e.g., base analogs, sugar analogs, and / or non-natural backbones, etc.), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in single or double stranded form. In one example, the nucleic acid is isolated. As used herein, the term "isolated nucleic acid" refers to a nucleic acid that is altered or removed from its natural state by human intervention.

[0075] The term "oligonucleotide" broadly refers to short nucleic acid molecules. Oligonucleotides can easily bind to their complementary oligonucleotides, DNA or RNA in a sequence-specific manner to form double strands. In one embodiment, oligonucleotides are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length or even longer.

[0076] In one embodiment, the oligonucleotide of the present disclosure is an inhibitory oligonucleotide. In one example, the term "inhibitory oligonucleotide" refers to any oligonucleotide that reduces the production, expression, or biological activity of one or more proteins. For example, an inhibitory oligonucleotide can prevent the translation of mRNA into a protein in a ribosome. In another example, an inhibitory oligonucleotide is sufficiently complementary to either a gene or mRNA that codes for one or more proteins to bind (hybridize) to the target gene(s) or mRNA, thereby reducing the expression or biological activity of the target protein. In another example, an inhibitory oligonucleotide inhibits the biological activity of intracellular nucleic acids that do not code for proteins. For example, an inhibitory oligonucleotide can inhibit the biological activity of non-coding RNA.

[0077] The term "antisense" as used herein refers to a sequence of nucleotides that is complementary to and can therefore bind to a coding sequence, which may be either the sequence of a DNA double helix undergoing transcription, or the sequence of a messenger RNA molecule. Antisense DNA is the non-coding strand that is complementary to the coding strand of a double-stranded DNA.

[0078] The term "short hairpin RNA" or "shRNA" refers to an RNA structure having a duplex region and a loop region.

[0079] The term small interfering RNA (siRNA), also called short interfering RNA or silencing RNA, is a class of double-stranded or single-stranded RNA molecules about 19-25 base pairs in length. siRNAs that inhibit or prevent translation into a specific protein are indicated by the protein name combined with the term siRNA. Typically, siRNAs in various embodiments are double-stranded or single-stranded nucleic acid molecules having about 19 to about 28 nucleotides (i.e., about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides).

[0080] The term "microRNA" (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses, which functions in RNA silencing and post-transcriptional regulation of gene expression. The prefix "miR" is followed by a dash and a number. The number often indicates the order of the nomenclature. Different miRNAs that have nearly identical sequences except for one or two nucleotides are annotated with additional lowercase letters. Many miRNAs are known in the art (see miRBase V.21 nomenclature; Kozomara et al. 2013; Griffiths-Jones, S. 2004). The sequences of these miRNAs are well known in the art and can be found, for example, at www.mirbase dot org.

[0081] In one embodiment, the "inhibitory oligonucleotide" mimics the activity of one or more miRNAs. As used herein, the term "miRNA mimic" refers to a small double-stranded RNA molecule designed to mimic endogenous mature miRNA molecules when introduced into cells. miRNA mimics are available from a variety of suppliers, including Sigma Aldrich and Thermo Fisher Scientific.

[0082] In one embodiment, an "inhibitory oligonucleotide" inhibits the activity of one or more miRNAs. A variety of miRNA species are suitable for this purpose. Examples include, but are not limited to, antagomirs, interfering RNAs, ribozymes, miRNA sponges, and miR masks. The term "antagomir" is used in the context of this disclosure to refer to a chemically modified antisense oligonucleotide that binds to a target miRNA and inhibits miRNA function by preventing the binding of the miRNA to its cognate gene target. An antagomir can include any base modification known in the art. In one example, the miRNA species referred to above is about 10-50 nucleotides in length. For example, the antagomir may have an antisense portion of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0083] In one embodiment, the miRNA species is a chimeric oligonucleotide that contains two or more chemically distinct regions, each composed of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confer one or more beneficial properties (e.g., increased nuclease resistance, increased uptake into cells, increased binding affinity for the target, etc.) and a region that is a substrate for an enzyme capable of cleaving RNA:DNA or RNA:RNA hybrids.

[0084] In one embodiment, the nucleic acids included in the present disclosure are synthetic. The term "synthetic nucleic acid" means that the nucleic acid does not have the chemical structure or sequence of a naturally occurring nucleic acid. Synthetic nucleotides include engineered nucleic acid molecules. In another example, the nucleic acid structure can be modified to become a locked nucleic acid (LNA) with a methylene bridge between the 2' oxygen and the 4' carbon, locking the ribose in a 3'-endo (North) conformation in the A-form conformation of the nucleic acid (Lennox et al 2011; Bader et al 2011). In the context of miRNA, such modifications can significantly improve both the target specificity and the hybridization properties of the molecule.

[0085] Nucleic acids for use in the methods disclosed herein may be designed using routine methods as needed. For example, in the context of inhibitory oligonucleotides, target segments of 5, 6, 7, 8, 9, 10 or more nucleotides in length, including a stretch of at least 5 consecutive nucleotides within or directly adjacent to the seed sequence, are believed to be suitable for targeting genes. Exemplary target segments include sequences that include at least 5 consecutive nucleotides from the 5' end of one of the seed sequences (the remaining nucleotides are a continuous stretch of the same RNA that begins immediately upstream of the 5' end of the seed sequence and continues until the nucleic acid includes about 5 to about 30 nucleotides). In another example, the target segment is represented by an RNA sequence that includes at least 5 consecutive nucleotides from the 3' end of one of the seed sequences (the remaining nucleotides are a continuous stretch of the same RNA that begins immediately downstream of the 3' end of the target segment and continues until the nucleic acid includes about 5 to about 30 nucleotides). The term "seed sequence" is used in the context of this disclosure to refer to a 6-8 nt long substring (i.e., seed sequence) within the first 8 nucleotides (nt) at the 5' end of a miRNA that is a key determinant of target specificity. Once one or more target regions, segments or sites are identified, inhibitory nucleic acid compounds are selected that are sufficiently complementary to the target, i.e., hybridize sufficiently well, and have sufficient specificity (i.e., do not substantially bind to other non-target nucleic acid sequences) to impart the desired effect. Antisense compounds against α4 integrin

[0086] In one embodiment, the method of the present disclosure relies on the use of antisense compounds against α4 integrin. Such antisense compounds target nucleic acids encoding the α4 integrin chain of VLA-4 (α4β1) or α4β7 integrin. In some embodiments, the antisense compounds are oligonucleotides. However, other oligomeric antisense compounds are contemplated, including, but not limited to, oligonucleotide mimetics.

[0087] The hybridization of an antisense compound with its target nucleic acid is generally referred to as "antisense". The hybridization of an antisense compound with its target nucleic acid inhibits the function of the target nucleic acid. Such "antisense inhibition" is typically based on hydrogen bond-based hybridization of the antisense compound to the target nucleic acid, such that the target nucleic acid is cleaved, degraded, or otherwise inoperable. The function of the target DNA to be interfered with can include replication and transcription. Replication and transcription can be, for example, from an endogenous cellular template, a vector, a plasmid construct, or otherwise. The function of the RNA to be interfered with can include translocation of the RNA to a protein translation site, translocation of the RNA to a site in the cell that is distant from the site of RNA synthesis, translation of a protein from the RNA, splicing of the RNA to produce one or more RNA species, catalytic activity or complex formation with the RNA that may be involved in or facilitated by the RNA.

[0088] "Hybridization" as used herein refers to the pairing of complementary bases of an oligonucleotide and a target nucleic acid. Base pairing typically involves hydrogen bonding between complementary nucleoside or nucleotide bases (nucleobases), which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds. Guanine (G) and cytosine (C) are examples of complementary nucleobases that pair through the formation of three hydrogen bonds. Adenine (A) and thymine (T) are examples of complementary nucleobases that pair through the formation of two hydrogen bonds. Hybridization can occur under a variety of circumstances.

[0089] A "nucleoside" is a base-sugar combination. The base portion of a nucleoside is usually a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. A "nucleotide" is a nucleoside that further includes a phosphate group covalently linked to the sugar portion of the nucleoside. In the case of nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3', or 5' hydroxyl moiety of the sugar.

[0090] "Specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity between antisense compounds and target nucleic acid so that stable and specific binding occurs.An antisense compound does not need to be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.An antisense compound is specifically hybridizable, and there is a sufficient degree of complementarity to avoid non-specific binding of antisense compounds to non-target sequences under conditions where specific binding is desired, such as physiological conditions in the case of therapeutic treatment, if the binding of antisense compounds to target nucleic acid interferes with the normal function of target molecule, causing loss of activity.

[0091] "Complementary" as used herein refers to the ability for precise pairing between the nucleobase of an antisense compound and a target nucleic acid.For example, if the nucleobase at a certain position of an antisense compound can hydrogen bond with the nucleobase at a certain position of a target nucleic acid, the position of hydrogen bonding between the antisense compound and the target nucleic acid is considered to be a complementary position.Antisense compounds may hybridize to one or more segments such that no intervening or adjacent segments are involved in the hybridization event (e.g., loop structure or hairpin structure).In one embodiment, antisense compounds comprise at least 70% sequence complementarity to the target region in the target nucleic acid.

[0092] For example, 18 nucleobases out of 20 nucleobases are complementary to the target region in the target nucleic acid, so that the specifically hybridized antisense compound exhibits 90% complementarity.In this example, the remaining non-complementary nucleobases may be clustered with complementary nucleobases or may be interspersed with complementary nucleobases, and do not need to be adjacent to each other or to complementary nucleobases.Thus, an 18 nucleobase long antisense compound has four non-complementary nucleobases adjacent to two regions that are completely complementary to the target nucleic acid, and thus has a total complementarity of 77.8% to the target nucleic acid, and is therefore within the scope of the present disclosure.The complementarity percentage of an antisense compound to a region of a target nucleic acid can be determined using the BLAST program (basic local alignment search tool) and PowerBLAST programs (Altschul et al., 1990; Zhang and Madden, 1997), which are generally known in the art. Antisense oligonucleotides

[0093] The present disclosure provides antisense oligonucleotides for inhibiting expression of α4 integrin, and / or VLA-4 and / or α4β7 integrin, such antisense oligonucleotides targeting nucleic acids encoding the α4 integrin chain of VLA-4 or α4β7 integrin.

[0094] As used herein, the term "inhibit" refers to any measurable decrease (eg, 10%, 20%, 50%, 90%, or 100%) in VLA-4 or α4β7 integrin expression.

[0095] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of RNA or DNA, or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, as well as oligonucleotides with non-naturally occurring portions that function similarly. Such modified or substituted oligonucleotides are often preferred over natural forms due to desired properties, such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acid, and increased stability in the presence of nucleases.

[0096] Oligonucleotides may contain chiral (asymmetric) centers, or the entire molecule may be chiral. Individual stereoisomers (enantiomers and diastereoisomers) and their mixtures are within the scope of this disclosure. See Wan et al. Nucleic Acids Research 42 (22:13456-13468, 2014, for disclosure of antisense oligonucleotides comprising chiral phosphorothioate linkages).

[0097] In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound. The respective ends of this linear polymeric compound can then be further linked to form a circular compound. However, linear compounds are generally preferred. Furthermore, linear compounds can have internal nucleobase complementarity and thus can fold to produce a fully or partially double-stranded compound. With respect to oligonucleotides, the phosphate groups are generally referred to as forming the internucleoside backbone of the oligonucleotide. The usual linkage or backbone of RNA and DNA is the 3' to 5' phosphodiester bond.

[0098] Antisense oligonucleotides of the present disclosure include, for example, ribozymes, siRNAs, external guide sequence (EGS) oligonucleotides, alternative splicers, primers, probes, and other oligonucleotides that hybridize to at least a portion of target nucleic acid.Antisense oligonucleotides of the present disclosure can be administered in the form of single strands, double strands, circular or hairpins, and can include structural elements such as internal or terminal bulges or loops.Antisense oligonucleotides, when administered, can induce the action of one or more enzymes or structural proteins, resulting in the modification of target nucleic acid.

[0099] One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds that are "DNA-like" induce RNAse H. Thus, the activation of RNase H leads to the cleavage of RNA targets, which greatly improves the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles are assumed for other ribonucleases, such as RNaseIII and ribonuclease L family enzymes.

[0100] The introduction of double-stranded RNA (dsRNA) molecules has been shown to induce potent and specific antisense-mediated reduction of the function of genes or their associated gene products. This phenomenon occurs in both plants and animals and is thought to have an evolutionary relationship to viral defense and transposon silencing. The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from studies in the nematode Caenorhabditis elegans (Guo and Kempheus, 1995). Montgomery et al. (1998) showed that the major interference effect of dsRNA is posttranscriptional. The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been called RNA interference (RNAi). The term has been generalized to mean antisense-mediated gene silencing that involves the introduction of dsRNA resulting in a sequence-specific reduction in the levels of endogenous target mRNA (Fire et al., 1998). Recently, it has been shown that it is indeed single-stranded RNA oligomers of antisense polarity of dsRNA that are the potent inducers of RNAi ( Tijsterman et al., 2002 ).

[0101] One of ordinary skill in the art would be able to identify antisense oligonucleotides that are useful in the methods of the present disclosure without undue experimentation. Modified internucleoside linkages (backbone)

[0102] The antisense compounds of the present disclosure include oligonucleotides having modified backbones or non-natural internucleoside linkages. Oligonucleotides with modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0103] Modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidate and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates having the normal 3'-5' linkages, 2'-5' linked analogs thereof, as well as those having inverted polarity where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linked. Oligonucleotides with polarity inversion contain a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e., a single inverted nucleoside residue that may be abasic (either lacking a nucleobase or having a hydroxyl group instead).Various salts, mixed salts, and free acid forms are also included.

[0104] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453, 5,547, 5,551, 5,560, 5,571, 5,580, 5,593, 5,607, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 630, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 670, 671, 672 ,496, US5,455,233, US5,466,677, US5,476,925, US5,519,126, US5,536,821, US5,541,306, US5,550,111, US5,563,253, US5,571,799, US5,587,361, US5,194,599, US5,565,555, US5,527,899, US5,721,218, US5,672,697, and US5,625,050.

[0105] Modified oligonucleotide backbones that do not contain phosphorus atoms include, for example, backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonic acid and sulfonamide backbones; amide backbones; and other components with mixed N, O, S, and CH2 component moieties.

[0106] Representative U.S. patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, , US5,541,307, US5,561,225, US5,596,086, US5,602,240, US5,610,289, US5,602,240, US5,608,046, US5,610,289, US5,618,704, US5,623,070, US5,663,312, US5,633,360, US5,677,437, US5,792,608, US5,646,269, and US5,677,439. Modified sugars and internucleoside linkages

[0107] Antisense compounds of the present disclosure include oligonucleotide mimetics in which both the sugar and the internucleoside linkage (i.e., backbone) of the nucleotide units are replaced with novel groups, while the nucleobase units are maintained for hybridization with a target nucleic acid.

[0108] The oligonucleotide mimics that have been shown to have excellent hybridization properties are called peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of oligonucleotides is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleobases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. No. 5,539,082; U.S. Pat. No. 5,714,331; and U.S. Pat. No. 5,719,262. Further teachings on PNA compounds can be found in Nielsen et al., 1991.

[0109] Antisense compounds of the present disclosure also include oligonucleotides having phosphorothioate backbones and oligonucleotides having heteroatom backbones, such as, -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- [known as the methylene (methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -ON(CH3)-CH2-CH2- [wherein the natural phosphodiester backbone is represented as -OPO-CH2-] (U.S. Pat. No. 5,489,677), and the amide backbones of U.S. Pat. No. 5,602,240.

[0110] Antisense compounds of the present disclosure also include oligonucleotides having morpholino backbone structures as described in US Pat. No. 5,034,506. modified sugar

[0111] Antisense compounds of the present disclosure include oligonucleotides with one or more substituted sugar moieties.

[0112] Examples include oligonucleotides containing any of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl.

[0113] In one embodiment, the oligonucleotide comprises one of the following at the 2' position: O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10.

[0114] Further examples of modified oligonucleotides include oligonucleotides containing at the 2' position any one of C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties.

[0115] In one embodiment, the modification comprises 2'-methoxyethoxy (also known as 2'-O-CH2CHOCH3 (2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995), i.e., an alkoxyalkoxy group. In a further embodiment, the modification comprises 2'-dimethylaminooxyethoxy, i.e., an O(CH2)2ON(CH3)2 group (also known as 2'-DMAOE), or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0116] Other modifications include 2'-methoxy (2'-O-CH), 2'-aminopropoxy (2'-OCHCHCHNH), 2'-allyl (2'-CH-CH=CH), 2'-O-allyl (2'-O-CH-CH=CH), and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) or ribo (down) position. In one embodiment, the 2'-arabino modification is 2'-F.

[0117] Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.

[0118] Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0119] Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, Nos. 5,567,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, 5,792,747, and 5,700,920.

[0120] Further modifications of the sugar include Locked Nucleic Acids (LNAs) in which a 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In one embodiment, the linkage is a methylene (-CH2-)n group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNAs and their preparation are described in WO98 / 39352 and WO99 / 14226. Natural and Modified Nucleobases

[0121] The antisense compounds of the present disclosure include oligonucleotides having nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0122] Modified nucleotides include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-CC-CH3) uracil, and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, 6-aminoadenine, 6-methyl- and other alkyl derivatives of adenine and guanine, 2-propyl- and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-CC-CH3) uracil, and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, 6-aminoadenine ... Examples of substituted uracils include aryl, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenines.

[0123] Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).

[0124] Modified nucleobases can also include those in which purine or pyrimidine bases are replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Furthermore, nucleobases include those disclosed in US 3,687,808, those disclosed in JI Kroschwitz (editor), The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, John Wiley and Sons (1990), and those disclosed by Englisch et al., (1991), and those disclosed by YS Sanghvi, Chapter 15: Antisense Research and Applications, pages 289-302, STCrooke, B. Lebleu (editors), CRC Press, 1993.

[0125] Some of these nucleobases are particularly useful for increasing the binding affinity of oligonucleotides. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6 to 1.2 degrees Celsius. In one embodiment, these nucleobase substitutions are combined with 2'-O-methoxyethyl sugar modifications.

[0126] Representative U.S. patents that teach the preparation of the above specific modified nucleobases and other modified nucleobases include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,48 US 4,908, US 5,502,177, US 5,525,711, US 5,552,540, US 5,587,469, US 5,594,121, US 5,596,091, US 5,614,617, US 5,645,985, US 5,830,653, US 5,763,588, US 6,005,096, US 5,681,941, and US 5,750,692. conjugate group

[0127] Antisense compounds of the disclosure may be conjugated to one or more moieties or groups that enhance the activity, cellular distribution or cellular uptake of the antisense compound.

[0128] These moieties or groups may be covalently attached to functional groups such as primary or secondary hydroxyl groups.

[0129] Exemplary moieties or groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that improve the pharmacodynamic properties of an oligomer, and groups that improve the pharmacokinetic properties of an oligomer.Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0130] Moieties or groups that enhance the pharmacodynamic properties include those that improve uptake, increase resistance to degradation, and / or enhance sequence-specific hybridization with the target nucleic acid.

[0131] Moieties or groups that improve the pharmacokinetic properties include those that improve the uptake, distribution, metabolism or excretion of the compounds of the present disclosure. Representative moieties or groups are disclosed in PCT / US92 / 09196 and US6,287,860. Moieties or groups include, but are not limited to, lipid moieties, such as cholesterol moieties, cholic acid, thioethers, such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains, such as dodecanediol or undecyl residues, phospholipids, such as dihexadecyl-rac-glycerol or triethylammonium, 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties. Chimeric Compounds

[0132] As will be understood by those of skill in the art, it is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide, or even a single nucleoside within an oligonucleotide.

[0133] Antisense compounds of the present disclosure include chimeric oligonucleotides. "Chimeric oligonucleotides" contain two or more chemically distinct regions, each composed of at least one monomer unit, i.e., nucleotides, in the case of oligonucleotide compounds. These oligonucleotides typically contain at least one region in which the oligonucleotide is modified to confer increased resistance to nuclease degradation, increased cellular uptake, increased stability, and / or increased binding affinity to the target nucleic acid. Additional regions of the oligonucleotide may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly improving the efficiency of oligonucleotide-mediated inhibition of gene expression. Cleavage of RNA:RNA hybrids can be achieved in a similar manner by the action of endoribonucleases such as RNAseL, which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

[0134] The chimeric antisense compound of the present disclosure can be formed as a composite structure of two or more oligonucleotides, modified oligonucleotides, and / or oligonucleotide mimetics.Such compounds are also called hybrids or gapmers in the art.Representative US patents that teach the preparation of such hybrid structures include, but are not limited to, US5,013,830, US5,149,797, US5,220,007, US5,256,775, US5,366,878, US5,403,711, US5,491,133, US5,565,350, US5,623,065, US5,652,355, US5,652,356, and US5,700,922. Exemplary Oligonucleotides

[0135] Exemplary antisense platforms known in the art include, but are not limited to, morpholino, first generation oligo, second generation oligo, gapmer, siRNA, LNA, BNA, or oligomimetic such as peptide nucleic acid. Oligonucleotides can be naked or formulated in liposomes. Oligonucleotides can be linked or not to a delivery means to cells. Oligonucleotides can be used or not with endosomal release agents.

[0136] In one embodiment, the antisense compounds are second generation phosphorothioate backbone 2'-MOE modified chimeric oligonucleotide gapmers designed to hybridize to the 3' untranslated region of VLA-4 mRNA. In one embodiment, the oligonucleotides selectively inhibit expression of VLA-4 in both primary human cells and several human cell lines by hybridizing to RNA encoding the α4 integrin subunits of VLA-4, CD49 and α4β7 integrin.

[0137] In one embodiment, the oligonucleotide is a 19 sodium salt of a 3'→5' phosphorothioate oligonucleotide 20mer, also referred to as a 3-9-8 MOE gapmer, having a molecular weight of 7230 daltons, in which the nucleotides at positions 1-3 from the 5' end are 2'-O-(2-methoxyethyl) (2'MOE) modified ribonucleosides (2'-O-(2-methoxyethyl ribose); the nucleotides at positions 4-12 from the 5' end are 2'-deoxyribonucleosides (all cytosines are 5-methylcytosines); and the nucleotides at positions 13-20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides.

[0138] In one embodiment, the sequence of the oligonucleotide is (SEQ ID NO:1): 5'- Me C Me UG AGT Me CTG TTT Me U Me CMe CA Me U Me U Me C Me U-3' The empirical formula of the oligonucleotide is: C 233 H 327 N 60 O 129 P 19 S 19 Na 19

[0139] The antisense oligonucleotide ATL1102 has previously been shown to be effective in the central nervous system disorder, MS, and at doses significantly higher than those proposed herein (Limmroth et al.). The ability of antisense oligonucleotides against the CD49d alpha chain of VLA-4 to selectively inhibit VLA-4 in immune cells prevents serious safety events such as PML. This is characterized by the administration of antibodies and small molecule inhibitors of VLA-4 that are pan-VLA-4 inhibitors, affecting all cells expressing VLA-4.

[0140] In one embodiment, all uracils are 5-methyluracil (MeU). Typically, oligonucleotides are synthesized using 2-methoxyethyl modified thymidines rather than 5-methyluracil.

[0141] In one embodiment, all pyrimidines are C5 methylated (ie, U, T, C are C5 methylated).

[0142] In one embodiment, the sequence of the oligonucleotides may be named according to accepted oligonucleotide nomenclature, indicating each O--O linked phosphorothioate internucleotide linkage: 2'-O-Methoxyethyl-5-methylcytidyl-(3'->5'O,O-phosphorothioyl)-2'-O-Methoxyethyl-5-methyluridyl-(3'->5'O,O-phosphorothioyl)-2'-O-Methoxyethylguanosilyl-(3'->5'O,O-phosphorothioyl)-2'-O-Deoxyadenosilyl-(3'->5'O,O-phosphorothioyl)-2'-O-Deoxyguanosilyl-(3'-> 5'O,O-phosphorothioyl)-thymidylyl-(3'->5'O,O-phosphorothioyl)-2'-deoxy-5-methylcytidylyl-(3'->5'O,O-phosphorothioyl)-thymidylyl-(3'->5'O,O-phosphorothioyl)-2'-deoxyguanosylyl-(3'->5'O,O-phosphorothioyl)-thymidylyl-(3'->5'O,O-phosphorothioyl)-thymidylyl-(3'->5'O,O-phosphorothioyl) -phosphorothioyl)-thymidylyl-(3'->5'O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methyluridyl-(3'->5'O,O-phosphorothioyl)-2'-methoxyethyl-5-methylcytidylyl-(3'->5'O,O-phosphorothioyl)-2'-methoxyethyl-5-methylcytidylyl-(3'->5'O,O-phosphorothioyl)-2'-O-methoxyethyl-5-adenosyl Lyl-(3'->5'O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methyluridyl-(3'->5'O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methyluridyl-(3'->5'O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methylcytosine, (3'->5'O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methyluridyl-19 sodium salt.

[0143] Oligonucleotides can be synthesized by a multi-step process that can be divided into two distinct operations: solid-phase synthesis and downstream processing. In the first operation, the nucleotide sequence of the oligonucleotide is assembled via a computer-controlled solid-phase synthesizer. Subsequent downstream processing includes a deprotection step, preparative reversed-phase chromatography purification, isolation, and drying to produce the oligonucleotide drug substance. Chemical synthesis of oligonucleoids utilizes phosphoramidite coupling chemistry followed by oxidative sulfurization and involves the sequential coupling of activated monomers to an elongating oligomer whose 3' end is covalently attached to a solid support. Detritylation (reaction a)

[0144] Each cycle of solid-phase synthesis begins with the removal of the acid-labile 5'-O-4,4'-dimethoxytrityl (DMT) protecting group of the 5'-terminal nucleoside of the support-bound oligonucleotide. This is accomplished by treatment with an acidic solution (e.g., dichloroacetic acid (DCA) in toluene). After detritylation, excess reagent is removed from the support by washing with acetonitrile in preparation for the next reaction. Coupling (reaction b)

[0145] Chain extension is achieved by reaction of the 5'-hydroxyl group of the support-bound oligonucleotide with a solution of a phosphoramidite corresponding to that particular base position (e.g., for base 2: MOE-MeC amidite) in the presence of an activator (e.g., 1H-tetrazole). This forms a phosphite triester linkage between the incoming nucleotide synthon and the support-bound oligonucleotide chain. After the coupling reaction, excess reagent is removed from the support by washing with acetonitrile in preparation for the next reaction. Sulfurization (reaction c)

[0146] The newly formed phosphite triester linkage is converted to the corresponding [O,O,O)-trialkylphosphorothioate triester by treatment with a solution of a sulfur transfer reagent (e.g., phenylacetyl disulfide). After sulfurization, excess reagent is removed from the support by washing with acetonitrile in preparation for the next reaction. Capping (reaction d)

[0147] A small fraction of the 5'-hydroxy groups available in any given cycle cannot be extended. If these groups are attached in any of the subsequent cycles, process-related impurities ("DMT-on(nl)-mers") are formed that are difficult to separate from the desired product. To prevent the formation of these impurities and to facilitate purification, a "capping reagent" (e.g., acetic anhydride and N-methylimidazole / acetonitrile / pyridine) is introduced into the reaction vessel to obtain capped sequences. The resulting failed sequences ("DMT-off shortmers") are separated from the desired product by reverse-phase HPLC purification. After the capping reaction, excess reagent is removed from the support by washing with acetonitrile in preparation for the next reaction.

[0148] Repeating this basic four-step cycle using appropriately protected nucleoside phosphoramidites allows the assembly of the entire protected oligonucleotide sequence. Deprotection of the backbone (reaction e)

[0149] After completion of the assembly portion of the process, the cyanoethyl groups protecting the (O,O,O)-trialkylphosphorothioate triester internucleotide linkages are removed by treatment with a solution of triethylamine (TEA) in acetonitrile. The reagents and acrylonitrile generated during this step are removed by washing the column with acetonitrile. Cleavage from the support and deprotection of the base (reaction f)

[0150] Deprotection of the exocyclic amino group and cleavage of the crude product from the support is achieved by incubation with aqueous ammonium hydroxide (reaction f). Purification of the crude 5'-O-DMT protected product is performed by reversed-phase HPLC. The reversed-phase HPLC step removes the DMT-off hindered sequences. The elution profile is monitored by UV absorption spectroscopy. Fractions containing the DMT-on oligonucleotide product are collected and analyzed. Acidic deprotection (reaction g)

[0151] The reverse-phase HPLC fractions containing the 5'-O-DMT protected oligonucleotide are pooled and transferred to a precipitation tank. Products from the purification of several syntheses are combined at this stage of the process. The purified DMT-on oligonucleotide is treated with acid (e.g., acetic acid) to remove the DMT group attached to the 5' end. After a period of acid exposure and neutralization, the oligonucleotide drug substance is isolated and dried.

[0152] After the final acidic deprotection step, the solution is neutralized by adding aqueous sodium hydroxide and the oligonucleotide drug substance is precipitated from the solution by adding ethanol. The precipitated material is allowed to settle to the bottom of the reaction vessel and the ethanolic supernatant is decanted. The precipitated material is redissolved in purified water and the pH of the solution is adjusted to pH 7.2-7.3. The precipitation step is repeated. The precipitated material is dissolved in water and the solution is filtered through a 0.45 micron filter, transferred to a disposable polypropylene tray and added to the lyophilizer. The solution is cooled to -50°C. Primary drying is performed at 25°C for 37 hours. The temperature is increased to 30°C and a secondary drying step was performed for 5.5 hours. After completion of the lyophilization process, the drug substance is transferred to high density polyethylene bottles and stored at -200°C. target nucleic acid

[0153] "Targeting" an antisense compound to a particular nucleic acid can be a multi-step process. This process usually begins with the identification of the target nucleic acid whose function is to be modulated. In the present disclosure, the target nucleic acid encodes the alpha 4 integrin chain of VLA-4 or α4β7 integrin.

[0154] The targeting process typically involves determining at least one target region, segment, or site within a target nucleic acid where antisense interactions will occur resulting in a desired effect (e.g., inhibition of expression). As used herein, a "region" is defined as a portion of a target nucleic acid that has at least one identifiable structure, function, or characteristic. Within a region of a target nucleic acid are segments. A "segment" is defined as a smaller or subpart of a region within a target nucleic acid. As used herein, a "site" refers to a location within a target nucleic acid.

[0155] A "translation initiation codon" is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), and thus the translation initiation codon is also referred to as an "AUG codon," "start codon," or "AUG start codon." A small number of genes have translation initiation codons with the RNA sequences 5'-GUG, 5'-UUG, or 5'-CUG, with 5'-AUA, 5'-ACG, and 5'-CUG having been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiating amino acid in each case is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes can have two or more alternative start codons, any of which may be preferentially utilized to initiate translation in a particular cell type or tissue, or under a particular set of conditions. As used herein, the terms "start codon" and "translation initiation codon" refer to one or more codons used in vivo to initiate translation of an mRNA transcribed from a gene encoding, for example, the α4 integrin chain of VLA-4 or α4β7 integrin, regardless of the sequence or sequences of such codons.

[0156] A "translation termination codon," also referred to as a "stop codon," can have one of the three RNA sequences 5'-UAA, 5'-UAG, and 5'-UGA (5'-TAA, 5'-TAG, and 5'-TGA, respectively, in the corresponding DNA molecule). As used herein, the terms "translation termination codon" and "stop codon" refer to one or more codons used in vivo to terminate the translation of an mRNA transcribed from a gene encoding VLA-4 or the α4 integrin chain of α4β7 integrin, regardless of the sequence or sequences of such codons.

[0157] The terms "start codon region" and "translation initiation codon region" refer to a portion of an mRNA or gene that encompasses about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from the translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of an mRNA or gene that encompasses about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from the translation termination codon. Thus, "start codon regions" or "translation initiation codon regions" and "stop codon regions" or "translation termination codon regions" are all regions that can be effectively targeted with the antisense compounds of the present disclosure.

[0158] "Open reading frames" (ORFs) or "coding regions", known in the art to refer to the region between a translation initiation codon and a translation termination codon, are also regions that can be effectively targeted. In one embodiment, the intragenic region that includes the translation initiation or termination codon of the ORF of a gene is targeted.

[0159] Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA 5' from the translation initiation codon, and thus the 5' cap site and translation initiation codon (or corresponding nucleotides on a gene) of the mRNA, and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA 3' from the translation termination codon, and thus the nucleotides between the translation termination codon and the 3' end of the mRNA (or corresponding nucleotides on a gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue attached to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself and the first 50 nucleotides adjacent to the cap site. In one embodiment, the 5' cap region is targeted.

[0160] Some eukaryotic mRNA transcripts are directly translated, but many contain one or more regions, known as "introns," that are excised from the transcript before it is translated. The remaining (and thus translated) regions are known as "exons" and are spliced ​​together to form a continuous mRNA sequence. mRNA transcripts generated through the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts." In one embodiment, introns, or splice sites, i.e., intron-exon or exon-intron junctions, or aberrant fusion junctions due to rearrangements or deletions, are targeted. Alternative RNA transcripts can be generated from the same genomic region of DNA. These alternative transcripts are commonly known as "variants."

[0161] A "pre-mRNA variant" is a transcript produced from the same genomic DNA that differs from other transcripts produced from the same genomic DNA in either the start or stop position and contains both intron and exon sequences. When one or more exon or intron regions or parts thereof are excised during splicing, the pre-mRNA variant produces a smaller "mRNA variant". As a result, the mRNA variant is a processed pre-mRNA variant, and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splicing variants". If no splicing of the pre-mRNA variant occurs, the pre-mRNA variant is identical to the mRNA variant.

[0162] Variants can be generated by using alternative signals to start or stop transcription, i.e., by using alternative start or stop codons. Variants derived from pre-mRNA or mRNA using alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. These transcripts using alternative stop codons are known as "alternative stop variants" of that pre-mRNA or mRNA. One particular type of alternative stop variant is the "polyA variant", where multiple internally generated transcripts result from alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby generating transcripts that terminate at unique polyA sites. In one embodiment, pre-mRNA or mRNA variants are targeted.

[0163] The position on target nucleic acid that antisense compound hybridizes to is called "target segment".As used herein, the term "target segment" is defined as at least 8 nucleobases of the target region that antisense compound targets.Without wishing to be bound by theory, it is currently believed that these target segments represent the part of target nucleic acid that is accessible for hybridization.

[0164] Once one or more target regions, segments or sites have been identified, antisense compounds are selected that are sufficiently complementary to the target segment, i.e., that hybridize sufficiently well and with sufficient specificity to impart the desired effect.

[0165] Target segments can also be combined with their respective complementary antisense compounds to form stabilized double-stranded (double-stranded) oligonucleotides. Such double-stranded oligonucleotide moieties have been shown in the art to regulate target expression and regulate translation, as well as RNA processing via antisense mechanisms. In addition, double-stranded moieties may undergo chemical modification (Fire et al., 1998; Timmons and Fire, 1998; Timmons et al., 2001; Tabara et al., 1998; Montgomery et al., 1998; Tuschl et al., 1999; Elbashir et al., 2001a; Elbashir et al., 2001b). For example, such double-stranded moieties have been shown to inhibit targets by classical hybridization of the antisense strand of the double strand to the target, thereby causing enzymatic degradation of the target (Tijsterman et al., 2002). composition

[0166] The antisense compounds of the present disclosure may be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds to aid in uptake, distribution, and / or absorption, resulting in, for example, liposomal, receptor-targeting molecules, oral, rectal, topical, or other formulations. Representative U.S. patents that teach the preparation of such uptake, distribution, and / or absorption-aiding formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921, 5,354,844, 5,416,016, 5,459,127, 5,521,291, 5,543,158, 5,547,932, 5,583,020, 5,591,721, 5,426,330, 5,534,899, 5,013,556, 5,1 US 5,512,295, US 5,527,528, US 5,534,259, US 5,543,152, US 5,556,948, US 5,580,575, and US 5,595,756.

[0167] The antisense compounds of the present disclosure can be administered in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a molecular entity that does not cause allergic, toxic or adverse reactions when administered to a subject, particularly a mammal, more particularly a human. The pharmaceutically acceptable carrier may be solid or liquid. Useful examples of pharmaceutically acceptable carriers include, but are not limited to, diluents, solvents, surfactants, excipients, suspending agents, buffers, lubricants, adjuvants, vehicles, emulsifiers, absorbents, dispersion media, coating agents, stabilizers, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, sequestering agents, isotonic and absorption retarding agents that do not affect the activity of the active agent of the present disclosure.

[0168] In one embodiment, the pharmaceutical carrier is water for injection (WFI) and the pharmaceutical composition is adjusted to pH 7.4, 7.2 to 7.6.

[0169] In one embodiment, the salt is a sodium or potassium salt.

[0170] Oligonucleotides may contain chiral (asymmetric) centers or the entire molecule may be chiral. The individual stereoisomers (enantiomers and diastereoisomers) and mixtures thereof are within the scope of this disclosure.

[0171] Antisense compounds of the disclosure may be pharma- ceutically acceptable salts, esters, or salts of esters, or any other compounds capable of providing (directly or indirectly) a biologically active metabolite upon administration.

[0172] As used herein, the term "pharmaceutical acceptable salt" refers to a physiologically and pharma- ceutical acceptable salt of an antisense compound that retains the desired biological activity of the parent compound and does not impart undesirable toxicological effects upon administration. Examples of pharma-ceutical acceptable salts and their uses are further described in US 6,287,860.

[0173] The antisense compounds of the present disclosure may be prodrugs or pharma- ceutically acceptable salts of prodrugs, or other bioequivalents. The term "prodrug" as used herein refers to a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) upon administration by the action of endogenous enzymes or other chemicals and / or conditions. In particular, the prodrug forms of the antisense compounds of the present disclosure are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO93 / 24510, WO94 / 26764, and US5,770,713.

[0174] A prodrug can be converted in the body to its active form, which has medical effects, for example, by hydrolysis in blood. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the ACS Symposium Series (1976); "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985; and Edward B. Roche, ed. Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, which are incorporated herein by reference. Those skilled in the art of organic chemistry will understand that many organic compounds can form complexes with the solvents in which they react or in which they are precipitated or crystallized. These complexes are known as "solvates". For example, complexes with water are known as "hydrates". Conventional treatment

[0175] Corticosteroid therapy is the mainstay of treatment for DMD in ambulatory patients. "Corticosteroid" refers to any one of several synthetic or naturally occurring substances with the general chemical structure of a steroid that mimics or augments the effects of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (prednisone, the precursor of prednisolone, methylprednisolone, etc.), dexamethasone triamcinolone, budesonide, and betamethasone.

[0176] In one embodiment, the present invention's treatment for MD in a human subject with MD comprises administering to the subject an effective amount of a therapeutic agent, such as an antisense oligonucleotide against CD49d (alpha chain of VLA-4), and further comprises administering to the subject an effective amount of a second pharmaceutical agent, i.e., a corticosteroid. In one embodiment, the corticosteroid is prednisone (or a prednisone equivalent), deflazacort (derivative or prednisolone). Other corticosteroids are known in the art as described above.

[0177] Concomitant administration herein includes coadministration using separate formulations (or a single pharmaceutical formulation) and sequential administration in any order, generally with a period of time during which both (or all) active agents exert their biological activities simultaneously.

[0178] Corticosteroid treatment at standard doses has been shown to have some effect in maintaining ambulation in some patients, and is therefore used in ambulatory DMD patients. However, long-term treatment at standard doses (0.75 mg / kg / day prednisone or 0.9 mg / kg / day deflazacort) may cause muscle atrophy and / or other side effects. Non-ambulatory DMD patients who may continue CS have no standard treatment, which may be a fixed dose of CS used when they lost ambulation, i.e., a reduced dose of CS (mg / kg / day), or are taken off CS treatment due to side effects and / or lack of benefit. As proposed and shown herein, antisense treatment against CD49d in combination with corticosteroid treatment, such as a reduction in the level of corticosteroid treatment, in non-ambulatory subjects reduced or delayed the progression of muscle function. This supports ATL1102 monotherapy or combination therapy, as it is unclear whether CS treatment provided any benefit in such subjects.

[0179] As used herein, the term "combination" in the context of administering a therapy refers to the use of multiple therapies or therapeutic agents.The use of the term "in combination" does not limit the order in which a therapy or therapeutic agent is administered to a subject.A therapy or therapeutic agent can be administered before, simultaneously with, or after administering a second therapy or therapeutic agent to a subject. Administration

[0180] In one embodiment, the antisense compounds of the present disclosure are administered systemically. As used herein, "systemic administration" refers to either enteral or parenteral routes of administration.

[0181] As used herein, "enteral" refers to a mode of administration involving any part of the gastrointestinal tract, including, for example, oral administration of antisense oligonucleotides in tablet, capsule, or drop form; gastric feeding tube, duodenal feeding tube, or gastrostomy; and rectal administration of antisense compounds, for example, in suppository or enema form.

[0182] As used herein, "parenteral" includes administration by injection or infusion. Examples include intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), subcutaneous (under the skin), intraosseous injection (into the bone marrow), intradermal (into the skin itself), intrathecal (into the spinal canal), intraperitoneal (injection or injection into the peritoneum), intravesical (injection into the bladder), transdermal (diffusion through intact skin), transmucosal (diffusion through mucous membranes), and inhalation.

[0183] In one embodiment, administration of the pharmaceutical composition is subcutaneous.

[0184] Antisense compounds can be administered as a single dose or on a periodic basis, for example, once daily, once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, once per week, twice per week, three times per week, or as repeated doses every two or three weeks.

[0185] In one embodiment, administration is 1-3 times per week, or once per week, every 2 weeks, 3 weeks, 4 weeks, or every 2 months.

[0186] In one embodiment, administration is once a week.

[0187] In one embodiment, a low dose is administered for 3-6 months, for example, about 25-50 mg / week for at least 3-6 months and then for up to 12 months, and chronically.

[0188] Exemplary doses are about 10 mg to 300 mg. Exemplary doses include 25, 50, 100, 150, 200 mg. Exemplary doses include 1.5 mg / kg (about 50 to 100 mg) and 3 mg / kg (100 to 200 mg) and 4.5 mg / kg (150 to 300 mg). In one embodiment, the dose is administered once a week. Thus, in one embodiment, a low dose of about 10 mg to 30 mg, or 20 mg to 40 mg, or 20 mg to 28 mg can be administered to a subject typically weighing about 25 kg to 65 kg. In one embodiment, the antisense oligonucleotide is administered at a dose of less than 50 mg, or less than 30 mg, or about 25 mg per dose to produce a therapeutic effect. In one embodiment, the antisense oligonucleotide, ATL1102, is administered at a dose of less than 50 mg, or less than 30 mg, or about 25 mg per dose to produce a therapeutic effect.

[0189] In one embodiment, therapeutic effect, such as delay of progression, is observed within about 3 months after administration of the first dose.The term "therapeutically effective amount" as used herein refers to the dose of antisense compound that is sufficient to improve, for example, one or more markers, signs or symptoms of muscular dystrophy, or to delay the progression of muscular dystrophy in a subject, or to improve one or more markers, signs or symptoms of dystrophic muscle fibers, or to delay the progression of muscular dystrophy in a subject under administration conditions.

[0190] In another embodiment, administration is effective to provide a Cmax of the oligonucleotide in the plasma of a human subject of greater than or equal to 2890 ng / mL, in one embodiment, about 10,000-11,000 ng / mL.

[0191] In another embodiment, the administration is effective to provide a Cmin or Ctrough of the oligonucleotide in the plasma of the human subject of at least 2.5 ng / mL, in one embodiment at least 20 ng / mL, or at least 45 ng / mL.

[0192] The study is carried out to demonstrate the treatment of muscular dystrophy with inhibitory oligonucleotides against VLA-4 integrin, which reduces the level of VLA-4 in the blood or muscle of human subjects.The reduction in the level of VLA-4 can be detected in a subset of cells in one or more organs, such as blood, muscle, or lung.In one embodiment, the subject is weaned off corticosteroids about 24 hours before administration.

[0193] This allows the evaluation of the effect of inhibitory antisense oligonucleotides against the CD49d alpha chain of the VLA-4 integrin on immune cells in the absence of corticosteroids, which are not present in significant levels in the bloodstream at 24 hours post-administration to have an effect on circulating immune cells. Example 1

[0194] In one embodiment, ATL1102 is administered with DMD to non-ambulatory young (or adolescent) boys aged 10 years or older at about 1.5 mg / kg (about 50-100 mg), 3 mg / kg (100-200 mg), and 4.5 mg / kg (150-300 mg) weekly for up to 12 weeks. For example, the effect of the administered oligonucleotide on motor / muscle function and inflammatory markers is measured. Markers of muscle degeneration-regeneration and fibrosis are also evaluated. Markers may be detected in situ or in samples such as plasma, urine, muscle biopsy, etc. Inspiratory and expiratory pressures, peak cough flow rate, and FVC are assessed to assess changes in respiratory performance. Muscle function is assessed using the percentage change in normalized upper limb reachable surface area, the percentage change in Performance of the Upper Limb Assessment score, and the percentage change in the (PROM-UL) Person-Reported Outcome Measure Upper Limb functional capacity score. Quality of life questionnaires are useful in determining the effectiveness of treatment. Corticosteroids may be administered once daily or less frequently. Prednisolone at 0.75 mg / kg / day and deflazacort at 0.9 mg / kg / day may be administered at two-thirds, half, or one-third of the standard dose as standard treatment for ambulatory DMD patients. Example 2

[0195] In one embodiment, ATL1102 is administered with DMD at about 1.5 mg / kg (about 10-100 mg) and 3 mg / kg (20-200 mg) and 4.5 mg / kg (30-300 mg) weekly for up to 12 weeks to non-ambulatory pediatric boys aged 4-11 years or older. For example, the effect of the administered oligonucleotide on motor / muscle function and inflammatory markers is determined. Ambulatory pediatric boys may have good or poor ambulatory ability. Maintenance or reduction of loss of ambulatory ability may be assessed by methods known to those skilled in the art. Example 3

[0196] In one embodiment, 10 non-ambulatory DMD patients aged 12-18 years receive ATL1102 at a starting dose of 3 mg / kg once a week for 4 weeks. The first 5 patients continue at 3 mg / kg / week for an additional 4 weeks, while the remaining 5 patients increase the dose to 4.5 mg / kg / week (2.25 mg / kg twice a week) for 4 weeks. After 8 weeks of treatment, a 4-week monitoring period is performed. During the treatment and monitoring period, evaluations are performed at baseline, 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 and 12 weeks. The primary activity outcomes are to evaluate safety, including changes in circulating lymphocytes, CD4+ and CD8+ T cells, and hi CD49d T cell counts, injection site reactions, platelet changes, liver enzymes GGT-bilirubin, CRP, and albumin, and A / G ratio at 4 and 8 weeks of treatment versus baseline. Secondary clinical endpoints include measures of strength, functional capacity, quality of life, and respiratory markers in upper limb function, as well as MRI assessments of muscle fibrosis, fatty inflammation-edema, atrophy, and pharmacokinetics.Exploratory outcome measures include muscle inflammation, muscle fibrosis, muscle apoptosis / degeneration, and those related to muscle regeneration including cytokines, and proteomic and serum / plasma biomarker responses including mononuclear cell RNA arrays and exosomal RNA. Example 4 Low doses of inhibitory oligonucleotides

[0197] In one embodiment, nine non-ambulatory DMD patients, age 10-18 years, weighing 25kg-60kg, receive ATL1102 at a starting dose of 25mg once weekly for 24 weeks. After 24 weeks of treatment, an 8-week monitoring period will be conducted. During the treatment and monitoring period, assessments will be at baseline, every 2 weeks during the treatment period, and every 4 weeks during the post-treatment monitoring period. The primary activity outcomes will be to evaluate safety, including circulating lymphocyte, CD4+ and CD8+ T cell, and hi CD49d T cell counts, injection site reactions, platelet changes, changes in liver enzymes GGT-bilirubin, CRP, and albumin, A / G ratio at 4 and 8 weeks of treatment versus baseline. Secondary endpoints for clinical evaluation are strength in upper limb function (measured by myoset), functional capacity PUL-2 (measured by performance of upper limb module for DMD2.0), quality of life, and respiratory markers, and MRI assessment of muscle fibrosis, lipoinflammation-edema, and atrophy, and pharmacokinetics. Exploratory outcome measures include muscle inflammation, muscle fibrosis, muscle apoptosis / degeneration, those related to muscle regeneration including cytokines (which may be one or more of the markers of muscle damage), and proteomics, and serum / plasma biomarker responses such as mononuclear cell RNA arrays, and potentially exosomal RNA. Example 5 result

[0198] A 13-year-old non-ambulatory subject weighing 62 kg was administered a low dose of ATL1102 at 25 mg / week for 12 weeks and the corticosteroid (CS) deflazacort (0.48 mg / kg / day) at 30 mg once daily. ATL1102 was effective in reducing the number of circulating CD8+ cells and CD8+ cells with high expression levels of CD49d per microliter observed in this patient at baseline (week 1) prior to daily administration of CS, markers of muscle damage as measured by biochemical discovery markers, muscle strength as measured by myoset, and importantly, function as measured by PUL-2. This subject lost ambulation approximately 2.5 years ago and was on approximately 50% of the standard of care 0.90 mg / kg / day dose of deflazacort used to treat ambulatory subjects with DMD. The equivalent prednisolone dose used as standard of care is 0.75 mg / kg / day in ambulatory DMD. immune cells

[0199] The effect of ATL1102 on immune cells was measured by flow cytometry and hematology at baseline (week 1), week 5 (3 days after ATL1103 administration), and weeks 8 and 12 (7 days after week 8 and 12 administration). The effect of ATL1102 on CD8+ cells was relatively selective compared to previous experience with higher doses of ATL1102 in multiple sclerosis (MS) (Limmroth et al. 2014). Also, as previously measured in MS studies, the effect lasted longer than 3 days after administration, with the first effect noted 7 days after ATL1102 administration. Specific immune cells were not affected at this dose, indicating that the ATL1102 effect was relatively specific at these low doses at the time of evaluation. For example, there was no significant reduction in neutrophils or platelets, as observed with higher doses of ATL1102 in the MS study (Limmroth et al. 2014). Myoset - Muscle Strength Data

[0200] Preliminary myoset data for subject 1 suggests that after 12 weeks of treatment compared to baseline, there was a loss of strength in the dominant hand as measured by myogrip, but no loss of strength in the thumb as measured by myopinch, and no loss of finger strength as judged by moviplate, with tap counts being the same after 12 weeks compared to baseline. Meanwhile, the data indicate that there was no decrease in hand or finger strength, and thumb strength was numerically greater at 12 weeks compared to baseline. Ricotti et al (2016) examined 15 patients (14 treated with CS) treated with CS and myoset for over 12 weeks and observed a mean trend decrease of 0.22 kg with myogrip, a mean trend decrease of 0.1 kg with myopinch, and an increase with moviplate compared to baseline. PUL-2-Functional Data

[0201] The PUL-2 for DMD is an updated version of the PUL-1 used to measure upper limb function. The PUL-2 measures high level shoulder function with a maximum score of 12, intermediate level elbow function with a maximum score of 17, and distal wrist and hand function with a maximum score of 13. An entry score of 3-6 means the subject is assessable for shoulder function (maximum is 6 and minimum is 1). Subject 1's entry level function is 5, indicating that there has been 12 weeks or more of treatment with no loss of shoulder function and a score of 8 at baseline and at week 12 in the recorded measurements. Subject 1 gained function at intermediate level elbow with a score of 14 at week 12 and 13 at baseline, and at distal wrist and hand with a score of 12 at week 12 and 10 at baseline. At 12 weeks of treatment compared to baseline, the PUL-2 score was 34 versus 31. The patient's entry score also increased from a level 5 at baseline to a level 6, consistent with the recorded PUL-2 results.

[0202] ATL1102 add-on therapy may help maintain muscle strength as measured by myoset, and even if it does not improve function in this subject, it is likely to maintain function as measured by PUL-2. Thus, ATL1102 therapy may slow disease progression.

[0203] The results validate the use of antisense against CD49d (the alpha chain of VLA-4) in general, and ATL1102 in particular, to treat DMD patients and slow muscular dystrophy disease progression as monotherapy or in combination with CS. Exploratory Pharmacodynamic Consequence Measures of Muscle Damage

[0204] Creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) are indicators of muscle damage in boys with DMD, primarily associated with having low levels or no dystrophin, which damages muscles when they contract, and secondarily associated with inflammation and other downstream damage to muscles. Creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) are indicators of muscle damage in young ambulatory patients with DMD, who have more muscle mass and inflammation than nonambulatory patients.

[0205] Nevertheless, blood and serum samples were collected to investigate changes in muscle damage markers in subject 1. In subject 1, CK, ALT, AST, and LDH decreased at weeks 8 and 12 compared to baseline and week 5. Baseline / week 5 levels (units / liter) of CK, ALT, AST, and LDH, respectively, were 5860 / 6881, 304 / 404, untested / 184, and 632 / 681, compared to week 8 / 12 levels of 4606 / 5358, 265 / 250, 116 / 134, and 564 / 498. LDH levels decreased from levels considered high to within the normal range. This suggests that signs of muscle damage associated with loss of dystrophin or inflammation or other damage were reduced in this non-ambulatory patient.

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Claims

1. 1. A pharmaceutical composition for use in a method for treating muscular dystrophy in a human subject in need thereof, comprising a therapeutically effective amount of an inhibitory oligonucleotide against human CD49d, the method comprising periodically administering a pharmaceutical composition to the subject to improve one or more markers, signs or symptoms of muscular dystrophy or dystrophic muscle fibers in the subject, or to slow the progression of muscular dystrophy in the subject, and the method is for improving muscle strength or function, or slowing the decline in muscle function or strength in a subject, The oligonucleotide has the following structure: 5'- Ｍｅ 4 Ｍｅ ________________ Ｍｅ ()) () Ｍｅ ( Ｍｅ 4 Ｍｅ pi? Ｍｅ ( Ｍｅ ( Ｍｅ 4 Ｍｅ !3' (In the formula, a) each of the 19 internucleotide linkages of said oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; c) the nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides; d) the nucleotides 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; e) All cytosines are 5-methylcytosines ( Ｍｅ C) is or a pharma- ceutically acceptable salt or stereoisomer thereof, the pharmaceutical composition is administered for a time and under conditions sufficient to ameliorate one or more markers, signs or symptoms of muscular dystrophy in the subject or slow the progression of muscular dystrophy in the subject; The administration is in combination with corticosteroid treatment. Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the regular administration is once or twice or three times weekly, semi-monthly, or monthly.

3. The pharmaceutical composition of claim 1 or 2, wherein the oligonucleotide is a sodium or potassium salt.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the pharmaceutical carrier is WFI and the pharmaceutical composition is adjusted to a pH of 7.2 to 7.

6.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the muscular dystrophy (MD) is selected from the group consisting of Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy (LGMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), Fukuyama CMD, CMD with myosin deficiency, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, and distal muscular dystrophy.

6. The pharmaceutical composition according to claim 5 , wherein the muscular dystrophy is associated with a mutation in the dystrophin gene.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the subject is diagnosed with muscular dystrophy and is ambulatory or non-ambulatory.

8. The pharmaceutical composition of claim 6, wherein the subject is an adolescent or adolescent male aged 10 years or older.

9. The pharmaceutical composition according to any one of claims 1 to 8, which is administered subcutaneously.

10. (i) when the corticosteroid is prednisolone, the prednisolone is administered at two-thirds, one-half, or one-third of the standard dose of 0.75 mg / kg / day of prednisolone; or (ii) when the corticosteroid is deflazacort, it is administered at two-thirds, one-half, or one-third of the standard dose of 0.9 mg / kg / day of deflazacort; The pharmaceutical composition according to any one of claims 1 to 9.

11. A pharmaceutical composition described in any one of claims 2 to 10, wherein the therapeutically effective amount of the inhibitory oligonucleotide is 25 mg to 100 mg per week.