Methods and compositions for treatment of epilepsy

Abstract

The present specification enables compositions and methods for treating epilepsy. In some embodiments the epilepsy to be treated is focal epilepsy, generalized epilepsy, or autoimmune epilepsy. In some embodiments, where the epilepsy is an autoimmune epilepsy, the autoimmune epilepsy is associated with autoimmune encephalitis, Hashimoto's disease, and Rasmussen's encephalitis.

Description

[0001] METHODS AND COMPOSITIONS FOR TREATMENT OF EPILEPSY

[0002] RELATED APPLICATION DATA

[0003] This application claims priority to Australian Provisional Patent Application 2024902731 filed on 30 August 2024, the contents of which is incorporated by reference in their entirety herein.

[0004] FIELD

[0005] The present specification enables compositions and methods for treating epilepsy, e.g., generalized epilepsy, focal epilepsy, and autoimmune epilepsy. In particular, where the epilepsy is autoimmune epilepsy, it is autoimmune epilepsy associated with autoimmune encephalitis, Hashimoto’s disease, and Rasmussen’s encephalitis.

[0006] BACKGROUND

[0007] Reference to any prior art in this specification is not, and should not be taken as, acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0008] Epilepsy, and particularly Autoimmune epilepsy (AE), e.g., autoimmune encephalitis (AEn) encompasses disorders with autoimmune blockade of neural signalling often leading to epilepsy. Many AE patients do not respond to current treatments such as antiepileptics, and are placed on anti-inflammatory and immune regulating treatments. There are several different forms of AE including Acute disseminated encephalomyelitis, anti -NMD AR receptor encephalitis, Hashimoto’s encephalopathy, LG11 / CASPR2- antibody encephalitis, limbic encephalitis, and Rasmussen’s encephalitis (RE). RE for instance is a rare AE and manifests mostly as focal encephalitis that affects children, adolescents and adults with 20-30% resistant to treatments. RE occurs in males and females, with onset during childhood up to middle age or later. Current treatments address seizures via antiepileptics or via hemispherectomy or hemispherectomy. However, in some cases AE is resistant to currently available drug treatments. Thus, there is an ongoing need for new treatments for AE particularly in the patient subpopulation that is resistant to conventional treatments.

[0009] Current treatments for AE include corticosteroids to treat brain inflammation, plasma exchange to remove antibodies and IVIG to introduce blocking antibodies to reduce immune mediated damage, immunosuppressants and / or surgery, but these treatments can be ineffective perhaps not effectively targeting underpinning immune damage initiated by circulating immune cells and their mediators in the periphery and / or in the CNS. These deficiencies in current therapy suggest the need for additional therapeutic approaches in AE, and in focal and other epilepsy.

[0010] SUMMARY OF THE INVENTION

[0011] As disclosed herein, it has been found, unexpectedly, that an antisense oligonucleotide (ASO) targeting the CD49d adhesion molecule expressed on a subset of immune cells is effective in the treatment of epilepsy, and particularly, autoimmune or inflammation- driven epilepsy by reducing expression of the CD49d.

[0012] Accordingly, in one aspect provided herein is a method for treating epilepsy in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide targeting CD49d; and a pharmaceutically acceptable excipient.

[0013] In some embodiments the epilepsy is a focal epilepsy, a generalized epilepsy, or an autoimmune epilepsy.

[0014] In some embodiments oligonucleotide the oligonucleotide targeting CD49d comprising the structure: wherein, a) each of the 19 internucleotide linkages of the oligonucleotide is an 0,0- linked phosphorothioate diester; b) the nucleotides at the positions 1 to 3 from the 5' end are 2'-O-(2- methoxy ethyl) modified ribonucleosides; c) the nucleotides at the positions 4 to 12 from the 5' end are 2'- deoxy rib onucl eosi des ; d) the nucleotides at the positions 13 to 20 from the 5' end are 2'-O-(2- methoxyethyl) modified ribonucleosides; and e) all cytosines are 5-methylcytosines (MeC).

[0015] In some embodiments the oligonucleotide consists of the structure.

[0016] In some embodiments the autoimmune epilepsy is associated with a condition in the subject selected from the group consisting of autoimmune encephalitis, Hashimoto’s disease, Rasmussen’s encephalitis, systemic lupus erythematosus, Sjorgren’s syndrome, Wegener’s granulomatosis, neurosarcoidosis, celiac disease, Chrohn’s disease, and Behcet’s. In some embodiments the condition is an autoimmune encephalitis, Hashimoto’s disease, or Rasmussen’s encephalitis.

[0017] In some embodiments the subject to be treated suffers from focal epilepsy.

[0018] In some embodiments the therapeutically effective amount of the oligonucleotide is about 10 mg to about 300 mg. In some embodiments the therapeutically effective amount of the oligonucleotide is about 25 mg to about 250 mg.

[0019] In some embodiments the administration is once, twice, or three times per week.

[0020] In some embodiments the oligonucleotide is a sodium or potassium salt.

[0021] In some embodiments the pharmaceutical composition is administered by a parenteral route. In some embodiments the pharmaceutical composition is administered subcutaneously.

[0022] In some embodiments any of the foregoing treatment methods also include administration of a corticosteroid. In other embodiments a treatment method also include administering an anti-epileptic agent.

[0023] In some embodiments the therapeutically effective amount of the oligonucleotide administered as monotherapy or in combination with an additional therapeutic agent is sufficient to result in one or more of: (i) reduced severity of status epilepticus (SE); (ii) seizure duration; or (iii) seizure frequency.

[0024] In some embodiments, administration is for a time and under conditions sufficient to improve one or more markers, signs or symptoms or to delay progression of autoimmune encephalitis (AE) and focal epilepsy (FE) in a subject.

[0025] In some embodiments, the markers, signs or symptoms of the AE or FE is epileptic seizure, and the onset is delayed, or duration, or severity of the seizure is reduced, or frequency of the seizures is reduced. In other embodiments the seizure may be severe so as to bring on convulsion and the onset is delayed, or duration, or severity, or frequency of the convulsion is reduced.

[0026] In some embodiments the subject to be treated does not or has not suffered from muscular dystrophy or multiple sclerosis. In some embodiments the subject to be treated is a human subject.

[0027] In another aspect provided herein is the use of an oligonucleotide targeting CD49d in the manufacture of a medicament for treatment of autoimmune epilepsy.

[0028] In a related aspect provided herein is an oligonucleotide targeting CD49d for treatment of autoimmune epilepsy. In some embodiments the oligonucleotide in the medicament or for the use is the oligonucleotide referred to as ATL1102 disclosed herein.

[0029] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise. For instance, as the skilled person would understand examples of inhibitors and health conditions outlined above for the methods of the invention equally apply to the use and pharmaceutical compositions of the invention.

[0030] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0031] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0032] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

[0033] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0034] Figure 1: Results immediately post pilocarpine onset of status epilepticus (SE). Onset to SE is delayed by antisense oligonucleotide (ASO) to CD49d compared to the negative control mismatch oligonucleotide (MM) and compared to pooled saline + MM groups. Analysis performed using ANOVA showed 4 minutes (-37%) delay to onset of SE with ASO compared to MM using ANOVA followed by Tukey’s correction for multiple comparisons, and 3 minutes delay (26%) to onset of SE with ASO compared to pooled saline and MM data using unpaired t-test. Figure 2: Result immediately post pilocarpine. ASO reduced seizure severity of SE compared to saline at between 90-120 minutes. Analysed using unpaired t-test of the AUC of seizure severity between 90 and 120 minutes. Decrease of lp=0.0072, 94.82 vs 88.93 was shown.

[0035] Figure 3: Early ASO effects post-onset of spontaneous recurring seizure rate (SRS). A) Results showed significantly reduced median # SRS at day 32 compared to saline and SRS onset. Data was non-parametric, and medians and 95%CI was used. Median number of SRS is zero for ASO in the combined days 31, 32, and 33. Median number of SRS for saline is 1 in the combined day 31-32. Median number of SRS for saline is 2.5 for day 32. B) ASO treatment decreased SR seizures / h early compared to saline day 31-32. Kruskal -Wallis non-parametric H-test with Dunn’s correction for multiple comparisons (p=0.0101) showed ASO treated mice had (median 0 seizures / hr) with a -100% significant decrease in seizure frequency / hr compared to saline (0.125 median seizures / hr) (p=0.0019) over the first 2 days (days 31-32). Median was 0.3125 (p=0.0006) at day 32 analysed by Mann Whitney (non-parametric) test. C) nonparametric data (medians). Kruskal -Wallis assessment of cumulative (combined) day 31, 32, 33 SRS (p=0.0121) suggests a difference in onset post-day 31 onset SRS: potential delay with ASO of three days (median 0) SRS compared to saline (0.5SRS) (0.0041) analysed by Mann Whitney. There was no significant delay in SRS onset with ASO compared to MM though ASO median is still 0 at day 34 compared to MM day 34 SRS of 1.

[0036] Figure 4: ASO treatment decreased SR seizures per hour compared to MM and pooled saline + MM. A) Day 31-38 (8 days), non-parametric data. Kruskal -Wallis nonparametric H-test with Dunn’s correction for multiple comparisons (p=0.0850) showed that ASO-treated mice had median (0.047 seizures / hr) with a trend 54% significant decrease in SRS frequency (seizures / hr) compared to saline (0.102 seizures / hr) and / or MM (1.02 seizures / hr) over the first 8 days (p=0.0868, 0.0361, Mann Whitney) and -54% decrease compared to pooled saline and MM (0.102 seizures / hr p=0.0256), Mann Whiteny. B) ASO treatment decreased SR seizures / hr compared to saline or MM at days 31-38 (8 days). According to one way ANOVA (p=0.0799) ASO treated mice had mean (0.06593 seizures / hr) with a trend 59% significant decrease in SRS frequence (seizures / hr) compared to saline (0.1593 seizures / hr) and / or MM (0.1420 seizures / hr) over the first 8 days using multiple comparisons (p=0.0692, 0.9519). According to t-test, ASO compared to saline gave (p=0.0412), ASO compared to MM gave (p=0.0356), ASO compared to pooled (mean = 0.1479) (p=0.0246).

[0037] Figure 5: ASO treatment significantly decreases SR seizures per hour compared to saline at days 31-43 (13 days). Kruskal -Wallin non-parametric H-test with Dunn’s correction for multiple comparisons (p=0.0381) showed ASO treated mice had (median 0.048 seizures / hr) with a -65% significant decrease in seizure frequency / hr compared to saline (median 0.1395 seizures / hr (p=0.0488) but not compared to MM (0.134 seizures / hr) (p=0.096) over the first 13 days (day 31-43). ASO treated mice had a median 0.048 seizures / hr with a -64.9% significant decrease in seizure frequency / hr compared to saline (median 0.1395 seizures / hr) (p=0.0307) and 54.5% compared to MM (median 0.1055 seizures / hr) (p=0.0258) at day 31-43 (13 days). ASO reduced median SRS -62.6% compared to pooled saline and MM (0.125 seizures / hr)(p=0.0096) Mann Whitney non-parametric.

[0038] Figure 6: ASO treatment reduced SRS vs MM and pooled saline + MM Days 31- 46. Kruskal -Wallin non-parametric H-test with Dunn’s correction for multiple comparisons (p=0.0642). ASO treated mice had (median 0.0705 seizures / hr) with a -62% decrease in seizure frequency compared to saline (median 0.1290 seizures / hr) (p=0.0508) and a significant -68.4% decrease compared to MM (median 0.1520 seizures / hr) (p=0.039) at day 31-46 (16 days). ASO reduce median SRS -41% compared to pooled saline and MM (median 0.1370 seizures / hr) (p=0.0182) Mann Whitney nonparametric t-test.

[0039] KEY TO NUCLEIC ACID SEQUENCES: human a4 integrin antisense sequence (ATL1102): cugagtctgt ttuccauucu murine a4 integrin antisense sequence: atatttttcc acctgtgccc

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] General Techniques and Definitions

[0042] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g. , in cell culture, cell biology, molecular genetics, cancer biology and treatment thereof, infectious disease especially acute infections, immunology, pharmacology, protein chemistry, and biochemistry). Unless otherwise indicated, the cell culture and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et cd.. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley -Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0043] As used herein, the term about, unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, of the designated value.

[0044] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply 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.

[0045] As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or”. That is, unless specified otherwise, or clear from context, “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then “X employs A or B” is satisfied under any of the foregoing instances. Further, at least one of A and B and / or the like generally means A or B or both A and B. In addition, the articles “a” and “an” as used in this application and the appended claims may generally be construed to mean “one or more” unless specified otherwise or clear from context to be directed to a singular form.

[0046] The terms “treating”, “treated”, or “treatment” as used herein, refer to both direct treatment of a subject by a medical professional (e.g., by administering a therapeutic agent to the subject), or indirect treatment, effected, by at least one party, (e.g., a medical doctor, a nurse, a pharmacist, or a pharmaceutical sales representative) by providing instructions, in any form, that (i) instruct a subject to self-treat according to a claimed method (e.g., self-administer a drug) or (ii) instruct a third party to treat a subject according to a claimed method. Also encompassed within the meaning of the term “treating”, “treated” or “treatment” are prevention or reduction of the disease to be treated, e.g., by administering a therapeutic at a sufficiently early phase of disease and / or in sufficient doses to prevent or slow its progression.

[0047] As used herein, the term “preventing”, “prevent” or “prevention” includes providing prophylaxis with respect to occurrence or recurrence of a specified disease or condition in an individual. An individual may be predisposed to or at risk of developing the disease but has not yet been diagnosed with the disease.

[0048] As used herein, the phrase “delaying progression of’ includes reducing or slowing down the progression of the disease or condition in an individual and / or at least one symptom of a disease or condition.

[0049] As used herein, the terms “disease”, “disorder” or “condition” refers to a disruption of or interference with normal function, and is not to be limited to any specific condition, and will include diseases or disorders.

[0050] As used herein, the term “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone. By convention, sequences are presented from the 5' end to the 3' end, unless otherwise specified.

[0051] Reduced epilepsy may be assessed by MRI or EEG.

[0052] Quality of life questionnaires are very useful in determining the effect of treatments.

[0053] Clinical outcomes may involve, for example, determining the epilepsy type, seizure type, and response to standard or investigational treatments.

[0054] Alternatively or in addition biomarkers and brain scan assessments, pharmacodynamics markers measuring change in plasma biomarker panel measured by ELISA or proteomics, or change in circulating immune cell markers are assessed. The term "antisense compound" as used herein refers to an oligomeric compound that hybridizes to a nucleic acid molecule encoding the a4 integrin chain of VLA-4 (a4pi) and / or a4p7 integrin. The a4 integrin chain in humans is CD49d. The antisense compound may interfere with expression of CD49d, pi integrin and / or P7 integrin.

[0055] The term "nucleic acid molecule encoding alpha4 integrin" as used herein encompasses DNA encoding the a4 integrin chain of VLA-4 or a4p7 integrin, RNA (including pre- mRNA and mRNA or portions thereof) transcribed from such DNA, and further, cDNA derived from such RNA.

[0056] The term "VLA-4" as used herein refers to a heterodimer of an a4 integrin and a pi integrin. VLA-4 is expressed at substantial levels on normal peripheral blood B and T cells, thymocytes, monocytes, and other cells, as well as on hematopoietic stem and progenitor cells. VLA-4 is also expressed on mesenchymal and endothelial progenitor cells and mesenchymal stem cells and potentially endothelial stem cells. Ligands for VLA-4 include vascular cell adhesion molecule-1 (VCAM-1) and CS-1, an alternately spliced domain within the Hep II region of fibronectin.

[0057] The term "a4p7 integrin" as used herein refers to a heterodimer of an a4 integrin and a P7 integrin. a4p7 integrin identifies a subset of memory T cells with a tropism for the intestinal tract. a4p7 integrin and is also expressed on a subset of mast, lymphocyte and NK progenitor cells. a4p7 integrin is expressed on some stem and progenitor cells. Ligands for a4p7 integrin include MAdCam-1 and VCAM-1.

[0058] Nucleic Acids

[0059] The present disclosure encompasses use of various oligonucleotides which are 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 analogues (e.g., containing base analogues, sugar analogues and / or a non-native backbone and the like), RNA / DNA hybrids and polyamide nucleic acids (PNAs), all of which can be in single- or double-stranded form. In an example, the nucleic acid is isolated. As used herein, the term "isolated nucleic acid" means a nucleic acid that is altered or removed from the natural state through human intervention. The term "oligonucleotide" broadly means a short nucleic acid molecule. Oligonucleotides readily bind, in a sequence-specific manner, to their respective complementary oligonucleotides, DNA, or RNA to form duplexes. In some embodiments, oligonucleotides are five, six, seven, eight, nine, 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 or more in length.

[0060] In some embodiments, oligonucleotides of the present disclosure are inhibitory oligonucleotides. In an 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 interfere with translation of mRNA into protein in a ribosome. In another example, an inhibitory oligonucleotide can be sufficiently complementary to either a gene or a mRNA encoding one or more proteins to bind to (hybridize with) a targeted gene(s) or mRNA thereby reducing expression or biological activity of the target protein. In another example, an inhibitory oligonucleotide inhibits the biological activity of an intracellular nucleic acid that does not code for a protein. For example, an inhibitory oligonucleotide can inhibit the biological activity of a non-coding RNA.

[0061] The term "antisense" as used herein means a sequence of nucleotides complementary to and therefore capable of binding to a coding sequence, which may be either that of the strand of a DNA double helix that undergoes transcription, or that of a messenger RNA molecule. Antisense DNA is the non-coding strand complementary to the coding strand in double-stranded DNA.

[0062] The terms "short hairpin RNA" or "shRNA" refer to an RNA structure having a duplex region and a loop region.

[0063] The term small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded or single stranded RNA molecules, about 19-25 base pairs in length. A siRNA that inhibits or prevents translation to a particular protein is indicated by the protein name coupled with the term siRNA. Typically, a siRNA in various embodiments is a double-stranded or single stranded nucleic acid molecule having about 19 to about 28 nucleotides (i.e. about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). The term “microRNA” (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression. The prefix "miR" is followed by a dash and a number, the latter often indicating order of naming. Different miRNAs with nearly identical sequences except for one or two nucleotides are annotated with an additional lower case letter. Numerous miRNAs are known in the art (miRBase V.21 nomenclature; see Kozomara et al. 2013; Griffiths- Jones, S. 2004). Sequences of these miRNAs are well known in the art and may be found, for example, on the world wide web at mirbase dot org.

[0064] In some embodiments, “inhibitory oligonucleotides” mimic the activity of one or more miRNA. The term “miRNA mimic”, as used herein, refers to small, double-stranded RNA molecules designed to mimic endogenous mature miRNA molecules when introduced into cells. miRNA mimics can be obtained from various suppliers such as Sigma Aldrich and Thermo Fisher Scientific.

[0065] In embodiment, “inhibitory oligonucleotides” inhibit the activity of one or more miRNA. Various miRNA species are suitable for this purpose. Examples include, without limitation, antagomirs, interfering RNA, ribozymes, miRNA sponges and miR-masks. The term “antagomir” is used in the context of the present disclosure to refer to chemically modified antisense oligonucleotides that bind to a target miRNA and inhibit miRNA function by preventing binding of the miRNA to its cognate gene target. Antagomirs can include any base modification known in the art. In an example, the above referenced miRNA species are about 10 to 50 nucleotides in length. For example, antagomirs can have antisense portions 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.

[0066] In some embodiments, the miRNA species are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. In some embodiments, nucleic acids encompassed by the present disclosure are synthetic. The term "synthetic nucleic acid" means that the nucleic acid does not have a chemical structure or sequence of a naturally occurring nucleic acid. Synthetic nucleotides include an engineered nucleic acid molecule. In another example, the nucleic acid structure can also be modified into a locked nucleic acid (LNA) with a methylene bridge between the 2' Oxygen and the 4' carbon to lock the ribose in the 3'-endo (North) conformation in the A- type conformation of nucleic acids (Lennox et al 2011; Bader et al 2011). In the context of miRNAs, this modification can significantly increase both target specificity and hybridization properties of the molecule.

[0067] Nucleic acids for use in the methods disclosed herein can be designed using routine methods as required. For example, in the context of inhibitory oligonucleotides, target segments of 5, 6, 7, 8, 9, 10 or more nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides within the seed sequence, or immediately adjacent thereto, are considered to be suitable for targeting a gene. Exemplary target segments can include sequences that comprise at least the 5 consecutive nucleotides from the 5'- terminus of one of the seed sequence (the remaining nucleotides being a consecutive stretch of the same RNA beginning immediately upstream of the 5'-terminus of the seed sequence and continuing until the nucleic acid contains about 5 to about 30 nucleotides). In another example, target segments are represented by RNA sequences that comprise at least the 5 consecutive nucleotides from the 3'-terminus of one of the seed sequence (the remaining nucleotides being a consecutive stretch of the same RNA beginning immediately downstream of the 3 '-terminus of the target segment and continuing until the nucleic acid contains about 5 to about 30 nucleotides). The term "seed sequence" is used in the context of the present disclosure to refer to a 6-8 nucleotide (nt) long substring within the first 8 nt at the 5 -end of the miRNA (i.e., seed sequence) that is an important determinant of target specificity. Once one or more target regions, segments or sites have been identified, inhibitory nucleic acid compounds are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity (i.e., do not substantially bind to other non-target nucleic acid sequences), to give the desired effect.

[0068] Antisense compounds to «4 integrin

[0069] In some embodiments the methods of the present disclosure rely on the use of an antisense compound to a4 integrin. Such antisense compounds are targeted to nucleic acids encoding the a4 integrin chain of VLA-4 (a4pi) or a4p7 integrin. In some embodiments, the antisense compound is an oligonucleotide. However, other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics are contemplated.

[0070] Hybridization of an antisense compound with its target nucleic acid is generally referred to as "antisense". Hybridization of the antisense compound with its target nucleic acid inhibits the function of the target nucleic acid. Such "antisense inhibition" is typically based upon hydrogen bonding-based hybridization of the antisense compound to the target nucleic acid such that the target nucleic acid is cleaved, degraded, or otherwise rendered inoperable. The functions of target DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA.

[0071] "Hybridization" as used herein means pairing of complementary bases of the oligonucleotide and target nucleic acid. Base pairing typically involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases). Guanine (G) and cytosine (C) are examples of complementary nucleobases which pair through the formation of 3 hydrogen bonds. Adenine (A) and thymine (T) are examples of complementary nucleobases which pair through the formation of 2 hydrogen bonds. Hybridization can occur under varying circumstances.

[0072] A "nucleoside" is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. "Nucleotides" are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.

[0073] "Specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the antisense compound and target nucleic acid. It is understood that the antisense compound need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the antisense compound to the target nucleic acid interferes with the normal function of the target molecule to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to nontarget sequences under conditions in which specific binding is desired, for example, under physiological conditions in the case of therapeutic treatment.

[0074] "Complementary" as used herein, refers to the capacity for precise pairing between a nucleobase of the antisense compound and the target nucleic acid. For example, if a nucleobase at a certain position of the antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of the target nucleic acid, then the position of hydrogen bonding between the antisense compound and the target nucleic acid is considered to be a complementary position. The antisense compound may hybridize over one or more segments, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). In some embodiments, the antisense compound comprises at least 70% sequence complementarity to a target region within the target nucleic acid.

[0075] For example, an antisense compound in which 18 of 20 nucleobases are complementary to a target region within the target nucleic acid, and would therefore specifically hybridize, would represent 90% complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other, or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 noncomplementary nucleobases which are flanked by 2 regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus, fall within the scope of the present disclosure. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., 1990; Zhang and Madden, 1997). Antisense oligonucleotides

[0076] The present disclosure provides antisense oligonucleotides for inhibiting expression of a4 integrin, and / or VLA-4 and / or a4p7 integrin. Such antisense oligonucleotides are targeted to nucleic acids encoding the a4 integrin chain of VLA-4 or a4p7 integrin.

[0077] The term "inhibits" as used herein means any measurable decrease (e.g., 10%, 20%, 50%, 90%, or 100%) in VLA-4 or a4p7integrin expression.

[0078] 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 having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for the target nucleic acid and increased stability in the presence of nucleases.

[0079] The oligonucleotides may contain chiral (asymmetric) centers or the molecule as a whole may be chiral. The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present disclosure. Reference may be made to Wan et al. Nucleic Acids Research 42 (22: 13456-13468, 2014 for a disclosure of antisense oligonucleotides containing chiral phosphorothioate linkages.

[0080] In forming oligonucleotides, phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner so as to produce a fully or partially double-stranded compound. With regard to oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

[0081] Antisense oligonucleotides of the disclosure include, for example, ribozymes, siRNA, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligonucleotides which hybridize to at least a portion of the target nucleic acid. Antisense oligonucleotides of the disclosure may be administered in the form of single stranded, double-stranded, circular or hairpin and may contain structural elements such as internal or terminal bulges or loops. Once administered, the antisense oligonucleotides may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.

[0082] One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that singlestranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H therefore results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases, such as those in the RNase III and ribonuclease L family of enzymes.

[0083] The introduction of double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing. The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, 1995). Montgomery et al. (1998) have shown that the primary interference effects of dsRNA are posttranscri phonal. The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., 1998). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., 2002).

[0084] A person having ordinary skill in the art could, without undue experimentation, identify antisense oligonucleotides useful in the methods of the present disclosure.

[0085] Modified internucleoside linkages (backbones)

[0086] Antisense compounds of the present disclosure include oligonucleotides having modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0087] Modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'- 5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more intemucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage, that is, a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0088] Representative United States patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, US 3,687,808, US 4,469,863, US 4,476,301, US 5,023,243, US 5,177,196, US 5,188,897, US 5,264,423, US 5,276,019, US 5,278,302, US 5,286,717, US 5,321,131, US 5,399,676, US 5,405,939, US 5,453,496, US 5,455,233, US 5,466,677, US 5,476,925, US 5,519,126, US 5,536,821, US 5,541,306, US 5,550,111, US 5,563,253, US 5,571,799, US 5,587,361, US 5,194,599, US 5,565,555, US 5,527,899, US 5,721,218, US 5,672,697 and US 5,625,050.

[0089] Modified oligonucleotide backbones that do not include a phosphorus atom therein 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 heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, US 5,034,506, US 5,166,315, US 5,185,444, US 5,214,134, US 5,216,141, US 5,235,033, US 5,264,562, US 5,264,564, US 5,405,938, US 5,434,257, US 5,466,677, US 5,470,967, US 5,489,677, US 5,541,307, US 5,561,225, US 5,596,086, US 5,602,240, US 5,610,289, US 5,602,240, US 5,608,046, US 5,610,289, US 5,618,704, US 5,623,070, US 5,663,312, US 5,633,360, US 5,677,437, US 5,792,608, US 5,646,269 and US 5,677,439.

[0090] Modified sugar and internucleoside linkages

[0091] Antisense compounds of the present disclosure include oligonucleotide mimetics where both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with the target nucleic acid.

[0092] An oligonucleotide mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, in particular, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, US 5,539,082, US 5,714,331, and US 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al., 1991.

[0093] The antisense compounds of the present disclosure also include oligonucleotides with phosphorothioate backbones and oligonucleotides with heteroatom backbones, for example, -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- [known as a methylene (methylimino) or MMI backbone], -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)- CH2- and -O-N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone is represented as -O-P-O-CH2-] of US 5,489,677, and the amide backbones of US 5,602,240.

[0094] The antisense compounds of the present disclosure also include oligonucleotides having morpholino backbone structures of US 5,034,506. Modified sugars

[0095] Antisense compounds of the present disclosure include oligonucleotides having one or more substituted sugar moieties.

[0096] Examples include oligonucleotides comprising one 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, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl.

[0097] In some embodiments, 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.

[0098] Further examples include of modified oligonucleotides include oligonucleotides comprising one of the following at the 2' position: Cl to CIO 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 having similar properties.

[0099] In some embodiments, the modification includes 2' -methoxy ethoxy (2'-O- CH2CH2OCH3 (also known as 2'-O-(2 -methoxy ethyl) or 2'-M0E) (Martin et al., 1995), that is, an alkoxyalkoxy group. In a further embodiment, the modification includes 2'- dimethylaminooxyethoxy, that is, a 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), that is, 2'-O-CH2-O-CH2-N(CH3)2.

[0100] Other modifications include 2'-methoxy (2'-O-CH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-O-CH2-CH=CH2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. In some embodiments a 2'-arabino modification is 2'-F. 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 the 5' terminal nucleotide.

[0101] Oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0102] Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, US 4,981,957, US 5,118,800, US 5,319,080, US 5,359,044, US 5,393,878, US 5,446,137, US 5,466,786, US 5,514,785, US 5,519,134, US 5,567,811, US 5,576,427, US 5,591,722, US 5,597,909, US 5,610,300, US 5,627,053, US 5,639,873, US 5,646,265, US 5,658,873, US 5,670,633, US 5,792,747, and US 5,700,920.

[0103] A further modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In some embodiments, the linkage is a methylene (-CH2-)n group bridging the 2' oxygen atom and the 4' carbon atom, wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98 / 39352 and WO 99 / 14226.

[0104] Natural and modified nucleobases

[0105] 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).

[0106] Modified nucleobases include other synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, 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-azo uracil, 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 particularly 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 -deazaadenine.

[0107] Further modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine(lH-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as, for example, a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4- b][l,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).

[0108] Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Further nucleobases include those disclosed in US 3,687,808, those disclosed in J.I. Kroschwitz (editor), The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, John Wiley and Sons (1990), those disclosed by Englisch et al. (1991), and those disclosed by Y.S. Sanghvi, Chapter 15: Antisense Research and Applications, pages 289-302, S.T. Crooke, B. Lebleu (editors), CRC Press, 1993.

[0109] Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligonucleotide. These include 5-substituted pyrimidines, 6-azapyrimidines and N- 2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 DC. In some embodiments, these nucleobase substitutions are combined with 2'-O-methoxy ethyl sugar modifications.

[0110] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, US 3,687,808, US 4,845,205, US 5,130,302, US 5,134,066, US 5,175,273, US 5,367,066, US 5,432,272, US 5,457,187, US 5,459,255, US 5,484,908, 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. Conjugates

[0111] Antisense compounds of the present disclosure may be conjugated to one or more moieties or groups which enhance the activity, cellular distribution or cellular uptake of the antisense compound.

[0112] These moieties or groups may be covalently bound to functional groups such as primary or secondary hydroxyl groups.

[0113] Exemplary moieties or groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins and dyes.

[0114] Moieties or groups that enhance the pharmacodynamic properties include those that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with the target nucleic acid.

[0115] Moieties or groups that enhance the pharmacokinetic properties include those that improve uptake, distribution, metabolism or excretion of the compounds of the present disclosure. Representative moieties or groups are disclosed in PCT / US92 / 09196 and US 6,287,860. Moieties or groups include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, for example, hexyl -S-tritylthiol, a thiocholesterol, an aliphatic chain, for example, dodecandiol or undecyl residues, a phospholipid, for example, di-hexadecyl-rac-glycerol or tri ethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety.

[0116] Chimeric compounds

[0117] As would be appreciated by those skilled 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 at a single nucleoside within an oligonucleotide. Antisense compounds of the disclosure include chimeric oligonucleotides. "Chimeric oligonucleotides" contain two or more chemically distinct regions, each made up of at least one monomer unit, that is, a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and / or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions 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, associated nucleic acid hybridization techniques known in the art.

[0118] Chimeric antisense compounds of the disclosure may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, and / or oligonucleotide mimetics. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, US 5,013,830, US 5,149,797, US 5,220,007, US 5,256,775, US 5,366,878, US 5,403,711, US 5,491,133, US 5,565,350, US 5,623,065, US 5,652,355, US 5,652,356, and US 5,700,922.

[0119] Exemplary oligonucleotides

[0120] Illustrative antisense platforms known in the art include without limitation, morpholino, Igen oligos, 2ndgen oligo’ s, gapmer, siRNA, LNA, BNA, or oligo mimetics like Peptide Nucleic acids. Oligonucleotides may be naked or formulated in liposomes. Oligonucleotides may be linked to a delivery means to cells or not. Oligonucleotides may use an endosome release agent or not.

[0121] In some embodiments, the antisense compound is a second generation phosphorothioate backbone 2'-MOE-modified chimeric oligonucleotide gapmer designed to hybridize to the 3 '-untranslated region of VLA-4 mRNA. In some embodiments, the oligonucleotide selectively inhibits VLA-4 expression in both primary human cells and in several human cell lines by hybridizing to RNA encoding CD49, which is the a4 integrin subunit of VLA-4 and a4p7 integrin.

[0122] In some embodiments, the oligonucleotide is the 19-sodium salt of a 3'— >5' phosphorothioate oligonucleotide 20mer also referred as a 3-9-8 MOE gapmer having a molecular weight of 7230 Daltons, in which the nucleotides at positions 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) (2'MOE) modified ribonucleosides (2'-O-(2- methoxyethyl ribose); the nucleotides at positions 4 to 12 from the 5' end are 2'- deoxyribonucleosides of which all cytosines are 5 -methylcytosines; the nucleotides at positions 13 to 20 from the 5' end are 2'-O-(2-methoxy ethyl) modified ribonucleosides.

[0123] In some embodiments, the sequence of the oligonucleotide is (SEQ ID NO: 1):

[0124] The empirical formula of the oligonucleotide as a sodium salt is: C233H3o8N60O129P19S 19Nai9.

[0125] The empirical formula of the oligonucleotide as a free acid is: C233H327N60O129P19S19

[0126] Antisense oligonucleotide ATL1102 has previously been shown to be effective in central nervous system disorder, MS and at significantly higher doses than proposed herein (Limmroth et al). The ability of antisense oligonucleotide to CD49d alpha chain of VLA- 4 to selectively inhibit VLA-4 in immune cells prevents significant safety events such as PML which have characterised administration of antibodies and small molecule inhibitors of VLA-4 which are pan VLA-4 inhibitors affecting all cells which express VLA-4.

[0127] In some embodiments, all uracils are 5 -methyluracils (MeU). Typically, the oligonucleotide is synthesized using 2-methoxyethyl modified thymidines not 5- methyluracils.

[0128] In some embodiments, all pyrimidines are C5 methylated (i.e., U, T, C are C5 methylated). In some embodiments, the sequence of the oligonucleotide may be named by accepted oligonucleotide nomenclature, showing each 0-0 linked phosphorothioate internucleotide linkage:

[0129] 2'-O-methoxyethyl-5-methylcytidylyl-(3'^-5' O, O-phosphorothioyl)-2'-O- methoxyethyl-5-methyluridylyl-(3'^5' O, O-phosphorothioyl)-2'-O- methoxyethylguanosylyl-(3'^-5' O, O-phosphorothioyl)-2'-O-deoxyadenosylyl-(3'^5' O, O-phosphorothioyl)-2'-O-deoxyguanosylyl-(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)-thymidylyl-(3'^5' O, O-phosphorothioyl)-2'-O- methoxyethyl-5-methyluridylyl-(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-adenosylyl-(3'^5' O, O- phosphorothioyl)-2'-O-methoxyethyl-5-methyluridylyl-(3'^5' O, O-phosphorothioyl)- 2'-O-methoxyethyl-5-methyluridylyl-(3'^5' O, O-phosphorothioyl)-2'-O- methoxyethyl-5-methylcytosine, (3'^5' O, O-phosphorothioyl)-2'-O-methoxyethyl-5- methyluridylyl-19 sodium salt.

[0130] The oligonucleotide may be synthesized by a multi-step process that may be divided into two distinct operations: solid-phase synthesis and downstream processing. In the first operation, the nucleotide sequence of the oligonucleotide is assembled through a computer-controlled solid-phase synthesizer. Subsequent downstream processing includes deprotection steps, preparative reversed-phase chromatographic purification, isolation and drying to yield the oligonucleotide drug substance. The chemical synthesis of the oligonucelotide utilizes phosphoramidite coupling chemistry followed by oxidative sulfurization and involves sequential coupling of activated monomers to an elongating oligomer, the 3'-terminus of which is covalently attached to the solid support.

[0131] Detritylation (reaction a)

[0132] Each cycle of the solid-phase synthesis commences with removal of the acid-labile 5'-0- 4, 4'-dimethoxytrityl (DMT) protecting group of the 5' terminal nucleoside of the support bound oligonucleotide. This is accomplished by treatment with an acid solution (for example di chloroacetic acid (DCA) in toluene). Following detritylation, excess reagent is removed from the support by washing with acetonitrile in preparation for the next reaction. Coupling (reaction b)

[0133] Chain elongation is achieved by reaction of the 5'-hydroxyl group of the support-bound oligonucleotide with a solution of the phosphoramidite corresponding to that particular base position (e.g., for base2: MOE-MeC amidite) in the presence of an activator (e.g., IH-tetrazole). This results in the formation of 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.

[0134] Sulfurization (reaction c)

[0135] The newly formed phosphite triester linkage is converted to the corresponding [O, O, O)- trialkyl phosphorothioate triester by treatment with a solution of a sulfur transfer reagent (e.g., phenylacetyl disulfide). Following sulfurization, excess reagent is removed from the support by washing with acetonitrile in preparation for the next reaction.

[0136] Capping (reaction d)

[0137] A small proportion of the 5'-hydroxy groups available in any given cycle fail to extend. Coupling of these groups in any of the subsequent cycles would result in formation of process-related impurities ("DMT-on (n-l)-mers") which are difficult to separate from the desired product. To prevent formation of these impurities and to facilitate purification, a "capping reagent" (e.g., acetic anhydride and N- methylimidazole / acetonitrile / pyridine) is introduced into the reactor vessel to give capped sequences. The resulting failure sequences ("DMT-off shortmers") are separated from the desired product by reversed phase HPLC purification. After the capping reaction, excess reagent is removed from the support by washing with acetonitrile in preparation of the next reaction.

[0138] Reiteration of this basic four-step cycle using the appropriate protected nucleoside phosphoramidite allows assembly of the entire protected oligonucleotide sequence.

[0139] Backbone deprotection (reaction e)

[0140] Following completion of the assembly portion of the process the cyanoethyl groups protecting the (O, O, O)-trialkyl phosphorothioate triester internucleotide linkages are removed by treatment with a solution of tri ethylamine (TEA) in acetonitrile. The reagent and acrylonitrile generated during this step are removed by washing the column with acetonitrile.

[0141] Cleavage from support and base deprotection (reaction j)

[0142] Deprotection of the exocyclic amino groups 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 accomplished by reversed phase HPLC. The reversed phase HPLC step removes DMT-off failure sequences. The elution profile is monitored by UV absorption spectroscopy. Fractions containing DMT- on oligonucleotide product are collected and analyzed.

[0143] Acidic deprotection (reaction g)

[0144] Reversed phase HPLC fractions containing 5'-O-DMT-protected oligonucleotide are pooled and transferred to a precipitation tank. The products obtained from the purification of several syntheses are combined at this stage of the process. Purified DMT-on oligonucleotide is treated with acid (e.g., acetic acid) to remove the DMT group attached to the 5' terminus. After acid exposure for the prescribed time and neutralization, the oligonucleotide drug substance is isolated and dried.

[0145] Following the final acidic deprotection step, the solution is neutralized by addition of aqueous sodium hydroxide and the oligonucleotide drug substance is precipitated from solution by adding ethanol. The precipitated material is allowed to settle at the bottom of the reaction vessel and the ethanolic supernatant decanted. The precipitated material is redissolved in purified water and the solution pH adjusted to between pH 7.2 and 7.3. The precipitation step is repeated. The precipitated material is dissolved in water and the solution filtered through a 0.45 micron filter and transferred into disposable polypropylene trays that are then loaded into a lyophilizer. The solution is cooled to - 50°C. Primary drying is carried out at 25°C for 37 hours. The temperature is increased to 30°C and a secondary drying step performed for 5.5 hours. Following completion of the lyophilization process, the drug substance is transferred to high density polyethylene bottles and stored at -200°C.

[0146] Target nucleic acid

[0147] "Targeting" an antisense compound to a particular nucleic acid can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. In the present disclosure, the target nucleic acid encodes the alpha4 integrin chain of VLA-4 or a4p7 integrin.

[0148] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, for example, inhibition of expression, will result. The term "region" as used herein is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of the target nucleic acids are segments. "Segments" are defined as smaller or sub-portions of regions within a target nucleic acid. "Sites" as used herein, means positions within the target nucleic acid.

[0149] Since the "translation initiation codon" is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon", the "start codon" or the "AUG start codon". A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG, or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. The terms "start codon" and "translation initiation codon" as used herein refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding, for example, a4 integrin chain of VLA-4 or a4p7 integrin, regardless of the sequence(s) of such codons.

[0150] A "translation termination codon" also referred to a "stop codon" may have one of three RNA sequences: 5'-UAA, 5'-UAG and 5'-UGA (5'-TAA, 5'-TAG and 5'-TGA, respectively in the corresponding DNA molecule). The terms "translation termination codon" and "stop codon" as used herein refer to the codon or codons that are used in vivo to terminate translation of an mRNA transcribed from a gene encoding the a4 integrin chain of VLA-4 or a4p7 integrin, regardless of the sequence(s) of such codons. The terms "start codon region" and "translation initiation codon region" refer to a portion of the mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from the translation initiation codon. Similarly, the terms and "stop codon region" and "translation termination codon region" refer to a portion of the mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from the translation termination codon. Consequently, the "start codon region" or "translation initiation codon region" and the "stop codon region" or "translation termination codon region" are all regions which may be targeted effectively with the antisense compounds of the present disclosure.

[0151] The "open reading frame" (ORF) or "coding region", which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. In some embodiments, the intragenic region encompassing the translation initiation or termination codon of the ORF of a gene is targeted.

[0152] Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of the mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of the mRNA (or corresponding nucleotides on the gene), and the 3' untranslated region (3'UTR), known in the art to refer to the portion of the mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of the mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue joined 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, as well as the first 50 nucleotides adjacent to the cap site. In some embodiments, the 5' cap region is targeted.

[0153] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". In some embodiments, introns, or splice sites, that is, intron-exon junctions or exon-intron junctions, or aberrant fusion junctions due to rearrangements or deletions are targeted. Alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants".

[0154] "Pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants 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 splice variants". If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0155] Variants can be produced through the use of alternative signals to start or stop transcription, that is through use of an alternative start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. In some embodiments, the pre-mRNA or mRNA variants are targeted.

[0156] The location on the target nucleic acid to which the antisense compound hybridizes is referred to as the "target segment". As used herein the term "target segment" is defined as at least an 8-nucleobase portion of a target region to which an antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0157] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to a target segment, that is, antisense compounds that hybridize sufficiently well and with sufficient specificity, to give the desired effect. The target segment may also be combined with its respective complementary antisense compound to form stabilized double-stranded (duplexed) oligonucleotides. Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation, as well as RNA processing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (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 the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., 2002).

[0158] Compositions

[0159] Antisense compounds of the disclosure may be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, resulting in, for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and / or absorption. Representative United States patents that teach the preparation of such uptake, distribution and / or absorption-assisting formulations include, but are not limited to, US 5,108,921, US 5,354,844, US 5,416,016, US 5,459,127, US 5,521,291, US 5,543,158, US 5,547,932, US 5,583,020, US 5,591,721, US 4,426,330, US 4,534,899, US 5,013,556, US 5,108,921, US 5,213,804, US 5,227,170, US 5,264,221, US 5,356,633, US 5,395,619, US 5,416,016, US 5,417,978, US 5,462,854, US 5,469,854, 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.

[0160] Antisense compounds of the disclosure may be administered in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to molecular entities that do not produce an allergic, toxic or otherwise adverse reaction when administered to a subject, particularly a mammal, and 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, buffering agents, lubricating agents, adjuvants, vehicles, emulsifiers, absorbants, dispersion media, coatings, stabilizers, protective colloids, adhesives, thickeners, thixotropic agents, penetration agents, sequestering agents, isotonic and absorption delaying agents that do not affect the activity of the active agents of the disclosure. In some embodiments, the pharmaceutical carrier is water for injection (WFI) and the pharmaceutical composition is adjusted to pH 7.4, 7.2-7.6.

[0161] In some embodiments, the salt is a sodium or potassium salt. In other embodiments, it is the free acid.

[0162] The oligonucleotides may contain chiral (asymmetric) centers or the molecule as a whole may be chiral. The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present disclosure.

[0163] Antisense compounds of the disclosure may be pharmaceutically acceptable salts, esters, or salts of the esters, or any other compounds which, upon administration are capable of providing (directly or indirectly) the biologically active metabolite.

[0164] The term "pharmaceutically acceptable salts" as used herein refers to physiologically and pharmaceutically acceptable salts of the antisense compounds that retain the desired biological activities of the parent compounds and do not impart undesired toxicological effects upon administration. Examples of pharmaceutically acceptable salts and their uses are further described in US 6,287,860.

[0165] Antisense compounds of the disclosure may be prodrugs or pharmaceutically acceptable salts of the prodrugs, or other bioequivalents. The term "prodrugs" as used herein refers to therapeutic agents that are 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, prodrug forms of the antisense compounds of the disclosure are prepared as SATE [(S acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93 / 24510, WO 94 / 26764 and US 5,770,713.

[0166] A prodrug may, for example, be converted within the body, e. g. by hydrolysis in the blood, into its active form that has medical effects. Pharmaceutical acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A. C. S. Symposium Series (1976); "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985; and in 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 appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate".

[0167] Methods of treatment

[0168] Autoimmune epilepsy

[0169] Epilepsy is a disease of the brain defined by any of the following conditions:

[0170] 1. At least two unprovoked (or reflex) seizures occurring >24 h apart;

[0171] 2. One unprovoked (or reflex) seizure and a probability of further seizures similar to the general recurrence risk (at least 60%) after two unprovoked seizures, occurring over the next 10 years; and

[0172] 3. Diagnosis of an epilepsy syndrome

[0173] Epilepsy types are categorized as:

[0174] 1. Focal

[0175] 2. Generalized

[0176] 3. Generalized and F ocal

[0177] 4. unknown

[0178] Different seizure types can result from genetic, structural, metabolic, immune, infectious, unknown causes and can involve

[0179] 1. Focal (one hemisphere)

[0180] 2. Generalized (bilateral)

[0181] 3. Unknown sites

[0182] Epilepsy is considered resolved for individuals who had an age-dependent epilepsy syndrome but are now past the applicable age or those who have remained seizure-free for the last 10 years, with no seizure medicines for the last 5 years.

[0183] Status epilepticus (SE) is a condition resulting either from the failure of the mechanisms responsible for seizure termination or from the initiation of mechanisms, which lead to abnormally, prolonged seizures (after a first time point). They can have long-term consequences (after a second time point), including neuronal death, injury, and alteration of neuronal networks, depending on type and duration of seizures.”

[0184] Drug-resistant epilepsy (DRE) can involve high IL- lb in blood innate immune cells, monocytes with high number of CNS glial cells which are difficult to reach with drugs administered systemically. Super refractory status epilepticus SRSE. SE continues for more than 24 h after the first administration of general anaesthesia.

[0185] New-onset refractory status epilepticus (NORSE) has a clinical presentation without cause; Febrile infection-related epilepsy syndrome (FIRES) is a NORSE Subgroup with prior febrile infection 2 weeks + 24 hours prior to onset refractory SE.

[0186] Electrical status epilepticus in sleep (ESES) is a childhood-onset epileptic encephalopathy, epilepsy, cognitive regression, and marked activation during non-rapid eye movement (NREM) sleep to produce an electroencephalography (EEG) pattern of near-continuous spike-wave discharges.

[0187] Autoimmune encephalopathy has an autoimmune component often targeting GABA, and NMDA receptor of glutamate signalling which are ligands for anti-epileptic drugs. Autoimmune encephalitis (AE) is often associated with the presence of antibodies targeting GABA and NMDA receptor of glutamate signaling channels. Rasmussen encephalitis (RE) is a type of AE with unilateral hemispheric encephalitis whose main clinical features include refractory focal epilepsy or epilepsia partialis continua, hemiparesis, and progressive cognitive decline. It is a progressive disease characterised by drug-resistant focal epilepsy, progressive hemiplegia, and cognitive decline, with unihemispheric brain atrophy. RE is a rare disorder and affects mostly children or young adults. A German study estimated the countrywide incidence at 2 4 cases per 10 million people <18 years. Current treatments address seizures via antiepileptics or via hemispherectomy or hemispherotomy._Some of the underpinning immune damage may be via T-cell toxicity with inflammatory T cells being CD8+ found in the CNS, and with CD8s and NK cells in the circulation.

[0188] Focal human epilepsy is common and the most common focal epilepsy is temporal Lobe Epilepsy (TLE), representing about 60% of focal epilepsy cases. Injection of Pilocarpine (CAS No. 54-71-7) in mice and rats is considered a good model of TLE because it produces characteristics observed in human epilepsy, including a seizure-free (latent) period prior to onset of (spontaneous) recurrent seizures and development of localized lesions within the hippocampus Of interest also, the chronic seizures induced by pilocarpine are largely refractory to anti -seizure drugs, a situation similar to that observed in patients with TLE and AE. Treatment of epilepsy with oligonucleotides against CD49d

[0189] As disclosed herein antisense oligonucleotides targeting CD49d, are effective in the treatment of epilepsy. In some embodiments the epilepsy is autoimmune epilepsy, focal epilepsy, or generalized epilepsy.

[0190] Accordingly, in some embodiments provided herein is a method for treating autoimmune epilepsy in a subject identified as in need thereof, where the method includes a step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide targeting CD49d and a pharmaceutically acceptable excipient.

[0191] In some embodiments the oligonucleotide targeting CD49d is an oligonucleotide comprising the structure: wherein, a) each of the 19 intemucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at the positions 1 to 3 from the 5' end are 2'-O-(2- methoxy ethyl) modified ribonucleosides; c) the nucleotides at the positions 4 to 12 from the 5' end are 2'- deoxy rib onucl eosi des ; d) the nucleotides at the positions 13 to 20 from the 5' end are 2'-O-(2- methoxyethyl) modified ribonucleosides; and e) all cytosines are 5-methylcytosines (MeC).

[0192] This oligonucleotide is referred to herein as “ATL1102” and has the sequence of SEQ ID NO: 1 disclosed herein.

[0193] In some embodiments the autoimmune epilepsy is associated with a condition in the subject selected from the group consisting of: autoimmune encephalitis, Hashimoto’s disease, Rasmussen’s encephalitis, systemic lupus erythematosus, Sjogren’s syndrome, Wegener’s granulomatosis, neurosarcoidosis, celiac disease, Chron’s disease, and Behcet’s. In some preferred embodiment the condition is an autoimmune encephalitis, Hashimoto’s disease, or Rasmussen’s encephalitis.

[0194] In some embodiments, the form of AE in a subject is selected from the group consisting of Acute disseminated encephalomyelitis, anti-NMDAR receptor encephalitis, Hashimoto’s encephalopathy, LG1 l / CASPR2-antibody encephalitis, limbic encephalitis, and Rasmussen’s encephalitis (RE). In some embodiments the AE is refractory to standard treatments.

[0195] In some embodiment the subject to be treated is suffering from focal epilepsy (FE).

[0196] In some preferred embodiments the subject to be treated is a human subject. In some embodiments, the subject has Rasmussen’s encephalitis and is pediatric. In some embodiments, the subject has Rasmussen’s encephalitis and is post-pubescent.

[0197] In some embodiments the subject to be treated does not suffer from muscular dystrophy or multiple sclerosis.

[0198] In other embodiments the subject is a rodent, a lagomorph, or a non-human primate.

[0199] In some embodiments the subject to be treated by administration of an oligonucleotide targeting CD49d suffers from focal epilepsy (FE).

[0200] In some embodiments the therapeutically effective amount is sufficient to result in one or more of: (i) reduced severity of status epilepticus (SE); (ii) seizure duration; or (iii) seizure frequency, as assessed by methods known in the art as described in, e.g., Handbook of Clinical Neurology, volume 107, pages 2-507.

[0201] In some embodiments a treatment comprising administration of a CD49d-targeting oligonucleotide as disclosed herein results in a reduction of CD49+ CD4+ or CD8+ T cells of at about 10% to about 70%, e.g., 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or another reduction in CD49+ CD4+ or CD8+ T cells (relative to an untreated subject) of about 10% to about 70%.

[0202] In some embodiments the method includes monitoring for circulating lymphocytes, CD4+ and / or CD8+ T cell and / or NK lymphocyte levels. In some embodiments the method includes monitoring for reduced CD8+ T cell levels in the circulation or CNS. In some embodiments, the method includes monitoring for microglia, or astrocytes in the CNS, or neural apoptosis, including via neurofilament light chain in the CNS, CSF, or the blood. In some embodiments, the method comprises determining the level or presence of one or more markers of AE or FE and include the level or number of immune cells or immunomodulatory factors produced thereby, including the level of antibody markers relevant to the AE.

[0203] In some embodiments, the one or more symptoms of AE include quality of life factors such as energy levels, happiness, perceived ease of daily functional activities etc.

[0204] In some embodiments, the subject in need thereof includes subjects with clinical diagnosis of AE based on antibodies, or immune cells and inflammatory mediators.

[0205] In some embodiments, the subject displays normal or only slightly elevated levels of inflammatory cells. Inflammatory cells include lymphocytes (T cells (CD4, CD8), NK cells, B-cells (CD-19)), granulocytes, (neutrophils, basophils, and eosinophils).

[0206] Suitable methods for cell surface markers (e.g., CD49d or VLA-4) including cell or protein / nucleic acid or lipid analysis are known in the art and include without limitation flow cytometry, bead technologies and ELISA-based methods, chromatographic and / or Mass Spectrometry methods. Likewise, expression of such markers at the nuclei acid level can be assessed by hybridization or sequencing based methods.

[0207] In a further embodiment, the subject diagnosed with AE or FE displays significantly elevated or acute levels of neural necrosis and inflammation as determined by immune cells or their mediators.

[0208] In some embodiments, the subject displays significantly elevated levels of CD49d T- cells relative to normal healthy controls.

[0209] Combination treatments

[0210] In some embodiments a pharmaceutical composition comprising a CD49d-targeting oligonucleotide disclosed herein can be used in combination with an additional therapeutic agent, e.g., a therapeutic agent used in conventional treatment of epilepsy or autoimmune epilepsy.

[0211] Conventional treatments for AE include anti-epileptics, and anti-inflammatory and immune regulating treatments, and may include surgery. Current anti-inflammatory immune regulating treatments for AE include corticosteroids to reduce brain inflammation and immune system response, plasma exchange to remove harmful antibodies and IVIG to introduce blocking antibodies from the plasma of healthy donors to block harmful antibody immune mediated damage and immunosuppressants if other treatments are not effective, but standard of care is often ineffective.

[0212] Accordingly, in some embodiments, treatment of a subject suffering from an autoimmune epilepsy includes administering, in addition to administration of a CD49d- targeting oligonucleotide as disclosed herein, a therapeutically effective amount of an additional therapeutic agent. In some embodiments the additional therapeutic agent is a corticosteroid. In some embodiments the corticosteroid is prednisone (or a prednisone equivalent), deflazacort (a derivative or prednisolone), dexamethasone, budesonide, betamethasone, deflazacort, and valmoralone. Other corticosteroids are known in the art.

[0213] In some embodiments, corticosteroid is administered at a low dose. Reference to a low dose corticosteroid includes 2 / 3rd, 1 / 2, 1 / 4, and a l / 3rdof a standard corticosteroid dose for treatment of epilepsy or seizures, e.g., 60 mg / day.

[0214] In other embodiments the additional therapeutic agent is an anti-epileptic. Suitable examples of anti-epileptics include, but are not limited to, Diazepam, Midazolam (as GABA receptor agonists), Valproate, Levetiracetam, Fosphenytoin, Lamotrigine, Oxcarbazepine, Pentobarbital, Gabapentin, and Lithium.

[0215] Combination treatments as disclosed herein include any of co-administration, using separate formulations (or a single pharmaceutical formulation), and consecutive administration in either order, wherein generally there is a time period while both (or all) active agents simultaneously exert their biological activities.

[0216] As used herein, the term "combination" in the context of the administration of a therapy refers to the use of more than one therapy or therapeutic agent. The use of the term "in combination" does not restrict the order in which the therapies or therapeutic agents are administered to a subject. A therapy or therapeutic agent can be administered prior to, concomitantly with, or subsequent to the administration of a second therapy or therapeutic agent to a subject. In some embodiments no additional therapeutic agents are administered in a treatment method disclosed herein, such that a subject is treated with a monotherapy of a pharmaceutical composition comprising a CD49d-targeting oligonucleotide as disclosed herein.

[0217] Administration

[0218] In some embodiments, the antisense compound of the disclosure is administered systemically. As used herein "systemic administration" is a route of administration that is either enteral or parenteral.

[0219] As used herein "enteral" refers to a form of administration that involves any part of the gastrointestinal tract and includes oral administration of, for example, the antisense oligonucleotide in tablet, capsule or drop form; gastric feeding tube, duodenal feeding tube, or gastrostomy; and rectal administration of, for example, the antisense compound in suppository or enema form.

[0220] As used herein "parenteral" includes administration by injection or infusion outside of the alimentary canal. In some embodiments administration of a pharmaceutical composition comprising a CD49d-targeting oligonucleotide is by a parenteral route. Examples of parenteral routes of administration include, intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), subcutaneous (under the skin), intraosseous infusion (into the bone marrow), intradermal, (into the skin itself), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical (infusion into the urinary bladder), transdermal (diffusion through the intact skin), transmucosal (diffusion through a mucous membrane), intranasal, and inhalational.

[0221] In some embodiments, administration of a pharmaceutical composition comprising a CD49d-targeting oligonucleotide as disclosed herein is subcutaneous.

[0222] The antisense composition may be administered as single dose or as repeated doses on a period basis, for example, daily, once every two days, three, four, five, six seven, eight, nine, ten, eleven, twelve, thirteen or fourteen days, once weekly, twice weekly, three times weekly, or every two weeks, or every three weeks. In some embodiments, administration is 1 to 3 times per week, or once every week, two weeks, three weeks, four weeks, or once every two months.

[0223] In some embodiments, administration is once weekly.

[0224] In some embodiments, a low dose is administered for 3 to 6 months, such as about 25- 50 mg / week for at least three to six months and then up to 12 months and chronically.

[0225] Illustrative doses are between about 10 to 300 mg. Illustrative doses include 10, 25, 50, 100, 150, 200 mg. Illustrative doses include 0.5 mg / kg (about 10-25 mg), 1 mg / kg (about 25-50 mg), 1.5 mg / kg (about 50 to 100 mg) and 3 mg / kg (about 100-200 mg) and 4.5 mg / kg (about 150-300 mg). In some embodiments doses are administered once per week. Thus in some embodiments, a low dose of approximately 10 to 30, or 20 to 40, or 20 to 28 mg may be administered to subjects typically weighing between about 25 kg and 65kg. In some embodiments the antisense oligonucleotide is administered at a dose of less than 50 mg, or less than 30 mg, or about 25 mg or about 10 mg per dose to produce a therapeutic effect. In some embodiments the antisense oligonucleotide, ATL1102 is administered at a dose of less than 75 mg or about 50 mg, or less than 30 mg, or about 25 mg per dose to produce a therapeutic effect.

[0226] In some embodiments, a therapeutic effect such as a delay in AE or FE progression is seen within about three months after administration of the first dose with absence of status epilepticus (SE) or severity of SE, or reduced seizure burden with lower frequency of recurrent seizures and / or seizure severity or duration. The term "therapeutically effective amount" as used herein refers to a dose of the antisense compound sufficient for example to improve one or more markers, signs or symptoms of AE or FE or to delay progression of AE or FE in a subject, or to improve one or more markers, signs or symptoms of AE or FE or to delay progression of AE or FE in a subject under the conditions of administration.

[0227] In other embodiments, the administration is effective to provide a Cmax of the oligonucleotide in the plasma of the human subject upwards of 2890ng / mL and in some embodiments, about 10,000-11,000 ng / mL In other embodiments, 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 some embodiments at least 20 ng / mL, or at least 45 ng / mL.

[0228] Studies in patients can be conducted to demonstrate the treatment of AE or FE with an inhibitory oligonucleotide to CD49b (a subunit of VLA-4 integrin) which reduces the level of VLA-4 in the blood of human subjects. Reduction in the level of VLA-4 may be detected in hematology assessments of lymphocytes or cytometry assessments of subsets of T cells or NK cells in one or more organ including blood, or CSF or CNS also looking at microglia, astrocytes. In some embodiments, where corticosteroids are used in a combination therapy, subjects are taken off corticosteroids approximately 24 hours prior to administration to assess effects on lymphocytes. This allows an assessment of the effects of inhibitory antisense oligonucleotide to the CD49d alpha chain of VLA-4 integrin in immune cells in the absence of corticosteroid, which is not present in significant levels in the blood stream at 24 hours after administration to be having an effect on circulating immune cells.

[0229] In other embodiments subjects are administered inhibitory oligonucleotide to VLA-4 integrin after corticosteroid with the corticosteroid used to reduce injection site reactions.

[0230] The following examples are intended to be illustrative and not limiting.

[0231] EXAMPLES

[0232] EXAMPLE 1

[0233] Pilocarpine injection in mice is considered a model of temporal lobe epilepsy (TLE) relevant for human focal encephalitis (FE) and autoimmune encephalitis (AE). Pilocarpine injection produces characteristics observed in human epilepsy, including a seizure-free (latent) period prior to onset of (spontaneous) recurrent seizures and development of localised lesions within the hippocampus.

[0234] Buckmaster et al (2017) showed post pilocarpine treatment onset of spontaneous epilepsy (SE) in FVB mice, and an average spontaneous recuring seizure rate (SRS) of 0.13 seizures per mouse per hour from day 31 to 45. Mice were video monitored for 30 days, from day 31 to 60. Days 31 to 45 were the first 15 days of the 30-day video monitoring, in which spontaneous recurrent seizure rate (SRS) was recorded over 9-hour monitoring sessions each day. Buckmaster et al (2011) showed that the immunosuppressant Rapamycin, targeting mTOR kinase to inhibit T and B cell response to IL2, when used at 3mg / kg, had no effect on SRS frequency or SRS severity in the FVB strain of mice, and immune modulating drugs have yet to show benefits in the FVB mouse TLE model.

[0235] In order to investigate the effect of ATL1102, an antisense oligonucleotide (ASO) to the human CD49d alpha-subunit of VLA-4 lymphocyte adhesion molecule in treating epilepsy induced in this translationally relevant model, FVB male mice (n=20 / group) were dosed with 20mg / kg / week ASO to mouse CD49d (ISIS348574), negative control mismatch oligonucleotide (MM), or saline, 2 days before seizures were induced with 315mg pilocarpine with two thirds of mice surviving as anticipated (14, 12, and 14 mice respectively).

[0236] Onset to status epilepticus (SE) and SE severity was assessed for 120 minutes postinduction. Mice were then recorded for spontaneous recuring seizures (SRS) 8hrs daily, starting 31 days post SE for 15 days, and seizure duration, severity and frequency recorded in a blinded fashion.

[0237] Results

[0238] ASO to CD49d delayed SE onset compared to MM (37%,p=0.0237) and pooled saline+MM controls (26%,p=0.0166) (Figure 1), and reduced SE severity compared to saline over 90-120 minutes after SE onset (p=0.0072) (Figure 2).

[0239] ASO delayed median SRS frequency compared to saline on days 31-32 (100%, p=0.0019) and median SRS onset significantly, by 3 days, compared to saline (p=0.0041) (Figures 3a and 3b). A trend towards delay was seen in ASO SRS onset compared to MM, the ASO median SRS is still zero at day 34 whereas the MM day 34 median SRS is 1 (Figure 3c).

[0240] ASO reduced median SRS frequency compared to both saline and MM from days 31-38 (53%, p=0.0868, 53%, p=0.0356 Figure 4), days 31-43 (65%, p=0.0307, 55%, p=0.0258 Figure 5) and days 31-46 (62%, p=0.0508, 68%, p=0.039 Figure 6), and compared to pooled controls (p=0.0256, p=0.0575, p=0.0182, Figures, 4, 5, and 6 right hand side, respectively).

[0241] Conclusion

[0242] The present inventors are the first to demonstrate use of ASO to CD49d in mouse pilocarpine AE models and to demonstrate support for the use of ATL1102, ASO to the human CD49d alpha-subunit of VLA-4 lymphocyte adhesion molecule (see, e.g. Limmroth et al.), in AE and FE patients, especially those resistant to current treatments.

[0243] EXAMPLE 2 (prophetic)

[0244] ATL1102 is administered to juvenile (or pubescent) subject(s) 10 years, or older subjects including adults of up to any age, with refractory epilepsy once weekly at about 0.5mg / kg (about 10-25mg), about Img / kg (about 25-50mg), about 1.5mg / kg (about 50-100mg), about 3mg / kg (about 100-200mg), and 4.5mg / kg (150-300mg) for up to 12 weeks, or in a dose escalation study. The effects of the administered oligonucleotide for example on seizure frequency and inflammatory markers are measured. Markers for neural regeneration such as neurofilament light chain are measured or epilepsy is monitored via EEG. Markers may be detected in situ or in any suitable biological sample, for example in plasma, urine, or cerebrospinal fluid biopsy. Quality of life questionnaires are useful in determining the effect of treatments, and may be combined and responses collected and analysed.

[0245] ATL1102 is suitable for administration to patients currently on other medications, for example anti-epileptic medications and therapeutics, and / or corticosteroids, which may continue to be dosed. Alternatively, ATL1102 may be the only current treatment a patient is on, or the patient may begin treatment with another medication, for example antiepileptic medications and therapeutics, and / or corticosteroids, during the period of time in which they are being administered ATL1102, for example during a period of up to 12 weeks. If patients have failed corticosteroids or IvIG, they may be kept on such treatment or taken off such treatment as recommended by the treating physician. ATL1102 administration is thus suitable for use in combination with other therapies, as determined by the skilled clinician.

[0246] EXAMPLE 3 (prophetic)

[0247] ATL1102 is administered to paediatric subjects aged 4-18 years old with AE, RE, or FE once weekly at about 0.5mg / kg (about 10-25mg), about Img / kg (about 25-50mg), about 1 ,5mg / kg (about 50-100mg) for up to 12 weeks or twice weekly at the above doses such that the total weekly dose does not exceed about 3mg / kg (about 100-200mg). twice weekly doses may be equal doses, or they may be unequal, for example, a larger first dose followed by a smaller top-up dose. The effects of the administered ATL1102 then assessed as in Example 1 above.

[0248] EXAMPLE 4 (prophetic)

[0249] Low-dose administration of inhibitory oligonucleotide

[0250] 16 RE patients aged 10 to 18 years of age, and of about 25 to 60kg in weight, receive ATL1102 at a starting dose of 25 mg once weekly for 24 weeks. After 24 weeks of treatment an 8-week monitoring period is performed. In the treatment and monitoring period, assessments are made: at baseline, and every 4 weeks during the treatment period, and every 4 weeks in the post-treatment monitoring period. The pharmacodynamic activity outcome is to assess the number of circulating lymphocytes, CD4+ and CD8+ T cells, at 8, 12 and 24 weeks of treatment compared to baseline and four weeks post the end of treatment. Safety is also assessed at these timepoints, including identifying, monitoring, recording, and analysing injection site reactions, platelet changes, liver enzyme GGT -bilirubin, CRP and albumin, A / G ratio changes measured. Secondary endpoints for clinical assessments are measures of status epilepticus (SE) onset and severity, including number of spontaneous recurrent seizures, and their duration and severity, number of convulsions, and quality of life, and pharmacokinetics. Exploratory outcome measures identified, monitored, measured, recorded, and analysed will include: serum / plasma biomarker response such as those related to inflammation and neural apoptosis / degeneration, for example nfl; and / or cytokines, one or more of which may be markers of CNS injury; and / or proteomics; and / or mononuclear cell RNA array; and / or exosome RNA.

Claims

1. CLAIMS1. A method for treating epilepsy in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide targeting CD49d, and a pharmaceutically acceptable excipient.

2. A method for treating autoimmune epilepsy in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide targeting CD49d; and a pharmaceutically acceptable excipient.

3. The method according to claim 1 or claim 2, wherein the oligonucleotide targeting CD49d is an oligonucleotide comprising the structure:wherein, a) each of the 19 intemucleotide linkages of the oligonucleotide is an 0,0- linked phosphorothioate diester; b) the nucleotides at the positions 1 to 3 from the 5' end are 2'-O-(2- methoxy ethyl) modified ribonucleosides; c) the nucleotides at the positions 4 to 12 from the 5' end are 2'- deoxy rib onucl eosi des ; d) the nucleotides at the positions 13 to 20 from the 5' end are 2'-O-(2- methoxyethyl) modified ribonucleosides; and e) all cytosines are 5-methylcytosines (MeC).

4. The method according to claim 3, wherein the oligonucleotide consists of the structure.

5. The method according to any one of claims 1 to 4, wherein the autoimmune epilepsy is associated with a condition in the subject selected from the group consisting of: autoimmune encephalitis, Hashimoto’s disease, Rasmussen’s encephalitis, systemic lupus erythematosus, Sjorgren’s syndrome, Wegener’s granulomatosis, neurosarcoidosis, celiac disease, Chrohn’s disease, and Behcet’s.

6. The method according to claim 5, wherein the condition is an autoimmune encephalitis, Hashimoto’s disease, or Rasmussen’s encephalitis.

7. The method according to any one of claims 1 to 6, wherein the subject suffers from focal epilepsy (FE).

8. The method according to any one of claims 1 to 7, wherein the therapeutically effective amount is a dose of 10 mg to 300 mg.

9. The method according to claim 8, wherein the therapeutically effective amount is a dose of about 25 mg to about 250 mg.

10. The method according to claim 8 or claim 9, wherein the administration is once, twice, or three times per week.

11. The method according to any one of claims 1 to 10, wherein the oligonucleotide is a sodium or potassium salt.

12. The method according to any one of claims 1 to 11, wherein the pharmaceutical composition is administered subcutaneously.

13. The method of according to any one of claims 1 to 12, further comprising administration of a corticosteroid.

14. The method according to any one of claims 1 to 13, wherein the therapeutically effective amount of the oligonucleotide administered as monotherapy or in combination with an additional therapeutic agent is sufficient to result in one or more of:(i) reduced severity of status epilepticus (SE);(ii) seizure duration; or(iii) seizure frequency.

15. The method according to any one of claims 1 to 14, wherein the subject does not suffer from muscular dystrophy or multiple sclerosis.

16. The method according to any one of claims 1 to 15, wherein the subject is a human subject.

17. Use of an oligonucleotide targeting CD49d in the manufacture of a medicament for treatment of epilepsy.

18. An oligonucleotide targeting CD49d for use in treatment of epilepsy.

19. The use according to claim 17 or the oligonucleotide for use according to claim 18, wherein the oligonucleotide (ATL1102) comprises the structure:wherein, a) each of the 19 intemucleotide linkages of the oligonucleotide is an 0,0- linked phosphorothioate diester; b) the nucleotides at the positions 1 to 3 from the 5' end are 2'-O-(2- methoxy ethyl) modified ribonucleosides; c) the nucleotides at the positions 4 to 12 from the 5' end are 2'- deoxy rib onucl eosi des ; d) the nucleotides at the positions 13 to 19 from the 5' end are 2'-O-(2- methoxyethyl) modified ribonucleosides; and e) all cytosines are 5-methylcytosines (MeC), or a pharmaceutically acceptable salt thereof.

20. The use or the oligonucleotide for use according to any one of claims 17 to 19, wherein the epilepsy is autoimmune epilepsy.

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