Treatment of amyotrophic lateral sclerosis

Abstract

The present disclosure provides the use of neurofilament light chain levels to select subjects with mutations in the superoxide dismutase 1 (SOD1) gene for treatment with an SOD1-targeted antisense oligonucleotide or a salt thereof. The method according to the present disclosure can be applied to the treatment of amyotrophic lateral sclerosis, including preclinical amyotrophic lateral sclerosis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 168,972, filed March 31, 2021. The entire contents of the aforementioned application are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on Mar. 24, 2022, is named 13751-0344WO1_SL.txt and is 7,948 bytes in size.

[0003] The present disclosure relates to biomarkers and treatments for amyotrophic lateral sclerosis. [Background technology]

[0004] Soluble superoxide dismutase 1 (SOD1) enzyme, also known as Cu / Zn superoxide dismutase, converts superoxide into hydrogen peroxide (H 2 O 2 ) to the superoxide anion (O 2- ) is a potentially harmful cellular by-product produced primarily by errors in mitochondrial oxidative phosphorylation (Turrens, J. Physiol., 552:335-344 (2003)).

[0005] Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) usually refers to a serious progressive neurodegenerative disease that affects about 17,000 Americans (Mehta, P., et al. (2018) "Prevalence of Amyotrophic Lateral Sclerosis-United States, 2015." MMWR Morb Mortal Wkly Rep 67(46):1285-1289). Approximately 2% of ALS cases are due to mutations in the gene that codes for SOD1 (Bunton-Stasyshyn RKA, et al. Neuroscientist. 2015; 21:519-29). Mutations in the SOD1 gene are usually associated with the overtly inherited form of ALS, a disease characterized by selective degeneration of upper and lower motor neurons (Rowland, N. Engl. J. Med., 2001, 344:1688-1700 (2001)).

[0006] The toxicity of mutant SOD1 is thought to result from early misfolding (gain of function) that reduces nuclear protection from active enzyme (loss of nuclear function), a process that may be involved in the pathogenesis of ALS (Sau, Hum. Mol. Genet., 16:1604-1618 (2007)). The progressive degeneration of motor neurons in ALS eventually leads to neuronal death. When motor neurons die, the brain loses its ability to initiate and control muscle movements. Voluntary muscle activity is gradually affected, and patients in the later stages of the disease may become completely paralyzed. There remains an unmet need for effective therapies to treat ALS. Accordingly, it is an object of the present disclosure to provide methods for treating the disease. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Fridovich, Annu.Rev.Biochem.,64:97-112(1995) [Non-Patent Document 2] Turrens, J.Physiol.,552:335-344(2003) [Non-Patent Document 3] Mehta, P., et al. (2018) “Prevalence of Amyotrophic Lateral Sclerosis-United States,2015.” MMWR Morb Mortal Wkly Rep 67(46):1285-1289 [Non-Patent Document 4] Bunton-Stasyshyn RKA, et al.Neuroscientist.2015;21:519-29 [Non-Patent Document 5] Rowland, N. Engl. J. Med., 2001, 344: 1688-1700 (2001) [Non-Patent Document 6] Sau,Hum.Mol.Genet.,16:1604-1618(2007) Summary of the Invention

[0008] The present disclosure relates, in part, to the treatment of amyotrophic lateral sclerosis associated with mutations in the SOD1 gene in subjects (e.g., adults) with clinical symptoms / signs and in pre-symptomatic subjects (e.g., adults) with evidence of biomarkers of disease (e.g., neurofilament light chain levels of at least 44 pg / mL).

[0009] Provided herein, in some aspects, is a treatment for amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene in a human subject having a neurofilament light chain level of at least 44 pg / ml, e.g., a human subject who is clinically presymptomatic with ALS.

[0010] In one aspect, the disclosure features a method of treating amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene in a human subject in need of treatment by administering to the human subject a pharmaceutical composition that includes a therapeutically effective amount of an antisense oligonucleotide according to the formula: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te (nucleic acid base sequence of SEQ ID NO: 1) During the ceremony, A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl ribose modified sugar; d is a 2'-deoxyribose sugar, s is a phosphorothioate internucleoside linkage, o is a phosphodiester internucleoside linkage, The human subject has a neurofilament light chain level of at least 44 pg / ml prior to the initiation of treatment.

[0011] In some embodiments, the human subject experiences an increase in neurofilament light chain levels of at least 10 pg / ml prior to the initiation of treatment.

[0012] In some embodiments, the neurofilament light chain level is a level in a biological sample from a human subject, e.g., a blood, serum, plasma, or cerebrospinal fluid sample. In some embodiments, the neurofilament light chain level corresponds to a plasma level, e.g., a plasma level of at least 44 pg / ml and / or an increase in plasma level of at least 10 pg / ml. In some embodiments, the neurofilament light chain level is a blood, serum, or cerebrospinal fluid level that corresponds to a corresponding plasma level (e.g., a plasma level of at least 44 pg / ml and / or an increase in plasma level of at least 10 pg / ml).

[0013] In some embodiments, the human subject has a neurofilament light chain level of at least 44 pg / ml prior to the initiation of treatment, and further, the human subject experiences an increase in neurofilament light chain levels of at least 10 pg / ml prior to the initiation of treatment.

[0014] In some embodiments, the human subject has a plasma neurofilament light chain level of at least 44 pg / ml prior to the initiation of treatment, and further, the human subject experiences an increase in plasma neurofilament light chain level of at least 10 pg / ml prior to the initiation of treatment.

[0015] In another aspect, the disclosure features a method of treating amyotrophic lateral sclerosis in a human subject in need of treatment, the method comprising: measuring a neurofilament light chain level of at least 44 pg / ml in a biological sample obtained from the human subject prior to initiation of treatment; administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of an antisense oligonucleotide according to the formula: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te (nucleic acid base sequence of SEQ ID NO: 1) During the ceremony, A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl ribose modified sugar; d is a 2'-deoxyribose sugar, s is a phosphorothioate internucleoside linkage, o is a phosphodiester internucleoside linkage.

[0016] In some embodiments, the biological sample is blood, serum, plasma, or cerebrospinal fluid.

[0017] In some embodiments, the biological sample is blood.

[0018] In some embodiments, the biological sample is serum.

[0019] In some embodiments, the biological sample is plasma.

[0020] In some embodiments, the method further comprises measuring an increase of at least 10 pg / ml in blood, serum, plasma, or cerebrospinal fluid neurofilament light chain levels in the human subject prior to administering the antisense oligonucleotide or a pharma- cerebrospinal fluid salt thereof.

[0021] In some embodiments, the method further comprises measuring an increase in plasma neurofilament light chain levels of at least 10 pg / ml in the human subject prior to administering the antisense oligonucleotide or a pharma- ceutically acceptable salt thereof.

[0022] In some embodiments, the method further comprises measuring, prior to administering the antisense oligonucleotide or a pharma- cerebrospinal fluid salt thereof, in the human subject an increase in neurofilament light chain levels at blood, serum, or cerebrospinal fluid levels corresponding to an increase in plasma neurofilament light chain levels of at least 10 pg / ml.

[0023] In some embodiments of any of the methods described herein, the pharmaceutical composition is administered by intrathecal administration.

[0024] In some embodiments of any of the methods described herein, the pharmaceutical composition delivers a fixed dose of about 100 mg of the antisense oligonucleotide.

[0025] In some embodiments of any of the methods described herein, the mutation in the SOD1 gene is A4V.

[0026] In some embodiments of any of the methods described herein, the mutation in the SOD1 gene is A4V, H46R, G93S, A4T, G141X, D133A, V148G, N139K, G85R, G93A, V14G, C6S, I113T, D49K, G37R, A89V, E100G, D90A, T137A, E100K, G41A, G41D, G41S, G13R, G7 2S, L8V, F20C, Q22L, H48R, T54R, S591, V87A, T88deltaTAD, A89T, V97M, S105deltaSL, V118L, D124G, L114F, D90A, G12R, G147R, C6F, C6G, D101G, D101H, G114A, G85S, H43R, L106F, L106V, L38V, or R115G.

[0027] In some embodiments of any of the methods described herein, the human subject has clinically pre-symptomatic amyotrophic lateral sclerosis.

[0028] In some embodiments of any of the methods described herein, the human subject is administered a loading dose of the pharmaceutical composition, followed by a maintenance dose of the pharmaceutical composition.

[0029] In some embodiments in which a human subject is administered a loading dose of the pharmaceutical composition followed by a maintenance dose of the pharmaceutical composition, the human subject is administered three loading doses, the loading doses being administered every 14 days.

[0030] In some embodiments in which a human subject is administered a loading dose of the pharmaceutical composition followed by a maintenance dose of the pharmaceutical composition, the maintenance dose is administered every 28 days starting 28 days after the third loading dose.

[0031] In some embodiments in which a human subject is administered a loading dose of a pharmaceutical composition followed by a maintenance dose of the pharmaceutical composition, the loading dose and maintenance dose of the pharmaceutical composition are administered to a human subject as follows. (i) administering a first loading dose of a pharmaceutical composition; (ii) administering a second loading dose of the pharmaceutical composition 14 days after the date of administration of the first loading dose; (iii) administering a third loading dose of the pharmaceutical composition 28 days after the date of administration of the first loading dose; (iv) administering a first maintenance dose of the pharmaceutical composition 28 days or one month after the date of administration of the third loading dose.

[0032] In some embodiments in which a human subject is administered a loading dose of a pharmaceutical composition followed by a maintenance dose of the pharmaceutical composition, the loading dose and maintenance dose of the pharmaceutical composition are administered to a human subject as follows. (i) administering a first loading dose sufficient to deliver a fixed dose of about 100 mg of antisense oligonucleotide; (ii) a second loading dose sufficient to deliver about 100 mg of a fixed dose of antisense oligonucleotide is administered 14 days after the date of administration of the first loading dose; (iii) a third loading dose sufficient to deliver about 100 mg of a fixed dose of antisense oligonucleotide is administered 28 days after the date of administration of the first loading dose; (iv) A first maintenance dose sufficient to deliver a fixed dose of about 100 mg of antisense oligonucleotide is administered 28 days after the date of administration of the third loading dose.

[0033] According to any of the methods described herein, in some embodiments, the human subject is an adult, e.g., the human subject is at least 18 years old, e.g., at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 years old or older.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In the practice or testing of the present invention, methods and materials similar or equivalent to those described herein can be used, among which exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present application will take precedence, including definitions. The materials, methods, and examples are merely illustrative and do not limit the present invention.

[0035] Other features and advantages of the invention will be apparent from the following detailed description and claims. [Brief description of the drawings]

[0036] [Figure 1] Graph showing model-based estimates of geometric mean NfL levels for participants aged 30 years or older at baseline and with clinically definite ALS harboring rapidly progressing mutations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure features the use of an antisense oligonucleotide or a salt thereof for the treatment of amyotrophic lateral sclerosis associated with mutations in the SOD1 gene in subjects (e.g., adults) with clinical symptoms / signs and in pre-symptomatic subjects (e.g., adults) with evidence of a biomarker of disease (e.g., a neurofilament light chain level of at least 44 pg / mL).

[0038] Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) refers to a rare neurodegenerative disease that causes the loss of motor neurons in the cortex, brainstem, and spinal cord. Patients suffer from progressive loss of muscle mass, strength, and function in bulbar, respiratory, and voluntary muscles. Debility is inevitable, and death usually occurs due to respiratory failure, on average, 2-5 years after diagnosis. Most patients suffer from sporadic ALS, but a small proportion of patients, approximately 2%, suffer from hereditary or familial ALS caused by various mutations in superoxide dismutase 1 (SOD1). Since its first discovery in 1993, more than 180 SOD1 mutations causing such ALS (called SOD1 ALS) have been reported to date. The Amyotrophic Lateral Sclerosis Online Genetics Database (ALSoD). Institute of Psychiatry, Psychology & Neuroscience. Published 2015; Rosen, Nature, 364(6435):362(1993)). Disease progression varies with each mutation, with the most severe mutation resulting in a survival time of less than 15 months.

[0039] Approved drugs for the treatment of ALS include riluzole (Rilutek®) and edaravone (Radicava™). Riluzole extends survival slightly (2-3 months) but does not result in significant improvement in muscle strength or disability. Edaravone reduces functional decline as measured by the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R). The effect of edaravone on survival remains unknown. There is still no SOD1-specific treatment for ALS.

[0040] Superoxide dismutase 1 Superoxide dismutase [Cu-Zn], also called superoxide dismutase 1 (SOD1), is an enzyme that in humans is encoded by the SOD1 gene, located on chromosome 21.

[0041] SOD1 is a 32 kDa homodimer that forms a β-barrel, with each subunit containing an intramolecular disulfide bond and a binuclear Cu / Zn site that holds copper and zinc ions and catalyzes the dismutation of superoxide to hydrogen peroxide and dioxygen.

[0042] SOD1 is one of three superoxide dismutases responsible for the destruction of free superoxide radicals in the body. The encoded isozyme is a soluble cytoplasmic and mitochondrial intermembrane space protein that acts as a homodimer to convert harmful natural superoxide radicals into molecular oxygen and hydrogen peroxide, which is broken down by another enzyme called catalase.

[0043] At least 180 mutations in the SOD1 gene are involved in familial ALS (Conwit RA, J Neurol Sci., 251(1-2): 1-2 (2006); Al-Chalabi A, Leigh PN, Curr. Opin. in Neurol., 13(4): 397-405 (2000); Redler RL, Dokholyan NV, Progress in Molecular Biology and Translational Science, 107: 215-62 (2012)). However, wild-type SOD1 is also thought to be involved in a significant number of sporadic ALS cases, which account for 90% of ALS patients, under conditions of cellular stress. The most frequent mutations in human SOD1 are A4V in the United States, H46R in Japan, and G93S in Iceland. Other well-known human SOD1 gene mutations include A4V, H46R, G93S, A4T, G141X, D133A, V148G, N139K, G85R, G93A, V14G, C6S, I113T, D49K, G37R, A89V, E100G, D90A, T137A, E100K, G41A, G41D, G41S, G13R, G72S, L8V, and F20C. , Q22L, H48R, T54R, S591, V87A, T88deltaTAD, A89T, V97M, S105deltaSL, V118L, D124G, L114F, D90A, G12R, G147R, C6F, C6G, D101G, D101H, G114A, G85S, H43R, L106F, L106V, L38V, and R115G. Virtually all known SOD1 mutations that cause ALS act dominantly; one mutated copy of the SOD1 gene is sufficient to induce the disease.

[0044] The amino acid sequence of human SOD1 can be found at UniProt P00441 and GENBANK accession number NP_000445, and is as follows: MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTS AGPHFNPLSR KHGGPKDEER HVGDLGNVTA DKDGVADVSI EDSVISLSGD HCIIGRTLVV HEKADDLGKG GNEESTKTGN AGSRLACGVI GIAQ (SEQ ID NO:2)

[0045] The nucleotide sequence encoding human SOD1 has GENBANK Accession No. NM_000454.4 and is as follows (the regions recognized by the antisense oligonucleotides of the present disclosure are underlined): [ka]

[0046] SOD1 antisense oligonucleotides "Antisense A" refers to a 5-10-5 MOE gapmer having the sequence (5' to 3' positions) of CAGGATACATTTCTACAGCT (SEQ ID NO:1). wherein each of nucleosides 1 to 5 and 16 to 20 is a 2'-O-methoxyethyl ribose modified nucleoside, each of nucleosides 6 to 15 is a 2'-deoxynucleoside, the internucleoside bonds present among nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 are phosphodiester bonds, the internucleoside bonds present among nucleosides 1 to 2, 3 to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 17 to 18, and 19 to 20 are phosphorothioate bonds, and each cytosine is 5-methylcytosine.

[0047] Antisense A is represented by chemical notation as follows: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te (SEQ ID NO: 1); During the ceremony, A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl ribose modified sugar; d is a 2'-deoxyribose sugar, s is a phosphorothioate internucleoside linkage, o is a phosphodiester internucleoside linkage.

[0048] "2'-O-methoxyethyl" (2'-MOE, 2'-OCH 2 CH 2 -OCH 3 , and MOE) refers to an O-methoxy-ethyl modification at the 2' position of the furanose ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0049] "5-methylcytosine" means a cytosine modified with a methyl group attached to the 5' position. 5-Methylcytosine is a modified nucleobase.

[0050] "Phosphorothioate linkage," or "phosphorothioate internucleoside linkage," refers to a linkage between nucleosides in which the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

[0051] The antisense A sequence may also be expressed in abbreviated form as follows: [ka] The underlined residues are 2'-MOE nucleosides. The P=O annotation reflects the position of the phosphodiester bond.

[0052] "2'-MOE nucleoside" (also known as 2'-O-methoxyethyl nucleoside) means a nucleoside that includes an MOE modified sugar moiety.

[0053] Antisense A is represented by the following chemical structure: [ka]

[0054] Antisense A is described in detail in US Pat. No. 10,385,341, the contents of which are incorporated herein by reference.

[0055] It will be understood that in solution (eg, in solution in a pharmaceutical composition), the antisense oligonucleotides may be present in a free acid form, a salt form, or a mixture thereof.

[0056] Conjugated antisense oligonucleotides The antisense oligonucleotides of the present disclosure can be covalently linked to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotide. Exemplary conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyes. Antisense oligonucleotides can also be modified to have one or more stabilizing groups that are typically attached to one or both ends of the antisense oligonucleotide to improve properties such as, for example, nuclease stability. Stabilizing groups include cap structures. These end modifications can protect antisense oligonucleotides with terminal nucleic acids from exonuclease degradation and aid in delivery and / or localization within cells. The cap can be present at the 5' end (5' cap) or 3' end (3' cap), or can be present at both ends. Cap structures are well known in the art and include, for example, reverse deoxy abasic caps. Additional 3'- and 5'-stabilizing groups that can be used to cap one or both ends of an antisense oligonucleotide to confer nuclease stability include those disclosed in WO03 / 004602.

[0057] Compositions and methods for formulating pharmaceutical compositions The antisense oligonucleotide or its salt of the present disclosure can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations.The composition and method for formulating pharmaceutical compositions are determined by several criteria, including but not limited to the route of administration, the extent of disease, or the dosage to be administered.

[0058] Antisense oligonucleotides or salts thereof targeted to SOD1 nucleic acid can be used in pharmaceutical compositions by combining antisense oligonucleotides or salts thereof with a suitable pharma- ceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate buffered saline (PBS). PBS is a suitable diluent for use in parenterally delivered compositions. Thus, in one embodiment, the pharmaceutical composition used in the methods described herein includes antisense oligonucleotides or salts thereof targeted to SOD1 nucleic acid and a pharma-ceutically acceptable diluent.

[0059] The antisense oligonucleotides or salts thereof described herein can be formulated as pharmaceutical compositions for intrathecal administration to a subject.

[0060] The pharmaceutical compositions comprising the antisense oligonucleotides of the present disclosure encompass any pharmaceutically acceptable salts, esters or salts of such esters, or any other oligonucleotides that can provide (directly or indirectly) a metabolite or residue thereof that has biological activity when administered to animals, including humans.Thus, for example, the present disclosure also covers the pharmaceutically acceptable salts of antisense oligonucleotides, as well as other bioequivalents.Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts.

[0061] treatment method The disclosure features a method of treating amyotrophic lateral sclerosis associated with a mutation in the human SOD1 gene (e.g., preclinical amyotrophic lateral sclerosis) in a human subject in need of such treatment. The method includes administering (e.g., intrathecally administering) an antisense oligonucleotide to the human subject, the antisense oligonucleotide having a nucleobase sequence of CAGGATACATTTCTACAGCT (SEQ ID NO:1), wherein each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyethyl ribose modified nucleoside, each of nucleosides 6-15 is a 2'-deoxynucleoside, and wherein The internucleoside bonds between nucleosides 2-3, 4-5, 16-17, and 18-19 are phosphodiester bonds, the internucleoside bonds between nucleosides 1-2, 3-4, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 17-18, and 19-20 are phosphorothioate bonds, and each cytosine is a 5-methylcytosine. In certain examples, the antisense oligonucleotide is administered at about 100 mg or a fixed dose of 100 mg.

[0062] "About" with respect to the amount of material means ±10% of the indicated value. "About" 100 mg of antisense oligonucleotide contains 90 mg to 110 mg of antisense oligonucleotide. For time units, e.g., about 10 days or about 1 week, "about" means ±3 days.

[0063] "Intrathecal or IT" refers to administration into the cerebrospinal fluid below the arachnoid membrane that covers the brain and spinal cord.

[0064] Also provided is a method of decreasing human SOD1 protein synthesis in a human subject having a mutation in the human SOD1 gene associated with amyotrophic lateral sclerosis. The method comprises administering (e.g., intrathecally administering) an antisense oligonucleotide to the human subject, the nucleobase sequence of the antisense oligonucleotide consisting of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), each of nucleosides 1-5 and 16-20 being a 2'-O-methoxyethyl ribose modified nucleoside, each of nucleosides 6-15 being a 2'-deoxynucleoside, and The internucleoside bonds between nucleosides 2-3, 4-5, 16-17, and 18-19 are phosphodiester bonds, the internucleoside bonds between nucleosides 1-2, 3-4, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 17-18, and 19-20 are phosphorothioate bonds, and each cytosine is a 5-methylcytosine. In certain examples, the antisense oligonucleotide is administered at about 100 mg or a fixed dose of 100 mg.

[0065] Also provided is a method of reducing human SOD1 mRNA levels in a human subject having a mutation in the human SOD1 gene associated with amyotrophic lateral sclerosis. The method comprises administering (e.g., intrathecally administering) an antisense oligonucleotide to the human subject, the nucleic acid sequence of the antisense oligonucleotide consisting of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), each of nucleosides 1 to 5 and 16 to 20 being a 2'-O-methoxyethyl ribose modified nucleoside, each of nucleosides 6 to 15 being a 2'-deoxynucleoside, and The internucleoside bonds between nucleosides 2-3, 4-5, 16-17, and 18-19 are phosphodiester bonds, the internucleoside bonds between nucleosides 1-2, 3-4, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 17-18, and 19-20 are phosphorothioate bonds, and each cytosine is a 5-methylcytosine. In certain examples, the antisense oligonucleotide is administered at about 100 mg or a fixed dose of 100 mg.

[0066] Also provided is a method of treating amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene (e.g., preclinical amyotrophic lateral sclerosis) in a human subject in need of such treatment, the method comprising administering (e.g., intrathecally) to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the structure: [ka]

[0067] In a particular example, the antisense oligonucleotide or a salt thereof is administered at a dose of about 100 mg or the equivalent of 100 mg of the antisense oligonucleotide.

[0068] Also provided is a method of decreasing human SOD1 protein synthesis in a human subject having a mutation in the human SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering (e.g., intrathecally) to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the structure: [ka]

[0069] In a particular example, the antisense oligonucleotide or a salt thereof is administered at a dose of about 100 mg or the equivalent of 100 mg of the antisense oligonucleotide.

[0070] Also provided is a method for reducing human SOD1 mRNA levels in a human subject having a mutation in the human SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering (e.g., intrathecally) to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the structure: [ka]

[0071] In a particular example, the antisense oligonucleotide or a salt thereof is administered at a dose of about 100 mg or the equivalent of 100 mg of the antisense oligonucleotide.

[0072] In some examples, the fixed dose of the antisense oligonucleotide or salt thereof is administered to a human subject once every week, once every two weeks, once every three weeks, or once every four weeks.

[0073] In some examples, the antisense oligonucleotide described herein is administered to a human subject as part of a pharmaceutical composition.In certain embodiments, the pharmaceutical composition is administered to a human subject in an amount sufficient to deliver a fixed dose of about 100 mg of antisense oligonucleotide.

[0074] In certain embodiments, the antisense oligonucleotide or its salt is administered in a loading dose(s). In some embodiments, the antisense oligonucleotide is administered in a maintenance dose(s). In certain examples, the antisense oligonucleotide is administered in a loading dose(s), followed by a maintenance dose(s). The loading dose(s) can be administered, for example, every week, every two weeks, every three weeks, or every four weeks. The maintenance dose(s) can be administered, for example, every week, every two weeks, every three weeks, or every four weeks after the last loading dose. In some examples, the maintenance dose(s) is administered monthly.

[0075] "Loading dose" refers to the dose administered during the dosing phase when dosing is initiated and a steady state concentration of drug (eg, antisense oligonucleotide) is achieved.

[0076] A "maintenance dose" refers to the dose administered during the administration phase after a steady state concentration of a drug (eg, an antisense oligonucleotide) has been achieved.

[0077] In certain embodiments, a human subject is administered three loading doses of the antisense oligonucleotide or salt thereof, followed by at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) maintenance doses. In some examples, the three loading doses are administered every two weeks. In some examples, the three loading doses are administered every 14 days. In some examples, the maintenance dose(s) are administered starting four weeks after the date of administration of the third loading dose. In some examples, the maintenance dose(s) are administered monthly, starting one month after the date of administration of the third loading dose. In some examples, the maintenance dose(s) are administered every 28 days, starting 28 days after the date of administration of the third loading dose.

[0078] The SOD1 mutation may be any mutation in the human SOD1 gene associated with ALS. In some examples, the mutation is a mutation in a slowly progressing ALS disease. In other examples, the mutation is a mutation in a rapidly progressing ALS disease. In certain examples, the mutation in the human SOD1 gene is A4V, H46R, G93S, A4T, G141X, D133A, V148G, N139K, G85R, G93A, V14G, C6S, I113T, D49K, G37R, A89V, E100G, D90A, T137A, E100K, G41A, G41D, G41S, G13R, G72S, L8V, F20C, The human subject may have one or more of Q22L, H48R, T54R, S591, V87A, T88deltaTAD, A89T, V97M, S105deltaSL, V118L, D124G, L114F, D90A, G12R, G147R, C6F, C6G, D101G, D101H, G114A, G85S, H43R, L106F, L106V, L38V, or R115G. In one particular embodiment, the human subject has an A4V mutation in the human SOD1 gene. In another particular embodiment, the human subject has an L106V mutation in the human SOD1 gene. In another particular embodiment, the human subject has an H46R mutation in the human SOD1 gene. In yet another particular embodiment, the human subject has a G93S mutation in the human SOD1 gene.

[0079] In certain examples, the mutation of SOD1 gene is identified by genetic testing.Therefore, the subject suffering from ALS or susceptible to ALS can be identified by genetic testing of the subject's SOD1 gene, for example, by using assays well known in the art, such as gene sequencing.

[0080] The subject's susceptibility to ALS can also be analyzed by analyzing the subject's family history of ALS, which may include a three-generation pedigree documenting the history of ALS, reviewing family medical records and autopsy studies, and identifying an autosomal dominant pattern of SOD1 mutations.

[0081] In certain embodiments, administering a therapeutically effective amount of an antisense oligonucleotide or a salt thereof to a human subject involves monitoring SOD1 levels in the human subject to determine the human subject's response to administration of the antisense oligonucleotide or a salt thereof. A physician may use the subject's response to administration of the antisense oligonucleotide or a salt thereof to define the extent and duration of therapeutic intervention. In certain embodiments, human SOD1 levels are monitored in CSF. In certain embodiments, human SOD1 levels are monitored in plasma. In certain embodiments, human SOD1 levels are monitored in blood. In certain embodiments, human SOD1 levels are monitored in serum.

[0082] In certain embodiments, administration of the antisense oligonucleotide or its salt results in a reduction in SOD1 protein expression. In certain embodiments, administration of the antisense oligonucleotide or its salt results in a reduction in SOD1 protein expression by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%, or a range separated by any two of these values. In certain embodiments, the reduction in SOD1 protein expression corresponds to a reduction in CSF. In certain embodiments, the reduction in SOD1 protein expression corresponds to a reduction in plasma. In certain embodiments, the reduction in SOD1 protein expression corresponds to a reduction in blood. In certain embodiments, the reduction in SOD1 protein expression corresponds to a reduction in serum.

[0083] In certain embodiments, administration of the antisense oligonucleotide or its salt results in improved motor function and respiration in a human subject. In certain embodiments, administration of the antisense oligonucleotide or its salt results in improved motor function and respiration by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%, or a range separated by any two of these values.

[0084] In certain embodiments, a pharmaceutical composition comprising the antisense oligonucleotide or a salt thereof is used for the preparation of a medicament for treating a human subject suffering from or susceptible to ALS (e.g., a human subject having a mutation in SOD1 associated with ALS).

[0085] Neurofilament The present disclosure provides for the use of neurofilament light chain levels as a marker for selecting subjects with mutations in the SOD1 gene for treatment with antisense oligonucleotides or salts thereof described herein. In some examples, if a subject has a neurofilament light chain level equal to or above a predetermined threshold, the subject is selected for treatment. In some examples, if a subject experiences an increase in neurofilament light chain levels corresponding to a predetermined minimum amount, the subject is selected for treatment. In some examples, if a subject has a neurofilament light chain level equal to or above a predetermined threshold and also experiences an increase in neurofilament light chain levels corresponding to a predetermined minimum amount, the subject is selected for treatment.

[0086] Assays for measuring neurofilament light chain in serum have been described (see, e.g., Gaiottino et al., PLoS ONE 8:e75091, 2013; Kuhle et al., J. Neurol. Neurosurg. Psychiatry 86(3):273-279, 2014). Neurofilament light chain (NfL) (e.g., plasma or serum NfL) concentrations can be measured, for example, using ready-to-use enzyme-linked immunosorbent assay (ELISA) diluents (Mabtech AB, Nacka Strand, Sweden), electrochemiluminescence (ECL) immunoassays as described in Gaiottino et al., PLoS ONE 8:e75091, 2013, or single molecule array (SIMOA) methods as described in Disanto et al., Ann. Neurol. 81(6):857-870, 2017. These three assays are compared in Kuhl et al., Clinical Chemistry and Laboratory Medicine 54(10):1655-1661, 2016. The SIMOA assay (Simoa NF-light Advantage Kit) is commercially available from Quanterix Corp. (Lexington, MA, USA). In some embodiments, NfL (e.g., plasma or serum NfL) concentrations are measured using a Siemens Healthineers (SHL; Erlangen, Germany) NfL assay. In some cases, total NfL (e.g., phosphorylated and non-phosphorylated) is measured. In some embodiments, phosphorylated NfL is measured, and in some embodiments, non-phosphorylated NfL is measured.

[0087] In some embodiments, a subject with a mutation in the SOD1 gene (e.g., a subject with preclinical amyotrophic lateral sclerosis) is selected for treatment if the subject has a neurofilament light chain level above a predetermined minimum threshold (e.g., a neurofilament light chain level of at least 44 pg / ml as determined by the Siemens Healthineers NfL assay, or an equivalent NfL level measured using another assay).

[0088] In some embodiments, a subject with a mutation in the SOD1 gene (e.g., a subject with preclinical amyotrophic lateral sclerosis) is selected for treatment if the subject experiences a predetermined minimum increase in neurofilament light chain levels (e.g., an increase in neurofilament light chain levels of at least 10 pg / ml as determined by the Siemens Healthineers NfL assay, or an equivalent increase in NfL levels measured using another assay).

[0089] In some embodiments, a subject with a mutation in the SOD1 gene (e.g., a subject who is clinically presymptomatic with amyotrophic lateral sclerosis) is selected for treatment if the subject has a neurofilament light chain level at or above a predetermined minimum threshold (e.g., a neurofilament light chain level of at least 44 pg / ml as determined by the Siemens Healthineers NfL assay, or an equivalent NfL level measured using another assay) and further experiences a predetermined minimum amount of increase in neurofilament light chain level (e.g., an increase in neurofilament light chain level of at least 10 pg / ml as determined by the Siemens Healthineers NfL assay, or an equivalent NfL level measured using another assay). In some embodiments, without being bound by theory, it is believed that a change (e.g., increase) of at least 10 pg / mL from baseline in plasma NfL levels can reliably account for the potential effect of aging on NfL levels.

[0090] The amino acid sequence of human NF-L is shown in SEQ ID NO:4, as well as in Julien et al., Biochimica et Biohysica Acta, 909:10-20 (1987), UniProtKB-P07196, NCBI Reference Sequence: NP_006149.2, and NCBI Reference Sequence: NG_008492.1.

[0091] SEQ ID NO:4 MSSFSYEPYYSTSYKRRYVETPRVHISSVRSGYSTARSAYSSYSAPVSSSLSVRRSYSSSSGSLMPSLENLDLSQVAAISNDLKSIRTQEKAQLQDLNDRFASFIERVHELEQQNKVLEAELLVLRQKHSEPSRF RALYEQEIRDLRLAAEDATNEKQALQGEREGLEETLRNLQARYEEEVLSREDAEGRLMEARKGADEAALARAELEKRIDSLMDEISFLKKVHEEEIAELQAQIQYAQISVEMDVTKPDLSAALKDIRAQYEKLAAK NMQNAEEWFKSRFTVLTESAAKNTDAVRAAKDEVSESRRLLKAKTLEIEACRGMNEALEKQLQELEDKQNADISAMQDTINKLENELRTTKSEMARYLKEYQDLLNVKMALDIEIAAYRKLLEGEETRLSFTSVGS ITSGYSQSSQVFGRSAYGGLQTSSYLMSTRSFPSYYTSHVQEEQIEVEETIEAAKAEEAKDEPPSEGEAEEEEKDKEEAEEEEAAEEEEAAKEESEEAKEEEEGGEGEEGEETKEAEEEEKKVEGAGEEQAAKKKD

[0092] Biological samples Biological samples suitable for the methods described herein include any biological fluid, cell, tissue, or fraction thereof that contains an analyte biomolecule of interest, such as NF protein or nucleic acid (e.g., RNA (mRNA)). Biological samples may be, for example, specimens obtained from a human subject or may be derived from such a subject. For example, the sample may be a tissue section obtained by biopsy, a stored biological fluid, or cells placed or adapted to tissue culture. In some cases, the biological sample is a biological fluid such as blood, serum, plasma, or cerebrospinal fluid (CSF), or such a sample absorbed onto a substrate (e.g., glass, polymer, paper). Biological samples may be further fractionated, if desired, into fractions containing specific cell types. For example, a blood sample may be fractionated into serum, or into fractions containing specific types of blood cells, such as red blood cells or white blood cells (leukocytes). If desired, the sample may be a combination of samples from a subject, such as a combination of tissue and liquid samples.

[0093] The biological sample can be obtained from a subject having a mutation in the SOD1 gene (e.g., an SOD1 mutation described herein). In certain embodiments, the subject has pre-symptomatic clinical amyotrophic lateral sclerosis.

[0094] Any suitable method for obtaining a biological sample can be used, but exemplary methods include, for example, phlebotomy, fine needle aspiration biopsy procedure. Samples can also be collected, for example, by microdissection (e.g., laser capture microdissection (LCM) or laser macrodissection (LMD)).

[0095] Methods for obtaining and / or storing samples that preserve the activity or integrity of molecules (e.g., nucleic acids or proteins) in the sample are well known to those skilled in the art. For example, the biological sample can be further contacted with one or more additional agents that preserve or minimize the alteration of the molecules (e.g., nucleic acids or proteins) in the sample, such as buffers and / or inhibitors (including one or more of nuclease, protease, and phosphatase inhibitors). Such inhibitors include, for example, chelating agents such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(P-aminoethyl ether) N,N,N1,N1-tetraacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antipain, and phosphatase inhibitors such as phosphate, sodium fluoride, vanadate, and the like. Suitable buffers and conditions for isolating molecules are well known to those of skill in the art and may vary, for example, depending on the type of molecule in the sample to be characterized (see, e.g., Ausubel et al. Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999); Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press (1988); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999); Tietz Textbook of Clinical Chemistry, 3rd ed. Burtis and Ashwood, eds. WB Saunders, Philadelphia, (1999)). Samples may also be treated to eliminate or minimize the presence of interfering substances. For example, biological samples may be fractionated or purified to remove one or more undesired substances.Methods for fractionating or purifying biological samples include, but are not limited to, chromatographic methods, such as liquid chromatography, ion exchange chromatography, size exclusion chromatography, or affinity chromatography. For use in the methods described herein, samples can be in various physical states. For example, samples can be liquid or solid, dissolved or suspended in a liquid, in an emulsion or gel, or absorbed into a substance.

[0096] The following examples are provided to more fully illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art will be able to readily develop equivalent means or reactants that do not depart from the scope of the invention by exercising their ordinary inventive skills. EXAMPLES

[0097] Example 1: Use of a neurofilament threshold to select patients to be treated with SOD1 antisense oligonucleotides A phase 3 randomized placebo-controlled trial of antisense A (described herein) was conducted in clinically presymptomatic adult SOD1 mutation carriers with a long-term longitudinal natural history run-in period. The SOD1 mutations included in this study are associated with high or complete penetrance, as well as rapid disease progression. SOD1 mutation carriers are considered to have clinically presymptomatic amyotrophic lateral sclerosis (ALS) if they do not have clinically definite ALS. Clinically definite ALS is defined as the emergence of clinical symptoms or signs, supported by EMG findings, conclusively indicating the emergence of ALS.

[0098] A plasma neurofilament light chain (NfL) threshold of 44 pg / mL was selected to identify study participants considered at high risk for developing overt clinical symptoms / signs of ALS within 12 months (after reaching the NfL threshold). The plasma NfL threshold of 44 pg / mL was determined based on simulations of an NF trajectory model using data from pre-symptomatic familial amyotrophic lateral sclerosis (Pre-fALS) samples and was selected to minimize false positive rates while allowing sufficient time between NF elevation and clinical onset for enrollment and intervention.

[0099] Given the frequency of blood sampling in pre-fALS, there are gaps between NfL levels, often making it impossible to identify how long before the onset of clear clinical symptoms / signs of ALS. To overcome this, we used a Bayesian method to fit an Emax model to natural log-transformed NfL concentration data in participants with clinically definite ALS but pre-fALS, age 30 years or older and with a rapidly progressive mutation at baseline. Separately, we fitted a Bayesian Weibull model to the time to emergence of clinically definite ALS data in participants with pre-fALS, age 30 years or older and with a rapidly progressive mutation at baseline. The NfL trajectory model allowed prediction of the time course from NfL elevation to the appearance of clinically definite ALS. Posterior predictive simulations from the fitted models were utilized to evaluate the performance of different NfL thresholds. Herein, analysis of plasma NfL was performed using the Siemens Healthineers (SHL) NfL assay.

[0100] The NfL threshold of 44 pg / mL was derived based on the following factors: 1. Low false positive rate (e.g., less than 5%). 2. An appropriate expected time period from NfL elevation to clinical onset (e.g., ≥2–3 months), taking into account time to treat NfL, screening, and randomization of the study. 3. Clinical experience of subjects who do not demonstrate onset of ALS symptoms 12 months after their NfL levels reach the 40 pg / mL threshold (but do not reach the 44 pg / mL threshold).

[0101] For a given NfL threshold, false-positive rates were assessed for presymptomatic carriers with NfL levels below the threshold at enrollment as well as for at least 12 months of follow-up. Participants were considered false-positive if their NfL levels were above the threshold at the postbaseline visit but remained clinically presymptomatic for at least 12 months thereafter. False-negative rates were assessed for all participants with clinically definite ALS. Participants were considered false-negative if clinically definite ALS emerged before the NfL threshold was reached.

[0102] An NfL threshold of 44 pg / mL resulted in a false positive rate of 0 / 24 (0%) and a false negative rate of 1 / 12 (8.3%). A threshold of 40 pg / mL was also considered but rejected due to a higher false positive rate (1 / 24, 4.1%), although it had the same false negative rate as 44 pg / mL. Minimizing the false positive rate with an NfL threshold of 44 pg / mL ensures that healthy subjects are not exposed to treatments that may not be necessary or that may not be clinically effective.

[0103] Based on the fitted NfL trajectory model, the geometric mean NfL levels before clinical onset were estimated by month and are illustrated in Figure 1. The expected geometric mean NfL level 3 months before the appearance of clinically definite ALS is approximately 44 pg / mL. For an NfL threshold of 44 pg / mL, and a change in NfL of at least 10 pg / mL from baseline, the posterior predicted probability of clinically definite ALS by 12 months in placebo patients is 77.66%.

[0104] This clinical study consists of four parts: Part A, Part B, Part C, and Part D.

[0105] Part A is the natural history run-in period, during which participants do not receive the study treatment antisense A or placebo. Participants in Part A, for example, are 18 years of age or older at the time of providing informed consent. Participants in Part A had plasma NfL levels less than 44 pg / mL and did not have clinically definite ALS during screening. Participants in Part A have one of the following SOD1 mutations identified during screening: p.Ala5Thr(A4T,A5T) p.Ala5Val(A4V,A5V) p.Cys7Phe(C6F,C7F) p.Cys7Gly(C6G,C7G) p.Asp102Gly(D101G,D102G) p.Asp102His(D101H,D102H) p.Gly115Ala(G114A,G115A) p.Gly42Ser(G41S,G42S) p.Gly86Arg(G85R,G86R) p.Gly86Ser(G85S,G86S) p.Gly94Ala(G93A,G94A) p.His44Arg(H43R,H44R) p.Leu107Phe(L106F,L107F) p.Leu107Val(L106V,L107V) p.Leu39Val(L38V,L39V) p.Arg116Gly(R115G,R116G) p.Val149Gly(V148G,V149G)

[0106] The brackets above indicate two alternative naming conventions for each amino acid substitution. When each of the above amino acid substitutions is referred to elsewhere in this specification, the first listed variant identifier within the brackets (e.g., A4T) is used.

[0107] Alternatively, participants in Part A may have an SOD1 mutation other than those listed above that has been adjudicated for inclusion in the study by an external Mutation Adjudication Committee. To be adjudicated as an additional SOD1 mutation in the study, the SOD1 mutation must be confirmed to have high or complete penetrance and be associated with rapid disease progression.

[0108] Part B is a double-blind, randomized, placebo-controlled period for presymptomatic participants with plasma NfL levels ≥ 44 pg / mL, a change from Part A baseline in NfL of at least 10 pg / mL, and no alternative identifiable cause for elevated NfL as determined by the investigator. Participants in Part A who achieve plasma NfL levels ≥ 44 pg / mL, a change from baseline in NfL of at least 10 pg / mL, and do not have clinically definite ALS may be eligible to enroll in Part B. In Part B, participants are randomly assigned to study arms in a 1:1 (antisense A:placebo) ratio and receive one of the treatments (antisense A 100 mg or placebo) via intrathecal injection. Participants in Part B will receive three loading doses approximately 14 days apart (i.e., on days 1, 15, and 29), followed by maintenance doses administered via blinded intrathecal injection approximately every 28 days.

[0109] The primary endpoint of the study was the proportion of participants who developed clinically definite ALS within 12 months of initiation of Part B. Secondary endpoints of the study included the proportion of participants who developed clinically definite ALS within 24 months of initiation of Part B, time to clinically definite ALS, change in Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) total score, change in predicted slow vital capacity (SVC), ventilatory support-free survival (VAFS) (defined as the time to death or permanent ventilation, whichever occurs first), and overall survival.

[0110] Part C is an open-label extension period in which participants will receive 100 mg of Antisense A via intrathecal injection. Participants in Part B who have clinically definite ALS will be enrolled and monitored.

[0111] Part D represents the randomized, double-blind, placebo-controlled study period in participants with clinically definite ALS in Part A. Participants will be randomly assigned in a 2:1 (antisense A:placebo) ratio to receive either 100 mg of antisense A or placebo via intrathecal injection.

[0112] Other embodiments Although the present invention has been described in conjunction with its detailed description, the above description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A pharmaceutical composition for treating amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 (SOD1) gene in a human subject in need of such treatment, the pharmaceutical composition comprising a compound of the formula: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te (nucleobase sequence of SEQ ID NO: 1), wherein A is adenine, mC is 5'-methylcytosine; G is guanine, T is thymine; e is a 2'-O-methoxyethyl ribose modified sugar; d is a 2'-deoxyribose sugar; s is a phosphorothioate internucleoside linkage; o is a phosphodiester internucleoside linkage; comprising an antisense oligonucleotide according to the formula above, or a pharma- ceutically acceptable salt thereof; The pharmaceutical composition, wherein the human subject has a neurofilament light chain level of at least 44 pg / ml prior to the initiation of the treatment.

2. 2. The pharmaceutical composition of claim 1, wherein the human subject experiences an increase in neurofilament light chain levels of at least 10 pg / ml prior to the initiation of the treatment.

3. The pharmaceutical composition of claim 2, wherein the human subject has a blood, serum, or cerebrospinal fluid neurofilament light chain level corresponding to a plasma neurofilament light chain level of at least 44 pg / ml prior to the initiation of the treatment, and further wherein the human subject experiences an increase in blood, serum, or cerebrospinal fluid neurofilament light chain level corresponding to an increase in plasma neurofilament light chain level of at least 10 pg / ml prior to the initiation of the treatment.

4. 3. The pharmaceutical composition of claim 2, wherein the human subject has a plasma neurofilament light chain level of at least 44 pg / ml prior to initiation of the treatment, and further wherein the human subject experiences an increase in plasma neurofilament light chain level of at least 10 pg / ml prior to initiation of the treatment.

5. 1. A pharmaceutical composition for use in a method of treating amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene in a human subject in need of such treatment, said pharmaceutical composition comprising an antisense oligonucleotide, or a pharma- ceutical acceptable salt thereof, said method comprising: measuring a neurofilament light chain level of at least 44 pg / ml in a biological sample obtained from said human subject prior to initiation of treatment; and administering the pharmaceutical composition to the human subject. The antisense oligonucleotide has the formula: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te (nucleobase sequence of SEQ ID NO: 1), wherein A is adenine, mC is 5'-methylcytosine; G is guanine, T is thymine; e is a 2'-O-methoxyethyl ribose modified sugar; d is a 2'-deoxyribose sugar; s is a phosphorothioate internucleoside linkage; o is a phosphodiester internucleoside linkage; The pharmaceutical composition according to the formula.

6. The pharmaceutical composition of claim 5 , wherein the biological sample is blood, serum, plasma, or cerebrospinal fluid.

7. The pharmaceutical composition of claim 5 , wherein the biological sample is plasma.

8. The pharmaceutical composition of claim 5 or 6, wherein the method further comprises measuring in the human subject an increase in neurofilament light chain levels in blood, serum, or cerebrospinal fluid levels corresponding to an increase in plasma neurofilament light chain levels of at least 10 pg / ml prior to administering the antisense oligonucleotide or a pharma- ceutically acceptable salt thereof.

9. The pharmaceutical composition described in any one of claims 5 to 7, wherein the method further comprises measuring an increase in plasma neurofilament light chain levels of at least 10 pg / ml in the human subject prior to administering the antisense oligonucleotide or a pharma- ceutically acceptable salt thereof.

10. 10. The pharmaceutical composition according to any one of the preceding claims, characterized in that it is administered by intrathecal administration.

11. 10. The pharmaceutical composition according to any one of the preceding claims, characterized in that it delivers a fixed dose of about 100 mg of antisense oligonucleotide.

12. 2. The pharmaceutical composition of any one of the preceding claims, wherein the mutation in the SOD1 gene is A4V.

13. The mutations in the SOD1 gene are A4V, H46R, G93S, A4T, G141X, D133A, V148G, N139K, G85R, G93A, V14G, C6S, I113T, D49K, G37R, A89V, E100G, D90A, T137A, E100K, G41A, G41D, G41S, G13R, G72S, L8V, F20C, Q22L, H48R, T5 4R, S59I, V87A, T88deltaTAD, A89T, V97M, S105deltaSL, V118L, D124G, L114F, D90A, G12R, G147R, C6F, C6G, D101G, D101H, G114A, G85S, H43R, L106F, L106V, L38V, or R115G.

14. 2. The pharmaceutical composition of any one of the preceding claims, wherein the human subject is clinically pre-symptomatic with amyotrophic lateral sclerosis.

15. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the human subject is administered a loading dose of the pharmaceutical composition followed by a maintenance dose of the pharmaceutical composition.

16. 16. The pharmaceutical composition of claim 15, wherein the human subject is administered three loading doses, the loading doses being administered every 14 days.

17. 17. The pharmaceutical composition of claim 16, wherein the maintenance dose is administered every 28 days starting 28 days after the third loading dose.

18. The loading dose and the maintenance dose of the pharmaceutical composition are (i) administering a first loading dose of the pharmaceutical composition; (ii) administering a second loading dose of the pharmaceutical composition 14 days after the date of administration of the first loading dose; (iii) administering a third loading dose of the pharmaceutical composition 28 days after the date of administration of the first loading dose; (iv) The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is administered to the human subject in a manner that comprises administering a first maintenance dose of the pharmaceutical composition 28 days or one month after the date of administration of the third loading dose.

19. The loading dose and the maintenance dose of the pharmaceutical composition are (i) administering a first loading dose sufficient to deliver about 100 mg of a fixed dose of antisense oligonucleotide; (ii) a second loading dose sufficient to deliver about 100 mg of a fixed dose of antisense oligonucleotide is administered 14 days after the date of administration of the first loading dose; (iii) a third loading dose sufficient to deliver about 100 mg of a fixed dose of antisense oligonucleotide is administered 28 days after the date of administration of the first loading dose; (iv) A first maintenance dose is administered to the human subject 28 days after the date of administration of the third loading dose in an amount sufficient to deliver a fixed dose of about 100 mg of antisense oligonucleotide.

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