Compositions and methods for treating and preventing amyotrophic lateral sclerosis

Abstract

To provide compositions and methods for treating and preventing amyotrophic lateral sclerosis.SOLUTION: Dosage regimens for SOD1-targeting antisense oligonucleotides and salts thereof are provided. These dosage regimens find use in the treatment of subjects having or at risk of developing amyotrophic lateral sclerosis. In a first aspect, the disclosure provides a method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the human superoxide dismutase 1 (SOD1) gene in a human subject in need thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 62 / 779,916, filed December 14, 2018; 62 / 807,603, filed February 19, 2019; and 62 / 840,879, filed April 30, 2019, the entire contents of which are incorporated herein by reference in their entireties.

[0002] This application generally relates to dosing regimens for clinical use of antisense oligonucleotides or salts thereof to reduce expression of superoxide dismutase 1 (SOD1) in human subjects in need thereof (e.g., adults with amyotrophic lateral sclerosis (ALS) who have a confirmed mutation in the human SOD1 gene). Such methods are useful for treating, preventing, or ameliorating ALS by inhibiting expression of SOD1. [Background technology]

[0003] The soluble SOD1 enzyme (also known as Cu / Zn superoxide dismutase) is one of the superoxide dismutases that provides protection against oxidative damage to biomolecules by catalyzing the dismutation of superoxide to hydrogen peroxide (HO) (Fridovich, Annu. Rev. Biochem., 64:97-112 (1995)). The superoxide anion (O 2- ) is a potentially harmful cellular by-product produced primarily by errors in oxidative phosphorylation in mitochondria (Turrens, J. Physiol., 552:335-344 (2003)).

[0004] Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) is a devastating progressive neurodegenerative disease that affects 30,000 Americans at any one time. Mutations in the SOD1 gene are associated with a dominantly inherited form of ALS, a disorder characterized by selective degeneration of upper and lower motor neurons (Rowland, N. Engl. J. Med., 2001, 344:1688-1700 (2001)). There is close genetic linkage between familial ALS and missense mutations in the SOD1 gene (Rosen, Nature, 362:59-62 (1993)).

[0005] The toxicity of mutant SOD1 is thought to result from early misfolding (gain of function) that reduces nuclear protection from active enzyme (loss of function in the nucleus), a process that may be involved in ALS pathogenesis (Sau, Hum. Mol. Genet., 16:1604-1618 (2007)). The progressive degeneration of motor neurons in ALS ultimately leads to their death. As motor neurons die, the brain's ability to initiate and control muscle movement is lost. Voluntary muscle activity is gradually affected, and patients in the later stages of the disease may become completely paralyzed.

[0006] Currently, there is a lack of acceptable options for treating such neurodegenerative diseases. It is therefore an object herein to provide methods for the treatment of such diseases. [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] Rowland, N. Engl. J. Med., 2001, 344: 1688-1700 (2001) [Non-Patent Document 4] Rosen, Nature, 362:59-62 (1993) [Non-Patent Document 5] Sau,Hum.Mol.Genet.,16:1604-1618(2007) Summary of the Invention [Means for solving the problem]

[0008] The present disclosure relates in part to dosing regimens of antisense oligonucleotides that reduce expression of superoxide dismutase 1 (SOD1), and to uses of such antisense oligonucleotides, or salts thereof, to inhibit expression of SOD1 and to treat, prevent or ameliorate ALS in human subjects having a mutation in the SOD1 gene.

[0009] In a first aspect, the disclosure features a method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the human superoxide dismutase 1 (SOD1) gene in a human subject in need thereof. The method includes administering (e.g., by intrathecal administration) a pharmaceutical composition to the human subject in an amount sufficient to deliver about 100 mg or a fixed dose of 100 mg of an antisense oligonucleotide, wherein the nucleobase sequence of the antisense oligonucleotide consists of CAGGATACATTTCTACAGCT (SEQ ID NO:1), each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyethyl ribose modified nucleoside, and each of nucleosides 6-15 is , 2'-deoxynucleosides, 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.

[0010] In a second aspect, the disclosure features a method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the human SOD1 gene in a human subject in need thereof. The method includes administering (e.g., by intrathecal administration) a pharmaceutical composition to the human subject in an amount sufficient to deliver about 60 mg or a fixed dose of 60 mg of an antisense oligonucleotide, wherein the nucleobase sequence of the antisense oligonucleotide consists of CAGGATACATTTCTACAGCT (SEQ ID NO:1), each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyethyl ribose modified nucleoside, and each of nucleosides 6-15 is It is a 2'-deoxynucleoside, 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.

[0011] In a third aspect, the disclosure features a method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the human SOD1 gene in a human subject in need thereof. The method includes administering (e.g., by intrathecal administration) a pharmaceutical composition to the human subject in an amount sufficient to deliver about 40 mg or a fixed dose of 40 mg of an antisense oligonucleotide, wherein the nucleobase sequence of the antisense oligonucleotide consists of CAGGATACATTTCTACAGCT (SEQ ID NO:1), each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyethyl ribose modified nucleoside, and each of nucleosides 6-15 is It is a 2'-deoxynucleoside, 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.

[0012] In a fourth aspect, the disclosure features a method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the human SOD1 gene in a human subject in need thereof. The method includes administering (e.g., by intrathecal administration) a pharmaceutical composition to the human subject in an amount sufficient to deliver about 20 mg or a fixed dose of 20 mg of an antisense oligonucleotide, wherein the nucleobase sequence of the antisense oligonucleotide consists of CAGGATACATTTCTACAGCT (SEQ ID NO:1), each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyethyl ribose modified nucleoside, and each of nucleosides 6-15 is It is a 2'-deoxynucleoside, 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.

[0013] In a fifth aspect, the present disclosure provides a method for determining whether human SOD1 protein synthesis or human SOD1 protein synthesis is increased in a human subject having a mutation in the human SOD1 gene associated with amyotrophic lateral sclerosis. A method of reducing mRNA levels is provided. The method comprises administering a pharmaceutical composition to a human subject by intrathecal administration in an amount sufficient to deliver about 100 mg or a fixed dose of 100 mg of an antisense oligonucleotide, wherein the nucleobase sequence of the antisense oligonucleotide consists of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), each of nucleosides 1-5 and 16-20 being a 2'-O-methoxyethyl ribose modified nucleoside, and each of nucleosides 6-15 being a 2'-O-methoxyethyl ribose modified nucleoside. -deoxynucleosides, 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.

[0014] In a sixth aspect, the present disclosure provides a method for determining whether human SOD1 protein synthesis or human SOD1 protein synthesis is increased in a human subject having a mutation in the human SOD1 gene associated with amyotrophic lateral sclerosis. A method of reducing mRNA levels is provided, the method comprising administering a pharmaceutical composition to a human subject by intrathecal administration in an amount sufficient to deliver about 60 mg or a fixed dose of 60 mg of an antisense oligonucleotide, 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'-O-methoxyethyl ribose modified nucleoside, 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.

[0015] In a seventh aspect, the present disclosure provides a method for determining whether or not human SOD1 protein synthesis or human SOD1 protein synthesis is increased in a human subject having a mutation in the human SOD1 gene associated with amyotrophic lateral sclerosis. A method of reducing mRNA levels is provided, the method comprising administering a pharmaceutical composition to a human subject by intrathecal administration in an amount sufficient to deliver about 40 mg or a fixed dose of 40 mg of an antisense oligonucleotide, 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 and each of nucleosides 6-15 being a 2'-O-methoxyethyl ribose modified nucleoside. 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.

[0016] In an eighth aspect, the present disclosure provides a method for determining whether human SOD1 protein synthesis or human SOD1 protein synthesis is increased in a human subject having a mutation in the human SOD1 gene associated with amyotrophic lateral sclerosis. A method of reducing mRNA levels is provided, the method comprising administering a pharmaceutical composition to a human subject by intrathecal administration in an amount sufficient to deliver about 20 mg or a fixed dose of 20 mg of an antisense oligonucleotide, 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'-O-methoxyethyl ribose modified nucleoside, 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.

[0017] In some embodiments of the above aspects, 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, G72S, L8V, F20C, Q22L, H48R, T54R, S591, V87A, T88 delta TAD, A89T, V97M, S105 delta SL, V118L, D124G, L114F, D90A, G12R, or G147R. In one embodiment, the mutation in the SOD1 gene is A4V. In another embodiment, the mutation in the SOD1 gene is H46R. In yet another embodiment, the mutation in the SOD1 gene is G93S.

[0018] In some embodiments, mutations in the human SOD1 gene are identified by genetic testing.

[0019] In certain embodiments, the above method further comprises identifying a mutation in the human SOD1 gene by genetic testing.

[0020] In some embodiments, the pharmaceutical composition is administered to a human subject at least 5 times (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 times) over the course of 4 months.

[0021] In certain embodiments, a human subject is administered a loading dose(s) followed by a maintenance dose(s) of the pharmaceutical composition. In some cases, three loading doses are administered, with a second loading dose administered about 2 weeks or 2 weeks after the first loading dose, and a third loading dose administered about 2 weeks or 2 weeks after the second loading dose (e.g., loading doses administered on days 1, 15, and 29). In some cases, a maintenance dose is administered about every 4 weeks or every 4 weeks, starting 4 weeks after the third loading dose (e.g., for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months).

[0022] In certain embodiments, a human subject is administered three loading doses of the pharmaceutical composition followed by at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) maintenance dose. In some cases, the three loading doses are administered at about 2 weeks or at 2 week intervals. In some cases, the three loading doses are administered at about 14 days or at 14 day intervals. In some cases, the maintenance dose(s) are administered starting at about 4 weeks or at 4 weeks after the third loading dose. In some cases, the maintenance dose(s) are administered monthly starting one month after the third loading dose. In some cases, the maintenance dose(s) are administered every 28 days starting 28 days after the third loading dose.

[0023] In certain embodiments, the loading dose and maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose of a pharmaceutical composition; (ii) a second loading dose of the pharmaceutical composition administered 14 days after the first loading dose; (iii) a third loading dose of the pharmaceutical composition administered 28 days after the first loading dose; and (iv) a first maintenance dose of the pharmaceutical composition administered 28 days or one month after the third loading dose. The drug is administered to a human subject as follows:

[0024] In certain embodiments, the loading dose and maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose sufficient to deliver a fixed dose of 100 mg of antisense oligonucleotide; (ii) a second loading dose administered 14 days after the first loading dose in an amount sufficient to deliver a fixed dose of 100 mg of the antisense oligonucleotide; (iii) a third loading dose administered 28 days after the first loading dose in an amount sufficient to deliver a fixed dose of 100 mg of the antisense oligonucleotide; and (iv) a first maintenance dose administered 28 days after the third loading dose in an amount sufficient to deliver a fixed dose of 100 mg of the antisense oligonucleotide. The drug is administered to a human subject as follows:

[0025] In certain embodiments, the loading dose and maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose sufficient to deliver a fixed dose of 100 mg of antisense oligonucleotide; (ii) a second loading dose administered 14 days after the first loading dose in an amount sufficient to deliver a fixed dose of 100 mg of the antisense oligonucleotide; (iii) a third loading dose administered 28 days after the first loading dose in an amount sufficient to deliver a fixed dose of 100 mg of the antisense oligonucleotide; and (iv) a first maintenance dose administered one month after the third loading dose in an amount sufficient to deliver a fixed dose of 100 mg of the antisense oligonucleotide. The drug is administered to a human subject as follows:

[0026] In a ninth aspect, the present disclosure provides a method for treating or preventing amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene in a human subject in need thereof. The method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof by intrathecal administration, wherein the antisense oligonucleotide has the following structure: [ka] and the antisense oligonucleotide or its salt is administered at a dose equivalent to 100 mg of antisense oligonucleotide. 105.9 mg of compound A (i.e., the nonadeca sodium salt of ISIS 666853) is equivalent to 100 mg of antisense oligonucleotide. The pharmaceutical composition administered may include the antisense oligonucleotide, one or more salts of the antisense oligonucleotide, or a mixture thereof.

[0027] In a tenth aspect, the present disclosure provides a method for treating or preventing amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene in a human subject in need thereof. The method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof by intrathecal administration, wherein the antisense oligonucleotide has the following structure: [ka] and the antisense oligonucleotide or its salt is administered at a dose equivalent to 60 mg of antisense oligonucleotide. 63.5 mg of Compound A is equivalent to 60 mg of antisense oligonucleotide. The pharmaceutical composition administered may include the antisense oligonucleotide, one or more salts of the antisense oligonucleotide, or a mixture thereof.

[0028] In an eleventh aspect, the present disclosure provides a method for treating or preventing amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene in a human subject in need thereof. The method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof by intrathecal administration, wherein the antisense oligonucleotide has the following structure: [ka] and the antisense oligonucleotide or its salt is administered at a dose equivalent to 40 mg of antisense oligonucleotide. 42.3 mg of Compound A is equivalent to 40 mg of antisense oligonucleotide. The administered pharmaceutical composition may include the antisense oligonucleotide, one or more salts of the antisense oligonucleotide, or a mixture thereof.

[0029] In a twelfth aspect, the present disclosure provides a method for treating or preventing amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene in a human subject in need thereof. The method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof by intrathecal administration, wherein the antisense oligonucleotide has the following structure: [ka] and the antisense oligonucleotide or its salt is administered at a dose equivalent to 20 mg of antisense oligonucleotide. 21.2 mg of Compound A is equivalent to 20 mg of antisense oligonucleotide. The pharmaceutical composition administered may include the antisense oligonucleotide, one or more salts of the antisense oligonucleotide, or a mixture thereof.

[0030] In a thirteenth aspect, the present disclosure provides a method for reducing human SOD1 protein synthesis or 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 to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof by intrathecal administration, wherein the antisense oligonucleotide has the following structure: [ka] and the antisense oligonucleotide or its salt is administered at a dose equivalent to 100 mg of antisense oligonucleotide. 105.9 mg of compound A is equivalent to 100 mg of antisense oligonucleotide. The pharmaceutical composition administered may include the antisense oligonucleotide, one or more salts of the antisense oligonucleotide, or a mixture thereof.

[0031] In a fourteenth aspect, the present disclosure provides a method for reducing human SOD1 protein synthesis or 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 to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof by intrathecal administration, wherein the antisense oligonucleotide has the following structure: [ka] and the antisense oligonucleotide or its salt is administered at a dose equivalent to 60 mg of antisense oligonucleotide. 63.5 mg of Compound A is equivalent to 60 mg of antisense oligonucleotide. The pharmaceutical composition administered may include the antisense oligonucleotide, one or more salts of the antisense oligonucleotide, or a mixture thereof.

[0032] In a fifteenth aspect, the present disclosure provides a method for reducing human SOD1 protein synthesis or 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 to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof by intrathecal administration, wherein the antisense oligonucleotide has the following structure: [ka] and the antisense oligonucleotide or its salt is administered at a dose equivalent to 40 mg of antisense oligonucleotide. 42.3 mg of Compound A is equivalent to 40 mg of antisense oligonucleotide. The administered pharmaceutical composition may include the antisense oligonucleotide, one or more salts of the antisense oligonucleotide, or a mixture thereof.

[0033] In a sixteenth aspect, the present disclosure provides a method for reducing human SOD1 protein synthesis or 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 to the human subject a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof by intrathecal administration, wherein the antisense oligonucleotide has the following structure: [ka] and the antisense oligonucleotide or its salt is administered at a dose equivalent to 20 mg of antisense oligonucleotide. 21.2 mg of Compound A is equivalent to 20 mg of antisense oligonucleotide. The pharmaceutical composition administered may include the antisense oligonucleotide, one or more salts of the antisense oligonucleotide, or a mixture thereof.

[0034] In some embodiments, the human subject is administered a salt of the antisense oligonucleotide. In some embodiments, the salt is a sodium salt. In some embodiments, the salt of the antisense oligonucleotide has the following structure: [ka] has.

[0035] In some embodiments of the above aspects, 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, G72S, L8V, F20C, Q22L, H48R, T54R, S591, V87A, T88 delta TAD, A89T, V97M, S105 delta SL, V118L, D124G, L114F, D90A, G12R, or G147R. In one embodiment, the mutation in the SOD1 gene is A4V. In another embodiment, the mutation in the SOD1 gene is H46R. In yet another embodiment, the mutation in the SOD1 gene is G93S.

[0036] In some embodiments, mutations in the human SOD1 gene are identified by genetic testing.

[0037] In certain embodiments, the above method further comprises identifying a mutation in the human SOD1 gene by genetic testing.

[0038] In some embodiments, the pharmaceutical composition is administered to a human subject at least five times over the course of four months.

[0039] In certain embodiments, a human subject is administered a loading dose(s) followed by a maintenance dose(s) of the pharmaceutical composition. In some cases, three loading doses are administered, the loading doses being separated by two weeks, for example, on days 1, 15, and 29. In some cases, the maintenance doses are administered every four weeks, starting four weeks after the third loading dose (for example, for one, two, three, four, five, six, seven, eight, nine, ten months).

[0040] In certain embodiments, a human subject is administered three loading doses of the pharmaceutical composition followed by at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) maintenance dose. In some cases, the three loading doses are administered at two-week intervals. In some cases, the three loading doses are administered at 14-day intervals. In some cases, the maintenance dose(s) are administered every four weeks, starting four weeks after the third loading dose. In some cases, the maintenance dose(s) are administered monthly, starting one month after the third loading dose. In some cases, the maintenance dose(s) are administered every 28 days, starting 28 days after the third loading dose.

[0041] In certain embodiments, the loading dose and maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose of a pharmaceutical composition; (ii) a second loading dose of the pharmaceutical composition administered 14 days after the first loading dose; (iii) a third loading dose of the pharmaceutical composition administered 28 days after the first loading dose; and (iv) a first maintenance dose of the pharmaceutical composition administered 28 days or one month after the third loading dose. The drug is administered to a human subject as follows:

[0042] In certain embodiments, the loading dose and maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose equivalent to 100 mg of the antisense oligonucleotide; (ii) a second loading dose equivalent to 100 mg of the antisense oligonucleotide administered 14 days after the first loading dose; (iii) a third loading dose equivalent to 100 mg of the antisense oligonucleotide administered 28 days after the first loading dose; and (iv) a first maintenance dose equivalent to 100 mg of the antisense oligonucleotide administered 28 days after the third loading dose. The drug is administered to a human subject as follows:

[0043] In certain embodiments, the loading dose and maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose equivalent to 100 mg of the antisense oligonucleotide; (ii) a second loading dose equivalent to 100 mg of the antisense oligonucleotide administered 14 days after the first loading dose; (iii) a third loading dose equivalent to 100 mg of the antisense oligonucleotide administered 28 days after the first loading dose; and (iv) a first maintenance dose equivalent to 100 mg of the antisense oligonucleotide administered one month after the third loading dose; The drug is administered to a human subject as follows:

[0044] In another embodiment, the disclosure features a syringe or pump containing a sterile preparation of an antisense oligonucleotide, the syringe or pump being adapted for intrathecal administration of the antisense oligonucleotide at a fixed dose of 20 mg, 40 mg, 60 mg, or 100 mg. The nucleic acid base sequence of the antisense oligonucleotide consists of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), 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 between nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 are phosphodiester bonds, the internucleoside bonds between 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 a 5-methylcytosine.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the practice or testing of the present invention, methods and materials similar or equivalent to those described herein can be used, but exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present application, including definitions, will control. The materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0046] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. The present invention provides, for example, the following items. (Item 1) 1. A method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 (SOD1) gene in a human subject in need thereof, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition in an amount sufficient to deliver a fixed dose of about 100 mg of an antisense oligonucleotide, the nucleobase sequence of which consists of CAGGATACATTTCTACAGCT (SEQ ID NO:1), wherein each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyphenyl 1,2'-diaminobenzyl 2,3 ... the internucleoside bond between nucleosides 2-3, 4-5, 16-17, and 18-19 is a phosphodiester bond; the internucleoside bond 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 is a phosphorothioate bond; and each cytosine is a 5-methylcytosine. (Item 2) 1. A method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 (SOD1) gene in a human subject in need thereof, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition in an amount sufficient to deliver a fixed dose of about 60 mg of an antisense oligonucleotide, the nucleobase sequence of which consists of CAGGATACATTTCTACAGCT (SEQ ID NO:1), wherein each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyphenyl 1,2'-diaminobenzyl 2,3 ... the internucleoside bond between nucleosides 2-3, 4-5, 16-17, and 18-19 is a phosphodiester bond; the internucleoside bond 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 is a phosphorothioate bond; and each cytosine is a 5-methylcytosine. (Item 3) 1. A method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 (SOD1) gene in a human subject in need thereof, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition in an amount sufficient to deliver a fixed dose of about 40 mg of an antisense oligonucleotide, the nucleobase sequence of which consists of CAGGATACATTTCTACAGCT (SEQ ID NO:1), wherein each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyphenyl 1,2'-diaminobenzyl 2,3 ... the internucleoside bond between nucleosides 2-3, 4-5, 16-17, and 18-19 is a phosphodiester bond; the internucleoside bond 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 is a phosphorothioate bond; and each cytosine is a 5-methylcytosine. (Item 4) 1. A method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 (SOD1) gene in a human subject in need thereof, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition in an amount sufficient to deliver a fixed dose of about 20 mg of an antisense oligonucleotide, the nucleobase sequence of which consists of CAGGATACATTTCTACAGCT (SEQ ID NO:1), wherein each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyphenyl 1,2'-diaminobenzyl 2,3 ... the internucleoside bond between nucleosides 2-3, 4-5, 16-17, and 18-19 is a phosphodiester bond; the internucleoside bond 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 is a phosphorothioate bond; and each cytosine is a 5-methylcytosine. (Item 5) 1. A method of reducing superoxide dismutase 1 (SOD1) protein synthesis in a human subject having a mutation in the SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition in an amount sufficient to deliver a fixed dose of about 100 mg of an antisense oligonucleotide, the nucleobase sequence of the antisense oligonucleotide consisting of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), wherein each of nucleosides 1-5 and 16-20 is a 2'-O-methoxy group. each of nucleosides 6 to 15 is a 2'-deoxynucleoside; the internucleoside bonds among nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 are phosphodiester bonds; the internucleoside bonds 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. (Item 6) 1. A method of reducing superoxide dismutase 1 (SOD1) protein synthesis in a human subject having a mutation in the SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition in an amount sufficient to deliver a fixed dose of about 60 mg of an antisense oligonucleotide, the nucleobase sequence of the antisense oligonucleotide consisting of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), wherein each of nucleosides 1-5 and 16-20 is a 2'-O-methoxy group. each of nucleosides 6 to 15 is a 2'-deoxynucleoside; the internucleoside bonds among nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 are phosphodiester bonds; the internucleoside bonds 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. (Item 7) 1. A method of reducing superoxide dismutase 1 (SOD1) protein synthesis in a human subject having a mutation in the SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition in an amount sufficient to deliver a fixed dose of about 40 mg of an antisense oligonucleotide, the nucleobase sequence of the antisense oligonucleotide consisting of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), wherein each of nucleosides 1-5 and 16-20 is a 2'-O-methoxy group. each of nucleosides 6 to 15 is a 2'-deoxynucleoside; the internucleoside bonds among nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 are phosphodiester bonds; the internucleoside bonds 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. (Item 8) 1. A method of reducing superoxide dismutase 1 (SOD1) protein synthesis in a human subject having a mutation in the SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition in an amount sufficient to deliver a fixed dose of about 20 mg of an antisense oligonucleotide, the nucleobase sequence of the antisense oligonucleotide consisting of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), wherein each of nucleosides 1-5 and 16-20 is a 2'-O-methoxy group. each of nucleosides 6 to 15 is a 2'-deoxynucleoside; the internucleoside bonds among nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 are phosphodiester bonds; the internucleoside bonds 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. (Item 9) 9. The method according to any one of items 1 to 8, wherein the mutation in the SOD1 gene is A4V. (Item 10) 9. The method of any one of items 1 to 8, wherein 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, G72S, L8V, F20C, Q22L, H48R, T54R, S591, V87A, T88 delta TAD, A89T, V97M, S105 delta SL, V118L, D124G, L114F, D90A, G12R, or G147R. (Item 11) 11. The method according to any one of items 1 to 10, wherein the mutation in the SOD1 gene is identified by genetic testing. (Item 12) 11. The method according to any one of items 1 to 10, comprising identifying the mutation in the SOD1 gene by genetic testing. (Item 13) 13. The method of any one of items 1 to 12, wherein the pharmaceutical composition is administered to the human subject at least five times over the course of four months. (Item 14) 14. The method of any one of items 1 to 13, wherein the human subject is administered a loading dose of the pharmaceutical composition followed by a maintenance dose of the pharmaceutical composition. (Item 15) 15. The method of item 14, wherein the human subject is administered three loading doses, the loading doses being administered two weeks apart. (Item 16) 15. The method of item 14, wherein the maintenance dose is administered every 4 weeks starting 4 weeks after the third loading dose. (Item 17) The loading dose and the maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose of the pharmaceutical composition; (ii) a second loading dose of the pharmaceutical composition administered 14 days after the first loading dose; (iii) a third loading dose of the pharmaceutical composition administered 28 days after the first loading dose; and (iv) a first maintenance dose of said pharmaceutical composition administered 28 days or one month after said third loading dose. 15. The method of claim 14, wherein the human subject is administered (Item 18) The loading dose and the maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose sufficient to deliver a fixed dose of about 100 mg of said antisense oligonucleotide; (ii) a second loading dose administered 14 days after the first loading dose in an amount sufficient to deliver a fixed dose of about 100 mg of the antisense oligonucleotide; (iii) a third loading dose administered 28 days after the first loading dose in an amount sufficient to deliver a fixed dose of about 100 mg of the antisense oligonucleotide; and (iv) a first maintenance dose administered 28 days after the third loading dose in an amount sufficient to deliver a fixed dose of about 100 mg of the antisense oligonucleotide. 15. The method of claim 14, wherein the human subject is administered (Item 19) The loading dose and the maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose sufficient to deliver a fixed dose of about 100 mg of said antisense oligonucleotide; (ii) a second loading dose administered 14 days after the first loading dose in an amount sufficient to deliver a fixed dose of about 100 mg of the antisense oligonucleotide; (iii) a third loading dose administered 28 days after the first loading dose in an amount sufficient to deliver a fixed dose of about 100 mg of the antisense oligonucleotide; and (iv) a first maintenance dose administered one month after the third loading dose in an amount sufficient to deliver a fixed dose of about 100 mg of the antisense oligonucleotide. 15. The method of claim 14, wherein the human subject is administered (Item 20) A syringe or pump containing a sterile preparation of an antisense oligonucleotide, said syringe or pump adapted for intrathecal administration of said antisense oligonucleotide at a fixed dose of about 20 mg, about 40 mg, about 60 mg, or about 100 mg, said antisense oligonucleotide having a nucleobase sequence consisting of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), each of nucleosides 1-5 and 16-20 being a 2'-O-methoxyethyl ribose modified nucleoside, and the syringe or the pump, wherein each of the nucleosides 6 to 15 is a 2'-deoxynucleoside, the internucleoside bonds between the nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 are phosphodiester bonds, the internucleoside bonds between the 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. (Item 21) 1. A method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 (SOD1) gene in a human subject in need thereof, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] wherein the antisense oligonucleotide or salt thereof is administered in a dose equivalent to about 100 mg of the antisense oligonucleotide. (Item 22) 1. A method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 (SOD1) gene in a human subject in need thereof, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] wherein the antisense oligonucleotide or salt thereof is administered in a dose equivalent to about 60 mg of the antisense oligonucleotide. (Item 23) 1. A method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 (SOD1) gene in a human subject in need thereof, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] wherein the antisense oligonucleotide or salt thereof is administered in a dose equivalent to about 40 mg of the antisense oligonucleotide. (Item 24) 1. A method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the superoxide dismutase 1 (SOD1) gene in a human subject in need thereof, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] wherein the antisense oligonucleotide or salt thereof is administered in a dose equivalent to about 20 mg of the antisense oligonucleotide. (Item 25) 1. A method of reducing superoxide dismutase 1 (SOD1) protein synthesis in a human subject having a mutation in the SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] wherein the antisense oligonucleotide or salt thereof is administered in a dose equivalent to about 100 mg of the antisense oligonucleotide. (Item 26) 1. A method of reducing superoxide dismutase 1 (SOD1) protein synthesis in a human subject having a mutation in the SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] wherein the antisense oligonucleotide or salt thereof is administered in a dose equivalent to about 60 mg of the antisense oligonucleotide. (Item 27) 1. A method of reducing superoxide dismutase 1 (SOD1) protein synthesis in a human subject having a mutation in the SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] wherein the antisense oligonucleotide or salt thereof is administered in a dose equivalent to about 40 mg of the antisense oligonucleotide. (Item 28) 1. A method of reducing superoxide dismutase 1 (SOD1) protein synthesis in a human subject having a mutation in the SOD1 gene associated with amyotrophic lateral sclerosis, the method comprising administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] wherein the antisense oligonucleotide or salt thereof is administered in a dose equivalent to about 20 mg of the antisense oligonucleotide. (Item 29) 29. The method of any one of items 21 to 28, wherein the mutation in the SOD1 gene is A4V. (Item 30) 29. The method of any one of items 21 to 28, wherein 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, G72S, L8V, F20C, Q22L, H48R, T54R, S591, V87A, T88 delta TAD, A89T, V97M, S105 delta SL, V118L, D124G, L114F, D90A, G12R, or G147R. (Item 31) 31. The method according to any one of items 21 to 30, wherein the mutation in the SOD1 gene is identified by genetic testing. (Item 32) 31. The method according to any one of items 21 to 30, comprising identifying the mutation in the SOD1 gene by genetic testing. (Item 33) 33. The method of any one of items 21 to 32, wherein the human subject is administered a salt of the antisense oligonucleotide. (Item 34) 34. The method of claim 33, wherein the salt is a sodium salt. (Item 35) The salt of the antisense oligonucleotide has the following structure: [ka] 34. The method according to item 33, comprising the steps of: (Item 36) 36. The method of any one of items 21 to 35, wherein the human subject is administered a loading dose of the pharmaceutical composition followed by a maintenance dose of the pharmaceutical composition. (Item 37) 37. The method of claim 36, wherein the human subject is administered three loading doses, the loading doses being administered at two week intervals. (Item 38) 37. The method of claim 36, wherein the maintenance dose is administered every 4 weeks starting 4 weeks after the third loading dose. (Item 39) The loading dose and the maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose of the pharmaceutical composition; (ii) a second loading dose of the pharmaceutical composition administered 14 days after the first loading dose; (iii) a third loading dose of the pharmaceutical composition administered 28 days after the first loading dose; and (iv) a first maintenance dose of said pharmaceutical composition administered 28 days or one month after said third loading dose. 37. The method of claim 36, wherein the human subject is administered (Item 40) The loading dose and the maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose equivalent to about 100 mg of the antisense oligonucleotide; (ii) a second loading dose equivalent to about 100 mg of the antisense oligonucleotide administered 14 days after the first loading dose; (iii) a third loading dose equivalent to about 100 mg of the antisense oligonucleotide administered 28 days after the first loading dose; and (iv) a first maintenance dose equivalent to about 100 mg of said antisense oligonucleotide administered 28 days after said third loading dose. 37. The method of claim 36, wherein the human subject is administered (Item 41) The loading dose and the maintenance dose of the pharmaceutical composition are as follows: (i) a first loading dose equivalent to about 100 mg of the antisense oligonucleotide; (ii) a second loading dose equivalent to about 100 mg of the antisense oligonucleotide administered 14 days after the first loading dose; (iii) a third loading dose equivalent to about 100 mg of the antisense oligonucleotide administered 28 days after the first loading dose; and (iv) a first maintenance dose equivalent to about 100 mg of said antisense oligonucleotide administered one month after said third loading dose. 37. The method of claim 36, wherein the human subject is administered [Brief description of the drawings]

[0047] [Figure 1] FIG. 1 is a graphical representation of the mean disease duration (years from onset of symptoms) for patients with different human SOD1 mutations. [Diagram 2] FIG. 1 illustrates the dose-dependent increase in cerebrospinal fluid (CSF) concentrations of Compound A observed in the multiple ascending dose (MAD) cohort. The top dashed line represents the 100 mg dose, the next two dashed lines represent the 60 and 40 mg doses, and the bottom dashed line represents the 20 mg dose. [Diagram 3] 1 shows the dose-dependent reduction in CSF SOD1 concentrations observed in the MAD cohort. On study day 85, the top circle corresponds to 20 mg, the next lower circle to placebo, the next lower circle to 60 mg, the next lower circle to 40 mg, and the bottom circle to 100 mg Compound A. [Figure 4A] 1 is a plot of the LS mean change from baseline in the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALFSFRS-R) for patients in the MAD cohort. On Study Day 85, the top circle corresponds to 40 mg Compound A, the next lower circle corresponds to 100 mg Compound A, the next lower circle corresponds to 60 mg Compound A, the next lower circle corresponds to 20 mg Compound A, and the bottom circle corresponds to placebo. [Figure 4B] Plot of LS mean change from baseline (with 90% confidence intervals) in predicted static vital capacity (SVC) by percentage of patients in the MAD cohort. On Study Day 85, the top circle corresponds to 60 mg Compound A, the next lower circle to 40 mg Compound A, the next lower circle to 100 mg Compound A, the next lower circle to 20 mg Compound A, and the bottom circle to placebo. [Figure 4C]Plot of LS mean change from baseline (with 90% confidence intervals) in handheld dynamometry (HHD) global megascore for patients in the MAD cohort. On Study Day 92, the top circle corresponds to 40 mg Compound A, the next lower circle corresponds to 100 mg Compound A, the next lower circle corresponds to 60 mg Compound A, the next lower circle corresponds to 20 mg Compound A, and the bottom circle corresponds to placebo. [Diagram 5] 1 shows the change from baseline in ALSFRS-R, SVC, HHD Megascore, and CSF SOD1 levels to Day 85 in patients with aggressive mutations receiving either placebo or 100 mg Compound A. [Figure 6] 1 shows the change from baseline in ALSFRS-R, SVC, HHD Megascore, and CSF SOD1 levels to Day 85 in patients with slow-growing mutations receiving either placebo or 100 mg of Compound A. [Figure 7] 1 shows the effect of treatment with 100 mg Compound A on pNFH levels from baseline to Day 85 in patients with aggressive SOD1 mutations. [Figure 8] 1 shows the effect of treatment with 100 mg Compound A on pNFH levels from baseline to day 85 in patients with slow-progressing SOD1 mutations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The disclosure features dosing regimens of antisense oligonucleotides or salts thereof that reduce expression of superoxide dismutase 1 (SOD1), and uses of such antisense oligonucleotides or salts thereof to treat, prevent, or ameliorate amyotrophic lateral sclerosis (ALS) in adults with a mutation in the human SOD1 gene.

[0049] definition "2'-O-Methoxyethyl" (also 2'-MOE and 2'-OCH2CH2-OCH3 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.

[0050] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside) means a nucleoside that includes an MOE modified sugar moiety.

[0051] "5-methylcytosine" means a cytosine modified with a methyl group attached to the 5' position. 5-Methylcytosine is a modified nucleobase.

[0052] "Phosphorothioate bond" means 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 bond is a modified internucleoside linkage.

[0053] "About" in the context of an amount of material means ±10% of the indicated value. Thus, about 100 mg of antisense oligonucleotide encompasses 90 mg to 110 mg of antisense oligonucleotide. In the context of a time unit (e.g., about 10 days or about 1 week), "about" means ±3 days.

[0054] "Intrathecal or IT" means administration into the cerebrospinal fluid beneath the arachnoid membrane that covers the brain and spinal cord.

[0055] "Loading dose" means the dose administered during the dosing phase when administration is initiated and a steady state concentration of drug (eg, an antisense oligonucleotide) is achieved.

[0056] "Maintenance dose" means the dose administered during a dosing phase after a steady state concentration of a drug (eg, an antisense oligonucleotide) has been achieved.

[0057] "Fixed dose" refers to a predetermined amount (e.g., 20 mg, 40 mg, 60 mg, 100 mg) of an antisense oligonucleotide intended to achieve a desired therapeutic concentration (e.g., steady state concentration) or effect in a subject.

[0058] Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease that results in 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. Decline is inevitable, and death typically occurs from respiratory failure, on average 2-5 years after diagnosis. The majority of patients suffer from sporadic ALS, but a small percentage of patients (approximately 2%) have an inherited or familial form of ALS caused by various mutations in superoxide dismutase 1 (SOD1). Since it was first identified in 1993, over 180 SOD1 mutations have been reported to cause this form of ALS, referred to as SOD1 ALS. The Amyotrophic Lateral Sclerosis Online Genetics Database (ALSoD). Institute of Psychiatry, Psychology & Neuroscience. Published 2015; Rosen, Nature, 364(6435): 362 (1993). Disease progression for individual mutations is variable, with the most severe mutations resulting in survival of less than 15 months. The mechanism by which mutations cause SOD1 ALS is unknown, but compelling data suggest that a toxic gain of function, rather than a loss of SOD1 activity, is the trigger that initiates a cascade of events that leads to motor neuron death (Bruijn et al., Science, 281(5384): 1851-4 (1998)).

[0059] Approved treatments for ALS are riluzole (Rilutek®) and edaravone (Radicava™). Riluzole provides a small increase in survival (2-3 months) without clear improvement in strength or disability. Edaravone reduces functional decline as measured by the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R). The effect of edaravone on survival is unknown. No SOD1-specific ALS treatment is available.

[0060] Superoxide dismutase 1 Superoxide dismutase [Cu-Zn], also known as superoxide dismutase 1 (SOD1), is an enzyme located on chromosome 21 and encoded in humans by the SOD1 gene.

[0061] SOD1 is a 32 kDa homodimer that forms a β-barrel and contains an intramolecular disulfide bond and a binuclear Cu / Zn site in each subunit that holds copper and zinc ions and is responsible for catalyzing the dismutation of superoxide to hydrogen peroxide and dioxygen.

[0062] 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 the naturally occurring but harmful superoxide radical into molecular oxygen and hydrogen peroxide. Hydrogen peroxide can then be degraded by another enzyme called catalase.

[0063] At least 180 mutations in the SOD1 gene have been associated with 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 has also been implicated in a significant proportion of sporadic ALS cases (corresponding to 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 known human SOD1 mutations include 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, T54R, S591, V87A, T88 delta TAD, A89T, V97M, S105 delta SL, V118L, D124G, L114F, D90A, G12R, and G147R. There is significant heterogeneity in disease duration based on SOD1 mutations (see Figure 1). Virtually all known ALS-causing SOD1 mutations act in a dominant manner; a single mutant copy of the SOD1 gene is sufficient to cause the disease.

[0064] The amino acid sequence of human SOD1 is found at UniProt P00441 and GENBANK accession number NP_000445, and is provided below. MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTS AGPHFNPLSR KHGGPKDEER HVGDLGNVTA DKDGVADVSI EDSVISLSGD HCIIGRTLVV HEKADDLGKG GNEESTKTGN AGSRLACGVI GIAQ (SEQ ID NO: 2)

[0065] The nucleotide sequence encoding human SOD1 is provided in GENBANK Accession No. NM_000454.4, and is also provided below (the region recognized by the antisense oligonucleotides of the disclosure is underlined): [ka] (SEQ ID NO:3)

[0066] ISIS 666853 ISIS 666853 is a 5-10-5 MOE gapmer having the sequence (5' to 3') of CAGGATACATTTCTACAGCT (SEQ ID NO:1), in which each of nucleosides 1-5 and 16-20 are 2'-O-methoxyethyl ribose modified nucleosides, each of nucleosides 6-15 are 2'-deoxynucleosides, and nucleosides 2-3, 4-5, 16-17, and 18-20 are 2'-O-methoxyethyl ribose modified nucleosides. The internucleoside linkages 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 linkages, and each cytosine is a 5-methylcytosine. ISIS 666853 has the following chemical notations: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te; A=adenine, mC=5-methylcytosine, G=guanine, T=thymine; e = 2'-O-methoxyethyl ribose modified sugar; d=2'-deoxyribose sugar, s=phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage. The ISIS 666853 sequence may also be written in abbreviation as follows: [ka]

[0067] The underlined residues are 2'-MOE nucleosides. The P=O annotation reflects the location of the phosphodiester bond.

[0068] ISIS 666853 has the following chemical structure: [ka] As illustrated by:

[0069] It will be understood that the antisense oligonucleotides may be present in solution in a free acid form, a salt form, or a mixture thereof.

[0070] Nonadeca sodium salt of ISIS 666853 ("Compound A") Compound A is the nonadeca sodium salt of ISIS 666853, an antisense oligonucleotide inhibitor of SOD1 messenger RNA (mRNA) that reduces levels of SOD1 protein in subjects with SOD1 ALS. Reducing SOD1 mRNA and subsequently toxic SOD1 protein may provide therapeutic benefit to subjects with SOD1 ALS.

[0071] The structure of Compound A is provided below. [ka]

[0072] The molecular formula of compound A is C 230 H 298 N 72 Na 19 O 123 P19 S 15 and has a molecular weight = 7545.59 amu.

[0073] Compound A is complementary to a portion of the 3' untranslated region (3'UTR) of human SOD1 mRNA and binds by Watson-Crick base pairing. Hybridization (binding) of Compound A to the cognate mRNA leads to RNase-H1-mediated degradation of SOD1 mRNA, thus reducing the amount of SOD1 protein synthesis. RNase H is a ubiquitously expressed enzyme (nuclease) that recognizes deoxyribonucleic acid-ribonucleic acid (DNA-RNA) heteroduplexes and cleaves the RNA strand of the double helix. By binding to the 3'-UTR region of SOD1 mRNA, Compound A inhibits RNase H1 from SOD1. It selectively targets the mRNA and enhances its cleavage, leading to reduced expression of both wild-type and mutant forms of SOD1.

[0074] Compound A significantly increased median survival in SOD1 G93A transgenic mice (Mantel-Cox, p<0.01). It also caused a dose-dependent protection of neuromuscular function as measured by compound muscle action potentials in SOD1 G93A transgenic mice.

[0075] Conjugated Antisense Oligonucleotides The antisense oligonucleotides of the present disclosure may 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 may also be modified to enhance properties (such as nuclease stability) with one or more stabilizing groups that are typically attached to one or both ends of the antisense oligonucleotide. Stabilizing groups include cap structures. These end modifications can protect antisense oligonucleotides with terminal nucleic acids from exonuclease degradation and aid in intracellular delivery and / or localization. Caps may be present at the 5' end (5' cap) or 3' end (3' cap), or may be present on both ends. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Additional 3' and 5' stabilising 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.

[0076] 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 the formulation of pharmaceutical compositions depend on a number of criteria, including but not limited to the route of administration, the extent of disease, or the dose to be administered.

[0077] Antisense oligonucleotides targeted to SOD1 nucleic acid or their salts can be used in pharmaceutical compositions by combining antisense oligonucleotides or their salts with suitable pharma- ceutically acceptable diluents or carriers. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in parenterally delivered compositions. Thus, in one embodiment, pharmaceutical compositions comprising antisense oligonucleotides targeted to SOD1 nucleic acid or their salts and pharma-ceutically acceptable diluents are used in the methods described herein.

[0078] The antisense oligonucleotides or salts thereof described herein can be formulated as pharmaceutical compositions for intrathecal administration to a subject.

[0079] The pharmaceutical composition comprising the antisense oligonucleotide of the present disclosure encompasses any pharmaceutically acceptable salt, ester or salt of such ester, or any other oligonucleotide that can provide its biologically active metabolite or residue (directly or indirectly) upon administration to animals, including humans.Thus, for example, the present disclosure also relates to the pharmaceutically acceptable salt and other bioequivalents of antisense oligonucleotide.Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salt and potassium salt.

[0080] Treatment methods The disclosure features a method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the human SOD1 gene in a human subject in need thereof. The method includes administering a fixed dose of an antisense oligonucleotide to the human subject by intrathecal administration, wherein the nucleobase sequence of the antisense oligonucleotide consists of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), 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 each of nucleosides 7-15 is a 2'-deoxynucleoside. The internucleoside bonds between nucleosides 2-3, 4-5, 16-17, and 18-19 are phosphodiester bonds, and 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 embodiments, the fixed dose of the antisense oligonucleotide is about 100 mg or 100 mg. In other embodiments, the fixed dose of the antisense oligonucleotide is about 60 mg or 60 mg. In yet other embodiments, the fixed dose of the antisense oligonucleotide is about 40 mg or 40 mg. In some other embodiments, the fixed dose of antisense oligonucleotide is about 20mg or 20mg. In certain embodiments, the fixed dose of the sodium salt of antisense oligonucleotide is about 105.9mg or 105.9mg. In other embodiments, the fixed dose of the sodium salt of antisense oligonucleotide is about 63.5mg or 63.5mg. In still other embodiments, the fixed dose of the sodium salt of antisense oligonucleotide is about 42.3mg or 42.3mg. In some other embodiments, the fixed dose of the sodium salt of antisense oligonucleotide is about 21.2mg or 21.2mg.

[0081] Also provided is a method of reducing 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 a fixed dose of an antisense oligonucleotide to the human subject by intrathecal administration, wherein the nucleobase sequence of the antisense oligonucleotide consists of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), 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 each of nucleosides 1-5 and 16-20 is a 2'-O-methoxyethyl ribose modified nucleoside. The internucleoside bonds between nucleosides 2-3, 4-5, 16-17, and 18-19 are phosphodiester bonds, and 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 embodiments, the fixed dose of the antisense oligonucleotide is about 100 mg or 100 mg. In other embodiments, the fixed dose of the antisense oligonucleotide is about 60 mg or 60 mg. In yet other embodiments, the fixed dose of the antisense oligonucleotide is about 40 mg or 40 mg. In other instances, the fixed dose of antisense oligonucleotide is about 20 mg or 20 mg.

[0082] 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 a fixed dose of an antisense oligonucleotide to the human subject by intrathecal administration, wherein the nucleobase sequence of the antisense oligonucleotide consists of CAGGATACATTTCTACAGCT (SEQ ID NO: 1), 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 each of nucleosides 1-5 and 16-20 is a 2'-deoxynucleoside. The internucleoside bonds between nucleosides 2-3, 4-5, 16-17, and 18-19 are phosphodiester bonds, and 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 embodiments, the fixed dose of the antisense oligonucleotide is about 100 mg or 100 mg. In other embodiments, the fixed dose of the antisense oligonucleotide is about 60 mg or 60 mg. In yet other embodiments, the fixed dose of the antisense oligonucleotide is about 40 mg or 40 mg. In other instances, the fixed dose of antisense oligonucleotide is about 20 mg or 20 mg.

[0083] Also provided is a method of treating or preventing amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene in a human subject in need thereof, the method involving administering by intrathecal administration to the human subject a pharmaceutical composition comprising an antisense oligonucleotide, or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] has.

[0084] In certain embodiments, antisense oligonucleotide or its salt is administered at about 100mg or equivalent to 100mg of antisense oligonucleotide.In other embodiments, antisense oligonucleotide or its salt is administered at about 60mg or equivalent to 60mg of antisense oligonucleotide.In other embodiments, antisense oligonucleotide or its salt is administered at about 40mg or equivalent to 40mg of antisense oligonucleotide.In other embodiments, antisense oligonucleotide or its salt is administered at about 20mg or equivalent to 20mg of antisense oligonucleotide.

[0085] Also provided is a method of reducing human SOD1 protein synthesis in a human subject having a mutation in the human SOD1 gene associated with amyotrophic lateral sclerosis, the method involving administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide, or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] has.

[0086] In certain embodiments, antisense oligonucleotide or its salt is administered at about 100mg or equivalent to 100mg of antisense oligonucleotide.In other embodiments, antisense oligonucleotide or its salt is administered at about 60mg or equivalent to 60mg of antisense oligonucleotide.In other embodiments, antisense oligonucleotide or its salt is administered at about 40mg or equivalent to 40mg of antisense oligonucleotide.In other embodiments, antisense oligonucleotide or its salt is administered at about 20mg or equivalent to 20mg of antisense oligonucleotide.

[0087] 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 involving administering to the human subject by intrathecal administration a pharmaceutical composition comprising an antisense oligonucleotide, or a salt thereof, wherein the antisense oligonucleotide has the following structure: [ka] has.

[0088] In certain embodiments, antisense oligonucleotide or its salt is administered at about 100mg or equivalent to 100mg of antisense oligonucleotide.In other embodiments, antisense oligonucleotide or its salt is administered at about 60mg or equivalent to 60mg of antisense oligonucleotide.In other embodiments, antisense oligonucleotide or its salt is administered at about 40mg or equivalent to 40mg of antisense oligonucleotide.In other embodiments, antisense oligonucleotide or its salt is administered at about 20mg or equivalent to 20mg of antisense oligonucleotide.

[0089] In some cases, 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.

[0090] In some cases, 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 about 20 mg of fixed dose (i.e., the equivalent amount) of antisense oligonucleotide.In certain embodiments, the pharmaceutical composition is administered to a human subject in an amount sufficient to deliver about 40 mg of fixed dose of antisense oligonucleotide.In certain embodiments, the pharmaceutical composition is administered to a human subject in an amount sufficient to deliver about 60 mg of fixed dose of antisense oligonucleotide.In certain embodiments, the pharmaceutical composition is administered to a human subject in an amount sufficient to deliver about 100 mg of fixed dose of antisense oligonucleotide.

[0091] In certain embodiments, the fixed dose of antisense oligonucleotide or its salt is administered as a loading dose(s). In some embodiments, the fixed dose of antisense oligonucleotide is administered as a maintenance dose(s). In certain examples, the fixed dose of antisense oligonucleotide is administered as 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 cases, the maintenance dose(s) is administered monthly.

[0092] In certain embodiments, a human subject is administered three loading doses of an 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 cases, the three loading doses are administered at two-week intervals. In some cases, the three loading doses are administered at 14-day intervals. In some cases, the maintenance dose(s) are administered starting 4 weeks after the third loading dose. In some cases, the maintenance dose(s) are administered monthly starting after the third loading dose. In some cases, the maintenance dose(s) are administered every 28 days starting after the third loading dose.

[0093] The mutation in SOD1 can be any mutation in the human SOD1 gene that is associated with ALS. In some cases, the mutation is a slow-progressing ALS disease mutation. In other instances, the mutation is a fast-progressing ALS disease mutation. In certain instances, the mutation in the human SOD1 gene is one or more of 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, T54R, S591, V87A, T88 delta TAD, A89T, V97M, S105 delta SL, V118L, D124G, L114F, D90A, G12R, or G147R. 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 H46R mutation in the human SOD1 gene. In yet another particular embodiment, the human subject has a G93S mutation in the human SOD1 gene.

[0094] In certain instances, mutations in the SOD1 gene are identified by genetic testing.

[0095] In some cases, the methods described above involve identifying a mutation in the SOD1 gene by genetic testing.

[0096] In certain embodiments, administering a therapeutically effective amount of antisense oligonucleotide or its salt (e.g., compound A) to a human subject is accompanied by monitoring the SOD1 level in the human subject to determine the response of the human subject to administering the antisense oligonucleotide or its salt.The response of the human subject to administering the antisense oligonucleotide or its salt can be used by a physician to determine the amount and duration of therapeutic intervention.In certain embodiments, the human SOD1 level is monitored in CSF.In certain embodiments, the human SOD1 level is monitored in plasma.

[0097] In certain embodiments, administration of antisense oligonucleotide or its salt (e.g., compound A) results in the reduction of SOD1 protein expression.In certain embodiments, administration of antisense oligonucleotide or its salt (e.g., compound A) results in the reduction of 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 defined by any two of these values.In certain embodiments, the reduction of SOD1 protein expression is in CSF.In certain embodiments, the reduction of SOD1 protein expression is in plasma.

[0098] In certain embodiments, administration of an antisense oligonucleotide or a salt thereof (e.g., Compound A) results in improved motor function and respiration in a human subject. In certain embodiments, administration of an antisense oligonucleotide or a salt thereof improves 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 defined by any two of these values.

[0099] In certain embodiments, a pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof (e.g., Compound A) is used for the preparation of a medicament for the treatment of a human subject suffering from or susceptible to ALS (e.g., a human subject having a mutation in SOD1 that is associated with ALS).

[0100] Delivery Device In certain embodiments, the antisense oligonucleotide or its salt (e.g., Compound A) is administered to the human subject by a syringe for intrathecal delivery. In another embodiment, the antisense oligonucleotide or its salt (e.g., Compound A) is administered to the human subject by a pump for intrathecal delivery. Thus, the present disclosure also provides a pump or syringe containing a sterile preparation of the antisense oligonucleotide or its salt (e.g., Compound A). The syringe or pump can be adapted for intrathecal administration of the antisense oligonucleotide or its salt. In some cases, the syringe or pump delivers a fixed dose(s) (e.g., about 20 mg or less, about 40 mg or less, about 60 mg or less, or about 100 mg or less) of the antisense oligonucleotide. The present disclosure also provides a pump or syringe containing a sterile preparation of a pharmaceutical composition comprising the antisense oligonucleotide or its salt (e.g., Compound A). The syringe or pump can be adapted for intrathecal administration of the pharmaceutical composition. In some cases, the syringe or pump delivers a fixed dose(s) (e.g., about or at 20 mg, about or at 40 mg, about or at 60 mg, or about or at 100 mg) of the antisense oligonucleotide of the pharmaceutical composition. In certain embodiments, the pump or syringe contains a sterile preparation of the antisense oligonucleotide or a salt thereof, and the syringe or pump is adapted for intrathecal administration of the antisense oligonucleotide or a salt thereof (e.g., Compound A) at a fixed dose of 20 mg, 40 mg, 60 mg, or 100 mg of the antisense oligonucleotide.

[0101] Assay RNA isolation RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. RNA is prepared using methods well known in the art, for example using TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocol.

[0102] Analysis of inhibition of target levels or expression The inhibition of the level or expression of SOD1 nucleic acid can be assayed by various techniques known in the art. For example, the target nucleic acid level can be quantified, for example, by Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routinely used in the art. Quantitative real-time PCR can be performed using commercially available ABI PRISM. This may be conveniently accomplished using a 7600, 7700, or 7900 Sequence Detection System (available from PE-Applied Biosystems, Foster City, Calif., used according to the manufacturer's instructions).

[0103] Quantitative real-time PCR analysis of target RNA levels Quantitation of SOD1 RNA level can be achieved by quantitative real-time PCR using ABI PRISM 7600, 7700 or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.Methods of quantitative real-time PCR are well known in the art.

[0104] Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction to generate complementary DNA (cDNA), which is then used as a substrate for real-time PCR amplification. The RT and real-time PCR reactions are carried out sequentially in the same sample well. The RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, CA). The RT real-time PCR reaction is carried out by methods well known to those skilled in the art.

[0105] The amount of gene (or RNA) target obtained by real-time PCR is normalized either by using the expression level of a gene whose expression is constant (such as cyclophilin A) or by quantifying total RNA using RIBOGREEN (Invitrogen, Inc., Carlsbad, CA). Cyclophilin A expression is quantified by real-time PCR by running simultaneously (multiplexed) or separately with the target. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invitrogen, Inc., Eugene, OR). The method of RNA quantification by RIBOGREEN is taught in Jones, LJ, et al (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence. Probes and primers are designed to hybridize to SOD1 nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art and can include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).

[0106] Protein level analysis Antisense inhibition of SOD1 nucleic acid can be assessed by measuring SOD1 protein level. Protein level of SOD1 can be evaluated or quantified in various ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblot), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g. caspase activity assay), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS). Antibodies directed against targets can be identified and obtained from various sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared by conventional monoclonal or polyclonal antibody production methods well known in the art. Antibodies useful for detecting human SOD1 are commercially available.

[0107] Testing for SOD1 mutations One underlying genetic cause of ALS is the mutation(s) in human SOD1 gene.Therefore, the identification of the subject suffering from or susceptible to ALS can be accomplished by genetic testing (such as gene sequencing) of the subject's SOD1 gene using assays known in the art.It is known in the art that at least 180 mutations in human SOD1 are associated with ALS.

[0108] The subject's susceptibility to ALS disease can also be assessed by analyzing the subject's family history for ALS. Family history analysis can include reviewing three-generation pedigrees in which ALS has been reported, medical records and autopsy studies of family members, and identifying SOD1 mutations in an autosomal dominant pattern.

[0109] The following examples should not be construed as limiting the scope of the invention in any manner. EXAMPLES

[0110] Example 1: Study Design Compound A is being investigated in ongoing clinical trials. The trials include randomized, placebo-controlled, single ascending dose (SAD) and multiple ascending dose (MAD) studies in SOD1 ALS patients. In the MAD portion, participants received five doses of study drug over approximately three months. Fifty participants were randomized (3:1 per cohort) to receive 20 mg, 40 mg, 60 mg, or 100 mg of Compound A, or placebo. Between 1 and 4 participants treated with Compound A per cohort had a reported SOD1 mutation that was a priori determined to be a fast-progressing one (mainly A4V).

[0111] Example 2: Safety Profile In the study, 66 of 70 patients (94%) experienced at least one adverse event (AE), the majority of which were mild or moderate grade. The most common AEs occurring in 3 or more participants who received Compound A were headache (n=16), procedural pain (n=14), and post-lumbar puncture syndrome (n=13). Seven patients experienced serious adverse events (SAEs), three of which were fatal events. No SAEs were reported in the highest dose group (100 mg Compound A). Six subjects had SAEs that were assessed as unrelated to Compound A and likely related to ALS or a comorbid condition. All fatal events were assessed as unrelated. One patient had an SAE of CSF leukocytosis and CSF protein increase that was assessed as related to Compound A. Laboratory abnormalities resolved despite continued dosing with Compound A, and the patient completed the study. No serious adverse events were reported at the highest dose tested, 100 mg.

[0112] Example 3: Pharmacokinetics and Pharmacodynamics Dose-dependent increases in plasma (not shown) and CSF concentrations of Compound A were observed in the SAD (not shown) and MAD cohorts (Figure 2). At measurements on days 1 and 85, Compound A plasma concentrations were dose-proportional, while CSF Compound A exposure showed a less than dose-proportional response. Reductions from baseline in CSF SOD1 concentrations were observed at multiple dose levels of 40 mg and above (i.e., 60 mg and 100 mg), which increased with dose, with a maximum mean reduction of 36% in the 100 mg multiple dose group (Figure 3). Reductions from baseline in CSF SOD1 were observed in all participants in the 100 mg dose group. The maximum SOD1 reduction was observed at or shortly after the last dose, and it was noted that consecutive dosing beyond 5 doses may result in further reductions. Models based on preclinical data suggest that 100 mg of Compound A effectively reduces SOD1 levels by >99% in the spinal cord and approximately 25-30% in the cortex.

[0113] Example 4: Clinical Observations Efficacy was assessed at multiple time points against multiple scales including the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALFSFRS-R), static vital capacity (SVC), and handheld dynamometry (HHD) scale. In all cohorts, the Compound A treatment group had higher (i.e., better) scores compared to the placebo treatment group (Figures 4A-4C). By day 85, the mean change from baseline in ALSFRS-R score for participants treated with Compound A at the 100 mg dose group (N=10) was -1.1 and for placebo-treated participants (N=12) was -5.6, a difference of 4.4 points. In participants with SOD1 mutations known to progress faster (e.g., A4V), a significant difference was observed between participants treated with Compound A at the 100 mg dose group and those treated with placebo. In these fast progressors, the mean difference in change from baseline in ALSFRS-R by day 85 approached 10 points (Figure 5). Treatment effects appeared consistent across multiple clinical scales and CSF SOD1 reduction in both fast progressors (Figure 5) and slow progressors (Figure 6). As a frame of reference, the difference in ALSFRS-R at 6 months was 2.5 points in the pivotal trial of edaravone in ALS.

[0114] Taken together, this study demonstrated that Compound A is a safe and effective treatment for subjects with SOD1 ALS. This is most strongly reinforced by the outcomes of subjects with fast-growing mutation subtypes (mainly A4V) treated with Compound A, especially in the 100mg dose group, who would be expected to decline rapidly in the absence of effective treatment. As noted above, efficacy was assessed at multiple time points against multiple scales, including the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R), static vital capacity (SVC), and handheld dynamometry (HHD). These results show much less decline in each of the three clinical functional endpoints in the 100mg Compound A dose group compared to the placebo group.

[0115] Example 5: Phosphorylated neurofilament heavy chain (pNFH) levels in CSF In patients with fast-progressing SOD1 mutations, treatment with Compound A resulted in a reduction in CSF pNFH levels and a slowing of clinical decline compared to placebo. A larger difference in pNFH levels at day 85 between the 100 mg Compound A group and the placebo group was observed in patients with fast-progressing SOD1 mutations (Figure 7) compared to patients with other SOD1 mutations (Figure 8).

[0116] Other embodiments Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, but not to limit, 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

[Claim 1] The invention described in the specification.

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