Methods for Treating or Preventing Spinal Muscular Atrophy

Abstract

To provide a method of treating or preventing spinal muscular atrophy.SOLUTION: There is provided a method of treating or preventing spinal muscular atrophy. The effective dosing regimen is described. A biomarker and a kit are also provided. For example, the present disclosure features dosing regimens for antisense compounds described herein that rapidly achieve and maintain higher antisense compound concentrations in patients with spinal muscular atrophy (SMA) (relative to the approved SPINRAZA(R) dosing regimen) and thus further enhance the efficacy of treatment.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 876,360, filed July 19, 2019, the contents of which are incorporated by reference in their entirety. Technical Field

[0002] The present disclosure relates generally to methods and effective dosing regimens for the treatment and prevention of spinal muscular atrophy. [Background technology]

[0003] Spinal muscular atrophy (SMA) is an inherited neurodegenerative disorder characterized by atrophy of voluntary muscles of the limbs and trunk as a result of loss of spinal motor neurons in the anterior horn of the spinal cord. SMA is an early-onset, autosomal recessive disease with an incidence of 8.5 to 10.3 per 100,000 live births, and is the most common monogenic cause of infant mortality and a major cause of childhood morbidity in the United States. The natural history of SMA includes four major recognized phenotypes that depend on the age of onset and the motor skills achieved. Type I SMA (infantile-onset SMA) is the most severe type that occurs at birth or within 6 months and generally results in death within 2 years. Children with Type I SMA are unable to sit or walk. Late-onset SMA can be divided into Type II and Type III SMA. Type II SMA (appears between 6 and 18 months of age) is an intermediate type, in which patients can sit but cannot stand or walk. Patients with Type III SMA generally develop SMA after 18 months of age and can sit and walk but become severely and progressively disabled.Patients with Adult-Onset SMA or Type IV SMA develop the disease after age 18 and have a normal life expectancy.

[0004] The molecular basis of SMA is the loss of both copies of the survival of motor neuron gene 1 (SMN1), which encodes a protein that is part of a multiprotein complex thought to be involved in snRNP biogenesis and recycling. A nearly identical gene, SMN2, resides in a duplicated region of chromosome 5q13 and regulates disease severity. Normal expression of the SMN1 gene alone results in expression of the survival of motor neuron (SMN) protein. Although SMN1 and SMN2 potentially encode the same protein, SMN2 contains a translationally silent mutation at position +6 of exon 7, which makes the inclusion of exon 7 in the SMN2 transcript inefficient. Thus, the predominant form of SMN2 is a truncated form lacking exon 7, which is unstable and inactive (Cartegni and Krainer, Nat. Genet., 30:377-384 (2002)). Expression of the SMN2 gene results in approximately 10-20% SMN protein and 80-90% unstable / non-functional SMN delta 7 protein. SMN protein has a well-established role in spliceosome assembly and may also be involved in mRNA transport in neuronal axons and nerve terminals.

[0005] Humans have a variable copy number of the SMN2 gene (from 0 to 8 copies). SMN2 is an important modifier of the disease phenotype, as the number of SMN2 copies expressed in SMA patients and the resulting amount of full-length SMN protein correlate with the severity of SMA disease. Antisense technology is an effective means for regulating the expression of one or more specific gene products, including alternative splicing products, and is uniquely useful in many therapeutic, diagnostic, and research applications. The principle behind antisense technology is that antisense compounds hybridizing to target nucleic acids regulate gene expression activities such as transcription, splicing, or translation by one of several antisense mechanisms. The sequence specificity of antisense compounds makes them highly attractive as tools for target validation and gene functioning, as well as therapeutic agents that selectively regulate the expression of genes involved in disease. Certain antisense compounds complementary to SMN2 pre-mRNA are known in the art. See, for example, WO2007 / 002390. SPINRAZA® is an antisense compound approved in the United States, Europe, and other countries and regions around the world at a recommended dosage of 12 mg for the treatment of SMA in pediatric and adult patients. At the approved dosage of 12 mg, SPINRAZA® has a robust safety profile and has provided significant benefit to patients with SMA. In light of the importance of early intervention, especially in patients with the most severe forms of SMA, Applicant believed that more rapid achievement and maintenance of higher concentrations of SPINRAZA® in patients with SMA would be beneficial. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2007 / 002390 [Non-patent literature]

[0007] [Non-Patent Document 1] Cartegni and Krainer, Nat. Genet., 30:377-384 (2002) Summary of the Invention

[0008] This application relates to methods of treating or preventing SMA. The disclosure features dosing regimens for the antisense compounds described herein that rapidly achieve and sustain higher antisense compound concentrations in patients with SMA (compared to the approved SPINRAZA® dosing regimen), thereby further improving the efficacy of treatment.

[0009] In one aspect, the disclosure features a method for treating spinal muscular atrophy (SMA) in a human subject in need thereof. The method comprises administering to the human subject at least two loading doses of the following structure: [ka] or a salt thereof. The administration comprises administering, by intrathecal injection (e.g., intrathecal bolus injection), a first loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 50 mg of the antisense compound; and a second loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days (e.g., about 10 to 18 days, about 12 to 16 days, or about 14 days) after administration of the first loading amount.

[0010] In another aspect, the disclosure features a method for increasing levels of motor neuron survival 2 (SMN2) messenger ribonucleic acid (mRNA) that includes exon 7 in a human subject having a mutation on chromosome 5q that is associated with SMN protein deficiency. The method comprises administering to the human subject at least two loading doses of the following structure: [ka] or a salt thereof. The administration comprises administering, by intrathecal injection (e.g., intrathecal bolus injection), a first loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 50 mg of the antisense compound; and a second loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days (e.g., about 10 to 18 days, about 12 to 16 days, or about 14 days) after administration of the first loading amount.

[0011] In some instances, the method further comprises administering to the human subject at least one maintenance dose equivalent to about 28 mg of the antisense compound via intrathecal injection (eg, intrathecal bolus injection).

[0012] In some examples, the above method further comprises administering to the human subject by intrathecal injection (e.g., intrathecal bolus injection) a maintenance dose of an antisense compound described herein or a salt thereof beginning about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the second loading dose, and continuing once every about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) thereafter, each equivalent to about 28 mg of the antisense compound.

[0013] In some instances, the second loading dose is administered about 14 days after administration of the first loading dose. In some instances, the second loading dose is administered 14 days after administration of the first loading dose.

[0014] In certain examples, the method further comprises administering to the human subject by intrathecal injection (e.g., intrathecal bolus injection) a third loading dose of an antisense compound described herein or a salt thereof equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days (e.g., about 10 to 18 days, about 12 to 16 days, or about 14 days) after administration of the second loading dose; and one or more maintenance doses of an antisense compound described herein or a salt thereof equivalent to about 28 mg of the antisense compound, beginning about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the third loading dose and continuing once every about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) thereafter. In some examples, the second loading dose is administered about 14 days after administration of the first loading dose, and the third loading dose is administered about 14 days after administration of the second loading dose. In certain instances, the second loading dose is administered 14 days after administration of the first loading dose, and the third loading dose is administered 14 days after administration of the second loading dose.

[0015] In some examples, the method further comprises administering to the human subject by intrathecal injection (e.g., intrathecal bolus injection) a third loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days (e.g., about 10 to 18 days, about 12 to 16 days, or about 14 days) after administration of the second loading amount; a fourth loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days (e.g., about 10 to 18 days, about 12 to 16 days, or about 14 days) after administration of the third loading amount; and one or more maintenance doses of an antisense compound described herein, or a salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the fourth loading amount and continuing once every about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) thereafter. In some cases, the second loading dose is administered about 14 days after administration of the first loading dose, the third loading dose is administered about 14 days after administration of the second loading dose, and the fourth loading dose is administered about 14 days after administration of the third loading dose. In other cases, the second loading dose is administered 14 days after administration of the first loading dose, the third loading dose is administered 14 days after administration of the second loading dose, and the fourth loading dose is administered 14 days after administration of the third loading dose.

[0016] In another embodiment, the disclosure provides a method for treating SMA in a human subject in need of such treatment. The method comprises administering to the human subject the following structure: [ka] The human subject to be treated is pre-administered with one or more doses of an antisense compound or salt thereof as described herein, the one or more pre-administered doses corresponding to about 12 mg of the antisense compound. The method includes administering to the human subject by intrathecal injection (e.g., intrathecal bolus injection) a loading dose of an antisense compound or salt thereof as described herein equivalent to about 50 mg of the antisense compound, administered about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the last pre-administered dose corresponding to about 12 mg of the antisense compound; and one or more maintenance doses of an antisense compound or salt thereof as described herein, each corresponding to about 28 mg of the antisense compound, beginning about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the loading dose and continuing once every about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) thereafter.

[0017] In another embodiment, the disclosure relates to a method for increasing the level of SMN2 mRNA comprising exon 7 in a human subject having a mutation on chromosome 5q associated with SMN protein deficiency. The method comprises administering to the human subject the following structure: [ka] The method includes administering to the human subject an antisense compound or salt thereof having the formula: embedded image wherein the human subject is pre-administered with one or more doses of an antisense compound or salt thereof described herein, the one or more pre-administered doses corresponding to about 12 mg of the antisense compound. The method includes administering to the human subject by intrathecal injection (e.g., intrathecal bolus injection) a loading dose of the antisense compound or salt described herein equivalent to about 50 mg of the antisense compound, administered about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the last pre-administered dose corresponding to about 12 mg of the antisense compound; and one or more maintenance doses of the antisense compound or salt described herein, each equivalent to about 28 mg of the antisense compound, beginning about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the loading dose and continuing once every about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) thereafter.

[0018] In another embodiment, a method for treating spinal muscular atrophy (SMA) in a human subject in need of such treatment is provided. The method comprises administering to the human subject the following structure: [ka] or a salt thereof. The human subject was pre-administered one or more doses of an antisense compound described herein, or a salt thereof, wherein the one or more pre-administered doses corresponded to about 12 mg of the antisense compound. The method includes administering to a human subject by intrathecal injection (e.g., intrathecal bolus injection) a first loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 28 mg of the antisense compound, administered about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of a last previously administered dose equivalent to about 12 mg of the antisense compound; a second loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 50 mg of the antisense compound, administered about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the first loading amount; and one or more maintenance doses of an antisense compound described herein, or a salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the second loading amount and continuing once every about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) thereafter.

[0019] In another embodiment, the disclosure provides a method for increasing the level of SMN2 mRNA comprising exon 7 in a human subject having a mutation on chromosome 5q associated with SMN protein deficiency. The method comprises administering to the human subject the following structure: [ka] or a salt thereof. The human subject was pre-administered one or more doses of an antisense compound described herein, or a salt thereof, wherein the one or more pre-administered doses corresponded to about 12 mg of the antisense compound. The method includes administering to a human subject by intrathecal injection (e.g., intrathecal bolus injection) a first loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 28 mg of the antisense compound, administered about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of a last previously administered dose equivalent to about 12 mg of the antisense compound; a second loading amount of an antisense compound described herein, or a salt thereof, equivalent to about 50 mg of the antisense compound, administered about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the first loading amount; and one or more maintenance doses of an antisense compound described herein, or a salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) after administration of the second loading amount and continuing once every about 3 to 6 months (e.g., about 4 to 5 months; or about 4 months) thereafter.

[0020] In some examples, the human subject has been pre-administered at least 4 (e.g., 4, 5, 6, 7, 8, 9, 10), at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 doses of an antisense compound described herein or a salt thereof, wherein each of the at least 4, 5, 6, 7, 8, 9, or 10 pre-administered doses corresponds to about 12 mg of the antisense compound.

[0021] In some examples of all of the above methods, the human subject has SMA type I, SMA type II, SMA type III, or SMA type IV. In other examples, the human subject is pre-symptomatic with SMA.

[0022] In some instances, a human subject is administered an antisense compound or salt thereof described herein using a spinal anesthesia needle.

[0023] In some examples, the antisense compounds described herein or salts thereof are administered in an injection volume of 5.0 mL.

[0024] In some examples, the antisense compounds described herein or salts thereof are dissolved in phosphate buffered saline (PBS).In other examples, the antisense compounds described herein or salts thereof are dissolved in artificial cerebrospinal fluid (aCSF).

[0025] In certain instances, the salt of the antisense compound is a sodium salt.

[0026] In one example, a salt of an antisense compound has the structure: [ka]

[0027] In some embodiments of the methods described herein, the methods further comprise detecting neurofilament levels in a human subject before and / or after the initiation of treatment. Neurofilament levels can be used, for example, to determine whether a subject administered an antisense compound or salt thereof described herein is responding to treatment and to determine appropriate subsequent treatments for the subject.

[0028] In one embodiment, the method involves (i) measuring a first neurofilament level in a first biological sample obtained from the human subject prior to the initiation of treatment; (ii) administering to the human subject an antisense compound described herein, or a salt thereof, according to any one of the methods described herein; and (iii) measuring neurofilament levels in a subsequent (e.g., second) biological sample obtained from the human subject after administration of at least one loading dose.

[0029] In one embodiment, the method involves (i) measuring a first neurofilament level in a first biological sample obtained from the human subject prior to the initiation of treatment; (ii) administering to the human subject an antisense compound described herein, or a salt thereof, according to any one of the methods described herein; and (iii) measuring neurofilament levels in a subsequent (e.g., second) biological sample obtained from the human subject after administration of the entire loading dose.

[0030] In one embodiment, the method involves (i) measuring a first neurofilament level in a first biological sample obtained from the human subject prior to the initiation of treatment; (ii) administering to the human subject an antisense compound or salt thereof described herein according to any one of the methods described herein; and (iii) measuring neurofilament levels in a subsequent (e.g., second) biological sample obtained from the human subject after administration of all loading doses and at least one maintenance dose of an antisense compound or salt thereof described herein.

[0031] In one example, the subsequent (e.g., second) neurofilament level is lower than the first neurofilament level (e.g., the second neurofilament level is reduced by at least 50% compared to the first neurofilament level), and the method further includes administering either another loading dose or another maintenance dose, each as described herein, at each dosing interval as described herein.

[0032] In another example, the subsequent (e.g., second) neurofilament level is equal to or higher than the first neurofilament level, and the method further comprises administering to the human subject additional doses of an antisense compound described herein, or a salt thereof, wherein each of the additional doses of the antisense compound, or a salt thereof, is an increased amount and / or a shorter dosing interval compared to the last dose administered prior to measuring the subsequent (e.g., second) neurofilament level.

[0033] In some embodiments, the neurofilament is a neurofilament heavy chain (NF-H, e.g., pNF-H), a medium / intermediate chain (NF-M), or a light chain (NF-L).

[0034] In some embodiments, the first and second biological samples are blood, serum, plasma, or cerebrospinal fluid.

[0035] The term "about" in connection with an amount, e.g., about X mg, means + / - 10%, so "about 50 mg" includes 45 mg to 55 mg. The term "about" in connection with X days means + / - 3 days, so "about 10 days" includes 7 to 13 days. The term "about" in connection with X months means + / - 1 week, so "about 4 months" includes 1 week before and after the 4 month point.

[0036] 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 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.

[0037] Other features and advantages of the invention will become apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0038] [Figure 1]Relationship between CSF C trough and CHOP-INTEND (change from baseline) with fixed E0 and Emax (E=efficacy) is shown. The (long) dashed line, solid line, and (short) dashed line represent the predicted PK / PD relationship when Emax is fixed at 10, 25, and 40, respectively. Observed data are divided based on deciles of CSF C trough values. Open and filled circles represent observed mean change and predicted change in CHOP-INTEND from baseline, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present disclosure relates to methods for treating or preventing SMA using effective dosing regimens of antisense compounds or salts thereof, each as described herein, which methods are useful for treating and / or preventing SMA in pediatric and adult patients.

[0040] SMA patients (Types I, II, III, IV and pre-symptomatic) may achieve greater improvements in motor function and motor milestone development with higher antisense drug exposure than is achieved with the current SPINRAZA® dosing regimen.

[0041] Antisense Compounds An exemplary antisense compound for use in the present invention has the nucleobase sequence: TCACTTTCATAATGCTGG (SEQ ID NO:1), in which each nucleoside has a 2'-O-(2-methoxyethyl) (MOE) modification, each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage, and each "C" in the sequence is a 5-methyl-cytosine.

[0042] The free acid form of this antisense compound (also called nusinersen) is shown below. [ka]

[0043] In some instances, a salt (e.g., a pharma- ceutically acceptable salt) of the antisense compound is used. In some instances, the salt may be sodium (also referred to as nusinersen sodium or nusinersen sodium salt). In other instances, the salt may be potassium.

[0044] Exemplary salts of the antisense compounds are shown below. [ka]

[0045] The antisense compound is nusinersen, and the sodium salt thereof is nusinersen sodium or nusinersen sodium salt. 2.40 mg of nusinersen is equivalent to 2.53 mg of nusinersen sodium salt. A dose of nusinersen sodium equivalent to 50 mg of nusinersen is 52.71 mg of nusinersen sodium salt. A dose of nusinersen sodium equivalent to 28 mg of nusinersen is 29.52 mg of nusinersen sodium salt. A dose of nusinersen sodium equivalent to 12 mg of nusinersen is 12.65 mg of nusinersen sodium salt.

[0046] The antisense compounds or their salts may be prepared as a solution for injection by intrathecal administration.

[0047] The antisense compounds described herein can be used in human subjects in need of treatment or prevention of SMA. In some instances, SMA is SMA type I. In other instances, SMA is SMA type II. In certain instances, SMA is SMA type III. In some instances, SMA is SMA type IV. In other instances, the human subject is pre-symptomatic of SMA.

[0048] Pharmaceutical Compositions The present disclosure also provides a pharmaceutical composition comprising the antisense compound or a salt thereof described herein. In certain instances, such a pharmaceutical composition comprises or consists of a sterile saline solution and the antisense compound or a salt thereof. In some instances, such a pharmaceutical composition is a sterile buffered isotonic solution. In some instances, the pharmaceutical composition does not contain a preservative.

[0049] The antisense compounds described herein or their salts may be mixed with pharma- ceutically acceptable active and / 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 the disease, or the dose to be administered.

[0050] Antisense compounds or their salts can be used in pharmaceutical compositions by mixing such compounds with suitable pharmaceutically acceptable diluent or carrier.In a specific example, the pharmaceutically acceptable diluent is phosphate buffered saline (PBS).In a specific embodiment, the pharmaceutically acceptable diluent is artificial cerebrospinal fluid (aCSF).

[0051] In certain embodiments, the aCSF formulation has a pH of 7.2. The pH of the composition may be adjusted with hydrochloric acid or sodium hydroxide during preparation, if necessary. In some examples, the pharmaceutical composition comprises an antisense compound or a salt thereof (e.g., sodium salt) described herein at a concentration of about 10.0 mg / mL. In certain examples, the pharmaceutical composition comprises an antisense compound or a salt thereof (e.g., sodium salt) described herein at a concentration of 10.0 mg / mL. In other examples, the pharmaceutical composition comprises an antisense compound or a salt thereof (e.g., sodium salt) described herein at a concentration of about 5.6 mg / mL. In some examples, the pharmaceutical composition comprises an antisense compound or a salt thereof (e.g., sodium salt) described herein at a concentration of 5.6 mg / mL. In certain examples, the pharmaceutical composition comprises an antisense compound or a salt thereof (e.g., sodium salt) described herein at a concentration of 3.6 mg / mL to 10.0 mg / mL. In other examples, the pharmaceutical composition comprises an antisense compound described herein or a salt thereof (e.g., a sodium salt) at a concentration of 4.8 mg / mL to 10.0 mg / mL. In yet other examples, the pharmaceutical composition comprises an antisense compound described herein or a salt thereof (e.g., a sodium salt) at a concentration of 5.6 mg / mL to 10.0 mg / mL.

[0052] In some examples, the pharmaceutical composition comprises about 50 mg of the antisense compound described herein or a salt thereof (e.g., sodium salt). In some examples, the pharmaceutical composition comprises 50 mg of the antisense compound described herein or a salt thereof (e.g., sodium salt). In other examples, the pharmaceutical composition comprises about 28 mg of the antisense compound described herein or a salt thereof (e.g., sodium salt). In some examples, the pharmaceutical composition comprises 28 mg of the antisense compound described herein or a salt thereof (e.g., sodium salt). In some examples, the pharmaceutical composition is formulated in PBS or aCSF. In certain examples, the pharmaceutical composition is formulated for intrathecal administration.

[0053] In one example, the disclosure features a pharmaceutical composition comprising 50 mg of an antisense compound disclosed herein or a salt thereof in a volume of 5 mL of pharmaceutical excipient (e.g., aCSF, PBS) such that the concentration of the antisense compound is 10 mg / mL. In certain examples, the pharmaceutical composition is formulated for intrathecal administration (e.g., intrathecal bolus injection).

[0054] In another example, the disclosure features a pharmaceutical composition comprising 28 mg of an antisense compound disclosed herein or a salt thereof in a volume of 5 mL of pharmaceutical excipient (e.g., aCSF, PBS) such that the antisense compound concentration is 5.6 mg / mL. In certain examples, the pharmaceutical composition is formulated for intrathecal administration (e.g., intrathecal bolus injection).

[0055] Pharmaceutical compositions that include antisense compounds include any pharmaceutically acceptable salts, esters, or salts of such esters.Thus, for example, the present disclosure also covers pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0056] Treatment Method The present disclosure features a method of treating or preventing spinal muscular atrophy (SMA) in a human subject. In one example, the SMA is SMA type I. In another example, the SMA is SMA type II. In yet another example, the SMA is SMA type III. In another example, the SMA is SMA type IV. In certain examples, the human subject is pre-symptomatic with SMA.

[0057] The disclosure also provides methods for increasing the inclusion of exon 7 in SMN2 messenger ribonucleic acid (mRNA) transcripts and production of full-length SMN protein in human subjects who have lost both functional copies of the SMN1 gene.

[0058] Also featured is a method for increasing exon 7 inclusion in an SMN2 messenger ribonucleic acid (mRNA) transcript in a human subject having a mutation in the SMN1 gene that leads to a lack of functional SMN protein.

[0059] Additionally, the disclosure relates to methods for increasing the level of SMN2 mRNA that includes exon 7 in a human subject that has a mutation on chromosome 5q that is associated with or leads to SMN protein deficiency. The human subject may have a homozygous gene deletion, substitution, or may be a compound heterozygote.

[0060] The method includes administering to a human subject in need thereof an antisense compound described herein, or a salt thereof (e.g., a sodium salt). The human subject may be an adult or a child.

[0061] In one example, a human subject is administered a dose of an antisense compound or a pharma- ceutically acceptable salt thereof that is useful for treating SMA by intrathecal injection of the human subject.Intrathecal administration can be by lumbar puncture (i.e., spinal tap).Administration can also be by bolus injection.In one example, administration is by intrathecal bolus injection.

[0062] In one example, the antisense compound administered to a human subject comprises or consists of the nucleic acid base sequence set forth in SEQ ID NO:1, wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage, each nucleoside of the oligonucleotide is a 2'-MOE nucleoside, and each C is a 5-methylcytosine.

[0063] In certain examples, when the subject is less than 1 week old, the first dose of the antisense compound or salt thereof described herein is administered. In certain examples, when the subject is less than 1 month old, the first dose of the antisense compound or salt thereof described herein is administered. In certain examples, when the subject is less than 3 months old, the first dose of the antisense compound or salt thereof described herein is administered. In certain examples, when the subject is 6 months old or less, the first dose of the antisense compound or salt thereof described herein is administered. In certain examples, when the subject is more than 6 months old, the first dose of the antisense compound or salt thereof described herein is administered. In certain examples, when the subject is less than 1 year old, the first dose of the antisense compound or salt thereof described herein is administered. In certain examples, when the subject is less than 2 years old, the first dose of the antisense compound or salt thereof described herein is administered. In certain examples, when the subject is less than 15 years old, the first dose of the antisense compound or salt thereof described herein is administered. In certain examples, if the subject is over 15 years old, the first dose of the antisense compound described herein or a salt thereof is administered. In certain examples, if the subject is between 1 year old and under 18 years old, the first dose of the antisense compound described herein or a salt thereof is administered. In certain examples, if the subject is 18 years old or over, the first dose of the antisense compound described herein or a salt thereof is administered.

[0064] In certain instances, the human subject has an SMN2 copy number of 1. In other instances, the human subject has an SMN2 copy number of 2. In some instances, the human subject has an SMN2 copy number of 3. In other instances, the human subject has an SMN2 copy number of 4. In certain instances, the human subject has an SMN2 copy number of 5.

[0065] In some embodiments, the antisense compound or its salt as described herein is administered as intrathecal bolus injection.In some embodiments, the antisense compound or its salt as described herein is administered as a single intrathecal bolus lumbar puncture injection.In certain embodiments, the volume of intrathecal bolus lumbar puncture injection is 5mL.

[0066] Dosing regimen The present disclosure features enhanced or high-dosing regimens of the antisense compounds or salts thereof described herein for treating or preventing SMA. In some instances, the dose of the antisense compounds or salts thereof (e.g., sodium salts) used for treating or preventing SMA corresponds to more than 12 mg and less than or equal to 50 mg of the antisense compounds described herein. In certain examples, the dose of an antisense compound or a salt thereof (e.g., a sodium salt) used to treat or prevent SMA corresponds to about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 mg of an antisense compound described herein. In other examples, the dose of antisense compound or its salt (e.g., sodium salt) used to treat or prevent SMA corresponds to 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg of the antisense compound described herein. In certain embodiments, the dose is administered (e.g., intrathecally) in a volume of 5 mL (e.g., of aCSF or PBS). In some examples, the dose is administered by bolus injection. In one example, the dose is administered by intrathecal bolus injection.

[0067] In some examples, a human subject in need thereof is administered an antisense compound or salt thereof as described herein at a dose equivalent to 18 to 50 mg of the antisense compound in a 5 mL dose volume. In other examples, a human subject in need thereof is administered an antisense compound or salt thereof as described herein at a dose equivalent to 24 to 50 mg of the antisense compound in a 5 mL dose volume. In certain examples, a human subject in need thereof is administered an antisense compound or salt thereof as described herein at a dose equivalent to 28 to 50 mg of the antisense compound in a 5 mL dose volume. In yet another example, a human subject in need thereof is administered an antisense compound or salt thereof as described herein at a dose equivalent to 50 mg of the antisense compound in a 5 mL dose volume. In some examples, the dose of the antisense compound or salt thereof as described herein is administered by bolus injection. In one example, the dose of the antisense compound or salt thereof as described herein is administered by intrathecal bolus injection. In certain cases, the antisense compound or salt thereof as described herein is administered in aCSF or PBS.

[0068] The dosing regimen may include administering to a human subject (e.g., intrathecally) at least two (e.g., 2, 3, 4, 5), about two, or two loading doses of an antisense compound described herein or a salt thereof (e.g., a sodium salt) equivalent to about 50 mg of an antisense compound used to treat or prevent SMA, followed by a maintenance dose of an antisense compound described herein or a salt thereof (e.g., a sodium salt) equivalent to about 28 mg of an antisense compound used to treat or prevent SMA. In some examples, each loading dose corresponds to 50 mg of an antisense compound described herein. In some examples, each maintenance dose corresponds to 28 mg of an antisense compound described herein. In some examples, the loading doses are administered 10 to 20 days apart (e.g., 10 to 18 days apart, 12 to 16 days apart, biweekly), and the maintenance doses are administered 3 to 6 months (e.g., 4 to 5 months or about 4 months) after the last loading dose, and every 3 to 6 months thereafter. In some cases, two loading doses are administered. In some cases, three loading doses are administered. In yet other cases, four loading doses are administered. In certain instances, the loading doses are administered 10 to 18 days apart, and the maintenance doses are administered 3 to 6 months after the last loading dose, and then 3 to 6 months. In some instances, the loading doses are administered 12 to 16 days apart, and the maintenance doses are administered 3 to 6 months after the last loading dose, and then 3 to 6 months. In other instances, the loading doses are administered 10 to 20 days apart (e.g., every other week), and the maintenance doses are administered 3 to 6 months after the last loading dose, and then 3 to 6 months. In certain instances, the loading doses are administered 14 days apart, and the maintenance doses are administered 3 to 6 months after the last loading dose, and then 3 to 6 months. In some instances, the maintenance dose(s) are administered 4 to 5 months after the last loading dose, and then 4 to 5 months. In some instances, the maintenance dose(s) are administered about 4 months after the last loading dose, and then about 4 months. In some instances, the maintenance dose(s) are administered four months after the final loading dose and then four months thereafter.In some instances, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 maintenance doses are administered.

[0069] In one example, a human subject in need thereof is administered an antisense compound shown below in a loading amount, or a salt thereof (e.g., sodium salt): [ka] at a dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound. In some examples, a human subject in need thereof is administered two loading doses of an antisense compound described herein or a salt thereof (e.g., sodium salt) 10-20 days, 10-18 days, 12-16 days, or about 14 days apart, each loading dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound. In some cases, a human subject in need thereof is administered two loading doses of an antisense compound described herein or a salt thereof (e.g., sodium salt) about 14 days apart, each loading dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound. In some cases, a human subject in need thereof is administered two loading doses of an antisense compound described herein or a salt thereof (e.g., sodium salt) every other week, each loading dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound. In one example, a human subject in need thereof is administered a first loading amount of an antisense compound described herein or a salt thereof (e.g., a sodium salt) on day 1 at a dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound, and a second loading amount of an antisense compound described herein or a salt thereof on day 15, the second loading amount corresponding to about 50 mg (e.g., 50 mg) of the antisense compound. In some examples, the human subject is further administered at least one maintenance dose of an antisense compound described herein or a salt thereof, the maintenance dose being administered 3 to 6 months, 4 to 5 months, or about 4 months after the administration of the second loading dose. In certain examples, the human subject is further administered one or more additional maintenance doses of an antisense compound described herein or a salt thereof 3 to 6 months, 4 to 5 months, or about 4 months after the administration of the first maintenance dose. In certain examples, the human subject is further administered one or more additional maintenance doses of an antisense compound described herein or a salt thereof 4 months after the administration of the first maintenance dose.

[0070] In another example, a human subject in need thereof is administered a salt of an antisense compound shown below in a loading amount: [ka] is administered at a dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound. In some examples, a human subject in need thereof is administered two loading doses of the salt of the antisense compound 10-20 days, 10-18 days, 12-16 days, or about 14 days apart, each loading dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound. In some cases, a human subject in need thereof is administered two loading doses of the salt of the antisense compound 14 days apart, each loading dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound. In some cases, a human subject in need thereof is administered two loading doses of the salt of the antisense compound every other week, each loading dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound. In one example, a human subject in need thereof is administered a first loading amount of a salt of an antisense compound on day 1 at a dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound, and a second loading amount of a salt of an antisense compound equivalent to about 50 mg (e.g., 50 mg) of the antisense compound on day 15. In some examples, the human subject is further administered at least one maintenance dose of a salt of an antisense compound, the maintenance dose being administered 3 to 6 months, 4 to 5 months, or about 4 months after the administration of the second loading dose. In certain examples, the human subject is further administered one or more additional maintenance doses of a salt of an antisense compound 3 to 6 months, 4 to 5 months, or about 4 months after the administration of the first maintenance dose.

[0071] In one embodiment, a human subject in need thereof (e.g., having or pre-symptomatic of SMA (any of types I through IV)) is administered a first loading amount of an antisense compound described herein or a salt thereof on day 1 at a dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound, a second loading amount of an antisense compound described herein or a salt thereof on about day 15 at a dose equivalent to about 50 mg (e.g., 50 mg) of the antisense compound, a first maintenance amount of an antisense compound described herein or a salt thereof on about day 135 at a dose equivalent to about 28 mg (e.g., 28 mg) of the antisense compound, and one or more further maintenance amounts of an antisense compound described herein or a salt thereof on about 120 days after the first maintenance amount. In one example, a human subject in need thereof (e.g., having or pre-symptomatic of SMA (any of types I through IV)) is administered a first loading amount of an antisense compound described herein or a salt thereof on day 1 at a dose equivalent to 50 mg of the antisense compound, a second loading amount of an antisense compound described herein or a salt thereof on day 15 at a dose equivalent to 50 mg of the antisense compound, a first maintenance amount of an antisense compound described herein or a salt thereof on any one of days 134-140 at a dose equivalent to 28 mg of the antisense compound, and one or more further maintenance amounts of an antisense compound described herein or a salt thereof on days 253 to 259 after the first maintenance amount. In some embodiments, the antisense compound has the structure: [ka]

[0072] In some embodiments, a salt of an antisense compound has the structure: [ka]

[0073] In another embodiment, the present disclosure features a dose-escalation administration regimen for a human subject in need thereof. This dose-escalation (bridging) regimen can effectively transition a subject receiving an approved 12 mg regimen of an antisense compound or salt thereof described herein to a regimen that allows increased CSF exposure. In one example, the dose-escalation regimen includes administering a loading dose of an antisense compound or salt thereof described herein equivalent to about 50 mg of the antisense compound to a subject who has previously been administered an antisense compound or salt thereof described herein at a dose equivalent to 12 mg of the antisense compound. In some cases, the loading dose is a single intrathecal bolus injection. In some cases, the 50 mg loading dose is administered 4 months after the last administration of the antisense compound or salt thereof described herein. In some cases, the 12 mg dose is followed by a 50 mg dose, followed by a 28 mg dose. In certain examples, one or more maintenance doses of an antisense compound or salt thereof described herein are administered to a subject at a dose equivalent to 28 mg of the antisense compound, beginning about 4 months after the loading dose. In some instances, the subject is given at least one, at least two, at least three, at least four, at least five, or one, two, three, four, or five doses equivalent to about 12 mg of the antisense compound described herein or a salt thereof prior to receiving a loading dose. In some embodiments, the antisense compound has the structure: [ka]

[0074] In some instances, the salt of the antisense compound has the following structure: [ka]

[0075] The doses described herein are generally provided in a volume of about 5.0 mL. In one example, the administration volume is 5.0 mL. In some cases, the diluent is PBS. In other examples, the diluent is aCSF. In certain cases, the loading dose is provided to a human subject at a concentration of 10.0 mg / mL. In some cases, the maintenance dose is provided to a human subject at a concentration of 5.6 mg / mL.

[0076] The dose is generally administered intrathecally, for example, by lumbar puncture. The dose is generally administered as an intrathecal injection (eg, an intrathecal bolus injection).

[0077] Biomarkers Biomarkers for SMA with diagnostic, prognostic, predictive, and pharmacodynamic value are useful for facilitating the timing of treatment initiation, determining response to intervention, determining therapeutic efficacy, and evaluating long-term outcomes. Such biomarkers include genetic, epigenetic, proteomic, electrophysiological, and imaging measurements. The present disclosure features the use of such biomarkers to facilitate detection of disease states when compared to healthy populations, provide information regarding likely health outcomes, disease progression or risk of recurrence, facilitate stratification of phenotypic severity, potentially identify responders and patient populations to treatment, confirm responses to therapy, and / or monitor therapeutic efficacy.

[0078] In one embodiment, the biomarker is a neurofilament protein. Neurofilaments are the major cytoskeletal elements in nerve cells and play a role in determining axon diameter as well as providing mechanical stability. Human neurofilaments are composed of three protein subunits, neurofilament light chain (NF-L), neurofilament medium / intermediate chain (NF-M), and neurofilament heavy chain (NF-H). These proteins share the same basic structure as other intermediate filament subunit proteins. Neurofilaments in the mammalian nervous system also contain the protein internexin, and neurofilaments in the peripheral nervous system may also contain the protein peripherin. Thus, as used herein, "neurofilament protein" refers to NF-H, NF-M, NF-L, internexin, or peripherin. The SMA biomarker may be one or more of NF-H, NF-M, NF-L, internexin, and peripherin. In a particular example, the SMA biomarker is phosphorylated NF-H (pNF-H). In certain instances, the SMA biomarker is phosphorylated NF-L.

[0079] In clinical trials of nusinersen with SMA types 1 and 2 and presymptomatic infants, plasma pNf-H was different between SMA individuals and healthy controls. Initiation of treatment with nusinersen was associated with a rapid decline in pNf-H followed by stabilization at levels close to those of healthy controls. Thus, assessment of neurofilament (e.g., pNf-H and / or NfL) levels may be a useful biomarker before or during treatment.

[0080] Neurofilament levels can be used to determine whether a subject (e.g., a human) administered an antisense compound or salt thereof described herein is responding to treatment. This can be assessed by obtaining a first biological sample from the subject before treatment and a second biological sample after the subject is treated, and measuring the levels of neurofilament (e.g., NF-H, NF-M, or NF-L) in such samples. In one example, the level of neurofilament is the level of pNF-H.

[0081] In certain examples, the first biological sample(s) may be taken from the subject at any time before the treatment, for example, one week, several days, one day, several hours, one hour, or less than one hour before administering the antisense compound or salt thereof described herein. Similarly, the second biological sample(s) may be taken from the subject at any time after the administration of the treatment, for example, less than one hour, one hour, several hours, one day, several days, one week, several weeks, one month, two months, three months, four months, five months, six months, seven months, or eight months after administering the antisense compound or salt thereof described herein. A decrease in neurofilament level after the start of the treatment indicates the effectiveness of the treatment. In such a case, the continuation of the treatment therapy is indicated (for example, administering an additional dose of the antisense compound at the same or lower amount as the last dose administered before measuring the second neurofilament level and at the same or longer dosing interval compared to the last dose administered before measuring the second neurofilament level). A lack of decrease in neurofilament levels after initiation of treatment indicates the need to alter the dose and / or dosing interval of the treatment (e.g., administering an additional dose of the antisense compound in an increased amount and / or at a shorter dosing interval compared to the last dose administered before measuring the second neurofilament level) or a lack of efficacy of the treatment. In the latter case, cessation of treatment may be indicated and the use of a different SMA therapy(es) considered.

[0082] Neurofilament levels can be assessed by measuring RNA or protein levels. In some examples, pNF-H levels are measured. The concentration of the neurofilament protein(s) of interest can be measured using any method known in the art, such as immunological assays. Non-limiting examples of such methods include enzyme immunoassays, radioimmunoassays, chemiluminescent immunoassays, electrochemiluminescent immunoassays, latex turbidimetric immunoassays, latex photometric immunoassays, immunochromatographic assays, and Western blotting. In certain embodiments, the concentration of the protein(s) of interest is measured by mass spectrometry.

[0083] In certain examples, the neurofilament level (e.g., pNF-H) in the first biological sample is greater than 300 pg / mL, greater than 400 pg / mL, greater than 500 pg / mL, greater than 600 pg / mL, greater than 700 pg / mL, greater than 800 pg / mL, greater than 900 pg / mL, greater than 1,000 pg / mL, greater than 1,500 pg / mL, greater than 2,000 pg / mL, greater than 3,000 pg / mL, greater than 4,000 pg / mL, or greater than 5,000 pg / mL. In certain embodiments, the neurofilament level measured in the next (e.g., second) biological sample is lower than the neurofilament level measured in the first biological sample. In some embodiments, neurofilament levels measured in a subsequent (e.g., second) biological sample exhibit a decrease of more than 30% (e.g., 31%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, or 95%) relative to neurofilament levels measured in the first biological sample.

[0084] In another embodiment, the SMA biomarker is any one of those described in WO2011 / 032109.

[0085] In another embodiment, the SMA biomarker is any one of those described in WO2019197954.

[0086] In another embodiment, the SMA biomarker is any one of those described in WO2019 / 122125.

[0087] In another embodiment, the SMA biomarker is any one of those described in WO2019 / 147960.

[0088] In yet another embodiment, the SMA biomarker is any one or more of COMP, DPP4, SPP1, CLEC3B, VTN, and AHSG.

[0089] In some embodiments, a combination or combinations of any of the foregoing biomarkers are assayed from a subject (eg, human) sample.

[0090] kit The present disclosure also features a kit for treating spinal muscular atrophy comprising an antisense compound or a salt thereof (e.g., a sodium salt) described herein. In some examples, the kit comprises at least one pharmaceutical composition comprising about 50 mg of an antisense compound or a salt thereof (e.g., a sodium salt) described herein and at least one pharmaceutical composition comprising about 28 mg of an antisense compound or a salt thereof (e.g., a sodium salt) described herein. In some examples, the kit comprises at least one pharmaceutical composition comprising 50 mg of an antisense compound or a salt thereof (e.g., a sodium salt) described herein and at least one pharmaceutical composition comprising 28 mg of an antisense compound or a salt thereof (e.g., a sodium salt) described herein. In some examples, the kit comprises a pharmaceutical composition comprising about 50 mg of an antisense compound or a salt thereof (e.g., a sodium salt) described herein. In some examples, the kit comprises a pharmaceutical composition comprising 50 mg of an antisense compound or a salt thereof (e.g., a sodium salt) described herein. In other examples, the kit comprises a pharmaceutical composition comprising about 28 mg of an antisense compound or a salt thereof (e.g., a sodium salt) described herein. In some examples, the kit comprises a pharmaceutical composition comprising 28 mg of an antisense compound or a salt thereof (e.g., sodium salt) as described herein. In some examples, the pharmaceutical composition is formulated in PBS or aCSF. In some examples, the kit comprises an antisense compound or a salt thereof (e.g., sodium salt) as described herein diluted in 5 mL of PBS or aCSF. In some examples, the kit comprises an antisense compound or a salt thereof (e.g., sodium salt) as described herein at a concentration of 10 mg / mL. In some examples, the kit comprises an antisense compound or a salt thereof (e.g., sodium salt) as described herein at a concentration of 5.6 mg / mL. In certain examples, the kit comprises a pharmaceutical composition formulated for intrathecal administration. In some examples, the kit comprises a pharmaceutical composition formulated for intrathecal bolus injection.

[0091] In one example, the kit may include an antisense compound described herein or a salt thereof (e.g., sodium salt) in lyophilized form, a volume (e.g., 5 to 10 mL) of diluent (e.g., PBS, aCSF), a syringe for injection, and / or instructions for use.

[0092] In one example, the kit may include at least one single-dose vial containing 5 mL of a pharmaceutical composition, the pharmaceutical composition containing 50 mg of an antisense compound described herein or a salt thereof, such that the concentration of the antisense compound in the single-dose vial is 10 mg / mL.

[0093] In one example, the kit may include at least one single-dose vial containing 5 mL of a pharmaceutical composition, the pharmaceutical composition containing 28 mg of an antisense compound described herein or a salt thereof, such that the concentration of the antisense compound in the single-dose vial is 5.6 mg / mL. In some examples, the kits described herein further include a syringe for injection and / or instructions for use.

[0094] In one example, the kit may include: (i) at least one single dose vial containing 5 mL of the pharmaceutical composition, the pharmaceutical composition containing 50 mg of an antisense compound described herein or a salt thereof, such that the concentration of the antisense compound in the single dose vial is 10 mg / mL; and (ii) at least one single dose vial containing 5 mL of the pharmaceutical composition, the pharmaceutical composition containing 28 mg of an antisense compound described herein or a salt thereof, such that the concentration of the antisense compound in the single dose vial is 5.6 mg / mL. In some examples, the kits described herein further include a syringe for injection and / or instructions for use.

[0095] 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 may develop equivalent means or reactants without the exercise of inventive ability and without departing from the scope of the invention. EXAMPLES

[0096] Example 1: Nusinersen Population PK / PD Analysis In nusinersen clinical trials in subjects with SMA, subjects who received treatment before or shortly after the onset and diagnosis of SMA symptoms generally had better outcomes than subjects who had a longer disease duration at the time of treatment initiation. In the infantile-onset SMA study, the majority of deaths among nusinersen-treated infants occurred within the first two months of the study, prior to completion of the loading dose regimen and achievement of steady-state nusinersen concentrations. Due to the rapid decline associated with type I SMA and the importance of early treatment, achieving higher nusinersen concentrations more rapidly may further improve the efficacy of nusinersen and prevent or further ameliorate disease progression in patients with SMA. The results of the infantile-onset SMA study, combined with the results of a sham-controlled study in subjects with later-onset SMA, support the idea that achieving therapeutic concentrations earlier in the disease process confers greater benefit, as subjects in both studies who had a shorter disease duration at the time of study enrollment had better outcomes than subjects who had a longer disease duration.

[0097] Example 2: Exposure-response relationships An exploratory exposure-response analysis was performed using data on 14 subjects with infantile-onset SMA who received nusinersen during the course of the nusinersen clinical development program. Results of this analysis showed a statistically significant positive correlation between nusinersen CSF exposure and motor function. Dose-related trends were observed in subjects' motor milestone performance, with earlier and greater improvements in subjects receiving a 12 mg loading dose compared to subjects receiving a 6 mg loading dose on days 1, 15, and 85. All subjects received a 12 mg maintenance dose every 4 months after day 85. Separation of the response curves for the two cohorts began around day 29 and became increasingly greater over the course of the study.

[0098] Greater improvements in motor function were also observed in subjects with infantile-onset SMA who received more frequent dosing (loading dose of 12 mg on days 1, 15, 29, and 64, and a maintenance dose of 12 mg every 4 months).

[0099] Data from subjects with infantile-onset SMA (subjects <6 months of age at diagnosis or symptom onset) were used in studies investigating the relationship between nusinersen exposure and clinical response. Analyses were performed to characterize the quantitative PK / PD relationship of nusinersen and to determine whether higher nusinersen exposure was associated with additive efficacy in the infantile-onset SMA population. Overall, the observed CSF C トラフ The PK / PD relationship between and motor function efficacy endpoints (change from baseline in CHOP-INTEND) appeared to follow an Emax relationship (Figure 1).

[0100] The model predicted a CSF C of 23 ng / mL. トラフ This estimate is consistent with the observed CSF C concentration achieved at steady state with the approved dosing regimen of 12 mg. トラフThese results suggest that increased CNS exposure (up to 20 ng / mL) may result in clinical benefits beyond those observed in clinical trials to date. Furthermore, this higher clinical exposure is consistent with existing clinical safety and tolerability data observed in other studies (maximum mean [SD] CSF C of 31.3 [24.8] ng / mL). トラフ ), as well as preclinical safety results in non-human primates (data not shown).

[0101] The above PK / PD relationships were demonstrated primarily in the infantile-onset SMA population due to limited data and the lower drug exposure of the less frequent dosing regimen used in the later-onset population (approximately 4.77 ng / mL on study day 273). However, subjects with later-onset SMA receiving the approved (more frequent) dosing regimen had similar CSF C concentrations as subjects with infantile-onset SMA in the same study (10.7 ng / mL on study day 302). トラフ The positive PK / PD relationship observed in subjects with infantile-onset SMA is expected to be similar across SMA types and patient age groups that share similar mechanisms of disease and drug action. This is consistent with the results of the PK / PD relationship observed in subjects with infantile-onset or late-onset SMA who received 12 mg nusinersen as four loading doses followed by a maintenance dose of 12 mg every four months. トラフ This is supported by a preliminary analysis of another study (data not shown) that showed a positive correlation between and total motor milestone scores.

[0102] Example 3: Loading Optimization PK simulations were performed with the goal of identifying a loading dosing regimen that would more rapidly result in higher drug exposure (approximately 2-fold increase) on target with fewer intrathecal (IT) doses. These simulations were performed after 2 years of treatment for both infantile-onset and late-onset SMA populations and were based on population PK models developed using data from patients with infantile-onset, late-onset, or pre-symptomatic SMA between the ages of ≦6 months and 18 years. No evaluation of maintenance dosing frequency was performed as previous modeling had shown that a 4-month dosing frequency best maintained CSF concentrations achieved at steady state.

[0103] Clinical CSF C トラフ Simulations were performed to evaluate additional dosing scenarios with higher doses and less frequent loading doses using the predicted CSF PK profile of 20 ng / mL as the target concentration and 24 mg nusinersen (four loading doses followed by a maintenance dose every four months) as the reference dosing regimen. As mentioned above, previous modeling indicated that a dosing frequency of every four months best maintained the CSF concentrations achieved at steady state, so no further evaluation of the maintenance dosing frequency was performed. Assuming linearity of PK, PK simulations were performed using population PK models developed from patients spanning the age range of ≦6 months to 18 years in both the infantile-onset and late-onset SMA populations after 2 years of treatment. Relative to the reference dosing regimen, both 28 mg administered as three loading doses (every other week) and 50 mg administered as two loading doses (every other week), followed by a maintenance dose of 28 mg every four months, respectively, resulted in a more rapid increase in the desired CSF concentration at the end of the loading dose period. トラフ Nusinersen 28 mg administered as three loading doses (every other week) had a predicted maximum CSF concentration (Cmax) similar to the reference dosing regimen, while the 50 mg dosing regimen surprisingly exceeded the predicted Cmax from the reference dosing regimen. Non-human primate safety studies evaluating the safety of both the 28 and 50 mg doses have been conducted to support the use of these doses.

[0104] Example 4: Exemplary Dosing Regimen • A first loading dose of nusinersen sodium administered at a dose equivalent to 50 mg of nusinersen; ● A second loading dose of nusinersen sodium administered at a dose equivalent to 50 mg of nusinersen 10 to 20 days; 10 to 18 days; 12 to 16 days; or 14 days after administration of the first loading dose; ● optionally, a third and / or fourth loading dose of nusinersen sodium at a dose equivalent to 50 mg of nusinersen administered 10 to 20 days; 10 to 18 days; 12 to 16 days; or 14 days after the administration of the second or third loading dose; ●One or more maintenance doses of nusinersen sodium administered at a dose equivalent to 28 mg of nusinersen starting approximately 3 to 6 months; 4 to 5 months; or 4 months after the last loading dose and continuing to be administered once every approximately 3 to 6 months, 4 to 5 months, or 4 months thereafter. Dosage volume: 5mL Diluent: aCSF or PBS Administration: Intrathecal bolus injection

[0105] Example 5: Exemplary Dosing Regimen Exemplary enhanced or high dosing regimens for the antisense compounds or salts thereof described herein for use in human subjects in need thereof (e.g., infantile-onset SMA, late-onset SMA, adult-onset SMA, pre-symptomatic subjects with SMA) are provided below. Loading dose: a dose of nusinersen sodium equivalent to 50 mg of nusinersen administered on days 1 and 15; Maintenance dose: A dose of nusinersen sodium equivalent to 28 mg of nusinersen administered every 4 months starting 4 months after the second loading dose and thereafter. Dosage volume: 5mL Vehicle: aCSF or PBS Administration: Intrathecal bolus injection

[0106] Example 6: Dose Escalation (Bridging) Dosing Regimen Potential dose escalation dosing regimens were also evaluated using PK modeling and simulation to identify an effective transition process for subjects already receiving the approved 12 mg dosing regimen of nusinersen. The goal of these evaluations is to increase CSF exposure in these subjects to the target concentration (approximately 20 ng / mL) provided by the intensive or high dosing regimen.

[0107] PK simulations demonstrated that the drug could be administered at a higher target C without the need for an additional loading dose period (maintaining a maintenance dosing interval of once every 4 months) and without increasing risk to patients. トラフ Various dosing regimens were evaluated with the goal of identifying a dose escalation scheme that would more rapidly achieve PK. All simulations were performed using an updated population PK model. Dose escalation regimens were simulated after 1 year of treatment with the approved dosing regimen of 12 mg nusinersen, which included four 12 mg loading doses (days 1, 15, 29, and 64) followed by two maintenance doses administered every 4 months. All dose escalation regimens followed a 4-month dosing frequency to match the 4-month maintenance dosing frequency of the approved 12 mg dosing regimen.

[0108] An exemplary dose escalation dosing regimen (capable of increasing CSF exposure in a subject to a target concentration of approximately 20 ng / mL) for the antisense compounds described herein (e.g., nusinersen or a sodium salt thereof) for use in a human subject previously administered 12 mg of nusinersen in need thereof (e.g., subjects with infantile-onset SMA, late-onset SMA, adult-onset SMA, pre-symptomatic SMA) is provided below. Loading dose: a dose of nusinersen sodium equivalent to 50 mg of nusinersen administered on day 1; Maintenance dose: A dose of nusinersen sodium equivalent to 28 mg of nusinersen administered starting 4 months after the loading dose and then every 4 months. Dosage volume: 5mL Vehicle: aCSF or PBS Administration: Intrathecal bolus injection

[0109] Example 7: Additional Dose Escalation Regimens Regimen 1 Four months after the last dose of nusinersen sodium equivalent to 12 mg of nusinersen, the patient receives a loading dose of nusinersen sodium equivalent to 50 mg of nusinersen; ●One or more maintenance doses of nusinersen sodium equivalent to 28 mg of nusinersen are then administered approximately once every 4 months. Dosage volume: 5mL Vehicle: aCSF or PBS Administration: Intrathecal bolus injection

[0110] Regimen 2 • Four months after the last dose of nusinersen sodium equivalent to 12 mg of nusinersen, patients receive a first loading dose equivalent to 28 mg of nusinersen; A second loading dose of nusinersen sodium equivalent to 50 mg of nusinersen is administered approximately 4 months after the first loading dose; and ●One or more maintenance doses of nusinersen sodium equivalent to 28 mg of nusinersen are then administered approximately every 4 months. Dosage volume: 5mL Vehicle: aCSF or PBS Administration: Intrathecal bolus injection

[0111] Other embodiments Although aspects of the invention have been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for treating spinal muscular atrophy (SMA) in a human subject in need thereof, the method comprising administering to the human subject at least two loading doses of a compound having the following structure: [ka] or a salt thereof, The method comprises administering to the human subject by intrathecal injection (i) a first loading amount of the antisense compound or a salt thereof equivalent to about 50 mg of the antisense compound; (ii) a second loading dose of the antisense compound or salt thereof equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days after administration of the first loading dose; The method comprises administering (Item 2) 1. A method for increasing a level of motor neuron survival 2 (SMN2) messenger ribonucleic acid (mRNA) comprising exon 7 in a human subject having a mutation on chromosome 5q associated with SMN protein deficiency, the method comprising administering to the human subject at least two loading doses of the following structure: [ka] or a salt thereof, The method comprises administering to the human subject by intrathecal injection (i) a first loading amount of the antisense compound or a salt thereof equivalent to about 50 mg of the antisense compound; (ii) a second loading dose of the antisense compound or salt thereof equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days after administration of the first loading dose; The method comprises administering (Item 3) 3. The method of claim 1 or 2, further comprising administering to the human subject at least one maintenance dose by intrathecal injection, wherein the at least one maintenance dose corresponds to about 28 mg of the antisense compound. (Item 4) 3. The method of claim 1 or 2, further comprising administering to the human subject by intrathecal injection a maintenance dose of the antisense compound or salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 4 months after administration of the second loading dose and continuing about every 4 months thereafter. (Item 5) 5. The method of claim 4, wherein the second loading dose is administered about 14 days after administration of the first loading dose. (Item 6) The method comprises administering to the human subject by intrathecal injection (iii) a third loading amount of the antisense compound or salt thereof equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days after administration of the second loading amount; (iv) administering a maintenance dose of the antisense compound or salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 4 months after administration of the third loading dose and continuing about every 4 months thereafter. 3. The method of claim 1 or 2, further comprising administering (Item 7) 7. The method of claim 6, wherein the second loading dose is administered about 14 days after administration of the first loading dose and the third loading dose is administered about 14 days after administration of the second loading dose. (Item 8) The method comprises administering to the human subject by intrathecal injection (iii) a third loading amount of the antisense compound or salt thereof equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days after administration of the second loading amount; (iv) a fourth loading amount of the antisense compound or salt thereof equivalent to about 50 mg of the antisense compound, administered about 10 to 20 days after administration of the third loading amount; (v) administering a maintenance dose of the antisense compound or salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 4 months after administration of the fourth loading dose and continuing about every 4 months thereafter. 3. The method of claim 1 or 2, further comprising administering (Item 9) 9. The method of claim 8, wherein the second loading dose is administered about 14 days after administration of the first loading dose, the third loading dose is administered about 14 days after administration of the second loading dose, and the fourth loading dose is administered about 14 days after administration of the third loading dose. (Item 10) 1. A method for treating spinal muscular atrophy (SMA) in a human subject in need thereof, the method comprising administering to the human subject a compound having the following structure: [ka] or a salt thereof, The human subject is pre-administered with one or more doses of the antisense compound or salt thereof, the one or more pre-administered doses corresponding to about 12 mg of the antisense compound, and the method includes administering to the human subject by intrathecal injection: (i) a loading amount of the antisense compound or salt thereof equivalent to about 50 mg of the antisense compound administered about 4 months after administration of the last previously administered dose equivalent to about 12 mg of the antisense compound; (ii) administering a maintenance dose of the antisense compound or salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 4 months after administration of the loading dose and continuing about every 4 months thereafter; The method comprises administering (Item 11) 1. A method for increasing the level of motor neuron survival 2 (SMN2) messenger ribonucleic acid (mRNA) comprising exon 7 in a human subject having a mutation on chromosome 5q associated with SMN protein deficiency, the method comprising administering to the human subject a gene having the following structure: [ka] or a salt thereof, The human subject is pre-administered with one or more doses of the antisense compound or salt thereof, the one or more pre-administered doses corresponding to about 12 mg of the antisense compound, and the method includes administering to the human subject by intrathecal injection: (i) a loading amount of the antisense compound or salt thereof equivalent to about 50 mg of the antisense compound administered about 4 months after administration of the last previously administered dose equivalent to about 12 mg of the antisense compound; (ii) administering a maintenance dose of the antisense compound or salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 4 months after administration of the loading dose and continuing about every 4 months thereafter; The method comprises administering (Item 12) 1. A method for treating spinal muscular atrophy (SMA) in a human subject in need thereof, the method comprising administering to the human subject a compound having the following structure: [ka] or a salt thereof, The human subject is pre-administered with one or more doses of the antisense compound or salt thereof, the one or more pre-administered doses corresponding to about 12 mg of the antisense compound, and the method includes administering to the human subject by intrathecal injection: (i) a first loading amount of the antisense compound or a salt thereof equivalent to about 28 mg of the antisense compound, administered about 4 months after administration of the last previously administered dose equivalent to about 12 mg of the antisense compound; (ii) a second loading amount of the antisense compound or salt thereof equivalent to about 50 mg of the antisense compound, administered about 4 months after administration of the first loading amount; (iii) a maintenance dose of the antisense compound or salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 4 months after administration of the second loading dose and continuing about every 4 months thereafter; The method comprises administering (Item 13) 1. A method for increasing the level of motor neuron survival 2 (SMN2) messenger ribonucleic acid (mRNA) comprising exon 7 in a human subject having a mutation on chromosome 5q associated with SMN protein deficiency, the method comprising administering to the human subject a gene having the following structure: [ka] or a salt thereof, The human subject is pre-administered with one or more doses of the antisense compound or salt thereof, the one or more pre-administered doses corresponding to about 12 mg of the antisense compound, and the method includes administering to the human subject by intrathecal injection: (i) a first loading amount of the antisense compound or a salt thereof equivalent to about 28 mg of the antisense compound, administered about 4 months after administration of the last previously administered dose equivalent to about 12 mg of the antisense compound; (ii) a second loading amount of the antisense compound or salt thereof equivalent to about 50 mg of the antisense compound, administered about 4 months after administration of the first loading amount; (iii) a maintenance dose of the antisense compound or salt thereof, each equivalent to about 28 mg of the antisense compound, beginning about 4 months after administration of the second loading dose and continuing about every 4 months thereafter; The method comprises administering (Item 14) 14. The method of any one of items 10 to 13, wherein the human subject was previously administered at least five doses of the antisense compound or salt thereof, each of the at least five previously administered doses corresponding to about 12 mg of the antisense compound. (Item 15) 15. The method of any one of items 1 to 14, wherein the human subject has: (i) SMA type I; (ii) SMA type II; (iii) SMA type III; or (iv) SMA type IV. (Item 16) 15. The method of any one of items 1 to 14, wherein the human subject is presymptomatic with SMA. (Item 17) 17. The method of any one of items 1 to 16, wherein the human subject is administered the antisense compound or salt thereof using a spinal anesthesia needle. (Item 18) 18. The method of any one of items 1 to 17, wherein the antisense compound or salt thereof is administered in an injection volume of 5.0 mL. (Item 19) 20. The method of claim 18, wherein the antisense compound or the salt thereof is dissolved in phosphate buffered saline (PBS). (Item 20) 19. The method of claim 18, wherein the antisense compound or the salt thereof is dissolved in artificial cerebrospinal fluid (aCSF). (Item 21) 21. The method of any one of items 1 to 20, wherein the salt of the antisense compound is a sodium salt. (Item 22) 21. The method of any one of items 1 to 20, wherein the salt of the antisense compound has the following structure: [ka] (Item 23) 23. The method of any one of items 1 to 22, wherein the method comprises detecting neurofilament levels in the human subject before and / or after initiation of treatment. (Item 24) measuring a first neurofilament level in a first biological sample obtained from the human subject prior to initiation of treatment; administering to the human subject the antisense compound or salt thereof according to any one of the preceding paragraphs; measuring at least one of the following neurofilament levels in a second biological sample obtained from the human subject after administration of all loading doses and at least one maintenance dose of the antisense compound or salt thereof; 24. The method according to item 23, comprising: (Item 25) measuring a first neurofilament level in a first biological sample obtained from the human subject prior to initiation of treatment; administering to the human subject a first loading amount of the antisense compound or salt thereof according to any one of the preceding paragraphs; measuring at least one of the following neurofilament levels in a biological sample obtained from the human subject after administering the first loading amount of the antisense compound or salt thereof: 24. The method according to item 23, comprising: (Item 26) measuring a first neurofilament level in a first biological sample obtained from the human subject prior to initiation of treatment; administering to the human subject a first loading amount and a second loading amount of the antisense compound or salt thereof according to any one of the preceding paragraphs; measuring at least one of the following neurofilament levels in a biological sample obtained from the human subject after administering the first loading amount and the second loading amount of the antisense compound or salt thereof; 24. The method according to item 23, comprising: (Item 27) 27. The method of any one of items 24 to 26, wherein the subsequent neurofilament level is reduced by at least 50% compared to the first neurofilament level. (Item 28) 27. The method of any one of items 25 to 26, wherein the subsequent neurofilament level is equal to or higher than the first neurofilament level, and the method further comprises administering to the human subject an additional dose of the antisense compound or salt thereof. (Item 29) 29. The method of any one of items 23 to 28, wherein the neurofilament is a neurofilament heavy chain (NF-H), a medium / intermediate chain (NF-M), or a light chain (NF-L). (Item 30) 30. The method according to items 23 to 29, wherein the first and second biological samples are blood, serum, plasma, or cerebrospinal fluid.

Claims

1. A composition for use in a method for increasing levels of motor neuron survival 2 (SMN2) messenger ribonucleic acid (mRNA) containing exon 7 in a human subject having a loss of a functional copy of motor neuron survival gene 1 (SMN1), said composition having the following structure: 【Chemistry 1A】 or a salt thereof, the method comprising administering to the human subject by intrathecal injection at least two loading doses of the antisense compound or salt thereof having the formula: (i) a first loading amount of the antisense compound or a salt thereof equivalent to 50 mg of the antisense compound; (ii) a second loading dose of the antisense compound or salt thereof equivalent to 50 mg of the antisense compound administered 10 to 20 days after administration of the first loading dose.

2. The composition of claim 1, wherein the method further comprises administering to the human subject at least one maintenance dose by intrathecal injection, the at least one maintenance dose equivalent to 28 mg of the antisense compound.

3. The composition of claim 1, wherein the method further comprises administering to the human subject by intrathecal injection a maintenance dose of the antisense compound or salt thereof, each equivalent to 28 mg of the antisense compound, beginning about 4 months after administration of the second loading dose and continuing about every 4 months thereafter.

4. The composition of claim 1, wherein the method further comprises administering to the human subject by intrathecal injection a maintenance dose of the antisense compound or salt thereof, each equivalent to 28 mg of the antisense compound, beginning four months after administration of the second loading dose and continuing every four months thereafter.

5. 5. The composition of claim 1, wherein the second loading dose is administered 14 days after administration of the first loading dose.

6. A composition for use in a method for increasing levels of motor neuron survival 2 (SMN2) messenger ribonucleic acid (mRNA) containing exon 7 in a human subject having a loss of a functional copy of motor neuron survival gene 1 (SMN1), said composition having the following structure: 【Chemistry 1B】 or a salt thereof, wherein the human subject has been pre-administered one or more doses of the antisense compound or salt thereof, wherein the one or more pre-administered doses correspond to 12 mg of the antisense compound; The method comprises administering to the human subject by intrathecal injection (i) a loading amount of the antisense compound or salt thereof equivalent to 50 mg of the antisense compound administered 3 to 6 months after administration of a last previously administered dose equivalent to 12 mg of the antisense compound; (ii) administering a maintenance dose of the antisense compound or salt thereof, each equivalent to 28 mg of the antisense compound, beginning about 4 months after administration of the loading dose and continuing about every 4 months thereafter; A composition comprising:

7. The composition of claim 6, wherein the human subject has been previously administered at least one, at least two, at least three, at least four, at least five, or one, two, three, four, or five doses of the antisense compound or salt thereof, each of the previously administered doses corresponding to 12 mg of the antisense compound.

8. 8. The composition of claim 6 or 7, wherein the loading dose of the antisense compound or salt thereof is administered 4 months after administration of the last pre-administered dose equivalent to 12 mg of the antisense compound.

9. 9. The composition of any one of claims 6 to 8, wherein the maintenance dose of the antisense compound or salt thereof begins four months after administration of the loading dose and continues to be administered once every four months thereafter.

10. The composition of claim 6, wherein the human subject was previously administered at least five doses of the antisense compound or salt thereof, each of the at least five previously administered doses corresponding to 12 mg of the antisense compound.

11. The composition of claim 6, wherein the human subject was previously administered at least four doses of the antisense compound or salt thereof, each of the at least four previously administered doses corresponding to 12 mg of the antisense compound.

12. The composition of claim 6, wherein the human subject was pre-administered four doses of the antisense compound or a salt thereof, each of the pre-administered doses corresponding to 12 mg of the antisense compound.

13. The composition of claim 6, wherein the human subject was pre-administered with one dose of the antisense compound or salt thereof, the pre-administered dose corresponding to 12 mg of the antisense compound.

14. The human subject comprises: (i) infantile-onset SMA (Type I SMA); (ii) late-onset SMA (Type II or Type III SMA); or (iii) Adult-onset SMA (Type IV SMA) The composition of any one of claims 1 to 13, having the formula:

15. 14. The composition of any one of claims 1 to 13, wherein the human subject is presymptomatic with SMA.

16. 16. The composition of any one of claims 1 to 15, wherein the human subject is administered the antisense compound or salt thereof using a spinal anesthesia needle.

17. 17. The composition of any one of claims 1 to 16, wherein the antisense compound or salt thereof is administered in an injection volume of 5.0 mL.

18. 18. The composition of any one of claims 1 to 17, wherein the antisense compound or the salt thereof is dissolved in phosphate buffered saline (PBS) or artificial cerebrospinal fluid (aCSF).

19. 19. The composition of any one of claims 1 to 18, wherein the salt of the antisense compound is a sodium salt.

20. The salts of the antisense compounds have the following structure: 【Chemistry 2】 19. The composition of any one of claims 1 to 18, having the formula:

21. The composition of any one of claims 1 to 5, wherein the first load and the second load of the antisense compound are each formulated in a single dose vial.

22. The composition of any one of claims 6 to 20, wherein the loading dose and the maintenance dose of the antisense compound are each formulated in a single-dose vial.

23. The composition described in any one of claims 1 to 22, wherein approximately 5 mL of cerebrospinal fluid is removed from the human subject prior to administration of each dose to the subject.

24. The composition of any one of claims 1 to 23, wherein the antisense compound or its salt is administered to the human subject by bolus injection.

25. The composition described in any one of claims 1 to 5, wherein the first loading amount and the second loading amount are provided to the human subject at a concentration of the antisense compound of about 10 mg / mL.

26. A composition described in any one of claims 2 to 5, wherein the at least one maintenance dose is provided to the human subject at a concentration of the antisense compound of about 5.6 mg / mL.

27. ​​A composition described in any one of claims 6 to 13, wherein the loading dose is provided to the human subject at a concentration of about 10 mg / mL and the maintenance dose is provided to the human subject at a concentration of about 5.6 mg / mL of the antisense compound.

28. The composition described in any one of claims 6 to 13, wherein the loading amount of the antisense compound or its salt is administered approximately 4 to 5 months after administration of the last previously administered dose equivalent to approximately 12 mg of the antisense compound.

29. The composition described in claim 28, wherein the loading amount of the antisense compound or its salt is administered approximately 4 months after administration of the last previously administered dose equivalent to approximately 12 mg of the antisense compound.

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