Compounds and methods for modulating smn2

Abstract

Compounds, methods, and pharmaceutical compositions for regulating SMN2 RNA and / or protein in cells or individuals are provided. These compounds, methods, and pharmaceutical compositions can be used to improve at least one symptom of neurodegenerative diseases. Such symptoms include: decreased muscle strength; inability to sit, stand, and / or walk or reduced ability; decreased neuromuscular activity; decreased electrical activity in one or more muscles; decreased respiration; inability to eat, drink, and / or breathe without assistance or reduced ability; weight loss or reduced weight gain; and / or decreased survival rate.

Description

Technical Field

[0001] Compounds, methods, and pharmaceutical compositions for modulating SMN2 RNA in a cell or an individual are provided. Such compounds, methods, and pharmaceutical compositions can be used to ameliorate at least one symptom of a neurodegenerative disorder. Such symptoms include: decreased muscle strength; inability or reduced ability to sit upright, stand, and / or walk; decreased neuromuscular activity; decreased electrical activity of one or more muscles; decreased respiration; inability or reduced ability to eat, drink, and / or breathe without assistance; weight loss or decreased weight gain; and / or decreased survival rate. Background Art

[0002] Spinal muscular atrophy (SMA) is a hereditary neurodegenerative disorder characterized by the loss of spinal motor neurons. SMA is an autosomal recessive disease with an early onset and is a leading genetic cause of infant death. The severity of SMA varies among patients and is thus classified into four types. Type I SMA is the most severe form, presenting at birth or within 6 months, and usually leading to death within 2 years. Children with type I SMA cannot sit or walk. Type II SMA is an intermediate form, and patients can sit but cannot stand or walk. Patients with type III SMA (the chronic form of the disease) usually develop SMA after 18 months (Lefebvre et al., Hum. Mol. Genet., 1998, 7, 1531-1536). Type IV SMA is a milder form and usually presents after 18 years of age, sometimes after 10 years of age; patients with type IV SMA experience limited mild motor impairment, can walk in adulthood, and usually have no respiratory or nutritional problems (Farrar et al., Ann. Neurol., 2017, 81, 355-368; D’Amico et al., Orphanet J. of Rare Diseases, 2011, 6: 71).

[0003] The molecular basis of SMA is two copies of the survival motor neuron gene 1 (SMN1) (also known as SMN telomeric) and encodes a protein that is part of a multi-protein complex thought to be involved in snRNP biogenesis and recycling. An almost identical gene, SMN2 (also known as SMN centromeric), is present in a duplicated region on chromosome 5q13 and modulates disease severity. Although SMN1 and SMN2 have the potential to encode the same protein, expression of the normal SMN1 gene results only in the expression of full-length survival motor neuron (SMN) protein, while expression of the SMN2 gene results in two different protein forms, full-length SMN2 protein and truncated SMN2 protein (SMNΔ7 protein). SMN2 contains a translational silent mutation at position +6 in exon 7, resulting in the inefficient inclusion of exon 7 in the SMN2 transcript. Thus, the predominant form of SMN2 is a truncated form lacking exon 7, which is unstable and non-functional (Cartegni and Krainer, Nat. Genet., 2002, 30, 377-384). Expression of the SMN2 gene results in approximately 10-20% full-length SMN protein and 80-90% unstable / non-functional SMNΔ7 protein. SMN protein plays a recognized role in the assembly of the spliceosome and can also mediate mRNA transport in the axons and nerve terminals of neurons.

[0004] The aim of this article is to provide compounds, methods and pharmaceutical compositions for the treatment of SMA. Summary of the Invention

[0005] This article provides compounds, methods and pharmaceutical compositions for modulating the splicing of SMN2 RNA in a cell or an individual. In certain embodiments, the compounds useful for modulating the splicing of SMN2 RNA are oligomeric compounds. In certain embodiments, the oligomeric compounds increase the amount of SMN2 RNA including exon 7. In certain embodiments, the oligomeric compounds increase the expression of full-length SMN2 protein. In certain embodiments, the oligomeric compounds comprise modified oligonucleotides. In certain embodiments, the individual suffers from a neurodegenerative disease. In certain embodiments, the individual suffers from spinal muscular atrophy (SMA).

[0006] Also provided are methods useful for ameliorating at least one symptom of a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is SMA. In certain embodiments, the symptoms include: reduced muscle strength; inability or reduced ability to sit upright, stand, and / or walk; reduced neuromuscular activity; reduced electrical activity of one or more muscles; reduced respiration; inability or reduced ability to eat, drink, and / or breathe without assistance; weight loss or reduced weight gain; and / or reduced survival rate. In certain embodiments, the present disclosure provides modified oligonucleotides for treating SMA. Embodiments

[0007] [Sequence Listing] [] This application is being filed together with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0367USLSEQ_ST25.txt, created on February 28, 2020, and having a size of 44 KB. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0008] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. As used herein, the singular forms include the plural unless otherwise specifically stated. As used herein, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "including" and other forms (such as "includes" and "included") is not restrictive. Similarly, unless otherwise specifically stated, terms such as "element" or "component" encompass elements and components that include a single unit and elements and components that include more than one sub-unit.

[0009] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and papers, as well as GenBank and NCBI reference sequence records, are hereby expressly incorporated by reference in their entirety for the portions of the documents and the full text relevant to what is discussed herein. [Definitions] []

[0010] Unless otherwise provided with specific definitions, the nomenclature, as well as the processes and techniques used in combination with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, are well-known and commonly used in the art. Where permitted, all patents, applications, published applications, and other publications and other materials cited throughout this disclosure are incorporated herein by reference in their entirety.

[0011] Unless otherwise indicated, the following terms have the following meanings: As used herein, "2'-deoxyribonucleoside" means a nucleoside containing a 2'-H (H) deoxyribosyl sugar moiety. In certain embodiments, the 2'-deoxyribonucleoside is 2'-β-D-deoxyribonucleoside and contains a 2'-β-D-deoxyribosyl sugar moiety having the β-D configuration as found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2'-deoxyribonucleoside may contain a modified nucleobase or may contain an RNA nucleobase (uracil).

[0012] As used herein, "2'-MOE" means that the 2'-OH group of the ribosyl sugar moiety is replaced by a 2'-OCH2CH2OCH3 group. The "2'-MOE sugar moiety" is a sugar moiety in which the 2'-OH group of the ribosyl sugar moiety is replaced by a 2'-OCH2CH2OCH3 group. Unless otherwise indicated, the 2'-MOE sugar moiety has a β-D configuration. "MOE" means O-methoxyethyl.

[0013] As used herein, "2'-MOE nucleoside" means a nucleoside containing a 2'-MOE sugar moiety.

[0014] As used herein, "2'-NMA" means that the 2'-OH group of the ribosyl sugar moiety is replaced by an -O-CH2-C(=O)-NH-CH3 group. The "2'-NMA sugar moiety" is a sugar moiety in which the 2'-OH group of the ribosyl sugar moiety is replaced by a 2'-O-CH2-C(=O)-NH-CH3 group. Unless otherwise indicated, the 2'-NMA sugar moiety has a β-D configuration. "NMA" means O-N-methylacetamide.

[0015] As used herein, "2'-NMA nucleoside" means a nucleoside containing a 2'-NMA sugar moiety.

[0016] As used herein, "2'-OMe" means that the 2'-OH group of the ribosyl sugar moiety is replaced by a 2'-OCH3 group. "2'-OMe sugar moiety" is a sugar moiety in which the 2'-OH group of the ribosyl sugar moiety is replaced by a 2'-OCH3 group. Unless otherwise indicated, the 2'-OMe sugar moiety is in the β-D configuration. "OMe" means O-methyl.

[0017] As used herein, "2'-OMe nucleoside" means a nucleoside containing a 2'-OMe sugar moiety.

[0018] As used herein, "2'-substituted nucleoside" means a nucleoside containing a 2'-substituted sugar moiety. As used herein, "2'-substituted" with respect to a sugar moiety means a sugar moiety containing at least one 2'-substituent other than H or OH.

[0019] As used herein, "5-methylcytosine" means cytosine modified with a methyl group attached to the 5-position. 5-methylcytosine is a modified nucleobase.

[0020] As used herein, "administer" means to provide an agent to an individual.

[0021] As used herein, "improvement" with respect to a treatment means a modification of at least one symptom relative to the same symptom in the absence of that treatment. In certain embodiments, the improvement is a decrease in the severity or frequency of a symptom or a delay in the onset or a slowdown in the progression of the severity or frequency of a symptom. In certain embodiments, the symptoms are: decreased muscle strength; decreased ability to sit upright, stand, and / or walk or inability to do so; decreased neuromuscular activity; decreased electrical activity of one or more muscles; decreased respiration; inability to eat, drink, and / or breathe without assistance or decreased ability to do so; weight loss or decreased weight gain; and / or decreased survival rate.

[0022] As used herein, "antisense activity" means any detectable and / or measurable change attributable to the hybridization of an antisense compound with its target nucleic acid.

[0023] As used herein, "antisense compound" means an oligomeric compound or oligomeric duplex capable of achieving at least one antisense activity.

[0024] As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside containing a bicyclic sugar moiety.

[0025] As used herein, "bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety containing two rings, wherein the second ring is formed by a bridge connecting two atoms in the first ring, thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the furanosyl moiety is a ribosyl moiety. In certain embodiments, the bicyclic sugar moiety does not contain a furanosyl moiety.

[0026] As used herein, "cerebrospinal fluid" or "CSF" means the fluid that fills the space around the brain and spinal cord. "Artificial cerebrospinal fluid" or "aCSF" means a fluid that is prepared or manufactured to have certain properties of cerebrospinal fluid.

[0027] As used herein, "cEt" means that the 4'-to-2' bridge replaces the 2'-OH group of a ribosyl sugar moiety, wherein the bridge has the formula 4'-CH(CH3)-O-2', and wherein the methyl group of the bridge is in the S configuration. "cEt sugar moiety" is a bicyclic sugar moiety in which the 4'-to-2' bridge replaces the 2'-OH group of a ribosyl sugar moiety, wherein the bridge has the formula 4'-CH(CH3)-O-2', and wherein the methyl group of the bridge is in the S configuration. "cEt" means constrained ethyl.

[0028] As used herein, "cEt nucleoside" means a nucleoside containing a cEt sugar moiety.

[0029] As used herein, "chiral enriched population" means a plurality of molecules having the same molecular formula, wherein the number or percentage of molecules in the population containing a specific stereochemical configuration at a specific chiral center is greater than the number or percentage of molecules that would be expected to contain the same specific stereochemical configuration at the same specific chiral center if the specific chiral center were racemic. A chiral enriched population of molecules having multiple chiral centers within each molecule may contain one or more racemic chiral centers. In certain embodiments, the molecule is a modified oligonucleotide. In certain embodiments, the molecule is a compound containing a modified oligonucleotide.

[0030] As used herein, "complementary" with respect to an oligonucleotide means that when the oligonucleotide is aligned with the nucleobase sequence of another nucleic acid in an opposite orientation, at least 70% of the nucleobases of the oligonucleotide or one or more of its portions are capable of hydrogen bonding with the nucleobases of the other nucleic acid or one or more of its portions. Complementary nucleobases mean nucleobases that are capable of forming hydrogen bonds with each other. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or target nucleic acids need not have nucleobase complementarity at every nucleotide. Instead, some mismatches are tolerated. As used herein, "fully complementary" or "100% complementary" with respect to an oligonucleotide or a portion thereof means that the oligonucleotide or the portion thereof is complementary to another oligonucleotide or target nucleic acid at each nucleobase of the shorter of the two oligonucleotides, or if the oligonucleotides are of the same length, at each nucleotide.

[0031] As used herein, "consecutive" in the context of an oligonucleotide refers to nucleotides, nucleobases, sugar moieties, or internucleoside linkages being adjacent to each other. For example, "consecutive nucleobases" means nucleobases that are adjacent to each other in a sequence.

[0032] As used herein, "hybridization" means the pairing or annealing of complementary oligonucleotides and / or nucleic acids. Although not limited to a specific mechanism, the most common hybridization mechanism involves hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding.

[0033] As used herein, "internucleoside linkage" means a covalent linkage between consecutive nucleotides in an oligonucleotide. As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a phosphodiester internucleoside linkage. A "phosphorothioate internucleoside linkage" is a modified internucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester internucleoside linkage is replaced by a sulfur atom.

[0034] As used herein, "mismatch" or "non-complementary" means that when a first oligonucleotide and a second oligonucleotide are aligned, the nucleobase of the first oligonucleotide is not complementary to the corresponding nucleobase of the second oligonucleotide or target nucleic acid.

[0035] As used herein, "motif" means a pattern of unmodified and / or modified sugar moieties, nucleobases, and / or internucleoside linkages in an oligonucleotide.

[0036] As used herein, "non-bicyclic modified sugar moiety" means a modified sugar moiety that contains a modification (such as a substituent) that does not form a bridge between two atoms of the sugar to form a second ring.

[0037] As used herein, "nucleobase" means an unmodified nucleobase or a modified nucleobase. As used herein, "unmodified nucleobase" is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, "modified nucleobase" is a group of atoms other than unmodified A, T, C, U, or G that is capable of pairing with at least one unmodified nucleobase. "5-Methylcytosine" is an unmodified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, "nucleobase sequence" means the order of consecutive nucleobases in a target nucleic acid or oligonucleotide, which is independent of any sugar or internucleoside linkage modification.

[0038] As used herein, "nucleoside" means a compound that contains a nucleobase and a sugar moiety. The nucleobase and the sugar moiety are each independently unmodified or modified. As used herein, "modified nucleoside" means a nucleoside that contains a modified nucleobase and / or a modified sugar moiety. "Linked nucleosides" are nucleosides that are linked in a continuous sequence (i.e., there are no additional nucleosides between the linked nucleosides).

[0039] As used herein, "oligomeric compound" means an oligonucleotide and optionally one or more other features, such as a conjugate group or a terminal group. The oligomeric compound can be paired with a second oligomeric compound that is complementary to the first oligomeric compound or can be unpaired. A "single-stranded oligomeric compound" is an unpaired oligomeric compound. The term "oligomeric double helix" means a double helix formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of the oligomeric double helix can be referred to as a "double helix oligomeric compound".

[0040] As used herein, "oligonucleotide" means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage can be modified or unmodified. Unless otherwise indicated, an oligonucleotide consists of 8-50 linked nucleosides. As used herein, "modified oligonucleotide" means an oligonucleotide in which at least one nucleoside or internucleoside linkage is modified. As used herein, "unmodified oligonucleotide" means an oligonucleotide that does not contain any nucleoside modification or internucleoside modification.

[0041] As used herein, "pharmaceutical composition" means a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition can comprise an oligomeric compound and a sterile aqueous solution.

[0042] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for administration to an individual. Some such carriers enable a pharmaceutical composition to be formulated into, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, serums, suspensions, and lozenges for oral ingestion by an individual. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, sterile buffer solution, or sterile artificial cerebrospinal fluid.

[0043] As used herein, "pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of a compound. The pharmaceutically acceptable salt retains the desired biological activity of the parent compound and does not impart undesired toxicological effects.

[0044] Unless otherwise specified, as used herein, "RNA" means an RNA transcript and includes precursor mRNA and mature mRNA.

[0045] As used herein, "random stereogenic center" means a stereogenic center having a random stereochemical configuration in the context of a population of molecules having the same molecular formula. For example, in a population of molecules containing a random stereogenic center, the number of molecules having an (S) configuration at the random stereogenic center may, but need not, be the same as the number of molecules having an (R) configuration at the random stereogenic center. When the stereochemical configuration of a stereogenic center is considered random because the synthetic method was not designed to control the stereochemical configuration. In certain embodiments, the random stereogenic center is a random stereoisomeric phosphorothioate nucleoside internucleoside linkage.

[0046] As used herein, "individual" means a human or non-human animal.

[0047] As used herein, "sugar moiety" means an unmodified sugar moiety or a modified sugar moiety. As used herein, "unmodified sugar moiety" means a 2'-OH(H) β-D-ribosyl moiety as found in RNA ("unmodified RNA sugar moiety") or a 2'-H(H) β-D-deoxyribosyl moiety as found in DNA ("unmodified DNA sugar moiety"). The unmodified sugar moiety has a hydrogen at each of the 1', 3' and 4' positions, an oxygen at the 3' position and two hydrogens at the 5' position. As used herein, "modified sugar moiety" or "modified sugar" means a modified furanosyl sugar moiety or a sugar substitute.

[0048] As used herein, "sugar substitute" means a modified sugar moiety other than a furanosyl moiety that can link a nucleobase to another group (such as an internucleoside linkage, a conjugating group or a terminal group) in an oligonucleotide. Modified nucleosides containing a sugar substitute can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to a complementary oligomeric compound or a target nucleic acid.

[0049] As used herein, "standard in vivo assay" means the assay described in Example 2 and reasonable variations thereof.

[0050] As used herein, "symptom" means any physical characteristic or test result that indicates the presence or degree of a disease or disorder. In certain embodiments, the symptom is apparent to the individual or to a medical professional examining or testing the individual.

[0051] As used herein, "target nucleic acid" means a nucleic acid that an antisense compound is designed to affect.

[0052] As used herein, "target region" means a portion of a target nucleic acid to which an oligomeric compound is designed to hybridize.

[0053] As used herein, "terminal group" means a chemical group or atomic group that is covalently linked to the terminus of an oligonucleotide.

[0054] As used herein, "therapeutically effective amount" means an amount of an agent that provides a therapeutic benefit to an individual. For example, a therapeutically effective amount ameliorates the symptoms of a disease. [Certain embodiments] [] This disclosure provides the following non-limiting numbered embodiments:

[0055] Example 1. An oligomeric compound comprising a modified oligonucleotide composed of 16, 17, 18, 19, or 20 linked nucleosides and having a nucleobase sequence comprising at least 15 or at least 16 consecutive nucleobases of any one of nucleobase sequences SEQ ID NO: 20-50, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0056] Example 2. An oligomeric compound comprising a modified oligonucleotide composed of 17, 18, 19, or 20 linked nucleosides and having a nucleobase sequence comprising at least 15, at least 16, or at least 17 consecutive nucleobases of any one of nucleobase sequences SEQ ID NO: 20-27, 29-30, or 32-50, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0057] Example 3. An oligomeric compound comprising a modified oligonucleotide composed of 18, 19, or 20 linked nucleosides and having a nucleobase sequence comprising at least 15, at least 16, or at least 17 or at least 18 consecutive nucleobases of any one of nucleobase sequences SEQ ID NO: 20-27, 30, or 33-50, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0058] Example 4. An oligomeric compound comprising a modified oligonucleotide composed of 19 or 20 linked nucleosides and having a nucleobase sequence comprising at least 15, at least 16, or at least 17, at least 18, or at least 19 consecutive nucleobases of any one of nucleobase sequences SEQ ID NO: 20, 22, 24-27, 30, 33-50, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0059] Example 5. An oligomeric compound comprising a modified oligonucleotide composed of 20 linked nucleosides and having a nucleobase sequence comprising at least 15, at least 16, or at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any one of nucleobase sequences SEQ ID NO: 20, 22, 25, 27, 35, 39-46, or 49, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0060] Example 6. An oligomeric compound according to any one of Examples 1-5, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%, 85%, 87.5%, 88.2%, 89%, 89.4%, 90%, 93.7%, 94%, 94.7%, 95% or 100% complementary to the nucleobase sequence SEQ ID NO: 1 when measured over the entire nucleobase sequence of the modified oligonucleotide.

[0061] Example 7. An oligomeric compound according to any one of Examples 1-6, wherein the modified oligonucleotide has a internucleoside linkage motif selected from the following (5' to 3'): sosososssssssssssss, ssosssssssssssoss, ssosssssosssssoss, ssosssosssosssoss, soossssssssssooss, sooosssssssssooss, sooossssssssoooss, ssssssssooosssssss, ssossssssssssssss, sssssossssssssssss, sssssssossssssssss, sssssssssossssssss, sssssssssssossssss, sssssssssssssossss, sssssssssssssssoss, sossssssssssssoss, sosssssssssosssss, sosssssssosssssss, sosssssosssssssss, sosssosssssssssss, sssssosssssssssoss, sssssssosssssssoss, sssssssssosssssoss, sssssssssssosssoss, sssssssssssssososs, soossssssssssssss, sssoossssssssssss, ssssssoossssssssss, ssssssssoossssssss, ssssssssssoossssss, ssssssssssssoossss, ssssssssssssssooss, ssssssssoooossssss, ssoooosssssssssss, ssssoooosssssssss, sssssssssoooosssss, sssssssssssoooosss, ssssssssssssooooss, ssssssooooossssss, ssssssoooooosssss, sooosssssssoooss, ssssssooooooosssss, sssssssssssssssoss, sssssssssssssssosss, ssssssssssssssooss, sssssssssssssososs, ssssssssssssosssss, ssssssssssssososss, sssssssssssossosss, sssssssssosssssss, sssssssssosssosss, sssssssssosssssoss,ssssssssosssss、ssssssssssss、ssssssssss、ssssssssoosssssssss、s ...、sosssssssssssssss、sosssssssssssss ss、soossssssssssss、osssssssssssssso、s ...、sssssssssssssssssss、sssssssssssssssssss、ssssssssssssssssssss、sss sssssssssss、sssssssssssss、ssssssssssss、ssssssssssss、sosssssssssssssssss、sossssssssssssssssssss、sosssssssssssssssssss、sosssssssssssssssss、sosssssssssssssssss、sossssssssssssssss、sosssssssssssssss、sosss sssssssssss、soosssssssssssss、ssssssssssssssss、sssssssssssssssss、ssssssssssssssss、osssssssssssssssssss、sssssssssssssss、sssssssssssssss、sssssssssssssss、ssssssssssssssss、ssssssssssssssss、sssssssssssssssssss、sssssssssssssssss ssssssssssssoss、ssssssssssssss、ssssssssssssssss、sssssssssssssssss、soss ...、sosssssssssssssssssss、sossssssssssssssssss、sosssssssssssssssss sossssssssssss、sssssssssooooss、ssssssssssss、ssssssssssss、ssssssssssss、sssssssssooossssssss、s ...ooooossssss、sssssssssss、ssssssssss ooooooossss、sssssssssss、ssossssssssss、ssosssssssssss、ssossssssssssss、ssossssssssssss、ssosssssssssss、ssossssssssss、ssoooosssssssssss、sooosssssssssssss、sooossssssssssssss、sooossssssssssssss、ssssssssssssssss、ssooos ...、sssssssssssssssss、sssssssssssssssss、sssssssssssssssss、ssssssoooosssss, ssssssooooosssss, ssssssoooooossss, ssssoooosssssss, ssssooooooossss, sssosssosssosss, ssosssssssssoss, ssossossossosss, ssossossosososs, sosososososososs, ssoooosssssssss, soossssssssooss, sooosssssssooss, sooossssssoooss and soooosssssoooss; wherein's' represents a phosphorothioate internucleoside linkage and 'o' represents a phosphodiester internucleoside linkage.

[0062] Example 8. An oligomeric compound as in any one of Examples 1-6, wherein the modified oligonucleotide has an internucleoside linkage selected from: ssssssssssssssssxs and ssssssssssssssssx; wherein's' represents a phosphorothioate internucleoside linkage, 'o' represents a phosphodiester internucleoside linkage and "x" represents a methoxypropylphosphonate internucleoside linkage.

[0063] Example 9. An oligomeric compound as in any one of Examples 1-6, wherein the modified oligonucleotide has an internucleoside linkage selected from: zzzzzzzzzzzzzzzzzz, sssssssssszzzzzz, sssszzzzzzssssss, zzo oooooooooooozz, zzzzo oooooooooozz, zzzzzzo oooooooooz z, zzzzzzzooooooozz and ssoooooooooooooss; wherein's' represents a phosphorothioate internucleoside linkage, 'o' represents a phosphodiester internucleoside linkage and "z" represents a methanesulfonamido phosphonate internucleoside linkage.

[0064] Example 10. An oligomeric compound according to any one of Examples 1-9, wherein the modified oligonucleotide has a sugar moiety selected from the following (5' to 3'): eeeeeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeee, nnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnnnnn, nennnnneneennnnnnn, nnnnnnnnnnnenneen, nennnnneneenenneen, nnnnnnnnnnnnnnnnnnne, nnnnnnnnnnnnnnnnnnd, nnnnnnnnnnnnnnnnnny, nnnnnnnnnnnnnnnnnndd, nnnnnnnnnnnnnnnnnned, nnnnnnnnnnnnnnnnnnde, nnnnnnnnnnnnnnnnnnee, eeeeeeeeeeeeeeeeeeedd, eeeeeeeeeeeeeeeeeeeed, eeeeeeeeeeeeeeeeeeede, nnnnnnnnnnnnnnnnnnnd, nnnnnnnnnnnnnnnnnnne, eeeeeeeeeeeeeeeeeeed, keekeekeekeekeeeek, keeeekeeekeeekeeeek, keeeeekeeeeekeeeek, keeeeeeekeeeeeeeek, keeeeeeeeeeeeeeeek, eeekeekeekeekeekek, eeekeekeekeekeekee, eeeeeeekeekeekeekee, eeeeeeekeekeekeeeee, eeeeeeekeeeeekeeeee, keekeekeekeeeeeeee, eeeeeeeeekeekeekeek, keekeekeeeeeeeeeee, eeeeeeeeeeeekeekeek, keekeeeeeeeeeeeeee, eeeeeeeeeeeeeeekeek, keekeekeekeekeeek, keeeekeeekeeekeeek, keeeekeeeeekeeeek, keeeeeeekeeeeeeek, keeeeeeeeeeeeeeek, eekeekeekeekeekek, eekeekeekeekeekee,eeeeekeekeekeekee, eeeeekeekeekeeeee, eeeeekeeeeeekeeeee, keekeekeekeeeeeee, eeeeeeekeekeekeek, keekeekeeeeeeeeee, eeeeeeeeekeekeek, keekeeeeeeeeeeee, eeee eeeeeeeekeek, keekeekeekeekeek, keeekeekeekeekeek, keeeekeeeekeeeek, keeeeeeekeeeeeek, keeeeeeeeeeeeek, kekeekeekeekeeke, eekeekeekeekeeke, eeeeekeekeekeeke 、eeeeekeekeekeeee、eeeeekeeeeekeeee、keekeekeekeeeeee、eeeeeeekeeek、keekeeeeeeeeeeeeee、eeeeeeeeeeeeeekeek、eeeeeeeeeeeeeeeeeed、eeeeeeeeeeeeeeeeeeeey、ennnnnnnnnnnnnnnnnnn and ennnnnnnnnnnnnnnnnnnne; wherein 'e' represents a 2'-MOE sugar moiety, 'n' represents a 2'-NMA sugar moiety, 'k' represents a cEt sugar moiety, 'd' represents a 2'-β-D-deoxyribosyl sugar moiety and 'y' represents a 2'-OMe sugar moiety. 、

[0065] Embodiment 11. An oligomeric compound as in any one of Embodiments 1-9, wherein the modified oligonucleotide has a sugar motif (5' to 3') selected from the following: nnnnnnnnnnnnnnenn and nnnnnnnnnnnnnnnnnenen, wherein 'e' represents a 2'-MOE sugar moiety and 'n' represents a 2'-NMA sugar moiety.

[0066] Embodiment 12. An oligomeric compound as described in any one of Embodiments 1-9, wherein the modified oligonucleotide has a sugar motif (5' to 3') qqnqqqqqnqnnqnqqnn, wherein each "n" represents a 2'-NMA sugar moiety, and each "q" is independently selected from a 2'-O-(N,N-dimethyl)acetamine sugar moiety, a 2'-O-(N-ethyl)acetamine sugar moiety, a 2'-O-(N-propyl)acetamine sugar moiety, a 2'-O-(N-cyclopropyl)acetamine sugar moiety, and a 2'-O-(N-cyclopropylmethyl)acetamine sugar moiety.

[0067] Example 13. An oligomeric compound according to any one of Examples 1-9, wherein the modified oligonucleotide comprises at least one modified sugar moiety.

[0068] Example 14. An oligomeric compound according to Example 13, wherein the modified oligonucleotide comprises at least one bicyclic sugar moiety.

[0069] Example 15. An oligomeric compound according to Example 14, wherein the bicyclic sugar moiety has a 4'-2' bridge, and the 4'-2' bridge is selected from -CH2-O- and -CH(CH3)-O.

[0070] Example 16. An oligomeric compound according to Example 13, wherein the modified oligonucleotide comprises at least one non-bicyclic modified sugar moiety.

[0071] Example 17. An oligomeric compound according to Example 16, wherein the non-bicyclic modified sugar moiety is any one of a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, a 2'-OMe sugar moiety, and a 2'-F sugar moiety.

[0072] Example 18. An oligomeric compound according to Example 13, wherein the modified oligonucleotide comprises at least one sugar substitute.

[0073] Example 19. An oligomeric compound according to Example 18, wherein the sugar substitute is any one of N-morpholino, a modified N-morpholino, PNA, THP, and F-HNA.

[0074] Example 20. An oligomeric compound according to any one of Examples 1-6 and 10-19, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

[0075] Example 21. An oligomeric compound according to Example 20, wherein each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.

[0076] Example 22. An oligomeric compound according to Example 20 or Example 21, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[0077] Example 23. An oligomeric compound as in any one of Examples 1-20 or 22, wherein the modified oligonucleotide comprises at least one phosphodiester internucleoside linkage.

[0078] Example 24. An oligomeric compound as in Example 20, 22 or 23, wherein each internucleoside linkage is independently selected from phosphodiester internucleoside linkages and phosphorothioate internucleoside linkages.

[0079] Example 25. An oligomeric compound as in any one of Examples 13-19, wherein the modified oligonucleotide has an internucleoside linkage motif (5' to 3') selected from: sosososssssssssssss, soossssssssssssss, sosssosssssssssss, sosssssosssssssss, sosssssssosssssss, sssoossssssssssss, sssssssoossssssss, ssssssssssoossssss and ssssssssssssoossss; where's' represents a phosphorothioate internucleoside linkage and 'o' represents a phosphodiester internucleoside linkage.

[0080] Example 26. An oligomeric compound as in any one of Examples 1-25, wherein the modified oligonucleotide comprises a modified nucleobase.

[0081] Example 27. An oligomeric compound as in Example 26, wherein the modified nucleobase is 5-methylcytosine.

[0082] Example 28. An oligomeric compound as in any one of Examples 1-27, wherein the modified oligonucleotide consists of 16, 17, 18, 19 or 20 linked nucleosides.

[0083] Example 29. An oligomeric compound as in any one of Examples 1-28, wherein the modified oligonucleotide comprises 1 or 2 non-complementary nucleobases.

[0084] Example 30. An oligomeric compound as in any one of Examples 1-29, wherein the modified oligonucleotide comprises 1 or 2 cleavable moieties.

[0085] Example 31. An oligomeric compound as in Example 30, wherein the cleavable moiety is a phosphodiester internucleoside linkage.

[0086] Example 32. An oligomeric compound as in any one of Examples 1-31, which is composed of the modified oligonucleotide.

[0087] Example 33. An oligomeric compound as in any one of Examples 1-32, wherein the oligomeric compound is a single-stranded oligomeric compound.

[0088] Example 34. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: mC esA eo mC esT eoT esT es mC esA esT esA esA esT esG es mC esT esG esG es mC e (SEQ ID NO: 21), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, e = 2'-MOE sugar moiety, s = phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage.

[0089] Example 35. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: T eoT es mC esA es mC esT esT esT es mC esA esT esA esA esT esG es mC esT esG esG eo mC e (SEQ ID NO: 22), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, e = 2'-MOE sugar moiety, s = phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage.

[0090] Example 36. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: TeoTnsmCnsAns mCnsTnsTnsTns mCnsAnsTnsAnsAnsTnsGns mCnsTnsGnsGnomCe (SEQ ID NO: 22), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, e = 2'-MOE sugar moiety, n = 2'-NMA sugar moiety, s = phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage.

[0091] Example 37. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: mCnsAns nomCnsTnoTnsTns mCnsAnsTnsAnsAnsTnsGns mCnsTnsGnsGns mCn (SEQ ID NO: 21), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n = 2'-NMA sugar moiety, s = phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage.

[0092] Example 38. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 21), or a salt thereof.

[0093] Example 39. The modified oligonucleotide of Example 38, which is a sodium salt or a potassium salt.

[0094] Example 40. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 21).

[0095] Example 41. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 22), or a salt thereof.

[0096] Example 42. The modified oligonucleotide of Example 41, which is a sodium salt or a potassium salt.

[0097] Example 43. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 22).

[0098] Example 44. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 22), or a salt thereof.

[0099] Example 45. The modified oligonucleotide of Example 44, which is a sodium salt or a potassium salt.

[0100] Example 46. A modified oligonucleotide corresponding to the following chemical structure: (SEQ ID NO: 22).

[0101] Example 47. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 21), or a salt thereof.

[0102] Example 48. The modified oligonucleotide of Example 47, which is a sodium salt or a potassium salt.

[0103] Example 49. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 21).

[0104] Example 50. A pharmaceutical composition comprising an oligomeric compound as in any one of Examples 1 - 36 or a modified oligonucleotide as in any one of Examples 38 - 49 and a pharmaceutically acceptable diluent or carrier.

[0105] Example 51. The pharmaceutical composition of Example 50, which comprises a pharmaceutically acceptable diluent, and wherein the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS.

[0106] Example 52. The pharmaceutical composition of Example 51, wherein the pharmaceutical composition consists essentially of the modified oligonucleotide and artificial CSF (aCSF).

[0107] Example 53. The pharmaceutical composition of Example 51, wherein the pharmaceutical composition consists essentially of the modified oligonucleotide and PBS.

[0108] Example 54. A chiral enriched population of a modified oligonucleotide as in any one of Examples 38 - 49, wherein the population is enriched in modified oligonucleotides comprising at least one specific phosphorothioate internucleoside linkage having a specific stereochemical configuration.

[0109] Example 55. The chiral enriched population of Example 54, wherein the population is enriched in modified oligonucleotides comprising at least one specific phosphorothioate internucleoside linkage having an (Sp) configuration.

[0110] Example 56. The chiral enriched population of Example 54, wherein the population is enriched in modified oligonucleotides comprising at least one specific phosphorothioate internucleoside linkage having an (Rp) configuration.

[0111] Example 57. The chiral enriched population of Example 54, wherein the population is enriched in modified oligonucleotides having a specific stereochemical configuration independently selected at each phosphorothioate internucleoside linkage.

[0112] Example 58. A chiral enriched population as in Example 57, wherein the population is enriched in modified oligonucleotides having an (Sp) configuration at each internucleoside phosphorothioate linkage or modified oligonucleotides having an (Rp) configuration at each internucleoside phosphorothioate linkage.

[0113] Example 59. A chiral enriched population as in Example 57, wherein the population is enriched in modified oligonucleotides having an (Rp) configuration at a specific internucleoside phosphorothioate linkage and an (Sp) configuration at each of the remaining internucleoside phosphorothioate linkages.

[0114] Example 60. A chiral enriched population as in Example 57, wherein the population is enriched in modified oligonucleotides having at least 3 consecutive internucleoside phosphorothioate linkages in the 5' to 3' direction having Sp, Sp, and Rp configurations.

[0115] Example 61. A population of modified oligonucleotides as in any one of Examples 38 - 49, wherein all internucleoside phosphorothioate linkages of the modified oligonucleotides are randomly configured.

[0116] Example 62. A method of treating a disease associated with SMN1 or SMN2, comprising administering to an individual suffering from or at risk of developing a disease associated with SMN1 or SMN2 a therapeutically effective amount of a pharmaceutical composition as in any one of Examples 50 - 53; and thereby treating the disease associated with SMN1 or SMN2.

[0117] Example 63. The method of Example 62, wherein the disease associated with SMN1 or SMN2 is a neurodegenerative disease.

[0118] Example 64. The method of Example 63, wherein the neurodegenerative disease is spinal muscular atrophy (SMA).

[0119] Example 65. The method of Example 64, wherein the SMA is any one of type I SMA, type II SMA, type III SMA, or type IV SMA.

[0120] Example 66. The method of Example 64 or Example 65, wherein at least one symptom of SMA is improved.

[0121] Example 67. The method of Example 66, wherein the symptom is any one of the following: reduced muscle strength; inability to sit upright, stand, and / or walk or reduced ability; reduced neuromuscular activity; reduced electrical activity of one or more muscles; reduced respiration; inability to eat, drink, and / or breathe without assistance or reduced ability; weight loss or reduced weight gain; and / or reduced survival rate.

[0122] Example 68. The method of any one of Examples 62 - 67, wherein the pharmaceutical composition is administered to the central nervous system or systemically.

[0123] Example 69. The method of Example 68, wherein the pharmaceutical composition is administered to the central nervous system and systemically.

[0124] Example 70. The method of any one of Examples 62 - 67, wherein the pharmaceutical composition is administered by any one of intrathecal, systemic, subcutaneous, or intramuscular routes.

[0125] Example 71. A method of increasing SMN2 RNA comprising exon 7, comprising contacting a cell, tissue, or organ with an oligomeric compound of any one of Examples 1 - 37, a modified oligonucleotide of any one of Examples 38 - 49, or a pharmaceutical composition of any one of Examples 50 - 53. [Certain oligonucleotides]

[0126] In certain embodiments, the present disclosure provides oligomeric compounds comprising oligonucleotides composed of linked nucleosides. The oligonucleotides can be unmodified oligonucleotides (RNA or DNA) or can be modified oligonucleotides. Relative to unmodified RNA or DNA, the modified oligonucleotides comprise at least one modification. That is, the modified oligonucleotides comprise at least one modified nucleoside (comprising a modified sugar moiety and / or a modified nucleobase) and / or at least one modified internucleoside linkage. [Certain modified nucleosides] []

[0127] The modified nucleosides comprise a modified sugar moiety, or a modified nucleobase, or both a modified sugar moiety and a modified nucleobase. [Certain sugar moieties] []

[0128] In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety. In certain embodiments, the modified sugar moiety is a bicyclic or tricyclic sugar moiety. In certain embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates can include one or more substitutions corresponding to other types of modified sugar moieties.

[0129] In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety that includes a furanosyl ring having one or more substituents, none of which bridge two atoms of the furanosyl ring to form a bicyclic structure. Such non-bridging substituents can be located at any position of the furanosyl, including but not limited to substituents at the 2’, 4’ and / or 5’ positions. In certain embodiments, one or more non-bridging substituents of the non-bicyclic modified sugar moiety are branched. Examples of suitable 2’-substituents for non-bicyclic modified sugar moieties include but are not limited to: 2’-F, 2’-OCH3 (“OMe” or “O-methyl”), 2’-O(CH2)2OCH3 (“MOE” or “O-methoxyethyl”), and 2’-O-N-alkylacetamides, e.g., 2’-O-N-methylacetamide (“NMA”), 2’-O-N-dimethylacetamide, 2’-O-N-ethylacetamide, or 2’-O-N-propylacetamide. See, e.g., U.S. 6,147,200, Prakash et al., 2003, Org. Lett., 5, 403-6. The “2’-O-N-methylacetamide nucleoside” or “2’-NMA nucleoside” is shown below: In certain embodiments, the 2'-substituent is selected from: halogen, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C10 alkoxy, O-C1-C10 substituted alkoxy, O-C1-C10 alkyl, O-C1-C10 substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkenyl-O-alkyl, alkynyl, aralkyl, arylalkyl, O-aralkyl, O-arylalkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H, an amino protecting group or a substituted or unsubstituted C1-C10 alkyl, and the 2'-substituents as described in Cook et al., U.S. 6,531,584; Cook et al., U.S. 5,859,221; and Cook et al., U.S. 6,005,087. Certain embodiments of these 2'-substituents may be further substituted with one or more substituents independently selected from: hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of suitable 4'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl and those described in Manoharan et al., WO 2015 / 106128. Examples of suitable 5'-substituents for non-bicyclic modified sugar moieties include, but are not limited to: 5'-methyl (R or S), 5'-vinyl and 5'-methoxy. In certain embodiments, the non-bicyclic modified sugar moiety contains more than one non-bridging sugar substituent, such as the 2'-F-5'-methyl sugar moiety and the modified sugar moieties and modified nucleosides as described in Migawa et al., WO 2008 / 101157 and Rajeev et al., US2013 / 0203836.

[0130] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from: F, NH₂, N₃, OCF₃, OCH₃, O(CH₂)₃NH₂, CH₂CH=CH₂, OCH₂CH=CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂ON(Rm)(Rn), O(CH₂), ON(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substituted acetamide (OCH₂C(=O)-N(Rm)(Rn)), wherein each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C₁-C₁₀ alkyl, such as OCH₂C(=O)-N(H)CH₃ (「NMA」).

[0131] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from: F, OCF₃, OCH₃, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, and OCH₂C(=O)-N(H)CH₃ (「NMA」).

[0132] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from: F, OCH₃, OCH₂CH₂OCH₃, and OCH₂C(=O)-N(H)CH₃.

[0133] Certain modified sugar moieties contain substituents that bridge two atoms of the furanosyl ring to form a second ring, thereby generating a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety contains a bridge between the 4' and 2' furanose ring atoms. Examples of such 4'-to-2' bridged sugar substituents include, but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "constrained ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("constrained MOE" or "cMOE") and analogs thereof (see, e.g., Seth et al., U.S. 7,399,845; Bhat et al., U.S. 7,569,686; Swayze et al., U.S. 7,741,457 and Swayze et al., U.S. 8,022,193), 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., Seth et al., U.S. 8,278,283), 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., Prakash et al., U.S. 8,278,425), 4'-CH2-O-N(CH3)-2' (see, e.g., Allerson et al., U.S. 7,696,345 and Allerson et al., U.S. 8,124,745), 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogs thereof (see, e.g., Seth et al., U.S. 8,278,426), 4'-C(RaRb)-N(R)-O-2', 4'-C(RaRb)-O-N(R)-2', 4'-CH2-O-N(R)-2' and 4'-CH2-N(R)-O-2', wherein each R, Ra and Rb is independently H, a protecting group or a C1-C12 alkyl (see, e.g., Imanishi et al., U.S. 7,427,672).

[0134] In certain embodiments, such 4’-to-2’ bridges independently comprise from 1 to 4 linking groups independently selected from the following: -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)x-, and -N(Ra)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Ra and Rb is independently H, a protecting group, a hydroxyl group, a C1-C12 alkyl group, a substituted C1-C12 alkyl group, a C2-C12 alkenyl group, a substituted C2-C12 alkenyl group, a C2-C12 alkynyl group, a substituted C2-C12 alkynyl group, a C5-C20 aryl group, a substituted C5-C20 aryl group, a heterocyclic group, a substituted heterocyclic group, a heteroaryl group, a substituted heteroaryl group, a C5-C7 cycloaliphatic group, a substituted C5-C7 cycloaliphatic group, a halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), a substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and each J1 and J2 is independently H, a C1-C12 alkyl group, a substituted C1-C12 alkyl group, a C2-C12 alkenyl group, a substituted C2-C12 alkenyl group, a C2-C12 alkynyl group, a substituted C2-C12 alkynyl group, a C5-C20 aryl group, a substituted C5-C20 aryl group, acyl (C(=O)-H), a substituted acyl, a heterocyclic group, a substituted heterocyclic group, a C1-C12 aminoalkyl group, a substituted C1-C12 aminoalkyl group, or a protecting group.

[0135] Other bicyclic sugar moieties are known in the art, see for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; Albaek et al., J. Org. Chem., 2006, 71, 7731-7740; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 8362-8379; Wengel et al., U.S. 7,053,207; Imanishi et al., U.S. 6,268,490; Imanishi et al., U.S. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. 6,794,499; Wengel et al., U.S. 6,670,461; Wengel et al., U.S. 7,034,133; Wengel et al., U.S. 8,080,644; Wengel et al., U.S. 8,034,909; Wengel et al., U.S. 8,153,365; Wengel et al., U.S. 7,572,582; and Ramasamy et al., U.S. 6,525,191; Torsten et al., WO 2004 / 106356; Wengel et al., WO 1999 / 014226; Seth et al., WO 2007 / 134181; Seth et al., U.S. 7,547,684; Seth et al., U.S. 7,666,854; Seth et al., U.S. 8,088,746; Seth et al., U.S. 7,750,131; Seth et al., U.S. 8,030,467; Seth et al., U.S. 8,268,980; Seth et al., U.S. 8,546,556; Seth et al., U.S. 8,530,640; Migawa et al., U.S. 9,012,421; Seth et al., U.S.8,501,805; and Allerson et al., U.S. Patent Publication No. US2008 / 0039618 and Migawa et al., U.S. Patent Publication No. US2015 / 0191727.

[0136] In certain embodiments, the bicyclic sugar moiety and the nucleoside incorporating such a bicyclic sugar moiety are further defined by the isomeric configuration. For example, an LNA nucleoside (described herein) can be in the α-L configuration or the β-D configuration.

[0137] α-L-methoxy (4'-CH2-O-2') or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides, demonstrating antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). In this context, the general description of a bicyclic nucleoside includes both isomeric configurations. Unless otherwise specified, when the position of a particular bicyclic nucleoside (e.g., LNA or cEt) is identified in the examples illustrated herein, it is in the β-D configuration.

[0138] In certain embodiments, the modified sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., a 5'-substituted and 4'-2'-bridged sugar).

[0139] In certain embodiments, the modified sugar moiety is a sugar surrogate. In certain such embodiments, the oxygen atoms of the sugar moiety are replaced by, for example, sulfur, carbon, or nitrogen atoms. In certain such embodiments, such modified sugar moieties also comprise bridging and / or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4'-sulfur atom and substitutions at the 2'-position (see, e.g., Bhat et al., U.S. 7,875,733 and Bhat et al., U.S. 7,939,677) and / or the 5'-position.

[0140] In certain embodiments, the sugar surrogate comprises a ring other than 5 atoms. For example, in certain embodiments, the sugar surrogate comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans can be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid ("HNA"), anitol nucleic acid ("ANA"), mannitol nucleic acid ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro-HNA: ("F-HNA", see, for example, Swayze et al., U.S. 8,088,904; Swayze et al., U.S. 8,440,803; Swayze et al., U.S. 8,796,437; and Swayze et al., U.S. 9,005,906; F-HNA may also be referred to as F-THP or 3'-fluorotetrahydropyran) and nucleosides containing other modified THP compounds having the following formula: wherein, independently, for each of the modified THP nucleosides: Bx is a nucleobase moiety; T3 and T4 are each independently a internucleoside linking group that links the modified THP nucleoside to the remainder of the oligonucleotide, or one of T3 and T4 is a internucleoside linking group that links the modified THP nucleoside to the remainder of the oligonucleotide, and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group; q1, q2, q3, q4, q5, q6 and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and R1 and R2 are each independently selected from: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, where X is O, S or NJ1, and J1, J2 and J3 are each independently H or C1-C6 alkyl.

[0141] In certain embodiments, modified THP nucleosides are provided, wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is not H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, modified THP nucleosides are provided, wherein one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and R2 is H.

[0142] In some embodiments, the sugar substitute comprises a ring having more than 5 atoms and more than one heteroatom. For example, nucleosides containing an N-morpholinyl sugar moiety and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5,698,685; Summerton et al., U.S. 5,166,315; Summerton et al., U.S. 5,185,444; and Summerton et al., U.S. 5,034,506). As used herein, the term "N-morpholinyl" means a sugar substitute having the following structure: .

[0143] In some embodiments, the N-morpholinyl can be modified, for example, by adding or changing various substituents compared to the above N-morpholinyl structure. Such sugar substitutes are referred to herein as "modified N-morpholinyls".

[0144] In some embodiments, the sugar substitute comprises an acyclic moiety. Examples of nucleosides and oligonucleotides containing such acyclic sugar substitutes include, but are not limited to: peptide nucleic acid ("PNA"), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865) and the nucleosides and oligonucleotides described in Manoharan et al., WO2011 / 133876.

[0145] Many other bicyclic and tricyclic sugar and sugar substitute ring systems are known in the art for use in modified nucleosides. [Certain modified nucleobases] []

[0146] In some embodiments, the modified oligonucleotide comprises one or more nucleosides containing an unmodified nucleobase. In some embodiments, the modified oligonucleotide comprises one or more nucleosides containing a modified nucleobase. In some embodiments, the modified oligonucleotide comprises one or more nucleosides that do not contain a nucleobase, referred to as abasic nucleosides.

[0147] In certain embodiments, the modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. In certain embodiments, the modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynylcytosine, 6-azauracil, 6-azacytosine, 6-azathymine, 5-ribosyluracil (pseudouracil), 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, 8-aza and other 8-substituted purines; 5-halo, especially 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine; 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, hybrid bases, size-expanded bases and fluorinated bases. Other modified nucleobases include tricyclic pyrimidines such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp). Modified nucleobases may also include nucleobases in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine and 2-pyridone.Other nucleobases include those disclosed below: Merigan et al., U.S. 3,687,808; The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I. ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebleu, B. eds., CRC Press, 1993, 273-288; and Chapters 6 and 15, Antisense Drug Technology, Crooke S.T. ed., CRC Press, 2008, 163-166 and 442-443.

[0148] Publications teaching the preparation of certain of the modified nucleobases described above, as well as other modified nucleobases, include but are not limited to: Manoharan et al., US2003 / 0158403; Manoharan et al., US2003 / 0175906; Dinh et al., U.S. 4,845,205; Spielvogel et al., U.S. 5,130,302; Rogers et al., U.S. 5,134,066; Bischofberger et al., U.S. 5,175,273; Urdea et al., U.S. 5,367,066; Benner et al., U.S. 5,432,272; Matteucci et al., U.S. 5,434,257; Gmeiner et al., U.S. 5,457,187; Cook et al., U.S. 5,459,255; Froehler et al., U.S. 5,484,908; Matteucci et al., U.S. 5,502,177; Hawkins et al., U.S. 5,525,711; Haralambidis et al., U.S. 5,552,540; Cook et al., U.S. 5,587,469; Froehler et al., U.S. 5,594,121; Switzer et al., U.S. 5,596,091; Cook et al., U.S. 5,614,617; Froehler et al., U.S. 5,645,985; Cook et al., U.S. 5,681,941; Cook et al., U.S. 5,811,534; Cook et al., U.S. 5,750,692; Cook et al., U.S. 5,948,903; Cook et al., U.S. 5,587,470; Cook et al., U.S. 5,457,191; Matteucci et al., U.S. 5,763,588; Froehler et al., U.S. 5,830,653; Cook et al., U.S. 5,808,027; Cook et al., 6,166,199; and Matteucci et al., U.S. 6,005,096. [Certain modified internucleoside linkages] []

[0149] In some embodiments, the nucleosides of the modified oligonucleotides can be linked together using any internucleoside linkage. Two major classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to: phosphodiester, which contains a phosphodiester bond (P(O2)=O) (also referred to as an unmodified or naturally occurring linkage); phosphotriester; methylphosphonate; methoxypropylphosphonate ("MOP"); phosphoramidate; methylsulfonamidate; phosphorothioate (P(O2)=S) and dithiophosphate (HS-P=S). Representative phosphorus-free internucleoside linking groups include, but are not limited to: methylenemethylimino (-CH2-N(CH3)-O-CH2-); phosphorothioate; thiocarbonylcarbamate (-O-C(=O)(NH)-S-); siloxane (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to the naturally occurring phosphoester linkage, modified internucleoside linkages can be used to alter (usually increase) the nuclease resistance of oligonucleotides. In some embodiments, internucleoside linkages having chiral atoms can be prepared as racemic mixtures or separate enantiomers. Methods for preparing phosphorus-containing and phosphorus-free internucleoside linkages are well known to those skilled in the art.

[0150] Representative internucleoside linkages having a chiral center include, but are not limited to, alkyl phosphonates and phosphorothioates. Modified oligonucleotides comprising internucleoside linkages having a chiral center can be prepared as a population of modified oligonucleotides comprising random stereoisomeric internucleoside linkages, or as a population of modified oligonucleotides comprising phosphorothioate internucleoside linkages in a specific stereochemical configuration. In certain embodiments, the population of modified oligonucleotides comprises phosphorothioate internucleoside linkages in which all of the phosphorothioate internucleoside linkages are random stereoisomers. Such modified oligonucleotides can be generated using synthetic methods that randomly select the stereochemical configuration of each phosphorothioate internucleoside linkage. Nevertheless, as is well understood by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, the population of modified oligonucleotides is enriched in modified oligonucleotides comprising one or more specific phosphorothioate internucleoside linkages in a specific independently selected stereochemical configuration. In certain embodiments, a specific phosphorothioate internucleoside linkage in a specific configuration is present in at least 65% of the molecules in the population. In certain embodiments, a specific phosphorothioate internucleoside linkage in a specific configuration is present in at least 70% of the molecules in the population. In certain embodiments, a specific phosphorothioate internucleoside linkage in a specific configuration is present in at least 80% of the molecules in the population. In certain embodiments, a specific phosphorothioate internucleoside linkage in a specific configuration is present in at least 90% of the molecules in the population. In certain embodiments, a specific phosphorothioate internucleoside linkage in a specific configuration is present in at least 99% of the molecules in the population. Such chiral enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, such as those described in Oka et al., JACS, 2003, 125, 8307; Wan et al., Nuc. Acid. Res., 2014, 42, 13456, and WO 2017 / 015555. In certain embodiments, the population of modified oligonucleotides is enriched in modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In certain embodiments, the population of modified oligonucleotides is enriched in modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration. In certain embodiments, modified oligonucleotides comprising (Rp) and / or (Sp) phosphorothioates comprise one or more of the following formulas, where "B" indicates a nucleobase: Unless otherwise indicated, the chiral internucleoside linkages of the modified oligonucleotides described herein can be random stereoisomers or in a specific stereochemical configuration.

[0151] In certain embodiments, the modified oligonucleotide comprises internucleoside moieties (5’ to 3’) sooosssssssssssssss. In certain embodiments, the specific stereochemical configuration of the modified oligonucleotide is (5’ to 3’) Sp-o-o-o- Sp- Sp- Sp- Rp- Sp- Sp- Rp- Sp- Sp- Sp- Sp- Sp- Sp- Sp- Sp or Sp-o-o-o- Sp- Sp- Sp- Rp- Sp- Sp- Sp- Sp- Sp- Sp- Sp- Sp- Sp- Sp- Sp; wherein each 'Sp' represents a phosphorothioate internucleoside linkage in the S configuration; Rp represents a phosphorothioate internucleoside linkage in the R configuration; and 'o' represents a phosphodiester internucleoside linkage.

[0152] Neutral internucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, MMI (3’-CH 2-N(CH 3)-O-5’), amide-3 (3’-CH 2-C(=O)-N(H)-5’), amide-4 (3’-CH 2-N(H)-C(=O)-5’), acetal (3’-O-CH 2-O-5’), methoxypropyl, and thioacetal (3’-S-CH 2-O-5’). Other neutral internucleoside linkages include nonionic linkages, which include siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates, and amides (see Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Other neutral internucleoside linkages include nonionic linkages that contain a mixture of N, O, S, and CH 2 components. [Certain motifs] []

[0153] In certain embodiments, the modified oligonucleotide comprises one or more modified nucleosides comprising a modified sugar moiety. In certain embodiments, the modified oligonucleotide comprises one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, the modified oligonucleotide comprises one or more modified internucleoside linkages. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and / or internucleoside linkages of the modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of the sugar moiety, nucleobase, and internucleoside linkage are each independent of one another. Thus, the modified oligonucleotide is described by its sugar motif, nucleobase motif, and / or internucleoside linkage motif (as used herein, the nucleobase motif describes modifications to the nucleobase that are independent of the sequence of the nucleobase). [Certain sugar motifs]

[0154] In certain embodiments, the oligonucleotide comprises one or more types of modified sugars and / or unmodified sugar moieties arranged in a defined pattern or sugar motif along the oligonucleotide or a portion thereof. In some cases, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.

[0155] In certain embodiments, the modified oligonucleotide has a gapmer motif defined by two outer regions or "flanks" and a central or inner region or "gap". The three regions of the gapmer motif (5'-flank, gap, and 3'-flank) form a continuous sequence of nucleosides, wherein at least some of the sugar moieties of the nucleosides in each flank are different from at least some of the sugar moieties of the nucleosides in the gap. Specifically, at least the sugar moiety of the nucleoside closest to the gap in each flank (the most 3' end nucleoside of the 5'-flank and the most 5' end nucleoside of the 3'-flank) is different from the sugar moiety of the adjacent gap nucleoside, thus defining the boundary between the flank and the gap (i.e., the flank / gap junction). In certain embodiments, the sugar moieties within the gap are the same as each other. In certain embodiments, the gap comprises one or more nucleosides having sugar moieties different from those of one or more other nucleosides in the gap. In certain embodiments, the sugar motifs of the two flanks are the same as each other (symmetric gapmer). In certain embodiments, the sugar motif of the 5'-flank is different from the sugar motif of the 3'-flank (asymmetric gapmer).

[0156] In certain embodiments, the flanks of the gapmer comprise 1-6 nucleosides. In certain embodiments, each nucleoside in each flank of the gapmer comprises a modified sugar moiety. In certain embodiments, at least two, at least three, at least four, at least five, or at least six nucleosides in each flank of the gapmer comprise a modified sugar moiety.

[0157] In certain embodiments, the gap between the spacers comprises 7 - 12 nucleosides. In certain embodiments, each nucleoside in the gap between the spacers comprises a 2'-deoxyribosyl sugar moiety. In certain embodiments, at least one nucleoside in the gap between the spacers comprises a modified sugar moiety and each of the remaining nucleosides comprises a 2'-deoxyribosyl sugar moiety.

[0158] Herein, the lengths (number of nucleosides) of the three regions of the spacer can be provided using the notation [number of nucleosides in the 5'-flank]-[number of nucleosides in the gap]-[number of nucleosides in the 3'-flank]. Thus, a 5-10-5 spacer consists of 5 linked nucleosides in each flank and 10 linked nucleosides in the gap. In the case where such nomenclature is subsequently followed by a specific modification, the modification is a modification in each sugar moiety of each flank and the gap nucleosides comprise 2'-deoxyribosyl sugar moieties. Thus, a 5-10-5 MOE spacer consists of 5 linked 2'-MOE nucleosides in the 5'-flank, 10 linked 2'-deoxyribonucleosides in the gap, and 5 linked 2'-MOE nucleosides in the 3'-flank.

[0159] In certain embodiments, each nucleoside or a portion thereof of the modified oligonucleotide comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, a sugar surrogate, or a 2'-deoxyribosyl sugar moiety. In certain embodiments, the 2'-substituted sugar moiety is selected from 2'-MOE sugar moiety, 2'-NMA sugar moiety, 2'-OMe sugar moiety, and 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from cEt sugar moiety and LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from N-morpholino, modified N-morpholino, PNA, THP, and F-HNA.

[0160] In certain embodiments, the modified oligonucleotide comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleosides comprising a modified sugar moiety. In certain embodiments, the modified sugar moiety is independently selected from a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2'-substituted sugar moiety is selected from 2'-MOE sugar moiety, 2'-NMA sugar moiety, 2'-OMe sugar moiety, and 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from cEt sugar moiety and LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from N-morpholino, modified N-morpholino, THP, and F-HNA.

[0161] In certain embodiments, each nucleoside of the modified oligonucleotide comprises a modified sugar moiety (a "fully modified oligonucleotide"). In certain embodiments, each nucleoside of the fully modified oligonucleotide comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2'-substituted sugar moiety is selected from a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, a 2'-OMe sugar moiety, and a 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from N-morpholino, a modified N-morpholino, THP, and F-HNA. In certain embodiments, each nucleoside of the fully modified oligonucleotide comprises the same modified sugar moiety (a "uniformly modified sugar motif"). In certain embodiments, the length of the uniformly modified sugar motif is from 7 to 20 nucleosides. In certain embodiments, each nucleoside of the uniformly modified sugar motif comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2'-substituted sugar moiety is selected from a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, a 2'-OMe sugar moiety, and a 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from N-morpholino, a modified N-morpholino, THP, and F-HNA. In certain embodiments, a modified oligonucleotide having at least one fully modified sugar motif may also comprise at least 1, at least 2, at least 3, or at least 4 2'-deoxyribonucleosides. [Certain nucleobase motifs] []

[0162] In certain embodiments, an oligonucleotide comprises modified and / or unmodified nucleobases arranged in a defined pattern or motif along the oligonucleotide or a portion thereof. In certain embodiments, each nucleobase is modified. In certain embodiments, no nucleobase is modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the pyrimidine nucleobases in the modified oligonucleotide are 5-methylcytosine. In certain embodiments, all cytosine nucleobases are 5-methylcytosine, and all other nucleobases of the modified oligonucleotide are unmodified nucleobases.

[0163] In certain embodiments, the modified oligonucleotide comprises a block of modified nucleobases. In certain such embodiments, the block is at the 3'-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleotides of the 3'-end of the oligonucleotide. In certain embodiments, the block is at the 5'-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleotides of the 5'-end of the oligonucleotide.

[0164] In certain embodiments, an oligonucleotide having a spacer motif comprises a nucleoside comprising a modified nucleobase. In certain such embodiments, a nucleoside comprising a modified nucleobase is in the central gap of the oligonucleotide having a spacer motif. In certain such embodiments, the sugar moiety of the nucleoside is a 2'-deoxyribosyl sugar moiety. In certain embodiments, the modified nucleobase is selected from: 2-thiopyrimidine and 5-propynylpyrimidine. [Certain internucleoside linkage motifs] []

[0165] In certain embodiments, the oligonucleotide comprises modified and / or unmodified internucleoside linkages arranged in a defined pattern or motif along the oligonucleotide or a portion thereof. In certain embodiments, each internucleoside linking group is a phosphodiester internucleoside linkage. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is independently selected from phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In certain embodiments, each phosphorothioate internucleoside linkage is independently selected from random stereoisomeric phosphorothioates, (Sp) phosphorothioates, and (Rp) phosphorothioates. In certain embodiments, the sugar moiety of the modified oligonucleotide is a spacer and all internucleoside linkages within the gap are modified. In some such embodiments, some or all of the internucleoside linkages in the flanks are unmodified phosphodiester internucleoside linkages. In certain embodiments, the terminal internucleoside linkage is modified. In certain embodiments, the sugar moiety of the modified oligonucleotide is a spacer and the internucleoside linkage motif comprises at least one phosphodiester internucleoside linkage in at least one flank, wherein the at least one phosphodiester internucleoside linkage is not a terminal internucleoside linkage and the remaining internucleoside linkages are phosphorothioate internucleoside linkages. In some such embodiments, all phosphorothioate internucleoside linkages are random stereoisomeric. In certain embodiments, all phosphorothioate internucleoside linkages in the flanks are (Sp) phosphorothioates and the gap comprises at least one Sp, Sp, Rp motif. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides comprising such internucleoside linkage motifs. In certain embodiments, one or more internucleoside linkages are methylsulfonamidate phosphonate internucleoside linkages. In certain embodiments, each internucleoside linkage is independently selected from phosphodiester internucleoside linkages, phosphorothioate internucleoside linkages, and methylsulfonamidate phosphonate internucleoside linkages. In certain embodiments, each internucleoside linkage is independently selected from phosphorothioate internucleoside linkages and methylsulfonamidate phosphonate internucleoside linkages. In certain embodiments, one or more internucleoside linkages are methoxypropylphosphonate internucleoside linkages. In certain embodiments, each internucleoside linkage is independently selected from phosphodiester internucleoside linkages, phosphorothioate internucleoside linkages, and methoxypropylphosphonate internucleoside linkages. In certain embodiments, each internucleoside linkage is independently selected from phosphorothioate internucleoside linkages and methoxypropylphosphonate internucleoside linkages.

[0166] In certain embodiments, the modified oligonucleotide comprises at least 1, 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, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 phosphodiester internucleoside linkages. In certain embodiments, the modified oligonucleotide comprises at least 1, 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, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 phosphorothioate internucleoside linkages. In certain embodiments, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, or at least 5 phosphodiester internucleoside linkages, and the remaining internucleoside linkages are phosphorothioate internucleoside linkages. [Certain lengths] []

[0167] It is possible to increase or decrease the length of the oligonucleotide without eliminating activity. For example, in Woolf et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 7305-7309, 1992), a series of oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target nucleic acid in an oocyte injection model. Oligonucleotides 25 nucleobases in length and having 8 or 11 mismatched bases near the ends of the oligonucleotide were able to direct specific cleavage of the target nucleic acid, although to a lesser extent than oligonucleotides without mismatches. Similarly, target-specific cleavage was achieved using 13-nucleobase oligonucleotides, including those having 1 or 3 mismatches.

[0168] In certain embodiments, oligonucleotides (including modified oligonucleotides) can have any of a variety of length ranges. In certain embodiments, an oligonucleotide consists of from X to Y linked nucleosides, where X represents the minimum number of nucleosides in the range and Y represents the maximum number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50; provided that X ≤ Y. For example, in certain embodiments, an oligonucleotide consists of from 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26,Composed of 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30 or 29 to 30 linked nucleosides.

[0169] In certain embodiments, the oligonucleotide is composed of 16 linked nucleosides. In certain embodiments, the oligonucleotide is composed of 17 linked nucleosides. In certain embodiments, the oligonucleotide is composed of 18 linked nucleosides. In certain embodiments, the oligonucleotide is composed of 19 linked nucleosides. In certain embodiments, the oligonucleotide is composed of 20 linked nucleosides. [Certain modified oligonucleotides] []

[0170] In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into the modified oligonucleotide. In certain embodiments, the modified oligonucleotide is characterized by its modification motif and overall length. In certain embodiments, such parameters are independent of each other. Thus, unless otherwise indicated, each internucleoside linkage of the oligonucleotide with a spacer motif can be modified or unmodified and can follow or not follow the spacer modification pattern of the sugar modification. For example, the internucleoside linkages within the flanking regions of the sugar spacer can be the same or different from each other and can be the same or different from the internucleoside linkages in the gap region of the sugar motif. Similarly, such sugar spacer oligonucleotides can contain one or more modified nucleobases that are not related to the spacer pattern of the sugar modification. Unless otherwise indicated, all modifications are independent of the nucleobase sequence. [Populations of certain modified oligonucleotides] []

[0171] A population of modified oligonucleotides, where all of the modified oligonucleotides in the population have the same molecular formula, can be a racemic population or an enantiomerically enriched population. In a racemic population, all of the chiral centers of all of the modified oligonucleotides are racemic. In an enantiomerically enriched population, at least one specific chiral center of the modified oligonucleotides of the population is not racemic. In certain embodiments, the modified oligonucleotides of the enantiomerically enriched population are enriched in β-D-ribosyl sugar moieties and all phosphorothioate internucleotide linkages are racemic. In certain embodiments, the modified oligonucleotides of the enantiomerically enriched population are enriched in β-D-ribosyl sugar moieties and at least one specific phosphorothioate internucleotide linkage having a specific stereochemical configuration. [Nucleobase sequence] []

[0172] In certain embodiments, an oligonucleotide (unmodified or modified) is further described by its nucleobase sequence. In certain embodiments, the nucleobase sequence of the oligonucleotide is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, the nucleobase sequence of a portion of the oligonucleotide is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a portion or the entire length of the oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a second oligonucleotide or nucleic acid, such as a target nucleic acid. [Certain oligomeric compounds] []

[0173] In certain embodiments, provided herein are oligomeric compounds that consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugating groups and / or terminal groups. A conjugating group consists of one or more conjugate moieties and a conjugation linker that connects the conjugate moiety to the oligonucleotide. The conjugating group can be attached to either or both ends of the oligonucleotide and / or at any internal position. In certain embodiments, the conjugating group is attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, a conjugating group attached to either or both ends of the oligonucleotide is a terminal group. In certain embodiments, the conjugating group or terminal group is attached to the 3'- and / or 5'-end of the oligonucleotide. In certain such embodiments, the conjugating group (or terminal group) is attached to the 3'-end of the oligonucleotide. In certain embodiments, the conjugating group is attached near the 3'-end of the oligonucleotide. In certain embodiments, the conjugating group (or terminal group) is attached to the 5'-end of the oligonucleotide. In certain embodiments, the conjugating group is attached near the 5'-end of the oligonucleotide.

[0174] Examples of end groups include, but are not limited to, conjugation groups, capping groups, phosphate moieties, protecting groups, abasic nucleosides, modified or unmodified nucleosides, and two or more independently modified or unmodified nucleosides. [Certain conjugation groups] []

[0175] In certain embodiments, the oligonucleotide is covalently linked to one or more conjugating groups. In certain embodiments, the conjugating group modifies one or more properties of the linked oligonucleotide, including but not limited to pharmacology, pharmacokinetics, stability, binding, absorption, tissue distribution, cell distribution, cell uptake, charge, and clearance. In certain embodiments, the conjugating group confers new properties on the linked oligonucleotide, such as a fluorophore or information group capable of detecting the oligonucleotide.Certain conjugating groups and conjugating moieties have been previously described, such as: cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556); cholanic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538); aliphatic chains, such as dodecane-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54); phospholipids, such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate triethyl-ammonium (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantane acetic acid palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237); octadecylamine or hexylamino-carbonyl-hydroxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937); tocopherol groups (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740) or GalNAc clusters (such as WO2014 / 179620). [Conjugation moiety] []

[0176] The conjugation moiety includes, but is not limited to, inserts, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterol, thiolcholesterol, bile acid moieties, folates, lipids, lipophilic groups, phospholipids, biotin, phenazines, phenanthridines, anthraquinones, adamantanes, acridines, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

[0177] In certain embodiments, the conjugation moiety contains an active pharmaceutical ingredient, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazine, chlorothiazide, diazepam, indomethicin, barbiturate, cephalosporin, sulfonamide, antidiabetic agent, antibacterial agent, or antibiotic. [Conjugation linker] []

[0178] The conjugation moiety is connected to the oligonucleotide through a conjugation linker. In certain oligomeric compounds, the conjugation linker is a single chemical bond (i.e., the conjugation moiety is directly connected to the oligonucleotide through a single bond). In certain oligomeric compounds, the conjugation moiety is connected to the oligonucleotide via a more complex conjugation linker that includes one or more conjugation linker moieties, which are sub-units that make up the conjugation linker. In certain embodiments, the conjugation linker includes an oligomer of a linking structure (such as a hydrocarbon chain) or repeating units (such as ethylene glycol, nucleoside, or amino acid units).

[0179] In certain embodiments, the conjugate linker comprises one or more groups selected from the following: alkyl, amine, side-oxy group, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamine group. In certain such embodiments, the conjugate linker comprises a group selected from the following: alkyl, amine, side-oxy group, amide, and ether group. In certain embodiments, the conjugate linker comprises a group selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate moiety. In certain embodiments, the conjugate linker includes at least one neutral linking group.

[0180] In certain embodiments, the conjugate linker, including the conjugate linker described above, is a bifunctional linking moiety, such as those known in the art for use in linking a conjugate group to a parent compound, such as the oligonucleotides provided herein. Generally, the bifunctional linking moiety comprises at least two functional groups. One functional group is selected to bind to a specific site on the parent compound, and the other functional group is selected to bind to the conjugate group. Examples of functional groups used in the bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, the bifunctional linking moiety comprises one or more groups selected from the following: amine, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[0181] Examples of conjugate linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid N-succinimidyl ester (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include, but are not limited to, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl, where a non-limiting list of preferred substituents includes hydroxyl, amine, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0182] In certain embodiments, the conjugate linker comprises 1 - 10 linker - nucleosides. In certain embodiments, the conjugate linker comprises 2 - 5 linker - nucleosides. In certain embodiments, the conjugate linker comprises exactly 3 linker - nucleosides. In certain embodiments, the conjugate linker comprises a TCA motif. In certain embodiments, such linker - nucleosides are modified nucleosides. In certain embodiments, such linker - nucleosides comprise a modified sugar moiety. In certain embodiments, the linker - nucleosides are unmodified. In certain embodiments, the linker - nucleosides comprise an optionally protected heterocyclic base selected from: purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside selected from: uracil, thymine, cytosine, 4 - N - benzoyl cytosine, 5 - methyl cytosine, 4 - N - benzoyl - 5 - methyl cytosine, adenine, 6 - N - benzoyl adenine, guanine and 2 - N - isobutyryl guanine. It is generally desirable for the linker - nucleosides to cleave from the oligomeric compound after the oligomeric compound reaches the target tissue. Thus, the linker - nucleosides are typically linked to each other and to the remainder of the oligomeric compound by cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

[0183] As used herein, linker - nucleosides are not considered part of an oligonucleotide. Thus, in embodiments where the oligomeric compound comprises an oligonucleotide composed of a specified number or range of linked nucleosides and / or having a specified percentage complementarity to a reference nucleic acid and / or the oligomeric compound also comprises a conjugate group comprising a conjugate linker having linker - nucleosides, those linker - nucleosides are not counted in the length of the oligonucleotide and are not used to determine the percentage complementarity of the oligonucleotide to the reference nucleic acid. For example, an oligomeric compound can comprise (1) a modified oligonucleotide composed of 8 - 30 nucleosides and (2) a conjugate group comprising 1 - 10 linker - nucleosides contiguous with the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such an oligomeric compound is more than 30. Alternatively, the oligomeric compound can comprise a modified oligonucleotide composed of 8 - 30 nucleosides and having no conjugate group. The total number of contiguous linked nucleosides in such an oligomeric compound is not more than 30. Unless otherwise indicated, the conjugate linker comprises no more than 10 linker - nucleosides. In certain embodiments, the conjugate linker comprises no more than 5 linker - nucleosides. In certain embodiments, the conjugate linker comprises no more than 3 linker - nucleosides. In certain embodiments, the conjugate linker comprises no more than 2 linker - nucleosides. In certain embodiments, the conjugate linker comprises no more than 1 linker - nucleosides.

[0184] In certain embodiments, it is desirable to cleave a conjugate group from an oligonucleotide. For example, in some cases, an oligomeric compound comprising a particular conjugate moiety may be better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable to cleave the conjugate group to release the unconjugated or parent oligonucleotide. Accordingly, certain conjugate linkers may comprise one or more cleavable moieties. In certain embodiments, the cleavable moiety is a cleavable bond. In certain embodiments, the cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, the cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, the cleavable moiety is selectively cleaved within a cell or subcellular compartment such as a lysosome. In certain embodiments, the cleavable moiety is selectively cleaved by an endogenous enzyme such as a nuclease.

[0185] In certain embodiments, the cleavable bond is selected from: an amide, an ester, an ether, one or two esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, the cleavable bond is one or two esters of a phosphodiester. In certain embodiments, the cleavable moiety comprises a phosphate ester or a phosphodiester. In certain embodiments, the cleavable moiety is a phosphoester linkage between the oligonucleotide and the conjugate moiety or conjugate group.

[0186] In certain embodiments, the cleavable moiety comprises one or more linker-nucleosides or consists thereof. In certain such embodiments, the one or more linker-nucleosides are connected to each other and / or to the remainder of the oligomeric compound by cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, the cleavable moiety is a 2'-deoxyribonucleoside that is linked to the 3' or 5'-terminal nucleoside of the oligonucleotide by a phosphoester nucleoside-to-nucleoside linkage and is covalently linked to the remainder of the conjugate linker or conjugate moiety by a phosphoester or phosphorothioate nucleoside-to-nucleoside linkage. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine. [Certain terminal groups] []

[0187] In certain embodiments, the oligomeric compound comprises one or more terminal groups. In certain such embodiments, the oligomeric compound comprises a stabilized 5'-phosphate ester. Stabilized 5'-phosphate esters include, but are not limited to, 5'-phosphonate esters, including, but not limited to, 5'-vinylphosphonate esters. In certain embodiments, the terminal group comprises one or more abasic nucleosides and / or inverted nucleosides. In certain embodiments, the terminal group comprises one or more 2'-linked nucleosides. In certain such embodiments, the 2'-linked nucleoside is an abasic nucleoside. [Oligomeric double helix] []

[0188] In certain embodiments, the oligomeric compounds described herein include oligonucleotides having nucleobase sequences complementary to the sequences of target nucleic acids. In certain embodiments, an oligomeric compound pairs with a second oligomeric compound to form an oligomeric double helix. Such an oligomeric double helix includes a first oligomeric compound having a portion complementary to the target nucleic acid and a second oligomeric compound having a portion complementary to the first oligomeric compound. In certain embodiments, the first oligomeric compound of the oligomeric double helix comprises (1) a modified or unmodified oligonucleotide and optionally conjugated groups and (2) a second modified or unmodified oligonucleotide and optionally conjugated groups, or consists of the foregoing. Either or both of the oligomeric compounds of the oligomeric double helix may include conjugated groups. The oligonucleotides of each oligomeric compound of the oligomeric double helix may include non-complementary overhanging nucleosides. [Antisense activity] []

[0189] In certain embodiments, the oligomeric compounds and oligomeric double helices are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity; such oligomeric compounds and oligomeric double helices are antisense compounds. In certain embodiments, an antisense compound has antisense activity when it reduces, modulates, or increases the amount or activity of a target nucleic acid by 25% or more in a standard cell assay. In certain embodiments, an antisense compound selectively affects one or more target nucleic acids. Such antisense compounds include nucleobase sequences that hybridize to one or more target nucleic acids to produce one or more desired antisense activities and do not hybridize to one or more non-target nucleic acids, or do not hybridize to one or more non-target nucleic acids in a manner that produces significant undesired antisense activities.

[0190] In certain antisense activities, hybridization of an antisense compound to a target nucleic acid results in the recruitment of a protein that cleaves the target nucleic acid. For example, certain antisense compounds result in RNase H-mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double helix. The DNA in such an RNA:DNA double helix need not be unmodified DNA. In certain embodiments, antisense compounds that are sufficiently "DNA-like" to elicit RNase H activity are provided herein. In certain embodiments, one or more non-DNA-like nucleosides in the spacer gap are tolerated.

[0191] In certain antisense activities, an antisense compound or a portion of the antisense compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain antisense compounds cause cleavage of the target nucleic acid by Argonaute. The antisense compound loaded into RISC is an RNAi compound. The RNAi compound can be double-stranded (siRNA) or single-stranded (ssRNA).

[0192] In certain embodiments, hybridization of the antisense compound with the target nucleic acid does not result in recruitment of a protein that cleaves the target nucleic acid. In certain embodiments, hybridization of the antisense compound with the target nucleic acid results in a change in splicing of the target nucleic acid. In certain embodiments, hybridization of the antisense compound with the target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain embodiments, hybridization of the antisense compound with the target nucleic acid results in a change in translation of the target nucleic acid. In certain embodiments, hybridization of the antisense compound with the target nucleic acid results in exon inclusion. In certain embodiments, hybridization of the antisense compound with the target nucleic acid results in an increase in the amount or activity of the target nucleic acid. In certain embodiments, hybridization of an antisense compound complementary to the target nucleic acid results in a change in splicing, thereby resulting in exon inclusion in the mRNA.

[0193] Antisense activity can be observed directly or indirectly. In certain embodiments, observing or detecting antisense activity involves observing or detecting a change in the amount of the target nucleic acid or the protein encoded by the target nucleic acid, a change in the ratio of splicing variants of the nucleic acid or protein, and / or a phenotypic change in a cell or an individual. [Certain target nucleic acids] []

[0194] In certain embodiments, the oligomeric compound comprises or consists of an oligonucleotide that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: mature mRNA and precursor mRNA, including introns, exons, and untranslated regions. In certain embodiments, the target nucleic acid is mature mRNA. In certain embodiments, the target nucleic acid is precursor mRNA. In certain embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron / exon junction. In certain embodiments, at least 50% of the target region is within an intron. [Complementary to the target nucleic acid] [ / ] [Mismatch]

[0195] It is possible to introduce mismatched bases without eliminating activity. For example, Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide that is 100% complementary to bcl-2 mRNA and has 3 mismatches with abcl-xL mRNA to reduce the expression of bcl-2 and bcl-xL in vivo and in vitro. In addition, this oligonucleotide showed effective antitumor activity in vivo. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested the ability of a series of tandem 14-nucleotide oligonucleotides and 28- and 42-nucleotide oligonucleotides containing sequences of two or three tandem oligonucleotides to inhibit the translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14-nucleotide oligonucleotides alone was able to inhibit translation, but at a more moderate level compared to the 28- or 42-nucleotide oligonucleotides.

[0196] In certain embodiments, the oligonucleotide is complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, the oligonucleotide is 99%, 95%, 90%, 85% or 80% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and a portion is 100% or fully complementary to the target nucleic acid. In certain embodiments, the length of the fully complementary portion is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0197] In certain embodiments, the oligonucleotide contains one or more mismatched bases relative to the target nucleic acid. In certain embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 from the 5' end of the oligonucleotide. [SMN2] []

[0198] In certain embodiments, the oligomeric compound comprises or consists of a modified oligonucleotide that is complementary to the target nucleic acid encoding SMN2 or a portion thereof. In certain embodiments, SMN2 has the sequence shown in SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777).

[0199] In certain embodiments, contacting a cell with an oligomeric compound complementary to SEQ ID NO: 1 modulates the splicing of SMN2 RNA in the cell. In certain embodiments, contacting a cell with an oligomeric compound complementary to SEQ ID NO: 1 increases the amount of SMN2 RNA including exon 7. In certain embodiments, contacting a cell with an oligomeric compound complementary to SEQ ID NO: 1 increases the expression of full-length SMN2 protein. In certain embodiments, the oligomeric compound consists of a modified oligonucleotide.

[0200] In certain embodiments, contacting a cell of an individual with an oligomeric compound complementary to SEQ ID NO: 1 ameliorates one or more symptoms of a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is SMA, including type I SMA, type II SMA, type III SMA, and type IV SMA. In certain embodiments, the symptoms are any of the following: reduced muscle strength; inability or reduced ability to sit upright, stand, and / or walk; reduced neuromuscular activity; reduced electrical activity of one or more muscles; reduced respiration; inability or reduced ability to eat, drink, and / or breathe without assistance; weight loss or reduced weight gain; and / or reduced survival rate.

[0201] In certain embodiments, when administered according to a standard in vivo assay, the oligomeric compound complementary to SEQ ID NO: 1 is capable of increasing SMN2 RNA including exon 7 in vivo by at least 1-fold, 2-fold, or 3-fold. In certain embodiments, when administered according to a standard in vivo assay, the oligomeric compound complementary to SEQ ID NO: 1 is capable of increasing full-length SMN2 protein in vivo by at least 1-fold, 2-fold, or 3-fold. [Certain target nucleic acids in certain tissues] []

[0202] In certain embodiments, the oligomeric compound comprises or consists of an oligonucleotide that is partially complementary to a target nucleic acid, wherein the target nucleic acid is expressed in a pharmacologically relevant tissue. In certain embodiments, the pharmacologically relevant tissue is cells and tissues that make up the central nervous system (CNS). Such tissues include brain tissues such as spinal cord, cortex, and corona cerebri. [Certain pharmaceutical compositions] []

[0203] In certain embodiments, pharmaceutical compositions are described herein that comprise one or more oligomeric compounds. In certain embodiments, each of the one or more oligomeric compounds is composed of a modified oligonucleotide. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutical composition comprises and consists of a sterile saline solution and one or more oligomeric compounds. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, the pharmaceutical composition comprises and consists of one or more oligomeric compounds and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, the pharmaceutical composition comprises and consists of one or more oligomeric compounds and phosphate buffered saline (PBS). In certain embodiments, the sterile PBS is pharmaceutical grade PBS. In certain embodiments, the pharmaceutical composition comprises and consists of one or more oligomeric compounds and artificial cerebrospinal fluid (“artificial CSF” or “aCSF”). In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.

[0204] In certain embodiments, the pharmaceutical composition comprises a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the pharmaceutical composition consists of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the pharmaceutical composition consists essentially of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.

[0205] In certain embodiments, the pharmaceutical composition comprises one or more oligomeric compounds and one or more excipients. In certain embodiments, the excipients are selected from water, saline solution, ethanol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silica, viscous paraffin, hydroxyethyl cellulose, and polyvinylpyrrolidone.

[0206] In certain embodiments, the oligomeric compounds can be mixed with pharmaceutically acceptable active and / or inert substances for the preparation of a pharmaceutical composition or formulation. The composition and method of formulation of the pharmaceutical composition depend on a number of criteria, including but not limited to the route of administration, the degree of the disease, or the dose to be administered.

[0207] In certain embodiments, a pharmaceutical composition comprising an oligomeric compound encompasses any pharmaceutically acceptable salt of the oligomeric compound, an ester of the oligomeric compound, or a salt of such an ester. In certain embodiments, a pharmaceutical composition comprising an oligomeric compound that comprises one or more oligonucleotides is capable of providing (directly or indirectly) a bioactive metabolite or a residue thereof upon administration to a subject, including a human. Accordingly, for example, the present disclosure also relates to pharmaceutically acceptable salts of oligomeric compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other equivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts. In certain embodiments, a prodrug comprises one or more conjugating groups attached to an oligonucleotide, wherein the conjugating group is cleaved in vivo by endogenous nucleases. In certain embodiments, a prodrug comprises one or more conjugating groups attached to an oligonucleotide, wherein the conjugating group is cleaved in vivo by endogenous nucleases.

[0208] Lipid moieties have been used in nucleic acid therapies in a variety of ways. In some such methods, a nucleic acid, such as an oligomeric compound, is introduced into preformed liposomes or a lipid complex (lipoplex) made from a mixture of cationic and neutral lipids. In some methods, DNA complexes are formed with a single cationic lipid or polycationic lipid in the absence of neutral lipid. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to a particular cell or tissue. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to adipose tissue. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to muscle tissue.

[0209] In certain embodiments, the pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems can be used to prepare certain pharmaceutical compositions, including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0210] In certain embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules that are designed to deliver one or more agents comprising the oligomeric compounds provided herein to a particular tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises liposomes coated with a tissue-specific antibody.

[0211] In certain embodiments, the pharmaceutical composition comprises a co-solvent system. Some of such co-solvent systems include, for example, benzyl alcohol, non-polar surfactants, water-miscible organic polymers, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. Non-limiting examples of such co-solvent systems are VPD co-solvent systems, which are absolute ethanol solutions containing 3% w / v benzyl alcohol, 8% w / v non-polar surfactant polysorbate 80™, and 65% w / v polyethylene glycol 300. The proportions of such co-solvent systems can vary significantly without significantly altering their solubility and toxicity characteristics. In addition, the identity of the co-solvent components can vary: for example, other surfactants can be used in place of polysorbate 80™; the fraction size of polyethylene glycol can vary; other biocompatible polymers can replace polyethylene glycol, such as, polyvinylpyrrolidone; and other sugars or polysaccharides can replace dextrose.

[0212] In certain embodiments, the pharmaceutical composition is prepared for oral administration. In certain embodiments, the pharmaceutical composition is prepared for buccal administration. In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), etc.). In some such embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution, such as water or a physiologically compatible buffer, such as Hanks's solution, Ringer's solution, or saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in dissolution or act as preservatives). In certain embodiments, injectable suspensions are prepared using suitable liquid carriers, suspending agents, and the like. Some injectable pharmaceutical compositions are in unit dosage forms, for example, in ampoules or multi-dose containers. Some injectable pharmaceutical compositions are suspensions, solutions, or emulsions in an oily or aqueous vehicle and may contain formulating agents, such as suspending agents, stabilizers, and / or dispersing agents. Some solvents suitable for injectable pharmaceutical compositions include, but are not limited to: lipophilic solvents and fatty oils, such as sesame oil; synthetic fatty acid esters, such as ethyl oleate or triglycerides; and liposomes.

[0213] Under certain conditions, certain compounds disclosed herein act as acids. Although such compounds may be drawn or described in protonated (free acid) form or ionized and associated with a cation (salt) form, the aqueous solutions of such compounds exist in equilibrium in these forms. For example, the phosphodiester linkages of oligonucleotides in aqueous solution exist in equilibrium as free acid, anion, and salt forms. Unless otherwise indicated, the compounds described herein are intended to include all such forms. In addition, certain oligonucleotides have several such linkages, each of which is in equilibrium. Thus, an oligonucleotide in solution exists in a series of forms at multiple positions, all in equilibrium. The term "oligonucleotide" is intended to include all such forms. The structures drawn must depict a single form. However, unless otherwise indicated, such schemes are also intended to include the corresponding forms. In this text, a structure depicting the free acid of a compound followed by the term "or its salt" explicitly includes all such forms that may be fully or partially protonated / deprotonated / associated with a cation. In some cases, one or more specific cations are identified.

[0214] In certain embodiments, the modified oligonucleotide or oligomeric compound is in an aqueous solution containing sodium. In certain embodiments, the modified oligonucleotide or oligomeric compound is in an aqueous solution containing potassium. In certain embodiments, the modified oligonucleotide or oligomeric compound is in PBS. In certain embodiments, the modified oligonucleotide or oligomeric compound is in water. In certain such embodiments, the pH of the solution is adjusted with NaOH and / or HCl to achieve the desired pH.

[0215] In this text, certain specific doses are described. The doses can be in the form of dose units. For clarity, the dose (or dose unit) (in milligrams) of a modified oligonucleotide or oligomeric compound indicates the mass of the modified oligonucleotide or oligomeric compound in the free acid form. As described above, in an aqueous solution, the free acid is in equilibrium with the anionic and salt forms. However, for the purpose of calculating the dose, it is assumed that the modified oligonucleotide or oligomeric compound exists in a solvent-free, sodium acetate-free, water-free, free acid form. For example, in the case where a modified oligonucleotide or oligomeric compound is in a solution containing sodium (such as saline), the modified oligonucleotide or oligomeric compound can be partially or fully deprotonated and associated with Na+ ions. However, the mass of the proton is still included in the weight of the dose, while the mass of the Na+ ion is not included in the weight of the dose. Thus, for example, a dose or dose unit of 10 mg of Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 is equal to the number of fully protonated molecules weighing 10 mg. This is equivalent to: 10.53 mg of solvent-free, sodium acetate-free, water-free sodiated Compound No. 1263789; 10.53 mg of solvent-free, sodium acetate-free, water-free sodiated Compound No. 1287717; 10.52 mg of solvent-free, sodium acetate-free, water-free sodiated Compound No. 1287745; and 10.51 mg of solvent-free, sodium acetate-free, water-free sodiated Compound No. 1358996. When an oligomeric compound contains a conjugate group, the mass of the conjugate group is included in the calculation of the dose of such oligomeric compounds. If the conjugate group also has an acid, for the purpose of calculating the dose, it is also assumed that the conjugate group is fully protonated. [Certain Compositions] [] Compound No.: 1263789

[0216] In certain embodiments, Compound No. 1263789 is characterized as a modified oligonucleotide having the sequence (5' to 3') CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside contains a 2'-MOE sugar moiety, wherein the internucleoside linkages between nucleosides 2 to 3 and 4 to 5 are phosphodiester internucleoside linkages, and the internucleoside linkages between nucleosides 1 to 2, 3 to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, and 17 to 18 are phosphorothioate internucleoside linkages, and wherein each cytosine is 5-methylcytosine.

[0217] In certain embodiments, Compound No. 1263789 is represented by the following chemical notation (5' to 3'): mC esA eo mC esT eoT esT es mC esA esT esA esA esT esG es mC esT esG esG es mC e (SEQ ID NO: 21), wherein, A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, e = 2'-MOE sugar moiety, s = phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage.

[0218] In certain embodiments, Compound No. 1263789 is represented by the following chemical structure: (SEQ ID NO: 21). [Structure] [1.] [Compound No.] [1263789] []

[0219] In certain embodiments, the sodium salt of Compound No. 1263789 is represented by the following chemical structure: (SEQ ID NO: 21). [Structure] [2.] [Compound No.] [1263789] [of the sodium salt] [] Compound No.: 1287717

[0220] In certain embodiments, Compound No. 1287717 is characterized as a modified oligonucleotide having the sequence (5' to 3') TTCACTTTCATAATGCTGGC (SEQ ID NO: 22), wherein each nucleoside comprises a 2'-MOE sugar moiety, wherein the internucleoside linkages between nucleosides 1 to 2 and 19 to 20 are phosphodiester internucleoside linkages, and the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 17 to 18, and 18 to 19 are phosphorothioate internucleoside linkages, and wherein each cytosine is 5-methylcytosine.

[0221] In certain embodiments, Compound No. 1287717 is represented by the following chemical notation (5' to 3'): T eoT es mC esA es mC esT esT esT es mC esA esT esA esA esT esG es mC esT esG esG eo mC e(SEQ ID NO: 22) wherein, A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, e = 2'-MOE sugar moiety, s = phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage.

[0222] In certain embodiments, Compound No. 1287717 is represented by the following chemical structure: (SEQ ID NO: 22). [Structure] [3.] [Compound No.] [1287717] []

[0223] In certain embodiments, the sodium salt of Compound No. 1287717 is represented by the following chemical structure: (SEQ ID NO: 22). [Structure] [4.] [Compound Number] [1287717] [Sodium Salt of] [] Compound Number: 1287745

[0224] In certain embodiments, Compound Number 1287745 is characterized as a modified oligonucleotide having the sequence (5' to 3') TTCACTTTCATAATGCTGGC (SEQ ID NO: 22), wherein each of nucleosides 1 and 20 comprises a 2'-MOE sugar moiety, each of nucleosides 2-19 comprises a 2'-NMA sugar moiety, wherein the internucleoside linkages between nucleosides 1 to 2 and 19 to 20 are phosphodiester internucleoside linkages, and the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 17 to 18, and 18 to 19 are phosphorothioate internucleoside linkages, and wherein each cytosine is 5-methylcytosine.

[0225] In certain embodiments, Compound Number 1287745 is represented by the following chemical notation (5' to 3'): T eoT ns mC nsA ns mC nsT nsT nsT ns mC nsA nsT nsA nsA nsT nsG ns mC nsT nsG nsG no mC e(SEQ ID NO: 22) wherein, A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, e = 2'-MOE sugar moiety, n = 2'-NMA sugar moiety, s = phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage.

[0226] In certain embodiments, Compound Number 1287745 is represented by the following chemical structure: (SEQ ID NO: 22). [Structure] [5.] [Compound number] [1287745]

[0227] In certain embodiments, the sodium salt of compound number 1287745 is represented by the following chemical structure: (SEQ ID NO: 22). [Structure] [6.] [Compound number] [1287745] [of the sodium salt] Compound number: 1358996

[0228] In certain embodiments, compound number 1358996 is characterized as a modified oligonucleotide having the sequence (5' to 3') CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside comprises a 2'-NMA sugar moiety, wherein the internucleoside linkages between nucleosides 2 to 3 and 4 to 5 are phosphodiester internucleoside linkages, and the internucleoside linkages between nucleosides 1 to 2, 3 to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, and 17 to 18 are phosphorothioate internucleoside linkages, and wherein each cytosine is 5-methylcytosine.

[0229] In certain embodiments, compound number 1358996 is represented by the following chemical notation (5' to 3'): mC nsA no mC nsT noT nsT ns mC nsA nsT nsA nsA nsT nsG ns mC nsT nsG nsG ns mC n(SEQ ID NO: 21) wherein, A = adenine nucleobase, mC = 5-methylcytosine nucleobase,​​ G = guanine base, T = thymine base, n = 2'-NMA sugar moiety, s = phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage.

[0230] In certain embodiments, Compound No. 1358996 is represented by the following chemical structure: (SEQ ID NO: 21). [Structure] [7.] [Compound No.] [1358996] []

[0231] In certain embodiments, the sodium salt of Compound No. 1358996 is represented by the following chemical structure: (SEQ ID NO: 21). [Structure] [8.] [Compound No.] [1358996] [of the sodium salt] [] [Certain comparative compositions] []

[0232] In certain embodiments, Spinraza® (generic name nusinersen; compound number 396443), which is approved for the treatment of SMA, is a comparative compound (see, for example, Chiroboga et al., Neurology, 86(10): 890-897, 2016; Finkel et al., Lancet¸ 338(10063): 3017-3026, 2016; Finkel et al., N. Engl. J. Med., 377(18):1723-1732 2017; Mercuri et al., N. Engl. J. Med., 378(7):625-635, 2018; Montes et al., Muscle Nerve. 60(4): 409-414, 2019; Darras et al., Neurology, 92(21):e2492-e2506, 2019). Spinraza® was previously described in WO2010120820, which is incorporated herein by reference, and has the sequence (5’ to 3’) TCACTTTCATAATGCTGG (SEQ ID NO: 23), where each nucleoside contains a 2’-MOE sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is 5-methylcytosine.

[0233] In certain embodiments, other previously described compounds, including compound numbers 387954, 396442, 443305, and 819735, are comparative compounds, although not approved for human treatment.

[0234] Compound number 387954 was previously described in WO 2014 / 179620, which is incorporated herein by reference. Compound number 387954 has the sequence (5’ to 3’) ATTCACTTTCATAATGCTGG (SEQ ID NO: 20), where each nucleoside contains a 2’-MOE sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is 5-methylcytosine.

[0235] Compound number 396442 was previously described in WO 2010 / 120820, which is incorporated herein by reference. Compound number 396442 has the sequence (5’ to 3’) CACTTTCATAATGCTGGC (SEQ ID NO: 21), where each nucleoside contains a 2’-MOE sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is 5-methylcytosine.

[0236] Compound number 443305 was previously described in WO 2018 / 014041, which is incorporated herein by reference. Compound number 443305 has the sequence (5’ to 3’) TCACTTTCATAATGCTGG (SEQ ID NO: 23), wherein each nucleoside contains a 2’-NMA sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is 5-methylcytosine.

[0237] Compound number 819735 was previously described in WO 2018 / 014041, which is incorporated herein by reference. Compound number 819735 has the sequence (5’ to 3’) CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside contains a 2’-NMA sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is 5-methylcytosine. [Table] [1] [Certain comparative compositions] [] [Compound] [Number] [Nucleobase sequence] [(5’] [to 3’] [)] [Sugar motif] [Internucleoside linkage motif] [SEQ ID NO:] [Reference number] 396443 TCACTTTCATAATGCTGG all 2’-MOE all PS 23 WO 2010 / 120820 387954 ATTCACTTTCATAATGCTGG all 2’-MOE Fully PS 20 WO 2014 / 179620 396442 CACTTTCATAATGCTGGC Fully 2'-MOE Fully PS 21 WO 2010 / 120820 443305 TCACTTTCATAATGCTGG Fully 2'-NMA Fully PS 23 WO 2018 / 014041 819735 CACTTTCATAATGCTGGC Fully 2'-NMA Fully PS 21 WO 2018 / 014041

[0238] In certain embodiments, the compounds described herein are superior to the compounds described in WO 2007 / 002390, WO 2010 / 120820, WO 2015 / 161170 and WO 2018 / 014041 because they exhibit one or more improved properties such as potency, efficacy and tolerability.

[0239] For example, Compound No. 1263789, Compound No. 1287745 and Compound No. 1358996 each exhibit improved in vivo potency compared to Compound No. 396443. As shown in Example 5, Compound No. 1263789, Compound No. 1287745 and Compound No. 1358996 achieved ED50s of 13.3, 8.8 and 7.4 in the spinal cord, respectively. In contrast, Compound No. 396443 achieved an ED50 of 22.0 in the spinal cord. Thus, each of Compound No. 1263789, Compound No. 1287745 and Compound No. 1358996 is more effective than Compound No. 396443 in this assay.

[0240] For example, Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 each showed improved 3-hour FOB scores compared to Compound No. 396443, Compound No. 387954, and Compound No. 443305. As shown in Example 6, at 700 µg, Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 achieved 3-hour FOB scores of 0, 3.25, 1, and 0, respectively. In contrast, at half the dose (350 µg), Compound No. 396443 achieved a 3-hour FOB score of 4.0; and at the same dose (700 µg), Compound No. 387954 and Compound No. 443305 achieved 3-hour FOB scores of 4.0 and 4.75, respectively. Thus, each of Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 was more tolerant in this assay than Compound No. 396443, Compound No. 387954, and Compound No. 443305.

[0241] For example, Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 each showed improved long-term tolerance compared to Compound No. 396442 and Compound No. 819735. As shown in Example 7, Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 showed no adverse effects, no Purkinje cell loss, and cortical GFAP mRNA less than 2-fold of the control. In contrast, 396442 and 819735 each showed adverse events, Purkinje cell loss, and cortical GFAP mRNA greater than 2-fold of the control in some of the treated animals. Thus, each of Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 was more tolerant in this assay than Compound No. 396442 and Compound No. 819735. Non-limiting disclosure and incorporation by reference

[0242] The various documents and patent publications listed herein are incorporated herein by reference in their entirety. Although certain compounds, compositions, and methods described herein have been specifically described in terms of certain embodiments, the following examples are for illustrative purposes only and are not intended to limit the compounds described herein. Each of the references, GenBank accession numbers, and the like cited in this application are incorporated herein by reference in their entirety.

[0243] Although the Sequence Listing accompanying this application identifies each sequence as "RNA" or "DNA" as required, in reality, those sequences can be modified with any combination of chemical modifications. Those skilled in the art will readily appreciate that such nomenclature as "RNA" or "DNA" for describing modified oligonucleotides is arbitrary in some cases. For example, an oligonucleotide containing a nucleoside with a 2'-OH sugar moiety and a thymine base can be described as DNA with a modified sugar moiety (2'-OH replacing a 2'-H of DNA) or as RNA with a modified base (thymine (methylated uracil) replacing uracil of RNA). Thus, the nucleic acid sequences provided herein, including but not limited to the nucleic acid sequences in the Sequence Listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to such nucleic acids with modified nucleobases. Further by way of example and not limitation, an oligomeric compound with a nucleobase sequence of "ATCGATCG" encompasses any oligomeric compound with such a nucleobase sequence, whether modified or unmodified, including but not limited to: such compounds containing RNA bases, such as a sequence with "AUCGAUCG"; and those with some DNA bases and some RNA bases, such as "AUCGATCG"; and oligomeric compounds with other modified nucleobases, such as "AT mCGAUCG", where mC represents a cytosine base containing a methyl group at the 5 position.

[0244] Certain compounds described herein (e.g., modified oligonucleotides) have one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations, which can be defined as (R) or (S), α or β (such as for sugar anomers) or (D) or (L) (such as for amino acids) etc. according to absolute stereochemistry. Compounds depicted or described herein as having certain stereoisomeric configurations include only the indicated compounds. Unless otherwise specified, compounds depicted or described herein as having indeterminate stereochemistry include all such possible isomers, including their racemic and optically pure forms. Similarly, unless otherwise indicated, all cis and trans isomers and tautomeric forms of the compounds herein are also included. The oligomeric compounds described herein include enantiomerically pure or enantiomerically enriched mixtures as well as racemic mixtures. For example, oligomeric compounds having plural phosphorothioate internucleoside linkages include such compounds with chirally controlled or random phosphorothioate internucleoside linkages. Unless otherwise indicated, compounds described herein are intended to include the corresponding salt forms.

[0245] Compounds described herein include variants in which one or more atoms are replaced by non-radioactive or radioactive isotopes of the indicated element. For example, compounds herein containing hydrogen atoms encompass all possible deuterium substitutions of each 1H hydrogen atom. Isotope substitutions encompassed by the compounds herein include, but are not limited to: 2H or 3H in place of 1H; 13C or 14C in place of 12C; 15N in place of 14N; 17O or 18O in place of 16O; and 33S, 34S, 35S or 36S in place of 32S. In certain embodiments, non-radioactive isotope substitutions can impart new properties to the oligomeric compounds that are beneficial for use as therapeutic or research tools. In certain embodiments, radioactive isotope substitutions can render the compounds suitable for research or diagnostic purposes, such as imaging. [Examples] []

[0246] The following examples illustrate certain embodiments of the disclosure but are not limiting. In addition, in providing specific embodiments, the inventors contemplated the general application of their specific embodiments. [Examples] [1] [: with humans] [SMN2] [Design of Modified Oligonucleotides Complementary to Nucleic Acids] []

[0247] Design and synthesize modified oligonucleotides complementary to human SMN2 nucleic acid as indicated in the table below.

[0248] The length of the modified oligonucleotides in the table below is 16, 17, 18, 19, or 20 nucleotides, as specified. The modified oligonucleotides contain 2'-MOE sugar moieties, 2'-NMA sugar moieties, cEt sugar moieties, 2'-OMe sugar moieties, and / or 2'-β-D-deoxyribosyl sugar moieties, as specified. The internucleoside linkage of the entire modified oligonucleotide is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage, as specified. Cytosine is non-methylated cytosine or 5-methylcytosine, as specified.

[0249] Unless otherwise specifically stated, each of the modified oligonucleotides listed in the table below is 100% complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). Non-complementary nucleobases are indicated in the nucleobase sequence column by [, Underline, Bold, Italic Font Specification , ]. Each of the modified oligonucleotides listed in the table below targets the active site of the SMN2 transcript to include exon 7. "Start site" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table] [2] []

[0250] The length of the modified oligonucleotides in Table 2 below is 16, 17, 18, 19, or 20 nucleotides. Each nucleotide contains a 2'-MOE sugar moiety. The sugar moieties of each modified oligonucleotide are provided in the sugar moiety column, where each 'e' represents a 2'-MOE sugar moiety. The internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide is provided in the internucleoside linkage motif column, where each's' represents a phosphorothioate internucleoside linkage and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0251] Unless otherwise specifically stated, each of the modified oligonucleotides listed in Table 2 below is 100% complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). Non-complementary nucleobases are indicated in the nucleobase sequence column by [, Underline, Bold, Italic Font Specification , ]. The "starting point" indicates the most 5'-nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending point" indicates the most 3'-nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table] [2] [Having] [PS] [or mixed] [PS / PO] [Internucleoside linkage of] 2' [-MOE] [Modified oligonucleotide] [] [Compound number] [Nucleobase sequence] [(5'] [to 3')] [Sugar moiety] [(5'] [to 3')] [Internucleoside linkage moiety (5'] [to 3')] [SEQ ID No: 1] [Starting point] [SEQ ID No: 1] [Ending point] [SEQ ID No.] 1287063 ACTTTCATAATGCTGGCAG eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27059 27077 24 1287048 CACTTTCATAATGCTGGCAG eeeeeeeeeeeeeeeeeeee sssssssssssssssssss 27059 27078 25 1287064 CACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27060 27078 26 1287049 TCACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeeee sssssssssssssssssss 27060 27079 27 1210340 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sssssssssssssss 27061 27076 28 1212868 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sssssssssooooss 27061 27076 28 1212867 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssssssssoooosss 27061 27076 28 1212863 CTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeeee s 27061 27076 28 1212866 CTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeeee s 27061 27076 28 1212861 CTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeeee s 27061 27076 28 1212860 CTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeeee s 27061 27076 28 1212865 CTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeeee sssssssssssssss 27061 27076 28 1212859 CTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeeee s 27061 27076 28 1212851 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sssosssosssosss 27061 27076 28 1212850 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssosssssssssoss 27061 27076 28 1212852 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssossossossosss 27061 27076 28 1212853 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssossossosososs 27061 27076 28 1212854 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssososososososs 27061 27076 28 1212864 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee ssoooosssssssss 27061 27076 28 1212855 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee soossssssssooss 27061 27076 28 1212856 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sooosssssssooss 27061 27076 28 1212857 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee sooossssssoooss 27061 27076 28 1212858 CTTTCATAATGCTGGC eeeeeeeeeeeeeeee soooosssssoooss 27061 27076 28 1210339 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssssssssssssss 27061 27077 29 1212849 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssssssssooooss 27061 27077 29 1212848 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssssssoooossss 27061 27077 29 1212845 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee sssssssooossssss 27061 27077 29 1212844 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssssoooossssss 27061 27077 29 1212843 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssssooooosssss 27061 27077 29 1212842 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee sssssoooooosssss 27061 27077 29 1212841 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee sssssooooooossss 27061 27077 29 1212847 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssssoooossssssss 27061 27077 29 1212832 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssossssssssssoss 27061 27077 29 1212833 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssosssssossssoss 27061 27077 29 1212834 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssosssosssossoss 27061 27077 29 1212835 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssossossossososs 27061 27077 29 1212836 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssososososososss 27061 27077 29 1212846 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee ssoooossssssssss 27061 27077 29 1212837 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee soosssssssssooss 27061 27077 29 1212838 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee sooossssssssooss 27061 27077 29 1212839 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee sooosssssssoooss 27061 27077 29 1212840 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeeee soooossssssoooss 27061 27077 29 1263814 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssossssssssssssss 27061 27078 21 1263816 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssossssssssssss 27061 27078 21 1263818 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssossssssssss 27061 27078 21 1263820 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssossssssss 27061 27078 21 1263822 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssossssss 27061 27078 21 1263824 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssssossss 27061 27078 21 1263826 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssssssoss 27061 27078 21 1210342 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssosssssssssssoss 27061 27078 21 1263778 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sossssssssssssoss 27061 27078 21 1263781 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sosssssssssosssss 27061 27078 21 1263783 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sosssssssosssssss 27061 27078 21 1263785 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sosssssosssssssss 27061 27078 21 1263787 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sosssosssssssssss 27061 27078 21 1263789 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sososssssssssssss 27061 27078 21 1263791 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssosssssssssoss 27061 27078 21 1263793 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssosssssssoss 27061 27078 21 1263795 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssosssssoss 27061 27078 21 1263797 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssosssoss 27061 27078 21 1263799 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssssososs 27061 27078 21 1263800 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee soossssssssssssss 27061 27078 21 1263802 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssoossssssssssss 27061 27078 21 1263804 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssoossssssssss 27061 27078 21 1263806 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssoossssssss 27061 27078 21 1263808 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssssoossssss 27061 27078 21 1263810 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssssssoossss 27061 27078 21 1263812 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssssssssooss 27061 27078 21 1210343 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssosssssosssssoss 27061 27078 21 1212825 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssooosssssss 27061 27078 21 1212817 CACTTTCATAATGCTGGC Eeeeeeeeeeeeeeeeeeeeeee sooss 27061 27078 21 1212824 CACTTTCATAATGCTGGC Eeeeeeeeeeeeeeeeeeeeeee s 27061 27078 21 1212826 CACTTTCATAATGCTGGC Eeeeeeeeeeeeeeeeeeeeeee ssoooosssssssssssss 27061 27078 21 1212827 CACTTTCATAATGCTGGC Eeeeeeeeeeeeeeeeeeeeeee s 27061 27078 21 1212828 CACTTTCATAATGCTGGC Eeeeeeeeeeeeeeeeeeeeeee sssssssoooosssssss 27061 27078 21 1212829 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssoooosssss 27061 27078 21 1212830 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssoooosss 27061 27078 21 1212831 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssssssssooooss 27061 27078 21 1212818 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sooosssssssssooss 27061 27078 21 1212823 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssooooossssss 27061 27078 21 1212819 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sooossssssssoooss 27061 27078 21 1212822 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssoooooosssss 27061 27078 21 1212820 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee soooosssssssoooss 27061 27078 21 1212821 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee sssssooooooosssss 27061 27078 21 1287065 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27061 27079 30 1210341 ACTTTCATAATGCTGG eeeeeeeeeeeeeeee sssssssssssssss 27062 27077 31 524403 CACTTTCATAATGCTGG eeeeeeeeeeeeeeeee ssssssssssssssss 27062 27078 32 1287121 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssssssssoss 27062 27079 23 1287120 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssssssosss 27062 27079 23 1287113 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssssssooss 27062 27079 23 1287110 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssssssososs 27062 27079 23 1287119 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssssosssss 27062 27079 23 1364782 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssssososss 27062 27079 23 1364777 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssssossosss 27062 27079 23 1287118 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssosssssss 27062 27079 23 1364783 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssssosssosss 27062 27079 23 1287109 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssosssssoss 27062 27079 23 1364784 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssossssosss 27062 27079 23 1287117 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssosssssssss 27062 27079 23 1287112 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssssoossssssss 27062 27079 23 1287116 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssssosssssssssss 27062 27079 23 1287115 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sssosssssssssssss 27062 27079 23 1287114 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sosssssssssssssss 27062 27079 23 1287106 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sossssssssssssoss 27062 27079 23 1287107 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sossssssossssssss 27062 27079 23 1287108 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee sososssssssssssss 27062 27079 23 1287111 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee soossssssssssssss 27062 27079 23 1287066 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27062 27080 33 1287074 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee sssssssssssssssoss 27062 27080 33 1287071 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee sssssssssssssososs 27062 27080 33 1287073 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee ssssssssosssssssss 27062 27080 33 1287070 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee ssssssssossssssoss 27062 27080 33 1287072 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee sossssssssssssssss 27062 27080 33 1287067 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee sosssssssssssssoss 27062 27080 33 1287068 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee sossssssosssssssss 27062 27080 33 1287069 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee sosossssssssssssss 27062 27080 33 1287060 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee ssssssssssssssssoss 27062 27081 20 1287057 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee sssssssssssssssooss 27062 27081 20 1287054 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee ssssssssssssssososs 27062 27081 20 1287059 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee sssssssssosssssssss 27062 27081 20 1287053 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee sssssssssossssssoss 27062 27081 20 1287056 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee ssssssssoosssssssss 27062 27081 20 1287058 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee sosssssssssssssssss 27062 27081 20 1287050 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee sossssssssssssssoss 27062 27081 20 1287051 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee sosssssssosssssssss 27062 27081 20 1287052 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee sosssssssssssss 27062 27081 20 1287055 ATTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeeee soosssssssssssss 27062 27081 20 1287075 ATTCACTTTCATAATGCTG eeeeeeeeeeeeeeeeeee sssssssssssssss 27063 27081 34 1287062 AGATTCACTTTCATAATGCT eeeeeeeeeeeeeeeeeeee ssssssssssssssss 27064 27083 35 1287061 GATTCACTTTCATAATGCTG eeeeeeeeeeeeeeeeeeee ssssssssssssssss 27063 27082 49 1287076 GATTCACTTTCATAATGCT eeeeeeeeeeeeeeeeeee sssssssssssssss 27064 27082 50 1287701 TCACTTTCATAATGCTGG [ , T , ] eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27062 27079 36 1287702 TCACTTTCATAATGCTGG [ , A , ] eeeeeeeeeeeeeeeeeee ssssssssssssssssss 27062 27079 37 [Table] [3] [ ]

[0252] The length of the modified oligonucleotides in Table 3 below is 16, 17, 18, 19 or 20 nucleotides. Each nucleotide contains a 2'-NMA sugar moiety. The sugar moiety of each modified oligonucleotide is provided in the sugar moiety column, where each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage moiety of each modified oligonucleotide is provided in the internucleoside linkage moiety column, where each's' represents a phosphorothioate internucleoside linkage and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0253] Each modified oligonucleotide listed in Table 3 below is 100% complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). "Start site" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table] [3] [Having] [PS] [or a mixture] [PS / PO] [2’ of the internucleoside linkage] [-NMA] [modified oligonucleotide] [] [Compound number] [Nucleobase sequence] [(5’] [to 3’)] [Sugar moiety] [(5’] [to 3’)] [Internucleoside linkage moiety (5’] [to 3’)] [SEQ ID No: 1] [Starting point] [SEQ ID No: 1] [Termination point] [SEQ ID No.] 1287127 CACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnnn ssssssssssssssssss 27060 27078 26 1287122 TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnnnn sssssssssssssssssss 27060 27079 27 1212871 CTTTCATAATGCTGGC nnnnnnnnnnnnnnnn sssssssssssssss 27061 27076 28 1212869 ACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnn ssssssssssssssss 27061 27077 29 1358996 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn sososssssssssssss 27061 27078 21 1212873 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn ssosssssssssssoss 27061 27078 21 1212874 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn ssosssssosssssoss 27061 27078 21 1212875 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn ssosssosssosssoss 27061 27078 21 1212879 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn soosssssssssooss 27061 27078 21 1212880 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn sooossssssssooss 27061 27078 21 1212881 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn sooosssssssoooss 27061 27078 21 1212885 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn ssssssooooosssss 27061 27078 21 1212887 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnn sssssssooossssss 27061 27078 21 1287128 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnn sssssssssssssss 27061 27079 30 1212870 CACTTTCATAATGCTGG nnnnnnnnnnnnnnnnn ssssssssssssssss 27062 27078 32 1287132 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn sos ... 27062 27079 23 1287133 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn s ... 27062 27079 23 1332246 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn s ... 27062 27079 23 1332265 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn s ... 27062 27079 23 1364778 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn s ... 27062 27079 23 1364779 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn s ... 27062 27079 23 1364780 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn s ... 27062 27079 23 1364781 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn s ... 27062 27079 23 1287129 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn ssssssssssssssssss 27062 27080 33 1287130 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn sos ... 27062 27080 33 1287131 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn sssssssssssss 27062 27080 33 1332263 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn sssssssssssss 27062 27080 33 1332264 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn ssssssssssssss 27062 27080 33 1332266 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn ssssssssssssoss 27062 27080 33 1332270 TTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnn ssssssssssssooss 27062 27080 33 1287124 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnnn sssssssssssssssssss 27062 27081 20 1287125 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnnn sos ... 27062 27081 20 1287126 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnnn s ... 27062 27081 20 1332267 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnnn s ... 27062 27081 20 1332268 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnnn s ... 27062 27081 20 1332269 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnnn s ... 27062 27081 20 1332271 ATTCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnnnn sssssssssossssssoss 27062 27081 20 1287123 TTCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnnn sssssssssssssssssss 27061 27080 22 [Table] [4] [ ]

[0254] The length of the modified oligonucleotides in Table 4 below is 18 or 19 nucleotides. Each nucleotide contains a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar moiety of each modified oligonucleotide is provided in the sugar moiety column, where each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. The internucleoside linkage is a phosphorothioate internucleoside linkage. The internucleoside linkage moiety of each modified oligonucleotide is provided in the internucleoside linkage moiety column, where each's' represents a phosphorothioate internucleoside linkage. Each cytosine is 5-methylcytosine.

[0255] Unless otherwise specifically stated, each modified oligonucleotide listed in Table 4 below is 100% complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). Non-complementary nucleobases are designated in the nucleobase sequence column in underlined, bold, and italic font. "Start site" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table] [4] [Having] [PS] [Mixture of internucleoside linkages] 2’ [-MOE / ] 2’ [-NMA] [Modified oligonucleotide] [] [Compound number] [Nucleobase sequence] [(5’] [to 3’)] [Sugar moiety] [(5’] [to 3’)] [Internucleoside linkage moiety] [(5’] [to 3’)] [SEQ ID No: 1] [Start site] [SEQ ID No: 1] [Stop site] [SEQ ID No.] 1212931 CACTTTCATAATGCTGGC nennnnneneennnnnnn sssssssssssssssss 27061 27078 21 1212936 CACTTTCATAATGCTGGC nnnnnnnnnnnnenneen sssssssssssssssss 27061 27078 21 1212941 CACTTTCATAATGCTGGC nennnnneneenenneen ssssssssssssss 27061 27078 21 1287728 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnnne sssssssssssssss 27061 27079 30 1287729 TCACTTTCATAATGCTGG , T , nnnnnnnnnnnnnnnnnnnne sssssssssssssss 27062 27079 36 1287730 TCACTTTCATAATGCTGG , A , nnnnnnnnnnnnnnnnnnnne sssssssssssssss 27062 27079 37 [table] [5] ,

[0256] The lengths of the modified oligonucleotides in Table 5 below are 16, 17, or 18 nucleotides. Each nucleotide contains a 2'-MOE sugar moiety or a cEt sugar moiety. The sugar moiety of each modified oligonucleotide is provided in the sugar moiety column, where each 'e' represents a 2'-MOE sugar moiety and each 'k' represents a cEt sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage. The internucleoside linkage moiety of each modified oligonucleotide is provided in the internucleoside linkage moiety column, where each's' represents a phosphorothioate internucleoside linkage. Each cytosine is 5-methylcytosine.

[0257] Each modified oligonucleotide listed in Table 5 below is 100% complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). "Start site" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table] [5] [With] [PS] [Mixture of internucleoside linkages] 2' [-MOE / cEt] [Modified oligonucleotide] [] [Compound number] [Nucleobase sequence] [(5'] [to 3')] [Sugar moiety] [(5'] [to 3')] [Internucleoside linkage moiety (5'] [to 3')] [SEQ ID No: 1] [Start site] [SEQ ID No: 1] [Stop site] [SEQ ID No.] 1212961 CACTTTCATAATGCTGGC keekeekeekeekeeeek ssssssssssssss 27061 27078 21 1212962 CACTTTCATAATGCTGGC keeekeeekeeekeeeek ssssssssssssss 27061 27078 21 1212963 CACTTTCATAATGCTGGC keeeeekeeeeeekeeeek ssssssssssssss 27061 27078 21 1212964 CACTTTCATAATGCTGGC keeeeeeeeeeeeeee ssssssssssssss 27061 27078 21 1212965 CACTTTCATAATGCTGGC keeeeeeeeeeeeeeeeeek ssssssssssssss 27061 27078 21 1212966 CACTTTCATAATGCTGGC eeekeekeekeekeekeek ssssssssssssss 27061 27078 21 1212967 CACTTTCATAATGCTGGC eeekeekeekeekeekee ssssssssssssss 27061 27078 21 1212968 CACTTTCATAATGCTGGC eeeeeekeekeekeekee ssssssssssssss 27061 27078 21 1212969 CACTTTCATAATGCTGGC eeeeeekeekeekeeeeee ssssssssssssss 27061 27078 21 1212970 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeee ssssssssssssss 27061 27078 21 1212971 CACTTTCATAATGCTGGC keekeekeekeeeeeeee ssssssssssssss 27061 27078 21 1212972 CACTTTCATAATGCTGGC eeeeeeeeeekeekeekeek ssssssssssssss 27061 27078 21 1212973 CACTTTCATAATGCTGGC keekeekeeeeeeeeeeee ssssssssssssss 27061 27078 21 1212974 CACTTTCATAATGCTGGC eeeeeeeeeeeeekeekeek ssssssssssssss 27061 27078 21 1212975 CACTTTCATAATGCTGGC keekeeeeeeeeeeeeee ssssssssssssss 27061 27078 21 1212976 CACTTTCATAATGCTGGC eeeeeeeeeeeeeeeekeek ssssssssssssss 27061 27078 21 1212977 ACTTTCATAATGCTGGC keekeekeekeekeekeek sssssssssssss 27061 27077 29 1212978 ACTTTCATAATGCTGGC keeekeeekeeekeeek sssssssssssss 27061 27077 29 1212979 ACTTTCATAATGCTGGC keeeekeeeeeekeeeek sssssssssssss 27061 27077 29 1212980 ACTTTCATAATGCTGGC keeeeeeeeeeeeeee sssssssssssss 27061 27077 29 1212981 ACTTTCATAATGCTGGC keeeeeeeeeeeeeeeeek sssssssssssss 27061 27077 29 1212982 ACTTTCATAATGCTGGC eekeekeekeekeekeek sssssssssssss 27061 27077 29 1212983 ACTTTCATAATGCTGGC eekeekeekeekeekee sssssssssssss 27061 27077 29 1212984 ACTTTCATAATGCTGGC eeeeekeekeekeekee sssssssssssss 27061 27077 29 1212985 ACTTTCATAATGCTGGC eeeeekeekeekeeeeee sssssssssssss 27061 27077 29 1212986 ACTTTCATAATGCTGGC eeeeekeeeeeeeeeeee sssssssssssss 27061 27077 29 1212987 ACTTTCATAATGCTGGC keekeekeekeeeeeeee sssssssssssss 27061 27077 29 1212988 ACTTTCATAATGCTGGC eeeeeeeeekeekeekeek sssssssssssss 27061 27077 29 1212989 ACTTTCATAATGCTGGC keekeekeeeeeeeeee sssssssssssss 27061 27077 29 1212990 ACTTTCATAATGCTGGC eeeeeeeeeeeekeekeek sssssssssssss 27061 27077 29 1212991 ACTTTCATAATGCTGGC keekeeeeeeeeeeeeee sssssssssssss 27061 27077 29 1212992 ACTTTCATAATGCTGGC eeeeeeeeeeeeeeekeek sssssssssssss 27061 27077 29 1212993 CTTTCATAATGCTGGC keekeekeekeekeek sssssssssssss 27061 27076 28 1212994 CTTTCATAATGCTGGC keeekeeekeeekeek sssssssssssss 27061 27076 28 1212995 CTTTCATAATGCTGGC keeeekeeeekeeeek sssssssssssss 27061 27076 28 1212996 CTTTCATAATGCTGGC keeeeeeeeeeeee sssssssssssss 27061 27076 28 1212997 CTTTCATAATGCTGGC keeeeeeeeeeeeeeeek sssssssssssss 27061 27076 28 1212998 CTTTCATAATGCTGGC kekeekeekeekeekee sssssssssssss 27061 27076 28 1212999 CTTTCATAATGCTGGC eekeekeekeekeekee sssssssssssss 27061 27076 28 1213000 CTTTCATAATGCTGGC eeeeekeekeekeekee sssssssssssss 27061 27076 28 1213001 CTTTCATAATGCTGGC eeeeekeekeekeeee sssssssssssss 27061 27076 28 1213002 CTTTCATAATGCTGGC eeeeekeeeeeeeeee sssssssssssss 27061 27076 28 1213003 CTTTCATAATGCTGGC keekeekeekeeeeee sssssssssssss 27061 27076 28 1213004 CTTTCATAATGCTGGC eeeeeekeekeekeek sssssssssssss 27061 27076 28 1213005 CTTTCATAATGCTGGC keekeekeeeeeeeeee sssssssssssss 27061 27076 28 1213006 CTTTCATAATGCTGGC eeeeeeeeeeekeekeek sssssssssssss 27061 27076 28 1213007 CTTTCATAATGCTGGC keekeeeeeeeeeeee sssssssssssssss 27061 27076 28 1213008 CTTTCATAATGCTGGC eeeeeeeeeeeekeek sssssssssssssss 27061 27076 28 [Table] [6] []

[0258] The length of the modified oligonucleotides in Table 6 below is 19 or 20 nucleotides. Each nucleotide contains a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, a 2'-OMe sugar moiety, or a 2'-β-D-deoxyribosyl sugar moiety. The sugar motif of each modified oligonucleotide is provided in the sugar motif column, where each 'e' represents a 2'-MOE sugar motif, each 'n' represents a 2'-NMA sugar motif, each 'y' represents a 2'-OMe sugar motif, and each 'd' represents a 2'-β-D-deoxyribosyl sugar moiety. The internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the internucleoside linkage motif column is (5' to 3'): sssssssssssssssssso; where each's' represents a phosphorothioate internucleoside linkage and each 'o' represents a phosphodiester internucleoside linkage. Cytosine is non-methylated cytosine or 5-methylcytosine, where each lowercase letter 'c' in the nucleobase sequence column represents non-methylated cytosine and each uppercase letter 'C' in the nucleobase sequence column represents 5-methylcytosine.

[0259] Unless otherwise specifically stated, each nucleobase in the modified oligonucleotides listed in Table 6 below is complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). Non-complementary nucleobases are indicated in the nucleobase sequence column by [, Underline, Bold, Italic Font Specification , ]. The "starting point" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending point" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table] [6] [Modified oligonucleotides with mixed] [PS / PO] [Internucleoside linkages] [] [Compound number] [Nucleobase sequence] [(5'] [to 3')] [Sugar moiety] [(5'] [to 3')] [Internucleoside linkage moiety] [(5'] [to 3')] [SEQ ID No: 1] [Starting point] [SEQ ID No: 1] [Ending point] [SEQ ID No.] 1287707 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeed ssssssssssssssssso 27061 27079 30 1287708 TCACTTTCATAATGCTGGc eeeeeeeeeeeeeeeeeed ssssssssssssssssso 27061 27079 30 1287709 TCACTTTCATAATGCTGG [ , T , ] eeeeeeeeeeeeeeeeeed ssssssssssssssssso 27062 27079 36 1287710 TCACTTTCATAATGCTGG [ , A , ] eeeeeeeeeeeeeeeeeed ssssssssssssssssso 27062 27079 37 1287711 TCACTTTCATAATGCTGGc eeeeeeeeeeeeeeeeeey ssssssssssssssssso 27061 27079 30 1287712 TCACTTTCATAATGCTGG [ , U , ] eeeeeeeeeeeeeeeeeey ssssssssssssssssso 27062 27079 38 1287713 TCACTTTCATAATGCTGG [ , A , ] eeeeeeeeeeeeeeeeeeeeey sssssssssssssssssso 27062 27079 37 1287731 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnnnn sssssssssssssssssso 27061 27079 30 1287732 TCACTTTCATAATGCTGGc nnnnnnnnnnnnnnnnnnnnn sssssssssssssssssso 27061 27079 30 1287733 TCACTTTCATAATGCTGG [ , T , ] nnnnnnnnnnnnnnnnnnnnn sssssssssssssssssso 27062 27079 36 1287734 TCACTTTCATAATGCTGG [ , A , ] nnnnnnnnnnnnnnnnnnnnn sssssssssssssssssso 27062 27079 37 1287735 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnnnd sssssssssssssso 27061 27079 30 1287736 TCACTTTCATAATGCTGGc nnnnnnnnnnnnnnnnnnnnd sssssssssssssso 27061 27079 30 1287737 TCACTTTCATAATGCTGG , T , nnnnnnnnnnnnnnnnnnnnd sssssssssssssso 27062 27079 36 1287738 TCACTTTCATAATGCTGG , A , nnnnnnnnnnnnnnnnnnnnd sssssssssssssso 27062 27079 37 1287739 TCACTTTCATAATGCTGGc nnnnnnnnnnnnnnnnnnnny sssssssssssssso 27061 27079 30 1287740 TCACTTTCATAATGCTGG [ , U , ] nnn ... sssssssssssssssssso 27062 27079 38 1287741 TCACTTTCATAATGCTGG [ , A , ] nnn ... sssssssssssssssssso 27062 27079 37 1287705 TCACTTTCATAATGCTGG [ , T , ] eeeeeeeeeeeeeeeeeeee sssssssssssssssssso 27062 27079 36 1287706 TCACTTTCATAATGCTGG [ , A , ] eeeeeeeeeeeeeeeeeeee sssssssssssssssssso 27062 27079 37 1287704 TCACTTTCATAATGCTGGc eeeeeeeeeeeeeeeeeeee sssssssssssssssssso 27061 27079 30 1287703 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeee ssssssssssssssssso 27061 27079 30 [Table] [7] []

[0260] The length of the modified oligonucleotides in Table 7 below is 19 or 20 nucleotides. Each nucleotide contains a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, or a 2'-β-D-deoxyribosyl sugar moiety. The sugar moiety of each modified oligonucleotide is provided in the sugar moiety column, where each 'e' represents a 2'-MOE sugar moiety, each 'n' represents a 2'-NMA sugar moiety, and each 'd' represents a 2'-β-D-deoxyribosyl sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the internucleoside linkage motif column is (5' to 3'): ssssssssssssssssssoo; where each's' represents a phosphorothioate internucleoside linkage, and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0261] Unless otherwise specifically stated, each nucleobase in the modified oligonucleotides listed in Table 6 below is complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). Non-complementary nucleobases are indicated in the nucleobase sequence column by [, Underline, Bold, Italic Font Specification , ]. The "starting point" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending point" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table] [7] [Modified oligonucleotides with mixed] [PS / PO] [Internucleoside linkages] [] [Compound Number] [Nucleobase Sequence] [(5’] [to 3’)] [Sugar Moiety] [(5’] [to 3’)] [Inter-Nucleoside Linkage Moiety] [(5’] [to 3’)] [SEQ ID No: 1] [Start Site] [SEQ ID No: 1] [Stop Site] [SEQ ID No.] 1318749 TCACTTTCATAATGCTGG [, A , ]A nnnnnnnnnnnnnnnnnndd sssssssssssssssssoo 27062 27079 39 1318750 TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnned sssssssssssssssssoo 27060 27079 27 1318751 TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnndd sssssssssssssssssoo 27060 27079 27 1318752 TCACTTTCATAATGCTGG , A , ]A nnnnnnnnnnnnnnnnnnnned sssssssssssssssoo 27062 27079 39 1318753 TCACTTTCATAATGCTGG , A , ]A nnnnnnnnnnnnnnnnnnde sssssssssssssssoo 27062 27079 39 1318754 TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnnde sssssssssssssssoo 27060 27079 27 1318755 TCACTTTCATAATGCTGG , A , ]A nnnnnnnnnnnnnnnnnnee sssssssssssssssoo 27062 27079 39 1318756 TCACTTTCATAATGCTGGCA nnnnnnnnnnnnnnnnnnee sssssssssssssssoo 27060 27079 27 1318757 TCACTTTCATAATGCTGG [ , AT , ] eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 40 1318758 TCACTTTCATAATGCTGG [ , AC , ] eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 41 1318759 TCACTTTCATAATGCTGG [ , TC , ] eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 42 1318760 TCACTTTCATAATGCTGG [ , A , ]A eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 39 1318761 TCACTTTCATAATGCTGG [ , TT , ] eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 43 1318762 TCACTTTCATAATGCTGG [ , T , ]A eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27062 27079 44 1318763 TCACTTTCATAATGCTGGC [ , C , ] eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27061 27079 45 1318764 TCACTTTCATAATGCTGG [ , A , ]A eeeeeeeeeeeeeeeeeeed sssssssssssssssssoo 27062 27079 39 1318765 TCACTTTCATAATGCTGG [ , AC , ] eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27062 27079 41 1318766 TCACTTTCATAATGCTGGC [ , T , ] eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27061 27079 46 1318767 TCACTTTCATAATGCTGG [ , TT , ] eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27062 27079 43 1318768 TCACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeedd sssssssssssssssssoo 27060 27079 27 1318769 TCACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeeed sssssssssssssssssoo 27060 27079 27 1318770 TCACTTTCATAATGCTGG [ , AT , ] eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27062 27079 40 1318771 TCACTTTCATAATGCTGG [ , T , ]A ee s 27062 27079 44 1318772 TCACTTTCATAATGCTGG [ , A , ]A ee s 27062 27079 39 1318773 TCACTTTCATAATGCTGG [ , TC , ] ee s 27062 27079 42 1318774 TCACTTTCATAATGCTGGC [ , C , ] ee s 27061 27079 45 1318775 TCACTTTCATAATGCTGGC [ , T , ] eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27061 27079 46 1318776 TCACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeede sssssssssssssssssoo 27060 27079 27 1333508 TCACTTTCATAATGCTGGC [ , T , ] nnnnnnnnnnnnnnnnnnee sssssssssssssssssoo 27061 27079 46 1318777 TCACTTTCATAATGCTGG [ , AC , ] eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 41 1318778 TCACTTTCATAATGCTGG [ , TT , ] eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 43 1318779 TCACTTTCATAATGCTGG [ , A , ]A eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 39 1318780 TCACTTTCATAATGCTGG [ , TC , ] eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 42 1318781 TCACTTTCATAATGCTGG [ , AT , ] eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 40 1318782 TCACTTTCATAATGCTGGC [ , T , ] eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27061 27079 46 1318783 TCACTTTCATAATGCTGG [ , T , ]A eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27062 27079 44 1318784 TCACTTTCATAATGCTGGC [ , C , ] eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27061 27079 45 1318748 TCACTTTCATAATGCTGGCA eeeeeeeeeeeeeeeeeeee sssssssssssssssssoo 27060 27079 27 [Table] [8] []

[0262] The lengths of the modified oligonucleotides in Table 8 below are each 19 nucleotides. Each nucleotide contains a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, or a 2'-β-D-deoxyribosyl sugar moiety. The sugar moiety of each modified oligonucleotide is provided in the sugar moiety column, where each 'e' represents a 2'-MOE sugar moiety, each 'n' represents a 2'-NMA sugar moiety, and each 'd' represents a 2'-β-D-deoxyribosyl sugar moiety. The internucleoside linkage for each is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif for each modified oligonucleotide provided in the internucleoside linkage motif column is (5' to 3'): sssssssssssssososso; where each's' represents a phosphorothioate internucleoside linkage and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0263] Unless otherwise specifically stated, each nucleobase in the modified oligonucleotides listed in Table 8 below is complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). Non-complementary nucleobases are shown in the nucleobase sequence column as [, Underline, Bold, Italic Font Specification, ]. The "starting point" indicates the most 5'-nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending point" indicates the most 3'-nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table] [8] [With mixture] [PS / PO] [Modified oligonucleotide with internucleoside linkage] [] [Compound number] [Nucleobase sequence] [(5’] [to 3’)] [Sugar moiety] [(5’] [to 3’)] [Internucleoside linkage moiety] [(5’] [to 3’)] [SEQ ID No: 1] [Starting point] [SEQ ID No: 1] [Ending point] [SEQ ID No.] 1332247 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnd ssssssssssssososso 27061 27079 30 1332248 TCACTTTCATAATGCTGG [, A , ] nnnnnnnnnnnnnnnnnnd ssssssssssssososso 27062 27079 37 1332249 TCACTTTCATAATGCTGG [ , A , ] nnnnnnnnnnnnnnnnnne ssssssssssssososso 27062 27079 37 1332251 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnne ssssssssssssososso 27061 27079 30 1332255 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeed ssssssssssssososso 27061 27079 30 1332257 TCACTTTCATAATGCTGG [ , A , ] eeeeeeeeeeeeeeeeeed ssssssssssssososso 27062 27079 37 1332256 TCACTTTCATAATGCTGG [ , A , ] eeeeeeeeeeeeeeeeeee ssssssssssssososso 27062 27079 37 1332258 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeee ssssssssssssososso 27061 27079 30 [Table] [9] [ ]

[0264] The lengths of the modified oligonucleotides in Table 9 below are each 19 nucleotides. Each nucleotide contains a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, or a 2'-β-D-deoxyribosyl sugar moiety. The sugar moiety of each modified oligonucleotide is provided in the sugar moiety column, where each 'e' represents a 2'-MOE sugar moiety, each 'n' represents a 2'-NMA sugar moiety, and each 'd' represents a 2'-β-D-deoxyribosyl sugar moiety. The internucleoside linkage for each modified oligonucleotide is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif for each modified oligonucleotide provided in the internucleoside linkage motif column is (5' to 3'): sssssssssssssssosso; where each's' represents a phosphorothioate internucleoside linkage and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0265] Unless otherwise specifically stated, each nucleobase in the modified oligonucleotides listed in Table 9 below is complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). Non-complementary nucleobases are indicated in the nucleobase sequence column by [, Underline, Bold, Italic Font Specification , ]. "Start site" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table] [9] [With mixed] [PS / PO] [Modified Oligonucleotides with Nucleoside Linkages] [] [Compound Number] [Nucleobase Sequence] [(5’] [to 3’)] [Sugar Moiety] [(5’] [to 3’)] [Inter-Nucleoside Linkage Moiety] [(5’] [to 3’)] [SEQ ID No: 1] [Starting Point] [SEQ ID No: 1] [Termination Point] [SEQ ID No.] 1332250 TCACTTTCATAATGCTGG [, A , ] nnnnnnnnnnnnnnnnnnd ssssssssssssssosso 27062 27079 37 1332252 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnnd ssssssssssssssosso 27061 27079 30 1332253 TCACTTTCATAATGCTGG [, A , ] nnnnnnnnnnnnnnnnnne ssssssssssssssosso 27062 27079 37 1332254 TCACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnnne ssssssssssssssosso 27061 27079 30 1332259 TCACTTTCATAATGCTGG [ , A , ] eeeeeeeeeeeeeeeeeed ssssssssssssssosso 27062 27079 37 1332260 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeed ssssssssssssssosso 27061 27079 30 1332261 TCACTTTCATAATGCTGG [ , A , ] eeeeeeeeeeeeeeeeeee ssssssssssssssosso 27062 27079 37 1332262 TCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeee ssssssssssssssosso 27061 27079 30 [Table]

[10] [ ]

[0266] The lengths of the modified oligonucleotides in Table 10 below are each 19 nucleotides. Each nucleotide contains a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar moiety of each modified oligonucleotide is provided in the sugar moiety column, where each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the internucleoside linkage motif column is (5' to 3'): osssssssssssssssss; where each's' represents a phosphorothioate internucleoside linkage and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0267] Unless otherwise specifically stated, each nucleobase in the modified oligonucleotides listed in Table 10 below is complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). Non-complementary nucleobases are shown in the nucleobase sequence column as [, Underline, Bold, Italic Font Specification , ]. "Start site" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table]

[10] [Modified oligonucleotides with mixed] [PS / PO] [internucleoside linkages] [ ] [Compound number] [Nucleobase sequence] [(5'] [to 3')] [Sugar moiety] [(5’] [to 3’)] [Nucleoside linkage motif] [(5’] [to 3’)] [SEQ ID No: 1] [Start point] [SEQ ID No: 1] [End point] [SEQ ID No.] 1287742 [, C , ]TCACTTTCATAATGCTGG ennnnnnnnnnnnnnnnnn osssssssssssssssss 27062 27079 47 1287743 TTCACTTTCATAATGCTGG ennnnnnnnnnnnnnnnnn osssssssssssssssss 27062 27080 33 1287744 [, A , ]TCACTTTCATAATGCTGG ennnnnnnnnnnnnnnnnn osssssssssssssssss 27062 27079 48 1287714 [, C , ]TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee osssssssssssssssss 27062 27079 47 1287716 [ , A , ]TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee osssssssssssssssss 27062 27079 48 1287715 TTCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeeee osssssssssssssssss 27062 27080 33 [surface]

[11] []

[0268] The length of each modified oligonucleotide in Table 11 below is 20 nucleosides. Each nucleoside comprises a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif of each modified oligonucleotide is provided in the Sugar Motif column, wherein each 'e' represents a 2'-MOE sugar motif and each 'n' represents a 2'-NMA sugar motif. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the Internucleoside Linkage Motif column is (5' to 3'): ossssssssssssssssso; wherein each 's' represents a phosphorothioate internucleoside linkage and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0269] Each of the modified oligonucleotides listed in Table 11 below is 100% complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). "Start site" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. [Table]

[11] [With mixed] [PS / PO] [Modified oligonucleotides with internucleoside linkages] [] [Compound number] [Nucleobase sequence] [(5'] [to 3')] [Sugar moiety] [(5'] [to 3')] [Internucleoside linkage moiety] [(5'] [to 3')] [SEQ ID No: 1] [Start site] [SEQ ID No: 1] [Stop site] [SEQ ID No.] 1287745 TTCACTTTCATAATGCTGGC ennnnnnnnnnnnnnnnnne ossssssssssssssssso 27061 27080 22 1287717 TTCACTTTCATAATGCTGGC eeeeeeeeeeeeeeeeeeee ossssssssssssssssssso 27061 27080 22 [Example] [2] [:] [In transgenic mice and humans] [SMN2] [Activity of modified oligonucleotides complementary to, single dose] [(35 µg)]

[0270] The activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice. The Taiwanese strain of SMA type III mice was obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). These mice lack murine SMN and are homozygous for human SMN2 (mSMN - / -; hSMN2 + / +; FVB.Cg-Tg(SMN2)2HungSMN1tm1Hung / J, stock number 005058; Bar Harbor, Maine), or are murine heterozygous and human SMN2 (mSMN + / -; hSMN2 + / -; FVB.Cg-Tg(SMN2)2HungSMN1tm1Hung / J) heterozygous obtained by breeding HOM / HOM (stock number 00005058) with FVB / NJ (stock number 001800). Treatment

[0271] Homozygous or heterozygous transgenic mice were grouped, with 4 mice in each group. Each mouse received a single ICV bolus of 35 µg of the modified oligonucleotide. Comparative compounds 387954, 396442, and 396443 were also tested in this assay. A group of 4 mice received PBS as a negative control. RNA Analysis

[0272] Two weeks after treatment, the mice were sacrificed and RNA was extracted from the cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. The primer-probe set hSMN2vd#4_LTS00216_MGB (forward sequence: GCTGATGCTTTGGGAAGTATGTTA (SEQ ID NO: 11); reverse sequence: CACCTTCCTTCTTTTTGATTTTGTC, herein designated SEQ ID NO: 12; probe sequence: TACATGAGTGGCTATCATACT (SEQ ID NO: 13)) was used to determine the amount of SMN2 RNA including exon 7 (exon 7+). The primer-probe set hSMN2_Sumner68_PPS50481 (forward sequence: CATGGTACATGAGTGGCTATCATACTG (SEQ ID NO: 14); reverse sequence: TGGTGTCATTTAGTGCTGCTCTATG (SEQ ID NO: 15); probe sequence CCAGCATTTCCATATAATAGC (SEQ ID NO: 16)) was used to determine the amount of SMN2 RNA not including exon 7 (exon 7-). The primer-probe set hSMN2_LTS00935 (forward sequence: CAGGAGGATTCCGTGCTGTT (SEQ ID NO: 17); reverse sequence: CAGTGCTGTATCATCCCAAATGTC, (SEQ ID NO: 18); probe sequence: ACAGGCCAGAGCGAT (SEQ ID NO: 19)) was used to measure the total SMN2 RNA level.

[0273] Results are presented as fold change of RNA levels normalized to SMN2 levels relative to the PBS control. Each of Tables 12 - 18 represents a different experiment. [Table]

[12] [Modified oligonucleotides in homozygous transgenic mice on human] [SMN2 RNA] [Effect on splicing] [] [Compound number] [Dose (µg)] [Cortex] [Spinal cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1 1 1 1 396442 35 3.3 0.3 3.4 0.3 396443 35 3.0 0.5 2.3 0.5 524403 35 3.3 0.4 2.5 0.5 1210339 35 2.5 0.5 3.0 0.3 1210340 35 2.1 0.6 2.6 0.4 1210341 35 1.8 0.7 2.0 0.6 1210342 35 2.5 0.5 2.9 0.3 1210343 35 3.0 0.4 2.4 0.5 1212817 35 2.4 0.6 2.2 0.6 1212818 35 2.4 0.5 2.1 0.6 1212823 35 2.0 0.6 2.0 0.6 1212824 35 2.1 0.6 2.1 0.6 1212825 35 2.9 0.4 2.5 0.5 1212826 35 2.5 0.6 2.2 0.7 1212827 35 2.5 0.6 2.6 0.5 1212828 35 2.9 0.5 2.4 0.6 1212830 35 2.8 0.7 2.1 0.8 1212831 35 2.5 0.7 2.3 0.7 1212832 35 2.9 0.6 2.9 0.5 1212833 35 2.4 0.7 2.7 0.5 1212837 35 2.5 0.6 2.7 0.5 1212838 35 2.1 0.7 2.5 0.6 1212844 35 2.6 0.6 2.4 0.7 1212845 35 2.3 0.7 2.5 0.7 1212846 35 2.8 0.6 2.6 0.6 1212849 35 2.1 0.7 2.3 0.6 1212850 35 1.8 0.8 2.2 0.7 1212855 35 2.0 0.7 2.1 0.8 [Table]

[13] [] [Effect of Modified Oligonucleotides on Human [SMN2 RNA] [Splicing in Homozygous Transgenic Mice] [] [Compound Number] [Dose (µg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1.0 1.0 1.0 1.0 396443 35 2.7 0.3 1.9 0.5 1210342 35 2.4 0.5 2.5 0.4 1212961 35 1.8 0.7 1.7 0.6 1212962 35 2.0 0.6 1.9 0.5 1212963 35 2.3 0.5 2.5 0.3 1212966 35 1.6 0.8 2.0 0.5 1212967 35 1.9 0.6 1.9 0.4 1212971 35 1.6 0.5 2.0 0.4 1212972 35 1.8 0.6 2.2 0.5 1212977 35 2.1 0.5 2.2 0.4 1212978 35 2.1 0.6 2.2 0.4 1212979 35 2.1 0.5 2.6 0.3 1212982 35 2.0 0.7 1.8 0.6 1212983 35 1.9 0.6 1.7 0.5 1212984 35 1.9 0.6 1.9 0.5 1212987 35 2.4 0.4 2.5 0.4 1212988 35 1.8 0.7 1.8 0.5 1212995 35 2.5 0.5 2.5 0.4 1212998 35 1.8 0.6 1.8 0.7 1212999 35 2.0 0.6 2.0 0.5 1213003 35 1.9 0.7 2.3 0.5 1213004 35 1.8 0.7 2.3 0.6 [Table]

[14] [] [Effect of Modified Oligonucleotides on Human] [SMN2 RNA] [Splicing in Homozygous Transgenic Mice] [] [Compound Number] [Dose (µg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7,- , ] PBS - 1.0 1.0 1.0 1.0 396443 35 2.6 0.5 3.1 0.5 1212964 35 2.5 0.6 3.6 0.4 1212965 35 2.9 0.5 3.3 0.4 1212968 35 2.2 0.6 2.3 0.6 1212973 35 2.6 0.5 3.2 0.4 1212974 35 2.3 0.6 2.8 0.5 1212975 35 2.9 0.3 3.1 0.4 1212976 35 2.5 0.5 2.8 0.5 1212980 35 2.6 0.5 3.2 0.4 1212981 35 2.9 0.4 3.6 0.3 1212985 35 2.4 0.6 2.9 0.5 1212986 35 2.8 0.4 3.3 0.4 1212989 35 3.3 0.3 3.6 0.2 1212990 35 1.8 0.8 2.1 0.7 1212991 35 3.2 0.3 3.8 0.3 1212992 35 2.4 0.5 2.2 0.6 1212996 35 2.2 0.6 3.2 0.5 1212997 35 2.9 0.4 3.9 0.4 1213001 35 2.1 0.5 2.8 0.6 1213002 35 2.0 0.6 2.9 0.6 1213005 35 2.8 0.5 3.2 0.3 1213006 35 1.9 0.9 2.0 0.8 1213007 35 3.3 0.2 2.9 0.5 1213008 35 2.3 0.7 2.2 0.7 [surface]

[15] [] [Modified oligonucleotides in heterozygous transgenic mice to humans] [SMN2 RNA] [The impact of editing] [] [Compound number] [Dose (µg)] [Cortex] [Spinal cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1.0 1.0 1.0 1.0 387954 35 2.3 0.6 2.2 0.5 396443 35 2.5 0.5 2.4 0.5 1287048 35 2.2 0.5 2.2 0.5 1287049 35 2.3 0.6 2.5 0.4 1287061 35 2.4 0.5 2.2 0.4 1287062 35 3.0 0.3 2.3 0.4 1287050 35 2.8 0.5 2.3 0.4 1287054 35 2.2 0.5 2.3 0.4 1287063 35 1.8 0.7 1.7 0.6 1287064 35 2.6 0.3 2.4 0.4 1287065 35 2.5 0.4 2.3 0.4 1287066 35 2.2 0.5 2 0.5 1287075 35 2.3 0.6 1.8 0.7 1287076 35 2.6 0.4 1.9 0.6 1287067 35 2.7 0.4 1.9 0.6 1287070 35 2.5 0.5 1.8 0.7 1287071 35 2.6 0.4 1.8 0.7 1287074 35 2.6 0.5 2 0.6 1287109 35 2.7 0.6 2.4 0.5 1287110 35 2.6 0.5 2.3 0.5 1287701 35 2.6 0.6 2.8 0.3 1287702 35 3 0.5 2.8 0.4 1287703 35 2.3 0.6 2.4 0.4 1287704 35 2.7 0.5 2.3 0.4 1287717 35 3.3 0.3 2.4 0.6 [Table]

[16] [] [Effect of Modified Oligonucleotides on Human [SMN2 RNA] [Splicing in Transgenic Mice with Aberrant Junctions] [] [Compound Number] [Dose (µg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1 1 1 1 396442 35 2.5 0.6 3.2 0.3 396443 35 3 0.5 2.8 0.5 1263783 35 3 0.3 2.6 0.5 1263785 35 3.1 0.4 2.9 0.5 1263787 35 2.4 0.6 2.9 0.4 1263789 35 3.8 0.2 2.6 0.5 1263800 35 3.6 0.2 2.6 0.5 1263802 35 3.4 0.3 2.9 0.4 1263806 35 3.5 0.2 2.7 0.5 1263808 35 3.2 0.4 2.7 0.5 1263810 35 2.8 0.5 2.4 0.5 [Table]

[17] [] [Modified Oligonucleotides in Transgenic Mice with Heterologous Junctions on Human] [SMN2 RNA] [Effect on Splicing] [] [Compound Number] [Dose (µg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1 1 1 1 396443 35 2.5 0.6 2.9 0.4 1364784 35 2.3 0.7 2.8 0.5 1364783 35 2.9 0.5 2.4 0.5 1364777 35 2.7 0.6 2.3 0.5 1364782 35 2.7 0.6 2.6 0.5 [Table]

[18] [] [Modified Oligonucleotides in Transgenic Mice with Aberrant Junctions on Human] [SMN2 RNA] [Effect on Splicing] [] [Compound Number] [Dose (µg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1 1 1 1 396443 35 2 0.7 2.7 0.5 1318748 35 2.1 0.7 2.5 0.6 1318782 35 2.2 0.8 2.5 0.6 1332262 35 3.3 0.4 2.9 0.5 1332258 35 2.4 0.7 2.3 0.6 [Example] [3] [: Activity of Modified Oligonucleotides Complementary to Human] [SMN2] [in Transgenic Mice, Single Dose] [(15 µg)]

[0274] Basically as described above in Example 2, the activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice. Comparative compounds Nos. 396443 and 819735 were also tested in this assay. The transgenic mice were grouped with 4 mice in each group. Each mouse received a single ICV bolus of 15 µg of the modified oligonucleotide. A group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed and RNA was extracted from the cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. The results are presented as the fold change in RNA levels normalized to the SMN2 level relative to the PBS control. Each of Tables 19 - 23 represents a different experiment. [Table]

[19] [] [Effect of Modified Oligonucleotides on Human] [SMN2 RNA] [Splicing in Homozygous Transgenic Mice] [] [Compound Number] [Dose (µg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1.0 1.0 1.0 1.0 819735 15 2.4 0.4 3.3 0.3 1212869 15 2.4 0.4 3.2 0.4 1212870 15 2.1 0.5 2.8 0.4 1212873 15 2.2 0.4 2.0 0.6 1212874 15 2.1 0.5 2.4 0.6 1212875 15 2.1 0.5 2.3 0.5 1212880 15 1.7 0.6 2.0 0.6 1212881 15 1.8 0.6 2.3 0.6 1212885 15 2.3 0.4 2.4 0.5 1212887 15 2.0 0.5 2.2 0.5 1212931 15 2.9 0.2 2.9 0.3 1212936 15 2.9 0.3 3.3 0.3 1212941 15 3.0 0.1 3.4 0.2 [surface]

[20] [] [Modified oligonucleotides in heterozygous transgenic mice to humans] [SMN2 RNA] [Effect on splicing] [] [Compound number] [Dose (µg)] [Cortex] [Spinal cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1.0 1.0 1.0 1.0 396443 15 2.2 0.5 2.2 0.7 819735 15 2.7 0.5 3.1 0.5 1287122 15 2.9 0.5 2.3 0.6 1287123 15 3.0 0.4 3.0 0.4 1287124 15 3.0 0.4 3.2 0.3 1287125 15 3.0 0.4 3.0 0.4 1287126 15 2.8 0.4 2.8 0.4 1287127 15 2.7 0.5 3.0 0.5 1287128 15 2.6 0.5 3.2 0.5 1287129 15 2.9 0.4 2.9 0.5 1287130 15 3.7 0.1 3.1 0.5 1287131 15 2.2 0.6 2.7 0.4 1287132 15 3.2 0.3 2.2 0.6 1287133 15 2.9 0.4 2.8 0.4 1287728 15 2.8 0.6 3.4 0.3 1287729 15 3.1 0.4 3.0 0.3 1287730 15 3.1 0.3 2.7 0.4 1287731 15 3.3 0.3 2.8 0.5 1287735 15 2.9 0.5 2.6 0.5 1287738 15 3.7 0.2 3.2 0.3 1287739 15 3.3 0.4 3.2 0.4 1287743 15 3.6 0.4 3.8 0.4 1287745 15 3.1 0.5 3.8 0.5 [Table]

[21] [] [Modified Oligonucleotides in Transgenic Mice with Heterologous Junctions on Human] [SMN2 RNA] [Effect on Splicing] [] [Compound Number] [Dose (µg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1 1 1 1.0 396443 15 1.9 0.6 1.7 0.7 819735 15 2.3 0.5 1.9 0.6 [Table]

[22] [] [Modified Oligonucleotides in Transgenic Mice with Aberrant Junctions to Human] [SMN2 RNA] [Effect on Splicing] [] [Compound Number] [Dose (µg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1 1 1 1 1364781 15 2.5 0.6 2.7 0.4 1364780 15 2.8 0.5 2.6 0.5 1364779 15 2.7 0.5 2.6 0.5 1364778 15 3 0.5 2.7 0.4 [Table]

[23] [] [Modified oligonucleotides in transgenic mice with heterozygous junctions for human] [SMN2 RNA] [Effect on splicing] [] [Compound number] [Dose (µg)] [Cortex] [Spinal cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1 1 1 1 819735 15 2.8 0.5 2.4 0.6 1332265 15 2.1 0.7 2.6 0.6 1332269 15 2.5 0.6 2.7 0.5 1332268 15 2.9 0.5 2.3 0.6 1318756 15 2.2 0.7 2.4 0.6 1333508 15 2 0.6 2.2 0.6 1332251 15 2.9 0.5 1.9 0.7 1332249 15 2.3 0.7 2.3 0.7 [Example] [4] [: Activity of Modified Oligonucleotides Complementary to Human] [SMN2] [in Transgenic Mice, Single Dose] [(70 µg)]

[0275] Essentially as described above in Example 2, the activity of modified oligonucleotides in human SMN2 transgenic mice was tested. The transgenic mice were grouped with 4 mice in each group. Each mouse received a single ICV bolus of 70 µg of the modified oligonucleotide. A group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed and RNA was extracted from the cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. The results were presented as the fold change in RNA levels normalized to SMN2 levels relative to the PBS control. [Table]

[24] [Activity of Modified Oligonucleotides in Homozygous Transgenic Mice against Human] [SMN2 RNA] [Effect of splicing] [] [Compound number] [Dose (µg)] [Cortex] [Spinal cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1 1 1 1 1212969 70 2.5 0.4 2.4 0.3 1212970 70 2.7 0.3 2.6 0.3 [Example] [5] [: Activity of modified oligonucleotides complementary to human] [SMN2] [Multiple doses] []

[0276] Basically as described above in Example 2, the activity of the selected modified oligonucleotides described above was tested in human SMN2 gene transgenic mice. Comparative compound number 396443 was also tested in this assay. The transgenic mice were grouped with 4 mice in each group. Each mouse received a single ICV bolus of the modified oligonucleotide at multiple doses as indicated in the table below. A group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed and RNA was extracted from the cerebral cortex and spinal cord for real-time qPCR analysis of SMN2 RNA. The results were presented as the fold change in RNA levels normalized to SMN2 levels relative to the PBS control. In GraphPad Prism 7, the ED50 for exon inclusion (exon 7+) was calculated using a non-linear regression 4-parameter dose-response curve [Y = Bottom + (Top - Bottom) / (1 + (10^logEC50 / X)^HillSlope)]. [Table]

[25] [] [Effect of Modified Oligonucleotides on Human] [SMN2 RNA] [Splicing in Homozygous Transgenic Mice] [] [Compound Number] [Dose (µg)] [Cerebral Cortex] [ED50 (µg)] [Spinal Cord] [ED50 (µg)] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1.0 1.0 1.0 1.0 396443 3 1.4 0.9 32.5 1.3 0.9 22.1 10 1.8 0.8 2.0 0.7 30 2.6 0.5 2.6 0.4 100 3.5 0.4 3.2 0.3 300 4.2 0.1 3.6 0.2 1263789 3 1.5 0.9 38.3 1.5 0.8 13.3 10 2.0 0.7 2.2 0.6 30 2.3 0.6 3.0 0.4 100 3.4 0.3 3.4 0.3 300 3.9 0.1 3.7 0.2 1287717 3 1.3 0.8 38.7 1.3 0.9 20.5 10 1.8 0.7 1.9 0.7 30 2.4 0.7 2.7 0.5 100 3.5 0.4 3.3 0.3 300 4.1 0.1 3.8 0.2 1358996 3 1.6 0.9 16.6 1.7 0.8 7.4 10 2.5 0.6 2.6 0.5 30 3.0 0.4 3.5 0.2 100 4.0 0.2 3.6 0.2 300 4.0 0.1 3.9 0.1 1287745 3 1.5 0.8 22.8 1.7 0.7 8.8 10 2.1 0.6 2.4 0.5 30 3.0 0.3 3.3 0.3 100 3.6 0.1 3.5 0.2 300 4.2 0.1 3.8 0.1 [Example] [6] [: Tolerance of modified oligonucleotides complementary to] [SMN2] [in wild-type mice,] [3] [hour study] [ ]

[0277] The modified oligonucleotides described above were tested in wild-type female C57 / Bl6 mice to assess tolerance. Wild-type female C57 / Bl6 mice each received a single ICV dose of 700 μg of the modified oligonucleotides listed in the table below. Comparative compound number 396443 was also tested at a dose of 350 μg in this assay. Comparative compound numbers 387954, 396442, 443305, and 819735 were also tested at a dose of 700 μg in this assay. Each treatment group consisted of 4 mice. A group of 4 mice received PBS as the negative control for each experiment (identified in the separate table below). Three hours after injection, the mice were evaluated according to seven different criteria. The criteria were: (1) the mouse was bright, alert, and responsive; (2) the mouse stood or arched its back without stimulation; (3) the mouse showed any movement without stimulation; (4) the mouse showed forward movement after it was picked up; (5) the mouse showed any movement after it was picked up; (6) the mouse responded to tail pinch; (7) even breathing. For each of the 7 criteria, if the mouse met the criterion, the mouse was given a subscore of 0, and if it did not meet the criterion, it was given a subscore of 1 (Functional Observation Battery score or FOB). After all 7 criteria were evaluated, the scores were summed and averaged within each treatment group. The results are presented in the table below. Each of Tables 26 - 49 represents a different experiment. [Table]

[26] [At] [350 μg] [Tolerance scores in mice at dose] [] [Compound number] [3 h FOB] PBS 0 396443 4.0 [Table]

[27] [] [At] [700 μg] [Tolerance scores in mice at dose] [] [Compound number] [3 h FOB] PBS 0.00 443305 4.75 [Table]

[28] [] [At] [700 μg] [Tolerance score in mice at the dose] [] Compound number 3 h FOB PBS 0 396442 2.5 524403 3.25 1210339 1.25 1210340 2.25 1210341 3.75 1210342 0 1210343 0 1212817 0 1212818 0 1212819 0 1212820 0 1212821 0 1212822 0 1212823 0 1212824 0 1212825 1 1212826 0 1212827 0 1212828 0 1212829 0 1212830 0 1212831 0 [Table]

[29] [] [At] [700 μg] [Tolerance scores in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 396442 2.50 1210340 3.50 1212850 0.50 1212851 0.75 1212852 0.00 1212853 0.00 1212854 0.25 1212855 0.25 1212856 0.00 1212857 0.00 1212858 0.00 1212859 0.00 1212860 0.75 1212861 1.00 1212863 2.00 1212864 0.00 1212866 0.75 1212867 0.00 1212868 0.00 [Table]

[30] [] [At] [700 μg] [Tolerance Score in Mice at the Dose] [] [Compound Number] [3 h FOB] PBS 0.00 396442 3.25 1212961 0.00 1212963 1.00 1212964 2.00 1212965 1.25 1212966 1.25 1212968 0.00 1212971 1.00 1212972 3.25 1212973 0.50 1212974 2.00 1212975 0.50 1212976 1.75 [Table]

[31] [ ] [At] [700 μg] [Tolerance scores in mice at the dose] [ ] [Compound number] [3 h FOB] PBS 0.00 1212977 0.75 1212978 0.00 1212979 1.75 1212980 1.50 1212981 0.00 1212982 0.50 1212983 0.75 1212984 2.75 1212985 0.00 1212986 1.00 1212987 1.75 1212988 4.50 1212989 1.75 1212990 4.50 1212991 1.25 1212992 3.75 [Table]

[32] [] [At] [700 μg] [Tolerance score in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 1212993 7.00 1212994 6.50 1212995 4.25 1212996 3.25 1212997 4.00 1212998 2.00 1212999 1.00 1213000 1.25 1213001 3.00 1213002 2.00 1213003 4.00 1213004 3.00 1213005 3.75 1213006 4.00 1213007 4.00 1213008 3.50 [Table]

[33] [] [At] [700 μg] [Tolerance score in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 1212832 0.00 1212833 0.00 1212834 0.00 1212835 0.00 1212836 0.00 1212837 0.00 1212838 0.00 1212839 0.00 1212840 0.00 1212841 0.00 1212842 0.00 1212843 0.00 1212844 0.25 1212845 1.00 1212846 0.00 1212847 0.00 1212848 0.00 1212849 0.00 [Table]

[34] [] At 700 μg Tolerance score in mice at the dose [] Compound number 3 h FOB PBS 0.00 396442 1.75 1210339 1.00 1212865 1.00 1212962 0.00 1212967 0.50 1212969 0.50 1212970 1.25 Table

[35] [] At 700 μg Tolerance score in mice at the dose [] Compound number 3 h FOB PBS 0.00 819735 2.00 1212869 2.00 1212870 4.75 1212871 1.00 1212873 0.00 1212874 0.00 1212875 0.00 1212879 3.00 1212880 0.00 1212881 4.00 1212885 1.00 1212887 2.25 1212931 2.00 1212936 2.00 1212941 1.25 [Table]

[36] [] [At] [700 μg] [Tolerance scores in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 1263778 0.00 1263781 0.00 1263783 0.00 1263785 1.00 1263787 0.00 1263789 0.00 1263791 0.00 1263793 0.00 1263795 0.00 1263797 0.00 1263799 0.00 1263800 0.00 1263802 0.00 1263804 0.00 1263806 0.00 1263808 1.00 1263810 0.00 1263812 0.00 1263814 1.00 1263816 0.50 1263818 0.00 1263820 0.00 1263822 0.25 1263824 0.00 [surface]

[37] [] [exist] [700 μg] [Tolerance score in mice at different doses] [] [Compound number] [3h FOB] PBS 0.00 1263826 0.00 [surface]

[38] [] In [700 μg] [tolerance scores in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 387954 4.00 1287048 0.00 1287049 0.00 1287050 2.00 1287051 3.25 1287052 3.50 1287053 2.75 1287054 2.00 1287055 3.25 1287056 4.00 1287057 3.00 1287058 4.00 1287059 4.00 1287060 4.00 1287061 4.00 1287062 3.50 Table

[39] [] In [700 μg] [Tolerance Score in Mice at the Dose] [] [Compound Number] [3 h FOB] PBS 0.00 1287106 3.50 1287107 4.00 1287108 3.75 1287109 3.25 1287110 3.00 1287111 4.75 1287112 4.00 1287113 3.50 1287114 3.25 1287115 3.50 1287116 4.00 1287117 4.25 1287118 3.00 1287119 3.50 1287120 3.75 1287121 2.75 [Table]

[40] [] [At] [700 μg] [Tolerance Score in Mice at the Dose] [] [Compound Number] [3h FOB] PBS 0.00 1287063 0.00 1287064 0.00 1287065 1.00 1287066 3.75 1287067 1.00 1287068 2.50 1287069 2.25 1287071 1.00 1287072 3.00 1287073 3.75 1287074 1.75 1287075 3.50 1287076 2.00 [surface]

[41] [] [exist] [700 μg] [Tolerance score in mice at different doses] [] [Compound number] [3h FOB] PBS 0.00 1287070 2.00 1287701 2.50 1287702 3.75 1287703 3.75 1287705 4.00 1287706 4.00 1287707 4.00 1287709 4.75 1287710 4.00 1287711 4.75 1287712 4.00 1287713 4.00 1287714 3.50 1287715 4.00 1287716 4.00 1287717 3.25 [surface]

[42] [] [exist] [700 μg] [Tolerance score in mice at different doses] [] [Compound number] [3h FOB] PBS 0.00 1287728 1.00 1287729 1.25 1287730 2.00 1287731 2.50 1287732 3.00 1287733 3.25 1287734 3.00 1287735 0.50 1287736 2.50 1287737 4.00 1287738 3.00 1287739 2.50 1287740 2.75 1287741 3.75 1287742 3.00 1287743 2.75 1287744 2.25 1287745 1.00 [surface]

[43] [] [exist] [700 μg] [Tolerance score in mice at different doses] [] [Compound number] [3h FOB] PBS 0.00 1287122 0.00 1287123 0.00 1287124 3.50 1287125 3.00 1287126 3.00 1287127 0.00 1287128 0.00 1287129 4.00 1287130 2.75 1287131 2.50 1287132 2.75 1287133 3.25 1287704 3.50 1287708 3.50 [Table]

[44] [] [At] [700 μg] [Tolerance scores in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 1318748 2.00 1318765 4.00 1318767 4.25 1318770 3.75 1318771 4.50 1318772 4.25 1318773 4.25 1318774 3.50 1318775 3.75 1318776 3.75 1318777 4.00 1318778 4.00 1318779 4.00 1318780 4.00 1318781 4.00 1318782 1.00 1318783 4.00 1318784 2.00 [Table]

[45] [] [At] [700 μg] [Tolerance scores in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 1318757 4.00 1318758 4.25 1318759 3.75 1318760 3.75 1318761 4.00 1318762 4.00 1318763 4.00 1318764 3.75 1318766 3.75 1318768 4.00 1318769 4.00 [Table]

[46] [] [At] [700 μg] [Tolerance scores in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 1318749 4.25 1318750 2.25 1318751 4.00 1318752 3.75 1318753 2.25 1318754 3.00 1318755 3.75 1318756 0.00 [Table]

[47] [] [At] [700 μg] [Tolerance scores in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 1332247 1.75 1332248 0.25 1332249 0.00 1332250 3.75 1332251 0.00 1332252 3.00 1332263 2.00 1332265 1.50 1332266 1.00 1332267 3.75 1332268 2.75 1332269 1.25 1332270 2.25 1332271 2.50 1333508 0.00 [surface]

[48] [] [exist] [700 μg] [Tolerance score in mice at different doses] [] [Compound number] [3h FOB] PBS 0.00 1332255 1.00 1332256 2.00 1332257 1.25 1332258 1.25 1332259 2.25 1332260 2.25 1332261 2.50 1332262 2.00 [Table]

[49] [] [At] [700 μg] [Tolerance scores in mice at the dose] [] [Compound number] [3 h FOB] PBS 0.00 1358996 0.00 1364777 2.00 1364778 3.00 1364779 3.50 1364780 3.50 1364781 5.25 1364782 2.50 1364783 3.50 1364784 3.50 [Example] [7] [: Tolerance of modified oligonucleotides complementary to human] [SMN2] [in rats, long-term assessment] []

[0278] In separate studies conducted under the same conditions, the selected modified oligonucleotides described above were tested in Sprague-Dawley rats to evaluate long-term tolerability. Comparative compounds Nos. 396442 and 819735 were also tested in this assay. Each Sprague-Dawley rat received a single intrathecal (IT) delivery dose of 3 mg of oligonucleotide or PBS. Starting 1 week after treatment, each animal was weighed and adverse events were evaluated weekly by trained observers. Adverse events were defined as neurological dysfunctions that were atypical in PBS-treated control animals, including but not limited to: abnormal limb splaying, abnormal gait, tremors, abnormal respiration, paralysis, and spasms. The onset of an adverse event was defined as the week in which dysfunction was first recorded after dosing. If no adverse event was achieved, there was no onset (-). The onset of adverse events was generally associated with growth impairment as defined by lack of weight gain / maintenance, similar to PBS-treated animals. Similar tolerability assessments are described in Oestergaard et al., Nucleic Acids Res., November 2013, 41(21), 9634-9650 and Southwell et al., Mol Ther., December 2014, 22(12), 2093-2106.

[0279] At the end of the study, the rats were sacrificed and tissues were collected. Histopathological examination was performed on cerebellar sections using calbindin staining. Purkinje cell loss was observed in calbindin-stained cerebellar sections as indicated in the table below. The cerebellum and spinal cord were also evaluated using an antibody specific for the modified oligonucleotide. Animals showing no oligonucleotide uptake were excluded from the histopathological analysis. For animals sacrificed prematurely due to adverse events, histology was not completed. In addition, cortical GFAP, a marker of astrogliosis (Abdelhak et al., Scientific Reports, 2018, 8, 14798), was measured using RT-PCR, and a >2-fold mean elevation is described below. [Table]

[50] [] [In] [3 mg] [Long-Term Tolerability in Rats at the Dose] [] Compound No. Onset of Adverse Event, Weeks Post-Treatment, Individual Animal Purkinje cell loss (number of animals with loss / number of animals tested) Cortical GFAP mRNA > 2-fold PBS control PBS None Not observed N / A 396442 6, 6, 2 2 / 3 Yes 819735 4, 6, 6, - 1 / 4 Yes 1263789 -,-,- 0 / 3 No 1287717 -,-,-,-,-,-,-,- 0 / 8 No 1287745 -,-,-,-,-,-,- 0 / 7 No 1358996 -,-,-,- 0 / 4 No 1263783 -,-,-,- 0 / 4 No 1263785 -,-,- 0 / 3 No 1263787 -,-,-,- 0 / 4 No 1263800 -,- 0 / 2 No 1263802 -,-,- 0 / 3 No 1263806 -,-,- 0 / 3 No 1263808 -,-,- 0 / 3 No 1263810 -,-,- 0 / 3 No [Example] [8] [: Tolerance and Pharmacokinetics of Modified Oligonucleotides in Non-Human Primates, Single or Repeated Dosing] []

[0280] Cynomolgus monkeys were treated with modified oligonucleotides to determine the local and systemic tolerance and pharmacokinetics of the modified oligonucleotides. Each group received artificial CSF or modified oligonucleotides administered as a single intrathecal lumbar bolus dose (IT), or for the repeated dosing group, the study day 1 was an IT bolus dose, followed by IT bolus doses at subsequent time points. Tissues were collected 1 week after the last injection.

[0281] In the single-dose study, monkeys were administered a single dose of modified oligonucleotides and tolerance was evaluated. Representative doses for the single-dose study in adult cynomolgus monkeys included 1 mg, 3 mg, 7 mg, and 35 mg.

[0282] In the repeated dosing study, monkeys were administered an IT bolus dose on study day 1, followed by IT bolus dosing weekly (e.g., on days 8, 15, and 22 of a four-week study) or monthly (e.g., on days 29, 57, and 84 of a 13-week study). Representative doses for the repeated-dose study in adult cynomolgus monkey studies included 1 mg, 3 mg, 7 mg, and 35 mg.

[0283] The assessment of tolerance was based on clinical observations, body weight, food consumption, physical and neurological examinations (including sensorimotor reflexes, cerebral reflexes and spinal reflexes), coagulation, hematology, clinical chemistry (blood and cerebrospinal fluid (CSF)), cell counts and anatomical pathology assessments. A complete necropsy was performed and any macroscopic abnormalities were recorded. Organs were weighed and microscopic examinations were carried out. Blood was collected for complementary analysis. Additionally, blood, CSF and tissues (at necropsy) were collected for pharmacokinetic assessments.

[0284] The tolerance of modified oligonucleotides in brain and spinal cord tissues was analyzed by measuring the Aif1 and Gfap levels in stone crab macaques treated with the modified oligonucleotides or controls. Brain and spinal cord samples were collected and snap-frozen in liquid nitrogen and stored frozen (-60 °C to -90 °C). At the time of sampling, 2 mm biopsy punches were used to collect samples from the frozen tissues for RNA analysis. Punching was performed in multiple brain and spinal cord regions. [Example] [9] [: Compound number] [1263789] [,] [1287717] [,] [1287745] [or] [1358996] [of] [Ia] [phase human clinical trial]

[0285] The safety, tolerance, pharmacokinetics, pharmacodynamics and efficacy of modified oligonucleotides complementary to human SMN2 were evaluated in a clinical trial setting. Single and / or multiple doses of the modified oligonucleotides were evaluated in patients with confirmed SMA such as type I SMA, type II SMA, type III SMA or type IV SMA.

[0286] ​Patient safety will be closely monitored during the study period. Safety and tolerability assessments include: physical examination and standard neurological assessment (including basal), vital signs (HR, BP, postural changes, weight), ECG, AE and concomitant medications, Columbia Suicide Severity Rating Scale (C-SSRS), CSF safety laboratory (cell count, protein, glucose), plasma laboratory tests (clinical chemistry, hematology), and urine analysis.

[0287] Efficacy assessments appropriate for age and type are selected and include, for example, the Hammersmith Motor Function Scale-Expanded (HFMSE), a reliable and validated tool for assessing motor function in children with SMA; the Pediatric Quality of Life Inventory (PedsQL™) Measure 4.0 Generic Core Scale; the Pediatric Quality of Life Inventory 3.0 Neuromuscular Module; Compound Muscle Action Potential (CMAP); Motor Unit Number Estimation (MUNE); Upper Limb Module (ULM); and 6 Minute Walk Test (6MWT) (Darras et al., Neurology, 2019, 92: e2492-e2506). [Example]

[10] [: with humans] [SMN2] [Design of Modified Oligonucleotides Complementary to Nucleic Acids] []

[0288] Design and synthesize modified oligonucleotides complementary to human SMN2 nucleic acids as indicated in the following table.

[0289] Each of the modified oligonucleotides listed in the following tables is 100% complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleotide 19939708 to 19967777). "Start site" indicates the most 5'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the most 3'-nucleotide in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0290] The length of the modified oligonucleotides in the table below is 18 nucleotides. Each nucleotide contains a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar moiety of each modified oligonucleotide is provided in the sugar moiety column, where each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage, a phosphodiester internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, or a methanesulfonamidate phosphate (MsP) internucleoside linkage. The internucleoside linkage moiety of each modified oligonucleotide is provided in the internucleoside linkage moiety column, where each's' represents a phosphorothioate internucleoside linkage, each 'o' represents a phosphodiester internucleoside linkage, each 'x' represents a methoxypropylphosphonate internucleoside linkage, and each 'z' represents a methanesulfonamidate phosphate (MsP) internucleoside linkage. Each cytosine is 5-methylcytosine. The modified oligonucleotide 449320 has been previously described in WO2015 / 161170 A2. [Table]

[51] [With mixed] [PO / PS] [,] [PO / MsP] [, uniform] [MsP] [or] [PS / MOP] [Of internucleoside linkage] [MOE] [and] [NMA] [Modified oligonucleotide] [] [Compound number] [Sequence (5'] [to 3')] [Sugar moiety] [(5'] [to 3')] [Internucleoside linkage moiety] [(5'] [to 3')] [SEQ ID No: 1] [Starting point] [SEQ ID No: 1] [Ending point] [SEQ ID No.] 449320 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssoooooooooooooss 27062 27079 23 1287723 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnenn sssssssssssssssxs 27062 27079 23 1287724 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnen ssssssssssssssssx 27062 27079 23 1287727 CACTTTCATAATGCTGGC nnnnnnnnnnnnnnnnen ssssssssssssssssx 27061 27078 21 1405549 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee zzzzzzzzzzzzzzzzz 27062 27079 23 1405552 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssssssssszzzzzz 27062 27079 23 1405553 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee ssssszzzzzzssssss 27062 27079 23 1545359 TCACTTTCATAATGCTGG nnnnnnnnnnnnnnnnnn ssoooooooooooooss 27062 27079 23 1547773 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee zzooooooooooooozz 27062 27079 23 1549028 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee zzzzooooooooooozz 27062 27079 23 1549029 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee zzzzzzooooooooozz 27062 27079 23 1549030 TCACTTTCATAATGCTGG eeeeeeeeeeeeeeeeee zzzzzzzzooooooozz 27062 27079 23

[0291] The modified oligonucleotides in the following table are all composed of the following sequence (5' to 3'): TCACTTTCATAATGCTGG (SEQ ID NO: 23). Each modified oligonucleotide listed in the following tables is 100% complementary to SEQ ID NO: 1 (described above in this text). "Start site" indicates the most 5'-nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the most 3'-nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0292] The modified oligonucleotides in the following table are 18 nucleosides in length. Each nucleoside contains a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar moiety of each modified oligonucleotide is provided in the sugar moiety column, where each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage, a phosphodiester internucleoside linkage, or a methanesulfonamidate phosphate (MsP) internucleoside linkage. The internucleoside linkage moiety of each modified oligonucleotide is provided in the internucleoside linkage moiety column, where each's' represents a phosphorothioate internucleoside linkage, each 'o' represents a phosphodiester internucleoside linkage, and each 'z' represents a methanesulfonamidate phosphate (MsP) internucleoside linkage. Each cytosine is 5-methylcytosine. The modified oligonucleotides in the following table are conjugated to a 6-palmitoylaminohexyl phosphate conjugate group attached to the 5'-OH of the oligonucleotide. The structure of the conjugate group is: 。 [Table]

[52] [] [6-] [Palmitoylaminohexyl phosphate conjugated with mixed] [PO / PS] [、] [PO / MsP] [或均勻] [MsP] [核苷間鍵聯之] [MOE] [及] [NMA] [修飾之寡核苷酸] [] [化合物編號] [糖模體(5’] [至3’)] [核苷間鍵聯模體(5’] [至3’)] [SEQ ID No: 1] [起始位點] [SEQ ID No: 1] [終止位點] [SEQ ID No.] 1545361 eeeeeeeeeeeeeeeeee ssoooooooooooooss 27062 27079 23 1545362 nnnnnnnnnnnnnnnnnn ssoooooooooooooss 27062 27079 23 1547772 eeeeeeeeeeeeeeeeee zzzzzzzzzzzzzzzzz 27062 27079 23 1547774 eeeeeeeeeeeeeeeeee zzooooooooooooozz 27062 27079 twenty three 1549031 eeeeeeeeeeeeeeeeee zzzzooooooooooozz 27062 27079 twenty three 1549032 eeeeeeeeeeeeeeeeee zzzzzzoooooooooozz 27062 27079 twenty three 1549033 eeeeeeeeeeeeeeeeee zzzzzzzzooooooozz 27062 27079 twenty three

[0293] The modified oligonucleotides in the following table all consist of the following sequence (5' to 3'): TCACTTTCATAATGCTGG (SEQ ID NO: 23), the start position is 27062 on SEQ ID No: 1 (described herein above) and the end position is 27079, wherein the "start position" indicates the 5'-nucleoside that is complementary to the modified oligonucleotide in the target nucleic acid sequence, and wherein the "end position" indicates the 3'-nucleoside that is complementary to the modified oligonucleotide in the target nucleic acid sequence.

[0294] The length of the modified oligonucleotides in the table below is 18 nucleotides. The sugar and internucleoside linkage motifs of each modified oligonucleotide are provided in the Sequence and Chemical Notation column, where each subscript 'n' represents a 2'-NMA sugar moiety, each subscript '[DMA]' represents a 2'-O-(N,N-dimethyl)acetamide moiety, each subscript '[NEA]' represents a 2'-O-(N-ethyl)acetamide moiety, each subscript '[NPA]' represents a 2'-O-(N-propyl)acetamide moiety, each subscript '[NcPA]' represents a 2'-O-(N-cyclopropyl)acetamide moiety, each subscript '[McPA]' represents a 2'-O-(N-cyclopropylmethyl)acetamide moiety, and each subscript's' represents a phosphorothioate internucleoside linkage. Each cytosine is 5-methylcytosine, where the subscript'm' before a cytosine residue (mC) represents 5-methylcytosine. The structure of each sugar represented in the table below is: [Table]

[53] [with uniform] [PS] [internucleoside linkages of] [NMA] [and] [NMA] [oligonucleotides modified with analogs] [] [Compound No.] [Sequence and Chemical Notation (5'] [to 3')] [SEQ ID No.] 1355763 T [DMA]s mC [DMA]sA ns mC [DMA]sT [DMA]sT [DMA]sT [DMA]s mC [DMA]sA nsT [DMA]sA nsA nsT [DMA]sG ns mC [DMA]sT [DMA]sG nsG n 23 1359463 T [NEA]s mC [NEA]sA ns mC [NEA]sT [NEA]sT [NEA]sT [NEA]s mC [NEA]sA nsT [NEA]sA nsA nsT [NEA]sG ns mC [NEA]sT [NEA]sG nsG n twenty three 1358995 T [NPA]s mC [NPA]sA ns mC [NPA]sT [NPA]sT [NPA]sT [NPA]s mC [NPA]sA nsT [NPA]sA nsA nsT [NPA]sG ns mC [NPA]sT [NPA]sG nsG n twenty three 1355776 T [NcPA]s mC [NcPA]sA ns mC [NcPA]sT [NcPA]sT [NcPA]sT [NcPA]s mC [NcPA]sA nsT [NcPA]sA nsA nsT [NcPA]sG ns mC [NcPA]sT [NcPA]sG nsG n twenty three 1355777 T [McPA]s mC [McPA]sA ns mC [McPA]sT [McPA]sT [McPA]sT [McPA]s mC [McPA]sA nsT [McPA]sA nsA nsT [McPA]sG ns mC [McPA]sT [McPA]sG nsG n twenty three [Example]

[11] [:In transgenic mice and humans] [SMN2] [Activity of complementary modified oligonucleotides, single dose] [(35 µg)]

[0295] Essentially as described above in Example 2, the activity of the selected modified oligonucleotides described above was tested in mice transfected with the human SMN2 gene. handle

[0296] The transgenic mice were grouped, with 4 mice in each group. Each mouse received a single ICV bolus injection of the modified oligonucleotide at the doses indicated in the following table. A group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed, and RNA was extracted from the cerebral cortex and spinal cord for real-time qPCR analysis of SMN2 RNA. The results were presented as the fold change of the RNA level normalized to the SMN2 level relative to the PBS control. In GraphPad Prism 7, the ED50 of exon inclusion (exon 7+) was calculated using a non-linear regression 4-parameter dose-response curve [Y = Bottom + (Top - Bottom) / (1 + (10^logEC50 / X)^HillSlope)]. RNA analysis

[0297] Two weeks after treatment, the mice were sacrificed, and RNA was extracted from the cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. The amount of SMN2 RNA including exon 7 (exon 7+) was determined using the primer-probe set hSMN2vd#4_LTS00216_MGB. The amount of SMN2 RNA not including exon 7 (exon 7-) was determined using the primer-probe set hSMN2_Sumner68_PPS50481. The total SMN2 RNA level was measured using the primer-probe set hSMN2_LTS00935. The results were presented as the fold change of the RNA level normalized to the SMN2 level relative to the PBS control. [Table]

[54] [Effect of Modified Oligonucleotides on Human] [SMN2 RNA] [Splicing in Heterozygous Transgenic Mice] [] [Compound Number] [Dose (μg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7,+ , ] [Exon 7, - , ] PBS - 1 1 1 1 396443 35 3 0.6 3 0.5 1405549 35 1.6 0.9 1.2 0.9 1405552 35 3.2 0.6 2.1 0.7 1405553 35 2.4 0.7 1.7 0.8 [Table]

[55] [] [Effect of Modified Oligonucleotides on Human] [SMN2 RNA] [Splicing in Heterozygous Transgenic Mice] [] [Compound Number] [Dose (µg)] [Cortex] [Spinal Cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7, + , ] [Exon 7, - , ] PBS - 1 1 1 N / A 396443 35 2.7 0.5 3 N / A 1405549 35 1.5 0.8 1.9 N / A 1547772 35 1.8 0.8 1.8 N / A 1547773 35 1.6 0.9 1.9 N / A 1547774 35 1.6 1 1.6 N / A 1549028 35 1.5 1 1.5 N / A 1549029 35 1.4 1 1.7 N / A 1549030 35 1.4 1 1.4 N / A 1549031 35 1.7 0.8 1.6 N / A 1549032 35 1.4† 0.9† 1.7† N / A 1549033 35 1.4 1 1.3 N / A † Indicates fewer than four samples available [Example]

[12] [: in transgenic mice with human] [SMN2] [Activity of modified oligonucleotides complementary to, single dose] [(15 µg)]

[0298] Essentially as described above in Example 2, the activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice. Treatment

[0299] The transgenic mice were grouped, with 4 mice in each group. Each mouse received a single ICV bolus injection of the modified oligonucleotide at the doses indicated in the following table. A group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed, and RNA was extracted from the cerebral cortex and spinal cord for real-time qPCR analysis of SMN2 RNA. The results were presented as the fold change in RNA levels normalized to the SMN2 level relative to the PBS control. In GraphPad Prism 7, the ED50 of exon inclusion (exon 7+) was calculated using a non-linear regression 4-parameter dose-response curve [Y = bottom + (top-bottom) / (1 + (10^logEC50 / X)^HillSlope)]. RNA analysis

[0300] Two weeks after treatment, the mice were sacrificed, and RNA was extracted from the cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. The amount of SMN2 RNA including exon 7 (exon 7+) was determined using the primer-probe set hSMN2vd#4_LTS00216_MGB. The amount of SMN2 RNA not including exon 7 (exon 7-) was determined using the primer-probe set hSMN2_Sumner68_PPS50481. The total SMN2 RNA level was measured using the primer-probe set hSMN2_LTS00935. The results were presented as the fold change in RNA levels normalized to the SMN2 level relative to the PBS control. [Table]

[56] [Effect of modified oligonucleotides on human] [SMN2 RNA] [in splicing] [] [Compound number] [Dose (µg)] [Cortex] [Spinal cord] [Exon 7, + , ] [Exon 7, - , ] [Exon 7,+ , ] [Exon 7, - , ] PBS - 1 1 1 1 443305 15 3.4 0.4 3.3 0.3 1287723 15 3.9 0.3 3.3 0.3 1287724 15 4.2 0.2 3.7 0.2 1287727 15 4.5 0.2 3.4 0.2 [Example]

[13] [: In transgenic mice with the human] [SMN2] [activity of complementary modified oligonucleotides, multiple doses]

[0301] Basically as described above in Example 2, the activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice. Treatment

[0302] The transgenic mice were grouped, with 4 mice in each group. Each mouse received a single ICV bolus injection of the modified oligonucleotide at multiple doses as indicated in the following table. A group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed, and RNA was extracted from the cerebral cortex and spinal cord for real-time qPCR analysis of SMN2 RNA. The results were presented as the fold change in RNA levels normalized to the SMN2 level relative to the PBS control. In GraphPad Prism 7, the ED50 of exon inclusion (exon 7+) was calculated using a non-linear regression 4-parameter dose-response curve [Y = bottom + (top - bottom) / (1 + (10^logEC50 / X)^HillSlope)]. RNA analysis

[0303] Two weeks after treatment, the mice were sacrificed, and RNA was extracted from the cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. The amount of SMN2 RNA including exon 7 (exon 7+) was determined using the primer-probe set hSMN2vd#4_LTS00216_MGB. The amount of SMN2 RNA not including exon 7 (exon 7-) was determined using the primer-probe set hSMN2_Sumner68_PPS50481. The total SMN2 RNA level was measured using the primer-probe set hSMN2_LTS00935. The results were presented as the fold change in RNA levels normalized to the SMN2 level relative to the PBS control. [Table]

[57] [Effect of Modified Oligonucleotides on Human] [SMN2 RNA] [Splicing in Heterozygous Transgenic Mice] [] [Compound Number] [Dose (µg)] [Cortex] [ED50 (µg)] [Spinal Cord] [ED50 (µg)] [] [] [Exon 7+] [Exon 7-] [] [Exon 7+] [Exon 7-] [] PBS - 1 1 - 1 1 - 396443 10 2.1 0.7 1.9 0.8 30 2.8 0.5 26 2.6 0.7 22 100 3.2 0.3 2.8 0.4 449320 10 1.2 1.0 1.2 1.2 30 1.5 1.0 >100 1.3 1.2 >100 100 1.5 0.9 1.3 0.9 1545361 10 1.4 1.0 1.2 1.1 30 1.8 1.1 >100 1.6 1.3 >100 100 1.3 0.9 1.4 0.9 443305 10 2.4 0.6 2.6 0.5 30 3.5 0.3 14 3.0 0.4 9 100 3.7 0.1 3.3 0.1 1545359 10 1.4 1.0 1.4 1.1 30 2.0 0.9 >100 1.7 1.1 >100 100 2.3 0.6 1.7 0.8 1545362 10 1.4 0.9 1.3 1.1 30 1.9 0.8 95 2.4 1.0 51 100 2.7 0.5 2.6 0.6 [surface]

[58] [] [Modified oligonucleotides in heterozygous transgenic mice to humans] [SMN2 RNA] [The impact of editing] [] [Compound number] [Dose (µg)] [Cortex] [ED50 (µg)] [Spinal cord] [ED50 (µg)] [Exon 7+] [Exon 7-] [Exon 7+] [Exon 7-] PBS - 1 1 - 1 1 - 1263789 3 1.5 0.9 35 1.6 0.8 16 10 1.9 0.8 2.2 0.6 30 2.7 0.6 2.9 0.5 100 3.8 0.3 3.5 0.3 300 4.3 0.2 3.9 0.2 1287703 3 1.5 0.7 43 1.5 0.8 28 10 1.6 0.7 2.0 0.7 30 2.7 0.5 2.6 0.5 100 3.7 0.3 3.2 0.4 300 4.0 0.2 3.5 0.2 1287717 3 1.4 0.8 31 1.4 0.9 30 10 1.7 0.7 1.8 0.7 30 3.1 0.4 2.4† 0.5† 100 3.8 0.3 3.3 0.3 300 4.3 0.2 3.8 0.2 1318768 3 1.3 0.8 49 1.2 0.8 32 10 1.8 0.7 1.8 0.8 30 2.4 0.6 2.4 0.6 100 3.5 0.3 3.3 0.4 300 4.1 0.2 3.9 0.2 1287731 3 1.5 0.8 29 1.5 1.0 13 10 2.0 0.6 2.4 0.6 30 2.7 0.4 3.2 0.4 100 4.3 0.2 3.8 0.2 300 4.2 0.1 3.7 0.1 1287735 3 1.7 0.7 22 1.5 0.8 15 10 2.4 0.6 2.2 0.6 30 3.0 0.3 3.1 0.3 100 4.0 0.2 3.5 0.2 300 4.2 0.1 3.9 0.1 1287745 3 1.5 0.7 35 1.4 0.8 13 10 2.0 0.6 2.4 0.6 30 2.9 0.4 3.2 0.4 100 3.7 0.2 3.6 0.2 300 3.7 0.2 3.8 0.2 396443 3 1.7 0.7 47 1.5 0.9 22 10 1.4 0.7 1.6 0.8 30 2.8 0.5 3.1 0.4 100 3.3 0.4 3.3 0.5 300 4.3 0.2 4.0 0.2 † Indicates less than four samples available [Table]

[59] [] [Modified oligonucleotides in heterozygous transgenic mice on human] [SMN2 RNA] [Effect on splicing] [] [Compound number] [Dose (µg)] [Cortex] [ED50 (µg)] [Spinal cord] [ED50 (µg)] [Exon 7+] [Exon 7-] [Exon 7+] [Exon 7-] PBS - 1 1 - 1 1 - 396443 3 1.1 0.9 39 1.2 0.9 33 30 2.0 0.6 2.3 0.5 100 2.3 0.4 2.7 0.3 443305 3 1.4 0.7 13 1.6 0.7 10 30 2.4 0.3 3.0 0.2 100 2.9 0.2 3.1 0.2 1355763 3 1.1 0.8 54 1.1 0.8 45 30 1.7 0.6 2.1 0.5 100 2.4 0.4 2.6 0.3 1359463 3 1.3 0.8 18 1.3 0.7 19 30 2.3 0.3 2.7 0.3 100 2.9 0.2 2.7 0.2 1358995 3 1.2 0.8 45 1.0 0.8 59 30 2.0 0.4 1.8 0.4 100 2.2 0.4 2.6 0.3 1355776 3 1.1 0.8 25 1.3 0.7 24 30 2.2 0.4 2.4 0.4 100 2.6 0.3 2.9 0.3 1355777 3 1.0 0.9 107 0.9 0.9 72 30 1.6 0.7 1.8 0.6 100 1.7 0.5 2.2 0.6 [Example]

[15] [: Tolerance of Modified Oligonucleotides Complementary to] [SMN2] [in Wild-Type Mice] []

[0304] The modified oligonucleotides described above were tested in wild-type female C57 / Bl6 mice to evaluate the tolerance of the oligonucleotides. Wild-type female C57 / Bl6 mice each received a single ICV dose of 700 μg of the modified oligonucleotides listed in the table below. Each treatment group consisted of 4 mice. A group of 4 mice received PBS as a negative control for each experiment (identified in the separate table below). Three hours after injection, the mice were evaluated according to seven different criteria. The criteria were (1) the mice were bright, alert, and responsive; (2) the mice stood or arched their backs without stimulation; (3) the mice showed any movement without stimulation; (4) the mice showed forward movement after they were picked up; (5) the mice showed any movement after they were picked up; (6) the mice responded to tail pinch; (7) even breathing. For each of the 7 criteria, if the mouse met the criterion, the mouse was given a subscore of 0, and if it did not meet the criterion, a subscore of 1 was given (Functional Observation Battery score or FOB). After all 7 criteria were evaluated, the scores for each mouse were summed and averaged within each treatment group. The results are presented in the table below. [Table]

[60] [At] [700 μg] [Tolerance score in mice at the dose] [ ] [Compound number] [3 h FOB] PBS 0.00 1287723 2.00 1287724 1.00 1287727 2.00

[0305] None

[0306] <![CDATA[<110> Ionis Pharmaceuticals, Inc.]]> <![CDATA[<120> Compounds and Methods for Modulating SMN2]]> <![CDATA[<130> BIOL0367US.L]]> <![CDATA[<150> US 62 / 983,545]]> <![CDATA[<151> 2020-02-28]]> <![CDATA[<160> 50 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211> 28070]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 1]]> ccacaaatgt gggagggcga taaccactcg tagaaagcgt gagaagttac tacaagcggt 60 cctcccggcc accgtactgt tccgctccca gaagccccgg gcggcggaag tcgtcactct 120 taagaaggga cggggcccca cgctgcgcac ccgcgggttt gctatggcga tgagcagcgg 180 cggcagtggt ggcggcgtcc cggagcagga ggattccgtg ctgttccggc gcggcacagg 240 ccaggtgagg tcgcagccag tgcagtctcc ctattagcgc tctcagcacc cttcttccgg 300 cccaactctc cttccgcagc ctcgggacag catcaagtcg atccgctcac tggagttgtg 360 gtccgcgttt ttctacgtct tttcccactc cgttccctgc gaaccacatc cgcaagctcc 420 ttcctcgagc agtttgggct ccttgatagc gttgagtgga ggccctgccg cgacttggca 480 gtagcttatt ttgttcactc ctctctggct ggtgtggggg aggtgggggc attaggccag 540 ggtgaagcag gggaaccact taggagtctg ttaagatgat ctgaacttca gaacaagatg 600 ttattaacag agtgaaagta tttggattct gggtatattt tgaaatcgga ggcaacaggt 660 ttttcagata gattcgataa cggaggttat cctgaatagt tgaaaagata aagttgcctt 720 ttgctgaggt gggaaagaga agattgccag tagagcaggt ttctcaggag ttcagtcttg 780 ggcatagcat ggtaggggtg aatttggctg gagtgagttg gagagtagga gaagagaaat 840 ccaaggcaac atttgaccag cctgggcaac atagtgtgac tccgagtctg caaaaattag acgggtgttg tggtgcgcgt ctgtggtctc agctacctgg aaggttcagg ccttggaagg 960 ctcagggagg tggaggctgc agtgatctgt gattgcgcct ctgcactcca gcctgggcga cagagccaga ccctgtctta aaacaaaata aacggccggg cgcggtggct caagcctgta atcccagcac tttgggaggc cgaggcggcc ggatcacaag gtcaggagat cgagaccatc ctggctaaca cggtgaacc ccgtctctac aaaaattagc cggggcgtggt gacgggcgcc tgtagtccca gctactcggg aggctgaggc aggagaatgt catgaagccg ggaggcggag cttgcagtga gccgagatcg cgccactgca ctccagcctg ggcgatagag 1320 caagactccg tctcaata aaaaataat aaaaataaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacacatcggtagg 1380 catatttcaa ggaattctat ttaaaaaaaa ttttttaga gandaagttcg ctctctgtgg 1440 cccaggctgg agtacagtgg catgatccta gcccatggca gcgttgatct cttggcctca 1500 agcgaccctc ctttggagtc gctggggccta aaggagtgag ccaccacgaa atttattat 1560 aaatggagggg tagagaattt gggcaataa tggaggggga agtgagtta gaggaattttt 1620 aattatgtgt gtgtggtttt aaagagggg ggtcttgctc tgttgcccag gctgctgggg 1680 tgccagtgc gcaatcatga atcactacag ccttggactc ctggcctca gctatcctcc 1740 cacctctgcc tcccaagta ctgggattac tagtgtgagc cactgcacta agataggagc 1800 aacatgtttc agcatgtttg tgggttgata ggaagatga gatgggaaa gttgatgtcg 1860 gaagaagac aatggctaga gcaatgtcct agagtaggta agaaggatg gatttggcct 1920 ttgttggaaa cattagcggt tcttttgtg acagctatat agttaacaca tctatgatac 1980 gtgaatgggc agataggatg gcaggagatt tgaaagttc tcttgattct tactgttctc 2040 tagtgaag aagcaggtt atcagctaga agctgggatg ggagagaa gagaagatgg 2100 gaagtagata gttctttaga agagtgggca agggttggac tagggatt taggtggaat 2160 attgctaggc aacataaga gcctacttga gattcgtggt catgagttga aggagaccag 2220 acagcaagat tgtgtatgag ggcacccaca gagtaaatgg agagttgaaa ttaatgcagt 2280 tgtgatttta ccacgtggat atgaagaagt gagggggaga agtacaaagg agttctctta 2340 atgattgacc atggaattta agctggctaa gaaaggaagt gagaggccgg gcgcggtggc 2400 tcacgcctgt aatcccagca ctttgggaga ctgaggtggg tggattacct gaggtcagga 2460 gtttgagacc aacctggccg atatggcgaa accccatctc tataaaaat acagaaaaat 2520 tagccgggaa tggtggcagg tgcctgtaat cccagctact caagaggctg tggcaggagt 2580 atcccttgga cccaggaggt ggaggttgca gtgagccgag atcacgccac tgtactccag 2640 cctggacgat atagtgagac ttcacctcaa aaaaaaaaa aaagaaagga agtgaggatt 2700 ttaagaccct gagacacagt ttaaaagtg ggaggatcgg ccggcgctg tggctgacac 2760 ctgtaatccc agcactttgg gaggccgagt tggcagatc acaggtcag gagttcgaga 2820 ccagcctggc caatagtg aaaccttgtc tctaaaa atacaaaat tagccgggca 2880 tggtgtcacg tgtctataat cccagctact cgggaggctg aggcagaaaa attgcttgaa 2940 cctgggaggc agaggttgca zgagctgag atcactccat tgcactccag cctgggcaac 3000 aagagcaaa ctttgtcttt aaaaaaaaaaaaaaaag atacaaaaa ttagccgggc 3060 gtggtggcgc gtgcctataa tcccagctac tgggaggct gaggcaggag atcagttga 3120 acacgggagg cgaggtttgc agtgagccga gattgcgcca ctgcactcca gcctgggcga 3180 cagagcagga ctcctcttgg aaaaaaaaa ttagctgggc atggtggcag gtgcctgtag 3240 tctcagctac tagggaggct gaggcaggaa aatcacttga acccgggatg tggagtttgc 3300 agtgacccga gatcgtgcca ctgtactcca tctgggcga caaaatgaga ctctgcctca 3360 aaaaaaaaa aaaaaaaaag tgggaggatc aatgtactgc cagtcctaat gaagtggaat 3420 gattgtcccc atcaaatcac tagtaggagt aagttgcaga gcctagaagg tgatggttaa 3480 gagagtggga ttcttgaaac tgcatttatg gagaggttgt ggttattggt tataataaat 3540 aaatacagtt gaagtgagtg agtagctgag atttggggat gtatcagttc attcttacac 3600 tgctacaaag acatacctga gaccaggtat ttataaagat aagaggttta atcagctcac 3660 agttctgctg cctgtacagg cttctctgt ggaggcctaa ggaacttac agtcatggtg 3720 gaggtgaag gggaaacaag cacagctctc acatggccag gaggagag aggaagggg 3780 gaagtgctac atactttaaaaaccagat cttgtgagaa cgcttatcag gaaacagcac 3840 ttgggatgg tgctaaatca ttagaatca ccccatgat ccagtcgcct cctaccatgc 3900 ccacctccaa cactggggat cacattcag catgagattt gggtaggaac acagagctgc 3960 accacatcag aggatgtaca agattgtggt ggagaggagt ttagagacct gcaaatag 4020 gggtattgaa gggatcatct acatggatat ttaaatcacc aaaaattatg acaggagtag 4080 tgttggag aggactgcga tgtaacatt aaggaatgag gagagtgac tcggtaggct 4140 gtaggtgact gcaataggaa acgataatag actgtgagtc tggtgacaag attttccttc 4200 tttctttttt tccccccccc cgagacaggg cctctttttg ttgcccaggt gggagtgcag 4260 tggcgcgatc acggctcact acaacctcct cccaagctca agggattctc ccacttcagc 4320 ctctcaagta gctggaacta caggtgctga ccaccatgcc tggctacttt ttgtcaggat 4380 tttcaaggct gggaattttg agaggggaat ggaggagaat aatctgaaag tgcaagtaag 4440 gagcagggaa gatttctttt ttcttttttt tttttttttt tgagtcggag tctggctcag 4500 tcgcccaggc tggagtgcag tggcgagatc tccgctcact gcaagctccg cctcccgtgt 4560 tcacgccatt ctcctccttc agcctcccga gtagctggga ctacaggcgc ccgccaccac 4620 gcccagctaa ttgttttttt gtatttttag tagagacggg gtttcaccgt gttagccagg 4680 atggtctcaa tctcctgact ttgtgatccg cccaccccgg cctcccaaag cgcttgggat 4740 tacaggcgtg agccaccgcg ccagccagag cagggaagat ttcttcccca catctccagt 4800 aggtacagtg atatgaagtg tgtggaggag aaaagaggaa acatctatca tttgagatgg 4860 ctgcgaaagg aaaaggcatc ctcagggagc tagattttac ttagagcaag aaatgaaggg 4920 atgattcaga ggttaaaaga gtggatttta tgaattactc aagggagcac agtggaagtt 4980 tcaggaagtg gtaggagaag gtagagaatg gcagggtgtt gggaataatt tgagaaatct 5040 gagctactgg aaatgactga gaatcagata taaaggcagt cctggtggtc cgttctggct 5100 gccgttgctg tgtaacgaat ctgccaaaac ttagtggctt gaaacaacaa agaacatttt 5160 attatctctc attgtttctg tgggttagga atttgtgaga gccgtgctgg gcagttttcg 5220 tgcggctgtc tcgtggttgc acctacatag ttgctagagc tacagtagct ggggactgag 5280 cagctaggga ttggcaggct atctcttttt ttcatgtagt ctcatgaaga tttctttatg 5340 tggtttcaat gtgtgggctg gtttggattt ccttatagca tggtggcctc agttggattg 5400 ctgttttgtg atccttttca tccctccttg tcctgtcccc agacaaccac tgatctactt 5460 tctgtcacca tagattagcc tgcattttta agaattttta taaacgtgga atgatagagt 5520 accttttttg tcacgtttct tttatttatc atagctattt tgattttcat ccattttatt 5580 gctgagtagt atcccattgc atgtatac tatactgtat tcattcgctt gcttgtgaac 5640 atttgggctt tttccagttt gggactgtta acaagtagag ccactatgaa tattagtgta 5700 taagacttca tatagccaag gctggcagat cgcttgagcc caggagtttg agaccagcct 5760 gggaaacatg gtgaaacctc tatttttatt ttaaaatcaa aaattaaaaa ttttctataa 5820 aaaattttaa agaagacttt gtatagacat acgctttcat ttttcttgag tgaatactta 5880 ggtctcaggg tagatgtatt ttaagtcttt aaggagctgt caaactcttc ctcaaagtgg 5940 tggttgtacc atgttacttt ttaatataac agagattaat tgagcaaaga aaaattcaaa 6000 agttggacag cccccacaac taataggtt cagaacagct cccccatttt gcattttgac 6060 cagcaatgta tgaaagttcc atttgctcag tgtccctgca aacacctggt atggtcagtc tttttaattt tagcattat aatagatata gtggcttctt gtgattttaa ttagcatttc ctaatgacca gtgctgctgt tgatcatttc atgagtgtat ttgccatccg tatatctttt ttggtgaagt gtctattcaa atcatttggg tttttttttt ttttgtttttt tttttttgga 6300. gacagtgtct cactctgtca cccaggctgt tgtgcagtgg tgcaatcaca cagcctactg cagcctccac ctcctgcgct cagtcttctt gtctcagcct tctgagtagc tgaaattacg 6420 agcacacgcc acaatgcctg gctaattttt taaaatttg tagaaacaag gtctcattat gttgcctggg cttgtcgtga actcctgggc tcaagcaatc ttcctgcctc agcctcccaa 6540 agattgggat tgcaagtatg agccactgca cccggccaac ttacccatct tttaattgaa 6600 tttttttgtt gttgaggttt gagagttctt catgtttgct gggtacaata tctttatcag 6660 ataggtaact tgcatgtatt ttctcccggt ttacactttg gtttttcatt ttgttaacaa 6720 cgtcttttta agaacagaaa atcttaattt tgctgaaatc taatttttca gttttttctt 6780 tgatggtttt gagagaggag gtaaaaaaag actaggtaag ccgatagtta gacagagtcc 6840 tcggtagaac ttcccttcta acaaaaagca gcccaagaaa tcacttctct tctaacaagg 6900 agcagcctgg aagatcgggc tgtaaacatg tataaggaag cagctctggc acagaggggg 6960 agcttcctgg gtaatcagca agcttcacat acgtaaggtg ggtatgtgaa gtaaacacag 7020 tatgtgaagt aaacacagtg gaccttagta catactcaga taaggaagct ggaagcttgc 7080 atgttgtgag ttgttggggt tgcctgcagc tgcacggaga gaaaggggta cctggggcca 7140 ggcatgtcca ccatggtggc tccacctccc cttatttagc acatgcacaa taggaaagag 7200 ataagcaatg tggagtagct caggccaagg acctgcctgc ataataaaag gttggggtgg 7260 gggatgccag agattcacgc tctgtgcaga tggcaacacc tggtcctaac tggttttttg 7320 ctccctatgt gtagataagc taccccttc ccattagctc atttataaaa atgcttgcat 7380 ttcactgtgg aatgggaact cttttcagga cctctctctg caggagagag ctagtctctt 7440 tctttgcct attaaacttc tgctctagcc tcacaccctt ggtgtgtcag cgtccttgat 7500 ttcctcagcg tgagaccaag aacctcgggt gccaccccag gcaacaaggc catttcagtt 7560 tgttctttg ttataggcaa tccatgatca cagatttttc tctcttttt ttttttacac 7620 agttagagt tttagtttta cacttaggtc tgtaatccat tttgtattaa ttcttatatg 7680 tggctcagtg taggtggaaa tttggtttgt ttttgcataa ggatttccaa tagttttacc 7740 accatttctt gaaactacta tgctttctct attaaaccac atttgtaact ttagttaaaa 7800 tcagtcacat atatcacagg gctatttctg actctcaatt ctgttacatt gtctattagt 7860 gtatattgat gtcagtacta cacttttaat tactattgct tcagggtatg tcttgtaaac 7920 caaaaataaa attataggcc ccccccgcc ctgcacaacc aactgaatgg acccatcctc 7980 tcagccaagg gcattccaaa attaacctga aaaactagtt caagccatga tgggaagggg 8040 gagttggaca tgtctcatca caccctacta ccttttggaa ttactgatag aacagactct 8100 taaagtctga aaagaaacat ttacaaccta ccctctctga agcctgctac ctgggagctt 8160 catctgcatg ataaaacctt ggtctccaca accccttatg gtaacccaaa cattcctttc 8220 tgttgataat aactctttca actagttgcc aattagaaaa tctttaaatc ttcctatgac 8280 ctagaaacct ccctaccccc actttgagtt gtcctgcctt tcctgacaga actcatgtac 8340 atcttacata tattgattga tgcctcatgt ctccctaaaa tgtataaaac aaagctgtac 8400 cccaccacct tggggacatg tcatcaggac ctcctgtggc tgtgtcatag gagcgtcttt 8460 aactttggca aaataaactt tctaaattga ttgaaacctg tcttagctac ttctggttta 8520 cagtcttaaa gttagataat gtaaattgtc cagctttggt ttatttttgt ccttagtagt 8580 tccatataaa ttttagaatc agcttttcaa tttaatacac tactttcctc ttagatccac 8640 aattaaatat atttgatgct aacaattctg ttttatgttt ttcgtttttt ttttttgaga 8700 caagagtttc gctcttgttg cccaggctgg agtgcagtgg cgcgatcttg gctcaccaca 8760 acctccacct cccaggttca agcaattctt ctgcctcagc ctcccgagta gctgggatta 8820 caggcatgcg ccaccacgcc cggctaattt tgtattttta gtagagacgg ggtttcacca 8880 tgttgatcag gctggtcttg aactcctgac ctcaggtgat ccacccacct cggcctccca 8940 aagtgttggg attacaggcg tgaaccacca tgcctggcca gttctgttat ttttaaaacc 9000 caagtttccc tggtcatatc ttggttggat gaagcgtatt ttcaatagat taccctggaa 9060 aggctagtga gtacggtatt cttctacatt ttagactttt cttagtcttg ctacttcaag 9120 gacagctagg ctgcatataa aattcttggc tcatactttt tccccataaa tttctatgag 9180 aaagtctaat gataactgat tttctttatt ttgtaactta gtctttttgc ttagaggctc 9240 tctgaggatg ggagggggtt cttcctccca tccctaggaa tttttctttt ttttaaattc 9300 ctaatcacta gaccaccagg aagattgttt gttttgtttt gtttttattc ttcagggacc 9360 ccatttatac atacgttaaa taaatactgt ttgccaatgt atcaaccatt ttgcttctta 9420 tttatttttg ttcctttggt tctttttcat ggctttgctt tggtgctcct tagattttca 9480 gtcagatgta tttgtccttg ggtaccttgt aatcagtatt accttttctt ctgtcgcttt 9540 gttttctgtt cgttttgaaa ttacttgttt cctggtctgg caataacagt tgagatatga 9600 ggagtttgag ctgccatctg tctatgtatc ttgctttaag actgcactct tctattgata 9660 tcactggcct tgattttgtg atttctttat ttcttcagga ccacccttca ttttctactg 9720 tttgcttcct ttttttttga gatggagtct cactctgtca ctcaggctgg agtgcagtga 9780 tcttggctca ttgcaacctc tgcctcccgg gttccagcaa ttctcctgcc tcagcctccc 9840 aagtatctgg gactacaggt gtgcaccacc atgcccggct aagttttgta tttttaatag 9900 agacggggtt ttgccacatt ggcaggctgg tctcaaactc ctgatgtcaa gtgatccacc 9960 caccccaccc acctctgcat cccaaagtgc tgggattaca ggaatgagct gccgtgccca 10020 gcctcccccc tacccccctt tttttctttc gagacagaga ttataggtgt gagccactgg 10080 acccagcctg tttttattcc ttttaccaaa tctccaagga atatcttccc ttccaagtgc 10140 gaatgtaacc ttaagtcagt taacctcttt gtgattactt ttcttatctg caaagtgact 10200 taatgatctt aagtactttt tttttttgag acagggtctc actgtcaccc tggctggagt 10260 gcagtggcac gatctctgat ctccactcac tgcaatctcc tcttccctgg ttcaagcggc 10320 cctcccacct tagccttctg ggtagctggg actacagatg tgaaccacca cgcccagcta 10380 atttttgtac ttttttgtaga gatggggttt tgccatgttg cccaggctgg gattattaag 10440 tacttttat catacagcaa gattgacatt ttatattgga atacatttgt ctctatataa 10500 cggagattaa caggaaaatg acaagcctgg gtgcggtggc tcatgcctgt aatcccagca 10560 ctttgggagg ctgaggtggg aggatcactt gaggtcagga gttcgagacc agttttgcca 10620 agatgatgaa agcccatgtc tactaaaaat acaaaaatta gcccagcttg atggtgggcg 10680 cctataatcc cagctatttg agagactgag gcaggagaat cacttgaacc tgggcagcag 10740 aggttgcagt gagccgagat catgccactg cactccagcc tgggtggcat agcgagactc 10800 ttgtctcaag agaaaacaaa acaaaacaaa aaaaaaacag gaaaatgaca aaaagtaata 10860 ttacaactca gtgaatttta taacaaactt ttttggaatt cattgactaa tactatacca 10920 aatccaaaat actctctagt ataccaaatc caactctacc ctatagtata aattggattc 10980 tatttggact tgtctcacta atccctcata cagtgtgttt tattttttat tgaagtaaaa 11040 aaatttgtca ttttaaccat ttttaagtat atagttcagt aatattaagt atgttcatgt 11100 tgttgcgcaa tagatcttcg gaagtttttc gtcttgcaac ctgaaactct acccattagc 11160 aaattcccat ttctccttac acttagccct tggtaatcat cattcttttt tttttttttt 11220 tgagatggag ttttactctt gttgcccagg ctggagtgca atggtgcaat ctcgactcac 11280 cacaacctcc gcctcccagg ttcaagcaat tctacctcag cctcccgagt agctgggatt 11340 acagtcatgc accaccacgc ccggctaatt ttgtattttt agtagagaag gggtttctcc 11400 atgttgaggc tggtctcgaa ctcctgacct caggtgatct gcccacctcg gcctcccaaa 11460 gtgctgggat tacaggcgtg agccactgcg cctggcccat tctttctaat tctataaatt 11520 tgactactta gttaccttac ataaataaat tcttatagtt agtgttattt ttgcttccat 11580 gccttttttg ttgttgttca tgctcttact tggaatgcgt tctattttgt ctacctatgc 11640 acatcctgtt gggttttttt tttttttggg ggtttttttt gttttttttt gttttttttt 11700 cccagacaag gtctcaattt gttacccagg ctggagtgca gcggcgccat ctccactcac 11760 tgcatcctca acttcctggg cccaggtgat cctctcgcct cagcccctgc aggtagctgg 11820 gactataggc atgtgccacc atgcccagct aaatttggtt tttttgttttg tttgttttg 11880. agacagagtc tcactctgtc acccaggctg gagtgcagtg gcacaatctc agctcactgc aatctctgcc gcccgggttc aagtgattct cctgcctcag cctcccaagc agctgggatt acaggtgact gccaccacgc cagctaagtt ttgtagtttt agtaggatg gggtttcacc 12060. ttgttggcca tgctggtctc gaactcctga cctcgtgatc tgcctgcttc tgcctcccaa agtgctggaa ttacaggcat gagccaccac gcccggccag aatttttgta tttttagtag acacaaggtt cttaccctgt tgcctaggct ggtctggaag tcctggactc aagcaattca cctgccttgg cctcccaaaa tgctgggatt acaagccacc atgcccggcc taaatcctgt tgttttgttt tgttttattt tgttttgttt tgttttgttt gttttttgag acagagtctc 12360 gctatgtctc tcaggctgta gtgcagtggc gcgatcttgg ctcactgcca cctctgcctc 12420 ccaggttcaa gtgattctcc tgcctcagcc tcccaagtag ctgggattac aggcatgtgc 12480 tactatgtcc ggctaatttt tgtattttta gtagagacag ggtttcacca tgttggccag 12540 gctggtctcg aactcctgac ctcgtgatcc acccacctcg gccacccaaa gtgctgggat 12600 tacaggcgtg agtggttttt attcttagg ccggttttcct ccatatgatc ttgcagtaga 12660 cattaatttc tttcctttt attaaaata ctgtttgtat ttcacatttt gatgtttgtt 12720 aagatttgtt ttatattgtt ttttgttttg tcttgtgtga tagtcttaaa tccctagtta 12780 gataataact ggagagtacc atgtttctat atactctca gtgacttgca cagtgctagc 12840 agatagtgct aaaaaattat ttattattat tattattttg ttattgttgt tgttgttgtt 12900 agacagggtc ttcctctgtc acccaggcta gagggcaatg ggatgatcat agcttactgc 12960 agcctccaac aactgggctc atgtaattct cctgcctcag cttcccaagt agctgggatt 13020 acaggcatga gccaccatgt ctggacaaaa atatttccag gtgcagtggc tcatgcctgt 13080 aattcccaca cttgggaggc cgagcgaggc tggaggatca cttgagccta ggagttcaag 13140 accagcttgg ctaagatggc gagaccccgt ccctacaaaa aattttaaaa actagccagg 13200 catggtggca tgcacctata ttcccaacta ctcagtgggc tgaggtggga gggtcatttg 13260 aacacagga tttgagggga gaaaaaaaga agagagaaag agaagtgaaag gaagaaga aggaaggagg gaggagaga agaaagaaac gaaagaaagg aaaagaaaag gaggaaaga aaattggtac caggaaagca ggaaaggga atggaagtaa aaaaataata aaatataa aatgaaaatt ggttagtcac father ttgtatcctt father aacatta 13560. tttcaaaaga aaaaatattc tttggatcat aggttctgag gtcagaacag cattcccgta gtctagatga agtcaagttt tatctgatct taattgaat aaatatagct ggccttgaac aaatctactc atggtatgtg gataggaatt aaattgtagg ggcattcact tgatggcatt cattcttaga acatttacct atgtctagct tttggagtaa agtcacataa cctctaacca ggtaagtttc ctgtggcttt atttaggatt ttaaatactc attttcagtg taattttgtt 13800 atgtgtggat taagatgact cttggtacta acatacattt tctgattaaa cctatctgaa 13860 catgagttgt ttttatttct taccctttcc agagcgatga ttctgacatt tgggatgata 13920 cagcactgat aaaagcatat gataaagctg tggcttcatt taaggtatga aatgcttgct 13980 tagtcgttt cttattttct cgttattcat ttggaaagga attgataaca tacgataaag 14040 tgttaaagta catgttattc agttttcatt ttgaagatta gatggtagta tgagttagtt 14100 aaatcaggtg atatcctcct ttagaagttg atagcctata tatgtcatcc tttgtggagg 14160 caatttaaat aaaatttaaa acatttattc ctggctgggt atggtggctc actcctgtaa 14220 tcccagcact ttgagaggct gaggcgggtg gatcacctga ggtcaggagt ttgagaccag cctggccaac atggtgaaac cccgtcttta ctaaaaatac aaaaattagc caagcatggt ggcacgtgcc tgtaatccca gctgcttggg acactgaggc aggagaattg cttgaacctg gggggcagag gttgcaatga ttgcaccact gcactccagc ctgggcgata gagtgagact 14460 ccatctcaga aaacgaacaa acaatgtatt ccttttagta tttttacatt gtatcaaact atggaagtcc tctaattgag attack aaaagacaat ctgaattata attack tttaacaagc atgtagtaaa attachment to the cattagtaca gcaattaata tttgtagcat gctgacagtg ctctgtgtgc gtttcatata ttaaattact ctaatcatcc caaatcctgt aagttgggta tcaattcaag tgttcctatt gggtaggaat atacagttct 14760 tttaggaaat gtagtatggt tctgtgtctc aaacaggaca cttacacagt tggccaacat 14820 catcaccttc tccattctct gagatgttta gtcttactga gcactaaata tgggtcatca 14880 atagtccaga ctaccttgag caaacaatag tccagactac cttgagcaaa cagagcatat 14940 actcatacag tgtataaaga gcaccaagca tacagatttc atgtctttct catagttact 15000 cttgtaacat gagctaaaga tcagacctct atgtcacctt tgtaactgat ttctagattt 15060 tttttttttt ttgagatggg gtcttgccct gtcacccagg ctggagtgta gtggcgtgat 15120 catgcctcat tggagccttc aactcatgag ctcaaacaat cctcctacct cagcttcctg 15180 agtagttggg accacaggtg tgtgccacca cacccagctc atttttgtat tctttgtaga 15240 gatgcagtct caccctgttg cccacgctgg cctggactc ctgagctcaa aagatccctc 15300 cgccttgacc ttccaagtg ctgggattac aagcatgac cactgcacccc ggcctagatt 15360 tttaaatgtg ctttccagta tacactgaaa ctagaagtcg actaaagaat taccagaga 15420 attcttaaa atagagattg aaatggggct cgatgtggga tggttggtg atattgcagg 15480 gagaagtaat ctgagtaag gaggaaag actgatttgg gaaaacgata gttttagtag 15540 tgagtttgag tatgattga gttgattg aatttgatt aagttgaggt tgaatatga 15600 ttaagttgag gttgagttg aggtagat tagatgtga attgatcat tggatgtt 15660 agattgagaa aagtcacagc tggattata gcttcagaag tgtgttgca gagagttgca 15720 actaaagtaa tagaataga tggccttggc cggggcgcggt ggctcacgcc tgtaatccca 15780 gtactttggg aggctgaggc gagcaatca cgaggtcagg agttcagac cagcctggcc 15840 cacatggtga aaccccgtct ttattaaaaa tacaaaatt agctgtgcac agtggtgcac 15900 gcctgtaatc ccagctactc gggaggctga gaggagaa tcgcttgaac ctgggaggtg 15960 gaggttgcag tgagctgaga tcagtgtgac tgcactccag cccggtgaca gagtgagact 16020 ctgtgtaaaaaaaaaaaaaaat tggccgtaag caagtaag aggatggcca 16080 gctcttattg ggaatgccta aatctaggc ttgatcagaa gtaatgaac cgttggggcc 16140 ctacattgct atgacatcca aagggccatg aatatcagga agaaagataa ttaacagggt 16200 ctaatgttac agagaggttg agagcaagga gatttgatta aaagggtctt tagagctgat 16260 gtcaggtgta tgatgccttt aagagcagtt tttatagtgc agggggtggt caaaagagaa 16320 aataggtgct ttctgaggtg acggagcctt gagactagct tatagtagta actgggttat 16380 gtcgtgactt ttattctgtg caccaccctg taacatgtac atttttattc ctattttcgt 16440 agcatgctct aaagaatggt gacatttgtg aaacttcggg taaaccaaaa accacaccta 16500 aaagaaaacc tgctaagaag aataaaagcc aaaagaagaa tactgcagct tccttacaac 16560 aggttatttt aaaatgttga gatttaactt caaaggatgt ctcattagtc cttatttaat 16620 agtgtaaaat gtctttaact tagtgatta gtacagtgtt tctattgaca tatactata 16680 caactcaa aacaactatt aaatttctg tattttagga acatgcatat tagtcatgaa 16740 agtataaaga attagatggg aatgaataat gctaaaatca ggacatgtgt tccattgtg 16800 aatggaaggc aggagagg tgccgttgg aaggagtacc aagagccgt aagctgaatt 16860 ggcagtgttt tacatcttaa gctgagagat agattttt ttccctttt tctttaaaaa 16920 ctctaaaact gttaattcca aggaacccag aagtctaggt agattatttc tgctagttaa 16980 aagcagtagt cctgaaagct gatatttg gtgtctttg agccaacttt agttcatca 17040 ttaccaaggg ggaagagagc tacagttga tgagcacttg ctctaggcca gtccagagtg 17100 ctgggcacca tacgcatttt atctccctcc cgctattcac aacaaatatg ggaggtagtt 17160 tatattatag ccatctaata agatggggaa actaagactc aaagagattc agaaacttgt 17220 ccatgattat aaatgtaaga gagttggaat tcagatttat gtatttagac cccaagcctt 17280 tctcattaca tcattttgcc ttccaaatct ctaccctcta tccttcacct ccccactgat 17340 caaaacgaga tgatagtttg ccctcttcaa aagaaatgtg tgcatgtata tatctttgat 17400 ttcttttgta gtggaaagtt ggggacaaat gttctgccat ttggtcagaa gacggttgca 17460 tttacccagc taccattgct tcaattgatt ttaagagaga aacctgtgtt gtggtttaca 17520 ctggatatgg aataagagag gagcaaaatc tgtccgatct actttcccca atctgtgaag 17580 tagctataa tatagaacaa aatgctcaag aggtaaggat acaaaaaaaa aaaaattcaa 17640 tttctggaag cagagactag atgagaaact gttaaacagt atacacagtt gtcagtttga 17700 tccaccgagg cattaatttt ttcttaatca cacccttata acaaaaacct gcatattttt 17760 tcttttaaa gaatgaaaat gaaagccaag tttcaacaga tgaaagtgag aactccaggt 17820 ctcctggaaa taaatcagat aacatcaagc ccaaatctgc tccatggaac tcttttctcc 17880 ctccaccacc ccccatgcca gggccaagac tgggaccagg aaaggtaaac cttctatgaa 17940 agttttccag aaaatagtta atgtcgggac atttaacctc tctgttaact aatttgtagc 18000 tctcccatga aacttttgta gcttaaatac acaagaattt tttgaaaagg aaataagata 18060 atgatgcaaa atagttaatt ttttaaaaaa atgttagaca ctgcagtgga tgcaacaaaa 18120 tactttatat gaaagattta tccagttaac ttttgtggag tattaggtat tagactaata 18180 attagcacac ttacttaagt tagaaagtat aataatgcgc cggacgcggt agctcacgcc 18240 tgtaatccca gcactttggg aggccaaggt gggcggatca caaggtcagg agatcgagac 18300 catcctggct aacacggtga aaccccatct ctactgaaaa tacaaaaaaa tttgccgggc 18360 gtgatggcgg gcacctgtag tcccagctac tcgggaggct gaggcaggag gatggtgtga 18420 accccggagg cagagcttgc agtgagtcaa gatcgtgcca ctgcactcca acctgggcga 18480 cagaatgaga ctccatctca aaaaaaaaa caaaaaaaaaa tgtaataata 18540 attatcatt agctggatga tatgctgttg tttcccatgt cacctgtata agatatgtaa 18600 aataagaaca cattatttac atctaatata gataaaatcc tgaggcgctc tcagattgtt 18660 ttgtagagtt caaatgtaaa tattgttttc attatggtc cttttggtta taagtaacag 18720 aaatcaactc taaaaagatt tttattatag gttagattat gtcatggaac cttaaggctt 18780 gtccctttct agttcttttg tgtaaagcgg tgatttcttc catggaggga atggtattta 18840 ggcaattttt ttttttttt cgagatggag tcttgctctg tcgctcaggc tggagtgcag 18900 tggcaccatt tcagctcact gcaacttcca cctcctgggt tcaagtgatt ctcctgcttc 18960 agcctcccaa gtagctgaga ttacaggcac ccgccaccac acccggctta ttttgtattt 19020 ttagtagaga tggggtttca ccatgttggc caggctggtc ttgaactcct gacctcaagt 19080 gatctcccca ccttggcctt ccaaagtgct aggattacag gcgcctagcc taggcagtca 19140 ttttcaaaaa acaagcatga ctcaccaaaa gttttaagat tttctgtgat aatgttctta 19200 ttgaggctta cattatatta cagtttcttg aatctaaaat gatgtaccct cttagaatat 19260 atacatcatg cttcattggt ctcagggggc tgatttttat aaggagagat ttgctagtttt 19320 tcacaatatg tcctctaagt tggcatgtat agctaaacag gctttcataa aatatacaa 19380 tttagttaat gaaatttggg atatagtctt ttatgattga aataattttg ctaaatagac 19440 tgtctctgat ttattaggta atcaccactc ttattttgtt ttacttcctt aatgtctaca 19500 tagaaaggaa atgagaaaaa tccagaggtt gtcatttgac ttatgagtct gtttgacttc 19560 aggatttggt acatgaaatt tcacttaatc tttttgatat gtataaaaca aatattctgg 19620 gtaattattt ttatcctttt ggttttgagt cctttttatt cctatcatat tgaaattggt 19680 aagttaattt tcctttgaaa tattccttat agccaggtct aaaattcaat ggcccaccac 19740 cgccaccgcc accaccacca ccccacttac tatcatgctg gctgcctcca tttccttctg 19800 gaccaccagt aagtaaaaa gaggataggt tagattttgc tttcacatac aatttgataa 19860 ttagcagaat agaggattgt aaaatgtcat tgtagaaacat cccttgggcc agattctaat 19920 gggtagaaat ttgaactaaa cctctgggtt ttgtttgttt ttaatgcctt tctgttaccc 19980 agatgcagtg ctcttgtagt cccaagtcta agctctaggt tgccttcttt cctggcagaa 20040 gttggtgtct atgccataag gaggtagttc ctgttagaag ggatttaatt ataccttata 20100 taaggaatta gtgtttgccc ttctaggtat agttggatgt tagcttctga tgtaaactgg 20160 atttcttttt ctttctctct cttttttttt ttttgttttg gaggcagagt tttgcccttg 20220 taccccaggc tggagtgcag tggtgtgatc tcagctcaca gcaacctccg cctcctgggt 20280 tcaagcaatt ctgcctcggc ctcccaagta gctgggatta caggcgactg ccaccacacc 20340 cggctaattt ttgttttatt agtagagatg gggtttcacc atgttggcca gactgatctt 20400 gaactcctga cctcaggtga tccacccgcc ttggcctccc aaagcgctgg gattacaggc 20460 gtgagctgcc gcacccagct gtaaactgga tttctaatgg tagattttta ggtattaaca 20520 atagataaaa agatactttt tggcatactg tgtattggga tggggttaga acaggtgttc 20580 tacccaagac attacttaa aatcgccctc gaaatgctat gtgagctgtg tgtgtgtgtg 20640 tgtgtgtgtg tgtattaagg aaaagcatga aagtatttat gcttgattttt ttttttttac 20700 tcatagcttc atagtggaac agatacatag tctaaatcaa aatgtttaaa ctttttatgt 20760 cacttgctgt cttttcgtcc tcgttaaatt taattttgtt ggtctttttgt tgttattggt 20820 tggttttctc caaatgctag ctatgttaag aaatttaagg ccaggtacag tggctcatgc 20880 ctgtaatccc ggcattttag aaggctgagg caggaggatc acttgagctc aggagtttga 20940 gaccagtctg ggcacatag cagacctcg tctttgtttta ggggaaaaa aagaaattta 21000 agtaggagat tatataagca aaatacaat taatttccag cattcactat ataataaa 21060 tctccagact ttacttttt gtttactgga tataaacaat atctttct gtctccagat 21120 aattccccca ccacctccca tatgtccaga ttctctgat gatgctgatg ctttgggaag 21180 tatgttaatt tcatgtaca tgagtggcta tcatactggc tattatatgg tagtaatca 21240 ctcagcatct tttcctgaca atttttttgt agttatgtga ctttgttttg taaatttata 21300 aaatactact tgcttctctc tttatac taaaaaataa aaataaaaa atacactgt 21360 ctgaggctta aattactctt gcattgtccc taagtataat tttagttaat tttaaaagc 21420 tttcatgcta ttgttagatt attttgatta tacacttttg aattgaaatt atactttttc 21480 taaataatgt tttaatctct gatttgaaat tgattgtagg gaatggaaaa gatgggataa 21540 tttttcataa atgaaaaatg aaattctttt tttttttttttttttttga gacggagtct 21600 tgctctgttg cccaggctgg agtgcaatgg cgtgatcttg gctcacagca agctctgcct 21660 cctggattca cgccattctc ctgcctcagc ctcagaggta gctgggacta caggtgcctg 21720 ccaccacgcc tgtctaattt tttgttttt tttgtaaaga cagggtttca ctgtgttagc 21780 caggatggtc tcaatctcct gaccccgtga tccacccgcc tcggccttcc aagagaaatg 21840 aaattttttt aatgcacaaa gatctggggt aatgtgtacc acattgaacc ttggggagta 21900 tggcttcaaa cttgtcactt tatacgttag tctcctacgg acatgttcta ttgtatttta 21960 gtcagaacat ttaaaattat tttattttat tttatttttt tttttttttt gagacggagt 22020 ctcgctctgt cacccaggct ggagtacagt ggcgcagtct cggctcactg caagctccgc 22080 ctcccgggtt cacgccattc tcctgcctca gcctctccga gtagctggga ctacaggcgc 22140 ccgccaccac gcccggctaa ttttttttta tttttagtag agacggggtt tcaccgtggt 22200 ctcgatctcc tgacctcgtg atccacccgc ctcggcctcc caaagtgctg ggattacaag 22260 cgtgagccac cgcgcccggc ctaaaattat ttttaaaagt aagctcttgt gccctgctaa 22320 aattatgatg tgatattgta ggcacttgta tttttagtaa attaatatag aagaaacaac 22380 tgacttaaag gtgtatgttt ttaaatgtat catctgtgtg tgcccccatt aatattctta 22440 tttaaaagtt aaggccagac atggtggctt acaactgtaa tcccaacagt ttgtgaggcc 22500 gaggcaggca gatcacttga ggtcaggagt ttgagaccag cctggccaac atgatgaaac 22560 cttgtctcta ctaaaaatac caaaaaaaat ttagccaggc atggtggcac atgcctgtaa 22620 tccgagctac ttgggaggct gtggcaggaa aattgcttta atctgggagg cagaggttgc 22680 agtgagttga gattgtgcca ctgcactcca cccttggtga cagagtgaga ttccatctca 22740 aaaaaagaaa aaggcctggc acggtggctc acacctataa tcccagtact ttgggaggta 22800 gaggcaggtg gatcacttga ggttaggagt tcaggaccag cctggccaac atggtgacta 22860 ctccatttct actaataca caaaacttag cccagtggcg ggcagttgta atcccagcta cttgagaggt tgaggcagga gaatcacttg aacctggggag gcagaggttg cagtgagccg agatcacacc gctgcactct agcctggcca acagagtgag aatttgcgga gggaaaaaaa agtcacgctt cagttgttgt agtataacct tggtatattg tatgtatcat gaattcctca ttttaatgac caaaagtaa taaatcaaca gcttgtaatt tgttttgaga tcagttatct gactgtaaca ctgtaggctt ttgtgttttt 23220. gactgtaaca ctgtaggctt ttgtgttttt aatgtatata taaagtattg gtataattta tgttctaat aactttcttg agaaataatt 23340. gcagtttacc tttgaaagta tacaagttgg ctgggcacaa tggctcacgc ctgtaatccc agcactttgg gaggccaggg caggtggatc acgaggtcag gagatcgaga 23400 ccatcctggc taacatggtg aaaccccgtc tctactaaaa gtacaaaaac aaattagccg 23460 ggcatgttgg cgggcacctt ttgtcccagc tgctcgggag gctgaggcag gagagtggcg 23520 tgaacccagg aggtggagct tgcagtgagc cgagattgtg ccagtgcact ccagcctggg 23580 cgacagagcg agactctgtc tcaaaaaata aaataaaaaa gaaagtatac aagtcagtgg 23640 ttttggtttt cagttatgca accatcacta caatttaaga acattttcat caccccaaaa 23700 agaaaccctg ttaccttcat tttccccagc cctaggcagt cagtacactt tctgtctcta 23760 tgaatttgtc tattttagat attatatata aacggaatta tacgatatgt ggtcttttgt 23820 gtctggcttc tttcacttag catgctattt tcaagattca tccatgctgt agaatgcacc 23880 agtactgcat tccttcttat tgctgaatat tctgttgttt ggttatatca cattttatcc 23940 attcatcagt tcatggacat ttaggttgtt tttatttttg ggctataatg aataatgttg 24000 ctatgaacat tcgtttgtgt tctttttgtt tttttggttt tttgggtttt ttttgttttg 24060 tttttgtttt tgagacagtc ttgctctgtc tcctaagctg gagtgcagtg gcatgatctt 24120 ggcttactgc aagctctgcc tcccgggttc acaccattct cctgcctcag cccgacaagt 24180 agctgggact acaggcgtgt gccaccatgc acggctaatt ttttgtattt ttagtagaga 24240 tggggtttca ccgtgttagc caggatggtc tcgatctcct gacctcgtga tctgcctgcc 24300 taggcctccc aaagtgctgg gattacaggc gtgagccact gcacctggcc ttaagtgttt 24360 ttaatacgtc attgccttaa gctaacaat cttaacctt gttctactga agccacgtgg 24420 ttgagatagg ctctgagtct agctttaac ctctacttt tgtcttaga aatctaagca 24480 gatgcaat gactaagaat atgttgttg aaatacata aaataggtta taactttgat 24540 actcattagt aacaatctt tcaatacatc ttacggtctg ttaggtgtag attagtaatg 24600 aagtgggaag ccactgcaag ctagtataca tgttaggaaa gatgaaagc attgaagcca 24660 gagagagac agaggacatt tgggtagat ctgacagaa aaaaaaatgt tttagtta 24720 atttttgact ttaaattttt ttttatta gtgaatactg gtgtttaatg gtctcatttt 24780 aataagtatg acacaggtag tttaaggtca tatattttt ttgatgaaaa taaggtatag 24840 gccgggcacg gtggctcaca cctgtaatcc cagcactttg ggaggccgag gcaggcggat 24900 cacctgaggt cgggagttag agactagcct caacatggag aaaccccgtc tctactaaaa 24960 aaaatacaaa attaggcggg cgtggtggtg catgcctgta atcccagcta ctcaggaggc 25020 tgaggcagga gaattgcttg aacctgggag gtggaggttg cggtgagccg agatcacctc 25080 attgcactcc agcctgggca acaagagcaa aactccatct caaaaaaaaa aaaataaggt 25140 ataagcgggc tcaggaacat cattggacat actgaaagaa gaaaaatcag ctgggcgcag 25200 tggctcacgc cggtaatccc aacactttgg gaggccaagg caggcgaatc acctgaagtc 25260 gggagttcca gatcagcctg accaacatgg agaaaccctg tctctactaa aaatacaaaa 25320 ctagccgggc atggtggcgc atgcctgtaa tcccagctac ttgggaggct gaggcaggag 25380 aattgcttga accgagaagg cggaggttgc ggtgagccaa gattgcacca ttgcactcca 25440 gcctgggcaa caagagcgaa actccgtctc aaaaaaaaaa ggaagaaaaa tattttttta 25500 aattaattag tttatttatt ttttaagatg gagttttgcc ctgtcaccca ggctggggtg 25560 caatggtgca atctcggctc actgcaacct ccgcctcctg ggttcaagtg attctcctgc 25620 ctcagcttcc cgagtagctg tgattacagc catatgccac cacgcccagc cagttttgtg 25680 ttttgttttg ttttttgttt tttttttg agagggtgtc ttgctctgtc ccccaagctg 25740 gagtgcagcg gcgcgatctt ggctcactgc aagctctgcc tcccaggttc acaccattct 25800 cttgcctcag cctcccgagt agctgggact acaggtgccc gccaccacac ccggctaatt 25860 tttttgtgtt tttagtagag atggggtttc actgtgttag ccaggatggt ctcgatctcc 25920 tgaccttttg atccacccgc ctcagcctcc ccaagtgctg ggattatagg cgtgagccac 25980 tgtgcccggc ctagtcttgt atttttagta gagtcgggat ttctccatgt tggtcaggct 26040 gttctccaaa tccgacctca ggtgatccgc ccgccttggc ctccaaaagt gcaaggcaag 26100 gcattacagg catgagccac tgtgaccggc aatgttttta aattttttac atttaaattt 26160 tattttttag agaccaggtc tcactctatt gctcaggctg gagtgcaagg gcacattcac 26220 agctcactgc agccttgacc tccagggctc aagcagtcct ctcacctcag tttcccgagt 26280 agctgggact acagtgataa tgccactgca cctggctaat ttttattttt atttatttat 26340 ttttttttga gacagagtct tgctctgtca cccaggctgg agtgcagtgg tgtaaatctc 26400 agctcactgc agcctccgcc tcctgggttc aagtgattct cctgcctcaa cctcccaagt 26460 agctgggatt agaggtcccc accaccatgc ctggctaatt ttttgtactt tcagtagaaa 26520 cggggttttg ccatgttggc caggctgttc tcgaactcct gagctcaggt gatccaactg 26580 tctcggcctc ccaaagtgct gggattacag gcgtgagcca ctgtgcctag cctgagccac 26640 cacgccggcc taatttttaa attttttgta gagacagggt ctcattatgt tgcccagggt 26700 ggtgtcaagc tccaggtctc aagtgatccc cctacctcg cctcccaaag ttgtgggatt 26760 gtaggcatga gccactgcaa gaaaacctta actgcagcct aataattgtt ttctttggga 26820 taactttaa agtacattaa aagactatca acttaatttc tgatcatatt ttgttgaata 26880 aaataagtaa aatgtctttgt gaaacaaaat gctttttaac atccatataa agctatctat 26940 atatagctat ctatatctat atagctattt ttttaactt cctttatttt ccttacaggg 27000 ttttagacaa aatcaaaaag aaggaaggtg ctcacattcc ttaaattaag gagtaagtct 27060 gccagcatta tgaaagtgaa tcttactttt gtaaaacttt atggtttgtg gaaaacaaat 27120 gttttgaac atttaaaaag ttcagatgtt agaaagttga aaggttaatg taaaacaatc 27180 aatattaaag aattttgatg ccaaaactat tagataaaag gttaatctac atccctacta 27240 gaattctcat acttaactgg ttggttgtgt ggaagaaaca tactttcaca ataaagagct 27300 ttaggatatg atgccatttt atatcactag taggcagacc agcagacttt tttttattgt 27360 gatatgggat aacctaggca tactgcactg tacactctga catatgaagt gctctagtca 27420 agtttaactg gtgtccacag aggacatggt ttaactggaa ttcgtcaagc ctctggttct 27480 aatttctcat ttgcaggaaa tgctggcata gagcagcact aaatgacacc actaaagaaa 27540 cgatcagaca gatctggaat gtgaagcgtt atagaagata actggcctca tttcttcaaa 27600 atatcaagtg ttgggaaaga aaaaaggaag tggaatgggt aactcttctt gattaaaagt 27660 tatgtaataa ccaaatgcaa tgtgaaatat tttactggac tctattttga aaaaccatct 27720 gtaaaagact gaggtggggg tgggaggcca gcacggtggt gaggcagttg agaaaatttg 27780 aatgtggatt agattttgaa tgatattgga taattattgg taattttatg agctgtgaga 27840 agggtgttgt agtttataaa agactgtctt aatttgcata cttaagcatt taggaatgaa 27900 gtgttagagt gtcttaaaat gtttcaaatg gtttaacaaa atgtatgtga ggcgtatgtg 27960 gcaaaatgtt acagaatcta actggtggac atggctgttc attgtactgt ttttttctat 28020 cttctatatg tttaaaagta tataataaa atatttaatt ttttttaaa 28070 <![CDATA[ <210> 2]]> <![CDATA[ <400> 2]]> 000 <![CDATA[<210> 3]]> <![CDATA[<400> 3]]> 000 <![CDATA[<210> 4]]> <![CDATA[<400> 4]]> 000 <![CDATA[<210> 5]]> <![CDATA[<400> 5]]> 000 <![CDATA[<210> 6]]> <![CDATA[<400> 6]]> 000 <![CDATA[<210> 7]]> <![CDATA[<400> 7]]> 000 <![CDATA[<210> 8]]> <![CDATA[ <400> 8]]> 000 <![CDATA[ <210> 9]]> <![CDATA[ <400> 9]]> 000 <![CDATA[ <210> 10]]> <![CDATA[ <400> 10]]> 000 <![CDATA[ <210> 11]]> <![CDATA[ <211> 24]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Introduction]]> <![CDATA[ <400> 11]]> gctgatgctt tgggaagtat gtta 24 <![CDATA[ <210> 12]]> <![CDATA[ <211> 25]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Introduction]]> <![CDATA[ <400> 12]]> caccttcctt ctttttgatt ttgtc 25 <![CDATA[ <210> 13]]> <![CDATA[ <211> 21]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Probe]]> <![CDATA[ <400> 13]]> tacatgagtgctatcatac t 21 <![CDATA[ <210> 14]]> <![CDATA[ <211> 27]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Introduction]]> <![CDATA[ <400> 14]]> catggtacat gagtggctat catactg 27 <![CDATA[ <210> 15]]> <![CDATA[ <211> 25]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Introduction]]> <![CDATA[ <400> 15]]> tggtgtcatt tagtgctgct ctatg 25 <![CDATA[<210> 16]]> <![CDATA[<211> 21]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Probe]]> <![CDATA[<400> 16]]> ccagcatttc catataatag c 21 <![CDATA[<210> 17]]> <![CDATA[<211> 20]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Primer]]> <![CDATA[<400> 17]]> caggaggatt ccgtgctgtt 20 <![CDATA[ <210> 18]]> <![CDATA[ <211> 24]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Introduction]]> <![CDATA[ <400> 18]]> cagtgctgta tcatcccaaa tgtc 24 <![CDATA[ <210> 19]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Probe]]> <![CDATA[ <400> 19]]> acaggccaga gcgat 15 <![CDATA[ <210> 20]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 20]]> attcactttc ataatgctgg 20 <![CDATA[ <210> 21]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 21]]> cactttcata atgctggc 18 <![CDATA[ <210> 22]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> ]]>Synthetic Oligonucleotides <![CDATA[ <400> 22]]> ttcactttca taatgctggc 20 <![CDATA[ <210> 23]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 23]]> tcactttcat aatgctgg 18 <![CDATA[ <210> 24]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 24]]> actttcataa tgctggcag 19 <![CDATA[ <210> 25]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 25]]> cactttcata atgctggcag 20 <![CDATA[ <210> 26]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 26]]> cactttcata atgctggca 19 <![CDATA[ <210> 27]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 27]]> tcactttcat aatgctggca 20 <![CDATA[ <210> 28]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 28]]> ctttcataat gctggc 16 <![CDATA[ <210> 29]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 29]]> actttcataa tgctggc 17 <![CDATA[ <210> 30]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic oligonucleotides <![CDATA[ <400> 30]]> tcactttcat aatgctggc 19 <![CDATA[ <210> 31]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 31]]> actttcataa tgctgg 16 <![CDATA[ <210> 32]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 32]]> cactttcata atgctgg 17 <![CDATA[ <210> 33]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 33]]> ttcactttca taatgctgg 19 <![CDATA[ <210> 34]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 34]]> attcactttc ataatgctg 19 <![CDATA[ <210> 35]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 35]]> agattcactt tcataatgct 20 <![CDATA[ <210> 36]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 36]]> tcactttcat aatgctggt 19 <![CDATA[ <210> 37]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 37]]> tcactttcat aatgctgga 19 <![CDATA[ <210> 38]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (19)..(19)]]> <![CDATA[ <223> The bases at these positions are RNA]]> <![CDATA[ <400> 38]]> tcactttcat aatgctggu 19 <![CDATA[ <210> 39]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 39]]> tcactttcat aatgctggaa 20 <![CDATA[ <210> 40]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[<2]]> 13> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 40]]> <![CDATA[tcactttcat aatgctggat 20 <![CDATA[ <210> 41]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 41]]> tcactttcat aatgctggac 20 <![CDATA[ <210> 42]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 42]]> tcactttcat aatgctggtc 20 <![CDATA[ <210> 43]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 43]]> tcactttcat aatgctggtt 20 <![CDATA[ <210> 44]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 44]]> tcactttcat aatgctggta 20 <![CDATA[ <210> 45]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 45]]> tcactttcat aatgctggcc 20 <![CDATA[ <210> 46]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 46]]> tcactttcat aatgctggct 20 <![CDATA[ <210> 47]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> DNA]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic Oligonucleotides]]> <![CDATA[ <400> 47]]> ctcactttca taatgctgg 19 <![CDATA[ <210>...

Claims

1. Use of a modified oligonucleotide (SEQ ID NO: 21) or a salt thereof according to the following chemical structure for the preparation of a medicament for the treatment of spinal muscular atrophy (SMA) or for improving at least one symptom of SMA: .

2. Use of a modified oligonucleotide or a salt thereof according to the following chemical structure for the preparation of a medicament for regulating SMN2 RNA in cells or individuals: (SEQ ID NO: 21).

3. As used in claim 1 or 2, wherein the salt of the modified oligonucleotide is a sodium or potassium salt.

4. Use of a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21), for the preparation of a medicament for treating spinal muscular atrophy (SMA) or improving at least one symptom of SMA.

5. Use of a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21), for the preparation of a drug for regulating SMN2 RNA in cells or individuals.

6. Use of an oligomeric compound for the preparation of a medicament for treating spinal muscular atrophy (SMA) or improving at least one symptom of SMA, the oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: mCns Ano mCns Tno Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gns mCn (SEQ ID NO: 21), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n = 2'-NMA sugar moiety, s = thiophosphate nucleoside linkage, and o = phosphodiester nucleoside linkage.

7. Use of an oligomeric compound for the preparation of a medicament for regulating SMN2 RNA in cells or individuals, the oligomeric compound comprising a modified oligonucleotide according to the following chemonotation: mCns Ano mCns Tno Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gns mCn (SEQ ID NO: 21), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n = 2'-NMA sugar moiety, s = thiophosphate nucleoside linkage, and o = phosphodiester nucleoside linkage.

8. Use of a group of (a) modified oligonucleotides or (b) oligomeric compounds for the preparation of a medicament for treating spinal muscular atrophy (SMA) or improving at least one symptom of SMA, wherein the (a) modified oligonucleotide comprises: (i) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21) or a salt thereof; and / or (ii) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21); or the (b) oligomeric compound comprises a modified oligonucleotide according to the following chemical notation: mCns Ano mCns Tno Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gns mCn (SEQ ID NO: 21), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n = 2'-NMA sugar moiety, s = thiophosphate nucleoside linkage, and o = phosphodiester nucleoside linkage, wherein all thiophosphate nucleoside linkages in the modified oligonucleotide are stereorandom.

9. Use of a group of (a) modified oligonucleotides or (b) oligomeric compounds for the preparation of a medicament for the preparation of SMN2 RNA for the regulation of cells or individuals, wherein the (a) modified oligonucleotide comprises: (i) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21) or a salt thereof; and / or (ii) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21); or the (b) oligomeric compound comprises a modified oligonucleotide according to the following chemical notation: mCns Ano mCns Tno Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gns mCn (SEQ ID NO: 21), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n = 2'-NMA sugar moiety, s = thiophosphate nucleoside linkage, and o = phosphodiester nucleoside linkage, wherein all thiophosphate nucleoside linkages in the modified oligonucleotide are stereorandom.

10. Use of a pharmaceutical composition comprising an oligonucleotide or oligomer and a pharmaceutically acceptable diluent for the preparation of a medicament for the treatment of spinal muscular atrophy (SMA) or for the improvement of at least one symptom of SMA: (a) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21) or a salt thereof; or (b) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21); or (c) a group comprising: (i) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21) or a salt thereof; and / or (ii) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21); or (d) an oligomer comprising a modified oligonucleotide according to the following chemical notation: mCns Ano mCns Tno Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gns mCn (SEQ ID NO: 21) 21), of which: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n = 2'-NMA glycosidic moiety, s = thiophosphate nucleoside linkage, and o = phosphodiester nucleoside linkage; or (e) a group of oligomeric compounds comprising modified oligonucleotides according to the following chemonotation: mCns Ano mCns Tno Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gns mCn (SEQ ID NO: 21), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n = 2'-NMA glycosidic moiety, s = thiophosphate nucleoside linkage, and o = phosphodiester nucleoside linkage.

11. Use of a pharmaceutical composition comprising an oligonucleotide or oligomer and a pharmaceutically acceptable diluent for the preparation of a medicament for regulating SMN2 RNA in cells or individuals: (a) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21) or a salt thereof; or (b) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21); or (c) a group comprising: (i) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21) or a salt thereof; and / or (ii) a modified oligonucleotide according to the following chemical structure, (SEQ ID NO: 21); or (d) an oligomer comprising a modified oligonucleotide according to the following chemical notation: mCns Ano mCns Tno Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gns mCn (SEQ ID NO: 21), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n = 2'-NMA glycosidic moiety, s = thiophosphate nucleoside linkage, and o = phosphodiester nucleoside linkage; or (e) a group of oligomeric compounds comprising modified oligonucleotides according to the following chemonotation: mCns Ano mCns Tno Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gns mCn (SEQ ID NO: 21), wherein: A = adenine nucleobase, mC = 5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n = 2'-NMA glycosidic moiety, s = thiophosphate nucleoside linkage, and o = phosphodiester nucleoside linkage.

12. As used in any of claims 8 to 11, wherein the salt of the modified oligonucleotide is a sodium or potassium salt.

13. As requested in item 10 or 11, wherein the medically acceptable diluent is artificial CSF (aCSF) or PBS.

14. As claimed in claim 13, wherein the pharmaceutical composition comprises the modified oligonucleotide or a group of the modified oligonucleotide and aCSF or PBS.

15. As claimed in claim 13, wherein the pharmaceutical composition comprises the oligomeric compound or a group of the oligomeric compounds and aCSF or PBS.

16. For the purposes of claims 1, 4, 6, 8 and 10, wherein at least one symptom includes decreased muscle strength; inability to sit, stand and / or walk upright or decreased ability; decreased neuromuscular activity; decreased electrical activity of one or more muscles; decreased respiration; inability to eat, drink and / or breathe without assistance or decreased ability; weight loss or decreased weight gain; and / or decreased survival rate.