BR122024003572A2 - MODIFIED OLIGONUCLEOTIDE, OLIGOMERIC COMPOUND, POPULATION OF MODIFIED OLIGONUCLEOTIDES, POPULATION OF OLIGOMERIC COMPOUNDS AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

The present invention relates to compounds, methods and pharmaceutical compositions for modulating SMN2 RNA and/or protein in a cell or subject. Such compounds, methods and pharmaceutical compositions are useful for attenuating at least one symptom of a neurodegenerative disorder. Such symptoms include reduced muscle strength; inability or reduced ability to sit upright, stand and/or walk; reduced neuromuscular activity; reduced electrical activity in one or more muscles; reduced breathing; inability or reduced ability to eat, drink and/or breathe without assistance; weight loss or reduced weight gain; and/or decreased survival.

Description

[001] This application is filed together with a Sequence Listing in electronic format. The Sequence Listing is provided as a file titled BIOL0367WOSEQ_ST25.txt, created on February 26, 2021, which is 44 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

[002] Compounds, methods, and pharmaceutical compositions for modulating SMN2 RNA in a cell or subject are provided. Such compounds, methods, and pharmaceutical compositions are useful for attenuating at least one symptom of a neurodegenerative disorder. Such symptoms include reduced muscle strength; inability or reduced ability to sit upright, stand, and/or walk; reduced neuromuscular activity; reduced electrical activity in one or more muscles; reduced breathing; inability or reduced ability to eat, drink, and/or breathe without assistance; weight loss or reduced weight gain; and/or decreased survival.

BACKGROUND

[003] Proximal spinal muscular atrophy (SMA) is a genetic neurodegenerative disorder characterized by the loss of spinal motor neurons. SMA is an autosomal recessive disease of early onset and is a leading genetic cause of death among children. The severity of SMA varies among patients and has been classified into four types. Type I SMA is the most severe form with onset at birth or within 6 months and typically results in death within 2 years. Children with Type I SMA are unable to sit or walk. Type II SMA is the intermediate form and patients are able to sit but cannot stand or walk. Patients with Type III SMA, a chronic form of the disease, typically develop SMA after 18 months of age (Lefebvre et al., Hum. Mol. Genet., 1998, 7, 1531-1536). AMS Type IV is a milder form and typically has an onset after 18 years of age, sometimes after 10 years of age; patients with AMS Type IV experience mild limited motor impairment, are able to walk in adulthood, and usually do not have 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).

[004] The molecular basis of AMS is the loss of both copies of the survival motor neuron 1 (SMN1) gene, which may also be known as SMN Telomeric, and encodes a protein that is part of a multi-protein complex believed to be involved in snRNP biogenesis and recycling. A nearly identical gene, SMN2, which may also be known as SMN Centromeric, exists 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 in only the expression of full-length survival motor neuron (SMN) protein, whereas expression of the SMN2 gene results in two different protein forms, full-length SMN2 protein and a truncated SMN2 protein, SMNΔ7 protein. SMN2 contains a translationally silent mutation at position +6 of exon 7, which results in inefficient inclusion of exon 7 in SMN2 transcripts. Thus, the predominant form of SMN2 is a truncated version lacking exon 7, which is unstable and inactive (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/nonfunctional SMNΔ7 protein. SMN protein has a well-established role in spliceosome assembly and may also mediate mRNA trafficking in the axon and nerve ending of neurons.

[005] It is an objective herein to provide compounds, methods and pharmaceutical compositions for the treatment of AMS.

SUMMARY OF THE INVENTION

[006] Provided herein are compounds, methods, and pharmaceutical compositions for modulating SMN2 RNA splicing in a cell or subject. In certain embodiments, compounds useful for modulating SMN2 RNA splicing are oligomeric compounds. In certain embodiments, oligomeric compounds increase the amount of SMN2 RNA that includes exon 7. In certain embodiments, oligomeric compounds increase the expression of full-length SMN2 protein. In certain embodiments, the oligomeric compound comprises a modified oligonucleotide. In certain embodiments, the subject has a neurodegenerative disease. In certain embodiments, the subject has Spinal Muscular Atrophy (SMA).

[007] Also provided are methods useful for ameliorating at least one symptom of a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is AMS. In certain embodiments, symptoms include reduced muscle strength; inability or reduced ability to sit upright, stand, and/or walk; reduced neuromuscular activity; reduced electrical activity in one or more muscles; reduced breathing; inability or reduced ability to eat, drink, and/or breathe without assistance; weight loss or reduced weight gain; and/or decreased survival. In certain embodiments, provided herein are oligonucleotides modified to target AMS.

DETAILED DESCRIPTION OF THE INVENTION

[008] It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. In this document, the use of the singular includes the plural unless specifically determined otherwise. As used herein, the use of "or" means "and/or" unless otherwise determined. In addition, the use of the term "include" as well as other forms such as "includes" and "included" is not limiting. In addition, terms such as "element" or "component" encompass both elements and components comprising a unit and elements comprising more than one subunit, unless specifically determined otherwise.

[009] 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 referenced in this application, including, but not limited to, patents, patent applications, articles, books, and dissertations, and the GenBank and NCBI reference sequence records, are expressly incorporated herein by reference by the portions of the document discussed herein as well as in their entirety.

DEFINITIONS

[0010] Unless specific definitions are provided, the nomenclature used in connection with and the procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein is that well known and commonly used in the art. Where permitted, all patents, applications, published applications and other publications and other data referenced throughout the disclosure are incorporated by reference herein in their entirety.

[0011] Unless otherwise indicated, the following terms have the following meanings:

[0012] As used herein, the term "2'-deoxyribonucleoside" means a nucleoside comprising a 2'-H(H) deoxyribosyl sugar moiety. In certain embodiments, a 2'-deoxyribonucleoside is a 2'-β-D-deoxyribonucleoside and comprises a 2'-β-D-deoxyribosyl sugar moiety, which has the β-D configuration as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2'-deoxyribonucleoside can comprise a modified nucleobase or can comprise an RNA nucleobase (uracil).

[0013] As used herein, "2'-MOE" means a 2'-OCH2CH2OCH3 group in place of the 2'-OH group of a ribosyl sugar moiety. A "2'-MOE sugar moiety" is a sugar moiety with a 2'-OCH2CH2OCH3 group in place of the 2'-OH group of a ribosyl sugar moiety. Unless otherwise indicated, a 2'-MOE sugar moiety is in the β-D configuration. "MOE" means O-methoxyethyl.

[0014] As used herein, "2'-MOE nucleoside" means a nucleoside comprising a 2'-MOE sugar moiety.

[0015] As used herein, "2'-NMA" means an -O-CH2-C(=O)-NH-CH3 group in place of the 2'-OH group of a ribosyl sugar moiety. A "2'-NMA sugar moiety" is a sugar moiety with a 2-O-CH2-C(=O)-NH-CH3 group in place of the 2'-OH group of a ribosyl sugar moiety. Unless otherwise indicated, a 2'-NMA sugar moiety is in the β-D configuration. "NMA" means O-N-methyl acetamide.

[0016] As used herein, "2'-NMA nucleoside" means a nucleoside comprising a 2'-NMA sugar moiety.

[0017] As used herein, "2'-OMe" means a 2'-OCH3 group in place of the 2'-OH group of a ribosyl sugar moiety. A "2'-OMe sugar moiety" is a sugar moiety with a 2'-OCH3 group in place of the 2'-OH group of a ribosyl sugar moiety. Unless otherwise indicated, a 2'-OMe sugar moiety is in the β-D configuration. "OMe" means O-methyl.

[0018] As used herein, "2'-OMe nucleoside" means a nucleoside comprising a 2'-OMe sugar moiety.

[0019] As used herein, "2'-substituted nucleoside" means a nucleoside comprising a 2'-substituted sugar moiety. As used herein, "2'-substituted" in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.

[0020] As used herein, "5-methyl cytosine" means a modified cytosine with a methyl group attached to the 5-position. A 5-methyl cytosine is a modified nucleobase.

[0021] As used herein, "administer" means to provide a pharmaceutical agent to an individual.

[0022] As used herein, "attenuate", in reference to a treatment, means improvement in at least one symptom relative to the same symptom in the absence of the treatment. In certain embodiments, attenuation is the reduction in severity or frequency of a symptom or the delayed onset or slowing of progression in severity or frequency of a symptom. In certain embodiments, the symptom is reduced muscle strength; inability or reduced ability to sit upright, stand, and/or walk; reduced neuromuscular activity; reduced electrical activity in one or more muscles; reduced breathing; inability or reduced ability to eat, drink, and/or breathe without assistance; weight loss or reduced weight gain; and/or decreased survival.

[0023] As used herein, "antisense activity" means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.

[0024] As used herein, "antisense compound" means an oligomeric compound or oligomeric duplex that has the ability to achieve at least one antisense activity.

[0025] As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety.

[0026] As used herein, "bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the 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 comprise a furanosyl moiety.

[0027] 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 prepared or manufactured fluid that has certain properties of cerebrospinal fluid.

[0028] As used herein, "cEt" means a 4' to 2' bridge in place of 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. A "cEt sugar moiety" is a bicyclic sugar moiety with a 4' to 2' bridge in place of 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 restricted ethyl.

[0029] As used herein, "cEt nucleoside" means a nucleoside comprising a cEt sugar moiety.

[0030] As used herein, "chirally enriched population" means a plurality of molecules of identical molecular formula, wherein the number or percentage of molecules within the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom. Chirally enriched populations of molecules that have multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In certain embodiments, the molecules are modified oligonucleotides. In certain embodiments, the molecules are compounds comprising modified oligonucleotides.

[0031] As used herein, "complementary" in reference to an oligonucleotide means that at least 70% of the nucleobases of the oligonucleotide or one or more portions thereof and the nucleobases of another nucleic acid or one or more portions thereof have the ability to hydrogen bond with each other when the nucleobase sequence of the oligonucleotide and another nucleic acid are aligned in opposite directions. Complementary nucleobases means nucleobases that have the ability to form hydrogen bonds with each other. Complementary nucleobase pairs include adenine (A) with thymine (T), adenine (A) with uracil (U), cytosine (C) with guanine (G), and 5-methylcytosine (mC) with guanine (G). Complementary oligonucleotide and/or target nucleic acids need not have nucleobase complementarity on each nucleoside. Instead, some mismatches are tolerated. As used herein, "completely complementary" or "100% complementary" in reference to an oligonucleotide, or a portion thereof, means that the oligonucleotide, or a portion thereof, is complementary to another oligonucleotide or target nucleic acid at each nucleobase of the shorter of the two oligonucleotides, or at each nucleoside if the oligonucleotides are the same length.

[0032] As used herein, "contiguous" in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside bonds that are immediately adjacent to one another. For example, "contiguous nucleobases" means nucleobases that are immediately adjacent to one another in a sequence.

[0033] As used herein, "hybridization" means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. Although not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding, between complementary nucleobases.

[0034] As used herein, "internucleoside linkage" means the covalent bond between contiguous nucleosides in an oligonucleotide. As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a phosphodiester internucleoside linkage. "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.

[0035] As used herein, "mismatch" or "non-complementary" means a nucleobase of a first oligonucleotide that is not complementary to the corresponding nucleobase of a second oligonucleotide or a target nucleic acid when the first and second oligonucleotides are aligned.

[0036] As used herein, "motif" means the pattern of unmodified and/or modified sugar chemical moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.

[0037] As used herein, "non-bicyclic modified sugar moiety" means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.

[0038] As used herein, "nucleobase" means an unmodified nucleobase or a modified nucleobase. As used herein, an "unmodified nucleobase" is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a "modified nucleobase" is a group of atoms other than unmodified A, T, C, U, or G with the ability to pair with at least one unmodified nucleobase. A "5-methyl cytosine" is a modified nucleobase. A universal base is a modified nucleobase that can pair with any of the five unmodified nucleobases. As used herein, "nucleobase sequence" means the order of contiguous nucleobases in a target nucleic acid or oligonucleotide independent of any internucleoside bond or sugar modification.

[0039] As used herein, "nucleoside" means a compound comprising 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 comprising a modified nucleobase and/or a modified sugar moiety. "Linked nucleosides" are nucleosides that are connected in a contiguous sequence (i.e., with no additional nucleosides being present between those that are linked).

[0040] As used herein, "oligomeric compound" means an oligonucleotide and optionally one or more additional features, such as a conjugating group or a terminal group. An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired. A "single-stranded oligomeric compound" is an unpaired oligomeric compound. The term "oligomeric duplex" means a duplex formed by two oligomeric compounds that have complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a "duplexed oligomeric compound".

[0041] As used herein, "oligonucleotide" means a strand of linked nucleosides connected by internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8 to 50 linked nucleosides. As used herein, "modified oligonucleotide" means an oligonucleotide in which at least one nucleoside or one internucleoside linkage is modified. As used herein, "unmodified oligonucleotide" means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.

[0042] As used herein, "pharmaceutical composition" means a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may comprise an oligomeric compound and a sterile aqueous solution.

[0043] As used herein, "pharmaceutically acceptable carrier or diluent" refers to any substance suitable for use in administration to a subject. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, sterile buffered solution, or sterile artificial cerebrospinal fluid.

[0044] As used herein, "pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of compounds. "Pharmaceutically acceptable salts" retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects to it.

[0045] As used herein, "RNA" means an RNA transcript and includes pre-mRNA and mature mRNA unless otherwise specified.

[0046] As used herein, "stereorandom chiral center" in the context of a population of molecules of identical molecular formula means a chiral center that has a random stereochemical configuration. For example, in a population of molecules comprising a stereorandom chiral center, the number of molecules that have the (S) configuration of the stereorandom chiral center may be, but is not necessarily, equal to the number of molecules that have the (R) configuration of the stereorandom chiral center. The stereochemical configuration of a chiral center is considered random when it is the result of a synthetic method that was not designed to control the stereochemical configuration. In certain embodiments, a stereorandom chiral center is a stereorandom phosphorothioate internucleoside bond.

[0047] As used herein, "individual" means a human or non-human animal.

[0048] 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 (an "unmodified RNA sugar moiety") or a 2'-H(H)β-D deoxyribosyl moiety as found in DNA (an "unmodified DNA sugar moiety"). Unmodified sugar moieties have one hydrogen at each of the 1', 3', and 4' positions, one oxygen at the 3' position, and two hydrogens at the 5' position. As used herein, "modified sugar moiety" or "modified sugar" means an unmodified furanosyl sugar moiety or a sugar substitute.

[0049] As used herein, "sugar substitute" means a modified sugar moiety having a non-furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugative group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar substitutes can be incorporated at one or more positions within an oligonucleotide, and such oligonucleotides have the ability to hybridize to complementary oligomeric compounds or target nucleic acids.

[0050] As used herein, "standard in vivo assay" means the assay described in Example 2 and reasonable variations thereof.

[0051] As used herein, "symptom" means any physical characteristic or test result that indicates the existence or extent of a disease or disorder. In certain embodiments, a symptom is evident to an individual or to a medical professional examining or testing the individual.

[0052] As used herein, "target nucleic acid" means a nucleic acid that an antisense compound is designed to affect.

[0053] As used herein, "target region" means a portion of a target nucleic acid to which an oligomeric compound is designed to hybridize.

[0054] As used herein, "terminal group" means a chemical group or group of atoms that is covalently attached to a terminus of an oligonucleotide.

[0055] As used herein, "therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual. For example, a therapeutically effective amount improves a symptom of a disease.

CERTAIN MODALITIES

[0056] The present disclosure provides the following numbered embodiments without limitation:

[0057] Embodiment 1. An oligomeric compound comprising a modified oligonucleotide consisting of 16, 17, 18, 19, or 20 linked nucleosides and having a nucleobase sequence comprising at least 15 or at least 16 contiguous nucleobases from any of the nucleobase sequences of SEQ ID NOs: 20-50, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0058] Embodiment 2. An oligomeric compound comprising a modified oligonucleotide consisting of 17, 18, 19, or 20 linked nucleosides and having a nucleobase sequence comprising at least 15, at least 16, or at least 17 contiguous nucleobases from any of the nucleobase sequences of SEQ ID NOs: 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.

[0059] Embodiment 3. An oligomeric compound comprising a modified oligonucleotide consisting of 18, 19, or 20 linked nucleosides and having a nucleobase sequence comprising at least 15, at least 16, at least 17, or at least 18 contiguous nucleobases from any of the nucleobase sequences of ID NOs: 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.

[0060] Embodiment 4. An oligomeric compound comprising a modified oligonucleotide consisting 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 contiguous nucleobases from any of the nucleobase sequences of SEQ ID NOs: 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.

[0061] Embodiment 5. An oligomeric compound comprising a modified oligonucleotide consisting 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 contiguous nucleobases from any of the nucleobase sequences of SEQ ID NOs: 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.

[0062] Embodiment 6. The oligomeric compound of any one of embodiments 1-5, wherein the modified oligonucleotide has a nucleobase sequence that is at least 80%, 85%, 87.5%, 88.2%, 89%, 89.4%, 90%, 93.7%, 94%, 94.7%, 95% or is 100% complementary to the nucleobase sequence of SEQ ID NO: 1 when measured across the entire nucleobase sequence of the modified oligonucleotide.

[0063] Embodiment 7. The oligomeric compound of any one of embodiments 1-6, wherein the modified oligonucleotide has an internucleoside linkage motif (5' to 3') selected from: sosossssssssssss, ssosssssssssss, ssossssssssss, ssossssssssss, soossssssssssooss, sooosssssssssooss, sooosssssssssoooss, ssssssssssss, sssssssssssss, sssssssssssss, sssssssssssss, sssssssssssss, ssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss but sssosssssssss, ssssssssssssss, sssssssssssssss, ssssssssssssss, sssssssssssssoss, ssssssssssssoss, sssssssssssssoss, soossssssssssss, sssoooosssss, ssoooossssssssss, ssssoooossssssss, ssssssssoooosssss, ssssssssssoooosss, sssssssssssooooss, ssssssooooossssss, ssssssoooooosssss, ssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss but sssososss, ssssssssssssssss, sssssssssssssss, ssssssssssssss, ssssssssssssss, sssssssssssssss, sssssssssssssss, sssssssoossssssss, ssssssssssssss, ssssssssssssss, sosssssssssssss, soossssssssssssss, ssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss, sossssssssssssss, sooossssss, sssssoooooossss, sssosssosssosss, ssossososososs, soossssssssooss, soooossssssoooss; ssssssssss, sossssss, ssssssssoooosss, sssssooooosssss, ssssooooooooossss, ssossossossoss, ssoooossssssss, sooosssssssoooss and where 's' represents a phosphorothioate internucleoside linkage and 'o' represents a phosphodiester internucleoside linkage.

[0064] Embodiment 8. The oligomeric compound of any of embodiments 1-6, wherein the modified oligonucleotide has an internucleoside linkage motif 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.

[0065] Embodiment 9. The oligomeric compound of any of embodiments 1-6, wherein the modified oligonucleotide has an internucleoside linkage motif selected from: zzzzzzzzzzzzzzzzz, ssssssssssszzzzzz, ssssszzzzzzsssss, zzooooooooooooozz, zzzzooooooooooooozz, zzzzzzooooooooozz, zzzzzzzzooooooooozz, and ssoooooooooooooss, wherein 's' represents a phosphorothioate internucleoside linkage, 'o' represents a phosphodiester internucleoside linkage, and 'z' represents a mesylphosphoramidate internucleoside linkage.

[0066] Embodiment 10. The oligomeric compound of any one of embodiments 1-9, wherein the modified oligonucleotide has a sugar motif (5' to 3') selected from: eeeeeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeee, eeeeeeeeeeeeeee, eeeeeeeeeeeeeee, nnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnn, nennnnneneennnnn, nnnnnnnnnnnnenneen, nennnnneneenenneen, nnnnnnnnnnnnnnnnnne, nnnnnnnnnnnnnnnnnnd, nnnnnnnnnnnnnnnnnnnnnd, nnnnnnnnnnnnnnnnnnnnd, nnnnnnnnnnnnnnnnnnnnd, nnnnnnnnnnnnnnnnnned, nnnnnnnnnnnnnnnnnnde, nnnnnnnnnnnnnnnnnnee, eeeeeeeeeeeeeeeeeedd, eeeeeeeeeeeeeeeey, ennnnnnnnnnnnnnnnnn and ennnnnnnnnnnnnnnnnne; where '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.

[0067] Embodiment 11. The oligomeric compound of any of embodiments 1-9, wherein the modified oligonucleotide has a sugar motif (5' to 3') selected from nnnnnnnnnnnnnnnnn and nnnnnnnnnnnnnnnnen, wherein 'e' represents a sugar moiety of 2'-MOE and 'n' represents a sugar moiety of 2'-NMA.

[0068] Embodiment 12. The oligomeric compound of any of embodiments 1-9, wherein the modified oligonucleotide has a sugar motif (5' to 3') of qqnqqqqqnqnnqnqqnn, wherein each 'n' represents a 2'-NMA sugar moiety and each 'q' is independently selected from a 2'-O-(N,N-dimethyl)acetamide sugar moiety, a 2'-O-(N-ethyl)acetamide sugar moiety, a 2'-O-(N-propyl)acetamide sugar moiety, a 2'O-(N-cyclopropyl)acetamide sugar moiety, and a 2'-O-(N-cyclopropylmethyl)acetamide sugar moiety.

[0069] Embodiment 13. The oligomeric compound of any one of embodiments 1-9, wherein the modified oligonucleotide comprises at least one modified sugar moiety.

[0070] Embodiment 14. The oligomeric compound of embodiment 13, wherein the modified oligonucleotide comprises at least one bicyclic sugar moiety.

[0071] Embodiment 15. The oligomeric compound of embodiment 14, wherein the bicyclic sugar moiety has a 4'-2' bridge, wherein the 4'-2' bridge is selected from -CH2-O-; and -CH(CH3)-O-.

[0072] Embodiment 16. The oligomeric compound of embodiment 13, wherein the modified oligonucleotide comprises at least one non-bicyclic modified sugar moiety.

[0073] Embodiment 17. The oligomeric compound of embodiment 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, or a 2'-F sugar moiety.

[0074] Embodiment 18. The oligomeric compound of embodiment 13, wherein the modified oligonucleotide comprises at least one sugar substitute.

[0075] Embodiment 19. The oligomeric compound of embodiment 18, wherein the sugar substitute is any of morpholino, modified morpholino, PNA, THP, and F-HNA.

[0076] Embodiment 20. The oligomeric compound of any one of embodiments 1-6 and 10-19, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

[0077] Embodiment 21. The oligomeric compound of embodiment 20, wherein each internucleoside bond of the modified oligonucleotide is a modified internucleoside bond.

[0078] Embodiment 22. The oligomeric compound of embodiment 20 or 21, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[0079] Embodiment 23. The oligomeric compound of any one of embodiments 1-20 or 22, wherein the modified oligonucleotide comprises at least one phosphodiester internucleoside linkage.

[0080] Embodiment 24. The oligomeric compound of any one of embodiments 20, 22, or 23, wherein each internucleoside linkage is independently selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.

[0081] Embodiment 25. The oligomeric compound of any of embodiments 13-19, wherein the modified oligonucleotide has an internucleoside linkage motif (5' to 3') selected from: sosossssssssssss, soosssssssssssss, sosssssssssss, sossssssssssss, sossssssssssss, sssoossssssssss, ssssssssoosssssss, sssssssssoosssssss, and sssssssssssoosssss; where 's' represents a phosphorothioate internucleoside bond and 'o' represents a phosphodiester internucleoside bond.

[0082] Embodiment 26. The oligomeric compound of any one of embodiments 1-25, wherein the modified oligonucleotide comprises a modified nucleobase.

[0083] Embodiment 27. The oligomeric compound of embodiment 26, wherein the modified nucleobase is a 5-methylcytosine.

[0084] Embodiment 28. The oligomeric compound of any one of embodiments 1-27, wherein the modified oligonucleotide consists of 16, 17, 18, 19, or 20 linked nucleosides.

[0085] Embodiment 29. The oligomeric compound of any one of embodiments 1-28, wherein the modified oligonucleotide comprises 1 or 2 non-complementary nucleobases.

[0086] Embodiment 30. The oligomeric compound of any one of embodiments 1-29, wherein the modified oligonucleotide comprises 1 or 2 cleavable chemical moieties.

[0087] Embodiment 31. The oligomeric compound of embodiment 30, wherein the cleavable chemical moiety is a phosphodiester internucleoside bond.

[0088] Embodiment 32. The oligomeric compound of any one of embodiments 1-31 consisting of the modified oligonucleotide.

[0089] Embodiment 33. The oligomeric compound of any of embodiments 1-32, wherein the oligomeric compound is a single-stranded oligomeric compound.

[0090] Embodiment 34. An oligomeric compound comprising an oligonucleotide modified according to the following chemical notation: mCes Aeo mCes Teo Tes Tes mCes Aes Tes Aes Aes Tes Ges mCes Tes Ges Ges mCe (SEQ ID NO: 21), wherein: A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2'-MOE sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage.

[0091] Embodiment 35. An oligomeric compound comprising an oligonucleotide modified according to the following chemical notation: Teo Tes mCes Aes mCes Tes Tes Tes mCes Aes Tes Aes Aes Tes Ges mCes Tes Ges Geo mCe (SEQ ID NO: 22), wherein: A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2'-MOE sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage.

[0092] Embodiment 36. An oligomeric compound comprising an oligonucleotide modified according to the following chemical notation: Teo Tns mCns Ans mCns Tns Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gno mCe (SEQ ID NO: 22), wherein: A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2'-MOE sugar moiety, n = a 2'-NMA sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage.

[0093] Embodiment 37. An oligomeric compound comprising an oligonucleotide modified 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 = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, n = a 2'-NMA sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage.

[0094] Embodiment 38. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 21) or a salt thereof.

[0095] Embodiment 39. The modified oligonucleotide of embodiment 38, which is the sodium salt or the potassium salt.

[0096] Embodiment 40. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 21).

[0097] Embodiment 41. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 22) or a salt thereof.

[0098] Embodiment 42. The modified oligonucleotide of embodiment 41, which is the sodium salt or the potassium salt.

[0099] Embodiment 43. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 22).

[00100] Embodiment 44. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 22) or a salt thereof.

[00101] Embodiment 45. The modified oligonucleotide of embodiment 44, which is the sodium salt or the potassium salt.

[00102] Embodiment 46. A modified oligonucleotide corresponding to the following chemical structure: (SEQ ID NO: 22).

[00103] Embodiment 47. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 21) or a salt thereof.

[00104] Embodiment 48. The modified oligonucleotide of embodiment 47, which is the sodium salt or the potassium salt.

[00105] Embodiment 49. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 21).

[00106] Embodiment 50. A pharmaceutical composition comprising the oligomeric compound of any one of embodiments 1-36 or the modified oligonucleotide of any one of embodiments 38-49 and a pharmaceutically acceptable diluent or carrier.

[00107] Embodiment 51. The pharmaceutical composition of embodiment 50 comprising a pharmaceutically acceptable diluent and wherein the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS.

[00108] Embodiment 52. The pharmaceutical composition of embodiment 51, wherein the pharmaceutical composition consists essentially of the modified oligonucleotide and artificial CSF (aCSF).

[00109] Embodiment 53. The pharmaceutical composition of embodiment 51, wherein the pharmaceutical composition consists essentially of the modified oligonucleotide and PBS.

[00110] Embodiment 54. A chirally enriched population of modified oligonucleotides according to any one of claims 38-49, wherein the population is enriched with modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having a particular stereochemical configuration.

[00111] Embodiment 55. The chirally enriched population of embodiment 54, wherein the population is enriched with modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having the (Sp) configuration.

[00112] Embodiment 56. The chirally enriched population of embodiment 54, wherein the population is enriched with modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having a (Rp) configuration.

[00113] Embodiment 57. The chirally enriched population of embodiment 54, wherein the population is enriched with modified oligonucleotides having a particular independently selected stereochemical configuration at each phosphorothioate internucleoside linkage.

[00114] Embodiment 58. The chirally enriched population of embodiment 57, wherein the population is enriched for modified oligonucleotides having the (Sp) configuration at each phosphorothioate internucleoside bond or for modified oligonucleotides having the (Rp) configuration at each phosphorothioate internucleoside bond.

[00115] Embodiment 59. The chirally enriched population of embodiment 57, wherein the population is enriched with modified oligonucleotides having the (Rp) configuration at a particular phosphorothioate internucleoside linkage and the (Sp) configuration at each of the remaining phosphorothioate internucleoside linkages.

[00116] Embodiment 60. The chirally enriched population of embodiment 57, wherein the population is enriched with modified oligonucleotides having at least 3 contiguous phosphorothioate internucleoside bonds in the Sp, Sp, and Rp configurations in the 5' to 3' direction.

[00117] Embodiment 61. A population of modified oligonucleotides according to any one of embodiments 38-49, wherein all of the phosphorothioate internucleoside linkages of the modified oligonucleotide are stereorandom.

[00118] Embodiment 62. A method of treating a disease associated with SMN1 or SMN2 comprising administering to a subject who has or is at risk of developing a disease associated with SMN1 or SMN2 a therapeutically effective amount of a pharmaceutical composition according to any one of embodiments 50-53; and thereby treating the disease associated with SMN1 or SMN2.

[00119] Embodiment 63. The method of embodiment 62, wherein the disease associated with SMN1 or SMN2 is a neurodegenerative disease.

[00120] Embodiment 64. The method of embodiment 63, wherein the neurodegenerative disease is Spinal Muscular Atrophy (SMA).

[00121] Embodiment 65. The method of embodiment 64, wherein the AMS is any of Type I AMS, Type II AMS, Type III AMS, or Type IV AMS.

[00122] Embodiment 66. The method of embodiment 64 or embodiment 65, wherein at least one symptom of AMS is alleviated.

[00123] Embodiment 67. The method of embodiment 66, wherein the symptom is any of reduced muscle strength; inability or reduced ability to sit upright, stand, and/or walk; reduced neuromuscular activity; reduced electrical activity in one or more muscles; reduced breathing; inability or reduced ability to eat, drink, and/or breathe without assistance; weight loss or reduced weight gain; and/or decreased survival.

[00124] Embodiment 68. The method of any of embodiments 62-67, wherein the pharmaceutical composition is administered to the central nervous system or systemically.

[00125] Embodiment 69. The method of embodiment 68, wherein the pharmaceutical composition is administered to the central nervous system or systemically.

[00126] Embodiment 70. The method of any of embodiments 62-67, wherein the pharmaceutical composition is administered by any route including intrathecal, systemic, subcutaneous, or intramuscular.

[00127] Embodiment 71. A method for increasing SMN2 RNA that includes exon 7 comprising contacting a cell, tissue, or organ with an oligomeric compound of any of embodiments 1-37, a modified oligonucleotide of any of embodiments 38-49, or a pharmaceutical composition of any of embodiments 50-53.

CERTAIN OLIGONUCLEOTIDES

[00128] In certain embodiments, provided herein are oligomeric compounds comprising oligonucleotides, which consist of linked nucleosides. The oligonucleotides can be unmodified oligonucleotides (RNA or DNA) or can be modified oligonucleotides. The modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA. 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

[00129] Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.

CERTAIN CHEMICAL MOTION OF SUGAR

[00130] In certain embodiments, the modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, the modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, the modified sugar moieties are sugar substitutes. Such sugar substitutes may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.

[00131] In certain embodiments, the modified sugar moieties are non-bicyclic modified sugar moieties comprising a furanosyl ring with one or more substituent groups, none of which bridge two atoms of the furanosyl ring to form a bicyclic structure. Such non-bridging substituents can be at any position on 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 non-bicyclic modified sugar moieties are branched. Examples of suitable 2'-substituent groups for non-bicyclic modified sugar moieties include, but are not limited to: 2'-F, 2'-OCH3 ("OMe" or "O-methyl") and 2'-O(CH2)2OCH3 ("MOE" or "O-methoxyethyl"), and 2'-ON-alkylacetamide, e.g., 2'-ON-methylacetamide ("NMA"), 2'-ON-dimethylacetamide, 2'-ON-ethylacetamide or 2'-ON-propylacetamide. For example, see U.S. Pat. No. 6,147,200, Prakash et al., 2003, Org. Lett., 5, 403-406. A "2'-ON-methylacetamide nucleoside" or "2'-NMA nucleoside" is shown below:

[00132] In certain embodiments, the 2'-substituent groups are selected from: halo, 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-alkylenyl-O- alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(=O )-N(Rm)(Rn), in that each Rm and Rn is, independently, H, a substituted or unsubstituted amino or C1 C10 alkyl protecting group, and the 2'-substituent groups 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'-substituent groups may be further substituted by one or more substituent groups independently selected from: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of suitable 4'-substituent groups 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 groups 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 modified sugar moieties non-bicyclic comprise more than one non-bridged sugar substituent, for example, 2'-F-5'-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.

[00133] In certain embodiments, a 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent group 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 substituted or unsubstituted C1-C10 alkyl, e.g., OCH2C(=O)-N(H)CH3 ("NMA").

[00134] In certain embodiments, a 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent group 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").

[00135] In certain embodiments, a 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent group selected from: F, OCH3, OCH2CH2OCH3, and OCH2C(=O)-N(H)CH3.

[00136] Certain modified sugar moieties comprise a substituent that bridges two furanosyl ring atoms to form a second ring, which results in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and 2' furanose ring atoms. Examples of such 4'-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' (termed "restricted ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("restricted MOE" or "cMOE"), and analogues thereof (see, e.g., Seth et al., U.S. 7,399,845 , Bhat et al., U.S. 7,399,845 , and others. 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 analogues thereof (see, e.g., Seth et al., U.S. 8,278,283 ), 4'-CH2-N(OCH3)-2' and analogues 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., English: e.g., Zhou, et al., J. Org. Chem.,2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogues 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 C1-C12 alkyl (see, e.g., Imanishi et al., U.S. 7,427,672).

[00137] In certain embodiments, such bridges between 4' and 2' independently comprise 1 to 4 independently linked groups selected from: -[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, hydroxyl, C1-C12 alkyl, C1-C12 substituted alkyl, C2-C12 substituted alkenyl, C2-C12 substituted alkenyl, C2-C12 substituted alkynyl, C2-C12 substituted alkynyl, C5-C20 aryl, C5-C20 substituted araryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, C5-C7 substituted alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1) or sulfoxyl (S(=O)-J1); and each J1 and J2 is independently H, C1-C12 alkyl, C1-C12 substituted alkyl, C2-C12 alkenyl, C2-C12 substituted alkenyl, C2-C12 alkynyl, C2-C12 substituted alkynyl, C5-C20 aryl, C5-C20 substituted aryl, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[00138] Additional 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, 2219-222; Singh et al., J. Org. Chem., 1998, 63, 10,035 to 10,039; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 8,362 to 8,379; 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 U.S. Patent Publications No. Allerson et al., US2008/0039618 and Migawa et al., US2015/0191727.

[00139] In certain embodiments, the bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the α-L configuration or the β-D configuration. α-L-methylene-oxy (4'-CH2-O-2') or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that have shown antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. Where positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in embodiments exemplified herein, they are in the β-D configuration unless otherwise specified.

[00140] In certain embodiments, the modified sugar moieties comprise one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5'-substituted and 4'-2'-bridged sugars).

[00141] In certain embodiments, the modified sugar moieties are sugar substitutes. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, for example, with a sulfur, carbon, or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridged and/or non-bridged substituents as described herein. For example, certain sugar substitutes comprise a 4'-sulfur atom and a substitution 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.

[00142] In certain embodiments, the sugar substitutes comprise rings having a number of atoms other than 5. For example, in certain embodiments, a sugar substitute comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may 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, e.g., Swayze et al., US 8,088,904; Swayze et al., US 8,440,803; Swayze et al., US 8,796,437; and Swayze et al., US 9,005,906; F-HNA may also be referred to as a F-THP or 3'-fluoro tetrahydropyran) and nucleosides comprising additional modified THP compounds having the formula: wherein, independently, for each said modified THP nucleoside: Bx is a nucleobase moiety; T3 and T4 are each, independently, an internucleoside linking group that attaches the modified THP nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an internucleoside linking group that attaches the modified THP nucleoside to the remainder of an 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, C1-C6 substituted alkyl, C2-C6 alkenyl, C2-C6 substituted alkenyl, C2-C6 alkynyl, or C2-C6 substituted alkynyl; and each of R1 and R2 is 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 each J1, J2, and J3 is independently H or C1-C6 alkyl.

[00143] In certain embodiments, provided are modified THP nucleosides 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 other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, provided are modified THP nucleosides 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.

[00144] In certain embodiments, the sugar substitutes comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., US 5,698,685; Summerton et al., US 5,166,315; Summerton et al., US 5,185,444; and Summerton et al., US 5,034,506). As used herein, the term "morpholino" means a sugar substitute having the following structure:

[00145] In certain embodiments, morpholinos can be modified, for example, by adding or altering various substituent groups of the above morpholino structure. Such sugar substitutes are referred to herein as "modified morpholinos."

[00146] In certain embodiments, the sugar substitutes comprise acyclic chemical moieties. Examples of nucleosides and oligonucleotides comprising 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 nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.

[00147] Many other bicyclic and tricyclic sugar and sugar substitute ring systems are known in the art that can be used in modified nucleosides.

CERTAIN MODIFIED NUCLEOBASES

[00148] In certain embodiments, the modified oligonucleotides comprise one or more nucleosides comprising an unmodified nucleobase. In certain embodiments, the modified oligonucleotides comprise one or more nucleosides comprising a modified nucleobase. In certain embodiments, the modified oligonucleotides comprise one or more nucleosides not comprising a nucleobase, referred to as an abasic nucleoside.

[00149] 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 (-CQC-CH3) uracil, 5-propynylcytosine, 6-azouracyl, 6-azocytosine, 6-azotimine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deaza-adenine, 3-deazaguanine, 3-deaza-adenine, 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, indiscriminate bases, size-expanded bases and fluorinated bases. Additional modified nucleobases include tricyclic pyrimidines such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, for example, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nucleobases include those disclosed in Merigan et al., U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858 to 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 those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S.T., Ed., CRC Press, 2008, 163–166 and 442–443.

[00150] Publications teaching the preparation of certain of the above-indicated modified nucleobases as well as other modified nucleobases include, without limitation, 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 BONDAGES

[00151] In certain embodiments, the modified oligonucleotide nucleosides can be linked to one another using any internucleoside linkage. The 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, phosphodiesters, which contain a phosphodiester linkage, P(O2)=O, (also referred to as unmodified or naturally occurring linkages); phosphotriesters; methylphosphonates; methoxypropylphosphonates ("MOP"); phosphoramidates; mesylphosphoramidates; phosphorothioates (P(O2)=S); and phosphorodithioates (HS-P=S). Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, as compared to naturally occurring phosphate linkages, can be used to alter, typically increase, the nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture or as separate enantiomers. Methods of preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those of skill in the art.

[00152] Representative internucleoside linkages having a chiral center include, but are not limited to, alkylphosphonates and phosphorothioates. Modified oligonucleotides comprising internucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom internucleoside linkages or as populations of modified oligonucleotides comprising phosphorothioate internucleoside linkages in particular stereochemical configurations. In certain embodiments, the populations of modified oligonucleotides comprise phosphorothioate internucleoside linkages, wherein all of the phosphorothioate internucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate internucleoside linkage. However, 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 modified oligonucleotide populations are enriched with modified oligonucleotides comprising one or more particular phosphorothioate internucleoside bonds in a particular independently selected stereochemical configuration. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside bond is present in at least 65% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside bond is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside bond is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside bond is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside bond is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., the methods 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, a population of modified oligonucleotides is enriched with modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In certain embodiments, a population of modified oligonucleotides is enriched with modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration. In certain embodiments, the modified oligonucleotides comprising phosphorothioates (Rp) and/or (Sp) comprise one or more of the following formulas, respectively, wherein "B" indicates a nucleobase:

[00153] Unless otherwise indicated, chiral internucleoside linkages of modified oligonucleotides described herein may be stereorandom or may be in a particular stereochemical configuration.

[00154] In certain embodiments, modified oligonucleotides comprise a (5' to 3') sooosssssssssssssss internucleoside motif. In certain embodiments, the particular stereochemical configuration of the modified oligonucleotides 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; 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.

[00155] Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and thioformacetal (3'-S-CH2-O-5'). Additional neutral internucleoside linkages include nonionic bonds comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester of amides (see, e.g., Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40–65). Additional neutral internucleoside linkages include nonionic bonds comprising mixed N, O, S, and CH2 component moieties.

[00156] In certain embodiments, a modified internucleoside linkage is any of those described in WO 2021/030778, incorporated by reference herein.

CERTAIN REASONS

[00157] In certain embodiments, the modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar moiety. In certain embodiments, the modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkages. In such embodiments, the modified, unmodified, or differently modified sugar, nucleobase, and/or internucleoside linkage moieties of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide can be described by its sugar motif, nucleobase motif, and/or internucleoside linkage motif (as used herein, nucleobase motif describes modifications to nucleobases independent of the nucleobase sequence).

CERTAIN SUGAR CRAVINGS

[00158] In certain embodiments, oligonucleotides comprise one or more types of modified sugar moiety and/or unmodified sugar arranged throughout the oligonucleotide, or portion thereof, in a defined sugar pattern or motif. In certain cases, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.

[00159] In certain embodiments, modified oligonucleotides have a gapmer motif, which is defined by two outer regions or "wings" and a central or inner region or "gap". The three regions of a gapmer motif (the 5' wing, the gap, and the 3' wing) form a contiguous sequence of nucleosides, wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the gap nucleosides. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3'-most nucleoside of the 5' wing and the 5'-most nucleoside of the 3' wing) differ from the sugar moiety of neighboring gap nucleosides, thereby defining the boundary between the wings and the gap (i.e., the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as each other. In certain embodiments, the gap includes one or more nucleosides that have a sugar moiety that differs from the sugar moiety of one or more other nucleosides in the gap. In certain embodiments, the sugar motifs of the two wings are the same as each other (symmetric gapmer). In certain embodiments, the sugar motif of the 5' wing differs from the sugar motif of the 3' wing (asymmetric gapmer).

[00160] In certain embodiments, the wings of a gapmer comprise 1-6 nucleosides. In certain embodiments, each nucleoside of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least one, at least two, at least three, at least four, at least five, or at least six nucleosides of each wing of a gapmer comprises a modified sugar moiety.

[00161] In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, each gap nucleoside of a gapmer comprises a 2'-deoxyribosyl sugar moiety. In certain embodiments, at least one gap nucleoside of a gapmer comprises a modified sugar moiety and each remaining nucleoside comprises a 2'-deoxyribosyl sugar moiety.

[00162] Herein, the lengths (number of nucleosides) of the three regions of a gapmer may be given using the notation [# of nucleosides in 5' wing] - [# of nucleosides in gap] - [# of nucleosides in 3' wing]. Thus, a 5-10-5 gapmer consists of 5 nucleosides linked in each wing and 10 nucleosides linked in the gap. When such nomenclature is followed by a specific modification, that modification is the modification in which the sugar moiety of each wing and the gap nucleosides comprise a 2'-deoxyribosyl sugar moiety. Thus, a 5-10-5 MOE gapmer consists of 5 2'-MOE nucleosides linked in the 5' wing, 10 2'-deoxyribonucleosides linked in the gap, and 5 2'-MOE nucleosides linked in the 3' wing.

[00163] In certain embodiments, each nucleoside of a modified oligonucleotide, or portion thereof, comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, a sugar substitute, or a 2'-deoxyribosyl sugar moiety. 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 a LNA sugar moiety. In certain embodiments, the sugar substitute is selected from morpholino, modified morpholino, PNA, THP, and F-HNA.

[00164] In certain embodiments, modified oligonucleotides comprise 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 substitute. 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 a LNA sugar moiety. In certain embodiments, the sugar substitute is selected from morpholino, modified morpholino, THP, and F-HNA.

[00165] In certain embodiments, each nucleoside of a modified oligonucleotide comprises a modified sugar moiety (“fully modified oligonucleotide”). In certain embodiments, each nucleoside of a fully modified oligonucleotide comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar substitute. 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 a LNA sugar moiety. In certain embodiments, the sugar substitute is selected from morpholino, modified morpholino, THP, and F-HNA. In certain embodiments, each nucleoside of a fully modified oligonucleotide comprises the same modified sugar moiety (“uniformly modified sugar motif”). In certain embodiments, the uniformly modified sugar motif is 7 to 20 nucleosides in length. In certain embodiments, each nucleoside of the uniformly modified sugar motif comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar substitute. 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 a LNA sugar moiety. In certain embodiments, the sugar substitute is selected from morpholino, modified morpholino, THP, and F-HNA. In certain embodiments, modified oligonucleotides having at least one fully modified sugar motif can also comprise at least 1, at least 2, at least 3, or at least 4 2'-deoxyribonucleosides.

CERTAIN NUCLEOBASE MOTIVES

[00166] In certain embodiments, the oligonucleotides comprise modified and/or unmodified nucleobases arranged throughout the oligonucleotide or portion thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are 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 cytosine nucleobases in a modified oligonucleotide are 5-methyl cytosines. In certain embodiments, all of the cytosine nucleobases are 5-methyl cytosines and all other nucleobases in the modified oligonucleotide are unmodified nucleobases.

[00167] In certain embodiments, the modified oligonucleotides comprise 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 nucleosides 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 nucleosides of the 5' end of the oligonucleotide.

[00168] In certain embodiments, the oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, a nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer 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: a 2-thiopyrimidine and a 5-propynepyrimidine.

CERTAIN INTERNUCLEOSIDE BINDING MOTIFS

[00169] In certain embodiments, the oligonucleotides comprise modified and/or unmodified internucleoside linkages arranged throughout the oligonucleotide or portion thereof in a defined pattern or motif. In certain embodiments, each internucleoside linking group is a phosphodiester internucleoside linkage. In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate internucleoside linkage and a phosphodiester internucleoside linkage. In certain embodiments, each phosphorothioate internucleoside linkage is independently selected from a stereorandom phosphorothioate, a phosphorothioate (Sp), and a phosphorothioate (Rp). In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside bonds within the gap are all modified. In certain such embodiments, some or all of the internucleoside bonds in the wings are unmodified phosphodiester internucleoside bonds. In certain embodiments, the terminal internucleoside bonds are modified. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside bond motif comprises at least one phosphodiester internucleoside bond in at least one wing, wherein the at least one phosphodiester internucleoside bond is not a terminal internucleoside bond, and the remaining internucleoside bonds are phosphorothioate internucleoside bonds. In certain such embodiments, all of the phosphorothioate internucleoside bonds are stereorandom. In certain embodiments, all of the phosphorothioate internucleoside bonds in the wings are phosphorothioates (Sp), and the gap comprises at least one Sp, Sp, Rp motif. In certain embodiments, the modified oligonucleotide populations are enriched with modified oligonucleotides comprising such internucleoside bond motifs. In certain embodiments, the one or more internucleoside bonds is a mesylphosphoramidate internucleoside bond. In certain embodiments, each internucleoside bond is independently selected from a phosphodiester internucleoside bond, a phosphorothioate internucleoside bond, and a mesylphosphoramidate internucleoside bond. In certain embodiments, each internucleoside bond is independently selected from a phosphorothioate internucleoside bond and a mesylphosphoramidate internucleoside bond. In certain embodiments, the one or more internucleoside linkages is a methoxypropylphosphonate internucleoside linkage. In certain embodiments, each internucleoside linkage is independently selected from a phosphodiester internucleoside linkage, a phosphorothioate internucleoside linkage, and a methoxypropylphosphonate internucleoside linkage. In certain embodiments, each internucleoside linkage is independently selected from a phosphorothioate internucleoside linkage and a methoxypropylphosphonate internucleoside linkage.

[00170] In certain embodiments, modified oligonucleotides comprise 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 bonds. In certain embodiments, modified oligonucleotides comprise 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, modified oligonucleotides comprise at least 1, at least 2, at least 3, at least 4, or at least 5 phosphodiester internucleoside linkages, and the remainder of the internucleoside linkages are phosphorothioate internucleoside linkages.

CERTAIN LENGTHS

[00171] It is possible to increase or decrease the length of an oligonucleotide without eliminating activity. For example, in Woolf et al., Proc. Natl. Acad. Sci. U.S.A., 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 with 8 or 11 mismatched bases near the ends of the oligonucleotides were able to direct specific cleavage of the target nucleic acid, although to a lesser extent than oligonucleotides that did not contain mismatches. Similarly, target-specific cleavage was achieved using oligonucleotides with 13 nucleobases, which include those with 1 or 3 mismatches.

[00172] In certain embodiments, the oligonucleotides (including modified oligonucleotides) can have any of a variety of length ranges. In certain embodiments, the oligonucleotides consist of X to Y linked nucleosides, wherein X represents the smallest number of nucleosides in the range, and Y represents the largest 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, the oligonucleotides consist of 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13- 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, 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 2 5, 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, 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, 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, to 29, 27 to 30, 28 to 29, 28 to 30 or 29 to 30 linked nucleosides.

[00173] In certain embodiments, the oligonucleotides consist of 16 linked nucleosides. In certain embodiments, the oligonucleotides consist of 17 linked nucleosides. In certain embodiments, the oligonucleotides consist of 18 linked nucleosides. In certain embodiments, the oligonucleotides consist of 19 linked nucleosides. In certain embodiments, the oligonucleotides consist of 20 linked nucleosides.

CERTAIN MODIFIED OLIGONUCLEOTIDES

[00174] In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, the modified oligonucleotides are characterized by their modification motifs and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region of the sugar motif. Similarly, such sugar gapmer oligonucleotides may comprise one or more modified nucleobases independent of the gapmer pattern of the sugar modifications. Unless otherwise noted, all modifications are independent of nucleobase sequence.

CERTAIN POPULATIONS OF MODIFIED OLIGONUCLEOTIDES

[00175] Modified oligonucleotide populations in which all of the modified oligonucleotides in the population have the same molecular formula may be stereorandom populations or chirally enriched populations. All of the chiral centers of all of the modified oligonucleotides are stereorandom in a stereorandom population. In a chirally enriched population, at least one particular chiral center is not stereorandom in the modified oligonucleotides in the population. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for β-D ribosyl sugar moieties, and all of the phosphorothioate internucleoside linkages are stereorandom. In certain embodiments, modified oligonucleotides from a chirally enriched population are enriched for both β-D ribosyl sugar moieties and at least one particular phosphorothioate internucleoside linkage in a particular stereochemical configuration.

NUCLEOBASE SEQUENCES

[00176] In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by their nucleobase sequence. In certain embodiments, oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, a portion of an oligonucleotide has a nucleobase sequence that 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 entire length of an 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 the second oligonucleotide or nucleic acid, such as a target nucleic acid.

CERTAIN OLIGOMERIC COMPOUNDS

[00177] In certain embodiments, provided herein are oligomeric compounds, which consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugated groups and/or terminal groups. The conjugated groups consist of one or more conjugated chemical moieties and a conjugated linker that attaches the conjugated chemical moiety to the oligonucleotide. The conjugated groups can be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, the conjugated groups are attached to the 2' position of a nucleoside of a modified oligonucleotide. In certain embodiments, the conjugated groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, the conjugated groups or terminal groups are attached at the 3' and/or 5' end of the oligonucleotides. In certain such embodiments, the conjugative groups (or terminal groups) are attached to the 3' end of the oligonucleotides. In certain embodiments, the conjugative groups are attached near the 3' end of the oligonucleotides. In certain embodiments, the conjugative groups (or terminal groups) are attached to the 5' end of the oligonucleotides. In certain embodiments, the conjugative groups are attached near the 5' end of the oligonucleotides.

[00178] Examples of terminal groups include, but are not limited to, conjugated groups, cap groups, phosphate moieties, protecting groups, abasic nucleosides, modified and unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.

CERTAIN CONJUGATED GROUPS

[00179] In certain embodiments, the oligonucleotides are covalently linked to one or more conjugating groups. In certain embodiments, the conjugating groups modify one or more properties of the linked oligonucleotide, including, but not limited to, pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance. In certain embodiments, the conjugating groups confer a novel property to the linked oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide. Certain conjugated groups and conjugated chemical moieties have been described previously, for example: cholesterol chemical moiety (Letsinger et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., 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), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-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), a phospholipid, e.g., dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or a palmityl moiety of adamantane acetic acid (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229–237), an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923–937), a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734–740) or a GalNAc group (e.g., WO2014/179620).

CONJUGATED CHEMICAL MOTION

[00180] Conjugated moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, lipophilic groups, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

[00181] In certain embodiments, a conjugated chemical moiety comprises an active pharmaceutical substance, e.g., aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fembufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial, or an antibiotic.

CONJUGATED LIGANDS

[00182] Conjugated chemical moieties are attached to oligonucleotides via conjugated linkers. In certain oligomeric compounds, the conjugated linker is a single chemical bond (i.e., the conjugated chemical moiety is attached directly to an oligonucleotide via a single bond). In certain oligomeric compounds, a conjugated chemical moiety is attached to an oligonucleotide via a more complex conjugated linker comprising one or more conjugated linker chemical moieties, which are subunits that make up a conjugated linker. In certain embodiments, the conjugated linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units, such as ethylene glycol, nucleoside, or amino acid units.

[00183] In certain embodiments, a conjugated linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugated linker comprises groups selected from alkyl, amino, oxo, amide, and ether groups. In certain embodiments, the conjugated linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugated linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugated linker comprises at least one phosphorus moiety. In certain embodiments, the conjugated linker comprises at least one phosphate group. In certain embodiments, the conjugated linker includes at least one neutral linking group.

[00184] In certain embodiments, the conjugated linkers, including the conjugated linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugated groups to precursor compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected for attachment to a particular site on a precursor compound and the other is selected for attachment to a conjugated group. Examples of functional groups used in a bifunctional linking moiety include, but are not limited to, electrophiles for reaction with nucleophilic groups and nucleophiles for reaction with electrophilic groups. In certain embodiments, the bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[00185] Examples of conjugated ligands include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other conjugated ligands 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, wherein an unlimited list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[00186] In certain embodiments, the conjugated linkers comprise 1 to 10 nucleoside linkers. In certain embodiments, the conjugated linkers comprise 2 to 5 nucleoside linkers. In certain embodiments, the conjugated linkers comprise exactly 3 nucleoside linkers. In certain embodiments, the conjugated linkers comprise the TCA motif. In certain embodiments, such nucleoside linkers are modified nucleosides. In certain embodiments, such nucleoside linkers comprise a modified sugar moiety. In certain embodiments, the nucleoside linkers are unmodified. In certain embodiments, the nucleoside linkers comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine, or substituted pyridine. In certain embodiments, a cleavable chemical moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methyl cytosine, 4-N-benzoyl-5-methyl cytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine. It is typically desirable for the linker nucleosides to be cleaved from the oligomeric compound upon reaching a target tissue. Accordingly, the linker nucleosides are typically linked to each other and to the remainder of the oligomeric compound via cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

[00187] Herein, linker nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments where an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate group comprising a conjugate linker comprising linker nucleosides, these linker nucleosides are not counted in the length direction of the oligonucleotide and are not used to determine the percent complementarity of the oligonucleotide to the reference nucleic acid. For example, an oligomeric compound can comprise (1) a modified oligonucleotide consisting of 8 to 30 nucleosides and (2) a conjugate group comprising 1 to 10 linker nucleosides that are contiguous to the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such an oligomeric compound is more than 30. Alternatively, an oligomeric compound can comprise a modified oligonucleotide consisting of 8 to 30 nucleosides and no conjugated groups. The total number of contiguous linked nucleosides in such an oligomeric compound is no greater than 30. Unless otherwise indicated, the conjugated linkers comprise no more than 10 nucleoside linkers. In certain embodiments, the conjugated linkers comprise no more than 5 nucleoside linkers. In certain embodiments, the conjugated linkers comprise no more than 3 nucleoside linkers. In certain embodiments, the conjugated linkers comprise no more than 2 nucleoside linkers. In certain embodiments, the conjugated linkers comprise no more than 1 nucleoside linker.

[00188] In certain embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide. For example, in certain circumstances, oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable for the conjugate group to be cleaved to release the unconjugated oligonucleotide or precursor. Thus, certain conjugate linkers may comprise one or more cleavable chemical moieties. In certain embodiments, a cleavable chemical moiety is a cleavable bond. In certain embodiments, a cleavable chemical moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable chemical moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable chemical moiety is selectively cleaved within a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.

[00189] In certain embodiments, a cleavable bond is selected from: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate bond between an oligonucleotide and a conjugate moiety or conjugate group.

[00190] In certain embodiments, a cleavable chemical moiety comprises or consists of one or more nucleoside linkers. In certain such embodiments, the one or more nucleoside linkers are linked to each other and/or to the remainder of the oligomeric compound via cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable chemical moiety is 2'-deoxyribonucleoside that is linked to the 3'- or 5'-terminal nucleoside of an oligonucleotide by a phosphate internucleoside linkage and covalently linked to the remainder of the conjugated linker or conjugated chemical moiety by a phosphate or phosphorothioate internucleoside linkage. In certain such embodiments, the cleavable chemical moiety is 2'-deoxyadenosine.

CERTAIN TERMINAL GROUPS

[00191] In certain embodiments, the oligomeric compounds comprise one or more terminal groups. In certain such embodiments, the oligomeric compounds comprise a stabilized 5'-phosphate. Stabilized 5'-phosphates include, but are not limited to, 5'-phosphanates, which include, but are not limited to, 5'-vinylphosphonates. In certain embodiments, the terminal groups comprise one or more abasic nucleosides and/or inverted nucleosides. In certain embodiments, the terminal groups comprise one or more 2'-linked nucleosides. In certain such embodiments, the 2'-linked nucleoside is an abasic nucleoside.

OLIGOMERIC DUPLEXES

[00192] In certain embodiments, the oligomeric compounds described herein comprise an oligonucleotide, which has a nucleobase sequence complementary to that of a target nucleic acid. In certain embodiments, an oligomeric compound is paired with a second oligomeric compound to form an oligomeric duplex. Such oligomeric duplexes comprise a first oligomeric compound having a moiety complementary to a target nucleic acid and a second oligomeric compound having a moiety complementary to the first oligomeric compound. In certain embodiments, the first oligomeric compound of an oligomeric duplex comprises or consists of (1) a modified or unmodified oligonucleotide and optionally a conjugate group and (2) a second modified or unmodified oligonucleotide and optionally a conjugate group. Either or both of the oligomeric compounds of an oligomeric duplex can comprise a conjugate group. The oligonucleotides of each oligomeric compound of an oligomeric duplex may include non-complementary overhang nucleosides.

ANTI-SENSE ACTIVITY

[00193] In certain embodiments, the oligomeric compounds and oligomeric duplexes have the ability to hybridize to a target nucleic acid, resulting in at least one antisense activity; such oligomeric compounds and oligomeric duplexes are antisense compounds. In certain embodiments, antisense compounds have antisense activity when they reduce, modulate, or increase the amount or activity of a target nucleic acid by 25% or more in the standard cellular assay. In certain embodiments, the antisense compounds selectively affect one or more target nucleic acids. Such antisense compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acids, resulting in one or more desired antisense activities and does not hybridize to one or more non-target nucleic acids or does not hybridize to one or more non-target nucleic acids in such a manner as to result in significant undesired antisense activity.

[00194] In certain antisense activities, hybridization of an antisense compound to a target nucleic acid results in 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 duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, antisense compounds are provided herein that are sufficiently "DNA-like" to elicit RNase H activity. In certain embodiments, one or more non-DNA-like nucleosides in the gap of a gapmer are tolerated.

[00195] In certain antisense activities, an antisense compound or a portion of an 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 result in cleavage of the target nucleic acid by Argonaute. Antisense compounds that are loaded into RISC are RNAi compounds. RNAi compounds can be double-stranded (siRNA) or single-stranded (ssRNA).

[00196] In certain embodiments, hybridization of an antisense compound to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain embodiments, hybridization of the antisense compound to the target nucleic acid results in altered splicing of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a 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 an antisense compound to a target nucleic acid results in altered translation of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in exon skipping. In certain embodiments, hybridization of an antisense compound to a 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 a target nucleic acid results in a splicing alteration that results in the inclusion of an exon in the mRNA.

[00197] Antisense activities can be observed directly or indirectly. In certain embodiments, observing or detecting an antisense activity involves observing or detecting an alternation in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein, and/or a phenotypic change in a cell or subject.

CERTAIN TARGET NUCLEIC ACIDS

[00198] In certain embodiments, the oligomeric compounds comprise or consist of an oligonucleotide comprising a moiety 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: a mature mRNA and a pre-mRNA, which includes intronic, exonic, and untranslated regions. In certain embodiments, the target nucleic acid is a mature mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain embodiments, the target region is completely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.

COMPLEMENTARITY/MISMATCH TO THE TARGET NUCLEIC ACID

[00199] 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 has 100% complementarity to bcl-2 mRNA and that has 3 mismatches to bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent antitumor activity in vivo. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of 14-nucleobase tandem oligonucleotides and 28- and 42-nucleobase oligonucleotides comprised of the sequence of two or three of the tandem oligonucleotides, respectively, for their ability to stop translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14-nucleobase oligonucleotides alone had the ability to inhibit translation, although to a more modest extent than the 28- or 42-nucleobase oligonucleotides.

[00200] In certain embodiments, the oligonucleotides are complementary to the target nucleic acid throughout the length of the oligonucleotide. In certain embodiments, the oligonucleotides are 99%, 95%, 90%, 85%, or 80% complementary to the target nucleic acid. In certain embodiments, the oligonucleotides are at least 80% complementary to the target nucleic acid throughout the length of the oligonucleotide and comprise a portion that is 100% or completely complementary to a target nucleic acid. In certain embodiments, the completely complementary portion is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length.

[00201] In certain embodiments, the oligonucleotides comprise one or more nucleobases that are mismatched 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

[00202] In certain embodiments, oligomeric compounds comprise or consist of a modified oligonucleotide that is complementary to a target nucleic acid encoding SMN2, or a portion thereof. In certain embodiments, SMN2 has the sequence set forth in SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777).

[00203] In certain embodiments, contacting a cell with the oligomeric compound complementary to SEQ ID NO: 1 modulates SMN2 RNA splicing in a cell. In certain embodiments, contacting a cell with the oligomeric compound complementary to SEQ ID NO: 1 increases the amount of SMN2 RNA that includes exon 7. In certain embodiments, contacting a cell with the 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.

[00204] In certain embodiments, contacting a cell in a subject with an oligomeric compound complementary to SEQ ID NO: 1 attenuates one or more symptoms of a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is AMS which includes AMS Type I, AMS Type II, AMS Type III, and AMS Type IV. In certain embodiments, the symptom is any of reduced muscle strength; inability or reduced ability to sit upright, stand, and/or walk; reduced neuromuscular activity; reduced electrical activity in one or more muscles; reduced breathing; inability or reduced ability to eat, drink, and/or breathe without assistance; weight loss or reduced weight gain; and/or decreased survival.

[00205] In certain embodiments, an oligomeric compound complementary to SEQ ID NO: 1 is capable of increasing SMN2 RNA that includes exon 7 in vivo by at least 1-fold, 2-fold, or 3-fold when administered according to a standard in vivo assay. In certain embodiments, an 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 when administered according to a standard in vivo assay.

CERTAIN NUCLEIC ACIDS TARGET CERTAIN TISSUES

[00206] In certain embodiments, the oligomeric compounds comprise or consist of an oligonucleotide comprising a moiety that is complementary to a target nucleic acid, wherein the target nucleic acid is expressed in a pharmacologically relevant tissue. In certain embodiments, the pharmacologically relevant tissues are the cells and tissues that comprise the central nervous system (CNS). Such tissues include brain tissues, such as spinal cord, cortex, and coronal cerebral tissue.

CERTAIN PHARMACEUTICAL COMPOSITIONS

[00207] In certain embodiments, pharmaceutical compositions comprising one or more oligomeric compounds are described herein. In certain embodiments, the one or more oligomeric compounds each consist of a modified oligonucleotide. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises or consists of a sterile saline solution and one or more oligomeric compounds. In certain embodiments, the sterile saline solution is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compounds and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises or 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, a pharmaceutical composition comprises or 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. In certain embodiments, a pharmaceutical composition comprises a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists essentially of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.

[00208] In certain embodiments, the pharmaceutical compositions comprise one or more oligomeric compounds and one or more excipients. In certain embodiments, the excipients are selected from water, saline solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, and polyvinylpyrrolidone.

[00209] In certain embodiments, the oligomeric compounds may be mixed by addition with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions are dependent on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

[00210] In certain embodiments, pharmaceutical compositions comprising an oligomeric compound encompass any pharmaceutically acceptable salts of the oligomeric compound, esters of the oligomeric compound, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising oligomeric compounds comprising one or more oligonucleotides, upon administration to a subject, including a human, have the ability to deliver (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure also relates to pharmaceutically acceptable salts of oligomeric compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, the prodrugs comprise one or more conjugate groups linked to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the compound. In certain embodiments, the prodrugs comprise one or more conjugate groups linked to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the compound.

[00211] Lipid moieties have been used in nucleic acid therapies in a variety of methods. In some such methods, the nucleic acid, such as an oligomeric compound, is introduced into preformed liposomes or lipoplexes produced from mixtures of cationic lipids and neutral lipids. In certain methods, complexes of DNA with monocationic or polycationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to enhance delivery of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to enhance delivery of a pharmaceutical agent to adipose tissue. In certain embodiments, a lipid moiety is selected to enhance delivery of a pharmaceutical agent to muscle tissue.

[00212] In certain embodiments, the pharmaceutical compositions comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions that include those comprising hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[00213] In certain embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents comprising an oligomeric compound provided herein to specific tissues or cell types. For example, in certain embodiments, the pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

[00214] In certain embodiments, the pharmaceutical compositions comprise a co-solvent system. Certain such co-solvent systems comprise, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is an absolute alcohol solution comprising 3% w/v benzyl alcohol, 8% w/v of the non-polar surfactant Polysorbate 80™, and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems can be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of the cosolvent components can be varied: for example, other surfactants can be used instead of Polysorbate 80™; the polyethylene glycol fraction size can be varied; other biocompatible polymers can replace polyethylene glycol, for example, polyvinyl pyrrolidone; and other sugars or polysaccharides can replace dextrose.

[00215] In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), etc.). In certain such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water, or physiologically compatible buffers, such as Hanks' solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using suitable liquid carriers, suspending agents, and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, for example, in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents, such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection 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.

[00216] 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 in association with a cationic (salt) form, aqueous solutions of such compounds exist in equilibrium between such forms. For example, a phosphate bond of an oligonucleotide in aqueous solution exists in equilibrium between the free acid, anion and salt forms. Unless otherwise indicated, the compounds described herein are intended to include all such forms. Furthermore, certain oligonucleotides have several such bonds, each of which is in equilibrium. Thus, oligonucleotides in solution exist in a set of forms at multiple positions, all in equilibrium. The term "oligonucleotide" is intended to include all such forms. The structures drawn necessarily represent a single form. However, unless otherwise indicated, such drawings are also intended to include the corresponding forms. In this document, a structure representing the free acid of a compound followed by the term "or a salt thereof" expressly includes all such forms that may be fully or partially protonated/deprotonated/in association with a cation. In certain cases, one or more specific cations are identified.

[00217] In certain embodiments, the modified oligomeric or oligonucleotide compounds are in aqueous solution with sodium. In certain embodiments, the modified oligomeric or oligonucleotide compounds are in aqueous solution with potassium. In certain embodiments, the modified oligomeric or oligonucleotide compounds are in PBS. In certain embodiments, the modified oligomeric or oligonucleotide compounds are in water. In certain embodiments, the pH of the solution is adjusted with NaOH and/or HCl to achieve a desired pH.

[00218] Certain specific doses are described herein. A dose may be in the form of a dosage unit. For clarity, a dose (or dosage unit) of a modified oligonucleotide or oligomeric compound in milligrams indicates the mass of the free acid form of the modified oligonucleotide or oligomeric compound. As described above, in 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 as an anhydrous, sodium acetate-free, solvent-free free acid. For example, when a modified oligonucleotide or oligomeric compound is in solution comprising sodium (e.g., saline), the modified oligonucleotide or oligomeric compound may be partially or fully deprotonated and may be in association with Na+ ions. However, the mass of the protons is nevertheless counted toward the dose weight, and the mass of the Na+ ions is not counted toward the dose weight. Thus, for example, a dose, or dosage 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 would be the equivalent of 10.53 mg of Compound Sodium Acetate Free Anhydrous and Solvent No. 1263789, 10.53 mg of Compound Sodium Acetate Free Anhydrous and Solvent No. 1287717, 10.52 mg of Compound Sodium Acetate Free Anhydrous and Solvent No. 1287745, and 10.51 mg of Compound Sodium Acetate Free Anhydrous and Solvent No. 1358996. When an oligomeric compound comprises a conjugated group, the mass of the conjugated group is included in calculating the dose for such oligomeric compound. If the conjugated group also contains an acid, the conjugated group is likewise assumed to be fully protonated for the purpose of calculating the dose.

CERTAIN COMPOSITIONS COMPOUND NO.: 1263789

[00219] In certain embodiments, Compound No. 1263789 is characterized as a modified oligonucleotide having a sequence of (from 5' to 3') CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside comprises a sugar moiety of 2'-MOE, wherein the internucleoside linkages between nucleosides 2 and 3 and 4 and 5 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 1 and 2, 3 and 4, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 15 and 16, 16 and 17 and 17 and 18 are phosphorothioate internucleoside linkages, and each cytosine is a 5-methylcytosine.

[00220] In certain embodiments, Compound No. 1263789 is represented by the following chemical notation (5' to 3'): mCes Aeo mCes Teo Tes Tes mCes Aes Tes Aes Aes Tes Ges mCes Tes Ges Ges mCe (SEQ ID NO: 21), wherein, A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2'-MOE sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage.

[00221] In certain embodiments, Compound No. 1263789 is represented by the following chemical structure: (SEQ ID NO: 21).

STRUCTURE 1. COMPOUND NO. 1263789

[00222] In certain embodiments, the sodium salt of Compound No. 1263789 is represented by the following chemical structure: (SEQ ID NO: 21).

STRUCTURE 2. THE SODIUM SALT OF COMPOUND NO. 1263789 COMPOUND NO.: 1287717

[00223] In certain embodiments, Compound No. 1287717 is characterized as a modified oligonucleotide having a sequence of (from 5' to 3') TTCACTTTCATAATGCTGGC (SEQ ID NO: 22), wherein each nucleoside comprises a sugar moiety of 2'-MOE, wherein the internucleoside linkages between nucleosides 1 and 2 and 19 and 20 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 15 and 16, 16 and 17, 17 and 18, and 18 and 19 are phosphorothioate internucleoside linkages, and where each cytosine is a 5-methylcytosine.

[00224] In certain embodiments, Compound No. 1287717 is represented by the following chemical notation (5' to 3'): Teo Tes mCes Aes mCes Tes Tes Tes mCes Aes Tes Aes Aes Tes Ges mCes Tes Ges Geo mCe (SEQ ID NO: 22) wherein, A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2'-MOE sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage.

[00225] In certain embodiments, Compound No. 1287717 is represented by the following chemical structure: (SEQ ID NO: 22).

STRUCTURE 3. COMPOUND NO. 1287717

[00226] In certain embodiments, the sodium salt of Compound No. 1287717 is represented by the following chemical structure: (SEQ ID NO: 22).

STRUCTURE 4. SODIUM SALT OF COMPOUND NO. 1287717 COMPOUND NO.: 1287745

[00227] In certain embodiments, Compound No. 1287745 is characterized as a modified oligonucleotide having a sequence of (from 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 internucleotide linkages between nucleosides 1 and 2 and 19 and 20 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 15 and 16, 16 and 17, 17 and 18, and 18 and 19 are phosphorothioate internucleoside linkages, and where each cytosine is a 5-methylcytosine.

[00228] In certain embodiments, Compound No. 1287745 is represented by the following chemical notation (5' to 3'): Teo Tns mCns Ans mCns Tns Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gno mCe (SEQ ID NO: 22) wherein, A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2'-MOE sugar moiety, n = a 2'-NMA sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage.

[00229] In certain embodiments, Compound No. 1287745 is represented by the following chemical structure: (SEQ ID NO: 22).

STRUCTURE 5. COMPOUND NO. 1287745

[00230] In certain embodiments, the sodium salt of Compound No. 1287745 is represented by the following chemical structure: the (SEQ ID NO: 22).

STRUCTURE 6. SODIUM SALT OF COMPOUND NO. 1287745 COMPOUND NO.: 1358996

[00231] In certain embodiments, Compound No. 1358996 is characterized as a modified oligonucleotide having a sequence of (from 5' to 3') CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside comprises a 2'-NMA sugar moiety, wherein the internucleoside linkages between nucleosides 2 and 3 and 4 and 5 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 1 and 2, 3 and 4, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 15 and 16, 16 and 17 and 17 and 18 are phosphorothioate internucleoside linkages, and where each cytosine is a 5-methylcytosine.

[00232] In certain embodiments, Compound No. 1358996 is represented by the following chemical notation (5' to 3'): mCns Ano mCns Tno Tns Tns mCns Ans Tns Ans Ans Tns Gns mCns Tns Gns Gns mCn (SEQ ID NO: 21) wherein, A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, n = a 2'-NMA sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage.

[00233] In certain embodiments, Compound No. 1358996 is represented by the following chemical structure: (SEQ ID NO: 21).

STRUCTURE 7. COMPOUND NO. 1358996

[00234] In certain embodiments, the sodium salt of Compound No. 1358996 is represented by the following chemical structure: (SEQ ID NO: 21).

STRUCTURE 8. THE SODIUM SALT OF COMPOUND NO. 1358996 CERTAIN COMPARATIVE COMPOSITIONS

[00235] In certain embodiments, Spinraza® (generic name nusinersen; Compound No. 396443), approved for the treatment of AMS, is a comparator compound (see, e.g., 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. al., Neurology, 92(21):e2492-e2506, 2019). Spinraza® was previously described in WO2010120820, which is incorporated herein by reference, and has a sequence (5' to 3') of TCACTTTCATAATGCTGG (SEQ ID NO: 23), wherein each nucleoside comprises a 2'-MOE sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is a 5-methylcytosine.

[00236] In certain embodiments, although not approved for human therapy, other previously described compounds, which include Compound Nos. 387954, 396442, 443305, and 819735, are comparator compounds.

[00237] Compound No. 387954 was previously described in WO 2014/179620, which is incorporated herein by reference. Compound No. 387954 has a sequence (5' to 3') of ATTCACTTTCATAATGCTGG (SEQ ID NO: 20), wherein each nucleoside comprises a 2'-MOE sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is a 5-methylcytosine.

[00238] Compound No. 396442 was previously described in WO 2010/120820, which is incorporated herein by reference. Compound No. 396442 has a sequence (5' to 3') of CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside comprises a 2'-MOE sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is a 5-methylcytosine.

[00239] Compound No. 443305 was previously described in WO 2018/014041, which is incorporated herein by reference. Compound No. 443305 has a sequence (5' to 3') of TCACTTTCATAATGCTGG (SEQ ID NO: 23), wherein each nucleoside comprises a 2'-NMA sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is a 5-methylcytosine.

[00240] Compound No. 819735 was previously described in WO 2018/014041, which is incorporated herein by reference. Compound No. 819735 has a sequence (5' to 3') of CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside comprises a 2'-NMA sugar moiety, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is a 5-methylcytosine. TABLE 1 CERTAIN COMPARATOR COMPOSITIONS

[00241] In certain embodiments, compounds described herein are superior to compounds described in WO 2007/002390, WO2010/120820, WO 2015/161170, and WO 2018/014041 as they demonstrate one or more improved properties such as potency, efficacy, and tolerability.

[00242] For example, Compound No. 1263789, Compound No. 1287745, and Compound No. 1358996 each demonstrated 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 a spinal cord ED50 of 13.3, 8.8, and 7.4, respectively. In comparison, Compound No. 396443 achieved a spinal cord ED50 of 22.0. Therefore, each of Compound #1263789, Compound #1287745, and Compound #1358996 are more potent than Compound #396443 in this assay.

[00243] For example, Compound #1263789, Compound #1287717, Compound #1287745, and Compound #1358996 each demonstrated improved 3-hour FOB scores compared to Compound #396443, Compound #387954, and Compound #443305. As shown in Example 6, at 700 μg, Compound #1263789, Compound #1287717, Compound #1287745, and Compound #1358996 achieved 3-hour FOB scores of 0, 3.25, 1, and 0, respectively. In comparison, at half the dose (350 μg) Compound #396443 achieved a 3-hour FOB score of 4.0; and at the same dose (700 μg) Compound #387954 and Compound #443305 achieved a 3-hour FOB score of 4.0 and 4.75, respectively. Therefore, each of Compound #1263789, Compound #1287717, Compound #1287745, and Compound #1358996 are more tolerable than Compound #396443, Compound #387954, and Compound #443305 in this trial.

[00244] For example, Compound #1263789, Compound #1287717, Compound #1287745, and Compound #1358996 each demonstrated improved long-term tolerability compared to Compound #396442 and Compound #819735. As shown in Example 7, Compound #1263789, Compound #1287717, Compound #1287745, and Compound #1358996 demonstrated no adverse events, no Purkinje cell loss, and cortex GFAP mRNA less than 2-fold of control. In comparison, 396442 and 819735 each demonstrated adverse events, Purkinje cell loss, and cortex GFAP mRNA greater than 2-fold of control in certain treated animals. Therefore, each of Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 are more tolerable than Compound No. 396442 and Compound No. 819735 in this assay.

NON-LIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

[00245] Each of the literature and patent publications listed herein is incorporated by reference in its entirety. Although certain compounds, compositions, and methods described herein have been described with specificity according to certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit them. Each of the references, GenBank accession numbers, and the like mentioned in this application is incorporated by reference in its entirety.

[00246] Although the sequence listing accompanying this filing identifies each sequence as "RNA" or "DNA" as needed, in reality, such sequences may be modified with any combination of chemical modifications. One of skill in the art will readily understand that such a designation as "RNA" or "DNA" to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2'-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar moiety (2'-OH in place of a DNA 2'-H) or as an RNA having a modified base (thymine (methylated uracil) in place of an RNA uracil). Accordingly, the nucleic acid sequences provided herein, including, but not limited to, those 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 that have modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence "ATCGATCG" encompasses any oligomeric compounds having such a nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having the sequence "AUCGAUCG" and those having some DNA bases and some RNA bases, such as "AUCGATCG", and oligomeric compounds having other modified nucleobases, such as "ATmCGAUCG", where mC denotes a cytosine base comprising a methyl group at position 5.

[00247] Certain compounds described herein (e.g., modified oligonucleotides) have one or more asymmetric centers and thus generate enantiomers, diastereomers, and other stereoisomeric configurations that can be defined, in terms of absolute stereochemistry, as (R) or (S), as α or β, such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are designed or described with certain stereoisomeric configurations include only the indicated compounds. Compounds provided herein that are designed and described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms, unless otherwise specified. Likewise, all cis- and trans-isomers and tautomeric forms of the compounds herein are also included unless otherwise indicated. Oligomeric compounds described herein include chirally pure or enriched mixtures, as well as racemic mixtures. For example, oligomeric compounds having a plurality of phosphorothioate internucleoside linkages include such compounds in which the chirality of the phosphorothioate internucleoside linkages is controlled or is random. Unless otherwise indicated, compounds described herein are intended to include corresponding salt forms.

[00248] The compounds described herein include variations in which one or more atoms are replaced by a non-radioactive isotope or radioactive isotope of the indicated element. For example, the compounds herein comprising hydrogen atoms encompass all possible deuterium substitutions for each of the 1H hydrogen atoms. Isotypic 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, the non-radioactive isotopic substitutions may impart novel properties to the oligomeric compound that are beneficial for use as a therapeutic agent or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research and diagnostic purposes, such as imaging.

EXAMPLES

[00249] The following examples illustrate certain embodiments of the present disclosure and are not limiting. Furthermore, if specific embodiments are provided, the inventors have contemplated a generic application of these specific embodiments.

EXAMPLE 1: DESIGN OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO A HUMAN SMN2 NUCLEIC ACID

[00250] Modified oligonucleotides complementary to a human SMN2 nucleic acid were designed and synthesized as indicated in the Tables below.

[00251] The modified oligonucleotides in the Tables below are 16, 17, 18, 19, or 20 nucleosides in length, as specified. The modified oligonucleotides comprise 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. Each internucleoside linkage along the modified oligonucleotides is either a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage, as specified. Cytosines are unmethylated cytosines or 5-methylcytosines, as specified.

[00252] Each modified oligonucleotide listed in the Tables below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777) unless specifically stated otherwise. Non-complementary nucleobases are specified in the Nucleobase Sequence column in underlined, bold, italic font. Each modified oligonucleotide listed in the Tables below targets an active site in the SMN2 transcript for exon 7 inclusion. "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 2

[00253] The modified oligonucleotides in Table 2 below are 16, 17, 18, 19, or 20 nucleosides in length. Each nucleoside comprises a 2'-MOE sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif column, where each 'e' represents a 2'-MOE sugar moiety. Each internucleoside bond is either a phosphorothioate internucleoside bond or a phosphodiester internucleoside bond. The internucleoside bond motif for each modified oligonucleotide is provided in the Internucleoside Bond Motif column, where each 's' represents a phosphorothioate internucleoside bond and each 'o' represents a phosphodiester internucleoside bond. Each cytosine is a 5-methylcytosine.

[00254] Each modified oligonucleotide listed in Table 2 below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777) unless specifically stated otherwise. Non-complementary nucleobases are specified in the Nucleobase Sequence column in bold, italicized, underlined font. "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 2 2'-MOE MODIFIED OLIGONUCLEOTIDES WITH PS OR MIXED PS/PO INTERNUCLEOSIDIC BONDINGS TABLE 3

[00255] The modified oligonucleotides in Table 3 below are 16, 17, 18, 19, or 20 nucleosides in length. Each nucleoside comprises a 2'-NMA sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif column, where each 'n' represents a 2'-NMA sugar moiety. Each internucleoside bond is either a phosphorothioate internucleoside bond or a phosphodiester internucleoside bond. The internucleoside bond motif for each modified oligonucleotide is provided in the Internucleoside Bond Motif column, where each 's' represents a phosphorothioate internucleoside bond and each 'o' represents a phosphodiester internucleoside bond. Each cytosine is a 5-methylcytosine.

[00256] Each modified oligonucleotide listed in Table 3 below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777). "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 3 2'-NMA MODIFIED OLIGONUCLEOTIDES WITH PS OR MIXED PS/PO INTERNUCLEOSIDIC LINKAGES TABLE 4

[00257] The modified oligonucleotides in Table 4 below are 18 or 19 nucleosides in length. Each nucleoside comprises either a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif column, where each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside bond is a phosphorothioate internucleoside bond. The internucleoside bonding motif for each modified oligonucleotide is provided in the Internucleoside Bonding Motif column, where each 's' represents a phosphorothioate internucleoside bond. Each cytosine is a 5-methylcytosine.

[00258] Each modified oligonucleotide listed in Table 4 below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777) unless specifically stated otherwise. Non-complementary nucleobases are specified in the Nucleobase Sequence column in bold, italicized, underlined font. "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 4 2'-MOE/2'-NMA MODIFIED OLIGONUCLEOTIDES MIXED WITH PS INTERNUCLEOSIDIC BONDS TABLE 5

[00259] The modified oligonucleotides in Table 5 below are 16, 17, or 18 nucleosides in length. Each nucleoside comprises either a 2'-MOE sugar moiety or a cEt sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif column, where each 'e' represents a 2'-MOE sugar moiety and each 'k' represents a cEt sugar moiety. Each internucleoside bond is a phosphorothioate internucleoside bond. The internucleoside bonding motif for each modified oligonucleotide is provided in the Internucleoside Bonding Motif column, where each 's' represents a phosphorothioate internucleoside bond. Each cytosine is a 5-methylcytosine.

[00260] Each modified oligonucleotide listed in Table 5 below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777). "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 5 2'-MOE/CET MODIFIED OLIGONUCLEOTIDES MIXED WITH PS INTERNUCLEOSIDIC BONDAGES TABLE 6

[00261] The modified oligonucleotides in Table 6 below are 19 or 20 nucleosides in length. Each nucleoside comprises 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 for each modified oligonucleotide is provided in the Sugar Motif column, where each 'e' represents a 2'-MOE sugar moiety, each 'n' represents a 2'-NMA sugar moiety, each 'y' represents a 2'-OMe sugar moiety, and each 'd' represents a 2'-β-D-deoxyribosyl sugar moiety. Each internucleoside bond is either a phosphorothioate internucleoside bond or a phosphodiester internucleoside bond. The internucleoside bond motif for each modified oligonucleotide given in the Internucleoside Bond Motif column is (from 5' to 3'): sssssssssssssssso; where each 's' represents a phosphorothioate internucleoside bond and each 'o' represents a phosphodiester internucleoside bond. Cytosines are either unmethylated cytosines or 5-methylcytosines, where each lowercase 'c' in the Nucleobase Sequence column represents an unmethylated cytosine and each uppercase 'C' in the Nucleobase Sequence column represents a 5-methylcytosine.

[00262] Each nucleobase in the modified oligonucleotides listed in Table 6 below is complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777) unless specifically stated otherwise. Non-complementary nucleobases are specified in the Nucleobase Sequence column in bold, italicized, underlined font. "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 6 MODIFIED OLIGONUCLEOTIDES WITH MIXED PS/PO INTERNUCLEOSIDIC BONDS TABLE 7

[00263] The modified oligonucleotides in Table 7 below are 19 or 20 nucleosides in length. Each nucleoside comprises a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, or a 2'-β-D-deoxyribosyl sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif 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 either a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside binding motif for each modified oligonucleotide given in the Internucleoside Binding Motif column is (from 5' to 3'): sssssssssssssssssoo; where each 's' represents a phosphorothioate internucleoside bond and each 'o' represents a phosphodiester internucleoside bond. Each cytosine is a 5-methylcytosine.

[00264] Each nucleobase in the modified oligonucleotide listed in Table 6 below is complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777) unless specifically stated otherwise. Non-complementary nucleobases are specified in the Nucleobase Sequence column in bold, italicized, underlined font. "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 7 MODIFIED OLIGONUCLEOTIDES WITH MIXED PS/PO INTERNUCLEOSIDIC BONDS TABLE 8

[00265] The modified oligonucleotides in Table 8 below are each 19 nucleosides in length. Each nucleoside comprises a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, or a 2'-β-D-deoxyribosyl sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif 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 either a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside binding motif for each modified oligonucleotide given in the Internucleoside Binding Motif column is (from 5' to 3'): ssssssssssssso; where each 's' represents a phosphorothioate internucleoside bond and each 'o' represents a phosphodiester internucleoside bond. Each cytosine is a 5-methylcytosine.

[00266] Each nucleobase in the modified oligonucleotide listed in Table 8 below is complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777) unless specifically stated otherwise. Non-complementary nucleobases are specified in the Nucleobase Sequence column in bold, italicized, underlined font. "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 8 MODIFIED OLIGONUCLEOTIDES WITH MIXED PS/PO INTERNUCLEOSIDIC BONDS TABLE 9

[00267] The modified oligonucleotides in Table 9 below are each 19 nucleosides in length. Each nucleoside comprises a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, or a 2'-β-D-deoxyribosyl sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif 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 either a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside binding motif for each modified oligonucleotide given in the Internucleoside Binding Motif column is (from 5' to 3'): s ...

[00268] Each nucleobase in the modified oligonucleotide listed in Table 9 below is complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777) unless specifically stated otherwise. Non-complementary nucleobases are specified in the Nucleobase Sequence column in bold, italicized, underlined font. "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 9 MODIFIED OLIGONUCLEOTIDES WITH MIXED PS/PO INTERNUCLEOSIDIC BONDS TABLE 10

[00269] The modified oligonucleotides in Table 10 below are each 19 nucleosides in length. Each nucleoside comprises either a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif column, where each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is either 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 (from 5' to 3'): ossssssssssssssssss; where each 's' represents a phosphorothioate internucleoside bond and each 'o' represents a phosphodiester internucleoside bond. Each cytosine is a 5-methylcytosine.

[00270] Each nucleobase in the modified oligonucleotide listed in Table 10 below is complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777) unless specifically stated otherwise. Non-complementary nucleobases are specified in the Nucleobase Sequence column in bold, italicized, underlined font. "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 10 MODIFIED OLIGONUCLEOTIDES WITH MIXED PS/PO INTERNUCLEOSIDIC BONDS TABLE 11

[00271] The modified oligonucleotides in Table 11 below are each 20 nucleosides in length. Each nucleoside comprises either a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif column, where each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is either 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 (from 5' to 3'): ossssssssssssssssso; where each 's' represents a phosphorothioate internucleoside bond and each 'o' represents a phosphodiester internucleoside bond. Each cytosine is a 5-methylcytosine.

[00272] Each modified oligonucleotide listed in Table 11 below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777). "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. TABLE 11 MODIFIED OLIGONUCLEOTIDES WITH MIXED PS/PO INTERNUCLEOSIDIC LINKAGES

EXAMPLE 2: ACTIVITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO HUMAN SMN2 IN TRANSGENIC MICE, SINGLE DOSE (35 μG)

[00273] Activity of selected modified oligonucleotides described above was tested in human SMN2 transgenic mice. Taiwanese strain of AMS Type III mice was obtained from the Jackson Laboratory (Bar Harbor, Maine). These mice lack mouse SMN and are either homozygous for human SMN2 (mSMN -/-; hSMN2 +/+; FVB.Cg- Tg(SMN2)2HungSMN1tm1Hung/J, stock number 005058; Bar Harbor, Maine), or are heterozygous for mouse SMN and heterozygous for human SMN2 (mSMN +/-; hSMN2 +/-; FVB.Cg- Tg(SMN2)2HungSMN1tm1Hung/J) obtained by breeding HOM/HOM (stock number 00005058) to FVB/NJ (stock number 001800).

TREATMENT

[00274] Homozygous or heterozygous transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of 35 μg of modified oligonucleotide. Comparator Compounds #387954, 396442, and 396443 were also tested in this assay. One group of 4 mice received PBS as a negative control.

RNA ANALYSIS

[00275] Two weeks after treatment, mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. Primer probe set hSMN2vd#4_LTS00216_MGB (forward sequence: GCTGATGCTTTGGGAAGTATGTTA (SEQ ID NO: 11); reverse sequence CACCTTCCTTCTTTTGATTTTGTC, designated herein as SEQ ID NO: 12; probe sequence TACATGAGTGGCTATCATACT (SEQ ID NO: 13)) was used to determine the amount of SMN2 RNA that includes exon 7 (exon 7+). 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 that excludes exon 7 (exon 7-). Total SMN2 RNA levels were measured using 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)).

[00276] Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. Each of Tables 12-18 represents a different experiment. TABLE 12 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HOMOZYGOUS TRANSGENIC MICE TABLE 13 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HOMOZYGOUS TRANSGENIC MICE TABLE 14 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HOMOZYGOUS TRANSGENIC MICE TABLE 15 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE TABLE 16 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE TABLE 17 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE TABLE 18 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE

EXAMPLE 3: ACTIVITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO HUMAN SMN2 IN TRANSGENIC MICE, SINGLE DOSE (15 μG)

[00277] Activity of selected modified oligonucleotides described above was tested in human SMN2 transgenic mice essentially as described above in Example 2. Comparator Compounds #396443 and 819735 were also tested in this assay. Transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of 15 μg of modified oligonucleotide. One group of 4 mice received PBS as a negative control. Two weeks after treatment, mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. Each of Tables 19-23 represents a different experiment. TABLE 19 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HOMOZYGOUS TRANSGENIC MICE TABLE 20 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE TABLE 21 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE TABLE 22 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN MICE HETEROZYGOTIC TRANSGENICS TABLE 23 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE

EXAMPLE 4: ACTIVITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO HUMAN SMN2 IN TRANSGENIC MICE, SINGLE DOSE (70 μG)

[00278] Activity of modified oligonucleotides was tested in human SMN2 transgenic mice essentially as described above in Example 2. Transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of 70 μg of modified oligonucleotide. One group of 4 mice received PBS as a negative control. Two weeks after treatment, mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. TABLE 24 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HOMOZYGOUS TRANSGENIC MICE

EXAMPLE 5: ACTIVITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO HUMAN SMN2 IN TRANSGENIC MICE, MULTIPLE DOSES

[00279] Activity of selected modified oligonucleotides described above was tested in human SMN2 transgenic mice essentially as described above in Example 2. Comparator Compound #396443 was also tested in this assay. Transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of modified oligonucleotide at multiple doses as indicated in the Tables below. One group of 4 mice received PBS as a negative control. Two weeks after treatment, mice were sacrificed and RNA was extracted from the coronal brain and spinal cord for real-time qPCR analysis of SMN2 RNA. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. ED50 for exon inclusion (exon 7+) was calculated in GraphPad Prism 7 using nonlinear regression, 4-parameter dose response curve [Y=Lower + (Upper-Lower)/(1+ (10AlogEC50 /X)AHillSlope)]. TABLE 25 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HOMOZYGOUS TRANSGENIC MICE

EXAMPLE 6: TOLERABILITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO SMN2 IN WILD-TYPE MICE, 3-HOUR STUDY

[00280] Modified oligonucleotides described above were tested in wild-type female C57/B16 mice to assess tolerability. Wild-type female C57/B16 mice each received a single ICV dose of 700 μg of modified oligonucleotide listed in the Tables below. Comparator Compound #396443 was also tested in this assay at a dose of 350 μg. Comparator Compound #387954, 396442, 443305, and 819735 were also tested in this assay at a dose of 700 μg. Each treatment group consisted of 4 mice. One group of 4 mice received PBS as a negative control for each experiment (identified in separate Tables below). At 3 hours post-injection, mice were evaluated according to seven different criteria. The criteria are (1) the mouse was radiant, alert, and responsive; (2) the mouse was standing or curled up without stimulation; (3) the mouse showed no movement without stimulation; (4) the mouse showed forward movement after being lifted; (5) the mouse showed no movement after being lifted; (6) the mouse responded by pinching its tail; (7) regular breathing. For each of the 7 criteria, a mouse received a subscore of 0 if it met the criteria and 1 if it did not (the Functional Observational Battery or FOB score). After all 7 criteria had been evaluated, the scores were summed and averaged within each treatment group. The results are presented in the Tables below. Each of Tables 26–49 represents a different experiment. TABLE 26 TOLERABILITY SCORES IN MICE AT A 350 μG DOSE TABLE 27 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 28 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 29 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 30 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 31 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 32 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 33 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 34 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 35 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 36 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 37 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 38 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 39 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 40 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 41 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 42 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 43 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 44 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 45 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 46 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 47 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 48 TOLERABILITY SCORES IN MICE AT 700 μG DOSE TABLE 49 TOLERABILITY SCORES IN MICE AT 700 μG DOSE

EXAMPLE 7: TOLERABILITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO HUMAN SMN2 IN RATS, LONG-TERM EVALUATION

[00281] In separate studies performed under the same conditions, selected modified oligonucleotides described above were tested in Sprague Dawley rats to evaluate long-term tolerability. Comparator Compounds #396442 and #819735 were also tested in this assay. Sprague Dawley rats each received a single intrathecally (IT) delivered dose of 3 mg oligonucleotide or PBS. Beginning 1 week after treatment, each animal was weighed and evaluated weekly by a trained observer for adverse events. Adverse events were defined as neurological dysfunction not typical of PBS-treated control animals, which included, but were not limited to: abnormal limb extension, abnormal gait, tremors, abnormal breathing, paralysis, and spasticity. Adverse event onset is defined as the week after dosing when the dysfunction was first recorded. If no adverse events were achieved, there is no onset (-). The onset of adverse events typically correlates with a failure to thrive as defined by a lack of body weight gain/maintenance similar to PBS-treated animals. Similar tolerability assessments were 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.

[00282] At the end of the study, rats were sacrificed and tissues were collected. Histopathology was performed on cerebellar sections using calbindin stain. Purkinje cell loss was observed in calbindin-stained cerebellar sections as indicated in the Table below. Cerebellum and spinal cord were also evaluated using an antibody specific for modified oligonucleotides. Animals that did not demonstrate oligonucleotide uptake were excluded from histopathology analysis. Histology was not completed for animals that were sacrificed early due to adverse events. Additionally, cortical GFAP, a marker of astrogliosis (Abdelhak, et al., Scientific Reports, 2018, 8, 14798), was measured using RT-PCR and mean elevations >2-fold are noted below. TABLE 50 LONG-TERM TOLERABILITY IN RATS AT 3 MG DOSE

EXAMPLE 8: TOLERABILITY AND PHARMACOKINETICS OF MODIFIED OLIGONUCLEOTIDES IN NON-HUMAN PRIMATES, SINGLE OR REPEATED DOSING

[00283] Cynomolgus monkeys are treated with modified oligonucleotides to determine the site and systemic tolerability and pharmacokinetics of the modified oligonucleotides. Each group receives artificial CSF or modified oligonucleotide as a single intrathecal (IT) lumbar bolus dose injection, or, for repeated dosing groups, an IT bolus dose on day 1 of the study, followed by IT bolus doses at later time points. Tissues are collected 1 week after the final injection.

[00284] In a single-dose study, monkeys are administered a single dose of modified oligonucleotide and tolerability is assessed. Representative doses for single-dose studies in adult cynomolgus monkeys include 1 mg, 3 mg, 7 mg, and 35 mg.

[00285] In a repeat-dose study, monkeys are administered an IT bolus dose on day 1 of the study, followed by IT bolus dosing weekly (e.g., days 8, 15, and 22 for a four-week study) or monthly (e.g., days 29, 57, and 84 for a 13-week study). Representative doses for repeat-dose studies in adult cynomolgus studies include 1 mg, 3 mg, 7 mg, and 35 mg.

[00286] Tolerability assessment is based on clinical observations, body weights, feed 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 anatomic pathology assessments. Complete necropsies are performed with a recording of any gross abnormalities. Organ weights are obtained and microscopic examinations are conducted. Blood is collected for supplemental analysis. Additionally, blood, CSF and tissues (at necropsy) are collected for toxicokinetic assessments.

[00287] Tolerability of modified oligonucleotides is analyzed in brain and spinal cord tissue by measuring Aif1 and Gfap levels in cynomolgus monkeys treated with the modified oligonucleotide or the control. Brain and spinal cord samples are collected and snap-frozen in liquid nitrogen and stored frozen (-60°C to -90°C). At the time of sampling, 2 mm biopsy punches are used to collect frozen tissue samples for RNA analysis. Punches are obtained from multiple regions of the brain and spinal cord.

EXAMPLE 9: PHASE IA HUMAN CLINICAL TRIAL WITH COMPOUND NO. 1263789, 1287717, 1287745 OR 1358996

[00288] Safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of modified oligonucleotide complementary to human SMN2 are evaluated in a clinical trial setting. Single and/or multiple doses of modified oligonucleotide are evaluated in patients with confirmed MSA, such as MSA Type I, MSA Type II, MSA Type III, or MSA Type IV.

[00289] Patient safety is closely monitored throughout the study. Safety and tolerability assessments include: physical examination and standard neurological assessment (which includes fundi), vital signs (HR, BP, orthostatic changes, weight), ECG, concurrent AEs and medications, Columbia-Suicide Severity Rating Scale (C-SSRS), CSF safety labs (cell counts, protein, glucose), plasma lab tests (clinical chemistry, hematology), and urinalysis.

[00290] Efficacy assessments that are age- and Type-appropriate are selected and include, for example, the Hammersmith Motor Functioning Scale—Expanded (HFMSE), which is a reliable and validated tool used to assess motor function in children with AMS; the Pediatric Quality of Life Inventory (PedsQL™) Measurement 4.0 Genetic Core Scale; the Pediatric Quality of Life Inventory 3.0 Neuromuscular Modules; the Composite Muscle Action Potential (CMAP); the Motor Unit Number Estimate (MUNE); the Upper Limb Module (ULM); and the 6-Minute Walk Test (6MWT) (Darras, et al., Neurology, 2019, 92: e2492- e2506).

EXAMPLE 10: DESIGN OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO A HUMAN SMN2 NUCLEIC ACID

[00291] Modified oligonucleotides complementary to a human SMN2 nucleic acid were designed and synthesized as indicated in the Tables below.

[00292] Each modified oligonucleotide listed in the tables below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to 19967777). "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence.

[00293] The modified oligonucleotides in the table below are 18 nucleosides in length. Each nucleoside comprises either a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif column, where each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is either a phosphorothioate internucleoside linkage, a phosphodiester internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, or a mesylphosphoramidate (MsP) internucleoside linkage. The internucleoside bonding motif for each modified oligonucleotide is provided in the Internucleoside Bonding Motif column, where each 's' represents a phosphorothioate internucleoside bond, each 'o' represents a phosphodiester internucleoside bond, each 'x' represents a methoxypropylphosphonate internucleoside bond, and each 'z' represents a mesylphosphoramidate (MsP) internucleoside bond. Each cytosine is a 5-methylcytosine. Modified oligonucleotide 449320 was previously described in WO2015/161170 A2. TABLE 51 MOE AND NMA MODIFIED OLIGONUCLEOTIDES WITH PO/PS, PO/MSP, UNIFORM MSP, OR MIXED PS/MOP INTERNUCLEOSIDIC BONDAGES

[00294] The modified oligonucleotides in the Table below all consist of the sequence (from 5' to 3'): TCACTTTCATAATGCTGG (SEQ ID NO: 23). Each modified oligonucleotide listed in the Tables below is 100% complementary to SEQ ID NO: 1 (described herein above). "Start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence.

[00295] The modified oligonucleotides in the table below are 18 nucleosides in length. Each nucleoside comprises either a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif for each modified oligonucleotide is provided in the Sugar Motif column, where each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is either a phosphorothioate internucleoside linkage, a phosphodiester internucleoside linkage, or a mesylphosphoramidate (MsP) internucleoside linkage. The internucleoside binding motif for each modified oligonucleotide is given in the Internucleoside Binding Motif column, where each 's' represents a phosphorothioate internucleoside bond, each 'o' represents a phosphodiester internucleoside bond, and each 'z' represents a mesylphosphoramidate (MsP) internucleoside bond. Each cytosine is a 5-methylcytosine. The modified oligonucleotides in the table below are conjugated at a 6-palmitamidohexyl phosphate conjugate group attached to the 5'-OH of the oligonucleotide. The structure for the conjugate group . TABLE 52 6-PALMITAMIDO-HEXYL CONJUGATED MOE AND NMA MODIFIED OLIGONUCLEOTIDES WITH MIXED UNIFORM PO/PS, PO/MSP OR MSP INTERNUCLEOSIDIC BONDS

[00296] The modified oligonucleotides in the table below all consist of the sequence (from 5' to 3'): TCACTTTCATAATGCTGG (SEQ ID NO: 23), with a start site of 27062 and a stop site of 27079 in SEQ ID NO: 1 (described above herein), wherein the "start site" indicates the 5'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence, and wherein the "stop site" indicates the 3'-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence.

[00297] The modified oligonucleotides in the table below are 18 nucleosides in length. The sugar and internucleoside linkage motifs for each modified oligonucleotide are provided in the Sequence and Chemical Notation column, where each subscript 'n' represents a sugar chemical moiety of 2'-NMA, each subscript '[DMA]' represents a chemical moiety of 2'-O-(N,N-dimethyl)acetamide, each subscript '[NEA]' represents a chemical moiety of 2'-O-(N-ethyl)acetamide, each subscript '[NPA]' represents a chemical moiety of 2'-O-(N-propyl)acetamide, each subscript '[NcPA]' represents a chemical moiety of 2'-O-(N-cyclopropyl)acetamide, each subscript '[McPA]' represents a chemical moiety of 2'-O-(N-cyclopropylmethyl)acetamide, and each subscript 's' represents an internucleoside linkage of phosphorothioate. Each cytosine is a 5-methylcytosine, where the superscript 'm' before the cytosine residue (mC) represents a 5-methylcytosine. The structures for each of the sugars represented in the table below are: TABLE 53 NMA AND NMA ANALOG MODIFIED OLIGONUCLEOTIDES WITH UNIFORM PS INTERNUCLEOSIDIC BONDS

EXAMPLE 11: ACTIVITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO HUMAN SMN2 IN TRANSGENIC MICE, SINGLE DOSE (35 μG)

[00298] Activity of selected modified oligonucleotides described above was tested in human SMN2 transgenic mice, essentially as described above in Example 2.

TREATMENT

[00299] Transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of modified oligonucleotide at doses as indicated in the Tables below. One group of 4 mice received PBS as a negative control. Two weeks after treatment, mice were sacrificed and RNA was extracted from the coronal brain and spinal cord for real-time qPCR analysis of SMN2 RNA. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. ED50 for exon inclusion (exon 7+) was calculated in GraphPad Prism 7 using nonlinear regression, 4-parameter dose response curve [Y=Lower + (Upper-Lower)/(1+ (10AlogEC50 /X)AHillSlope)].

RNA ANALYSIS

[00300] Two weeks after treatment, mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. Primer probe set hSMN2vd#4_LTS00216_MGB was used to determine the amount of SMN2 RNA that includes exon 7 (exon 7+). Primer probe set hSMN2_Sumner68_PPS50481 was used to determine the amount of SMN2 RNA that excludes exon 7 (exon 7-). Total SMN2 RNA levels were measured using primer probe set hSMN2_LTS00935. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. TABLE 54 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE TABLE 55 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE t indicates less than four samples available

EXAMPLE 12: ACTIVITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO HUMAN SMN2 IN TRANSGENIC MICE, SINGLE DOSE (15 μG)

[00301] Activity of selected modified oligonucleotides described above was tested in human SMN2 transgenic mice, essentially as described above in Example 2.

TREATMENT

[00302] Transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of modified oligonucleotide at doses as indicated in the Tables below. One group of 4 mice received PBS as a negative control. Two weeks after treatment, mice were sacrificed and RNA was extracted from the coronal brain and spinal cord for real-time qPCR analysis of SMN2 RNA. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. ED50 for exon inclusion (exon 7+) was calculated in GraphPad Prism 7 using nonlinear regression, 4-parameter dose response curve [Y=Lower + (Upper-Lower)/(1+ (10AlogEC50 /X)AHillSlope)].

RNA ANALYSIS

[00303] Two weeks after treatment, mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. Primer probe set hSMN2vd#4_LTS00216_MGB was used to determine the amount of SMN2 RNA that includes exon 7 (exon 7+). Primer probe set hSMN2_Sumner68_PPS50481 was used to determine the amount of SMN2 RNA that excludes exon 7 (exon 7-). Total SMN2 RNA levels were measured using primer probe set hSMN2_LTS00935. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. TABLE 56 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE

EXAMPLE 13: ACTIVITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO HUMAN SMN2 IN TRANSGENIC MICE, MULTIPLE DOSES

[00304] Activity of selected modified oligonucleotides described above was tested in human SMN2 transgenic mice, essentially as described above in Example 2.

TREATMENT

[00305] Transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of modified oligonucleotide at multiple doses as indicated in the Tables below. One group of 4 mice received PBS as a negative control. Two weeks after treatment, mice were sacrificed and RNA was extracted from the coronal brain and spinal cord for real-time qPCR analysis of SMN2 RNA. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. ED50 for exon inclusion (exon 7+) was calculated in GraphPad Prism 7 using nonlinear regression, 4-parameter dose response curve [Y=Lower + (Upper-Lower)/(1+ (10AlogEC50 /X)AHillSlope)].

RNA ANALYSIS

[00306] Two weeks after treatment, mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. Primer probe set hSMN2vd#4_LTS00216_MGB was used to determine the amount of SMN2 RNA that includes exon 7 (exon 7+). Primer probe set hSMN2_Sumner68_PPS50481 was used to determine the amount of SMN2 RNA that excludes exon 7 (exon 7-). Total SMN2 RNA levels were measured using primer probe set hSMN2_LTS00935. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. TABLE 57 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE TABLE 58 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOTIC TRANSGENIC MICE † indicates fewer than four samples available TABLE 59 EFFECT OF MODIFIED OLIGONUCLEOTIDES ON HUMAN SMN2 RNA SPLICING IN HETEROZYGOUS TRANSGENIC MICE

EXAMPLE 15: TOLERABILITY OF MODIFIED OLIGONUCLEOTIDES COMPLEMENTARY TO SMN2 IN WILD-TYPE MICE

[00307] Modified oligonucleotides described above were tested in wild-type female C57/Bl6 mice to assess tolerability of the oligonucleotides. Wild-type female C57/Bl6 mice each received a single ICV dose of 700 μg of modified oligonucleotide listed in the Table below. Each treatment group consisted of 4 mice. One group of 4 mice received PBS as a negative control for each experiment (identified in separate Tables below). At 3 hours post-injection, mice were evaluated according to seven different criteria. The criteria are (1) the mouse was radiant, alert, and responsive; (2) the mouse was standing or hunched over without stimulation; (3) the mouse showed no movement without stimulation; (4) the mouse showed forward movement after being lifted; (5) the mouse showed no movement after being lifted; (6) the mouse responded to tail pinching; (7) Regular breathing. For each of the 7 criteria, a mouse received a subscore of 0 if it met the criteria and 1 if it did not (the Functional Observational Battery or FOB score). After all 7 criteria had been assessed, the scores were summed for each mouse and averaged within each treatment group. The results are presented in the Tables below. TABLE 60 TOLERABILITY SCORES IN MICE AT A 700 μG DOSE

Claims (32)

1. Modified oligonucleotide, characterized by the fact that it conforms to the following chemical structure: (SEQ ID NO: 21) or a salt thereof. 2. Modified oligonucleotide, according to claim 1, characterized by the fact that it is the sodium salt or the potassium salt. 3. Modified oligonucleotide, characterized by the fact that it conforms to the following chemical structure: (SEQ ID NO: 21). 4. An oligomeric compound comprising an oligonucleotide modified 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 = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, n = a 2'-NMA sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage. 5. A population of modified oligonucleotides as defined in claim 1, wherein all phosphorothioate internucleoside bonds of the modified oligonucleotide are stereorandom. 6. A population of modified oligonucleotides as defined in claim 3, wherein all phosphorothioate internucleoside bonds of the modified oligonucleotide are stereorandom. 7. A population of oligomeric compounds as defined in claim 4, wherein all phosphorothioate internucleoside bonds of the modified oligonucleotide are stereorandom. 8. Pharmaceutical composition, characterized by the fact that it comprises the modified oligonucleotide, as defined in claim 1, and a pharmaceutically acceptable diluent. 9. Pharmaceutical composition according to claim 8, characterized in that the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS. 10. Pharmaceutical composition according to claim 9, characterized in that it consists essentially of the modified oligonucleotide and artificial CSF (aCSF) or PBS. 11. Pharmaceutical composition, characterized by the fact that it comprises the modified oligonucleotide, as defined in claim 3, and a pharmaceutically acceptable diluent. 12. Pharmaceutical composition according to claim 11, characterized in that the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS. 13. Pharmaceutical composition according to claim 12, characterized in that it consists essentially of the modified oligonucleotide and artificial CSF (aCSF) or PBS. 14. Pharmaceutical composition, characterized by the fact that it comprises the modified oligonucleotide, as defined in claim 4, and a pharmaceutically acceptable diluent. 15. Pharmaceutical composition according to claim 14, characterized in that the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS. 16. Pharmaceutical composition according to claim 15, characterized in that it consists essentially of the modified oligonucleotide and artificial CSF (aCSF) or PBS. 17. Pharmaceutical composition, characterized by the fact that it comprises the population of modified oligonucleotides, as defined in claim 5, and a pharmaceutically acceptable diluent. 18. Pharmaceutical composition according to claim 17, characterized in that the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS. 19. Pharmaceutical composition according to claim 15, characterized in that it consists essentially of the modified oligonucleotide population and artificial CSF (aCSF) or PBS. 20. Pharmaceutical composition, characterized by the fact that it comprises the population of modified oligonucleotides, as defined in claim 6, and a pharmaceutically acceptable diluent. 21. Pharmaceutical composition according to claim 20, characterized in that the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS. 22. Pharmaceutical composition according to claim 21, characterized in that it consists essentially of the modified oligonucleotide population and artificial CSF (aCSF) or PBS. 23. Pharmaceutical composition, characterized by the fact that it comprises the population of oligomeric compounds, as defined in claim 7, and a pharmaceutically acceptable diluent. 24. Pharmaceutical composition according to claim 23, characterized in that the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS. 25. Pharmaceutical composition according to claim 24, characterized in that it consists essentially of the population of oligomeric compounds and artificial CSF (aCSF) or PBS. 26. Pharmaceutical composition, characterized by the fact that it comprises the modified oligonucleotide, as defined in claim 2, and a pharmaceutically acceptable diluent. 27. Pharmaceutical composition according to claim 26, characterized in that the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS. 28. Pharmaceutical composition according to claim 27, characterized in that it consists essentially of the modified oligonucleotide and artificial CSF (aCSF) or PBS. 29. A population of modified oligonucleotides as defined in claim 2, wherein all phosphorothioate internucleoside bonds of the modified oligonucleotide are stereorandom. 30. Pharmaceutical composition, characterized by the fact that it comprises the population of modified oligonucleotides, as defined in claim 29, and a pharmaceutically acceptable diluent. 31. Pharmaceutical composition according to claim 30, characterized in that the pharmaceutically acceptable diluent is artificial CSF (aCSF) or PBS. 32. Pharmaceutical composition according to claim 31, characterized in that it consists essentially of the modified oligonucleotide population and artificial CSF (aCSF) or PBS.